key: cord-269013-ge7nmgsa authors: vui, dam thi; tung, nguyen; inui, ken; slater, steven; nilubol, dachrit title: complete genome sequence of porcine epidemic diarrhea virus in vietnam date: 2014-08-14 journal: genome announc doi: 10.1128/genomea.00753-14 sha: doc_id: 269013 cord_uid: ge7nmgsa porcine epidemic diarrhea virus (pedv) has emerged in vietnam since 2009. herein, full-length genome sequences are reported for three pedv isolates from pigs displaying severe diarrhea from farms located in northern and southern provinces of vietnam. the results provide more understanding of the molecular characteristics of pedv in vietnam. stranded positive-sense rna virus in the genus alphacoronavirus, family coronaviridae, order nidovirales, is a causative agent of porcine epidemic diarrhea (ped) (1), a disease characterized by severe watery diarrhea with high mortality in young pigs (2) . ped is endemic in asia (3) (4) (5) (6) (7) (8) , has been reported in europe (2, 9) , and its recent emergence in north america suggests a threat to the swine industry worldwide (10) . ped has been observed in vietnam since 2009, but no vietnamese strains have previously been sequenced. intestinal samples were collected in vietnam in 2013 from 3-day-old pigs displaying severe watery diarrhea. three pedv variants, designated vn/kchy-310113, vap1113_1, and jfp1013_1, were isolated using the vero cell line (11) . two came from swine farms in southern vietnam and one came from the north. total rna was extracted from culture supernatant, and twelve overlapping regions of each genome were amplified, cloned in pgem-t easy vector (promega, usa), and sequenced in both directions in triplicate. the 5= terminal sequences were determined by 5= rapid amplification of cdna ends (race) (12) . all three vietnamese pedv isolates are 28,035 nucleotides (nt) in length, and are greater than 99.8% and 99.6% identical at the nucleotide and amino acid levels, respectively. genome organization resembles that of others pedv genomes previously reported a comparison to pedv sequences available in genbank demonstrated that the three new pedv isolates share high similarity (98.6% to 98.7% and 97.7% to 98.0% at nucleotide and amino acid levels, respectively) with more recent isolates from china (lc, zjcz4, chgd-01, ch/gdgz/2012, gd-1, gd-a, and aj1102) responsible for 2010 -2012 outbreaks (14) . all have unique characteristics including deletion and insertion in spike genes, which make them genetically distinct from cv777 and other earlier chinese isolates (8) . specifically, spike genes in the vietnamese strains have 2 insertions of 4 ( 56 genq 59 ) and 1 ( 140 n) amino acids at amino acid positions 55 to 60 and 140, and 1 deletion of 2 ( 160 dg 161 ) amino acids at amino acid positions 160 to 161. the insertions and deletions are located in the hypervariable domain in the n-terminus of the s1 region. this is the first report of full-length pedv genomes from vietnam. the full-length genome sequence suggests that pedv variants circulating in vietnam swine farms are novel variants with changes in the spike structure. the data provide valuable information on the molecular epidemiology of pedv in vietnam and will promote further investigation on genetic evolution and the selection of pedv variants for vaccines that can be used to successfully control ped in vietnam. nucleotide sequence accession numbers. the complete genome sequences of vn/kchy-310113, vap1113_1 and jfp1013_1 isolates have been deposited in genbank under accession numbers kj960178 through kj960180. completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence a new coronavirus-like particle associated with diarrhea in swine mutations in the spike gene of porcine epidemic diarrhea with growth adaptation in vitro and attenuation of virulence in vivo sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field strains in south china phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china genetic diversity of orf3 and spike genes of porcine epidemic diarrhea virus in thailand epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states propagation of the virus of porcine epidemic diarrhea in cell culture rapid amplification of 5' cdna ends porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines new variants of porcine epidemic diarrhea virus, china we are grateful to national research council of thailand for funding this research.this research has been supported by the ratchadaphiseksomphot endowment fund 2013 of chulalongkorn university (cu-56-527-hr). key: cord-001658-algzczs8 authors: theuns, sebastiaan; conceição-neto, nádia; christiaens, isaura; zeller, mark; desmarets, lowiese m. b.; roukaerts, inge d. m.; acar, delphine d.; heylen, elisabeth; matthijnssens, jelle; nauwynck, hans j. title: complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium, january 2015 date: 2015-05-21 journal: genome announc doi: 10.1128/genomea.00506-15 sha: doc_id: 1658 cord_uid: algzczs8 porcine epidemic diarrhea virus (pedv) is a member of the family coronaviridae and can cause severe outbreaks of diarrhea in piglets from different age groups. here, we report the complete genome sequence (28,028 nt) of a pedv strain isolated during a novel outbreak in belgium. diarrhea in europe from the 1970s to the 1990s, but has since been reported only sporadically in the last few decades (1, 2) . from 2010, severe outbreaks with mortality have been identified in asia, and since the spring of 2013, the virus was detected for the first time in u.s. swine herds (3) (4) (5) (6) (7) (8) (9) . genetically, these american isolates were highly related (99.5% nucleotide similarity) to asian pedv strains (8, 10) . in january 2014, a novel pedv variant strain oh851 was isolated in the united states, which was associated with milder disease symptoms (11) . similar strains contained several deletions and insertions in their spike genes and were therefore called indel strains (6) . in may 2014, an outbreak of diarrhea occurred in fattening pigs on a german farm, and the isolated strains were genetically almost identical to u.s. strain oh851 (12) . an outbreak of diarrhea in fattening pigs occurred on a belgian pig farm at the end of january 2015, and the presence of pedv rna in the feces of these pigs was demonstrated using an in house rt-qpcr assay targeting the nucleocapsid gene. this was the first confirmed pedv case in belgium in decades. therefore, the complete genome of this novel isolate, bel/15v010/2015, was unraveled by next-generation sequencing, in order to assess its genetic relation to other pedv isolates circulating around the globe. viral particles were purified, rna was extracted using the qiaamp viral rna mini kit (qiagen), and whole transcriptome amplification was performed (wta2 kit, sigma aldrich) to generate cdna. the sequencing library was prepared using the nextera xt dna library preparation kit (illumina). sequencing was performed on a hiseq 2500 platform (illumina) for 301 cycles (150-bp pairedend reads). raw reads were trimmed for quality and adapters using trimmomatic (13) , and reads were mapped using bwa against the german pedv strain l00721 (genbank lm645057) (14) . remaining gaps were closed using sanger sequencing. isolate 15v010 contained a genome with a size of 28,028 nucleotides (nt), excluding the poly-a tail. open reading frame 1 (orf1), encoding the replicase polyprotein, was located between nt 293 and 12601 and between nt 12601 and 20637, with a ribosomal frameshift between both parts. the other genes were arranged as follows: spike (s), nt 20634 to 24785; accessory membrane protein (mp, orf3), nt 24785 to 25459; envelope (e), nt 25440 and 25670); membrane (m), 25678 to 26358; and nucleocapsid (n), nt 26370 to 27695. at the whole-genome level, strain 15v010 was most closely related to strain l00721 (99.9% nucleotide similarity) isolated in germany in 2014, and the prototype u.s. indel strain oh851 (99.4%). strain 15v010 was less closely related to the former prototype european strain cv777 (97.0%). in conclusion, a pedv strain from a novel outbreak in belgium was characterized and showed high relatedness to recent german pedv isolates and u.s. pedv indel strains. our findings indicate that pedv is still or again circulating in europe. surveillance and development of prophylactic measures are urgently needed. nucleotide sequence accession number. the complete genome sequence of pedv bel/15v010/2015 has been deposited at genbank under the accession number kr003452. epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy a new coronavirus-like particle associated with diarrhea in swine complete genome sequence of a highly prevalent isolate of porcine epidemic diarrhea virus in south china phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china complete genome sequence of a novel porcine epidemic diarrhea virus in south china distinct characteristics and complex evolution of pedv strains pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs complete genome sequence of porcine epidemic diarrhea virus strain usa/colorado/2013 from the united states emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states new variant of porcine epidemic diarrhea virus comparison of porcine epidemic diarrhea viruses from germany and the united states trimmomatic: a flexible trimmer for illumina sequence data fast and accurate short read alignment with burrows-wheeler transform key: cord-001956-jpk854i3 authors: choe, se-eun; park, kee-hwan; lim, seong-in; le, van phan; hien, nguyen ba; thach, pham ngoc; phuong, le huynh thanh; an, byung-hyun; han, song hee; cho, in-soo; an, dong-jun title: complete genome sequence of a porcine epidemic diarrhea virus strain from vietnam, hua-14ped96, with a large genomic deletion date: 2016-02-18 journal: genome announc doi: 10.1128/genomea.00002-16 sha: doc_id: 1956 cord_uid: jpk854i3 a highly virulent strain of porcine epidemic diarrhea virus (pedv) causing severe diarrhea has recently emerged in vietnam. genomic sequences from a novel strain, hua-14ped96, isolated from a vietnamese piglet with serious diarrhea show relatively high identity with u.s.-like pedv strains, and have a 72-nt deletion in the open reading frame 1a (orf1a) gene. coronaviridae and is an enveloped, single stranded rna virus (1) . pedv was first reported in belgium and the united kingdom in 1971 (2) , and has since been identified in many european, asian, and north and south american countries (3) . two genetic types of pedv are also circulating in the united states; the highly virulent u.s. pedv strains and the mildly virulent indel pedv strains, the latter of which contain insertions and deletions in the n-terminal region of the spike protein (4, 5) . vietnamese pedvs detected in 2013 are closely related to chinese strains (6) , and of the two vietnamese pedv strains detected in northern and central vietnam in 2014, one lineage is closely related to chinese strains and the other to the mildly virulent indel pedv group of strains (7) . in this study, we sequenced the complete genome of a vietnamese strain of pedv, hua-14ped96, and analyzed it to understand the molecular characteristics and diversity of pedvs in vietnam. in october 2014, fecal samples were collected from piglets with watery diarrhea in hung yen province, vietnam. viral rna was extracted using the rneasy minikit (qiagen, germany) and cdna synthesized with the onestep reverse transcription-pcr kit (qiagen, germany). twenty sets of primers were designed (based on the usa/iowa/18984/2013 strain) to facilitate amplification of the full-length pedv genome. pcr products amplified using the primers were cloned into pgem-t easy vector (promega, usa) and sequenced using an abi prism 3730xi dna sequencer. multiple pedv sequences were aligned using the program bioedit 7.053 and maximum-likelihood estimation, using the general time reversible nucleotide substitution model, was performed using mega 6.0 software (8) . the length of the hua-14ped96 genome is 27,966 nucleotides (nt), excluding the 3= poly (a) tail, and is arranged as follows: the complete genome sequence of hua-14ped96 shows high nucleotide sequence identity (98.6%) with the highly virulent u.s. pedv strain (usa/iowa/18984/2013) and relatively lower identity (97.5%) with a vietnamese strain (vn/kchy-310113/2013). interestingly, sequence analyses of the hua-14ped96 genome demonstrated a 72-nt deletion in the orf1a gene, corresponding to a 24 amino acid deletion. phylogenetic analysis using the nucleotide sequences of the full-length genomes of pedvs revealed that hua-14ped96 belonged to the g2 group. the 72-nt deletion could cause a conformational change in the orf1a protein, resulting in altered antigenicity and immunogenicity of this pedv variant strain. further study is required to analyze additional genomic sequences and determine the biological properties of the novel strain, including pathogenicity, tissue tropism, and transmissibility. nucleotide sequence accession number. the complete genome sequence of pedv strain hua-14ped96 has been deposited in genbank under accession no. kt941120. sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhea virus confirms that this virus is a coronavirus related to human coronavirus 229e and porcine transmissible gastroenteritis virus a new coronavirus-like particle associated with diarrhea in swine molecular characterization of pig epidemic diarrhea viruses isolated in japan from distinct characteristics and complex evolution of pedv strains new variant of porcine epidemic diarrhea virus complete genome characterization of porcine epidemic diarrhea virus in vietnam a novel strain of porcine epidemic diarrhea virus in vietnamese pigs mega6: molecular evolutionary genetics analysis version 6.0 key: cord-014932-web2tdef authors: li, jian-qiang; liu, ji-xing; lan, xi; cheng, jie; wu, run; lou, zhong-zi; yin, xiang-ping; li, xue-rui; li, bao-yu; yang, bin; li, zhi-yong title: cloning the structure genes and expression the n gene of porcine epidemic diarrhea virus dx date: 2009-05-28 journal: virol sin doi: 10.1007/s12250-009-2982-y sha: doc_id: 14932 cord_uid: web2tdef the structure genes spike (s), nucleocapsid (n), membrane (m), small membrane (sm) of a porcine epidemic diarrhea virus (pedv) strain dx isolated in gansu province, north-west of china, were cloned, sequenced and compared with published sequences of pedv strains. the nucleotide sequences encoding the entire s, sm, m and n genes open reading frame (orf) of dx were 4 152, 231, 681 and 1 326 bases long respectively. there were transcription regulatory sequences (trss) upstream of the initiator atg of the s, n and m genes. the amino acids sequences of s, m and n contained 30, 3 and 7 potential asparagine (n)-linked glycosylation sites. homologous analysis and phylogenetic trees showed that dx had the closest relationship with strains ljb/06, js-2004-2z and ch/hljh/06 that were also isolated from china and indicated the prevalence of some pedv isolates in china were widespread since the js-2004-2z strain originated from the south of the china, and ljb/06 and ch/hljh/06 were isolated from northeast china. the n gene was cloned using two primers which contained nco i and bamh i restriction enzyme sites and subcloned into expression vector pet30a. the recombinant plasmid was then transformed into e.coli rossta. sds-page showed there was a protein of about 55kda as expected and western blot indicated the n protein had biological activity. several subgenomic mrnas for the production of structure and non-structure proteins (6, 18) . (1, 5) . the m and sm proteins are essential for viral envelope formation and release, the m protein also can stimulate the production of interferon (ifn) (1, 7) . the n protein participates in transcription of the viral genome, the formation of the viral core, and packaging of viral rna. in the early stage of pedv infection, the pig produces high levels of antibodies against the protein n. since the n protein is highly conservative in the coronaviruses, it has a good response and immunogenicity, so it is the best candidate protein for early diagnosis reagents and vaccine development. although some nucleotides sequences of pedv isolated from china has been reported, the data of dx structure genes are useful for furthering the study of the molecular biology of pedv strains that are prevalent in china, especially in the north-west. in this study, the structure genes have been cloned and the n protein has been expressed. the pedv dx strain was collected from the feces of piglets suffering from severe diarrhea in gansu north-west china. total rna was isolated from purified feces samples and extracted using a rna extraction kit (qiagene, germany) following the manufacturer's instructions. the rt-pcr amplifications were carried out using five primer sets (table 1) and an rt-pcr amplification kit (toyobo, japan). the products were ligated with the pmd18-t vector (takara) and transformed into the competent e. coli jm109. positive clones were sequenced by the takara biotechology (dalian) co.ltd. phylogenetic analysis was performed for the amino acids sequence of peev dx strain structure genes and compared to the pedv reference strains retrieved from genbank. the sequence data were aligned using two pcr primers of pedv n, which contain specific restriction enzyme digestion sites depending on the multiple cloning sites contained in the expression vector pet30a were used. the sense primer the positive recombinant transformant was grown in lb media containing 100μg/ml amp while shaking at 220 r/min at 37℃ and then induced with iptg. cells were harvested by centrifugation at 12000 r/min for 1 min. total cellular pellets were analyzed by 10% sds-page and western blotting. using rt-pcr, two overlapping products of the s gene of approximately 4.2 kb were amplified ( fig.1 to analyze the phylogenetic relationships between dx and other pedv strains isolated in various parts of the world, we constructed 3 neighbor-joining phylogenetic trees (fig.2, fig.3, fig.4) the recombinant plasmid pet30-pn was digested with ncol i and bamh i producing two fragments of 5 400bp (pet30a) and 1 400bp. pcr identification confirmed the presence of the 1 400bp fragment (fig.6) . to obtain the expressed recombinant protein, the time course expression of recombinant plasmid pet-pn was induced by iptg with ranging from 0.5-1 mmol/l at 2, 3, 4, 5 and 6 h respectively. sds-page revealed that the best condition was the iptg with 1 mmol/l at 6 h. the expressed proteins had a molecular weight of 55kda as expected (fig.7) . western blot showed the the n protein is a rna binding protein, it contains basic amino acids such as arginine and lysine and shows a high degree of alkalinity, it is also the only phosphorylated structure protein in the coronavirus. the serine residue is a potential phosphorylation site (2). thedx n protein contained 37 arginine, 33 lysine and 36 serine. the n protein is the most highly expressed protein in coronavirus infected cells and is about 45kda-60kda (12) . the pedv n protein is about 49kda-58kda in different expression systems (2). in this study, an n protein of about 50kda (the expressed vector was abou 5kda) was obtained using the e. coli system, the result is consistent with other e. coli expression system, although there are differences in the observed weights (16) . western blot results showed the expressed n protein reacted with the antibodies, which indicated that the expressed n protein has the biological activity. our results provide a basis for further development of a diagnosis method. the production of recombinant infectious di-particles of a sequence of the spike protein of the porcine epidemic diarrhea virus characterization of the structural proteins of porcine epidemic diarrhea virus, strain cv777 cloning and sequence analysis of the m gene of porcine epidemic diarrhea virus ljb/03 cloning and sequence analysis of the n gene of porcine epidemic diarrhea virus ljb/03 serologic study of the occurrence of epizootic viral diarrhea in swine in switzerland cloning and sequence analysis of the korean strain of spike gene of porcine epidemic diarrhea virus and expression of its neutralizing epitope in plants cloning and sequence analysis of the nucleocapsid gene of porcine epidemic diarrhea virus chinju99 the molecular biology of coronaviruses erhebungen uber porcine coronaviren in osterreich ii porcine epidemic diarrhea virus (pedv) der schweine complete genome sequence of transmissible gastroenteritis coronavirus pur46-mad clone and evolution of the purdue virus cluster isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages a novel internal open reading frame product expressed from a polycistronic of porcine edpidemic diarrhea virus may not contribute to virus attenuation disease of swine a cell culture vaccine against bovine ephemeral fever cloning and sequence analysis of the spike gene of porcine epidemic diarrhea virus chiju99 the nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein a1 in vitro and in vivo key: cord-299345-2i48ld8d authors: nefedeva, mariia; titov, ilya; malogolovkin, alexander title: molecular characteristics of a novel recombinant of porcine epidemic diarrhea virus date: 2019-02-06 journal: arch virol doi: 10.1007/s00705-019-04166-4 sha: doc_id: 299345 cord_uid: 2i48ld8d porcine epidemic diarrhea (ped) is a contagious viral disease in pigs, caused by the coronavirus porcine epidemic diarrhea virus (pedv). pedv infection results in significant mortality in piglets in unvaccinated herds. like many others rna viruses, pedv has high evolutionary rate and is prone to genetic mutations. in this study, we analyzed the complete genome sequence of the recently sequenced isolate pedv/belgorod/dom/2008. a recombination event in s gene of pedv/belgorod/dom/2008 was detected. pairwise identity analysis of the whole genome sequences revealed that pedv/belgorod/dom/2008 is an intermediate between pedv and transmissible gastroenteritis virus (tgev) strains. these results can be used for further analysis of the evolutionary variability, prevalence, and epidemiology of the porcine epidemic diarrhea virus. the spike (s) protein of pedv is the viral protein that is subjected to the greatest immunological pressure and variability. deletions (s-indels) or small insertions have been observed in the s gene nucleotide sequences of many pedv isolates [9] . the pedv strains that are currently circulating in the european union are similar to the american s-indel strains [10] [11] [12] . the phylogenetic classification of the pedv strains is based on the analysis of complete genomes sequences obtained worldwide [13] or individual genes such as s, m, n, or orf3 [9, 11, 14] . in this study, we analyzed the genome sequence of recently sequenced pedv isolate, pedv/belgorod/ dom/2008 (genbank accession number mf577027) [15] . pathological samples from the intestine and stomach were taken from one-month-old sick piglets from the belgorod region of russia in 2008 [15] . total rna was extracted from a 10% organ suspension using trizol reagent (ther-mofisher scientific) according to the manufacturer's instructions. next-generation sequencing was done using an illumina miseq instrument with a miseq reagent kit v3 in 2-× 300-bp pe mode (illumina, san diego, ca, usa) [15] . the pedv/belgorod/dom/2008 isolate was subsequently isolated from the small-intestine tissue in vero cell culture. prediction of homologous recombination events was carried out using rdp4 (recombination detection program) and simplot [16, 17] . pairwise identity analysis [19] . phylogenetic trees were constructed based on pedv m and s gene sequences using the maximum-likelihood method in mega 6.0. [20] . bootstrap values were estimated for 1000 replicates. the complete coding sequence of the pedv/belgorod/ dom/2008 is 28,315 nucleotides (nt) in length (genbank accession number mf577027) [15] . two putative recombination sites were detected in the genome of recombinant pedv/belgorod/dom/2008 at nt 20476 in orf1b and nt 24403 in the s gene ( fig. 1 ). pedv strain lzc (ef185992) and pedv strain slo/jh-11/2015 (ku297956) were identified as the major and minor parental viruses, respectively. the recombinant event was identified by six modules (rdp, maxchi, chimaera, geneconv, bootscan, siscan) with high confidence (average p-value, 2.77 × 10 −23 ). a similarity plot showed high overall sequence similarity between the pedv/belgorod/dom/2008 strain and the parental pedv strains, but with a marked drop in the nucleotide sequence similarity in the s gene region (fig. 1) . phylogenetic analysis of the complete genomes showed that pedv/belgorod/dom/2008 has a distant relationship to known pedv strains. the pedv/belgorod/dom/2008 isolate does not belong to any groups formed by the american or chinese strains and forms a separate cluster together with the secov-ita09 recombinant strain isolated in italy (fig. 2) . since only m gene sequences are available in the gen-bank database for the russian pedv isolates, we rebuilt the phylogenetic tree to refine the analysis. based on the phylogenetic analysis of the m gene, the pedv/belgorod/ dom/2008 isolate belongs to the same clade as other virulent russian pedv strains, indicating a high degree of sequence homogeneity in the m gene (fig. 3a) isolate is genetically distinct and does not belong to any group (fig. 3b ). this robust incongruence between the mand s-gene-based trees may be explained by a recombination event within the genome of the pedv/belgorod/dom/2008 isolate. such variability can lead to dramatic changes in viral virulence, pathogenicity, and antigenicity. pairwise identity analysis based on the spike amino acid sequences revealed that pedv/belgorod/dom/2008 is an intermediate between pedv and tgev and is also distantly related to other pedv strains (fig. 4) . pedv/belgorod/dom/2008 has a unique spike protein sequence and low similarity to other pedv isolates. changes in the s glycoprotein gene play an important role, since this protein is important for tissue tropism and virulence [5] . a preliminary animal study with pedv/belgorod/dom/2008 demonstrated that this recombinant is highly virulent in unvaccinated suckling piglets [21, 22] . recombination events are possible and can sometimes be observed in cases where pigs have been vaccinated or infected with a mixture of tgev and pedv. such recombination events can potentially result in a loss of vaccine efficacy. boniotti et al. reported a virus possessing a tgev genome sequence in which the s protein sequence was identical to that of a pedv isolate (secov-ita09) [23] . this chimeric virus was probably generated by recombination between tgev and pedv. similar chimeric viruses have been found by other research groups in germany [24] and eastern europe [25] . the genome sequence of one pedv isolate (ch/hnqx-3/14) from china shows that this strain appeared due to naturally occurring recombination of the attenuated strains cv777 and dr13 with the circulating field strain ch/ zmdzy/11. the recombination events occurred in the s, orf3, and n-structural protein-coding region and the replicase orf1a region [26] . the results of phylogenetic and recombination analysis revealed a discrepancy between the s gene sequence of pedv/belgorod/dom/2008 and the sequences of other isolates available in the genbank database. our results indicate that pedv/belgorod/dom/2008 is a new recombinant strain. interestingly, pedv/belgorod/dom/2008 and secov-ita09 (a recombinant strain from italy) form a unique phylogenetic group. pairwise identity analysis demonstrated that the amino acid sequence of the s gene of pedv/belgorod/dom/2008 is 60% identical to the s gene of other pedv strains and 50% identical to those of tgev strains. these data argue that pedv/belgorod/dom/2008 occupies an intermediate position between tgev and pedv. the identification of recombinant regions in pedv/belgorod/dom/2008 can be useful for further analysis of evolutionary variability, epidemiology, and development of a new diagnostic gene-based assay for porcine epidemic diarrhea virus. porcine epidemic diarrhea isolation and characterization of a variant porcine epidemic diarrhea virus in china nc_028806.1_secov_strain_italy/213306/2009 kr011756 kr003452.1_pedv_isolate_15v010/bel/2015 mf737355.1:pedv_isolate_kupe21_2001 kx791060.1:pedv_isolate_chsd2014 kc140102.1_pedv_strain_ch/fjzz-9 ef185992.1_pedv_strain_lzc_from_china_2006 kc109141.1_pedv_isolate_js2008 jq023162.1_pedv_strain_aʃenuated_dr13_2003 jq023161.1_pedv_strain_virulent_dr13_1999 mf577027.1_pedv_strain_pedv/belgorod/dom/2008 kc962433 kx499468.1_tgev_strain_tgev_ahhf_2015 dq201447.1_tgev_strain_ts_2006 mf737355.1:pedv_isolate_kupe21_2001 kx791060.1:pedv_isolate_chsd2014 kc140102.1_pedv_strain_ch/fjzz-9 mf782687.1_pedv_strain_nw8_2015 kp403954.1_pedv_strain_ukraine kj645707.1_pedv_strain_usa/minnesota af353511.1_pedv_strain_cv777_1978 ef185992.1_pedv_strain_lzc_from_china_2006 kc109141.1_pedv_isolate_js2008 jq023162.1_pedv_strain_aʃenuated_dr13_2003 jq023161.1_pedv_strain_virulent_dr13_1999 mf577027.1_pedv_strain_pedv/belgorod/dom kx499468.1_tgev_strain_tgev_ahhf_2015 dq201447.1_tgev_strain_ts_2006 new variants of porcine epidemic diarrhea virus, china emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china genomic and epidemiological characteristics provide new insights into the phylogeographical and spatiotemporal spread of porcine epidemic diarrhea virus in asia sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea detection of porcine epidemic diarrhea virus using polymerase chain reaction and comparison of the nucleocapsid protein genes among strains of the virus porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene new variant of porcine epidemic diarrhea virus comparison of porcine epidemic diarrhea viruses from germany and the united states genomic and evolutionary inferences between american and global strains of porcine epidemic diarrhea virus distinct characteristics and complex evolution of pedv strains complete genome sequence of a porcine epidemic diarrhea virus rdp4: detection and analysis of recombination patterns in virus genomes full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination sdt: a virus classification tool based on pairwise sequence alignment and identity calculation muscle: multiple sequence alignment with high accuracy and high throughput isolation and identification of porcine epidemic diarrhea virus in pigs under the outbreak at a large farm biological characteristics of an epizootic isolate bs-08 of porcine epidemic diarrhea virus porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus new chimeric porcine coronavirus in swine feces characterization of a novel chimeric swine enteric coronavirus from diseased pigs in central eastern europe in 2016 genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from henan, china acknowledgements we thank olga strizhakova for providing pedv/ belgorod/dom/2008. we also acknowledge yegor bazykin and alexey neverov for fruitful discussions of virus evolution and recombination analysis. we appreciate sandra blome and dennis hanke for pedv/ belgorod/dom/2008 complete genome sequencing. funding our work was supported by the ministry of science and higher education of the russian federation. the authors declare no competing interests. key: cord-258546-1tf5ggfo authors: chung, hee-chun; lee, jee-hoon; nguyen, van giap; huynh, thi my le; lee, ga-eun; moon, hyoung-joon; park, seong-jun; kim, hye-kwon; park, bong kyun title: new emergence pattern with variant porcine epidemic diarrhea viruses, south korea, 2012–2015 date: 2016-12-02 journal: virus res doi: 10.1016/j.virusres.2016.06.013 sha: doc_id: 258546 cord_uid: 1tf5ggfo since outbreaks of porcine epidemic diarrhea virus (pedv) in the united states in 2013, explosive outbreaks of ped in south korea have infected all age groups of pigs in 2014–2015 year. this study analyzed a large collection of the spike protein coding gene to infer the spatial-temporal diffusion history of pedv. the studying results suggested that pedvs in korea belonged to different genogroups. while classical g1 was continuingly circulating between provinces of korea, the pandemic g2a were recently introduced from china and usa. by the application of bayesian phylogeographical analysis, this study demonstrated the spatial-temporal transmission of pedvs within korea. of the recent emerged g2a viruses, j3142 strains showed potential recombination breakpoint (376–2,143nt) of s1 gene between knu1303_korea strain_g2a (kj451046) and 45rwvcf0712_thailand strain_g2b (kf724935). the pandemic g2a virus was partial neutralized by the antibodies invoked by the g1based ped vaccine virus. porcine epidemic diarrhea virus (pedv) is an acute contagious diarrhea disease caused in pigs (song and park, 2012; jung et al., 2014) . the virus belongs to genus alpha-coronavirus, family coronaviridae, which includes other genera; beta-, gamma-, and delta-coronavirus, and has positive-sense single stranded rna genome with envelope (woo et al., 2012) . pedv is transmitted mainly through the fecal-oral route, infecting all age groups of pigs but the most severe form of diseases occurs in suckling piglets (song and park, 2012) . in 2013, pedv was reported for the first time in north american and now pedvs are present in all of swine producing countries (song and park, 2012; huang et al., 2013 ; mole, 2013) . in korea, pedv was first reported in 1992 (kweon et al., 1993) , and continuingly circulated and exhibited significant genetic diversity * corresponding author. e-mail address: parkx026@snu.ac.kr (b.k. park). 1 these authors have contributed equally to this study. (choi et al., 2014; lee and lee, 2014) . ped outbreaks re-occurred in korea in 2013, however, it was demonstrated that the emerging pedvs were not variants of old korean isolates or attenuated vaccine strains (chung et al., 2015) . though a recent publication characterized the genetic evolution of pedvs in korea from 1998 to 2013, to our knowledge, the transmission patterns of the virus in korea are largely unknown. using a large dataset collected globally and locally, this study aimed to trace the spatial-temporal dynamics of pedvs in korea. the spike protein coding gene (s gene) was selected for genetic analysis. aimed at provide a detail about the evolution of pedv in korea, we obtained from the previous study 31 partial sequences of the s gene which were collected in years 2002-2005. additionally, this study sequenced complete s genes from pedv positive samples which were stored in our laboratory from october 2012 fig. 1 . bayesian time-scaled phylogeny of pedv with inferred geographical location states. the branches of maximum clade credibility tree were colored according to the most probable location state of their descendent nodes. the color codes are defined in the insert legend. the s gene-based phylogeny clearly divided pedvs into genogroup 1 (g1) and two subgroups of genogroup 2 (g2a, g2b). for clarity, only the leaves of korean pedvs were highlighted. it was obvious that pedvs circulating in korea were within g1. to march 2015. to infer the spatial-temporal diffusion history of pedv, sequences of the s gene which had known collection date and country of origin were achieved from genbank. overall, the final dataset contained 805 sequences originating from asia (china, korea, vietnam, thailand, japan, taiwan), europe (belgium, slovenia, france), and american (usa, canada, mexico, colombia), and covering a sampling period from 1986 to 2015. the details of the dataset are summarized in supplementary table s1. a bayesian framework (lemey et al., 2009 ) was applied to reconstruct the spatial-temporal diffusion history of pedvs. in brief, the spatial diffusion of the time-scaled genealogy is modeled as a standard continuous-time markov chain (ctmc) process over discrete sampling locations. a bayesian stochastic search variable selection (bssvs) approach, which allows the exchange rates in the ctmc to be zero with some prior probability, was used to find a parsimonious set of rates explaining the diffusions in the phylogeny. the analysis was performed using beast package v1.8.2 under assumptions of (i) a codon based srd06 nucleotide substitution model, (ii) a constant population size for the coalescent prior, and (iii) the molecular clock model of uncorrelated lognormal distribution. the analysis was run for 100 million chains, sampling every 10,000 generations. pedv 411 s complete gene sequences aligned were used by recombination detection program v.4.46 (tian et al., 2014 and vlasova et al., 2014) ; x-over automated rdp analysis was used to identify recombination points within the pedv genome. in the previous study, it was noted that mutation occurred at ss6 neutralizing epitope of the s gene of an isolate collected from 2013 to 2014 outbreak. this study tested whether pedv isolated recently was crossed neutralized by serum of pigs which were vaccinated with korean ped oral vaccine (attenuated dr13 strain, green cross veterinary product co., ltd., yong-in, korea). the serum neutralizing test (sn test) was conducted using the previous method (song et al., 2007) , with some modifications. in brief, sera of pigs (n = 25) were heat inactivated at 56 • c for 30 min and stored at −20 • c until use. the sera were serially two-fold diluted. subsequently either bm3 (a recent pedv isolate) or dr13 (ped vaccine strain) of 200 tcid 50 /0.1 ml were mixed at equal volume of the diluted sera. the mixtures were incubated for 1 h at 37 • c. after incubation, 0.1 ml of each virus-serum mixture was infected to monolayer of vero cells. the presence/absence of cpe were monitored daily for 5 days. the sn titers were expressed as the reciprocal of the highest serum dilution which resulted in the inhibition of cpe. the result of the bayesian phylogeographic analysis is shown in fig. 1 . it was obvious that pedvs are circulating in korea from the earliest to the latest sequences (years 1997-2015) , the virus were classified into genogroup 1 (g1) and genogroup 2 (subgroups 2a and 2b). for the korean pedvs of g1, the viruses were found spanning from 1997 until 2012. meanwhile, the viruses of g2 (g2b and g2a) were detected from 2008 until 2015. as shown in fig. 1 , it was clear that the root of korean g1 and g2b viruses were inferred to be originated from korea, while the recent korean pedvs were derived from china and usa. the result implied that the transmission histories of pedvs in korea were diverse, which reflecting both the continuingly circulation of classical g1 and the introduction of the recent pandemic g2a. the detail of spatial-temporal dynamics of pedvs between provinces of korea is shown in figs. 2-4 . among the korean g1 viruses, the result of fig. 2 suggested the dispersal of the virus between different provinces, of which gyeonggi was the main source in the viral migration network. among the korean g2b viruses (fig. 3) , another province (chungnam) was predicted to be the initial source for the spreading of virus into gyeongbuk, and then from gyeongbuk to jeonnam. the picture was totally different from the korean g2a viruses, of which the sources were predicted to be china and usa (fig. 4) . in order to detect potential recombinant strains of j3142 and bm3, we used recombination detection program with 411 references pedv strains in this study. it has not been found that bm3 has recombinant region. interestingly, two potential strains of j3142 had recombination which had breakpoint from 376nt (aligned; 391nt) to 2143nt (aligned; 2206nt). one had major patent similarity of 95.3% with knu1303 korea strain (kj451046) which belongs to the subgroup g2a, and the other had minor patent similarity of 96.4% with 45rwvcf0712 thailand strain (kf724935) which belongs to the subgroup g2b. in this study, j3142 had a variant s1 (duarte and laude, 1994; gallagher and buchmeier, 2001; lee et al., 2010) region, suggesting that the recombination between south korea and thailand strains had occurred. in fig. 5 , we used only 411 references for the recombination analysis; thus, it might be possible that in south korea, there were strains, which are similar to those of thailand such as 45rwvcf0712, leading to the recombinant event. fig. 3 . bayesian time-scaled phylogeny of sub-genogroup 2b of pedvs. the branches of maximum clade credibility tree were colored according to the most probable location state of their descendent nodes. for clarity, the annotations of leaves were omitted. shown in every internal nodes of the phylogeny were the inferred locations. it was observed that chungnam was predicted to be the initial source for the spreading of virus into several provinces. in the sn test (table 1) , 52% (13/25) of the bm3 strain were positive and 88% (22/25) of the dr13 strain were positive. the average titer of the dr13 (14.53 value) was 5 times more than that of the bm3 (2.9 value). the results implied that, the specific antibodies invoked by ped vaccine of g1 inefficiently conferred cross neutralizing activities to the pandemic g2a isolate. our result was inline with the previous report that suggests that limited cross-reactivity was detected between ped vaccine (of genogroup 1) and field viruses (of genogroup 2). the attenuated pedv vaccines based on the cv777 strains or dr13-derived strains might be antigenically less related to the newly emergent pedv strains; thus, the development of new vaccines based on current strains are needed (chen et al., 2014; chung et al., 2015; song and park, 2012; . in summary, this study suggested that pedvs in korea belonged to different genogroups. while classical g1 was continuingly circulating between provinces of korea, the pandemic g2a were recently introduced from china and usa. by the application of bayesian phylogeographical analysis, this study demonstrated the spatial-temporal transmission of pedvs within korea. of the recent emerged g2a viruses, j3142 strains showed potential recombination breakpoint (376-2,143nt) of s1 gene between knu1303 korea strain g2a (kj451046) and 45rwvcf0712 thailand strain g2b (kf724935). the pandemic g2a virus was partial neutralized by the antibodies invoked by the g1-based ped vaccine virus. isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states comparative genome analysis and molecular epidemiology of the reemerging porcine epidemic diarrhea virus strains isolated in korea isolation of porcine epidemic diarrhea virus during outbreaks in south korea sequence of the spike protein of the porcine epidemic diarrhoea virus coronavirus spike proteins in viral entry and pathogenesis origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs isolation of porcine epidemic diarrhea virus (pedv) in korea genetic characterization of porcine epidemic diarrhea virus in korea from outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea suchard. bayesian phylogeography finds its roots novel porcine epidemic diarrhea virus variant with large genomic deletion porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr13 strain molecular characterization and phylogenetic analysis of new variants of the porcine epidemic diarrhea virus in gansu evidence of recombinant strains of porcine epidemic diarrhea virus distinct characteristics and complex evolution of pedv strains discovery of seven novel mammalian and avian coronaviruses in deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus the authors would like to thank hye jung yang, and jung ah kim for excellent technical assistance. this study was supported by the biogreen 21 program, rural development administration (grant no. pj011184), and by the bio-industry technology development program (grant no. 114055031sb010), ministry of agriculture, food and rural affairs, south korea. the authors declare that there are no conflicts of interest. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2016.06. 013. key: cord-016858-pbjj50bx authors: jung, kwonil title: immunohistochemical staining for detection of porcine epidemic diarrhea virus in tissues date: 2015-09-10 journal: animal coronaviruses doi: 10.1007/978-1-4939-3414-0_2 sha: doc_id: 16858 cord_uid: pbjj50bx porcine epidemic diarrhea virus (pedv), a member of the genus alphacoronavirus, has resulted in significant economic losses in the european, asian, and north american swine industries in previous years. pedv infection causes acute diarrhea/vomiting, dehydration, and high morbidity and mortality in seronegative neonatal piglets. in this chapter, materials and methods for performing immunohistochemistry (ihc) for the detection of pedv antigens in frozen or formalin-fixed, paraffin-embedded (ffpe) tissues are provided. in ihc of frozen tissues where viral antigens are well preserved, the use of specific antibodies labeled with fluorescence dyes provides excellent advantages and convenience, resulting in high sensitivity and specificity of ihc and reduction of operation time. in ihc of ffpe tissues where tissue or cell morphology is well preserved, the use of specific antibodies labeled with enzymes, such as alkaline phosphatase, also gives rise to significant advantages in defining the correlation of viral antigens with histopathologic lesions. pedv antigens in frozen tissues are visualized as green staining in the cytoplasm of infected cells by fluorescent dyes conjugated with antibodies when activated by exciting light of a specific wavelength under a fluorescence microscope. in ffpe tissues, pedv antigens are visualized as red staining in the cytoplasm of infected cells by the deposition of the substrate chromogen, fast red. 2013 to present, has led to a substantial loss of piglets (more than 10 % of us swine population). because of similar clinical and pathogenic features between pedv and another alphacoronavirus , transmissible gastroenteritis virus (tgev), or a deltacoronavirus , porcine deltacoronavirus (pdcov), differential laboratory tests are required for their diagnosis [ 3 -5 ] . reverse transcriptionpolymerase chain reaction ( rt-pcr ) or quantitative rt-pcr (qrt-pcr) is useful for the rapid differential diagnosis; however, detection of viral antigens in tissues is essential for confi rming each viral infection . viral antigens in frozen, fi xed cells, or tissues can be detected by immunohistochemistry (ihc) (or immunohistochemical staining) using specifi c antibodies labeled with fl uorescent dyes, such as alexa fluor ® 488 and fl uorescein isothiocyanate (fitc), or enzymes, such as alkaline phosphatase (ap) and peroxidase. immunofl uorescence (if) staining is the fi rst immunohistochemical staining method but is still widely used in veterinary and medical diagnosis . with fundamentality of antigen -antibody and antibody-antibody binding reactions, antigens are visualized by fl uorescent dyes conjugated with antibodies when activated by an exciting light of a specifi c wavelength (499-519 nm for alexa fluor ® 488 and 494-521 nm for fitc), under a fl uorescence microscope. due to the high sensitivity, specifi city, and convenience in using alexa fluor ® 488 in frozen tissues, this chapter details an if staining method for the rapid and precise detection of pedv antigens in pedv-infected, frozen tissues, contributing to verifi cation of the tissue sites of pedv replication in infected pigs. a combination of pedv-specifi c anti-sera as antigen detection antibody and secondary antibody conjugated with alexa fluor ® 488 is applied in the if staining method. however, because of suboptimal conditions of tissue or cell morphology in frozen tissues, this staining method limits investigation of the correlation of pedv antigens with histopathologic lesions. to compensate for the limitation of if staining in frozen tissues, additional ihc staining method using enzyme-labeled antibodies, i.e., immunoenzymological staining, in formalin-fi xed, paraffi n-embedded (ffpe) tissues is also provided in this chapter. after adding a substrate of enzyme, such as fast red, it generates insoluble particles that can be localized in cells or tissues under light microscope. compared to the if staining in frozen tissues, the ihc in ffpe tissues has more accurate localization of antigens with a better contrast ratio, contributing to defi ning the correlation of pedv antigen -positive cells with severity of histopathologic lesions caused by pedv, such as intestinal villous atrophy. only one serotype of pedv has been reported from different countries [ 4 ] . there has been no evidence of cross-reactivity of pedv with tgev [ 6 , 7 ] . the use of hyperimmune anti-sera or polyclonal antibodies against pedv in ihc staining is likely able to detect geographically different strains of pedv and differentiate them from tgev in tissues [ 8 , 9 ] , but with a potential disadvantage of inducing background or false signals. to improve the sensitivity and specifi city of ihc for the detection of pedv antigens in tissues, the use of monoclonal antibodies to structural proteins of pedv, such as spike (s) or membrane (m) protein, has been preferred [ 10 , 11 ] . prior to their application on the tissues, potential cross-reactivity of monoclonal antibodies to tgev and pdcov can be tested by more sensitive assays, such as enzyme-linked immunosorbent assay, immunoblotting, and immunoprecipitation, compared to ihc. tissue tropism of pedv is related to the expression of aminopeptidase n (apn), a 150 kda glycosylated transmembrane protein identifi ed as the cellular receptor , on porcine small intestinal villous enterocytes [ 12 ] . pedv-infected enterocytes rapidly undergo acute necrosis, leading to marked villous atrophy in the small but not large intestine [ 10 ] . in pedv-infected nursing pigs, major histologic lesions include acute diffuse, severe atrophic enteritis , and mild vacuolation of superfi cial epithelial cells and subepithelial edema in cecum and colon [ 8 -10 , 13 ] . pedv antigens are observed mainly in villous enterocytes of the small (duodenum to ileum) and large intestines (except rectum) [ 1 , 8 , 10 ] . occasionally, a few pedv-positive cells were detected in the intestinal crypt cells or the peyer's patches [ 1 , 8 -10 ] . no lesions were seen in the spleen, liver, lung, kidney, and mesenteric lymph node of orally and/or intranasally infected piglets [ 8 ] . lung tissue of oronasally infected pigs was negative for pedv antigen [ 1 , 8 -10 ] . pedv antigens were not detected in other major organs, such as the pylorus, tonsils, liver, and kidneys. however, a recent study reported the replication of pedv in swine pulmonary macrophages in vitro and in vivo [ 14 ] . epidemic pedv strains are highly enteropathogenic and acutely infect villous epithelial cells of the entire small and large intestines, but the jejunum and ileum are the primary sites of infection . to detect pedv antigen in tissues and evaluate the pathogenicity of pedv strains in pigs, the jejunum and ileum are the most critical tissue samples for performing ihc. 2. place the slides in the rack into xylene for 20 min at rt to remove the paraffi n. 3. place the slides in 100 % ethanol for 5 min to rehydrate tissues through a graded ethanol series (100-50 %; the following steps only) (200-300 μl) and incubate in a humidifi ed chamber at 37 °c for 1 h ( see note 3 ). 10 . rinse the slides gently with pbs on a rocker platform shaker at rt for 5 min. repeat through three changes of fresh pbs, 5 min for each. 11 . gently blot the slides with kimwipes around the tissue and place horizontally. 12. apply 3-5 drops of prolong ® gold antifade mountant (with dapi) to the tissue section using a glass dropping pipette and immediately put a cover slip on ( see note 4 ). 13. the slides are ready to be evaluated under fl uorescence microscope. they need to be stored in a dark area until evaluation. pedv antigens will appear to be green or as fl uorescent staining in the cytoplasm of infected cells (fig. 1 ) . cell nuclei are stained blue with dapi. 1. when antigen retrieval procedure is completed (sect. 3.3 ), drop 300-500 μl of 1× universal blocking reagent on the tissue within the hydrophobic barrier and incubate at 37 °c for 30 min. 2. drain the slides and place them on a horizontal surface. 8. drain the slides and place them on a horizontal surface. 9. apply the fast red solution enough to cover the tissue section (300-500 μl) and incubate in a humidifi ed chamber at rt for 30-60 min ( see note 6 ). 10. place the slides in a rack and rinse well in distilled water. three changes, 2 min each. 11. tissue sections are counterstained in a glass dish with gill's hematoxylin at rt for 10 min. 12. rinse the slides thoroughly in tap water for 5 min, and move into deionized water. 13. drain the slides and place them on a horizontal surface. 14. apply 2-4 drops of permanent aqueous mounting medium to the tissue section ( see note 7 ), and immediately put a cover slip on ( see note 8 ). 15. the slides are ready to be evaluated under light microscope. pedv antigens will appear as a red precipitate in the cytoplasm of infected cells (fig. 2 ) . cell nuclei are stained blue with hematoxylin. 1. use of fresh reagents is recommended. a large amount of washing buffer, 1× pbs, is needed, because complete washing is critical to reduce background and increase true signals. 2. throughout immunostaining procedures, the tissues should stay rehydrated. adequate antigen -antibody or antibody-antibody binding reaction is not expected in dried tissues, resulting in poor or weak staining results or a high level of background staining. it is also critical for a comparative immunostaining study in multiple different tissues. 3. the optimal dilutions of primary and secondary antibodies should be tested and selected in both frozen and ffpe tissue conditions. 4. instead of plastic pipette tips, the use of glass dropping pipette will reduce the number of bubbles in the mounting medium as applied to the tissue sections. 5. when the fast red tablet is completely dissolved, the solution can be fi ltered via 0.9 μm syringe fi lter and used to reduce an irregular deposition of fast red on the tissues or background. 6. the color development, including intensity of true or false signals, in all tissue slides tested should be frequently monitored under the microscope. wipe the non-charged slide surface with kimwipes before putting the tissue slides on the microscope. 7. gently drop the mounting medium so as not to create bubbles. the mounted slides need to be evaluated as soon as possible, because bubbles can be created spontaneously in the mounted medium. detection of pedv antigens ( red staining) in the cytoplasm of enterocytes lining atrophied villi by immunohistochemical staining in formalin-fi xed, paraffi nembedded jejunal tissues using a monoclonal antibody specifi c for the spike protein of pedv and secondary antibody conjugated with alkaline phosphatase. original magnifi cation ×200. immunohistochemistry. fast red. gill's hematoxylin counterstaining 8. to make the stained slides permanent, a large amount of mounting medium can be applied to the tissues so that the entire section is covered. place slides horizontally in a 60 °c oven for 30 min to allow the medium to harden. remove the slides from the oven, and allow them to cool at rt. dip the slides in xylene and cover slip with permount permanent mounting medium (fisher scientifi c). 10% (w/v) sucrose solution antifade mountant with 4′,6-diamidino-2-phenylindole, dihydrochloride (dapi) (invitrogen) 1× phosphate-buffered saline (pbs) (ph 7.4) 1× pbs (ph 7.4) containing tween 20, 0.1 % 100, 95, 70, and 50 % ethanol % (v/v) glacial acetic acid in deionized water 1× pbs 1× pbs (ph 7.4) containing tween 20, 0.1 % m tris buffer gill's or mayer's hematoxylin ultramount permanent aqueous mounting medium (dako) glass slide-staining dishes or jars. 2. slide racks and trays adjustable pipettors with tips barrier dako pen (dako) microscope slides: superfrost™ plus gold slides glass cover slip (fisher scientifi c) emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences fighting a deadly pig disease porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis diseases of swine pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (pedv) an immunoelectron microscopic and immunofl uorescent study on the antigenic relationship between the coronavirus-like agent, cv 777, and several coronaviruses the pathogenesis of an enteric infection in pigs, experimentally induced by the coronavirus-like agent, cv-777 an immunohistochemical investigation of porcine epidemic diarrhoea pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs monoclonal antibody-based immunohistochemical detection of porcine epidemic diarrhea virus antigen in formalin-fi xed, paraffi n-embedded intestinal tissues porcine aminopeptidase n is a functional receptor for the pedv coronavirus pathology of experimental cv777 coronavirus enteritis in piglets. i. histological and histochemical study porcine epidemic diarrhea virus infects and replicates in porcine alveolar macrophages salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development center, the ohio state university. this work was supported by a grant from the oardc seeds, grant # oaoh1536. key: cord-279903-z0wf1wli authors: wang, leyi; zhang, yan; byrum, beverly title: development and evaluation of a duplex real-time rt-pcr for detection and differentiation of virulent and variant strains of porcine epidemic diarrhea viruses from the united states date: 2014-07-12 journal: j virol methods doi: 10.1016/j.jviromet.2014.07.005 sha: doc_id: 279903 cord_uid: z0wf1wli porcine epidemic diarrhea virus (pedv) has caused significant economic losses in the us swine industry since may 2013. a new variant strain of pedv emerged in the us in the late december, 2013. this variant strain of pedv differs from the virulent strain of pedv currently circulating in the us in 1170 nt of the 5’end of the s1 domain in the spike gene. importantly, the variant pedv caused significantly less mortality in piglets than the virulent pedv, based on clinical observations. this suggests it may be a potential vaccine candidate for ped. variant pedv has been detected in samples from multiple states by our laboratory as well as other laboratories in the us. it is critical to detect and differentiate variant pedv from the virulent pedv during outbreaks to enhance control and to prevent ped associated disease. in this study, the development and validation of a duplex real-time rt-pcr assay for detection and differentiation of the variant and the virulent strains of pedv currently circulating in the us was reported. porcine epidemic diarrhea (ped) virus is a member of the order nidovirales, family coronaviridae, subfamily coronavirinae, genus alphacoronavirus. ped is a highly contagious diarrheal disease, characterized by severe watery diarrhea and high mortality in piglets. ped was originally identified in england in 1971 (oldham, 1972) . since then, it has been reported in several european and asian countries including china and korea. since 2010, a highly virulent strain of pedv emerged in china and caused significant loss in the pig industry (sun et al., 2012) . in may 2013, this virulent strain of pedv was recognized in the united states (us). by march 8 of 2014, ped had been detected in 27 us states and a total 4458 cases were confirmed (http://www.aasv.org/pedv/pedv weekly report 140312.pdf). the disease has caused severe economic losses to the swine industry in the us. recently the virus was found in canada. studies have shown that pigs either naturally or experimentally infected with virulent strain of pedv developed characteristic gross (thin and dilated intestinal walls) and histologic lesions (severe atrophy of villi) (stevenson et al., 2013; jung et al., 2014) . complete genomic analysis of the virulent pedv strains from the us showed * corresponding author. tel.: +1 6147286220; fax: +1 6147286310. e-mail address: yzhang@agri.ohio.gov (y. zhang). that they cluster in a single clade and are closely related with the ah2012 strain reported in china (stevenson et al., 2013; huang et al., 2013) . recently, we reported a new variant pedv detected in ohio . clinical observation indicated that this new variant virus (oh 851) caused mild clinical disease with low mortality in newborn piglets (unpublished data), making it a potential vaccine candidate. strain oh 851 is distinct from the virulent strains of pedv in the us and is most closely related to ch/hbqx/10 reported in central china (zheng et al., 2013) , based on the phylogenetic analysis of the full-length spike gene . further analysis showed that the strain oh851 differs from the virulent strains of pedv in the first 1170 nucleotides (nt) of spike gene, indicating that at least two genotypes of pedv are circulating in the us pigs (stevenson et al., 2013; huang et al., 2013; wang et al., 2014) . since effective vaccines are not currently available in north america, accurate diagnosis combined with biosecurity is the only reliable method for control and prevention of ped. electron microscopy was widely used in the diagnosis of the initial outbreaks of pedv (pospischil et al., 1981) . since then, several methods have been developed for laboratory diagnosis of pedv, including the direct immunofluorescence test for detection of pedv antigen (guscetti et al., 1998) , enzyme-linked immunosorbant assays (elisa) for detection of either pedv antigen or antibodies (carvajal et al., 1995) , and reverse transcription-polymerase chain reaction (rt-pcr) (kwon et al., 1997; ishikawa et al., 1997 years, real-time rt-pcr has increasingly been used to detect viral pathogens because of its advantages including high specificity and sensitivity, fast turnaround, and quantification of pathogen loads. currently, the animal disease diagnostic laboratory in the ohio department of agriculture uses a real-time rt-pcr method which targets the membrane (m) gene for detection of pedv. however, since the m gene is highly conserved between the virulent and the variant strains of pedv in the us, this real-time rt-pcr assay cannot differentiate between the two strains of pedv. therefore, the present study sought to develop and evaluate a duplex real-time rt-pcr method to distinct between the virulent and the variant strains of pedvs. multiple-sequence alignments with the virulent and the variant strains of pedv sequences detected in the us were carried out with the mega 6.05 program. primers were designed by targeting the conserved regions between virulent and variant pedv, while the probes were designed by targeting the location where variant pedvs have two deletions. for multiplexing, the probe for the virulent pedv was labeled with the 5 -reported dye cy5 and the 3 -quencher bhq2, and the probe for the variant pedv was labeled with the 5 -reported dye 6-carboxyfluorescein (fam) and double quencher of the internal zen and 3'iowa black ® fq (3iabkfq). the sequences and amplicon sizes of the primers and probes are listed in table 1 and fig. 1 . sequences of primers and probe for the real-time rt-pcr targeting m gene were listed in table 1 . rna was extracted with the trizol reagent (invitrogen, carlsbad, ca, usa). amplification was performed with the qiagen one step rt-pcr kit (valencia, ca, usa) in a smartcycler ii instrument. the amplification conditions were 50 • c for 30 min; 95 • c for 15 min; and 45 cycles of 94 • c, 10 s, 54 • c, 30 s, and 72 • c, 12 s. primers (integrated dna technologies, coralville, iowa, usa) at 240 nm and each probe (integrated dna technologies, coralville, iowa, usa) at 240 nm were used for one reaction. the primer set and individual probe were tested first in a single real-time rt-pcr assay and then in a duplex real-time rt-pcr assay. intra-specificity of the duplex rt-pcr assay was determined using single probe of either virulent pedv probe for variant pedv strain or variant pedv probe for virulent pedv strain and two probes for both types of pedvs for positive control. inter-specificity of duplex rt-pcr assay was examined using various swine virus strains available in our lab. these viruses include porcine reproductive and respiratory syndrome virus, swine influenza virus (h3n2), transmissible gastroenteritis virus, encephalomyocarditis virus, porcine coronavirus hku15, porcine parvovirus, and pseudorabies virus. for dna virus porcine parvovirus and pseudorabies virus, dna samples were extracted using dneasy blood & tissue kit (qiagen, valencia, ca, usa). in the assay, 2.5 l rna or dna samples were used, 2.5 l each of virulent pedv oh1715 strain and variant pedv oh851 were used as positive control in the duplex rt-pcr and 2.5 l distilled water was used as negative control. the pcr products amplified by using rnas from oh851 (variant pedv) and oh1715 (virulent pedv) and the primer set p160-p161 covering the region where contains the majority of sequence variations between the virulent and variant pedvs were cloned into the pcr 2.1 vector (invitrogen, carlsbad, ca, usa). the plasmids with the oh851 (pcr 2.1-oh851) or oh1715 (pcr 2.1-oh1715) genes were confirmed by sequencing. the detection limit of the real-time rt-pcr assay was determined through serial dilutions of each plasmid. duplicates for each dilution were examined for separate and duplex reactions. clinical fecal and intestinal samples submitted to the animal disease diagnostic laboratory in ohio department of agriculture were processed for rna extraction. rna samples were first tested for pedv by a real-time rt-pcr targeting the m gene. if positive, the duplex real-time rt-pcr was then used to differentiate the variant and the virulent strains of pedv. based on the sequence alignment and analysis of both virulent and variant pedv partial s1 region, in addition to several sequence variations, there were 3 deletions and one insertion present in the variant pedv as compared with virulent pedv (fig. 1) . the primers were designed by targeting the conserved regions between the two viruses whereas the probes targeting the region where the first two-deletion regions were located in the variant strain of pedv. the duplex rt-pcr assay can detect specifically the virulent strain of pedv by the cy5 probe or the variant strain of pedv by the fam probe. in contract, the duplex rt-pcr did not cross-react with any other pig viruses used in the study, the cy5 probe did not cross-react with variant pedv strain, and the fam probe did not cross-react with virulent pedv strain (table 2) . table 1 sequences of primers and probes used in this study. primer/probe sequence amplicon size (bp) pedv s1 forward 5 -aggcggttcttttcaaaatttaatg-3 pedv s1 reverse 5 -gaaatgccaatctcaaagcc-3 191 for virulent pedv virulent pedv s1 probe 5 -/5cy5/tattggtgaaaaccagggtgtcaat/3bhq 2/-3 179 for variant pedv variant pedv s1 probe 5 the sensitivity of the duplex real-time rt-pcr assay was validated through serial dilutions of pcr 2.1-oh851 and pcr 2.1-oh1715 constructs. the detection limit was 1 copy for both variant and virulent strains of pedvs. standard curves were plotted using 10-fold serial dilutions of plasmid dna of virulent and variant pedv for the duplex real-time rt-pcr. as shown in fig. 2 , there is a strong linear correlation (r 2 > 0.99) between c t values and the corresponding amount of plasmid copy numbers for both virulent and variant pedv. the standard curves of virulent and variant pedv were plotted with slopes of −3.40 and −3.31, respectively ( fig. 2a and b) . the duplex real-time rt-pcr detected 1 genomic copy for both virulent and variant strain of pedvs. a total of 295 positive samples tested by the real-time rt-pcr targeting on m gene were run again by the duplex real-time rt-pcr. forty five samples tested positive for the variant pedv and the remaining 250 samples were positive for the virulent pedv. the results were confirmed by sequencing using p160-p161 primer set. pedv causes diarrhea in pigs and high mortality in piglets. since may of 2013, pedv has been identified in the us resulting in severe economic losses to the us swine industry. data on ped outbreaks have been collected and complied by the us national animal health laboratory network each week since june 17 of 2013. since there is no pedv vaccine available in north america, it is important to take biosecurity strategies to control ped. the findings of a recent study demonstrated that pedv was found in 5.2% of trailers used to transport pigs, highlighting the importance of strict biosecurity (lowe et al., 2014) . in the late december 2013, a variant strain of pedv was detected in ohio by our laboratory . genetic analysis showed that this virus differed from the virulent strains of pedv currently circulating in the us in the 5 end of the s1 domain (mainly located in the first 1170 nt of spike gene), thus the real-time rt-pcr targeting on the m gene does not provide differentiation of the variant pedv from virulent strain of pedv. therefore, the aim of this study was to develop a duplex real-time rt-pcr which would detect and differentiate the virulent strain from the variant strain of pedv. to optimize the ability of the assay to detect both variant and virulent strains of pedv, the two primers were located in the conserved region of s1 region. to differentiate specifically the variant pedv from the virulent strains of pedv, probes were designed by targeting the highly variable region containing the first two deletions present in the variant pedv (fig. 1) . it is possible that the third deletion and insertion site may also be used as a target for designing primers and probes (fig. 1) . the assay is specific for pedvs, since cross-reaction with non-ped viral genomes used in this study was not detected. in addition, the assay is highly sensitive, being able to detect 1 copy in 25 l reaction of either variant or virulent strains of pedv. the efficiency of the duplex real-time rt-pcr assay was determined by testing clinical samples which were positive by the real-time rt-pcr assay targeting the m gene. of the 45 clinical samples that tested positive for the variant pedv, all of them were confirmed by sequencing of the spike gene. in conclusion, we have developed a duplex real-time rt-pcr assay that reliably detects and differentiates the virulent strain and variant strain of pedv. this assay may be used by veterinary diagnostic laboratories to detect the new variant strain and the virulent strains of pedv currently circulating in the us. evaluation of a blocking elisa using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies immuno-histochemical detection of porcine epidemic diarrhea virus compared to other methods origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states direct and rapid detection of porcine epidemic diarrhea virus by rt-pcr pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs rapid diagnosis of porcine epidemic diarrhea virus infection by polymerase chain reaction role of transportation in spread of porcine epidemic diarrhea virus infection, united states letter to the editor. pig farming light microscopy and ultrahistology of intestinal changes in pigs infected with epizootic diarrhoea virus (edv): comparison with transmissible gastroenteritis (tge) virus and porcine rotavirus infections emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences outbreak of porcine epidemic diarrhea in suckling piglets new variant of porcine epidemic diarrhea virus, united states molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus field strains in central china during 2010-2012 outbreaks key: cord-279794-hn5vmic0 authors: guo, jiahui; fang, liurong; ye, xu; chen, jiyao; xu, shangen; zhu, xinyu; miao, yimin; wang, dang; xiao, shaobo title: evolutionary and genotypic analyses of global porcine epidemic diarrhea virus strains date: 2018-08-27 journal: transbound emerg dis doi: 10.1111/tbed.12991 sha: doc_id: 279794 cord_uid: hn5vmic0 porcine epidemic diarrhea virus (pedv), which re‐emerged in china in october 2010, has spread rapidly worldwide. detailed analyses of the complete genomes of different pedv strains are essential to understand the relationships among re‐emerging and historic strains worldwide. here, we analysed the complete genomes of 409 strains from different countries, which were classified into five subgroup strains (i.e., gi‐a, gi‐b, gii‐a, gii‐b, and gii‐c). phylogenetic study of different genes in the pedv strains revealed that the newly discovered subgroup gii‐c exhibited inconsistent topologies between the spike gene and other genes. furthermore, recombination analysis indicated that gii‐c viruses evolved from a recombinant virus that acquired the 5′ part of the spike gene from the gi‐a subgroup and the remaining genomic regions from the gii‐a subgroup. molecular clock analysis showed that divergence of the gii‐c subgroup spike gene occurred in april 2010, suggesting that the subgroup originated from recombination events before the pedv re‐emergence outbreaks. interestingly, ascaris suum, a large roundworm occurring in pigs, was found to be an unusual pedv host, providing potential support for cross‐host transmission. this study has significant implications for understanding ongoing global pedv outbreaks and will guide future efforts to develop effective preventative measures against pedv. porcine epidemic diarrhoea (ped) is a devastating enteric disease in pigs that results in severe diarrhoea, vomiting, and dehydration, with very high mortality observed in suckling pigs (pensaert & de bouck, 1978) . porcine epidemic diarrhea virus (pedv), which is the causative agent of ped, belongs to the genus alphacoronavirus within the family coronaviridae and is an enveloped single-stranded positivesense rna virus (woo et al., 2012) . the pedv genome consists of seven open reading frames (orfs) organized in the order orf1a, orf1b, spike (s) glycoprotein gene, orf3 hypothetical protein gene, envelope (e) gene, membrane (m) gene, and nucleocapsid (n) gene . among the proteins encoded by the orfs, the s glycoprotein is located on the envelope of the virus in the large surface projections of the virion and plays an important role in the attachment of viral particles to host cell receptors (lee, park, kim, & lee, 2010) . thus, the s gene is considered important for understanding the genetic relatedness and epidemiological status of pedv field isolates, as well as for advancing vaccine development (chen, liu, lang, et al., 2013) . the ped disease was first discovered in pig farms in belgium and the united kingdom in 1976 (pensaert & de bouck, 1978) , with reports of its occurrence in china as early as the 1980s (li et al., 2012) . with the emergence of new pedv strains, however, serious disease epidemics have been observed in china since october 2010 (sun et al., 2012) . beyond china, the disease has rapidly spread to more than 38 states in the usa following its first outbreak in may 2013, affecting more than 4,000 farms accounting for more than 7 million piglets (cima, 2014) . japan, canada, mexico, and colombia have also experienced successive outbreaks, with considerable economic losses to the global pig industry (lara-romero et al., 2018; ojkic et al., 2015; takahashi, okada, & ohshima, 1983; valko et al., 2017) . major global outbreaks since 2012 have renewed concerns about the potential changes in the mode of pedv transfer (chen, liu, lang, et al., 2013; li et al., 2012; sun et al., 2012) . although increasing evidence suggests that pedv routinely undergoes significant changes, especially in spike proteins (lara-romero et al., 2018; stott et al., 2017) , the prevalence and evolution of pedv strains is not well-defined and limited knowledge is known regarding the ways in which pedv subgroups circulate among themselves and how they might influence the evolution of pedv. to better understand the molecular epidemiology and genetic diversity of pedv field isolates, we investigated the genetic characterization, origin, and evolution of emergent pedv strains worldwide, which will provide much needed information for the effective prevention and control of this disease. the complete pedv genome was selected for genetic analysis. to clarify the evolution of pedvs, we obtained four complete genome sequences of pedv from our own lab (i.e., zl29, aj1102, hub1-2017, and hub7-2017) (bi, zeng, xiao, chen, & fang, 2012) firstly, we performed multiple sequence alignment of the 409 complete pedv genomes, as well as the orf1ab, s, orf3-e-m-n genes, and applied the bat coronavirus btcov/512/2005 (genbank accession no. dq648858) sequence as an outgroup (tang et al., 2006) . a maximum-likelihood (ml) phylogenetic tree was constructed using iq-tree v.1.6.5 (nguyen, schmidt, von haeseler, & minh, 2015) , with the best fitting evolutionary model suggested by the program following 1,000 bootstrap replicates. the phylogenetic tree was rooted against the pedv-related bat coronavirus, with removal of the long-branch leading to greater resolution of the viruses of interest. nucleotide and deduced amino acid sequences were aligned using mafft v.7.402 (katoh & standley, 2013) . the resulting tree was visualized using itol v.4 (interactive tree of life, http://itol.e mbl.de/). we preliminarily screened the pedv sequence data set for recombination using rdp, geneconv, chimaera, maxchi, and 3seq, followed by secondary scanning and recombination using bootscan and siscan in recombination detection program version.4.95 (rdp v.4 .95) (martin, murrell, golden, khoosal, & muhire, 2015) . sequences with significant signals for recombination determined by more than two methods were analysed in greater detail. nucleotide sequence similarity was assessed by simplot v.3.5.1 (lole et al., 1999) , with a sliding window size of 500 bp, step size of 100 nucleotides, and 1,000 bootstrap replicates, using gap-stripped alignments and the f84 (ml) distance model. all data were analysed using graphpad prism software (v.5.03, san diego, ca, usa). all s protein sequences from the pedv sample strains were analysed using the meta data-driven comparative analysis tool (meta-cats) (pickett et al., 2013) , with a p-value threshold of 0.05 (this threshold f i g u r e 1 genotyping and origin of the 409 pedv strains based on full-length genomic sequence analyses. (a) phylogram was tested by 1,000 bootstrap replicates, branch lengths were measured by the number of substitutions per site (see scale bars). names of strains, years, places of isolation, genbank accession numbers, genogroups, and subgroups are shown. (b) line chart shows the number of pedv sequences obtained by gene subgroup and year of sampling. yearly percentages of samples positive for pedv are indicated by different coloured lines respectively. data are indicated below sampling years. (c) subgroup distribution of all available complete or partial pedv genome sequences from countries reporting pedv infections (n.a., sequence not available). in the bar charts, counts are shown by country or region. data are indicated below bar charts [colour figure can be viewed at wileyonlinelibrary.com] was the maximum probability level for the likelihood that the position differed among groups simply by chance), to identify significantly different sites between the five subgroups. to characterize the genetic diversity of pedvs circulating globally, we constructed a phylogenetic tree using iq-tree based on the 409 complete pedv genomes (see materials and methods 2.2). consistent with our previous research (wang, fang, & xiao, 2016a) , the phylogenetic tree indicated that the complete pedv genomes evolved into two separate genogroups, gi (classical) and gii (variant), as presented in figure 1a . furthermore, genogroup gi evolved into two subgroups (gi-a and gi-b) and genogroup gii evolved into three subgroups (gii-a, gii-b, and gii-c). the gi-a subgroup mainly included the earlier pedv strains found in europe and belgium (virulent we identified the geographical and temporal distributions of the pedv strains to clarify the evolution of the virus. as shown in figure 1b , only sporadic outbreaks of pedv were reported before 2010, with the pathogens involved in these outbreaks found within the gi genogroup. the pedvs were primarily located in the gi-a (virulent cv777) and gi-b (attenuated dr13) subgroups and were predominantly from asia (figure 1b ). however, considerable pedv outbreaks were reported in asia and the united states after 2010, even for vaccinated piglets (lin, saif, marthaler, & wang, 2016) . based on our examination and assembly of public data, we identified that the pedv strains were primarily from the gii genogroup. interestingly, gii-b subgroup strains were reported predominantly in 2011, whereas gii-a strains were reported more prevalently after 2011 and occupied a larger proportion of strains. moreover, reports of the newly discovered gii-c strains showed a significant increase after 2012 due to further sequencing from europe. the above results indicate that the epidemic strains from different periods were from the five different subgroups. based on the geographical distribution of pedvs (figure 1c) , the gi genogroup (classical and cell culture-adapted vaccine strains) largely originated from the earlier pedv-threatened areas, such as china, south korea, and europe. the different subgroups from the gii genogroup also showed characteristic geographical distribution. while most gii-a subgroup strains were from the americas, a small number were from china and japan or from sporadic outbreaks in a few other isolated areas. the gii-b subgroup strains were mostly endemic to asia, especially china, south korea, and japan. the gii-c subgroup strains were primarily from europe, with some from the usa and china. our study showed that pedv strains from different subgroups were prevalent within the same areas, implying that the coincident "hot spots" in pedv-endemic areas (e.g., china and south korea, figure 1c critical for determining the sources of some pedv variations. these "hot spot" areas have the potential to be important reservoirs for the genetic variation of pedvs, resulting in recombination between different pedv subgroups. we also examined the potential hosts of the 409 pedv strains. results showed an unusual pedv strain (genbank accession no. kx883635) hosted by ascaris suum (supporting information table s1 ) (shi et al., 2016) , a large roundworm in pigs, thus providing novel insight into the possible epidemiology of pedv infection. indeed, parasites have long been regarded as a harmful factor to the pig industry as sources for a variety of infectious agents (jesudoss chelladurai et al., 2017) . for example, metastrongylus larvae are considered a reservoir for various porcine viral pathogens, primarily swine fever virus and swine flu virus (sen, kelley, underdahl, & young, 1961) . however, whether ascaris suum plays a critical role as a pedv reservoir requires further investigation. to further explore the evolution of pedvs, we constructed three phylogenetic trees based on the orf1ab, s, and orf3-e-m-n gene sequences of the 409 pedv strains. the orf1ab and orf3-e-m-n gene alignments confirmed that the gii-c subgroup was deeply nested within the gii-a subgroup (figure 3a and b) . strikingly, the phylogeny of the s gene suggested an entirely different evolutionary history for the gii-c subgroup compared to the other subgroups (figure 3c ). all gii-c subgroup strains showed inconsistent topology in the s gene phylogenetic tree, differing from the orf1ab gene phylogenetic tree. this inconsistent topology, in which outlier sequences were found between two well-defined subgroups in a phylogenetic tree, was attributed to (95%-99% genetic identity to the orf1ab and orf3-e-m-n genes). in contrast, the genetic identity between the s gene sequences of these viruses was only 85% and showed strong similarity with virulent cv777 (95% gene identity). in the s gene phylogeny, virulent dr13 did not cluster with kupe21 but instead with virulent cv777, suggesting that the pedv s gene was subjected to relatively frequent recombination, even between divergent subgroups (figure 3c and supporting information figure s2a ). moreover, the italy/7239/2009 pedv strain intragenogroup recombination provides a mechanism for amalgamation among these distinct subgroups and increases the genetic repertoire of co-circulating pedv strains. the s protein attaches to the cellular receptors of a host, resulting in virus entry by membrane fusion, and contains the domain that stimulates the production of neutralizing antibodies. variations in the s protein are important for understanding the genetic relatedness of pedv field strains (chang et al., 2002; lara-romero et al., 2018; wang et al., 2016b) . to determine the significant s protein sequences among the five different pedv subgroups, meta-cats analysis was performed for spike protein sequences of all 409 pedv strains. we identified 257 amino acid positions with significant variation among the isolates from the five subgroups (supporting information table s2 ). as shown in figure 4 , the gii genogroup contained 11 distinct patterns of aa mutations (i116t, i356t, e365q, t549s, g594s, n724s, a959v, s1044a, g1173d, s1232r, and r1298q), distinguishing it from isolates in the gi genogroup. although the gii-c subgroup s genes shared some aa substitutions with the gi-a and gii-a subgroups (two parents of recombination), they also exhibited three unique patterns (l76, a/s92, and h/t113), which clearly distinguished these isolates from those in the other subgroups, suggesting that the gii-c subgroup s gene evolved gradually through antigenic drift. it is well established that the s protein of coronaviruses induces high levels of neutralizing antibodies. four neutralizing epitopes (499-638, 748-755, 764-771 , and 1,368-1,374 amino acids) have also been identified in the pedv s protein (chang et al., 2002) . to explore whether the gii genogroup acquired substitutions in neutralizing epitopes characteristic of the gi genogroup, we mapped the significantly different positions to the equivalent positions in representative sequences (figure 4) . results identified seven substitutions in the neutralizing epitopes of the s protein in the gii genogroup (l521h, s523g, v527i, t549s, g594s, a605e, and l612f), which may explain why traditional inactivated vaccines and attenuated vaccines against the gi genogroup cannot effectively protect piglets threatened by a pandemic strain from the gii genogroup. in summary, this study revealed the genetic diversity and evolutionary dynamics of pedv strains. our genetic analyses showed that the pedv strains could be categorized into two groups, namely, gi (classical) and gii (variant) . we also discovered a new subgroup (gii-c) with novel genetic, molecular, and phylogenic characteristics. the gii-c subgroup evolved from a recombination event between the gi-a and gii-a subgroups, and we further found recombination in two relatively early strains: virulent dr13 and italy/7239/2009. these recombination events occurred prior to the re-emergence of pedv in 2010. additionally, to explore the potential link between s protein amino acid sequence variations and recombination, we performed a series of comparative analyses of the pedv s protein sequences. we found 10 positions that were localized in a well-known neutralizing epitope and revealed several unique amino acids that could easily distinguish the different subgroups. this study provides critical information to help trace the sources of pedv variants and identify the evolutionary mechanisms involved. furthermore, this research will hopefully facilitate the development of diagnostic kits, vaccines, and new therapeutic strategies, which are expected to turn the tide in the prevention of pandemic outbreaks of pedv. none. http://orcid.org/0000-0003-3394-3702 baele, g., lemey, p., rambaut, a., & suchard, m. a. (2017) . adaptive mcmc in bayesian phylogenetics: an application to analyzing partitioned data in beast. bioinformatics, 33, 1798-1805. https://doi.org/ 10.1093/bioinformatics/btx088 bi, j., zeng, s., xiao, s., chen, h., & fang, l. 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virosphere evolutionary and epidemiological analyses based on spike genes of porcine epidemic diarrhea virus circulating in thailand outbreak of porcine epidemic diarrhea in suckling piglets an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan. nihon juigaku zasshi prevalence and genetic diversity of coronaviruses in bats from china porcine epidemic diarrhoea virus with a recombinant s gene detected in hungary immunogenicity and antigenic relationships among spike proteins of porcine epidemic diarrhea virus subtypes g1 and g2 porcine epidemic diarrhea in china 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 key: cord-004713-gzts5h0y authors: fennestad, k. l. title: pathogenetic observations on pleural effusion disease in rabbits date: 1985 journal: arch virol doi: 10.1007/bf01378969 sha: doc_id: 4713 cord_uid: gzts5h0y a pathogenetic study of pleural effusion disease (ped) in rabbits was made, using the virulent ped agent or virus (pedv) and an avirulent derivate of this isolate. independent of infective dose within the range examined, the virulent isolate caused fatal clinical disease, whereas the avirulent isolate caused subclinical infection. the two isolates differed in rapidity of initial spread of infection and in the maximum virus titres in serum, but they both resulted in a similar low level persisting viraemia. circulating virulent virus gradually became avirulent during the viraemia. avirulent infection induced protective immunity to virulent challenge during the first week after primary infection, but full clinical protection was not established until after the fourth week. the findings, corrobated with other closely comparable observations, suggest that the emergence of ped as an intercurrent mortality problem during rabbit passage of pathogenictreponema pallidum is the result of a specific selective pressure on a benign passenger virus. the expression of virulence of pedv appears to be dependent on length of interval between passages. pleural effusion disease (ped) is a rabbit infection probably caused by a host-specific, small, enveloped virus which measures 25--50 nm by filtration (6) . although known since 1970 the agent has not been convincingly demonstrated by tissue culture, electron microscopy or specific serological technique (8, i3, 16) . ped is considered to be a new-disease with characteristic clinical signs and pleural effusion as the typical post mortem finding (1, 16) . the infection is characterized by a long lasting viraemia and formation of antibodies which appear to have only little neutralizing capacity (4, 16) . however, there is clinical protection to re-infection after the fourth week (1) . ped emerged during the sixties as a serious intereurrent mortality problem among rabbits inoculated with treponema pcdlidum (7, 9, 10, 18) . subsequently, it was shown that. the causative agent was being passed from rabbit to rabbit as a passenger of the rabbit testieular suspensions of trepoheroes (3, 8) . as such the infection has undoubtedly been spread from laboratory to laboratory, but the disease per se appears to be confined to this experimental situation (1) . there is evidence to suggest that the serial propagation of treponemes in rabbits, as practised in a number of laboratories at intervals of 6 to 10--14 days, may have been instrumental in the emergence of disease. by such propagation a normally harmless virus may enter the passages and gradually acquire virulence through a selective pressure on a heterogeneous virus population during hundreds of passages. support for this contention is the enhancement of virulence of ped virus (pedv) observed in the "natural" disease and --after its isolation --by continued serial propagation in rabbits at intervals of 3---10 days (fig. 1) . however, there is also evidence which might suggest that increasing time of persistence of virulent virus in the individual rabbit host may operate in the reverse direction by selecting a population of predominantly avirulent pedv particles, which appear to be unable to cause disease unless undergoing new rabbit passages at short intervals (t, 6). the present pathogenetic study on virulence, viral growth and the development of protection was carried out with the original pedv isolate and an avirulent progeny of this isolate obtained from a rabbit six months after experimental infection. conventional albino rabbits (new zealand-white type), aged 3--5 months, were used, and all inoculations were made by the subcutaneous (se.) route in 1 ml amounts. before use, these animals had been employed once for pyrogen testing of protein fractions of human blood. all rabbbits came from the same colony (sse:cph) which has been dosed since 1950. the colony is considered free of pedv, since convincing observations to the contrary have never been made (3) . the rabbits used for propagation of treponemes at statens seruminstitut sinee 1951 also came from this colony (ef. fig. 1 ). virus isolates the highly virulent pedv was isolated in 1970 from a freeze-dried rabbit testicular suspension of t. pallidum (1) . following isolation the virus was passed serially at short intervals through more than 124 rabbits before preparing a virus stock. the stock consisted of pooled rabbit serum obtained 48 hours after inoculation with blood or pleural fluid. i t will appear from fig. 1 that the virulence of virus had not become "fixed" at time of preparation of the stock pool. the avirulent derivate of p e d v was obtained from blood of a healthy rabbit six months after experimentm neonatm infection with the above mentioned stock virus. this isolate was passed serimly through 61 rabbits at weekly intervals by inoculation of 0.2 ml serum mixed with 0.8 ml pbs (ph 7.0). during these ahnost subctinicm passages no mortality occurred and typical clinical signs of p e d were seen in only two rabbits (passages nos. 46 and 55). the stock pool of the isolate consisted of serum from the 1 l t h rabbit passage obtained 72 hours after inoculation (6) . both stocks were stored in 1 ml miquots at --70 o c until required for experiments. lack of in vitro methods necessitated the use of rabbits for demonstration and quantitation of pedv. for these purposes the previously described rabbit test was mortmity among several hundreds of rabbits annumly inoculated with treponemat suspensions at intervals of 6--10 days. about 1961 p e d was recognized as a disease entity causing intercurrent death among the treponema-inoeulated rabbits, and in october 1970 the causative passenger virus was isolated. after isolation p e d v was initimly carried through 20 passages in femme rabbits by sc. inoculation of blood or pleural fluid at intervals of 2--14 days. the virus was then passed serimly through t70 male rabbits in the same manner, but at intervals of 3--10 days. the black dots indicate the annual mortmity during the 3-year period of these passages. the intercurrent disease problem among the treponema-infected rabbits was solved by removing the passenger virus from the rabbit testicular suspensions of treponemes by p~ssing the contaminated treponemes through hamsters arid back to rabbits. this procedure was first used in 1970, but due to a mistake not suecesfully accomplished until 1976. until now (1983) these passages have remained free of p e d v k . l . fennes~ad : used (3) . briefly, the inoculum to be examined was given sc. to a rabbit. fever together with uveitis or death with necropsy findings characteristic of ped, or both, was considered as evidence of p e d v in the inoculum. animals failing to show these signs were challenged 30 days after inoculation with the highly virulent pedv. presence of clinical protection after challenge was considered as evidence of p e d v in the inoculum. the number of rabbit-infective doses (rid) per mi of the virus stocks was estimated by inoculating 10-fold dilutions in pbs, using 2~4 rabbits per dilution. the term r i d refers to a dose capable of producing typical signs of f e d and/or clinical protection against challenge. the highest dilution producing these responses was taken as endpoint titre of the virus. i n this way the stock of virulent f e d v was found to contain 106 i~id per ml, while the stock of the avirulent derivate contained 102 i~id per ml. uniform groups of rabbits were inoculated with decimal dilutions of the two stocks. during a period of 10 days the rabbits were then observed twice daily for clinical signs of disease. dead animals were examined for gross lesions and bacteriologically in order to establish the cause of death (1) . surviving rabbits failing to show the typical signs of f e d were challenged with 104 i~td of p e d v and observed for a period of 10 days. in a preliminary experiment six rabbits were inoculated with approximately 105 r i d of fedv. two rabbits were killed for the study of infectivities of tissues and body fluids at 72 hours, 10 and 20 days after inoculation. from each rabbit was obtained serum and heparinized blood. erythrocytes and buffy coat were washed 6--8 times in pbs (ph 7.2). lung, thymus, liver, spleen, kidney, popliteal lymph nodes, and brain were frozen at --70 ° c. a t0 per cent suspension (wt/vol) in hanks' solution was prepared from the frozen organs by grinding with sand. serial 10-fold dilutions of the preparations were made in pbs using one rabbit per dilution. in a succeeding experiment two groups of rabbits were inoculated, respectively, with p e d v and the avirulent derivate. these rabbits were followed by serum samples. endpoint virus titres of these samples were determined as for the virus stocks, using i --3 rabbits per dilution. to observe the temporal development of clinical protection rabbits were inoculated with 101 i~id of the avirulent derivate of pedv. groups of rabbits were then challenged with 10 ~ i~id of f e d v at selected intervals after the primary infection. a control group of animals were inoculated with 105 i~id of f e d v without primary infection. four groups of animals were examined for viraemia immediately prior to challenge, using 0.2 ml of serum mixed with 0.8 ml fbs as inoculum. and fever, together with uveitis. in assessing the infectivity of the two isolates all surviving rabbits failing to show clinical signs were challenged with pedv to determine whether or not the primary inoculation had induced protection (table 1) . after inoculation with pedv 30 of 40 rabbits developed clinical disease and 18 of 20 rabbits died with pleural effusion. the severity of the clinical response did not appear to be dose dependent, but the incubation period, i.e. the time from inoculation until onset of fever, and the mean death time (mdt) appeared to be influenced by the dose. the i0 rabbits failing to showany clinical signs were fully susceptible to challenge. after inoculation with the avirulent derivate none of the 32 rabbits showed typical clinical signs of ped, but ephemeral fever occurred in 3 of 8 rabbits receiving the highest dose. when challenged with pedv 20 of the 32 rabbits were clinically protected, indicating that the primary inoculation had induced clinical immunity. the remaining 12 rabbits were fully susceptible to challenge. these data indicate a dose-independent difference in virulence between the two isolates. pedv infection resulted in clinical disease, whereas infection with the avirulent derivate almost exclusively resulted in a subclinical response. from the total incidence of typical clinical responses and the development of clinical protection the median infective doses (id50) per ml was calculated to be 106.3 for pedv and t08-0 for the avirulent derivate. virus in blood did not appear to be cell associated, but it was demonstrable in serum and almost all tissues examined on days 3, 10 and 20 after inoculation with pedv ( table 2 ). the highest concentration of virus, i.e. about 106 rid was found in serum and lungs on p.i. day 3. in other experiments (data not shown) the same virus concentration was found in pleural fluid from animals dying about this time. pooled fluid from the anterior chamber of the eye examined at 60 hours after inoculation, i.e. before uveitis became clinically detectable, had a titre of 105 and on p.i. day 20, i.e. after the clinical uveitis had disappeared, the titre of this fluid was about 101 rid. from p.i. days 3 to 20 there was a generm decrease in titre of all materim examined. the significance of the lower titres of various tissues as compared with serum is difficult to evaluate, particularly since no attempts were made to remove the blood by perfusion prior to the titrations of tissues and because of the method of quantitation. to examine viral growth and persistence of viraemia, serum was assayed for virus content at various times after rabbit infection with the two isolates ( figs. 2 and 3 ). after infection with 104 rid of pedv, virus was demonstrable already at 6 hours and then the titre increased rapidly to a maximum of about 106 at 72 hours p. i. inoculation of a 1000-fold lower dose delayed the appearance of virus in serum until between 24 and 48 hours, but a titre of about 106 was still reached at 72 hours. this suggests that the dose of virus had no effects on the maximum titre attained. from p.i. day 5 the titre slowly declined to about l0 s on p.i. day 20 and on p.i. day 30 it ranged from <101 to 108. the same range in titres was present during the following five months. one rabbit was consistently negative after p.i. day 20, and one was negative at 60 and 90 days after inoculation. fig. 3 the results show that for comparable doses of the two isolates the virulent pedv became detectable earlier and reached a t00-to 1000-fold higher maximum titre than the avirulent isolate. the in vivo determination of virus concentration in serum at various times after infection offered an opportunity to compare virulence of circulating virus on primary rabbit inoculation (table 3) . following infection with the virulent pedv, infectious serum diluted 10 -1 to 10 -7 almost invariably resulted in typical clinical disease provided that the serum was obtained during the first 20 days of viraemia. serum from p.i. day 30 diluted t0 -1 to 10 -3 caused typical clinical response in two of six rabbits, but from day 60 of viraemia serum diluted 10 -1 to 10 -a never resulted in clinical signs of ped. this suggests that the population of virulent pedv particles after p.i. day 20 gradually was being replaced by a population of low or avirulent particles. after infection with the avirulent derivate, the isolates from serum obtained during the entire period of viraemia were demonstrable in serum dilution 10 -1 to 10 -a, but the primary rabbit inoculation of these dilutions never resulted in clinical signs of ped. the protective response was tested by sequential infection with the avirulent isolate and pedv. for this purpose groups of 8 rabbits were inoculated with the avirulent isolate. ephemeral fever occurred on p.i. days 3--6 (average 3.6 days) in 13 of 64 animals observed for 6--10 days, but no other ciinical signs of disease were seen. four of the groups of rabbits were examined for presence of viraemia on p. i. days 30, 36, 60 and 90 and the proportion of animals with demonstrable viraemia was 4/8, 7/8, 8/8, and 7/8. at selected intervals after the primary infection, the various groups of rabbits were challenged with pedv. in addition, a control group received the same inoculum (fig. 4) . no clinical signs of protection were present 24 hours after chmlenge, i. e. at a time when viraemia after avirulent infection had not yet become detectable (fig. 3) . six days after challenge there was clear evidence of protection, but typical clinical signs occurred in single animals challenged on days 12, 18 and 24, indicating individual differences in immunogenic or protective potential. at 30 days the only clinical sign of disease was transient fever in 2 of 8 rabbits and at all challenges later on there was a subclinical response. the results show that 101 r i d of the avirulent virus induced full clinical protection against challenge with 105 r i d of the virulent p e d v from the 4th--5th week after infection. the two isolates of the same origin tested in the study were selected for excessive, but not ultimate virulence and avirulence. the serial rabbit passages of the isolates before and after preparation of the virus stocks indicate stability of their virulence properties within a relative large number of passages. isolates of p e d v from other laboratories propagating contaminated treponemes in rabbits have varied widely in virulence and behaved similarly in serial passages, but concerning virulence they all appear to fall within the range observed in this study (3, 6) . the difference in virulence was correlated with a difference in rapidity of the initial multiplication and in the maximum titres attained, but already after about three weeks the titre level in the two infections was similar. ped is accompanied by interferon production during the first days after infection and there is evidence to suggest that the interferon response also is correlated with the virulence of isolates (2, 5) . from about one month after the virulent infection, circulating virus appeared to lose virulence, i.e. upon primary rabbit inoculation the various dilutions of serum induced mainly a subclinical response as observed after avirulent infection. a similar observation was made after infection of baby rabbits with virulent pedv (4). in these experiments it was also observed that isolates from serum obtained on p.i. days 90 and 120 regained their virulence after 2--6 serial transfers in rabbits, whereas isolates from p.i. day 180 required from 7 to more than 39 serial passages before virulence appeared restored. these data suggest that throughout the course of infection the original virulent virus becomes avirulent and that the latter property becomes increasingly stable. an early change in virulence during residence in the host is suggested by other observations. thus serial passages of virulent pedv isolates preserved or enhanced virulence provided that the passages were carried out at intervals of 3--10 days (mortahty: 51 per cent,), whereas virulent isolates became avirulent when the passage interval was prolonged to 30 days. passage at intervals of 20 days resulted in a mortality of 36 per cent (1, 6) . this selection mechanism for virulence depending on the interval between passages may explain why ped was first recognized in laboratories using the t. pallidum immobilization (tpi) test. this test was introduced in 1949 (12) and became widely popular from the fifties, particularly in europe. the test requires propagation of treponemes in rabbits at short intervals. ped emerged between 10 and 20 years later as a cause of intercurrent rabbit mortality in a number of tpi laboratories. this may suggest that the change in virulence by selection during passages was very slowly imposed, but besides the time of contamination of the treponemal suspensions, other factors such as the number of animals used per passage, passage interval, and the breed of rabbits have probably also been instrumental in the development of virulence. it has previously been shown that acute ped is followed by a rise of electrophoretic gamma globulin and that the concentration of serum igg in viraemic rabbits 180 days after virulent infection is significantly higher than in uninfected controls (1, 4) . failure of such antibodies to eliminate circulating virus is in accordance with the observation of poor protective value of non-infectious immune or hyperimmune sera in passive protection experiments (4) . the development of clinical protection as seen in the present study may be explained by a cooperation between antibody and cellular factors. persisting viraemia is a characteristic of a number of infections caused by different viruses and it has been suggested that invasion of the lymphoreticular tissues is a common feature of such infections (11, 17) . in the present study the titrations of various organs and tissues did not suggest any particular site of virus replication, but this does not exclude replication in the lymphoreticuiar tissues. pedv infection resembles lactate dehydrogenase-elevating virus (ldv) infection, which cause persisting viraemia in mice, at least with respect to discovery and laboratory transmission (15) . both were first found as biological contaminants of commonly used biological preparations. ldv occurs as a contaminant of transplantable tumours without causing disease, and pedv occurs as a contaminant of treponemal suspensions causing intercurrent deaths or subclinical infections. ldv contaminated material may interfere with the interpretation of experimental results (14) , and the same may well apply to pedv contaminated treponemal suspensions. ldv is probably larger than pedv and reaches a maximum plasma titre of 10 l° which is considerably higher than that of pedv. this may perhaps explain why pedv, in contrast to ldv, has not yet been demonstrated by electron microscopy of serum. pleural effusion disease in rabbits. clinical and post mortem observations. acta path. microbiol, scand pteural effusion disease in rabbits. interferon in body fluids and tissues after experimental infection. acta path. microbiol, scand pleural effusion disease agent as passenger of treponema pallidum suspensions from rabbits pleural effusion disease in rabbits. observations on viraemia, immunity and transmissibility interferon in rabbit sera after inoculation with treponeraa patlidum suspensions contaminated with p e d virus pleural effusion disease in rabbits. properties of the aetiological agent fever after inoculation of rabbits with treponema pallidum screening out a virus-like agent from the testicular suspension of the nichols pathogenic t. pauidum. with observations on certain characteristics of the agent spontaneous deaths among rabbits inoculated with treponema pauidum less than 2 weeks before intercurrent death of rabbits after inoculation with treponema pallidum in the netherlands. 7th iclas syrup general features of persistent virus infections immobilization of treponema pallidura in vitro by antibody produced in syphilitic infection coronavirus-like particles in laboratory rabbits -with different syndromes in the netherlands biological contaminants and scientific misinterpretations lactic dehydrogenase virus. (virology monographs rabbit cardiomyopathy associated with a virus antigenically related to human coronavirus strain 229e persistent, slow and latent viral infections entretien au laboratoire des souches de tr@pongmes pathog@nes authors' address: dr. k. l. fennestad, statens seruminstitut, amager boulevard 80, dk-2300 copenhagen s., denmark.received june 2, i984 key: cord-003587-zminzrov authors: wang, xueyu; xu, xin; hu, wen; zuo, kejing; li, zhili; kan, yunchao; yao, lunguang; ji, jun; bi, yingzuo title: visual detection of porcine epidemic diarrhea virus using a novel reverse transcription polymerase spiral reaction method date: 2019-04-15 journal: bmc vet res doi: 10.1186/s12917-019-1851-7 sha: doc_id: 3587 cord_uid: zminzrov background: porcine epidemic diarrhea virus (pedv) is a major etiological agent of porcine epidemic diarrhea around the world. point-of-care testing in the field is lacking owing to the requirement for a simple, robust field applicable test that does not require professional laboratory equipment. the aim of this study was to establish a novel reverse transcription polymerase spiral reaction (rt-psr) assay for the rapid detection of porcine epidemic diarrhea virus (pedv). for the assay, a specific rt-psr primer pair was designed against a conserved region in pedv orf3. results: the rt-psr was optimized, and pedv could be detected after a 50 min incubation at 62 °c, in addition to the 15 min required for reverse transcription. no cross-reaction with other porcine infectious viruses was observed. this new method for pedv detection was 10 times more sensitive than the conventional reverse transcription-polymerase chain reaction (rt-pcr) assay. the positive rates for 65 clinical samples using the new rt-psr assay and the conventional rt-pcr assay were 58.46% (38/65) and 53.84% (35/65), respectively. in the rt-psr assay, the addition of a mixture of dyes allowed a positive reaction to be directly observed by the naked eye. conclusions: these results indicate that this rt-psr assay is capable of accurately detecting pedv, and has the advantages of high specificity and sensitivity for the detection of pedv. porcine epidemic diarrhea (ped) is a highly contagious swine enteritis accompanied by vomiting, watery diarrhea, dehydration, and other symptoms [1] . ped is caused by the porcine epidemic diarrhea virus (pedv), which belongs to the order nidovirales and the family coronaviridae [2] . pedv infections are more frequent in the winter [3] , and although pedv can infect swine of all ages, it causes the most serious harm to suckling piglets. since the discovery of ped in england in 1971, the disease has expanded to many countries in europe and asia, especially china and south korea, which has caused huge economic losses [4] . in china, pedv is widely distributed, and outbreaks have a strong negative impact on the swine industry, in part due to the acute onset and fast spread of the virus,the incidence rate in piglets can be very prevalent [5] [6] [7] . the methods currently used to diagnose ped in clinical samples are mainly divided into two types: 1) immunological methods including immunochromatography and enzyme-linked immunosorbent assay (elisa) [8, 9] , and 2) molecular biology assays including conventional reverse transcription-polymerase chain reaction (rt-pcr), reverse transcription quantitative pcr (rt-qpcr), multiplex pcr, and reverse transcription loop-mediated isothermal amplification (rt-lamp) [10] [11] [12] [13] . although immunological methods are generally low cost and easy to perform, they have several disadvantages, including inconclusive results and the long time required to perform the assays. to decrease the time required for pedv detection, pcr-related methods focused on the amplification of viral nucleic acids have been developed, which have been shown to be more efficient, highly sensitive and specific, even at different stages of the disease, when compared to immunological diagnostic methods. however, these molecular diagnostic methods cannot be widely used because of their complex operation, time-consuming nature, and the requirement for expensive instrumentation. detection methods based on isothermal amplification of nucleic acids, which can rapidly synthesize large amounts of dna without any specific requirements for precision instruments, have been widely used. the polymerase spiral reaction (psr) [14] is a novel nucleic acid isothermal amplification method that has the advantages of simplicity, rapidity, accuracy, and low cost when compared to conventional pcr. in addition, less primer is required, and primer design is simpler than for loop-mediated isothermal amplification (lamp). due to the high efficiency of amplification, product formation is accompanied by high levels of pyrophosphate ion by-product, leading to a change in ph. therefore, a ph-sensitive dye can be used to detect the product of the reaction with the naked eye [15] . psr detection methods have been used for numerous human and veterinary pathogens [16] [17] [18] [19] . therefore, we evaluated clinical samples using the newly developed rt-psr method to determine the method's utility for early detection of pedv. optimum reaction temperature and time for the diagnosis of pedv by rt-psr electrophoretograms showed no obvious difference in the gradient bands produced at temperatures ranging from 60°c to 64°c; however, the bands were slightly more obvious at 62°c. with increasing reaction time, the bands become more visible, and reached a peak at 50 min. therefore, the optimum temperature and time for detecting pedv by rt-psr was 62°c and 50 min, respectively. the rt-psr assay for pedv detection was optimized as follows: reverse transcription at 42°c for 15 min and spiral amplification at 62°c for 50 min. samples were serially diluted tenfold (10 − 1 , 10 − 2 , 10 − 3 , 10 − 4 , 10 − 5 , and 10 − 6 ) and were used in both the rt-psr and conventional pcr assays for pedv detection, and the results are shown in fig. 1 at the 10 − 4 dilution, conventional pcr yielded the clear bands. however, at dilutions below 10 − 4 , there was no obvious band (fig. 1c) . the results in fig. 1a demonstrated that when the concentration was 10 − 5 , the psr produced a clear ladder banding pattern; however, there were no obvious bands when the concentration was below 10 − 5 . therefore, the rt-psr assay is more sensitive than the conventional rt-pcr assay. based on the results of this experiment, the rt-psr method can be used to detect pedv, and with the addition of a colorimetric dye, positive clinical samples containing amplified product were orange-yellow, while negative samples were purple under natural light. thus, confirmation of a positive result can be visually confirmed with the naked eye (fig. 1b) . the specificity of the rt-psr method was tested using selected reference swine viruses. figure 2a shows that only the reaction containing pedv yielded an obvious ladder, which demonstrates that the rt-psr assay specifically detected pedv, and no other tested viral pathogen was detected. figure 2b shows the corresponding reactions containing a ph indicator dye. the product was digested with ecor i, and the results are shown in fig. 2c . based on the primer design, digestion of the amplified pedv product with ecor i should yield three main bands of 220, 182, and 42 bp. the results of the experiment are in good agreement with these fig. 1 sensitivity of the rt-psr and rt-pcr assays for the detection of pedv. ten-fold serial dilutions of pedv rna were subjected to the rt-psr and rt-pcr assays and analyzed. lane m, bases pair (bp) marker dl2000. lanes 1-7, dilutions of pedv rna (10 0 , 10 − 1 , 10 − 2 , 10 − 3 , 10 − 4 , 10 − 5 , and 10 − 6 ). a agarose gel electrophoresis demonstrating the sensitivity of the rt-psr assay. b colorimetric analysis demonstrating the sensitivity of the rt-psr assay. c agarose gel electrophoresis demonstrating the sensitivity of the rt-pcr assay theoretical values, thus verifying the identity of the amplification products. this further confirms that the rt-psr method is highly accurate for pedv detection at the molecular level. the results of the rt-psr and conventional pcr assays for clinical isolates detection are shown in table 1 , the positive rates for the rt-psr and conventional rt-pcr methods were 58.46% (38/65) and 53.84% (35/65), respectively. the rt-pcr-negative samples were confirmed the presence of pedv by virus isolation. the rt-psr and rt-pcr assay for detecting pedv possessed an analytical specificity of 100% (0 false negative) and 92.1% (3 false negative), respectively. these results demonstrate that conventional pcr is not adequately sensitive to detect pedv in samples with low viral loads or the psr assay is less affected by potential inhibitors within the samples. moreover, the efficiency of the developed detection method is not affected by co-infection. based on the above results, the accuracy of the rt-psr method is well suited for the detection of early viral infections. pedv, a predominant cause of acute enteric infection in swine, leads to severe dehydrating diarrhea and economic losses in the swine industry worldwide. in recent years, the incidence rates of various infectious diseases in swine have been rapidly increasing, and co-infection with pedv and a variety of other porcine diseases is becoming more and more common [20] . moreover, the cure rate for pedv is significantly higher in the early stage of infection than in the late stage of infection. therefore, it is necessary to establish a rapid, sensitive, and specific method for detecting pedv early in primary veterinary clinics, which would be a major breakthrough for this disease. at present, numerous established laboratory diagnostic techniques are used to identify pedv. kim [21] compared pedv detection using rt-pcr, immunohistochemistry, and in situ hybridization; zhou [22] developed and evaluated three assays, including conventional rt-pcr, sybr green i real-time rt-pcr, and taqman real-time rt-pcr assays. their results indicated that the taqman real-time rt-pcr could be a useful tool for clinical diagnosis, epidemiological surveys, and outbreak investigations of pedv. in addition, although rt-pcr detected pedv more frequently than serological techniques, when only tissues are submitted, immunohistochemistry and in situ hybridization would be useful methods for the detection of pedv antigen and nucleic acids. a comprehensive analysis showed that although immunological techniques can rapidly and quantitatively detect pedv in samples, molecular technologies have become more popular for their superior specificity and sensitivity. by combining current research and technological progress, we developed a more suitable molecular-based assay for the detection of pedv for early diagnosis. because of the difficulties in clinical diagnosis, the low accuracy of serological diagnosis, and the complicated operation of diagnosis via viral isolation methods, psr technology has been widely used for the differential diagnosis of animal epidemics, such as canine parvovirus 2 (cpv-2) [18] , african swine fever virus (asfv) [19] , and bovine herpesvirus 1 (bhv-1) [23] . psr not only has the advantages of traditional pcr, as it can also detect pathogenic genes efficiently and specifically, it also has a shorter detection time, is easier to perform, and uses less reagents, when compared to traditional pcr. specific nucleic acids can be amplified by the psr method under isothermal conditions, without the need for template pre-denaturation, or sacrificing amplification efficiency. in a positive reaction, the large amount of products, and white pyrophosphoric acid precipitates make a positive result easy to detect. in this experiment, by using the pedv orf3 sequence in genbank, a highly conserved region of the sequence was analyzed and selected for primer design, and a fast, simple, and accurate pedv rt-psr method was successfully developed for the detection of pedv. it offers many advantages compared to conventional pcr, and the most obvious advantage of this assay is that it is sensitive for pedv. rt-psr is more accurate than rt-pcr for the detection of clinical samples, and its sensitivity is about 10 times higher than that of conventional pcr. further analysis of the rt-psr-positive samples that were not detected by rt-pcr by viral isolation and identification confirmed the presence of pedv. we speculated that, for such samples, the viral load may be low or it may have been collected early in the course of the infection. besides limiting by viral load, psr assay is less affected by potential inhibitors within the samples, which indicated that rna extraction of from samples could be omitted [24] . so far, extraction of nucleic acid from clinical samples was consumedly increased the time and cost for diagnosis on farm or in typical veterinary clinics. therefore, nucleic acid adsorption or other simple strategy needed study and evaluate for application of rt-psr assay. in addition, by using the developed rt-psr detection method, pedv can be amplified across a wide range of temperatures from 60°c to 65°c; therefore, it can be performed in a water bath. in addition, expensive electrophoresis equipment and gel imaging systems are required to analyze the results of conventional pcr, which increases the detection costs. in contrast, using the psr method established in this study, a positive result can be directly determined by the naked eye when mixed dyes are added before the reaction, which do not affect the amplification efficiency, nor agarose gel electrophoresis. these advantages facilitate the rapid detection of pedv. through the aforementioned experiments and analyses, we concluded that the developed rt-psr method offers multiple advantages compared to conventional pcr, including shorter time and higher sensitivity. the popularization of this technology for pedv detection will be an important development in the study of ped in china, which should be beneficial for the prevention and treatment of pedv in the swine industry. tissue samples were collected from pigs died from diarrhea symptoms on farms, the small intestine was immersed in phosphate-buffered saline (pbs), washed several times to eliminate residual blood, and then placed in a centrifuge tube and stored at − 80°c until use. the experimental procedure for virus isolation is in african green monkey kidney (vero) cells according to reference [25] . the isolates were determined by more propagation until apparent cytopathic effects appeared. classical swine fever virus (csfv), porcine reproductive and respiratory syndrome virus (prrsv), transmissible gastroenteritis virus (tgev), porcine circovirus type 2 (pcv2), porcine parvovirus (ppv), pseudorabies virus (prv), and the clinical samples suspected of pedv described above included in this study were stored in the china-uk-nynu-rres joint laboratory of insect biology, nanyang normal university. prior to rna extraction, frozen small intestine samples (~20 mg) were homogenized in liquid nitrogen. then, total rna was extracted by using a commercial extraction kit (easypure viral dna/rna kit; transgen biotechnology, inc., beijing, china) according to the manufacturer's instructions. the rna was dissolved in depc-treated water for cdna synthesis. the extracted rnas were suspended in 100 μl of elution buffer and stored at − 80°c until use. using the published pedv sequences in genbank (ncbi), the conserved genes of the virus were analyzed, specific primer pairs were designed for the rt-psr and rt-pcr reaction based on a conserved region of the orf3 gene using primer premier 5.0 software. a pair of primer (p1 and p2) for rt-pcr were also designed. the obtained primers included a forward and reverse primer (table 2) , the 5′ sequences of the psr-s1 and psr-s2 primers were obtained from a botanical gene to avoid nonspecific reactions with pedv. the rt-psr products with repeat target sequences displayed multiple banding patterns were produced due to different spiral amplification stages by the aid of simultaneous bst dna polymerase extension at 3′ end and strand displacement at 5′ end. [14] . based on our previous experimental data, the rt-psr reaction was performed in a volume of 25 μl, containing primers for reverse transcription and the initial amplification (0.2 mm each psr-1 and psr-2), the forward and reverse primers for the psr reaction (0.8 mm each psr-s1 and psr-s2), dntps (1.5 mm), 2 u of amv reverse transcriptase (new england biolabs, hitchin, uk), 8 u of bst dna polymerase (new england biolabs), 10 mm (nh4) 2 so 4 , 50 mm kcl, 0.1% v/v tween-20, dye mix (0.025 mm phenol red and 0.08 mm cresol red), 1 μl of template rna, and nuclease free water to 25 μl. the reaction mixture was covered with mineral oil to prevent aerosol cross contamination. psr-amplified products were observed by the naked eye and analyzed by 2% agarose gel electrophoresis. determination of the optimum temperature and time for the rt-psr assay total pedv rna was used as the template in this reaction. after reverse transcription at 42°c for 15 min. the psr was conducted at various temperatures (60°c, 61°c, 62°c, 63°c, 64°c, 65°c, and 66°c) to determine the optimum reaction temperature. then, the optimum time for rt-psr was evaluated at 30, 40, 50, and 60 min. the analytical sensitivity of the rt-psr was determined by its ability to detect a low concentration of pedv and therefore expressed as a concentration (ng/assay) [26] . ten-fold serial dilutions of pedv total rna in nuclease free water (diluted from 10 − 1 to 10 − 6 ; minimum concentration, 0.1 ng/ml) were used to calculate the sensitivity of the newly developed rt-psr assay and compare it to that of the rt-pcr assay. the products were analyzed by separation via electrophoresis on a 2% agarose gel and directly visualized with a colorimetric ph indicator dye. to determine the specificity of the rt-psr assay, rna or dna samples from different porcine viruses, including csfv, prrsv, tgev, prv, pcv2, and ppv were tested in the assay, and ddh 2 o was used as a negative control. all viruses, except pedv, are reference swine viruses. the specificity of the pedv rt-psr assay was further evaluated by enzyme digestion of amplified products. the primers were designed with ecor i restriction sites, and digestion of the amplified products should yield fragments of the expected sizes by agarose gel electrophoresis. the text shown in italics in psr-s1 and psr-s2 is the central sequence, which is the same in psr-1 and psr-2, respectively, and the bold text is the ecor i restriction site a the primer position is based on the sequence of the hen/my/2015 strain, genbank accession number: ku641647 porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus an apparently new syndrome of porcine epidemic diarrhoea genetic properties of endemic chinese porcine epidemic diarrhea virus strains isolated since 2010 comparative proteome analysis of porcine jejunum tissues in response to a virulent strain of porcine epidemic diarrhea virus and its attenuated strain complete genome sequence of porcine epidemic diarrhea virus strain aj1102 isolated from a suckling piglet with acute diarrhea in china isolation and characterization of a variant porcine epidemic diarrhea virus in china complete genome sequence of porcine epidemic diarrhea virus from an outbreak in a vaccinated farm in shandong an elisa optimized for porcine epidemic diarrhoea virus detection in faeces a novel diagnostic approach to detecting porcine epidemic diarrhea virus: the lateral immunochromatography assay direct and rapid detection of porcine epidemic diarrhea virus by rt-pcr multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus a novel duplex taqman probe-based real-time rt-qpcr for detecting and differentiating classical and variant porcine epidemic diarrhea viruses polymerase spiral reaction (psr): a novel isothermal nucleic acid amplification method visual detection of isothermal nucleic acid amplification using ph-sensitive dyes rapid detection of pseudomonas aeruginosa targeting the toxa gene in intensive care unit patients from beijing rapid detection of candida albicans by polymerase spiral reaction assay in clinical blood samples polymerase spiral reaction (psr): a novel, visual isothermal amplification method for detection of canine parvovirus 2 genomic dna polymerase cross-linking spiral reaction (pclsr) for detection of african swine fever virus (asfv) in pigs and wild boars porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis comparison of reverse transcription polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine epidemic diarrhea virus in pigs comparison and evaluation of conventional rt-pcr, sybr green i and taqman real-time rt-pcr assays for the detection of porcine epidemic diarrhea virus novel polymerase spiral reaction (psr) for rapid visual detection of bovine herpesvirus 1 genomic dna from aborted bovine fetus and semen tolerance of loop-mediated isothermal amplification to a culture medium and biological substances isolation, molecular characterization and an artificial infection model for a variant porcine epidemic diarrhea virus strain from jiangsu province sensitivity" and "specificity" reconsidered: the meaning of these terms in analytical and diagnostic settings not applicable. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. the newly developed rt-psr assay was further evaluated using 65 clinical samples obtained from pigs with diarrhea located in jiangxi, jiangsu, hunan, hubei, and anhui, china. both the new rt-psr and conventional rt-pcr assays were performed to determine the positive rate compared with virus isolation. the analytical specificity of the assays was calculated using the following definition for specificity as the percentage of false negative samples/ the number of true positive samples [26] . all products of rt-psr were visualized after separation by electrophoresis on a 2% agarose gel. the other prevalent porcine viruses were also investigated. the autopsy protocols for dead pigs were conducted in accordance with the recommendations of the guide for the care and use of laboratory animals of the national institutes of health. the approval of using animals during the process of this study was obtained from south china agricultural university committee for animal experiments (approved id: syxk-2014-0136). not applicable. 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-290833-m0wodqr3 authors: yuan, lvfeng; zhang, shuai; peng, jie; li, yuchen; yang, qian title: synthetic surfactin analogues have improved anti-pedv properties date: 2019-04-11 journal: plos one doi: 10.1371/journal.pone.0215227 sha: doc_id: 290833 cord_uid: m0wodqr3 surfactin has antiviral activity against various enveloped viruses by inhibiting viral membrane fusion. however, the potential utility of surfactin as an antiviral drug is limited by its cytotoxicity. in this study, 10 surfactin analogues were obtained by chemical synthesis and evaluated to determine their anti-pedv activities, hemolytic activities, and critical micelle concentrations. the main goal of our study was to develop a safer drug; a surfactin analogue with high anti-pedv activity and low hemolytic activity. compared with surfactin, one of the analogues we developed, slp5, has lower hemolytic activity, with the same antiviral activity. the selectivity index of slp5 is 52, while the si for surfactin is 4, in other words, the safe and effective concentration range of slp5 is 12 times greater than that of surfactin. like surfactin, slp5 has a direct antiviral effect on pedv. structurally, slp5 is a linear lipopeptide with three carboxyl groups. surfactin derivatives similar to slp5 could be obtained by lactone bond hydrolyzation of surfactin, as well as total synthesis. surfactin has antiviral activity against a variety of enveloped viruses, including herpes simplex virus (hsv-1, hsv-2), vesicular stomatitis virus (vsv), simian immunodeficiency virus (siv) and newcastle disease virus (ndv) [1, 2] . we recently demonstrated that surfactin exerts its antiviral effects by inhibiting viral membrane fusion [3] . membrane fusion between the viral envelope and the cell membrane is essential for enveloped viruses to invade host cells. surfactin can act directly on virus particles by insertion into the viral envelopes' lipid bilayer and thereby reduce the membrane fusion rate. in addition, since the lipid components of viral envelopes are provided by the host cell, their composition, structure, and function are widely similar in the enveloped viruses. surfactin has antiviral activity against numerous enveloped viruses, and it has promise as a broad-spectrum antiviral reagent, however, the effective dose range of surfactin is narrow, merely 4 x the antiviral concentration causes hemolysis and cytotoxicity. in this study we compared chemically synthesized surfactin analogues to determine future directions for surfactin modification. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 surfactin is a cyclic lipopeptide naturally produced by various strains of bacillus subtilis, the structure consists of a seven amino acid peptide loop and a hydrophobic fatty acid chain. the production of designer surfactins, made by changing the number and composition of amino acids and fatty acids has proven to be an effective strategy for screening large numbers of lipopeptides for biological activity, but most current research focuses on their anticancer [4] , antimicrobial [5] and insulin delivery [6] properties but not on their antiviral potential. fatty acid chain length is critical to the antimicrobial effect of synthetic polymyxin analogues [7] , while the specific antimicrobial spectrum of these analogues depends the amino acids in the lipopeptide [8] . additionally, in synthetic daptomycin analogues, the introduction of aromatic groups in the fatty acid moiety also affects antibacterial activity [9] . in this study, a series of similar lipopeptides were designed and synthesized using surfactin as a template. these analogues differ from each other in the number of hydrophobic amino acids, number of hydrophilic groups, charge properties, amino acid chirality, position of hydrophilic amino acids, and aromatic groups in the fatty chain. porcine epidemic diarrhea virus (pedv), a coronavirus, can infect pigs of all ages, but is especially virulent in newborn piglets, causing diarrhea, dehydration and even death [10] . outbreaks of pedv have been reported in many countries [11] [12] [13] [14] , and have caused immeasurable losses to the global swine industry. recent research in our lab showed that surfactin acts directly on the pedv envelope and inhibits the fusion process with the host cell membrane. in this study, pedv was used as a target to screen chemically synthesized surfactin analogues for enhanced antiviral properties. our results provide a theoretical basis for the development of new surfactin-derivative antiviral drugs. all lipopeptides were synthesized by synpeptide co., ltd (shanghai, china). mass spectrometry was used to determination molecular weight and to confirm product sequences. the purity of all synthetic lipopeptide samples was over 90% by hplc. ten synthetic lipopeptide samples were named slp1 to slp10. and their sequences as table 1 . cercopithecus aethiops kidney epithelial cells (vero, atcc, ccl-81) were cultured in high glucose dmem (gibco, us), supplemented with 10% fetal bovine serum (fbs, gibco), at 37 c in a 5% co 2 humidified atmosphere. cells were routinely seeded at a density of 2×10 5 /ml vero cells were seeded at 2 × 10 5 cells/well in 24-well tissue culture plates and incubated 18-24 h at 37˚c until approximately 95% confluency was reached. 100 pfu of pedv mixed with an equal volume of slp in dmem or dmem alone were incubated 10 minutes at 37˚c, then added into the wells of the vero cells and incubated 30 minutes at 4˚c. cells were washed 3 times with dmem then overlaid with dmem/1% agar and incubated 72 hours at 37˚c. the cells were fixed with 4% formaldehyde, then stained with 0.1% crystal violet after the agar overlay was removed. the data are representative of 3 biological replicates and each plaque assay was performed in triplicate and values are expressed as means ± the standard deviation. curve fitting and ec 50 were calculated with graphpad prism 6 using the log[inhibitor] vs. response equation. hemolytic activity was measured according to the methods described in jingdan [15] , with some modifications. briefly, serial dilutions of slps were added to 200 ul of a 1% suspension of porcine rbcs in pbs, followed by incubation for 1 h at 37˚c. cells were centrifuged at 1000 g for 10 min, then 100 ul of each supernatant was transferred to wells of a 96-well plate. the optical density at 540 nm was measured using a microplate reader (infinite 200 pro, tecan group ltd., switzerland) and % hemolysis was calculated using the formula: where od s , od b , and od p represent the optical density of the slp-treated samples, negative control and positive control, respectively. 1% triton x-100 was used for the positive control. each sample was run in triplicate. surface tensions were measured using the ring method [16] . samples were freshly prepared in a testing flask and allowed to stand for 30 minutes at 22˚c. surface tension measurements were made using a surface tensiometer (hld-lst-ii, henlida co., ltd, china). linear regressions of the drop and the flat area were performed separately for the surface tension-concentration curve. the concentration at the intersection of the two lines is the critical micelle concentration (cmc). time of addition assay was performed according to the procedures described [17] , with modifications. confluent vero cells in 12-well plates were infected with 1000 pfu of pedv and incubated for 1 hr at 4˚c to synchronize infection. the inoculum was removed and 1 ml of 37˚c dmem was added to each well, cells were then placed at 37˚c in a humidified incubator. at the indicated time points, slp5, surfactin, or dielaidoyl-phosphatidylethanolamine (depe, avantipolarlipids, alabaster, al) was added dropwise to cells or virus to a final concentration of 50 μg/ml, 20 μg/ml, or 5 μg/ml and incubated a further 12 hours at 37˚c. viral nucleic acid and protein levels in the cells were measured by qrt-pcr and western-blot respectively. the data shown is representative of 3 independent experiments. total rna was extracted from cells using trizol reagent (invitrogen) according to the manufacturer's instructions. cdna was generated by reverse transcription using hiscript tm qrt supermix for qpcr (vazyme) according to the manufacturer's instructions. pedv nucleic acid levels were assessed by measuring the viral nucleoprotein (n) using qrt-pcr with the takara sybr green qpcr kit (takara). primer sequences were as follows: gene level was calculated using the comparative ct method and normalized to the endogenous levels of gapdh. boiled cell lysates were subjected to sds-page then transferred to pvdf membrane (roche, basel, switzerland) using a semi-dry transfer apparatus (ge, little chalfont, buckinghamshire, uk). membranes were blocked in 5% non-fat milk in tbs containing 0.01% tween-20 (tbst) then incubated overnight at 4˚c with anti-pedv n-protein monoclonal antibody (median diagnostics, south korea) diluted in tbst with 1% bsa. membranes were washed 30 min in tbst followed by incubation for 1 h at rt with 1:5000 hrp goat anti-mouse igg antibody (abgent, san diego, ca, usa) then thrice washed for 15 min in tbst. membranes were incubated with ecl reagent (thermo, waltham, ma, usa) and imaged using a chemidoc™ system (bio-rad, hercules, ca, usa). native surfactin is an amphiphilic cyclic lipopeptide, it consists of a heptadine interlinked with fatty acid. using the native structure as a template, we designed 10 surfactin analogues that were then commercially synthesized ( fig 1a) . slp1 consists of a heptadiene identical to the peptide of native surfactin, and a fatty acid chain the same length as in native surfactin. the n' of heptadine is linked to the fatty acid and the c'-is aminated. slp2 and slp 3 have one and two fewer hydrophobic amino acids respectively, than slp1. relative to spl3, slp4 has one fewer hydrophilic amino acids, while slp5 has a free carboxyl at its c'-end that acts as an additional hydrophilic group. slp6 replaces the two acidic amino acids of slp 3 with lysine (a basic amino acid). slp7 is a version of slp3 that contains only l-amino acids. the two hydrophilic amino acids of slp8 are distant from each other, but are in close proximity in slp9. slp 10 is identical to slp3 except for a diphenyl group in the fatty acid moiety. the mass spectra of the slps are shown in fig 1b and are consistent with the expected molecular weights. in all cases, slp purity exceeded 90%. the slps were tested for anti-pedv activity using a plaque reduction assay. serial dilutions of slps were incubated with an equal volume of pedv then aliquoted onto precooled cells in 12-well plates. after the virus has adsorbed to the cells at 4˚c, slps were washed away. the adsorbed virus particles were then counted by plaque forming assays (fig 2a) . the results were fitted to a sigmoid-curve and plotted fig 2b. all slps have potent anti-pedv effects above 50 μg/ml, but differences in their anti-pedv effect. slp2, slp4, slp6, and slp8 have a higher potency than surfactin. hemolysis is commonly used to assess the biosafety of lipopeptides [18, 19] . as shown in fig 3, slps have a range hemolytic activities. those of slp1, slp2, slp3, and surfactin are similar, suggesting that the number of hydrophobic amino acids has little effect on hemolytic activity. however, other modifications significantly affect activity; slp4, slp3 and slp5 have one, two and three carboxyl groups respectively, and the hemolytic activity is reduced in turn. in addition, the chiral differences that distinguish slp7 and slp3 at two amino acid residues, and the differences in the arrangement of amino acids between slp8 and slp9 also affect hemolytic activity. these results indicate that amino acid composition by itself does not completely determine the characteristics of slps. it is noteworthy that slp8 has an unexpected effect on porcine erythrocytes. although slp8 has a low hemolytic activity, it prevents the precipitation of red bloods under our assay conditions at concentrations above 50 μg/ml. the selectivity index (si) was determined as the ratio of the 50% cytotoxicity concentration (cc 50 ) to the 50% effective concentration (ec 50 ). as shown in table 2 , slp8 and slp5 have the highest and second-highest si values respectively. given the anti-sedimentation effect of slp8 on porcine blood cells, slp5 was selected as the most promising candidate. although slp4 and slp6 have stronger antiviral activity than surfactin, their si is lower due to their high hemolytic activity. the cmc was calculated by measuring the surface tension of each slp over a range of concentrations. as shown in fig 4, surface tension decreases as slp concentration increases, and as it approaches its lowest value, the curve exhibits an inflection point when the slp concentration reaches cmc. linear regressions were performed independently using the data on the descending part of the curve below the cmc and the equilibrium part above the cmc. the intersection of the linear regression lines corresponds to the cmc (table 3) . in order to explore the relationship between cmc, anti-pedv activity, and hemolytic activity, scatter plots for pairs of assays (fig 5) . each plot contains 11 points representing the characteristic indices of slp1 to 10 and surfactin. the distribution of points has no obvious regularity in any of the three plots, and the linear regression results are not significant. we conclude that the anti-pedv and hemolytic activities of the slps examined in this study are not related to their surfactant activity (represented by the cmc). in addition, there is no significant correlation between anti-pedv activity and hemolytic activity. synthetic lipopeptides that combine greater antiviral activity with lower cytotoxicity are still to be found, and the specific structure-activity relationship needs to be further elucidated. since slp5 was the most promising surfactin analogue, we chose it for further study. time of addition assays were performed to determine whether the slp5 exerts its anti-pedv effect at the same stage during infection as surfactin. since surfactin acts directly on virions, the experiment was designed to examine events early in the infection process. fig 6a summarizes the experimental plan and the eight treatments tested. briefly, slp5, surfactin, or depe was added to virus alone, cells alone, or to cells and virus together prior to infection, during virus adsorption, during virus invasion (1 h post infection at 37˚c), or during replication (1-12 hpi). samples were harvested 12 hours after infection, and analyzed to measure cellular levels of viral protein (fig 6b and 6c ) and viral rna ( fig 6d) . as expected for a normal component of the cell membrane, depe did not affect pedv replication at any stage, while slp5 and surfactin exhibited antiviral activity at specific stages. for example, when slp5 or surfactin are present throughout the experiment (treatment group 2), little pedv replication occurs, as indicated by nucleic acid and protein levels. the same is true in treatment group 5, indicating that both compounds act directly on the virus. other antiviral mechanisms of slp5 and surfactin cannot be ruled out since pedv replication is inhibited to different degrees in some of the other treatment groups. wedge-shaped lipids in which the hydrophilic head has a larger cross-sectional area than the hydrophobic tail, are potential membrane fusion inhibitors [20, 21] . in a recent study of rigid amphipathic fusion inhibitors, compounds with deoxyribose or acetate as hydrophilic moieties had anti-hsv effects [22] . in addition, a recent study on antimicrobial activity of synthetic lipopeptides reported that lipopeptides with two to four positive charges and 16 carbon atoms in the lipid chain have potent antimicrobial activity [23] . fatty acid chain length from 8 to 16 carbon atoms is positively correlated with antimicrobial activity, but is also positively correlated with hemolytic activity and membrane selectivity [24] . these factors must be considered in the design of new lipopeptides, and the structure-function relationships warrant further study. here we investigated lipopeptides containing two and three negative charges, and two positive charges, all with a fatty acid chain of 16 carbon atoms. all of these lipopeptides had antiviral activity. in a previously published study we demonstrated that surfactin has antiviral activity as membrane fusion inhibitor [3] . in order to improve the viral envelope selectivity of surfactin, we designed 10 analogues with altered peptide amino acids, but kept the length of fatty acid chain of 16 carbon atoms. compared with slp3, slp5 has one additional carboxyl group at the end of the peptide, but the antiviral effect is similar. although slp4 has one less hydrophilic amino acid than slp3, its antiviral activity increases about 3-fold. this result indicates that the hydrophilic portion of the wedge-shaped lipid needs to be sized appropriately for better antiviral function. in addition, slp8 differs from slp3 only in the order of two amino acids, but the antiviral potency of slp8 is 6.5 times greater, indicating the complexity of the relationship between molecular structure and antiviral effects. the cationic properties of synthetic lipopeptides are thought to be related to their antibacterial and hemolytic activities [25] . slp6, the only cationic lipopeptide examined in this study, has the strongest hemolytic activity, consistent with this hypothesis. cyclic lipopeptides are reported to more readily lyse bacterial membranes than linear or branched lipopeptides [26] . in agreement, we found that the hemolytic activity of slp1 (a linear molecule, identical in amino acid sequence to surfactin) is slightly weaker than that of surfactin, which has a cyclic structure. it has also been reported that the total hydrophobicity of synthetic lipopeptides is inversely related to hemolytic activity, although this conclusion was from data on only 3 related samples [27] . our results do not support this conclusion. the relative hydrophobicity of slp1, 2, and 3 is slp1>slp2>slp3, as indicated by their hplc retention times (17.6, 16.7, and 14.5 min, respectively). however, their hemolytic activity is not significantly related to the total hydrophobicity. in summary, we synthesized 10 surfactin analogues and characterized them to identify those with enhanced anti-viral properties. slp5 equaled and slp8 exceeded surfactin's anti-pedv activity and both compounds had greatly reduced hemolytic activity, making their selection indexes 13 and 21 times larger respectively than surfactin's. however, slp8 exhibited a peculiar inhibition of red blood cell sedimentation. slp5 is therefore a more promising synthetic surfactin analogue. compared with surfactin, the structure of slp5 is distinguished by its linear lipopeptide and the additional carboxyl group at the c' of the peptide. slp5 also has two fewer hydrophobic amino acids than surfactin, this reduces the cost of synthesis while having little effect on antiviral activity. since surfactin has been shown to protect piglets from pedv challenge [3] , the in vivo antiviral properties of surfactin analogues needs to be tested. furthermore, with respect to the future use of synthetic surfactins to control or ameliorate infection in pigs, the broader spectrum physiological activities of surfactin analogues needs further study and side effects beyond hemolytic toxicity need to be explored. our study suggests that slp5, and other surfactin analogues, should be the object of in-depth study in order to develop a safer broad-spectrum family of surfactin antiviral agents. synthetic surfactin analogues have improved anti-pedv properties conceptualization: lvfeng yuan, qian yang. the pedv genome was detected by qrt-pcr. the experiment was repeated three times, normalized against group 1, and plotted as mean ± sd. � , p < 0.05; �� , p < 0.01 in two-tail t-test, for each reagent, compared with group 1. https://doi.org/10.1371/journal.pone.0215227.g006 synthetic surfactin analogues have improved anti-pedv properties mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from bacillus subtilis antiviral activity of antimicrobial lipopeptide from bacillus subtilis fmbj against pseudorabies virus, porcine parvovirus, newcastle disease virus and infectious bursal disease virus in vitro surfactin inhibits membrane fusion during invasion of epithelial cells by enveloped viruses a multicomponent macrocyclization strategy to natural product-like cyclic lipopeptides: synthesis and anticancer evaluation of surfactin and mycosubtilin analogues semisynthetic lipopeptides derived from nisin display antibacterial activity and lipid ii binding on par with that of the parent compound surfactin variants for intra-intestinal delivery of insulin probing the penetration of antimicrobial polymyxin lipopeptides into gram-negative bacteria a bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity discovery and development of surotomycin for the treatment of clostridium difficile outbreak of porcine epidemic diarrhea in suckling piglets, china. emerging infectious diseases porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus, italy. emerging infectious diseases pig jejunum protein profile changes in response to a porcine epidemic diarrhea virus challenge comparison of porcine epidemic diarrhea viruses from germany and the united states a potential biopreservative: chemical composition, antibacterial and hemolytic activities of leaves essential oil from alpinia guinanensis surface tension examination of various liquid oral, nasal, and ophthalmic dosage forms discovery of cyclosporine a and its analogs as broad-spectrum anti-influenza drugs with a high in vitro genetic barrier of drug resistance improving the biological activity of the antimicrobial peptide anoplin by membrane anchoring through a lipophilic amino acid derivative interaction of acylated and substituted antimicrobial peptide analogs with phospholipid-polydiacetylene vesicles. correlation with their biological properties. chemical biology & drug design 5-(perylen-3-yl)ethynyl-arabino-uridine (auy11), an arabino-based rigid amphipathic fusion inhibitor, targets virion envelope lipids to inhibit fusion of influenza virus, hepatitis c virus, and other enveloped viruses rigid amphipathic fusion inhibitors, small molecule antiviral compounds against enveloped viruses antivirals acting on viral envelopes via biophysical mechanisms of action cationic net charge and counter ion type as antimicrobial activity determinant factors of short lipopeptides characterization of antimicrobial and hemolytic properties of short synthetic cationic lipopeptides based on qsar/qstr approach roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in peptide-membrane interactions short cationic lipopeptides as effective antibacterial agents: design, physicochemical properties and biological evaluation identification of novel cyclic lipopeptides from a positional scanning combinatorial library with enhanced antibacterial and antibiofilm activities key: cord-010053-kniq2mbw authors: lee, sunhee; lee, dong‐uk; noh, yun‐hee; lee, seung‐chul; choi, hwan‐won; yang, hyoung‐seok; seol, jun‐ho; mun, seong hwan; kang, won‐myoung; yoo, hyekyung; lee, changhee title: molecular characteristics and pathogenic assessment of porcine epidemic diarrhoea virus isolates from the 2018 endemic outbreaks on jeju island, south korea date: 2019-05-20 journal: transbound emerg dis doi: 10.1111/tbed.13219 sha: doc_id: 10053 cord_uid: kniq2mbw since the 2013–2014 incursion of the virulent g2b porcine epidemic diarrhoea virus (pedv) pandemic strains in south korea, frequent moderate‐scale regional outbreaks have recurred. in particular, areas of jeju island with extensive swine production have faced repeated epidemics since the re‐emergence in 2014. the current study reports the complete genome sequences and molecular characterization of the representative pedv strains responsible for the 2018 endemic outbreaks on jeju island. all isolates were determined to belong genetically to the highly pathogenic pandemic g2b group. full‐length genome sizes of four isolates differed from that of the g2b epidemic field strain due to insertion or deletion (del) mutations in the non‐structural protein (nsp)‐ or spike (s) protein‐coding regions. the 2018 jeju isolates shared 96.7%–98.7% and 98.5%–99.4% identity at the s gene and whole‐genome levels, respectively, compared to global g2b pedv strains. genetic and phylogenetic analyses indicated that the 2018 isolates were closest to the 2014 g2b re‐emergent jeju strains, but appeared to have undergone substantial rapid independent evolution. among the isolates, a notable nsp3 del variant strain, kor/knu‐1807/2018, was isolated and propagated by continuous passages in vero cells, and displayed typical pedv‐induced syncytia formation. genomic sequencing identified a unique 8‐nt del in the extreme c‐terminal region of the s gene at the 4th passage (knu‐1807‐p4) compared to its original sample. this del resulted in the premature termination of s by nine amino acid residues (evfekvhvq), which contained a kxhxx motif that is a potential endoplasmic reticulum retrieval signal. in vivo animal studies showed that variant strain knu‐1807 had decreased virulence in suckling piglets. these results advance our knowledge regarding the genetic variation and pathogenicity of the g2b pedv endemic strains prevalent in jeju swine herds in south korea. since its emergence in south korea in 1992, small-to large-scale pedv epizootics have occurred annually throughout the nation, leading to substantial economic losses in domestic pig production (lee, 2015 (lee, , 2019 . the 2013 ped pandemic that ravaged the united states (stevenson et al., 2013) also struck the korean peninsula and decimated more than 40% of the pig farms across the country during 2013-2014 (lee, 2015 (lee, , 2019 . subsequently, in late march 2014, the virus invaded jeju island located 80 km from the south korea mainland at the closest point, which had maintained pedv-naïve status for a decade, causing massive neonatal mortality in provincial herds. genetic and phylogenetic analyses revealed that the re-emergent jeju island pedv isolates were most closely related to the pandemic genogroup 2b (g2b) strains that were responsible for the 2013-2014 global outbreaks, suggesting a direct introduction of the virus from the mainland of south korea via unknown contaminating sources . even with province-wide vaccination or intentional virus-exposure practices being implemented in order to provide herd immunity around the areas that contain dense swine populations, pedv has continued to plague the provincial pork industry. since several pig farms have experienced recurrent pedv outbreaks within a single year, ped has become endemic on jeju island . in this study, we determined the complete genome sequences of field isolates on jeju island to investigate the diversity of the pedvs responsible for the ongoing endemic outbreaks. additionally, we isolated and serially cultured a novel pedv strain, kor/knu-1807/2018, in cell culture and investigated its genotypic and phenotypic characteristics in vitro and in vivo. in early 2018, mild sporadic suspect-pedv outbreaks with low mortality rates in newborn piglets occurred on several farms in the hallim and daejeong areas of jeju province. small intestine (si) specimens were collected at 18 different pig farms located in those districts from january through june 2018 from dead piglets that had acute diarrhoea. intestinal homogenates were prepared as 10% (wt/vol) suspensions in phosphate-buffered saline (pbs) using a magna lyser instrument (roche diagnostics, mannheim, germany) with three rounds of 15 s at a force of 8,000 g. the suspensions were then vortexed and centrifuged for 10 min at 4,500 g (hanil centrifuge fleta5, incheon, south korea). the clarified supernatants were initially subjected to rt-pcr analysis using an i-tge/ped detection kit (intron biotechnology) according to the manufacturer's instructions. pedv-positive samples were filtered through a 0.22-μm-pore syringe filter (millipore) and stored at −80°c until subsequent sequencing analysis and virus isolation were performed. the s glycoprotein gene sequences of the virus isolates were determined by traditional sanger methods. two overlapping cdna fragments spanning the entire s gene of each isolate were amplified by rt-pcr as previously described (lee, park, kim, & lee, 2010) . the individual cdna amplicons were gel-purified, cloned using the pgem-t easy vector system (promega) and sequenced in both directions using two commercial vector-specific t7 and sp6 primers and gene-specific primers. in addition, the complete genomes of representative pedv field strains were also sequenced. ten overlapping cdna fragments spanning the entire genome of each virus strain were rt-pcr-amplified as described previously (lee, kim, & lee, 2015; and each pcr product was sequenced as described above. the 5′ and 3′ ends of the genomes of the individual isolates were determined by rapid amplification of cdna ends (race) as described previously . the full-length s gene or whole-genome sequences of the 2018 viruses have been deposited in the genbank database under the accession numbers shown in figure 2a. the sequences of 66 fully sequenced s genes and 39 complete genomes of global pedv isolates were independently used in sequence alignments and phylogenetic analyses. multiple sequence alignments were generated using the clustalx 2.0 program (thompson, gibson, plewniak, jeanmougin, & higgins, 1997) and the percentages of nucleotide sequence divergences were assessed using the same software. phylogenetic trees were constructed from the aligned nucleotide or amino acid sequences using the neighbourjoining method and subsequently subjected to bootstrap analysis with 1,000 replicates to determine the percentage reliability values of each internal node of the tree (saitou & nei, 1987) . all phylogenetic trees were generated using mega 4.0 software (tamura, dudley, nei, & kumar, 2007) . porcine epidemic diarrhoea virus isolation was performed using vero cells in the presence of trypsin (usb) as described previously . briefly, inocula were prepared by adding trypsin (usb) to intestinal suspensions to a final concentration of 10 μg/ml. confluent vero cells grown in 6-well plates were washed with pbs and inoculated with 400 μl of each trypsincontaining inoculum. after incubating at 37°c for 1 hr to allow for viral adsorption, 2 ml of virus growth medium consisting of alpha minimum essential medium (α-mem; invitrogen) supplemented with antibiotic-antimycotic solutions (100×; invitrogen), 0.3% tryptose phosphate broth (tpb; sigma), 0.02% yeast extract (difco), 10 mm hepes (invitrogen) and 5 μg/ml of trypsin was added to each well. the inoculated cells were maintained at 37°c under 5% co 2 and monitored daily for cytopathic effects (cpe). when ~70% of cells showed cpe, the infected cells were subjected to three rounds of freezing and thawing. the culture supernatants were then collected and centrifuged for 10 min at 400 g and filtered through a 0.22-μm pore filter. the clarified supernatants were aliquoted and stored at −80°c as passage 1 (p1) viral stocks for use in plaque purification and subsequent serial passaging. if cpe and rt-pcr results were negative after five blind passages, virus isolation was considered negative for those samples. vero cells grown on microscope coverslips placed in 6-well tissue culture plates were mock infected or infected with pedv at a multiplicity of infection (moi) of 0.1. the virus-infected cells were cultured until 24 hr, fixed with 4% paraformaldehyde for 10 min at room temperature (rt) and permeabilized with 0.2% triton x-100 in pbs at rt for 10 min. the cells were blocked with 1% bovine serum albumin (bsa) in pbs for 30 min at rt and then incubated for 2 hr with a monoclonal antibody (mab) specific for pedv n protein (choogang vaccine laboratories). after being washed five times with pbs, the cells were incubated for 1 hr at rt with a goat anti-mouse secondary antibody conjugated to alexa fluor 488 (invitrogen) followed by counterstaining with 4′,6-diamidino-2phenylindole (dapi; sigma). the coverslips were mounted onto glass microscope slides using mounting buffer and the stained cells were visualized using a fluorescence leica dm il led microscope (leica). vero cells were infected with each passage of knu-1807 virus stock in the presence of trypsin as described above. the culture supernatants were collected at 24 or 48 hr post-infection (hpi) when 70% cpe had commonly developed. for growth kinetics experiments, supernatants were harvested from cells infected with each selected passage virus at various time points (6, 12, 24, 36 and 48 hpi) and stored at −80°c. virus titres were measured by end-point titration in 96-well plates using 10-fold serial dilutions of the samples in triplicate for each dilution to determine the amount of virus required to produce cpe in 50% of the inoculated vero cells. the 50% tissue culture infectious dose (tcid 50 ) per ml of virus stock was calculated using the reed-muench method (reed & muench, 1938) . viral rna was extracted from virus supernatants from infected vero cells and faecal specimens using an i-tge/ped detection kit according to the manufacturer's protocol. quantitative real-time rt-pcr was performed using a one step primescript rt-pcr kit (takara) and primers (forward primer 5′-acgtccctttactttcaattcaca-3′, reverse primer 5′-tatacttggtacacacatccagagtca-3′) and a probe (5′-fam-tgagttgattactggcacgcctaaaccac-bhq1-3′) described elsewhere (kim et al., 2007; sagong & lee, 2011) . amplification of the reaction mixtures was performed using a thermal cycler dice real time system (takara) and the results were analysed using software as described previously sagong & lee, 2011 ). the in vivo swine studies were performed at the choongang vaccine laboratory animal facility under the guidelines established by its institutional animal care and use committee. a total of nine 3-dayold suckling piglets were obtained from commercial cross-bred sows (great yorkshire × dutch landrace) at a conventional breeding farm with a good health record and either vaccinated against pedv or no known prior ped outbreak. all animals were confirmed negative for pedv, transmissible gastroenteritis virus (tgev), porcine deltacoronavirus and porcine rotaviruses by virus-specific rt-pcr analysis of rectal swabs and determined to be free of antibodies to pedv, tgev and porcine reproductive and respiratory syndrome virus (prrsv) by serum neutralization tests as described previously and a commercial prrsv antibody elisa kit (herdchek prrs x3; idexx laboratories). pigs were randomly assigned to three experimental groups: the highly virulent knu-141112-p5-inoculated group (n = 3) , the knu-1807-p10-inoculated group (n = 4) and the sham-inoculated control group (n = 2). animals were fed commercial milk replacer (3-4 times daily) and had ad libitum access to water for the 5-day duration of the study. following a 2-day acclimation period, piglets (5-day old) in the virus-inoculated groups received an oral 1-ml dose of 10 3.0 tcid 50 /ml of the appropriate virus, which was equivalent to 100 median pig diarrhoea dose pdd 50 of knu-141112 (baek et al., 2016; lee et al., 2015 . the sham-inoculated pigs were administered with cell culture media as a placebo. animals were monitored three times daily throughout the experiment for clinical signs of vomiting and diarrhoea and for mortality. stool samples from the pigs in all groups were collected prior to inoculation and thereon daily using 16-inch cotton-tipped swabs. the pedv faecal shedding titres were determined by real-time rt-qpcr as described above. a pedv isolate with a known infectivity titre was 10-fold serially diluted to generate a standard curve in each pcr at necropsy, small intestine tissue specimens (<3 mm thick) were collected from each piglet, fixed in 10% formalin for 24 hr at rt, and embedded in paraffin according to standard laboratory procedures. the formalin-fixed paraffin-embedded tissues were cut at 5-8-μm thick sections using a microtome (leica), floated in a 40°c water bath containing distilled water, and transferred to glass slides. the tissues were then deparaffinized in xylene for 5 min and rehydrated in decreasing concentrations of ethanol (100%, 95%, 90%, 80% and 70%, respectively) for 3 min each. the deparaffinized intestinal tissue sections were stained with haematoxylin and eosin (sigma) for histopathology or subjected to immunohistochemistry (ihc) using pedv n-specific mab as described previously . villous height and crypt depth were also measured throughout the h&e-stained jejunal sections and the mean ratio of jejunal villous height to crypt depth (vh:cd) was calculated as described previously (jung, kim, ha, choi, & chae, 2006 ). all values are expressed as the means ± standard deviation of the means (sdm). all statistical significances were evaluated by a we in vero cells, reaching a titre >10 6 tcid 50 /ml by 12 hpi (figure 3b ). in addition, we sequenced the coding region of nsp3 and the entire s gene of strain knu-1807 at the first productive passages of p4 and table 4 ). these genetic alterations that occurred in p4 remained unchanged through p10 and no additional variations emerged in the s-coding region during the subsequent serial passages. the partial or complete genome sequences of all the cell culture-passaged viruses were compared to the original knu-1807-si and the results are summarized in table 4 . since the knu-1807 strain with the truncated s cytoplasmic tail (0) 100 (0) nsp10 (405) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) 99.2 (1) nsp11 (54) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) nsp12 ( the number of individual differences in the 5′-utr, protein-coding region, and 3′-utr, respectively. f i g u r e 2 phylogenetic analyses based on the nucleotide sequences of the spike (s) genes (a) and full-length genomes (b) of the pedv strains. a region of the s gene and the complete genome sequence of tgev were included as the outgroups in each tree. multiple sequence alignments were performed using clustalx software and phylogenetic trees were constructed from the aligned nucleotide sequences using the neighbour-joining method. numbers at each branch are bootstrap values greater than 50% based on 1,000 replicates. the names of the strains, countries and dates ( in the current study, determined the complete genome sequences similar to other coronavirus replicase-encoded nsps, many pedv nsps (nsp1, nsp3, nsp5, nsp7, nsp14, nsp15 and nsp16) function as interferon (ifn) antagonists that modulate the innate immune response (wang et al., 2016; zhang, shi, & yoo, 2016) . along with variations extensively dispersed throughout the s gene, it would be interesting to identify mutations in these nsp genes, including indels that possibly contribute to the pathogenesis of pedv. a majority of the non-silent point mutations, which appeared to have resulted from their continuous accumulation in the field over the past 3-4 years, occurred in the orf1ab region encoding 16 nsps, particularly in nsp3. these 29-41 variations seem to be significant since the cell culture-attenuated g2b strain contained only 4-aa changes over 100 serial passages when compared to the virulent parental strain . although no indels arose in the attenuated g2b virus s del5/orf3 , the attenuated g1a-derived vaccine strains possess an 8-aa del in nsp3, which overlaps or is located 12 or 18 aa downstream of those found in the recent g2b field ta b l e 4 nucleotide and amino acid changes of knu-1807 during serial passages in cell culture strains, implicating the potential involvement of the nsp3 del in attenuation ( figure 1b) . although nsp3 critically acts as a pl pro that post-transcriptionally cleaves replicase polyproteins into functional nsps, the dels present in the glu-rich acidic region of nsp3 have no effect on its own roles and therefore are dispensable for the replication of coronaviruses, including pedv (lee & lee, 2018; lei, kusov, & hilgenfeld, 2018) . consistent with our previous study (lee & lee, 2018) , this tolerance for the nsp3 del during pedv replication was reproduced in the current study as shown in to contain a homologous 3-aa del in nsp3, consistent with a typical farm-to-farm transmission of pedv via lax biosecurity. in addition to genetic drift under field conditions, an outstanding del in the cytosolic endodomain of s was identified in the novel nsp3-del pedv knu-1807 isolate since the first productive passage in cell culture (p4) and subsequently retained, thereby resulting in a 9-aa del at the end of the knu-1807-p4 s protein. like other coronaviruses, the pedv s glycoprotein can be functionally divided into two subdomains, s1 responsible for binding to cell receptor(s) and s2 involved in direct fusion between the viral and cellular membranes (lee, 2015) . the last 5-aa (kvhvq) of the cytoplasmic tail of s is known to be a potential er retrieval signal with its kxhxx motif and loss of this motif increases cell fusion activity by defecting the er retention of s protein and promoting their transport to the cell surface lin et al., 2017; shirato et al., 2011) . since knu-1807-p4 exhibited a premature termination of the s protein by 9-aa residues (evfekvhvq) that included the er retrieval motif, it would be anticipated that this strain would demonstrate increased fusion activity in vitro. our data revealed that the knu-1807 strain in the s gene, resulting in a stop codon and a 9-aa (evfekvhvq) del. the s c-del9 identified in this study differed from aforementioned strains in that the early termination was caused by a combination of a novel nt del and a −1 frameshift event that occurred in the most primitive cell culture passage p4. it is possible that the s del naturally emerged during the initial culturing in the vero cell via an unknown mechanism. indeed, the occurrence of a large 197-aa s del in the n-terminus of the s protein that was absent in the original sample has been reported during the first cell culture passage of the us g2b pc177 strain (oka et al., 2014) . we were consistently unable to detect the same s c-del9 in the original knu-1807-si sample by deep genome sequencing (data not shown). however, since coronaviruses are known to innately exist as quasispecies or as mixed populations of several strains (zhang et al., 2007) , we cannot exclude the possibility that this variant was initially present as a minor proportion in the clinical sample and was able to replicate more competently in cell culture instead of the emergence of the extraordinarily del during the earliest stages of cell adaption. similarly, a chinese field g2b strain, fl2013, that naturally contains a unique 21-nt del in the extreme c-terminus of its s protein, leading to a 7-aa (fekvhvq ) s c-del pattern, has been found to have reduced virulence to newborn piglets (zhang et al., 2015) . thus, it is important to further surveil whether s c-del variant strains naturally circulate in korean pig populations. since a growing body of evidence proposes the early termination of the s protein and the extinction of its er retention signal as being a marker for pedv attenuation, we investigated whether the virulence of the knu-1807 virus would be markedly less than that previously reported for pedv pathogenesis. our data revealed that despite similarities of macroscopic and microscopic small intestine lesions, the knu-1807 virus possessed weakened pathogenicity in experimentally inoculated piglets compared to the virulent knu-141112 strain in terms of disease severity of clinical presentation, including the mortality rate and onset of virus shedding, indicating the involvement of the extreme c-terminal region of the s protein in virulence. however, we were unable to observe a fully attenuated phenotypes for knu-1807 in inoculated animals compared with those seen in our previous study . this suggested that del mutations in several parts of the s protein in conclusion, pedv continues to endemically affect pig farms on jeju island, giving rise to moderate-to-severe clinical disease associated with ped in infected piglets. however, the loss of neonates to death varies among litters and between farms and is less than that reported for severe g2b epidemics, which approach 100% mortality in newborn piglets. this mild-to-moderate outbreak scenario seen on jeju island may be due to herd immunity developed from vaccination and intentional infection since the re-emergence of pedv in the area. another possibility is that genetic variation and/or del in nsps and s protein have arisen under field circumstances leading to the evasion of host immune defenses, such as ifn and neutralizing antibodies, and consequently alter viral pathogenicity, leading to endemic and low-pathogenic outbreaks in the field. thus, cutting-edge research using reverse genetics will be necessary to provide fundamental insights into the specific role of nsp and s gene mutations in pedv pathogenesis. more importantly, the current study confirmed that the contemporary field g2b isolates have nearly 4% amino acid sequence divergence in the s protein compared to the 2013-2014 domestic g2b strains. this mutation rate is approximately twice as high as that in recent mainland strains of pedv (lee & lee, 2018) . furthermore, the evolutionary rate estimated for the s gene of pedv g2b jeju island strains was 14.80 × 10 -4 substitutions/site/ year, whereas that for the g2b mainland strains was 7.18 × 10 -4 substitutions/site/year (lee & lee, 2018) . these results indicated that ongoing genetic drift appears to be faster on jeju island than the authors declare that they have no conflict of interest. changhee lee https://orcid.org/0000-0002-5930-5461 efficacy of an inactivated genotype 2b porcine epidemic diarrhea virus vaccine in neonatal piglets nidovirales: evolving the largest rna virus genome the effects of transplacental porcine circovirus type 2 infection on porcine epidemic diarrhoea virus-induced enteritis in preweaning piglets multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus porcine epidemic diarrhea virus: an emerging and reemerging epizootic swine virus porcine epidemic 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and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene a simple method of estimating fifty percent endpoints porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-β production by inhibiting irf3 activation in immortalized porcine alveolar macrophages coronaviruses the neighbor-joining method: a new method for reconstructing phylogenetic trees mutation in the cytoplasmic retrieval signal of porcine epidemic diarrhea virus spike (s) protein is responsible for enhanced fusion activity emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences mega4: molecular evolutionary genetics analysis (mega) software version 4.0 the clustalx windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools porcine epidemic diarrhea virus 3c-like protease regulates its interferon antagonism by cleaving nemo an economic evaluation of intervention strategies for porcine epidemic diarrhea (ped) suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp1 quasispecies of bovine enteric and respiratory coronaviruses based on complete genome sequences and genetic changes after tissue culture adaptation identification and pathogenicity of a variant porcine epidemic diarrhea virus field strain with reduced virulence key: cord-275499-25dp6u68 authors: tan, zhen; dong, wanting; ding, yaqun; ding, xiangdong; zhang, qin; jiang, li title: porcine epidemic diarrhea altered colonic microbiota communities in suckling piglets date: 2019-12-30 journal: genes (basel) doi: 10.3390/genes11010044 sha: doc_id: 275499 cord_uid: 25dp6u68 porcine epidemic diarrhea (ped) is a major gastrointestinal disease afflicting suckling pigs that causes huge industrial economic losses. in this study, we investigated microbiota from the colonic mucosa and content in healthy and ped piglets. high-throughput 16s rrna gene sequencing was performed to identify inter-group differences. firmicutes, fusobacteria, proteobacteria, and bacteroidetes were the top four affected phyla. the proportion of proteobacteria was higher in infected than in healthy piglets, and the opposite was observed for bacteroidetes (more than four-fold higher in the healthy group). in the infected group, fusobacterium accounted for 36.56% and 21.61% in the colonic mucosa and contents, respectively, while in the healthy group, they comprised 22.53% and 12.67%, respectively. the percentage of lactobacillus in healthy colons (15.63%) was considerably higher than that in the disease group (<10%). in both the colonic mucosa and contents, functional enrichment differed significantly between healthy and diseased groups. overall, infection with the ped virus increased the proportion of harmful bacteria and decreased the proportion of beneficial bacteria in the colons of piglets. targeting intestinal microbiota could be a promising method for ped prevention, thus opening new avenues for future research. to minimize the effects of random sequencing errors, raw fastq files were demultiplexed, quality-filtered using trimmomatic version 0.33, and merged using flash version 1.2.7 [25, 26] . to obtain high-quality tag sequences, initial base sites with phred score <20 were truncated, tags were filtered out if their continuous high-quality base length was less than three-quarters of the whole sequence, and chimeric sequences were removed in uchime version 4.2 [27] . sequences with ≥97% similarity were assigned to the same operational taxonomic units (otus) using usearch version 10.0 [28] . a representative sequence for each otu was screened and taxonomically analyzed against the 16s rrna database silva, using ribosomal database project classifier version 2.2 [29] . representative sequences were subjected to multiple sequence alignment and a phylogenetic tree was constructed. next, the composition of each sample community was determined at the phylum, class, order, family, genus, and species level. the alpha diversity indices were evaluated using mothur version 1.30 [30] and compared with student's t-test. the beta diversity per group was calculated using principal coordinate analysis (pcoa) based on weighted unifrac distances [31, 32] in qiime version 1.8.0 [25] . between-sample differences in evolutionary information were determined using the unweighted pair-group method with arithmetic mean (upgma) [33] . images were drawn and statistical analyses were performed using r software. linear discriminant analysis (lda) effect size (lefse) was used to identify biomarkers that differed significantly between groups. analysis of variance (anova) was used to determine between-group differences in gut microbiota. statistical significance level was set at p < 0.05. functional predictions for all otus were performed using the kyoto encyclopedia of genes and genomes (kegg) and clusters of orthologous groups (cog) databases, based on the picrust-established structure of gastrointestinal microbiota [34] . we collected 1,616,525 quality-filtered and chimera-checked sequences with an average length of 440.25 bp. the mean number of reads per sample was 42,540, ranging from 31,732 to 56,822 reads. a good coverage of more than 99.9% was obtained for all samples, indicating reliable sequencing accuracy. we obtained a total of 316 otus, and 297 of these were observed in all four groups (supplementary figure s2) . the chao1 index was the highest in comh, while the shannon index was higher in coch and comh than in cocd and comd (p < 0.05) (figure 1 ). dynamic changes in gut microbial alpha diversity (a, chao1 index; b, shannon index) of pedv-infected piglets. different letters denote significant differences in alpha diversity indices between groups based on the student's t-test and adjusted for false discovery rate (fdr) (* p < 0.05, ** p < 0.01). taxonomic analysis indicated that firmicutes, fusobacteria, proteobacteria, and bacteroidetes were the top four phyla in all groups, representing approximately 95% of the sequences (figure 2a ). microorganism structure differed more between healthy and infected groups than between colonic content and mucosa. the proportions of firmicutes in colonic mucosa and content in the healthy piglets were 30.74% and 41.08%, respectively; while they were 17.86% and 35.00%, respectively, in the infected piglets. the proportions of fusobacteria in colonic mucosa and content in the infected piglets were 37.15% and 22.05%, respectively; and 23.32% and 13.01%, respectively, in the healthy piglets. the proportions of proteobacteria were higher in the infected colonic mucosa and content (35.02% and 34.15%, respectively) than that in the healthy groups (7.95% and 9.79%, respectively). in contrast to this general trend, the proportions of bacteroidetes were more than four-fold greater in healthy groups (33.17% in coch and 32.04% in comh) than in the infected groups (7.99% in cocd and 7.27% in comd). taxonomic analysis indicated that firmicutes, fusobacteria, proteobacteria, and bacteroidetes were the top four phyla in all groups, representing approximately 95% of the sequences (figure 2a ). microorganism structure differed more between healthy and infected groups than between colonic content and mucosa. the proportions of firmicutes in colonic mucosa and content in the healthy piglets were 30.74% and 41.08%, respectively; while they were 17.86% and 35.00%, respectively, in the infected piglets. the proportions of fusobacteria in colonic mucosa and content in the infected piglets were 37.15% and 22.05%, respectively; and 23.32% and 13.01%, respectively, in the healthy piglets. the proportions of proteobacteria were higher in the infected colonic mucosa and content (35.02% and 34.15%, respectively) than that in the healthy groups (7.95% and 9.79%, respectively). in contrast to this general trend, the proportions of bacteroidetes were more than four-fold greater in healthy groups (33.17% in coch and 32.04% in comh) than in the infected groups (7.99% in cocd and 7.27% in comd). taxonomic analysis indicated that firmicutes, fusobacteria, proteobacteria, and bacteroidetes were the top four phyla in all groups, representing approximately 95% of the sequences (figure 2a ). microorganism structure differed more between healthy and infected groups than between colonic content and mucosa. the proportions of firmicutes in colonic mucosa and content in the healthy piglets were 30.74% and 41.08%, respectively; while they were 17.86% and 35.00%, respectively, in the infected piglets. the proportions of fusobacteria in colonic mucosa and content in the infected piglets were 37.15% and 22.05%, respectively; and 23.32% and 13.01%, respectively, in the healthy piglets. the proportions of proteobacteria were higher in the infected colonic mucosa and content (35.02% and 34.15%, respectively) than that in the healthy groups (7.95% and 9.79%, respectively). in contrast to this general trend, the proportions of bacteroidetes were more than four-fold greater in healthy groups (33.17% in coch and 32.04% in comh) than in the infected groups (7.99% in cocd and 7.27% in comd). ten bacterial genera accounted for 70% of the total reads in all groups except coch ( figure 2b ). among healthy piglets, fusobacterium, bacteroides, and lactobacillus were dominant, while in the infected piglets, fusobacterium, actinobacillus, and escheria-shigella showed higher relative abundances. the proportions of fusobacterium in the infected groups were 36.56% (comd) and 21.61% (cocd), whereas in the healthy groups they were 22.53% (comh) and 12.67% (coch). the bacteroides percentages in the healthy piglets were 17.35% (comh) and 14.03% (coch), but only 4.54% (comd) and 4.49% (cocd) in the infected piglets. the lactobacillus proportion in coch (15.63) was considerably higher than that in the other groups. actinobacillus, however, showed noticeably higher abundance in the infected piglets (17.21% in cocd; 15.86% in comd) than in the healthy piglets (1.66% in coch; 2.26% in comh). the escherichia-shigella proportions were also higher in the infected groups (12.86% in cocd; 12.61% in comd) than in the healthy groups (2.62% in coch; 1.58% in comh). at each taxonomic level, we selected the most abundant otu sequence as representative and performed multiple sequence alignment. we then constructed a phylogenetic tree from these data ( figure 3 ). our analysis revealed that firmicutes was the most well-represented phylum (by number of genera), followed by proteobacteria and bacteroidetes. genes 2020, 11, x for peer review 5 of 13 ten bacterial genera accounted for 70% of the total reads in all groups except coch ( figure 2b ). among healthy piglets, fusobacterium, bacteroides, and lactobacillus were dominant, while in the infected piglets, fusobacterium, actinobacillus, and escheria-shigella showed higher relative abundances. the proportions of fusobacterium in the infected groups were 36.56% (comd) and 21.61% (cocd), whereas in the healthy groups they were 22.53% (comh) and 12.67% (coch). the bacteroides percentages in the healthy piglets were 17.35% (comh) and 14.03% (coch), but only 4.54% (comd) and 4.49% (cocd) in the infected piglets. the lactobacillus proportion in coch (15.63) was considerably higher than that in the other groups. actinobacillus, however, showed noticeably higher abundance in the infected piglets (17.21% in cocd; 15.86% in comd) than in the healthy piglets (1.66% in coch; 2.26% in comh). the escherichia-shigella proportions were also higher in the infected groups (12.86% in cocd; 12.61% in comd) than in the healthy groups (2.62% in coch; 1.58% in comh). at each taxonomic level, we selected the most abundant otu sequence as representative and performed multiple sequence alignment. we then constructed a phylogenetic tree from these data ( figure 3 ). our analysis revealed that firmicutes was the most well-represented phylum (by number of genera), followed by proteobacteria and bacteroidetes. pcoa revealed variations between the microbiome profiles within each group based upon bray curtis dissimilarity ( figure 4a ). coordinate 1 (45.14% of variation) was associated with disease status (healthy or infected) and sample location (colonic content or mucosa). samples from healthy and pcoa revealed variations between the microbiome profiles within each group based upon bray curtis dissimilarity ( figure 4a ). coordinate 1 (45.14% of variation) was associated with disease status (healthy or infected) and sample location (colonic content or mucosa). samples from healthy and pedv infected pigs showed more distant separation, while samples from the same group were more similar. based on bray curtis distances, upgma showed that the microbes in healthy piglets were grouped in one cluster, while microbes in infected piglets were grouped in another in both colonic contents and mucosa samples ( figure 4b ). thus, the healthy samples were clearly distinguishable from the infected samples in the clustering tree, likely due to variations in their microbial compositions. genes 2020, 11, x for peer review 6 of 13 pedv infected pigs showed more distant separation, while samples from the same group were more similar. based on bray curtis distances, upgma showed that the microbes in healthy piglets were grouped in one cluster, while microbes in infected piglets were grouped in another in both colonic contents and mucosa samples ( figure 4b ). thus, the healthy samples were clearly distinguishable from the infected samples in the clustering tree, likely due to variations in their microbial compositions. next, the lefse-identified biomarkers ( figure 5a ) were found to be different between the four groups, as shown in the microbial cladogram ( figure 5b) . we identified 52 different taxonomic groups (15 in comh, 6 in comd, 23 in coch, and 8 in cocd) that could be used as biomarkers. more biomarkers were present in healthy groups than in infected groups. microorganisms that differed between groups mainly included firmicutes and bacteroidetes in the coch and comh groups, proteobacteria in the comd group, and class gammaproteobacteria in the cocd group. eight phyla were shared between the four groups, and four phyla differed significantly between groups (p < 0.05, wilcoxon rank-sum test; supplementary figure 3 ). the proportion of proteobacteria was 3.92 fold higher in the infected groups than that in the healthy groups, while the proportion of bacteroidetes was 4.25 fold higher in the healthy groups than in the infected groups. next, the lefse-identified biomarkers ( figure 5a ) were found to be different between the four groups, as shown in the microbial cladogram ( figure 5b) . we identified 52 different taxonomic groups (15 in comh, 6 in comd, 23 in coch, and 8 in cocd) that could be used as biomarkers. more biomarkers were present in healthy groups than in infected groups. microorganisms that differed between groups mainly included firmicutes and bacteroidetes in the coch and comh groups, proteobacteria in the comd group, and class gammaproteobacteria in the cocd group. eight phyla were shared between the four groups, and four phyla differed significantly between groups (p < 0.05, wilcoxon rank-sum test; supplementary figure s3 ). the proportion of proteobacteria was 3.92 fold higher in the infected groups than that in the healthy groups, while the proportion of bacteroidetes was 4.25 fold higher in the healthy groups than in the infected groups. a total of 43 second-level kegg pathways were verified. carbohydrate metabolism, global and overview maps, and amino acid metabolism were the top three functional annotations in all four groups, followed with energy metabolism, and metabolism of cofactors and vitamins. 14 pathways were identified for the colonic content ( figure 6a ) and 11 were identified for the colonic mucosa ( figure 6b ) between the healthy and pedv-infected groups. among them, 6 pathways were identified for both tissues. additionally, membrane transport and transcription were more enriched in the disease groups than in the healthy groups. a total of 43 second-level kegg pathways were verified. carbohydrate metabolism, global and overview maps, and amino acid metabolism were the top three functional annotations in all four groups, followed with energy metabolism, and metabolism of cofactors and vitamins. 14 pathways were identified for the colonic content ( figure 6a ) and 11 were identified for the colonic mucosa ( figure 6b ) between the healthy and pedv-infected groups. among them, 6 pathways were identified for both tissues. additionally, membrane transport and transcription were more enriched in the disease groups than in the healthy groups. through cog analysis, we obtained 24 second-level classifications across the four groups; the top annotations were general function prediction only, carbohydrate transport and metabolism, amino acid transport and metabolism, and transcription. six annotations differed between the cocd and coch groups ( figure 7a ), while five differed between the comd and comh groups ( figure 7b ). in both colonic content and mucosa, amino acid transport and metabolism and cell wall/membrane/envelope biogenesis enrichment differed between the healthy and diseased groups. genes 2020, 11, x for peer review 8 of 13 through cog analysis, we obtained 24 second-level classifications across the four groups; the top annotations were general function prediction only, carbohydrate transport and metabolism, amino acid transport and metabolism, and transcription. six annotations differed between the cocd and coch groups ( figure 7a ), while five differed between the comd and comh groups ( figure 7b ). in both colonic content and mucosa, amino acid transport and metabolism and cell wall/membrane/envelope biogenesis enrichment differed between the healthy and diseased groups. through cog analysis, we obtained 24 second-level classifications across the four groups; the top annotations were general function prediction only, carbohydrate transport and metabolism, amino acid transport and metabolism, and transcription. six annotations differed between the cocd and coch groups ( figure 7a ), while five differed between the comd and comh groups ( figure 7b ). in both colonic content and mucosa, amino acid transport and metabolism and cell wall/membrane/envelope biogenesis enrichment differed between the healthy and diseased groups. several microorganisms, including rotavirus, coronavirus, escherichia coli, clostridium perfringens, c. difficile, cryptosporidium spp., giardia spp., cystoisospora suis, and strongyloides ransomi, have been linked to enteritis and diarrhea in suckling pigs. in this study, we successfully demonstrated that the microbial community structure of colonic mucosa and content differed significantly between healthy and pedv-infected piglets. likewise, previous research has shown that the proportions of escherichia-shigella, enterococcus, fusobacterium, and veillonella increased significantly in pedv-infected piglets, while those of short-chain fatty acid (scfa)-producing bacteria (e.g., rikenellaceae_rc9_gut_group, butyricimonas, and alistipes) underwent a decrease [21] . moreover, the gut microbiota of pedv-infected piglets was shown to exhibit significant changes at the genus and phylum levels [22] that are likely to disrupt microecological homeostasis. the intestinal microbial ecosystem of suckling piglets is unstable and sensitive to different external and internal factors in first week of birth. this sensitivity may generate individual differences in microorganisms, even in piglets from the same cohort [35] . here, we found that pedv infection significantly altered the gut microbiota in piglets, an outcome that was confirmed in several other studies [20] [21] [22] [23] . although the small intestine is the main site of pedv infection, the large intestines are the main metabolic and absorption sites for microbial fermentation, and contain considerably greater microbial abundance than the small intestines. therefore, studying the cecum and colon is necessary to obtain useful information for understanding the overall gut responses to pedv infection. microorganisms in the cecum and colon produce scfas that are beneficial to host health [36] . in our previous study, we compared the cecal microbial diversity between the healthy and pedv-infected piglets and found that the diseases associated bacteria were increased along with a decrease of beneficial bacteria in the pedv-infected piglets [37] . similar results were observed for the colonic microbial diversity. according to the analysis of lefse, more bacteria were significantly enriched in cecal mucosa and cecal contents in the infected group than in the healthy group, while it was the opposite in the colon. in addition, differences existed in the distribution of some bacteria in the cecum and colon of the infected piglets. for example, the fusobacteria was enriched in the colonic mucosa of the infected group in colonic position, while in cecum, this bacteria was higher in the cecal contents of the infected group than other three groups. we observed a clear increase in the relative abundance of proteobacteria and fusobacteria in pedv-infected suckling pigs. fusobacteria increases have previously been observed in the feces of pedv-infected suckling pigs [1, 20, 21] . furthermore, many studies suggested that this phylum is associated with various clinical anaerobic infections (including in the mouth, teeth, gut, and brain) in humans, and is positively correlated with catarrhal appendicitis on the mucosal surface [38, 39] . within this phylum, the genus fusobacterium plays a key role in promoting human colorectal cancer and various diseases in animals [40, 41] . fusobacterium nucleatum is important for the pathogenesis of various gut diseases, including intestinal inflammation and colon cancer [42] . a previous report showed that the abundance of fusobacterium decreases sharply as the porcine gut microbiota matures from suckling to weaning [15] . the elevated fusobacterium proportions in pedv-infected piglets is likely correlated to colon inflammation. the proportion of bacteroides decreased in the colonic microbiota of pedv-infected piglets. bacteroides are important for carbohydrate fermentation and polysaccharide catabolism, as well as amino acid and protein utilization [43] . several genera, including bacteroides, prevotella, and parabacteroides, are important producers of scfas and are dominant in healthy piglet intestines [15, 44] . scfas are rapidly absorbed by colonic epithelial cells [15] , protect the host against colonic diseases, exhibit anti-inflammatory effects, and promote energy intake by intestinal fibers [45, 46] . thus, scfa-producing bacteria contribute to carbohydrate fermentation, mucosal defense mechanisms, adipogenesis, and lipid oxidation [22, 43] . decreased bacteroides abundance can result in reduced scfa concentrations among pedv-infected piglets, a link that could compromise intestinal and immune homeostasis. here, lefse showed that each experimental group differed significantly in terms of microbial enrichment, suggesting that microbial profiles are useful as specific biomarkers for distinguishing between healthy and infected piglets ( figure 5) . a number of the microbes identified as biomarkers are associated with intestinal diseases and piglet health. the relevant microbes mainly belonged to firmicutes, bacteroidetes, proteobacteria, fusobacteria, verrucomicrobia, and epsilonbacteraeota. some bacteria from firmicutes and bacteroidetes were enriched in healthy piglets, while some from proteobacteria and fusobacteria were more abundant in pedv-infected groups. we observed similar outcomes when comparing cecal microbial diversity between healthy and pedv-infected piglets. the abundance of butyrate-producing clostridium butyricum was greater in the comh group than in the other groups. another study found that butyricimonas was present in all healthy piglets, but not in pedv-infected piglets [23] . a reduction in this genus is linked to numerous autoimmune and inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis, and type 1 diabetes. overall, these changes in biomarker bacteria compositions partly explained the low relative abundance of microflora related to energy metabolism, secondary metabolite synthesis, and amino acid metabolism in the intestines of pedv-infected piglets. collectively, our findings suggest that providing supplements to suckling piglets based on deficiencies of individual beneficial bacteria may prevent or alleviate pedv-induced diarrhea. colonic microorganisms that differ in abundance between healthy and diseased groups are mainly involved in transport and energy metabolism. therefore, pedv likely causes negative effects by disrupting microecosystem homeostasis in porcine intestines. the timely treatment of pedv-induced diarrhea and vomiting can effectively reduce mortality in newborn piglets [2] . many previous researches [1, [21] [22] [23] linking specific bacteria to healthy versus infected piglets suggests that regulating intestinal microbiota is a promising method for ped treatment. indeed, gut microbiota have been successfully used to prevent diarrhea [47] . however, we noted that all infected samples in this study were pedv-positive, so we could not determine the clinical importance of other detected viruses. to better characterize the intestinal microbiomes of healthy versus pedv-infected piglets, we recommend that future studies fully examine virome diversity using a larger sample size and metagenomic de novo sequencing of the gut microbial genome. our study revealed the dysbiosis of colonic microbiota in pedv-infected piglets. infection increased the abundance of bacteria associated with diarrhea and other conditions, while decreased the abundance of beneficial scfa-producing bacteria. our results suggest that the gut microbiota is crucial for ped pathology and physiology. overall, our findings greatly enhance the current understanding of pedv-associated gut microbiota. supplementary materials: the following are available online at http://www.mdpi.com/2073-4425/11/1/44/s1, figure s1 : detection of pedv in healthy groups and diarrhea groups using qpcr, figure s2 : shared otu analysis of the different piglet groups using venn diagrams, figure s3 : significant differences in microbe compositions between piglet groups, (* p < 0.05, ** p < 0.01). author contributions: l.j., q.z. and x.d. planned the project and designed the experiments. z.t. and w.d. conducted the experiments and carried out the data analysis with help from y.d. and l.j., z.t., w.d. and l.j. contributed reagents preparation and samples collection. z.t. wrote the manuscript, which was critically reviewed by z.t. and l.j. all authors have read and agreed to the published version of the manuscript. changes in the swine gut microbiota in response to porcine epidemic diarrhea infection. microbes environ porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences evidence of porcine epidemic diarrhea virus (pedv) shedding in semen from infected specific pathogen-free boars animal disease deadly pig virus slips through us borders porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines re-emergence of porcine epidemic diarrhea virus in the global pig population porcine epidemic diarrhea: a review of 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fusobacterium gastrosuis sp tlr2/tlr4 activation induces tregs and suppresses intestinal inflammation caused by fusobacterium nucleatum in vivo fusobacterium nucleatum increases proliferation of colorectal cancer cells and tumor development in mice by activating toll-like receptor 4 signaling to nuclear factor-kappa b, and up-regulating expression of microrna-21 how host-microbial interactions shape the nutrient environment of the mammalian intestine gut microbiome, short-chain fatty acids, and mucosa injury in young adults with human immunodeficiency virus infection regulation of inflammatory responses by gut microbiota and chemoattractant receptor gpr43 bifidobacteria can protect from enteropathogenic infection through production of acetate immune responses to the microbiota at the intestinal mucosal surface this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we are grateful to the reviewers of this manuscript for their constructive suggestions. the authors are also indebted to the molecular quantitative genetics team at the china agricultural university for their expertise. the authors declare no conflict of interest.data availability: data were deposited in the ncbi short read archive (accession no. srp162202). key: cord-301347-22lt6h40 authors: jarvis, matthew c.; lam, ham ching; zhang, yan; wang, leyi; hesse, richard a.; hause, ben m.; vlasova, anastasia; wang, qiuhong; zhang, jianqiang; nelson, martha i.; murtaugh, michael p.; marthaler, douglas title: genomic and evolutionary inferences between american and global strains of porcine epidemic diarrhea virus date: 2016-01-01 journal: prev vet med doi: 10.1016/j.prevetmed.2015.10.020 sha: doc_id: 301347 cord_uid: 22lt6h40 porcine epidemic diarrhea virus (pedv) has caused severe economic losses both recently in the united states (us) and historically throughout europe and asia. traditionally, analysis of the spike gene has been used to determine phylogenetic relationships between pedv strains. we determined the complete genomes of 93 pedv field samples from us swine and analyzed the data in conjunction with complete genome sequences available from genbank (n = 126) to determine the most variable genomic areas. our results indicate high levels of variation within the orf1 and spike regions while the c-terminal domains of structural genes were highly conserved. analysis of the receptor binding domains in the spike gene revealed a limited number of amino acid substitutions in us strains compared to asian strains. phylogenetic analysis of the complete genome sequence data revealed high rates of recombination, resulting in differing evolutionary patterns in phylogenies inferred for the spike region versus whole genomes. these finding suggest that significant genetic events outside of the spike region have contributed to the evolution of pedv. porcine epidemic diarrhea virus (pedv) causes diarrhea, vomiting, and dehydration, leading to high mortality (up to 100%) in suckling piglets. pedv was first discovered in the united kingdom in 1971, and later was found in belgium, hungary, france, italy, and the czech republic (chasey and cartwright, 1978; fan et al., 2012) . in 1986, pedv was first reported in china, and proceeded to spread throughout asia (cui, 1990; song and park, 2012) . in late 2010, a "variant" pedv strain with increased pathogenesis compared to the pedv is a single-stranded, positive sense rna virus belonging to the family coronaviridae, genus alphacoronavirus. the pedv genome is approximately 28 kb in length and roughly two-thirds of the genome consists of open reading frame (orf) 1, which encodes 16 non-structural proteins (nsps) (lai et al., 2013) . these nsps play important roles in viral replication, post-translational processing, and immune evasion (lai et al., 2013) . the virus produces various structural proteins, including spike, membrane, and nucleocapsid (lai et al., 2013) . the spike protein is crucial to cell attachment and infection, and the envelope is an integral membrane protein, aiding in membrane fusion while the nucleocapsid protein is necessary for genomic packaging (hagemeijer and de haan, 2015) . in addition, the pedv genome includes orf3, located between the spike and membrane genes, that encodes an ion channel protein possibly associated with pedv pathogenesis (park et al., 2008; wang et al., 2012) . researchers have explored various regions of the coronavirus (cov) genome to link specific areas with virulence and host cell attachment. for example, the spike gene codes for a viral attachment protein that can be divided into the s1 (1-789 aa) and s2 (790-1383 aa) regions (song and park, 2012) . comparative analysis of transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), and murine hepatitis virus (mhv) revealed two main antigenic sites in the s1 region: the n-terminal domain (ntd) and the c-terminal receptor binding domain (rbd) (li et al., 2007) . while both domains can influence virus infectivity, such as in tgev, one domain tends to be central to a cov's tropism: the ntd is important for mhv tropism, and the rbd is central to pedv infectivity and virulence (reguera et al., 2012) . the ntd can bind to various sialic acids on the host cell surface (reguera et al., 2012) . the rbd contains residues that bind to the porcine aminopeptidase-n (papn), the host receptor utilized by tgev and pedv (delmas et al., 1992) . since the last large-scale north american pedv outbreak ended in the spring of 2014, the complete genomes of 93 pedv strains from the us were sequenced and analyzed to further understand the origin and phylogenetic relationships among the american and global pedv strains. in-depth nucleotide and amino acid analysis was conducted to identify genes of high diversity. bayesian analysis was performed to understand the evolution of pedv and the emergence of different clades within us strains. in addition, the rbd was modeled to visualize the differences between american and asian strains to better understand how changes in the rbd might affect vaccine efficacy and development. samples were routinely submitted to the university of minnesota veterinary diagnostic laboratory (umvdl) for pathogen detection. between january 2014 and december 2014 samples were screened for pedv by real time rt-pcr . samples for complete genome sequencing were selected based on the criteria of a high viral concentration from the rt-pcr results and geographical diversity within the us. a total of 83 samples, including fecal (n = 38), intestinal homogenate (n = 21), fecal swab (n = 10), oral fluid (n = 5), feedback (n = 4), and environmental (n = 5) samples were selected for complete genome sequencing using next generation sequencing (ngs) techniques as previously described (genbank numbers kr265759-kr265834, kr265840-kr265846) (marthaler et al., 2013; marthaler et al., 2014) . whole genomic pedv sequences obtained using ngs techniques were also generously supplied from iowa state university (n = 7, genbank numbers km975735-km975741) and the ohio department of agriculture (n = 3, genbank numbers kp641661-kp641663), using previously described methods chen et al., 2014) . using the complete pedv genome sequences from this study (n = 93) and the available pedv sequences from genbank (n = 126), two nucleotide alignments were created and analyzed to determine the phylogenetic relationships between american and global pedv sequences: the concatenation of all orfs (orf1, s, orf3, envelope, membrane, and nucleocapsid), and a s1 alignment. vaccine and cell-passaged strains were excluded from the analysis (table s1 ). nucleotide and amino acid entropy analyses were performed using the matlab software (matlab v8.0 and statistics toolbox v8.1, the mathworks, inc., natick, ma, usa). threshold values were determined using previously published methods (shannon, 1948; litwin and jores, 1992) . recombination analysis was performed using the recombination detection program (rdp) v4, which uses multiple detection algorithms, including the rdp method, genecov, and maxchi, to check for the presence of recombinant sequences in the sequence dataset (martin et al., 2015) . window size was set to 100 bp. breakpoints, the presence of major/minor donor sequences, and confidence intervals were used to determine regions that required excision from the alignment, or if entire sequences needed to be removed from the analysis due to multiple recombination events within the sequence. recombinant sequences were removed only prior to the bayesian analysis, but remained in the alignments for all entropy analysis and molecular modeling. bayesian markov chain monte carlo (mcmc) approach using beast v1.8.1, with a relaxed molecular clock and bayesian skyline population (bsp) prior, with a general-time reversible nucleotide substitution and gamma distributed among-site rate variation was used to infer time-scaled phylogeny (drummond et al., 2002 , 2006 , 2012drummond and rambaut, 2007 minin et al., 2008; drummond and suchard, 2010) . the mcmc chain was run for 800 million generations, with sub-sampling every 80,000 iterations. a maximum clade credibility (mcc) tree was created by discarding the initial 10% of the chains and summarized in treeannotator (v.1.8.0). key nodes were identified using figtree (v.1.4 .2) to determine time to most recent common ancestor (tmrca). the putative papn receptor-binding residues were analyzed to determine residue trends between classical and pandemic strains (reguera et al., 2012) . the c-terminal rbd within the s1 region of the spike gene was modeled using the open-source modeling server swiss-model provided by the swiss institute of bioinformatics (biasini et al., 2014) . predicted tertiary structure of the pedv papn rbd was modeled using prcv as a template since a pedv template was not available. spike monomer and trimer models were developed using a theoretical sars-cov model as a template (bernini et al., 2004) . illustrations were created using the open-source javabased molecular viewer jmol (herraez, 2006) and the python-based molecular viewer pymol (the pymol molecular graphics system, version 1.7.4 schrödinger, llc.). 1932, 1951, 1976, 2467, 2517, 2652, 2658, 2664, 2667, 2679 2720, 2809, 2856, 2883, 2940, 3003, 3021, 3025, 3078, 3105, 3110, 3171, 3516, 3708, 3876 the pedv nucleotide sequences ranged from 27,529 to 28,061 bases in length. two pedv genomes from our study had insertions or deletions. ohio249 had a 3-nt insertion between positions 22,039 and 22,041 in the spike gene while minnesota309 with a 4-nt deletion from positions 27,768 to 27,771 in the 3 utr compared to the original us strain, usa/colorado/2013. entropy analysis was conducted with 219 whole nucleotide and amino acid sequences, containing the concatenated orfs excluding 5 and 3 utrs. entropy values greater than 0.8 and 0.6 were considered highly variable for the nucleotide and amino acid alignments, respectively, based on the level of diversity in the dataset and previously determined entropy values (litwin and jores, 1992) . within the nucleotide alignment, 15 of the 20 pedv regions lacked positions with entropy levels above 0.8 (nsp1, nsp5-nsp10, nsp13, nsp15, nsp16, s2, orf3, envelope, membrane, and nucleocapsid) while 5 regions had entropy levels above 0.8 (nsp2, nsp3, nsp12, nsp14, and s1) (fig. 1) . the nsp2 and 3 were the most divergent regions containing 10 and 16 diverse nucleotide positions, respectively (table 1) . interestingly, the nsp12 gene contained 4 diverse nucleotide positions, which were absent in the amino acid sequence (fig. 1a) . inversely, high amino acid diversity was observed in the nsp4, nsp13, nsp16, and s2 genes, which were absent in the nucleotide alignment (fig. 1b) . higher entropy levels were present in the nsp2, nsp3, and s1 regions in both the nucleotide and amino acid alignments. overall, the orf1a entropy levels were higher compared to orf1b in the amino acid analysis. of the structural genes, the s gene had the highest entropy levels compared to the envelope, membrane, and nucleocapsid genes. recombination was detected in 7 main areas of the concatenated full genome, including the nsp2, nsp3, nsp14-16, s1 domain, and nucleocapsid gene ( fig. 2a) . in these areas, recombination was present in the majority of the sequences, so the entire region was excised from the alignment prior to bayesian analysis. in addition, 35 sequences (23 from asia, 12 from the americas) were omitted from the bayesian analysis due to evidence of widespread recombination throughout the genome (table s1 ). for example, the pandemic sequence minnesota211 contained a recombinant region with the characteristic s-indel deletions and insertions in the s1 domain, indicating a recombinant event occurred between an s-indel strain and a non s-indel pathogenic strain in the us (fig. 2b) . a maximum clade credibility (mcc) phylogeny was inferred for both the concatenated genomic sequences excluding the recombinant regions (12,999 nt) and the spike s1 gene (2142 nt). the analysis was run independently twice until convergence was reached, with high agreement between the two runs. in the concatenated alignment tree, the classical and pandemic asian strains were positioned as basal to the us strains, consistent with an asian origin for the us outbreak (fig. 3) . importantly, the concatenated alignment tree suggests that the us epidemic may have resulted from two independent pedv introductions into the us, including minor and major clade of viruses. the minor clade contained the american and european s-indels, and a small subclade of non s-indel sequences from the ohio, including ohio249/2014, pc21a/2013, and oh15962/2013. the major clade of us pedv strains was supported by high posterior probability (100%) and appears to have diverged further into two highly supported sublineages (99% and 100% posterior probability). the phylogeny is consistent with multiple incursions of the major clade of us pedv viruses into mexico, canada, and south korea. the minor clade includes sequences from late 2013 to early 2014 that are localized to the midwestern and eastern us regions. the estimated tmrca of the minor clade of us pedv strains is july 2009-2011, and the estimated tmrca of the major clade of us pedv strains is september 2010-august 2012. the estimated evolutionary rate for the complete genome (excluding recombinant regions and sequences) is 6.2 × 10 −4 substitutions/site/year (4.8 × 10 −4 -7.6 × 10 −4 , 95% highest posterior density (hpd)). the rate estimate for the us strains is slightly higher, but not significantly: 5.5 × 10 −3 substitutions/site/year (4.6 × 10 −3 -6.5 × 10 −3 , 95% hpd). the spike tree illustrates the evolutionary relationship between the classical strains and the s-indels, which suggests a classical origin for the s-indel genotype (fig. 4) . the pathogenic strains form a highly diverged major clade (fig. 4b) , which braches into 2 large american clades. in addition, the bayesian analysis of the spike gene might suggest 2 separate introductions of pedv into the us. the evolutionary rate for the s1 gene is 1.5 × 10 −3 substitutions/site/year (1.1 × 10 −3 -1.9 × 10 −3 , 95% hpd). considering the high entropy levels in the spike gene and the evolutionary rate determined from the s1 bayesian spike tree, the rbd within the s1 was further examined. the s-indel and classical pedv strains shared similar amino acid substitutions, specifically in the ntd of the s1 region (fig. s1) . furthermore, the pandemic pedv strains from china had an increased number of substitutions within the s1 domain when compared to the american strains due to the longer circulation time in china. compared to the attenuated vaccine strain dr13, 29 of 185 (16%) american strains and 19 of 34 (56%) asian strains had at least one amino acid substitution in the papn rbd ( table 2 ). the majority of the american strains (n = 156) did not represent any amino acid differences in the papn rbd. in this region, 8 positions in the american strains had amino acid differences compared to the vaccine strain dr13 (fig. 5a) . the most common substitutions were in the fourth region of the papn rbd at positions e594d (n = 10) and g598d (n = 9), which were substituted with aspartic acid. more substitutions (n = 13) occurred in the papn rbd of the asian strains (table 2) , with the most common substitutions at position h515l (n = 5). the rbd regions were three-dimensionally modeled to illustrate the 8 and 13 amino acid positions at which substitutions occurred in the north american and asian strains, respectively. the modeling of the spike protein suggests that the papn rbd residues cluster around the inner pore created by the trimer molecule, while the ntd is oriented around the outer surface of the s1 domain ( fig. 5b through 5f). genomic analysis depends critically on complete sequence data to conduct accurate research on phylogeny, evolution, and gene regulation. in the past, it was more economically and time effective to sequence smaller pieces of a genome and develop evolutionary conclusions from these relatively small genomic pieces. however, without full genomic sequences, it is impossible to compare variations within a genome to determine selective pressures on specific genes or regions. because of ngs technology, tools like site-specific entropy analysis can be used to examine variability throughout the genome of many pedv sequences. sun and collaborators reported four regions of diversity within the pedv genome, including v1 in the nsp2 and nsp3, v2 in the s1, v3 in the s2 and orf3, and v4 in the nucleocapsid (sun et al., 2015) . while our results support the nucleotide variance in the nsp2, nsp3, and spike genes, high levels of diversity were not present in the s2, orf3, and the nucleocapsid. this could be due to the omission of pedv isolates in our analysis, as well as the comparatively large number of new us sequences in our dataset. our variance results may more accurately represent variance within american pedv strains, and underrepresent variance within chinese strains. the diversity in the s1 region is comprehensible since it is under strong immunological pressure while the s2 region is more conserved throughout covs (aydin et al., 2014) . the functions of nsp2 and nsp3 remain relatively ambiguous. despite being involved in viral growth and propagation, nsp2 is dispensable for viral replication because cov strains can replicate in absence of the nsp2 (graham et al., 2006) . the function of nsp3 may be related to innate immune evasion since it encodes proteases that facilitate proteasome degradation, changes in intracellular destination, signaling, protein interactivity, and host type i interferon (ifn) antagonist activities (xing et al., 2013) . due to the multifaceted nature of nsp3, other nsp regions could produce proteins with novel effects not yet understood that mediate the virulence of cov species. acquired nucleotide differences throughout the nsp2 and nsp3 regions could contribute to the evasion of host immunity. thus, future research should focus on the functionality and importance of all pedv genes to further understand cov pathogenesis. recombination plays a pivotal role in the evolution of covs by creating new strains with altered virulence. the minnesota211 strain originated from a recombination even between an s-indel and a us pandemic strain, which has been associated with altered pathogenesis. while recombination may occur more often during an epidemic, recombination events occurred in most of the asian strains. recombination events can affect the phylogenetic analy-sis because different regions of the genome may have different evolutionary histories (spade et al., 2015) . our recombination analysis resulted in a significant portion of the complete genome being removed prior to more detailed phylogenetic analysis. at this time, the beast program cannot accommodate genetic data that includes recombined regions. our analysis supported an asian origin for the us outbreak while the inference is biased by the lack of background sequences from other regions. although over 100 genomes were from the us, interpretation of the evolutionary and spatial history of this data is limited by the lack of genomic pedv data from other regions, including europe and asia. us strains had a higher evolutionary rate compared to the global strains, but the bayesian skyline plot did not show any significant increase in evolutionary rate, possibly due to the lack of temporal sampling in the us dataset. the evolutionary rate for the spike gene was higher compared to the rest of the genome, reflecting greater selective pressure. the overall evolutionary rate of pedv (6.2 × 10 −4 substitutions/site/year) is similar to that of tgev and wild animal covs (6.08 × 10 −3 substitutions/site/year, 1.3 × 10 −5 -1.4 × 10 −2 95% hpd), but lower than that of sars-cov (2.3 × 10 −3 nucleotide substitutions/site/year), except during the time of the us pedv 2013 epidemic (5.5 × 10 −3 sub-stitutions/site/year) (song et al., 2005; vijaykrishna et al., 2007) . surprisingly, 3 pandemic strains were positioned within the s-indel clade. possibly, a pandemic and s-indel strain were introduced into the americas, and a recombination event occurred in the ntd that removed the characteristic insertions and deletions of an s-indel strain, as indicated in the minnesota211 sequence. the relationship between the us minor clade and the recent pedv strains from europe is less clear. while these viral populations are closely related (posterior probability of 100%), the direction of transmission is unclear at this time. additional sequences from europe might help to resolve the origins of these recent european pedv cases. (e) a monomer model of the pedv spike protein, with the c-terminal rbd represented in green, dark blue represents the s2 region, light blue represents the s1 region, and yellow represents the n-terminal rbd. (f) a theoretical tertiary structure model of the pedv spike protein. blue represents the s1 region, with the specific n-and c-terminal rbds highlighted in yellow and green, respectively. the papn-rbd is shown in violet.(for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) examining the spike gene can reveal interesting conclusions about the papn rbd. while the ntd spans a larger region of the s1 domain, it has not been directly linked to pedv tropism and functionality, as in tgev and mhv (reguera et al., 2012) . the korean attenuated vaccine strain and the us strains share similar residues in the papn rbd, with numerous differences compared to the older classical strains, suggesting this vaccine may protect against the american strains. however, developing a consistently and longitudinally efficacious vaccine may prove challenging, considering the high evolutionary rate of the s1 region. failures in the development of an efficacious vaccine have been reported, further supporting the difficulty in generating vaccines for pedv . despite some uncertainties in vaccine efficacy, a recent study demonstrated that prior exposure of sows to the s-indel strain provided a level of protective immunity when their piglets were challenged with the more virulent original us pedv strain, which is probably due to conservation within the c-terminal region of the viral genome (goede et al., 2015) . while the exact functionality of all the genes of pedv and other covs is unknown, adding the complete genomes of diverse strains to the global database promotes better understanding of evolutionary and phylogenetic relationships. multiple regions within the genome are variable, and recombination is common between pedv strains. despite excising a large portion of the genome prior to analysis, the bayesian trees illustrate two distinct entries of pedv into the us and characterize the evolution of pedv compared to other covs. modeling of the papn rbd region has revealed that asian strains have increasing diversity compared to previously developed vaccines, and the variability in both the american and asian strains needs to be considered for future vaccine development. as the us swine industry recovers from the pedv epidemic of 2013-2014, research is maturing to understand the regions of diversity, evolution, and the rbd of pedv to prevent future outbreaks and foster vaccine development. influence of hydrophobic and electrostatic residues on sars-coronavirus s2 protein stability: insights into mechanisms of general viral fusion and inhibitor design prediction of quaternary assembly of sars coronavirus peplomer swiss-model: modelling protein tertiary and quaternary structure using evolutionary information virus-like particles associated with porcine epidemic diarrhoea isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the 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diarrhea in suckling piglets evolutionary insights into the ecology of coronaviruses distinct characteristics and complex evolution of pedv strains new variant of porcine epidemic diarrhea virus the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase this study was supported partially by the rapid agricultural response fund, established by the minnesota legislature and administered by the university of minnesota agricultural experiment station, and by boehringer ingelheim vetmedica, inc.the authors thank the faculty and personal at the umvdl for their technical services. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prevetmed.2015. 10.020. key: cord-290819-zhywlf6r authors: wu, jiaqi; chi, heng; fu, yali; cao, aiping; shi, jingxuan; zhu, min; zhang, lilin; hua, deping; huang, jinhai title: the antiviral protein viperin interacts with the viral n protein to inhibit proliferation of porcine epidemic diarrhea virus date: 2020-07-27 journal: arch virol doi: 10.1007/s00705-020-04747-8 sha: doc_id: 290819 cord_uid: zhywlf6r in the early stage of virus infection, the pattern recognition receptor (prr) signaling pathway of the host cell is activated to induce interferon production, activating interferon-stimulated genes (isgs) that encode antiviral proteins that exert antiviral effects. viperin is one of the innate antiviral proteins that exert broad-spectrum antiviral effects by various mechanisms. porcine epidemic diarrhea virus (pedv) is a coronavirus that causes huge losses to the pig industry. research on early antiviral responses in the gastrointestinal tract is essential for developing strategies to prevent the spread of pedv. in this study, we investigated the mechanisms of viperin in pedv-infected ipej-c2 cells. increased expression of interferon and viperin and decreased replication of pedv with a clear reduction in the viral load were observed in pedv-infected ipec-j2 cells. amino acids 1–50 of porcine viperin contain an endoplasmic reticulum signal sequence that allows viperin to be anchored to the endoplasmic reticulum and are necessary for its function in inhibiting pedv proliferation. the interaction of the viperin s-adenosylmethionine domain with the n protein of pedv was confirmed via confocal laser scanning microscopy and co-immunoprecipitation. this interaction might interfere with viral replication or assembly to reduce virus proliferation. our results highlight a potential mechanism whereby viperin is able to inhibit pedv replication and play an antiviral role in innate immunity. porcine epidemic diarrhea virus (pedv), the causative agent of ped, is a member of the genus alphacoronavirus, family coronaviridae [27] . in pigs, pedv first infects the peyer's patch, a small area of intestinal lymph nodes [31] . it then proliferates and spreads to intestinal epithelial cells, eventually leading to infection of the entire small intestine [37] . injury of intestinal organelles causes cell dysfunction and a reduction or loss of related enzyme activities. impaired nutrient absorption due to enzyme inactivation can lead to osmotic diarrhea, dehydration, and death [18] . pedv is a common coronavirus that has caused huge economic losses to the pig industry and its related peripheral industries worldwide [32] . research on the natural immune responses to pedv is still in its infancy, and there have been few reports in the literature about this topic [6] . viperin is a broad-spectrum antiviral protein that has important antiviral effects, and its full potential remains to be explored. its role in pedv infection remains unclear, but its possible involvement in preventing ped is potentially significant for the stable and healthy development of the pig industry. previous studies have shown that virus-infected cells activate various signaling pathways to produce interferons [19] . when type i interferon is released, it binds to specific receptors on the cell surface and activates more than 300 downstream ifn-stimulated genes (isgs) through a signal cascade reaction [11] . many isgs have been found to significantly limit viral replication and participate in a variety of antiviral processes, including presentation of viral antigens, apoptosis, and interference with viral replication and assembly [22] . products of interferonstimulated genes with antiviral activity are also known as "innate immune factors". at present, only a small number of proteins encoded by interferon-stimulated genes have been reported, such as protein kinase r (pkr), ribonuclease l (rnase l), and viperin [9] . host stress or overexpression of certain proteins can inhibit virus proliferation during viral infection [34] . the antiviral effect of ifn is usually indirect. it induces host cells to produce antiviral proteins and then exerts antiviral effects on transcription and translation through protein kinases, 2'-5'a synthase, and 2-phospholipase [15] . viperin is a broad-spectrum antiviral protein that is usually overexpressed at a low level in many types of normal healthy cells [5, 40] . however, when induced by interferon, double-stranded dna, double-stranded rna, lipopolysaccharide, poly(i:c), or various viruses, the expression of viperin increases significantly [4, 42] . there are two main pathways by which the expression of viperin is induced. sendai virus, pseudorabies virus, and sindbis virus are all capable of inducing the expression of interferon-stimulated genes [8, 16, 35] . these viruses are first recognized by pattern recognition receptors (prrs), such as the toll-like receptors tlr3 and tlr4, the retinoic-acid-inducible gene rig-1, and the cytoplasmic dna sensor. the interferon regulatory factors irf3 and irf7 are then activated to produce ifn-β, which binds to type i interferon receptors on the cell surface through autocrine or paracrine pathways, resulting in the synthesis of the complex isgf3, which binds to the viperin promoter to activate its expression [36] . in addition, there are some other viruses, such as vesicular stomatitis virus and human cytomegalovirus, whose dsrna stimulates rlrs and interaction with the adaptor protein mavs can activate the production of irf1 and irf3, which in turn can induce viperin expression [10] . viperin plays an important role in the production of type i interferon in plasmacytoid dendritic cells (pdcs) [30] , which are immune cells derived from bone marrow. these cells are capable of rapidly activating responses to non-self nucleic acids to produce interferons in large amounts [23] . the main reason for this ability is that pdcs continuously produce the endogenous toll-like receptors tlr7 and tlr9. activated tlr7/9, combined with irak1 and traf6, can induce viperin expression [21] . in pdcs, viperin is necessary for producing type i interferon [28] . in viperin-deleted mouse cells, activation of the toll-like receptors tlr7 and tlr9 does not lead to the production of interferon. recruiting irak1 and traf6 in lipid droplets can activate irf7, and viperin is also crucial for this process. thus, the induction of viperin in pdcs depends on the activation of the tlr7 and tlr9 signaling pathways. irak1 and traf6 need to be recruited to lipid droplets through viperin, and irf7 is then activated to induce the expression of type i interferon [29] . the role of porcine viperin in pedv infection is still not well understood. in this study, the mechanisms by which porcine viperin regulates pedv proliferation were investigated in detail. the study was approved by tianjin university institutional animal care and use committee (tjiacuc) (protocol number: syxk-jin 2014-0004). all balb/c mice were kept in well-ventilated cages and were anesthetized for blood collection. the guide for the care and use of laboratory animals of the tianjin government authority was strictly followed. balb/c mice were obtained from the tianjin laboratory animals center. cells of the porcine alveolar macrophage (pam) cell line crl2843-cd163 (3d4/21 cells) were kindly donated by professor han jun of china agricultural university. hek-293t cells were cryopreserved in our laboratory. cells of the porcine intestinal epithelial cell line ipec-j2 and human embryonic kidney (hek) 293t cells were cultured in high-sugar dmem medium supplemented with 10% (v/v) fetal bovine serum (fbs) and antibiotic-antifungal solution. both cell lines were cultured in a humidified 5% co 2 incubator at 37 °c. pedv strain cv777 was used in our study, and the titer was 10 4 pfu/ml [41] . polyclonal antibodies against viperin were prepared by immunizing balb/c mice with recombinant his-viperin with a mineral oil adjuvant. labeled antibodies were purchased from cell signaling technology (cst, danvers, ma, usa) and applied biological materials inc. (abm, vancouver, canada). the internal reference antibody and hrp-conjugated antibody were purchased from invitrogen (thermo fisher scientific, waltham, ma, usa). total rna was extracted from pams using trizol reagent (takara, china) and transcribed into cdna using reverse transcriptase (takara). viperin was amplified using oligonucleotide primers (table 1 ) designed based on a porcine viperin sequence (nm_213817.1) obtained from the genbank database. the amplified fragment was cloned into pgem-t easy vector (transgen, beijing). a flag-viperin construct for expression in eukaryotic cells was constructed as follows: the viperin gene was amplified using a specific primer pair (table 2 ) with a sequence in common with the vector was ligated to the pflag-cmv2 vector using a one-step cloning kit (vazyme, nanjing, china). the prokaryotic expression plasmid pet-28a-viperin was constructed using the same method to express the recombinant protein his-viperin. the 50% tissue culture infective dose (tcid 50 ) method was used to determine virus titers. ipec-j2 cells were seeded in 96-well plates (1 × 10 4 cells/well). after the cells had adhered to the plate and grown to about 50% confluency, a serial dilution (1-10 −6 ) of pedv was inoculated onto the cells. mock-infected cells were used as a control. the cells were incubated at 37°c for 7 days, and tcid 50 values were calculated by the reed-muench method [41] . primers were designed based on available sequences in the genbank database (table 3) . relative gene expression was analyzed using transstart top green qpcr supermix (dye i + dye ii) reagents on a real-time pcr instrument (abi 7500) with the following cycling conditions: 95 °c for 5 min, 40 cycles of 95 °c for 15 s, 58 °c for 30 s, and 72 °c for 45 s; 95 °c for 15 s, 60 °c for 1 min, 95 °c for 15 s, and 60°c for 15 s. the cdna was obtained by rna extraction and reverse transcription. three replicates were performed for each sample. the relative transcription level of each gene was calculated using graphpad prism software with β-actin as an internal reference. the procedure used for confocal microscopy was described previously [20] . ipec-j2 cells were cultured overnight at 37 °c in 12-well plates (corning inc., corning, ny, usa) and transfected either with plasmids containing the viperin gene or with empty vector (0.5 g). after the cells had adhered to the plate and grown to about 50% confluency, pedv was inoculated onto the cells (100 ml/well), and an uninfected blank control (nc) was also included. twenty-four hours later, cells were fixed with 4% paraformaldehyde solution and permeabilized with pbs containing 0.3% triton x-100. five hundred microliters of 0.5% triton x-100 was then added to each well to permeabilize the cells for 20 minutes at room temperature. the cells were blocked with 1% bovine serum albumin (bsa) at room temperature for 30 minutes and incubated with anti-flag and anti-myc or anti-gfp antibody at 4°c overnight. secondary antibodies (fluorescein-isothiocyanateconjugated anti-mouse igg or phycoerythrin-conjugated anti-rabbit igg) were used, and nuclear dna was stained with 4,6-diamidino-2-phenylindole (dapi). the cells were examined under an inverted fluorescence and phase-contrast microscope (olympus) to determine the subcellular localization of viperin protein. cells were lysed and homogenized in ripa cell lysate in the presence of 1% phenylmethylsulfonyl fluoride (pmsf) to inhibit proteases. the protein samples were separated by sds-polyacrylamide gel electrophoresis, transferred to nitrocellulose (nc) filter membranes (expro), and blocked. the nc membrane was treated overnight at 4°c with antibodies against flag (1:5000), myc (1:5000), viperin (1:500) or β-actin (1:5000) and then washed and incubated with hrp-conjugated antibody for 1 h at room temperature. immunoreactive bands were visualized using a chemiluminescence detection kit (thermo scientific, waltham, ma, usa) and a gel doc xr imaging system (bio-rad, usa). ipec-j2 cells cultivated in 60-mm-diameter plates were transfected with flag-viperin and myc-n, or related expression plasmids. twenty-four hours after transfection, the cells in each plate were treated with 400 μl of lysis buffer containing 1% protease inhibitor (pmsf). the supernatant of the cell lysate was incubated overnight with anti-myclabeled or anti-flag-labeled beads (sigma) at 4 °c. the beads were washed three times for 10 min with lysis buffer and boiled for 5 min. the proteins bound to the beads were separated by sds-page and detected by western blot. a small interfering rna (sirna) against viperin was synthesized by genepharm (table 3) . a tube containing the rna oligo dry powder was centrifuged at 3000 rpm for 2 min and diluted with diethyl pyrocarbonate (depc)-treated water. when the cells in a 12-well plate had grown to 70% confluency, they were transfected with the sirna using lipofectamine 3000 at a final concentration of 50 nmol. after transfection with sirna-viperin for 16 hours, the cells were collected to measure the level of interference and then infected with pedv. the cells were then collected for realtime quantitative rt-pcr and western blotting (table 4 ). statistical analysis was performed using graphpad prism software, and the significance of changes in gene expression was analyzed by two-way anova. statistical results are presented as mean ± standard deviation (mean ± sem). p < 0.05 (*) indicates a statistically significant difference. through cell culture and inoculation, we found that pedv can stably proliferate in porcine intestinal epithelial cells (ipec-j2), providing a cell model for further studies. at 36 h postinfection, the cells gradually became detached from the culture vessel, indicating that infection had occurred (fig. 1a) . fluorescence-based quantitative analysis also showed that the number of pedv virus particles stabilized and peaked at 24-36 h postinfection (fig. 1b) . we selected the structural protein n and the non-structural protein nsp1 of pedv for immunofluorescence experiments and observed their expression to reflect the replication of the virus. in the early stage of pedv replication, genes encoding viral non-structural proteins were transcribed first, which could reflect viral replication to some extent. the amount of nsp1 protein gradually increased with time (fig. 1c) . in the late stage of pedv replication, the viral structural protein genes began to be transcribed and expressed. expression of the structural protein n was also be used to trace the proliferation of pedv (fig. 1d) , and the pedv cv777 strain was found to stably proliferate in ipec-j2 cells, reaching a peak at 36 hours postinfection. the innate immune system is evolutionarily conserved to defend against pathogens [26] . after virus infection, pattern recognition receptors recognize released viral nucleic acids and activate downstream signaling pathways to promote the production of specific transcription factors and type i interferons [25] . to measure interferon levels during pedv infection, we infected ipec-j2 cells with pedv at a multiplicity of infection (moi) of 0.5. using quantitative reverse transcription pcr (qrt-pcr), we found that pedv infection resulted in a significant increase in the level of ifn-β mrna compared to mock-infected cells. interferon production was lower in pedv-infected cells than in cells that were stimulated with poly(i:c) (fig. 2a) . when ipec-j2 cells were infected with pedv, the transcription level of viperin increased significantly and reached a peak after 12 hours. after that, as the virus continued to replicate, the cells began to die and the transcription level of viperin began to decline. however, compared to control cells, the transcription level of viperin remained high in the infected cells (fig. 2b ). an immunofluorescence assay also showed that the expression of viperin protein increased significantly infection (fig. 2c) . these results indicate that pedv infection upregulates the expression of interferon and viperin in ipec-j2 cells. we constructed a flag-viperin eukaryotic expression plasmid and verified the expression of this protein in 293t cells (fig. 3a) . to investigate the effect of viperin on pedv replication, the viperin gene was overexpressed in ipec-j2 cells, and the cells were infected with pedv 24 h later. the viral load was measured to investigate the effect of viperin overexpression on pedv proliferation. overexpression of the viperin gene was found to reduce the levels of pedv produced (p < 0.05) (fig. 3b) . for further validation, a specific sirna targeting viperin was designed. transfection of j2 cells with this sirna resulted in a decrease in the expression of viperin (fig. 3c) , which was accompanied by an increase in pedv proliferation that was proportional to the degree of interference of viperin expression (fig. 3d) . this suggests that viperin inhibits the proliferation of pedv in ipec-j2 cells. it has been reported that human viperin can be localized in the endoplasmic reticulum [38] . it has been proposed that viperin forms a dimer through its c-terminal domain and its n-terminal alpha helical domain to mediate the endoplasmic reticulum crystallization [13] . since the amino acid sequences of porcine and human viperin are 83% identical, we investigated the location of porcine viperin in ipec-j2 cells. fluorescence microscopy showed that the viperin protein was distributed in the cytoplasmic region (fig. 4a) and had significant aggregation and colocalization with an endoplasmic reticulum marker protein. at the same time, no co-localization of viperin with golgi and lysosome markers was observed (fig. 4b) . when pedv infects cells, structural proteins and nonstructural proteins encoded by the virus use the endoplasmic reticulum for their synthesis, processing, and modification, which makes us suspect that viperin interacts with certain proteins in pedv. we therefore chose the major structural protein n and two non-structural proteins, nsp13 and nsp16, for experiments. immunofluorescence images of ipec-j2 cells 24 hours after infection with pedv showed that viperin and n showed clear co-localization, but no obvious co-localization with nsp13 and nsp16 was observed (fig. 5a) . considering that the antibodies used in previous experiments were polyclonal antibodies, we cotransfected ipec-j2 cells with recombinant plasmids encoding viperin and n, nsp13 or nsp16 and carried out the immunofluorescence experiments again. the results showed that there was significant co-localization between viperin and n, but no colocalization between viperin and nsp13 or nsp16 (fig. 5b) . a co-immunoprecipitation assay further demonstrated that viperin interacted with n ( fig. 5c) but not nsp13 or nsp16 (fig. 5d) . these experiments showed that porcine viperin interacts with the n protein, but not with the non-structural proteins nsp13 and nsp16. studies have shown that the n-terminal alpha-helical structure of human viperin protein helps to anchor it to the endoplasmic reticulum. in order to explore the specific mechanism of interaction between viperin and the pedv n protein, the domains of viperin was analyzed and a truncated form of the gene was constructed. viperin is approximately 42 kda in size and can be divided into three distinct domains [12] , an n-terminal alpha-helical domain, a c-terminal conserved domain, and a middle s-adenosyl methionine domain (sam domain) (fig. 6a) . we made three ??truncated forms of the viperin gene, vip1-50, vipδ1-50, and vipδ343-362??. these truncated genes were overexpressed in ipec-j2 cells, which were then infected with pedv. the transcription level of n was measured by fluorescence quantification (fig. 6b) . as described previously, viperin inhibited the proliferation of pedv. ??this inhibition was not observed with the n-terminal fragment vip1-50 alone, but inhibition was still observed with the deletion mutants vipδ1-50, and vipδ343-362?? (fig. 6c) , suggesting that the interaction region should be the middle part, and this was confirmed by immunoprecipitation (fig. 6d) . these data indicated that aa 1-50 of viperin play an important role in its anchoring to the endoplasmic reticulum, while the middle sam domain of viperin the specific region interacts with n protein. this study showed that the expression of type i interferon increases and that of viperin is also upregulated in ipec-j2 cells infected with pedv. overexpression of viperin and interference with its expression showed that viperin inhibits pedv proliferation. porcine viperin was found to be localized in the endoplasmic reticulum, and aa 1-50 of this protein were found to be indispensable for its ability to anchor itself to the endoplasmic reticulum. furthermore, there was colocalization and interaction between porcine viperin and the structural n protein of pedv. the s-adenosyl methionine domain in the middle of the viperin protein was found to be the key region for its interaction with the n protein. viperin may interfere with the assembly and proliferation of the virus by interacting with the n protein, thereby exerting an antiviral effect. viperin is a broad-spectrum antiviral protein that can inhibit the proliferation of multiple viruses [17] . previous studies have shown that human viperin relies on its n-terminal amphipathic alpha-helical domain to anchor itself to the endoplasmic reticulum membrane, with its c-terminus protruding into the cytoplasm. overexpression of viperin results in the formation of homodimers that crystallize the endoplasmic reticulum and destroy its structure [14] . viperin can also be localized on lipid droplets [1] . it is noteworthy that lipid droplets have been shown to be sites of replication of many viruses, such as hepatitis c virus (hcv) and dengue virus (denv). the amphiphilic alpha-helical domain at the n-terminus of viperin is also necessary for its localization to lipid droplets [39] . consistent with previous studies, we demonstrated that porcine viperin was also localized in the endoplasmic reticulum. after entering the host cell by membrane fusion, pedv releases its positive-strand rna genome in the cytoplasm [25] . the viral polymerase uses the positive-strand genomic rna as a template to generate negative-strand rna, which in turn is used as a template for synthesis of subgenomic rna and subgenomic mrna of different lengths by the polymerase. mrnas of different lengths are translated to produce viral proteins of different sizes [43] . the newly synthesized structural n protein forms a nucleocapsid by binding to the genomic rna in a helical pattern [24] . the nucleocapsid then binds to the e and m proteins and to the modified s protein to form a complete mature virus particle in the endoplasmic reticulum-golgi interaction between truncated viperin and the pedv n protein in ipec-j2 cells transfected with constructs with a truncated viperin gene and pedv n overexpression plasmids, detected by immunoprecipitation intermediate compartment (ergic), which is then released by exocytosis [3] . our experiments showed that viperin can inhibit the proliferation of pedv. in order to investigate the specific mechanism of this inhibition, we analyzed interactions between viperin and different viral proteins of pedv. the experiments showed that viperin colocalized and interacted with the n protein, but there was no obvious co-localization or interaction with the nsp13 or nsp16 protein. viperin differs slightly among different species although it is highly conserved. in porcine viperin, the n-terminal amphipathic alpha-helical domain consists of approximately aa 1-50. the highly conserved c-terminal domain consists of approximately aa 340-361. the middle part consists of aa 71 to 182 in which there are four relatively conserved modules (m1, m2, m3, m4). the m1 module contains a conserved cxxxcxxc sequence, a feature of enzymes that use s-adenosyl methionine as a cofactor that binds to iron-sulfur clusters [2, 7] . viperin decreases flavivirus virulence by promoting the secretion of unproductive noninfectious virus particles via a gbf1-dependent mechanism [33] . the iron-sulfur clusters are also important for its antiviral function. in order to identify the specific site of interaction between viperin and n, we constructed truncation forms of viperin. coimmunoprecipitation assays showed that the loss of the n-terminus and c-terminus did not affect the interaction between viperin and the n protein, indicating that the key residues responsible for interaction between viperin and n are in the middle region of the s-adenosyl methionine domain. in conclusion, this study showed that viperin regulates the proliferation of pedv, and our results suggest a possible role of viperin in pedv infection, and its antiviral activity was confirmed. this provides new ideas for research on the natural immune response to pedv infection and strategies for prevention 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cells a diverse range of gene products are effectors of the type i interferon antiviral response porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines outbreak of porcine epidemic diarrhea in suckling piglets viperin targets flavivirus virulence by inducing assembly of noninfectious capsid particles porcine epidemic diarrhea virus 3c-like protease regulates its interferon antagonism by cleaving nemo viperin inhibits classical swine fever virus replication by interacting with viral nonstructural 5a protein promyelocytic leukemia zinc finger protein regulates interferon-mediated innate immunity porcine epidemic diarrhea virus infections induce apoptosis in vero cells via a reactive oxygen species (ros)/p53, but not p38 mapk and sapk/jnk signalling pathways formation of crystalloid endoplasmic reticulum in cos cells upon overexpression of microsomal aldehyde dehydrogenase by cdna transfection cloning and sequence analysis of the spike gene of porcine epidemic diarrhea virus chinju99 targeting ube4a revives viperin protein in epithelium to enhance host antiviral defense evaluation of reed-muench method in determination of activity of biological preparations grouper viperin acts as a crucial antiviral molecule against iridovirus transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j2 cells conflict of interest the authors declare no competing interests. key: cord-272031-o2hx667i authors: carvajal, ana; argüello, héctor; martínez-lobo, f. javier; costillas, sara; miranda, rubén; g. de nova, pedro j.; rubio, pedro title: porcine epidemic diarrhoea: new insights into an old disease date: 2015-09-29 journal: porcine health manag doi: 10.1186/s40813-015-0007-9 sha: doc_id: 272031 cord_uid: o2hx667i porcine epidemic diarrhea (ped) is an enteric disease in swine caused by an alphacoronavirus. it affects swine of all ages causing acute diarrhoea and can lead to severe dehydration and death in suckling piglets. being recognized for the first time in europe and asia during the seventies and the eighties, respectively, it has remained a relevant cause of diarrhea outbreaks in asia for years and to the present. it has become a major concern in swine production since 2013 when the virus was detected for first time in the usa and in other american countries causing a high number of pig deaths and significant economic losses. the present review aims at approaching the reader to the state of the art of ped giving answer to some of the most recent questions which have arisen related to this disease. porcine epidemic diarrhoea (ped) is a highly contagious infectious disease caused by a coronavirus, porcine epidemic diarrhoea virus (pedv). it causes acute and watery diarrhoea in pigs of all ages although the most severe signs are reported in piglets less than two weeks old, in which diarrhoea leads to severe dehydration and is associated with mortalities which can reach up to 100 % in affected litters. the first clinical description of ped occurred in the uk and belgium in the early seventies. however, it was not until 1978 when the etiological agent of these diarrhoeal outbreaks, a new coronavirus, was identified [1, 2] . soon afterwards, studies by the research group led by professor pensaert in ghent, belgium, demonstrated that there were no specific antibodies against pedv in sera collected from sows prior to 1971, confirming that pedv was a new virus in the european swine population. up to now, there is no information available on the potential origin of this virus. distribution after its first description in uk and belgium, pedv spread throughout european countries causing diarrhoeal outbreaks in a relevant number of pig herds [3] [4] [5] . pedv or specific antibodies against pedv were reported in several european countries (belgium, the uk, the netherlands, germany, hungary, bulgaria, france, switzerland and spain) in the seventies and the eighties [3] [4] [5] . mortality in piglets less than two weeks old varied from 0 to 100 %, but it was usually lower than that described in outbreaks of diarrhoea caused by transmissible gastroenteritis virus (tgev) which is another porcine coronavirus classically recognized as a cause of diarrhoea disease in swine. however, for unknown reasons, ped outbreaks markedly decreased in the nineties and in subsequent years in europe. isolated outbreaks associated with low mortality in piglets were reported in some countries, i.e. spain [6] , hungary [7] , the uk [8] or the czech republic [9, 10] . the only well-documented ped epidemic over the last 10 years in europe occurred during the winter of 2005-2006 in northern italy. on average, pre-weaning mortality raised from 8.3 to 11.9 %, peaking at 34.5 % in one particular farm [11] . recent ped outbreaks have been reported in germany [12] [13] [14] , italy [15, 16] , the netherlands [17] , belgium [18] , france [19] , the ukraine [20] and other european countries (unpublished data). in contrast to the situation in europe, pedv has remained as a major cause of diarrhoea outbreaks on swine farms in asia for over 30 years. viral-like diarrhoea outbreaks were reported on pig farms in shanghai, china in 1973 and despite the fact that tgev and other known enteropathogenic agents were ruled out, the aetiology could not be determined. pedv was firstly demonstrated in the area in 1983 in china [21] and japan [22] . in the nineties the virus spread to neighbouring countries such as korea [23] , the philippines and thailand [24] . later on, it was reported in taiwan in 2007 [25] and vietnam in 2009 [26] . due to its relevance in this area, attenuated or killed vaccines, which confer partial protection against pedv, have been used in several asian countries. these vaccines have been used in china since 1995 and also introduced in japan in 1997, south korea in 2004 and the philippines in 2011 [27] . it is relevant to point out that in october 2010, a large-scale and severe ped outbreak was reported in several provinces in southern china and spread to other provinces within this country as well as to other neighbouring countries [28, 29] . the outbreak caused high mortality among suckling piglets, between 50 and 90 % and given the fact that china was clearly not a naïve country regarding pedv infection, it was proposed that probably a new variant of pedv with a higher virulence was circulating. recently, in april 2013, pedv was identified for the first time in the usa, on pig farms located in ohio [30] . the virus spread quickly within the country and 1 year after the first description, the number of ped affected farms exceeded 5000 spreading to over 25 states. in addition, the virus spread to other countries in north, central and south america and ped outbreaks were reported for the first time on pig farms in mexico (july 2013), peru (october 2013), the dominican republic (november 2013), canada (january 2014), colombia (march 2014) and ecuador (july 2014) [31] . based on genomic analysis, the coronaviridae family has been recently divided into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus [32] . pedv is a member of the genus alphacoronavirus together with other coronaviruses which infect pigs (tgev and its respiratory variant, porcine respiratory coronavirus or prcv), dogs (canine coronavirus), cats (feline infectious peritonitis virus), humans (human coronavirus 229e or human coronavirus nl-63) or bats. there are also other swine coronaviruses (table 1) . porcine hemagglutinating encephalomyelitis virus (phev) is a betacoronavirus which causes an infection associated with chronic emaciation and death in young pigs (vomiting and wasting disease) while porcine deltacoronavirus (pdcov), a member of the genus deltacoronavirus, has recently been identified as the etiological agent of an enteric disease similar to ped or tge [33] . coronaviruses are enveloped viruses which possess a positive-sense single-stranded rna genome. they are morphologically characterized by the presence of projections or peplomers on their surface. like other members of the alphacoronavirus genus, pedv possesses four structural proteins: three membrane proteins identified as s protein or spike protein, m protein or membrane protein and e protein (previously sm or small membrane protein) and a nucleocapsid protein or n protein which encapsidates viral rna. s protein is particularly relevant among the structural proteins. it is a glycoprotein which induces neutralizing antibodies and interacts with cell receptor in the host. there are also three nonstructural proteins: two of them are encoded in open reading frames (orf) 1a and 1b and are involved in genome replication and transcription while the third, encoded in orf3, has been reported to be an ionchannel protein [3] [4] [5] . antigenic relationships in pedv and other coronavirus have been researched into [34] . although some crossreactivity between pedv and tgev associated with one epitope on the n-terminal region of n protein was recently reported, pig tgev antisera do not neutralized pedv and vice versa. no cross-reactivity has been reported between pedv and any other coronavirus of the beta, gamma or delta genera. direct and indirect pedv transmission occurs mainly by faecal-oral route. viral shedding in faeces starts on postinfection day one or two and continues for a period of 7 to 10 days [35, 36] , although it can extend up to 36 weeks in some animals [37, 38] . the transmission of the infection is facilitated by the high viral load in faeces from infected animals [39, 40] as well as by the minimum infectious dose required to infect naïve pigs [31] . moreover, the resistance of the virus in the environment facilitates the faecal-oral transmission. pedv is stable under low temperatures, while it is adversely affected by high temperatures. it survives between ph 5.0-9.0 at 4°c while only between ph 6.5-7.5 at 37°c. it can survive for at least 28 days in slurry at 4°c, 7 days in contaminated dry feed at 25°c or 14 days in contaminated wet feed at 25°c [31] . this fact favours the indirect transmission by different faeces-contaminated fomites such as transport vehicles [41] , feed [42] , clothing or footwear. genetic and phylogenetic analyses of american pedv isolates revealed a close relationship with chinese isolates and their likely chinese origin [43] . however, how the virus might have travelled from china to the usa is a matter of speculation. the rapid spread of pedv on swine farms in the usa raised questions regarding the possibility of airborne transmission of this infection. although undoubtedly the faecal-oral route is the main source of pedv transmission, it has been suggested [44] that pedv may travel through the air for short distances on faecal dust particles, at least under certain conditions. however, airborne transmission of pedv has only been shown under experimental conditions and up to now infectious pedv has not been demonstrated in field air samples containing pedv genetic material [44, 45] . the role that vectors play in the transmission of pedv has also been investigated. so far, there has been no evidence of pedv replication in non-porcine hosts, including rodents and starlings [46] [47] [48] . however, the potential role of vectors in the mechanic transmission of the virus from one farm to another cannot be ruled out, as has been described for tgev [4] . using highly sensitive molecular assays the presence of viral rna has been reported in milk samples from infected lactating sows [28, 29] as well as in semen samples [29, 31] . however, infectious pedv in these samples has not been demonstrated and their contamination with faecal material in the sampling cannot be excluded. moreover, viral rna has been detected in the serum fraction of whole blood samples from infected pigs [40, 49] . the role of spray-dried porcine plasma (sdpp), normally used as feed additive, as a potential vehicle of transmission of pedv has been researched into. a number of experimental studies have demonstrated that spray-drying process as well as storage conditions are sufficient to inactivate infectious pedv in sdpp [50, 51] . the infectivity of commercial sdpp positive for pedv-rna has also been investigated. a research group from canada managed to reproduce pedv infection in sdpp-inoculated piglets, although they failed to reproduce the infection in animals receiving feed supplemented with the same pedv-positive sdpp [52] . similarly, neither clinical signs nor pedv rna in faeces or pedv specific antibodies were detected in pigs which were fed a diet containing 5 % sdpp confirmed positive for pedv, in a bioassay experiment conducted by opriessnig et al. [53] . according to this, there is no experimental evidence of pedv transmission through pcr positive sdpp supplemented feed. this experimental data is corroborated by the fact that despite the use of large amounts of pedv positive sdpp from the usa to feed pigs in brazil or western canada, these areas remained free of pedv infection [54] . pedv replicates in the cytoplasm of villous enterocytes of the small intestine and causes villous shortening and reduced enzymatic and absorptive capacity in the small intestine causing profuse watery diarrhoea, which lasts about a week [37, 55, 56] . other clinical signs which are frequently associated to pedv infection include vomiting, anorexia and fever. although pigs of all ages are affected, the severity of ped is higher in suckling piglets of less than one week old which may die due to severe dehydration. the slower turnover of enterocytes in neonatal piglets (5-7 days) compared to three weeks-old piglets (2-3 days) could explain, at least partially, the higher susceptibility of these young piglets to pedv [4] . pedv has also been detected in epithelial cells of the colon in both experimentally and naturally infected pigs, although villous atrophy has not been demonstrated in the large intestine [40] . replication of pedv was classically circumscribed to the intestinal tract [3] , until a recent research showed pedv replication in alveolar macrophages of 3 day-old-colostrum-free piglets, which were experimentally inoculated with a korean wild-type pedv isolate [57] . further studies are needed to confirm whether extra-intestinal replication also occurs with other pedv isolates as well as to determine their clinical and epidemiological relevance. two epidemiologic presentations of ped have been described on the farms. (a) epidemic ped outbreaks occur when pedv is introduced into a naïve farm (where most of the animals are pedv seronegative). the disease spreads rapidly affecting pigs of all ages with morbidity approaching 100 %. moreover, pedv can persist and become (b) endemic on the farm affecting post-weaning piglets that have lost their lactogenic immunity as well as newly introduced seronegative gilts. mortality associated with ped outbreaks is highly dependent on the age of the infected animals. mortality can reach up to 80-100 % in suckling piglets of less than one week old, while in weaned pigs mortality rates are typically only 1 to 3 % [11, 30] . no mortality associated with ped is usually observed among adult pigs. as has already been mentioned, differences in the severity of ped outbreaks have been reported. particularly severe ped outbreaks have been described in asia since 2010 and also in the usa. differences in the virulence of pedv isolates have been proposed to explain this variability [28, 58, 59] . from our point of view, this is one of the most relevant questions to face regarding ped nowadays: the reason or reasons which could explain variations in the clinical outcome of an outbreak. although some reports have suggested that they could be associated with differences in the virulence of pedv isolates, exhaustive challenge studies using pig adapted virus (not cell culture adapted isolates) in suckling piglets are needed to elucidate the role of the strain. some insights have been obtained related to the virulence of different strains. in the usa, at least two main variants of pedv have been recently identified using molecular methods. the first one seems to be a highly virulent virus and similar to viruses described in several asian countries after 2010 while the second, the s indel variant, has been associated to mild clinical outbreaks [59] . this s indel variant includes some particular insertions and deletions in the s gene and is also similar to some asian isolates, part of which were recovered before 2010. the classical european reference strain of pedv cv777 is also an s indel isolate although it is located in a different cluster and well differentiated from american indel isolates ( fig. 1a and b) . pedv isolates recovered in european countries (germany, italy, belgium, the netherlands and france) in 2014 and 2015 have been characterized and all of them were found to be indel isolates similar to the variant described in the usa [13] [14] [15] [16] [17] [18] [19] . most of these recent ped outbreaks in europe occurred in fattening farms and, as expected, no mortality was observed. however, pedv isolates recently recovered in severe outbreaks of pedv in ukraine have shown a genome nucleotide similarity reaching 99.8 % with non-indel isolates from the united states and mexico [20] . so far, this has been the only report of pedv non-indel isolates in europe. apart from differences in the virulence of the pedv strains, many other parameters including management, immune status of the population and herd sanitary status could also explain variations in the clinical outcome of ped outbreaks [31] . thus, the contribution of co-infections with other viruses, particularly with other enteric viruses such as porcine delta coronavirus (pdcov) or the recently described mammalian orthoreovirus 3 (mrv3) has also been pointed out. both viruses have been detected in faecal samples collected from pedv positive farms in the usa. pdcov has been associated with mild to moderate diarrhoea in experimentally inoculated naïve suckling piglets [33] while mrv3 caused severe diarrhoea with 100 % mortality in 3-day-old piglets [60] . although the rapid spread of a disease characterized by profuse watery diarrhoea affecting pigs of all ages allows the clinician to suspect that a viral agent is involved in the infection, differential diagnosis to identify pedv at the laboratory would be needed. direct detection of pedv in faecal samples by conventional or real-time pcr, are the most frequent assays used at present [27] . pcr-assays are generally based on the amplification of fragments within the m, n or s protein genes and are associated with a high sensitivity and specificity. there are also some elisas, which are usually based on the use of monoclonal antibodies against pedv. although their analytical sensitivity is generally lower than pcr assays [61] , they are useful under field conditions as the amount of virus in faecal samples from diseased animals in epidemic outbreaks of ped is very high. immunohistochemistry (ihc) is also a very useful tool based on the detection of pedv antigens within infected cells in formalin-fixed sections of small intestine. it is less sensitive than molecular diagnostic methods but, in contrast, it allows for the evaluation of tissue lesions [62] . in order to increase the sensitivity of ihc assays, several sections of the small intestine of affected pigs sacrificed in the acute phase of the infection should be investigated. indirect methods are focused on the detection of antibodies. the detection of pedv specific antibodies is very useful, not for the investigation of diarrhoea outbreaks, but to determine whether an animal or a herd has previously been infected by this virus. taking this approach into account, serology is a good tool for surveillance as it provides useful information regarding viral spread in a region or a country. however, the number of tests for the detection of pedv specific antibodies is limited to elisas, indirect immunofluorescence assays (ifa), immunoperoxidase monolayer assays (ipma) and seroneutralization. most of these tests are in-house assays and information regarding their sensitivity and specificity is usually scarce. in general, the elisa tests have proven to be capable of detecting pedv specific antibodies a little earlier and for longer periods of time than ifa tests [35] . there is no specific treatment for pedv other than supportive care and symptomatic treatment. mortality occurs in suckling piglets as a result of dehydration which should be corrected using oral electrolyte solutions. in adult pigs, dry feed should be withdrawn for a period of 12-24 h and then, carefully reintroduced while water should be kept freely available [3, 4] . in order to increase passive immunity to piglets and minimize losses, sows due to farrow in at least 2 weeks can be deliberately exposed to virulent virus by the oral route. a recent study revealed that morbidity was reduced from 100 to 43 % in litters exposed to virulent pedv when their sows were previously exposed to a mild virulent strain (s indel variant) of pedv [63] . oral administration of chicken egg-yolk or cow colostrum containing pedv immunoglobulins could offer an immunoprophilactic defence [64, 65] . the increase in lactogenic immunity is also the aim of pedv vaccines which are used in pregnant sows. attenuated or killed vaccines against pedv have been used in several asian countries for years [66] . however, it has been suggested that live vaccines can revert to virulence and their use and usefulness under field conditions have been questioned [5, 27, 67] . recently, a pedv subunit vaccine based on the s protein gene of pedv as well as a vaccine with killed virus have been licensed in the usa [68] , although there are still no studies which prove their efficacy. however, pedv vaccines have never been used in europe as the disease was not of sufficient economic importance in this area. in general, pedv vaccines have been reported to be useful to booster antibody response in animals that have already been infected by pedv. as there are no specific treatments for the control and potential eradication of the disease from the herd, preventive measures which preclude the introduction of the virus or new pedv strains in the area, country or farm are of paramount importance. supported by the detection methods mentioned in the diagnosis, surveillance should be used to certify that trading of swine or related derivatives do not cause the spread of new strains of the virus. lorries used in transport have been highlighted as a relevant source of transmission [41] and special attention should be paid in the effectiveness of the cleaning and disinfecting protocols to inactivate and remove the virus. at herd level, basic external biosecurity rules such as quarantine of reposition, ban the entrance of unwashed vehicles, strict visitor policies (time interval between visiting two farms, provide footwear and appropriate clothing, showers and so on) should be carried out without exception and internal biosecurity such as controlling the slurry level, carcasses disposal and carcass bin cleaning, movement of the caretakers on the farm and so on could prevent the establishment of an endemic form of the disease. finally, many virucidal disinfectants have been shown to be effective in inactivating pedv. phenol, quaternary ammonium compounds, glutaraldehyde and bleach are examples of such disinfectants. water temperature is a crucial factor and temperatures over 60°c help to inactivate the virus. proper cleaning and disinfecting of facilities and equipment is crucial to control pedv. the emergence and spread of pedv on us pig farms has aroused growing interest in this coronavirus. the main areas of recent research on this disease have been focused on the molecular characterization of the isolates as well as the sources of infection and means of transmission. despite the fact that relevant knowledge has increased, there are still a number of questions to be answered. on one hand, any difference in virulence among the pedv variants described needs to be clarified. on the other hand, the rapid spread of this virus in the usa has raised concerns about its transmission mechanisms. pedv is mainly spreads by the faecal-oral route either by direct or indirect contact (feed or fomites such as vehicles). other routes or sources for its transmission such as air-transmission, vectors or sdpp have been investigated although their implication has not been clearly demonstrated. the recent ped outbreak in the american continent also shows that more research is needed for the control of the disease, based on the development of useful vaccines and surveillance of the virus, standardising its detection in laboratories with the final goal being the limiting of its spread. the authors declare that they have no competing interests. all the authors helped to draft the manuscript, read and approved the final manuscript. submit your next manuscript to biomed central and take full advantage of: virus-like particles associated with porcine epidemic diarrhoea a new coronavirus-like particle associated with diarrhoea in swine update on porcine epidemic diarrhoea diseases of swine porcine epidemic diarrhoea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis evaluation of an elisa for the 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viral genomic sequences scientific opinion on porcine epidemic diarrhoea and emerging pig deltacoronavirus discovery of seven novel mammalian and avian coronaviruses in deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus pathogenicity and pathogenesis of a united states porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains evaluation of a blocking elisa using monoclonal antibodies for the detection of porcine epidemic diarrhoea virus and its antibodies use of an internal control in a quantitative rt-pcr assay for quantitation of porcine epidemic diarrhoea virus shedding in pigs pathogenesis of porcine epidemic diarrhoea virus isolate (us/iowa/18984/ 2013) in 3-week-old weaned pigs evaluation of porcine epidemic diarrhea virus transmission and the immune response in growing pigs multiple factors contribute to persistent porcine epidemic diarrhoea infection in the field: an investigation on porcine epidemic diarrhoea repeated outbreaks in the same herd pathology of us porcine epidemic diarrhoea virus strain pc21a in gnotobiotic pigs role of transportation in spread of porcine epidemic diarrhoea virus infection, united states an evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhoea virus infection of naive pigs following consumption via natural feeding behaviour: proof of concept origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states evidence of infectivity of airborne porcine epidemic diarrhoea virus and detection of airborne viral rna at long distances from infected herds concentration, size distribution and infectitivy of airborne particles carrying swine viruses susceptibility of mice to porcine epidemic diarrhoea virus prevalence of swine viral and bacterial pathogens in rodents and stray cats captured around pig farms in korea experimental inoculation with pedv in sparrows and mice oral/nasal inoculation of four-week-old pigs with pedv: tissue tropism, shedding, carriage, antibody response, and aerosol transmission the spray-drying process is sufficient to inactivate infectious porcine epidemic diarrhoea virus in plasma survivability of porcine epidemic diarrhoea virus (pedv) in bovine plasma submitted to spray drying processing and held at different time by temperature storage conditions investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhoea in canada porcine epidemic diarrhoea virus rna present in commercial spray-dried porcine plasma is not infectious to naïve pigs analysis of spray dried porcine plasma (sdpp) produced in brazil and western canada confirm negative porcine epidemic diarrhoea virus (pedv) status of pigs in these regions experimental infection of pigs with a new porcine enteric coronavirus, cv-777 in situ hybridization for the detection and localization of porcine epidemic diarrhea virus in the intestinal tissues from naturally intected piglets porcine epidemic diarrhoea virus infects and replicates in porcine alveolar macrophages porcine epidemic diarrhoea virus variants with high pathogenicity distinct characteristics and complex evolution of pedv strains a novel pathogenic mammalian orthoreovirus from diarrheic pigs and swine blood meal in the united states comparison of enzyme-linked immunosorbent assay and rt-pcr for the detection of porcine epidemic diarrhoea virus immunohistochemical detection of porcine epidemic diarrhoea virus compared to other methods previous infection of sows with a "mild" strain of porcine epidemic diarrhea virus confers protection against infection with a "severe" strain immunoprophylactic effect of chicken egg yolk immunoglobulin (igy) against porcine epidemic diarrhea virus (pedv) in piglets isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages porcine epidemic diarrhea: a review of current epidemiology and available vaccines molecular epidemiology of porcine epidemic diarrhoea virus in china an inactivated vaccine made from a u.s. field isolate of porcine epidemic disease virus is immunogenic in pigs as demonstrated by a dose-titration we gratefully acknowledge the pig farmers and veterinarians, their organisations and the feed companies for their active cooperation in the development of our research. g.f. bayón provided excellent technical assistance. our research on pedv has been funded by the ministerio de agricultura, pesca y alimentación. key: cord-266571-qbskh1uu authors: de arriba, m.l; carvajal, a; pozo, j; rubio, p title: lymphoproliferative responses and protection in conventional piglets inoculated orally with virulent or attenuated porcine epidemic diarrhoea virus date: 2002-06-04 journal: j virol methods doi: 10.1016/s0166-0934(02)00063-0 sha: doc_id: 266571 cord_uid: qbskh1uu lymphocyte proliferative responses were evaluated in mucosal (mesenteric lymph nodes) and systemic (spleen and blood) lymphoid tissues of conventional piglets inoculated with the virulent or attenuated isolates of porcine epidemic diarrhoea virus (pedv) strain cv-777 and challenged 21 days later with the virulent isolate of the same virus. a lymphoproliferative assay was developed in which mononuclear cells isolated from lymphoid tissues at different postinoculation and postchallenge days underwent a secondary in vitro stimulation with semipurified antigen obtained from pedv-infected cell cultures. vigorous lymphocyte proliferative responses were detected in the pigs inoculated with the virulent pedv at postinoculation days 4–21, especially in the mesenteric lymph nodes and the blood; however, in the spleen this response was lower and less regular. the pigs inoculated with the attenuated virus showed a less intense response, the higher lymphocyte proliferation also corresponded to the mononuclear cells from mesenteric lymph nodes. lymphocyte proliferation responses showed high correlations with protection against homologous challenge with virulent pedv, and this correlation was higher in the gut associated lymphoid tissues (mesenteric lymph nodes). the cell proliferation response detected in blood mirrored that detected in the mesenteric lymph nodes, and showed also good correlation with protection. the results confirm that t-cell-helper function, assessed by lymphocyte proliferation responses, contributes to establishing a protective immune response against pedv infections. porcine epidemic diarrhoea is an enteric disease in pigs caused by a member of the coronaviridae family, porcine epidemic diarrhoea virus (pedv) (cavanagh et al., 1994; murphy et al., 1999) . because of the high prevalence and the severity of clinical symptoms, porcine epidemic diarrhoea represents the most important viral infection of swine intestinal tract in spain and in many other european and asean countries (carvajal et al., 1995b; pensaert, 1999; sueyoshi et al., 1995; van reeth and pensaert, 1994) , although not in the usa where the pedv is not present but a high prevalence of transmissible gastroenteritis virus infections is still present (saif, 1998; saif and wesley, 1999) . the disease is characterized by acute watery diarrhoea, depression and anorexia. although morbidity is high and most of the animals in the herd can be affected within a week, mortality is usually low in adult animals (3%), which can recover in 7-10 days. however, when piglets less than 4-5 weeks are infected, mortality is usually 50% and can reach 90% in severe outbreaks, as usually happens in transmissible gastroenteritis. besides the mortality, pedv infection causes important economic loses due to the diminishing of the productive indexes (pensaert, 1999) . as in many other viral infections of food animals, the lack of effective treatment makes immunity the main key for prevention and control of the disease. however, the development of candidate vaccines needs previous knowledge of immunological aspects related to the infection. due to the enteric nature of the disease and the special configuration of the mucosal immune system, protection depends mostly on the local immune response (corthesy and kraehenbuhl, 1999; kagnoff, 1996; saif et al., 1994; van cott et al., 1994) , and is the reason why these studies cannot be limited to blood but require organs containing gut associated lymphoid tissues, in which local immune response can be measured. in spite of the fact that the importance of humoral immune response in gastroenteric viral infections of porcine is well recognized (corthesy and kraehenbuhl, 1999; saif et al., 1994; tô et al., 1998; van cott et al., 1994; yuan et al., 1996) , little is known about cell-mediated immunity, particularly in pedv infections. however, it has been proposed that cellular immune response may play an important role in the protection and recovery from infection, besides the fact that production of antibodies is regulated by cytokines produced by activated t lymphocytes and other mononuclear cells (corthesy and kraehenbuhl, 1999; kraehenbuhl and neutra, 1992; saif, 1999; totterdell et al., 1988) . the aim of this study was to assess the cellular immune response following natural infection with pedv and also after inoculation of an attenuated virus, and the contribution to the establishment of a protective response. virus-specific lymphoproliferative responses of systemic tissues (spleen and blood) and mesenteric lymph nodes were studied in conventional piglets after primary inoculation with the virulent, wild type, strain cv-777 of pedv or its cell culture attenuated form and after challenge, 3 weeks later, with a high dose of the virulent virus. vero cells were grown with eagle's minimum essential medium (gibco, life technologies) buffered with bicarbonate and supplemented with 5% (v/v) fetal calf serum (gibco), 0.04% (w/v) yeast extract (difco, mi, usa), streptomycin (10 mg/l) and penicillin (10,000 ui/l) (penicillin-streptomycin, gibco). the cell culture adapted strain of pedv cv-777, attenuated by many passages, was propagated in vero cells as described by hofmann and wyler (1989) , infecting confluent monolayers of cells after removing the growth medium and adding the viral inoculum diluted in medium without fetal calf serum but containing 10 ml/ml of trypsin (difco). a pedv-infected cell lysate was used as attenuated pedv inoculum. the wild type isolated of the cv-777 strain of pedv, kindly provided by dr peansert (gent, belgium), was amplified by passages in conventional 1-week-old piglets without antibodies against pedv and prepared in pbs for use as virulent pedv inoculum. after oral inoculation, the animals were killed in the acute phase of diarrhoea, and the intestinal contents and the small intestine were collected at necropsy. the small intestine from each animal was macerated in pbs (1:2 (w/v)) and, like the intestinal contents, clarified by centrifugation at 5000× g for 20 min at 4°c. the richest fractions were pooled and stored at − 70°c. a total of 62 conventional 11-day-old piglets, seronegative to pedv and from a herd with no previous history of the disease were assigned to three different experimental groups which were maintained in isolation facilities to prevent virus circulation. group 1 (n = 29) was inoculated with a low dose of the virulent isolate of pedv strain cv-777 that was adjusted in a previous experiment to produce a high morbidity without causing severe disease in the animals. pigs from group 2 (n =20) were inoculated with 2.55×10 5 fluorescent focus-forming units per pig of the attenuated isolate of the same pedv strain. the third group (n = 13) was mock-inoculated and served as control. twenty-one days after inoculation, the three groups of pigs were challenged with the virulent pedv, using a two times higher dose than that used to inoculate the group 1. animals were observed daily for clinical symptoms and rectal swabs were taken for 11 days after inoculation and for 9 days after challenge. faecal scores were recorded as normal faeces, pasty faeces, semiliquid (moderate diarrhoea) and liquid (watery diarrhoea). at different postinoculation and postchallenge days (postinoculation days 4, 7 and 14, and postinoculation/postchallenge days 21/0, 25/4 and 33/12) subsets of each group of pigs (n= 2-5) were killed by injection of barbiturate overdose (eutalender, normon, madrid, spain). hundred millilitre of blood were collected from cardiac cavities in 25% (v/v) acid citrate glucose. spleen and mesenteric lymph nodes were also collected aseptically and placed in ice-cold wash medium (rpmi 1640 containing 10 mm hepes and 200 mg of gentamicin and 20 mg ampicillin per ml). three pedv-seronegative, unexposed pigs served as negative control and were killed to obtain the background values for the lymphoproliferative assay. mononuclear cells from blood and tissues were isolated as described previously (de arriba et al., 2001a) . briefly, peripheral blood lymphocytes were obtained by density gradient centrifugation in ficoll-paque (ficoll-paque research grade, pharmacia biotech, upsala, sweden). lymphocytes collected from the interface were washed twice in hanks' balanced salt solution and suspended in rpmi 1640 containing 8% fetal calf serum, 2 mm l-glutamine, 1 mm sodium pyruvate, 0.1 mm nonessential aminoacids, 20 mm hepes and 20 mg of ampicillin and 100 mg of gentamicin per ml (enriched medium). mononuclear cells from spleen and mesenteric lymph nodes were obtained by pressing the tissues through stainless steel screens (80 mesh) of a cell collector (cellecter; e-c apparatus corp., fla, usa). cell suspensions were centrifuged, and the mononuclear cells were removed from the pellet by continuous and discontinuous gradient centrifugation in percoll (pharmacia biotech.), washed twice with wash medium and resuspended in enriched medium. viability of all mononuclear cells preparations was confirmed by the trypan blue exclusion test, in every case being \ 95%. the lymphoproliferative assay for detection of pedv-specific t cells was adapted from methods published previously (brim et al., , 1995 ward et al., 1996) . semipurified pedv antigen for in vitro stimulation of mononuclear cells cultures was obtained from lysates of pedv-infected cell cultures that were concentrated 50 times by ultracentrifugation at 100,000×g for 2 h at 4°c and then semipurified by ultracentrifugation through 20% sucrose under the same conditions. the most favourable concentration of antigen for optimal antigenic stimulation of the mononuclear cells was established by dose-response curves. a pedvcontrol antigen was obtained giving the same treatment to mock-infected cultures. the t-cell mitogen phytohaemagglutinin (gibco) was used as positive control at a final concentration of 10 ml/ml, following the manufacturer's instructions. optimal conditions of number of cells and duration of incubation were determined by preliminary studies. mononuclear cells at a concentration of 5×10 5 cells per 100 ml and per well were placed in 96-well culture plates and stimulated in triplicate with the pedv antigen or the control antigen or the phytohaemagglutinin. cells were incubated for 72 h at 37°c in 5% co 2 and 18 h prior to harvest each well was labelled by pulsing 1 mci of [ 3 h]thymidine (amersham pharmacia biotech). harvesting of cells was carried out on glass fiber filters (filtermat, skatron inc., va, usa) and [ 3 h]thymidine incorporation was determined by liquid scintillation spectrophotometry. the lymphocyte proliferative responses for each mononuclear cells sample assayed was expressed as the stimulation index (si), calculated as si= mean cpm of pedv stimulated wells/mean cpm of control antigen stimulated wells, being cpm counts per minute. viral antigen was detected in faecal samples by the double antibody sandwich elisa described by carvajal et al. (1995a) . this elisa is based on the use of two monoclonal antibodies (lelsytad cvi-66.31 and lelystad cvi-66.49) directed specifically against the s protein of the virus. a blocking step with rabbit-anti pedv hyperimmune serum was included to increase the specificity. twofold serial dilutions of the samples were assayed starting at 1:2 and titres were expressed as the inverse of the lowest positive dilution. for calculation of the geometric mean titre (gmt), negative samples were given a titre of 1. one-way analysis of variance followed by the paired student's t-test was used to determine the nature of differences observed in virus shedding and lymphocyte proliferation responses among inoculated groups, tissues and days. correlation between proliferation responses at challenge day and protection against the infection was established by spearman's correlation coefficient (z). significance was assessed at pb 0.05. for the analysis the systat for windows v.5.03 (sys-tat inc.) and the spreadsheet microsoft excel v.7.0 (microsoft comp.) were used. a summary of faecal virus shedding and clinical disease after inoculation and challenge is given in table 1 . after primary inoculation of pigs from group 1 with virulent pedv, moderate to severe diarrhoea was observed in 33% of the animals and virus shedding in 100%. the onset of diarrhoea was observed between postinoculation days 2 and 4 and the average duration was 1.7 days. virus shedding was detected in some of the pigs at postinoculation day 1 but most shed pedv in faeces from postinoculation day 2 to 6. the average duration of the shedding period was 5.4 days. the gmt of viral antigen in faecal samples was measured using elisa; it increased strongly from postinoculation day 2 and reached a peak at postinoculation day 5. conversely, pigs from group 2 inoculated with the attenuated pedv did not show typical signs of the disease and only one pig had moderate diarrhoea for 1 day and virus shedding was detected in only one sample at postinoculation day 5 and with a low titre. differences between the gmt of antigen in the faeces of the two groups were significant statistically. on the challenge day, at postinoculation day 21, pigs from group 1 were protected against infection and disease, none developed diarrhoea and not viral antigen was detected in the samples taken after challenge. diarrhoea was not observed in pigs from group 2 after challenge either, but viral detection in rectal swab samples revealed that protection against the infection was only partial (25%), and the antigen was detected in 75% of the challenged animals (9 out of 12). in the control group moderate diarrhoea was seen in 46% of the pigs starting between postchallenge days 3 and 4 and with an average duration of 1.5 days. viral antigen was detected in faeces of all of the challenged pigs from this group in which the average duration of viral shedding (4.6 days) was significantly higher than in group 2 (2.6 days). the gmt of viral antigen detected in the control group was also significantly higher than in group 2. in order to obtain the optimal secondary antigenic stimulation for the lymphoproliferative assay, two different doses of antigen were used, 25.5 ng of the semipurified antigen were added to each 5× 10 5 mononuclear cells from spleen and blood, whereas in the mesenteric lymph nodes a minor dose, 12.8 ng, yielded the optimal stimulation of the cells. mean cpm obtained after stimulation of each tissue with the different antigens and the mitogen are shown in table 2 . background cpm, obtained after stimulation of each mononuclear cells culture with the control antigen, were low in mesenteric lymph nodes and blood (usually b 4000) but not in spleen, where these counts sometimes reached values close to 22,000. after inoculation of group 1, a specific lymphocyte proliferation response was detected for the first time in the mesenteric lymph nodes at postinoculation day 4 (fig. 1) and was maintained until challenge day (postinoculation day 21), when the maximum value of si was found (18.82). si in this tissue was significantly higher from postinoculation day 4 to 21 than that observed in unexposed pigs. the lymphoproliferative responses in mesenteric lymph nodes of pigs from group 2 increased significantly at postinoculation day 14 compared with their responses in previous days ( fig. 1) and si reached its peak that day at 5.73. values of the si between postinoculation days 14 and 21 were significantly higher compared with responses for mononuclear cells from mesenteric lymph nodes in unexposed pigs. group 1 si was greater than group 2 at any pid, however statistical significance was only detected at postinoculation day 4. group 3 was mock inoculated and served as control at challenge. statistically significant differences among groups are denoted by the letters: 'a' when differences are between groups 1 and 2, 'b' when differences are between groups 1 and 3 and 'c' for differences between groups 2 and 3. level of significance is defined by *p50.5, **p50.01. a pid: postinoculation days. b pcd: postchallenge days. c gmt: geometric mean titre of antigen detected in faeces by elisa. mononuclear cells were stimulated with positive, negative antigen or the t-cell mitogen phytohaemagglutinin (pha). background values correspond to pigs with no previous contact to pedv. pid, postinoculation day; pcd, postchallenge day. fig. 2 . correlations between lymphocyte proliferative responses in mononuclear cells collected from mesenteric lymph nodes, blood and spleen from pigs inoculated with virulent or attenuated pedv or mock-inoculated and protection against challenge 21 days later with virulent pedv. correlations were assessed by spearman rank correlation test. lymphocyte proliferative responses were expressed as mean cpm of pedv stimulated wells versus mean cpm of control antigen stimulated wells, being cpm counts per minute. fig. 1 . course of the virus-specific lymphocyte proliferative responses represented by si for mononuclear cells from mesenteric lymph nodes, spleen and blood from pigs after inoculation with virulent (group 1) or attenuated (group 2) pedv or mock-inoculation (group 3) and after challenge with virulent pedv. the si are the mean cpm of virus-specific stimulated wells versus mean cpm of control antigen stimulated wells, being cpm counts per minute. the mean value of the si obtained from the group of unexposed pigs is represented by a line crossing the y-axis. statistically significant differences (p 50.05) with values obtained in nonexposed pigs are noted as *. differences between groups are nodded as: 'a' when differences are between groups 1 and 2, 'b' between groups 1 and 3 and 'c' for differences between groups 2 and 3. mononuclear cells purified from blood of group 1 showed a vigorous proliferative response after inoculation starting at postinoculation day 7 with significant increases over the following days. the si obtained for this group in blood were significantly higher than values in blood of unexposed pigs between postinoculation days 7 and 21 and as in the mesenteric lymph nodes, the peak value occurred at postinoculation day 21 (fig. 2) . virusspecific lymphoproliferative responses in blood from group 2 occurred at postinoculation day 14, the only day in which si value was significantly higher than that in unexposed animals (si= 9.58, pb0.001). likewise in mesenteric lymph nodes, the si of group 2 were minor than the indexes of group 1, although the difference was statistical significant only at postinoculation day 21. the magnitude of the virus-specific proliferation in the spleen of group 1 after inoculation was lower than in the other tissues, being also less regular. at postinoculation days 4 and 7, the response of this group was low and similar to the proliferation shown by unexposed pigs. in the following days there was an increase in the si that peaked at postinoculation day 14, however, the value of si was not significantly higher than the background values at any postinoculation time. group 2 did not show a virus-specific proliferative response in mononuclear cells from spleen after inoculation, with an si similar to that in unexposed pigs. after challenge at postinoculation day 21, the lymphoproliferative responses in mesenteric lymph nodes of group 1 underwent an important increase and even though at postchallenge day 4 (postinoculation day 25) the si was lower than on challenge day (although not significantly), this value again reached its peak at postchallenge day 7 (postinoculation day 28) with a value of 29.25 (fig. 1) . in group 2 lymphocyte proliferation responses after challenge were low and only at postchallenge day 7 (postinoculation day 28) was the si significantly higher than that in unexposed animals. responses in group 3, the mock-inoculated control group, after challenge were similar to responses described in mesenteric lymph nodes of pigs from group 1 after inoculation with virulent pedv, but showed a higher intensity. the si in this group was significantly higher than in unexposed animals from postchallenge day 7 (postinoculation day 28) and reached 5 days later (at postchallenge day 12, postinoculation day 33) the highest value detected in the mesenteric lymph nodes of all the groups. when the si of the three groups were compared, statistically significant differences were found at postchallenge day 4 (postinoculation day 25) between group 1 and groups 2 and 3 and at postchallenge day 12 (postinoculation day 33) the si of groups 1 and 3 were significantly higher than index in group 2. lymphocyte proliferative responses in blood after challenge in group 1 were significantly lower at postchallenge day 4 (postinoculation day 25) compared to the challenge day, however, from postchallenge day 7 (postinoculation day 28) there were significant increases, reaching maximum value at postchallenge day 12 (postinoculation day 33) (fig. 2) , the only day that this value could be demonstrated significantly higher than that in unexposed pigs. similarly to mesenteric lymph nodes, the response detected after challenge in blood from group 2 pigs was low, only the si at postchallenge day 12 (postinoculation day 33) was significantly higher than unexposed animals index. in the control group, lymphoproliferative responses were low up to postchallenge day 12 (postinoculation day 33) and there was not statistical significance in the differences observed with regard to the unexposed animals. comparisons between the si in the blood after challenge in the different groups showed a higher response in group 1, although statistical significance was only shown at postchallenge day 12 (postinoculation day 33). the si was lower in group 3 than in group 2, but not significantly. in the spleen, responses after challenge of group 1 increased significantly at postchallenge day 7 (postinoculation day 28) with regard to the previous day, similar to the mesenteric lymph nodes. group 2 response after challenge was maximum at postchallenge day 4 (postinoculation day 25), this being the only time in which the si of mononuclear cells of spleen from this group was significantly higher than the si of unexposed animals. the control group underwent for the first time a specific proliferation response at postchallenge day 12 (postinoculation day 33). the si obtained in each group at each point in time were compared and no statistically significant differences were found. correlations between lymphoproliferative responses detected in each tissue and group at the challenge day and protection against challenge, represented by the protection rate against infection, were established by the spearman rank correlation test and are shown in fig. 2 . the magnitude of the response in all tissues examined at postinoculation day 21 correlated positively with protection against challenge, although statistical significance were not attained. the highest correlation was detected in the mesenteric lymph nodes (z= 0.99, p= 0.08). protection in swine gastroenteric viral infections, as ped, has been related almost exclusively to the antibody immune responses. however, cell-mediated immunity must play an important role in protecting and recovery from infection, besides the control function of the b cell-humoral responses carried out by t cell populations (corthesy and kraehenbuhl, 1999; kraehenbuhl and neutra, 1992; mcghee et al., 1992; saif, 1999; totterdell et al., 1988) . thus, without any b cell population deficiency in humans the lack of antibody and specific t-cell responses, resulting in rotavirus persistent infection with viral excretion in faeces for 15 months (totterdell et al., 1988) . moreover, welch et al. (1988) , in pigs inoculated with transmissible gastroenteritis virus, related peaks of lymphoproliferative responses ending up with final virus shedding in faeces and the beginning of recovery from the disease. in this study, an in vitro virus-specific proliferation assay was carried out as a method to estimate the cell-mediated immune response since this antigen-induced proliferation has been recognised as a property of cd4 +(t helper) cells in studies undertaken on pigs, mice and humans with rotavirus and coronavirus offit et al., 1992; ward et al., 1996) . the specific proliferative response after inoculation of pigs with virulent pedv was detected immediately in the mesenteric lymph nodes, the organs directly associated with the mucosal immune system. the maximum values were found around postinoculation day 21, just when a strong response of virus-specific antibody-secreting cells was detected in this organ and also in the duodenum and ileum lamina propria (de arriba et al., 2001b) . in pigs inoculated with the attenuated strain of pedv this specific lymphoproliferative response was detected later, at postinoculation day 14 and it was lower that in pigs inoculated with the wild virus. this minor response of group 2 also corresponded to a low response of pedv-specific antibody-secreting cells (de arriba et al., 2001b) . the difference observed between the lymphoproliferative responses of the two inoculated groups has also been described by other researchers (brim et al., , 1995 ward et al., 1996) in other gastroenteric viruses of swine, describing that the lymphocyte proliferative responses induced by attenuated strains of transmissible gastroenteritis and rotavirus were significantly lower than that induced by the homologous virulent virus. these results suggest that a protective antibody response to the virulent pedv could be associated with previous development of a strong specific cell-mediated immune response. this consideration could be reinforced by the fact that antibody production by specialised b cells requires t cell help (corthesy and kraehenbuhl, 1999) . the virus-specific lymphocyte proliferative response in the systemic lymph tissues (blood and spleen) was observed later than in mesenteric lymph nodes, as well as being considerably lower in the spleen than in other tissues. this delay could be explained if it is considered that the pedv-specific t cells located in blood and spleen originate in the inductive sites from the gut-associated lymphoid tissues, like the mesenteric lymph nodes, and its presence in systemic tissues is due to the homing process necessary for its maturation (corthesy and kraehenbuhl, 1999; kagnoff, 1996; kantele et al., 1997; salmi and jalkanen, 1997) . the specific cell proliferation response in the blood was more similar to that observed in the mesenteric lymph nodes than in the spleen, especially in the group 1, in spite of both, blood and spleen, being linked to the systemic immune system. however, the blood, together with the lymphatic system, is the main vehicle for lymphocyte migration (salmi and jalkanen, 1997) , and its lymphoid population may reflect primed t cells migrating to the gut for some time after an infection. the lymphocyte proliferative responses at the challenge day showed high correlation with protection against challenge. pigs from group 1 inoculated initially with the virulent pedv, were 100% protected against infection 21 days later with a higher dose of the same virus whereas protection in group 2, inoculated with the attenuated pedv, was just partial and only 25% of pigs were protected against infection with the virulent virus. the highest correlation was observed in mesenteric lymph nodes. this result again suggests that t-cell response, especially in the gut associated lymphoid tissues, contributes in an important way to the development of a protective immune response in pedv infections. the highly attenuated pedv conferred partial protection against challenge with virulent virus in conventional pigs, this protection is related to the inoculated dose and increases when a higher dose is used (de arriba et al., 2001b) . kweon et al. (1999) also described the induction of protective immunity by a attenuated strain of pedv inoculated intramuscularly. after the challenge, there was an increase in the lymphocyte proliferative response in pigs from group 1, however this increase was not reflected either by an enhancement of the virus-specific antibody secreting cell response or the gmt of pedv-specific serum igg and iga (de arriba et al., 2001b) . thus, ward et al. (1996) also reported that pigs inoculated and challenged with virulent rotavirus strains showed after challenge lymphoproliferative responses similar or poorer than after inoculation. although there is no clear explanation in this, the possibility remains that this secondary response after challenge could be related to the proliferation of cell clones involved in immune regulatory functions different to providing help for antibody production, such as t suppressor populations. in this study the development of cell-mediated immunity occurred in systemic and lymphoid tissues after inoculation with virulent and attenuated strains of the pedv. the results suggest that cell-mediated immune responses contribute significantly to the instauration of protective immune status against homologous virulent virus challenge. the lymphoproliferative responses both in gut associated lymphoid tissues and systemic tissues had a higher magnitude when virulent pedv was used versus attenuated pedv to 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respiratory coronavirus how do lymphocytes know where to go: current concepts and enigmas of lymphocyte homing an immunohistochemical investigation of porcine epidemic diarrhoea serum and intestinal isotype antibody responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease systemic lymphoproliferative responses to rotavirus contribution of antibody-secreting cells induced in mucosal lymphoid tissues of pigs inoculated with respiratory or enteric strains of coronavirus to immunity against enteric coronavirus challenge prevalence of infections with enzootic respiratory and enteric viruses in feeder pigs entering fattening herds development of mucosal and systemic lymphoproliferative responses and protective immunity to human group a rotavirus in a gnotobiotic pig model cell-mediated immune responses of suckling pigs inoculated with attenuated or virulent transmissible gastroenteritis virus systemic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease we wish to thank dr m.b. pensaert for providing the wild type isolated of the pedv strain cv-777 and dr l.j. saif and dr l.a. ward for laboratory training. we also wish to thank g.f. bayó n and b. escudero for their excellent techni-cal assistance. this work was funded by the comisió n interministerial de ciencia y tecnología (cicyt) project no. agf-960486. salaries were provided by the excelentisima diputació n provincial de leó n. key: cord-255238-adpn5fb9 authors: pan, yongfei; tian, xiaoyan; qin, pan; wang, bin; zhao, pengwei; yang, yong-le; wang, lianxiang; wang, dongdong; song, yanhua; zhang, xiangbin; huang, yao-wei title: discovery of a novel swine enteric alphacoronavirus (seacov) in southern china date: 2017-09-28 journal: vet microbiol doi: 10.1016/j.vetmic.2017.09.020 sha: doc_id: 255238 cord_uid: adpn5fb9 outbreaks of diarrhea in newborn piglets without detection of transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv) and porcine deltacoronavirus (pdcov), have been recorded in a pig farm in southern china since february 2017. isolation and propagation of the pathogen in cell culture resulted in discovery of a novel swine enteric alphacoronavirus (tentatively named seacov) related to the bat coronavirus hku2 identified in the same region a decade ago. specific fluorescence signal was detected in vero cells infected with seacov by using a positive sow serum collected in the same farm, but not by using tgev-, pedvor pdcov-specific antibody. electron microscopy observation demonstrated that the virus particle with surface projections was 100–120 nm in diameter. complete genomic sequencing and analyses of seacov indicated that the extreme amino-terminal domain of the seacov spike (s) glycoprotein structurally similar to the domain 0 of the alphacoronavirus nl63, whereas the rest part of s structurally resembles domains b to d of the betacoronavirus. the seacov-s domain 0 associated with enteric tropism had an extremely high variability, harboring 75-amino-acid (aa) substitutions and a 2-aa insertion, compared to that of hku2, which is likely responsible for the extended host range or cross-species transmission. the isolated virus was infectious in pigs when inoculated orally into 3-day-old newborn piglets, leading to clinical signs of diarrhea and fecal virus shedding. these results confirmed that it is a novel swine enteric coronavirus representing the fifth porcine coronavirus. coronavirus (cov) is an enveloped, single-stranded, positive-sense rna virus of the order nidovirales, family coronaviridae, subfamily coronavirinae, which comprises four genera, alpha-, beta-, gamma-, and delta-cov. covs infect humans, other mammals, and birds, causing subclinical or respiratory and gastrointestinal diseases (de groot et al., 2011; woo et al., 2012) . as of date, three types of swine enteric covs (secovs): transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv) and porcine deltacoronavirus (pdcov), have been identified to induce clinical diarrhea in young pigs (jung et al., 2016; pensaert and de bouck, 1978) . in particular, emergences of variant pedv fatal to newborn piglets in china in late 2010 (pan et al., 2012) , and later in the united states in 2013 tian et al., 2014) , have posed a serious threat to the pork industry. most recently, several chimeric secov strains with a tgev genomic backbone replaced by a pedv spike (s) gene were identified from swine fecal samples in europe (akimkin et al., 2016; belsham et al., 2016; boniotti et al., 2016) , implying that novel secov pathogens could emerge by inter-cov recombination under co-infection. the s gene encodes a glycoprotein, forming trimer projections on the viral surface, which is a major structural protein critical for cov attachment and entry into the host cell (hulswit et al., 2016) . in addition to recombination events between two distinct covs, amino acid (aa) mutations in the s protein may alter the tropism of the virus. for example, 21-aa substitutions and a 7-aa insertion in the amino-terminal domain (ntd) of the s glycoprotein of a murine hepatitis cov (mhv) variant confer the ability to bind and in some cases infect cells of nonmurine species including swine cells (schickli et al., 2004) . in this study, we report the isolation and genetic characterization of a novel swine enteric alphacoronavirus (tentatively named seacov), related to a bat enteric coronavirus, from a pig farm that reported newborn-piglet diarrhea in southern china in 2017. this is yet another example to corroborate that the extended host range of cov, here from bat to pig, is likely associated with aa substitutions at the ntd of the s glycoprotein. furthermore, we conducted a pilot experimental infection study with this novel seacov, confirming its infectivity and ability to induced clinical signs of diarrhea in piglets. baby hamster kidney fibroblast cell line bhk-21 (atcc ccl-10), swine testis cell line st (atcc crl-1746), porcine kidney epithelial cell line llc-pk1 (atcc cl-101), and african green monkey kidney epithelial vero cell (atcc ccl-81) were individually grown in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs) and 1% antibiotics (penicillin, streptomycin, w/v). a vero cell line stably expressing the tgev receptor porcine aminopeptidase n (vero-papn) was cultured in dmem supplemented with 10 μg/ml puromycin and antibiotics (unpublished data). all cells were grown at 37°c with 5% co 2 . a pan-cov rt-pcr assay was used to detect the unknown pathogen with a pair of primers: cor-fw (5′-acwcarhtvaayytnaartaygc-3′) and cor-rv (5′-tcrcayttdggrtartccca-3′) as described (moes et al., 2005) . after the pathogen (seacov) was identified, specific primers targeting the seacov-nucleocapsid (n) gene (the forward primer seaf: 5′-atggataaacctgaatggaagcg-3′, and the reverse primer sear: 5′-caccatctcaacctcttcctcag-3′) were used for virus detection during isolation and subsequent passages. fecal specimens collected from diarrheic piglets and positive for seacov rna were homogenized in dmem containing antibiotics followed by centrifugation at 4000 × g for 15 min. the supernatants was inoculated onto confluent monolayers of bhk-21, st, llc-pk1 or vero cells cultured with the maintenance medium plus trypsin (mmt) at 37°c and 5% co 2 . the mmt consisted of dmem supplemented with 10% fbs, 1% antibiotics and 5 μg/ml trypsin (sigma). cells were observed daily to record the development of cytopathic effect (cpe) as described previously (pan et al., 2012) . the virus strain isolated in vero cells with mmt, designated as ch/ gd-01/2017, was plaque-purified in the presence of trypsin using neutral red staining as described (qin et al., 2017) . it was passaged serially using the culture supernatant and the viral titer was determined by plaque assay. supernatant from purified seacov-infected cell cultures showing cpes was negatively-stained. grids were stained with 2% sodium phosphotungstic acid (ph 6.8) for 1.5 min and examined using a hitachi model h-7650 tem. vero cells infected with seacov on 24-well plates were washed twice with phosphate-buffered saline (pbs) and fixed with acetone. one hundred and fifty microliters of the collected sow serum samples at a 1:100 dilution in pbs was added to the cells in each well and incubated for 1 h at room temperature. cells were washed thrice with pbs followed by addition of 150 μl fitc-labeled rabbit anti-pig igg (thermo fisher scientific, usa) at 1:500 dilution. after incubation for 1 h at room temperature, the cells were washed with pbs, stained with 150 μl 4′, 6-diamidino-2-phenylindole (dapi) at 1:1000 dilution and visualized under a fluorescence microscope. for antibody cross-reactivity test, vero cells infected with seacov or pedv (zju/g2/2013 strain; genbank accession no. ku558701), vero-papn cells infected with tgev (purdue strain; a gift from dr. rong ye at shanghai medical college of fudan university), and llc-pk1 cells infected with pdcov (hunan strain; genbank accession no. ky513724) were stained with the anti-pedv-n, anti-tgev-n and anti-pdcov-n monoclonal antibody (purchased from medgene labs, brookings, sd, usa), respectively. the fitc-conjugated goat anti-mice igg (thermo fisher scientific, usa) was used as the secondary antibody followed by dapi staining. total rna was extracted from the isolated virus with trizol reagent, and cdnas were subsequently amplified by superscript ii with specific primers according to the manufacturer's instructions (thermo fisher scientific). a total of 16 primer pairs based upon the bat cov hku2 strain gd430-2006 (genbank accession no. ef203064; supplemental table s1 ) were designed to amplify the complete genome of seacov. pcr products were purified and cloned into a pcr-blunt vector (thermo fisher scientific). for each amplicon, three to five individual clones were sequenced to determine the consensus sequence. the sequences were assembled and analyzed using the dnastar program. multiple alignments of the full-length genomes, non-structural protein genes and s genes with representative cov sequences and phylogenetic analyses were performed using the neighbor-joining method in mega5.2, respectively. structure homology-modeling of seacov s glycoprotein was performed by the swiss-model server (https://www.swissmodel.expasy.org/). a pilot animal experiment was approved by the experimental animal ethics committee of zhejiang university (approval no. zju20170026). briefly, ten 3-day-old conventional piglets, free of seacov, pedv, tgev, and pdcov rna in the feces, were assigned into two groups with 5 in each. piglets in each group were housed with their mothers (seacov rna and serum antibody negative as determined by ifa) without any artificially supplemental colostrum or milk. piglets in group one were each challenged orally with a seacov/ch/gd-01/ 2017/p3 isolate at a dose of 1 × 10 5 plaque-forming units (pfu)/ml (3 ml per pig), whereas piglets in group two each received 3 ml of dmem orally as negative controls. all the piglets were monitored daily for any signs of illness. two piglets in each group were euthanized at 3 days post-infection (dpi) while the remaining three in each group were necropsied at 5 dpi. the duodenum, jejunum and ileum samples were subjected to histological examinations by hematoxylin and eosin (he) staining, respectively. the villous height (vh) and the crypt depth (cd) were measured on a minimum of eight different sites per small intestinal segment, and the ratios of vh to cd were then calculated to quantify the villous atrophy according to previously described (jung et al., 2014) . fecal swabs for viral rna detection were collected at 0, 1, 2, 3, 4 and 5 dpi from all five pigs until they were alive. beginning from february 2017, a remarkable increase in outbreaks of newborn-piglet diarrhea occurred in a commercial pig farm located in guangdong province of southern china. clinical signs of affected pigs were characterized by acute vomiting and watery diarrhea (fig. 1a) . the mortality rate was over 35% in piglets less than 10 days old during february-may 2017. in addition, the small intestine of the diseased pigs y. pan et al. veterinary microbiology 211 (2017) 15-21 displayed thin walls and contained yellow watery feces (fig. 1b) , which was indistinguishable from that of pedv infection described previously pan et al., 2012) . fecal and small intestinal samples collected from affected piglets in this farm were submitted to our labs at zhejiang university and hog production division of wen's foodstuffs group, respectively, for routine laboratory diagnostics. upon laboratory analysis by rt-pcr, rna of pedv, tgev, pdcov or porcine hemagglutinating encephalomyelitis virus (phev), was not detected in these samples (data not shown). other possibly known viral pathogens associated with piglet diarrhea such as porcine enterovirus, rotavirus or mammalian orthoreovirus (qin et al., 2017) also could not be detected. subsequently, samples were tested by a pan-cov rt-pcr assay designed to amplify a conserved region of 251-bp in the orf1b gene for all cov members (moes et al., 2005) . this test was positive for all the selected samples collected during february to may (data not shown). sequencing of the pcr products revealed that they were 100% identical to the corresponding region (nucleotide [nt] positions 14024-14274) of four known bat enteric alphacoronavirus hku2 strains (genbank accession nos. ef203064 to ef203067) identified from guangdong province and hong kong in 2004 and 2006 (lau et al., 2007) . the prevalence rate of bat cov hku2 from these two regions was reported to be 10.9% (7/64) and 8.3% (29/348) in chinese horseshoe bats (rhinolophus sinicus), respectively (lau et al., 2007) . hku2 infection associated with the other animal species has never been investigated. the results from pan-cov rt-pcr detection indicated that an hku2like viral pathogen might be responsible for outbreaks of diarrhea in the pig farm. in an effort to isolate the novel swine enteric hku2-related cov (seacov), suspension supernatants of selected hku2-positive samples were prepared and inoculated in a panel of bhk-21, st, llc-pk1 and vero cell lines routinely used to isolate porcine covs. cultured supernatants from each inoculated cell line were blind-passaged serially. from vero cell culture, we successfully isolated one seacov strain with cpe characterized by syncytia formation at 48 h post-infection, beginning from passage two (p2) and in the following passages after plaque purification (fig. 1c) . furthermore, viral antigens were demonstrated in seacov-infected vero cells by ifa, with a serum sample collected from a sow mothering the diseased piglets (fig. 1d ), but not with the specific monoclonal antibodies against the n protein of pedv, tgev or pdcov (fig. 2) , suggesting that seacov are probably antigenetically distinct from the three known porcine covs. seacov antibody-negative sera from the same farm were also found, as staining with these sera in seacov-infected cells displayed no fluorescent signal (fig. 1e) . electron microscopy of a negatively stained sample from the supernatant of virus-infected vero cells demonstrated that the virus particle was 100 to 120 nm in diameter, and had surface projections typical of cov (fig. 1f) . seacov rna was detected in supernatants from all virus passages to date (p2 to p8) by rt-pcr with primers seaf and sear. the virus titer reached up to 1 × 10 6 pfu/ml at p8. this isolated cov strain was designated as seacov/ch/gd-01/2017. we next determined the complete genome of p2 of ch/gd-01 strain by rt-pcr amplification of 16 regions covering the entire seacov, as described previously for pedv or pdcov genomic cloning wang et al., 2015b) . the complete genome sequence of the ch/ gd-01/2017/p2 strain has been deposited in genbank under accession no. mf370205. the genomic sequence of ch/gd-01/2017/p2 is 27,155 nt in length, excluding the poly(a) tail. the genome organization is similar to those of the four hku2 strains and a bat cov identified in yunnan province in southwestern china (btrf-alphacov/yn2012, genbank no. kj473808), with the typical gene order 5′-orf1a/1b (orf1ab)-s-orf3-e-m-n-ns7a-3′ (fig. 3) . the ch/gd-01/2017/p2 strain is 6-nt longer than hku2 (27,149 nt), including a 3-nt (ttg) insertion at nt 4,554-4,555 (corresponding to the hku2/gd430 sequence) in the nonstructural protein (nsp) 3 region, a 6-nt (ggcctc) insertion at nt 20,504-20,505 in the s gene, and a 3-nt (gta) deletion at nt 24,772-24,775 in the m gene (fig. 3) . however, these insertions/deletion are not unique for seacov since they are also present in the btrf-al-phacov/yn2012 genome in comparison with hku2. seacov shared 94.9% nt sequence identity with the four hku2 strains, and exhibited 88.3% nt identity with btrf-alphacov/yn2012. accordingly, seacov is phylogenetically located between hku2 and btrf-alphacov/yn2012, together forming a sublineage closely related to the proposed alphacov group-1b lineage, including pedv and human covs nl63 and 229e, at the complete genome level (fig. 4a) . however, analysis of the phylogenetic tree constructed based on the s genes (fig. 4b ) indicated that these six hku2-related cov strains along with a newly identified rat alphacov, lrnv (wang et al., 2015a) , formed a separate lineage clustered within the betacovs. the previous studies have suggested that hku2 and the related lrnv probably resulted from an ancient recombination event with an alphacov genomic fig. 2 . ifa results of vero cells infected with seacov or pedv, vero cells stably expressing porcine aminopeptidase n (vero-papn) infected with tgev, and llc-pk1 cells infected with pdcov at 48 h post-infection. seacov-infected vero cells were stained with the anti-pedv-n, anti-tgev-n or anti-pdcov-n monoclonal antibody, respectively (left panels). cells infected with pedv, tgev or pdcov were stained with the respective virus-specific antibody as the controls (right panels). the fitc-conjugated goat anti-mice igg was used as the secondary ab in ifa. magnification = 200×. fig. 3 . schematic diagram of the genomic structure of seacov and the proposed domain organization of the seacov spike protein s1 subunits according to the structure similarity analysis with nl63 and mhv that are both structure available. numbers indicate amino acid positions in s glycoprotein of seacov, nl63 or mhv, respectively. see supplemental fig. s1 for the detailed sequence alignment. nucleotide insertion/deletion at three locations (nsp3, s and m genes) in seacov compared to the consensus sequences of four bat-cov hku2 strains (genbank accession nos. ef203064 to ef203067) are marked by "*". y. pan et al. veterinary microbiology 211 (2017) 15-21 backbone replaced by a betacov s gene (lau et al., 2007; wang et al., 2015a) . furthermore, pairwise comparison of seacov genomic sequence with hku2 indicated that the most dissimilar region was in the s gene, particularly, in the extreme ntd (aa 1-238). the entire seacov s protein had 86.4% aa identical with s of the hku2/gd430 strain, but there was only a 67.4% identity in the extreme ntd of the s protein (s-ntd) between seacov and hku2. we identified a total of 75-aa substitutions plus a 2-aa insertion (resulting from a 6-nt insertion as mentioned above) within the seacov s-ntd compared to hku2. in contrast, only 78 aa substitutions were found in the remaining part of the s protein. the extreme ntd changes in seacov are likely to be associated with the extended host range, similar to a previously reported mhv variant that was able to expand nonmurine-species tropism, with the phenotype mapped to 21 substitutions and a 7-aa insert in ntd of s1 subunit (schickli et al., 2004) . during the time of this manuscript preparation, a sequence of another hku2-related seacov strain gds04, identified in the same region, was reported online but it did not give in-depth analyses (gong et al., 2017) . it remains unknown if gds04 can be isolated in cell culture. moreover, neither detection of serum anti-seacov antibodies nor observation of virus morphology was demonstrated. nevertheless, comparative sequence analysis showed that the gds04 strain, having the same genomic size (27,155 nt), shared 99.8% nt homology with gd-01/2017/p2 at the complete genome level. however, the gds04 sequence was determined by the next generation sequencing, which should theoretically be less accurate than the gd-01/2017/p2 sequence determined based upon the consensus sequences from different short rt-pcr fragments covering the full-length genome. the s-ntd of gds04 also contains 75-aa substitutions and a 2-aa insertion compared to that of hku2. there are only three aa differences at the positions 86 (d/g), 166 (m/r) and 208 (a/v) in the s-ntd between gds04 and gd-01/2017/p2. the corresponding aa in bat cov hku2 at these positions are g, m and m. for nonstructural protein genes analysis, the seacov gd-01/2017/p2 exhibited 99.8% and 97.7% nt identities, 99.8% and 97.5% nt identities, or 99.9% and 98.0% nt identities with gds04 and hku2 based on the orf1ab, the orf1a, or the orf1b genes, respectively. these sequence analyses suggested that the seacov strains gd-01/2017 and gds04 could have the same origin. the s glycoprotein of seacov or hku2 is unique and not related to any currently known betacovs at the aa sequence level. most recently, the structures of several cov s glycoprotein trimmers have been resolved (walls et al., 2016a (walls et al., , 2016b . the betacovs comprise of four domains (domains a-d) in the s1 subunit whereas human alphacov nl63 shows an additional domain, named domain 0 (equivalent to the extreme ntd) compared to betacovs (fig. 3) . therefore, a structure homology-modeling was performed in order to better understand the evolutionary origin of the s glycoprotein of seacov/hku2 in the protein structure level. surprisingly, the result suggested a hybrid structure of seacov-s: the extreme ntd (domain 0) of seacov-s is structurally similar to that of nl63, whereas the rest part in s1 structurally resembles domains b to d of the betacov mhv ( fig. 3 and supplemental fig. s1 ). the s2 subunits of seacov and mhv also have a similar structure (supplemental fig. s1 ). the deduced structure of the linking region between domain 0 and domain b of seacov is uncertain. we hypothesize that the domain a is likely not present, which may be a unique feature of seacov/hku2 s1 subunit. since domain 0 and domain a are structurally similar and might come from a gene-duplication event (walls et al., 2016b) , we also hypothesize that either of them is likely dispensable in the s1 of covs. in addition, the presence of domain 0 in seacov/hku2 is in line with the enteric tropism of these viruses since pedv and tgev also possess this domain (hulswit et al., 2016) . future study on developing the seacov infectious clone and resolving the alphacov/betacov-hybrid seacov-s glycoprotein structure are warranted to confirm these findings. in order to test whether or not, seacov is able to infect pigs, we performed a pilot challenge experiment using the cell-cultured seacov/ch/gd-01/2017 isolate. as expected, the five piglets in dmem-inoculated group neither showed clinical sign nor shedding y. pan et al. veterinary microbiology 211 (2017) 15-21 virus in the feces throughout the experimental period (data not shown). in contrast, clinical signs characterized by acute vomiting and watery diarrhea (similar to fig. 1a) were observed in the five seacov-infected piglets at 27 to 40 h post-infection, and thereafter lasted until necropsy. fecal virus shedding was detected in five seacov-infected pigs at 1, 2 and 3 dpi, and in three remaining pigs at 4 and 5 dpi by rt-pcr with the primers seaf and sear (data not shown). sequencing of the pcr products indicated that they were identical with the seacov n gene sequence, confirming that the infectious virus was originated from the seacov isolate. upon histopathological analysis, no intestinal lesions were observed in control pigs (fig. 5) ; the mean duodenal, jejunal and ileal vh/cd were 3.58 ( ± 0.82), 6.14 ( ± 1.55) and 4.70 ( ± 1.73), respectively. typical microscopic lesions, showing gradual atrophy with significantly reduced vh/cd (the mean jejunal or ileal value was 2.26 [ ± 0.44] or 0.65 [ ± 0.37]), diminishing capillaries and central lacteals of the intestinal villous (fig. 5) , were detected in the jejunum and ileum of seacov-infected piglets. the duodenal sections displayed only mild microscopic lesions (the mean duodenal vh/cd = 2.59 [ ± 0.27]) in all seacov-infected pigs (fig. 5) . it was different from the result observed for the experimental infection using the virulent chinese pedv strain, in which marked microscopic lesions in all the three parts of the small intestine were found (zhang et al., 2015) . the results indicated that the seacov isolate is actually infectious and causes diarrhea in pigs. since the specific non-swine antibodies against the structural proteins of seacov are not available currently, further comprehensively pathological studies by immunohistochemistry and serological assays, which is not the scope of this study, are warranted to provide more information on seacov infection. in summary, we have isolated, sequenced and genetically characterized a novel swine enteric alphacoronavirus, which is probably distinct from pedv, tgev and pdcov antigenetically, from diarrheal samples in a pig farm of southern china in 2017. the isolated seacov can actually infect and cause diarrhea in pigs, and should represent the fifth porcine coronavirus in addition to pedv, tgev (considering that porcine respiratory virus, prcv, is a variant of tgev), pdcov and phev. to our knowledge, this is also the first study describing seacov related to the bat coronavirus hku2 that could be isolated and propagated in cell culture. however, infection of vero cells (a monkey cell line) with seacov also raises concerns about its potential host range other than swine. we also identified that the extreme ntd (aa 1-238) of seacov spike protein consists of 75-aa substitutions and a 2-aa insertion compared to that of hku2, which is likely to be responsible for the cross-species transmission. moreover, this region but not the other betacov-related domains of seacov s1 subunit is structurally similar to the alphacov domain 0, implying that these viruses gained enteric tropism through this domain. the results provide much needed information on seacov and hku2 evolution, and the availability of seacov in cell culture will guide future efforts to develop effective vaccines against seacov. 5 . representative histological examinations of the duodenum, jejunum and ileum samples collected at 3 days post-infection from piglets inoculated with seacov or dmem in the animal challenge experiment. sections of jejunum and ileum in the seacovinfected group showed scattered areas of villi atrophy, whereas the section of duodenum showed mild microscopic lesions as compared to the dmem control group. y. pan et al. veterinary microbiology 211 (2017) 15-21 new chimeric porcine coronavirus in swine feces characterization of a novel chimeric swine enteric coronavirus from diseased pigs in central eastern europe in 2016 porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus virus taxonomy: ninth report of the international committee on taxonomy of viruses a new bat-hku2-like coronavirus in swine origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states coronavirus spike protein and tropism changes pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs porcine deltacoronavirus infection: etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis complete genome sequence of bat coronavirus hku2 from chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl63 in children hospitalized with respiratory tract infections in belgium isolation and characterization of a variant porcine epidemic diarrhea virus in china a new coronavirus-like particle associated with diarrhea in swine genetic and pathogenic characterization of a novel reassortant mammalian orthoreovirus 3 (mrv3) from a diarrheic piglet and seroepidemiological survey of mrv3 in diarrheic pigs from east china the n-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells evidence of recombinant strains of porcine epidemic diarrhea virus cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy discovery, diversity and evolution of novel coronaviruses sampled from rodents in china complete genome sequence of porcine deltacoronavirus strain ch/sichuan/s27/2012 from mainland china 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 identification and pathogenicity of a variant porcine epidemic diarrhea virus field strain with reduced virulence this work was supported by the national key research and developmentprogram of china (2016yfd0500102 and 2017yfd0500103), and the national natural science foundation of china (31572518). we thank dr. narayan paudyal for conducting english language review. supplementary data associated with this article can be found, in the online version, at https://doi.org/http://dx.doi.org/10.1016/j.vetmic. 2017.09.020. key: cord-265679-7gzont7l authors: guo, nan; zhang, bingzhou; hu, han; ye, shiyi; chen, fangzhou; li, zhonghua; chen, pin; wang, chunmei; he, qigai title: caerin1.1 suppresses the growth of porcine epidemic diarrhea virus in vitro via direct binding to the virus date: 2018-09-18 journal: viruses doi: 10.3390/v10090507 sha: doc_id: 265679 cord_uid: 7gzont7l porcine epidemic diarrhea (ped) has re-emerged in recent years and has already caused huge economic losses to the porcine industry all over the world. therefore, it is urgent for us to find out efficient ways to prevent and control this disease. in this study, the antiviral activity of a cationic amphibian antimicrobial peptide caerin1.1 against porcine epidemic diarrhea virus (pedv) was evaluated by an in vitro system using vero cells. we found that even at a very low concentration, caerin1.1 has the ability to destroy the integrity of the virus particles to block the release of the viruses, resulting in a considerable decrease in pedv infections. in addition, caerin1.1 showed powerful antiviral activity without interfering with the binding progress between pedv and the receptor of the cells, therefore, it could be used as a potential antiviral drug or as a microbicide compound for prevention and control of pedv. alphacoronavirus of the family coronaviridae with single-stranded positive-sense rna [1] . it is the causative agent of an acute infectious enteric disease known as porcine epidemic diarrhea (ped) that is clinically manifested by severe watery diarrhea, vomiting, and dehydration in the suckling piglets [2] . with the high mortality in the piglets, ped infection finally caused enormous economic losses to the global swine industry, especially after its recent re-emergence caused by variant pedv strain throughout the world [3, 4] . however, the classical ped vaccines could not provide appropriate protection against the variant pedv infection. considering this, relevant studies of new antiviral materials are needed to prevent and control emerging or re-emerging infectious diseases such as ped [5] . antimicrobial peptides (amps) are important components of the nonspecific immune system of animals to eradicate invaders [6] , and the skin secretion of anuran amphibians are rich sources for collecting amps [7] [8] [9] . amps have a wide spectrum of antimicrobial activity against microorganisms such as bacteria, viruses, fungi, and parasites [10, 11] , but are very friendly to host cells [12] [13] [14] . moreover, amps can work as growth and health promoters to improve the performance of pigs by enhancing the immune status, improving the intestinal health, and alleviating the toxic effects of deoxynivalenol in pigs [15] . there are also research findings that amps can modulate immune responses like chemokines, cytokine production, and pro-inflammatory responses [16, 17] . caerin1.1 is a peptide from the granular glands within the skin of the australian green tree frog with 25-residues (gllsv lgsva khvlp hvvpv iaehlnh2). nuclear magnetic resonance (nmr) of caerin1.1 in the membrane mimetic environments showed that it has two α-helices and a flexible hinge region composed of two prolines [18] . both prolines are essential for the antimicrobial activities [19] . some studies have reported that caerin1.1 exhibits antibacterial and antiviral properties by forming pores on the membrane to destroy the integrity of the particles [20, 21] . meanwhile, it has been confirmed that caerin1.1 has a complete inhibitory effect against hiv by preventing viral fusion to target cells and disrupting the hiv envelope, and, remarkably, that caerin1.1 is also highly effective in inhibiting the transfer of hiv from dendritic cells (dcs) to t cells even when dcs are continuously exposed to peptides for 8 h after virus capture, and that caerin1.1 has a bacteriostatic activity against escherichia coli and bacillus subtilis [8, 21] . although the mechanism that suppresses the activities of some bacteria and viruses has been illustrated clearly, it remains unknown whether caerin1.1 can inhibit the growth of pedv. so, this study is aimed to investigate the function of caerin1.1. for this purpose, we investigated the inhibitory ability and antiviral mechanisms of caerin1.1 against different pedv strains in vero cells, which will provide an insight into amps' antiviral mechanisms and its application as antiviral drugs or as drug loading compounds. vero cells (african green monkey kidney cell lines) were propagated at 37 • c in a 5% co 2 humidified incubator using dulbecco's modified eagle medium ((dmem), gibco, langley, va, usa) containing 10% fetal bovine serum (invitrogen, carlsbad, ca, usa). cells were infected with three different pedv strains: yn (accession no. kf761675), cv777 (accession no. kt323979), and dr13 (accession no. jq023161). the yn strain ch/ynkm-8/2013 was isolated from a suckling piglet suffering from acute diarrhea. the cells infected with pedv strains were cultured in dmem supplemented with 10 µg/ml trypsin. caerin1.1 and n-terminus fitc labeled caerin1.1 (purity: both >95%) were chemically synthesized by bioyeargene (wuhan, china). caerin1.1 was initially dissolved in 0.01% acetic acid to reach the concentrations of 1 mg/ml and 5 mg/ml as stock solutions, respectively and stored at −80 • c until further use [22] . the cytotoxicity of caerin1.1 in vero cells grown in 96-well culture plates was assessed by mtt (3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/ml) assay. the cells were incubated with caerin1.1 at different concentrations for 72 h, then 20 µl/well mtt was added and cultured for additional 4 h. the supernatant was then removed and dimethylsulfoxide (dmso) was added to culture plates (150 µl per well), then the plates were shaken at room temperature (rt) for 10 min. finally, the optical density (od) value was measured at the wavelength of 490 nm [23] . the plaque forming assay was performed on vero cells cultured in 12-well plates. pedv (200 pfu/well) was incubated using different concentrations of caerin1.1 for 1 h at 37 • c before infection and then incubated pedv was added on vero cells covering 80% monolayer. after 1 h of viral adsorption, the supernatant was removed, and the cells were rinsed with pbs and overlaid with 10 µg/ml trypsin supplemented with sodium carboxymethyl cellulose-containing medium. the plates were fixed with 10% formaldehyde after 72 h infection, and then stained with crystal violet solution [24] . vero cells cultured in 24-well plates were washed with pbs for 3 times and inoculated respectively with single medium, or single pedv, or pedv pre-incubated with different concentrations of caerin1.1. the cells were rinsed with pbs after 24 h infection, the cells were fixed with 4% formaldehyde for 15 min at rt. then the cells were incubated with an anti-pedv monoclonal antibody (made in our laboratory) for 1 h and fluorescein isothiocyanate (fitc)-conjugated goat anti-mouse antibody (1:60 dilution) for 1 h at 37 • c. the immunofluorescence images were taken with a nikon eclipse ti microscope (nikon, tokyo, japan) [25] . vero cells were cultured in 6-well plates and infected with pedv, and treated with caerin1.1 at different time points. the cells were rinsed three times with pbs at 20 h post infection (hpi), and treated with 120 µl/well lysis solution containing protease inhibitors (pmsf). then 30 µl of sodium dodecyl sulfate (sds) loading buffer was added to the cell extracts and the samples were boiled for 10min. the proteins were separated by 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) and then transferred into pvdf membranes (millipore, mississauga, on, canada). membranes were blocked with 5% skimmed milk for 2 h at 37 • c and then incubated with primary antibodies over night at 4 • c. the blots were then incubated with corresponding horseradish peroxidase (hrp)-conjugated secondary antibodies (abclonal, wuhan, china). the membranes were washed 3 times at each step. the protein bands were visualized using the clarity™ western ecl blotting substrate (bio-rad, hercules, ca, usa). the protein blots were quantified by image j software (national institutes of health, bethesda, md, usa). pedv-infected cell culture was prepared and centrifuged at 4 • c for 30 min at 2770× g to get rid of the cell debris. the supernatants were ultracentrifuged at 4 • c for 2 h at 200,500× g. then the purified virus pellets were re-suspended with pbs and treated with caerin1.1 (10 µg/ml) for 1 h at 37 • c and the viruses treated in the same manner were used as positive control. afterwards, the cocktail was dripped on the copper grid and negatively stained with phosphotungstic acid (pta). finally, the samples were examined using the electron microscope (hitachi h7500, tokyo, japan) [26] . pedv suspensions containing different concentrations of caerin1.1 were pre-incubated for 1 h at 37 • c, and were serially diluted before they were inoculated on the 80% confluent vero cell monolayers grown in the 96-well plates, followed by washing 3 times with pbs. about 1hpi, the inoculates were removed and the cells were washed again with pbs for 3 times and incubated for another 3 days with dmem containing trypsin (10 µg/ml). the infectivity was calculated by tcid 50 (tissue culture infectious dose 50) following the reed-muench method established by l. j. reed and h. muench [27] . the vero cell monolayers were infected with pedv (200 pfu). at 1 hpi, the cells were washed twice with pbs and then treated with different concentrations of caerin1.1, and at 12 hpi, the culture supernatants and the cells were collected respectively [28] . the cell samples were frozen and thawed 3 times to completely release the intracellular virus particles. both the extracellular and intracellular virus yields were determined by tcid 50 . the vero cells in 12-well plates were inoculated with pedv in the presence or in the absence of caerin1.1 at different time points: a. 1 h before infection (pre); b. incubation of caerin1.1 and pedv for 1 h before infection (co); c. 1 hpi (post). the supernatants and the cells were collected together at different time points and the samples were treated in the same way mentioned above in an infectious virus yield reduction assay. on the other hand, the inhibitory ability of caerin1.1 against pedv was examined using a 10-fold higher concentration for further understanding antiviral activities in the pre and post treatment situations. finally, the cell lysates were collected in a parallel set for western blot assay. cells were pre-chilled at 4 • c for 1 h before infection and were chilled again after inoculated with pedv for 3 h, then the cells were washed with ice cold pbs 3 times, replenished with dmem containing trypsin, were incubated at 37 • c for 72 h, and the inhibitory effects were determined by tcid 50 . caerin1.1 was separately added at two time points: together with pedv at the time of viral absorption, and after the cells were washed at the time of viral entry to reach a highest final concentration of 10 µg/ml. the cell lysates were processed in the same procedures with western blot analysis [29] . vero cells were seeded onto some glass coverslips and were separately treated with fitc-caerin1.1 (100 µg/ml), pedv (200 pfu), and both fitc-caerin1.1 (100 µg/ml) and pedv (200 pfu), then incubated at 37 • c for 12 h. cells were fixed with 4% formaldehyde followed by permeabilization for 30 min with 0.1% triton x-100, and were blocked with 1% bsa for 1 h. the cells were washed 3 times with pbs at each step. cell nuclei were counterstained with 0.01% 4 ,6-diamidino-2-phenylindole ((dapi), invitrogen), and the antibodies used here were: pedv-s monoclonal antibody (made in our laboratory) and alexa fluor 594-conjugated affinity pure donkey anti-mouse igg (h+l) (ant gene, wuhan, china). the samples were examined using a confocal microscope (lsm 510 meta, carl zeiss, munich, germany) [30] . all experiments were performed with three independent experiments, and the calculated results were presented as mean ± standard deviation (sd). statistical analyses were performed using student's t-test. graph pad prism5.0 was used to analyze the statistics in this study. the statistical significances were defined as p < 0.05 (*), and the higher significance was denoted by p < 0.01 (**) and p < 0.001 (***). the result of cytotoxicity assay indicated that cell viability increased with the decrease of the concentration of caerin1.1, that the viability of cells remained over 80% when treated with caerin1.1 at the concentrations not exceeding 110 µg/ml, and that the cell morphology was not affected even at the concentration 140 µg/ml with a cell viability value of 76%, as seen in figure 1 . thus, 10 µg/ml caerin1.1 was chosen as the highest final concentration in most of the subsequent experiments. we used 20 µg/ml, 10 µg/ml, 5 µg/ml, and 2.5 µg/ml of caerin1.1 in the ifa assay and in the addition assay. different incubating conditions of pedv in the presence of caerin1.1 can directly affect the antiviral activity of caerin1.1. in this experiment, we determined the titer of pedv under different incubating temperatures and time durations. the results showed that the most suitable incubating temperature was 37 °c, shown in figure 2a , and time length was 1 h, shown in figure 2b . low temperature and inadequate incubation time would obviously affect the inhibition activity of caerin1.1 against pedv. different incubating conditions of pedv in the presence of caerin1.1 can directly affect the antiviral activity of caerin1.1. in this experiment, we determined the titer of pedv under different incubating temperatures and time durations. the results showed that the most suitable incubating temperature was 37 °c, shown in figure 2a the results are presented as the mean ± sd of three independent experiments. the statistical analysis was performed using graph pad prism5.0. significance was defined as p < 0.05 (*), and higher significance was defined as p < 0.01(**). to fully understand the inhibitory effect of caerin1.1 against pedv-yn strain, several experiments were performed including cytopathic effects (cpe) observation, plaque reduction assay, tcid50, and ifa. the cpe observation results showed that there were remarkable differences between the cells infected with pedv in the absence or presence of caerin1.1. caerin1.1-treated cells were in a relatively normal status, and the cpe appeared in caerin1.1-treated group about 30 h later than in the pedv control group, as seen in figure 3a . with the addition of caerin1.1, the number of plaques declined at a surprising speed and the diameters decreased notably. similar results can be observed even with 2000 pfu of pedv treated by caerin1.1. the cells infected with 200 pfu pedv exactly remained the same as the cells in negative control group, as seen in figure 3b . caerin1.1 significantly inhibited the multiplication of pedv in a dose-dependent manner, demonstrating that pedv infection to vero cells was blocked to a large extent. the results of tcid50, shown in figure 3c , and ifa, in figure 3d , indicated that the titer and the fluorescence intensity of pedv decreased significantly with the increase in the concentration of caerin1.1. to fully understand the inhibitory effect of caerin1.1 against pedv-yn strain, several experiments were performed including cytopathic effects (cpe) observation, plaque reduction assay, tcid 50 , and ifa. the cpe observation results showed that there were remarkable differences between the cells infected with pedv in the absence or presence of caerin1.1. caerin1.1-treated cells were in a relatively normal status, and the cpe appeared in caerin1.1-treated group about 30 h later than in the pedv control group, as seen in figure 3a . with the addition of caerin1.1, the number of plaques declined at a surprising speed and the diameters decreased notably. similar results can be observed even with 2000 pfu of pedv treated by caerin1.1. the cells infected with 200 pfu pedv exactly remained the same as the cells in negative control group, as seen in figure 3b . caerin1.1 significantly inhibited the multiplication of pedv in a dose-dependent manner, demonstrating that pedv infection to vero cells was blocked to a large extent. the results of tcid 50 , shown in figure 3c , and ifa, in figure 3d , indicated that the titer and the fluorescence intensity of pedv decreased significantly with the increase in the concentration of caerin1.1. the inhibitory effects of caerin1.1 against another two different pedv strains were examined to evaluate the anti-pedv potential of caerin1.1 based on ifa. as shown in figure 4 , vero cells were infected with pedv (200 pfu) pre-incubated with different concentrations of caerin1.1. compared with the virus control, treatment group exhibited excellent inhibitory effects in a dose dependent manner even at extremely low concentrations of caerin1.1. these results revealed that the intracellular viruses evidently reduced in the presence of caerin1.1 in a dose dependent manner. groups (pedv) were not treated by caerin1.1. the results of three independent experiments are presented as the mean ± sd. the statistical analysis was performed using graph pad prism5.0. the significance was defined as p < 0.05 (*), and the higher significance was defined as p < 0.01(**), p < 0.001(***); (d) fluorescence intensity of pedv in the presence of caerin1.1 at different concentration. the inhibitory effects of caerin1.1 against another two different pedv strains were examined to evaluate the anti-pedv potential of caerin1.1 based on ifa. as shown in figure 4 , vero cells were infected with pedv (200 pfu) pre-incubated with different concentrations of caerin1.1. compared with the virus control, treatment group exhibited excellent inhibitory effects in a dose dependent manner even at extremely low concentrations of caerin1.1. these results revealed that the intracellular viruses evidently reduced in the presence of caerin1.1 in a dose dependent manner. to better understand the inhibitory effects of caerin1.1 against the progeny virus production of pedv, we added caerin1.1 at 1 hpi to pedv infected cells. and both the intracellular and extracellular virus titers were determined separately by using tcid 50, as shown in figure 5a . the progeny virus titer decreased significantly as compared to that of virus control in a dose dependent manner, as seen in figure 5a . on the other hand, extracellular virus titers were also determined at different time points and different caerin1.1 concentrations. the results showed that the increase of extracellular virus titer slowed down under the treatment of caerin1.1, as seen in figure 5b . incubated with caerin1.1 at the concentrations of 2.5 (c), 5 (d), 10 (e) and 20 (f) μg/ml for 1 h. scale bar, 20 μm. to better understand the inhibitory effects of caerin1.1 against the progeny virus production of pedv, we added caerin1.1 at 1 hpi to pedv infected cells. and both the intracellular and extracellular virus titers were determined separately by using tcid50, as shown in figure 5a . the progeny virus titer decreased significantly as compared to that of virus control in a dose dependent manner, as seen in figure 5a . on the other hand, extracellular virus titers were also determined at different time points and different caerin1.1 concentrations. the results showed that the increase of extracellular virus titer slowed down under the treatment of caerin1.1, as seen in figure 5b . in the fluorescent confocal experiment, the fluorescence of caerin1.1 and pedv around cell nucleus could be observed when we incubated them with vero cells respectively. additionally, the distribution of caerin1.1 and pedv were almost the same, so the results of confocal laser scanning microscopy verified that caerin1.1 could combine with pedv, and that the antiviral function of caerin1.1 in the cell plasma could be observed especially in the areas showing severe cpe, as seen in figure 6 . in the fluorescent confocal experiment, the fluorescence of caerin1.1 and pedv around cell nucleus could be observed when we incubated them with vero cells respectively. additionally, the distribution of caerin1.1 and pedv were almost the same, so the results of confocal laser scanning microscopy verified that caerin1.1 could combine with pedv, and that the antiviral function of caerin1.1 in the cell plasma could be observed especially in the areas showing severe cpe, as seen in figure 6 . in caerin1.1-pedv pre-incubation process, the morphological changes of pedv particles were observed clearly. so caerin1.1 could destroy the structure of the virus to block the infection of the host cells, as seen in figure 7a , resulting in the inhibition of viral release, which in turn could reduce the transmission of the progeny virus among the adjacent cells. as shown in the growth curve, pedv co-incubated with caerin1.1 showed the lowest titer, while pedv was only slightly inhibited in preincubation process of caerin1.1-cells and the delayed usage of caerin1.1, as seen in figure 7b . in a western blot experiment, the protein band could barely be observed in pedv-caerin1.1 co-incubation group. however, no obvious differences in pedv-n protein expression were observed among the cell-caerin1.1 pre-incubation group, the post treatment group, and pedv control group, as seen in figure 7c . to further explore whether caerin1.1 could bind to some virus receptors in host cells to interfere with the attachment and entry process, we kept the cells at 4 °c to maintain the attaching status. caerin1.1 did not show obvious inhibitory effects during the attachment and entry process. there were no significant differences in virus titers between the virus control groups and caerin1.1 treated groups during both attachment period and entry period, even if the concentration of caerin1.1 was increased tenfold, as seen in figure 7d . the western blot assay did not exhibit any obvious differences in protein expression among attachment period treated group, entry period treated group, and the pedv control group, shown in figure 7e , either. in caerin1.1-pedv pre-incubation process, the morphological changes of pedv particles were observed clearly. so caerin1.1 could destroy the structure of the virus to block the infection of the host cells, as seen in figure 7a , resulting in the inhibition of viral release, which in turn could reduce the transmission of the progeny virus among the adjacent cells. as shown in the growth curve, pedv co-incubated with caerin1.1 showed the lowest titer, while pedv was only slightly inhibited in pre-incubation process of caerin1.1-cells and the delayed usage of caerin1.1, as seen in figure 7b . in a western blot experiment, the protein band could barely be observed in pedv-caerin1.1 co-incubation group. however, no obvious differences in pedv-n protein expression were observed among the cell-caerin1.1 pre-incubation group, the post treatment group, and pedv control group, as seen in figure 7c . to further explore whether caerin1.1 could bind to some virus receptors in host cells to interfere with the attachment and entry process, we kept the cells at 4 • c to maintain the attaching status. caerin1.1 did not show obvious inhibitory effects during the attachment and entry process. there were no significant differences in virus titers between the virus control groups and caerin1.1 treated groups during both attachment period and entry period, even if the concentration of caerin1.1 was increased tenfold, as seen in figure 7d . the western blot assay did not exhibit any obvious differences in protein expression among attachment period treated group, entry period treated group, and the pedv control group, shown in figure 7e , either. figure 7b . gapdh (glyceraldehyde-3phosphate dehydrogenase) was used as a loading control; (d) tcid50 showed that caerin1.1 had no obvious inhibitory effects during the attachment and entry process. caerin1.1 was added at pedv attachment and entry periods, then the titers of pedv were detected; (e) western blot analysis of the expression level of pedv-n protein when caerin1.1 was added in different ways as shown in figure 7d . gapdh was used as a loading control. pedv is one of the most important pathogens leading to diarrhea in pig industry. the aim of this study is to find whether caerin1.1 has the antiviral activity against the three pedv strains and to reveal the antiviral mechanism of caerin1.1. according to published data, caerin1.1 is a cationic peptide with two α-helices and has proved to be an effective agent against hiv by damaging the virus envelope and stopping the infection of hiv to t cells [8, 21] . some α-helical peptides such as melittin and cecropin have been reported to exert antiviral activities by direct disruption of virus membranes or inhibition of the virus replication [31] . the current study found that caerin1.1 was able to destroy the structure of viral particles and reduce the viruses capable of infecting the cells and decrease their titers almost up to 3 logs. this is the first attempt to reveal that caerin1.1 has strong antiviral activity against pedv infection. figure 7b . gapdh (glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control; (d) tcid 50 showed that caerin1.1 had no obvious inhibitory effects during the attachment and entry process. caerin1.1 was added at pedv attachment and entry periods, then the titers of pedv were detected; (e) western blot analysis of the expression level of pedv-n protein when caerin1.1 was added in different ways as shown in figure 7d . gapdh was used as a loading control. pedv is one of the most important pathogens leading to diarrhea in pig industry. the aim of this study is to find whether caerin1.1 has the antiviral activity against the three pedv strains and to reveal the antiviral mechanism of caerin1.1. according to published data, caerin1.1 is a cationic peptide with two α-helices and has proved to be an effective agent against hiv by damaging the virus envelope and stopping the infection of hiv to t cells [8, 21] . some α-helical peptides such as melittin and cecropin have been reported to exert antiviral activities by direct disruption of virus membranes or inhibition of the virus replication [31] . the current study found that caerin1.1 was able to destroy the structure of viral particles and reduce the viruses capable of infecting the cells and decrease their titers almost up to 3 logs. this is the first attempt to reveal that caerin1.1 has strong antiviral activity against pedv infection. some amps including cecropin d (cd-prrsv, salps-aiv) were reported to have blocked viral attachment and invasion by interacting with the receptors of the host cells [32] . based on these findings, so we tried to keep pedv infection within the periods of the attachment and entry through the temperature control, and the results showed no evident differences in protein expression among attachment period treated group, entry period treated group, and the pedv control group. the number of virus infecting cells did not diminish. this means that caerin1.1 did not interfere with the attachment and entry process. considering the fact that caerin1.1 does not compete with the virus to conjugate with the cells, and the fact that viral attachment-entry processes are very fast and caerin1.1 will not have enough time to act against the viral membrane, it can be concluded that caerin1.1 has less significant antiviral activities in the pre-incubation of caerin1.1-cells progress. to further understand the mechanism in the post-treatment process, we examined the antiviral activity during the replication period. the results showed that caerin1.1 did not have obvious antiviral effect during viral replication period, but it can control the infection progress by blocking the release of pedv particles to reduce viral transmission among the adjacent cells. antiviral activities of caerin1.1 is expected to be improved through the combination usages with other different amps or other antiviral agents like graphene oxide (go), which could make caerin1.1 a good weapon to deal with the diseases caused by viruses including pedv [33] . as a matter of fact, we did a combination treatment with piscidin which was also proved to be effective against pedv [34] , but the results were not as good as expected based on our unpublished work. the mechanisms still remain to be further investigated. although the joint use of piscidin and caerin1.1 did not work as well as expected, other candidates can be explored for the prevention of pedv and other pathogens in drug combination studies. on the other hand, some related research work has been done in our laboratory. our other unpublished work has preliminarily testified the extraordinary ability of caerin1.1 against some other viruses including prv (pseudorabies virus) which is a herpesvirus with double-stranded dna that can infect many mammal species. therefore, many potential functions of caerin1.1 are still to be explored in the future. in this study, we investigated the antiviral activities of caerin1.1 against pedv strains and its antiviral mechanism. the results show that caerin1.1 can maintain the integrity of the host cells since it has very low cytotoxicity, and that it exhibits excellent virucidal activity in a dose-dependent manner even at very low concentrations. caerin1.1 can reduce the viruses infecting the cells by destroying the integrity of the viral membrane, resulting in the decrease in the virus replication and protein expression. caerin1.1 can also interfere with the virus release process essential for the inhibition of intracellular infection. therefore, caerin1.1 is an excellent antiviral material against pedv. in conclusion, our study testifies the outstanding antiviral activities of caerin1.1 and lays a foundation for future research and for the application of amphibian antimicrobial peptides. the authors have declared that no competing financial interests exist. comparative genomic analysis of classical and variant virulent parental/attenuated strains of porcine epidemic diarrhea virus a new coronavirus-like particle associated with diarrhea in swine isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states new variants of porcine epidemic diarrhea virus, china phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china the evolution and genetics 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activity of the antimicrobial peptide subtilosin inhibitory activity and mechanism of two scorpion venom peptides against herpes simplex virus type 1 porcine epidemic diarrhea virus induces autophagy to benefit its replication antimicrobial peptides as new recognition molecules for screening challenging species inhibition of porcine reproductive and respiratory syndrome virus by cecropin d in vitro antiviral activity of graphene oxide: how sharp edged structure and charge matter comparative pharmacokinetics and preliminary pharmacodynamics evaluation of piscidin 1 against prv and pedv in rats antimicrobial peptide ifa indirect immunofluorescent assay nmr nuclear magnetic resonance mtt 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide ped porcine epidemic diarrhea pedv porcine epidemic diarrhea virus tcid 50 tissue culture infectious dose 50 key: cord-263439-oquk4t96 authors: park, jung-eun; cruz, deu john m.; shin, hyun-jin title: clathrinand serine proteases-dependent uptake of porcine epidemic diarrhea virus into vero cells date: 2014-10-13 journal: virus res doi: 10.1016/j.virusres.2014.07.022 sha: doc_id: 263439 cord_uid: oquk4t96 porcine epidemic diarrhea virus (pedv), a member of the genus alphacoronavirus, is a causative agent of porcine enteric disease characterized by acute watery diarrhea and dehydration in sucking piglet. similar to other coronaviruses, pedv spike protein mediates its cell entry by binding to cellular receptors and inducing membrane fusion between viral envelopes and cellular membranes. however, the entry mechanism of pedv is not studied. here, we determined the entry mechanism of pedv into vero cells. our data confirmed that pedv entry followed clathrin-mediated endocytosis independence of caveolae-coated pit assembly. the internalized pedv was co-localized with the clathrin-mediated endocytic marker, but not with the caveolae-mediated endocytic marker. in addition, cells treated with lysosomotropic agents and serine protease inhibitors were resistant to pedv. our data revealed that pedv entry followed clathrin-mediated endocytosis and was dependent on a low ph and serine proteolysis for successful entry into cells. infection of enveloped viruses is initiated by binding of surface proteins with specific receptor(s) on the surface of the cell membrane, which leads to internalization of the virus into cells. the second step of infection following virus attachment is the uncoating of the viral genome into the cytoplasm after the viral envelope has fused with the host membrane. there are two major routes for enveloped viruses to enter host cells; the non-endosomal and the endosomal pathways (pelkmans and helenius, 2003; smith and helenius, 2004) . both pathways require the release of the viral genome by fusion of the viral envelope with the respective target membrane of the host cells such as the plasma or endosomal membrane, respectively (matlin et al., 1981) . in the non-endosomal pathway, the viral envelope directly fuses with the plasma membrane. membrane fusion is mediated by a conformational change of the viral glycoprotein, which is induced by its interaction with the corresponding receptor on the host cell surface and/or proteolytic processing (blumenthal et al., 2002) . the endocytic pathway can further differentiate into two well-characterized pathways; those acting via the clathrin-coated pit and the caveolae-mediated lipid raft (brodsky et al., 2001; pelkmans and helenius, 2002) . after internalization, viruses require a low-ph environment in the endosome to trigger conformational changes in the viral glycoproteins. the acidic ph environment is also important for proteolytic activation of viral glycoproteins by endosomal proteases (qiu et al., 2006; simmons et al., 2005) . the porcine epidemic diarrhea virus (pedv) is classified as alphacoronavirus together with transmissible gastroenteritis virus (tgev), feline infectious peritonitis virus (fipv), and human coronavirus 229e (hcov-229e). pedv causes an acute watery diarrhea in suckling piglets, which results in approximately 50% mortality among suckling piglets and reduces the weight among fattening pigs (debouck and pensaert, 1980) . porcine epidemic diarrhea (ped) is first recognized in pigs in the united kingdoms in 1971 (wood, 1977) . although no evidence of ped is currently reported from canada, similar coronavirus-like particles were reported from herds in quebec in 1980 (turgeon et al., 1980) . since then, outbreaks of ped have been documented in many european and asian countries such as czech republic, hungary, korea, the philippines, china, italy, thailand, germany, spain, and japan (song and park, 2012) . recently, pedv is spreading rapidly in swine farms in the united states, resulting in high mortality in piglets in more than 17 states (mole, 2013 as typical for the alphacoronavirus, the pedv spike (s) protein encounters virus entry into host cells by interacting with its receptor, porcine aminopeptidase n (apn), in porcine enterocytes and by mediating membrane fusion with host cell membranes (li et al., 2007; oh et al., 2003) . upon receptor binding, several coronaviruses in alphacoronavirus enter cells via endocytosis. for example, extensive studies on hcov-229e have shown that upon binding with the human apn receptor, it is taken up in lipid rafts and enters via caveolae-dependent endocytosis (nomura et al., 2004) . inside the endosome, cellular proteases that are active in a low-ph environment facilitate membrane fusion (kawase et al., 2009) . similarly, tgev binds to porcine apn (weingartl and derbyshire, 1994) , and has been shown to enter mdck cells over-expressing porcine apn via endocytosis and acidification of the intracellular compartment facilitated membrane fusion (hansen et al., 1998) . fipv also requires acidification of endosomes for successful entry (takano et al., 2008) . inhibition of fipv infection with nystatin, a pharmacological reagent that causes caveolae to flatten and disrupt the coat structure, and dynamin 2 inhibitor suggests that fipv entry might actually involve some types of caveolae-dependent endocytosis (van hamme et al., 2008) . although several studies have examined the mechanism of entry of other coronaviruses, the mechanism of pedv entry is still unknown. in this study, we studied the entry mechanism of pedv by measuring virus infectivity in the presence of chemical inhibitors and co-localization of pedv with endocytic pathway markers. we found that pedv infection was diminished by treatment with chloropromazine (cpz) and lysosomotropic agents. in addition, we also investigated that pedv required serine-like proteases for their entry through endocytosis and for cell-cell fusion. taken together, our findings reveal that pedv enters vero cells via clathrin-mediated endocytosis and requires serine proteolysis during infection. vero cells were maintained in eagle's minimum essential medium (mem, gibco) containing with 10% heat-inactivated fetal bovine serum (fbs, gibco), 100 u/ml penicillin, 100 g/ml streptomycin and 34 g/ml amphotericin b. kpedv-9, a vero cell-adapted korean strain, was propagated in vero cells as described previously (hofmann and wyler, 1988) . briefly, vero cells were inoculated with the kpedv-9 at a multiplicity of infection (moi) of 10 and cultured in serum-free mem for 72 h at 37 • c with 5% co 2 . the progeny viruses were titrated using the focus formation assay following a method described previously (cruz and shin, 2007) . kpedv-9 infection in vero cells under trypsin and non-trypsin conditions was compared for 48 h. vero cells in 4-well tissue culture (tc) plate (spl labware) were inoculated with kpedv-9 and cultured in either serum-free mem or mem supplemented with trypsin (10 g/ml). infection was stopped by addition of 5% paraformaldehyde (pfa) at the indicated times for immunocytochemistry. vero cells were treated with various concentrations of either cpz for 30 min or 0.45 m sucrose for 10 min to inhibit the formation of clathrin-coated pits. to block the caveolae-dependent pathway, cells were incubated with various concentrations of nystatin for 30 min. control cells were incubated with or without dimethyl sulfoxide (dmso). cells were inoculated with kpedv-9 at a moi of 10 for 2 h, and then overlaid with 0.5% methyl cellulose in mem containing trypsin for 10 h. at 10 hpi, pedv-infected cells were detected by immunocytochemistry. to prepare ultra-purified trypsin-free viruses, vero cells were inoculated with the kpedv-9 at a moi of 10 and cultured in serumfree mem for 72 h. supernatant was clarified by centrifugation at 20,000 × g for 20 min at 4 • c, followed by ultra-centrifugation using a 20% sucrose cushion at 150,000 × g for 3.5 h. following resuspension in buffer a (1 m tris, ph 8, 5 m nacl, 0.1 m cacl 2 ), protein concentration of purified virus stock was determined by the bradford assay. fluorochrome conjugation of kpedv-9 with alexa fluor 594 (af594) carboxylic acid-succinimidyl ester (molecular probes) was performed according to manufacturer's instructions. briefly, 5.0 mg of ultrapurified kpedv-9 was dialyzed in labeling buffer (0.1 m nahco 3 , ph 8.3) at 4 • c overnight. virus was then incubated for 1 h on a platform rocker at room temperature with 1 g of af594 succinimidyl ester in 100 l of dmso. the af594-labeled kpedv-9 was extensively dialyzed in buffer a. vero cells were prepared on cover glasses a day before assay. for af594-kpedv-9 co-localization with endocytic markers, the cells were incubated with af594-kpedv-9 combination with 10 g/ml of alexa fluor 488-conjugated transferrin (af488-tf) or 5.0 g/ml of alexa fluor 488-cholera toxin subunit b (af488-ct-b) for 30 min on ice to synchronize entry, and then shifted to 37 • c. unbound viruses were removed, and the cells were fixed in 2% pfa at indicated times and analyzed at magnification of 63× on the laser scanning confocal microscope. vero cells were treated with either 50 mm nh 4 cl or 1 g/ml baf-a1 to neutralize the intracellular ph. the cells were then inoculated with kpedv-9 at a moi of 10 for 2 h in the presence of lysosomotropic agents. the virus inoculums were removed by washing with pbs. cells were then overlaid with 0.5% methyl cellulose in mem containing trypsin for 10 h. at 10 hpi, pedv-infected cells were detected by immunocytochemistry. the effect of low ph on the fusion activity of the s protein was investigated by subjecting pedv-infected vero cells to a low ph range. vero cells were inoculated with kpedv-9 and cultured in trypsin-free mem for 20 h. afterwards, the cell monolayer was washed thrice with pbs and replenished with serum-free mem adjusted to ph 3, 4, 5, 6, 7. mem containing trypsin (10 g/ml) at ph 7 was used as positive control. the cultures were further incubated at 37 • c for 4 h, and then fixed with 5% pfa. pedv-infected cells were detected by immunocytochemistry. cells were pretreated with various protease inhibitors such as e-64 (10 m), aebsf-hcl (500 m), pepstatin a (10 g/ml), and phosphoramidon (10 m) for 1 h. for examination the synergistic antiviral activity of aebsf-hcl and lysosomotropic agent, cells were treated with aebsf-hcl and/or nh 4 cl for 1 h. treated cells were then infected with kpedv-9 at a moi of 1 for 1 h in the presence of inhibitors. after 1 h adsorption, virus inoculums were removed by washing with pbs. cells were then overlaid with 0.5% methyl cellulose in mem containing trypsin for 10 h. at 10 hpi, pedv-infected cells were detected by immunocytochemistry. vero cells were pretreated with either 50 mm nh 4 cl or 10 g/ml cpz and then inoculated with kpedv-9 at a moi of 10 for 2 h. after adsorption, virus inoculums were removed by washing with pbs. cells were then overlaid with 0.5% methyl cellulose in mem containing trypsin for 10 h. at 10 hpi, pedv-infected cells were detected by immunocytochemistry. to detect expression of viral proteins, kpedv-9 infected vero cells were fixed with 5% pfa for 5 min and permeabilized with 1% np40. following three washes with pbs, cells were incubated with 1:5000 dilution of mouse anti-pedv polyclonal antibody for 1 h. cells were washed three times with pbs and then incubated with 1:1000 dilution of goat anti-mouse igg conjugated with horseradish peroxidase. kpedv-9 infected vero cells were stained using a 3, 3 -diaminobenzidine tetrahydrochloride solution containing nicl 2 and h 2 o 2 (vector laboratories). clusters of immunostained cells were observed under the inverted microscope (zeiss) and were presented as the ratio between mock-treated and dmso treated cells. vero cells prepared in 6-well tc plates were treated with various chemicals to inhibit each endocytic pathway as described above. cells were inoculated with kpedv-9 at a moi of 10 in the presence of drugs. at 2 hpi, unbound viruses were washed out and then cells were incubated in serum-free mem. infected cells were harvested and lysed in pro-prep protein extraction solution (intron) at 36 hpi. the extracted proteins were diluted in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) sample buffer and electrophoresed in 10% sds-page system. separated proteins were electrically transferred onto a polyvinyl difluoride membrane and pedv n protein was traced using anti-pedv polyclonal antibodies. bands were visualized using supersignal west dura with las-1000plus. it has been reported that pedv requires an extracellular trypsin for its successful infection in vitro (kusanagi et al., 1992; lai and cavanagh, 1997; park et al., 2011) . early data on the infection of pedv were first provided by hofmann and wyler when they demonstrated the formation of multinucleated cells in pedvinfected vero cells by supplementing the culture media with trypsin (hofmann and wyler, 1988) . the trypsin-induced syncytium formation is corroborated by li et al. when papn-expressing mdck cells were infected with pedv in the presence of trypsin (li et al., 2007) . previous findings suggest that proteolytic processing of the s protein is required to facilitate viral membrane fusion with cellular membranes. and, it also raises the possibility that pedv entry by direct fusion with the plasma membrane could take place in the presence of trypsin. to confirm the role of exogenous proteases in pedv infection, pedv infected cells were treated with trypsin and evaluated viral infectivity by immunocytochemistry. as shown in fig. 1 , we found that pedv could infect vero cells even without trypsin treatment. initial infection was confirmed as early as 8 hpi on both trypsin and non-trypsin conditions. the number of infected cells in both conditions was similar but the spreading into adjacent cells was faster in the presence of trypsin. at 24 hpi, more than 95% of the cell monolayer had formed large multi-nucleated cells. in sharp contrast, pedv growth without exogenous trypsin did not involve syncytial spread. at 48 hpi, more than 99% of the cells showed signs of infection, but still no cytopathic effect (cpe) was found. these results confirm that trypsin catalyzes pedv s protein-mediated cell-cell fusion as demonstrated previously (hofmann and wyler, 1988; park et al., 2011) . based on these observations, we concluded that an exogenous protease, like trypsin, was necessary to induce cell-cell fusion in pedv-infected vero cells but not essentially required for virus-cell entry. so, we hypothesized that pedv entry into vero cells under the trypsin-free condition most likely occurred inside endosomal compartments where cellular proteases might operate similar to trypsin, facilitating s-mediated fusion of pedv with the endosomal membrane. the enveloped virus entry through endocytosis can differentiate into two well-characterized pathways; those acting via the clathrin-coated pit and the caveolae-mediated lipid raft (brodsky et al., 2001; pelkmans and helenius, 2002) . to explore whether the endocytic pathway supports pedv entry into vero cells and which endocytic pathway alters for pedv infection, we inhibited pathway by using substances interfering either clathrin-mediated endocytosis or caveolae-mediated endocytosis. for the inhibition of clathrin-mediated endocytosis, we used either (i) cpz, which is known to abolish the formation of clathrin-coated vesicles by interfering with the interaction of the adapter protein ap-2 with the clathrin-coated pit lattice and thus inhibiting clathrindependent endocytosis or (ii) hypertonic 0.45% sucrose, which inhibit clathrin-mediated endocytosis by inducing dispersion of clathrin lattices on the plasma membrane. for the inhibition of caveolae-mediated endocytosis, we used nystatin, a polyene antifungal agent that interacts with cholesterol and inhibits the lipid raft/caveolin pathway. concentrations of substances were chosen according to previous studies showing the inhibition of other enveloped viruses entering the cells via thee these endocytic pathways. to access the inhibitory effect of cpz on pedv infection, vero cells were treated with cpz and infected with kpedv-9. to measure the inhibitory effect of virus entry, cells were overlaid with 0.5% methyl cellulose in mem containing trypsin for 10 h. media containing methyl cellulose and trypsin blocks second-cycle infection, but allows the formation of syncytium to visualize infected cells. infectivity was determined by measuring infected cells by immunocytochemistry staining at 10 hpi and normalized with "untreated cells" control. as shown in fig. 2a , pedv infection remarkably diminished (>90%) by cpz treatment, and the inhibition rate was positively related with concentration of cpz. to confirm decreased replication of pedv, we determined the expression of pedv nucleocapsid (n) proteins by western blotting. the pedv n proteins were far less expressed in cpz-treated cells compared to untreated cells (fig. 2b) , whereas ␤-actin expression was same. consistent with previous results, pedv infection with hypertonic sucrose treated was also significantly inhibited (fig. 2c ). our result strongly suggested that pedv uses clathrin-mediated endocytosis pathway for their entry into vero cells. similar experiments were performed with nystatin treatment to determine whether pedv also uses caveolae-mediated endocytosis. unlike cpz treatment, pedv infection was only slightly decreased in highest concentrations (fig. 3a) . likewise, the levels of pedv n protein synthesized virtually identical in both presence and absence of nystatin (fig. 3b) . to verify and confirm our results, several other markers specific to the clathrin-mediated pathway or caveolae-mediated endocytosis were used to provide direct evidence that pedv uses this pathway for entry. we traced and visualized pedv location in vero tf is transported into the cells in a vesicle by receptor-mediated clathrin-dependent endocytosis pathway. ct-b specifically binds to glycolipid monoganglioside at the plasma membrane and is internalized and delivered to the golgi complex through caveolaemediated endocytosis pathway (nichols, 2002) . vero cells were incubated with fluorescence-labeled pedv along with each marker, and then evaluated their subcellular localizations by confocal microscopy. at 4 h later, both endocytic markers, ct-b and tf, were located in the cytoplasm. co-localization was observed only between pedv and tf (fig. 4a ), but not with ct-b (fig. 4b) . to further confirm these findings, vero cells were treated with cpz and nystatin prior to pedv inoculation. as shown in fig. 4c , pedv and tf were co-localized in the cytoplasm in both nystatin and mock-treated, but not in cpz-treated vero cells. both pedv and tf were found only on cell surface indicating that clathrin-mediated and tf (green, a), but not between kpedv-9 and ct-b (green, b), was observed in the merged images. (c) colocalization of (yellow) kpedv-9 and tf was observed inside cells in the mock-and nystatin-treated vero cells. by contrast, cpz treatment inhibited internalization of both kpedv-9 and tf. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) endocytosis was successfully (or completely) inhibited by cpz treatment. taken together, we confirmed and concluded that the clathrin-mediated endocytosis pathway was important for pedv entry. we next explored whether pedv infection requires the acidic environment in endosomal compartments. for the inhibition of endosomal acidification, vero cells were treated with either nh 4 cl, a relatively weak base accumulating inside endosomal vesicles, or baf-a1, specific inhibitors of the vacuolar h+-atpase in animal and other eukaryotic cells. neutralization of ph in acidic organelles was confirmed by a fluorescent ph indicator probe, lysosensor (data not shown). we evaluated the inhibitory activities of different lysosomotropic reagents by measuring infected cells. both lysosomotropic reagents showed strong inhibitory effects on production of progeny pedv, especially with 60% reduction at 10 hpi output titer by nh 4 cl (fig. 5a) . pedv replication was about 80% inhibited at concentrations as low as 100 nm of baf-a1. these results indicated that pedv entry was very sensitive to low ph and acidic condition in endosome and/or late endosome might be critical for its entry. for further observation whether acidification solely induce pedv s-mediated fusion, we evaluated pedv s-mediated cell-cell fusion in acidic conditions. as shown in fig. 5b , low ph did not induce cell-cell fusion. syncytium formation was not observed in any cells under the condition between ph 3 and ph 6 (panels a-d). similarly, no syncytium formation was found under neutral ph condition (panel e). this is in stark contrast to the cell-cell fusion observed after the addition of trypsin in the culture media at neutral ph (panel f). collectively, we concluded that the acidic condition is important but still not sufficient for pedv s-mediated membrane fusion. and also, the fusogenic property of s protein could be activated by proteolytic processing. to evaluate the role and effect of proteases other than trypsin in pedv entry, we used protease inhibitors. first of all, we checked cytotoxicity of all those inhibitors to exclude false results with recommended concentrations, and confirmed no cell damaged by them (data not shown). as shown in fig. 6a , we found aebsf-hcl induced the strongest inhibitory activity with more than 90% inhibition. in contrast, e-64, pepstatin a, and phosphoramidon showed relatively lower inhibition with about 5-10% inhibition. these results suggested that serine proteases were importantly involved in pedv entry into vero cells. for the cases of severe acute respiratory syndrome-associated coronavirus (sars-cov) and mhv-2, it has been reported that phdependent endosomal cellular factors were required for proteolytic activation of s proteins, rather than the virus requiring an acidic trigger itself (qiu et al., 2006; simmons et al., 2005) . to evaluate this for pedv s case, cells were treated with aebsf-hcl in combination with nh4cl prior to infection. as shown in previous results, pedv infection was significantly inhibited either by aebsf-hcl or nh 4 cl treatment (fig. 6b) . the inhibitory effect on combination treatments by both was similar to that of aebsf-hcl single treatment. s protein activation by exogenous proteases renders coronavirus s mediated virus-cell fusion independent of cathepsin activity. finally, we determine whether trypsin treatment bypass entry inhibition by endocytosis inhibitors. vero cells were pretreated with either cpz (10 g/ml for 30 min) or nh 4 cl (50 mm for 2 h) prior to virus infection and then infected with pedv in the absence or presence of trypsin. pedv infectivity was only slightly facilitated by trypsin treatment and trypsin treatment does not overcome the inhibitory effect of nh 4 cl and cpz. the small increases of pedv infectivity might be obtained by rapid spreading of virus infection via cell-cell fusion. these results suggested that trypsin did not support proteases-mediated virus-cell entry. the entry mechanism of pedv, a coronavirus, is largely unknown. here, to examine the entry pathway of pedv into vero cells, we used essentially two independent and complementary approaches: (i) the focus formation assay to assess the level of infection by viruses in cells treated with various inhibitors, and (ii) fluorescence microscopy to monitor the entry of viruses into cells along with well-established markers. both approaches provided similar conclusions on the mechanism of pedv cell entry. the infection inhibition assay using various substrates that interfere with endocytosis or lysosomotropic agents revealed that pedv enters vero cells via clathrin-mediated endocytic uptake and delivery of virus to an acidic intracellular compartment. more interestingly, we found that pedv requires serine proteolytic processes in early stages of infection. the serine proteolysis activates pedv entry in independent manner with acidic ph environment, but did not bypass the infection reduction by lysosomotropic agents. in the cell entry of many coronaviruses, the proteolytic activation of s proteins triggers viral membrane fusion and essentially required for virus entry (huang et al., 2006; qiu et al., 2006; simmons et al., 2005) . cathepsins, which is a family of cysteine proteinases commonly found in acidified endosomes, have been associated with the proteolytic processing of s glycoproteins in sars-cov, mhv-2, and hcov-229e and mediated viral membrane fusion with endosomal membranes within endosomes (kawase et al., 2009; qiu et al., 2006; regan et al., 2008; simmons et al., 2005; turk et al., 1999) . similarly, various exogenous and cellular proteases such as trypsin, transmembrane proteases serine 2 enhance sars-cov entry by inducing virus-cell fusion at cell surface (glowacka et al., 2011; matsuyama et al., 2005 matsuyama et al., , 2010 shulla et al., 2011) . the block to infection mediated by lysosomotropic agents could be bypassed by treating with exogenous or cellular proteases (matsuyama et al., 2005; simmons et al., 2005) . pedv infection in vitro is also largely dependent on trypsin supplement (hofmann and wyler, 1988; park et al., 2011) . it raises possibility that pedv s could fuse with plasma membrane by activation with proteases to deliver their genomes into cells. however, our study demonstrated that pedv entry occurs without exogenous proteases. although pedv infection was likely facilitated by trypsin treatment as demonstrated earlier, pedv also propagated even without trypsin (fig. 1) . based on our results, we could confirm that exogenous protease, especially trypsin, might be critical factor for cell-cell fusion but not for viral envelope-cell membrane fusion. and also, we could conclude that trypsin is not essentially required for virus entry into vero cells. our results encouraged us to hypothesize that pedv penetration must have been facilitated by fusion of its envelope with the host membrane in a fusion-permissive environment, which most likely occurs inside endosomal compartments. pedv might take endosomal entry pathway rather than direct fusion. the experiment using various inhibitors supported our hypothesis that pedv alters endosomal pathway for their entry. pedv infection was greatly diminished by pre-treatment with cpz and hypotonic sucrose (fig. 2) . it suggests that pedv enters vero cells via clathrin-mediated pathway. furthermore, co-localization between endocytosed tf and fluorochrome-labeled pedv may support conclusion that clathrin mediated endocytic uptake is major pathway for entry (fig. 4) . our data collectively propose that pedv enters vero cells via clathrin-mediated endocytosis similar to other coronaviruses. vero cells were treated with lysosomotropic agents, either nh4cl or baf-a1, and then infected with kpedv-9. pedv entry was scored by immunocytochemistry at 10 hpi. the relative infectivity was showed as percentages of infected cells to untreated cells. the error bars represent standard deviations of the mean values. (b) low ph does not convert the pedv s protein to its fusogenic form. pedv infected vero cells were exposed to various ph conditions, and then incubated in serum-free media for 4 h. low to neutral ph range (ph 3-7) did not induce cell-cell fusion of pedv-infected vero cells. in contrast, the addition of trypsin at neutral ph readily induced cell-cell fusion within 4 h after treatment (lower right). in order to mediate membrane fusion enveloped viruses, such as influenza and dengue, the low ph of acidified endosomal compartments is sufficient for their conformational changes (plemper, 2011) . unlike these viruses, ph-dependent endosomal cellular factors were required for activation of low ph-dependent proteases, rather than the virus requiring an acidic trigger itself (huang et al., 2006; simmons et al., 2005) . similar to other coronaviruses, pedv infection was significantly inhibited by both nh 4 cl and baf-a1 (fig. 5a ), but acidic ph did not induce pedv s-mediated fusion (fig. 5b) , suggesting that low ph did not differently mediate virus fusion and another triggering factors such as proteolytic activation might be required for successful fusion. while trypsin induce the fusion activity of pedv s proteins on the plasma membrane as demonstrated by formation of syncytia ( fig. 1) , it was not clear whether similar conditions are required for fusion between its envelope and endosomal membrane. pedv entry into vero cells was specifically inhibited by serine proteases inhibitor, but not by other proteases (fig. 6a ). it suggests that serine or serine-like proteases in the cytoplasm are involved in facilitating the fusion between pedv envelope and host endosomal membrane. it is not clear which serine proteases are involved in pedv entry, however, we are assuming that intracellular serine proteases may cleave the s protein so that induce membrane fusion during infection. as shown in fig. 6b , inhibitory effect of serine proteases inhibitors was observed when the lysosomotropic reagents were treated, indicating that novel serine protease(s) is involved in pedv entry with acidic ph independent manner unlike low ph-dependent endosomal cathepsins. in addition, our finding that trypsin treatment is not bypass the infection inhibition with lysosomotropic agents and cpz as shown in fig. 7 , clearly indicates that pedv entry requires ph-dependent step rather than the presence of ph-dependent proteolytic processing. recently, similar possibilities were proposed by others in mhv-a59 s mediated infection study (wicht et al., 2014) . another question is how trypsin sufficiently mediates cell-cell fusion at neutral ph. it was reported that the furin cleavage of s on mhv and sars-cov is required for virus-cell fusion, but not for cell-cell fusion (de haan et al., 2004; follis et al., 2006) . it seems like coronavirus s-mediated cell-cell fusion is regulated by different manner from virus-cell fusion. acidic ph and serine proteolysis are sufficient for virus-cell fusion, in contrast, serine proteolysis and/or unknown other factor(s) which may provide similar condition to low ph, are required for cell-cell fusion at plasma membranes; thus s induces membrane fusion without low ph. all our data confirmed that pedv entry followed clathrinmediated endocytosis independence of caveolae-coated pit assembly. the internalized pedv was co-localized with the clathrin-mediated endocytic marker, but not with the caveolaemediated endocytic marker. in addition, cells treated with lysosomotropic agents and serine protease inhibitors were resistant to pedv. our data revealed that pedv entry followed clathrin-mediated endocytosis and was dependent on an acidic ph and serine proteolysis for successful entry into cells. fluorescent lipid probes in the study of viral membrane fusion biological basket weaving: formation and function of clathrin-coated vesicles application of a focus formation assay for detection and titration of porcine epidemic diarrhea virus cleavage inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion experimental infection of pigs with a new porcine enteric coronavirus, cv 777 furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response the coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment propagation of the virus of porcine epidemic diarrhea in cell culture sars coronavirus, but not human coronavirus nl63, utilizes cathepsin l to infect ace2-expressing cells protease-mediated entry via the endosome of human coronavirus 229e isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate the molecular biology of coronaviruses porcine aminopeptidase n is a functional receptor for the pedv coronavirus infectious entry pathway of influenza virus in a canine kidney cell line efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 proteasemediated enhancement of severe acute respiratory syndrome coronavirus infection a distinct class of endosome mediates clathrin-independent endocytosis to the golgi complex human coronavirus 229e binds to cd13 in rafts and enters the cell through caveolae identification of a putative cellular receptor 150 kda polypeptide for porcine epidemic diarrhea virus in porcine enterocytes receptor-bound porcine epidemic diarrhea virus spike protein cleaved by trypsin induces membrane fusion endocytosis via caveolae insider information: what viruses tell us about endocytosis cell entry of enveloped viruses endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry differential role for low ph and cathepsin-mediated cleavage of the viral spike protein during entry of serotype ii feline coronaviruses a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry how viruses enter animal cells porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines analysis of the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection: aminopeptidase n is not important and a process of acidification of the endosome is necessary coronavirus-like particles associated with diarrhea in baby pigs in quebec acidic ph as a physiological regulator of human cathepsin l activity clathrin-and caveolae-independent entry of feline infectious peritonitis virus in monocytes depends on dynamin evidence for a putative second receptor for porcine transmissible gastroenteritis virus on the villous enterocytes of newborn pigs identification and characterization of a proteolytically primed form of the murine coronavirus spike proteins after fusion with the target cell an apparently new syndrome of porcine epidemic diarrhoea this research was supported by grant from the korea research foundation grant funded by the korean government (no. 20120008358, 2011(no. 20120008358, -0023942, 211-2006. we really appreciate for all the advices and technical supports from dr. fumihiro taguchi in nippon veterinary and life science university. key: cord-273745-mwjh5se7 authors: meng, fandan; suo, siqingaowa; zarlenga, dante s; cong, yingying; ma, xiaowei; zhao, qiong; ren, xiaofeng title: a phage-displayed peptide recognizing porcine aminopeptidase n is a potent small molecule inhibitor of pedv entry date: 2014-03-25 journal: virology doi: 10.1016/j.virol.2014.01.010 sha: doc_id: 273745 cord_uid: mwjh5se7 three phage-displayed peptides designated h, s and f that recognize porcine aminopeptidase n (papn), the cellular receptor of porcine transmissible gastroenteritis virus (tgev) were able to inhibit cell infection by tgev. these same peptides had no inhibitory effects on infection of vero cells by porcine epidemic diarrhea virus (pedv). however, when pedv, tgev and porcine pseudorabies virus were incubated with peptide h (hvtttfappppr), only infection of vero cells by pedv was inhibited. immunofluoresence assays indicated that inhibition of pedv infection by peptide h was independent of papn. western blots demonstrated that peptide h interacted with pedv spike protein and that pre-treatment of pedv with peptide h led to a higher inhibition than synchronous incubation with cells. these results indicate direct interaction with the virus is necessary to inhibit infectivity. temperature shift assays demonstrated that peptide h inhibited pre-attachment of the virus to the cells. coronaviruses belong to the family of coronaviridae and commonly cause respiratory or gastroenteric diseases (weiss and navas-martin, 2005; lai et al., 2007) . three groups of coronaviruses have been identified, based on differences in serology and genotyping (cavanagh, 1997; spaan et al., 2005) . these are enveloped viruses and consist of four major structural proteins: spike (s), membrane (m), nucleocapsid (n) and minor small envelop (e) protein (lai et al., 2007) . the host range and tissue tropism of coronaviruses depend on interactions between the viral s glycoprotein and receptors on susceptible cells (bosch et al., 2003; gallagher and buchmeier, 2001) . porcine transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) are swine-specific enteric coronaviruses that are antigenically distinguishable (lai et al., 2007; pensaert and yeo, 2006) . however, they replicate in the differentiated enterocytes of the small intestine resulting in similar clinical symptoms including lethal watery diarrhea and dehydration in piglets (pensaert and yeo, 2006; sanchez et al., 1992) . two decades ago, porcine aminopeptidase n (papn) was identified as a cellular receptor for tgev (delmas et al., 1992) . since that time, several limited reports have showed that addition of exogenous papn facilitates cell infection by pedv (li et al., 2007; oh et al., 2003.) . recent evidence also indicates that increased papn receptor density on the surface of st cells contributes to cell infection by pedv (nam and lee, 2010) . the available data supports the hypothesis that blockage of papn is a good strategy for preventing cell infection by tgev or pedv. using the papn as a target protein, we identified three 12-mer peptides (designated as h, s or f) by phage display which bind to papn and competitively inhibit cell infection by tgev (ren et al., 2011a) . the initial purpose of this study was to investigate the role of papn-binding peptides h, s and f on cell infection by pedv. interestingly, although there was no surface expression of papn on vero cells, peptide h decreased the infectivity of pedv in vitro. western blots indicated that peptide h (hvtttfappppr) interacted with the s protein of pedv. altering incubation temperatures further demonstrated that peptide h affected pre-attachment of pedv to cells. it is important to identify small molecules such as peptides that prevent infection by pedv, inasmuch as highly effective pedv vaccines which are currently not available. the peptide h identified herein may be one such candidate. concentration of compound that decreased the percentage of formazan produced in uninfected, peptide-treated cells to 50% of that produced in uninfected, peptide-free cells. the cc 50 values were greater than 1000 μg/ml. all subsequent antiviral experiments were performed at peptide concentrations below the experimentally-determined cc 50 value. in order to test the abilities of the three peptides to prevent attachment of pedv to cells, all combinations of peptide, virus and cell treatment were performed. cell post-treatment assays (fig. 1a) were performed to evaluate whether the three peptides were able to inhibit replication of pedv after infecting vero cells. plaque assays indicated that none of the three peptides inhibited pedv infection; however, rabbit anti-pedv decreased vial infectivity by more than 50% when the dilutions were reduced from 1:64 to 1:4. when vero cells were pre-treated with peptide (cell pretreatment assay) prior to virus infection (fig. 1b) , little changes in virus titers were observed between the control and peptide treatment groups; some small effects were observed with the rabbit anti-pedv neutralizing antibodies. finally, in the virus pretreatment assays where pedv was incubated with peptides prior to cell infection (fig. 1c) , the results indicated that both peptides h and s inhibited pedv infectivity where ec 50 values were approximately 1 μg/ml and 62.5 μg/ml, respectively. the antiviral activity of peptide h was dose-dependent and at 250 μg/ml it exhibited greater than 95% anti-pedv activity which is significantly higher than peptides s or f (p o0.01); at 15.6 μg/ml, inhibition was greater than 70%. the selectivity indices si of peptides h and s were 1000 and 16, respectively. peptide f showed little inhibitory activity against pedv infection even at concentrations z 1000 μg/ml. inasmuch as papn may be involved in cell infection by pedv, the existence of papn on st, vero and mdck cells was analyzed by ifa. as shown in fig. 2 , the endogenous papn expressed only on the surface of st cells, a porcine cell line. no expression was found on the surfaces of vero cells or mdck cells suggesting that the inhibitory activity of peptide h on pedv infection in vitro did not involve papn. the specificity of the inhibition of peptide h on pedv infection was assessed by comparing antiviral activities of peptide h on tgev and prv. further, peptide-induced cytotoxicity in st cells was also evaluated. results clearly show that peptide h had no demonstrable effects on tgev or prv even at very high peptide concentrations (1 mm/ml) (fig. 3) suggesting that a non-specific reactivity with virus envelopes is unlikely to be the cause for attenuating pedv infectivity. the effect of peptide h on the level of virus rna was quantified by real-time rt-pcr. the results demonstrated a dose-dependent decrease of viral rna synthesized in pedv-infected cells (fig. 4) . at 31.25 μg/ml and 15.625 μg/ml, peptide h showed reduction of viral rna synthesis (po 0.05). however, at 250 μg/ml, 125 μg/ml and 62.5 μg/ml it significantly decreased viral rna synthesis (p o0.01) when compared to the no peptide treatment group. the inhibitory activity of peptide h against pedv infection was confirmed by conventional rt-pcr. analysis of the pedv-rnas indicated that the density of the amplified sequences decreased with increasing concentrations of peptide h (data not shown). binding characteristics of the phage h (phage encoding peptide h) to pedv was analyzed by western blot. as shown in fig. 5 , the phage h reacted with a protein with an approximate molecular mass of 220 kda which is coincident with the molecular mass of the pedv s protein. antibody against the pedv s protein was used as a positive control. other controls including the m13 phage library and two phages bearing non-selected peptides did not react with the pedv s protein (fig. 5) . these results indicate that the peptide h binds to the s protein of pedv. to further examine the mechanism of action of peptide h on cell infection by pedv, we investigated the effects of incubation temperature on cell infectivity. plaque assays showed that peptide infected with pedv at an pfu of 5 â 10 3 /ml. (c) peptides h, s or f were first incubated with pedv at 37 1c for 1 h, and then the peptide treated viruses (pfu ¼ 5 â 10 3 /ml) were used to infect vero cells at 37 1c. plaque assays were performed at the end of each experiment. serially-diluted polyclonal antibody against pedv and pbs were used as positive and negative controls, respectively. peptide concentrations 1, 2, 3, 4, and 5 are 250 μg/ml, 125 μg/ml, 62.5 μg/ml, 31.25 μg/ml, and 15.625 μg/ml, respectively. anti-pedv antibody dilutions 1, 2, 3, 4, and 5 are 1:4, 1:8; 1:16; 1:32; and 1:64, respectively. bars show the standard deviation from three independent assays. h exhibited significantly higher inhibitory effects (po 0.01) than peptide f or pbs on the pre-attachment of pedv to vero cells ( fig. 6a ) when virus was incubated with peptide h at 4 1c for 1 h prior to incubation with the cells. when peptide h and pedv were co-incubated with vero cell at 4 1c for 1 h before shifting to 37 1c (binding only), this allowed us to measure peptide h effects on early-and pre-attachment of the virus. results showed that pedv pre-treated with peptide h exhibited slightly higher inhibition (p 40.05) than when peptide h and pedv were co-incubated with cells absent a pre-incubation step (fig. 6b) suggesting that there is a direct effect of peptide h on pre-attachment. little to no inhibition of infection was observed once the virus became attached to the cell surface. furthermore, when the peptides and pedv were co-incubated with vero cells at 37 1c absent any preincubation step, no effective inhibition of cell infection was observed (fig. 6c ). to characterize the temperature effect on interactions between peptide h and pedv as well as cell infection, various concentrations of the peptides were incubated with pedv (pfu ¼5 â 10 3 /ml) at 4 1c or 37 1c for 1 h, then the peptide-treated viruses were used to infect cells at 37 1c. the results showed that the inhibition rate at 37 1c was significantly higher (p o0.01) than that at 4 1c when the lower concentrations of peptide h were applied. the inhibition ratio reached 61.86% at 37 1c but only 1.25% at 4 1c at the lowest concentration (15.625 μg/ml). in contrast, high concentrations of peptide h gave rise to a similar inhibition of pedv infectivity (fig. 6d ). infection with tgev and pedv can cause high mortality in piglets and therefore enormous economic loss in the pig industry. the prevalence of pedv and tgev in asian countries such as china and korea has been documented (ren et al., 2011b; li and ren, 2011) . at present, live vaccines against the both viruses are extensively used in china which in turn decreases the occurrence of diseases to some extent. however, small molecule inhibitors to tgev or pedv are alternative approaches to controlling swine viral diarrhea diseases. using combinatorial phage-display peptide libraries can be a powerful tool for selecting ligands that bind target proteins. phage display techniques have been used to generate diagnostic and therapeutic peptides for bacteria (bishop-hurley et al., 2005 carnazza et al., 2008) , fungi (bishop-hurley et al., 2002; fang et al., 2006) and viruses (ren et al., 2011a ferrer and harrison, 1999; welch et al., 2007; wu et al., 2011; yang et al., 2003) . the papn is a member of a membrane-bound metalloprotease family and predominantly expressed on the surface of epithelial cells of the kidney, small intestine, and respiratory tract (nam and lee, 2010; kenny and maroux, 1982; lendeckel et al., 2000) . it is known that papn is a cellular receptor for tgev and that anti-papn antibody efficiently decreases cell infection by this virus (delmas et al., 1992; liu et al., 2009) . recently, three papn-binding peptides h, s, and f were identified using papn as an immobilized target for panning a 12-mer phage display peptide library. these peptides exhibited high affinity binding to papn and inhibited cell infection by tgev completely . as a member of group i coronaviruses, tgev and pedv have similar infection characterizations and as such it is difficult to differentiate these pathogens based only upon clinical symptoms. recent evidence indicates that pedv may also bind papn, a type ii glycoprotein, as a functional receptor (li et al., 2007; oh et al., 2003) . interestingly, tgev can be easily propagated in swine-originated cells such as st cells (delmas et al., 1992; hofmann and wyler, 1988) whereas pedv is adapted and cultivated in african green monkey kidney (vero) cells rather swine cells. given the stark similarities as well as differences between tgev and pedv, we were interested in evaluating the antiviral effects of the h, s and f peptides on cell infection by pedv. we first analyzed potential blockage of the papn-binding peptides on vero cell infection by pedv. plaque assays indicated no significant decrease in the infectivity of pdev even though prior studies showed that both anti-papn antibody and peptides h, s or f were capable of inhibiting tgev infection in vitro (ren et al., 2011a; liu et al., 2009) if pre-incubated with tgev permissive cells. there were limited reports indicating that papn may serve as a functional receptor of pedv; however, studies herein clearly demonstrated that papn is only expressed on the surface of st cells and is not present on vero or mdck cells. as such, the inhibitory activities of peptide h were not due to binding papn. further, we showed that only pre-treatment with peptide h inhibited infection by pedv. the prv is a swine neurotropic herpesvirus with a dna genome that can be propagated in many cell lines including vero cells. therefore, we used prv as an additional control to further confirm and evaluate the inhibition specificity of the h peptide. peptide h did not prevent infection of pretreated tgev or prv suggesting its inhibitory activity was specific and not due to virucidal effects of amphipathic peptides. clearly, peptide h was able to interact with pedv; however, it was unclear as to the mechanism of its antiviral activity. as such, the binding characteristics between peptide h (using the phage bearing the h peptide) and pedv were further examined by western blot. results clearly showed that phage h reacted with a protein with a molecular mass of 220 kda closely approximating the molecular mass of the pedv s protein. this supposition was corroborated using antibody against the pedv s protein. in contrast, control phages bearing other peptides did not show such reactivity. these results demonstrate that the peptide h abrogates infectivity in part by binding to the pedv s protein. this is consistent with the function of the coronavirus s protein that mediates cell infection. further, cell post-treatment assays evaluating the effects of each peptide on the replication of pedv in vitro relative amplification of the pedv x-n gene in the infected cells was normalized to that of beta-actin and calculated using the 2 à δδct method. peptide concentrations 1, 2, 3, 4, 5, and 6 are 0 μg/ml, 15.625 μg/ml, 31.25 μg/ml, 62.5 μg/ml, 125 μg/ml, 250 μg/ml, respectively; line 7 is cell control group. displayed results are averages of three independent experiments. clearly demonstrated that the peptides do not interfere with the intracellular replication of pedv. our results showed that only peptide h and not peptides s or f exhibited very high, dose-dependent inhibitory activity against pedv where as little as 1 μg/ml needed to achieve ec 50 . this was confirmed by real-time rt-pcr which showed decreasing amounts of viral rna in pedv-infected cells. this corroborated the relationship between the antiviral activity of peptide h and either blockage of the viral attachment or entry into vero cells. the impact of peptide h on the entry of pedv was first investigated by performing the cell post-treatment and co-incubation assays. when pedv was incubated with cells prior to treatment with peptide h no significant effects on pedv infection were observed. similar results were seen when peptides, pedv and the cells were combined at the same time and co-incubated at 37 1c suggesting that peptide h did not affect pedv entry into the cell postadsorption. however, when pedv was pre-incubated with peptide h prior to incubation with vero cells, peptide h blocked the attachment of pedv as determined from plaque assays and rt-pcr analysis. it became clear that peptide h did not interact with vero cells directly. rather, it exhibits its inhibitory activity via the interplay between the peptide h and pedv. it is accepted that virus adsorption occurs at 4 1c and internalization does not happen until the temperature is raised to 37 1c (baldick et al., 2010) . our results clearly showed that the effects of peptide h occurred during the incubation step at 4 1c rather than the 37 1c internalization step again targeting a specific interaction between peptide h and pedv that affects binding to the cell surface. both pedv and tgev are group i coronaviruses (bridgen et al., 1993) and propagate in vero and st cells, respectively; prv is a dna virus that also propagates in vero cells. as such, we selected tgev and prv as control viruses to examine any specificity in the inhibitory activity of peptide h on cell infection by these viruses. as expected, the results showed no significant inhibitory activity of peptide h on either tgev or prv infection. further, peptide h was not cytotoxic to either st or vero cells. these results corroborate our hypothesis that peptide h functions in part, by interacting with the s protein of pedv and affecting the ability of the virus to bind to the cell surface. future studies will focus on identifying the specific site of interaction of peptide h and whether or not such a peptide can be used in vivo to abrogate or attenuate pedv infections. swine testis (st), monkey kidney cell lines (vero) and madin-darby canine kidney (mdck) cells were maintained in dulbecco's modified eagle medium (dmem) (invitrogen, us) supplemented with 10% fetal bovine serum, (fbs, gibco, us), 100 units/ml of penicillin and 100 units/ml of streptomycin. all cells were purchased from american type culture collection (atcc) and kept in our laboratory. pedv isolate hljby was propagated in vero cells in the presence of 10 μg/ml trypsin (gibco, us) (ren et al., 2011b) . tgev strain pur46-mad was propagated in st cells (ren et al., 2008; yin et al., 2010) . porcine pseudorabies virus (prv) strain kaplan was propagated in vero cells (ren et al., 2011c) . st or vero cells were seeded in 96-well plates at a density of 5 â 10 4 cells/well and cultured in dmem containing 10% fbs at 37 1c under 5% co 2 for 24 h followed by addition of serial dilutions (62.5-1000 μg/ml) of the tested peptides. the cells were allowed to grow for 48 h at 37 1c and proliferation was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) method. briefly, the medium was removed and 10 μl of mtt solution (0.5 mg/ml, invitrogen, us) was added and incubated at 37 1c for 4 h. then 100 μl of dimethyl sulfoxide (dmso) was added and incubated for 15 min to solubilize the formazan crystals. cell survival rate was calculated as (od 490 treatment)/(od 490 control) (paeshuyse et al., 2006) . the 50% cytostatic concentration (cc 50 ) was defined as the concentration inhibiting the proliferation of (b) pedv (pfu ¼ 5 â 10 3 /ml), peptides h or f, and vero cells were co-incubated at 4 1c for 1 h, washed, then shifted to 37 1c. (c) pedv (pfu ¼ 5 â 10 3 /ml), peptides, and vero cells were co-incubated at 37 1c for 1 h then washed prior to assaying. (d) pedv was treated with various concentrations of peptides h or f at 4 1c or 37 1c for 1 h then used to infect vero cells (pfu ¼5 â 10 3 /ml) at 37 1c. peptide concentrations 1, 2, 3, 4, and 5 are 250 μg/ml, 125 μg/ml, 62.5 μg/ml, 31.25 μg/ml, and 15.625 μg/ml, respectively. anti-pedv antibody dilutions 1, 2, 3, 4, and 5 are 1:4, 1:8, 1:16, 1:32 and 1:64, respectively. plaque assays were further performed at 72 h post-infection. bars show the standard deviation from three independent assays. exponentially growing cells by 50%. non-cytotoxic concentrations of peptides (r cc 50 ) were used for antiviral assays. three different treatment approaches were used to analyze the antiviral action of the selected peptides h (hvtttfappppr), s (svvpskatwgfa) and f (fkpssppsitlw) as previously defined (kwon et al., 2010) . in the first method, i.e., cell post-treatment assay, vero cells were grown in 24-well plates at a density of 5 â 10 5 cells/well for 24 h. pedv at a pfu (plaque-forming unit) of 5 â 10 3 /ml was inoculated onto confluent cells for 1 h followed by removal of the medium and incubation of the infected cells with various noncytotoxic concentrations (r cc 50 ) of peptides for 1 h at 37 1c. the cells were then overlaid with 1% methylcellulose in dmem and incubated for 72 h at 37 1c followed by crystal violet staining and plaque assays as previously described with modifications (ren et al., 2011a (ren et al., , 2011d . briefly, after the medium was removed, the cells were fixed with 4% formaldehyde in pbs for 1 h at room temperature followed by staining with 1% crystal violet solution for 20 min. the staining solution was removed, the cells were washed with pbs and the plaques were counted. decreases in virus infectivity were calculated from the plaque assay as follows: 100 â [1 à (treatment wells/control wells)]. average ec 50 values (concentration inducing 50% inhibition of virus replication) were calculated and the effectiveness of each peptide were expressed using the selectivity index (si) (si¼ cc 50 /ec 50 ) (paeshuyse et al., 2006; müller et al., 2007) . in the second method, i.e., cell pre-treatment assay, vero cells were first grown in 24-well plates at a density of 5 â 10 5 cells/well for 24 h then treated with non-cytotoxic concentrations of peptide for 1 h prior to incubation with virus. the peptides were removed and the cells were washed twice with pbs. pedv at a pfu of 5 â 10 3 /ml was inoculated onto the pre-treated vero cells for 1 h. after the virus was removed, the cells were overlaid with 1% methylcellulose in dmem and incubated for 72 h at 37 1c followed by plaque assays. in the third experiment i.e., virus pre-treatment, various concentrations of the peptides were mixed with pedv (pfu ¼ 5 â 10 3 /ml) at 37 1c for 1 h prior to incubation with cells. vero cells were grown in 24-well plates at 5 â 10 5 cells/well for 24 h then the peptide/virus mixture was added to the cultured cells for 1 h. after the mixture was removed and the cells washed with pbs, the cells were overlaid with 1% methylcellulose in dmem and cultured for an additional 72 h at 37 1c followed by plaque assays as described above. in parallel, pedv-neutralizing, rabbit antiserum serially-diluted 1:2 and pbs were used as positive and negative controls, respectively, for the above-mentioned experiments. each concentration of the peptide and antibody was assayed in triplicate. immunofluorescence assays to identify papn on cell lines from different species st, vero and mdck cells were seed into the 24-well plates and incubated at 37 1c for 24 h. indirect immunofluorescence assays (ifa) were performed (ren et al., 2011d; baldick et al., 2010) with modification. the cells were washed twice with pre-chilled pbs then fixed with 4% paraformaldehyde in pbs for 30 min at room temperature. following two washes with pbs, they were quenched with 0.1 m glycine for 5 min then blocked with 2% bsa (sigma, us) in pbs for 30 min. samples were incubated for 1 h with anti-papn polyclonal antibody (1:1500 in pbs) (liu et al., 2009) , washed three times with pbs, and incubated with fluorescein isothiocyanate (fitc) conjugated goat anti-rabbit igg (1:1500 in pbs, zhongshan, china) for 1 h in the dark. the samples were washed three times with pbs and the fluorescence signals and phase contrast images were detected by fluorescence microscopy (leica, wetzlar, germany). various concentrations of peptide h were incubated with the tgev, pedv and prv (pfu¼5 â 10 3 /ml) at 37 1c for 1 h then added to confluent vero or st cell monolayers for 1 h. the mixtures were removed and the cultured cells washed twice with pbs then incubated with 1% methylcellulose in dmem for 72 h at 37 1c. the cells were then stained with crystal violet staining and plaque assays were performed as described above. the effect of peptide h on pedv infection of vero cells was confirmed by semi-quantitative real-time reverse transcription (rt-pcr) (ren et al., 2011d) . vero cells in 6-well plates were infected with pedv (pfu ¼5 â 10 3 /ml) pretreated with different concentrations of peptide h at 37 1c for 1 h. the culture was replaced with dmem at 37 1c for 24 h then washed 3 times with pbs. the virus-containing cultures were frozen and thawed three times followed by addition of an equal volume of 20% polyethylene glycol (pge) 8000 at room temperature and incubation for 30 min. the samples were centrifuged at 12,000 rpm for 5 min and the pellets were re-suspended in rnase-free water. total rna was extracted with a commercial kit (fastgene, china) according to the manufacturer's instructions. reverse transcription was performed in a total of 20 μl consisting of 5 μl total rna (2.5 μg), 1 μl oligo dt, 1 μl dntp (10 mm), 0.5 μl rnase inhibitor, 7.5 μl sterile water, 1 μl mlv, and 4 μl 5 â rt-pcr buffer. the mixture was incubated at 30 1c for 10 min, 42 1c for 60 min and 95 1c for 5 min. real-time pcr was performed using abi prism 7500 real-time pcr machine (applied biosystems, usa). the information regarding primers and rt-pcr products is provided in table 1 . the real-time pcr mixture included 0.5 μl (0.5 μg) of cdna template, 10 μl of sybr taq polymerase, 0.4 μl of rox pge 2, 0.5 μl (10 pmol) of each primer, and 8.1 μl of sterile water. the reactions were incubated at 95 1c for 10 s followed by 40 cycles of 95 1c for 5 s and 60 1c for 34 s. we examined virus rna levels using primers that specifically amplify a 244 bp fragment encompassing the 3' end of a small, non-structural gene (x) and the 5' end of the pedv-n gene ( table 1 ). the expression of pedv x-n in pedv-infected vero cells was normalized to that of beta-actin and taken as 100% compared to expression of the peptide h treatment group. data analysis is based on the measurement of the cycle threshold (ct). the difference in δct (ct sample fragment mean ct value-beta-actin fragment mean ct value) was used as a measure of the relative fragment with the 2 à δδct method in correlation to the amplification size of pedv x-n fragment. for each experimental condition, real-time pcr was conducted in quadruplicate and the resulting average ct values for the pedv x-n fragment was used to quantify viral load. the experiment was performed in triplicate. information on primers and real-time rt-pcr products. sense 5' cactggttgggctttctatgtc pedvx-n antisense 5'tgttagtgggtacagcgttgtt 244 sense 5' ggctcagagcaagagaggtatcc β-actin antisense 5' ggtctcaaacatgatctgagtcatct 208 western blot analysis of peptide h binding to pedv the pedv (1 â 10 6 pfu/ml) was harvested from vero cells and clarified by centrifugation at 1300g for 15 min followed by ultracentrifugation at 150,000g for 1.5 h to collect the virus. the pellets were suspended in pbs, subjected to 10% sds-page then blotted to a nitrocellulose (nc) membrane. the nc membranes were blocked overnight at 4 1c with 5% non-fat dry milk in pbs. after three washes with pbs, membranes were sliced and incubated with phage h (1 â 10 11 pfu), anti-pedv s polyclonal antibody (1:1000 in pbs), m13 phage library (1 â 10 11 pfu), and control phage bearing either the peptides svsvgmkpsprp or mscndtlcllpn. the membranes were then washed with pbs and successively incubated with anti-m13 polyclonal antibody (abcam, 1:600 in pbs) and horseradish peroxidase hrp-conjugated goat anti-rabbit igg (1:1500 in pbs) at room temperature for 1 h. protein bands were visualized using 3,3'-diaminodbenzidine (dab, the thermo scientific) to examine the effect of temperature on the binding of virus to the cells, four experiments were performed. first, various concentrations of peptide h and control peptide f were incubated with pedv (pfu¼ 5 â 10 3 /ml) at 4 1c for 1 h then added to confluent vero cells seeded in 24-well plates at 4 1c for 1 h followed by infection at 37 1c for 72 h. second, the peptides, pedv and vero cells were co-incubated at 4 1c for 1 h, after which the incubation temperature was raised to 37 1c for 72 h. third, the peptides, pedv and vero cells were co-incubated at 37 1c for 1 h then assayed without prior incubation at 4 1c. finally, peptides were pre-incubated with pedv at 4 1c or 37 1c for 1 h followed by cell infection at 37 1c for 72 h. all experiments were terminated by extensive washing of the cells and plaque assays to quantify the infection. statistical analysis of the data was performed using spss 11.5 software; p o0.05 and p o0.01 were defined as statistically significant and highly statistically significant, respectively. a novel small molecule inhibitor of hepatitis c virus entry phage-displayed peptides as developmental agonists for phytophthora capsici zoospores phage-displayed peptides selected for binding to campylobacter jejuni are antimicrobial peptides selected for binding to a virulent strain of haemophilus influenzae by phage display are bactericidal the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhoea virus confirms that this virus is a coronavirus related to human coronavirus 229e and porcine transmissible gastroenteritis virus specific and selective probes for pseudomonas aeruginosa from phage-displayed random peptide libraries nidovirales, a new order comprising coronaviridae and arteriviridae aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev combinatorially selected defense peptides protect plant roots from pathogen infection peptide ligands to human immunodeficiency virus type 1 gp120 identified from phage display libraries coronavirus spike proteins in viral entry and pathogenesis propagation of the virus of porcine epidemic diarrhea in cell culture topology of microvillar membrance hydrolases of kidney and intestine in vitro inhibitory activity of alpinia katsumadai extracts against influenza virus infection and hemagglutination coronaviridae review: the role of membrane peptidases in immune functions porcine aminopeptidase n is a functional receptor for the pedv coronavirus reverse transcription loop-mediated isothermal amplification for rapid detection of transmissible gastroenteritis virus expression and functional analysis of porcine aminopeptidase n produced in prokaryotic expression system evaluation of antiviral activity of south american plant extracts against herpes simplex virus type 1 and rabies virus contribution of the porcine aminopeptidase n (cd13) receptor density to porcine epidemic diarrhea virus infection identification of a putative cellular receptor 150 kda polypeptide for porcine epidemic diarrhea virus in porcine enterocytes a novel, highly selective inhibitor of pestivirus replication that targets the viral rna-dependent rna polymerase porcine epidemic diarrhea importance of cholesterol for infection of cells by transmissible gastroenteritis virus phage displayed peptides recognizing porcine aminopeptidase n inhibit transmissible gastroenteritis coronavirus infection in vitro action mechanisms of lithium chloride on cell infection by transmissible gastroenteritis coronavirus development of a porcine epidemic diarrhea virus m protein-based elisa for virus detection phages harboring specific peptides that recognize the n protein of the porcine reproductive and respiratory syndrome virus distinguish the virus from other viruses cholesterol dependence of pseudorabies herpesvirus entry genetic evolution and tropism of transmissible gastroenteritis coronavirus virus taxonomy, eighth report of the international committee on taxonomy of viruses coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol potent dpeptide inhibitors of hiv-1 entry phage displayed peptides to avian h5n1 virus distinguished the virus from other viruses potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type i (hiv-1) vif proteins cholesterol is important for a post-adsorption step in the entry process of transmissible gastroenteritis virus this work was supported by national natural science foundation of china (31340003 and 31372438), sponsored by chang jiang scholar candidates program for provincial universities in heilongjiang (2013cjhb002), the program for new century excellent talents in university of ministry of education of p.r. china (ncet-10-0144). key: cord-279598-xzionafe authors: chang, chia-yu; cheng, ivan-chen; chang, yen-chen; tsai, pei-shiue; lai, seiu-yu; huang, yu-liang; jeng, chian-ren; pang, victor fei; chang, hui-wen title: identification of neutralizing monoclonal antibodies targeting novel conformational epitopes of the porcine epidemic diarrhoea virus spike protein date: 2019-02-21 journal: sci rep doi: 10.1038/s41598-019-39844-5 sha: doc_id: 279598 cord_uid: xzionafe since 2010, newly identified variants of porcine epidemic diarrhoea virus (pedv) have caused high mortality in neonatal piglets which has devastated the swine industry. the spike (s) glycoprotein of pedv contains multiple neutralizing epitopes and is a major target for pedv neutralization and vaccine development. to understand the antigenicity of the new pedv variant, we characterized the neutralizing epitopes of a new genotype 2b pedv isolate from taiwan, pedv pintung 52 (pedv-pt), by the generation of neutralizing monoclonal antibodies (nmabs). two nmabs, p4b-1, and e10e-1–10 that recognized the ectodomain of the full-length recombinant pedv s protein and exhibited neutralizing ability against the pedv-pt virus were selected. recombinant truncated s proteins were used to identify the target sequences for the nmabs and p4b-1 was shown to recognize the c-terminus of co-26k equivalent epitope (coe) at amino acids (a.a.) 575–639 of the pedv s. interestingly, e10e-1–10 could recognize a novel neutralizing epitope at a.a. 435–485 within the s1(a) domain of the pedv s protein, whose importance and function are yet to be determined. moreover, both nmabs could not bind to linearized s proteins, indicating that only conformational epitopes are recognized. this data could improve our understanding of the antigenic structures of the pedv s protein and facilitate future development of novel epitope-based vaccines. determination of neutralizing antibodies. after purification and quantification of mabs, the neutralizing ability of each mab against the g2b taiwan pedv-pt strain was assessed using the virus neutralizing assay. the starting concentration for each mab in the neutralizing assay was 20 μg/ml, with a two-fold serial dilution to obtain a final neutralizing concentration of 2.5 μg/ml. the mabs, p4b-1, and e10e-1-10, possessed potent neutralizing ability that completely blocked the cpe of the taiwan pedv-pt strain under 5 μg/ml. on the contrary, the mabs, r7h-2, and unk-1, showed no neutralization ability against pedv-pt even at 20 μg/ml. synthesized codon-optimized complete gene sequence of the pedv-pintung 52 s protein (pedv-pt; genbank accession no. kp276252) was used to amplify dna amplicons of varying lengths that encode different truncated forms of the s protein genes. using the primer pairs listed in supplementary table 1 , dna sequences of different lengths were amplified namely s 1-435 (1305 bp), s 1-485 (1455 bp), s 1-501 (1503 bp), s 1-509 (1527 bp), s 1-575 (1725 bp), s 1-639 (1917 bp) and the full-length s ectodomain (4086 bp) of the pedv-pt strain. these dna fragments were digested with restriction enzymes and ligated into the pcdna3.1-v5-his vector. seven plasmids containing the v5-tag, namely pcdna3.1-pedv s 1-435 -v5-his, pcdna3.1-pedv s 1-485 -v5-his, pcdna3.1-pedv s 1-501 -v5-his, pcdna3.1-pedv s 1-509 -v5-his, pcdna3.1-pedv s 1-575 -v5-his, pcdna3.1-pedv s 1-639 -v5-his, and pcdna3.1-pedv s-v5-his were constructed. we have shown the construction of the full-length spike ectodomain of the taiwan pedv-pt strain in a previous publication 19 . the expression levels of various truncated recombinant s proteins, s 1-435 , s 1-485 , s 1-501 , s 1-509 , s 1-575 , and s 1-639 of the pedv-pt strain, were successfully detected using icc staining with the anti-v5 tag antibody ( fig. 1) , as well as western blotting (fig. 2) . the v5 tag-positive hek293 cells for each construct occupied 70% to 90% of the total population. the positive ratio of each construct was adjusted between 60% and 70% by mixing with untransfected hek293 cells, which were used as internal negative control in the icc staining. the corresponding molecular weights of s 1-435 , s 1-485 , s 1-501 , s 1-509 , s 1-575 , s 1-639 , and the full-length spike protein were detected to be approximately 60, 75, 75, 80, 85, 90, and 250 kda, respectively (fig. 2) . mapping the epitopes of the pedv-pt by icc staining. as summarized in table 1 , the two nmabs, p4b-1 and e10e-1-10 and the non-neutralizing mab, r7h-2, all exhibited the ability to recognize full-length pedv spike-expressing hek293 cells but were unable to recognize pedv s 1-435 -expressing hek293 cells in icc staining. more precisely, p4b-1 recognized pedv s 1-639 expressing hek293 cells but did not recognize the hek293 cells expressing s 1-575 , s 1-509 , s 1-501 , s 1-485 , s 1-435 of pedv-pt (fig. 3) . in other words, p4b-1 was unable to bind to the amino acids upstream of a.a. 575 of the spike protein of pedv-pt. therefore, the targeting epitope of p4b-1 must be located between a.a. 575-639 on the pedv spike protein. surprisingly, e10e-1-10 exhibited good binding affinity for hek293 cells expressing pedv proteins s 1-639 , s 1-575 , s 1-509 , s 1-501 , and s 1-485 , but with reduced binding affinity for the pedv s 1-435 . this evidence obtained from the icc staining confirmed this nmab recognizing the novel epitope present within the 435-485 a.a. region of pedv-pt spike protein (fig. 3) . in contrast, the non-neutralizing mab, r7h-2, that recognized only the full-length pedv spike-expressing hek293 cells, was unable to bind to the pedv spike protein upstream of a.a. 639 (fig. 3) . these results suggest that the targeting epitope of r7h-2 is downstream of a.a. 639 of the pedv spike protein. elisa results showing reactivity of mabs to various truncated pedv s proteins. to further confirm the reactivity of each mab to the targeting epitopes, elisas were performed using plates coated with www.nature.com/scientificreports www.nature.com/scientificreports/ various truncated pedv spike proteins. the pedv nmabs (p4b-1 and e10e-1-10) and the non-pedv recognizing mab (unk-1) were all serially diluted to conduct the elisa. as shown in fig. 4 , p4b-1 had a binding affinity with appropriate dilution effects toward the purified full-length pedv spike protein, as well as pedv s 1-639 , but had its affinity towards purified pedv proteins, s 1-575 , s 1-509 , s 1-501 , s 1-485 and s 1-435 was weak. however, e10e-1-10, which targets a novel neutralizing epitope as shown with icc staining, was also capable of binding to the full-length pedv spike protein as well as truncated pedv proteins, s 1-639 , s 1-575 , s 1-509 , s 1-501 , and s 1-485 with appropriate dilution effects. similar to the results obtained by icc staining, e10e-1-10 showed no binding ability www.nature.com/scientificreports www.nature.com/scientificreports/ toward pedv s 1-435 by elisa and therefore, an nmab targeting a novel epitope of the new variant of pedv specifically at a.a. 435-485 in the s1 region was confirmed. the non-pedv recognizing mab, unk-1 showed no binding affinity towards the various truncations of the spike protein and hence, it was used as the external control for the elisa assay. binding affinity of nmabs to linearized epitopes. to further determine whether the binding of neutralizing epitopes require conformational integrity, immunodot blotting was performed by denaturing the various truncated pedv spike proteins. the conformational proteins as well as the denatured proteins of s 1-435 , s 1-485 , s 1-501 , s 1-509 , s 1-575 , s 1-639 , and the full-length spike of pedv-pt were dotted onto the membrane. as shown in fig. 5 (a-c), p4b-1 was able to detect the conformational s 1-639 and the full-length spike protein on the membrane but was unable to detect the proteins upstream of a.a. 575 of spike, consistent with the findings from the elisa and icc staining. the other nmab, e10e-1-10, could bind to s 1-485 , s 1-501 , s 1-509 , s 1-575 , s 1-639 and the full-length spike proteins in conformational structures. the r7h-2, a spike-recognizing but non-neutralizing mab, was only capable of binding to the conformational full-length spike protein. p4b-1, e10e-1-10, and r7h-2 were unable to stain the denatured target proteins on the membrane, indicating the loss of binding affinity after protein linearization. moreover, to ensure all the proteins were equally dotted on the membrane, the results of mab-stripping and re-probing with anti-v5 tag antibodies are shown in fig. 5 (d-f). a molecular model of recombinant pedv s ectodomain protein was established using swiss model and re-edited using ucsf chimerax molecular modelling system. as shown in fig. 6 , the monomer of pedv s protein was divided into s1 (a.a. 1-735, in light-blue) and s2 (a.a. 736-1378, in grey). the novel neutralizing epitope (a.a. 435-485 of the s protein), characterized by the e10e-1-10 nmab labelled in yellow in fig. 6(a) , was inferred to be at the c terminal region of the s1 a domain. the two mutations, v 441 i, and mab s 1-435 s 1-485 s 1-501 s 1-509 s 1-575 s 1-639 s www.nature.com/scientificreports www.nature.com/scientificreports/ s 477 a that were different from the cv777 strain were highlighted in red sphere; and the one mutation, e 459 q that was different from the taiwan historic pedv hc070225-s strain was highlighted with a green sphere. on the other hand, the region of the neutralizing epitope (a.a. 575-639 of the s protein; labelled in yellow) characterized by the p4b-1 nmab was deduced at the c-terminal region of the s1 b domain in fig. 6(b) . the a.a. mutation (g 596 s) different from the cv777 strain was highlighted with a purple sphere; and the a.a. mutations, d 608 e and l 615 f, different from the taiwan historic pedv strain hc070225-s were highlighted with pink spheres. the mutation, q 636 e that was different from both cv777 and taiwan historic pedv strain hc070225 was highlighted with an orange sphere. the six truncated as well as full-length spike protein of pedv-pt were coated into separate wells and probed with two-fold serially diluted mabs, p4b-1 and e10e-1-10. the non-pedv recognizing mab, unk-1, was added as the external control for elisa under the same dilution conditions. the two-fold serially diluted anti-v5 tag antibody was used as the standard to show the dilution effect of the binding affinity assay. the data is represented as the s/p ratio. the wells incubated with only secondary antibodies and the wells incubated with anti-v5 tag antibody at 1,250 ng/ml were used as negative and positive controls for s/p ratio respectively. the results of the elisa for the various truncations of the spike proteins are shown as follows: www.nature.com/scientificreports www.nature.com/scientificreports/ since 2010, outbreaks of infections caused by new pedv variants have been correlated to viral mutations in critical neutralizing epitopes of the spike protein 8, 20 . although several sequences of the new variant pedv spike genes have been decoded 6, 21 and multiple domain architectures of the pedv s protein have been predicted by three-dimensional structures of alphacoronaviruses 16 , the effects of genetic mutations on the antigenicity of pedv remains poorly understood. a thorough evaluation of the neutralizing epitopes and antigenicity of the new pedv variants is therefore needed. in the present study, we generated a panel of mabs using pedv-pt viral particles. by selecting mabs that exhibited high binding affinity to full-length spike ectodomain-expressing hek293 cells, we successfully identified two representative nmab against pedv-pt. the targeting epitopes identified by these two nmabs were located at a.a. 575-639 and a.a. 435-485 of the pedv spike protein, respectively, as shown by icc staining and elisa. further, none of these nmabs were able to bind to the linearized forms of the recombinant ectodomain of s protein of pedv-pt strain was modelled using swiss model and edited using ucsf chimerax. the s protein was present as monomer and the s1 and s2 regions of pedv were displayed in light-blue and grey, respectively. the conformational neutralizing epitope regions characterized by the mabs are given as follows: (a) e10e-1-10 mab labelled in yellow; two mutations (v 441 i and s 477 a) on the epitope different from the cv777 strain were highlighted in red sphere and one mutation (e 459 q) on the epitope different from the taiwan historic pedv strain (hc070225-s) was highlighted in green sphere. (b) p4b-1 mab was labelled in yellow; the g 596 s mutation on the epitope different from the cv777 strain was highlighted in a purple sphere; mutations, d 608 e and l 615 f, on the epitope different from the taiwan historic pedv strain (hc070225-s) were highlighted in pink sphere. the orange sphere represented the common mutation (q 636 e) noted on the epitope of pedv-pt s protein that was different from both of cv777 and taiwan historic pedv (hc070225-s) strains. (2019) 9:2529 | https://doi.org/10.1038/s41598-019-39844-5 www.nature.com/scientificreports www.nature.com/scientificreports/ the truncated spike proteins, indicating that they recognize the integral epitopes with appropriate conformation. taken together, we discovered a novel conformational neutralizing epitope in the s1 a domain at a.a. 435-485, whose function has not been clearly determined in coronaviruses. in addition, we also discovered a conformational neutralizing epitope close to the c-terminus of the coe domain, at a.a. 575-639. the neutralizing epitopes of the historic strains of pedv on the s protein that have been previously published include the co-26k equivalent epitope (coe epitope; a.a. 499-638 in the brl strain; a.a. 501-640 in the pedv-pt strain) 12 , 2c10 epitope (a.a. 1368-1374 in the cv777 strain; a.a. 1373-1379 in the pedv-pt strain) 13 , and s1d (a.a. 636-789 in the cv777 strain; a.a. 640-794 in the pedv-pt strain) 14 . in case of the new variants of pedv, the domain b (a.a. 510-640 in the pedv gdu and the pedv-pt strain) and domain 0 in the s1 region (a.a. 1-219 in the pedv gdu and the pedv-pt strain) are demonstrated to have neutralizing epitopes 17 . furthermore, okda et al. also discovered several linear epitopes at the n-terminus of s2 18 . previously, the identification of neutralizing epitopes of the historic pedvs led to the proposal that several single nucleotide polymorphisms (snps), such as three serine amino acid substitutions in the coe epitope and two serine amino acid substitutions in the s1d epitope, may be related to newly-occurring global outbreaks 6, 22 . in the present study, the nmab, p4b-1, was able to recognize the epitope within a.a. 575-639 on the s protein, which overlaps with the c-terminus of the coe epitope, echoing the findings of previous studies 12 . as the s1 b domain consists of the main coe neutralizing epitope of pedv and is the receptor binding domain for the majority of the coronaviruses, its importance has been emphasized in many studies 11, 23, 24 . li et al. have demonstrated that the binding affinity of neutralizing antibodies could be dramatically altered by exchanging one amino acid on the s protein of pedv 17 . hence, mutations in s1 b domain, especially in neutralizing epitopes of the pedv s protein, are speculated to represent the evolution of viral escape from antibodies, like the one evoked by cv777-based vaccine immunization of a historic pedv strain infection 4, 25 . interestingly, the other nmab, e10e-1-10, presented a high binding affinity toward a.a. 435-485 of the s protein that precludes the coe epitope and locates in the s1 a domain has also been identified in our study. to our knowledge, the function of the s1 a domain of the pedv s protein has not been well studied. the s1 a domain of human coronavirus hcov-nl63 is speculated to interact with heparan sulfate proteoglycans on the host cells to mediate the virus anchoring and infection 26 . the n-terminus of s1 a domain of bovine coronavirus has been shown to have two sugar binding loops which can recognize the carbohydrate receptor on the host cells 27 . moreover, heparan sulfate has also been demonstrated as an attachment factor for pedv 28 . however, as the actual topography of the pedv trimeric s protein remains to be solved and the function of s1 a domain is yet to be determined, the actual interaction of the s1 a domain targeting mab with these sugars or the heparan sulfate proteoglycans-binding domains on the pedv s glycoprotein remains to be further evaluated. visualization of the topology and interaction of pedv s glycoprotein with these neutralizing antibodies for guiding future immunogen and therapeutics design is an important future goal. epitopes for antibody recognition can generally be divided into two main classes: linear and conformational forms. linear epitopes are formed by a continuous sequence of amino acid in a protein, while conformational epitopes consist of amino acid sequences that are discontinuous in the protein but are brought together upon three-dimensional protein folding. it has been demonstrated that 90% of b cell-recognizing epitopes are conformational epitopes that result from the antigen internalizing process and special antigen-recognizing ability 29, 30 . many conformational epitope-targeting antibodies, including neutralizing antibodies, are particularly difficult to determine because the antibody-antigen complex is formed solely in the native structure of the protein. over the past several decades, with the use of phage-display peptide probing, the neutralizing epitope of cv777, named 2c10 has been identified 13 . using elisa or pepscan assays, several linear neutralizing epitopes have been discovered on the s2 glycoprotein subunit of new pedv variants 18 . additionally, two b cell epitopes, named ss2 and ss6, in the region of the s1d neutralizing epitope were evaluated using a combination of phage-display peptide probing and pepscan 14, 15 . however, these epitope mapping approaches focus mainly on linear epitopes 31 . using icc staining, elisa, and denatured immunodot blotting, all of the b cell-recognizing epitopes identified in the present study were found to be conformational epitopes. thus, these affinity-binding assays could serve as valuable platforms for studying the conformational epitopes of nmabs. in conclusion, the nmabs and various truncated s proteins constructed generated in the present study could contribute to a better understanding of the antigenicity and immunogenicity of highly virulent pedvs, especially in revealing the antigenic role of s1 a domain, and the pathogenesis of immune escape that leads to an outbreak of pedv. moreover, our study may provide important fundamental information for the future development of novel epitope-based vaccines. collection (atcc) no. crl-1586) as previously described 32 . after freezing and thawing, the harvested viral supernatant was centrifuged at 3000 rpm for 30 min to remove cell debris and filtered through a 0.22 µm ultrafiltration cup (corning, new york, usa). the viral supernatant was then added onto a 20% sucrose (sigma, missouri, usa) cushion, purified by ultracentrifugation at 75,000 × g for 2.5 h. the viral pellet was re-suspended in phosphate buffered saline (pbs) (gibco, gaithersburg, usa), and then applied to a 20-60% sucrose-tne (20 mm tris-hcl (ph 7) (sigma), 100 mm nacl, 2 mm edta (sigma)) gradient, and centrifuged at 75,000 × g for 2.5 h in an optima ™ l-100xp preparative ultracentrifuge using an avanti j-25 rotor (beckman coulter, sykesville, usa). purified virions were diluted in tne buffer, pelleted by centrifugation at 75,000 × g for 1.5 h to remove the sucrose and then, re-suspended in tne buffer. www.nature.com/scientificreports www.nature.com/scientificreports/ mab production. three balb/c mice were intramuscularly (im) immunized with 20 μg purified pedv viral particles mixed with 100 μl complete freund's adjuvant (sigma). after two weeks, two im booster injections were administered using 20 μg purified pedv viral particles with 100 μl incomplete freund's adjuvant (sigma) at intervals of 3 weeks. three days before sacrifice, mice were immunized with 20 μg purified pedv viral particles in pbs (gibco) via intrasplenic (is) injection. serum antibody titres at each immunization were monitored using a complete pedv viral particle elisa and the mouse with the highest titre was sacrificed for hybridoma preparations. hybridoma preparation. splenocytes were isolated from the mice immunized with purified pedv particles. after gentle washing with brief centrifugation, splenocytes were fused with sp2 myeloma cells at a cell ratio of approximately 10:1 using 50% polyethylene glycol (sigma). hybridomas were seeded onto 96-well culture plates in rpmi-1640 medium supplemented with 20% foetal bovine serum (gibco), 100 mg/ml streptomycin, and 100 iu/ml penicillin (sigma), and incubated overnight at 37 °c in a humidified incubator with 5% co 2 . after incubation, approximately 50% medium was removed from each well, and a selective hat rpmi-1640 medium (hat-rpmi) (sigma) was added to achieve a final concentration of 20% foetal bovine serum (gibco), 100 mg/ml streptomycin, 100 iu/ml penicillin, 100 mm hypoxanthine (sigma), 400 mm aminopterin (sigma), and 16 mm thymidine (sigma). wells containing growing hybridoma cells were screened for antibody production by icc staining using pedv-infected vero cells or hek293 cells (atcc crl-1573 ™ ) expressing the full-length pedv s protein. positive clones were isolated for limiting dilution and incubated in selective ht rpmi-1640 medium without aminopterin. after two limiting dilutions, the supernatant from each line was further tested for anti-pedv s-specific antibodies by icc staining using pedv-infected vero cells or hek293 cells expressing the full-length recombinant pedv s protein. mab purification and quantification. to purify mabs from cultured supernatants, pierce ™ protein l magnetic beads (thermo fisher scientific, waltham, usa), which selectively bind to mouse immunoglobulin were utilized following the manufacturer's instructions. the beads were mixed with 40 ml supernatants of each mab and incubated for 1 h after which the antibody-bound beads were collected using a magnetic stand. after washing thrice wash buffer (tris-buffered saline (tbs), 0.05% tween-20 detergent), the mabs were eluted with 60 μl elution buffer (0.1 m glycine, ph 2.0) for 10 min and then, alkalized pbs buffer (ph 8.5) was added for neutralization. the concentrations of each purified mab were determined by the pierce ™ bca protein assay kit (thermo fisher scientific). of the full-length pedv s protein of the taiwan pedv-pintung-52 (pedv-pt) strain (genbank: ky929405.1) was performed as previously described 33 . to prepare recombinant truncated pedv s proteins, regions of the s gene coding for amino acids 1-435 (s 1-435 ), 1-485 (s 1-485 ), 1-501 (s 1-501 ), 1-509 (s 1-509 ), 1-575 (s 1-575 ), 1-639 (s 1-639 ) were amplified using specific primers (supplementary table s1 online) and the resultant proteins were purified as described previously 19 . the amplicons encoding different truncated genes were subcloned into a bamhi-noti restriction site in the pcdna tm 3.1/v5-his topo ® vector (invitrogen, carlsbad, ca, usa), transfected into hek293 cells and selected by using 750 μg/ml geneticin (g418, gibco). after two weeks of selection, cells stably expressing the truncated pedv s proteins were subjected to icc staining and western blotting. purification and western blot detection of truncated spike proteins. large-scale purification of the truncated s proteins of pedv-pt was performed as previously described 19 . the protein expressing hek-293 cells were harvested, resuspended, and cultured in freestyle 293 expression medium (gibco) for one week. after supernatant collection and removal of cell debris through a 0.22 μm filter, the proteins were banded with hispur cobalt resin (thermo fisher scientific) following the manufacturer's protocol. the purified proteins were subsequently concentrated with a 30 kda vivaspin ® 20 (ge healthcare life sciences, ny, usa), complete ™ edta-free protease inhibitor cocktail (roche molecular biochemicals, quebec, canada) was added and the concentrations were measured using the pierce ™ bca protein assay kit. the purified proteins were denatured in a buffer containing nupage ® lds sample buffer and nupage ® reducing agent, and heated for 5 min at 95 °c. using bio-rad mini-protein ® electrophoresis system(bio-rad, hercules, ca, usa), the samples were separated using 10% sodium dodecyl sulfate (sds)-polyacrylamide gel electrophoresis (page) cast in a gradient t-pro ez gel solution (t-pro biotechnology, taiwan) according to the manufacturer's protocol. following this, proteins were wet-blotted onto a polyvinylidene difluoride (pvdf) membranes (bio-rad)blocked with 5% skim milk in tris-buffered saline with 0.05% tween 20 (tbs-t) buffer at room temperature (rt) for 1 h. membranes were incubated with a 1:5000 diluted anti-v5 tag antibody for 1 h at rt. after washing with tbs-t, horseradish peroxidase (hrp)-conjugated goat anti-mouse igg secondary antibody (1:10,000 dilution in blocking buffer; jackson immunoresearch laboratories, philadelphia, usa) was added and incubated at rt for 1 h, the signals were visualized using clarity ™ western ecl blotting substrates (bio-rad) and detected with a chemidoc ™ xrs+ imaging system (bio-rad). neutralizing assay for the pedv-pt strain. a neutralizing assay was conducted as previously described with some modifications 32 . next, 100 μl suspended vero cells (atcc no. crl-1586) were seeded onto 96-well culture plates at 3 × 10 5 cells/ml and incubated overnight at 37 °c in a humidified incubator with 5% co 2 until cells reached 90% confluence. purified mabs were two-fold serially diluted from 20 μg/ml to 2.5 μg/ml in post-inoculation medium (pi medium) containing dulbecco's modified eagle medium (gibco) supplemented with tryptose phosphate broth (0.3%) (sigma, missouri, usa), yeast extract (0.02%) (acumedia, ca, usa), and 10 μg/ml trypsin (gibco). fifty microliters of diluted culture supernatant from each hybridoma clone were mixed and incubated with an equal volume of 100 tcid 50 /ml pedv-pt passage 5 (pedv-pt-p5) at 37 °c in 5% co 2 porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis journal of veterinary diagnostic investigation: official publication of the american association of veterinary laboratory diagnosticians a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea phylogenetic analysis of the spike (s) gene of the new variants of 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virus spike protein are key targets of neutralizing antibodies the s2 glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes efficacy of heat-labile enterotoxin b subunit-adjuvanted parenteral porcine epidemic diarrhea virus trimeric spike subunit vaccine in piglets antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains new variant of porcine epidemic diarrhea virus evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains receptor recognition mechanisms of coronaviruses: a decade of structural studies structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies epidemiology and vaccine of porcine epidemic diarrhea virus in china: a mini-review glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy crystal structure of bovine coronavirus spike protein lectin domain porcine epidemic diarrhea virus uses cell-surface heparan sulfate as an attachment factor mapping epitope structure and activity: from one-dimensional prediction to four-dimensional description of antigenic specificity continuous and discontinuous protein antigenic determinants prediction of residues in discontinuous b-cell epitopes using protein 3d structures evaluation and comparison of the pathogenicity and host immune responses induced by a g2b taiwan porcine epidemic diarrhea virus (strain pintung 52) and its highly cell-culture passaged strain in conventional 5-week-old pigs display of porcine epidemic diarrhea virus spike protein on baculovirus to improve immunogenicity and protective efficacy ucsf chimera-a visualization system for exploratory research and analysis washing six times, 50 μl of abts ® peroxidase substrate system (kpl, seracare) was used to obtain a positive signal. the coloration procedure was stopped after 20 min by adding 50 μl stopping solution (kpl, seracare). the signals were detected at 405 nm wavelength by emax plus microplate reader (molecular devices, crawley, uk). the final data is shown as a sample to positive ratio (s/p ratio), representing the difference between the od values of the sample and the negative control divided by the difference between the od value of the positive and negative controls. the od value of the positive control was obtained from the result of 80,000x diluted anti-v5 tag antibody, and the od value of the negative control was obtained from the result of the secondary antibody only wells.linear epitope mapping by immunodot blotting and western blotting. to confirm the conformational significance of the neutralizing epitopes, we also denatured the six purified truncated spike and the full-length spike protein of pedv-pt by mixing them with nupage ® reducing agent (thermo fisher scientific) and heating for 5 min at 95 °c. the purified proteins and the denatured proteins were separately dotted on nitrocellulose membranes (merck millipore, ma, usa) at 50 ng. the reducing agent mixed with water was also dotted on the membrane as a negative control. following a 1-h block in 5% skim milk, the membranes were stained with nmabs (p4b-1 and e10e-1-10) and non-neutralizing mab (r7h-2) at 2.5 μg/ml diluted in tbs-t buffer at rt for 1 h. after washing three times, hrp-conjugated goat anti-mouse igg secondary antibody (1:10,000 dilution, jackson immunoresearch laboratories) was added and incubated at rt for 1 h. protein signals were visualized using the clarity ™ western ecl blotting substrates (bio-rad) and detected with a chemidoc ™ xrs+ imaging system (bio-rad) as previously described. to ensure the denatured proteins were correctly dotted on the membrane, after the first run of probing, the mabs were stripped with stripping buffer (thermo fisher scientific) and the membranes were separately re-probed by anti-v5 tag antibodies (invitrogen). a full-length homology model of the ectodomain of recombinant pedv s protein was generated by swiss-model and built with promod3 version 1.0.0 (https://swissmodel.expasy. org). the manipulated protein sequence of the pedv s ectodomain was constructed using the trimeric human coronavirus nl63 spike structure (pdb accession no. 5szs) as a template. the images were edited using ucsf chimerax molecular modelling system 34 . the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-39844-5.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-298685-qxkxjxsz authors: pensaert, maurice b.; martelli, paolo title: porcine epidemic diarrhea: a retrospect from europe and matters of debate date: 2016-12-02 journal: virus research doi: 10.1016/j.virusres.2016.05.030 sha: doc_id: 298685 cord_uid: qxkxjxsz abstract a retrospect is given on the emergence of porcine epidemic diarrhea (ped) during the early seventies in europe. while, at first, it appeared as a disease affecting feeder pigs, fatteningand adult swine, it later also became pathogenic for neonatal and suckling pigs hereby drastically increasing its economic impact. isolation of the causative virus revealed a new porcine coronavirus, the origin of which has never been clarified. pathogenesis studies with the prototype strain cv777 showed severe villous atrophy in neonatal pigs and the virus-animal interactions showed many similarities with transmissible gastro-enteritis virus (tgev), another porcine coronavirus. disease patterns in field outbreaks showed muchvariation but, while farm related factors played a role, possible genetic variations of virus strains in europe have not been examined and are thus unknown. cv777 in experimental pigs caused diarrheal disease and mortality rates similar to those later encountered in asia and more recently with the “original” us strains even though genomic typing of the prototype european strain have shown that it belongs to the s-indel strains. in europe, ped has become endemic during the eighties and nineties and subsequently regressed so that, after 2000, swine populations in many countries have largely become seronegative. sporadic outbreaks have recently reappeared showing a large variety of clinical outcomes. one" or also "tge2" and both these denominations referred to its clinical similarity to tge, a common cause of viral diarrhea in pigs in europe at that time. an important difference with tge was, however, that neonatal pigs were not affected. since these denominations were considered unsatisfactory from a scientific point of view, the name of the new syndrome was quickly changed to "epidemic diarrhea-ed". the first ed outbreak occurred on a farm in the spring of 1971 and the second 6 months later, at a distance of 2 miles from the http://dx.doi.org/10.1016/j.virusres.2016.05.030 0168-1702/© 2016 elsevier b.v. all rights reserved. former. suckling piglets were not affected but pigs of 10 weeks and older and also adults showed an acute diarrhea lasting one week. the outbreak lasted 3-4 weeks on the farm. during the autumn of 1971 and also the following winter, several new outbreaks were reported. a clinical diagnosis and possibility for differentiation from tge was thus based on the high morbidity in fatteners and adult animals in the absence of disease in neonatal and freshly weaned pigs. in most of these diarrheal cases, tgev was excluded by laboratory examination. the fact that ed was observed on farms with a recent history of a tge outbreak, and thus in tgev immune animals, increased the conviction that tgev was not involved. during 1972, ed spread rapidly between pig farms, particulary fattening herds. mortality was rare and the effect of an outbreak was estimated at about 2 weeks feed cost. a similar disease pattern was observed during the early seventies in belgium and a rapid spread occurred to neighbouring countries in western europe. here also, suckling pigs were not affected and remained free of diarrhea even when their mothers suffered from a watery diarrhea during several days. some neonatal pig mortality could occur by starvation because the sick mothers often suffered from agalactia. an additional sign observed in belgium and later also reported in gemany, but not mentioned in england, was that some fatteners were found dead, particularly towards the end of the fattening period, and this occurred repeatedly in some farms but not in others. a mortality rate as high as 3% could be encountered. this was not due to dehydration accompanying the diarrhea but animals suddenly died from acute back muscle necrosis. while the clinical link with an ed infection was clear, the pathogenetic background has never been revealed. belgian pigs were highly stress positive at that time and a severe belly ache often observed in adult animals during an outbreak of ed may have been a trigger. in general, as no baby pig mortality occurred due to the ed agent, not very much attention was given to this new diarrheal syndrome and the etiology was not intensively investigated. however, it was assumed that a viral agent was involved since bacteriological examination of faecal material did not reveal a specific bacterial cause. none of the known porcine viruses could be associated and a new virus was, therefore, suspected. much changed in 1976 when wood (1977) from the veterinary investigation centre in norwich (england) described a new diarrheic syndrome. it differed from ed in that it now affected pigs of all ages, including neonatal and suckling pigs. mortality was variable, restricted to young piglets and averaged around 30%. this new disease now resembled tge more closely than ed did, but tgev was again excluded using direct immunofluorescence on intestines and applying established serological techniques available for detection of anti-tgev antibodies. now, a differentiation with tge on a clinical basis only became difficult, often impossible. this new syndrome was called ed2 to differentiate it from the 1971 ed1 where no baby pigs were involved. ed2 was economically much more important than ed1. ed2 also quickly spread to the european continent and was recognised in belgium in 1977. reports from other countries including the netherlands, germany, france, bulgaria, hungary and switzerland, followed soon. in belgium, mortality rates in neonates on breeding farms varied considerably. they could be as high as 80% (large variation from 30 to 80%) and the average was 50%. variation in mortality in neonatal pigs was litter bound, not explained at that time, but also farm bound where it appeared to be associated with farm size (still many small family farms at that time), farm struc-ture (one or several farrowing units), number of neonatal pig litters present at the start of the outbreak, number of pregnant sows due to farrow within one week of the appearance of disease signs and possibly other factors. differences in virulence of virus isolates was not given any attention. this new evolution to ed2 with the involvement of baby pigs and its larger economic impact yielded better opportunities for collecting material for etiological studies, for experimental reproduction of the disease and for the development of virological and serological techniques. in 1978, chasey and cartwright (1978) reported the detection of virus like particles, and pensaert and debouck (1978) described the isolation of a new coronavirus-like agent (cvla) from diarrheic pigs, with both research groups succeeding in reproducing diarrhea in experimental pigs. soon after the isolation of this new coronavirus, extensive pathogenesis studies were performed in colostrum deprived pigs with one of the belgian isolates, designated coronavirus cv777, (isolated in month 7 of 1977) which became the prototype strain for pedv in europe (debouck and pensaert, 1980; debouck et al., 1981) . ed2 was soon named "porcine epidemic diarrhea" (ped) caused by ped virus (pedv) a denomination which still stands at present. from the early studies with pedv in neonates (debouck and pensaert, 1980; debouck et al., 1981) it was soon clear that the pathogenesis resembled very much that of tgev. experience gathered from research with tgev helped much in the approach to study this new enteric disease. lack of success to cultivate the virus in cell cultures forced to produce clean virus stocks by oral inoculation of colostrum deprived pigs, performing surgery 18 h later and rinsing the in vivo produced virus from the lumen of the infected small intestines during 12 h while keeping the pig in the incubator (debouck and pensaert, 1980) . such "clean" pig adapted virus stocks served for experimental pig inoculation experiments and to produce an hyperimmune serum for the preparation of a conjugate for an immunofluorescence (if) conjugate to detect the virus in tissues. serological tests were developed to detect antibodies by elisa and to study possible relationship with other coronaviruses by immuno-electron microscopy . genome analysis of the ped isolate(s) was not available at that time. by immuno-electron microscopy and if, pedv was not related to any of the known porcine coronaviruses (tgev, haemagglutinating encephalo-myelitis virus) . some discrete relationship with members of the genus alpha-coronavirus was later demonstrated using other and more sensitive tests. the origin of pedv was thus unknown and no potential parent coronavirus could be indicated. an elisa test was soon used for routine serology . a crucial question was whether or not the ed1 agent and ed2/pedv were related or whether ed2/pedv was totally new. infectious material containing the ed1 agent from earlier outbreaks was not available. a retrospective serological survey was carried out on sow sera that had been collected in slaughterhouses in belgium starting in 1969 and thus prior to the emergence of ed1 in 1972 on the european continent. antibodies to pedv were not found in sera collected in 1969 but were present in 7% of the sows collected in 1971, in 42% of the sows in 1975 and in 32% of the sows in 1980. these results indicated that the coronavirus ped had been responsible for first ed1 outbreaks, for the ed2 outbreaks and thus it can be accepted that pedv emerged in 1971 but later widened its host tropism from growing and adult swine towards neonatal pigs. this finding was interesting from an evolutionary point of view. thus, pedv that presumably started as a cause of diarrhea in 1971 in feeders, fatteners and adult swine, had suddenly acquired tropism for neonatal pigs and now became a rather devastating disease. but even after the emergence of ed2/pedv, some outbreaks on breeding-finishing farms still did not involve neonatal pigs. while it was assumed that both ed1 and pedv were co-circulating in the swine population, it is also possible that some farms had experienced an earlier ed1 infection and that immune sows protected their offspring against pedv by lactogenic immunity while groups of fattening pigs had become susceptible. it must, however, be mentioned that cross-protection between ed1 and pedv has never been studied. also, after a first epidemic phase of the new pedv, the virus often persisted on breeding-finishing farms in weaned and feeder pigs (endemic ped). the sow population was immune, protecting its offspring, while feeder pigs became susceptible after loosing their maternal protection. the highly variable and mixed clinical picture was, at that time, ascribed to the possible co-circulation of the original ed1 agent and its presumed variant pedv in the population. genome analysis was not available and ed1 infectious material is also now no longer available to retrospectively examine this issue. ed2/pedv is likely a variant of ed1. that variants of pedv relatively easy emerge is not unusual as animal coronaviruses are known to easily undergo genetic alterations. recombination and insertions and deletions have repeatedly been demonstrated in pedv by genome analyses of isolates during more recent outbreaks in asia and in the usa (fan et al., 2012; li et al., 2016; jarvis et al., 2016; vlasova et al., 2014; oka et al., 2014) . even now (2016) in recent cases of ped in europe, varying types of clinical manifestations, either with or without affection of neonatal pigs, are observed (see later). the question on the origin of pedv (and thus of its presumed ancestor ed1 agent) in 1971 is unanswered. there are no indications for a possible evolution from another known so called "parental" coronavirus, even after comparative studies with the known coronaviruses using detailed genome analyses of different genes including the s gene. so far, only discrete antigenic relationship involving the n protein but without any cross protection was detected with some of the other animal members of the genus alphacoronavirus such as feline infectious peritonitis virus (fipv), tgev, porcine respiratory coronavirus (prcv), canine coronavirus (ccov) and mink coronavirus (mcv). by the use of monoclonal antibodies to the n proteins of the human alphacoronaviruses nl63 and 229e, no cross reactivity was detected with pedv (sastre et al., 2011) . the only alphacoronavirus in which also the m proteins cross reacts with pedv is mcv (have et al., 1992) . the nucleotide sequence of the pedv nucleocapsid gene and of typical coronavirus motifs show that pedv, within the region of the genome sequenced, shows indeed greatest homology to the human 229e, tgev, prcv, fipv, ccov and feline enteric coronavirus (fecv) (bridgen et al., 1993) . it is interesting to mention that, similar to pedv, several of the other alphacoronaviruses, including the human 229e, tgev, prcv, ccov, fecv and fipv use the cellular receptor aminopeptidase n (apn) for virus entry into cells in their host (weiss and navas-martin., 2005) and this seems to be a common evolutionary characteristic. still, the genetic and antigenic diversity between pedv and the other alphacoronaviruses is very largealso, no cross-reactivity has been reported between pedv and the coronaviruses belonging to the beta, gamma or delta genera.the genomic data presented above and the use of the same cellular receptor suggest a common origin of some of some of these alphacoronaviruses. a carrier-wild animal species as source of the virus, as often described with other coronaviruses, cannot be excluded. soon after its detection, experimental studies in neonatal pigs revealed that target cells of pedv were limited to the epithelium covering the intestinal villi and the pathogenesis was thus highly similar to that of tgev (debouck and pensaert, 1980; debouck et al., 1981) . cv777 virus infection in the villous enterocytes in neonatal pigs caused rapid cell desquamation throughout the small intestine within 24-36 h after inoculation which was somewhat slower than observed with tgev. still, the villous atrophy induced by pedv was so abrupt and extensive that rapid and severe dehydration occurred leading to death in neonatal pigs. due to this similarity in pathogenesis with tgev, much of the scientific knowledge acquired on tge diagnosis, −immunity, −prevention could be almost invariably applied to ped. an apparent difference with tgev, probably of minor importance from a clinical point of view, was that epithelial cells on colonic villi were also infected but desquamation was not observed. still now, it is a question if this colonic site of replication contributes to disease severity. ped diarrhea in fatteners and sows is often accompanied by an apparent belly ache, a clinical sign not seen with tge, and the question arises if the colon infection may contribute to this clinical manifestation. results of pathogenesis studies obtained in caesarean derived, colostrum deprived neonatal pigs upon inoculation with the european prototype strain cv777 of the seventies, were practically identical to those observed more recently in asia and in the usa epidemics with the so-called "original us pedv strains" stevenson et al., 2013; kim and chae, 2003) . a point of debate in the pedv evolution, particularly since its occurrence in asia and its emergence the usa, is the arising of pedv genetic variants influencing virulence. the history in europe, here presented, allows to assume that ed2/pedv was a variant of ed1 which had acquired tropism for intestinal enterocytes in neonatal pigs. this new tropism expanded and increased the virus virulence since a vulnerable age became affected and piglets mortality became an important economic aspect of the disease. currently, two major pedv variants are described in the usa upon routine genome analyses of usa isolates. the first, also called "original us pedv ", appears to be "highly virulent" while the second, the so called s-indel strains, standing for insertions and deletions in the s gene of the virus, are associated with mild(er) clinical outbreaks. similar genotypic variants have been detected in asia, the s-indels already before 2010 and the highly virulent since 2010. when adopting this genomic identification, it appears that cv777 is classified as a s-indel isolate apparently belonging to a different cluster compared to the us indels (carvajal et al., 2015) . considering the pathogenesis and virulence of the european prototype strain cv777 of the seventies as evaluated by the sites and degree of replication and the degree of villous atrophy, no real difference exists with the more recent highly virulent (original us pedv) isolates from the usa. for example, the pig adapted c777 when experimentally inoculated in neonatal piglets, caused villous atrophy with villous length reduction from the normal value of 700-900 m to as low as 200-300 m throughout the small intestine and within 6-36 h after the start of the diarrhea coussement et al., 1982) . much depends on how virulence of pedv is determined. if virulence of a pedv isolate is considered merely from the point of view of virus-neonatal pig interaction with parameters such as duration of incubation period, rapidity and severity of enterocyte desquamation, degree and extent of atrophy of villi, production of virulent virus quantities and severity of diarrhea, then cv777 can be classified as highly virulent despite its identification with s-indel isolates. that s-indels isolates do not systematically mean low virulence was recently shown in a us study (chun-ming et al., 2015) in which 4 litters of 3-4 days old suckling pigs were inoculated with the s-indel iowa 106 strain in the presence of their ped negative mothers. the severity of clinical signs and the mortality of the pigs varied between the 4 liters (from 0% to 75%). severe clinical signs were observed in 2 of the 4 litters. two of the 4 sows developed diarrhea. it was observed that, despite similar background of sows and environment in this experiment, the severity of disease was rather variable. it appeared that the pigs' body weight at birth and the sows health conditions and lactation were influential factors. in the same experiment mentioned above (chun-ming et al., 2015) , one litter was also inoculated with an original us pedv strain of high virulence. it was concluded that virulence of the s-indel isolate was generally lower based on the longer incubation period, the shorter duration of diarrhea, more limited regions of virus infection, overall lower pig mortality (18% vs 55% for "original") and some other additional parameters. the sites and extent of the deletions or insertions and the seqence differences in the s gene may play an important role. in a recent publication (chen et al., 2016 ) the pathogenicity differences between 3 u.s.pedv prototype strains and a s-indel-variant stain were compared in conventional neonatal pigs under experimental infections and enteric disease, as evidenced by clinical signs,fecal virus shedding, gross and histopathological intestinal lesions, were significantly lower for the s-indel strain.however,the molecular basis for the virulence differences were not elucidated. since the early beginning in europe, it was clear that ped disease can show much variability even in different litters of pigs particularly when suckling their mother. such differences and the high variation in pig mortality in different litters (from 30 to 80%) was an observation also made in the seventies in europe when the first epidemic occurred and the reason was never unravelled. even more variability is experienced when virulence and severity of ped disease is related to the interaction virus-farm population and thus in field outbreaks. the result of a ped outbreak will be much more difficult to predict, to evaluate and to define since, next to possible virulence differences of the isolates and next to variation among litters in suckling pigs, many additional factors play a role in determining the clinical outcome of the infection. they include immune status of sows, dose of virus exposure on the farm, herd size and pig farming management and others, all of which may be interacting in a different way. moreover, the procedures applied for intentional infection (feed back) of the sow population to speed up the induction of immunity to be passively transfered to the litter could be considered as a potential cause of worsening of the clinical status of the suckling pigs. in fact, that practice can also be a source of other pathogens for gilts/sows and/or for newborn piglets. it is thus possible that, particularly in a fully susceptible pig population and even with pedv strains of similar virulence, the mortality rates and losses are much higher in some continents or regions or farms with extensive and highly industrialised pig farming. the overall health status of the population apparently also plays an important role. while genomic changes surely will occur in ped virus isolates, it is advised to be careful when associating them with virulence changes. when a different clinical picture is observed on farms, it is often too hastily concluded that variants with varying virulence have arisen based on genome analysis only and without testing for virulence factors in experimental pigs. while genome analysis is certainly useful and may be directional, repeated comparative animal inoculation experiments with so called new isolates, clinically denominated as candidate "virulence-variants", need to be carried out in a standardized way before solid conclusions about virulence are made. this is indicated by the large variations very often observed with one and the same isolate. the neonatal, non suckling pig, preferably colostrum deprived, is reliable and even essential for this purpose. parameters as duration of incubation period, a timewise follow up of site and degree of villous affection in the small intestine are needed and must be repeated before calling a pedv isolate a variant with impact on virulence. it should be stressed that pig adapted virus strains should be used as it is known that major genomic modifications can arise when pedv is cell culture passaged, as well in porcine as in non-porcine cells, such as in vero cells. the epidemiology of ped in europe has been and still is quite puzzling. pedv outbreaks in the late seventies and early eighties occurred both on breeding and fattening swine farms. acute outbreaks with neonatal pig mortality were encountered in the breeding-fattening farms which became infected for the first time. pedv often became endemic. in farrowing-finishing farms, successive groups of pigs became infected upon weaning and after losing their lactogenic protection from their immune mothers, so that the virus could persist. whether the virus persisted or not after the original outbreak was somewhat unpredictable, as it could also disappear from the farm. the farm size (number of sows) and its structure (number of units) played a role. also ped persistence regularly occurred in fattening farms using the system of continuous introduction of feeder pigs originating from numerous and different breeders. a typical case of persistent diarrhea caused by pedv lasting 10 months on a breeding-finishing farm was described in the netherlands (pijpers et al.,1993) and this was a feature regularly observed in europe in the eighties. recent experience in the usa (2013) (2014) (2015) , has shown that management practices adopted in the epidemic phase of the infection can turn ped to a endemic/enzootic and long lasting form (jung and saif, 2015) . pedv infections were a regular cause associated with viral diarrheal picture in weaned and feeder pigs. in a serological study in belgium in 1986, pedv was associated with diarrhea in 13 out of 16 groups of feeder pigs after arrival in fattening farms (callebaut et al., 1986) but, the virus remained prevalent in the swine populations of western europe during the eighties. a serosurvey in belgian sows using sera collected in slaughterhouses, and thus mostly originating from different farms, showed pedv antibodies 32% in 1980 and 19% in 1984 . similar percentages of sows were positive in germany (on 3 regional locations), france, spain, the netherlands and bulgaria while no antibodies were found in scandinavia, northern ireland, usa or australia . in 1982, antibodies were detected in sera received from taiwan (the first evidence of the presence of pedv in asia). as the eighties advanced, fewer outbreaks on breeding farms were seen even though the virus was still detected but the general economic impact of ped had become lower. in belgium in 1992, 17 groups of feeder pigs from 15 commercial finishing herds, using the all in-all out production system, were examined for serocoversion to pedv and tgev. none of the groups seroconverted to tgev while 7 seroconverted to pedv with diarrhea observed in all 7 (van reeth and pensaert, 1994) . in an hungarian study published in 1996, 5.5% of 92 faecal samples from weaned pigs with diarrhea tested positively for pedv (nagy et al., 1996) . during the nineties, an acute ped outbreak which was described in spain involving a fattening unit of 5000 pigs with diarrhea starting in 7-9 weeks old pigs in one barn affecting pigs from 20 to 90 kg and subsequently spreading to the other barns (carvajal et al., 1995) . an isolated outbreak was described, in 1998 in england, in a large finishing herd where weaners were brought in over a 2 month period and positive sows were found in the breeding herds supply-ing the weaners (pritchard et al., 1999) . but, no further epidemic of ped occurred despite a very low pedv seroprevalence as only 1.9% of fatteners from 64 different finishing units were positive for antibodies to pedv(may 1996 -january 1997 . interest from a disease and economic point of view became very low in europe and no further research was performed on ped. practically no serosurveys were carried out. a serological survey in sows from farrow to finish herds carried out in belgium in 1996 revealed that gilts were positive for pedv antibodies in only 2 of the 144 considered farms, and in 1997, 72 fattening farms were examined for pev antibodies and none were positive (pensaert, unpublished) . it appeared that pedv, except for a focal case, was disappearing from the european swine population towards the turn of the century. for this reason, no attention or follow up was given anymore to this viral infection while its field of interest had fully moved to asia. however, a somewhat atypical ped outbreak occurred unexpectedly in the po valley in northern italy in 2006, (martelli et al., 2008) . it occurred between may 2005 and june 2006 in an area densely populated with pigs. the outbreak started with four cases occurring in fattening farms from may to july. no clinical cases were detected during august and september. in october, two new cases appeared: the first in a fattening unit and the second in the nursery of a three-site production unit. the disease spread during the winter of 2005-2006, affecting more than 60 farms including fattening units as well as farrow-to-finish or farrow-to-weaner farms. some pedv positive farms were still detected between mid-2006 and the end of 2007, but the disease progressively disappeared (sozzi et al., 2014) . from 2008-2014, only sporadic outbreaks were observed in grower and finisher herds (efsa, 2014) . this epidemic in italy in 2006-2007 inclined us to forecast a new episode of ped epidemics in europe but it did not occur. recently and due to an increased attention following the 2013 epidemic in the usa, single or limited ped outbreaks have sporadically been diagnosed in europe. one case in ukraine, (dastjerdi et al., 2015) occurred in a 5000-sow farm (240 farrowings a week) and mortality in pigs less than 10 days old approached 100%. the virus was closely related to "original us" strains reported form north america (sequence identity of 99.8%). isolates from other cases reported from belgium (theuns et al., 2015) , holland (van der wolf et al., 2015) , france (grasland et al., 2015) , germany hanke et al., 2015) and portugal (mesquita et al., 2015) and italy were, on the basis of genetic sequence, closely related to each other. when sequenced, they were classified as s-indel strains, and the german isolate showed 99.4% identity to the oh851 strain isolated in the usa in january 2014 . the size and clinical disease in these outbreaks were very variable. the outbreak involved in belgium: 1 fattening farm (no mortality), in france: 1 farrow to finish farm (mortality 12% in pigs at one week and 25% at weaning), in germany: 4 farms (2 fattening with 1.5% and 2% mortality, 2 breeding with 70% and no mortality, respectively) and in portugal where it started in one farm (with pig mortality, but not further defined) and where the virus spread to 43 other pig farms during a period of 3 months. from these data, it can be seen that, again, there was much and unexplained variation in ped clinical disease and outcome. except for the possibility of the outbreak in ukraine, it is very doubtful that the other european isolates have anything to do with those involved in the us epidemic. similar focal cases must have occurred in europe before the us outbreak but were, most likely, neither recognized nor diagnosed nor reported as ped. s-indel strains have been present in europe as cv777 appears to be the earliest known representative (carvajal et al., 2015) . it is remarkable that, in many european countries, no large epidemic occurred despite several indications that the breeding population in their densely populated swine raising regions is negative for antibodies to pedv and thus presumably fully susceptible. it is difficult to understand why a virus such as pedv has gradually regressed in the swine population in europe in the absence of any special control measures. vaccination has not been applied and no control programmes have been installed. the puzzling aspect is that, during the last decennia, pedv was and still is focally present in europe and did not cause an epidemic despite the high numbers of susceptible-seronegative farms and despite the very dense swine populations in some regions. one would expect that a virus such as pedv, which replicates to very high titers in swine and which can easily and rapidly spread from one swine farm to another, would be able to maintain itself in the swine population. as previously explained, once an outbreak has occurred on a breeding farm, pedv virus could persist easily when successive litters of pigs, after losing their lactogenic protection at weaning, become a susceptible target for infection. in fact, persistence for a virus such as pedv would be almost as a "natural" feature similar to the endemic character observed with other porcine enzootic enteric viruses such as swine rotaviruses, swine enteroviruses and others. tgev cannot serve as an example here because, in europe, it has largely been eliminated from the swine population due to the emergence, in the early eighties, of the closely related porcine respiratory coronavirus (prcv). prcv is a tgev deletion mutant which has acquired respiratory tropism and shows an epidemiological advantage of rapid aerogenic spreading while causing a protective immunity to tgev. endemic prcv has thus "replaced" tgev in europe. it would be interesting to study the mechanisms behind the regression/waning of pedv in europe. could it be that the virus has a non swine ancestor which has temporarily become adapted to swine but which is not really swine-borne? such evolution would not be unique for animal coronaviruses. could it be that the virus can maintain itself in the population only when present at a sufficient high dose allowing it to continue the infection chain but once reaching a low level quantity,e.g on a farm basis, is not longer able to do so? the waning of pedv has apparently not occurred in parts of asia within its 2-3 decennia of presence on that continent to the same degree as it did in europe, and it will be intriguing to closely follow the epidemiological course and evolution of pedv in the usa, once the epidemic phase has passed. surveillance and control of ped coronavirus in pig in italy seqence determination of the nucleocapsid protein gene of the porceni epidemic diarhoea viris confirms that this virusis a coronavirus relatyed to human coronavirus 229 e and porcine transmissible gastroenteritis virus enzyme-linked immunosorbent assay for the detection of the coronaviruslike agent and its antibodies in pigs with porcine epidemic diarrhea prevalence of influenza-, aujeszky, transmissible gastroenteritis and procine epizootic diarrhea virus in feeder pigs evaluation of a blocking elisa using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its 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diarrhea virus in south china complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france comparison of porcine epidemic diarrhoea viruses from germany and the united states serological evidence of infection with a coronavirus related to transmissible gastroenteritis virus and porcine epidemic diarrhea virus genomic and evolutionary inferences between american and global strains of porcine epidemic diarrhea virus pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis experimental infection of piglets with a korean strain of porcine epidemic diarrhoea virus genome sequencing and analysis of a novelrecombinant porcine epidemic diarrhea virus strain from henan epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy outbreak of porcine epidemic diarreha virus in portugal enterotoxigenic eschirichia coli, rotavirus, porcine epidemic diarrhoea virus, adenovirus and calici-like virus in porcine postweaning diarrohea in hungary cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene letter to the editor. pig farm a new coronaviruslike particle associated with diarrhea in swine an immunoelectron and immunofluorescent study on the antigenic relationship between the coronavirus-like agent cv777 and several coronaviruses porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs transmissible gastroenteritis and porcine epidemic diarrhoea in britain diagnosis and investigations on ped in northern italy differentiation betyween human coronaviruses nl63 and 229 e using a novel double-antibody sandwich enzyme-linked immunosorbent assay based on specific monoclonal antibodies emergence of porcine epidemic diarrhea virus in southern germany emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium first case of porcine epidemic diarrhea (ped) caused by a new variant of ped virus in the netherlands prevalence of infections with enzootic respiratory and enteric viruses in feeder pigs entering fattening units distinct characteristics and complex evolution of pedv strains, north america coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol an apparent new syndrome of porcine epidemic diarrhea key: cord-000699-2mfbqs8i authors: sun, rui-qin; cai, ru-jian; chen, ya-qiang; liang, peng-shuai; chen, de-kun; song, chang-xu title: outbreak of porcine epidemic diarrhea in suckling piglets, china date: 2012-01-17 journal: emerg infect dis doi: 10.3201/eid1801.111259 sha: doc_id: 699 cord_uid: 2mfbqs8i nan the genera eptesicus and rhinolophus in south korea. however, nucleotide sequencing showed the presence of prototypical hantaan virus indicating a spillover infection or laboratory contamination (7) . further screening is necessary to confi rm n. hispida as a natural reservoir host of the virus. although the presented bat-associated sequence is obviously distinct from other hantaviruses, which suggests association with a novel natural host, a spillover infection from another, yet unrecognized host cannot be ruled out. however, detection of the virus exclusively in 1 organ (lung but not in liver, kidney, and spleen; data not shown) suggests a persistent infection that is typically observed in natural hosts of hantaviruses (8) . to date, only a few reports exist on cases of hemorrhagic fever with renal syndrome in africa (9,10). however, underreporting must be assumed because the symptoms resemble those of many other febrile infections. moreover, in cases of infections by non-rodent-associated hantaviruses, cross-reactivity with routinely used rodent-borne virus antigens should be limited and may hamper human serodiagnostics (1). the results suggest that bats, which are hosts of many emerging pathogens (5), may act as natural reservoirs for hantavirus. the effect of this virus on public health remains to be determined. to the editor: beginning in october 2010, porcine epidemic diarrhea (ped), caused by a coronaviral infection affecting pigs, emerged in china in an outbreak characterized by high mortality rates among suckling piglets. the outbreak overwhelmed >10 provinces in southern china, and >1,000,000 piglets died. this outbreak was distinguished by ≈100% illness among piglets after birth (predominantly within 7 days and sometimes within only a few hours) and death rates of 80%-100% (online technical appendix table 1 , wwwnc. cdc.gov/eid/pdfs/11-1259-techapp. pdf). few sows or boars showed any clinical signs during the outbreak, which is not consistent with a recent report from thailand (1). in that outbreak during late 2007, pigs of all ages were affected, exhibiting different degrees of diarrhea and no appetite. we characterized the genetic variation of the ped virus (pedv) that caused a large-scale outbreak in china during 2010-2011 and compared it with viruses in other outbreaks. we also report a possible novel transmission pathway for pedv. a total of 177 samples (intestine, stool, and maternal milk) were collected from pigs from different farms who had diarrhea; 100% of farms had >1 porcine sample positive for pedv. a total of 125/177 porcine samples were confi rmed as positive for pedv by reverse transcription pcr using primers as described (2) . pedv was detected in 105 (82.0%) of 128 fecal samples and 20 (40.8%) of 49 sow milk samples. piglets infected with pedv showed mild hemorrhage, undigested curdled milk in the stomach, and thin-walled intestines with severe mucosal atrophy and foamy fl uid (data not shown). the spike (s) gene of the family coronaviridae has a high degree of variation and can induce neutralizing antibody (3). reverse transcription pcr products of the 651-bp partial s gene of pedv and the deduced amino acid sequences were aligned by using clustalw (www.genome. jp/tools/clustalw), and a neighborjoining tree with 1,000 bootstraps was constructed. sequences of the s genes from this outbreak were 99.1%-100.0% homologous and had 88.7%-98.9% nt identity with all reference strains (online technical appendix table 2 ), 98.5%-98.9% with thailand strains, and 94.5%-95.1% with vaccine strain cv777. the partial s gene deduced amino acid sequences were compared and also showed a high degree of homology (98.0%-100.0%); they had 85.3%-98.7% identity with all reference strains listed in online technical appendix table 2 , 98.0%-98.7% with thailand strains, and 93.3%-94.7% with vaccine strain cv777 (data not shown). phylogenetic analysis indicated that the pedv in the china outbreak was different from foreign and other domestic strains on the basis of the reported partial s gene sequences. all new strains were clustered in the same branch, close to the cluster of thailand strains, and far from the cluster of vaccine strain cv777 (figure) . in the china outbreak, pedv caused severe diarrheal disease in piglets; heavy economic losses in many provinces resulted, despite use of commercial vaccines (inactivated phylogenic analysis showed that strain cv777 did not cluster with current common strains and showed 4) . to our knowledge, fecal-oral transmission is probably the main or only route of pedv transmission (5) (6) (7) . in our study, if a fecal sample from a sick piglet was found to be positive for pedv, we also collected and studied milk from its mother. these results showed that pedv was present in sow milk (online technical appendix table 3 ), but the detection rate was lower for these samples (40.8%) than for the fecal samples (82.0%). on the basis of these results, we hypothesize that sow milk could represent a possible (and potentially major) route for the vertical transmission of pedv from sow to suckling piglet. this hypothesis could be indirectly verifi ed by our fi eld observation that piglet death rates decreased as a result of fostering (data not shown). our fi ndings show that pedv was identifi ed not only in fecal samples from sick piglets, as expected, but also in the milk of the sow, which suggests vertical transmission of the virus. human pathogenic hantaviruses and prevention of infection hantavirus in african wood mouse, guinea novel hantavirus sequences in shrew, guinea molecular evolution of azagny virus, a newfound hantavirus harbored by the west african pygmy shrew (crocidura obscurior bats: important reservoir hosts of emerging viruses molecular phylogenetics and the origins of placental mammals genomic characterization of m and s rna segments of hantaviruses isolated from bats hantavirus-induced immunity in rodent reservoirs and humans first human case of haemorrhagic fever with renal syndrome in the central african republic serological evidence of human hantavirus infections in guinea, west africa chinese-like strain of porcine epidemic diarrhea virus differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex rt-pcr critical epitopes in transmissible gastroenteritis virus neutralization light microscopy and ultrahistology of intestinal changes in pigs infected with epizootic virus diarrhoea (evd): comparison with transmissible gastroenteritis (tge) virus and porcine rotavirus infections large-scale spatial-transmission models of infectious disease identifi cation of the membrane protein of porcine epidemic diarrhea virus coronavirus-like particles associated with diarrhea in baby pigs in quebec we thank the technicians from the pig farms for assistance in sample collection and bin wu for assistance in fi gure preparation. we also thank the guangdong province pig industry innovation projects for their support. to the editor: urban trench fever caused by bartonella quintana has been reported in persons who abuse alcohol and in homeless persons in large cities worldwide. symptoms vary from asymptomatic intermittent bacteremia to serious complications (1) . pediculus humanus mites, the known vector of the infection, are not always identifi ed, which raises the possibility that other vectors might also be involved (2) . we report on an outbreak of b. quintana infection among a young family of high socioeconomic status and their visiting relatives.the family resides in a regional city (population 104,000) in northern czech republic in an old, renovated apartment located on the top fl oor, just under the roof. in the summer of 2007, hundreds of ectoparasitic mites migrated from a whole in the roof and settled on the inner side of a permanently open window before infesting family members. two weeks later (day 1 of symptom onset), a papular rash and pruritic vesicular lesions were noted by the parents on the body and legs of their 2 children, a 1-year-old girl and a 3-year-old boy. on day 3, the girl's body temperature rose to 38.0°c, and the boy's temperature rose to 39.5°c. the rash resolved in ≈10 days in both children. vesicular lesions on the girl's buccal mucosal membrane resolved in 5 days. excoriated areas resulting from spontaneous rupture of lesions or scratching were still visible on day 14.on day 4, a fever (temperature, 38.5°c) and intense tibialgia, which persisted for 5 days, developed in the 33-year-old father of the infected children. on day 5, a vesicular rash, which resolved in 10 days, developed in the 33-year-old mother. the children's key: cord-255607-dbexsugq authors: wu, yang; zhang, hongling; shi, zhaorong; chen, jianfei; li, mingwei; shi, hongyan; shi, da; guo, longjun; feng, li title: porcine epidemic diarrhea virus nsp15 antagonizes interferon signaling by rna degradation of tbk1 and irf3 date: 2020-05-31 journal: viruses doi: 10.3390/v12060599 sha: doc_id: 255607 cord_uid: dbexsugq porcine epidemic diarrhea virus (pedv) causes a porcine disease associated with swine epidemic diarrhea. the type i interferon (ifn-i or ifn α/β) is a key mediator of innate antiviral response during virus infection. different antagonistic strategies have been identified and determined as to how pedv infection inhibits the host’s ifn responses to escape the host innate immune pathway, but the pathogenic mechanisms of pedv infection are not fully elucidated. our preliminary results revealed that endogenous tank-binding kinase 1 (tbk1) and interferon regulatory factor 3 (irf3), the key components in the ifn signaling pathway were downregulated in pedv infected ipec-j2 cells by itraq analysis. in this study, we screened nsp15 as the most important viral encoded protein involved in tbk1 and irf3 reduction. endoribonuclease (endou) activity has been well determined for coronavirus nsp15. three residues (h226, h241, and k282) of pedv nsp15 were identified as critical amino acids for pedv endou but not d265, which was not well correlated with published results of other coronaviruses, such as severe acute respiratory syndrome virus (sars-cov). moreover, pedv nsp15 can directly degrade the rna levels of tbk1 and irf3 dependent on its endou activity to suppress ifn production and constrain the induction of ifn stimulated genes (isgs), by which pedv antagonizes the host innate response to facilitate its replication. collectively, these results have confirmed that pedv nsp15 was capable of subverting the ifn response by the rna degradation of tbk1 and irf3. porcine epidemic diarrhea (ped) is caused by porcine epidemic diarrhea virus (pedv), which has a positive-strand rna genome of 28 kb in length in the genus alphacoronavirus, family coronaviridae and order nidovirales [1] [2] [3] . the disease is characterized by severe enteritis, vomiting, watery diarrhea, dehydration, and a high mortality rate among swine [4] . two-thirds of the genome (orf1a and 1b) encode a large replicase polyprotein, whereas the remainder of the genome encodes for structural proteins and accessory proteins [5, 6] . after pedv virus entry into the host cells, orf1a codes for a large polyprotein 1a (pp1a), while orf1b is expressed as the pp1ab fusion protein via the ribosomal frameshifting. by the proteinase activity of nsp3 (a papain-like protease) and nsp5 (a main protease), these polyproteins are proteolytically processed to 16 nonstructural proteins (nsp1 to 16) , which mediate the replication of the viral rna genome and synthesis of a nested set of subgenomic mrnas [6] . in late 2010, a newly emerging pedv variant was reported with more virulence and higher mortality in suckling piglets, compared to the classical pedv that was first discovered in europe in 1971 [7] . to date, the newly emerging pedv variant is recognized as the major infectious pathogen for swine diarrhea-associated diseases in swine-raising farms in china and it has spread to other countries worldwide, causing a high number of pig deaths and significant economic impacts [8] [9] [10] [11] [12] . during viral infection, the innate immune response is activated, leading to the induction of the type i interferon (ifn-i or ifn α/β). ifn-i is the potent cytokine of critical importance in controlling viral infections and priming adaptive immune responses [13] . following production, ifn-i initiates a positive feed-back loop by binding to their cognate receptors on the cell surface in an autocrine and paracrine manner [14, 15] and activating jak protein tyrosine kinases (jak1 and tyk2) which phosphorylate signal transducers and activators of transcription stat1 and stat2. stat1 and stat2 together with interferon regulatory factor 9 (irf9) form a transcription factor complex termed ifn-stimulated gene factor 3 (isgf3). then, isgf3 is translocated into the nucleus and binds to the ifn-stimulated response elements (isre) to induce the expression of ifn-stimulated genes (isgs), which establish an antiviral state [15, 16] . however, many viruses, including coronaviruses, have evolved mechanisms to evade the host immune system [17] [18] [19] [20] [21] [22] [23] [24] . previous studies suggest that pedv can restrain host innate immune response by different strategies, such as by degradation or cleavage of key factors essential the ifn signaling pathway [25, 26] , competitive interaction between viral encoded proteins and modulators for ifn production [27, 28] , or localization changes of antiviral components [29] . whether there are other mechanisms utilized by pedv to circumvent the host response remains unclear. our previous results have demonstrated that differentially expressed proteins were identified in pedv infected ipec-j2 cells by the analysis of isobaric tags for relative and absolute quantitation (itraq). we identified 49 differentially expressed cellular proteins, of which eight were upregulated and 41 downregulated. these differentially expressed proteins were involved in apoptosis, signal transduction, and stress responses. in our analysis, tbk1 and irf3, two important modulators in the activation of the interferon signaling pathway were downregulated post pedv infection. in this study, we screened the pedv viral proteins involved in the reduction of tbk1 and irf3 expression. among the identified viral proteins, nsp15 was recognized as the most effective viral protein contributing to the reduction of both tbk1 and irf3 expression after the co-transfection of tbk1 or irf3 with individually encoded pedv proteins in vitro. it was confirmed that the two major well-known degradation systems, namely the ubiquitin-proteasome system or autophagy, were not involved in pedv nsp15 mediated reduction of tbk1 and irf3. in contrast, pedv nsp15 was capable of suppressing tbk1 and irf3 expression by endoribonuclease-dependent degradation of tbk1 and irf3 rna. this resulted in a decrease of ifn and isg production, resulting in pedv host innate immune escape. ipec-j2 cells (porcine small intestine epithelial cell clone j2; atcc), vero e6 (african green monkey kidney cell line; atcc), and hek293 cells (human embryonic kidney epithelial cells; atcc) were cultured in dulbecco's minimum essential medium (dmem) (life technologies, usa) supplemented with 10% heat-inactivated fetal bovine serum (fbs) (gibco, usa), 100 u/ml penicillin, 100 µg/ml streptomycin at 37 • c in an incubator with 5% co2 (thermo scientific, usa). pedv strain cv777 (genbank accession number kt323979) was prepared and titrated as previously described [30] . vesicular stomatitis virus that expresses the green fluorescence protein (vsv-gfp) was preserved in harbin veterinary research institute, harbin, and stored at −80 • c. the full-length sequence of tbk1 and irf3 were constructed into the pcaggs-ha vector to obtain recombinant plasmids, pcaggs/ha-tbk1 and pcaggs/ha-irf3, respectively. the recombinant pcaggs plasmids containing individual pedv viral protein (nsp1-10, nsp12-16, s, e, m, and n) with a flag fusion tag were kindly provided by prof. yue wang from harbin veterinary research institute. mutagenesis of the pedv nsp15 constructs (h226a, h241a, d265a and k282a) were performed by using site-directed mutagenesis kit (takara, china). recombinant prokaryotic expression plasmids were obtained following cloning of the individual gene of pedv nsp15 and its mutant into the ecor i and xho i sites of pgex-6p-1 plasmid vector (ge healthcare life sciences). recombinant pgem-t/tbk1 and pgem-t/irf3 plasmids were generated to serve as dna templates for an rna transcription assay in vitro by amplifying the full length sequence of tbk1 or irf3 into pgem-t easy vector by the t-a ligation method. the specific primers used for the construction of target plasmids are listed in table 1 and all the constructed plasmids were verified by dna sequencing. the listed antibodies were used in this study including tbk1 rabbit monoclonal antibody (mab) (cell signaling technology), irf3 rabbit mab (cell signaling technology), and phospho-irf3 (ser396) (4d4g), rabbit mab (cell signaling technology), anti-flag mouse mab (sigma), anti-ha mouse mab (sigma), irdye-conjugated secondary antibody (li-cor biosciences) , and β-actin mouse mab (sigma). monolayers of vero e6 and ipec-j2 cells were infected with pedv strain cv777 at multiplicity of infection (moi) of 0.1 for 1 h at 37 • c. unbound virus was removed, and cells were maintained in complete medium for various time points until samples had been harvested. some cell samples were treated with proteasome inhibitor mg132 (sigma) at the concentration of 2 µm, autophagy inhibitor 3-methyladenine (3-ma, sigma) at 5 mm, or carrier control dmso during some transfection assays as previously described [25] . hek293 cells were transfected with indicated plasmids using x-tremegene transfection reagent according to manufacturer's instruction (roche, usa). at 36 h post transfection, cell samples were collected and lysed in ripa buffer (beyotime, nantong, china) for the western blot analysis of targeted proteins. immunofluorescence assays (ifa) were performed as described previously with slight modification [25] . briefly, hek293 cells were co-transfected with tbk1, irf3 together with nsp15, nsp15 mutants, or empty vector control followed by the collection of supernatants for each treatment at 30 h post transfection. ipec-j2 cells were treated with the collected supernatants with three-fold dilution for 12 h followed by inoculation of vsv-gfp at moi of 0.1. the fluorescence was visualized at 10 hpi with an olympus inverted fluorescence microscope equipped with a camera. western blot analysis was performed as previously described with a slight modification [31] . treated samples were lysed in radioimmunoprecipitation assay (ripa) buffer (haigene, china) containing protease inhibitor cocktail and phosphatase inhibitors (roche, switzerland), separated by sds-page under reducing conditions, and transferred onto a pvdf membrane (merck millipore, usa). after blocking, the membranes were incubated with a primary antibody and then incubated with an appropriate irdye-conjugated secondary antibody (li-cor biosciences, lincoln, ne). the membranes were scanned using an odyssey instrument (li-cor biosciences) according to the manufacturer's instructions. linearized dna was prepared by digestion with restriction endonuclease sal i prior to in vitro transcription to produce rna of defined length. in vitro transcribed rna of tbk1 and irf3 were generated from recombinant pgem-t/tbk1 or pgem-t/irf3 plasmid as template, respectively, using the ribomax™ large scale rna production systems (promega, usa). transcribed rna was purified by removal of the dna template and proteases following transcription reaction as the manufacturer's instruction (promega, usa) and stored for nuclease assay at -80 • c. for protein expression, individual plasmid of pgex-6p-1-pedv nsp15, pgex-6p-1-pedv nsp15 mutant derivatives (h226a, h241a, d265a, and k282a), or pgex-6p-1 empty vector was transformed to escherichia coli bl21 (de3) cells, respectively. the glutathione s-transferase (gst) fusion proteins were expressed following isopropyl-β-d-thiogalactopyranoside (iptg) inductions and purified by affinity chromatography using glutathione immobilized to a sepharose matrix per the manufacturer's instruction (ge healthcare life sciences, usa). the endoribonuclease activity assay was done as previously described [32] . briefly, nuclease reactions contained 4 µg of purified wild-type pedv nsp15 protein, pedv nsp15 mutant protein, or gst tag protein as control, and 6 µg tbk1 or irf3 rna transcribed and purified in vitro. reactions were performed in 25 mm hepes-koh (ph 7.4)/50 mm nacl/5 mm mncl 2 /1 mm dtt. following incubation at 37 • c for 1 h, the reactions were extracted using phenol-chloroform-isoamyl alcohol and analyzed by agarose-formaldehyde gel electrophoresis. for northern blot, total rna was harvested by using trizol reagent (invitrogen, usa) and analyzed by agarose-formaldehyde gel electrophoresis. rnas were transferred to a 0.45-µm nylon membrane and probed with biotin-labeled dna probes generated with the specific primers (table 1) using the north2south tm biotin random prime dna labeling kit (thermo scientific). the membrane was imaged an odyssey instrument (li-cor biosciences) followed by incubation with irdye 800-conjugated streptavidin. quantitative rt-pcr analyses were carried out as described previously with a slight modification [33] . at indicated time points post transfection or pedv infection, total rna was extracted from cells and subjected to quantitative rt-pcr using specific primers as listed in table 1 . relative gene quantification was performed by the 2(-delta delta c(t)) method [34] . collected virus samples were frozen and thawed three times and clarified by centrifugation at 8000× g for 10 min prior to titration. tcid 50 assays were performed in vero e6 cells following the method of reed & muench as previously described [34] . briefly, cell monolayers were inoculated with serial dilutions of each virus stock and incubated for 4 days prior to observation of the presence of cytopathic effect. variables are expressed as mean ±s.d. data were statistically analyzed by using graphpad prism v5.0 software. statistical analyses were performed using student's t test. a p value of <0.05 was considered significant. ipec-j2 cells were infected with pedv or left untreated as control and cell samples were collected at 24 h and 36 h for itraq analysis as previously described [35] . our preliminary results revealed that endogenous tbk1 and irf3 were downregulated at 24 h and 36 h in pedv infected ipec-j2 cells by itraq analysis (data not shown). subsequently, ipec-j2 cells were infected with pedv at moi of 1.0 and the mrna levels were determined by quantitative pcr. as shown in figure 1a , tbk1 mrna levels were significantly decreased at 24 h and 36 h post infection (hpi). in contrast, irf3 mrna levels were first increased at 24 hpi and then decreased at 36 hpi, indicating no obvious changes in irf3 mrna levels following pedv infection ( figure 1b) . to further confirm the results, hek293 cells were inoculated with pedv at moi of 0.1 and cell samples were collected for endogenous tbk1 and irf3 detection at indicated time points post infection. consistent with the results from ipec-j2 cells by itraq, endogenous tbk1 and irf3 were evidently reduced at 24 hpi and 36 hpi in hek293 cells ( figure 1c ). these findings suggest that a reduction of endogenous tbk1 and irf3 may be achieved by downregulating the mrna transcriptional levels of tbk1 and irf3 post pedv infection. to explore which viral protein contributes to the reduction of tbk1 and irf3, hek293t cells were co-transfected with tbk1 or irf3 and each pedv encoded protein. at 36 h post transfection, cell samples were collected and lysed for detection of tbk1 or irf3 expression. several viral proteins were involved in the reduction of tbk1 or irf3 expressions to varying extents, e.g., nsp1, nsp14, and nsp15 for tbk1 and nsp1, nsp4, nsp5, and nsp15 for irf3, among which nsp15 can evidently downregulate the expression of either tbk1 or irf3 compared with other viral proteins (figure 2a,b) . moreover, endogenous tbk1 and irf3 were also reduced followed by the ectopic overexpression of nsp15 at 36 h post transfection ( figure 2c ). therefore, we mainly focused on pedv nsp15 as the research target in this study to investigate its role in modulating tbk1 and irf3 expressions. table 1 . three independent experiments were performed in triplicate, and values are the means ± sd for all three experiments. *, p < 0.05. (c) hek293 cells were inoculated with pedv at moi of 0.1 for 24 h and 36 h followed by verification of endogenous tbk1 and irf3 proteins by western blot analysis. within eukaryotic cells, there are two major intracellular protein degradation pathways: the ubiquitin-proteasome system and autophagy [36] . the proteasomal degradation pathway has high selectivity and the proteasome generally recognizes ubiquitinated substrates [37] . by contrast, autophagy is a highly conserved process for degrading redundant cellular components by encircling them with membrane followed by a fusion of the vesicle with lysosomes [38] . therefore, to determine the mechanism that might be responsible for the depletion of tbk1 and irf3 by nsp15, the expression levels of tbk1 and irf3 proteins were examined in cells treated with a protease inhibitor mg132 [39, 40] . as shown in figure 3a ,c, treatment with mg132 cannot block the downregulation of tbk1 and irf3 in hek293t cells with co-transfection of tbk1 or irf3 together with nsp15, thus not suggesting the proteasome-mediated degradation of tbk1 and irf3 by nsp15. additionally, we tested the possible role of autophagy in the reduction of tbk1 and irf3 by treating cells with 3-ma, which is commonly used to inhibit autophagy [38, 41] . we observed that 3-ma treatment did not inhibit tbk1 or irf3 downregulation in hek293t cells co-transfected with tbk1 or irf3 along with nsp15 ( figure 3b,d) . these data indicate that downregulation of tbk1 and irf3 by nsp15 is not through the ubiquitin-proteasome system and autophagy. coronavirus nsp15 has been reported as a uridine-specific endoribonuclease and nuclease activities as well as crystal structure have been well identified as previously described [32, [42] [43] [44] [45] . four inactive mutants of sars-cov nsp15, including h234a, h249a, d272a, and k289a, have been identified to lose the cleavage activity for substrate rna due to loss of endoribonuclease activity, indicating that these four residues were critical for maintaining the nuclease activity of sars-cov nsp15 [42] . based on amino acid sequence alignment of pedv nsp15 with other coronavirus orthologs as well as xendou from x. laevis, the four mentioned residues were also conserved in pedv nsp15 and were denoted in red with the corresponding position in pedv nsp15 sequence below ( figure 3e ). here, we asked whether tbk1 or irf3 downregulation by pedv nsp15 is dependent on its endoribonuclease activity. to determine whether the amino acid residues are also required for pedv nsp15 endonuclease activity, four mutants (h226a, h241a, d265a, and k282a) were constructed by mutating corresponding residue of pedv nsp15 to alanine. hek293t cells were co-transfected with tbk1 or irf3 together with nsp15 or constructed mutants following the detection of tbk1 and irf3 by western blot. it was demonstrated that nsp15 and d265a mutant can obviously reduce the expression levels of tbk1 and irf3 post transfection. in contrast, the reduction of tbk1 or irf3 expression was blocked when hek293t cells were co-transfected with tbk1 or irf3 and the remaining mutants (h226a, h241a, and k282a), indicating that residues of h 226 , h 241 , and k 282 but not d 265 are critical for the endoribonuclease activity of pedv nsp15 ( figure 3f,g) . . pedv-induced reduction in tbk1 and irf3 expression is due to pedv nsp15 endoribonuclease activity, but not proteasome or autophagy-mediated mechanisms. (a,c) hek293t cells were co-transfected with nsp15 (flag tagged) and tbk1 or irf3 (ha tagged) and were treated with the proteasome inhibitor mg132 (2 µm) or carrier control dmso. detergent lysates were collected and subjected to reducing sds-page and immunoblotting with anti-flag and ha antibodies. (b,d) hek293t cells were co-transfected with nsp15 (flag tagged) and tbk1 or irf3 (ha tagged) and were treated with 3-ma (5 mm) for further culture. at 36 h post transfection, cell lysates were subjected to blotting with corresponding antibody. (e) alignments of the nsp15 orthologs from several coronaviruses and x. laevis endou. the amino acid sequence of the pedv nsp15 (pedv, kt323979) was aligned with orthologs of severe acute respiratory syndrome coronavirus (sars, kf514422), murine hepatitis virus (mhv, kf268339), avian infectious bronchitis virus (aibv, nc_001451) and xenopus laevis (xendou, bc169902) using dnastar software. gaps in the sequence alignment are denoted by hyphens. residues with red are the conserved residues critical for nsp15 activity as previously described. the number below the red residue indicates its corresponding position at pedv nsp15 protein, respectively. (f,g) four mutants (h226a, h241a, d265a and k282a) of pedv nsp15 were constructed by mutating the corresponding conserved residue mentioned in (e) into alanine using site-directed mutagenesis kit. hek293t cells were then co-transfected with tbk1 or irf3 and wild-type pedv nsp15, the constructed nsp15 mutant (h226a, h241a, d265a or k282a) or empty vector. at 36 h post transfection, cell samples were subjected to immunoblotting with antibodies to flag, ha or β-actin (loading control). combined with the previous results, we hypothesized that pedv nsp15 contributed to reduction of tbk1 and irf3 expression by targeted mrna level degradation in an endou activity dependent manner. to test this hypothesis, hek293t cells were co-transfected with tbk1 or irf3 and pedv nsp15 as well as the constructed mutants followed by quantitative analysis of tbk1 or irf3 mrna level with the primers listed in table 1 . as shown in figure 4a , the relative mrna level of tbk1 was significantly more decreased in pedv nsp15 and d265a transfected cells than in other mutants and empty vector transfected cells, which suggested that pedv nsp15 can reduce tbk1 expression by downregulating the tbk1 mrna levels dependent on its endou activity. similar results were obtained that nsp15 can reduce irf3 expression by decreasing the irf3 mrna levels in an endou dependent manner ( figure 4b ). in addition, the mechanism was further verified by northern blot assay using the specific probe as designed in table 1 , following co-transfection with tbk1 or irf3 and pedv nsp15 as well as the constructed mutants in hek293t cells. consistent with the quantitative results by real time pcr assay, tbk1 or irf3 rna was evidently more reduced in nsp15 and d265a mutant transfected cells than in mutant h226a, h241a, k282a, or empty vector control transfected cells ( figure 4c,d) . these data demonstrate that pedv nsp15 can reduce tbk1 and irf3 expression by the targeted degradation of tbk1 and irf3 mrna. table 1 . three independent experiments were performed in triplicate, and values are the means ± sd for all three experiments. *, p < 0.05. (c,d) hek293t cells were co-transfected with tbk1 or irf3 and pedv nsp15 expression plasmid (pedv nsp15, h226a, h241a, d265a and k282a) or empty vector. at 36h post transfection, rna were extracted from the collected cell samples followed by rna detection by northern blot assay using the designed specific probes (table 1) as described in the materials and methods. we next investigated whether pedv nsp15 can directly degrade mrna of tbk1 and irf3. to this end, wild-type and four mutant versions of pedv nsp15 were produced as gst-tagged proteins and purified under mild conditions by the addition of reduced glutathione to the elution buffer as the manufacture's instruction (ge healthcare life sciences). as shown in figure 5a , recombinant wild-type pedv nsp15 and four mutants (h226a, h241a, d265a, and k282a) were purified successfully at the expected molecular weight of 66 kda by sds-page analysis. the tbk1 and irf3 sequences were amplified by pcr and subsequently cloned into the pgem-t easy vector containing a t7 rna polymerase promoter upstream of the multiple cloning region to construct the recombinant plasmids of pgem-t/tbk1 and pgem-t/irf3, respectively. in vitro-transcribed rnas of tbk1 and irf3 were synthesized from the constructed recombinant dna templates (pgem-t/tbk1 and pgem-t/irf3) by the ribomax™ large scale rna production systems (promega, usa). the gst-purified pedv nsp15 proteins were incubated with the generated tbk1 and irf3 mrna as substrate in presence of mn 2+ , a known cofactor for the endoribonuclease activity. [32, 46] . it was revealed that synthesized tbk1 and irf3 mrna were effectively reduced post incubation with wild-type pedv nsp15 and d265a mutant, but not with the other mutant derivatives (h226a, h241a, and k282a) , demonstrating the pedv nsp15 can directly degrade tbk1 and irf3 mrna dependent on its endoribonuclease activity ( figure 5b,c) . (b,c) targeted rna was transcribed and purified in vitro as described in the materials and methods. endoribonuclease activity assay was performed by incubating the transcriptional rna (tbk1 or irf3) with purified recombinant pedv nsp15 proteins (pedv nsp15, h226a, h241a, d265a or k282a) or gst tag protein as a control at 37 • c for 1 h followed by rna detection of tbk1 or irf3 rna as described in the materials and methods. type i ifns are transcriptionally regulated, and are induced following recognition of pathogen components during infection. tbk1 and irf3 are the key effectors during viral infections to induce ifn production [47] . following stimulation with virus components including dsrna, irf3 becomes phosphorylated by the serine-threonine kinases tank-binding kinase-1 (tbk1) or the inducible iκb kinase (ikk-i/ikkε) [48, 49] . irf-3 then dimerizes, translocates into the nucleus, and combines with the co-activator cbp/p300 to activate the expression of ifnβ [15] . to determine whether pedv nsp15 modulates the phosphorylated irf3, cells were co-transfected with tbk1 and irf3 along with pedv nsp15 as well as its mutant derivatives (h226a, h241a, d265a, and k282a) and then collected to examine the phosphorylated irf3 levels by western blot. as anticipated, the tbk1 stimulation led to the irf3 phosphorylation in empty vector transfected cells. however, the irf3 phosphorylations were significantly inhibited in nsp15 and d265a mutant transfected cells compared to the remaining mutants (h226a, h241a, and k282a) transfected cells, suggesting that pedv nsp15 impeded irf3 expression as well as irf3 phosphorylation dependent on its endou activity ( figure 6a ). vesicular stomatitis virus (vsv) is frequently used for the assessment assay of ifn activity [50] . to determine the role of nsp15 in regulation of ifn production, hek293t cells were co-transfected with tbk1 and irf3 together with wild-type nsp15, individual nsp15 mutant or empty vector and cell supernatants were collected at 30 h post co-transfection for determining the status of ifn secretion. ipec-j2 cells were treated with the supernatant followed by inoculation of vsv-gfp at moi of 0.1. fluorescence was visualized at 10 h post infection with an olympus inverted fluorescence microscope equipped with a camera. vsv-gfp infection was evident in treatments with supernatant from nsp15 and d265a mutant transfected cells, and conversely vsv-gfp infection was obviously inhibited in treatments with supernatant from cells transfected with the remaining mutants (h226a, h241a, and k282a) or empty vector, demonstrating that nsp15 was an antagonistic protein in ifn production ( figure 6b ). moreover, we continued to investigate the effects of collected supernatant on pedv infection in vero e6 and ipec-j2 cells. vero e6 cells were treated with the individual collected supernatant as mentioned above prior to pedv infection, cell samples were then collected and subjected to virus titration by tcid 50 assay. pedv infection was significantly enhanced in cells treated with supernatant from wild-type nsp15 and d265a mutant transfected cells than that from mutant h226a, h241a, k282a or empty vector transfected cells ( figure 6c ), confirming that pedv nsp15 can evade the host antiviral response by antagonizing ifn production. meanwhile, a pedv infection assay was further performed in ipec-j2 cells, a target cell line for pedv infection in vivo. similar results were obtained in that pedv nsp15 can facilitate pedv infection, based on pedv genomic quantitation by real-time pcr assay instead of by tcid 50 assay due to its low susceptibility to ipec-j2 cells ( figure 6d ). these data collectively demonstrate that nsp15 can promote pedv infection by limiting ifn secretion dependent on its endoribonuclease activity. ifn-i is the key innate immune cytokine produced by cells to trigger antiviral function [51, 52] . therefore, we assessed the effect of nsp15 on the ifn mediated antiviral response signaling pathway. here, hek293 cells were co-transfected with tbk1 and irf3 together with wild-type nsp15, nsp15 mutants, or empty vector control and cells samples were collected to investigate the effects of nsp15 on induction of innate antiviral molecules. quantitative rt-pcr showed that mrna levels of immune related molecules, such as ifnβ, tnfα, oas1, isg15, isg54, and isg56, were significantly disrupted by nsp15 and mutant d265a post transfection compared to that by empty vector control. however, the disruptions were impeded by the other nsp15 mutants that impaired the endoribonuclease activities (figure 7) , suggesting that pedv nsp15 restrains cellular antiviral activity and thus facilitates pedv infection. table 1 . the results are representative of three independent experiments (mean ± sd). *, p < 0.05. the p value is calculated using student's t-test. the host innate immune system is the first line of defense against virus invasion through production of ifns as well as various other cytokines. innate immune responses are activated through host pattern recognition receptors (prrs), which recognize pathogen-associated molecular patterns [53] . ifns exert antiviral effects through inducing the expression of hundreds of isgs [52, 54, 55] . however, during coevolution with their host, viruses always evolve diverse strategies to escape and even inhibit host ifn responses [17, 19, [26] [27] [28] [29] 47, 56] . pedv has acquired multiple mechanisms that avoid the action of ifn by preventing the binding of viral products to cellular sensors. it was revealed that pedv n protein antagonized ifn production by preventing tbk1 from interaction with irf3 [27] . of the several known viral evasion strategies, the cleavage of crucial innate immune molecules, including adaptors, kinases, and transcriptional factors, are considered to be a particularly powerful way for viruses to escape the innate immune response. for example, the 3c-like protease of pedv and porcine delta coronavirus (pdcov), disrupts type i ifn signaling by cleaving the nf-κb essential modulator (nemo) [26, 57] . in addition, pdcov nsp5 antagonizes type i ifn signaling by cleaving stat2, an essential factor for ifn responses [19] . furthermore, the ubiquitination and deubiquitination are highly regulated post-translational modification processes in modulating the antiviral innate immune response. within the cells, polyubiquitination plays several different roles depending upon the attachment position on the target proteins, and linked polyubiquitin chains regulate the proteasomal degradation of target proteins [25, 58, 59] . multiple ubiquitin ligases and ubiquitin-binding scaffold proteins contribute to the positive regulation of the ifn response, such as rig-i, traf2, traf6, and tbk1. previous studies have indicated that pedv plp2 significantly inhibits the ubiquitination of rig-i and sting, which is essential for the activation of type i ifn signaling [58] . meanwhile, the proteasomal degradation of target proteins for the ifn response can also be achieved by viruses through the removal of k48 polyubiquitin chains [59] [60] [61] . pedv-induced stat1 degradation inhibits type i interferon signalling in a proteasome-dependent manner [25] . however, viruses are not just limited to the mentioned strategies to antagonize ifn responses. in this study, we first identified that pedv nsp15 was capable of subverting the ifn response by the rna degradation of tbk1 and irf3, which differentiated from the strategies utilized by other coronavirus orthologues previously described. the functions of nsp15 of coronaviruses (nsp11 in arteriviruses), an endoribonuclease encoded by nidoviruses, have received more attention. previous studies showed that the nsp15 encoded by sars-cov [32] , mhv [62, 63] , pedv [45] , pdcov [64] , and the nsp11 encoded by prrsv [65, 66] can antagonize antiviral innate immune responses by utilizing the different mechanisms involved, e.g., by mediating the evasion of viral dsrna by host for mhv and hcov-229e [62, 63] , by suppressing both mavs and rig-i expression for prrsv [66] , by impairing the activation of transcription factor nf-κb for pdcov [64] , or by inhibiting mavs-induced apoptosis for sars-cov [67] . pedv nsp15 of is a 339-residue polypeptide that results from the cleavage of pp1ab at sites 6139 nlq↓gle 6144 and 6478 qlq↓ase 6483 by the main protease nsp5. several recent studies have focused on the structural and functional characterization of coronavirus nsp15 due to its potential importance as a drug target. it has been reported that the endou activity of pedv nsp15 is not required for pedv replication in vero cells. however, the endou activity is involved in the suppression of host ifn response in epithelial cells and macrophages in vitro, and subsequently can facilitate pathogenesis development in vivo by enhancing viral replication and shedding [45] . although previous studies have reported pedv nsp15 as a key virulence factor that suppressed ifn responses in vitro and facilitated pedv replication, the underlying mechanism remains unknown. in this study, we found that endogenous tbk1 and irf3 were downregulated post pedv by previous itraq assay and western blot analysis. nsp15 was selected as the investigation candidate due to its evident effect on tbk1 and irf3 reduction post co-transfection with each viral encoded protein. it was exhibited that pedv nsp15 was capable of downregulating the expression of tbk1 and irf3 proteins by the degradation of the rna of tbk1 and irf3 in an endoribonuclease activity dependent manner, while residues of h226a, h241a, and k282a were critical for the endoribonuclease activity of pedv nsp15, but not d265a. the reason that results in these differences remains unclear. whether these structure differences result in different mechanisms used to antagonize ifn production remains a subject of further study (figure 8) , elucidating a novel antagonistic mechanism utilized by pedv to counter the antiviral response. in summary, our data reveal that pedv nsp15 acts as an ifn antagonist to inhibit immune response by the rna level degradation of tbk1 and irf3, key ingredients involved in the ifn signaling pathway dependent on its endoribonuclease activity, which will facilitate pedv replication and the development of virus induced pathogenesis. molecular epidemiology of porcine epidemic diarrhea virus in china immunogenicity and antigenic relationships among spike proteins of porcine epidemic diarrhea virus subtypes g1 and g2 emerging and re-emerging coronaviruses in pigs porcine epidemic diarrhea virus: an overview of current virological and serological diagnostic methods origin and evolution of pathogenic coronaviruses immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling new variants of porcine epidemic diarrhea virus, china origin, evolution, and genotyping of emergent porcine epidemic 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of the mechanisms to antagonize type i interferon production mavs-mediated apoptosis and its inhibition by viral proteins this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we want to thank jin tian for his excellent technical assistance on this project. the authors declare no conflict of interest. key: cord-280564-kgoczioe authors: conceição-neto, nádia; theuns, sebastiaan; cui, tingting; zeller, mark; yinda, claude kwe; christiaens, isaura; heylen, elisabeth; van ranst, marc; carpentier, sebastien; nauwynck, hans j.; matthijnssens, jelle title: identification of an enterovirus recombinant with a torovirus-like gene insertion during a diarrhea outbreak in fattening pigs date: 2017-09-08 journal: virus evol doi: 10.1093/ve/vex024 sha: doc_id: 280564 cord_uid: kgoczioe diarrhea outbreaks in pig farms have raised major concerns in europe and usa, as they can lead to dramatic pig losses. during a suspected outbreak in belgium of porcine epidemic diarrhea virus (pedv), we performed viral metagenomics to assess other potential viral pathogens. although pedv was detected, its low abundance indicated that other viruses were involved in the outbreak. interestingly, a porcine bocavirus and several enteroviruses were most abundant in the sample. we also observed the presence of a porcine enterovirus genome with a gene insertion, resembling a c28 peptidase gene found in toroviruses, which was confirmed using re-sequencing, bioinformatics, and proteomics approaches. moreover, the predicted cleavage sites for the insertion suggest that this gene was being expressed as a single protein, rather than a fused protein. recombination in enteroviruses has been reported as a major mechanism to generate genetic diversity, but gene insertions across viral families are rather uncommon. although such inter-family recombinations are rare, our finding suggests that these events may significantly contribute to viral evolution. diarrhea is an important health problem affecting piglets, as well as an important cause of production losses in fattening pigs. typical causes of clinical and subclinical enteric problems in the latter age group are bacteria such as brachyspira spp., lawsonia intracellularis and/or salmonella spp., whereas viral causes at this age are generally less common (stå hl et al. 2011; collins and barchia 2014; de ridder et al. 2014) . in a minority of cases, fattening pigs and sows can be affected by porcine epidemic diarrhea virus (pedv), a re-emerging enteric coronavirus (stevenson et al. 2013; pensaert and martelli 2016) . classical diagnostic methods such as specific antigen-, gene-, or antibodydetection assays are currently in use in veterinary practice in order to reach an etiological diagnosis and to put into practice targeted prophylactic and therapeutic measures. however, in recent years, a considerable progress has been made in the field of viral metagenomics, which makes it more affordable to apply next-generation sequencing (ngs) technologies to analyze the entire fecal viral content (the fecal virome) of a sample. using this approach, the most frequently detected mammalian viruses in viral metagenomics studies were kobuviruses, rotaviruses, pig stool-associated ssdna viruses, astroviruses, sapoviruses, and enteroviruses (shan et al. 2011; sachsenrö der et al. 2012; zhang et al. 2013) . in january 2015, a case of diarrhea in fattening pigs occurred on a belgian pig farm. diarrhea emerged 2 days upon arrival after transport and clinical signs in the herd (1,000 fattening pigs) lasted for 21 days, affecting a total of 20 fattening pigs. the veterinarian suspected an infection with the re-emerging pedv. this virus belongs to the genus alphacoronavirus within the family of coronaviridae. the virus replicates in enterocytes and leads to sloughing of the gut villi, which causes diarrhea. it was widely spread across europe between the 1970s and 1990s, causing epidemics of diarrhea on pig farms. however, since then it has only sporadically been detected (pensaert and de bouck 1978) . in 2010, this virus was detected during severe outbreaks in asia, and for the first time in the us swine population during spring 2013, causing mortality and severe economic losses (song et al. 2015) . in 2014, a milder variant of the virus (oh 855) was detected in the us swine herds (wang et al. 2014 ). these strains were later also detected in german swineherds in 2014, as well as in other european countries (grasland et al. 2015; mesquita et al. 2015; stadler et al. 2015) . however, it is unclear if these genomic changes are correlating with an increased virulence of pedv (pensaert and martelli 2016) . at the beginning of 2015 and following the outbreaks of pedv in usa, there was an increased awareness in europe, including belgium, as this virus could also emerge in the local swine population at any given moment. therefore, when the outbreak of diarrhea occurred on the above-mentioned farm, a mixed fecal sample (n ¼ 12 pigs) was sent to the laboratory of virology at ghent university. pedv rna was detected using an in-house rt-qpcr, but high cq values (cq > 30) suggested a rather low viral load, whereas typical clinical infections in gnotobiotic piglets results in shedding of the virus at loads >10 10 copies per milliliter of feces (jung et al. 2014 ). still, a full genome characterization was performed and this indicated that the strain was genetically highly similar to the indel strains circulating in germany and usa, as reported elsewhere (theuns et al. 2015) . given the low abundance of pedv in the fecal sample, and the large number of other viruses which can also cause gastro-intestinal disease in pigs, it was questioned in the present study if other viruses might have been present in the pig's fecal virome and if they could have contributed to the emergence of diarrhea on this farm. a well-established virus often found in the feces of pigs is the porcine enterovirus (also known as enterovirus g), belonging to the picornaviridae family and genus enterovirus. enterovirus g encloses 16 viral types and has been detected in north america, europe, and asia (boros et al. 2012; anbalagan et al. 2014; van dung et al. 2014 . picornaviruses are positive-sense single-stranded rna viruses, which have one open reading frame (orf) that encodes for a single polyprotein. this polyprotein yields typically a leader (l) protein, four structural (vp1-vp4), and seven non-structural proteins (2apro, 2b, 2 c, 3 a, 3b, 3c pro , and 3d pol ) upon cleavage by proteases (ehrenfeld et al. 2004) . a chymotrypsin-like cysteine proteinase, 3c pro , is the main cleavage protein found in all picornaviruses and contains a conserved cleavage site aiding in the identification of cleavage sites of newly discovered viruses (lendeckel and hooper 2009) . the 2a pro is another protease encoded by entero-and rhinoviruses, that folds spontaneously into an active form, performing a primary cleavage (ehrenfeld et al. 2004; lendeckel and hooper 2009) . picornavirus evolution is determined by their high mutation rate, which is predicted to range between 10 à3 and 10 à5 mutations per nucleotide, per genomic replication (domingo and holland 1997) . in addition, the often reported recombination events are crucial to shape their genomic architecture (simmonds 2006) . another example highlighting the importance of proteases for viral pathogenesis can be found in the coronaviridae family. specially studied for severe acute respiratory syndrome coronavirus, its pl pro is also a deubiquitinating enzyme (ratia et al. 2006) . therefore, it is important in disrupting the host cellular ubiquitination machinery, which leads to enhanced viral replication (ratia et al. 2006) . in this study, we focus initially on unraveling the gut virome of the diarrhea outbreak in a fattening pig farm, and as a consequence of our findings we also further characterized an unusual recombinant enterovirus genome. a mixed diagnostic fecal sample of fattening pigs (n ¼ 12) from belgium was sent to the laboratory of virology (ghent university, merelbeke, belgium) in january 2015. no healthy controls were collected at the moment of the outbreak. the sample was prepared using a slightly modified version of the netovir protocol (conceição-neto et al. 2015) . a 10% weight/volume fecal suspension in viral transport medium (dmem, 10% p/s, 5% gentamycin, and 0.01% fungizone) was prepared from sample 15v010 and filtered through 0.45 lm membrane filters (millipore). the filtrate was treated with a cocktail of benzonase (novagen) and micrococcal nuclease (new england biolabs) at 37 c for 2 h to digest free-floating nucleic acids, in homemade buffer (1 m tris, 100 mm cacl 2 , and 30 mm mgcl 2 , ph ¼ 8). rna and dna were extracted using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's instructions but without addition of carrier rna to the lysis buffer. first-and second-strand synthesis and random pcr amplification were performed for 17 cycles using a modified whole transcriptome amplification (wta2) kit procedure (sigma-aldrich). denaturation temperature was increased to 95 c to allow for the denaturation of dsdna and dsrna. wta2 products were purified with msb spin pcrapace spin columns (stratec) and were prepared for illumina sequencing using the nextera xt library preparation kit (illumina). libraries were quantified with the kapa library quantification kit (kapa biosystems) and sequencing of the samples was performed on a hiseq tm 2500 platform (illumina) for 300 cycles (150 bp paired ends), generating 67,251,870 reads. raw reads were filtered and trimmed for quality and adapters using trimmomatic (bolger et al. 2014 ) and assembled using spades assembler version 3.5.0 (bankevich et al. 2012) . scaffolds were taxonomically classified using diamond (sensitive option) (buchfink et al. 2015) . orfs were identified with orf finder analysis tools, pfam was used to help predict enterovirus proteins and hmmer to infer insertion similarities (zhang and wood 2003; finn et al. 2011) . amino-acid alignments of the viral sequences were performed with muscle implemented in mega6.0 (edgar 2004) . substitution models for maximum likelihood phylogenetic trees were calculated using mega6.0 (tamura et al. 2013) , and the appropriate best substitution model (with the lowest aic) was used to build phylogenetic trees with 500 bootstrap replicates. a reverse-transcription polymerase chain reaction (rt-pcr) was performed on the original sample using the qiagen onestep rt-pcr kit (qiagen) using primer sequences shown in table 1 (primers were designed to cover the breakpoint between the enterovirus and the torovirus-like sequence). the reaction was performed as follows: 50 c for 30 min followed by a pcr activation step at 95 c for 15 min, 40 cycles of amplification: 30 s at 94 c, 30 s at 55 c, and 2 min at 72 c, and a final extension step for 10 min at 72 c in a biometra t3000 thermocycler (biometra). pcr products were run on a polyacrylamide gel, stained with ethidium bromide, and visualized under uv light. samples were then purified with exosap-it (affymetrix), and positive products were sanger sequenced with the abi prism bigdye terminator cycle sequencing reaction kit (applied biosystems). the raw chromatograms are provided in supplementary files s1 and s2, and the alignments used to generate fig. 1 are also provided as supplementary data. to be able to perform targeted analysis of the proteins, synthetic peptides were designed to determine the preferred m/z and retention time. synthetic peptides covering the enterovirus-torovirus breakpoint, the torovirus insertion and the enterovirus were designed using skyline 3.5.0 (maclean et al. 2010). predicted peptides containing methionine and cysteine were excluded. extraction of proteins from the fecal sample was carried out as previously described by carpentier et al. (2005) and buts et al. (2014) . in short, 350 ll of fecal suspension were resuspended in 350 ll of ice-cold extraction buffer [50 mm tris-hcl ph 8.5, 5 mm edta, 100 mm kcl, 1% w/v dtt, 30% w/v sucrose; complete protease inhibitor cocktail (roche applied science)] and vortexed for 30 s. seven hundred microliters of ice-cold tris buffered phenol (ph 8.0) were added and the sample was vortexed for 10 min at 4 c. after centrifugation (10 min, 12,000âg, 4 c), the phenolic phase was collected, re-extracted with 350 ll of extraction buffer and vortexed for 30 s. after centrifugation (5 min, 12,000âg, 4 c), the phenolic phase was collected and precipitated overnight with five volumes 100 mm ammonium acetate in methanol at à20 c. after centrifugation at 16,000âg for 30 min at 4 c, the supernatant was removed and the pellet was rinsed twice in ice-cold acetone/0.2% dtt. between the two rinsing steps, the sample was incubated for 60 min at à20 c. the pellet was airdried, resuspended in 75 ll of lysis buffer (8 m urea, 5 mm ddt, 30 mm tris dtt), and vortexed for 5 min at room temperature. then the protein concentration was determined using the 2-dquant kit from amersham biosciences. dtt was added to 20 lg of protein extract up to 20 mm of final concentration and incubated for 15 min. then, iodoacetamide was added to the mixture up to a final concentration of 50 mm and incubated for 30 min in the dark. then the sample was diluted 3 times in 150 mm ammonium bicarbonate. for protein digestion, 0.2 lg/ll trypsin was added and incubated overnight at 37 c. samples were acidified with trifluoroacetic acid (0.1% final concentration) and purified with pierce c18 spin columns (thermo scientific) according to the manufacturers' instructions. peptides were eluted with 40 ll and then evaporated using a speedvac. lyophilized peptide samples from the speedvac were then dissolved in a 0.1% v/v formic acid (fa) and 5% v/v acn solution. this was followed by liquid chromatography (lc) coupled to a q exactive hybrid quadrupole-orbitrap mass spectrometer (thermo scientific) in positive ion mode through a nanoelectrospray ion source (thermo scientific). peptides were separated on an ultimate 3000 uplc system (dionex, thermoscientific) equipped with an acclaim pepmap100 pre column (c18 3 lm-100 å , thermo scientific) and an c18 pepmap rslc (2 lm, 50 lm_15 cm, thermoscientific) using a linear gradient (300 ll/min) of 0-4% buffer b (80% acn, 0.08% fa) in 3 min, 4-10% in 12 min, 10-35% in 20 min, 35-65% in 5 min, 65-95% in 1 min 95% for 10 min, 95-5% in 1 min, 5% 10 min. then the separated peptides were analyzed in the orbitrap qe operated in positive ion mode (nanospray voltage 1.5 kv, source temperature 250 c). the instrument was first operated in data-dependent acquisition (dda) mode on the pool of synthetic peptides ordered (pepotec srm custom peptide libraries, thermo scientific) and an inclusion list based on the m/z and rt of the synthetic peptides detected was created (supplementary tables s1 and s2 ). for dda, ms scans were performed at a resolution of 70,000 fwhm for the mass range of m/z 400-1,600 for precursor ions, followed by ms/ ms scans of the top 10 most intense peaks with þ2, þ 3, and þ4 charged ions above a threshold ion count of 16,000 at 35,000 of resolution. ms/ms was performed using normalized collision energy (nce) of 25% with an isolation window of 3.0 m/z, an apex trigger 5-15 s, and a dynamic exclusion of 10 s. data were acquired with xcalibur 2.2 software (thermo scientific). then the sample and synthetic peptides were re-ran in mrm/msms mode (supplementary table s2 ). for peptide identification, raw ms files were converted into mgf.files by proteome discover version 1.4 (thermo scientific) and processed using sequest (eng et al. 1994 ) (ht version 1.3) against a customized in-house built database with sequences from all enterovirus sequences in uniprot and the recombinant enterovirus sequence. a parent mass tolerance of 10 ppm, a fragment tolerance of 0.02 da, a variable modification oxidation of m, a fixed modification with carbamidomethyl c and up to one missed cleavage for trypsin were used. common ms contaminants, such as human keratin and pig trypsin were used as decoy. the amino acid sequence of the novel inserted torovirus-like protein was analyzed using phyre2 by comparing it against the homologous c28 protein of fmdv and the torovirus sh1 protein (kelley et al. 2015) . the three-dimensional predicted models of the latter two proteins were derived from the protein data bank (pbd) and confidence of prediction was retrieved. at the end of january 2015, pedv was identified in a pooled diarrheic fecal sample of fattening pigs from a belgian pig farm. using an in house rt-qpcr assay, a low viral load of pedv was detected, indicating that other viruses may have played a more important role in the pathogenesis of diarrhea in these pigs. therefore, it was aimed to unravel the complete fecal virome of this pooled sample using an ngs approach. after trimming, a total of 57,164,223 ngs reads were assembled de novo into contigs (bankevich et al. 2012; bolger et al. 2014) . a total of 38,118,443 reads were assigned with diamond (buchfink et al. 2015) , of which 5,937,747 were assigned as viruses (16%). from these reads, 2,454,031 could be assigned to the order of the caudovirales (41%) and 537,279 (9%) to the family microviridae, which both contain exclusively bacteriophages. only 2,780 ngs reads (0.1%) were obtained for pedv, which corroborates the low viral load (cq > 30) found by rt-qpcr. a total of 2,246,215 reads could be attributed to (near) complete mammalian porcine virus genomes (table 2) . of these, reads matching the porcine bocavirus (2,110,362 reads, 34%) were far most dominant, followed by 63,317 reads matching porcine enteroviruses. all viruses described hereafter were deposited in genbank and accession numbers are provided in table 2 . in this sample, 71,088 reads were assigned to the genus enterovirus, of which 2 complete genomes could be retrieved. interestingly, a contig of 8,043 nt in length was identified and assigned to enterovirus, with a partial in-frame torovirus-like gene insertion. remapping of curated reads against this recombinant sequence using bwa (li and durbin 2009 ) as well as resequencing the insertion and breakpoints using sanger sequencing on the original sample, confirmed that the obtained sequence was genuine and neither an artifact of the de novo assembly nor a result of random amplification. in fig. 1b , a description of the genome organization of this novel recombinant enterovirus genome (porcine enterovirus b 15v010/bel/2015) is shown. as depicted in fig. 1b , the torovirus-like gene insertion of 636 nt was present between the enterovirus 2 c and 3 a non-structural proteins. the inserted region formed a phylogenetic outgroup most closely related to several toroviruses. in the sample, we observed the presence of a small torovirus contig of 256 bp, which was included in the phylogenetic tree (fig. 1a , torovirus/bel/2015), showing very low similarity with the gene insertion found in the recombinant virus. initial blastp searches attributed the highest aa similarity of this torovirus-like insertion to a porcine torovirus (66% aa similarity with nc_022787), but with the most recent reports of 2 similar recombinant viruses, it now shares 92.0% aa identity with the insertion of the recombinant enterovirus g from north carolina (08/nc_usa/2015) (shang et al. 2017 ) and 90.7% aa identity with the strain from texas (evg/porcine/usa/texas1/ 2014) (knutson et al. 2017) . to infer whether the insertion was produced as a separate moiety or fused with the upstream or downstream enterovirus protein, we identified potential polyprotein cleavage sites, which were present up-and downstream of the insertion (fig. 1b) . pfam searches for motifs using hidden markov models, revealed that the insertion seems to enclose an l-protease from the c28 family (e-value ¼ 1.3e à05 ). we also attempted to infer the function of the insertion using phyre2 (kelley et al. 2015) , which predicted a cysteine proteinase function (99.1% confidence, fig. 1e ). the same function was also predicted for the fmdv l pro , but not for the porcine torovirus that showed the highest sequence homology with the insertion (fig. 1e) . the enterovirus genome region before the torovirus insertion (vp1-vp4, 2 a, 2b, and 2 c) showed its highest similarity on the amino-acid level (94.5%) with evg/porcine/usa/texas1/ 2014 (fig. 1c) . downstream of the torovirus insertion (3 a, 3b, 3cpro, and 3dpol), the virus showed its highest similarity (98.0%) on the amino-acid level with evg 08/nc_usa/2015 (fig. 1d) . even though enteroviruses and toroviruses possess linear ssrna(þ) genomes, they belong to two different viral orders, namely picornavirales and nidovirales, respectively. to further investigate the presence of this highly unusual recombinant virus, we designed synthetic peptides from the 5 0 -agtcttctctcatctactggg-3 0 5016 enterovirus recombinant virus, which we then used for selected reaction monitoring (srm). these synthetic peptides were predicted to be generated after trypsin digestion. this frequently used approach in mass-spectrometry, allows to focus on the detection of a preselected group of peptides. the instrument was run on srm mode on synthetic peptides of the insertion and on the peptide extracted fecal sample. using sequest, we were able to identify two peptides from the insertion region of the enterovirus-torovirus recombinant (table 3 and supplementary tables). table 3 shows the retention time and mass-over-charge ratio (m/z) of both the synthetic peptides and the homologues found in the original sample. bocaviruses are ssdna viruses belonging to the family parvoviridae, which encode an additional orf, named np1, which is absent in the genome of other parvoviruses. in this study, a porcine bocavirus (bel/15v010) that showed 99% identity to the south korean strain pbov-ku14 was identified (fig. 2) . the latter porcine bocavirus was identified in pigs with respiratory problems, which revealed a truncated np1 gene, resulting in the shortest described porcine bocavirus genome thus far (yoo et al. 2015) . the novel bocavirus identified in this study also presented a truncated np1 gene, thought to be caused by cross-over recombination (yoo et al. 2015) . this suggests that this virus strain might be more widespread than initially thought. furthermore, the high number of reads matching to this bocavirus in the sample (%2.1 m) suggests an acute and active replication. apart from the recombinant enterovirus found in the sample, another 12,246 viral reads could be attributed to other enteroviruses. from these, 4,475 reads could be used to assemble a complete porcine enterovirus genome (porcine enterovirus a 15v010/bel/2015). the complete viral polyprotein showed its highest similarity on the amino-acid level (94.8%) with the porcine enterovirus 9 isolate ch-ah-f1 (zhang et al. 2012 ), which was found in 8.3% of screened pigs in china from 2007 to 2009. using recombination detection approaches (program rdp v4) (martin et al. 2015) , no recombination event was detected for this strain, when comparing to all complete sequences of porcine enteroviruses (data not shown). however, the previously described belgian strain 12r021 (theuns et al. 2016 ) seems to have gone through a recombination event, which could explain the distinct clustering in fig. 1c . in this study, we also report the presence of one porcine astrovirus type 2 and one astrovirus type 4 (fig. 3a) . these single-stranded positive sense rna viruses, encode for a capsid polyprotein and a non-structural polyprotein. association of porcine astroviruses with gastrointestinal disease is yet to be made, as they have been often reported as coinfections with rotavirus, coronavirus, and calicivirus (laurin et al. 2011) . the porcine astrovirus 2 (bel/15v010) rna-dependent rna polymerase (rdrp) showed its highest similarity (94.2%) on the amino acid level with a recently described astrovirus in a non-diarrheic belgian piglet co-infected with rotavirus (theuns et al. 2016) . for the porcine astrovirus 4 (bel/15v010), the highest similarity (96.0%) was observed with a hungarian wild boar astrovirus (reuter et al. 2012) , which also forms a common genetic lineage with other porcine astrovirus type 4 strains. furthermore, we identified 4 picobirnavirus capsid segments (fig. 3e ) and 3 rdrp segments (fig. 3d) . picobirnaviruses are bisegmented double-stranded rna viruses, belonging to the picobirnaviridae family. segment 1 encodes for a capsid protein and orf1 with unknown function, while segment 2 encodes for the rdrp gene (ganesh et al. 2012) . up to date, only seven complete sequences for porcine picobirnaviruses are available in (fig. 3b) . figure 3b shows the genome organization of the novel gemycircularvirus and the phylogenetic analysis of known gemycircularviruses based on the amino acid level of the rep gene. the porcine gemycircularvirus bel15v015 presented the typical nonanucleotide stem loop motif (tataaatag) and rolling circle replication motifs i (lftys), ii (hlhvfad), iii (yatkd), grs (rkfdvegfhpnivpsl) and helicase motifs walker-a (grsrtgkt) and walker-b (vfddi). the novel porcine gemycircularvirus replicase shares its highest similarity on the amino-acid level (76%) with mongoose fecesassociated gemycircularvirus b (conceição-neto et al. 2015) (fig. 3b) . the divergent ssdna circular virus (fig. 3c ) encodes for two orfs, bidirectionally transcribed: porcine stool associated circular virus bel/15v010. the complete genome of the virus identified showed its highest similarity with porcine stool associated circular virus 5 isolate cp3 (82.9% nt). interestingly, the capsid gene of the virus shows 91.3% nt similarity with isolate cp3 but only 65.2% similarity with the replicase. however, these two viruses did not cluster closely with other circular single-stranded dna viruses and seem to belong to a new isolated clade (fig. 3c) (cheung et al. 2014) . even though only two viruses from this clade were described so far, both were isolated from diarrheic porcine feces. diarrhea is a major concern in farms leading to growth impairment. in the last years, major awareness was raised in europe due to the emergence of pedv in usa (stevenson et al. 2013) . another recent study characterized a completely novel mammalian orthoreovirus, which was able to induce 100% mortality in experimentally infected piglets, further raising awareness that other viruses than those from the typical diagnostic lists can cause severe problems (thimmasandra narayanappa et al. 2015) . in fact, ngs forms a powerful tool for diagnostics. its application will lead to a better identification of viral enteric disease complexes and will allow investigating the relevance of coinfections of different enteric viruses, which may have remained unnoticed using traditional diagnostic techniques. however, it should be noted that this will lead to an abundance of information generated, which veterinarians and farmers may not be able to cope with without proper guidance. here, an outbreak of diarrhea in a belgian pig farm was reported, 2 days after the arrival of new pigs, suggesting that these novel pigs were the source of infection. since pedv was being reported in europe at the time, it was first diagnostically tested for. even though the sample was positive for pedv (rt-qpcr), the viral loads were rather low (cq > 30). our study does not provide evidence for direct causality of disease by a single viral agent, and the high number of viral reads attributed to a porcine bocavirus, the presence of other possible causative agents of diarrhea such as astroviruses, enteroviruses, and picobirnaviruses, merely indicate that more work needs to be performed to elucidate the role of coinfections in gastrointestinal disease. moreover, none of these viral species are routinely tested for at diagnostic laboratories in cases of diarrhea on pig farms. alongside these findings, an enterovirus with an insertion of a torovirus was described. due to the large number of viral reads attributed to bocavirus and the enterovirus-torovirus recombinant, we attempted to isolate them using primary porcine kidney epithelial cells and st (swine testis) cells (data not shown). although cpe was observed 2 days after inoculation on porcine kidney epithelial cells, neither bocavirus nor the enterovirus-torovirus recombinant could be detected using pcr assays on any of the passaged cells. this is in contrast with the recently described recombinant virus from north carolina, usa, where shang et al. (2017) could isolate the virus using the st cell line. we speculate that due to the large amount of additional non-recombinant enteroviruses in the sample, this might have put the recombinant enterovirus in an in vitro disadvantage. using, pfam and hmmer searches for motifs, the insertion was predicted to enclose an l-protease from the c28 family. this protein has been best studied for foot-and-mouth disease virus (fmdv), a member of the picornaviridae and is known to cleave host cell proteins, namely the p220 subunit of eukaryotic initiation factor 4 f (eif-4 f). the cleavage of this initiation factor 4 f results in the shutoff of cap-dependent host cell protein synthesis, without affecting viral protein synthesis which can occur in the presence of cleaved p220 (piccone et al. 1995) . moreover, our structural analysis confirmed that the function prediction from the structure was a cysteine proteinase (for both our insertion and l pro of fmdv), even though their relatedness in sequence identity is low (fig. 1e ). structure prediction is often important to determine function when sequence similarities are lower. the predicted cleavage sites for the virus could be identified, suggesting that the inserted gene is producing a separate protein. even though a small contig of 256 bp of torovirus from the same insertion region could be detected in our pooled fecal sample, it clustered very distantly from the gene inserted (fig. 1a) . since the fecal sample resulted of a pool of 12 pig feces, this is not surprising and likely has a different host origin. in addition to confirming the presence of the enterovirus-torovirus recombinant using sanger sequencing, we used proteomics to infer whether the protein of the insertion could be found in the sample. this is specially challenging since fecal samples are a very complex matrix. in addition, the torovirus insertion codes for a non-structural protein, which are only present inside infected cells, which might explain why only a few of the predicted peptides could be identified. in recent years, the field of proteomics has evolved greatly, in fact, srm has proven to be a powerful technique to detect and quantify proteins (picotti and aebersold 2012) . this is especially suitable for detecting low abundant peptides, since the mass spectrometer focuses on detecting a preselected group of peptides. taking the metagenomics data, the sanger sequence confirmation and the proteomics results all together we can conclude that this recombinant virus is present in the sample and that the inserted protein is being expressed. interestingly, two recent studies described porcine enteroviruses with a similar insertion (fig. 1a) (knutson et al. 2017; shang et al. 2017 ). one of these viruses was isolated and a knockout mutant virus without the insertion yielded impaired growth and higher expression levels of innate immune genes in infected cells (shang et al. 2017 ). our analyses suggests that these viruses have a common ancestor (knutson et al. 2017) even though a significant diversity was noted among them (>90% aa identity) (fig. 1a) . moreover, the fact that our strain before the insertion clusters with the virus isolated in texas and after the insertion with the virus isolated from both texas and north carolina, can hint that an additional recombination occurred after the insertion event. even though our recombinant strain is the first reported in europe, it is very likely that such viruses might be more widely spread in the pig population, as the in vitro experiments of shang et al. (2017) indicate that the virus might induce higher pathogenesis. while recombinations between enteroviruses are frequently being described (ren et al. 2012) , recombination events between different viral families are more scarcely reported among the virosphere. for example, it is established that coronaviruses encode a gene derived from ancestral influenza c virus (zhang et al. 1992) . with advances and democratization of ngs, these events are more likely to be picked up, as was also recently shown in a study in bats identifying a recombinant bat coronavirus with an inserted reovirus gene (huang et al. 2016) . not unexpectedly, a great number of other eukaryotic viruses were identified using viral metagenomics. however, even though the pathogenic role of pedv has been well demonstrated, for other viruses this link is less clear. for instance, the novel porcine circular virus described in this study is highly similar to another virus also identified in diarrheic pigs (cheung et al. 2014) . therefore, it still remains a possibility that these viruses infect pigs and might play a role in diarrhea, which needs further elucidation. gemycircularviruses, on the other hand, have been identified in a great variety of hosts, and no link with diarrhea has been yet demonstrated. it is likely that these viruses have a diet or plant origin, especially because the virus found in the pig sample clusters together with two gemycircularviruses found in the feces of a healthy mongoose (conceição-neto et al. 2015) . as for the picobirnaviruses, in animals they have been identified in diarrheic and non-diarrheic samples (ganesh et al. 2014) . the main issue with segmented viruses is to link the different segments to the same virus. in our case, we identified four capsid and three rdrp genes, and further research is needed to link the different segments to their respective rdrp. as capsid segments are more divergent than rdrp, it is unexpected that more capsid than rdrp segments were identified. however, it cannot be excluded that there were more picobirnaviruses present in the pool and/or a reassortment event occurred, as previously described (conceição-neto et al. 2016) . as a conclusion, this study raises awareness for the presence of many viruses in a porcine diarrheic fecal sample. in fact, it is important to consider the possibility of diarrhea as a result of the replication of a viral intestinal disease complex, where more than one agent might play a role. given the availability and democratization of next-and third-generation sequencing technologies, this will certainly change the way diagnostics are being performed in the coming decades. however, one should bear in mind that the abundance of information generated with these methods is not easy to interpret and appropriate tools should be developed to help farmers and the pig industry in translating this information into useful management information. supplementary data are available at virus evolution 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orthoreovirus from diarrheic pigs and swine blood meal in the united states', mbio, 6. van dung new variant of porcine epidemic diarrhea virus a novel porcine bocavirus harbors a variant np gene occurrence and investigation of enteric viral infections in pigs with diarrhea in china complete genome sequence of a novel porcine enterovirus strain in china the hemagglutinin/esterase gene of human coronavirus strain oc43: phylogenetic relationships to bovine and murine coronaviruses and influenza c virus a profile hidden markov model for signal peptides generated by hmmer key: cord-259794-6qoksn00 authors: shi, da; shi, hongyan; sun, dongbo; chen, jianfei; zhang, xin; wang, xiaobo; zhang, jialin; ji, zhaoyang; liu, jianbo; cao, liyan; zhu, xiangdong; yuan, jing; dong, hui; wang, xin; chang, tiecheng; liu, ye; feng, li title: nucleocapsid interacts with npm1 and protects it from proteolytic cleavage, enhancing cell survival, and is involved in pedv growth date: 2017-01-03 journal: sci rep doi: 10.1038/srep39700 sha: doc_id: 259794 cord_uid: 6qoksn00 porcine epidemic diarrhea virus (pedv) replicates in the cytoplasm of infected cells, but its nucleocapsid (n) protein localizes specifically to the nucleolus. the mechanism of nuclear translocation, and whether n protein associates with particular nucleolar components, is unknown. in this study, we confirm that a nucleolar phosphoprotein nucleophosmin (npm1) interacts and co-localizes with the n protein in the nucleolus. in vitro binding studies indicated that aa 148–294 of n and aa 118–188 of npm1 were required for binding. interestingly, n protein importation into the nucleolus is independent of the ability of npm1 to shuttle between the nucleus and the cytoplasm. furthermore, overexpression of npm1 promoted pedv growth, while knockdown of npm1 suppressed pedv growth. in addition, binding of n protein to npm1 protects it from proteolytic degradation by caspase-3, leading to increased cell survival. taken together, our studies demonstrate a specific interaction of the n protein with the host cell protein npm1 in the nucleolus. the results suggest potential linkages among viral strategies for the regulation of cell survival activities, possibly through an interaction of n protein with npm1 which prevents its proteolytic cleavage and enhances cell survival, thus ultimately promoting the replication of pedv. how these interactions may lead to porcine disease. the coronavirus n protein is abundantly produced within infected cells. n protein has multiple functions, including as a structural protein that forms complexes with genomic rna, and plays an important role in enhancing the efficiency of virus transcription and assembly. the identification of host proteins targeted by viral proteins during the infection process provides important insights into the mechanisms of viral protein function. to date, interactions of n protein with numerous host cell proteins have been identified, including hcypa 6 , proteasome subunit p42 7 , smad3 8 , hnrnp-a1 9 , the chemokine cxcl16 10 , translation elongation factor-1 alpha 11 , cellular pyruvate kinase protein 12 and 14-3-3 13 . comparative studies among various coronavirus n proteins could aid the development of novel antiviral therapeutics that target interactions between host cell proteins and the n protein 14 . manipulation of multiple host cell factors by a relatively small number of viral proteins is critical for virus replication and spread. given the limited coding capacity of the pedv genome, its protein products must be multifunctional in order to counter host cell antiviral defenses. although originally thought to serve purely structural roles, n proteins of coronavirus are emerging as important players at the virus-host interface. our research group has shown that the pedv n protein localizes not only in the cytoplasm, but also in the nucleolus in infected cells and cells expressing the n protein alone 15 ; however, the factors that determine the nucleolar localization of pedv n protein and the effect of this localization on virus replication are not clearly understood. during infection, a number of viral proteins interact with the nucleolus and are able to reorganize nucleolar antigens 16 , with examples from rna viruses, dna viruses and retroviruses. these include porcine reproductive and respiratory syndrome virus nucleocapsid protein 17 , hepatitis d virus large-delta antigen 18 , marek's disease virus meq protein 19 , the adenovirus iva2 gene product 20 and v protein 21 , newcastle disease virus matrix protein 22 , and human immunodeficiency virus type 1 (hiv-1) rev 23 and tat 24 . the nucleolus is a highly structured and dynamic nuclear organelle that is involved in the transcription of rrna and in ribosome biogenesis 25 . it contains many proteins, including nucleophosmin (npm1/b23), fibrillarin, nucleolin, spectrin, and the ribosomal proteins s5 and l9 26 . in the nucleolus, npm1 plays a role in centrosome duplication, ribosome biogenesis, intracellular transport, apoptosis and mrna splicing 27 . npm1 has been ascribed both growth promoting and tumor suppressive functions 28, 29 . loss of npm1 results in genome instability, which is manifested by aneuploidy, increase in centrosome numbers, and dna damage checkpoint activation [30] [31] [32] . several different types of cancer cell with elevated levels of npm1 are more resistant to uv-or hypoxia-induced apoptosis than those with low expression 33 . the ability of npm1 to suppress apoptosis may play a significant pro-survival role during tumor development 34 . to date, various studies have focused on the nuclear/nucleolar localization properties of the n protein of coronaviruses 15, 35 , but information on the interactions of n protein with nucleolar proteins, and their impact on the outcome of pedv infection, is limited. here, we show that nucleolus protein npm1 interacts specifically with the pedv n protein and positively modulates pedv growth. shown that n protein is localized in the cytoplasm and nucleolus 15 . in this study, to determine the intracellular distribution of n protein at the protein level, pedv-infected vero e6 cells were lysed, separated into nuclear and cytoplasmic fractions, and analyzed by western blotting. as shown in fig. 1a and b, pcna protein was detected only in the nuclear fraction, whereas gapdh was mostly present in the cytoplasmic fraction, confirming the successful separation of the nuclear and cytoplasmic fractions. the n protein was detected in the virus-infected nuclear and cytoplasmic fractions (fig. 1c , lanes 3, 4 and 5). in addition, multiple bands appeared on the western blots, because the n proteins of coronaviruses are phosphorylated in virus-infected cells (fig. 1d ). these results indicate that n is a protein that shuttles between the nucleus and the cytoplasm, which is consistent with the role of n protein in viral replication. n protein interacts and co-localizes with npm1. the nucleolus is structurally divided into three major subcompartments: the fibrillar center, a dense fibrillar component, and a granular component 36 . three distinct proteins that are mainly located in the nucleolus have been identified 37, 38 : the major nucleolar protein fibrillarin, which is a component of a nucleolar small nuclear ribonucleoprotein that is involved in rrna processing; npm1, which is a putative ribosome assembly factor; and nucleolin, which is involved in the processing of precursor rrna. to characterize the level of expression of n, npm1, fibrillarin and nucleolin, encoding plasmids were constructed with myc or 3× flag at the n-terminus of each protein. individual expression plasmids were transiently transfected into hek293t cells, and their expression was assessed using western blotting (fig. s1 ). as measured by western blotting using a monoclonal antibody (mab) against n (see supplementary fig. s1a ), myc (see supplementary fig. s1b ) or flag (see supplementary fig. s1c ) tag, individual expression constructs encoding n, npm1 and fibrillarin proteins were expressed at different levels at the appropriate size, however, the construct encoding nucleolin failed to be expressed. to investigate the possible molecular target of n in the nucleolar, co-immunoprecipitation (co-ip) experiments were performed. the result indicated that n protein showed an interaction with npm1 in the co-ip assay ( fig. 2a,b ). to investigate whether n protein is able to interact with endogenous npm1 in the context of pedv infection, virus-infected vero e6 cell lysates were immunoprecipitated with an anti-npm1 mab and probed for the presence of n protein with anti-n mab. n protein was readily detected in pedv-infected vero e6 cells (fig. 2c) , indicating that n protein indeed interacts with endogenous npm1 protein in pedv-infected vero e6 cells. however, the interaction of n protein with fibrillarin or nucleolin was not observed in the co-ip assay (see supplementary fig. s2a and b) . to verify and extend the binding data obtained in the co-ip assay, we performed glutathione s-transferase (gst)-pull down experiments. the gst or gst-npm1 protein was expressed in escherichia coli and immobilized on glutathione-conjugated sepharose beads. beads carrying gst or gst-npm1 were incubated with lysates from hek293t cells transfected with pcmv-myc-n. after thorough rinsing, the protein complex captured on the beads was solubilized, subjected to electrophoresis in a denaturing gel, and immunoblotted with anti-myc or anti-gst antibody. as shown in fig. 2d , the gst-npm1 protein could pull down myc-n. in contrast, gst alone did not pull down myc-n. these data indicate that n protein can specifically interact with npm1. to examine the co-localization of n protein with npm1, vero e6 cells were co-transfected with plasmids expressing acgfp-n and dsred-npm1 proteins, and the subcellular localization of n protein and npm1 was examined by confocal microscopy (fig. 2e ). imaging indicated that, as previously shown, the acgfp-n protein localized to both the cytoplasm and the nucleolus, but not to the nucleus, in vero e6 cells; dsred-npm1 protein localized to the nucleolus; and co-localization result showed 70.5 ± 14.2% of n protein positive cells were npm1 positive in the nucleolus. moreover, the interaction of n protein with porcine npm1 protein also was validated in our studies (fig. 2f ). in total, these data indicated that n protein is able to interact with npm1 protein. amino acids 148-294 of n protein are responsible for binding to npm1. to define the specific region of n protein required for the interaction with npm1, we used a series of gfp-tagged n protein deletions 15 to map the npm1 binding site on n protein (fig. 3a) . the gst-pull down assay revealed that gst-npm1 bound to gfp-nr2, gfp-nr1+ 2, gfp-nr2+ 3 and gfp-n (fig. 3b ), but not to gfp-nr1 and gfp-nr3. in contrast, gst alone did not pull down gfp-nr2 (fig. 3c) . furthermore, constructs lacking the nr2 domain (gfp-nδ 148-294 ) failed to interact with npm1, suggesting that the nr2 domain of n protein is critical in binding to npm1 (fig. 3d ). the c-terminal of npm1 mediates its interaction with n protein. various functional domains have been identified within npm1, including an n-terminal oligomerization domain (oligod) bearing chaperone activity, the c-terminal nucleic acid binding domain (nbd), and two central acid domains for histone binding (histond). to characterize further the interaction of npm1 and n protein, we mapped the domains of npm1 necessary for its association with n protein, based on the well-known functional domain of npm1, and using a series of gst-tagged npm1 deletion mutants (1-294, 1-117, 118-188, 189-294, 1-188, and 118-294) fused to gst (fig. 3e) . the results indicated that the c-terminal (aa 189-294) is essential for the association of npm1 with n protein (fig. 3f) . after separation by sds-page, proteins were detected by immunoblotting with the indicated antibodies. a 5% aliquot of wcl was also probed to confirm protein expression. the identities of the protein bands are indicated on the right. (c) co-ip of pedv n protein with endogenous npm1. pedv-infected (+ ) or mock-infected (− ) vero e6 cells were used for ip with anti-npm1 protein mab and immunoblotted with the indicated antibodies. the identities of the bands are shown on the right. (d) gst-pull down assay. glutathione beads conjugated to gst or the gst-npm1 fusion protein were incubated with recombinant myc-n. after washing, proteins were eluted from the beads and sds-page was performed. the presence of n protein was detected by immunoblotting with anti-myc mab. gst and gst-npm1 protein expression was confirmed by immunoblotting with mouse anti-gst mab. (e) co-localization of n protein with npm1. vero e6 cells were co-transfected with pacgfp-n and pdsred-npm1. the pedv n protein is colored green and the npm1 fusion protein colored red. merged images are also presented, and the position of the nucleus is indicated by dapi (blue) staining in the merged images. the nucleolus (no) is arrowed where appropriate. lower panels show boxed regions at high magnification. (f) co-ip of hek293t cells co-transfected with recombinant constructs encoding myc-n and 3× flag-tagged porcine npm1. scientific reports | 7:39700 | doi: 10.1038/srep39700 npm1 phosphorylation or sumoylation has no effect in mediating its binding to pedv n protein. it has been shown previously that cdk2/cyclin e-mediated phosphorylation of npm1 on thr-199 promotes dissociation of npm1 from centrosomes, allowing the initiation of centrosome duplication 39 . thus, when the t199a unphosphorylatable mutant is ectopically expressed, t199a binds continuously to centrosomes, resulting in suppression of centrosome duplication 40 . a recent study 41 showed that npm1 can be sumoylated on both lys-230 and lys-263 residues, although lys 263 is the major sumoylation site. mutation of k263 alters its subcellular distribution, and k263r mutation makes npm1 susceptible to caspase-3 cleavage and decreases cell proliferation. intriguingly, thr-199, lys-230 and lys-263 are all located in the c-terminal domain of the npm1 protein, which is essential for the interaction of npm1 with n protein. to explore whether thr-199, lys-230 and lys-263 have a role in the association between npm1 and n protein, we co-transfected various 3× flag-npm1 constructs into hek293t cells with myc-n. co-ip assays demonstrated that the unphosphorylated t199a, unsumoylated k230r and k263r were able to bind myc-n, and had either moderate effects or no effect on n protein binding (fig. 4 ). regions of the nucleolus 42 , is associated with preribosomal particles 43 , and forms pentamers that may be important for the assembly of ribosomes 44 . npm1 has the ability to shuttle between the nucleus and the cytoplasm 45 ; it binds to nuclear/nucleolar localization signal containing peptides 45 , and thus serves as a shuttle protein in nuclear/nucleolar import. interestingly, in this study we also found that the npm1 interacts with n protein, suggesting that npm1 may serve as a shuttle protein for transport of n protein into the nucleolus, although the interaction domain of npm1-n (aa 148-294) is not within the fragment of n protein that contains the nucleolus localization signal (aa 70-90). it is hypothesized that transport of n protein into the nucleolus depends on the movement of npm1 between the nucleus and the cytoplasm. to examine this hypothesis, we co-transfected hek293t cells with pacgfp-n and pdsred-npm1, and time-lapse images were acquired at 12-72 hpt at 5 min intervals. the transport into the nucleolus, interaction with npm1 and export of n protein in transfected cells were clearly observed at 42-43 hpt. as shown in fig. 5a , npm1 protein appeared first in the nucleolus (t = 10 min) and then a small amount of n was observed in the nucleolus at 42 hpt (t = 30 min). n protein accumulated continuously in the nucleolus of transfected cells and interacted with npm1 until t = 30-55 min, and was exported from the nucleolus at t = 55-60 min. real-time visualization of the kinetics of nucleolar import, interaction with npm1 and export of n protein indicated that the process was rapid, taking only 30 min in total, thereby ruling out the possibility that the nucleolar localization of n protein is npm1 independent. the time-lapse video showing the kinetics of nucleolar translocation of n protein and its interaction with npm1 is provided as supplementary material (video s3 in the supplementary materials). to corroborate our findings, we first tested whether depletion of npm1 by specific sirnas resulted in reduced nuclear import of n. for this purpose, three pairs of npm1 sirnas were synthesized. these sirnas were transfected into vero e6 cells, and it was found that npm1 sirna (2+ 3) reduced the level of expression of npm1 (fig. 5b ). in addition, we examined the viability of cells receiving sirna using the cck-8 assay. the results showed that there was no difference between npm1 rnai and rnai control in terms of the viability of transfected cells (see supplementary fig. s4 ). as shown in fig. 5c , transport of n protein was hardly affected in cells with reduced npm1 levels in comparison with that in the cells treated with a scramble sirna (siscr), mock-treated cells. as a control, we also co-transfected vero e6 cells with myc-n and 3× flag-npm1, but did not observe a stimulation of nuclear import of myc-n with the increased expression of the npm1 protein (fig. 5d ). the observation that npm1 interacts with pedv n protein prompted investigation of the relevance of this interaction to the pedv life cycle. vero e6 cells were transfected transiently with 3× flag-npm1 and subsequently . npm1 phosphorylation or sumoylation has no effect in mediating its binding to pedv n protein. hek293t cells were co-transfected with the indicated plasmids, and the wcl obtained at 48 hpt were immunoprecipitated with anti-flag mab. after separation by sds-page, proteins were detected by immunoblotting with the indicated antibodies. a 5% aliquot of wcl was also probed to confirm protein expression. the identities of the protein bands are indicated on the right. densitometric data for myc-n/3× flag-npm1 and mutants from three independent experiments are expressed as mean ± sd. infected with pedv. in these cells, 3× flag-npm1 could be readily detected, and the expression of n protein was increased ( fig. 6a and b) . the results showed that overexpression of npm1 results in upregulation of n protein expression when compared with the empty vector. an increase in viral titer was also observed in the supernatants of these cells (fig. 6c) . furthermore, the expression of npm1 in vero e6 cells infected with pedv was assessed, and the results indicated that pedv infection increases the expression of endogenous npm1 (fig. 6d and e) . given that overexpression of npm1 significantly affected pedv replication in vero e6 cells, it was interesting to investigate whether knockdown of npm1 also affected pedv replication. to this end, sirna-mediated knockdown of npm1 in vero e6 cells infected with pedv was investigated. as shown in fig. 7a and b, knockdown of npm1 resulted in downregulation of n protein expression at 48 or 60 hpi, accompanied by a significant reduction of viral load in the cell culture supernatants in comparison with that in the cells treated with a scramble sirna (siscr), mock-treated cells, or normal vero e6 cells (no treatment) (fig. 7c ). this indicated that growth of pedv was arrested in cells with a reduced level of npm1. together with the results of the overexpression experiments, these data highlight the synergistic action of cellular npm1 expression on pedv replication and n protein expression. programmed cell death, or apoptosis, is an essential event in animal development and is observed in many developing tissues in both invertebrates and vertebrates. the activation of the caspase family is a central event in apoptosis. downstream caspases include caspase-3, the precursor form of which is predominantly synthesized in the cytosol 46 . activated caspase-3 can be translocated from the cytoplasm into the nucleus 47 . caspase-3 is activated by upstream caspases and then cleaves many intracellular target proteins to induce apoptotic cell death; for example, npm1 is a substrate of caspase-3 48 . to explore whether n protein binding has any role in mediating the apoptotic cleavage of npm1, we transiently transfected myc-n and empty vector into vero e6 cells and then treated with or without 100 m m of ac-devd-cho (caspase-3 inhibitor). the cells induced apoptotic cleavage of npm1 by treatment with an apoptosis inducer, staurosporine (sts), or not. apoptotic stimulus clearly demonstrated that npm1 protein cleavage and occurred in empty vector cells following sts treatment. in contrast, npm1 was almost intact when n was overexpressed in vero e6 cells with or no sts treatment, highlighting that nucleolus-targeted n prevents apoptotic degradation of npm1 (fig. 8a) . meanwhile, cleaved caspase-3 was found in sts treated cells (fig. 8a) . furthermore, ac-devd-cho, a caspase-3 inhibitor, was able to restore the sts-induced npm1 and caspase-3 protein cleavage (fig. 8b ). this suggests that interaction with n protein protects npm1 from apoptotic degradation. a number of studies have indicated that npm1, one of the major nucleolar phosphoproteins, is involved in the regulation of nucleolar function during cellular differentiation 49 and in antiapoptosis 48 . to evaluate whether the n-npm1 complex is important in prevention of apoptosis, we studied vero e6 cells transiently expressing myc or myc-n and induced apoptosis by sts. compared with cells with no sts treatment, a dna fragmentation assay revealed that overexpression of pedv n protein slightly diminished dna degradation. by contrast, robust dna fragmentation was detected in cells transiently expressing myc tag (fig. 8c ). to evaluate further the antiapoptotic effect of n protein in vivo, we examined the sensitivity of vero e6 cells transfected with myc-n or empty vector and with induction of apoptosis by treatment with sts or not. dapi and tunel staining of the nucleus revealed that myc-n transfected cells displayed greater viability after stimulation by sts and were markedly less sensitive to sts-induced apoptosis than empty vector transfected cells (fig. 8d) . collectively, these data demonstrate that the n-npm1 interaction plays an essential role in protecting cells from apoptotic degradation, thus promoting cell survival. the interaction of viral proteins with nucleolar antigens may explain why viral proteins have been observed in the nucleolus and may also explain the viral exploitation of nucleolar function, leading to alterations in host cell transcription and translation, and disruption of the host cell cycle to facilitate viral replication. our previous 15 and current studies (fig. 1) indicate that the pedv n protein is actively transported to the nucleolus during the time course of pedv infection. the function of n protein during pedv infection is thought to require interaction with cellular proteins, therefore in this study we investigated whether the pedv n protein interacts with three major nucleolar antigens: npm1, fibrillarin and nucleolin. interaction with one or all of these antigens may explain our previous observations that pedv n protein is localized to the nucleolus 15 . proteins that localize to the nucleolus have been reported to be involved in cell growth, the cell cycle and cell survival 25, 41 . in the current studies, we also wished to investigate whether interaction of the n protein with nucleolar proteins affects pedv replication. this study was based on different lines of evidence, reflecting both in vivo and in vitro situations, and demonstrated that pedv n protein is able to associate with the major nucleolar protein npm1 of vero e6 cells (fig. 2) ; we failed to detect an interaction with the fibrillarin or nucleolin (see supplementary fig. s2 ). the n protein also interacted with porcine npm1 (fig. 2f ). in the immunoprecipitation ( fig. 2a,b ) and the gst-npm1 fusion protein pull down assay (fig. 2d) , both in vitro translated and cellular npm1 were shown to interact with n protein. the immunoprecipitation experiment in pedv-infected vero e6 cells provided further support for the in vivo binding of npm1 and n protein (fig. 2c) . the confocal microscopy analysis showed the co-localization of npm1 and n in the nucleolus (fig. 2e) , and the in vitro binding studies utilizing deletion mutants of n or npm1 defined the binding sites of these two proteins. as shown in fig. 3b,3c and 3d , apart from the full-length protein, only the n variants containing the nr2 fragment, nr2, nr1+ 2 and nr2+ 3, but not the c-terminal or n-terminal fragment, were able to bind to npm1, suggesting that the domain that interacts with npm1 is within amino acid residues 148-294 of n. interestingly, this region of n consists of an sr-domain, containing serine and arginine residues 50 , and is involved in cell signaling and post-translational modifications such as phosphorylation 51 . npm1 scientific reports | 7:39700 | doi: 10.1038/srep39700 has been reported to bind to the arginine-rich basic region of the human t-cell leukemia virus protein rex 52 , and to the hiv proteins rev 23 and tat 53 . however, further studies will be required to determine more precisely the location of the interaction domain and the specific amino acid residues that participate in the interaction. the protein npm1 is multifunctional and exhibits nucleic acid binding, ribonuclease activity, and molecular chaperone activity 54, 55 . these three activities reside in nearly independent but partially overlapping segments of the polypeptide chain 56 . the n-terminal nonpolar region and the acidic region of the middle portion of npm1 are important for its chaperone activity, and the c-terminal is essential for nucleic acid binding 56 . interestingly, analysis of the binding sites of targeting proteins on npm1 has revealed that most of them reside in the c-terminal portion of the molecule. for example, npm1 binds to the nucleolar proteins p120 57 , nucleolin 58 , and tat 53 through a fragment of npm1 containing amino acids 187-215 or 194-239. this is in accordance with the interaction of n protein with npm1, which occurs at the c-terminal portion of npm1 (fig. 3f) . the binding region of npm1 for viral proteins hdag 59 and rex 52 has been localized to these acidic regions. therefore, these results suggest that the interaction of npm1 with n protein is similar to the interactions with nucleolar protein p120, nucleolin, and tat, but is different from the interaction of npm1 with the viral proteins hdag and rex. the localization of viral proteins to the nucleolus generally occurs through the interactions of basic regions on the viral protein with stretches of acidic residues on nucleolar proteins such as npm1 and nucleolin 60, 61 . upon binding to viral protein in the cytoplasm or in the nucleus, npm1 and nucleolin function as shuttle proteins, directing the transport of viral proteins across the nuclear pore complex into the nucleoplasm and then to the nucleolus. however, the transport of n protein from the cytoplasm into the nucleolus was not dependent on the shuttle protein npm1 in this study (fig. 5 and video s3 in the supplementary materials). one possible reason is that the n protein may bind to importin α and importin β ; both play essential roles in the nuclear transport of proteins through the nuclear pore complex. whether other viral proteins or host factors are involved in the nuclear transport of pedv n protein needs to be determined in the future. the role of n protein during its interaction with npm1 may represent a unique function of n protein in the nucleolus. npm1 is a multifunctional protein involved in many cellular and viral activities. in particular, npm1 interacts with viral proteins from several different viruses and promotes viral replication cycles. interaction between npm1 and adenoviral protein v promotes virus assembly during virion maturation 21 . npm1 also forms a complex with hepatitis delta virus (hdv) antigens to enhance replication of hdv rna 59 . in this study, we also demonstrated the synergistic action of cellular npm1 expression on pedv replication and n protein expression (figs 6 and 7) . these interactions link npm1 with the viral life cycle as an important protein for viral replication. apoptosis is an important mechanism by which virus-infected cells are eliminated from the host. accordingly, many viruses have evolved strategies to prevent or delay apoptosis in order to provide a window of opportunity in which virus replication, assembly and egress can take place. interfering with apoptosis may also be important for establishment and/or maintenance of persistent infections. with few exceptions, most studies of virus-encoded antiapoptotic proteins have focused on dna viruses. the known exceptions are the picornavirus-encoded proteins leader and 2bc 62, 63 , as well as the rubella virus capsid protein 64 . although infection with these viruses induces apoptosis in many cell lines, this is generally observed late in the infection process. in this report, we have demonstrated that the major isoform of the pedv n protein in infected cells functions to block apoptosis. the protective capacity of n protein is dependent on interaction with npm1; it protects it from proteolytic cleavage, enhancing cell survival, and positively regulates pedv replication and growth. in summary, the key findings of this study are the identification of nucleolus protein npm1 as a novel interacting partner of the pedv n protein. that npm1 promotes pedv growth is due to n protein inhibition of caspase-3-mediated cleavage of npm1, which prevents proteolytic cleavage of npm1 and enhances host cell survival. the identification and characterization of the interaction of pedv n protein with npm1 with the resultant alteration in host cell survival may facilitate the development of vaccines and therapeutics for use in pigs. cells were purchased from atcc, grown in dulbeccos modified eagle's medium (dmem) supplemented with 10% heat-inactivated fetal bovine serum (fbs) and penicillin-streptomycin, and incubated at 37 °c in 5% co 2 . the pedv strain cv777 was propagated in pedv-infected vero e6 cells. virus titers in the culture supernatants of pedv-infected vero e6 cells were determined by the reed-muench method. plasmids. the plasmids expressing gfp-tagged n, nr1, nr2, nr3, nr1+ 2 and nr2+ 3 have been described previously 15 . the npm1 and fibrillarin genes were amplified from pdsred-npm1 15 and the genome of vero e6 cells, respectively. both were cloned into a p3× flag-cmv-10 vector (e7658; sigma) with the ecori and kpni restriction enzymes. the pedv n protein gene was cloned into the pcmv-myc vector (631604; clontech) with the sali and kpni restriction enzymes to generate the pmyc-n plasmid. for bacterial expression of the gst-tagged npm1 protein, the npm1 protein gene region was subcloned into the pgex-6p-1 vector (28-9546-48; ge healthcare), creating pgex-npm1. a series of mutant forms of npm1 was generated from pdsred-npm1 by conventional pcr with the mutagenesis primers listed in table 1 . additionally, δ nr2, npm1 (t199a), npm1 (k230r) and npm1 (k263r) were generated by overlapping pcr. all plasmids were verified by sequencing. plasmid dna transfection. cells in six-well plates (corning) cultured at 37 °c in a humidified incubator with 5% co 2 were transfected with the respective plasmids (3 m g each) using the attractene transfection reagent (301005; qiagen) according to the manufacturer's instructions. at 6 h post-transfection (hpt), the transfection mixture was replaced with complete growth medium and incubated for an additional 48 h before being used for assays. after dna or small interfering rna (sirna) transfection, cells were infected with pedv strain cv777 at a multiplicity of infection (moi) of 0.1. after 1 h, the viral inoculum was removed and the infected cells were washed three times with phosphate-buffered saline (pbs; ph7.4) and re-fed with dmem containing 1 m g/ml trypsin. at various time points post-infection, cell-free culture supernatants and cell lysates were harvested and stored at − 80 °c until use. preparation of nuclear and cytoplasmic fractions. nuclear and cytoplasmic fractions were prepared as described previously 65 . briefly, treated vero e6 cells were scraped into ice-cold pbs, centrifuged at 3000 × g, and resuspended in ice-cold buffer a (10 mm hepes [ph 7.9], 10 mm kcl, 0.1 mm edta, 0.1 mm egta, 1 mm dithiothreitol), and then nonidet p-40 (final concentration, 0.1%) was added. the cells were lysed by five strokes of a dounce tissue homogenizer (bellco glass). the nuclear fraction was pelleted by centrifugation at 12,000 × g for 30 s at 4 °c. the supernatant was used as the cytoplasmic fraction. to ensure that the subcellular fractions were separated properly, subcellular lysates were verified by the antibodies against the corresponding fractions. these antibodies were anti-glyceraldehyde-3-phosphate dehydrogenase (gapdh) for the cytoplasm and anti-proliferating cell nuclear antigen (pcna) for the nucleus. gst-pull down assays. for the gst-pull down assays, gst or gst-npm1 protein produced in escherichia coli bl21 (de3) cells was conjugated to glutathione beads (10049253; ge biosciences) and blocked for 1 h in 5% bovine serum albumin. the beads were then washed three times with tif buffer (20 mm tris-hcl [ph 8.0], 150 mm nacl, 1 mm mgcl 2 , 0.1% nonidet p-40, 10% glycerol, 0.1 mm dithiothreitol, 1 mg/ml protease inhibitor) and incubated for 6 h at 4 °c with recombinant myc-tagged n harvested from transfected hek293t cells. the beads were washed at least five times with tif buffer, followed by elution and detection of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and immunoblotting. hek293t cells were transfected with the indicated constructs as described above. the transfected cells were harvested at 48 hpt, washed three times with cold pbs (ph 7.4), and lysed with ip lysis buffer (87788; thermo) containing 1 mm phenylmethylsulfonyl fluoride (pmsf) and 1 mg/ml protease inhibitor cocktail (04693132001;roche) at 4 °c for 30 min. clarified extracts were precleared with protein a/g beads (sc-2003; santa cruz) and then incubated with protein a/g beads plus anti-flag (f3165; sigma), myc (c3956; sigma) or npm1 (pla0253; sigma) mab for 6-8 h. the beads were then washed with ip lysis buffer and boiled in sample buffer, and the proteins were subjected to sds-page, followed by immunoblotting analysis with anti-flag, anti-myc and anti-n mab. table 1 . primers used in this study. objective. all images were acquired with 500-ms exposures under the same illumination conditions and analyzed using las af 1.8.2 software (leica laser technik; germany). sirnas targeting npm1 were used at a final concentration of 200 nm, unless otherwise stated. cells were transfected with sirnas with x-tremegene sirna transfection reagent (4476093001; roche) as described previously 66 the sirna target sequences of npm1 were ggaagatgcagagtcagaatt (sinpm1-2) and ggaagccaagttcatcaattt (sinpm1-3). western blotting was used to analyze endogenous npm1 protein production with anti-npm1 mab. confocal imaging. vero e6 cells were seeded on microscope slide coverslips, which were set in 35-mm diameter dishes, and grown to a confluence of ~50%. at 48 hpt, the cells were fixed with 4% paraformaldehyde for 30 min. then the nucleus was stained with 4′ ,6-diamidino-2-phenylindole (dapi) (0.05 m g/ml) (d9542; sigma) for 15 min and analyzed by laser confocal scanning microscopy (leica laser technik; germany). western blotting analysis. total cellular proteins were extracted with the ripa lysis buffer (r0278; sigma) and the concentrations were determined with a pierce ® bca protein assay kit (23225; thermo). the total proteins (100 m g) were subjected to sds-page, and separated protein bands were electro-transferred onto a nitrocellulose membrane (66485; pall) using a semidry blotter (bio-rad). the membrane was soaked in blocking buffer (pbs containing 5% nonfat milk) for 2 h and then reacted with the indicated antibodies: licor biosciences), and thereafter the blots were visualized using an odyssey infrared imaging system (licor biosciences). quantification of band intensities by densitometry was carried out using the image j software. dna fragmentation assay. oligonucleosomal fragmentation of genomic dna was investigated as described below. briefly, 3 × 10 6 cells in 10 ml of medium were incubated with 250 nm of staurosporine (sts) for 18 h. after incubation, the cells were lysed on ice for 60 min in 500 m l lysis buffer (0.02% sds/1% nonidet p-40/0.2 mg/ml proteinase k in pbs). genomic dna was extracted by the phenol/chloroform method. the pellet was dissolved in 50 m l of te buffer (10 mg/ml rnase) for 2 h at 37 °c. a total of 10 m g of dna was loaded on a 2% agarose gel and visualized under uv light. terminal deoxynucleotidyl transferase-mediated dutp-biotin nick end labelling (tunel) assay. the tunel assay was performed using the in situ cell death detection kit, fluorescein (11684795910;roche), following the manufacturer's instructions. in brief, vero e6 cells treated or untreated with sts were fixed in 4% paraformaldehyde for 20 min at room temperature, washed with pbs, and permeabilized with freshly prepared 0.1% triton x-100 and 0.1% sodium citrate for 2 min on ice. after washing with pbs, the cells were overlaid with 100 m l of tunel reaction mixture, according to the manufacturer's instruction, and incubated for 1 h at 37 °c. finally, the cells were washed with pbs, then the nucleus was stained with dapi for 15 min and directly analyzed under a fluorescence microscope using an exciting wavelength in the range of 450-500 nm (488 was used in this experiment) and detection in the range of 515-565 nm. quantification of the green fluorescence-positive cells was performed by taking the average of at least six fields of view. cell viability assay. the cell viability assay was performed using the cell counting kit-8 (cck-8) (ck04; dojindo) according to the manufacture's protocol. in brief, vero e6 cells were seeded in a 96-well plate at a density of 10,000 per well and incubated at 37 °c for 24 h. the cells were transfected with sirnas (or not), and the plates were incubated for 48 h. subsequently, 10 m l of cck-8 was added to each well, and the cells were further incubated for 2 h. the optical density at 450 nm was measured. the viability of the treated cells was expressed as a percentage relative to the untreated cells. statistical analysis. statistical analysis was performed using spss 19.0 software. variables are expressed as mean ± sd. student's t test and one-way analysis of variance 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the nucleolar protein no38 in amphibian oocytes translocation of nucleolar phosphoprotein b23 (37 kda/pi 5.1) induced by selective inhibitors of ribosome synthesis function of homo-and hetero-oligomers of human nucleoplasmin/nucleophosmin family proteins npm1, npm2 and npm3 during sperm chromatin remodeling major nucleolar proteins shuttle between nucleus and cytoplasm caspases disrupt the nuclear-cytoplasmic barrier spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects increased stability of nucleophosmin/b23 in anti-apoptotic effect of ras during serum deprivation down-regulation of nucleophosmin/b23 during retinoic acid-induced differentiation of human promyelocytic leukemia hl-60 cells identification of in vivo-interacting domains of the murine coronavirus nucleocapsid protein intrinsically unstructured proteins and their functions nucleolar targeting signal of rex protein of human t-cell leukemia virus type i specifically binds to nucleolar shuttle protein b-23 protein b23 is an important human factor for the nucleolar localization of the human immunodeficiency virus protein tat preferential cleavage in pre-ribosomal rna byprotein b23 endoribonuclease nucleolar protein b23 has molecular chaperone activities mapping the functional domains of nucleolar protein b23 identification of the nuclear and nucleolar localization signals of the protein p120. interaction with translocation protein b23 c23 interacts with b23, a putative nucleolar-localization-signal-binding protein the nucleolar phosphoprotein b23 interacts with hepatitis delta antigens and modulates the hepatitis delta virus rna replication structure and functions of nucleolin molecular dissection of nucleolin's role in growth and cell proliferation: new insights antiapoptotic activity of the cardiovirus leader protein, a viral "security coxsackievirus protein 2bc blocks host cell apoptosis by inhibiting caspase-3 the rubella virus capsid is an anti-apoptotic protein that attenuates the pore-forming ability of bax a high-efficiency hela cell nuclear transcription extract antagonistic effects of cellular poly(c) binding proteins on vesicular stomatitis virus gene expression key: cord-254192-86ksgl5t authors: li, liang; xue, mei; fu, fang; yin, lingdan; feng, li; liu, pinghuang title: ifn-lambda 3 mediates antiviral protection against porcine epidemic diarrhea virus by inducing a distinct antiviral transcript profile in porcine intestinal epithelia date: 2019-10-17 journal: front immunol doi: 10.3389/fimmu.2019.02394 sha: doc_id: 254192 cord_uid: 86ksgl5t type iii interferon-lambda (ifn-λ) plays a critical role against infection, particularly in mucosal infection in the respiratory and gastrointestinal tract. our study and other previous studies have shown that porcine ifn-λ more efficiently curtails the infection of porcine epidemic diarrhea virus (pedv) in the intestine epithelia than type i ifn, whereas ifn-λ3 exerts a more potent effect than ifn-λ1. however, the underlying mechanism remains elusive, and in particular, the transcriptional profile induced by ifn-λ3 has not been reported. here, to resolve the mechanism responsible for the disparity between ifn-λ3 and type i ifn in anti-mucosal virus infection, we compared the transcription profiles induced by the two ifns in porcine intestinal epithelial (ipec-j2) cells by rna-seq. our results showed that the pretreatment of ipec-j2 cells with ifn-λ3 resulted in the differential expression of 983 genes. in contrast, ifn-α only modified the expression of 134 genes, and 110 of these genes were also observed in the response to ifn-λ3. a transcriptional enrichment analysis indicated that ifn-λ3 or ifn-α regulates multiple cellular processes and that ifn-λ3 activates more robust signaling pathways, particularly the antiviral jak-stat signaling pathway, than ifn-α. furthermore, we verified the rna-seq results through an rt-qpcr analysis of ipec-j2 cells and porcine enteroids. moreover, transient expression of the porcine rsad2 and mx2 genes among the top 10 genes induced by ifn-λ3 significantly inhibited pedv infection. collectively, the data showed that ifn-λ3 induces a unique transcriptional profile that does not completely overlap with that induced by ifn-α and strongly elicits a set of genes responsible for the antiviral activity of ifn-λ3. these findings provide important knowledge regarding the elicited isgs of type i and iii ifns in restricting porcine intestinal viral infection. type iii interferon-lambda (ifn-λ) plays a critical role against infection, particularly in mucosal infection in the respiratory and gastrointestinal tract. our study and other previous studies have shown that porcine ifn-λ more efficiently curtails the infection of porcine epidemic diarrhea virus (pedv) in the intestine epithelia than type i ifn, whereas ifn-λ3 exerts a more potent effect than ifn-λ1. however, the underlying mechanism remains elusive, and in particular, the transcriptional profile induced by ifn-λ3 has not been reported. here, to resolve the mechanism responsible for the disparity between ifn-λ3 and type i ifn in anti-mucosal virus infection, we compared the transcription profiles induced by the two ifns in porcine intestinal epithelial (ipec-j2) cells by rna-seq. our results showed that the pretreatment of ipec-j2 cells with ifn-λ3 resulted in the differential expression of 983 genes. in contrast, ifn-α only modified the expression of 134 genes, and 110 of these genes were also observed in the response to ifn-λ3. a transcriptional enrichment analysis indicated that ifn-λ3 or ifn-α regulates multiple cellular processes and that ifn-λ3 activates more robust signaling pathways, particularly the antiviral jak-stat signaling pathway, than ifn-α. furthermore, we verified the rna-seq results through an rt-qpcr analysis of ipec-j2 cells and porcine enteroids. moreover, transient expression of the porcine rsad2 and mx2 genes among the top 10 genes induced by ifn-λ3 significantly inhibited pedv infection. collectively, the data showed that ifn-λ3 induces a unique transcriptional profile that does not completely overlap with that induced by ifn-α and strongly elicits a set of genes responsible for the antiviral activity of ifn-λ3. these findings provide important knowledge regarding the elicited isgs of type i and iii ifns in restricting porcine intestinal viral infection. the surface epithelia of the mucosa are the major entry site of most pathogens in the host and serve as a first line of defense against invading pathogens. one of the most important antiviral cytokines in the host is interferons (ifns), which perform key roles in inhibiting viral infection (1, 2) . the ifn family is categorized into three different types: type i ifn (ifn-α/β), type ii ifn (ifn-γ), and type iii ifn (ifn-λ). type ii ifn, which is primarily produced by t cells and natural killer cells, exerts limited direct antiviral activity and plays a key role in modulating the host immune response (3) , whereas type i ifns (α/β) and the more recently discovered type iii ifns induce a strong antiviral state in responsive cells and play crucial roles in controlling viral infection (4) (5) (6) (7) (8) . although type i ifns have generally been thought to be a key element against systemic infections, recent research has shown that ifn-λ plays a critical role in mucosal infections, such as enteric infection (9, 10) . unlike type i ifns that are secreted by a wide range of different cell types upon stimulation, type iii ifns are primarily produced by epithelial cells, nk cells, and dendritic cells (dcs) (8, (11) (12) (13) . ifn-λ acts primarily on the mucosal epithelium, which might result in fewer side effects compared with type i ifn treatment (8) . these features make ifn-λ a potentially superior antiviral therapeutic candidate against local mucosal infection (7) . although the receptors for type i and iii ifns are different, the binding of both type i and iii ifns to their corresponding receptors stimulates a janus kinase (jak)-signal transducer of transcription (stat) pathway, and the stimulation of this pathways subsequently drives the transcription of ifnstimulated genes (isgs) and prompts cells toward an antiviral status (14) . consistent with the similarity of the induced signaling pathways, the spectrum of genes elicited by the two types of ifns show a high overlap (2) . however, recent studies have demonstrated that type iii ifns are critical non-redundant antiviral mediators of type i ifns in the gi tract (2) . to date, numerous studies in humans or mice have taken advantage of rna-seq or chip assays to show that ifn-λ and ifn-α elicit distinct downstream signaling events, even though many genes are induced by both type i and iii ifns (15, 16) . mice with type i ifn or iii ifn receptor knockout experience more severe viral intestinal infections, but ifnl −/− mice show higher viral loads and more serious clinical symptoms than ifnar −/− mice (17, 18) . studies conducted by pott et al. showed that intestinal epithelial cells exhibit stronger responses to ifn-λ compared with ifn-α/β in vivo (19, 20) . a comprehensive understanding of the unique signaling profiles of type i and iii ifns has become increasingly important for understanding host-virus interactions and the development of ifn-λ therapeutics. however, thus far, no direct comparative analyses of the transcriptional profiles induced by porcine type i vs. type iii ifns in swine intestinal epithelia have been performed. the piglet diarrhea caused by enteric coronavirus porcine epidemic diarrhea virus (pedv) is a highly contagious disease characterized by watery diarrhea, dehydration, and causes up to 100% mortality in neonatal piglets. we and other research groups previously reported that porcine ifn-λ results in better suppression against pedv infection compared with ifn-α and that ifn-λ3 more efficiently inhibits pedv than ifn-λ1 (21) (22) (23) . however, the mechanisms underlying the difference among ifn-λ1, ifn-λ3, and ifn-α in inhibiting enteric coronavirus remain less clear. previous studies have largely focused on the gene profiles induced by human or mouse ifn-λ1 and ifn-α, but the ifn-λ3-and ifn-α-elicited genes have not been compared. in this study, we comprehensively compared the transcriptional profiling of ifn-λ3-and ifn-α-induced genes in a porcine intestinal epithelial cell line (ipec-j2) and verified the rna-seq results by reverse transcriptase quantitative pcr (rt-qpcr) in vitro, and further confirmed the transcriptional profile difference in crypt-derived porcine enteroids. the intestinal porcine epithelial cell line j2 (ipec-j2; kindly provided by dr. anthony blikslager, north carolina state university, raleigh, nc, usa) was maintained in dulbecco's modified eagle's medium nutrient mixture f-12 (dmem/f12) supplemented with antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin), 0.1 mm hepes (gibco, usa), and 10% heat-inactivated fetal bovine serum (fbs) (gibco). african green monkey kidney cells (vero e6) were grown and maintained in dmem supplemented with antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin) and 10% heat-inactivated fbs (gibco). pedv strain cv777 of genotype 1 (genbank accession no. kt323979) was maintained at the harbin veterinary research institute of the chinese academy of agricultural sciences, harbin. the biological antiviral activity of e. coli-derived recombinant porcine ifn-lambda 3 was prepared in our laboratory and evaluated in mdbk cells using a recombinant vesicular stomatitis virus (vsv) with a gfp reporter as described previously (22, 24) . the weight-activity unit (u/ml) of samples was calculated using porcine prokaryotic-derived ifn-α (4.0 × 10 8 u/mg) (prosit sole biotechnology, co., ltd., beijing, china) as a reference. porcine intestinal crypts were prepared from specific pathogenfree piglets using previously described protocols (21) . in brief, the intestine was flushed with cold pbs with antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin), cut into 2mm segments, and washed with cold pbs with antibiotics until the supernatant was clear. the washed intestinal pieces were suspended in 15 ml of gentle cell dissociation reagent (stemcell, canada) and shaken at 100 rpm for 25 min to disassociate the crypts at room temperature (rt). the pellets of the intestinal pieces were suspended in 10 ml of cold pbs with 0.1% bovine serum albumin (bsa) and antibiotics (pen-strep) and passed through a 70-µm cell mesh. the crypt pellets were harvested by centrifugation at 200 × g at 4 • c for 5 min and resuspended in 10 ml of cold dmem/f12. after counting, the intestine crypts were resuspended in 25 µl of intesticult organoid growth medium (stemcell, canada) and 25 µl of matrigel (bd biosciences, usa) per 50 crypts and seeded into a 48-well plate at 50 crypts per well. the plate was incubated at 37 • c for 10 min until the matrigel solidified. the plate was filled with complete intesticult organoid growth medium and then incubated at 37 • c in a 5% co 2 incubator. the culture medium was exchanged every 3-4 days. the institutional animal care and use committee of the harbin veterinary research institute approved all the protocols related to the animal experiments performed in this study. expanded 3d enteroids were recovered from the matrigel after 7-11 days of growth by the addition of ice-cold dmem/f12 medium, transferred into 15-ml tubes, and centrifuged at 250 × g at 4 • c for 5 min. the pellet of enteroids was incubated in 0.25% trypsin (gibco) for 5 min at 37 • c and dissociated by repeated pipetting to obtain a single-cell suspension. dmem-f12 with 10% (v/v) fbs was added into the single-cell suspension, and the mixture was centrifuged at 800 × g for 5 min. the cell pellets were resuspended in complete intesticult organoid growth medium at rt and seeded at 50 enteroids per well in a matrigel-precoated 96-well plate. after differentiation for about 3-4 days, planar monolayers of 2d enteroids were ready for use in experiments. total cellular rna was extracted using the simply p total rna extraction kit (bioflux, china) according to the manufacturer's instructions. total rna (1 µg) was reverse-transcribed to cdna using the primescript tm ii first-strand cdna synthesis kit (takara, china). the synthesized cdna was subjected to qpcr performed in triplicate using a lightcycler r 480 ii real-time pcr instrument (roche, switzerland) and sybr green pcr mix (life technologies, usa) according to the manufacturer's instructions. all the data were acquired and analyzed using lightcycler r 480 ii software 1.5 based on the cycle threshold ( ct) method (25) . gapdh served as the internal control. the amplification efficiency of qpcr primers ranged from 85.83 to 106.38%. the primers used in this assay were designed using primer premier 5 software and are listed in table 1 . three biological replicates of each of the three groups, namely, untreated ipec-j2 cells (mock control), ipec-j2 cells treated with ifn-λ3 (1,000 ng/ml) for 24 h, or ipec-j2 cells treated with ifn-α (1,000 ng/ml) for 24 h, were prepared for rna sequencing. total rna was purified using the trizol reagent according to the manufacturer's instructions (thermo fisher scientific, usa). the total rna from each sample was quantified and qualified using an agilent 2100 bioanalyzer (agilent technologies, usa), a nanodrop instrument (thermo fisher scientific, inc.), and a 1% agarose gel. one microgram of total rna with a rin value >7 was used for subsequent library construction. next-generation enzyme mix to repair both ends and add a da tail through one reaction, and the product was subjected to t-a ligation to add adaptors to both ends. size selection of adaptor-ligated dna was then performed using an axyprep mag pcr clean-up kit (axygen), and fragments of ∼360 bp (with an approximate insert size of 300 bp) were recovered. each sample was then amplified by pcr for 11 cycles using the p5 and p7 primers carrying sequences that can anneal during bridge pcr with a flowcell and the p7 primer carrying a six-base index to allow multiplexing. the pcr products were cleaned up using the axyprep mag pcr clean-up kit (axygen), validated using an agilent 2100 bioanalyzer (agilent technologies, usa), and quantified with a qubit 2.0 fluorometer (invitrogen, usa). libraries with different indices were then multiplexed and loaded on an illumina hiseq instrument (illumina, usa) according to the manufacturer's instructions. sequencing was performed using a 2 × 150-bp paired-end (pe) configuration and image analysis and base calling were conducted using hiseq control software (hcs) + olb + gapipeline-1.6 (illumina) provided with the hiseq instrument. rna-seq was performed and analyzed using genewiz9 (suzhou, china). the analysis of the microarray data to identify differentially expressed genes was performed using edger software. the analysis starts with the count, and the data are standardized by tmm and then subjected to differential expression analysis. we selected | log 2 (fold change) (logfc) | > 1 and fdr < 0.05 as the screening criteria for the identification of differentially expressed genes. ipec-j2 or vero e6 cells were fixed with 4% paraformaldehyde for 30 min at rt. the fixed cells were permeabilized with 0.2% triton x-100 for 20 min at rt and then blocked with blocking buffer (pbs with 5% fbs and 5% skim milk) for 30 min at 37 • c. the cells were then incubated with pedv n protein antibody at 37 • c for 2 h and then labeled with alexa fluor 546 goat anti-mouse igg antibody (thermo fisher scientific, usa) at 37 • c for 1 h. dapi (sigma, usa) was used for the staining of cellular nuclei. the stained cells were visualized using an amg evos f1 fluorescence microscope (thermo fisher scientific, usa). the porcine rsad2 or mx2 coding region was amplified by rt-pcr, and cdna was prepared using the primescript tm ii 1st strand cdna synthesis kit (takara, china). rsad2 or mx2 was then amplified using a pair of primers specific for porcine rsad2 or mx2 (table 1) , respectively. the pcr products were purified, digested, and cloned into pcaggs-ha through ecor i and kpn i. construction of the prsad2/pmx2-ha expression plasmid was confirmed by sequencing. vero e6 cells were grown in 48-well plates to 70-80% confluence and then transfected with the prsad2/pmx2-ha expression plasmid using the attractene transfection reagent (qiagen), and the expression of prsad2/pmx2 was verified by anti-ha ifa. graphpad prism software version 7 was used to analyze the experimental results. the results are expressed as scatter plots in which the mean is indicated by a line. the differences between groups were compared using an unpaired mann-whitney test. p < 0.05 was considered significant, and the p-values are expressed as follows: * p < 0.05, * * p < 0.01, * * * p < 0.005, and * * * * p < 0.001. the inhibition of pedv in ipec-j2 cells by exogenous ifn-λ3 is superior to that achieved with ifn-α according to our study and other previous studies (21) (22) (23) , ifn-λ1, ifn-λ3, and ifn-α all significantly inhibit pedv infection, but ifn-λ3 shows the strongest antiviral activity against pedv. to confirm this disparity in the antiviral activities of ifn-λ3 and ifn-α against pedv, ipec-j2 cells were primed with ifn-α (1,000 ng/ml) or ifn-λ3 (1,000 ng/ml) for 24 h and then infected with pedv (moi = 1). consistent with our previous results, both ifn-α and ifn-λ3 substantially suppressed pedv infection in ipec-j2 cells, as demonstrated by measurements of the viral genomes and titers by rt-qpcr ( figure 1a ) and tcid 50 titration (figure 1b) , respectively. ifn-α reduced the number of pedv genomes by 24-fold, whereas ifn-λ3 decreased the number of pedv genomes by approximately 342-fold. the virus titers were consistent with results of pedv genomes: pedv titers decreased 10and 100-fold after pretreatment with ifn-α or ifn-λ3, respectively. the inhibition of pedv infection by both ifnα and ifn-λ3 was further verified by pedv n protein ifa ( figure 1c) . the virus-infected cells were significantly decreased after pretreatment with ifn-α or ifn-λ3, whereas the number of pedv-infected cells in the ifn-λ3-pretreated group was significantly decreased, as demonstrated by only a few sporadically distributed cells, compared with those in the ifnα-pretreated group. thus, ifn-λ3 restricted pedv infection in ipec-j2 cells more effectively than ifn-α, regardless of the quantification of viral rna, infectivity, or viral protein (figure 1 ). to assess the underlying mechanisms of the disparity between the anti-pedv responses induced by ifn-α or ifn-λ3, we performed an rna-sequencing (rna-seq) analysis of total cellular rna isolated from ipec-j2 cell cultures stimulated with ifn-α or ifn-λ3 for 24 h and ifn untreated ipec-j2 mock control. this duration of stimulation (24 h) was selected based on the efficacy of viral inhibition after exposure to ifn-λ3 and ifn-α (figure 1 ) (22) . each of the triple replicate yielded more than 3.89 × 10 7 clean reads and has more than 95% q20 (%), indicating the reliability of the rna-seq data. the ifn-λ3stimulated cells showed a total of 997 differentially expressed genes, including 983 upregulated genes and 14 downregulated genes, compared with the mock control, whereas ifn-α only upregulated the expression of 122 genes among the total of 126 differentially expressed genes and reduced the expression of four genes (figure 2a) . the number of ifn-λ3-modified genes was approximately 10-fold higher than that of ifn-αmodified genes. these results indicate that the intestine epithelia respond better to ifn-λ3 than to ifn-α. we further grouped the corresponding genes through supervised partitioning clustering and combined the differentially expressed genes induced by ifn-λ3 and ifn-α to obtain the heat map ( figure 2b ) and the venn map ( figure 2c ). ifn-λ3 yielded different gene expression profiles compared with that induced by ifn-α, even though these showed substantial overlap ( figure 2b) . one hundred ten genes were upregulated in both the ifn-λ3-and ifn-α-treated cells, whereas 873 and 12 genes were uniquely upregulated in the presence of ifn-λ3 and ifn-α, respectively ( figure 2c) . none of the coexpression genes were downregulated in both the ifn-λ3-and ifn-α-treated cells, whereas 14 and 4 genes were only downregulated in the presence of ifn-λ3 and ifnα, respectively. these results demonstrated that ifn-λ3 induces a unique gene transcriptional profile in the intestine epithelia compared with ifn-α. to further clarify the functional consequences of the gene profiles elicited by either ifn-λ3 or ifn-α, we performed a gene ontology (go) enrichment analysis using a database established by the gene ontology consortium, which aims to define and describe the functions of genes and proteins in various species (figure 3 ) (26) . among the 983 genes upregulated by ifn-λ3, 221 and 134 genes (22.17 and 13.44% of the total genes, respectively) were associated with a binding function and catalysis, respectively, and these two functions account for the main molecular functional changes. the dominant functions of the ifn-α-regulated genes are cellular transporter activity and nucleic acid-binding transcription factor activity, and these functions were associated with 40 genes (31.75% of total genes) and 22 genes (17.46% of total genes), respectively. the analysis of cellular components revealed that 221 differentially expressed genes in the ifn-λ3-treated group affected the cell part, and 36 genes associated with this cellular component were differentially expressed in the ifn-α-treated group. with respect to biological processes, the ifn-λ3-treated group included 200, 173, 169, 142, and 120 differentially expressed genes associated with the cellular process, the single-organism process, biological regulation, the metabolic process, and response to stimulus, respectively. the pattern obtained for the ifn-α-treated group was similar to that of the ifn-λ3-treated group, and the differentially expressed genes were also concentrated on the following five functions: the cellular process, the single-organism process, the biological regulation, the metabolic process, and the response to stimulus. however, the numbers of differentially expressed genes after ifn-α treatment were notably lower than those obtained after ifn-λ3 treatment. these results indicated that both ifn-λ3 and ifn-α are involved in the regulation of multiple cellular processes in ipec-j2 cells, such as cellular components and molecular functions, but ifn-λ3 exerts more potent effects than ifn-α. to further investigate the function of genes specifically induced by ifn-λ3, we extracted the transcriptional profiles of ifn-λ3 and ifn-α regarding biological processes (p < 0.01) and combined them based on the -log 10 (p) values to obtain a heat map that showed the enrichment of biological processes ( figure s1 ). the data demonstrated that in ipec-j2 cells, ifn-λ3 stimulation triggered more biological reaction processes than ifn-α, and these processes mainly involved the modulation of metabolic processes, including cellular metabolic processes, macromolecular metabolic processes, and primary metabolic regulation. taken together, these results indicate that the differentially expressed genes induced by ifn-λ3 are involved in more intracellular biological processes than those induced by ifn-α. to explore the clustering of the ifn-induced differential expression genes in anti-viral signaling pathways, we performed a kegg enrichment analysis of the differentially expressed genes using kobas 3.0. the kegg enrichment analysis revealed that the differentially expressed genes induced by ifn-λ3 involved in much broader signal pathways compared with ifn-α though they both primarily modified the nf-κb signaling pathway, the jak-stat signaling pathway, the phosphoinositide-3-kinase-akt (pi3k-akt) signaling pathway, the mitogen-activated protein kinase (mapk) signaling pathway, and the cgmp-pkg signaling pathway ( figure 4a) . to determine the signaling pathways involving the differentially expressed genes induced by ifn-λ3 or ifn-α, we combined the differentially expressed genes and analyzed their expression patterns ( figure 4b) . the results showed that the differentially expressed genes were most abundantly involved in the jak-stat signaling pathway, followed by the pi3k-akt signaling pathway and the mapk signaling pathway, which is consistent with the finding that the jak-stat signaling pathway primarily mediates ifn-induced antiviral responses. among the proteins encoded by the differentially expressed genes, sos2, pik3ca, jak2, il-6, socs4, il-28b, stat1, il22ra1, and socs1 play a major role in the jak-stat signaling pathway, and kras, pik3ap1, prkaa1, enssscg00000021148, enssscg00000022362, and map3k2 play a major role in the pi3k-akt signaling pathway. to predict the protein interactions between differentially expressed genes, we extracted the union of differentially expressed genes and introduced these into the web-based tool string (http:// www.string-db.org/) to generate protein-protein interaction networks ( figure 4c) . the results of the string analysis indicated that jak2, stat2, pten, pik3ca, irs1, kras, and il6st are closely related to other proteins and displayed significant differential expression compared with the ifn-αinduced proteins, which play critical roles in the innate immune response induced by ifn. collectively, the results showed that the ifn-λ3-induced differentially expressed genes are involved in more signaling pathways, particularly those associated with innate immunity, than those induced by ifn-α, which indicates that ifn-λ3 exhibits stronger antiviral activity in intestinal epithelial cells. to further elucidate the mechanisms underlying the antiviral activity discrepancy between ifn-λ3 and ifn-α, we focused on the top 100 ifn-λ3-induced genes (fold change compared with the mock control) and divided them into three subgroups (classical isgs, weakly ifn-λ3-induced genes, and strongly ifn-λ3-induced genes) as in a previous study (16) . the expression of the 100 top genes in all three subgroups induced by ifn-λ3 was significantly higher compared with that induced by ifn-α ( figure 5) . the isgs in the classic isg subgroup are primarily the classical isgs induced by ifn reported in the literature (16, 27) . the levels of isgs in this subgroup induced by ifn-λ3 were from 3-to 23-fold higher than those induced by ifnα. the weakly ifn-λ3-induced gene subgroup contained 25 genes, including three unknown genes (being denoted un1, un2, and un3). the analysis of this subgroup showed that both ifn-λ3 and ifn-α induced a more than 4-fold increase in the expression of isgs compared with mock control. the ifn-λ3-induced expression levels of ifit3, oasl, oas1, and gbp4, which are important innate immunity factors, were 10fold higher than those induced by ifn-α. the expression of 16 of the genes in the strongly ifn-λ3-induced gene subgroup was strongly induced by ifn-λ3, whereas ifn-α did not induce a substantial increase in expression (<2-fold compared with the mock control). interestingly, radical s-adenosyl methionine domain containing 2 (rsad2), a multifunctional protein with broad antiviral activity that can inhibit both dna and rna viruses, is the top gene upregulated by ifn-λ3 (28) . in contrast, ifn-α did not substantially upregulate rsad2 expression. protein interaction prediction analysis of differentially expressed genes by string database. the relevant genes were input into string (http://www.string-db. org/), the active interaction sources were selected as homology, and the experimentally determined interaction and database annotated were used as predictive conditions for protein interaction prediction. the thickness of the line represents combined score, which is the combined score of the prediction results of the three prediction conditions, indicating the strength of data support. several other genes (ifit2, parp14, and gbp6) in this subgroup are also important innate viral restriction factors (27, (29) (30) (31) . thus, ifn-λ3 induces a stronger antiviral innate immune response compared with ifn-α. to verify the unique antiviral gene expression profile induced by ifn-λ3 that was detected by rna-seq, as shown in figure 5 , we randomly selected three genes from each group to be verified by rt-qpcr. the analysis of the three subgroups confirmed that ifn-λ3 most substantially upregulated the expression of isgs (figure 6) . specifically, ifn-λ3 upregulated the expression of large distinct classes of isgs in the classical isg subgroup, such as mx2, isg15, and ifit3, the isg fold changes induced by ifn-λ3 were up to nearly 10-fold higher than those induced by ifn-α ( figure 6a ). the analysis of the weakly ifn-λ3induced gene subgroup showed that the expression of oasl, oas1, and isg12 (a) was induced by both ifn-λ3 and ifnα to significantly different levels. the oasl and oas1 fold changes induced by ifn-λ3 were up to 8-fold greater than those induced by ifn-α ( figure 6b ). more substantial fold changes in the expression of isgs in the strongly ifn-λ3-induced gene subgroup were obtained with ifn-λ3 compared with ifn-α, which resulted in only slight changes in expression ( figure 6c ). the differences between groups were compared using an unpaired mann-whitney test. p < 0.05 was considered significant, and the p values are expressed as follows: *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001. rsad2, plac8, and ifit2 were more substantially upregulated by ifn-λ3 than by ifn-α. collectively, the in vitro rt-qpcr results confirmed that ifn-λ3 induces stronger isg expression compared with ifn-α. enteroids derived from intestinal crypt stem cells, which mimic the diverse cellular nature and physiological activity of the small intestine while also maintaining the genetic identity of the host, constitute a unique ex vivo model for studying the intestine (21, 27) . to further confirm whether the ifn-λ3induced gene expression profile in enteroids was the same as that in ipec-j2 cells, we stimulated enteroids with ifn-λ3 or ifn-α under the same conditions as ipec-j2 cells and evaluated their gene expression by rt-qpcr. in all three groups, the expression pattern of the genes induced by ifn-λ3 and ifn-α in porcine enteroids was consistent with that found in the ipec-j2 cells (figure 7) . in the classical isg subgroup, the upregulated levels of mx2 and ifit3 elicited by ifn-λ3 were ∼3-to 5-fold higher than those induced by ifn-α ( figure 7a ). in contrast, the upregulated levels of oasl and oas1, which belong to the weakly ifn-λ3-induced gene subgroup, induced by ifn-λ3 were 2-fold higher than those induced by ifn-α, and the fold change difference was moderate ( figure 7b) . for the strongly ifn-λ3-induced gene rsad2 and plac8, just as we observed in the other subgroups, enteroids were or were not primed with ifn-λ3 (1,000 ng/ml) or ifn-α (1,000 ng/ml) for 24 h, and the total rna from the enteroids was extracted and used for rt-qpcr. the differences between groups were compared using an unpaired mann-whitney test. p < 0.05 was considered significant, and the p values are expressed as follows: *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001. ifn-λ3 significantly induced higher expression than ifn-α does ( figure 7c ). in summary, ifn-λ3 induces higher expression of isgs than ifn-α in porcine enteroids, which suggests that ifn-λ3 exerts a greater effect in gastrointestinal epithelial cells than ifn-α. pmx2 and prsad2 inhibit pedv infection in vero e6 cells we selected mx2 (classical isg) and rsad2 (strongly ifn-λ3induced gene) among the top 10 genes induced by ifn-λ3 to evaluate the antiviral effect of the ifn-λ3-induced expression of isgs against pedv infection. we cloned porcine mx2 or rsad2 into the eukaryotic expression vector pcaggs-ha, and the transient expression of pmx2 or prsad2 in vero e6 cells was verified by ifa (data not shown). as expected, pmx2 or prsad2 transient overexpression significantly inhibited pedv infection in vero e6 cells, as demonstrated by measuring the viral rna (figures 8a,c) and pedv n protein expression by ifa (figures 8b,d) . thus, these data indicate that the differentially upregulated pmx2 or prsad2 serves as an important ifn-λ3elicited antiviral host factor against pedv. type i and type iii ifns, which establish a cellular antiviral state and restrict viral infection in the host, are key players at the earliest stages of innate immunity against viral infection. despite the similarities between the effects of the two types of ifns, increasing evidence demonstrates that each class of ifn is essential for antiviral host defense and is not functionally redundant (2) . a previous study in mice demonstrated that the role of ifn-λ in functionally redundant intestinal viral infections cannot be compensated by ifn-α/β (20) . therefore, clarification of the induction of cell-specific ifn signaling profiles is essential for understanding the non-redundant roles of ifn-λ and ifn-α in viral infection at the tissue and organism levels. in this study, we found that the transcriptional profile induced by type iii ifn in the intestinal epithelia the differences between groups were compared using an unpaired mann-whitney test. p < 0.05 was considered significant, and the p values are expressed as follows: *p <0.05, **p <0.01, ***p <0.005, and ****p < 0.001. was unique compared with that induced by type i ifn. compared with ifn-α, stimulation with ifn-λ3 not only resulted in higher levels of most isgs but also substantially increased diversity in the gene profiles involved in various cellular functions. type i and iii ifns each signal through different heterodimeric complex receptors to initiate multiple downstream signaling pathways. in addition to activation of the jak-stat signaling pathway, ifns also activate the pi3k and mapk signaling cascades (5, 32) . the combined use of these signaling pathways by ifns and many other cytokines might help explain the different roles of ifns in regulating the antiviral and immune responses in a variety of environments and locations. we have very limited knowledge on the pathways activated by ifn-λ, which are important for understanding the immune-modulating activities of ifn-λ (8) . in this study, we found that ifn-λ3 not only activates the classical antiviral response jak-stat signaling pathway but also primarily activated the nf-κb signaling pathway, the camp signal pathway, the pi3k-akt signaling pathway, and mapk signaling pathway (figure 4) . ifn-λ3 activated much more signaling pathways in epithelia than ifn-α, and this finding might provide new insights into the mechanism through which ifn-λ modulates other cellular functions beyond its direct antiviral activity. ifn-λ3 largely stimulated wnt signaling pathway in iec compared with ifn-α, which plays critical roles in maintaining the homeostasis of intestinal epithelia in vivo (33) . further studies of different signaling pathways induced by ifn-λ3 or ifn-α will help dissect the complex interaction of ifn-induced signaling pathways with similar or overlapping intracellular signaling pathways stimulated by other cytokines. most somatic cells can induce and respond to type i ifns, but certain specialized cells, such as those in the mucosal epithelia, selectively produce and respond to type iii ifns during various virus infections. we demonstrated that the porcine intestinal epithelia respond to both type i and iii ifns, even though these ifns induced different isg expression levels. type iii ifns comprise four functional and highly homologous subtypes, namely, ifn-λ1, ifn-λ2, ifn-λ3, and ifn-λ4, in humans. our study and other studies have revealed differences in antiviral activity among different ifn-λ subtypes (22, 23, 34) . ifn-λ3 is superior or equal to ifn-λ1 in terms of its antiviral activity in the intestinal epithelia (22) and the immortalized liver cell line hepg2 in vitro (34) . the different kinetics and magnitude of isg induction after stimulation might account for the variation in the antiviral activity among different types or subtypes ifns. previous studies that attempted to identify the gene profiles induced by type iii ifns primarily focused on the comparison of ifn-λ1 with type i ifn (15) , and most of these studies were performed in immortalized human or mouse cell lines. the gene transcription profile induced by ifn-λ3, particularly the gene transcription profile induced by ifn-λ3 in porcine intestinal epithelial cells, has not been reported. here, we performed comparative analyses of the transcriptional profiles induced by ifn-λ3 and ifn-α in porcine intestinal epithelia cells. more importantly, in this study, we verified the rna-seq results in primary swine crypt-derived enteroids, an in vitro system that well recapitulates the complicated cellularity of the gi tract, which exhibits a potent response to both type i and iii ifns (21, 27, 35) . the differential expression in ipec-j2 or enteroids detected by qpcr is not related to the selection of housekeeping gene gapdh since we observed the same pattern when using another housekeeping gene actin (data not shown). therefore, studying the differences among ifn-λ3-and ifn-α-induced gene expression in enteroids is more realistic and clinically significant. the different antiviral activities of ifn-λ3 and ifn-α are likely due to the different degrees of isg induction by these two cytokines, and ifn-λ3 induced increased isg expression. the functions of some of the genes that were strongly induced by ifn-λ3 have been reported, but the functions and mechanisms of more genes remain unclear. rsad2, a host viral restrictor gene, showed the highest upregulated levels after ifn-λ3 rather than ifn-α stimulation, whereas mx1 was the most highly upregulated gene after ifn-α stimulation. a previous study demonstrated that porcine rsad2 effectively inhibited csfv replication in vitro, and this effect might occur via the interaction of rsad2 with csfv e2 protein in the cytoplasm (28) . our study found that rsad2 transient expression also substantially curtailed pedv infection in vero e6 cells in vitro, but the mechanism is unclear. mx2, an ifn-induced gtpbinding protein (36, 37) , reportedly inhibits the replication of lentivirus hiv-1, simian immunodeficiency virus (siv), and equine infectious anemia virus (eiav) in vitro (38, 39) . the function of porcine mx2 has been poorly studied. we confirmed that porcine mx2 inhibits pedv infection in vero e6 cells. we also discovered some unknown genes that were differentially expressed in response to ifn-λ3 or ifn-α stimulation, and this finding might provide a foundation for further elucidation of the different mechanisms of action of ifn-λ3 and ifn-α. in summary, we compared the transcriptional profiles of ifn-λ3 and ifn-α in ipec-j2 cells and identified a unique set of genes that were strongly induced by ifn-λ3 compared with ifn-α. the transcriptional enrichment analysis indicated that ifn-λ3 or ifn-α is involved in the regulation of cellular processes, such as cellular components and molecular functions, in ipec-j2 cells, and that ifn-λ3 activates more robust signaling pathways, particularly the antiviral jak-stat signaling pathway, compared with ifn-α. ifn-λ3 preferentially upregulates antiviral genes in epithelial cells compared with ifn-α. these results indicate that ifn-λ3 selectively targets small intestinal epithelial cells and induces a non-redundant antiviral host response at the enteric mucosal site. all datasets generated in this study are included in the manuscript/supplementary material. the animal study was reviewed and approved by the harbin veterinary research institute of the chinese academy of agricultural sciences, harbin. written informed consent was obtained from the owners for the participation of their animals in this study. pl, ll, and lf designed the research studies and analyzed and interpreted the data. ll, mx, ff, and ly conducted experiments and acquired data. ll and pl drafted the manuscript and all authors contributed revisions. funding support for this work was provided by grants from the national key r&d program of china (2017yfd0501102) and the national natural science fund (31772718). the content is solely the responsibility of the authors and does not necessarily represent the official views of the funding resources. ifn-lambda decreases murid herpesvirus-4 infection of the olfactory epithelium but fails to prevent virus reactivation in the vaginal mucosa type iii interferons in antiviral defenses at barrier 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signaling pathways involving both the jak-stat pathway and the mitogen-activated protein kinases distinct and overlapping genomic profiles and antiviral effects of interferon-lambda andalpha on hcv-infected and noninfected hepatoma cells identification of a predominantly interferon-lambda-induced transcriptional profile in murine intestinal epithelial cells commensal microbes and interferon-lambda determine persistence of enteric murine norovirus infection type iii interferon-mediated signaling is critical for controlling live attenuated yellow fever virus infection in vivo diverse intracellular pathogens activate type iii interferon expression from peroxisomes ifn-lambda determines the intestinal epithelial antiviral host defense porcine intestinal enteroids: a new model for studying enteric coronavirus porcine epidemic diarrhea virus infection and the host innate response ifn-lambda preferably inhibits pedv infection of porcine intestinal epithelial cells compared with ifn-alpha type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp1 in irf1 signaling establishment of a stable cho cell line with high level expression of recombinant porcine ifn-beta analyzing real-time pcr data by the comparative c(t) method gene ontology: tool for the unification of biology. the gene ontology consortium a family of ifn-gamma-inducible 65-kd gtpases protects against bacterial infection porcine viperin protein inhibits the replication of classical swine fever virus (csfv) in vitro decreased ifit2 expression promotes gastric cancer progression and predicts poor prognosis of the patients ifit2 is a restriction factor in rabies virus pathogenicity structure, function and inhibition of poly(adp-ribose)polymerase, member 14 (parp14) regulation of type i interferon responses sustained in vitro intestinal epithelial culture within a wnt-dependent stem cell niche human interferon-lambda3 is a potent member of the type iii interferon family expression of ifnlr1 on intestinal epithelial cells is critical to the antiviral effects of interferon lambda against norovirus and reovirus cdna structures and regulation of two interferon-induced human mx proteins human mxb protein, an interferon-alpha-inducible gtpase, contains a nuclear targeting signal and is localized in the heterochromatin region beneath the nuclear envelope equine myxovirus resistance protein 2 restricts lentiviral replication by blocking nuclear uptake of capsid protein the interferon-inducible mxb protein inhibits hiv-1 infection the authors thank dr. xiangxi pei (northeast agricultural university, harbin, china) for assistance and advice in rna-seq data analysis. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.02394/full#supplementary-material figure s1 | the heat map of ifn-λ3 and ifn-α transcriptional profile biological process enrichment analysis. 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 li, xue, fu, yin, feng and liu. 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-292958-k5d5fo3i authors: sekhon, simranjeet singh; nguyen, phat-loc; ahn, ji-young; lee, kyeong-ah; lee, lyon; kim, sang yong; yoon, hobaek; park, jihoo; ko, jung ho; kim, yang-hoon title: porcine epidemic diarrhea (ped) infection, diagnosis and vaccination: a mini review date: 2017-01-04 journal: toxicol environ health sci doi: 10.1007/s13530-016-0287-8 sha: doc_id: 292958 cord_uid: k5d5fo3i porcine epidemic diarrhea virus (pedv) is a main etiology causing severe enteric disease in piglets with clinical signs of anorexia, vomiting, diarrhea and dehydration resulting in loss of condition and death within a few days. historically, ped is one of major causes of loss in swine and remains prevalent in some parts of the world. even with increase in the available tests for ped diagnosis, which include histological diagnosis; virological diagnosis and serological diagnosis, there is no vaccine or specific treatment for this disease yet. in this mini review, the overview and current situation of ped is described with updated techniques, in an effort to comprehensively discuss and understand the disease characteristics. porcine epidemic diarrhea (ped) is a non-zoonotic viral disease of swine caused by a coronavirus and characterized by dramatic watery diarrhea and weight loss in swine with high mortality to neonatal piglets 1,2 . pigs of all age can be infected by ped virus from neonates to sows or boars but the fatality rate of ped in swine depends on age 3 . the morbidity and mortality of ped in 10-day-old piglets are less than piglets under 5 days old that may reach 100% in death because of severe diarrhea and dehydration 4 . the older pigs are able to recover within 7 to 10 days but can re-infect within five months 4 . the fecal-oral route is perhaps only transmission of ped and no other vector or reservoir in disease spreading 5 . however, the disease is highly contagious and rapidly infect throughout the year to both industrial and small pig farms. swine (sus scrofa) are the only natural hosts for ped virus infection. kamau et al. (2010) investigated the ability of using specific pathogen free mice as a potential vector in ped virus transmission. nevertheless, molecular tests and serological data showed no antibodies detection so mice and rat were not susceptible vectors of the ped transmission 6 . there are 2 forms of ped disease: the ped type i, which is only infected in weaning pigs, and the ped type ii affects pigs of all age 7 . the main etiologic agent of the disease is porcine epidemic diarrhea virus (pedv) which was firstly reported in europe in 1971 and was identified as coronaviruslike strain cv777 from pigs with watery diarrhea in belgium and united kingdom in 1978 [8] [9] [10] . after that the disease widely spread throughout many swine-raising countries in western europe: hungary (1981); germany (1981); to asian countries: japan (1982); south korea (2000) ; taiwan (2013) and north america (2013) 11 . during the 1970s and 1990s, a few severe ped outbreaks have been reported in europe 12 . nevertheless, pedv infection nowadays has become epidemic in asia pig industries, consist of china, japan, south korea, vietnam, thailand, the philippines and taiwan 13 . in china, a large-scale diarrhea outbreak was reported in the end of 2010 with the confirmation of pedv in pig population that over 1,000,000 piglets died with a mortality rate of 80%-100% and resulted enormous economic losses 14, 15 . in south korea, the pedv was firstly described in 1992 and re-emerged as a severe outbreak during 2013 with considerable variants that were different from previous korean isolates or vaccine strains 16, 17 . a massive ped outbreak suddenly occurred in the north american pig farm in april 2013 and rapidly spread across the country also to countries sharing the same border: canada (2014) and mexico (2013); causing high rates of mortality and huge economic losses 2, 3 . although the ped disease has similarly clinical signs to transmissible gastro-enteritis (tge) including anorexia; vomiting; diarrhea and dehydration, the causative agent ped virus is antigenically distinguishable from tge virus (tgev) and haemagglutinating encephalomyelitis virus (hev) 4 . the ped virus belongs to the member of genus alphacoronavirus in the family coronaviridae which constitutes the order nidoviracles and causes acute diarrhea in human and animals, especially fatal to newborn individuals 18, 19 . under the electron microscopy (em), the characteristic appearance of pedv contains an opaque and pleomorphic body about 90 to 190 nm diameter in range, with an electron-dense core, a large fringe and bulb-shaped projections of approximately 20 nm but the detailed internal structure of ped virus remains unknown 1, 7 . the ped virus adapts culture to be stable at wide range of ph from 5.0 to 9.0 at 4℃ and still keeps infectivity between ph 6.5 and 7.5 at 37℃ 1,20 . however, this virus is sensitive to high temperature when heating to or over 60℃ for 30 minutes and to chemical reagents such as ether and chloroform 1 . besides, most disinfectants are effective to against pedv, consisting of cresol, formalin (1%), anhydrous sodium carbonate (4%), sodium hydroxide (2%), iodophors (1%) in phosphoric acid, ionic and non-ionic detergents. more than 10 years after the first identification of pedv, the effort of growing pedv in cell culture was successfully proved in the study of hofmann m. and wyler r. (1988) 21, 22 . the vero cell (derived from african green monkey kidney) culture with the presence of trypsin was susceptible for ped virus propagation. in another research, shibata et al. (1999) described additional methods to isolate pedv using pig bladder cell-and kidney cell-derived cultures, also in the presence of trypsin in the medium because trypsin allowed the in vitro propagation of several enteric viruses and facilitated coronavirus growth as an important ingredient in the cell culture media 23 . the pedv is an enveloped virus with a single-stranded positive-sense rna genome with approximately 28,000 bases in size with a 5′ cap and a 3′ polyadenylated tail (poly a) 9, 24 . the pedv genome comprises at least 7 open reading frames (orfs) that encode three non-structural proteins (orf1a, orf1b and orf3) and four structural proteins (orf2, orf4-6) 1,8,25 (figure 1 ). the viral genome organization followed a conserved gene order: an untranslated region (utr) at 5′-end; the large orf1a and 1b that cover 70% at the 5′-end of genome for polymerase genes; a set of four genes encoding the structural proteins: 150-220 kda glycosylated spike protein (s), 7 kda envelope protein (e), 27-36 kda membrane protein (m) and 58 kda nucleocapsid protein (n); and a 3′utr 1, 8, 26 . table 1 based on all sequences of coronaviruses, the orf1a (nt 297-12650) and orf1b (nt 12605-20641) shared slightly overlapping sequences at a possible ribosomal frameshift (rfs) site 7, 8 . the orf1a translated for a replicase polyprotein 1a (pp1a) which could be extended into pp1ab at c-terminal of rfs site and encoded for several functional motifs such as three protease: papain-like proteinase (plp), x domain (x), poliovirus 3c-like proteinase (3c1) and one growth factor-like motif (gfl), whereas there were a metal ion binding domain (mb), a rna-dependent rna polymerase domain (rdrp) and a helicase motif (hel) that could be encoded within orf1b by analogy to amino acid sequences of other coronaviruses 25 . the post-translational cleavage of pp1a and pp1ab resulted in a group of 16 non-structural proteins (nsp1-16) by internal proteases. the orf3 is a functional accessory gene which located between s and e structural protein. it is conserved among canine coronavirus type i and encodes a glycoprotein gp3 that is concerned to virulence of pedv in the natural host and to be functional as an ion channel but not generally related to viral replication in cultured cells 1,27,28 . song et al. (2002) analyzed orf3 in restriction fragment length polymorphism (rflp) to differentiate the korean pedv, kpedv-9 field strains from wild-type strains for application in vaccine against pedv infection 28 . this study suggested that the loss of orf3 production or the changes in sequence of orf3 resulted in unexpected consequence of the adaption of pedv in cell culture. the e proteins, one of four structural components in pedv, are recognized as a small, hydrophobic transmembrane protein of coronaviruses with 76-109 amino acids (aa) in length that are rather divergent among different coronavirus strains 29, 30 . it was found that they play a remarkable role on the morphogenesis and viral assembly where they could prompt the curvature of membrane as well as assist in membrane scission 31, 32 . besides, the e proteins without modifications are also integrated into the endoplasmic reticulum (er) membrane where they are independent of inducing er stress 33 . another investigation of e protein expression in escherichia coli and mammalian cells demonstrate the crucial function in altering membrane permeability 34 and apoptosis promotion 35, 36 . additionally the e proteins are observed the interaction with host-cell proteins and in vitro ion channel activity which is more selective for monovalent cations and is blocked by hexamethylene amiloride in other coronaviruses 37 . the co-expression of e protein and a major membrane m glycoprotein, via their cytoplasmic domains localizing to pre-golgi membranes, allows the generation of coronavirus-like pseudoparticles which were identified same size with tgev virions 33, 38 . in recent studies of other coronaviruses, the absence of e proteins leads to two actions: non-infectious virions and the inhibition of full virus growth 39 . the membrane m proteins, the most abundant component of coronavirus envelope, have potential role in virus assembly. it is a glycoprotein type iii with molecular weight in 27-36 kda, consisting of triple-spanning transmembrane segments which flanked by one short n-terminal ectodomain on the outside of virus and one long c-terminal domain in the cytoplasm 40, 41 . when expressed in the absence of other viral proteins, m protein tends to accumulate in the golgi apparatus as detergent-insoluble, heterogeneous complexes in polymeric structures 42, 43 . by employing coimmunoprecipitation and immunofluorescence colocalization analysis in mouse hepatitis virus (mhv), m protein complexes appeared to immediately interact with the spike (s) proteins after their synthesis, forming heteromultimeric complexes in the pre-golgi membranes 42, 44, 45 . additional studies recently provided proof for the existence of m-m interactions which were observed that assembly-incompetent m could be rescued into viruslike particles (vlps) by assembly-competent one 42 . it is indicated that the self-association of coronaviruses envelope glycoproteins plays an essential role in arranging of the viral membrane proteins and is thought to drive virion envelope assembly but do not determine the viral budding site at the pre-golgi membrane. another function of pedv m protein is useful in developing epitope-based vaccines. zhang et al. (2012) identified epitopes on the ped virus m protein, highly conserved 4d4-defined epitope, to study the antigenic properties of this protein 40 . this epitope could be a candidate for development of protective antibodies for ped because it was recognized by positive serum of different pedv strains, comparing to tgev-positive serum. among pedv structural proteins, the s protein is a major type i membrane glycoprotein on virion surface, with average 1,300aa in length and 150-220 kda in weight 46 . this trimeric spike protein forms the characteristic peplomers (surface antigen) and consists of a signal peptide (1-18aa), four neutralizing epitopes (499-638, 748-755, 764-711, and 1,368-1,374aa), a transmembrane domain (1,334-1,356aa), and a short cytoplasmic tail 2 . the s protein can also have potential n-glycosylation sites and can be cleaved by exogenous or host cell protease into s1 (1-789aa) and s2 (790-1,383aa) domains 47 . the s1 domain is composed of a binding domain for host cell receptors whilst the s2 domain, involved in virus-cell attachment and in both virus-cell and cell-cell fusion, could be divided into three domains: a large ectodomain, a single transmembrane (tm) region and a cytoplasmic tail (ct) region 48 . the large ectodomain of s2 subunit was an identified role in membrane fusion activity and composed of a protease cleavage site, a putative fusion peptide and two heptad repeat (hr) region [48] [49] [50] . a few studies reported that the tm region with aromatic domain and the ct region carrying the cysteine-rich domain were concerned to regulate the fusogenic activities 51, 52 . further, another important role of ct region of the pedv spike protein, which was determined by a signal: a dibasic (kxxhxx-cooh), was accountable to retrieve this protein in the endoplasmic reticulum-golgi intermediate compartment (ergic), whereas the s protein with a mutation (h → r) or lack of this motif resulted in enhanced cell surfaces expression of s protein. nevertheless the role of a potential tyrosine-based (yxxf motif) signal of ct region remains unknown [53] [54] [55] . the interaction between s proteins and m or e proteins (as mentioned before), which is essential for virus assembly into those intracellular vesicles, involved in the localization of s protein in the ergic. some studies showed that the s protein were not essential for budding of coronavirus particle but needed for infectivity. by culturing coronaviruses mhv a59 in the presence of antibiotic tunicamycin, rottier et al. (1982) found that the spikeless and noninfectious virions were produced 56 . additionally, luytjes et al. (1996) conducted biochemical analysis and electron microscopy to survey the characteristic of temperature-sensitive mutants of coronavirus mhv a59 and found that the temperature-sensitive mutant s protein at 39.5℃ (non-permissive temperature) were fail to incorporate into virion particles and to mature to golgi membrane 57 . however, the synthesis of the m protein and the nucleocapsid n protein of mutant viruses was not sensitive to temperature. it is presumed that the s protein is the most antigenic of the pedv proteins because of containing virusneutralizing epitopes 58 . chang et al. (2002) targeted the s protein as a primary antigen to identify alternative epitope region for designing an effective vaccine against coronaviruses 59 . although the ped virus and tge virus are serological distinct, this study based on the co-26k, a collagenase-digested fragment, of the tgev s protein to find the critical virus-neutralizing epitope located in the s protein of pedv. by analyzing the neutralizing activity of the polyclonal antisera and comparing the sequences of coe (co-26k equivalent) gene of spike protein among pedv strains (pedv cv777 strain, korean isolate and pedv brl/87), this study suggested that the coe region of pedv spike protein contained an important virus-neutralizing epitope with no cross-neutralization (based on very low homology in the same region comparing with feline infectious peritonitis virus, fipv and tgev). the pedv n protein is a phosphoprotein which is associated with rna genome and indicates a basic structure for the helical nucleocapsid 1 . the encoded polypeptides range of pedv n protein is from 377 to 455 amino acids and has the similar physical properties with the other members of the family coronaviridae 60 . nevertheless one unique sequence with a 40-residuelong insertion in the central position of pedv n protein molecule could reflect recombinant event or stuttering of the viral polymerase. this sequence is particularly rich in arginine (arg), serine (ser) and asparagine (asp) residues, and presents in ped virus n protein with no counterpart in the remainder of coronaviruses. the n protein can be an alternative target to accurately diagnosis pedv in early infection. furthermore, xu et al. (2013) generated a specific cell line iec (porcine intestinal epithelial cell) expressed pedv n protein as well as investigated the function and localization of n protein 60, 61 . the study suggested that pedv n protein affected on cycle progression, interleukin-8 (il-8) expression, cell growth and survival. particularly the pedv n protein was identified in the er membrane, induced cell cycle prolongation at the s-phage, which is associated with a low level of cyclin a transcription and increased in cyclin a degradation, and was responsible for er stress, which included the initiation of an inflammatory react via activation of nf-kb, as well as up-regulation interleukin-8 expression 60,61 . this study was the first report to demonstrate novel functions of pedv n protein that was useful in investigating the molecular mechanisms and pedv pathogenesis. based on the partial sequence analysis of s, m and orf3 genes, the genetic and phylogenetic relation of pedv isolates were determined 26, 28, 46 . by phylogenetic analysis of s glycoprotein genes including epitope region, the diversity of koreans pedv strains were reported and divided into groups and subgroups. there were three groups of korean pedv isolates: the g1 group were highly homologous to reference pedv strains (cv777, js-2004-2, p-5v, sm98-1, kped-9, parent and attenuated dr13) but did not have specific nucleotides and amino acids comparing with other groups g2 and g3 korean pedv isolates, including highly homologous spk1 and chinju99 29 . otherwise the subgroup g1-1 korean pedv isolates had unique specific nucleotide sequences and had the dna sequence identities 95.3%-97.9%, 93.6-96.6%, 93.5-96.6%, and 88.7-90.7% with the subgroup g1-2, g1-3 and the group g2, g3 korean pedv isolates respectively. the study with analysis of s glycoprotein genes demonstrated the korean pedv strains were genetically diverse both among themselves and to other foreign reference pedv strains. the ped diagnosis cannot be determined only on the epidemiological investigation, the basic of clinical signs and the histopathological lesions. due to the dependence of ped clinical signs on the age of swine, the presence of secondary infection and the immunological status of the pigs, as well as the similarities in characteristics of ped syndromes from other causative agents of diarrhea such as tgev, rotavirus, bacteria (e. coli, salmonella sp., clostridium spp., etc.) or by parasites, the laboratory examinations are required to identify and confirm the pedv infection. many techniques are available for the detection of pedv from fecal materials, small infected intestines sample, including reverse transcript polymerase chain reaction (rt-pcr), direct immunofluorescence tests (if), indirect fluorescence antibody tests (ifa), immunohistochemistry techniques (ihc), in situ hydridization, electron microscopy and enzyme-linked immunosorbent assays (elisa) 2 . the popular diagnostic methods used to detect ped viral antigen, are immunofluorescence test while the most commonly applicable tests to detect pedv antibodies in serum and antigen in feces are enzyme-linked immunosorbent assays. of all available techniques to detect the causative agents of ped, electron microscopy (em) is the least sensitive technique. sueyoshi et al. (1995) evaluated the pedv-infected lesions using transmission em (tem) technique, nevertheless the result showed a mix of coronaviruses within the cytoplasm and microvilli of epithelial cells in small intestine 63 . after that they carried out a streptavidin-biotin (sab) technique to detect the specific viral antigen of ped in the cytoplasm of enterocytes, with light microscopy. this method examined formalin-fixed materials with a serial of washing step and no cryostat that demonstrated adequately detailed diagnosis methods for outbreak of pedv occurred in japan. in case the spikes of virus were lost or not clearly visible, the tem results were inconclusive to confirm that the causative agents were pedv or viruses with the similar size and morphology, especially tgev. for the reason that the em technique is not sensitive and specific, it could not be applied to differentiate ped virus from the remainder of coronaviruses, especially tge virus. shibata et al. (1999) employed virus neutralization (vn) test and immunofluorescence assay (ifa) to detect antibodies against pedv as well as the immunohistochemical examination for the detection of pedv antigens in the enterocytes of infected pigs by avidin-biotin (ab) technique 12 . the paper determined the isolation of pedv using porcine cell cultures (as mentioned before), whereas established the sb1 and sb2 cells derived from the epithelial cells of cesarean-derived colostrum-deprived (cdcd) pig bladder and the sk cells prepared from the cdcd pig kidney for different passage state, instead of using popular methods with vero cells. furthermore, this study discovered the effect of different pig age on the disease, in particular, the severe clinical signs were only found in two-day and one-week old piglets. so this research concluded that there was an age-dependent resistance to ped virus in swine. until now, the vero cell lines are the most popular and effective to isolate ped virus. pan et al. (2012) used the vero cell cultures to isolate chgd-01 pedv strain as well as employed direct immunofluorescence assay and em technique to investigate its specific cytopathic effects in the outbreak of diarrhea in guangdong (south china) swine 18 . the study also reported the susceptibility of vero cells to different pedv strains because the characteristic cpe caused by pedv isolate in this research was not confirmed until seven passages of vero cells. based on phylogenetic analysis of the whole genome, the chgd-01 isolates were placed in a cluster with two other chinese strains, sharing almost 98% of nucleotide sequence identities. however, this recent chinese strain might have originated from the korean pedv strains knu 0802, based on amino acid sequence analysis of viral spike protein gene. the rt-pcr could be applied for a rapid detection of pedv infection without showing any cross reaction with tgev or porcine rotavirus. kweon et al. (1996) designed three primer sequences from the membrane protein (m) gene of pedv for rt-pcr test 64 . the result of this study proved that a positive dna band of kpedv-9 m gene could be early detected from fecal specimens at 24 h post-infection in experimentally inoculated pigs. the research from ishikawa et al. (1997) was in the close agreement with the conclusion of rt-pcr as a practical, rapid and specific method for detecting pedv in swine 65 . the study developed a rt-pcr detection system by using primers to amplify an 854 bp fragment of m protein gene of pedv. the rt-pcr system in this study could effectively detect pedv rna from viral mixtures or small intestinal/ fecal samples in very low number of virus within short time. the virus isolation and the sab technique were employed to confirm pedv positive results by rt-pcr, based on virus and its antigen detection. moreover, a serial dilution of pedv jme2 strain for rt-pcr was conducted to evaluate the ability of this assay to amplify viral rna from small intestinal samples and fecal specimens as well as to eliminate the inhibitors of rt-pcr in specimens. previous studies have showed difference in the sensitivity of rt-pcr assay. this could be explained by the difference in rna extract processes, rt-pcr conditions or the presence of inhibitors in the intestinal and fecal samples that could affect the sensitivity of rt-pcr. there were many factors in the intestinal and fecal specimen that could inhibit the activity of thermostable polymerase in enzymatic reaction of pcr amplification, especially the presence of bilirubin and bile salts 66, 67 . to enhance the specificity and sensitivity of rt-pcr, the extraction process of viral rna and pcr conditions need to be optimized and chemical reagents that prevented the inhibition factors in raw materials, were advised to use. in the reverse transcription step, the rt-pcr also revealed high potential in cross contamination during mass screening, however. kim et al. (2007) improved the molecular detection methods of pedv by generating a multiplex real-time rt-pcr employing 2 sets of primers and different probes labeling with reporter dyes in a single reaction tube 68 . the primers and probes were designed and synthesized base on conserved sequence of the nucleocapsid n genes from a number of strains of tgev and pedv, and were labeled with specific dyes for each virus: 5′-reporter dye fam and 3′-quencher bhq1 for tgev, 5′-reporter dye cy-5 and 3′-quencher bhq2 for pedv. the selection of primers and probes which based on the highly conversed regions of nuclecapsid gene of the cv777 strain of pedv and the purdue 46-mad strain of pedv as well as using the same concentrations for both of them could optimize this assay. the multiplex real-time rt-pcr was able to differentially detect and qualify the pedv and tgev rapidly from both experimentally and naturally infected pigs, with no cross-reaction between diarrhea-causing viruses. this assay also saved the time-consuming comparing to complete rt-pcr-based dot blot hybridization and minimized the rate of contamination by reducing the number of experimental steps. hence, the rt-pcrbased technique was considered as a useful and practical method for rapid, sensitive and specific detection of pedv infection in fecal samples from live pigs, with out killing. the study of carvajal et al. (1995) was one of the first researches that focused on detection of pedv antigen and antibodies 69 . the study generated simultaneously 2 separate blocking enzyme-linked immunosorbent assays (elisas) and an indirect fluorescence test (ift) in order to detect both pedv antigen in fecal samples and pedv antibodies in serum, using monoclonal antibodies (mab) as capture and reporter agents. the results indicated that the antibodies in both natural and experimental pedv infections were detected by elisa in shorter time than by ift (3-5 days sooner). hence the mab-based elisa was considered higher sensitive method allowing the diagnosis of pedv infection in diarrhea endemic and on farm. however, there was unknown reason between elisa-negative but ift-positive results that remained in this study. in another research, the competitive blocking elisa (cb-elisa) was generated to identify pedv in culture medium and fecal samples, evolving non-conjugated monoclonal antibodies to membrane m protein of pedv. this study also showed the low correlation of sensitivity between the em technique and the cb-elisa that only 3/15 fecal samples was positive with coronavirus by em examination while the negative results of cb-elisa was 14% (9/65 fecal samples). hou et al. (2007) based on recombinant 48 kda nucleocapsid (n) protein of pedv as a useful antigen to develop an elisa (rnelisa) for detecting antibodies to pedv 18 . the rt-pcr was conducted to amplify nucleocapsid n protein gene from the pedv korean strain, using forward and reverse primer containing restriction enzyme sites (bamhi and saci). after subcloning into the prokaryotic expression vector pqe-30, the recombinant plasmid pqe30-pn was transformed into host cell (escherichia coli) to produce the pedv soluble nucleocapsid proteins with an n-terminal his6-tag of 442 amino acids. the recombinant n proteins were purified by affinity chromatography in ni-nitrilotriacetic acid (ni-nta) agarose and were analyzed by sds-page and western blotting with anti-his-tag monoclonal antibody before applying to rnel-isa for pedv detection. besides, the limitation of rnelisa was determined by a cut-off value that was calculated as absorbance values among 80 serum samples from field, based on the mean value plus two sds (standard deviation). in the additional confirmation from rt-pcr and the serum neutralization (sn) tests, the recombinant n protein igg elisa obtained 98% sensitivity (among 103 clinical pedv-infected sam-ples) and 98.7% specificity (among 80 pedv-free samples) compared to rt-pcr in methodologies as well as this recombinant protein-based serological test only reached 8.5% false-positive results by antibody detection in 18 of 213 sn negative results. the study indicated that the nucleocapsid n protein might be a sensitive antigen for the serological diagnosis of pedv and the rnelisa had high sensitivity and specificity for a rapid and simple method for the large-scale detection of pedv. the correlation between the das-elisa and the rt-pcr was also demonstrated by examining 506 specimens of pig herd from different farms in the po valley (italy), according to the study of sozzi et al. (2009) 70 . the double antibody sandwich (das) elisa was generated for detection of pedv in both swine intestinal and fecal samples, using monoclonal antibodies. the six mabs specific for pedv, which were generated and characterized from the screening of forty hybridomas by indirect if and indirect elisa using non-infected and pedv-infected cells, were purified and modified with horseradish peroxidase (hrpo) conjugation before applying to das-elisa. the six selected mabs were surveyed the intensity and specificity in different combination, i.e. 1f12 as conjugated mab and mab 4c3 as antigen catching antibody, for the best catcher and tracer in das-elisa. the comparison of das-elisa and rt-pcr indicated the complicated correlation between intestinal and fecal samples. for the fecal examining, the high kappa statistic value suggested an almost perfect agreement in two methods that only 2 of 215 samples were elisa-negative but rt-pcr-positive identification. this could be explained that the clinical samples were collected in the recovery period of disease with the low viral titres and the forms of specific antibody in faeces were immunocomplexes. however the intestinal examination by two methods gave a disagreement because there were 7 samples of rt-pcr negative but elisa positive. the disagreement might be due to the non-specific binding of antibodies in elisa reaction and the available inhibitors of pcr assay. these finding agreed with previous studies that serological test-elisas were rapid and sensitive for the screening of a large number of specimens so the pedv-elisas were suitable and effective in controlling ped disease during epizootic outbreaks. in a research by guscetti et al. (1998) , four methods were compared for diagnosis of wild-type pedv: an immunohistochemical detection using formalin-fixed tissues, a direct immunofluorescence assay using cryo-stat sections, an elisa and a pcr method 71 . this study showed that the elisa results confirmed a sensitive and reliable detection in fecal material for pedv but the presence of false positive reactions might affect the results when using for large number of specimen samples or samples from different infected animals in clinical practice. the pcr methods were ineffective in this study because wrong negative results were indicated by the other methods but the virus purification step before rna extraction could enhance this method. according to conclusion of this research, the ihc and if methods, which were used to detect viral antigen from gut tissues, demonstrated very high sensitivity and reliability, allowing detection for more than 75% enterocytes were positive for viral antigen by ihc in the mid-jejunum particularly. in contrast, the rt-pcr used in the recent study was indicated to be a simple, rapid, specific and sensitive method for the identification of pedv from fecal samples. kim and chae (2012) used and compared reverse-transcription polymerase chain reaction (rt-pcr), immunohistochemistry (ih) technique, and in situ hybridization for detection of pedv in fifteen experimental pedv-infected pigs and 94 samples of diarrheic piglets 72 . for the intestinal and fecal samples from experimental pedv-inoculated pigs, the rt-pcr could detect viral antigen and nucleic acid in all samples (15/15) while the ih and in situ hybridization could not identify the causative agent in 1/15 and 2/15 samples, respectively. for pedv detection of diarrheic pigs in fecal samples, 63/94 samples were positive and 15/94 samples were negative for causative pedv by all 3 methods, indicating a high agreement and correlation (83%) among these methods. the high rate of agreement demonstrated that the 3 methods could be applied independently to accumulate an accurate diagnosis for pedv infection. this study also indicated how to choose the suitable technique to diagnose pedv depends on the available types of sample. the rt-pcr test was recommended for virus detection if only fecal samples were provided whilst the immunohistochemistry (figure 2 ) and in situ hybridization were suggested for more sensitive detection of pedv in formalin-fixed intestinal samples 72 . due to the special features such as rapid detection, high sensitivity and specificity, and cost-effectiveness, the rt-pcr is considered as an important and successful test for detection of pedv both laboratory and field virus strains. vaccination is one of the traditional and required methods to prevent and control ped virus infection in swine. nowadays, the ped epidemic outbreaks, which neonatal pigs are involved, tend to be more serious and dangerous in asia countries than in europe and others with the sufficient economic loss. therefore, several trials of pedv vaccine were mainly researched and developed in asia countries such as attenuated vaccine ppedv-9 strain in korea, p-5v and 96-p4c6 strain in japan, cv777-attenuated or inactivated vaccines in china [73] [74] [75] . in sow, neonatal pigs are normally protected until they are 13-day-old by a transfer of igg maternal antibodies from colostrum and milk of immune mothers 1 . beside the 60% of igg accounting in colostrum immunoglobulin content, there are igm and especially iga which is more resistant and has high virus neutralizing activity than igg 76 . however, these antibodies from passive immunity are not able to prevent the intestinal infection of pedv. in south korea, researchers investigated attenuation of kpedv-9 isolate via serial passages in vero cell cultures (up to 93 passages) and its immunoprophylactic effects in pregnant sows 74 . the piglets intramuscularly or orally inoculated with attenuated virus of high passage level did not reveal any severe symptoms of diarrhea or death comparing to the animal with wild pedv-infection, so an alternative vaccine might be developed from attenuated virus deriving from serial passage of pedv. this study also indicated that the vaccinated pregnant sows by intramuscular inoculation had the low mortality rate of piglets. another researcher group generated a vero cell attenuated pedv dr13 which was differed from wild-type pedvs by a restriction fragment length polymorphism (rflp) pattern 77 . the study compared the ability of the cell attenuated dr13 virus after inoculation via oral (o) and intramuscular (im) routes in late-term pregnant sows to protect neonatal pigs against ped, employing the sn test and elisa. the experiment of the immunoprophylactic effect in pregnant pigs showed the similar agreement with the research by kweon et al. (1999) in decreasing mortality of piglets but cell-adapted dr13 vaccination in this study through oral route 74 . previously, pedv dr13 was also isolated in vero cells through serial passage at level 100 and was analyzed for differentiation from other korean field strain via rt-pcr rflp with hindiii and xhoii enzymes. moreover, the investigation of cell-adapted dr13 indicated the reduced virulence after high serial passage as well as its immune response in 14-day-old piglets and pregnant sows. the attenuation and safety of high serial passage dr13 after oral vaccination were also discussed in this study. hou et al. (2007) targeted the nucleocapsid n protein of pedv to develop a recombinant vaccine trial by employing a surface antigen display system on lactobacilli 78 . this system used the poly-γ-glutamate a (pgsa) protein, which is a synthetase complex of bacillus subtilis, as the transmembrane anchor to express heterologous antigens in the surface of lactobacillus casei525. the live and dead l. casei anchoring nucleocapsid protein of pedv were vaccinated to two animal models: intranasal and oral inoculation of mouse, oral inoculation in pregnant sows that induced the systemic and local immune responses in both. the rneli-sa were carried out to indicate the high levels of serum igg and mucosal iga after inoculation. moreover, the igg levels of piglets were highly increased after receiving colostrum secreted from vaccinated sows with recombinant l. casei. the surface antigen expression system on lab (lactic acid bacteria) was potential for vaccine applications to against ped but limited in the display size of foreign antigens. the pedv infection cause acute and severe enteritis with clinical symptoms: diarrhea and vomit, followed by extensive dehydration which is the main reason of death in neonatal pigs. this viral infection had a significant impact on the economy of the european and asia pig industries for the last three decades. the disappearance and re-emergence of epidemic ped de monstrated that the current vaccine application, the detection methods, the biosecurity system are old-fashioned and not effective in control and treatment of pedv infection. comprehensive knowledge of the pathogenic characteristics of epidemic and endemic pedv strains is required to generate effective tests for pedv detection in all affected countries and to develop suitable vaccines to different pedv strain for all regions. over the geographic limitation, a number of ped discoveries about the epidemiology, the virology, the pathogenesis, the immunology, the vaccinology as well as several detection methods such as histological examination, virological and serological diagnosis have been gained and upgraded 1, 2, 20 . the potential diagnosis of pedv should not only be rapid and robust but also cost-effective and able to detect a large number of samples in field. new researches and experiments are re quired to investigate cross-protection between field viruses and vaccine strains. more significant measures (such as physicochemical, genetic and antigenic analyses) need to be established to control and prevent the pedv infection. lastly the combined applications of early detection, vaccination, biosecurity programs and management play a major role in prevent and control ped. this review will provide basic and applied understanding of pathogenic pedv as well as the extensive discussion about the recent ped circumstance with modern and popular methods to against pedv. porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines 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characteristics of quebec isolates associated with acute and chronic outbreaks of porcine reproductive and respiratory syndrome this study was performed by the support of the "coo key: cord-266660-0wq77k6y authors: choi, jong-chul; lee, kun-kyu; pi, jae ho; park, seung-yong; song, chang-seon; choi, in-soo; lee, joong-bok; lee, dong-hun; lee, sang-won title: comparative genome analysis and molecular epidemiology of the reemerging porcine epidemic diarrhea virus strains isolated in korea date: 2014-06-19 journal: infect genet evol doi: 10.1016/j.meegid.2014.06.005 sha: doc_id: 266660 cord_uid: 0wq77k6y porcine epidemic diarrhea virus (pedv), a member of the coronaviridae family, is an enveloped, positive-sense, single-stranded rna virus, which causes severe diarrhea and dehydration in suckling pigs. we detected three pedv strains from ten small intestine samples from piglets with acute diarrhea and we determined the complete genome sequences of the reemerging korean pedv field isolates, except for the noncoding regions from both ends. the complete genome sequences of the strains were identical or almost identical (one synonymous single-nucleotide polymorphism (snp) in the orf1a/1b genomic sequence). interestingly, comparative genome analysis of recent korean pedv isolates and other strains revealed that the complete genome sequences of recent korean strains were almost identical (99.9%) to those of the us pedv strains isolated in 2013. these results suggest that the three reemerging korean strains are distinct from previous endemic korean pedv strains and has been recently introduced into korea from oversea with high likelihood. porcine epidemic diarrhea virus (pedv) is a member of the family coronaviridae, subfamily coronavirinae, and genus alphacoronavirus, which include some human and bat coronaviruses. pedv containing a positive-sense, single-stranded rna genome, causes severe diarrhea and dehydration in suckling piglets (song and park, 2012) . since the first report of isolation in europe in 1978 (pensaert and de bouck, 1978) , pedv has become an economic concern in the swine industry in europe and asia (song and park, 2012) . in late 2010, various chinese strains of pedv that were clinically more severe than the classical strains, with 80-100% morbidity and 50-90% mortality in suckling piglets, were detected (li et al., 2012) . in april 2013, pedv outbreaks were confirmed in the us for the first time and the isolates showed very close relationship with the chinese isolate ah2012. a previous study showed that the emergent us pedv strains were likely introduced into the us through intercontinental transmission from china (huang et al., 2013) . in korea, pedv was first isolated in 1992, followed by a large two-year-long outbreak. despite the use of vaccines, frequent occurrence of pedv was detected across the country, mainly during the winter season kweon et al., 1993) . since late november 2013, pedv has reemerged in korea and caused significant economic losses in the swine industry. this study aimed to determine the complete genome sequence of the reemerging korean pedv strain and to investigate their genetic relationship with other strains using comparative genome analysis and phylogenetic analysis. ten small intestine samples were collected from dead piglets from two commercial pig farms in korea. the piglets died following acute watery diarrhea. the macroscopic features of the intestines were typical of pedv infections, including yellowish contents and distended appearance. to detect pedv genome, m gene-targeted rt-pcr was performed using total rna from mucosal scrapings. three of ten samples were positive in the pedv specific rt-pcr. to investigate the origin of the reemerging korean pedv strain, complete genome sequences of the three reemerging korean pedv strains were determined using sanger sequencing. for sanger sequencing, 18 primer pairs were designed for the highly conserved sites of the pedv genome using primer3 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 (koressaar and remm, 2007; untergasser et al., 2012) or designed manually when primer3 failed to identify optimal primer sites near the appropriate genomic regions (table 1) . eighteen dna fragments, which covered the entire genome of pedv except the noncoding regions from both ends, were amplified using the superscript ò one-step rt-pcr system. sequencing reactions were performed using the bigdye ò terminator v3.1 cycle sequencing kit (applied biosystems). the products were analyzed using abi 3730xl dna analyzer (applied biosystems). the sequencing results were assembled using geneious v5.6.7 software. complete genome sequences of the three reemerging korean pedv strains have been submitted into the genebank database under the accession numbers kj588062, kj588063, and kj588064. complete genome alignment between the reemerging korean strains and ten other available strains were performed using multiple alignment with fast fourier transformation (mafft) v6 (katoh and toh, 2008) . to study the relationship between the us and korean pedv outbreaks, us and chinese strains (which were epidemic in 2012) were included in genome alignment. in addition, all available complete genome sequences of pedv isolates from korea were included in the alignment to compare the recent korean strains with previous endemic korean pedv strains. cv777 strain was included in the alignment as pedv reference strain. the maximum likelihood phylogenetic trees for the complete genome and the s gene sequence alignments were generated using phyml version 2.4.4 (guindon and gascuel, 2003) with the generalized time reversible (gtr) substitution model (rodriguez et al., 1990) . the best nucleotide substitution model for analysis was confirmed using mega 5.2.2 (tamura et al., 2011). the complete genome sequences of the reemerging korean strains showed a typical pedv gene order of 5 0 utr-orf1a/1b-s-orf3-e-m-n-3 0 utr and were identical or almost identical (one synonymous single-nucleotide polymorphism (snp) in the orf1a/1b gene) to each other. multiple alignment with other pedv complete genomes indicated that the reemerging korean strains possess genome sequences, which are distinct from those of previous korean field strains (fig. 1) . a previous study had discussed evidence of frequent recombination events between different genetic lineages or sublineages of pedv (huang et al., 2013) . however, genomic sequences of the reemerging korean strains did not show any regions recombined with those of the previous korean strains during the recombination analysis performed using simplot 3.5.1 (lole et al., 1999 ) (data not shown). in addition, phylogenetic analysis of the s gene between the reemerging and previous korean strains of pedv indicated that the reemerging korean strains were included into a genetic lineage different from those of previous endemic korean pedv strains (fig. 2) . these results suggest that the reemerging strains have been recently introduced into korea from another country. interestingly, comparative genome analysis of the reemerging korean pedv isolates and other strains revealed that the complete genome sequences of the recent korean strains were almost identical (99.9%) to those of the us pedv strains isolated in 2013 (fig. 1) . compared with the complete genome of the reemerging korean isolates, genomes of the us strains, usa/iowa/18984/2013 and usa/indiana/17846/2013 showed five (three non-synonymous table 1 primers used for the amplification of full-length genomes of the reemerging korean pedv strains. sequence (5 0 -3 0 ) pcr product size ( fig. 1 . nucleotide sequence alignment and phylogenetic tree analysis for complete genomes of pedv strains. (a) alignment of the complete genome sequences of the reemerging korean pedv field strains and other strains was performed using mafft. one of the reemerging korean strain sequences was set as the reference sequence. vertical lines indicate the snps compared to the reference sequence and dashes indicate sequence gaps. protein-coding regions are indicated with arrows. (b) a maximum likelihood phylogenetic tree was generated using the alignment. one-hundred bootstrap replicates were used to assess the significance of the tree topology. a bar indicates nucleotide substitutions per site. fig. 2 . phylogenetic tree showing the relationship between the reemerging korean strains from previous endemic korean strains and strains from overseas, based on the analysis of the s gene. a maximum likelihood phylogenetic tree was generated from the alignment of complete s gene sequences. one-hundred bootstrap replicates were used to assess the significance of the tree topology. a bar indicates nucleotide substitutions per site. korean pedv strains are denoted using bold characters. and one synonymous in the orf1a/1b gene and one nonsynonymous in the s gene) and seven (four non-synonymous and two synonymous in the orf1a/1b gene and one non-synonymous in the orf3 gene) snps, respectively. both us strains have one insertion sequence causing early termination of the translation of polyprotein encoded in the orf1a/1b gene. on the other hand, the complete genome of the us strain usa/ia2/2013 did not show any indels, but nine (three non-synonymous and three synonymous in the orf1a/1b gene, one non-synonymous in the s gene, and one non-synonymous in the n gene) snps, when compared with the complete genome of the reemerging korean isolates. according to the phylogenetic analysis, the reemerging korean pedv isolates were closely clustered with the us strains isolated in 2013 and chinese strains isolated in 2012 (fig. 1) . comparative genome analysis and phylogenetic analysis revealed that the reemerging korean pedv strains are practically identical to the us strains. a previous study suggested that the three emergent us strains were most closely related to a strain isolated in 2012 from anhui province in china. in addition, the genomes of the reemerging korean pedv strains did not possess any genetic feature from the genomes of the previously sequenced korean field and attenuated vaccine strains. these results suggest that the reemerging korean pedv strains are not variant strains of old korean field or attenuated vaccine strains. there are two possible sources of origin of the reemerging korean pedv strains. first, the same source of origin of the us strains containing chinese pedv-like virus could have been introduced into korea slightly later than the us outbreak events. this hypothesis can explain why the reemerging korean pedv strains are identical to the us strain. another possibility is that us strain has been directly transmitted into korea. during the us outbreak of pedv in 2013, two genetic sublineages of the us strains were isolated. in a previous report, the authors stated that the us strain of pedv diverged during evolution and that evolution generated two genetic sublineages, namely, ia1-co/13 and mn-ia2 (huang et al., 2013) . during complete genome alignment as part of this study, one of the us strains, ia1, showed a recombined genomic region in the orf1a/ 1b gene, which closely matched that of the chinese strain js-hz2012. all reemerging korean pedv strains isolated in this study showed a close relationship with only one of the genetic sublineages of the us strains, namely, mn-ia2. we could not detect a pedv strain with a close relationship with the ia1-co/13 sublineage. this possibly suggests that only one sublineage of the us strain has been directly introduced into korea from the us. to identify the exact source of origin of the reemerging korean strain, further investigation and surveillance are required. furthermore, to prevent the introduction of pedv into korea from overseas in future, the quarantine policy on feed ingredients should be reinforced. prevalence of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus infection in korean pigs a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states recent developments in the mafft multiple sequence alignment program detection and differentiation of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus in clinical samples by multiplex rt-pcr enhancements and modifications of primer design program primer3 isolation of porcine epidemic diarrhea virus (pedv) in korea new variants of porcine epidemic diarrhea virus full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination a new coronavirus-like particle associated with diarrhea in swine the general stochastic model of nucleotide substitution porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods primer3 -new capabilities and interfaces key: cord-254405-yc1q20fz authors: jie, tao; benqiang, li; jinghua, cheng; ying, shi; huili, liu title: preparation and characterization of an attenuated porcine epidemic diarrhea virus strain by serial passaging date: 2018-07-31 journal: arch virol doi: 10.1007/s00705-018-3968-6 sha: doc_id: 254405 cord_uid: yc1q20fz porcine epidemic diarrhea virus (pedv) is prevalent in most parts of the world. owing to its antigenic variation, prevention of the diseases caused by this virus is difficult. in this study, two pedv isolates with similar growth kinetics were successfully propagated in vero cells. complete genome sequence analysis showed that they have a 49nt deletion in the orf3 gene and were classified into group 1, the same group that includes the classical cv777 strain. recombination analysis revealed that the event had occurred in the orf1a gene, at 3596–6819 nt, among the two pedv isolates and the cv777 and dr13 strains. during their continuous propagation, 14 nonsynonymous mutations occurred in the spike (s) gene of strain js-2/2014 between generations g5 and g90, but there were no changes between g90 and g100. we assumed that strain js-2/2014 might be attenuated by the 90th generation. piglets orally fed with js-2/2014 g90 showed no clinical symptoms, and no virus was detected in the feces and nasal fluid. in conclusion, js-2/2014 was successfully identified by screening, was attenuated after propagation in vero cells, and may serve as a candidate virus for vaccine preparations. porcine epidemic diarrhea virus (pedv) is the causative agent of porcine epidemic diarrhea, an enteric disease characterized by acute watery diarrhea, dehydration, and vomiting and which affects pigs of all ages but has a high mortality rate among neonatal piglets [1] . pedv was first described in england in 1971, and until the end of the 1990s, sporadic cases were reported throughout europe [2] [3] [4] [5] . since october 2010, severe pedv outbreaks have occurred in the domestic pig population in china and caused enormous economic losses [6] [7] [8] [9] . pedv vaccines based on the classical pedv strain cv777, namely the inactivated bivalent transmissible gastroenteritis virus (tgev) & pedv vaccine (1999 -present) and the attenuated bivalent tgev & pedv vaccine (2003) (2004) (2005) (2006) were widely used in china and played an important role in controlling the disease. nonetheless, an outbreak of diarrheal disease in 2010 and the high mortality rate among neonatal piglets indicated that the epidemic virus may have changed and escaped the immune response specific to the vaccine [10] . it was subsequently confirmed that variants were responsible for the large-scale outbreak of the disease [11] . the spike (s) protein of coronaviruses performs an important function: binding to cellular receptors and initiating the infection. it also induces neutralizing antibodies in vivo. mutations, including deletions and/or insertions, in the s protein may change the pathogenicity and tissue tropism of coronaviruses; and this mechanism may be the main reason for the virus escaping the immune response generated by vaccination [12] . therefore, it is necessary to analyze the handling editor: sheela ramamoorthy. * liu huili huilil@163.com sequence and characteristics of the prevalent pedv strains for further exploration of these pathogenic mechanisms. in this study, we first determined the complete genome sequences and biological characteristics of pedv strains that have emerged in domestic pigs. subsequently, the genetic relationship between these and other pedv strains was analyzed. furthermore, we cultured the virus and screened for attenuated pedv strains by analyzing the genovariations that occurred during passaging -confirming the results in animal experiments. our results provide useful insights into the preparation of an effective and safe pedv vaccine. all animal experiments were conducted under the guidance of the institutional animal care and use committee at centers for disease control and prevention (cdc) and the laboratory animal care international accredited facility. strains jsls-1/2014 and js-2/2014 were isolated from the intestinal tissues of piglets with watery diarrhea and were sent to our laboratory for virus detection. sows were all inoculated with the bivalent killed or attenuated vaccines against tgev & pedv. as many as 50% of piglets died within 2 days of birth. pigs from another farm died at ~1 month of age following the development of watery diarrhea in the clinic. pedv-positive fecal samples were diluted 10-fold in pbs and then vortexed briefly, followed by centrifugation at 10,000×g for 10 min. the supernatants were passed through 0.22 µm syringe filters and then inoculated onto confluent vero cells. after adsorption at 37 °c for 1 h, the cells were incubated in dulbecco's modified eagle's medium (dmem). the pedv strains were identified by rt-pcr and an indirect immunofluorescence assay (ifa). for the ifa, the vero cells were fixed with 70% ethanol and incubated with an anti-pedv monoclonal antibody for 1 h, followed by incubation with a fluorescein isothiocyanate (fitc)-labeled goat anti-mouse igg antibody. finally, cell staining was examined under a fluorescence microscope. the pedv isolates were purified by a plaque method in vero cells. in brief, confluent cell monolayers in 6-well plates were infected with 10-fold dilutions of each virus isolate (to a total volume of 0.4 ml of pbs) for 1 h at 37 °c. the cells were washed twice with pbs and overlaid with modified eagle's medium containing 0.9% of agar. the plates were then incubated at 37 °c and 5% co 2 . after 5 days of incubation, the cells were stained with 0.1% crystal violet [13] . viral rna was extracted from a 250 μl sample using the trizol reagent (takara, shiga, japan). the pedv cdna was synthesized by means of the primescript high fidelity rt-pcr kit (takara). full-length pedv genome amplification was performed with primers described previously [14] . the 5′ and 3′ end sequences were determined with the 5′ and 3′ race kit (takara). for each amplicon, more than three independent clones were sequenced to determine the consensus sequence of a given genomic region. the clustalx (ver.1.81) software was used to align the full-length genome sequences. phylogenetic analyses based on the s and open reading frame 3 (orf3) genes or the entire genome were performed by the maximum-likelihood method with the general time-reversible nucleotide substitution model, where 1000 bootstrap replicates were implemented in the mega6.0 software [15] . growth kinetics of the pedv strains were measured in vero cells and compared. the viruses were added at a multiplicity of infection (moi) of 1 to vero cells in a 24-well plate. after 1 h, the cell medium was replaced with fresh dmem containing 2.5 µg/ml trypsin. culture samples collected at 12, 24, 48, 60, 72, and 96 h postinfection were freeze-thawed twice and then centrifuged at 12,000×g for 10 min at 4 °c. the supernatants were collected and analyzed for the virus titer (50% tissue culture infectious dose [tcid 50 ]/ml) following the reed-muench method [16] . to evaluate the pathogenicity of a pedv strain, we analyzed and purchased pedv-negative pregnant sows. negativity for known enteric pathogens was confirmed by quantitative rt-pcr. pigs were orally inoculated with the js-2/2014 strain (different passages), 1-day-old neonatal piglets were given access to milk and water ad libitum (three piglets per group). the virus inoculum was 10 6 tcid 50 per animal. clinical symptoms, survival, and virus shedding (daily rectal and throat swabs) were then examined. virus shedding was quantified by rt-pcr with the following primers, pedv-f: 5′-ttc ccg ttg atg agg tga t-3′, pedv-r: 5′-aag cat tga ctg aac gac c-3′. serum and feces samples were collected from each piglet weekly and tested for igg and iga antibodies by elisa. we isolated six pedv strains in this study. due to their high identity, only two strains -jsls-1/2014 and js-2/2014were chosen for genome analysis. they proliferated successfully in vero cells without trypsin (fig. 1a , b) and induced extensive cell death (lysis) without cell fusion (fig. 1c) . the plaque assay confirmed that they had a similar morphology (fig. 1d) . furthermore, their growth characteristics indicated that they had similar growth kinetics, with replication peaking 72 h after infection (fig. 1e ). the genomes of the pedv isolates were sequenced after the 5th passage. the details for each gene are listed in table 1 . complete sequence homology analysis of the pedv isolates and other pedv reference strains revealed that they shared high identity (99.0%-99.9%) with domestic strains oh851 and fl2013. the phylogenetic tree based on the complete genomic sequences indicated that jsls-1/2014 and js-2/2014 isolates clustered in the same group together with classical strains cv777 and dr13 (fig. 2a) . next, analysis of the n-glycosylation sites within the s, membrane (m), and nucleocapsid (n) proteins indicated (table 2 ). in the m protein, our two pedv strains and attenuated dr13 had three n-glycosylation sites, whereas cv777 had one more site. in the n protein, all the strains had six n-glycosylation sites, except for jsls-1/2014 which had one less. the full s sequences of our pedv isolates and of 33 reference strains from korea, germany, the us, and china were compared. the phylogenetic tree, constructed by the neighbor-joining method, showed that the two pedv isolates from this study clustered within the same group as the classical strains, and genetically distant from the chinese pedv strains isolated during 2011-2014 (fig. 2b) . further comparison indicated that the s1 genes from our two pedv isolates were conserved, with amino acid identities of 99.4-99.8%. nevertheless, they had obvious variation, relative to the domestic pedv strains isolated before 2014, with amino acid identities of 89.6-91.3%. analysis of the orf3 genes revealed 99.2% homology in the encoded amino acid sequence between jsls-1/2014 and js-2/2014 isolates, whereas with other strains it reached 100% (e.g., ah/hf/2015, heb/2015/121, and hlj/ qqhr/2015). this meant that there is a certain degree of variation between our two pedv isolates even though their identity is high. in addition, a 49nt sequence was found to be deleted in the orf3 gene in our two pedv isolates -a feature also found in the attenuated strains dr13, hljby, ah-m, and js2008. this deletion is a typical characteristic of cell-adapted pedv strains [17] . on the other hand, the orf3 gene of other domestic pedv strains isolated in recent years is intact in the same way as cv777. it was therefore speculated that the pathogenicity of our pedv isolates was likely attenuated. the m protein is one of the important proteins of pedv for activating host immunity. amino acid comparison indicated that there were only three amino acid mutations between our pedv isolates, cv777, and the attenuated dr13: at amino acid positions 5, 56, and 167 (data not shown). the sequence between amino acid residues 1 and 50 is a potential epitope, and the fact that most of the site mutations are located in this area implies antigenic diversity. by analyzing the amino acid sequences of the n protein, we found great differences among the pedv isolates, cv777, and attenuated dr13. compared with cv777, strain js-2/2014 has 14 amino acid mutations (data not shown). compared with attenuated dr13, strain js-2/2014 has five amino acid mutations, whereas jsls-1/2014 has no changes. to determine the involvement of recombination events in the evolution of the isolates, we performed recombination analysis, using the rdp4 software, to compare our two isolates with representative chinese historical pedv strains from different clusters in the phylogenetic tree. the findings indicated recombination among the two pedv isolates and cv777 and dr13 had occurred in the orf1a gene at site 3596-6819 (nt positions; fig. 3a) , with a higher recombination probability being detected for cv777 (fig. 3b) . the average p values for jsls-1/2014 and js-2/2014 were 3.677 × 10 −3 and 9.426 × 10 −3 , respectively (fig. 3c ). it has been reported that the pedv s gene demonstrates the biggest variability during evolution, prompting us to explore its key genovariation during cell propagation. these data will provide important information for researchers regarding cellular adaptation, virus attenuation, and pathogenicity. strain js-2/2014 was serially passaged in vero cells, and the 5th (g5), 20th (g20), 50th (g50), and 90th (g90) generations of the virus were selected and sequenced. comparison of the amino acid sequences revealed that there were 14 missense mutations in the s gene (table 3 ). three amino acid residues (at sites 288, 330, and 973) were mutated by g20, six (at sites 360, 784, 816, 901, 983, and 1033) more were mutated at g50, and five more (at sites 15, 56, 132, 1204, and 1308) were mutated by g90. it is worth noting that the amino acid variations at passages g20 and g50 also participated in the dr13 attenuation process. nevertheless, the five other amino acid mutations were not seen in other pedv strains and may be specific to strain js-2/2014. strains js-2/2014 g20, js-2/2014 g60, and js-2/2014 g90 at a titer of 10 6 tcid 50 were orally fed to pedv antibodynegative weaned piglets. after the virus challenge, the piglets fed with strain js-2/2014 g90 appeared asymptomatic, and no virus was detected in the feces and nasal fluid. by contrast, piglets infected with the js-2/2014 g20 strain developed watery diarrhea and died at 5 days post-infection (dpi) (fig. 4a ). in addition, the piglets infected with the js-2/2014 g60 strain did not develop diarrhea but had to be detoxified for a period (fig. 4b) . we found that piglets inoculated with js-2/2014 g60 grew slowly before 6 dpi, whereas the weight gain of piglets inoculated with js-2/2014 g90 was normal relative to that of control piglets (fig. 4c) . furthermore, infection with js-2/2014 g90 activated strong igg and iga antibody responses (fig. 4d) . these results confirmed that the js-2/2014 g90 strain was attenuated, and this change was attributed to its repeated propagation in tissue culture. porcine diarrheal disease has become epidemic in china in recent years, causing huge financial losses in the pig industry [18] . pedv, tgev, and porcine rotavirus (porv) are the three main pathogens of this disease [19, 20] . to trace the epidemiology of the recent outbreaks, porcine fecal samples were collected; our results revealing that pedv had a higher infection rate than both tgev and porv. this result is consistent with data from other reports [21, 22] . to explore the evolution and pathogenicity of this virus, we next identified two pedv variants in porcine fecal samples from the shanghai and jiangsu provinces. of note, unlike the pedv variants isolated in china in recent years, our two isolates could propagate in vero cells successfully without trypsin. nevertheless, most of the pedv variants isolated in recent years can proliferate in mammalian cells successfully only in the presence of trypsin [23] . it is unknown whether this phenomenon is related to the s gene, which is important for cell adaptation [24] , or to the orf3 gene, which is a recognized marker for attenuated and virulent strains [25] . our sequence analysis revealed that these two isolates clustered in the same group as classical strain cv777, and all had a 49nt deletion in the orf3 gene. sun et al. [14] reported that all newly isolated strains had intact orfs that could yield translation of a 224aa protein. these studies suggest that this area of the genome may be involved in determining cell tropism and pathogenicity of the virus [26] . park et al. [27] analyzed a cell culture-adapted pedv (passage 100) strain with a smaller orf3 gene and found it to have lower virulence relative to the wild-type virus. this may mean that the pathogenicity of our two pedv isolates is different from that of the pedv variants isolated in recent years. the s protein of pedv has always been used as a marker of viral variation. under the pressure of herd immunity, the s gene of pedv mutates frequently, with some of the missense mutations altering viral antigenicity to aid in the virus's escape from preexisting immunity. thus, periodic vaccine updates may be required to ensure sufficient efficacy against emerging virus variants [28] . in the present study, our data revealed that there is little variation in the s gene among the two pedv isolates, and that they are most similar to the attenuated dr13 strain. to further explore the role of the s gene in virulence, the js-2/2014 strain was successively cultured, and s gene variation during cultivation was analysed. of note, we found that there were three amino acid mutations between js-2/2014 g5 and js-2/2014 g20, six between js-2/2014 g20 and js-2/2014 g50, and five between js-2/2014 g50 and js-2/2014 g90. it is worth noting that the amino acid variations between js-2/2014 g5 and js-2/2014 g50 were also identified in the dr13 attenuation process. this finding indicates that these missense mutations in the s gene may be related to viral pathogenicity. furthermore, animal experiments confirmed that piglets orally fed js-2/2014 g5 (10 6 tcid 50 ) had mild diarrhea and long-term excretion. on the other hand, piglets challenged with js-2/2014 g90 (10 6 tcid 50 ) appeared asymptomatic, with no virus detected in the feces and nasal fluid, indicating that g90 was attenuated and therefore is potentially safe for vaccine preparations. moreover, recombination analysis confirmed that this strain has a high probability of being recombined with cv777 and dr13. most recently, the ch/hnqx-3/14 strain was reported as a cv777 and dr13 strain recombinant with the highly virulent chn/ zmdzy/11 strain. not surprisingly, this strain was detected on a swine farm that is located in the same province as the chn/zmdzy/11 strain; both cv777 and dr13 pedv vaccines were used on this farm [29] . therefore, it is possible that the virulence-enhanced cv777 and dr13 strains had been present in some areas, resulting in virus recombination. in conclusion, this study investigated the epidemiology behind recent outbreaks of porcine diarrheal disease in china and identified two pedv isolates from shanghai and jiangsu. furthermore, we successfully obtained an attenuated pedv strain by screening. the results should help our shanghai agriculture applied technology development program of china (no. t20170111), and the youth talent development plan of shanghai municipal agricultural system of china porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis new 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diarrhea virus in 2014 the epidemiological investigation on viral diarrhea of piglets in guizhou region isolation and identification of porcine epidemic diarrhea virus strain hbmc2012 and its pathogenicity in piglet cellular entry of the porcine epidemic diarrhea virus cloning and further sequence analysis of the orf3 gene of wild-and attenuated-type porcine epidemic diarrhea viruses characterisation of a recent virulent transmissible gastroenteritis virus from britain with a deleted orf 3a cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus dr13 evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from henan, china conflict of interest there are no conflicts of interest associated with this article.ethical approval all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. furthermore, this article does not contain any experiments with human subjects or animals performed by any of the authors. key: cord-260107-gqbtkf0x authors: lee, sunhee; kim, youngnam; lee, changhee title: isolation and characterization of a korean porcine epidemic diarrhea virus strain knu-141112 date: 2015-10-02 journal: virus res doi: 10.1016/j.virusres.2015.07.010 sha: doc_id: 260107 cord_uid: gqbtkf0x severe outbreaks of porcine epidemic diarrhea virus (pedv) have re-emerged in korea and rapidly swept across the country, causing tremendous economic losses to producers and customers. despite the availability of pedv vaccines in the domestic market, the disease continues to plague the korean pork industry, raising issues regarding their protective efficacy and new vaccine development. therefore, pedv isolation in cell culture is urgently needed to develop efficacious vaccines and diagnostic assays and to conduct further studies on the virus biology. in the present study, one korean pedv strain, kor/knu-141112/2014, was successfully isolated and serially propagated in vero cells for over 30 passages. the in vitro and in vivo characteristics of the korean pedv isolate were investigated. virus production in cell culture was confirmed by cytopathology, immunofluorescence, and real-time rt-pcr. the infectious virus titers of the viruses during the first 30 passages ranged from 10(5.1) to 10(8.2) tcid(50) per ml. the inactivated knu-141112 virus was found to mediate potent neutralizing antibody responses in immunized guinea pigs. animal studies showed that knu-141112 virus causes severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis, indicating that strain knu-141112 is highly enteropathogenic in the natural host. in addition, the entire genomes or complete s genes of knu-141112 viruses at selected cell culture passages were sequenced to assess the genetic stability and relatedness. our genomic analyses indicated that the korean isolate knu-141112 is genetically stable during the first 30 passages in cell culture and is grouped within subgroup g2b together with the recent re-emergent korean strains. porcine epidemic diarrhea (ped) is a devastating disease in pigs that is characterized by acute enteritis and lethal watery diarrhea followed by dehydration with high mortality in suckling piglets (debouck and pensaert, 1980; saif et al., 2012; pijpers et al., 1993) . the disease was initially recognized in england in 1971 and has then spread to swine-producing european countries (oldham, 1972; pensaert et al., 1981) . since the 1990s, ped has become rare in europe and is more often associated with post-weaning diarrhea in adult pigs (saif et al., 2012) . ped was first reported in asia in 1982 and has since had a great economic impact on the asian pork industry (chen et al., 2008; kweon et al., 1993; li et al., 2012; puranaveja et al., 2009; takahashi et al., 1983) . in may 2013, ped outbreaks suddenly appeared in the united states and have rapidly spread nationwide as well as to canada and mexico, causing high mortality in newborn piglets and significant financial concerns (mole, 2013; stevenson et al., 2013 vlasova et al., 2014 . the etiological agent of ped, ped virus (pedv), was identified as a coronavirus in 1978, which is a member of the genus alphacoronavirus within the family coronaviridae of the order nidovirales (lai et al., 2007; pensaert and de bouck, 1978; saif et al., 2012) . pedv is a large, enveloped virus possessing a single-stranded positivesense rna genome of approximately 28 kb with a 5 cap and a 3 polyadenylated tail (pensaert and de bouck, 1978; saif et al., 2012) . the spike (s) protein of pedv is the major envelope glycoprotein of the virion and plays pivotal roles in interacting with the cellular receptor for virus entry and mediating neutralizing antibodies in the natural host (jackwood et al., 2001; lai et al., 2007; lee et al., 2010) . therefore, the pedv s glycoprotein is known to be an appropriate viral gene for determining the genetic relatedness among pedv isolates and for developing diagnostic assays and effective vaccines (chen et al., 2014; gerber et al., 2014; lee et al., 2010; lee and lee, 2014; oh et al., 2014) . the first ped epizootic in korea was confirmed in 1992 (kweon et al., 1993) . however, a retrospective study revealed that pedv already existed as early as 1987 (park and lee, 1997) . since the emergence, ped outbreaks occurred every year, resulting in substantial economic losses to the korean swine industry until early 2010. after severe outbreaks of foot-and-mouth disease (fmd) during 2010 to 2011, however, the prevalence of pedv infections was occasional with only sporadic outbreaks in korea. this epidemic situation probably resulted from the mass culling of more than one-third of the entire domestic pig population in korea during the 2010-2011 fmd outbreaks. however, starting in november 2013, severe ped epidemics re-emerged in korea and swept more than 40% of pig farms lee et al., 2014a,b) . although both modified live and killed vaccines against ped are commercially available in korea, continuous ped epidemics indicate a low effectiveness of the domestic vaccines. this result appears to be due to genetic and antigenic differences between s proteins of vaccine and field strains (lee et al., 2010; oh et al., 2014; lee and lee, 2014) . thus, the lack of effective vaccines enhances the need for the development of next-generation vaccines to control ped. pedv isolation in cell culture is critical for developing effective vaccines for ped prevention as well as performing various pedv research. however, the cell culture isolation of pedv has shown to be difficult and even the isolated virus may be unable to maintain infectivity upon further passages in cell culture (chen et al., 2014) . to date, there have only been two reports in more than two decades on the cultivation of the korean pedv strain that is genetically divergent from field pedvs (kweon et al., 1999; song et al., 2003) , while a number of pedv strains have been recently isolated in the us and successfully grown in cell culture for a year (chen et al., 2014; oka et al., 2014) , in the present study, we attempted to isolate pedv from various pedv-positive samples using vero cells. at this time, one highly virulent korean strain kor/knu-141112/2014 has been successfully isolated and serially propagated in cell culture for over 30 passages. we aimed to characterize the growth and titer of the pedv isolate knu-141112 during the serial passages and the pathogenicity of the virus in suckling piglets. our in vivo assessment demonstrated that knu-141112 is highly entero-pathogenic in piglets, exhibiting severe clinical symptoms as well as macroscopic and microscopic lesions typical for pedv infection. in addition, the complete genome or full-length s gene sequences of knu-141112 were determined at selected passages to study the genetic stability and relationship. our data indicated that knu-141112 isolate is relatively stable during the first 30 passages in cell culture and is classified into subgroup g2b that includes pedv strains responsible for recent severe outbreaks in korea and the us. vero cells (atcc ccl-81) were cultured in alpha minimum essential medium (␣-mem; invitrogen, carlsbad, ca) with 5% fetal bovine serum (fbs; invitrogen) and antibiotic-antimycotic solutions (100×; invitrogen) and maintained at 37 • c in a humidified 5% co 2 incubator. seven small intestinal homogenates and 50 stool specimens that tested positive by rt-pcr using an i-tge/ped detection kit (intron biotechnology, seongnam, south korea) were selected for virus isolation experiments. intestinal homogenates were prepared to 10% (wt/vol) suspensions with phosphate-buffered saline (pbs) using a magna lyser (roche diagnostics, mannheim, germany) by three repetitions of 15 s at a speed of 7000 rpm. fecal samples were diluted with pbs to be 10% (wt/vol) suspensions. the suspensions were then vortexed and centrifuged for 10 min at 4500 × g (hanil centrifuge fleta5, incheon, south korea). the supernatant was filtered through a 0.22-m-pore-size syringe filter (millipore, billerica, ma) and stored at −80 • c as an inoculum for virus isolation until use. virus isolation of pedv was attempted on vero cells as described previously with some modifications (hofmann and wyler, 1988) . briefly, prior to inoculation, inocula were prepared by adding trypsin (usb, cleveland, oh) to intestinal or fecal suspensions prepared above to give a final concentration of 10 g/ml. confluent vero cells grown in 6-well plates were washed with pbs and inoculated with 400 l of each inoculum containing trypsin. after incubating at 37 • c for 1 h, 2 ml of virus growth medium [␣-mem supplemented with antibioticantimycotic solutions, 0.3% tryptose phosphate broth (tpb; sigma, st. louis, mo), 0.02% yeast extract (difco, detroit, mi), 10 mm hepes (invitrogen), and 5 g/ml of trypsin] was added. the inoculated cells were maintained at 37 • c under 5% co 2 and monitored daily for cytopathic effects (cpe). when 70% cpe appeared, inoculated cells were subjected to three rounds of freezing and thawing. the culture supernatants were then collected and centrifuged at 400 × g for 10 min. the clarified supernatants were aliquoted and stored at −80 • c as 'passage 1 (p1)' viral stocks until use. one hundred millimeter diameter tissue culture dishes were used for serial passages of the isolate. if no cpe was shown in inoculated cells for 7 days, the plates were frozen and thawed three times, and the supernatants were harvested by centrifugation and inoculated on fresh vero cells for the next passage. if cpe and rt-pcr results were negative after 6 blind passages, the virus isolation was considered negative. the pedv n protein-specific monoclonal antibody (mab) was obtained from choogang vaccine laboratory (cavac; daejeon, south korea). vero cells were infected with each passage knu-141112 virus stock in the presence of trypsin as described above. the culture supernatants were collected at 24 or 48 h postinfection (hpi) at which a 70% cpe is commonly developed. for growth kinetics experiments, the supernatants were harvested from cells infected with each selected passage virus at different time points (6, 12, 24, 36, and 48 hpi) and stored at −80 • c. virus titers were measured in 96-well plates by 10-fold serial dilution of the samples in triplicate per dilution to determine the quantity of viruses required to produce cpe in 50% of inoculated vero cells and calculated as tcid 50 per ml using the reed-muench method (reed and muench, 1938) . the pedv titer was also determined by a plaque assay using vero cells and quantified as plaque-forming units (pfu) per ml. vero cells grown on microscope coverslips placed in 6-well tissue culture plates were mock infected or infected with pedv at a multiplicity of infection (moi) of 0.1. the virus-infected cells were subsequently propagated until 24 hpi, fixed with 4% paraformaldehyde for 10 min at room temperature (rt) and permeabilized with 0.2% triton x-100 in pbs at rt for 10 min. the cells were blocked with 1% bovine serum albumin (bsa) in pbs for 30 min at rt and then incubated with n-specific mab for 2 h. after being washed five times in pbs, the cells were incubated for 1 h at rt with a goat anti-mouse secondary antibody conjugated to alexa fluor 488 (invitrogen), followed by counterstaining with 4 ,6-diamidino-2-phenylindole (dapi; sigma). the coverslips were mounted on microscope glass slides in mounting buffer and cell staining was visualized using a fluorescence leica dm il led microscope (leica, wetzlar, germany). viral rna was extracted from virus isolates or fecal samples prepared as described above using an i-tge/ped detection kit according to the manufacturer's protocol. quantitative real-time rt-pcr was performed using a one step sybr primescript rt-pcr kit (takara, otsu, japan) as described elsewhere (kim et al., 2007; sagong and lee, 2011) . the reaction took place using a thermal cycler dice real time system (takara) and the results were analyzed by the system software as described previously (sagong and lee, 2011) . eight 3-4 month-old guinea pigs (weighing 300-350 g) were randomly allocated into inoculated (n = 6) and control (n = 2) groups. six guinea pigs were immunized subcutaneously with 0.5 ml of binary ethylenimine (bei)-inactivated knu-141112-p10 virus in the presence of freund's complete adjuvant (sigma) and boosted once with a freshly prepared emulsion of the inactivated virus and freund's incomplete adjuvant (sigma) at a 2-week interval. two sham-inoculated guinea pigs were administered and boosted with cell culture media in the presence of the respective adjuvant. pre-immune sera were collected before starting the immunization and antisera were collected at 2 weeks after the final boost. the presence of pedv-specific neutralizing antibodies in serum samples collected from guinea pigs in all groups was determined using a serum neutralization (sn) test in 96-well microtiter plates using pedv isolate knu-141112 or vaccine strain sm98-1 as previously described (oh et al., 2014) with minor modifications. briefly, vero cells were grown at 2 × 10 4 /well in 96-well tissue culture plates for 1 day. the knu-141112-p10 virus stock was diluted in serum-free ␣-mem to make 200 tcid in a 50 l volume. the diluted virus was then mixed with 50 l of 2-fold serial dilutions of individual inactivated sera in 96-well plates and incubated at 37 • c for 1 h. the mixture was inoculated into vero cells and incubated at 37 • c for 1 h. after removing the mixture, the cells were thoroughly rinsed 5 times with pbs and maintained in virus growth medium at 37 • c in a 5% co 2 incubator for 2 days. for the sn test using the vaccine strain, pedv strain sm98-1 propagated in the absence of trypsin was diluted in serum-free ␣-mem to make 200 pfu in a 50 l volume. the diluted virus was then mixed with each serum sample in 96-well plates as described above and incubated at 37 • c for 1 h. subsequently, approximately 1 × 10 4 vero cells in 100 l of ␣-mem containing 5% fbs were added to each well and the mixture was maintained at 37 • c in a 5% co 2 incubator for 3 days. the neutralization titer was calculated as the reciprocal of the highest dilution of serum that inhibited virus-specific cpe in all of the duplicate wells. the in vivo swine studies described here were performed at the improah animal facility under the guidelines established by its institutional animal care and use committee. a total of 8 suckling piglets of 7 days of age were obtained from a commercial pig farm with no known prior ped outbreak or vaccination with pedv. all animals were determined to be free of antibodies to pedv as well as to transmissible gastroenteritis virus (tgev) and porcine reproductive and respiratory syndrome virus. pigs were randomly assigned to 3 experimental groups housed in 2 separated rooms: pedv-inoculated (n = 4) and contact control (n = 2) in room 1 and sham-inoculated control (n = 2) group in room 2. following a 1 day acclimation period, only piglets in the pedv-inoculated group orally received a 1 ml dose of 10 3 tcid 50 /ml of knu-141112-p10 virus. two piglets were exposed to the virus by direct contact with inoculated piglets in the same room. the sham-inoculated pigs were administered with cell culture media as a placebo. clinical signs of diarrhea and mortality were monitored daily for the duration of the study. stool samples from all groups were collected prior to inoculation and daily with 16 inch, cotton-tipped swabs and subjected to rt-pcr using an i-tge/ped detection kit and real-time rt-pcr as described above. pedv-inoculated piglets were necropsied daily (days 1, 2, 3, and 4) after challenge for post-mortem examinations throughout the study, whereas all pigs from the contact and control groups were euthanized at 4 days post-challenge for post-mortem examinations. small intestinal tissue specimens (<3 mm thick) collected from each piglet were fixed with 10% formalin for 24 h at rt and embedded in paraffin according to standard laboratory procedures. the formalin-fixed paraffin-embedded tissues were cut at 5-8 m thick on a microtome (leica), floated on a 40 • c water bath containing distilled water, and transferred onto glass slides. the tissues were then deparaffinized in xylene for 5 min and washed in decreasing concentrations of ethanol (100%, 95%, 90%, 80%, and 70%) for 3 min each. the deparaffinized intestinal tissues sections were stained with hematoxylin and eosin (sigma) for histopathology or subjected to immunofluorescence assay using pedv n-specific mab as described above. the paraffin-embedded tissue sections were deparaffinized, treated with 0.01 m citrate buffer (ph 6.0) in a microwave oven for 5 min, chilled at rt for 20 min, and then incubated with 0.3% hydrogen peroxide in dw for 20 min to block endogenous peroxidase. after being washed three times in pbs, the sections were blocked with normal horse serum (vectastain abc kit; vector laboratories, burlingame, ca) and incubated 1 h at rt with n-specific mab. after rinsing in pbs, the samples were reacted for 45 min at rt with a horse anti-mouse secondary antibody (vectastain abc kit), incubated with avidin-biotin peroxidase complex for 45 min (vectastain abc kit), and developed using the dab substrate kit (vector laboratories) according to the manufacturer's instructions. the slides were then counterstained with hematoxylin, dehydrated, cleared with xylene, and mounted on microscope glass slides in mounting buffer and tissue staining was visualized using a microscope. the complete genomic sequences of the original fecal sample as well as those of the p5 and p10 isolates were determined by nextgeneration sequencing (ngs) technology. total rna was extracted from the feces as well as p5 and p10 isolates using a rneasy mini kit (qiagen, hilden, germany) according to the manufacturer's instructions and used as a template to amplify cdna fragments as described elsewhere lee et al., 2014b) . ten overlapping cdna fragments were generated to encompass the entire genome of each sample, pooled in equimolar amounts, and subjected to ngs using the ion torrent personal genome machine (pgm) sequencer system (life technologies, carlsbad, ca) and a 316 v2 sequencing chip (life technologies) as described previously lee et al., 2014b; rothberg et al., 2011) . the single-nucleotide variants (snvs) were analyzed using the clc genomic workbench version 7.0 (clc bio, cambridge, ma) and the individual ngs reads were assembled using the complete genome of pedv reference strain kor/knu-1305/2013 (genbank accession no. kj662670). the 5 and 3 ends of the genomes of the original feces and the p5 and p10 isolates were determined by rapid amplification of cdna ends (race) as described previously . the full-length genomic nucleotide sequences of the knu-141112-feces, knu-141112-p5, and knu-141112-p10 were deposited in genbank under accession numbers kr873431, kr873434, and kr873435, respectively. the s glycoprotein gene sequences of the virus isolates were also determined by the traditional sanger method. two overlapping cdna fragments spanning the entire s gene of each isolate were rt-pcr amplified as described previously (lee et al., 2010) . the individual cdna amplicons were gel-purified, cloned into pgem-t easy (promega, madison, wi), and sequenced in both directions using two commercial vector-specific t7 and sp6 primers and the s gene-specific primers. in addition, the complete structural gene sequences of the virus isolate at selected passages (knu-141112-p3, knu-141112-p4, knu-141112-p20, and knu-141112-p30) were determined by the sanger method as described above and deposited to the gen-bank database under their accession numbers shown in fig. 5 . the sequences of 42 fully sequenced s genes and 25 complete genomes of pedv isolates were independently used in sequence alignments and phylogenetic analyses. the multiplesequencing alignments were generated with the clustalx 2.0 program (thompson et al., 1997) and the percentages of nucleotide sequence divergences were further assessed with the same software program. phylogenetic trees were constructed from the aligned nucleotide or amino acid sequences by using the neighborjoining method and subsequently subjected to bootstrap analysis with 1000 replicates in order to determine percentage reliability values on each internal node of the tree (saitou and nei, 1987) . all tree figures were produced using mega 4.0 software (tamura et al., 2007) . student's t test was used for all statistical analyses, and p-values of less than 0.05 were considered statistically significant. we attempted to isolate pedv from pcr-positive clinical samples, including 50 feces and 7 intestinal homogenates on vero cells. one pedv isolate designated knu-141112 was successfully isolated from the feces of a naturally infected piglet from a commercial farm located in kyungpook province obtained on september 29, 2014. the knu-141112 virus was capable of producing distinct cpes typical for pedv infection, such as cell fusion, syncytium, and detachment, in infected vero cells from passage 3 (p3). we then investigated whether the isolate can be efficiently cultivated and maintained in cell culture. thus, the isolated pedv strain knu-141112 was further serially passaged in vero cells for a total of 30 passages (p1 to p30). the time of cpe onset was at 24 hpi and, accordingly, prominent cpe was observed within 48 hpi in the first 2 productive passages (p3 and p4). in the later passages, visible cpe appeared at 12 hpi and became predominant by 24 hpi. virus propagation was confirmed by detecting pedv antigens by ifa using a pedv n protein-specific mab. the distinct staining was distributed in the cytoplasm of typical syncytial cells. in contrast, no cpe and n-specific staining was evident in mock-inoculated vero cells. examples of cpe and corresponding ifa images in selected passages are shown in fig. 1 . the level of viral genome in each selected passage was further assessed by real-time rt-pcr and the mean cycle threshold (ct) value was determined to be 16.7, ranging from 15.3 (p10) to 18.7 (p5). the infectious titer of the isolate ranged from 10 5.1 to 10 6.1 tcid 50 /ml up to p5, whereas it was determined to be approximately 10 7 tcid 50 /ml in the later passages. the peak viral titer reached 10 7.8 tcid 50 /ml (equivalent to 10 7.5 pfu/ml) or more since passage 10 ( fig. 2a) . the growth kinetics study further showed that knu-141112 replicated rapidly and efficiently in vero cells, reaching a maximum titer >10 7 tcid 50 /ml by 24 dpi (fig. 2b) . guinea pig antisera were collected before immunization (preimmune) and at 2 weeks after the final boost and tested for their neutralizing activity against the isolate knu-141112 or vaccine strain. as shown in fig. 3 , the guinea pig antisera were highly effective in inhibiting knu-141112 infection with mean neutralizing antibody (na) titers of 1:112, whereas the antisera at relatively low dilution fully protect vero cells from sm98-1 infection with mean na titers of 1:37. in contrast, none of the pre-immune and nonimmunized sera showed neutralizing activity against either strain. taken together, our data indicated that the isolate knu-141112 elicits potent antibody responses in immunized animals. however, the antisera strongly recognized the homologous field isolate, but inefficiently the heterologous pedv vaccine strain, suggesting the antigenic variations between the vaccine strain and field pedvs. four piglets were challenged orally with the knu-141112 virus, while 2 control animals were inoculated with cell culture media. two piglets were housed together with inoculated piglets in the same room for direct contact exposure. during the acclimation period, all piglets were active and had normal fecal consistency. pedv-challenged piglets exhibited clinical signs including lethargy and diarrheic feces by 1 day post-inoculation (dpi) and experienced severe watery diarrhea with vomiting thereafter. pedv-associated mortality occurred in one of the inoculated pigs at 1 dpi. contact piglets housed with the inoculated group exhibited diarrheic feces with vomiting by 2 dpi and the progression of clinical signs was similar to that of the inoculated animals. furthermore, all inoculated and contact piglets were positive for pedv, as determined by rt-pcr, by 1 or 2 dpi and shed pedv in feces with mean ct values of 18.7 (range 16.4-21.0) and 20.1 (range 15.4-23.1), respectively. negative control pigs remained active with normal feces and continued to be undetectable for fecal shedding of pedv throughout the study period. one piglet died from pedv and was subsequently necropsied at 1 dpi and the remaining inoculated piglets were randomly selected for necropsy thereafter. all contact and control animals were euthanized at the end of the study for postmortem assessments. all inoculated and naïve contact pigs macroscopically displayed typical ped-like lesions. the small intestine was dilated and accumulated with yellow fluid and its wall was thin and transparent, due to the villous atrophy (fig. 4, panel a) . the stomach was distended and filled with curdled and undigested milk. in contrast, the other intestinal organs appeared grossly normal. microscopic intestinal observations consistent with viral enteritis were developed in all inoculated and contact piglets, which included vacuolation of small intestinal enterocytes and shortening and fusion of small intestinal villi (fig. 4, panels b and c) . similar microscopic lesions were continuously present in the challenged and contact pigs through 4 dpi. furthermore, both ifa and ihc staining revealed that the viral antigen was predominantly detected in the cytoplasm of epithelial cells on atrophied villi in all segments of the small intestines (fig. 4 , panels d-f). these finding are comparable to those in pigs naturally or experimentally infected with virulent strains of pedv (debouck et isolates. pedv-specific cpes were observed daily and were photographed at 24 hpi using an inverted microscope at a magnification of 200× (first panels). for immunostaining, infected cells were fixed at 24 hpi and incubated with mab against the n protein, followed by alexa green-conjugated goat anti-mouse secondary antibody (second panels). the cells were then counterstained with dapi (third panels) and examined using a fluorescence microscope at 200× magnification. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 2013). neither macroscopic nor microscopic intestinal lesions were observed in the negative control piglets during the experiment. entire genome sequence data of the original fecal sample (knu-141112-feces) and cell culture-passaged knu-141112-p5 and -p10 viruses were determined using the ngs technology. the full-length genome of kor/knu-1305/2013 pedv was used as the initial reference for each ngs read and the individual complete genome sequences were successfully obtained by the assembly of respective ngs reads. the 5 and 3 ends of their genomes were also sequenced by race. all three genomes were 28,038 nucleotides (nt) in length, excluding the 3 poly(a) tail and exhibited the genomic organization typical of all previously sequenced pedv strains, consisting of the 292-nt 5 utr, the 20,345-nt orf1a/1b (nt 293 to 12,601 for 1a and nt 12,601 to 20,637 for 1b), the 4161-nt s gene (nt 20,634 to 24,794), the 675-nt orf3 (nt 24,794 to 25,468), the 231-nt e gene (nt 25,449 to 25,679) , the 681-nt membrane (m) gene (nt 25,687 to 26,367), the 1326-nt n gene (nt 26,379 to 27,704), and the 334fig. 3 . virus-neutralizing antibody titers in the sera of guinea pigs inoculated with a pedv knu-141112 isolate. guinea pigs were immunized twice with inactivated knu-141112-p10 virus by subcutaneous injection. blood samples were collected prior to immunization and at 2 weeks after the second immunization and subjected to the virus neutralization assay using homologous (knu-141112; solid circles) and heterologous (sm98-1; solid diamonds) strains. neutralizing antibody titers for individual infected animals were spotted as a log2 scale. values are representative of the mean from three independent experiments in duplicate and error bars denote standard deviations. nt 3 utr. the slippery heptamer sequence, tttaaac, followed by a stem-loop structure was present at the 3 end of orf1a in the pedv genome, which is a potential signal for a ribosomal frame shift during translation to generate the pp1ab. the complete genome sequences of all three viruses were compared, and the results are summarized in table 1 . compared to the knu-141112-feces, knu-141112-p5 showed one different nt at position 21,756 resulting in one amino acid (aa) change (leu to phe) in the s protein, while knu-141112-p10 gained two additional nt changes at positions 21,448 and 24,492 causing two aa mutations located in the s protein. the full-length s genes of the original feces and knu-141112 viruses at selected passages (p3, p4, p5, p10, p20, and p30) were also sequenced using the traditional sequencing method. the s protein sequences of knu-141112-feces, -p5, and -p10 were completely identical to those determined by ngs. one nt change present in the s gene of knu-141112-p5 had been acquired since passage 3 (knu-141112-p3). a total of three nt mutations, identified at passage 10 compared to the feces, were sustained through passage 30 (knu-141112-30) . in addition, compared to knu-141112-p10, we were able to detect two independent mutations at positions 24,869 and 25,656, resulting in amino acid changes in orf3 (asp to tyr) and e (pro to ser) of knu-141112-p20, respectively, which were maintained at passage 30. altogether, our results revealed that the pedv isolate knu-141112 is genetically stable during serial passages in cell culture. the entire genome sequences of pedvs determined in this study were further compared to those of 3 other korean and 8 non-korean pedv strains available in genbank, and the nucleotide homology and difference data are described in table 2 . knu-141112 isolates (feces, p5, and p10) had the highest nucleotide identity (99.9%) with the korean re-emergent strain kor-knu-1305 and us strains, in17846 and mn, showing 8 to 46 different nt at the genomic level. all three viruses were genetically distinct from the korean vaccine strains, sm98-1 and dr-13, and the prototype cv777 strain and exhibited relatively low nucleotide identity ranging from 96.3% to 96.8%. by alignment of the global pedv strains, a single-nucleotide insertion between nucleotides 20,204 and 20,205 has been previously identified in one chinese ah2012 and three us strains, table 1 nucleotide and amino acid changes of knu-141112 during serial passages in cell culture. nucleotide amino acid position feces p3 p4 p5 p10 p20 p30 position feces p3 p4 p5 p10 p20 p30 5 utr (292) -a -n d b nd --nd nd --nd nd --nd nd orf1a (12,354) --nd nd --nd nd --nd nd --nd nd orf1ab (20,345) --n d nd --nd nd --nd nd --nd nd s (4,161) 21448 a a a a c c c 272 k k k k t t t 21756 c t t t t t t resulting in the shorter pp1ab protein in length (chen et al., 2014) . however, none of the genomes of the three pedvs included such an insertion, and this result was further confirmed by the traditional sanger sequencing method (data not shown). in agreement with previous results (lee et al., 2010; lee and lee, 2014; lee et al., 2014a,b) , the full-length s gene-based phylogenetic tree revealed that the global pedv strains were clearly defined into 2 separate clusters, designated genogroup 1 (g1; classical) and genogroup 2 (g2; field epidemic). each genogroup can be further divided into subgroups 1a, 1b, 2a, and 2b (fig. 5a ). all original feces and passaged pedv viruses through passages 30 were classified into subgroup 2b along with the recent korean field isolates, which were most closely clustered together with the emergent us strains in an adjacent clade with the same subgroup. subsequent phylogenetic analysis of the s1 region showed the same grouping structure as the s gene-based tree (data not shown). in addition, phylogenetic analysis based on the entire genome sequences demonstrated that strain knu-141112 is grouped within the same cluster with the recent korean and us stains (fig. 5b ). in korea, three pedv strains, sm98-1, dr-13, and chinju99, were initially isolated almost two decades ago in korea. genetic and phylogenetic analyses revealed that sm98-1 and dr-13 belong to the classical group 1, whereas chinju99 is classified into the field epidemic group 2. of these, only sm98-1 and dr-13 isolates can be grown in cell culture and, accordingly, have been used as commercial vaccine seeds. since 2008, we have been monitoring the genetic diversity of pedv prevalent in the field based on the s gene. our data demonstrated the existence of antigenic and genetic variations between vaccine and field strains, exhibiting a more than 10% amino acid difference in the s1 domain (lee et al., 2010; lee and lee, 2014; oh et al., 2014) . this may be one of the reasons for the incomplete efficacy of current vaccines in korea, suggesting that the isolation of field pedv is required for the development of a next-generation vaccine. although virus isolation in cell culture from clinical samples of naturally or experimentally infected pigs is fastidious, recent studies reported the successful isolation and propagation of several us original pedv strains using vero cells (chen et al., 2014; oka et al., 2014) . in this study, we initially sought to isolate pedv efficiently propagated in vitro from 7 intestine homogenates and 50 fecal samples of naturally infected piglets (field cases) and were able to obtain only 1 isolate from feces in vero cell culture. the virus isolation rate, in the present study, was less than 2% and was relatively lower than that in recent us studies ranging from 5 to 10% (chen et al., 2014; oka et al., 2014) . in previous studies, all isolated pedv strains originated from intestinal contents of naturally infected or experimentally inoculated pigs, suggesting that intestine samples may be a better source for virus isolation (chen et al., 2014; oka et al., 2014) . however, we failed to isolate pedv from intestine homogenates, which may be due to the number of intestinal contents (n = 7) included in our study and be responsible for the low isolation rate. although pedv isolation might be affected by multiple factors, it appears to depend on the number of samples with good quality rather than the type of samples (intestinal contents or feces). further studies are needed to improve the isolation methodology or to determine the contributing factor(s) to enhance the success rate of pedv isolation in cell culture. the pedv isolate, knu-141112, was cytopathogenic in vero cells from passage 3 and after passage 5, exhibited more severe and rapid cpe characterized by fusion of infected cells (syncytium or polykaryon formation). the initial viral infectious titers ranged from approximately 5 to 6 log 10 tcid 50 /ml and increased after several more passages reaching to 8 log 10 tcid 50 /ml or more. these growth characteristics, including cytopathology, infectious titer, and growth kinetics were unchanged or even more efficient throughout the experiment (30 passages), indicating that the isolate knu-141112 is phenotypically stable during serial passages in vero cells. since the antibody response is a critical indicator to prove the cause of viral infection, we immunized guinea pigs twice with the inactivated isolate (knu-141112-p10) and determined whether the animals developed humoral immunity using an sn test. the guinea pig sera raised against the isolate contained high levels of na (6.4 log 2 ), demonstrating the ability of knu-141112 to elicit immune responses. on the other hand, the antisera showed a relatively weak neutralizing response (an almost 2-log 2 reduction), when the heterologous vaccine strain sm98-1 was used for an sn test. this weak neutralizing activity of the anti-knu-141112 guinea pig sera against sm98-1 was somewhat expected because of a high degree of genetic diversity between the s proteins of the vaccine strain and field isolates (lee et al., 2010; lee and lee, 2014; oh et al., 2014) . experimental oral inoculation of nursery pigs with pedv isolate knu-141112 induced severe clinical disease typical of acute enteritis throughout the study, demonstrating that the isolate was highly enteropathogenic in neonatal piglets. onset of clinical signs including lethargy and watery diarrhea in inoculated pigs was found as early as 1 dpi. the pedv genome was detected in feces in 100% of challenged pigs on dpi 1 and virus quantities from fecal shedding were maintained thereafter in all challenged pigs throughout the study period, leading to the source for direct transmission of virus to other animals. this result is inconsistent with a previous study using a us pedv strain that demonstrated virus fecal shedding on dpi 1 in only one-fourth of the challenged pigs (madson fig. 5 . phylogenetic analyses based on the nucleotide sequences of the spike genes (a) and the full-length genomes (b) of pedv strains. a putative similar region of the spike protein and the complete genome sequence of tgev was included as an outgroup in each panel. multiple-sequencing alignments were performed using clustalx program and the phylogenetic tree was constructed from the aligned nucleotide sequences by using the neighbor-joining method. numbers at each branch represent bootstrap values greater than 50% of 1000 replicates. names of the strains, countries and years of isolation, genbank accession numbers, and genogroups and subgroups proposed in this study are shown. the pedv isolates identified in this study are indicated by solid circles. scale bars indicate nucleotide substitutions per site. et al., 2014) . the difference between the current and previous studies is the age of the pigs: 1-week-old neonatal pigs and 3-week-old weaned pigs used in the present and previous experiments, respectively. therefore, younger piglets in this study were more sensitive to pedv infection shedding virus in feces earlier than older pigs, probably due to an age-dependent disease severity as previously described (shibata et al., 2000) . ihc and ifa revealed that viral antigen in villous enterocytes were observed at 1 dpi in all segments of the small intestine of inoculated piglets. the onset of clinical signs and viral fecal shedding and the detection time of viral antigen in the target tissue were similar to recent independent reports using different us pedv strains jung et al., 2014) . two non-challenged piglets were included in this study for direct contact to inoculated piglets in the same space. all contact piglets displayed ped-like symptoms within 24 h after the onset of clinical signs in inoculated piglets. the presence of pedv in feces and infected tissues was further verified in contact piglets, showing 100% morbidity in our study. mortality averages 50% in suckling piglets up to 1 week of age, often approaching 100% in 1-to 3-dayold piglets, and decreases to 10% thereafter (saif et al., 2012) . in our study, mortality was observed only in one out of four inoculated piglets, resulting in 25% mortality in the current study involving 8-day-old piglets. for reproducible challenge studies in vivo using the isolate in the future, a neonatal swine bioassay will be needed to determine either the median pig diarrhea dose or lethal dose as a standardized dose and this aspect is currently under investigation. whole-genome sequences of 3 korean pedv strains (knu-141112-feces, p5, and p10) were determined using ngs approaches coupled with race experiments. regions covering the structural genes were also sequenced at the selected passages by the sanger method for confirmation. compared to the original feces (knu-141112-feces), only one nt difference at position 21,756 was identified for the first 5 passages (knu-141112-p5) in cell culture, which led to a non-synonymous mutation at the corresponding position 375 of the s protein. this nt change was initially found at passage 3 and further maintained through passage 30. however, we were unable to investigate whether this mutation had been acquired at the beginning of the vero cell culture since infectious virus was not obtained during the first two passages. interestingly, the identical c21756t mutation at the whole-genome level (l375f at the aa level of s) has been previously reported in a us pedv isolate isu-19338e during cell culture passage (chen et al., 2014) , suggesting its potential importance for adaption of the field virus to growth in vitro. at passage 10, two more nt differences at positions 21,448 and 24,492 of the genome (a21448c and g24492c) were detected when compared to knu-141112-feces. all of the changes acquired in knu-141112-p10 led to non-synonymous mutations at the respective positions 272 and 1287 of the s protein (k272t and e1287q). these findings were similar to recent data reported by chen et al. (2014) that two us isolates individually gained the 4 mutations located in orf1b, s, and e through passage 9 in cell culture. however, no nucleotide changes were identified in orf1b and e for the first 10 passages in vero cells. the 3 mutations in the s protein were persistent for 30 passages in cell culture. celladapted pedv vaccine strains, sm98-1 and dr-13, are known to contain a 52-nt deletion spanned from the end of s to the start of orf3 and a 49-nt deletion in the middle of orf3. thus, we further sequenced structural genes of knu-141112-p20 and -p30 and identified two additional nucleotide changes (g24869t and c25656t) acquiring non-synonymous amino acid mutations in orf3 and e, respectively. except for those nucleotide substitutions, the isolate had no extra change, including such large deletions in structural genes through passage 30, indicating the genetic stability of knu-141112 during serial passages in vero cells. sequence comparisons with other pedv strains showed that knu-141112 isolates are genetically most similar to recent korean and us epidemic strains with 99.8-99.9% identities, but most distinctly related to classical strains, cv777, sm98-1, and dr-13, with 96.3-96.8% identities at the genomic level. all phylogenetic analyses based on the complete genome, the full-length s gene, and the s1 portion formed the similar tree structure, revealing that the korean knu-141112 strains are clustered together with the re-emergent korean strains in subgroup 2b that also includes 2013 emergent us strains and 2011-2012 field epidemic chinese strains. in conclusion, we isolated and serially propagated pedv in cell culture that is phenotypically and genotypically identical to field strains responsible for the recent severe ped outbreaks in korea. to our knowledge, this is the first report describing the isolation and in vitro and in vivo characterization of korean pedv associated with the field epidemic. with the availability of the korean isolate, we are now able to spur the development of new effective and safe vaccines for ped prevention. indeed, our pedv isolate has been used for the development of an inactivated vaccine that is currently being evaluated under experimental and field conditions. furthermore, we are continuing to passage the isolate in vero cells to develop a live attenuated vaccine that generally prove to provide a more efficient protective immunity than killed viral vaccines. for this purpose, pathogenic and molecular characterization of the isolates at selected passages will be assessed to determine their phenotypes in pigs and to identify the genetic changes involved in pedv attenuation. molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states experimental infection of pigs with a new porcine enteric coronavirus, cv777 the pathogenesis of an enteric infection in pigs, experimentally induced by the coronavirus-like agent, cv777 detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa propagation of the virus of porcine epidemic diarrhea in cell culture spike glycoprotein cleavage recognition site analysis of infectious bronchitis virus pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus isolation of porcine epidemic diarrhea virus (pedv) in korea derivation of attenuated porcine epidemic diarrhea virus (pedv) as vaccine candidate proteomic analysis of up-regulated proteins in human promonocyte cells expressing severe acute respiratory syndrome coronavirus 3c-like protease heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea reemergence of porcine epidemic diarrhea virus on jeju island full-genome sequence analysis of a variant strain of porcine epidemic diarrhea virus in south korea complete genome sequence of a novel porcine parainfluenza virus 5 isolate in korea new variants of porcine epidemic diarrhea virus pathogenesis of porcine epidemic diarrhea virus isolate (us/iowa/18984/2013) in 3-week-old weaned pigs immunogenicity and protective efficacy of recombinant s1 domain of the porcine epidemic diarrhea virus spike protein cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene letter to the editor retrospective study of porcine epidemic diarrhea virus (pedv) in korea by in situ hybridization a new coronavirus-like particle associated with diarrhea in swine an immunoelectron microscopic and immunofluorescent study on the antigenic relationship between the coronavirus-like agent, cv777, and several coronaviruses porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs chinese-like strain of porcine epidemic diarrhea virus a simple method for estimating fifty percent endpoints an integrated semiconductor device enabling non-optical genome sequencing porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-b production by inhibiting irf3 activation in immortalized porcine alveolar macrophages coronaviruses the neighbor-joining method: a new method for reconstructing phylogenetic trees isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf 3 emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan mega4: molecular evolutionary genetics analysis (mega) software version 4.0 the clustal x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools distinct characteristics and complex evolution of pedv strains we gratefully thank gun-seok park and jae-ho shin from kyungpook national university for their help in ngs analysis. this research was supported by basic science research program through the national research foundation of korea (nrf) funded by the ministry of education, science and technology(nrf-2012r1a1a2039746). key: cord-003899-a4w2nnos authors: yang, jiwen; tian, gang; chen, daiwen; zheng, ping; yu, jie; mao, xiangbing; he, jun; luo, yuheng; luo, junqiu; huang, zhiqing; wu, aimin; yu, bing title: dietary 25-hydroxyvitamin d(3) supplementation alleviates porcine epidemic diarrhea virus infection by improving intestinal structure and immune response in weaned pigs date: 2019-08-29 journal: animals (basel) doi: 10.3390/ani9090627 sha: doc_id: 3899 cord_uid: a4w2nnos simple summary: porcine epidemic diarrhea is one of the major problems in current swine husbandry worldwide, and effective measures for prevention and treatment are scarce. we found that high dose 25-hydroxyvitamin d(3) supplementation could ease intestinal injury and inhibit intestinal immune response induced by porcine epidemic diarrhea virus (pedv), suggesting that feeding a high dose of 25-hydroxyvitamin d(3) could be used as an approach against pedv infection. abstract: we conducted this experiment to determine if feeding 25-hydroxyvitamin d(3) (25(oh)d(3)) to weaned pigs would alleviate porcine epidemic diarrhea virus (pedv) infection and immune response. forty-two weaned pigs were allotted to 1 of 6 dietary 25(oh)d(3) treatments (5.5, 5.5, 43.0, 80.5, 118.0, 155.5 μg 25(oh)d(3)/kg diet) for 26 days. on day 22 of the trial, all the treatments were orally administrated with pedv except for one of the 5.5 μg 25(oh)d(3)/kg treatments, which was challenged with the same volume of sterile saline and served as control. another 5.5 μg 25(oh)d(3)/kg group for pedv challenge was named con-pedv. average daily gain (p < 0.05) was reduced by pedv infection. pedv administration also induced severe diarrhea (p < 0.05), reduction of villous height and the ratio of villous height to crypt depth, and increase of crypt depth and serum diamine oxidase activity (p < 0.05). serum igm and complement component 4 levels were increased by pedv challenge. however, 155.5 μg 25(oh)d(3)/kg supplementation alleviated intestinal damage (p < 0.05) compared with con-pedv. furthermore, 155.5 μg 25(oh)d(3)/kg supplementation downregulated the mrna abundance of inflammatory cytokines and interferon signal pathway-related genes (p < 0.05) compared with con-pedv. these results suggested that dietary supplementation of 155.5 μg 25(oh)d(3)/kg could alleviate intestinal damage and protect against pedv-induced inflammatory status. porcine epidemic diarrhea virus (pedv) infection causes severe damage to the intestinal function and barrier integrity of pigs [1] , leading to diarrhea, vomiting, dehydration, and high mortality in piglets [2] . recently, it was shown that the villus height and transepithelial resistance were decreased in pedv-infected pigs [3] . pedv also induced intestinal mucosa and systemic proinflammatory cytokine responses in pigs [4, 5] . currently, vaccination is the main means for preventing pedv infection. however, vaccines are poorly effective because of genetic variants of the viruses [6, 7] . vitamin d not only enhances calcium and phosphate absorption but also regulates immune function [8] . previous studies have shown that vitamin d can inhibit rotavirus replication and alleviate infection symptoms in piglets [9, 10] . pedv infection has shown similar symptoms to rotavirus and, therefore, we speculated that 25(oh)d 3 might ease the infection of pedv in pigs. however, studies on the feasibility of vitamin d 3 as anti-pedv infection agent in piglets are scarce. it is generally believed that the bioavailability of 25(oh)d 3 is higher than vitamin d 3 [11] [12] [13] . in this study, 25(oh)d 3 was used to investigate whether it could alleviate pevd-infected diarrhea and intestinal injury. the experimental protocol involved in the present study was approved by the animal care advisory committee of sichuan agricultural university (animal ethics committee approval number is cd-syxk-2017-015). forty-two crossbred healthy weaned pigs (duroc × landrace × yorkshire, 24 days old) with an initially body weight (bw) of 6.61 ± 0.41 kg were used in the 26 days trial. on the first day of the trial, all pigs were allotted on the basis of bw to six groups and each fed diets supplemented with either 5.5, 5.5, 43.0, 80.5, 118.0, or 155.5 µg 25(oh)d 3 /kg. each treatment consisted of four gilts and three barrows. at day 22 of this study, all the treatments were orally administrated with 35 ml of pedv (5.6 × 10 3 tcid 50 /ml) except for one of the 5.5 µg 25(oh)d 3 /kg (220 iu vitamin d/kg equivalent) treatment, which was served as control (con) and administrated with the same volume of sterile saline. another 5.5 µg 25(oh)d 3 /kg treatment for pedv challenge was named by con-pedv. in order to prevent infection, the con groups were housed in the next room of challenge groups. the settings for the two rooms were the same. pigs in the con group were negative for pedv throughout the trial period. as shown in table 1 , the basal diet was formulated to meet or exceed nutrient requirement for weaned piglets [14] , except for vitamin d 3 , which was not prepared in the vitamin premix. each treatment was formed by supplementing with indicated 25(oh)d 3 levels in the basal diet. all pigs had ad libitum access to water and experimental diets throughout the trial. 25(oh)d 3 (hy-d) was kindly provided by dsm nutritional products ltd. shanghai, china. the pedv was kindly presented by professor zhiwen xu, college of veterinary medicine, sichuan agricultural university. average daily gain (adg) and average daily feed intake (adfi) were determined via weighing to determine body weight and recording of feed intake. after the pedv challenge, the fecal consistency and diarrhea incidence were assessed every day according to hu et al. [15] : 1 = hard feces, 2 = firm well formed, 3 = soft and partially formed feces, 4 = loose, semi-liquid feces, and 5 = watery feces. diarrhea rate (%) = (a/5d) × 100, in which a = total days per pig with diarrhea after pedv challenge. mean cumulative score = a/b, in which a = the sum of daily scores, and b = pigs per treatment. all pigs were bled via anterior vein on 27 day. the blood was used for extracting serum via centrifugation at 3000 g for 15 min, and the serum samples were stored at −20 • c until analysis. all pigs were then euthanized by intramuscular injection of shumianning (comprised of ketamine, xylazine, and midazolam, nanjing agricultural university, 0.08 ml/kg body weight). about 2 cm jejunal tissue sample was stored in 4% paraformaldehyde solution for histological analysis. mucosal samples from the middle jejunum were scraped and rapidly frozen in liquid nitrogen, and then stored at −80 • c for further analysis. the concentration of igg, igm, and complement component 3 (c3) and c4 (sichuan maker biotechnology co. ltd. chengdu, china) in serum were detected by automatic biochemical analyzer (model 3100; hitachi, tokyo, japan). immunology multiple control were performed before sample determination to ensure the outcomes were correct. after being embedded in paraffin, the jejunal samples were stained with hematoxylin and eosin for intestinal morphology measurement. a minimum of 20 well-orientated villi and crypts from each intestinal sample of pigs were measured using image-pro plus 6.0 software. as a measurement of intestinal permeability, serum diamine oxidase activity (dao) was detected using commercial assay kits (nanjing jiancheng institute of bioengineering, jiangsu, china) following the protocols of the manufacturer. total rna was extracted from the mucosa of jejunum tissue using trizol reagent (invitrogen, shanghai, china). reverse transcription was performed with rna using a primescript rt reagent kit (takara, dalian, china). the mrna expression of genes of interest were quantified using an abi 7900ht detection system (applied biosystems, foster, ca, usa) and the sybr premix ex taq ii with rox reagents (takara, dalian, china). the primer sequences used for rt-pcr are listed in table 2 . all primer pairs were designed to have melting temperatures of approximately 60 • c. cycling conditions were as follows: 95 • c for 30 s, followed by 40 cycles of 95 • c for 5 s and 60 • c for 30 s. the relative mrna expression of each gene was calculated according to a previous publication [16] . expression levels were normalized to β-actin. protein sample processing was performed according to zhang et al. [17] . briefly, jejunal mucosa protein was extracted with lysis buffer (beyotime, shanghai, china). after centrifugation for 20 min at 12,000 rpm, the supernatants were harvested for bicinchoninic acid assay to detect protein concentration. then, the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes, and incubated with the corresponding antibodies: anti-sucrase-isomaltase (si), anti-siga, anti-occludin (abcam, shanghai, china), and anti-β-actin (santa cruz, shanghai, china). following washing, the samples were incubated with secondary antibodies, and then proteins were incubated with electro-chemi-luminescence reagent for chemiluminescence. the protein expression was analyzed by image lab and normalized to β-actin. in this study, each pig was used as the experimental unit. the differences between con and con-pedv group were assayed by student's t-test. on the condition of pedv challenge, the data were performed using the proc mixed sas 9.4 procedure according to the following model: where y = dependent variable; µ = mean; α = effect of treatment; β = block effect of bw, and ε = error. orthogonal comparisons were also applied for linear and quadratic responses of increasing dietary 25(oh)d3 levels (5.5, 43.0, 80.5, 118.0, 155.5 µg 25(oh)d3/kg). data of the innate immune gene expression were analyzed by tukey's test for post hoc comparisons. the significance was declared at p < 0.05 and trends at p < 0.10. as shown in table 3 , pedv challenge (con-pedv) decreased adg (p < 0.05) and adfi (p = 0.08) compared with con. however, the performance was not influenced among the different 25(oh)d 3 supplementation groups. pedv infection induced severe diarrhea in piglets (table 3 , p < 0.05). however, the dietary supplementation of 25(oh)d 3 decrease diarrhea scores (p < 0.05) and diarrhea rate ( table 3 , p < 0.1). table 3 . growth performance and diarrhea parameter of weaned piglets fed 25(oh)d 3 as indicated with porcine epidemic diarrhea virus (pedv) challenge. pedv infection increased serum igm and c4 concentrations compared with con (p < 0.05, table 4 ). however, different levels of 25(oh)d 3 supplementation had no effect on serum igm, igg, and c4 levels on the condition of pedv challenge. in addition, dietary supplementation of 155.5 µg 25(oh)d 3 /kg decreased serum c3 levels compared with the 118.0 µg 25(oh)d 3 /kg group. compared with con, pedv challenge decreased villous height and the ratio of villous height to crypt depth (vcr), and increased the crypt depth of jejunum in the con-pedv group ( table 5 , p < 0.05). however, 118.0 and 155.5 µg 25(oh)d 3 /kg treatments significantly increased villous height and vcr and decreased crypt depth compared with the con-pedv group ( table 5 , p < 0.05). furthermore, dietary supplementation of 25(oh)d 3 increased villous height and vcr and decreased crypt depth in a linear way (p < 0.05). in addition, serum dao activity of pigs in the con-pedv group was significantly increased compared with con ( figure 1 , p < 0.05). however, the dietary supplementation of 25(oh)d 3 tended to decrease serum dao activity (p < 0.1). animals 2019, 9, x 7 of 12 in addition, serum dao activity of pigs in the con-pedv group was significantly increased compared with con ( figure 1 , p < 0.05). however, the dietary supplementation of 25(oh)d3 tended to decrease serum dao activity (p < 0.1). compared with con, the gene expression of intestinal barrier-related genes was not influenced by pedv (table 6) . upon pedv challenge, the dietary supplementation of 25(oh)d3 increased jejunal claudin-2 gene expression significantly and this treatment effect was linear (p < 0.05). 25(oh)d3 also tended to increase muc2 and mrna expression (p < 0.1). interestingly, pedv increased sucraseisomaltase (si) and occludin protein levels, but high doses 25(oh)d3 (155.5 μg 25(oh)d3/kg) compared with con, the gene expression of intestinal barrier-related genes was not influenced by pedv (table 6) . upon pedv challenge, the dietary supplementation of 25(oh)d 3 increased jejunal claudin-2 gene expression significantly and this treatment effect was linear (p < 0.05). 25(oh)d 3 also tended to increase muc2 and mrna expression (p < 0.1). interestingly, pedv increased sucrase-isomaltase (si) and occludin protein levels, but high doses 25(oh)d 3 (155.5 µg 25(oh)d 3 /kg) inhibited these increase (figure 2 ). pedv induced a significant increase in the mrna expression of rigi, tlr2, myd88, il6, il8, ifnλ1, and mxa compared to con (table 7 , p < 0.05). however, a high dose of dietary 25(oh)d 3 supplementation decreased tlr2, tlr9, myd88, il6, il8, ifnλ1, stat1, mxa, ifnar1, and trif mrna expression compared with con-pedv (table 7 , p < 0.05). means not sharing the same superscript differ at p < 0.05. the p 1 means t-test between con and con-pedv, and p 2 value means multiple comparisons among the pedv challenge groups. early studies suggested that vitamin d exerts broad-spectrum antiviral effects, including inhibiting the replication of dengue virus [18] , hepatitis c virus [19] , rotavirus [9] , and others. due to the similarity in symptoms after rotavirus and pedv infection in pigs, we carried out this study to investigate whether 25(oh)d 3 could alleviate pedv infection. we have previously demonstrated that increasing dietary 25(oh)d 3 levels linearly increased serum 25(oh)d 3 concentrations, but no treatment effects were observed in the growth performance of weaned pigs [20] . in the current study, pedv challenge decreased adg and adfi, and resulted in severe diarrhea of the piglets. previous studies have reported that pedv infection reduced growth performance and resulted in severe diarrhea [21] [22] [23] . we have analyzed the pedv-n gene in jejunum mucosa by pcr, and the results revealed that pedv was prevalent in the pedv-inoculated pigs, whereas con treatments were negative for pedv (supplementary materials). this suggested that the pedv infection model was successfully established. in the current study, dietary 25(oh)d 3 supplementation decreased diarrhea scores, and 155.5 µg 25(oh)d 3 /kg treatment showed the minimum diarrhea scores and diarrhea rate, which indicated that supplementation with a high dose of 25(oh)d 3 might alleviate the symptoms of pedv infection. the complement system is a part of the innate immune system, and not only participates in inflammation but also enhances the adaptive immune response [24] . in this study, serum c4 level was increased with pedv challenge as compared with con. we also found that serum igm levels were increased by pedv challenge. immunoglobulins are the major secretory products of humoral immunity [25] . these results indicated that the pedv infection activated the innate and humoral immune response. however, dietary 25(oh)d 3 supplementation had no effect on serum c4 and igm levels under the conditions of pedv challenge. we inferred that 25(oh)d 3 might exert an antiviral effect on intestinal mucosa, which is the main target of pedv replication [26] . pedv infection always leads to morphological changes of the small intestine with a reduction in villus height and damage to intestinal integrity [3, 27] . in this study, we also found that villus height and vcr were decreased, and crypt depth was increased with pedv challenge. in addition, serum dao activity was also increased by pedv challenge. serum dao activity is a marker of mucosal integrity [28] . these results indicated that pedv induced morphological changes of jejunum and an increase in intestinal permeability. the 155.5 µg 25(oh)d 3 /kg 25(oh)d 3 treatment was shown to be optimal in alleviating intestinal injury induced by pedv. therefore, this may be the reason why the 155.5 µg 25(oh)d 3 /kg treatment showed the minimum diarrhea scores and diarrhea rate among the treatments. moreover, we found that claudin2 gene expression showed a linear response with increasing dietary 25(oh)d 3 concentration, and 25(oh)d 3 tended to increase muc2 expression under the conditions of pedv challenge. it suggested that high dose 25(oh)d 3 supplementation could improve tight junction protein expression to maintain intestinal barrier integrity. interestingly, levels of si, an intestinal absorptive cell marker, were significantly increased by pedv challenge. a previous study has shown that pedv infection decreased goblet cells in intestinal villous [29] . hence, we infer that pedv infection could cause disorder differentiation of intestinal cells, which promotes the differentiation of intestinal stem cells to intestinal absorption cells, while decreasing differentiation to intestinal secretion cells. however, high dose 25(oh)d 3 supplementation significantly reduced the si expression induced by pedv, which might be beneficial for maintaining normal intestinal function. we also found occludin expression was increased with pedv challenge compared with con, but 118.0 and 155.5 µg 25(oh)d 3 /kg groups inhibited this increase. luo et al. [30] demonstrated that overexpression of occludin in target cells makes them more susceptible to pedv infection, which indicated that occludin plays an essential role in pedv infection. in the present study, we speculated that 155.5 µg 25(oh)d 3 /kg supplementation might ease pedv infection through decreasing occludin expression. innate immune response plays an important role in defense against viral infections in mammalian cells. during viral infection, the virus is recognized by pattern-recognition receptors (prrs) including toll-like receptors (tlrs) and retinoic acid-inducible gene i (rigi) or melanoma differentiation gene 5 (mda5), then infs and proinflammatory cytokines are produced for initiation of the inherent antiviral immune response [31, 32] . different types of ifns bind to different receptors. type i ifns (ifn-α and ifn-β) signal through ifnar1 and ifnar2 to activate the jak-stat signaling pathway. and type iii ifns (ifn-λs) signal through ifnlr1 and il10r2 to activate jak-stat signaling pathway to induce the expression of hundreds of interferon stimulating genes [31] . unlike type i interferon receptors, which are seemingly ubiquitous, type iii ifn receptors are confined to the mucosal epithelium [33] . thus, ifn-λs mainly play an antiviral role in mucosal epithelial cells. in the current study, pedv increased the prr, inflammatory cytokine, and ifnλ expression in the jejunum mucosa. this indicates that the ifns signaling pathway was activated by pedv in the intestine of piglets. since the high dose of 25(oh)d 3 supplementation showed a better protective effect than the low dose groups, we investigated whether high dose supplementation could alleviate pedv infection by regulating immunity. we found that a high dose of 25(oh)d 3 inhibited the prr, ifnλ, stat1, and mxa expression. it was suggested that dietary 25(oh)d 3 supplementation inhibited the activation of intestinal immunity induced by pedv. previous studies have shown that vitamin d attenuated rotavirus infection and reduced the viability of mycobacterium tuberculosis through regulating autophagy and cathelicidin [9, 34] . therefore, we speculated that the suppression of the ifn signaling pathway from high dose supplementation of 25(oh)d 3 might be due to decreased pedv replication. in addition, we also found that high doses of 25(oh)d 3 inhibited jejunal mucosa il6 and il8 mrna expression compared with con-pedv. this suggested that high dose 25(oh)d 3 supplementation might inhibit intestinal inflammation induced by pedv. reducing the expression of intestinal inflammatory cytokines is also beneficial in maintaining normal intestinal function. in summary, the results of the current study indicate that dietary supplementation of 155.5 µg/kg 25(oh)d 3 alleviated the severity of diarrhea of piglets infected with pedv by improving the intestinal structure and immune response, and maintaining regular intestinal function. supplementary materials: the following are available online at http://www.mdpi.com/2076-2615/9/9/627/s1. figure s1 . porcine epidemic diarrhea virus induces caspase-independent apoptosis through activation of mitochondrial apoptosis-inducing factor porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis effects of porcine epidemic diarrhea virus infection on nursery pig intestinal function and barrier integrity immunohistochemical detection of the vomiting-inducing monoamine neurotransmitter serotonin and enterochromaffin cells in the intestines of conventional or gnotobiotic (gn) pigs infected with porcine epidemic diarrhea virus (pedv) and serum cytokine responses of gn pigs to acute pedv infection differential gene modulation of pattern-recognition receptor tlr and rig-i-like and downstream mediators on intestinal mucosa of pigs infected with pedv non s-indel and pedv s-indel strains outbreak of porcine epidemic diarrhea in suckling piglets porcine epidemic diarrhea virus: an 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article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we sincerely acknowledge the assistance of runqi fu, huifen wang and linyan jia for their assistance during the animal experiments and laboratory analyses. the authors declare no conflict of interest. the funder 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-003328-vvle1q1e authors: altawaty, tawfeek; liu, lulu; zhang, hongyong; tao, cong; hou, shaohua; li, kui; wang, yanfang title: lack of ltβr increases susceptibility of ipec-j2 cells to porcine epidemic diarrhea virus date: 2018-11-21 journal: cells doi: 10.3390/cells7110222 sha: doc_id: 3328 cord_uid: vvle1q1e the essential requirement of the lymphotoxin beta receptor (ltβr) in the development and maintenance of peripheral lymphoid organs is well recognized. evidence shows that ltβr is involved in various cellular processes; however, whether it plays a role in maintaining the cellular function of intestinal porcine enterocytes (ipec-j2), specifically during porcine epidemic diarrhea virus (pedv) infection, remains unknown. in this study, we generated ltβr null ipec-j2 cells using crispr/cas9 to examine the importance of ltβr in cell proliferation, apoptosis, and the response to pedv infection. our results showed that the lack of ltβr leads to significantly decreased cell proliferation, potentially due to s phase arrest in ltβr(−/−) ipec-j2 cells. label-free digital holographic microscopy was used to record the three-dimensional morphology of both cell types for up to 72 hours and revealed significantly increased numbers of ltβr(−/−) cells undergoing apoptosis. furthermore, we found that pedv-infected ltβr(−/−) null ipec-j2 cells exhibited significant suppression of nuclear factor kappa-light-chain-enhancer of activated b cells (nfκb) target genes (interleukin (il)-6 and il-8) and mucosal barrier integrity-related genes (vascular cell adhesion molecule 1 (vcam1) and il-22), which may explain why ltβr(−/−) cells are more susceptible to pedv infection. collectively, our data not only demonstrate the key role of ltβr in intestinal porcine enterocytes, but also provide data for the improved understanding of the cellular response to pedv infection. lymphotoxin beta receptor (ltβr) belongs to the tumor necrosis factor (tnf) receptor superfamily (tnfrsf), which includes more than 25 receptors that interact with nearly 20 ligands to regulate the immune response, and is activated by the proinflammatory cytokines lymphotoxin α 1 β 2 or tnf superfamily member 14 (tnfsf14, also named as light) [1] . ltβr is expressed on the surface of most cell types, with the highest expression on cells of epithelial and myeloid lineages [2] . the critical roles of ltβr in the development and maintenance of peripheral lymphoid organs were illustrated in ltβr-deficient mice two decades ago [3] . over the past twenty years, ltβr signaling has been reported to be crucially involved in many cellular processes and molecular events. the activation of ltβr by functional antibodies induces nuclear factor kappa-light-chain-enhancer of activated b cells 15% fetal bovine serum (fbs, hyclone, logan, ut, usa) and 1% penicillin-streptomycin. both cell types were incubated at 37 • c with 5% co 2 . the vero cell-adapted pedv cv777 strain, kept in hou's lab from ias, was propagated as previously described [16] . the px330 vector was used to construct the targeting genomic sequences, which were created by the laboratory of feng zhang and obtained from addgene (plasmid 42230). guide rnas were designed using an online tool provided by feng zhang's laboratory at the mit/broad institute as described previously [17] . the l1 and l3 small guide rna (sgrna) genome targeting sequences, (l1: 5 -gggaatggcgggcctcttggttt-3 ; l3: 5 -gaaggtgctcccttaccgcccgg-3 ), were cloned into the px330 vector as previously described [16] . ipec-j2 cells were transfected by nucleofection using an amaxa tm nucleofector tm kit (lonza, cologne, germany). the pcag-gfp plasmid was cotransfected with the px330 plasmid as an indicator for fluorescence activated cell sorting (facs). twenty-four hours after transfection, cells were subjected to facs sorting based on the expression of enhanced green fluorescent protein (egfp) fluorescence. single cells were plated into each well of 96-well plates and cultured for approximately 10 days in cell culture medium supplemented with 2.5 ng/ml basic fibroblast growth factor (sigma, st. louis, mo, usa). the medium was replaced every 3 days. confluent cell colonies were propagated and subjected to pcr-restriction fragment length polymorphism (rflp) assays. pcr products (411 bp) were digested with acii (new england biolabs, ipswich, ma, usa) and the restriction fragments were analyzed on a 2% agarose gel. the identified biallelic mutant clones were subjected for sequencing analysis. two different rt-pcrs were used in this study, real-time pcr and semi-quantitative pcr. total rna from tissues and cells was isolated by trizol reagent, and rna concentrations were determined with a nanodrop apparatus (nanodrop technologies, wilmington, de, usa). two milligrams of total rna was reverse transcribed using a first strand cdna synthesis kit (thermo fisher scientific, waltham, ma, usa). real-time pcr was performed using sybr green master mix (applied biosystems, foster city, ca, usa) and a 7500 fast real time pcr system (applied biosystems, foster city, ca, usa). expression levels were normalized to those of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gapdh). primers used for real-time pcr are shown in table 1 . the relative gene expression was calculated using the comparative cycle threshold (2 −ddct ) method. the parameter for semi-quantitative pcr was 4 min at 94 • c followed by 26 cycles of 45 s at 94 • c, 30 s at 60 • c, 45 s at 72 • c and a final extension of 5 min at 72 • c. pcr products (10 µl) were used to detect the expression. cells were washed twice with cold phosphate-buffered saline (pbs), and lysate samples were prepared in 350 µl t-per tissue protein extraction reagent (thermo scientific pierce, rockford, il, usa) in the presence of a protease inhibitor cocktail (roche, indianapolis, in, usa) and centrifuged at 20,000× g for 20 min at 4 • c. proteins (20-50 µg) and protein markers were separated by sds-polyacrylamide electrophoresis in 10% polyacrylamide slab gels and transferred to polyvinylidene difluoride (pvdf) membranes (millipore, madison, wi, usa). blots were blocked in 5% milk in 0.1% tris-buffered saline-tween 20 (tbst) for 1 h at room temperature. then, blots were incubated with antibodies against ltβr (1:1000, abcam, cambridge, ma, usa) and β-actin (1:2000, cst, danvers, ma, usa) overnight at 4 • c. immunoreactive bands were detected using pierce enhanced chemiluminescence (ecl) western blotting substrate (thermo scientific pierce, rockford, il, usa). to examine cell proliferation, ltβr +/+ and ltβr −/− cells were plated in 96-well plates at 5 × 10 3 cells per well in 100 µl cell culture medium and maintained at 37 • c in a humidified incubator containing 5% co 2 . proliferation was analyzed every 24 h with the cell counting kit-8 (cck-8 kit, beyotime biotechnology, shanghai, china) following the manufacturer's protocol. ltβr +/+ and ltβr −/− cells were plated in 6-well plates and serum-starved overnight for synchronization. the next day, serum was added to the cells and after 24 h of stimulation, cells were trypsinized and fixed in cold 70% ethanol. cells were then incubated at 4 • c for a minimum of 45 min to a maximum of overnight. subsequently, they were centrifuged at 1500× g for 10 min at 4 • c and re-suspended in 0.4% propidium iodide (pi: containing 50 µg/ml propidum iodide with 100 µg/ml rnase a) for staining. cells were then analyzed with an lsr ii cytometer (bd biosciences, san diego, ca, usa), and the pi staining intensity was determined by modfit software (bd biosciences, san diego, ca, usa). this analysis gave the percentage of cells in the g1, s, and g2 phases. holomonitor m4 microscopy (phase holographic imaging ab, lund, sweden) is an imaging time-lapse cytometer based on holographic microscopy, providing imaging and quantification of unstained living cells directly in their culture vessels. ltβr +/+ and ltβr −/− cells were seeded in 6-well plates and monitored for 72 h. apoptosis was analyzed by hstudio m4 tracking software (scheelevägen, sweden). statistical analyses were performed with graphpad prism 5.0 (graphpad software, la jolla, ca, usa). an unpaired t-test was used to compare values (means ± standard error means (sems)) between wild-type and mutant cells as described in the figure legends. * p < 0.05, ** p < 0.01 and *** p < 0.001 were considered statistically significant. we first examined expression profiles of ltβr in different sections of gut tissues, including the duodenum, jejunum, ileum, appendix, colon, rectum, and lymph nodes, by real-time pcr. our data showed that ltβr was expressed in all sections of gut tissues and lymph nodes from adult large white pigs. the highest expression level of ltβr was observed in the jejunum, whereas the duodenum exhibited significantly lower expression compared to other gut sections ( figure 1 ). figure 1 . expression of the lymphotoxin beta receptor (ltβr) in different porcine gut tissues. tissues, including the duodenum, jejunum, ileum, appendix, colon, rectum and lymph nodes, were collected from adult male large white pigs (n = 4), and real-time pcr was used to measure the expression level of ltβr. * p < 0.05 was considered statistically significant. note that the duodenum showed significantly lower expression of ltβr than other gut tissues. to generate ltβr knockout ipec-j2 cells, we designed two different sgrnas (l1 and l3) that target 32 bp regions in exon 2 of the porcine ltβr gene (figure 2a ). the pcag-gfp plasmid was co-transfected with the px330-l1 and px330-l3 plasmids, and single cells were sorted into 96-well plates by flow cytometry. to determine crispr-cas9-mediated mutations, 96 colonies were selected and subjected to rflp analysis ( figure 2b ). our data showed that 10 cell clones were biallelically mutated, and the targeting efficiency was 10.4% ( figure 2b ,c). to further validate the biallelic mutation, five cell clones, 1-10#, 1-19#, 1-22#, 2-3# and 6-18#, were randomly selected for dna sequencing (supplementary figure s1) , and the results confirmed those of rflp. further, the amino acid sequences from the wild-type 1-10# cell clone were compared, and our results demonstrated the shifted mutation in both alleles (supplementary figure s2) . to examine whether crispr/cas9-mediated gene editing could generate ltβr null alleles in ipec-j2 cells, we randomly selected two biallelic mutation clones (1-10# and 1-22#) and compared their ltβr expression levels to those of wild-type ipec-j2 cells (hereafter designated ltβr +/+ ) by real-time pcr. our data showed that the expression level of ltβr was significantly decreased in both 1-10# and 1-22# ( figure 2d ). in addition, the inactivation of ltβr in clone 1-10# was further confirmed by western blotting ( figure 2e ). the non-detectable ltβr expression in 1-10# at both the rna and protein levels suggested that ltβr was successfully knocked out and these cells are hereafter referred to as ltβr −/− cells and used for the following studies. to examine the potential effect of ltβr on cell proliferation, a cck-8 kit was used to analyze cell proliferation in both ltβr +/+ and ltβr −/− cells. as shown in figure 3a , the in vitro proliferation of ltβr −/− cells was significantly inhibited at 48 h (0.387 ± 0.023 vs. 0.189 ± 0.018 for ltβr +/+ and ltβr −/− cells, respectively, p < 0.01) and 72 h (0.633 ± 0.062 vs. 0.370 ± 0.027 for ltβr +/+ and ltβr −/− cells, respectively, p < 0.05), suggesting the ablation of ltβr inhibited ipec-j2 cell proliferation. in addition, semi-quantitative pcr was used to detect the expression level of proliferating cell nuclear antigen (pcna), a cell proliferation marker. consistently, semi-quantitative pcr results showed that pcna was down-regulated in ltβr −/− cells ( figure 3b ). these results suggest that the knockout of ltβr reduces cell growth in vitro. since cell growth is tightly regulated by a series of regulators of the cell cycle [18] , the effects of ltβr on cell cycle progression were analyzed by flow cytometry using propidium iodide staining. our data demonstrated s phase arrest in ltβr −/− cells, which resulted in a significant population increase of s phase cells (16.715% ± 0.345 vs. 24.09% ± 0.045 for ltβr +/+ and ltβr −/− cells, respectively, p < 0.01) and a dramatic decrease of g2 phase cells (28.12% ± 0.33 vs. 22.74% ± 0.36 for ltβr +/+ and ltβr −/− cells, respectively, p < 0.01) ( figure 3c,d) . cell cycle progression is regulated by a complex network of cell cycle-related genes [19] . since ltβr −/− cells were arrested at the s phase, the expression of cyclin e1, a key gene in the g1 to s phase transition, was measured by semi-quantitative pcr. the results revealed substantially higher expression levels of cyclin e1 in ltβr −/− cells than in ltβr +/+ cells ( figure 3e ). digital holographic microscopy offers an advantage in studying real-time observations of critical events by exhibiting a continuous two-dimensional (2d) and 3d visual picture of cellular activity in second intervals. a large portfolio of quantitative morphological parameters, including optical cell volume, thickness, area, irregularity, eccentricity, and single-cell tracking, can be recorded and analyzed [20] . here, digital holographic microscopy was used to monitor dynamic activities and morphological changes of ltβr +/+ and ltβr −/− cells in real-time for up to 72 hours. the results showed that these cells displayed distinct growth characteristics. specifically, many more ltβr null cells than ltβr +/+ cells exhibited increased cell volume (vertical axis) and decreased cell membrane thickness (horizontal axis) ( figure 4a , cells between the red lines). figure 4b shows the 3d structures of observed living ltβr +/+ cells ( figure 4b , left) and ltβr −/− cells ( figure 4b, right) . clearly, ltβr −/− cells with a white color are apoptotic because the flow of liquids through apoptotic cell membranes (permeability malfunction) leads to increased cell volume, and the lightened cell membranes therefore reflect different colors. next, because we found that the knockout of ltβr induces apoptosis, we investigated apoptosis-related genes in both types of cells. q-pcr was performed to examine the expression levels of apoptosis-related genes, including tnf superfamily member 10 (tnfsf10) and caspase 3 (casp3), in both cell lines. our data revealed that these two genes were significantly increased in ltβr −/− cells ( figure 4c ). ltβr is reportedly highly expressed in the lymph nodes, duodenum and jejunum of eight-day-old newborn piglets, which may be beneficial for developing resistance to e. coli f18 in pigs [21] , and ipec-j2 cells are susceptible to pedv infection [22] . as such, we investigated the effects of ltβr on pedv infections in ipec-j2 cells. we challenged ltβr +/+ and ltβr −/− cells with the pedv cv777 strain (at a multiplicity of infection (moi) of 1), and cells were harvested 48 h later to determine the relative viral expression level by real-time pcr using pedv-specific primers. our data showed that rna levels of pedv in ltβr −/− ipec-j2 cells were significantly higher than those in wild-type ipec-j2 cells ( figure 5a ). since pedv infection destroys epithelial barrier integrity [22] , and ltβr signaling was reported to limit mucosal damage through the il-22-il-23 pathway [10] , we examined the expression levels of ltβr downstream genes by semi-quantitative pcr, including vcam1, il-22 and il-23, in pedv-infected cells. our data revealed that vcam1 and il-22 expression was substantially decreased in ltβr −/− ipec-j2 cells, while il-23 was intact ( figure 5b ). the nfκb-dependent genes il-6 and il-8 are reported to be induced by activation of ltβr signaling [4, 23] . we detected expression levels of il-6 and il-8 in infected cells, and the data showed that both genes were significantly decreased in infected ltβr −/− ipec-j2 cells ( figure 5c ). ltβr plays a key role in lymphoid organogenesis; however, increasing studies show that ltβr signaling is involved in many cellular processes. herein, we showed that ltβr is ubiquitously expressed in all sections of gut tissues from adult pigs, indicating its potential function in the intestine. specifically, ltβr expression was significantly lower in the duodenum than in other intestinal sections. therefore, we used ipec-j2 cells, a non-transformed, non-tumorigenic porcine cell line derived from jejunal intestinal regions, to investigate the effects of ltβr on cell proliferation, apoptosis and viral infection. the blockade of ltβr can be achieved either by using the functional inhibitor ltβr-igg or by congenital deletion of ltβr or lymphotoxin α1β2. in this study, we successfully generated ltβr null cells using the crispr/cas9 technique, as evidenced by the low expression at both the mrna and protein levels. the gene targeting efficiency was determined, and the biallelic mutated clone reached 10.4%. further, our data showed that cell proliferation was significantly inhibited in ltβr null cells, suggesting that ltβr-mediated signaling is required to maintain ipec-j2 cell proliferation. in agreement with this result, the increased viability of light-stimulated human bone marrow-derived mesenchymal stem cells (bm-mscs) was observed [24] . cell proliferation is closely associated with the cell cycle. we performed cell cycle analysis with both cell lines using propidium iodide staining, and the ablation of ltβr prevents cells from completing the g2/s phase transition, so the cells in the s phase significantly increased. consistent with these results, expression levels of the s phase marker cyclin e1 were high in ltβr null cells. from these results, we speculate that ltβr knockout prevents cells from exiting the s phase, which leads to growth inhibition in ipec-j2 cells. it has been demonstrated that ltβr plays an important role in cell death via caspase-dependent and -independent pathways [25, 26] . in this study, we video monitored ltβr +/+ and ltβr −/− cells for up to 72 h by digital holographic microscopy. compared to other strategies that measure cell death and apoptosis, such as the terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay, digital holographic microscopy allows the visualization of real-time morphological alterations in cells and has become a powerful tool for the evaluation of cell responses to various stimuli with no labeling required [20] . the 3d images allowed the visualization of apoptotic cells, and the number of tall cells was increased in the ltβr −/− cell population, indicating that more ltβr −/− cells than ltβr +/+ cells underwent apoptosis. furthermore, we observed that the apoptotic-related genes tnfsf10 and casp3 were significantly increased in ltβr −/− cells, further confirming that the knockout of ltβr induced the apoptosis of ipec-j2 cells. interestingly, in contrast from our observations, wu et al. reported that the overexpression of either ltβr or the cytoplasmic domain of ltβr induces apoptosis in hela cells [26] . this opposite observation might be due to the distinct role of ltβr in different cell types. ltβr signaling has been reported to be crucially involved in many cellular processes and molecular events, and we are particularly interested in its role in bacterial and viral infection. several reports have demonstrated the critical role of ltβr signaling in bacterial infection of intestinal epithelial cells. for example, ltβr −/− mice are sensitive to bacterial infection due to the absence of lymphoid organs in these mice, and ltβr signaling in intestinal epithelial cells is required for the recruitment of neutrophils to the site of infection during early infection via the production of cxcl1 and cxcl2 [6, 27] . ltβr signaling is required for clearance of salmonella typhimurium in infected gut lumen [28] . additionally, very recent data showed that ltβr is highly expressed in the lymph nodes, duodenum and jejunum of eight-day-old newborn piglets, which may be beneficial for developing resistance to e. coli f18 in pigs [21] . in addition, the role of ltβr in viral infection was investigated. zhu et al. reported that the ltβr rs12345 polymorphism is related to the spontaneous resolution of hepatitis b virus infection [8] . all these observations revealed the function of ltβr in infection. since ipec-j2 cells have been reported to be susceptible to pedv infection [22] , we investigated the impact of ltβr on pedv infection in ipec-j2 cells. our data revealed that the levels of ped virus in ltβr −/− cells was significantly higher than in ltβr +/+ cells, indicating that ltβr-mediated signaling plays a key role in protecting ipec-j2 cells from pedv infection. since it has been demonstrated that pedv infection destroys epithelial barrier integrity [22] and ltβr signaling limits mucosal damage through the il-22-il-23 pathway [10] , we detected expression levels of vcam1, il-22 and il-23 in both cell types by semi-quantitative pcr. vcam1 and il-22 were significantly decreased in rna expression in ltβr −/− ipec-j2 cells, in agreement with the previous observation that an abundance of il-22 is significantly reduced in ltβr −/− mice fed a high-fat-diet [6] . il-22, a member of the il-10 superfamily, plays essential roles in fighting against mucosal microbial infection and maintaining mucosal barrier integrity within the intestine. the downregulation of il-22 and vcam1 in ltβr −/− ipec-j2 cells indicates that the epithelial barrier integrity of cells might be impaired, which leads to increased susceptibility to pedv infection, though further investigation is needed. surprisingly, il-23 was found to be intact in ltβr −/− cells, which suggested that il-22 and il-23 may play individual roles in ipec-j2 cells. in addition, the s-phase arrest might also contribute to the higher viral production in ltβr −/− cells. the overexpression of ltβr, or the activation of its mediated signaling by its functional antibody or cellular receptors such as light, will also be needed to positively determine its role in preventing infection. in summary, this study explored the effect of ltβr on ipec-j2 proliferation and apoptosis, as well as its role in pedv infection. the absence of ltβr increased susceptibility to pedv infection in ipec-j2 cells, which might be caused by significantly suppressed nfκb target genes (il-6 and il-8) and mucosal barrier integrity-related genes (vcam1 and il-22). our in vitro cellular model will be helpful for better understanding the biological function of ltβr and the cellular responses to pedv infection. the following are available online at http://www.mdpi.com/2073-4409/7/11/222/s1, figure s1 : sequencing analysis of cell clones 1-10#, 1-19#, 1-22#, 2-3# and 6-18#, figure s2 : amino acid sequences alignment for wild-type, 1-10# and 1-19# cell clones. the authors declare no conflicts of interest. mouse lymphotoxin-beta receptor. molecular genetics, ligand binding, and expression visualization of lymphotoxin-beta and lymphotoxin-beta receptor expression in mouse embryos the lymphotoxin beta receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues activation of the lymphotoxin-beta receptor induces nfkappab-dependent interleukin-6 and mip-2 secretion in mouse fibrosarcoma cells the lymphotoxin beta receptor is critically 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signaling in intestinal epithelial cells orchestrates innate immune responses against mucosal bacterial infection analysis of protein expression changes of the vero e6 cells infected with classic pedv strain cv777 by using quantitative proteomic technique efficient crispr/cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgrnas in pigs a gene regulatory network controls the binary fate decision of rod and bipolar cells in the vertebrate retina cyclins and cdks in development and cancer: a perspective label-free observation of three-dimensional morphology change of a single pc12 cell by digital holographic microscopy developmental expression of ltbetar and differential expression in escherichia coli f18 resistant/sensitive piglets transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j2 cells lymphotoxin beta receptor induces interleukin 8 gene expression via nf-kappab and ap-1 activation light (tnfsf14) increases the survival and proliferation of human bone marrow-derived mesenchymal stem cells the lymphotoxin-beta receptor is necessary and sufficient for light-mediated apoptosis of tumor cells the cytoplasmic domain of the lymphotoxin-beta receptor mediates cell death in hela cells the lymphotoxin-beta receptor induces different patterns of gene expression via two nf-kappab pathways cutting edge: lymphotoxin signaling is essential for clearance of salmonella from the gut lumen and generation of anti-salmonella protective immunity this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-254317-n2knqj4z authors: su, yunfang; hou, yixuan; wang, qiuhong title: the enhanced replication of an s-intact pedv during coinfection with an s1 ntd-del pedv in piglets date: 2018-11-27 journal: vet microbiol doi: 10.1016/j.vetmic.2018.11.025 sha: doc_id: 254317 cord_uid: n2knqj4z porcine epidemic diarrhea virus (pedv) variants having a large deletion in the n-terminal domain of the s1 subunit of spike (s) protein were designated as s1 ntd-del pedvs. they replicate well in experimentally infected pigs. however, on farms they often co-infect pigs with the pedv containing an intact s protein (s-intact pedv). we aimed to characterize viral replication and pathogenesis in neonatal gnotobiotic pigs infected simultaneously with the two types of pedv using two recombinant pedvs: icpc22a and its s1 ntd-del form icpc22a-s1δ197. additionally, viral replication was compared in vero and ipec-dq cells at the presence of bovine mucin (bm), porcine gastric mucin (pgm), swine bile and bile acids during inoculation. in the pigs coinfected with icpc22a and icpc22a-s1δ197, icpc22a replicated to a higher peak titer than its infection of pigs without the presence of icpc22a-s1δ197. the severity of diarrhea and intestinal atrophy were similar between icpc22a and the coinfection groups, but were significantly higher than icpc22a-s1δ197 group. in vero and ipec-dq cells, certain concentrations of bm, pgm, bile and bile acids increased significantly the infectivity of icpc22a but had no or negative effects on icpc22a-s1δ197. these results indicated that the replication of the s-intact pedv was enhanced during coinfection in piglets. this observation may be explained partially by the fact that mucin, bile and bile acids in gastrointestinal tract had facilitating effects on the infection of s-intact pedv, but no/inhibition effects on s1 ntd-del pedv. porcine epidemic diarrhea virus (pedv) belongs to the genus alphacoronavirus in the family coronaviridae. it causes severe gastroenteritis in neonatal pigs worldwide. the genome size of pedv is 28 kb. it contains orf1a and orf1b encoding 14-16 non-structural proteins, an orf3 encoding an accessory protein, and four orfs encoding structural proteins: spike (s), membrane (m), envelop (e), and nucleocapsid (n) proteins. pedv s protein mediates the essential functions of receptor binding (via s1 subunit) and subsequent fusion of the viral and cellular membranes (via s2 subunit) (li, 2016) . s1 subunit contains two domains, the n-terminal domain (s1 ntd, residues 21-324 based on pedv cv777) and the c-terminal domain (s1 ctd, residues 253-638). however, the cellular receptor of pedv is still unknown (li et al., 2017b; shirato et al., 2016) . although the sialic acid binding activity of s1-ntd was observed in many coronaviuses and facilitated virus replication (schwegmann-wessels et al., 2011; li et al., 2016; desmarets et al., 2014) , s1-ntd is dispensable for some mutants of transmissible gastroenteritis coronavirus (tgev) and pedv (schwegmann-wessels et al., 2011; oka et al., 2014; suzuki et al., 2016; hou et al., 2017) . pedv variants with a big deletion in the s1-ntd have been designated as s1 ntd-del pedvs (hou et al., 2017) . they have been detected in field pig samples, indicating sufficient replication of these s1 ntd-del pedvs in vivo (suzuki et al., 2015; diep et al., 2017; zhang et al., 2018a; su et al., 2018) . interestingly, most field s1 ntd-del pedvs exist as a member of coinfection of pigs with the s-intact pedv (diep et al., 2017; su et al., 2018) . however, the mechanisms and consequences of the coinfection have not been investigated. studies showed that under unfavorable conditions as encountered in the intestinal tract, sialic acid-binding of tgev played an important role in viral replication. when absorption time was reduced, tgev infectivity was also reduced in the cells whose sialic acids were removed (schwegmann-wessels et al., 2011) . consequently, sialic acid binding activity may facilitate the infection of tgev by helping the virus resist detergent-like substances encountered during passing through the gastrointestinal tract (krempl et al., 2000) . therefore, we hypothesized that mucin, rich in sialic acid, may enhance the infection of pedv. interestingly, bile acids can induce or promote the replication of some enteric viruses like a porcine sapovirus (chang et al., 2004) and human noroviruses (ettayebi et al., 2016) . since intestinal epithelial cells are exposed to bile acids, we hypothesized that bile acids promote pedv infection. previously, we generated two recombinant pedvs, pedv icpc22a and icpc22a-s1δ197 (hou et al., 2017) . pedv icpc22a is an infectious cdna clone-derived virus of the emerging highly virulent pedv pc22a strain. on the other hand, pedv icpc22a-s1δ197 is a mutant version of icpc22a bearing a 197-aa deletion (residues 34-230) in the s1 ntd region, corresponding to the s1 domain 0 (s1°) of the spike protein (li et al., 2017a; hou et al., 2017) . in this study, we investigated the replication and pathogenesis of the two viruses during coinfection in neonatal gnotobiotic (gn) piglets. some intestinal substances (mucin, bile and bile acids) were tested for their effects on the infection of the individual viruses in vitro. we selected two cell lines, vero and ipec-dq cells. vero cells are the most commonly used cell line for pedv isolation and propagation (teeravechyan et al., 2016) . ipec-dq cell is a subclone of porcine small intestinal cell line ipec-j2 (zhang et al., 2018b) . although pedv grows less efficiently in ipec-dq cells than in vero cells, pedv infection of ipec-dq cells is more relevant physiologically to pedv natural infection in the intestines of pigs. vero cells (atcc number ccl81) were cultured in dulbecco's modified eagle's medium (dmem, gibco, carlsbad, ca, usa) supplemented with 5% fetal bovine serum (fbs, hyclone, logan, ut, usa) and 1% penicillin/streptomycin (gibco). for virus propagation in vero cells, the maintenance medium was dmem supplemented with 0.3% tryptose phosphate broth solution (tpb, sigma, st. louis, mo, usa), 1% penicillin/streptomycin (gibco) and 10 μg/ml trypsin (2.5% trypsin, gibco). ipec-dq cell line was a gift from dr. dongwan yoo, university of illinois at urbana-champaign, urbana il, usa. it was a subclone of ipec-j2 cells, a porcine small intestinal cell line, and maintained in roswell park memorial institute 1640 medium (rpmi-1640, gibco) supplemented with 10% fbs and 1% penicillin/streptomycin (zhang et al., 2018b) . for virus propagation in ipec-dq cells, maintenance medium was rpmi-1640 containing 0.3% tpb, 1% penicillin/streptomycin and 1 μg/ml trypsin. two recombinant pedv, icpc22a and icpc22a-s1δ197, were generated previously by our lab using the infectious clone of pedv pc22a strain (hou et al., 2017) . neuraminidase (na, from clostridium welchii), porcine gastric mucin (pgm) and several bile acids [(glycochenodeoxycholic acid (gcdca), chenodeoxycholic acid (cdca), deoxycholic acid (dca), and ursodeoxycholic acid (udca)] were all purchased from sigma. bovine mucin (bm) was purchased from worthington (worthington biochemical corp., lakewood, nj, usa). swine bile was collected from a pregnant sow after c-section surgery. the mouse monoclonal antibodies (mab) 56h4, 60f11 and 72 a8 targeting the s1°domain of pedv s protein (fig. s1a ) was provided by dr. berend jan bosch, utrecht university, utrecht, netherlands (li et al., 2017a) . the mab sd6-29 targeting pedv nucleocapsid (n) protein was provided by drs. eric nelson and steven lawson, south dakota state university. the guinea pig polyclonal antibody (pab) gp17 targeting the s1 subunit of the s protein of pedv pc177 strain (s1δ197) (fig. s1a) , rabbit pab rb9 targeting pedv n protein and swine pedv-positive serum (virus neutralization titer 1:1024) were generated by our laboratory previously (hou et al., 2019) . 2.2. duplex rt-qpcr method for the detection and differentiation of pedv icpc22a and icpc22a-s1δ197 based on the nucleotide sequence differences in the s1 ntd coding region between the icpc22 a and icpc22 a-s1δ197, a duplex taqman real-time reverse-transcription quantitative pcr (rt-qpcr) was developed. we designed the primers and probes (table 1 , fig. s1b ) and had them synthesized by integrated dna technologies (skokie, il, usa). the rna of the two viruses was extracted using rneasy mini kit (qiagen, valencia, ca, usa). to generate a standard dna, a kit of superscript™ iii first-strand synthesis supermix (thermo fisher scientific, carlsbad, ca, usa) was used for cdna synthesis following manufacturer's instructions. primer set pedv-20320f and pedv-21816r (oka et al., 2014) was used to amplify a 1.5 kb or a 0.9 kb fragment covering pedv partial nsp16 gene and s1 gene of icpc22 a and icpc22 a-s1δ197, respectively. the pcr products were purified using gel extraction kit (qiagen) and used as standard dna. the reaction consisted with 4 μl 5 × pcr buffer, 0.8 μl 10 mm dntps (qiagen), 1.2 μl each primer (10 μm), 0.4 μl each probe (5 μm), 0.2 μl rnasin (promega, wi, usa), 0.8 μl enzyme mix (qiagen), 1.6 μl each dna/rna and 6.6 μl sterilized water. the rt-qpcr procedure was: 50°c for 30 min, 95°c for 15 min and 45 cycles of 95°c for 15 s and 60°c for 60 s. we generated two standard curves using light-cycler® 96 system (roche life science, indianapolis, in, usa). the two standard curves for the serially diluted dna fragments of icpc22a and icpc22a-s1δ197 showed regressions with coefficients of -0.2873 and -3.009, respectively, and the corresponding correlation coefficient (r 2 ) of 0.9987 and 0.9963, respectively. the primers and probes were further tested using individual and the mixture of two viruses. we tested the correlation between s gene titers and infectious titers of pedv icpc22 a and icpc22 a-s1δ197 using serially diluted, cell cultured viruses (table s1 and s2). for the samples of mixed viruses, the duplex rt-qpcr was performed to differentiate the s gene of each virus. the detection limit was 5 log 10 s gene copies/ml for both icpc22 a and icpc22 a-s1δ197. the gn piglets were derived and raised as described previously (saif et al., 1996) . thirty-one gn piglets were assigned to five experimental groups: (1) icpc22 a (n = 14; 100 pfu/pig); (2) icpc22a-s1δ197 (n = 7; 100 pfu/pig); (3) coinfection of icpc22a and icpc22 a-s1δ197 (n = 7; 100 pfu/pig of each virus); (4) mock (n = 2; pbs); (5) second passage of coinfection [n = 1; small intestinal contents (sic) (sample #pe1158) of one piglet in group 3 collected at 2 day post inoculation (dpi), at a dose of 10 log 10 copies/ml based on the n gene, 9 log 10 copies/ml and 7 log 10 copies/ml based on the icpc22a-specific s gene and icpc22a-s1δ197-specific s gene, respectively, corresponding to 4 table 1 primers and probes of the duplex rt-qpcr. log 10 pfu/ml of infectious pedv] . at 5 days of age, piglets in groups 1-4 were orally inoculated with individual or the mixture of the viruses. the group 5 pig was inoculated at 12 days of age and was euthanized at 1 dpi. to examine histopathological changes, one or two piglets of groups 1-4 were euthanized at 0.5 dpi and 2-3 dpi, respectively. after inoculation, all piglets were evaluated daily for clinical signs, such as diarrhea, vomiting, anorexia, and depression. fecal consistency was examined by collecting rectal swabs and scored as follows: 0, solid; 1, pasty; 2, semiliquid (mild diarrhea); and 3, liquid (severe diarrhea). total fecal rna was extracted using magmax-96 rna isolation kit (thermo fisher scientific), and the viral rna was titrated by the rt-qpcr targeting the n gene and the duplex rt-qpcr targeting the s gene of each virus. fecal infectious pedv shedding titers were tested using vero cells in 96-well plates and determined as 50% tissue culture infective doses (tcid 50 ) (reed and muench, 1938) . at necropsy, three sections of the jejunum (proximal, middle, and distal) and one section of the ileum were collected and fixed in 3.7% formalin (fisher scientific co llc, florence, ky, usa). all tissues were trimmed, processed, embedded in paraffin, and sectioned using routine procedures . to compare the pedv-specific histopathological changes among different groups of pigs, ihc staining was performed as described previously for the detection of pedv n proteins using mab sd6-29 as the primary antibody (hou et al., 2017) . the ihc staining was carried out using a nonbiotin polymerized horseradish peroxidase system (biogenex laboratories, san ramon, ca, usa). finally, tissues were counterstained with hematoxylin. images of tissues were observed and captured by olympus ix-70 fluorescent microscope (center valley, pa, usa). ratios of villous height to crypt depth (vh/ cd) of the jejunum and ileum of individual piglets were measured using a computerized imaging system (metamorph, olympus, japan) as described previously . for each intestinal section, 10 villi and crypts were measured. to detect and differentiate icpc22 a-and icpc22 a-s1δ197-infected cells in the coinfected pigs, if staining was performed. the slide preparation steps were as same as those of the above ihc procedure. the mixture of mouse mabs 56h4, 60f11 and 72a8 (1: 100 in pbs) (li et al., 2017a) targeting the s1°domain of pedv s protein was used as the primary antibody for the staining of icpc22 a-infected cells only. tissues were incubated at 4℃ overnight, after three washings with pbst (pbs with 0.1% tween20), alexa fluor 594-conjugated goat antimouse igg (h + l) (thermo fisher scientific) (1: 500 diluted in pbs) was used as the second antibody and tissues were incubated at room temperature for 2 h. after washing with pbst, guinea pig pab gp17 (1: 200 diluted in pbs), targeting the s1δ197 and was for the staining of both viruses, was added. then, the slides were incubated at room temperature for 2 h. after washing three times with pbst, alexa fluor 488-conjugated goat anti-guinea pig igg (h + l) (thermo fisher scientific) (1: 500 diluted in pbs) was used as the 2nd antibody and the slides were incubated at room temperature for 2 h. after washing with pbst, 1 mg/ml dapi (4′,6-diamidino-2-phenylindole, dihydrochloride, roche life science, indianapolis, in, usa) was added for nucleic acid staining. finally, an autofluorescence quenching kit (vector® trueview™, burlingame, ca, usa) was used to quench the autofluorescence of the tissues. images of stained tissues were captured by olympus ix-70 fluorescent microscope. red, green and blue fluorescence staining was merged by imagej software (https://imagej.nih. gov). 2.5. isolation of the pig-passaged icpc22 a and icpc22 a-s1δ197 from the sic of a pig coinfected with the viruses the sic (sample #pe1158) of a coinfected piglet in group 3 was positive for both viruses by the duplex rt-qpcr. virus isolation was performed using plague assays (oka et al., 2014) . the sic was diluted in dmem and the 10% suspension was vortexed briefly followed by centrifugation at 15,000 ×g for 3 min at 4℃. a series of 10-fold dilutions (10 −1 -10 −5 ) of the supernatants were prepared in dmem (containing 7.5 μg/ml trypsin) and were used immediately for inoculation of vero cell monolayers in 6-well plates (200 μl per well). after incubating at 37℃ for 1 h, the inoculum was removed and the monolayers were washed once with pbs. then 2 ml/well of agarose overlay was added. the plate was kept for 3 days before picking up clones. for propagating each clone, vero cell monolayers in 96-well plates were prepared and individual plagues were picked and added into individual wells. at 3 dpi, rna was extracted and the duplex rt-qpcr method specific for both viruses was performed. 2.6. multi-step growth kinetics and if staining of coinfection of pedv icpc22a and icpc22a-s1δ197 in vero and ipec-dq cells the growth kinetics of icpc22a-s1δ197 and icpc22a during the coinfection of the two viruses were investigated in vero and ipec-dq cells. cell monolayers on 96-well plates were inoculated with one of the three inocula: (1) 0.01 multiplicity of infection (moi) of icpc22a; (2) 0.01 moi of icpc22a-s1δ197; and (3) 0.01 moi of icpc22a and 0.01 moi of icpc22 a-s1δ197. viruses were diluted in dmem containing 10 μg/ml trypsin and were added to cell monolayers. after incubating at 37℃ for 2 h, the inoculum was removed and the cell monolayers were washed for three times. then the plates were added with the maintenance medium and incubated at 37℃. the samples were collected at different time points (2 h, 8 h, 12 h, 36 h, 48 h) and frozen (-80℃) and thawed once before testing. rna was extracted using the magmax-96 rna isolation kit (thermo fisher scientific) and virus titers were tested by the duplex rt-qpcr specific for both viruses. for if staining of s proteins at 8 h post infection (hpi), the procedure in 2.4 section was performed. 2.7. infection of pedv icpc22a and icpc22a-s1δ197 in na-treated vero and ipec-dq cells vero/ipec-dq cell monolayers in 96-well plates were treated with 250 mu na in dmem/rpmi-1640 or mock dmem/rpmi-1640 at 37℃ for 2 h. after three washings with dmem/rpmi-1640, cells were inoculated with icpc22 a or icpc22 a-s1δ197 at a moi of 0.02 with 10 μg/ml trypsin for 2 h. next, the inoculum was removed followed by three washings. dmem/rpmi-1640 containing 50 μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer 1:1024, 1:500 dilution) was added to prevent virus spreading. cells were fixed with acetone-methanol (1:4) at 6 hpi. the numbers of pedv-infected cell foci were determined by if assays as described for the staining of pedv n proteins (hou et al., 2017) . briefly, rabbit pab rb9 (1:1000) was used as the primary antibody. after incubating at 4℃ overnight, cells were washed three times. then alexa fluor 488-conjugated goat anti-rabbit igg (h + l) (thermo fisher scientific) (1:1000) was used as the 2 nd antibody and the cells were incubated at room temperature for 1 h. after washing and adding mounting medium, cells were observed and picture were captured by using olympus ix-70 fluorescent microscope. the numbers of fluorescent focus units (ffu) were counted by imagej software. the percentages of ffu were calculated by the value of mock group as 100%. 2.8. effect of mucin, bile and bile acids on the infection of pedv icpc22a and icpc22a-s1δ197 in vero and ipec-dq cells viruses (icpc22a or icpc22a-s1δ197) were mixed with different concentrations of bm (0, 0.1, 0.3, 0.5 mg/ml) or pgm (0, 0.5, 1.0, 2.5, 5.0 mg/ml). then 10 μg/ml trypsin were added to the mixture. monolayer of vero/ipec-dq cells in 96-well plates were inoculated with each virus mixture at a moi of 0.02. the plates were incubated at 37℃ for 2 h, then the inoculum was removed. after three washings, dmem/rpmi-1640 containing 50 μg/ml sbti and swine pedv positive serum (1:500 dilution) was added. cells were fixed at 6 hpi. the numbers of ffu were determined by if assay for the staining of pedv n proteins (see section 2.7). the percentages of ffu were calculated by the value of mock group as 100%. similarly, different concentration of swine bile (0, 0.1, 0.3, 0.5, 1.0%) and bile acids (gcdca, cdca, dca, and udca) at 0, 25, 50, 100, 200 μm were tested. statistical analysis was performed using graphpad prism 7 software (graphpad software, la jolla, ca, usa). the experimental data were analyzed by one-way anova and student`s t-test. data are shown as m ± sd, and a p < 0.05 was considered as significant. 3.1. the replication of icpc22a was eventually enhanced during coinfection with icpc22a-s1δ197 in neonatal gn piglets as shown in fig. 1 , all pedv-inoculated pigs but no pigs in the mock group had diarrhea and shed pedv. the fecal consistency scores and peak fecal pedv n and s rna shedding titers of piglets in the coinfection group reached the peak at 1.5 dpi, which was delayed half a day compared with the icpc22a group (1 dpi) but earlier than the icpc22a-s1δ197 group (> 4 dpi). by 4 dpi, all pigs in the icpc22a and coinfection groups but no pigs in the icpc22a-s1δ197 group died or showed moribund. compared with the peak fecal pedv n gene shedding titer (11.6 ± 0.2 log 10 copies/ml) of piglets in the icpc22a group (1 dpi), pigs in the coinfection group had a significantly higher peak titer (13.6 ± 0.7 log 10 copies/ml) ( fig. 1b and table 2 ) at a delayed time point (1.5 dpi). in addition, the peak fecal infectious pedv shedding titer of the coinfection group (5.8 ± 0.4 log 10 tcid 50 /ml) were significantly higher than that of the single infection groups (4.9 ± 0.1 and 1.8 ± 1.8 log 10 tcid 50 /ml of icpc22 a and icpc22 a-s1δ197 groups, respectively) ( table 2) . interestingly, the peak s gene shedding titers of icpc22 a (11.4 ± 0.5 log 10 copies/ml) in the coinfection group at 1.5 dpi was significantly higher than that of icpc22 a group (10.5 ± 0.5 log 10 copies/ml) at 1 dpi (fig. 1c, table 2 ). however, the peak s gene shedding titers (5.8 ± 1.2 log 10 copies/ml) of icpc22 a-s1δ197 in the coinfection group was significantly lower than that of the icpc22 a-s1δ197 group (7.6 ± 1.7 log 10 copies/ml) (fig. 1c , table 2 ). in the second passage of coinfection, only icpc22 a but not icpc22 a-s1δ197 s gene was detected in the large intestinal contents (lic) of the piglet, which was orally inoculated with the sic (sample #pe1158) of one pig in the coinfection group and was positive for both viruses. the lic had a virus titer of 8.8 log 10 tcid 50 /ml for infectious pedv, 12.3 log 10 s gene copies/ml and 14.6 log 10 n gene copies/ml for icpc22a. histopathological examination was performed and villous atrophy was observed in the jejunum and ileum of piglets in all pedv-inoculated groups ( fig. 2a and b ). the small intestinal villi in the icpc22a-s1δ197 group had a milder villous atrophy than those in the icpc22a and coinfection groups. the vh/cd ratios of jejunum and ileum of all the pedv-inoculated groups were significantly lower than those of the mock group (fig. 2b) . the ratios of the icpc22 a and coinfection groups were similar and were significantly lower than that of the icpc22 a-s1δ197 group. ihc staining showed that no pedv-positive intestinal epithelial cells were observed in all pedv-inoculated pigs fig. 1 . evaluation of the replication of icpc22a and icpc22a-s1δ197 in gn pigs infected with individual or both viruses. (a) fecal consistency scores of individual gn piglets and the mean of each group were shown. the scores were named as follows: 0, solid (normal); 1, pasty (normal); 2, semiliquid (mild diarrhea); and 3, liquid (severe diarrhea). two piglets from icpc22a, icpc22a-s1δ197 and coinfection groups, and one pig from mock group were euthanized at 12 hpi and 2-3 dpi, respectively, for histopathological examination. (b) fecal pedv n gene rna shedding titers (for both icpc22a and icpc22a-s1δ197) of each group. data are shown as mean (m) ± standard deviations (sd) of pigs in each group. (c) fecal pedv s gene rna shedding titles of each virus. data are shown as m ± sd. at 0.5 dpi (data not shown). at 2-3 dpi, a few pedv n antigens were observed in the small intestine of the icpc22a-s1δ197-infected piglets, while extensive antigens were stained in those of the icpc22a and coinfection piglets ( fig. 2a) . by if staining for the pedv s1 proteins, a few pedv antigens were observed in the jejunum of the icpc22a-s1δ197-inoculated piglets (green alone, positive for s1δ197 only), while many antigens were observed in those of the icpc22 a-inoculated piglets [yellow, representing positive for both s1δ197 (green) and s1°d omain (red)] and the coinfection piglets (yellow) (fig. 2c) . 3.2. icpc22a alone, but not icpc22a-s1δ197 alone was isolated from the sic of one of the pigs infected with the two viruses simultaneously the sic of the one piglet in the coinfection group, which was positive for both viruses by the duplex rt-qpcr were used for virus isolation in vero cells using plague assays. we picked up 72 plagues and found that 55 plagues were positive for icpc22a and 17 plagues were positive for both viruses by the duplex rt-qpcr. no plagues were positive for s1 ntd-del pedv alone. during coinfection in vero cells, icpc22a replicated to a titer of 10.10 ± 0.05 log 10 copies/ml at 72 hpi, which was similar to that of the single infection (10.20 ± 0.09 log 10 copies/ml) (fig. 3a) . however, icpc22a-s1δ197 s gene peak titer was 9.80 ± 0.03 log 10 copies/ ml, which was significantly lower than that of the single infection (10.30 ± 0.04 log 10 copies/ml). in vero cells, icpc22a alone and icpc22a-s1δ197 alone replicated to similar titers. similarly, during coinfection in ipec-dq cells, icpc22a replicated to a similar titer to its single infection (8.60 ± 0.18 vs. 8.70 ± 0.11 log 10 copies/ml at 48 hpi (fig. 3c ). icpc22a-s1δ197 s gene peak titer was 7.03 ± 0.12 log 10 copies/ml, which was significantly lower than that of the single infection (7.49 ± 0.25 log 10 copies/ml). in contrast to those results in vero cells, icpc22a alone replicated to significantly higher titers than icpc22a-s1δ197 alone in ipec-dq cells. by if staining, more icpc22a-s1δ197-infected cells (green) were observed in vero cells than in iepc-dq cells in both icpc22a-s1δ197 alone and co-infection conditions ( fig. 3b and d) . at the second passage of the coinfection, icpc22 a rna and antigens were detected in both cells; however, icpc22 a-s1δ197 rna and antigens were detected exclusively in vero cells but not in ipec-dq cells (data not shown). 3.4. the percentage of infectivity of icpc22a was significantly lower than that of icpc22a-s1δ197 in na-treated vero and ipec-dq cells after removing sialic acids from the cell surface using 250 mu na in vero and ipec-dq cells, the percentages of infectivity of icpc22a and icpc22a-s1δ197 were reduced significantly (fig. 4) . however, in both cells treated with na, the percentages of infectivity of icpc22a were significantly lower than those of icpc22a-s1δ197. 3.5. mucin enhanced the infectivity of icpc22a but had no or inhibition effects on icpc22a-s1δ197 in both vero and ipec-dq cells in vero cells, 0.1-0.3, 0.5 and 1.0 mg/ml bm increased, had no effects and decreased the infectivity of icpc22a, respectively (fig. 5a ). 0.5-5.0 mg/ml pgm increased the infectivity of icpc22a 1.7-2.5 fold (fig. 5b) . however, the infectivity of icpc22a-s1δ197 was unaffected or inhibited significantly by bm and pgm at the tested concentration ( fig. 5a and 5b) . in ipec-dq cells, the infectivity of icpc22a was increased 1.7-2.6 fold and 2.6-3.8 fold by bm (0.1-1.0 mg/ml) and pgm (0.5-2.5 mg/ ml), respectively. however, there was no effects at the highest pgm concentration tested (5.0 mg/ml). on the other hand, low to medium concentrations of bm (0.1-0.5 mg/ml) and pgm (0.5-1.0 mg/ml) had no effects on the infectivity of icpc22 a-s1δ197. only high concentrations of bm (1.0 mg/ml) and pgm (2.5-5.0 mg/ml) reduced the infectivity of icpc22 a-s1δ197 significantly ( fig. 5c and 5d ). 3.6. bile and bile acids enhanced the infectivity of icpc22a but had no or inhibition effects on icpc22a-s1δ197 in vero cells, low to medium concentrations of swine bile (0.1-0.5%) increased 2.4-3.3 fold of the infectivity of icpc22a (fig. 6a) . (fig. 6b-e) . however, the infectivity of icpc22a-s1δ197 was unaffected (0.1-0.3%) or inhibited significantly by bile (0.5-1.0%) by bile (fig. 6a) . bile acids a piglets in group 1 were inoculated with 100 pfu/pig of icpc22 a; piglets in group 2 were inoculated with 100 pfu/pig of icpc22a-s1δ197; piglets in group 3 were inoculated with 100 pfu/pig of each virus; piglets in group 4 were inoculated with pbs; piglets in group 5 were inoculated with the sic of piglet in group 3 with a dose of 4 log 10 pfu/ml targeting n gene of pedv. b fecal consistency (fc) was scored as follows: 0, solid, 1, pasty, 2, semiliquid; and 3, liquid. an fc score of 2 and 3 were considered diarrhea and severe diarrhea, respectively. c values in parentheses are the number of positive results/number of animals tested. d the detection limit of pedv rt-qpcr targeting the s gene of icpc22a or icpc22 a-s1δ197 is 5 log 10 copies/ml. e the detection limit of pedv rt-qpcr targeting the n gene of both icpc22a and icpc22 a-s1δ197 is 4.8 log 10 copies/ml . f the pig number for diarrhea observation is 1 or 2 less than the total pig number because 1 or 2 pigs of each group were euthanized at 12 hpi. g the experimental data were analyzed by student`s t-test. letters 'a, b and c' indicate a mean significant difference between groups (p < 0.05). '-', not detected. y. su et al. veterinary microbiology 228 (2019) 202-212 at the tested concentrations had no effects on the infection of icpc22 a-s1δ197 (fig. 6b-e) . similarly, in ipec-dq cells, the infectivity of icpc22a was increased significantly by 0.1-0.3% bile but was decreased significantly by the concentration of 1.0% (fig. 6f) . however, 0.1-1.0% bile decreased the infectivity of icpc22a-s1δ197 significantly. bile acids gcdca (fig. 6g-j) . no significant effects of bile acids on icpc22a-s1δ197 were observed except for that the highest concentration (200 μm) of gcdca and dca decreased its infectivity significantly (fig. 6g-j) . fig. 2 . histopathological examination of the gn piglets infected with individual or both icpc22a and icpc22a-s1δ197. (a) ihc staining of pedv n proteins in the jejunal and ileal sections of piglets that died or were euthanized at 2-3 dpi (magnification, 200×) . the brown signals represented the pedv n antigens in enterocytes. (b) villous height to crypt depth (vh/cd) ratios of jejunum and ileum of the gn piglets euthanized at 2-3 dpi. for each intestinal section, 10 villi and crypts were measured. data are shown as the m ± sd. number of asterisks indicate significant difference between groups (*, p < 0.05; **, p < 0.01; ***, p < 0.001). 'ns', not significant. (c) if staining of pedv s proteins in the jejunal sections of piglets that died or were euthanized at 2-3 dpi (magnification, 200 x). tissue sections were stained for the detection of icpc22a and icpc22a-s1δ197 antigens targeting the s1δ197 protein (green), and for icpc22a antigens targeting the s1°domain (red), and counterstained for cell nuclei (blue). in the merged images, yellow dots represented icpc22a-infected (or co-infected in coinfection group) cells (merged from red and green) and the green dots represented icpc22a-s1δ197 infection alone. (magnification, 200 x) . cells were stained for the detection of icpc22a and icpc22 a-s1δ197 antigens targeting the s1δ197 protein (green), and for icpc22a antigens targeting the s1°domain (red), and counterstained for cell nuclei (blue). in the merged images, yellow dots represented icpc22a-infected (or co-infected in coinfection condition) cells and the green dots represented icpc22a-s1δ197 infection alone. the experimental data were analyzed by student`s t-test. letters 'a, b and c' indicate a mean significant difference between groups (p < 0.05). previously, our laboratory isolated the first s1 ntd-del pedv strain, pc177, from vero cell culture (oka et al., 2014) . recent studies revealed that s1 ntd-del pedvs naturally evolved in the field and often coinfected pigs with the s-intact pedv (suzuki et al., 2015; diep et al., 2017; zhang et al., 2018a; su et al., 2018) . the s1 ntd-del pedv had no tissue tropism change compared with the s-intact pedv (suzuki et al., 2016; hou et al., 2017) . this differs from porcine respiratory coronavirus (prcv) that is a s1 ntd-del version of tgev and changes the major tissue tropism from intestines to respiratory tract (zhang et al., 2007) . in this study, we investigated the replication of s1 ntdfig. 4 . infection of pedv icpc22a and icpc22a-s1δ197 in neuraminidase (na)-treated vero and ipec-dq cells. vero or ipec-dq cell monolayers in 96-well plates were treated with 250 mu na or mock at 37℃ for 2 h. after three washings with dmem/rpmi-1640, cells were inoculated with equal amounts of virus at a moi of 0.02 diluted in the medium containing 10 μg/ml trypsin. after incubation at 37℃ for 2 h, the inoculum was removed followed by three washings. dmem/rpmi-1640 containing 50 μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer 1:1024, 1:500 dilution) was added. at 8 hpi, cells were fixed with acetone-methanol (1:4) and infectivity was tested by if assay. each experiment was performed three times. data are shown as the m ± sd, and number of asterisks indicate significant difference between groups (*, p < 0.05; **, p < 0.01; ***, p < 0.001). fig. 5 . effect of bovine mucin (bm) and porcine gastric mucin (pgm) on the infection of pedv icpc22a and icpc22a-s1δ197 in vero (a and b) and ipec-dq cells (c and d). monolayers of vero or ipec-dq cells in 96-well plates were inoculated with a moi of 0.02 icpc22a or icpc22a-s1δ197 in medium containing 10 μg/ml trypsin and different concentrations of bm or pgm. after incubation at 37℃ for 2 h, the inoculum was removed followed by three washings. dmem/rpmi-1640 containing 50 μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer 1:1024, 1:500 dilution) was added. at 8 hpi, cells were fixed with acetone-methanol (1:4) and infectivity was tested by if assay. each experiment was performed three times. data are shown as m ± sd, and number of asterisks indicate significant difference between groups (*, p < 0.05; **, p < 0.01; ***, p < 0.001). del and s-intact pedvs during coinfection in pigs. we developed a duplex rt-qpcr targeting the s gene of pedv to differentiate the two viruses. we found that the duplex rt-qpcr assay had different sensitivities for the detection of infectious icpc22 a and icpc22 a-s1δ197 (table s1) although it had a similar detection limit (5 log 10 copies/ml) for both viruses. for example, 9 log 10 s gene copies/ ml corresponded to 6.2 and 4.2 log 10 ffu/ml for icpc22a and icpc22a-s1δ197, respectively. it may reflect the fact that icpc22a-s1δ197 replicates to lower peak infectious titers (∼5.0 log 10 ffu/ml) than that (∼7.0 log 10 ffu/ml) of icpc22a in vero cells although both viruses replicated to similar peak titers based on the s gene (∼10.0 log 10 copies/ml) (fig. 3a) . in the mixture of two viruses, the detection fig. 6 . effect of bile and bile acids on the infection of pedv icpc22a and icpc22a-s1δ197 in vero (a-e) and ipec-dq cells (f-j). monolayers of vero or ipec-dq cells in 96-well plates were inoculated with a moi of 0.02 icpc22 a or icpc22 a-s1δ197 in medium containing 10 μg/ml trypsin and different concentrations of bile or bile acids. after incubation at 37℃ for 2 h, the inoculum was removed followed by three washings. dmem/rpmi-1640 containing 50 μg/ml sbti and swine pedv positive serum (virus neutralization titer 1:1024, 1:500 dilution) was added. at 8 hpi, cells were fixed with acetone-methanol (1:4) and infectivity was tested by if assay. each experiment was performed three times. data are shown as m ± sd, and number of asterisks indicate significant difference between groups (*, p < 0.05; **, p < 0.01; ***, p < 0.001). sensitivity of each virus differed significantly (tables s2). for the detection of icpc22 a at high titers (6.2 log 10 ffu/ml) and as the predominant virus in the mixture of two viruses (icpc22a: icpc22 a-s1δ197 = 10 2 -10 5 :1), the s gene titers (∼8.8-9.1 log 10 s gene copies/ ml) (table s2 ) were similar to that (∼9.0 log 10 s gene copies/ml) in the single virus alone (table s1 ). because icpc22 a replicated to high titers and was the predominant virus during the coinfection in pigs, we can predict that the peak infectious titer of icpc22a in coinfection was ∼1.0 log 10 -higher than that in its single infection based on the duplex rt-qpcr results (∼11.4 vs ∼10.5 log 10 s gene copies/ml) (table 2) . so, we conclude that the replication of icpc22a was interfered at the beginning based on delayed timing (1.5 vs 1.0 dpi) to reach peak titers but enhanced eventually in coinfection compared with its single infection in pigs. on the other hand, the sensitivity for the detection of icpc22a-s1δ197 decreased significantly and icpc22a-s1δ197 became undetectable when icpc22a was high (6.2 log 10 ffu/ml) and icpc22 a-s1δ197 infectious titers were lower than 3.2 log 10 ffu/ml in the mixture of two viruses (table s2 ). therefore, the decreased peak s gene titer (∼5.8 log 10 copies/ml) of icpc22a-s1δ197 in coinfection of pigs compared with that (∼7.6 log 10 copies/ml) in its single infection may be due to inhibition of replication or the decreased detection sensitivity of the duplex rt-qpcr for the detection of icpc22a-s1δ197 during coinfection (table 2 ). in the first passage of coinfection, we observed much lower numbers of s1 ntd-del pedv-infected cells than the s-intact pedv-infected cells in the small intestines of pigs infected with the same dose of both viruses simultaneously. in addition, plaque assays were performed to isolate and purify individual viruses from the sic of one of the coinfection pigs. however, only s-intact pedv was isolated alone. in contrast, s1 ntd-del pedv was exclusively detected together with the sintact pedv from the plagues. in the second passage of the coinfection in pigs, s-intact pedv but not s1 ntd-del pedv rna was detected in the lic of the pig inoculated with the sic of the pig inoculated with both viruses. this finding suggests that s1 ntd-del pedv had no replication advantage and was either outcompeted or coexisted with sintact pedv in pigs. this conclusion is in agreement with what were observed in the field (diep et al., 2017; su et al., 2018) . s1 ntd-del pedv replicated to a lower peak titer in coinfection than that in single virus infection in both vero cells and ipec-dq cells. these in vitro results were similar to those in the pig studies. in the second passage of coinfection, s1 ntd-del pedv rna was detected in vero cells but not in ipec-dq cells, probably due to the lower replication efficiency of the s1 ntd-del pedv in ipec-dq cells than in vero cells ( fig. 3a and c) . similar to other sialic acid-binding coronaviruses, the s1 ntd of pedv has a sialic acid binding activity, and the sugar-binding activities of a field isolate pedv variant chgd-01 was stronger than that of the prototype strain pedv cv777 using bm (deng et al., 2016) . the sialic acid binding activity occurred within the n-terminal 249 residues and the capacity of sialic acid binding differs among pedv strains using an hemagglutination assay . the recombinant virus used in this study, pedv icpc22 a-s1δ197, is a s1 ntd-del version of icpc22a. it lost most sialic acid binding activity tested in vero cells (hou et al., 2017) . in this study, we comparatively tested icpc22 a-s1δ197 and icpc22 a in vero and ipec-dq cells treated with na. similar to the previous reports, our results confirmed that the binding of s1 ntd of pedv to sialic acids on cell surface enhanced virus infectivity. bm is a mixture of highly glycosylated proteins containing sugar moieties, such as 5-n-acetyl-9-o-acetylneuraminic acid (neu5,9ac2), 5-n-glycolylneuraminic acid (neu5gc), and 5-n-acetylneuraminic acid (neu5ac). among them, neu5gc and neu5ac can serve as receptors or co-receptors for some alphacoronaviruses (e.g., tgev) and gammacoronaviruses [e.g., infectious bronchitis virus (ibv)] (cavanagh and davis, 1986; krempl et al., 1997) . similar to tgev, which uses neu5gc and neu5 ac as co-receptors (krempl et al., 1997; schultze et al., 1996; schwegmann-wessels and herrler, 2006) , the s1 ntd of pedv interacted with sugar (deng et al., 2016) . pgm contains 0.5-1.5% n-acetylneuraminic acid (neuac). our data indicated that low-medium concentrations of bm or pgm enhanced the infectivity of s-intact pedv in both vero and ipec-dq cells. in contrast, the s1 ntd-del pedv was not affected or inhibited, probably due to the covered mucin that blocked virus binding to the receptors. the intestinal epithelial cells are covered by a layer of mucus that is rich in sialic acids. these results suggest that low-medium amount of mucin binding to s-intact pedv may help the virus particles approach the receptors via sticking to the sialic acids on the cell surface. however, the receptor binding domain (rbd) on the s protein of s1 ntd-del pedv, which lacks most sialic acid binding activity, is probably covered with mucin and blocked its binding to the receptors. when high concentration of mucin saturates the s1 ntd of s-intact pedv, it can also cover viral rbd and block its binding to the receptors, resulting in decreased infectivity as observed in 1.0 mg/ml bm effects on s-intact pedv in vero cells (fig. 5a) . the major components of bile include bile acids, cholesterol, lipids, bilirubin, proteins and carbohydrates. the bile acids are stored in the gall bladder (at ∼300 mm) and released into the small intestine (mcleod and wiggins, 1968) . in the small intestine, the total bile acids have a concentration of 2-30 mm (dowling, 1973) . we selected two primary bile acids (gcdca and cdca) and two secondary bile acids (dca and udca) to test the effects of bile acids on pedv infection. the secondary bile acids are dehydroxylated ones from the primary bile acids by intestinal bacteria (björkhem, 1985) . our data indicated that a broad range of concentrations of bile and bile acids enhanced the infectivity of s-intact pedv in both vero and ipec-dq cells. however, bile and bile acids cannot increase the infectivity of s1 ntd-del pedv. these results suggest that the bile-and bile acid-mediated promotion of pedv infection is related to the s1 ntd. it was reported that gcdca may facilitate the adaptation of a s-intact pedv to trypsin-free growth in vero cells at the early passages (< p10) (kim et al., 2017) . however, the mechanisms of the bile acid function on the pedv infection is unknown and consequently need to be further investigated. the inability of the s1 ntd-del pedv to outcompete the s-intact pedv may account for the fact that s1 ntd-del pedvs were exclusively detected from coinfection with the s-intact pedv (diep et al., 2017; su et al., 2018) . consequently, the disease caused by the coinfection was as severe as that by the highly virulent s-intact pedv alone. this situation is quite different from what occurred for prcv and tgev: the wild spread of clinically mild prcv, which often infects pigs asymptomatically and repeatedly, inducing protective herd immunity against tgev outbreaks (vancott et al., 1994; brim et al., 1995) . prcv functions as a natural effective vaccine against tgev. however, for pedv, the clinically mild s1 ntd-del pedv variants do not infect pigs alone. therefore, safe and effective vaccines are still desired to control the deadly pedv infection in neonatal piglets. due to the 100% mortality rates of piglets in the icpc22 a-infected pigs (hou et al., 2017) and coinfection groups, we currently cannot determine whether the pathogenicity of pedv icpc22 a is enhanced during the coinfection. that should be investigated in older pigs (weaned pigs, sows and boars) which are more resistant to pedv infection and diseases (niederwerder and hesse, 2018) . in addition, the emergence of s1 ntd-del pedv variants may complicate pedv disease pattern on farms, which needs to be further investigated. in this study, we showed that the replication of the s-intact pedv was enhanced during coinfection with an s1 ntd-del pedv in pigs. we found that mucin, bile and bile acids can all increase the infection of sintact pedv but not the s1 ntd-del pedv. this feature may help explain why s-intact pedv outcompetes s1 ntd-del pedv in vivo. further studies are needed to understand the mechanisms of mucin-, bile-and bile acid-mediated enhancement or inhibition effects on the infection of different pedv variants. all animal experiments were performed according to the protocols approved by institutional animal care and use committee (iacuc) of the ohio state university (osu). mechanism of bile acid biosynthesis in mammalian liver cellular immune responses of pigs after primary inoculation with porcine respiratory coronavirus or transmissible gastroenteritis virus and challenge with transmissible gastroenteritis virus coronavirus ibv: removal of spike glycopolypeptide s1 by urea abolishes infectivity and haemagglutination but not attachment to cells bile acids are 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of pig epidemic diarrhoea viruses isolated in japan from pig epidemic diarrhoea virus s gene variant with a large deletion non-lethal to colostrum-deprived newborn piglets deciphering the biology of porcine epidemic diarrhea virus in the era of reverse genetics contribution of antibody-secreting cells induced in mucosal lymphoid tissues of pigs inoculated with respiratory or enteric strains of coronavirus to immunity against enteric coronavirus challenge complete genomic sequences, a key residue in the spike protein and deletions in nonstructural protein 3b of us strains of the virulent and attenuated coronaviruses, transmissible gastroenteritis virus and porcine respiratory coronavirus identification of porcine epidemic diarrhea virus variant with a large spike gene deletion from a clinical swine sample in the united states type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp1 in irf1 signaling the authors declare no conflict of interest. none. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetmic.2018.11.025. key: cord-004727-9sniu39j authors: fennestad, k. l.; mansa, b.; larsen, s. title: pleural effusion disease in rabbits: observations on viraemia, immunity and transmissibility date: 1981 journal: arch virol doi: 10.1007/bf01320789 sha: doc_id: 4727 cord_uid: 9sniu39j baby rabbits surviving infection with pleural effusion disease virus (pedv) developed viraemia persisting for at least six months. only the infectious serum samples collected during the first 2 months of disease could transfer the typical ped. six months after neonatal infection, virus concentration in serum was 10(2) to 10(4) rabbit-infectious doses per ml, the level of igg appeared elevated, and serum rendered non-infectious by ether-treatment had a protective effect in passive immunisation experiments. no evidence of glomerulonephritis or deposits of immunoglobulins could be demonstrated in the kidneys. during the nursing period pedv was transmitted from infected baby rabbits to two out of four dams, but not to control litter-mates. after the nursing period control rabbits, caged together with the viraemic rabbits for 60 to 150 days, remained free from pedv infection. the demonstration of pleural effusion disease (ped) virus as passenger of rabbit testicular suspensions of treponema pallidum in several laboratories shows that this virus can be transmitted from rabbit to rabbit by testicular fluids at intervals of 7--14 days, i.e. the customary time between intratesticular inoculation and harvest of treponemes from the testes (3, 6, 7) . ped is a little known rabbit disease first described in 1968 from the scandinavian countries as intercurrent death among rabbits inoculated with t. pallidum (4, 5) . the viral nature of the aetiological agent was reported in 1970 (6) , but as yet pedv has not been demonstrated convincingly by culture, electron microscopy, 0304-8608/81/0070/0011 / $ 01.80 or by a specific serological method (2, 3, 7, 9, 13) . recently, it has been suggested that the virus may be antigenically related to human coronavirus strain 229 e (13) . in experimental studies ped virus (pedv) freed from treponemes showed a remarkable persistence in rabbits. by serial passages of blood or pteural fluid pedv could readily be maintained in rabbits by serial passage at intervals of 2--10, 3--20, and 30 days. this increase in passage intervals reduced morbidity from fatal disease to almost subclinical infection. rabbits from the 30-day passages failed to develop clinical signs of ped on re-inoculation with virus (1) . the present experiments were carried out to study the persistence of pedv in blood of neonatally infected rabbits and the transmissibility of the infection by contact. the experimental design also permitted observations on the nature of immunity to ped. all animals were obtained from the closed colony belonging to statens seruminstitut. (sse:cph); they were fed a pelleted diet and water was given in bottles. cardboard trays with wood shavings were used as bedding material and changed once a week. male albino rabbits, aged 3--4 months, were used for demonstration of the pedv, for serial passages, titration and protection experiments. before use, these rabbits had been employed once for pyrogen testing of protein fractions of human blood. each of four dams with offspring was placed in a metal cage with a floor area of approximately 0.32 m 2. two to four baby rabbits in each litter were infected with pedv, while the remaining animals in the cage served as uninfected controls (of. table 1 ). the dam was removed from the cage 30 days after infection of her offspring and, after an additional period of 15--30 days in a separate cage, challenged with the pedv. after removal of the dam, an age-matched control baby rabbit was introduced into each of two cages (nos. 2 and 3) without uninfected cage mates. infected and uninfected rabbits remained together in the same cage for a period of 90--180 days, and their blood was examined for pedv at 30-day intervals from time of inoculation. the origin of the highly virulent copenhagen strain of pedv and the stock pool of infectious serum used for inoculation have been described previously (2) . each baby rabbit received subcutaneously 0.5 ml virus pool diluted 1 : 10 in pbs (ph 7.0) corresponding to approximately 0.5 × 105 rabbit-infectious doses of pedv (2) . for the subcutaneous challenge we used t ml of i : 10 diluted pleural fluid, corresponding to approximately 10 a rabbit-infectious doses. this fluid came from two virus stock pools of pleura] fluid kept at minus 70 ° c. each pool originated from 1~12 rabbits succumbing 3--5 days after inoculation during serial passages of the copenhagen strain of pedv (1). rabbit test ]or ped v the rabbit test described previously was used for the demonstration of pedv (3). briefly, the inoeulum to be examined was given subcutaneously to a 3--4 month-old rabbit. fever together with iridocyclitis or death with necropsy findings characteristic of ped or both were considered as evidence of the presence of :pedv in the inoculum. challenge 30 days after inoculation served to demonstrate presence or absence of immunity to pedv. baby rabbits and other rabbits that died were examined as described previously (1) . for the demonstration of pedv in dead baby rabbits 0.5--1 ml blood, pleural, or peritoneal fluid was used as inoculum in testing for pedv. at 30-day intervals blood samples were obtained from all infected and control animals. an amount of 0.2 ml blood from each sample, mixed with 0.8 ml pbs (ph 7.0), was used as inoculum. from post inoculation (p.i.) day 90 and at the subsequent 30-day intervals 0.2 ml serum from each of the infected rabbits was also examined in the same way. the infected rabbits were sacrificed and examined 180 days after infection. the control animals were placed in individual cages after 90--180 days of contact with infected cage-mates and challenged with pedv 30 days later. serial passages in rabbits by subcutaneous inoculation of infectious serum from p.i. days 90, 120, 150 and 180 days were carried out at 7-day intervals using one rabbit per passage. inoculmn for the passages was 0.2 ml serum mixed with 0.8 ml pbs. each rabbit used for passage was challenged with pedv 30 days after inoculation to study immunity to the original virus. serum samples were obtained h'om the infected rabbits 180 days after infection. each sample was divided into two portions and one portion was treated with ether to destroy infectivity of pedv (3). this was done by shaking undiluted serum with an equal volume of diethyl ether at room temperature for about 3 minutes. the ether phase was removed and the aqueous phase was re-extracted similarly with ether a total of four times. the residual ether was removed by aeration with nitrogen. ethertreated serum and untreated serum from each rabbit were diluted tenfold in pbs, and 5 to 1 ml of serum dilution 10-s were given intravenously to series of rabbits. rabbits receiving ether-treated serum were challenged subcutaneously 24 hours later with 108 rabbitdnfectious doses of pedv. rabbits receiving untreated serum were challenged in the same manner 60 days after serum injection. after challenge the animals were observed for clinical signs of ped for up to 10 days. serum from infected and control rabbits was examined 180 days after infection or after 150--180 days of contact. protein concentration was measured by refractometry (american optical hand refraetometer). the igg concentration was determined by the single radial immunodiffusion method (10) . the agarose plates contained 3.5 izl swine anti-rabbit immunoglobulins (dako, denmark) per cm 2. a purified igg preparation ( ~95 per cent igg) from normal rabbit serum was used as reference preparation. the serum samples were diluted with pbs (ph 7.38) to a concentration of 10--12 g protein per 1 and sedimentation analysis was carried out at 60,000 rpm and at 20 ° c in the beckman model e ultracentrifuge with schlieren optics. the photographic plates from the eentrifugation were enlarged for drawings, which were used for calculation of the sedimentation rates and for the planimetric determination of the relative concentrations of the 4s, 7s and 19s components. the kidneys from four infected rabbits were removed at necropsy on p.i. day 180. tissue blocks, fixed in lillies neutral buffered formalin, were embedded in paraplast ® for light microscopy. sections were cut at 2 and 3 lira and stained with h & e, pas ~ h, and silver methenamine -~ i-i & e. tissue for immunofluoreseence microscopy (ifm) was frozen in a dry ice-alcohol mixture and stored at minus 80 ° c. blocks were embedded in tissue teek ®-gelatine (ames lab.) ai~d cut into 1 fm sections at minus 24°c on a leitz histoeryotome. the details of preparation of specimens and the ifm procedures have been given previously (8) . fluorescein isothiocynate conjugated antisera (nordic hnmunol. lab.) from goat, specific for rabbit immunoglobulins (g1, g2, iga, igm, fe ~, fab) and from swine, specific for rabbit igg (fc + fab), were used in diiution 1 : 20, during the nursing period, i.e. from inoculation of the baby rabbits until post inoculation (p.i.) day 30, seven of the 13 inoculated baby rabbits died. one died within 24 hours after inoculation, another was devoured by the dam, and a third was killed on p.i. day 23 because of enterocolitis. two died with pleural and peritoneal effusions on p.i. days 11 and 26, respectively, but in the latter a haemorrhagie intussusception of the colon was also present. no cause of death could be established in the remaining two rabbits dying on p.i. days 9 and 12. table 1 shows the results of examination of the baby rabbits and their dams during nursing period. pedv was demonstrated in two dead baby rabbits examined on p.i. days 23 and 26, but not in two others dying on p.i. days 9 and 11. on p.i. day 30 pedv was demonstrable in blood of all six surviving infected . 1 and 4) . two of the four dams (nos. 1 and 3) were found to be protected when challenged with pedv, indicating that these two dams had become infected during the nursing period. after the nursing period two baby rabbits died on p.i. days 34 and 41 with pleural and peritoneal effusions. the remaining four infected baby rabbits, the five litter-mates, and two additional controls (introduced in cage nos. 2 and 3) appeared clinically normal until sacrifice or challenge. no gross lesions were observed at necropsy of the infected rabbits. this indicates that viraemia was present for six months in all four infected rabbits and that transmission of pedv infection from these rabbits to their agematched cage-mates did not take place. all rabbits inoculated with blood specimens obtained 30 days after infection responded with clinical disease typical of ped. this response was also seen in two of the four rabbits inoculated with blood from p.i. day 60. inoculation with blood or serum specimens from p.i. day 90 or later never resulted in clinical disease typical of ped; at most a transient fever was observed. to observe if the virus present in blood would retain inability to provoke clinical disease, serial rabbit passages of infectious serum from p.i. days 90, t20, 150 and 180 were carried out at 7-day intervals. table 3 shows the number of rabbit passages required before infectious serum regained a capacity to produce disease corresponding to that of t/he early isolates and the miginm virus. evidently, with continued viraemia ~n increasing number of passages was needed, and with two late isolates from the same rabbit clinical disease was not produced in spite of many passages. to determine virus concentration in serum obtained 180 days after infection, decreasing amounts of serum were given intravenously to series of rabbits, which were then challenged 60 days later, i.e. at a time when homologous antibody in the inoculum no longer could be expected to exert a protective effect. as seen from table 4 , rabbits receiving from 5 ml serum to 1 ml of serum dilutions 10 -2 to 10 -4 failed to develop clinical disease typical of ped on challenge. this indicates a virus concentration per ml of serum of 10 ~ to 104 rabbit-infectious doses. the protective effect of the same sera, after infectivity had been destroyed by ether-treatment, was studied by challenge one day after serum injection. table 4 shows that two of four rabbits, receiving the largest dose of serum, failed to develop clinical disease typical of ped. the table also shows the day of death from ped. using mortality rather than the full clinical picture as evidence of ped, it would appear that rabbits receiving 1 ml or more of ether-treated serum had a lower mortality (1 out of 8) than rabbits receiving 0.t ml or less (11 out of 14). this suggests a protective effect of all four ether-treated sera. table 5 shows the protein and igg concentra$ions in serum from the neonatally infected rabbits and their control cage-mates 180 days after infection. the concentrations of the 4s, 7s and 19s components are also listed. the higher contents of igg and the 7s component in serum fl'om infected rabbits as compared with the controls suggest that the infected rabbits developed an igg antibody response during the pedv infection. no pathological changes were found in the kidneys of the four rabbits with persisting viraemia. examination for deposits of immunoglobulins in the kidney tissues also gave negative results. the presence of pleural and peritoneal effusions in four baby rabbits, dying on p.i. days 11, 26, 34 and 41, respectively, provides strong evidence of death from ped. accepting only these four deaths as caused by pedv, the time of death was considerably delayed in baby rabbits as compared with older animals. k.l. fens;estad, b. mansa, and s. larse~: in 3--1 month-old rabbits of the same stock, infected by the same route and with the same highly virulent pedv strain, most deaths occm~red within the first week after infection and pleural effusion was the characteristic finding. the remaining deaths occurred in ttle 2nd or 3rd week with characteristic pleurat and peritoneal effusions (1) . the delay in occurrence of death from ped among the baby rabbits probably reflects a low reactivity between virus and host rather than resistance to infection, since pedv was demonstrated in all baby rabbits surviving infection. the persisting viraemia after neonatal infection is perhaps less surprising. in young adult rabbits pedv could regularly be transmitted from rabbit to rabbit. at intervals of 30 days, using only one animal per passage (1) . the reported failure of one attempt to extend this interval to 40 and 60 days, respectively, does not exclude the possibility that longer persisting viraemia may also exist in the adult rabbit. the few failures to demonstrate chronic viraemia in the present experiments may be explained by variation in virus concentration or perhaps differences in susceptibility to infection of the animals used in the rabbit test. the lack of clinical response in rabbits inoculated with infectious serum from p.i. day 90 or later may indicate a simultaneous presence of virus and virus antibody in the inoculum. however, when serial rabbit passages were carried out at 7-day intervals clinical disease was produced only in a single instance after the first passage. it may also be argued that the lack of response was due to a low virus concentration in serum. the virus concentration on p.i. day 180 was measured to 102 to 104 rabbit-infectious doses. the same concentrations of the original virus regularly produce clinical disease (unpubl. observations). this may suggest a change in virulence of the virus, production of defective interfering particles etc. further investigations of the virus-host interactions are required. the increased level of igg in infectious serum from p.i. day 180 is considered a sign of formation of humoral antibodies to pedv. apparently, these antibodies were unable to neutralize circulating virus. nevertheless, the antibodies had a protective effect when pedv in serum was destroyed by ether. the question whether virus was attached to immunoglobulins, forming circulating infectious virus-antibody complexes, was not examined. in other viral infections associated with circulating infectious virus-antibody complexes, glomerutonephritis and deposits of host immunoglobulins have been demonstrated (11) . the failure to demonstrate such kidney lesions in the present expei~ments may be due to the age of the animals at time of infection as observed in experiments with aleutian disease virus (i2). cross-infection between infected and uninfected rabbits in the same cage occurred from offspring to dam in two of four cases during the early stage of infection. this transmission is explained by the intimate contact between dam and offspring during the acute stage of infection. the lack of cross-infection by contact between baby rabbits before and after weaning indicates that direct transmission of ped is probably a rare occurrence. this assumption is in accordance with observations on ped among adult rabbits inoculated with treponemes contaminated with pedv. ped was present as an intercurrent disease at statens seruminstitut from 1961 to 1977. during this period evidence indicating cross-infection to adjacent or other cages was never found and ped remained strictly confined to animals inoculated with contaminated treponemal suspensions. pleural effusion disease in rabbits. clinical and post mortem observations. acta path. microbiol, seand pleural effusion disease in rabbits. interferon in body fluids and tissues after experimental infection. acta path. microbiol, scand pleural effusion disease agent as passenger of treponema pallidura suspensions from rabbits spontaneous deaths among rabbits inoculated with treponema paltidu.m less than 2 weeks before fever after inoculation of rabbits with treponema pallidum demonstration of a virus-like agent contaminating material containing the stockholm substraln of the nichols pathogenic treponema patlidum screening out a virus-like agent from the testicular suspension of the nichols pathogenic t. pallidum. with observations on certain characteristics of the agent immunofluorescent microscopy findings in minimal or no change disease and slight generalized mesangioproliferative glomerulonephritis. acta path. microbiol, scand intcreurrent death of rabbits after inoculation with treponema pallidum in the netherlands. 7th iclas syrup immunochemical quantitation of antigens by single radial immunodiffusion immune complex disease associated with viral infections reduced severity of lesions in minks infected transplacentally with aleutian disease virus rabbit cardiomyopathy associated with a virus antigenically related to human coronavirus strain 229 e key: cord-003973-pnareltx authors: hulst, m.m.; heres, l.; hakze‐van der honing, r.w.; pelser, m.; fox, m.; van der poel, w.h.m. title: study on inactivation of porcine epidemic diarrhoea virus, porcine sapelovirus 1 and adenovirus in the production and storage of laboratory spray‐dried porcine plasma date: 2019-04-01 journal: j appl microbiol doi: 10.1111/jam.14235 sha: doc_id: 3973 cord_uid: pnareltx aim: evaluation of the thermal and physical conditions for inactivation of adenovirus (adv), porcine sapelovirus 1 (psv1) and the economically important viruses porcine epidemic diarrhoea virus (pedv) and porcine circovirus 2 (pcv2) in the production of spray‐dried porcine plasma (sdpp). methods and results: citrate‐treated porcine plasma of ph 7·5, 9·8 and 10·2 (8·5% dry‐matter) was spiked with pedv, psv1, pcv2 and adv and incubated at 3°c for maximum 24 h, and at 44 or 48°c for maximum 10 min (experiment 1). spiked citrate‐treated concentrated plasma of ph 7·5 and 9·8 (24% dry‐matter) was spray dried in a laboratory scale apparatus (experiment 2). aliquots of sdpp were stored over a period of 0–10 weeks at 11 and 20°c (experiment 3). reverse transcription(rt)‐quantitative pcr detected no notable reduction in viral genomes in treated plasma and sdpp samples. no infectious psv1 was re‐isolated from plasma and sdpp samples in cell culture. at ph 10·2 and 3°c, infectivity of pedv in plasma was reduced with a reduction factor of 4·2 log 10 (lrf) at 10 h contact time, whereas heating to 44°c for at least 1 min at alkali ph was needed to achieve a lrf of 4·2 for adv. spray drying at an outlet temperature of 80°c reduced adv infectivity effectively (lrf = 5·2) and pedv infectivity for 95% (lrf = 1·4). after storage at 20°c for 2 weeks no infectious pedv was re‐isolated from sdpp anymore (lrf ≥4·0). due to growth of antibiotic‐resistant bacteria from plasma in cell cultures used for pcv2 isolation, no data regarding inactivation of pcv2 were obtained. conclusions: five percent of pedv stayed infectious after our spray drying conditions. spray drying in combination with storage for ≥2 weeks at 20°c eliminated infectivity of pedv effectively. significance and impact of the study: the conditions for inactivation of virus in plasma and sdpp determined are important for producers to inactivate pedv during production of sdpp. porcine epidemic diarrhoea virus (pedv) is singlestranded, positive-sense rna virus belonging to the family coronaviridae (pensaert and de bouck 1978) . the genome of approximately 28 kb is protected by a capsid that is surrounded by a membrane (envelope) in which the surface glycoproteins (so-called 'spike proteins') are anchored (bridgen et al. 1993) . pedv-diseased piglets shed high concentrations of infection-competent pedv particles in their faeces . as reported for other coronaviridae this suggests that the surface structure of these pedv particles stays intact in environments other than faeces (hofmann and wyler 1989; lai et al. 2015) . rescue of infectious pedv from several feed ingredients and additives was reported recently, showing that pedv stays viable in other environments than faeces (dee et al. 2014) . spray-dried plasma proteins (sdpp) are used worldwide as feed-ingredient in the pig industry for their bioactive function (pettigrew 2006) . it is mainly used in starter diets of weaned piglets, but also in milk replacers (hansen et al. 1993) . a multitude of studies showed that supplementation with sdpp reduces the period and severity of postweaning diarrhoea in piglets, indicating that sdpp may be a good alternative for growth-promoting antibiotics in the pig industry (pettigrew 2006; ferreira et al. 2009 ). however, the introduction of pedv in canada in 2014, following the severe outbreak of pedv in north america, starting in the spring of 2013, lead to suggestion that sdpp could be the cause of transmission of pedv among pig herds pasick et al. 2014) . after the start of the pedv outbreak in 2013 in the us, it became clear that pedv poses a serious threat for the profitability of the pig industry worldwide. although there was no substantial evidence for the belief that pedv was spread through plasma, the producers of sdpp commissioned a number of research projects to prove the virus-safety of their product. in animal trials piglets were fed with diets supplemented with sdpp produced from plasma of pigs obtained from pedv-infected farms and produced from plasma that was deliberately contaminated (spiked) with infectious pedv before spray drying opriessnig et al. 2014; schumacher et al. 2016) . in these trials piglets did not show any signs of disease and also no faecal shedding of pedv was detected. more detailed knowledge about the kinetics of inactivation by the thermal and ph conditions used in each the different steps of sdpp production should elucidate whether sdpp is a microbiological safe product. besides pedv, also new and re-emerging viruses pose a potential risk. to obtain a broader picture of virus inactivation in the different steps of production, inactivation of three other infectious viruses were measured in addition to pedv; namely, the naked rna virus porcine sapelovirus 1 (psv1, alias porcine enterovirus serotype 8), the economically important naked dna virus porcine circovirus 2 (pcv2) and adeno virus (adv), a virus frequently used to monitor virus inactivation in feed and food products (maunula et al. 2013) . to reflect the industrial production of sdpp as much as possible (see for a production scheme fig. 1 ) in this study raw porcine blood was collected at a regular slaughterhouse in the netherlands, preprocessed to plasma in a similar manner as is done in industrial production of sdpp and spiked with a mixture of pedv, adv, pcv2 and psv1. portions of unconcentrated plasma (as is) were used to test the thermal and physical stability at neutral and alkali ph of these viruses during prestorage (fig. 1) and after concentration by ultrafiltration, portions of concentrated plasma were used for spray drying using a laboratory apparatus (b€ uchi spray drying; see fig. 1 ). aliquots of the produced sdpp were stored at 11 and 20°c for up to 10 weeks (poststorage, fig. 1 ). samples collected were analysed by reverse transcription (rt)-quantitative pcr (qpcr) to monitor the concentration of viral genomes and dilutions of these samples were subjected to virus isolation/titration using susceptible cell lines to determine the degree of inactivation of each step in the production process of sdpp. african green monkey kidney epithelial cells (vero cells) were obtained from the american 109 type culture collection (atcc â ccl-81). vero cells were cultured and maintained in eagle's minimum essential medium (emem) with 10% v/v foetal bovine serum (fbs; bodinco bv, alkmaar, the netherlands) and 1% v/v antibiotic-antimycotic mixture (anti-anti, gibco â ), 1% l-glutamine (gibco â ), 1% nonessential amino acids (gibco â ) and 1% sodium bicarbonate (gibco â ). for preparation of pedv virus stocks and virus isolation assays vero cells were grown in serum-free medium, i.e. 1 : 1 mixture of emem and ultramcdk (lonza group ltd, bazel, switzerland) supplemented with 10 lg ml à1 trypsin and 1% v/v anti-anti. after each day of growth, 75% of this serum-free medium was replaced with fresh medium containing 10 lg ml à1 trypsin (hofmann and wyler 1988) . a549 and pk15 cells were obtained from atcc (a549-atcc â ccl-185 tm , pk15-atcc â ccl-33 tm ) and were cultured and maintained in dulbecco's modified eagle medium and emem, respectively, with 10% v/v fbs and 1% v/v anti-anti. psv1 strain po 5116 (nucleotide acc. number ay392538.1) was kindly provided by professor dr. roland zell (university of jena, germany). pedv strain cv777 was obtained from pensaert and de bouck in the late 1970s and induced diarrhoea in experimental pigs (pensaert and de bouck 1978) . pcv2 strain 35 was isolated from pigs with pmws in the netherlands (isolation number 35; wellenberg et al. 2000) . adv strain 2 was isolated from humans and is frequently used as internal control virus (index virus) to monitor virus inactivation in feed and food products (maunula et al. 2013 ). to prepare virus stocks for spiking, nearly confluent monolayers of vero (pedv), a549 (adv) or pk15 (psv1 and pcv2) cells were infected with a multiplicity of infection of about 0á1 and grown for 5 days (pvc2) or until 75% of the cells showed cytopathogenic effect (cpe) induced by pedv, adv or psv1. virus-infected cultures were freeze-thawed twice at à70°c and the suspension was centrifuged at 4000 g for 10 min to remove cell debris. the supernatant was harvested, divided in aliquots and stored at à70°c. aliquots of each virus stock were thawed and used to determine the titre of the virus stocks. for industrial production of sdpp, blood from healthy pigs is collected at slaughterhouses, citrate is added as anticoagulant and the blood cells are separated from the plasma by centrifugation. the liquid plasma is concentrated by ultra-or nano-filtration to remove water and to reduce the salt content, and subsequently spray dried (boxes with solid lines in fig. 1 ). for the experiments conducted on a laboratory scale in this study, raw porcine blood was collected at a regular slaughterhouse in the netherlands. per 1000 ml of blood 80 ml of a 10% w/v trisodium citrate solution was added on top as anti-coagulation and the blood was cooled to 4°c, centrifuged for 25 min at 1500 g to remove blood cells. for spray drying experiments 2á5 l of this plasma batch was concentrated by filtration using a hollow fibre membrane of 0á28 m 2 (general electric healthcare type ufp-10-e-5a). for heat-treatment experiments the plasma was not concentrated. before spiking of unconcentrated and concentrated plasma with viruses, four aliquots of 0á5 ml (negative control samples) were directly frozen on dry-ice and stored at à70°c until analysed with qpcr or subjected to virus isolation/titration (see below). similar was done for the portions of spiked unconcentrated plasma and culture medium that were incubated at different temperatures at different ph for different periods, and for the spiked concentrated plasma used to produce sdpp using the b€ uchi spray drier (spiked reference samples; see below). portions of 8á1 ml unconcentrated plasma (~8á5% dry matter) and culture medium were alkalized to ph 9á8 or 10á2 by slowly adding small volumes of naoh solution (1á65 n = 6á6% w/v) under constant agitation, or not alkalized (plasma ph = ae7á5). subsequently, these portions of plasma and culture medium were spiked on ice under constant agitation with 900 ll of a mixture of the four virus stocks (see the legends of table 3 for final concentration of viruses). an aliquot of 2á1 ml alkalized medium and plasma was divided over four reaction vessels (spiked reference samples; 0 h or 0 m) before samples were stored at à70°c. three aliquots of 2á1 ml were transferred to custom made stainless steel incubation tubes with screw cap and incubated in a water bath at various temperatures. the temperature inside the centre of the tubes was recorded using a calibrated thermocouple. at temperatures of 3, 44 and 48°c, aliquots were incubated for 1, 10 and 24 h (3°c), for 1, 5 and 10 m figure 1 flow scheme of spray drying process of plasma. solid lines; existing steps in the production process of sdpp in industrial plants. dotted lines: optional steps tested in this study, to increase inactivation of viruses in sdpp production plants. dashed lines: viruses were spiked in unconcentrated plasma (prestorage experiment) in a ratio virus stock to plasma of 1 : 9 and in concentrated plasma (buchi runs 1, 2, 3 and 4) in a ratio virus stock to plasma of 1 : 9 (run 1 and 2) or 1 : 12 (run 3 and 4). inserted table: overview of b€ uchi spray drying conditions tested. (44°c) and for 1, 2 and 10 m (48°c) respectively. after incubation, the content of the each tube was divided over four reaction vessels and stored at à70°c. concentrated plasma batches (24% dry matter) were kept on ice and alkalized to ph 9á8 by adding a 10 n naoh solution under constant agitation or not alkalized (as is; ph 7á5). subsequently, pcv2, adv, psv1 and pedv virus stocks were added slowly to these batches under constant agitation. immediately after spiking the plasma batches were spray dried in a b€ uchi 290 mini spray dryer (b€ uchi labortechnik ag, flawil, switzerland) using an inlet temperature of 190°c and an outlet temperature of 80 or 90°c at the maximum airflow. during the spray drying the spiked plasma batch was kept on ice. in total four batches of spiked plasma were spray dried. details about the volume of plasma spray dried, the volume of virus stocks spiked and the outlet temperature of spray drying for each run are listed in fig. 1 . sdpp powders produced in b€ uchi runs 1 and 2 were thawed on ice and 0á250 g portions were transferred to 12 ml polypropylene tubes with grip-stop (greiner 160282). two tubes with 0á250 g from run 1 and 2 were directly frozen at à70°c (0 weeks controls). all other tubes were stored in the dark in an incubator at 11 and 20°c for 1, 2, 3, 4, 5 and 10 weeks. for each time point and temperature of storage, duplicate portions were prepared. after the indicated weeks of storage both portions were transferred to à70°c. one of the duplicate portions was thawed and subjected to pedv isolation/titration in serial dilutions as described beneath (see methods section 'virus isolation/titration assays'). each dilution was tested in duplicate on cells. in case one of the duplicate dilutions around the end-point (the highest dilution scoring virus positive) scored positive and the other negative, the duplicate portion of sdpp kept at à70°c was thawed and tested in similar serial dilutions to confirm the endpoint of the titration the relative quantity of viral genomes in sdpp, plasma samples, or culture medium was determined using (rt)-qpcr. total nucleic acids were extracted from 100 ll samples (virus stocks, culture medium, or plasma) or 100 ll of the 5% w/v solution sdpp in culture medium using 300 ll trizol ls (invitrogen, thermo fisher scientific, naarden, the netherlands) followed by further purification using silica-based columns of the direct-zol tm rna miniprep kit (baseclear lab products, leiden, the netherlands). the nucleic acids were eluted in 50 ll of rnase-free water. five ll of eluate was used for quantification of adv and pcv2 dna by qpcr using the applied biosystems tm taqman universal pcr master mix (thermo fisher scientific, naarden, the netherlands) and the 'faststart dna master hybprobe' kit (roche, woerden, the netherlands) respectively. five ll of eluate was used as rna template for rt and quantification using the invitrogen ultra sense tm one-step quantitative rt-pcr kit (thermo fisher scientific, naarden, the netherlands) for psv1 and the taqman fas-tvirus-1-step master mix for pedv. qpcr tests were carried out with a lightcycler instrument (lc480; roche applied science, mannheim, germany) or applied biosystems 7500 fast real time pcr system using fluorescent labelled internal primers (probes) for detection of amplicons. the sequence of the forward, reverse primer and probe, their positions on the viral genome and the length of the dna fragments amplified in pcr reactions are listed in table 1 . in each qpcr run dilutions of virus stocks with a known titre (qpcr positive control) and culture medium of uninfected cells (qpcr negative control) were isolated and analysed along with the samples. the negative controls did not produce an s-shaped melting curve and scored a ct-value of ≥35, and the ct-value of the positive controls measured in all runs passed the sensitivity criteria documented for each virus-specific qpcr. details about the conditions used for amplification are provided in table s1 for each virus-specific qpcr. plasma, culture medium and sdpp samples were thawed on ice just before preparing dilutions in the appropriate, ice-cold culture medium for cell lines used in virus isolation/titration tests. the starting dilution of medium and plasma samples was 20-fold. a weighted amount of sdpp was dissolved in the ice-cold culture medium for each type of cell line to obtain a final concentration of 5% w/v sdpp in culture medium. it was necessary to dilute plasma samples 20-fold and test solutions of sdpp with a concentration that did not exceeded 5% w/v (1 : 20) in order to prevent coagulation and formation of clots. routinely, this 1 : 20 dilutions of plasma or sdpp was further diluted in fivefold steps to 1 : 100, 1 : 500, 1 : 2500, 1 : 12 500 and 1 : 62 500 dilutions. occasionally, pedv isolation/titration from specific samples was repeated with additional twofold dilutions around the 'end-point' determined in the first pedv isolation/titration assay (e.g. 1 : 25 000 in case 1 : 12 500 was the 'end-point'). nearly confluent monolayers of cells, grown in culture plates (2 cm 2 wells for pk15 and a549 cells) or flasks (25 cm 2 or 175 cm 2 flasks for vero cells), were inoculated with these dilution and grown as specified beneath for each virus. after growth, the 'end-point' was determined by detection of the characteristic cpe induced by adv, psv1 (rounded cells detaching from the monolayer and formation of plaques) and pedv (syncytia formation) by inspecting wells/flasks with a microscope. in case it was doubted whether cells were affected by cpe (e.g. in wells/flasks inoculated with dilutions around the 'end-point'), 100 ll of the culture medium was harvested and analysed by qpcr to confirm virus infection/replication. for determination of the 'endpoint' for pcv2, 100 ll of culture medium was harvested from all wells and analysed by qpcr. wells in which viral rna or dna was detected with a ct-value ≤30 and produced an s-shaped melting curve were considered as 'virus positive'. adv, pcv2 and psv1; the culture medium was removed and wells were washed with emem plus 2% w/v fbs and 1% anti-anti. a volume of 0á5 ml of samples diluted in prewarmed emem plus 2% w/v fbs and 1% anti-anti (hereafter denoted as infection medium), was applied to 2 cm 2 wells and plates were incubated for 2 h at 37°c and 5% co 2 . the dilutions were removed and wells were washed once with 0á5 ml of infection medium and supplied with the standard growth medium used for each cell line (see above). after 6-7 days (a549 cells) or 4-5 days (pk15) of growth the end-point for adv and psv1 was determined as described above. after an additional incubation period of 2-3 days, 100 ll of culture medium, was harvested from wells, and analysed by qpcr to determine the endpoint for pcv2. pk15 cells inoculated with a 20fold dilution of spiked plasma or with 5% w/v sdpp solutions in which no psv1 and/or pcv2 virus infection/ replication was detected were passaged blindly to 10 cm 2 tissue culture wells and grown for a second period of 7 days. psv1 and pcv2 infection/replication in pk15 cells was detected similar as described above. pedv; the medium with 10% fbs was discarded from 25 or 175 cm 2 culture flasks with vero cells, and monolayers were washed with serum-free medium (1 : 1 emem-ultramcdk plus 1% v/v anti-anti; hereafter denoted as pedv-infection medium). serial dilutions of samples prepared in pedv-infection medium supplemented with 10 lg ml à1 trypsin were applied to flasks (1á5 ml for a 25 cm 2 and 9 ml for a 175 cm 2 flasks) and flasks were incubated for 2 h at 37°c and 5% co 2 . dilutions were removed and monolayers were washed once with pedvinfection medium (5 ml per 25 cm 2 ) and provided with 4á2 ml (25 cm 2 ) or 25 ml of fresh pedv-infection medium containing 10 lg ml à1 trypsin. each day 75% of the medium was replaced with fresh pedv-infection medium containing 10 lg ml à1 trypsin. cultures were grown for 7-9 days at 37°c and 5% co 2 and were daily inspected with a microscope for cpe. in case no cpe was visible after 7-9 days, 100 ll of culture medium was analysed with (rt)-qpcr to detect pedv replication. in addition, 100 ll of culture medium of flasks inoculated with the 'next serial dilution' in which no cpe was observed, was analysed with (rt)-qpcr to confirm that no pedv replication had occurred in these cultures inoculated with a 'one step further' diluted sample (flasks scoring a ct-value ≤30 were considered as 'virus positive'). the dilution factor of the end-point dilution determined for untreated plasma (spiked reference samples; aliquots table s1 . †ncbi genbank accession numbers. of plasma or culture medium directly frozen after spiking with viruses) df r and of treated plasma and sdpp samples, df t , were used to calculate the virus reduction factor (lrf) on a log 10 scale, using the formula lrf = log 10 (df r /df t ). for each heat-treatment of plasma, for spray drying, and for the stored samples of sdpp an lrf was calculated. in case 25 cm 2 flasks of vero cells inoculated with 1á5 ml of a 1 : 20 dilution of plasma or dissolved sdpp (0á075 ml plasma or 0á075 g sdpp) scored virus negative for pedv, fresh aliquots of 0á45 ml plasma or 0á45 g sdpp were re-tested in a 1 : 20 dilution in a 175 cm 2 flasks (9 ml of inoculum per 175 cm 2 flask). the df t for these re-tested samples was corrected for the larger volume or amount (g) of inoculum using the formula, df t = 20/((n 9 0á45)/0á075), in which n stands for 1 or 2 175 cm 2 flasks. due to conversion of liquid plasma to powder by spray drying, the concentration of spiked virus per g sdpp can be up to 6á7-fold higher than the concentration per ml in concentrated plasma (see fig. 1 ). for sdpp samples, the df r measured to calculate the lrf was multiplied with 6á7 to account for this. the maximum measurable rf max (log 10 scale) was calculated for each experiment using the formula; rf max = log 10 (df r /20), in which 20 stands for the df of the 1 : 20 start dilution (see above). for pedv virus isolation in 175 cm 2 flasks the denominator in this rf max formula was corrected for the volume or grams tested, and for sdpp powders the nominator multiplied with 6á7 as described above. in the pilot experiments, in which untreated preprocessed and heat-treated plasma and dissolved sdpp powders were subjected to titration in culture plates with 2 cm 2 wells, large parts (up to 90-100%) of the monolayers of the vero cells detached, cells clotted and a substantial part of the cells were lost due to the daily refreshment of 75% of the volume of the pedv-infection medium during the 7-9 days growth. this loss of cells resulted in missing data points and partly unreliable results of titrations and demanded repeated re-testing of plasma and sdpp samples. therefore, the use of plates with 2 or 10 cm 2 culture wells for titration and isolation of infectious virus for large numbers of samples was inefficient and laborious. using vero cells grown in 25 and 175 cm 2 flasks, less cells detached. in addition, these larger cell surfaces allowed testing of larger quantities of plasma and sdpp, thereby improving the sensitivity of pedv isolation. to determine the sensitivity of this virus isolation assay for plasma samples, a known amount of pedv was spiked in preprocessed plasma of ph 7á5 and in pedvinfection medium. serial dilution of these spiked samples was subjected to virus isolation using 25 cm 2 flasks. in this assay, the df measured for spiked plasma was equal to that of spiked medium, and 100% of the spiked pedv was recovered (table 2) . to test this for sdpp, duplicate portions of powders from b€ uchi run 1 and 2 (note that these powders were produced from spiked plasma (see fig. 1 ) were dissolved in pedv-infection medium and one of the duplicates was re-spiked with a known amount of pedv stock and both were tested in dilutions in 25 cm 2 flasks. at a df of 12 500, typical pedv cpe was observed and qpcr analysis detected pedv replication for these re-spiked powders dissolved in infection medium. not re-spiked powders scored negative for cpe and in the qpcr analysis at a df of 12 500, and positive at a df of 2500 (results not shown). this showed that an additional amount of infectious pedv added to infection medium with sdpp was recovered efficiently and showed that components in sdpp did not inhibit pedv infection in our virus isolation/titration test. based on the concentration of pedv detected in 25 cm 2 flasks, at the measured df, the sensitivity for detection of infectious pedv in plasma or dissolved sdpp was calculated when larger quantities were subjected to virus isolation in 175 cm 2 flasks. these results showed that spiked pedv could be efficiently recovered from plasma and dissolved sdpp samples and an amount of 0á75 pfu per ml plasma or 0á73 pfu per g sdpp could be detected in this assay (table 2) . to obtain uniform data, testing of dilutions in fivefold steps, similar as was performed for pedv, was also used for the three other viruses in this study (see materials and methods). aliquots of spiked preprocessed unconcentrated plasma of ph 7á5 and alkalized to ph 9á8 and 10á2 that were incubated at different temperatures for the periods indicated in table 3 , including aliquots of the unconcentrated plasma before spiking (negative control plasma samples), were analysed with (rt)-qpcr. these latter negative control plasma samples scored a ct value of >35 in the (rt)-qpcr tests for pedv, psv1, adv and pcv2. for pedv, adv and pcv2 no decline in the amount of viral genomes was observed after heat treatment compared with the positive control medium and plasma samples directly frozen at à70°c after spiking (0 m or 0 h). the average ct value and standard deviation (aesd, n = 36 analysed in duplicate by (rt)-qpcr) for pedv, adv, psv1 and pcv2 was 21á4 (1á3), 25á0 (1á3), 27 (1á4) and 21á6 (1á1) respectively. for psv1 one plasma sample, which was alkalized to ph 10á2 and heated to 48°c for 10 min, was identified as a 'furthest from the rest' value in a grubbs outlier test (ct = 30á1, p > 0á05). this showed that the spiked viruses were uniformly dispersed in portions of plasma at neutral and alkaline ph. in addition, it indicated that heating at high ph did not resulted in extensive hydrolysis/fragmentation of the viral dna (adv and pcv2) and rna (pedv and psv1) genomes. dilutions of 'not heat-treated' plasma samples were subjected to virus isolation. in 2 cm 2 wells with pk15 cells inoculated with serial dilutions of these 'not heattreated' plasma samples no typical cpe induced by psv1 was observed, and no pcv2 and psv1 replication was detected by qpcr after growth for 5 (psv1) or 7 days (pcv2). after blind passage of the pk15 cells from wells inoculated with 1 : 20 dilutions to 10 cm 2 wells and growth for another period of 5 or 7 days also no typical psv1 cpe was observed and no replication of psv1 and pcv2 was detected by qpcr. because psv1 and pcv2 could not be re-isolated from these 'not heat-treated' plasma samples even not from those kept on ice at ph 7á5, no further efforts were undertaken to test the bulk of heat-treated plasma samples in the psv1 and pcv2 in the virus isolation/titration assays. the reduction factor (lrf, see materials and methods) for psv1 was calculated using the titre of the virus stock used for spiking and was equal to the maximum reduction factor (rf max = 3á3) measurable in the virus isolation assay for psv1. in the discussion it is explained why no lrf was calculated for pcv2. hundred percent of the amount of pedv spiked in plasma of ph 7á5 was recovered from 'not heat-treated' plasma samples (0m and 0h samples at ph 7á5; see table 3 ), indicating that no neutralizing antibodies directed against pedv were present in the pool of blood collected from the slaughterhouse. in addition, this showed that the presence of infectious adv or psv1 did not interfere with the detection/isolation of pedv when vero cells were infected and grown in the presence of 10 lg ml à1 trypsin. for all treatments of spiked plasma and culture medium listed in table 3 , the lrf was calculated. prolonged incubation at 3°c of alkalized plasma and culture medium lead to an efficient reduction in pedv-infectivity. after incubation for 24 h at 3°c no infectious pedv could be detected anymore in plasma alkalized to ph 10á2. (lrf = 4á2), whereas adv infectivity was reduced not more than fivefold (lrf = 0á7). compared with the relatively long period (24 h) at 3°c, heating of plasma at ph 10á2 to 44 or 48°c reduced the time needed to inactivate pedv with an lrf of 4á2 to 3 and 1 min respectively. at neutral ph, infectivity of adv in plasma was reduced effectively (lrf = 4á2) when plasma was heated to 48°c for more than 5 min. in contrast, under these conditions only a slight reduction in adv infectivity was observed in culture medium (lrf = 1á4). these large differences between culture medium and plasma were not observed for pedv after incubation at neutral ph, nor under alkaline conditions (table 3) . by heating of plasma to 44°c for at least 3 min at ph 10á2 a rf max (4á2) was achieved for both pedv and adv (table 3) . samples of sdpp produced in b€ uchi runs 1, 2, 3 and 4 (see fig. 1 ), and the spiked reference samples of concentrated plasma batches from which these sdpp samples were produced, were analysed by qpcr and dilutions 1á3 9 10 4 2á5 9 10 4 /2á5 9 10 4 1á7 9 10 4 0á68 0á75 pfu per ml culture medium (re-spiked sdpp) 1á3 9 10 4 1á25 9 10 4 /2á5 9 10 4 /2á5 9 10 4 1á7 9 10 4 0á68 na run 1 ph 7á5 8 á3 9 10 3 1á25 9 10 4 /1á25 9 10 4 8á3 9 10 3 0á66 0á73 pfu per g run 2 ph 9á8 8 á3 9 10 3 1á25 9 10 4 /1á25 9 10 4 8á3 9 10 3 0á66 0á73 pfu per g *concentration pedv spiked in preprocessed plasma (ph 7á5), culture medium and dissolved sdpp (5% w/v). †the highest dilution scoring virus positive (df): 1á5 ml of serial dilutions prepared from spiked samples were inoculated in duplicate in 25 cm 2 flasks. note that duplicates for 'culture medium' scored different df's in a first test. in a confirmation test (triplicate: see materials and methods) this sample scored a df of 2á5 9 10 4 . 2á5 9 10 4 was used to calculate the recovered pfu per ml. ‡recovered re-spiked pfu per ml in samples calculated from the df (df/1á5 ml). §concentration (pfu per ml) in the highest dilution that scored virus positive (df). ¶sensitivity: concentration of pedv in sdpp (pfu per g) and plasma (pfu per ml) detectable after inoculation of 2 175 cm 2 flask, each with 9 ml of dissolved sdpp (5% w/v) or 20-fold diluted plasma. values were calculated from the 'detected pfu per ml at df' ( §) determined in 25 cm 2 flasks. were subjected to virus isolation. aliquots of concentrated plasma before spiking (negative control plasma) scored a ct value of >35 in the (rt)-qpcr tests for pedv, psv1, adv and pcv2. the amount of pedv (note that pedv was not spiked in run 3 and 4), pcv2 and adv genomes detected in the produced sdpp powders was comparable to that detected in the spiked reference samples of concentrated plasma (results not shown). in contrast, less psv1 genomes were detected in produced plasma powder than in the spiked reference sample (dct = 4á9). repeatedly, growth of an unknown bacterium, resistant to the antibiotic cocktail used in pk15 cell cultures, was observed when spiked reference samples of the concentrated plasma of all four runs were tested in the virus isolation assays. whether this bacterium present in raw plasma was introduced in the collected blood by contamination from faeces, the environment in the slaughterhouse, or was present in the blood remains unclear. therefore, no valid dilution factors for these spiked reference samples (df r ) could be calculated. we used the df of the virus stock used for spiking for calculation of the lrf's of spray-dried samples. no typical cpe induced by psv1 and adv was observed, and no psv1, pcv2, or adv replication was detected by qpcr in the medium, in pk15 cells (psv1 and pcv2) or a549 cells (adv) infected with 1 : 20 dilutions of sdpp samples produced in b€ uchi runs at 80 and 90°c, also not after an additional blind passage of infected cultures. in contrast, it was calculated that~5% of the pedv spiked in plasma stayed infectious after spray drying at 80°c (note that pedv was not tested at 90°c; see fig. 1 ), irrespective of the ph (7á5 or 9á8) of the concentrated plasma used for spray drying. in table 4 the lrf calculated for spray drying at 80°c is provided for psv1, adv and pedv. similar as observed at 80°c, spray drying at 90°c of plasma of ph 7á5 and ph 9á8 at 90°c also achieved the maximum lrf for adv and psv1 of 5á2 and 4á2 respectively (results not shown). in the discussion it is explained why for pcv2 no lrf was calculated. aliquots of the sdpp batches produced in b€ uchi run 1 and 2 were immediately frozen after spray drying (0 weeks control samples) or were stored for 1, 2, 3, 4, 5 and 10 weeks at 11 and 20°c were subjected to pedvisolation/titration. in addition, these sdpp aliquots were analysed by qpcr. only for one sample, i.e. sdpp produced in b€ uchi run 2 (ph 7á5) and stored for 10 weeks at 20°c, a decline in the amount of pedv rna (dct = 5) was observed compared with not stored samples (0 weeks; ct avg = 22á8 ae 0á8 sd). the amount of *concentration of virus spiked in preprocessed plasma and medium: pedv 1á7 9 10 4 and adv 2á1 9 10 5 pfu per ml. †lrf max calculated using the formula lrf max = log 10 (df r /20), in which 20 stands for the df of the start dilution subjected to virus isolation. lrf max values are underlined. ‡lrf's were calculated using the formula lrf = log 10 (df r /df t ) in which df r is the measured dilution factor for the medium sample at ph 7á5 which was spiked at 3°c and directly frozen at à80°c (spiked reference sample) and df t is the dilution factor measured for treated medium or plasma samples. lrf's above 3á1 for pedv were measured in 1 (lrf = 3á9) or 2 (lrf = 4·2) 175 cm 2 flasks. psv1 and pcv2 could not be re-isolated from spiked 'not heattreated' (0 m and 0 h) plasma samples (see for details the results and discussion sections). §duplicate virus isolations that scored a different df in a first test. in a confirmation test (see materials and methods) the df established for a triplicate isolation, scoring similar to one of the duplicates of the first test, was used to calculate the listed lrf. pedv rna detected in all other samples stored at 11 and 20°c was comparable to that of not stored sdpp. the lrf as function of the storage time is displayed in fig. 2 . after storage for 2 weeks at 20°c, no infectious pedv was re-isolated anymore in sdpp powders produced in both runs (ph 7á5 and 9á8), whereas powders stored at 11°c still contained traces of infectious pedv (i.e. approximately 1% of the amount of infectious pedv spiked in the concentrated plasma). however, after 4 weeks of storage, no infectious pedv could be detected anymore in any of the stored sdpp powders. based on the amount of sdpp tested (0á25 g) and the sensitivity of this assay (~1 pfu per g; see table 2 ), it was calculated that sdpp powders scoring negative in the virus isolation assay contained less than 4 pfu per g of infectious pedv. conditions of pretreatment/storage of plasma before it is injected in an industrial size spray dryer differ in production plants around the world. we selected the conditions most commonly used in these plants and listed the lrf values for pedv, adv and/or psv1 infectivity in table 4 . combinations of temperature, ph and incubation periods for pretreatment and storage of sdpp may be extracted from table 4 to evaluate the safety of produced sdpp in different production plants for the viruses tested in this study. for pedv, our results show that an lrf of ≥4 can be achieved when a combination of heat-treatment of plasma before spray drying and a 'postproduction' storage period is applied. a number of virus-safety studies was performed in the last 2 years measuring inactivation of spiked pedv in bovine and porcine plasma and in laboratory spray-dried proteins fractions produced from plasma dee et al. 2014; pujols and segal es 2014; quist-rybachuk et al. 2015) . in contrast to these studies, we were able to rescue infectious pedv virus from sdpp produced in two independent b€ uchi runs at 80°c. despite the ph of the concentrated plasma spray dried in these runs were different and our experiments were no true duplicates, repeated rescue of infectious pedv irrefutable proved that the infectivity of pedv is not completely inactivated by the physical and thermal conditions of spray drying that were tested in this study. it has to be noted that the residence time of plasma in a b€ uchi laboratory spray dryer is shorter than in an industrial spray dryer (ranging from 10 to 60 s), suggesting that a higher lrf than 1á3 for pedv may be achieved in industrial production of sdpp. to obtain a broader knowledge of virus inactivation, we also measured inactivation of psv1, pcv2 and adv in experiments reflecting the main steps in industrial production of sdpp, prestorage/heat treatment of plasma, spray drying and storage of produced sdpp before dispatch. our results showed that conditions of thermal and ph-dependent inactivation of infectious virus were different for the four viruses under study. heating to 48°c was sufficient for effective reduction in adv infectivity in plasma of ph 7á5, but was insufficient for complete reduction in pedv infectivity. in line with this, the heat of the spray dryer was, most likely, also responsible for effective reduction in adv infectivity, whereas the residence time of plasma in the laboratory spray dryer was probably too short (0á2-0á35 s) (kemp et al. 2016) to completely reduce infectivity of pedv. in contrast, overnight incubation in alkaline condition at 3°c hardly affected adv infectivity, but reduced pedv infectivity efficiently. in a recent study, alkalization of plasma also potentiated inactivation of pedv infectivity at 3°c and during heat treatment (quist-rybachuk et al. 2015) . different conditions to inactivate pedv, psv1 and adv infectivity were observed in this study. in general, enveloped viruses (pedv) are more heat-labile than nd, not determined. *calculated concentration (pfu per ml) of spiked viruses in plasma batches spray dried at 80°c: pedv 1á7 9 10 4 , adv 2á0 9 10 5 . †average residence time (ctd) in b€ uchi spray drier. ‡lrf max for spray drying was calculated using the formula lrf max = log 10 ((df r 9 6á7]/20), in which df r represent the dilution factor of the virus stock used for spiking of the plasma batches, 20 stands for the 1 : 20 start dilution (5% w/v sdpp dissolved in pedvinfection medium) subjected to virus isolation, and 6á7 for the 'spray drying concentration factor' (see fig. 1 ). naked viruses (psv1 and adv). however, pedv showed to be less heat-labile than adv in plasma. with respect to psv1, infectivity was inactivated to an undetectable level in our tests immediately after spiking in chilled plasma of ph 7á5. as observed for foot and mouth disease virus (like psv1 also a picornavirus), addition of citrate (here used as anticoagulant) may have inactivated infectivity of psv1 in plasma by chelating calcium (hong et al. 2015) . pedv and psv1 particles are secreted by cells of the intestinal mucosa and both stay infectious in the content of the intestines and in faeces, both rough environments containing a broad diversity of proteases and glucanases from the host and microbiota. the resistance of the pedv surface structure to active components in plasma (e.g. chemicals, protease and other enzymes) or to added chemicals (e.g. citrate), in comparison to the damage imposed to the surface of psv1 in this environment, indicates that for each family/taxa of viruses, or even for specific virus strains, inactivation of infectivity is dependent of specific (structural) features of these viruses. no notable decline in the amount of viral rna and dna genomes was observed after heat-treatment of plasma, and in the b€ uchi produced sdpp. only at the most extreme conditions applied (10 min incubation at ph 10á2 and 48°c) some breakdown of psv1 rna was observed in plasma. a similar effect was observed for pedv rna in sdpp that was stored for 10 weeks at 20°c. for pedv and psv1 this suggests that their rna genomes (note that rna is more vulnerable to digestion/ hydrolysis than dna) were not readily accessible for degradation by enzymes present in plasma (proteases and rnases) or by alkali hydrolysis, indicating that the rna genomes were protected by the viral envelop and/or capsid under all tested conditions. in a recent study na€ ıve pigs were fed for nearly a month with sdpp produced from blood of pigs seropositive for pedv . although this sdpp contained a relative high concentration of pedv rna (5á1 ae 0á1 log 10 copies/g), it did not induce clinical signs of a pedv-infection, and also no seroconversion was observed. this also suggested that a considerable fraction of rna genomes were protected from rna degradation by rnases by the pedv envelop and/or capsid. failure of these pedv particles to mediate infection of cells in the intestine of pigs suggests that the surface structure of these pedv particles was damaged or altered. we observed that spray drying was more effective for inactivation of pedv than prestorage at ph 7á5 (at its natural state) at 3°c for 24 h (see table 3 ). however, no conclusion can be drawn whether this damage/alterations to the surface of virions was imposed by the heat or mechanical shear in the spray drier. we used a plasma batch that scored negative in the (rt)-qpcr test for pedv and fully recovered spiked infectious pedv from this plasma batch at~ph 7á5, indicating that no pedv antibodies in our plasma batch inhibited the infection of vero cells with pedv. this in contrast to the study of opriessnig et al. (2014) , in which plasma derived from pcr-positive pigs was used for production of sdpp in an industrial plant. this commercial sdpp contained igg antibodies directed against pedv, which could have contributed to the loss of infectivity of pedv in this commercial batch. we were unable to re-isolate infectious pcv2 from plasma and sdpp samples, even from the spiked reference samples with a ph of 7á5. partly, the growth of bacterial contaminations in pk15 cultures inoculated with plasma samples, interfered in our attempts to re-isolate and quantify infectious pcv2. also, it is known that isolation of infectious pcv2 from tissues and body fluids . the lrf max was calculated using the formula lrf max = log 10 (df r /(20/(0á25/0á075)), in which df r represent the dilution factor of the sdpp batches immediately frozen at à70°c after spray drying (0 weeks samples), 20 stands for the 1 : 20 start dilution (5% w/v sdpp dissolved in infection medium) and 0á25 for the maximum amount (g) of sdpp subjected to virus isolation. (including blood) is a difficult and time-consuming process and is often unsuccessful for unknown reasons (opriessnig et al. 2007) . given the high prevalence of pcv2 among pig herds in the netherlands (wellenberg et al. 2004) , it is also likely that the raw blood collected in a regular slaughterhouse contains antibodies directed against pcv2 (polo et al. 2013) . the presence of these antibodies may also have hampered re-isolation of infectious pcv2 from our plasma samples. because of these uncertain factors, we found it unjustified to calculate lrf values for pcv2 in our inactivation experiments. nevertheless, the results of our qpcr analysis were consistent with that of pujols et al. (2008) and shen et al. (2011) . in these studies it was also demonstrated that the integrity of the dna genomes of pcv2 was not affected by spray drying of plasma. in addition, we showed that heating of plasma to 48°c at ph 10á2, the most extreme condition tested, did not affect the integrity of the pcv2 dna. when used as ingredient in feed, the sdpp batches produced in the studies pujols et al. (2008) and shen et al. (2011) , containing a high concentration of pcv2 dna, were not able to transmit pcv2 to healthy pigs. recently pedv was spiked in bovine plasma and spray dried using practically the same b€ uchi apparatus and temperature settings as in this study (pujols and segal es 2014) . however, they used sodium phosphate as anticoagulant instead of citrate, and mixed a pedv virus stock with plasma in a 1 : 1 ratio instead of the 1 : 9 ratio we used. a ratio of 1 : 9 (or below 10%) is strongly recommended by the 'european agency for the evaluation of medicinal products' for studies validating inactivation and removal of viruses in production processes of human medicines (cpmp/bwp/268/95 report, 1996). although they almost fully recovered infectious pedv and pedv rna genomes from the 1 : 1 mixture of virus stock and plasma, they did not detect infectious pedv in produced spray-dried bovine plasma (sdbp), whereas we did in sdpp in two independent runs of spray drying with plasma of ph 7á5 and 9á8. in addition, in sdbp only a small fraction of the pedv rna present in the liquid mixture (ct = 13) was recovered in sdbp (ct = 23), whereas we fully recovered pedv rna after spray drying. the only plausible explanation we can think for these conflicting results is a difference in physical, chemical and biological conditions between the plasma mixtures used for spray drying. this may have imposed a different distribution of pedv particles between the watery phase (e.g. aerosol particles) and solid phase in the spray dryer or affected the droplet size and moisture content of the produced sdbp powders (see below). the moisture content of the spiked sdpp we produced in the b€ uchi apparatus was 6-7% and comparable to industrial produced sdpp. it was reported that the infectivity of a human adv vector in spray-dried powders decreased more rapidly when the moisture content was higher (tested in a range of 2-20%; leclair et al. 2016) . therefore, it is likely that the moisture in the sdpp batches we produced also favoured inactivation of pedv during storage of sdpp at 11 and 20°c, probably by facilitating a higher level of protease and/or rnase activity in the sdpp matrix (townsend and deluca 1990) or reactivity of chemicals. in line with this, we observed a faster inactivation of pedv infectivity under conditions that chemicals, proteases and rnases from blood display more activity, that is more at 20°c than at 11°c. also, a decline in the amount of pedv rna was detected after storage for 10 weeks at 20°c and ph 7á5, but not at 11°c. this suggested that the pedv rna became accessible for degradation by rnases, implying that the protective shell (capsid and envelop) of the pedv particles was (further) damaged during storage. this damage could have destroyed the capability of the pedv particles to bind to a receptor on the surface of cells to mediate infection. no clinical signs of pedv-infection were observed in na€ ıve pigs fed for 4 weeks with feed supplemented with sdpp produced from pedv-spiked plasma . in another study, pcr positive sdpp with an unknown origin of contamination induced clinical signs of pedv when piglets were inoculated with sdpp using a gastric tube (pasick et al. 2014 ), but not when sdpp was mixed in their feed. this latter study suggested that pedv particles lose their capability to infect cells in the small intestine, most likely, due to the passage through the stomach, an environment with low ph and high concentrations of digestive enzymes. however, pedv was rescued from various feed formulations (dee et al. 2014) indicating that the surface of the pedv particle endures the impact of various physical conditions and milieus. we demonstrated that infectivity of pedv particles also endured specific physical conditions and different milieus, irrespective of the matrix in which these particles were enclosed, liquid plasma, or solid sdpp. the fact that a fraction of pedv spiked in plasma stayed infectious after the heat imposed by spray drying gave us the opportunity to test the effect of storage on pedv infectivity in a nonartificial manner. storage proved to be an effective step to completely inactivate residual pedv infectivity in sdpp. in combination with spray drying, a storage period of 2 or 4 weeks at 20 or 11°c, respectively, reduced infectivity of pedv with an lrf of at least 4á0. the calculated level of residual infectious pedv present in the sdpp we produced from spiked plasma was approximately 4 pfu per g. when mixed in a 19 : 1 ratio starter diet-sdpp, a ratio routinely applied, this would be ≤0á2 pfu per g (≤1 pfu/5 g). this is 280-fold lower than the minimum dose of spiked pedv (56 pfu per g) in feed that led to infection in 10-day-old pigs under experimental conditions (schumacher et al. 2016) . based on a combined lrf of spray drying at ph 7á5 and 2 weeks storage of 4á0, it can be calculated that a plasma batch used for sdpp production must contain at least 6á2 log 10 infectious pedv particles per ml to prepare a starter diet batch with a concentration of 56 pfu per g pedv. in the opriessnig study of 2014, 5á1 log 10 pedv genome copies per g were detected in the sdpp commercially produced from plasma of seroconverted piglets . given a moisture content of 7á4%, we calculated that for production of 1 g sdpp approximately 7 ml of plasma was used with a concentration of 4á1 log 10 pedv genome copies per ml , i.e. plasma with a 100-fold lower concentration of pedv particles that would be needed to produce a starter diet batch with a minimum infectious dose of 56 pfu per g pedv (schumacher et al. 2016) . assuming that this concentration of 4á1 log 10 /ml is representative for the concentration of infectious pedv particles in the blood of pigs with viraemia, the combined lrf of 4á0 would be more than sufficient to formulate a 19 : 1 starter diet-sdpp batch with lower dose than 56 pfu per g. to our knowledge, no data about titres of infectious pedv in blood of viraemic pigs have been reported yet. likely, this is due to the lack of a cell line supporting efficient infection of native pedv. however, when data about pedv titres in blood of viraemic pigs become available they can easily be integrated with the results of this inactivation study to validate pedv safety of sdpp products in the future. we conclude that spray drying of plasma as is at an outlet temperature of 80°c in combination with a storage period at ambient temperature of 2 weeks or longer before dispatch, minimalizes the risk that adv, psv1 and pedv particles stay infectious in the feed-ingredient sdpp. sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhoea virus confirms that this virus is a coronavirus related to human coronavirus 229e and porcine transmissible gastroenteritis virus isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states an evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of na€ ıve pigs following consumption via natural feeding behavior: proof of concept spray dried plasma for pigs weaned at different ages the spray-drying process is sufficient to inactivate infectious porcine epidemic diarrhea virus in plasma evaluation of animal protein supplements in diets of early-weaned pigs environmental factors influencing human viral pathogens and their potential indicator organisms in the blue mussel propagation of the virus of porcine epidemic diarrhea in cell culture quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (pedv) inactivation of foot-and-mouth disease virus by citric acid and sodium carbonate with deicers spray drying with a two-fluid nozzle to produce fine particles: atomization, scale-up, and modelling survival of severe acute respiratory syndrome coronavirus evaluation of excipients for enhanced thermal stabilization of a human type 5 adenoviral vector through spray drying tracing enteric viruses in the european berry fruit supply chain porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies porcine epidemic diarrhea virus rna present in commercial spray-dried porcine plasma is not infectious to naive pigs investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in canada a new coronavirus-like particle associated with diarrhea in swine reduced use of antibiotic growth promoters in diets fed to weaning pigs: dietary tools, part 1 neutralizing antibodies against porcine circovirus type 2 in liquid pooled plasma contribute to the biosafety of commercially manufactured spray-dried porcine plasma survivability of porcine epidemic diarrhea virus (pedv) in bovine plasma submitted to spray drying processing and held at different time by temperature storage conditions lack of transmission of porcine circovirus type 2 to weanling pigs by feeding them spray-dried porcine plasma sensitivity of porcine epidemic diarrhea virus (pedv) to ph and heat treatment in the presence or absence of porcine plasma evaluation of the minimum infectious dose of porcine epidemic diarrhea virus in virus-inoculated feed commercially produced spray-dried porcine plasma contains increased concentrations of porcine circovirus type 2 dna but does not transmit porcine circovirus type 2 when fed to naive pigs stability of ribonuclease a in solution and the freeze-dried state isolation and characterisation of porcine circovirus type 2 from pigs showing signs of postweaning multisystemic wasting syndrome in the netherlands the presence of coinfections in pigs with clinical signs of pmws in the netherlands: a case-control study detection of porcine enteroviruses by nrt-pcr: differentiation of cpe groups i-iii with specific primer sets the authors thank jan van haren en arjen wagendorp of the nizo for conducting the inactivation and spray drying experiments and jet kant of wbvr for performing the qpcr analyses. we also thank jeanet van der goot of wbvr for critical reading of the manuscript and advice, and soumya kar of wageningen livestock research for his comments regarding english grammar. this research was commissioned and funded by sonac bv. lourens heres and meindert pelser are employees of darling/sonac ingredients inc. they were responsible for designing and arranging the virus inactivation experiments and the coordination of these experiments conducted at the nizo. lourens heres and meindert pelser provided technical information for the manuscript but were not involved in execution of the experiments and evaluations of the results of the virus isolation/ titration tests and pcr analysis conducted at wbvr. additional supporting information may be found in the online version of this article: table s1 . pcr conditions used for psv1, adv, pcv2 and pedv.1943 key: cord-273705-0oyzg5tq authors: duffy, mark a; chen, qi; zhang, jianqiang; halbur, patrick g; opriessnig, tanja title: impact of dietary spray-dried bovine plasma addition on pigs infected with porcine epidemic diarrhea virus date: 2018-08-29 journal: transl anim sci doi: 10.1093/tas/txy088 sha: doc_id: 273705 cord_uid: 0oyzg5tq experimental data suggest that the addition of spray-dried plasma (sdp) to pig feed may enhance antibody responses against certain pathogens and negatively impact virus survival. the benefit of sdp on escherichia coli infection is well documented. the aim of this study was to determine the effect of bovine sdp (bovsdp) in the pig diet on acute porcine epidemic diarrhea virus (pedv) infection. a total of 16 3-wk-old conventional crossbred pigs were used and divided into three groups. treatments included 1) a negative control group fed a commercial diet and sham inoculated with commercial liquid porcine plasma (n = 3), 2) a positive control group fed a commercial diet and inoculated with pedv-spiked porcine plasma (pedv; n = 8), and 3) a third group of pigs fed the commercial diet with inclusion of 5% spray-dried bovine plasma and inoculated with pedv-spiked porcine plasma (bovsdp; n = 5). although clinical signs associated with pedv infection were mild in the bovsdp group, two of eight pigs in the pedv group developed moderate clinical disease and had to be euthanized. the pedv igg and iga antibody levels and prevalence rates were significantly (p < 0.05) higher in the pedv–bovsdp group compared with the pedv group at 7 d postinoculation. the average fecal pedv rna shedding time was 7.2 ± 1.0 d for the pedv–bovsdp group and 9.3 ± 1.1 d for the pedv group with an overall time to clearance of pedv shedding of 11 d for pedv–bovsdp pigs and at least 14 d for pedv pigs, which was not different (p = 0.215). the results indicate that addition of bovsdp induced an earlier anti-pedv antibody response in pigs experimentally infected with pedv thereby reducing clinical disease and the amount and duration of viral shedding during acute pedv infection. coronaviridae. pedv isolates can be divided into two genogroups: g1, which encompasses a majority of strains isolated before 2010, and g2, which contains more recently identified strains (huang et al., 2013) . pedv was first discovered in north america in april 2013 and since its emergence has caused substantial losses to the u.s. pork industry. the clinical disease associated with pedv is characterized by watery diarrhea and vomiting in all ages of pigs and high mortality rates in young pigs (pensaert and debouck, 1978) . an intact intestinal mucosa, which prevents the entrance of agents across the epithelium, is a first line of protection with great importance in weaned pigs that, due to separation from their dam and comingling with other pigs, are under high stress and exposed to numerous pathogens they have not encountered before (pitman and blumberg, 2000; boudry et al., 2004; pie et al., 2004; bailey et al., 2005; moretó and pérez-bosque, 2009 ). producers commonly add spraydried plasma (sdp) of porcine or bovine origin to weaned pig diets as it has been shown to promote growth (grinstead et al., 2000) and aids in combating common postweaning pathogens such as escherichia coli (torrallardona et al., 2003; bosi et al., 2004) . it is well recognized that the addition of sdp to pig feed enhances the immune response and decreases pathogen loads compared with pigs without plasma access through feed. for instance, plasma protein supplements modulated the mucosal immune response in organized and diffuse gut-associated lymphoid tissue, which is accompanied by a reduction of proinflammatory cytokine production (bosi et al., 2004; nofrarias et al., 2006; pérez-bosque et al., 2008) . a canadian case-control study investigating factors associated with mortality due to porcine circovirus type 2 (pcv2) found that nursery rations were more likely to contain sdp in control herds compared with clinically affected case herds (dewey et al., 2006) ; however, alternatively the observed differences may be completely unrelated to the sdp inclusion in the diets. in addition, the biological neutralization activity of antibodies against common pig pathogens present in sdp was conserved and may contribute to the biosafety of commercially available sdp (polo et al., 2013) . previously, it has also been shown that sdp has an intrinsic effect reducing survival of pedv under in vitro conditions (quist-rybachuk et al., 2015) . after the introduction of pedv into the united states, concerns on possible contribution of porcine-based sdp in rapid farm-to-farm transmission were raised (pasick et al., 2014) despite studies clearly demonstrating that pedv is inactivated during the spray-drying process (opriessnig et al., 2014; pujols and segales, 2014; gerber et al., 2014b) . to eliminate any possible risk of pig pathogen spread via sdp while retaining its benefits as part of a pig diet, pork producers may switch from porcine-origin sdp toward bovine-origin sdp (bovsdp). the purpose of this study was to determine whether there is any benefit of adding bovsdp to a diet of pigs during acute pedv infection. the experiment was approved by the iowa state university institutional animal care and use committee (iacuc approval number 2-14-7742-s). sixteen, 2-wk-old, crossbred, colostrum-fed pigs were selected from a commercial pedv-free herd. the farm of origin was tested on a monthly basis for pedv rna on representative fecal samples and for pedv antibodies on selected serum samples and had no history of clinical diarrhea. the farm of origin was also free of porcine reproductive and respiratory syndrome virus (prrsv), influenza a virus (iav), and mycoplasma hyopneumoniae. before shipment to the research facility, the pigs received a one dose commercial pcv2 vaccine (merck animal health, inc.). the pigs were transported to the research facility at iowa state university, randomly assigned to one of three groups of three to eight pigs, and housed in separate rooms on solid concrete floors (table 1) . upon arrival of the pigs in the research facility, they were tested negative for pedv antibodies in serum and pedv rna in fecal samples. the experimental design is summarized in figure 1 . briefly, the pigs were inoculated with pedv at 3 wk of age (day postinoculation or dpi 0), and all pigs were necropsied at dpi 14. starting with arrival in the research facility and for the duration of the study, all pigs were fed the same standard commercial corn-soybean meal-dried whey-based diet ( table 2 ) except for the diet of the pedv-bovsdp group, which was supplemented with 5% spray-dried commercial bovine plasma replacing soy protein concentrate on an equal total lysine basis ( figure 1 ). the commercial spray-dried bovine plasma (lot #s510011001) was produced in a manufacturing plant located in kansas, united states, and submitted to commercial spray-drying conditions including a minimum outlet temperature of 80 °c throughout substance (throughout the entire particle's mass). the control diet did not contain any bovsdp. for the pedv inoculation, the passage 7 pedv g2b isolate 13-19338e (chen et al., 2014) at viral concentration of 10 3.0 50% tissue culture infectious dose per milliliter was used. in brief, pedv was propagated on vero cells (atcc ccl-81) with minimal essential medium supplemented with tryptose phosphate broth (0.3%), yeast extract (0.02%), trypsin 250 (5 µg/ml), gentamicin (0.05 mg/ml), penicillin (100 unit/ml), streptomycin (100 µg/ ml), and amphotericin (0.25 µg/ml) as previously described (chen et al., 2014) . commercial liquid porcine plasma negative for pedv rna was used as diluent to adjust the pedv inoculum (pedv and pedv-bovsdp groups) or saline (negative control [neg] group) to a total volume of 10 ml for each pig. inoculation was performed orally by the same team for all pigs. the pigs were held in vertical recumbence between the legs of the holder while the second person administered the material by slowly dripping the inoculum into the mouth of the pig using a syringe on dpi 0 when the pigs were 3 wk old. after pedv inoculation, all pigs were monitored daily for signs of illness such as lethargy, vomiting, or diarrhea. rectal temperatures were taken every other day on each pig, and the fecal consistency score ranging from 0 = normal, 1 = semisolid, 2 = pasty, and 3 = liquid was determined (gerber et al., 2016) . all pigs were weighed at dpi −6 and again at dpi 14, and the adg was calculated. to verify the presence of pedv iga and igg antibodies, blood samples were collected in serum separator tubes on dpi 0, 7, and 14 and centrifuged at 4,000 rpm for 10 min at 4 °c. all serum samples were tested for the presence of anti-pedv igg or iga by an "in-house" indirect elisa based on the spike 1 gene of a prototype u.s. pedv strain similar to the one used as challenge virus in this study (gerber et al., 2014a; gerber and opriessnig, 2015) . a sample with a sample-to-positive (s/p) ratio equal to or greater than 0.2 was considered positive for pedv igg, and a sample with an s/p ratio equal to or greater than 0.13 was considered positive for pedv iga. to determine fecal pedv shedding, rectal swabs were collected from all pigs on dpi 6 and from dpi 1 to 14 using polyester swabs and stored in 5-ml plastic tubes containing 1-ml sterile saline. total nucleic acids were extracted from fecal swab suspensions using the magmax pathogen rna/dna kit and an automated nucleic acid extraction system (thermo scientific kingfisher flex, thermo fisher scientific, pittsburgh, pa) according to the instructions of the manufacturer figure 1 . experimental design. blood was collected at arrival and dpi 0, 7, and 14, and rectal swabs were collected 6 d before challenge, on dpi 1 and daily thereafter. rectal temperatures were taken every other day starting with 1 d before pedv inoculation. translate basic science to industry innovation (opriessnig et al., 2014) . presence of pedv rna was determined by using a quantitative real time rt pcr that was set up using path-id multiplex one-step rt-pcr kit (thermo fisher scientific), and amplifications were performed on the applied biosystems 7500 fast real-time pcr system thermocycler and the accompanying software (opriessnig et al., 2014) . on dpi 14, all pigs were killed by intravenous pentobarbital overdose (fatal-plus, vortech pharmaceuticals ltd, dearborn, mi) and necropsied. tissues collected from pigs at necropsy included eight sections of small intestines, three sections of large intestine, and one section of mesenteric lymph node. tissues were immediately put in 10% buffered formalin and routinely processed for histopathology and assessed for lesions by a veterinary pathologist blinded to the treatment status. presence and degree of atrophic enteritis were scored ranging from 0 = normal to 3 = severe. to determine presence and amount of pedv antigen in tissue sections, a pedv immunohistochemical stain was performed on intestines from all pigs as described (stevenson et al., 2013) . the amount of pedv antigen was scored by a veterinary pathologist blinded to the treatment status with 0 = no signal, 1 = 1% to 10% staining, 2 = 11% to 50% staining, and 3 = greater than 50% staining. the statistical software used was jmp pro 11. analysis of variance (anova) was used for cross-sectional assessment of the average daily weight gain and continuous measures including viral shedding, iga, and igg antibody levels. the pedv genomic copy numbers per milliliter of fecal suspension were log transformed before statistical analysis. if a significant (p < 0.05) difference was detected, pairwise testing using the tukey-kramer adjustment was performed to determine which groups were different. daily rectal temperature data were analyzed with multivariate anova. non-repeated measures of necropsy and histopathology data were assessed using nonparametric kruskal-wallis anova. if a nonparametric anova test was significant (p < 0.05), then wilcoxon tests were used to assess the differences between pairs of groups. differences in incidence of clinical scores were evaluated by using the fisher's exact test. for fecal shedding of pedv rna, an area under the curve (auc) was calculated for each animal individually and differences between groups were assessed by unpaired t-test (data passed shapiro-wilks normality test). there was no difference in rectal temperatures among the groups over time, and none of the pigs developed elevated rectal temperatures during the experiment. diarrhea was not observed in the neg group while individual pigs in the pedv group and pedv-bovsdp group had fluid-to-pasty-to-semisolid feces between dpi 2 and 6 with no significant differences among groups. in addition, vomiting was occasionally observed in single pigs during this time. by dpi 9, two pedv pigs became lethargic and had reduced appetite. one of these pigs developed mild pasty diarrhea. due to welfare concerns, these pigs were euthanized between dpi 11 and 13. the adg is summarized in table 1 . overall, there were numerical differences among groups for the adg: neg pigs had the highest adg, and pedv pigs had the lowest adg; however, the differences were not significant. all pigs were negative for pedv iga and igg antibodies at arrival, and the neg pigs remained seronegative for the duration of the study (figures 2 and 3) . three of five pedv-bovsdp pigs had detectable anti-pedv iga antibodies in serum by dpi 7, and all pigs in this group and five per eight pedv pigs were positive by dpi 14. group mean iga levels were higher (p < 0.011) for pedv-bovsdp pigs compared with pedv pigs at dpi 7 ( figure 2 ). group mean igg levels were also higher (p < 0.021) in pedv-bovsdp pigs at dpi 7 compared with the pedv pigs ( figure 3 ). all pedv-infected pigs in both groups had anti-igg antibodies by dpi 14 (figure 3 ). all pigs were pedv rna negative at dpi −6, and pedv rna was not detected in any of the neg pigs throughout the study. fecal shedding of pedv rna was first detected in a pedv pig at dpi 1 and shedding in this group lasted until termination of the study at dpi 14 (figures 4 and 5) . pigs in the pedv-bovsdp group shed pedv from dpi 2 to 11 (figures 4 and 5) . the prevalence of pcr positive pedv-bovsdp pigs compared with the pedv group was higher (p = 0.021) at dpi 2, while it was lower (p = 0.028) at dpi 12. the average shedding period was 9.3 ± 1.1 for pedv pigs and 7.2 ± 1.0 for pedv-bovsdp pigs, which was not different (p = 0.215). similarly, the cumulative fecal viral rna shedding was not different (p = 0.384) between pedv pigs and pedv-bovsdp pigs. in the two pigs that were euthanized due to welfare reasons, in addition to the standard set of enteric tissues, sections of liver, lung, spleen, tonsil, heart, and kidney were also collected and assessed to rule out any concurrent systemic infection. one of these two pigs had moderate diffuse atrophic enteritis associated with pedv antigen as determined by immunohistochemical stains (score 2). this pig also had the highest amount of pedv rna measured (log 10 9.5 genomic copies per fecal swab at dpi 6 and figure 2 . mean group anti-pedv iga elisa s/p ratios ± sem at days 0, 7, and 14 after pedv inoculation (dpi) in the different treatment groups and number of positive pigs/total number of pigs in the group for each day. the results of the two pigs that were killed on dpi 11 and 13 were included at dpi 14. an s/p ratio greater than 0.13 was considered positive. different superscripts ( a,b ) indicate significantly different means at a given day. statistical analysis was performed by one-way anova followed by pairwise testing using the tukey-kramer adjustment if p < 0.05. the statistical software used was jmp pro 11. the number of elisa positive pigs per total number of pigs per group is listed next to each group mean. log 10 8.9 at dpi 11) when it was killed. the other pig had its peak pedv shedding at dpi 6 (log 10 6.3 pedv genomic copies), but its shedding decreased to log 10 5.6 genomic copies by dpi 13. no other lesions were seen in these two pigs. the remaining pigs were necropsied at the scheduled time at dpi 14, and lesions or pedv antigen were not seen in any of these pigs suggesting that the pedv infection and associated lesions had resolved. pedv has become a major economic concern for north american pig producers since it was first . mean group log 10 amount of pedv rna fecal samples in pedv and pedv-bovsdp pigs at different days after pedv infection and number of positive pigs per total number of pigs in the group for each day. an "a" on a given day indicates significant (p < 0.05) different group means. statistical analysis was performed by one-way anova followed by pairwise testing using the tukey-kramer adjustment if p < 0.05. the statistical software used was jmp pro 11. 443. mean group anti-pedv igg elisa s/p ratios ± sem at days 0, 7, and 14 after pedv inoculation in the different treatment groups and number of positive pigs per total number of pigs in the group for each day. the results of the two pigs that were killed on dpi 11 and 13 were included at dpi 14. an s/p ratio greater than 0.2 was considered positive. different superscripts ( a,b ) indicate significantly different means at a given day. the number of elisa positive pigs per total number of pigs per group is listed next to each group mean. identified in the united states in 2013 (stevenson et al., 2013; chen et al., 2014) . the lack of highly effective vaccines and the relative ineffectiveness of common treatment methods have led to a search for alternative methods of treating and preventing outbreaks. the inclusion of sdp into the diets of young pigs has been shown to have health benefits for other diseases. this study was designed to determine whether there is any benefit to adding bovsdp to the diet during pedv infection. supplementing feed rations of pigs, fish, poultry, cats, and dogs with sdp as protein source is performed on a regular basis. sdp is a protein-rich product obtained from blood from healthy animals (cattle or pigs) at slaughter (torrallardona, 2010; pérez-bosque et al., 2016) . in pet food, sdp is a preferred binder in canned food products due to its high-protein content and its physicochemical properties (polo et al., 2005; rodriguez et al., 2016) . porcine sdp was first introduced as protein source for pigs during the early 1990s (cole and sprent, 2001 ) and since has been used widely in the diet of weaned pigs (torrallardona, 2010) . benefits of adding porcine sdp include improvement of weight gain mainly due to increased feed intake and reduction of incidence and severity of diarrhea after weaning (adewole et al., 2016) . comprehensive information on the effect of sdp obtained in 75 trials involving over 12,000 pigs has been summarized (torrallardona, 2010) . in this study, the pedv challenge was performed after an acclimation period of 1 wk to minimize stress from weaning and transport to the new facility. successful pedv challenge was confirmed by detecting pedv rna in fecal swabs. all pigs (100%) from the bovsdp-pedv group shed pedv in fecal samples from dpi 2 to 6, whereas between 25% and approximately 87% of the animals in pedv group shed the virus during that time. these results highlight that a higher number of animals excreted pedv during the early stages of infection in bovsdp-pedv group. differences in the challenge dose or challenge process can be ruled out. the pedv inoculum was prepared in a similar manner and at the same time for both groups by a single person and stored on ice until challenge of each group. the challenge was conducted by the same personnel for both groups with approximately 20 min between the two groups. the obtained results could have been by chance due to the group sizes. alternatively, the bovsdp-pedv animals could be more prone to excrete pedv in the early stages in the infection. unlike in suckling pigs that are very susceptible to pedv infection, only 2 per 13 pedv-infected pigs (both from the pedv challenged groups) had to be killed due to the severity of clinical signs. this is expected and comparable with other trials infecting 3-wk-old pigs (crawford et al., 2015) . in the current study, the pedv igg and iga antibody responses were more rapid in the bovsdp group compared with the pedv group. specifically, by dpi 7, 60% of the bovsdp pigs were anti-igg and figure 5 . mean group log 10 amount of pedv rna in pcr positive pigs in fecal samples at different days after pedv infection. an "a" at a given day indicates significant (p < 0.05) different group means. statistical analysis was performed by one-way anova followed by pairwise testing using the tukey-kramer adjustment if p < 0.05. the statistical software used was jmp pro 11. iga positive compared with 12.5% of the pedv pigs. of note, while systemic iga antibodies were measured, mucosal iga levels were not determined, and it is therefore unknown how the addition of the bovsdp affected the gut immunity. in a previous study, a good correlation of iga levels in serum and feces was found , and as fecal samples and gut mucosa are more difficult to process during routine lab work, serum iga levels are commonly tested. explanations for the earlier humoral immune response in the bovsdp group may include acceleration of the clinical course by the dietary supplement; however, a more rapid immune response due to earlier replication of the virus in more pigs unrelated to the diet modification is also possible. serum anti-pedv ig in pig serum has been demonstrated to neutralize infectivity of pedv (hofmann and wyler, 1989) , and bovine plasma could have a similar neutralizing activity, which was not further assessed in this study. besides the presence of possible neutralizing antibodies in sdp, other plasma compounds such as peptides (anderson and anderson, 2002) could contribute to the benefits seen with sdp addition to a diet. virus shedding in the bovsdp group was 2.1 d shorter than in the pedv group. the fecal pedv rna shedding in this group was terminated by dpi 11. a previous study has shown that pedvinfected pigs shed infectious pedv capable of horizontal transmission for 14 to 16 d after infection (crawford et al., 2015) . this is similar to what was observed in the pedv-infected pigs without bovsdp in the diet in this study. the data from this trial indicate a beneficial effect of bovsdp on acute pedv infection, which is similar to previous reports using the prrsv infection model (pujols et al., 2011) . specifically, pigs fed bovsdp were able to clear virus shedding 2.1 d sooner than non-bovsdp pigs. the addition of bovsdp to the diet resulted in faster and stronger pedv antibody responses and reduced pedv shedding time compared with pigs with no bovsdp in the diet. limitations of this study include the usage of one single virus titer and the low numbers of pigs tested. in addition, two pedv pigs had to be removed early from the study, which could have impacted the outcomes. a larger study with higher numbers of pigs per group comparing porcine and bovine-derived sdp with multiple necropsy days should be conducted to further confirm the possible benefits of sdp in pedv-infected pigs. gut health of pigs: challenge models and response criteria with a critical analysis of the effectiveness of selected feed additives-a review the human plasma proteome: history, character, and diagnostic prospects the development of the mucosal immune system pre-and post-weaning: balancing regulatory and effector function spray-dried plasma improves growth performance and reduces inflammatory status of weaned pigs challenged with enterotoxigenic escherichia coli k88 weaning induces both 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enterotoxin spray dried plasma as an alternative to antibiotics in piglet feeds, mode of action and biosafety weaning is associated with an upregulation of expression of inflammatory cytokines in the intestine of piglets first line of defense: the role of the intestinal epithelium as an active component of the mucosal immune system neutralizing antibodies against porcine circovirus type 2 in liquid pooled plasma contribute to the biosafety of commercially manufactured spray-dried porcine plasma functional properties of spray-dried animal plasma in canned petfood commercial spray-dried porcine plasma does not transmit porcine circovirus type 2 in weaned pigs challenged with porcine reproductive and respiratory syndrome virus survivability of porcine epidemic diarrhea virus (pedv) in bovine plasma submitted to spray drying processing and held at different time by temperature storage conditions sensitivity of porcine epidemic diarrhea virus (pedv) to ph and heat treatment in the presence or absence of porcine plasma effect of spray-dried plasma on food intake and apparent nutrient digestibility by cats when added to a wet pet food recipe emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences spray dried animal plasma as an alternative to antibiotics in weanling pigs-a review. asian-aust effect of fishmeal replacement with spray-dried animal plasma and colistin on intestinal structure, intestinal microbiology, and performance of weanling pigs challenged with escherichia coli k99 this study was funded by the apc, inc. additional support was provided by the biotechnology and biological sciences research council (bbsrc) institute strategic programme grant awarded to the roslin institute (bb/j004324/1; bbs/e/d/20241864). we thank dr. huigang shen for assistance with sample testing and dr. priscilla gerber for assistance with the statistical analysis. conflict of interest statement. none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. key: cord-279784-o80x8nj7 authors: wu, yu; li, wei; zhou, qingfeng; li, qunhui; xu, zhichao; shen, hanqin; chen, feng title: characterization and pathogenicity of vero cell-attenuated porcine epidemic diarrhea virus ct strain date: 2019-10-28 journal: virol j doi: 10.1186/s12985-019-1232-7 sha: doc_id: 279784 cord_uid: o80x8nj7 background: porcine epidemic diarrhea virus (pedv) has caused enormous economic losses to the global pig industry. currently available pedv vaccine strains have limited protective effects against pedv variant strains. methods: in this study, the highly virulent epidemic virus strain ct was serially passaged in vero cells for up to 120 generations (p120). characterization of the different passages revealed that compared with p10 and p64, p120 had a higher viral titer and more obvious cytopathic effects, thereby demonstrating better cell adaptability. results: pathogenicity experiments using p120 in piglets revealed significant reductions in clinical symptoms, histopathological lesions, and intestinal pedv antigen distribution; the piglet survival rate in the p120 group was 100%. furthermore, whole-genome sequencing identified 13 amino acid changes in p120, which might be responsible for the attenuated virulence of p120. conclusions: thus, an attenuated strain was obtained via cell passaging and that this strain could be used in preparing attenuated vaccines. porcine epidemic diarrhea (ped) is an acute and highly infectious intestinal disease in pigs; it is caused by porcine epidemic diarrhea virus (pedv), which has caused enormous economic losses to the global pig industry [1, 2] . ped is characterized by diarrhea, vomiting, dehydration, anorexia, weight loss, and high mortality in suckling pigs. although ped also appears in summer, it mainly occurs in winter. pedv infection causes different symptoms according to the pigs' ages; however, in piglets, the symptoms of pedv infection are particularly serious, including a high mortality rate [3] . pedv belongs to the order nidovirales, family coronaviridae, and genus alphacoronavirus and is an enveloped virus with a single-stranded, positive-sense rna genome [2] . the whole pedv genome is approximately 28 kb nucleotides (nts) long and has a 5′-cap and 3′-polyadenosyl tail; the genome also includes 5′-and 3′-untranslated regions and at least 7 open reading frames (orf1a, orf1b, and orf2-6) [4, 5] . orf1a and orf1b encode the replicase polyproteins 1a and 1ab, respectively, which undergo autoproteolysis by viral proteases to form 16 nonstructural proteins (nspl-16) [6] , which participate in the basic mechanisms of viral rna transcription and replication. orf2-6 encode four structural proteins [fibrin (s), membrane protein (m), envelope protein (e), and nucleocapsid protein (n)] as well as coprotein orf3 [7, 8] ; these proteins are arranged in the genome in the following order: 5′-orf (la/lb)-s-orf3-e-m-n-3′ [9] . in 1978, the pedv strain cv777 was identified as the cause of the ped outbreak in belgium [10] . in october 2010, a highly pathogenic pedv was discovered in china, which caused the worst outbreak on record and quickly swept across the country [11, 12] . the variant then caused a pandemic in the united states in spring 2013 and spread to canada and mexico. in addition, severe ped outbreaks occurred almost simultaneously in many asian and european countries, such as korea, japan, belgium, and france [13, 14] . vaccination is considered effective in the prevention of pedv infection on farms [15] . several attenuated activated and inactivated vaccines for classical pedv strains, such as cv777 [4] , dr13 [16] , and kpedv-9 [17] , have been developed and made commercially available in many countries [18] ; however, the efficacy of these traditional vaccines against emerging pedv strains is questionable because of the antigenic and genetic differences between the vaccine strains and the prevalent strains [13] . therefore, there is an urgent need for a new pedv vaccine against new variant strains. in the present study, the ct strain was serially passaged in vero cells. the growth kinetics and biological characteristics of the different passages were determined. in addition, 6-day-old piglets were used to assess the pathogenicity of these strains. finally, the whole-genome sequences of the different passages were determined. a safe attenuated pedv strain was developed in this study, thereby providing an important basis for the preparation of an attenuated vaccine. the pedv ct strain, which belongs to the g2b subgroup in china, was previously isolated by and stored at our laboratory [19] . vero cells were obtained from the american type culture collection (atcc: ccl-81), regularly cultured in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (invitrogen, australia) and 1% antibiotics (100 u/ml penicillin, 100 μg/ml streptomycin, and 25 μg/ml fungizone®; gibco™, usa), and maintained at 37°c in a humidified 5% co 2 incubator. mouse anti pedv s monoclonal antibody and y3-labeled goat anti-mouse igg antibody were prepared and stored at our laboratory. vero cells were grown in a t25 flask and washed thrice with phosphate-buffered saline (pbs) at 90% confluency. the cells were then incubated with 1 ml of the pedv ct strain diluted 1:3000 in virus growth medium {dmem supplemented with antibiotics (100 u/ml penicillin, 100 μg/ml streptomycin, and 7.5 μg/ml trypsin [gibco])} for 1 h at 37°c in a humidified 5% co 2 incubator. then, 2 ml of the virus growth medium was added to the t25 flask, which was monitored daily for cytopathic effects (cpes). when cpes were observed in > 90% of the vero cells, the flask was subjected to three cycles of freezethawing. the cells and supernatants were mixed by pipetting, aliquoted, and stored at − 80°c. these harvested cells were then used as seed stock for the next passage. finally, the cells were further passaged under each condition for up to 120 passages [5, 20] . immunofluorescence assay (ifa) the vero cells were inoculated with pedv in 6-well plates at a multiplicity of infection (moi) of 0.01. at 12 h post infection (hpi), the cells were washed thrice with pbs, fixed at room temperature (rt) with 4% paraformaldehyde for 15 min, and then permeabilized with 0.2% triton x-100 (solarbio, china) for 15 min. after washing as described previously, the cells were blocked with 1% bovine serum albumin (solarbio) for 30 min at rt. the mouse anti pedv s monoclonal antibody and y3-labeled goat anti-mouse igg antibody were then used as the primary and secondary antibodies, respectively. finally, the cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (vectorlabs, usa) for 5 min at rt in the dark. the cells were then washed thrice with pbs and observed under a fluorescence microscope. vero cell monolayers were separately inoculated with pedv p10, p64, or p120 or were mock infected at an moi of 0.01 in 6-well plates. the culture supernatants and cell lysates were collected at 6, 12, 18, 24, and 30 hpi. after one round of freeze-thawing, following the reed-muench method, the cell culture samples were analyzed in 96-well plates to determine the titration for 50% tissue culture infectious dose (tcid 50 /ml) [21] . total rna was extracted from the cell culture samples using an rneasy kit (magen, china). all primers used to amplify pedv genomic fragments were designed and preserved at our laboratory [22] . reverse transcription and polymerase chain reaction (pcr) were performed using the primescript™ rt-pcr kit (promega, usa) and primestar® gxl dna polymerase, respectively. the 5′-and 3′-end sequences were determined using a 5′and 3′-competition kit (takara). the pcr products were cloned into a pmd19-t vector using the topo® ta cloning® kit (invitrogen, usa) and sent to sangon biotech (shanghai, china) for sequencing. for each amplicon, more than three independent clones were sequenced to determine the accurate sequence of a specific genomic region. all sequencing results were then spliced and aligned using lasergene seqman and lasergene megalign, respectively. all reference sequences were obtained from genbank and used for sequence alignment and phylogenetic analysis. all phylogenetic trees were constructed using mega7.0 software. twenty-two 6-day-old crossbred (duroc × landrace × big white) conventional piglets were obtained from wen's foodstuffs group co., ltd. (guangdong, china). the piglets were determined to be free of antibodies against pedv and confirmed to be negative for the major porcine enteric viruses pdcov, pedv, tgev, and prov [23] via virus-specific rt-pcr of fecal swab samples [23, 24] . the piglets were then placed in separate cages and randomly divided into four experimental groups. each group was placed in a separate room (biosafety level 2). the piglets in each group were orally inoculated with 1 ml of the virus culture medium (1 × 10 6 tcid 50 ) or dmem. the piglets were monitored daily for clinical signs of vomiting, diarrhea, and mortality throughout the experiment [25] . fecal consistency was scored as follows: 0, solid; 1, paste; 2, semi-liquid; 3, liquid; and 4, death [26] . one piglet from each group was euthanized for histopathological examination at 3 days post infection (dpi). the remaining piglets were euthanized at the end of the study (10 dpi). fresh fecal swabs were collected daily, diluted with pbs, stored at − 80°c, and subjected to three freeze-thaw cycles. viral rna was extracted from the supernatant of the fecal swab samples as described previously. the amount of viral rna in the fecal swab samples was determined using taqman real-time rt-qpcr with the following primers: sense, 5′-gaattcccaagggc gaaaat-3′; antisense, 5′-ttttcgacaaattc cgcatct-3′. a probe targeting the pedv n gene (5′-fam-cgtagcaggcttgcttcggaccca-bhq-3′) was also employed. the thermal cycling parameters were as follows: 95°c for 20 s followed by 40 cycles at 95°c for 3 s and 60°c for 30 s. the gross intestinal tract of each piglet was photographed at necropsy, and the intestinal tissue was collected and fixed with 4% paraformaldehyde. the fixed samples were sent to guangzhou saville for histopathology or ihc with pedc s-specific monoclonal antibodies. in each experimental group, statistical significance was measured using one-way analysis of variance. two-sided probability values < 0.05 (p < 0.05) were considered to indicate statistical significance. correlations between the fc scores and fecal pedv rna drop titers were analyzed using spearman's rank correlation [26] . the animal study protocol was approved by the south china agricultural university committee of animal experiments (approval id: syxk-2014-0136). all experiments were performed in accordance with the recommendations of the guide for the care and use of laboratory animals of the national institutes of health. biological characteristics of pedv p10, p64, and p120 our results indicated that syncytial, vacuole, and cell exfoliation were more obvious after infection with p120 than with p10 and p64 (fig. 1a) . the three different passage strains were confirmed by ifa using a specific monoclonal antibody against the pedv s protein. red signals were observed in the strain-infected vero cells for all the three passage strains but not in the uninfected vero cells at 12 hpi. obviously, the cells infected with p120 produced more red pedv antigens (fig. 1b) . in addition, growth kinetics indicated that p120 had a higher viral titer than the other two strains at all time points, indicating that p120 had better cell adaptability (fig. 1c) . fecal swabs were collected from the piglets and scored for diarrhea during the experiment; the clinical signs of the piglets in each group were also recorded. at 1 dpi, severe diarrhea and vomiting were observed in the p10 group. in contrast, slight diarrhea and vomiting were observed in the p64 group. interestingly, there were no obvious clinical symptoms in the p120 and mock groups (fig. 2a) . regarding the lesion onset times in the groups, lesions appeared at 16, 24, and 48 hpi in the p10, p64, and p120 groups, respectively. throughout the experiment, a high diarrhea score was maintained in the p10 group. in contrast, in the p64 and p120 groups, the diarrhea scores began to rise at 4 dpi, peaked at 5 dpi, and gradually returned to normal by 7 dpi (fig. 2b) . differences in pedv rna shedding in the four experimental groups pedv rna shedding was detected in fecal swab samples using rt-qpcr (fig. 3) . during the entire experiment, the p10 group maintained a high level of pedv rna shedding. in the p64 group, pedv rna shedding appeared at 1 dpi, peaked at 4 dpi, and gradually recovered by 7 dpi. pedv rna shedding was not observed in the p120 group until the second day after challenge; the viral shedding peaked at 5 dpi and gradually improved after 7 dpi. the entire experiment lasted for 10 days. the health of the piglets in the p120 and p64 groups had recovered by 10 dpi. at 3 dpi, one piglet was selected from each group for dissection and sampling. at necropsy, intestinal tissue, particularly the small intestinal tissue, of the the p64 group piglet exhibited dilatation and transparency (fig. 4a-d) . in the p10 group piglet, the intestinal tissue lesions were more obvious and the intestine was filled with yellow fluid and mesenteric hyperemia (fig. 4d) . in contrast, the p120 group piglet exhibited only slight changes in the intestinal tract, and the control animal was extremely healthy with no pathological changes (fig. 4b) . the jejunum and ileum of each piglet were examined via histopathological and ihc analyses. histopathological analysis revealed severe atrophy and shedding of the intestinal villi of the p10 group piglet (fig. 4h and l) ; in contrast, in the p64 group piglet, the intestinal villi were shortened and fused ( fig. 4f and j) . intestinal villus atrophy was mild in the p120 group piglet, and the examined control group piglet exhibited normal histopathology ( fig. 4e and i) . in contrast, ihc revealed that the pedv antigen was dominant in the cytoplasm of some segments of small intestinal villi. in the p10 and p64 group piglets, large amounts of the pedv antigen were detected in severe lesions; in contrast, the antigen was hardly detected in the p120 group piglet (fig. 4n and r) ; further, no pedv antigen was found in the small intestine of the control group piglet (fig. 4m and q) . in the p10 group, one piglet died at 3 dpi and two others died at 4 dpi. in addition, one piglet each died at 9 and 10 dpi, respectively; thus, the survival rate in the p10 group was 0%. in the p64 group, the survival rate was 83.3% because only one piglet died at 9 dpi. no animals died in the p120 and control groups (100% survival rate for both groups) (fig. 5 ). the genome length of the pedv ct strain was determined to be 28,038 nts. previous studies conducted at fig. 1 biological characteristics of porcine epidemic diarrhea virus strains after 10, 64, or 120 passages. a cytopathic effects of the three strains 12 hpi at an moi of 0.01. b immunofluorescence detection results for the p10, p64, and p120 strains in vero cells infected at an moi of 0.01 at 12 hpi. c growth kinetics of the p10, p64, and p120 strains in vero cells at an moi of 0.01. cell lysates were sampled at the designated time points and titrated using the titration for 50% tissue culture infectious dose infectivity assay. the asterisk means significant difference (***p < 0.001, **p < 0.01, and *p < 0.05). p10, pedv after 10 passages; p64, pedv after 64 passages; and p120, pedv after 120 passages; moi, multiplicity of infection; hpi, hours post infection our laboratory have reported that the pedv ct strain belongs to clade 6 of the g2b subgroup (fig. 6) . table 1 lists the changes in the amino acids and nts of the different passage strains. p120 was observed to contain 14 base mutations, resulting in 13 amino acid mutations. the s gene is a determining feature of pedv's virulence and evolution [27] . amino acid comparison indicated that there were only four amino acid mutations (d265a, f635r, s887r and c1362g) between p10 and p120. in addition, several nucleotide point mutations resulted in aa substitutions in orf1ab (a1538s, t1945 n, d2813g, h2925y, y3302s and v4566i), orf3 (t45 m), e (p70l) and m (g159d) proteins. we found that p64 had 10 base mutations (thus, 9 amino acid mutations). moreover, compared with p64, p120 contained 4 base mutations (thus, 4 amino acid mutations) ( table 1 ). in addition, p120 had high homology with the cv777 vaccine strain (96.9%) and the attenuated dr13 strain (97.9%). the complete genome sequences of the ct strains described here have been deposited in genbank under accession no.mn114121. ped outbreaks have caused huge economic losses to the pig industry in not only china but also the united states, japan, south korea, and other countries [28, 29] . currently, vaccination is an effective solution to tackling the virus; however, owing to variations in the virus [18] , the classical attenuated vaccine does not provide effective protection. thus, the epidemic strains of the virus must be urgently studied to prepare a new attenuated vaccine. in the classical method, attenuated vaccines are prepared via cell passaging [16, 17, 29, 30] . some previous studies have reported that increased viral titers and enhanced cell adaptability are the characteristics of viruses with weakened virulence [4, 13, 26] . in the present study, we passaged the epidemic pedv ct strain continuously in vero cells for up to 120 generations and observed that p120 had better cell adaptability than the other two strains; thus, our findings illustrated that increased passaging increases the cell adaptability and titers. finding balance between cell and animal adaptability and then determining the best generation is the key to the preparation of attenuated vaccine candidate strains via continuous passaging [5, 25, 29] . moreover, the adaptability of the strain to the host animal is characterized by the virulence of the strain, which is usually evaluated via pathogenicity testing of the animal infected with the virus. to prove that . e-h h&e-stained jejunum tissue sections of the different groups at 3 dpi. i-l h&e-stained ileum tissue sections of the different groups at 3 dpi. m-p immunohistochemically stained jejunum tissue sections of the different groups at 3 dpi pedv antigen is indicated by arrows). q-t immunohistochemically stained ileum tissue sections of the different groups at 3 dpi (pedv antigen is indicated by arrows). p10, pedv after 10 passages; p64, pedv after 64 passages; and p120, pedv after 120 passages; h&e, hematoxylin and eosin; pedv, porcine epidemic diarrhea virus fig. 5 survival rates of piglets infected with different passages of porcine epidemic diarrhea virus ct strain. **p < 0.01, and *p < 0.05 the strain virulence has been weakened, animal experiments were designed to confirm the strain [6, 8, 26] . thus, the virulence of the three pedv strains was assessed to confirm whether the p120 strain was weakened. according to data from animal experiments, the p120 strain could significantly reduce clinical symptoms, viral shedding, and histopathological changes in piglets. therefore, our results illustrated that the p120 strain had relatively weak virulence and that compared with the p10 and p64 strains, it caused lesser damage to the piglets. thus, it can be concluded that the p120 strain is safe and potentially useful as a candidate strain for the preparation of an attenuated vaccine. to further assess the attenuation of the p120 strain, we sequenced the whole genomes of the three passage strains. and the method to prepare attenuated vaccine by cell passage can also get clues to the key virulence factors of the virus [29] . the pedv s protein is involved in receptor binding, viral entry, neutralizing antibody production induction, and host cell fusion [9] . the s protein of pedv has always been used as a marker of viral variation. under the pressure of herd immunity, the s gene of pedv mutates frequently, with some of the missense mutations altering viral antigenicity to aid in the virus's escape from preexisting immunity. thus, periodic vaccine updates may be required to ensure sufficient efficacy against emerging virus variants [31] . in this study, our result revealed that there is little variation in the s gene among these pedv strains. moreover, s protein is implicated in virulence, which was found out in the other studies that s protein can be variant readily while receiving immune pressure [32] . mutations, including deletions and/or insertions, in the s protein may change the pathogenicity and tissue tropism of coronaviruses [33] . comparing to the p10 strain, there was no nucleotides insertion and deletion in the s gene of the p120 strain. however, we found that there were four amino acid mutations (d265a, f635r, s887r and c1362g) between the p10 strain and the p120 strain. fig. 6 phylogenetic tree analysis of the whole genomes of different pedv strains. phylogenetic analysis conducted for the whole-genome nucleotide sequence of the pedv strains reported in our study and the reference pedv strain. using the mega7.0 software, the adjacency method was used to construct the tree. the numbers at the branch are the guided values (%) after 100 copies. the front ends of the three different sub-strains have been labeled. pedv, porcine epidemic diarrhea virus these mutations have not been reported previously. this finding indicates that these mutations in the s gene may be related to viral pathogenicity. the orf3 protein was reported to function as an ion channel and can prolongs s-phase, facilitates formation of vesicles and thus to regulate virus production [34] . it has been speculated that the deletion or mutation of a base in the orf3 region promotes the adaptation of viral cells and changes in viral titers [35] . in this study, the t45 m mutation was detected in orf3 of p120. furthermore, mutations of amino acids in the orf1ab, m, and e regions of p120 indicate that multiple mutation combinations in the genome cause complete decay of the pedv strain via various molecular mechanisms [5] . these mutations found in orf1ab, m, orf3 and e proteins have not been reported previously. further studies using reverse genetics are required to determine whether a particular or a combination of genetic changes (point mutations, deletions, and insertions) in pedv strains alter viral infectivity, pathogenicity, and replication efficiency [26] . in conclusion, an attenuated strain of pedv with better cell adaptability and higher titers than the virulent strain was obtained via cell passaging. the results of our animal experiments illustrated that the pathogenicity of the p120 strain was reduced and that piglets infected with that strain had a 100% survival rate. moreover, the results of whole-genome analysis revealed amino acid mutations in the p120 strain, which may contribute to the weakened virulence of the virus. thus, the attenuated strain could be suitable for use in vaccine preparation. porcine epidemic diarrhea virus: an emerging and reemerging epizootic swine virus epidemic strain yc2014 of porcine epidemic diarrhea virus could provide piglets against 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using a newly isolated strain from korea new emergence pattern with variant porcine epidemic diarrhea viruses mucosal and systemic isotypespecific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr13 strain derivation of attenuated porcine epidemic diarrhea virus (pedv) as vaccine candidate vaccines for porcine epidemic diarrhea virus and other swine coronaviruses genetic epidemiology of porcine epidemic diarrhea virus circulating in china in 2012-2017 based on spike gene trypsin-independent porcine epidemic diarrhea virus us strain with altered virus entry mechanism improvements in methods for calculating virus titer estimates from tcid50 and plaque assays isolation and characterization of a variant porcine epidemic diarrhea virus in china a highly pathogenic strain of porcine deltacoronavirus caused watery diarrhea in newborn piglets oral administration of coated pedv-loaded microspheres elicited pedv-specific immunity in weaned piglets genetic characteristics, pathogenicity, and immunogenicity associated with cell adaptation of a virulent genotype 2b porcine epidemic diarrhea virus attenuation of an original us porcine epidemic diarrhea virus strain pc22a via serial cell culture passage sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis cell attenuated porcine epidemic diarrhea virus strain zhejiang08 provides effective immune protection attributed to dendritic cell stimulation cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa comparative genomic analysis of classical and variant virulent parental/attenuated strains of porcine epidemic diarrhea virus pedv orf3 encodes an ion channel protein and regulates virus production preparation and characterization of an attenuated porcine epidemic diarrhea virus strain by serial passaging publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank wen's group academy, wen's foodstuffs group. authors' contributions fc and yw conceptualized the study. yw and wl devised the experimental methods. yw and zcx curated the data. fc, yw, hqs, qhl, and qfz provided resources. yw prepared the original manuscript draft. fc reviewed the manuscript and edited it. all authors read and approved the final manuscript. the datasets analyzed during this study are available from the corresponding author on reasonable request. the animal study protocol was approved by the south china agricultural university committee of animal experiments (approval id: syxk-2014-0136). all experiments were performed in accordance with the recommendations of the guide for the care and use of laboratory animals of the national institutes of health. the authors declare that they have no competing interests. key: cord-261036-zdhg4axx authors: shirato, kazuya; maejima, madoka; hirai, asuka; ami, yasushi; takeyama, natsumi; tsuchiya, kotaro; kusanagi, kouich; nunoya, tetsuo; taguchi, fumihiro title: enhanced cell fusion activity in porcine epidemic diarrhea virus adapted to suckling mice date: 2010-09-09 journal: arch virol doi: 10.1007/s00705-010-0790-1 sha: doc_id: 261036 cord_uid: zdhg4axx porcine epidemic diarrhea virus (pedv) is the major causative agent of fatal diarrhea in piglets. to study the pathogenic features of pedv using a mouse model, pedv with virulence in mice is required. in pursuit of this, we adapted a tissue-culture-passed pedv mk strain to suckling mouse brains. pedv obtained after ten passages through the brains (mk-p10) had increased virulence for mice, and its fusion activity in cultured cells exceeded that of the original strain. however, the replication kinetics of mk and mk-p10 did not differ from each other in the brain and in cultured cells. the spike (s) protein of mk-p10 had four amino acid substitutions relative to the original strain. one of these (an h-to-r substitution at residue 1,381) was first detected in pedv isolated after eight passages, and both this virus (mk-p8) and mk-p10 showed enhanced syncytium formation relative to the original mk strain and viruses isolated after two, four, and six passages, suggesting the possibility that the h-to-r mutation was responsible for this activity. this mutation could be also involved in the increased virulence of pedv observed for mk-p10. the group i coronavirus porcine epidemic diarrhea virus (pedv) is an enveloped rna virus with a single, positive-stranded genome about 30 kb in length [9] . pedv is the causative agent of pig diarrhea and induces a loss of appetite and weight in adult pigs, whereas it is lethal in piglets [16] . however, our understanding of the pathogenesis of diseases caused by pedv is limited due to a lack of appropriate small-animal models. coronaviruses cause various diseases in a species-specific manner, and they grow in cultured cells established from susceptible host species; human, feline, and porcine coronaviruses grow only in cells isolated from the respective species [24] . one of the major factors determining species specificity is the cellular receptor, which interacts with a given virus, but not with other viruses, when the virus initially encounters cells [5] . some of the group i coronaviruses, such as porcine transmissible gastroenteritis virus (tgev) and human coronavirus 229e (hcov-229e), utilize aminopeptidase n (apn) [1, 19, 22, 23] . however, the receptor of pedv has not yet been identified, although there was a controversial report on a pedv receptor protein [10] . expression of the receptor protein for mouse hepatitis virus (mhv) and severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) in cultured cells confers susceptibility to each of these viruses [2, 4, 25] . expression of the tgev receptor in mice from a transgene rendered otherwise resistant mice susceptible to tgev [1] . however, this approach is not available for pedv. an alternative approach is to isolate viruses that have adapted for growth within the animals and which exhibit virulence. such adaptations have been reported for two other coronaviruses, ibv and oc43 [3] . in the present study, we isolated a strain of pedv that was more virulent than the original tissue-culture-adapted virus after passage through mouse brain cells. the adapted virus showed enhanced cytopathology compared with the original virus. vero cells were obtained from the american type culture collection (atcc; manassass, va, usa). the cells were maintained in dulbecco's modified eagle's medium (dmem; sigma, st. louis, mo, usa) containing 5% fetal calf serum (fcs). the pedv strain used in this study was an mk strain isolated from the jejunum of a pig that had exhibited diarrhea in 1996 in japan, and which had been passed nine times in vero cell cultures. pedv was maintained in vero cells using a slightly modified version of a reported method [8] . briefly, vero cells were infected with the virus. after 1 h, the cells were washed with phosphate-buffered saline (pbs) and cultured in dmem containing 10% tryptose phosphate broth (tpb) and 2.5 lg/ml trypsin (sigma) for 15 h. next, the cells and supernatant were pooled, ultrasonicated, and stored at -80°c until use. the pedv infectious titer was determined by a plaque assay using vero cells. briefly, vero cell monolayers grown in 24-well plates coated with type i collagen (agc techno glass, chiba, japan) were inoculated with serially diluted virus samples. after 1 h to allow for adsorption of the virus, the inoculum was removed, the cells were washed with pbs, and dmem containing 10% tpb and 1.25 lg/ml trypsin was added. for titration of the virus, half of the concentration of trypsin was used to drive cell fusion in a procedure that is gentle compared with viral propagation. after a 15-h incubation, the cells were fixed and stained with pbs containing 0.1% crystal violet and 20% formalin under ultraviolet light. after staining for 2 h, the cells were washed with water and dried, and the plaques were counted under a light microscope as described for the infectious titration of human coronavirus 229e [6] . the viral titer in the brains of the mice is given as plaqueforming units (pfu)/50 ll of 10% homogenate, whereas the titer in the supernatant is given as pfu/ml of supernatant; the cell titer is given as pfu/well of a 24-well plate. isolation of a murine-adapted variant of pedv specific-pathogen-free (spf) pregnant icr mice were obtained from japan slc (shizuoka, japan). all animal experiments were approved by the committee on experimental animals, national institute of infectious diseases, japan, and all experimental animals were handled according to the guidelines of the same committee. all spf mice were confirmed by seromonitoring to be free from infection with mhv, lactate dehydrogenase virus, and other pathogenic microorganisms. newborn (within 24 h of birth; 0-day-old) mice were used in this study. pedv strain mk (2,500 pfu) was inoculated intracerebrally (i.c.) into the brains of 0-day-old mice (n = 12-16). five days post-infection (p.i.), the mice were sacrificed and their brains collected, pooled, and homogenized in pbs to yield a 10% suspension, which was centrifuged at 2,000 rpm for 10 min at 4°c. next, 10 ll of the supernatant from the 10% brain homogenate was inoculated i.c. into suckling mouse brains, and the brains were collected as described above. this cycle was repeated ten times, and the virus obtained was designated mk-p10. mk-p10 was amplified once by vero cell culture to match its condition to that of the original virus, then stored at -80°c until use. to investigate viral virulence and replication in mouse brains, 0-day-old mice were infected i.c. with 5,000 pfu of pedv and monitored daily for clinical features; they were also weighed daily. pbs was used in a mock infection. to examine viral replication in the brains, the mice were euthanized on the indicated day p.i., their brains were collected, and 10% homogenates were prepared as described above. to determine the replication kinetics in cultured cells, vero cells were infected with pedv at a multiplicity of infection (m.o.i.) of 0.1. after 1 h, the cells were washed with pbs and cultured in dmem containing 10% tpb and 2.5 lg/ml trypsin. at the indicated hour p.i., the supernatant and cells were collected separately, the cells were ultrasonicated, and the viral titers were determined using a plaque assay as described above. sequence analysis viral rna was extracted with trizol ls reagent (invitrogen, carlsbad, ca, usa) following the manufacturer's protocol. first-strand cdna was synthesized using m-mlv reverse transcriptase (takara-bio, shiga, japan) and oligod(t) 16 to determine the statistical significance of the survival curves, log-rank and generalized wilcoxon tests were performed. significance was determined in all other cases using unpaired t tests. p \ 0.05 was considered to be statistically significant. to obtain a murine-adapted variant of pedv, passage through suckling mice was performed. zero-day-old mice were inoculated i.c. with the mk strain of pedv, and the brains of the infected mice were collected four or 5 days later. the brains were pooled and used for the next round of mouse inoculation. surprisingly, mice infected with the non-adapted, original strain (mk) showed clinical signs and mortality; these observations differ from those reported for hcov oc43 and ibv [3] . however, in subsequent passages, clinical signs in the infected mice, such as trembling, tremors, and growth retardation, became more obvious. the process was stopped after ten passages, at which point the variant was designated mk-p10. the original mk strain and the mouse-adapted variant mk-p10 were inoculated onto vero cells in the presence of trypsin and cultured for 15 h. as shown in fig. 1 , more syncytium formation was seen in the cells infected with mk-p10 than in those infected with the original strain. the mean diameter of the mk-p10 syncytia was significantly greater than that of the mk syncytia (874 ± 177 vs. 348 ± 104 lm; n = 10, p \ 0.0001). the neurovirulence of mk-p10 in suckling mice was examined. zero-day-old mice were infected i.c. with 5,000 pfu of virus and monitored daily for clinical symptoms and mortality. all mice infected with mk and mk-p10 succumbed to infection; however, the mice infected with mk-p10 died more quickly than the mk-infected mice ( fig. 2a ; n = 25, p \ 0.01). the mice were also weighed daily ( fig. 2b; n = 12 ). the weights of the mice infected with mk or mk-p10 increased for 3-4 days after infection. subsequently, the weight of the mice decreased beginning 4 days p.i., whereas the mice infected with mk began to lose weight 8 days p.i.-4 days later than the mk-p10infected mice. the body weight of the mice infected with mk-p10 was determined 7 days p.i. because most of the mice had succumbed by 7-11 days p.i. the mock-infected mice did not exhibit weight loss or clinical symptoms. as described above, a mouse-adapted variant of pedv was successfully isolated. the growth kinetics of the original strain and mk-p10 were examined in suckling mouse brains. mk and mk-p10 exhibited similar growth kinetics in suckling mouse brain, peaking on day 4 p.i. (fig. 3a) . their growth kinetics in cultured vero cells was also determined. some coronaviruses tend to remain intracellular, and viral stocks are prepared by ultrasonication or repeated freeze-thawing cycles. to compare the amounts of budded and intracellular virus, the titers in the supernatant (fig. 3b) and cells (fig. 3c) were determined separately. similar to the growth kinetics in the brains of the mice, the growth pattern in vero cells did not differ significantly between mk and mk-p10 (fig. 3b, c) . these results suggest that the increased virulence in suckling mice and syncytium formation in cultured cells is not due to increased growth in the brain or cultured cells. because the s proteins of coronaviruses are reported to be associated with viral pathogenicity [20] , table 1) . among these, the amino acid substitutions at positions 804 and 1,381 resulted in a polarity change (hydrophobic to hydrophilic). to determine at which brain passage the amino acid changes occurred, we determined the primary sequences of s protein from the viruses isolated after two, four, six, and eight passages in mouse brain. as shown in table 2 , the amino acid at position 804 differed at the second passage, while those at positions 496 and 774 changed simultaneously at the fourth passage. the amino acid change at position 1,381 occurred at the eighth passage. as described above, the plaque size of mk-p10 was larger than that of the parental strain (fig. 1) . this larger plaque morphology was also detected in pedv after eight passages, whereas the viruses isolated after four and six passages showed syncytium formation similar to that in mk (fig. 4) . these results suggest that the amino acid change at position 1,381 (h to r) first detected at the eighth passage, which is located in the cytoplasmic tail (ct) region, contributed to the larger plaques of mk-p10, though other genes must be considered. two different coronaviruses, oc43 and ibv, have successfully adapted to grow in mice and show virulence after brain-to-brain passage in suckling mice [3, 12] . these viruses initially showed low virulence (i.e., clinical symptoms such as tremor and rigidity and lethargy in suckling mice), but the virulence increased with repeated passage in mouse brains. in this study, the passage of pedv in suckling mouse brains clearly strengthened its virulence, similar to the adaptation seen in oc43 and ibv. unexpectedly, the original mouse non-adapted pedv showed significant virulence for suckling mice. the primary sequences of s protein from the viruses isolated after repeated passages were determined. pedv passed in mouse brains showed mutations in the s protein, which had accumulated after ten passages. the h-to-r mutation at residue 1,381, which was first detected after eight passages, is likely responsible for the enhanced cell-cell fusion activity, as only those viruses with this mutation showed significantly larger syncytia compared with the original pedv; the s protein is a major determinant of the fusion activity of coronaviruses [5, 20] . it would be interesting to find out whether this substitution in the s protein is responsible for the enhanced fusion activity by expressing s protein alone. those mutations found in the mouseadapted viral s proteins could be responsible for the increased virulence; however, other genes have been reported to be involved in the virulence of such coronaviruses as mhv and sars-cov [7, 15, 17, 21] . to identify the genes responsible for the increase in virulence, we would need the entire genomic sequences of the original and mouse-adapted pdevs; additionally, reverse-genetic analyses would be essential. pedv differs from most coronaviruses in that it grows in cultures of cells derived from animal species not susceptible to pedv; the virus grows efficiently in vero cells derived from the kidneys of green monkey, which is not a natural host of the virus [8] . few cases are known in which coronaviruses multiply in cells derived from non-susceptible host animals. sars-cov can grow in a variety of cells established from several animal species [13, 14, 18, 24] , and it is somewhat capable of growing in the same animals; however, efficient infection is restricted to cells from animals that support sars-cov infection [24] . the growth potential of sars-cov in a variety of animal cells correlated with the utilization of its receptor molecule, ace2 [11] . most known coronaviruses become infective in cells when their functional receptor molecule is expressed by transfection with a plasmid encoding the receptor molecule [5] . this suggests that vero cells susceptible to pedv infection express a functional receptor. in our study, both mouse-adapted and non-adapted mk strains of pedv could infect and grow in suckling mouse brains, suggesting that a receptor molecule utilized by both viruses exists in suckling mouse brain. to determine which cell types in the brain express the receptor, we are attempting to identify the cell types that are infected by pedv in suckling mouse brain. this will provide valuable information related to the receptor molecule utilized by pedv. in summary, a highly neurovirulent variant of pedv was successfully isolated by passage following the i.c. infection of 0-day-old suckling mice. the s protein of the variant had four amino acid substitutions, which might be responsible for its enhanced cell-cell fusion activity. of these substitutions, the h-to-r substitution at position 1,381 in the ct region of the s protein is likely to be the determinant of the high fusion activity. to address this possibility, the expression of wild-type and mutant s proteins in cultured cells and an analysis of their fusion activity are necessary. reverse-genetic analyses of the entire pedv genome are also required to assess the effect of s protein mutations on the virulence and pathogenesis of pedv. p2 p4 p6 p8 p10 fig. 4 plaque morphology of pedv isolates after mouse brain passage pedv passed in mouse brains two (p2), four (p4), six (p6), eight (p8), or ten (p10) times as well as the original mk (p0) was inoculated onto vero cells. cell fusion assays were performed as described in the legend to fig. 1 aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv growth of infectious bronchitis virus in suckling mice brain susceptibility to sars coronavirus s protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor coronavirus receptors. in: siddell sg (ed) the coronaviridae proteasemediated entry via the endosome of human coronavirus 229e severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate the molecular biology of coronaviruses porcine aminopeptidase n is a functional receptor for the pedv coronavirus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus growth in suckling-mouse brain of ''ibv-like'' viruses from patients with upper respiratory tract disease pathology and virus dispersion in cynomolgus monkeys experimentally infected with severe acute respiratory syndrome coronavirus via different inoculation routes mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice inactivation of expression of gene 4 of mouse hepatitis virus strain jhm does not affect virulence in the murine cns a new coronavirus-like particle associated with diarrhea in swine reverse genetic characterization of the natural genomic deletion in sars-coronavirus strain frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in vitro and in vivo severe acute respiratory syndrome coronavirus infection of golden syrian hamsters coronavirus 229e susceptibility in man-mouse hybrids is located on human chromosome 15 coronaviruses: structure and genome expression single-amino-acid substitutions in open reading frame (orf) 1b-nsp14 and orf 2a proteins of the coronavirus mouse hepatitis virus are attenuating in mice feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i feline aminopeptidase n is a receptor for all group i coronaviruses coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins acknowledgments we thank shutoku matsuyama, miyuki kawase, and makoto ujike for their valuable comments and encouragement. this work was supported by a grant-in-aid for scientific research (b; no. 19390135) from the ministry of education, culture, sports, science, and technology of japan. passage number 0 2 4 6 8 1 0 496 i i t t t t 774 t t m m m m 804 a d d d d d 1,381 h h h h r r fig. 3 the replication kinetics of mk (squares) and mk-p10 (circles). a viral replication in suckling mouse brain. zero-day-old mice were infected i.c. with 5,000 pfu of pedv. at the indicated days p.i., the brains were collected and 10% homogenates were prepared. the viral titer in the brains was determined using a plaque assay with vero cells and is expressed as pfu/50 ll of 10% homogenates (n = 3). b, c viral replication in cultured cells. vero cells were infected with pedv at an m.o.i. of 0.1. viral titers in the supernatants (b) and cells (c) were determined separately using plaque assays (n = 6). the viral titer in the supernatant is given as pfu/ml of supernatant, while that in the cells is given as pfu per well of a 24-well plate key: cord-257136-zpeh8pmc authors: huang, xin; chen, jianing; yao, gang; guo, qingyong; wang, jinquan; liu, guangliang title: a taqman-probe-based multiplex real-time rt-qpcr for simultaneous detection of porcine enteric coronaviruses date: 2019-04-26 journal: appl microbiol biotechnol doi: 10.1007/s00253-019-09835-7 sha: doc_id: 257136 cord_uid: zpeh8pmc swine enteric coronaviruses are a group of most significant pathogens causing diarrhea in piglets with similar clinical symptoms and pathological changes. to develop a simple, rapid, accurate, and high-throughput detection method for diagnosis and differential diagnosis on swine enteric coronaviruses, specific primers and probes were designed based on the highly conserved regions of transmissible gastroenteritis virus (tgev) n, porcine epidemic diarrhea virus (pedv) m, porcine deltacoronavirus (pdcov) m, and porcine enteric alphacoronavirus (peav) n genes respectively. a taqman-probe-based multiplex real-time rt-qpcr assay was developed and optimized to simultaneously detect these swine enteric coronaviruses. the results showed that the limit of detection can reach as low as 10 copies in singular real-time rt-qpcr assays and 100 copies in multiplex real-time rt-qpcr assay, with all correlation coefficients (r(2)) at above 0.99, and the amplification efficiency at between 90 and 120%. this multiplex real-time rt-qpcr assay demonstrated high sensitivity, extreme specificity, and excellent repeatability. the multiplex real-time rt-qpcr assay was then employed to detect the swine enteric coronavirus from 354 field diarrheal samples. the results manifested that tgev and pdcov were the main pathogens in these samples, accompanied by co-infections. this well-established multiplex real-time rt-qpcr assay provided a rapid, efficient, specific, and sensitive tool for detection of swine enteric coronaviruses. electronic supplementary material: the online version of this article (10.1007/s00253-019-09835-7) contains supplementary material, which is available to authorized users. porcine enteric viruses, the pathogens of viral diarrhea in pigs, caused huge economic losses in the swine industry in recent years ). there were more than ten enteric viruses discovered from swine gut in the past, including but not limited to transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), porcine deltacoronavirus within these swine enteric viruses, coronaviruses are the most devastating pathogens responsible for acute diarrhea, vomiting, dehydration, and high mortality in neonatal and suckling piglets. according to the genetic and antigenic characters, coronavirus was divided into four genera: alpha-, beta-, gamma-, and delta-cov (woo et al. 2010) . during the past years, some alphacoronaviruses (pedv, tgev, and peav) and a deltacoronavirus (pdcov) emerged or reemerged in the pig farms and resulted in severe diarrhea and mortality in the early stage of suckling piglets. the tgev and pedv were the traditional causal agents responsible for diarrhea in pigs for the past decades. however, the variant strains of pedv were discovered since 2010 and circulating in pig herds thereafter, showing up to 100% death rate for piglets younger than 1-week-old (lyoo et al. 2017; sun et al. 2012) . another swine enteric virus, porcine deltacoronavirus (pdcov), was firstly discovered from healthy pig herds by a electronic supplementary material the online version of this article (https://doi.org/10.1007/s00253-019-09835-7) contains supplementary material, which is available to authorized users. research team of hong kong in 2012 when they did a molecular epidemiology study in 3137 mammals and 3519 birds (woo et al. 2012) . two years later, this virus was reported to cause severe diarrhea and/or vomiting and atrophic enteritis in the usa ) and later on in china (song et al. 2015) . subsequently, a novel porcine enteric alphacoronavirus peav was discovered in 2017 from some pig farms located in southern china, causing more than 24,000 piglets' death (gong et al. 2017) . this virus was also named as seacov (pan et al. 2017) or sads-cov (zhou et al. 2018b ) by the other research groups. all these four swine enteric coronaviruses, causing similar clinical symptoms and pathological changes in piglets, circulate in the pig herds and result in huge economic losses across the world in recent years. to develop a simple, rapid, accurate, and high-throughput detection method for diagnosis and differential diagnosis on swine enteric coronaviruses, a taqman-probe-based multiplex real-time rt-qpcr assay was established to simultaneously detect tgev, pedv, peav, and pdcov from the same reaction vial. to our knowledge, this is the first multiplex real-time rt-qpcr method for swine enteric coronaviruses detection. the tgev was previously propagated and preserved in our laboratory. the pedv and pdcov were isolated from clinical samples and confirmed by conventional pcr and dna sequencing (genewiz, suzhou, china). the nucleoprotein gene of peav (genbank access number: mf370205) was synthesized from genewiz biotech company (suzhou, china). field samples were collected from diarrheal piglets between 2015 and 2018 from liaoning, shandong, chongqing, shaanxi, ningxia, and gansu provinces. all samples were stored at − 80°c until use. the primers and taqman probes for real-time qpcr assay were designed by the software beacon designer 7 (premier biosoft international, palo alto, ca, usa). the detailed information of primers and probes were listed in table 1 . all clinical samples were resuspended with phosphatebuffer saline (pbs), vortexed and centrifuged at 12,000×g at 4°c for 10 min. an aliquot of 250 μl supernatant was applied for total rna extraction with rnaiso reagent (takara, dalian, china) following the manufacturer's instruction. the total rna in rnasefree water was reversely transcribed into cdna using revertaid first strand cdna synthesis kit (thermo scientific, waltham, usa). the cdna was subjected to real-time qpcr analysis with the multiplex rt-qpcr method established in this study. the m genes of pedvand pdcov, and n genes of tgevand peav were constructed into pet-30a(+) vector. the recombinant plasmids were linearized by smai enzyme digestion and recovered by pcr purification kit. the purified recombinant plasmids were quantified by spectrophotometric analysis. the copy number of recombination plasmids was calculated by using the following formula (huang et al. 2009 ): to establish the standard curves for single coronavirus, each plasmid was diluted in a tenfold series, from 10 7 copies/μl to 10 1 copies/μl. for multiplex standard curves, each of the four linearization plasmids was adjusted to 4 × 10 9 copies/μl and pooled with equal volume to made 1 × 10 9 copies/μl of each plasmid. the pooled plasmid was then diluted serially by tenfold to establish multiplex standard curves. all real-time qpcr reaction systems were set to a volume of 20 μl. for single qpcr amplifying tgev, pedv, and pdcov, 10 μl 2 × transstart probe qpcr supermix (transgene, beijing, china), 200 nm primers and probe each, 1 μl plasmid dna template, and 6.8 μl nuclease-free water were pooled and mixed. for peav amplification, 10 μl 2 × transstart probe qpcr supermix (transgene, beijing, china), 500 nm each primer, 100 nm probe, 1 μl plasmid dna template, and 6.8 μl nuclease-free water were pooled and mixed. all reactions were amplified on a bio-rad cfx96™ real-time system (bio-rad, hercules, ca, usa) at 94°c for 30s, followed by 40 cycles of 94°c for 5 s and 60°c for 30s. for reaction system of multiplex real-time pcr, 10 μl 2 × transstart probe qpcr supermix combined with all primers, probes, templates, and nuclease-free water to a final volume of 20 μl. the concentrations of each primer and probe of pedv, pdcov, tgev, and peav were optimized for better outputs. the amplifying cycles of multiplex qpcr were carried out as same as singular real-time qpcr. the cq value higher than 35 was considered negative. all qpcr results were analyzed by cfx manager™ software. to analyze the sensitivity of established multiplex rt-qpcr, the linearization standard plasmids prepared above were diluted tenfold serially to a final concentration between 1.1 × 10 7 copies/μl and 1.1 × 10 1 copies/μl in nuclease-free water. the diluted standard plasmids were used as templates for realtime qpcr amplification. to estimate the specificity of this established multiplex rt-qpcr, standard dnas, or cdnas of major swine viruses, including porcine astrovirus (pastv), porcine kobuvirus (pkv), type o foot-and-mouth disease virus (fmdv-o), type a foot-and-mouth disease virus (fmdv-a), porcine reproductive and respiratory syndrome virus (prrsv), classical swine fever virus (csfv), porcine rotavirus (prv), porcine circovirus 2(pcv2), and pseudorabies virus (prv) were used as templates for amplification. the nuclease-free water was served as negative template control. to evaluate its repeatability, tenfold serially diluted standard template between 1.1 × 10 7 copies/μl to 1.1 × 10 1 copies/μl were used to test the coefficients of variation of real-time pcr. for intra-assay repeatability, all samples were triplicated. for inter-assay repeatability, the assays were repeated three times individually at different locations. a total of 354 fecal samples were collected from diarrheal pig farms located in liaoning, shandong, chongqing, shaanxi, ningxia, and gansu provinces of china between 2015 and 2018. all samples were diluted fivefold with sterile phosphate-buffered saline (pbs), vortexed, and centrifuged at 1847×g at 4°c for 20 min. the supernatant was collected and used to extract viral rna with trizol reagent (invitrogen, carlsbad, ca, usa). the cdnas were generated by reverse transcript system (promega, madison, wi, usa) using extracted total rna as templates and hexamer random primers. all cdna from clinical samples were measured by the multiplex rt-qpcr assay developed in this study. to develop a multiplex real-time rt-qpcr, the single real-time rt-qpcrs for detection of the individual virus were firstly established with different fluorescence-labeled target probes, for details, fam for pedv m gene, cy5 for pdcov m gene, hex for tgev n gene, and texasred for peav n gene. the standard curves for each virus were generated using 1.1 × 10 7 copies to 1.1 × 10 1 copies of tenfold serially diluted linearized plasmids conceiving target genes. the results demonstrated that the single real-time rt-qpcr assays for each virus were successfully established at the limit of detection at approximately 10 copies (fig. 1 ). all the standard curves showed an excellent correlation coefficient and amplification efficacy, for instance, pedv (r 2 = 1; eff% = 95.7), pdcov (r 2 = 1; eff% = 100.9), tgev (r 2 = 0.999; eff% = 106.0), and peav (r 2 = 0.995; eff% = 98.9), indicating that the single real-time rt-qpcr for each virus was valid and reliable. to establish the multiplex real-time rt-qpcr method, all primer sets, probes, and serially diluted standard plasmids for detection of pedv, pdcov, tgev, and peav were mixed with 2 × transstart probe qpcr supermix and nuclease-free water. the concentrations of each primer and probe were optimized for the optimum output. the optimal final concentrations of primers and probes were as follows: 300 nm primer and 100 nm probe for pedv, 200 nm primer and 200 nm probe for pdcov, 500 nm primer and 100 nm probe for tgev, and 500 nm primer and c the amplification curves (top) and a standard curve (bottom) for detection of tgev n gene were generated. the probe was labeled with hex at 5′-end and bhq1 at 3′-end. d the amplification curves (top) and a standard curve (bottom) for detection of pedv n gene were generated. the probe was labeled with texasred at 5′-end and bhq2 at 3′-end 50 nm probe for peav. the results demonstrated that the multiplex real-time rt-qpcr could detect all target genes of these four viruses efficiently with high correlation values (fig. 2) . all the standard curves showed excellent correlation coefficient and amplification efficacy, for details, pedv (r 2 = 0.999; eff% = 103.5), pdcov (r 2 = 0.999; eff% = 104.7), tgev (r 2 = 0.999; eff% = 105.6), and peav (r 2 = 1; eff% = 100.4) (fig. 2) . the limit of detection of this multiplex rt-qpcr was approximately 100 copies of each virus per reaction (fig. 2) . the specificity of multiplex real-time rt-qpcr assay to evaluate the specificity of the multiplex rt-qpcr developed in this study for swine enteric coronavirus detection, the dnas/cdnas of nine other major swine viruses were used as templates for amplification with this multiplex system. the cdnas of pedv, pdcov, tgev, and peav were served as positive control while the nuclease-free water was used as a negative control. the results displayed that all swine enteric coronaviruses were successfully detected. however, there was no positive signal detected from other nine swine viruses, pastv, pkv, fmdv-o, fmdv-a, prrsv, csfv, prv, pcv2, prv, and negative control using this multiplex rt-qpcr system (fig. 3) , demonstrating that the taqmanprobe-based multiplex rt-qpcr system was highly specific. to determine the sensitivity of this multiplex real-time rt-qpcr, tenfold serial dilution of linearized plasmid mixtures were added to the amplification system. the results showed that 1.1 × 10 2 copies of tgev, pedv, pdcov, and peav were detectable with the ct values at 28.75, 28.94, 30.03, and 28.4 respectively ( table 2 ). however, 1.1 × 10 1 copies of each target were not detectable in the same amplification system (table 2) . also, the amplification exhibited reliable and high efficacy, pedv (r 2 = 0.998; eff% = 96.04), pdcov (r 2 = 0.992; eff% = 91.38), tgev (r 2 = 0.998; eff% = 94.57), and peav (r 2 = 0.999; eff% = 98.60). this multiplex real-time rt-qpcr demonstrated the same sensitivity level when using viral rnas as templates (supplemental fig. s1 ). these results implied that the taqman fig. 2 establishment of multiplex real-time rt-qpcr. a amplification curves and b standard curves of optimized multiplex taqman-probe-based realtime rt-qpcr for detection of pedv, pdcov, tgev, and peav were generated at the optimum amplification conditions. the correlation coefficient and amplification efficacy of the standards curves, pedv (r 2 = 0.999; eff% = 103.5), pdcov (r 2 = 0.999; eff% = 104.7), tgev (r 2 = 0.999; eff% = 105.6), and peav (r 2 = 1; eff% = 100.4), were ideal for detecting the target genes enteric coronaviruses (pedv, tgev, pdcov, and peav) as less as 10 to 100 copies. to estimate the reproducibility of the multiplex real-time pcr, tenfold serial dilution of pooled linearization plasmids were in a triplicate manner for both intra-assay and inter-assay. for intra-assay, the standard plasmids were amplified three times simultaneously. for inter-assay, standard curves were done at three individual times and using a different batch of a standard substance. as shown in table 3 , the coefficient of variation for pedv intra-assay was 0.30-1.56% and interassay was 0.44-1.79%. the pdcov amplification exhibited the coefficients of variation at 0.09-5.51% and 1.91-3.78% for inter-assay and intra-assay respectively. as for tgev, the coefficients of variation were 0.33-1.29% and 0.95-3.70% corresponding to intra-assay and inter-assay. the amplification of peav showed relative higher coefficients of variation, which was 0.39-7.32% for intra-assay and 1.08-4.52% for inter-assay. these results indicated that the taqman-probe-based multiplex real-time rt-qpcr assay established in this study was repeatable and reliable. the cdnas originated from 354 clinical samples were subjected to amplification by this multiplex real-time rt-qpcr and confirmed by conventional rt-pcr (supplemental fig. s2 ). the results demonstrated that the samples from henan and shaanxi provinces were swine enteric coronavirusnegative while the gansu province suffered a high prevalence of coronaviruses, showing 83.9% (26/31) tgev, 48.4% (15/ 31) pdcov, and 38.7% (12/31) pedv positive (table 4 ). in ningxia province, tgev was also highly prevalent but the positive rates for the other three swine enteric coronaviruses were quite low or not detectable (table 4 ). only pedv (15/ 62, 24.2%) and pdcov (8/62, 12.9%) among these four coronaviruses were detectable from clinical samples of chongqing. all these four swine enteric coronaviruses were detectable in samples of liaoning province, with tgev positive rate at 12.6% (18/143) and the other three were less than 10% (table 4) . overall, pdcov (121/354, 34.2%) and tgev (72/354, 20.3%) were the main coronaviruses detected from all these clinical samples. the positive rate for the newly emerged coronavirus in guangdong, china, peav, was rarely found from these provinces. we further analyzed the co-infection of coronaviruses for all these positive samples. the results elucidated that the main co-infection were caused by a dual coronavirus, for example, 13 pdcov and tgev co-infection samples, five pedv and pdcov co-infection samples, and four pedv and tgev co-infection samples (fig. 4) . some clinical samples were co-infected by three (9 pedv, pdcov, and tgev co-infection) or all four coronaviruses (fig. 4) . these results indicated the pathogens of viral diarrhea disease in chinese pig farms were complicated. porcine diarrheal disease is one of the most severe diseases in pig farms. swine enteric coronaviruses are the most significant pathogens causing diarrhea in piglets, especially for newborn piglets (butler et al. 2014 ). all swine enteric coronaviruses, including traditional pedv, tgev, and newly emerged pdcov, peav, could cause serious diarrhea, vomiting, dehydration, weight loss, and up to 100% death in suckling piglets (gong et al. 2017; hsu et al. 2018) . previously, some investigators developed a serial of elisa assays to detect pedv, tgev, or pdcov infections based on m, n proteins or whole virus (luo et al. 2017; ma et al. 2016; su et al. 2016 ). however, molecular diagnostic tools, rather than serological methods, for swine enteric coronaviruses are urgently needed due to their similar and high pathogenicity to suckling piglets, lacking a mature immune system. for swine enteric coronavirus detection, conventional rt-pcr, multiplex rt-qpcr, or pan-coronaviruses rt-pcr methods targeting m, n, s, or polymerase genes were developed in recent years (hsu et al. 2018; song et al. 2006) . additionally, zhou et al. (2018a) recently reported a specific real-time pcr for the detection of peav. to our knowledge, there was no such method that could simultaneously detect and differentiate tgev, pedv, pdcov, and peav from the same detection vial. to solve this urgent and important issue, we developed this multiplex real-time rt-qpcr for the detection and differential diagnosis of the swine enteric coronaviruses circulating in pig herds. taqman-probe-based real-time qpcr is a rapid, high specificity, high sensitivity, and great reproducibility detection tool for identification of viruses (chang et al. 2014 ). among the seven open reading frames (orfs) and four structural protein genes, s (spike), e (envelope), m (membrane), n (nucleocapsid) of coronaviruses (su et al. 2018 ), the m and n genes are highly conserved within the same antigenic group but less homologous between each of these four swine enteric coronaviruses (yang et al. 2019) . to establish a high-specific multiplex real-time rt-qpcr for the detection and differential diagnosis of swine enteric coronaviruses, primer sets, and taqman probes were designed targeting the highly conserved regions of pedv or pdcov m genes and tgev or peav n genes, based on bioinformatics analysis of each virus. our results demonstrated that each primer and probe set can only detect target gene itself and could not bind with any other targets, indicating high specificity. our results showed that the singular real-time rt-qpcrs were capable to detect as less as 10 copies of pedv, pdcov, tgev, and peav templates. however, the detection limit of each target gene in the multiplex real-time rt-qpcr was approximately 100 copies, implying that the sensitivity of multiplex real-time rt-qpcr was lower than that of singular realtime rt-qpcr probably due to the competition between primers, probes, templates, and reagents. the analysis of clinical diarrhea samples using this developed multiplex rt-qpcr elucidated that the co-infection of swine enteric coronaviruses commonly existed in some pig farms. co-infection of swine enteric coronaviruses may cause recombination between co-infected viruses. during 2012 and 2016, some new swine enteric coronaviruses were generated by recombination with pedv and tgev and spread across the central eastern european countries (akimkin et al. 2016; belsham et al. 2016; boniotti et al. 2016) . the recombination might create more virulent enteric virus strains or new viruses, leading to potential outbreaks or pandemics of swine viral diarrhea. additionally, co-infections may promote the evolution of non-pathogenic enteric viruses into high virulent and pathogenic viruses. a recent example showed the existence of pdcov in pig herds for many years in china mainland and hong kong but without observed clinical symptoms (pan et al. 2017; woo et al. 2012) . unfortunately, this virus caused severe outbreaks in some states of the usa since 2014 (ma et al. 2015; wang et al. 2014 ). this virulence change was most probably caused by the previous exposure to pedv or other co-infected swine enteric viruses. therefore, swine enteric coronaviruses co-infection may bring risks for the prevention and control of swine diarrhea. in summary, we developed a taqman-probe-based multiplex real-time rt-qpcr with high specificity and sensitivity for simultaneous detection and differential diagnosis of swine enteric coronaviruses, pedv, tgev, pdcov, and peav. this real-time rt-qpcr assay is of great significance for the prevention and control and epidemiological investigation of swine viral diarrhea. conflict of interest the authors declare that they have no competing interests. ethical statement this article does not contain any studies with human participants or animals performed by any of the authors. new chimeric porcine coronavirus in swine feces characterization of a novel chimeric swine enteric coronavirus from diseased pigs in central eastern europe in 2016 porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus porcine reproductive and respiratory syndrome (prrs): an immune dysregulatory pandemic the application of a duplex reverse transcription real-time pcr for the surveillance of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 a new bat-hku2-like coronavirus in swine detection, sequence analysis, and antibody prevalence of porcine deltacoronavirus in taiwan development of a reverse transcription multiplex real-time pcr for the detection and genotyping of classical swine fever virus development and application of a recombinant m protein-based indirect elisa for the detection of porcine deltacoronavirus igg antibodies development of rapid immunochromatographic strip test for the detection of porcine epidemic diarrhoea virus origin, evolution, and virulence of porcine deltacoronaviruses in the united states two-way antigenic cross-reactivity between porcine epidemic diarrhea virus and porcine deltacoronavirus discovery of a novel swine enteric alphacoronavirus (seacov) in southern china multiplex reverse transcription-pcr for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group a rotavirus newly emerged porcine deltacoronavirus associated with diarrhoea in swine in china: identification, prevalence and full-length genome sequence analysis a recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus a novel duplex taqman probebased real-time rt-qpcr for detecting and differentiating classical and variant porcine epidemic diarrhea viruses outbreak of porcine epidemic diarrhea in suckling piglets detection and genetic characterization of deltacoronavirus in pigs coronavirus genomics and bioinformatics analysis 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 generation, identification and functional analysis of monoclonal antibodies against porcine epidemic diarrhea virus nucleocapsid evaluation of two singleplex reverse transcription-insulated isothermal pcr tests and a duplex real-time rt-pcr test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus development of a taqman-based real-time rt-pcr assay for the detection of sads-cov associated with severe diarrhea disease in pigs fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-009526-ghm1hvei authors: bertolini, f.; harding, j. c. s.; mote, b.; ladinig, a.; plastow, g. s.; rothschild, m. f. title: genomic investigation of piglet resilience following porcine epidemic diarrhea outbreaks date: 2016-12-12 journal: anim genet doi: 10.1111/age.12522 sha: doc_id: 9526 cord_uid: ghm1hvei porcine epidemic diarrhea virus (pedv) belongs to the coronaviridae family and causes malabsorptive watery diarrhea, vomiting, dehydration and imbalanced blood electrolytes in pigs. since the 1970s, ped outbreaks have become a source of problems in pig producing countries all over the world, causing large economic losses for pig producers. although the infection in adults is not fatal, in naïve suckling piglets mortality is close to 100%. in this study, we investigated genome‐wide differences between dead and recovered suckling piglets from commercial farms after ped outbreaks. samples from 262 animals (156 dead and 106 recovered) belonging to several commercial lines were collected from five different farms in three different countries (usa, canada and germany) and genotyped with the porcine 80k snp chip. mean f (st) value was calculated in 1‐mb non‐overlapping windows between dead and recovered individuals, and the results were normalized to find differences within the comparison. seven windows with high divergence between dead and recovered were detected—five on chromosome 2, one on chromosome 4 and one on chromosome 15—in total encompassing 152 genes. several of these genes are either under‐ or overexpressed in many virus infections, including coronaviridae (such as sars‐cov). a total of 32 genes are included in one or more gene ontology terms that can be related to ped development, such as golgi apparatus, as well as mechanisms generally linked to resilience or diarrhea development (cell proliferation, ion transport, atpase activity). taken together this information provides a first genomic picture of pedv resilience in suckling piglets. porcine epidemic diarrhea virus (pedv) belongs to the coronaviridae family; this family includes a wide variety of viruses that affect humans and other animals, causing respiratory and gastroenteric diseases (weiss & navas-martin 2005) . as with many coronaviruses, pedv has a limited host range and tropism, infecting only the small intestine of pigs (song & park 2012; jung et al. 2014 ). this infection causes acute intestinal disease during which the infected enterocytes rapidly develop necrosis, leading to a villous atrophy (debouck et al. 1981; ducatelle et al. 1982; jung et al. 2014 ) and development of malabsorptive watery diarrhea, vomiting, dehydration and imbalanced blood electrolytes (jung & saif 2015) . the major route of transmission for pedv infection is fecal-oral (turgeon et al. 1980; utiger et al. 1995; riley 2007 ) even if aerosolized pedv remains infectious (alonso et al. 2014) . it is known that pedv, as well as other swine coronaviruses, such as porcine tgev (transmissible gastroenteritis virus), gains entry in the host cells through interaction with alanyl (membrane) aminopeptidase, encoded by the anpep gene (oh et al. 2003; li et al. 2007 ). this receptor is largely expressed by enterocyte cells, and its density has been correlated to the replication rate of the virus in vitro (nam & lee 2010). since discovery of the virus during the 1970s in europe (pensaert et al. 1978) , ped outbreaks have become an increasing source of problems in many swine breeding countries all over the world, causing severe problems as well as large economic losses for pork producers (song & park 2012; snelson 2014) . it is widely understood that, although the infection in adults may not be fatal, pedv infection in unprotected piglets under 3 weeks of age results in severe disease with mortality reaching 100% on many farms (pensaert et al. 1978; shibata et al. 2000) . the degree to which the small percentage of na€ ıve suckling piglets recover during ped outbreaks in the wider industry is unknown, as is the biological mechanisms involved, but two main hypothesis can be suggested: (i) survival can be related to variation in the intestinal receptor used by pedv to gain entry to intestinal epithelial cells (the anpep gene) or (ii) as summarized by schneider & ayres (2008) and ayres & schneider (2012) , survival and recovery can be related to particular host immune responses that enhance viral clearance, increase cell epithelial regeneration rate or reduce viral replication rate. in this study, our aim was to investigate genome-wide differences between dead and recovered suckling piglets from commercial farms during ped outbreaks. the study was conducted using piglets younger than 3 weeks of age from farms during the acute phase of a ped outbreak (as soon after herd diagnosis as physically possible). as depopulation is one of the main approaches used to deal with outbreaks, it is difficult to obtain good information on survival rates, or indeed samples, to test these hypotheses. to address this, we collected samples from 262 animals belonging to several commercial terminal crossbred lines from five different farms in three different countries (usa, canada and germany). among these animals, 156 animals died as a result of severe diarrhea and dehydration and 106 animals successfully recovered (table s1 ). specific piglets were collected to ensure that balanced numbers of dead and recovered piglets were collected from each farm, with the exception of one farm in the usa from which the numbers were not balanced. moreover, in three of the five farms (one farm located in usa and the farms located in germany) dead and recovering piglets belonged to the same litters (1-2 dead and 1-2 recovered per litter). each animal was genotyped with the geneseek genomic profiler 80k snp chip (geneseek-neogen), which contains 68 517 snps. for the analysis, only snps mapped to the autosomes and with a call rate >90 were considered. in total, 57 422 snps passed the filtering steps, and missing snps were imputed using beagle 3.3.3 (browning & browning 2007) . admixture software (alexander et al. 2009 ) was used to investigate population stratification. following the software guidelines, the cross-validation (cv) procedure was applied to choose the best k for the model, where k is the number of (sub) populations that was assumed for the analysis. within a range of 10 k values, 1-10, k = 8 showed the lowest cv error and therefore was considered a sensible model. the admixture plot considering k = 8 is reported in fig. s1 . as expected, the samples appeared to be stratified, with more similarity between the two us farms. due to the expected low number of recovering piglets from each farm, detailed analysis by farm or within breed or population strata could not be considered. therefore, we focused our attention on a comparison of all dead with all recovered, attempting to detect common windows of divergence. a multidimensional scaling plot was obtained using plink 1.7 (purcell et al. 2007 ), comparing dead and recovered piglets (fig. s2 ). the plot clearly shows a lack of clustering between the two groups. to find windows that were more divergent within the two groups, f st was calculated for each snp, adapting the formula reported by karlsson et al. (2007) : where k is the snp marker k, with frequency p ½k 1 , p ½k 2 ; then, a mean f st value (f stm ) was calculated in 1-mb nonoverlapping windows, and the result was z transformed using a derived formula provided by rubin et al. (2010) : where i is the 1-mb window, lf stm is the mean and rf stm is the standard deviation. the distribution of the frequency for the normalized values is reported in fig. s3 . values lower than 6 represent 0.2% of the empirical distribution of all the normalized values and were the most divergent between the two groups and, therefore, were considered as significant. the normalized plot is reported in fig. 1 105-106 mb). the number of snps included in the selected windows ranged from eight to 14 (table s2 ). the anpep gene, known to be the receptor of pedv and a possible candidate for pedv resistance (nam & lee 2010), is located on chromosome 7 at position 60 240 145-60 262 914. the window that contains this gene had a zf stm score of 0.2, which is far below the threshold chosen for significance. therefore this gene, which has previously been suggested to be linked to the replication rate of the virus (nam & lee 2010), did not appear to be related to pedv recovery in this study. more specific analyses need to be conducted to establish if this gene does play a role in the recovery of suckling piglets after acute infection, because the number of markers and the linkage disequilibrium in the considered windows can affect the significance above all when only one gene in the window is involved. after the completion of the genomic analysis, immunoglobulin g (igg) in the serum of all animals was measured using either an indirect elisa (swinecheck â ped; biovet inc.) or an indirect immunofluorescence assay (iowa veterinary diagnostic laboratory), depending on the farm (data not shown). half (52/100) of the german pigs, including both dead (n = 11) and recovered (n = 41), tested elisa positive. because all dead pigs were 8 days old or younger at sampling, positive elisa tests were indicative of maternally derived antibodies. in spite of the fact that the recovered pigs were exposed to pedv during their first week of life, serum pedv igg was not measured in german pigs until 14-21 days of age. at that age, the majority of the piglets likely had naturally seroconverted (opriessnig et al. 2014) ; hence, it was not possible to distinguish whether a positive response was the result of maternal antibodies or an adaptive antibody-mediated immune response. furthermore, it is known that the swinecheck pedv elisa cross-reacts with porcine deltacoronavirus (personal communication, a. ambagala, june 24, 2016). re-analysis of the genomic data with and without the elisa positive pigs still identified the most interesting regions on the same chromosomes (chromosomes 2: 55-56 mb and 86-90 mb, 4: 53-54 mb and 15: 105-106 mb; for more details see fig. s4 ), and hence, the results obtained appear reliable. moreover, for this specific case, the imbalance between dead and survived and across farms (germany contributed with 39 dead piglets and nine recovered) could influence the results. as both dead and recovered pigs in these cohorts were elisa positive, it still suggests they responded differentially to the infection during the acute phase of the disease and are useful in this genomic analysis. the seven selected windows were then screened to find annotated genes located 200 kb before and after the windows, according to sscrofa 10.2 and the annotation provided by ensembl (http://www.ensembl.org/). to avoid ambiguous windows, we considered only protein-coding genes (not pseudogenes or mirnas) that were officially named, uniquely orthologous in mammalian species or named in the uniprot database. a total of 152 genes fit these requirements, with only one gene located in the window on chromosome 4 and the majority (40 genes) located on chromosome 2: 74.8-76.2 mb. the list of the annotated genes for each window is reported in table s2 . the detected genes were analyzed to investigate their role during virus infection and proliferation. all genes were screened to find evidence of gene perturbation and differential gene expression after virushost protein interaction using the online tool enrichr (chen et al. 2013 ). this analysis was performed because it is known that host targets of viral proteins reside in networks in proximity to products of disease susceptibility genes (goh et al. 2007; zhong et al. 2009; gulbahce et al. 2012) . then, enrichr and the gorilla tool (eden et al. 2007 (eden et al. , 2009 , with homo sapiens as a reference, were used to find gene ontology (go) terms of functions, processes or components for the considered genes. only go terms with p ≤ 0.001 were considered, which is the minimum threshold allowed by gorilla. several genes that have been found to be underexpressed or overexpressed in many viral infections, including coronaviruses (such as sars-cov), are shown in table s3 . a total of 32 genes were included in one or more go terms (fig. 2) . the go term outputs are related mainly to regulation of cell proliferation, ion transport, golgi apparatus, atpase activity, hydrolase activities and pyrophosphatase activity. interestingly, several of these go terms could be directly linked to pedv proliferation and diarrhea development, as follows: (i) cell proliferation has been linked to the ability of the organism to overcome virus or bacterial tissue destruction (schneider & ayres 2008 ); (ii) ion channels, particularly k+ and ca++ channels, are necessary for intestinal homeostasis, and their functions are altered during diarrhea in several species including humans (field 2003 ) and pigs (moeser & blikslager 2007) ; (iii) atpase activity is required for the function of several ion channels (reviewed by gouaux & mackinnon 2005) ; and (iv) the assembly of the ped virus occurs by budding through intra-cytoplasmic membranes, such as the endoplasmic reticulum and golgi apparatus (ducatelle et al. 1982) . in conclusion, we did not find evidence of a direct association between the anpep gene and pedv resilience in na€ ıve piglets. instead, the picture provided by the f st analyses seems to support the associations between recovery and host responses that could influence cell epithelial regeneration rate, virus replication rate and the general consequences of virus infection. the authors declare they do not have any conflicts of interest. data reported in this work will be available one year after the publication of the manuscript at the following link: http://www.animalgenome.org/repository/ pub/isu2016.0617/. additional supporting information may be found online in the supporting information tab for this article: figure s1 admixture plot considering k = 8 for the analyzed data. figure s2 multidimensional scaling plot of all dead and recovered piglets considering the first two dimensions. figure s3 distribution of the frequency of the zf stm values in 1-mb windows calculated comparing the dead with the recovered suckling piglets. figure s4 normalized f st plot without the samples that were positive based on the elisa test. table s1 number of dead and recovered piglets from the pedv outbreak in each considered farm. table s2 information about the windows with zf stm > 6. table s3 virus changes, upregulated and downregulated from the gene expression omnibus (geo). fast model-based estimation of ancestry in unrelated individuals evidence of infectivity of airborne porcine epidemic diarrhea virus and detection of airborne viral rna at long distances from infected herds tolerance of infections rapid and accurate haplotype phasing and missing data inference for whole genome association studies using localized haplotype clustering enrichr: interactive and collaborative html5 gene list enrichment analysis tool the pathogenesis of an enteric infection in pigs, experimentally induced by the coronavirus-like agent, cv-777 pathology of experimental cv777 coronavirus enteritis in piglets ii: electron microscopic study discovering motifs in ranked lists of dna sequences gorilla: a tool for discovery and visualization of enriched go terms in ranked gene lists intestinal ion transport and the pathophysiology of diarrhea the human disease network principles of selective ion transport in channels and pumps viral perturbations of host networks reflect disease etiology porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs efficient mapping of mendelian traits in dogs through genome-wide association porcine aminopeptidase n is a functional receptor for the pedv coronavirus mechanisms of porcine diarrheal disease contribution of the porcine aminopeptidase n (cd13) receptor density to porcine epidemic diarrhea virus infection identification of a putative cellular receptor 150 kda polypeptide for porcine epidemic diarrhea virus in porcine enterocytes porcine epidemic diarrhea virus rna present in commercial spray-dried porcine plasma is not infectious to na€ ıve pigs a new coronavirus-like particle associated with diarrhea in swine plink: a toolset for whole-genome association and population-based linkage analysis large-scale spatial-transmission models of infectious disease whole-genome resequencing reveals loci under selection during chicken domestication two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages pedv update porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines coronavirus-like particles associated with diarrhea in baby pigs in quebec identification of the membrane protein of porcine epidemic diarrhea virus coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus edgetic perturbation models of human inherited disorders the authors appreciate the financial support of the us pig genome coordination program, particularly chris tuggle and cathy ernst, and the state of iowa. funding supporting the collection and processing of samples from ped outbreak farms was provided by the genome alberta's 2014 program on research and innovation leading to a rapid genomics response to the porcine epidemic diarrhea virus (pedv). key: cord-252772-f3fctcru authors: wang, changlin; shan, lingling; qu, shuxin; xue, mei; wang, keliang; fu, fang; wang, lu; wang, ziqi; feng, li; xu, wanhai; liu, pinghuang title: the coronavirus pedv evades type iii interferon response through the mir-30c-5p/socs1 axis date: 2020-05-22 journal: front microbiol doi: 10.3389/fmicb.2020.01180 sha: doc_id: 252772 cord_uid: f3fctcru porcine epidemic diarrhea virus (pedv) is an economically important pathogen that has evolved several mechanisms to evade type i ifn responses. type iii interferon (ifn-λ), an innate cytokine that primarily targets the mucosal epithelia, is critical in fighting mucosal infection in the host and has been reported to potently inhibit pedv infection in vitro. however, how pedv escapes ifn-λ antiviral response remains unclear. in this study, we found that pedv infection induced significant ifn-λ expression in type i ifn-defective vero e6 cells, but virus-induced endogenous ifn-λ did not reduce pedv titers. moreover, we demonstrated that pedv escaped ifn-λ responses by substantially upregulating the suppressor of cytokine signaling protein 1 (socs1) expression, which impaired the induction of ifn-stimulated genes (isgs) and dampened the ifn-λ antiviral response and facilitated pedv replication in vero e6 cells. we further showed that pedv infection increased socs1 expression by decreasing host mir-30c-5p expression. mir-30c-5p suppressed socs1 expression through targeting the 3′ untranslated region (utr) of socs1. the inhibition of ifn-λ elicited isgs expression by socs1 was specifically rescued by overexpression of mir-30c-5p. collectively, our findings identify a new strategy by pedv to escape ifn-λ-mediated antiviral immune responses by engaging the socs1/mir-30c axis, thus improving our understanding of its pathogenesis. porcine epidemic diarrhea virus (pedv), a member of the alphacoronavirus family, is an enteropathogenic coronavirus with economic importance (madson et al., 2014; wang et al., 2014; zhang and yoo, 2016) . pedv infection in newborn piglets is characterized by vomiting, anorexia, watery diarrhea, and dehydration (song and park, 2012) . the virus primarily infects small intestinal epithelial cells in vivo and causes high morbidity and mortality in piglets (li et al., 2012) . interferons (ifns) are the key components of innate immunity in response to viral infection . among three types of ifns (types i, ii, and iii), type iii ifn-lambda (ifn-λ) primarily acts on mucosal surfaces, including epithelial surfaces of the liver, respiratory, and gastrointestinal systems, and plays vital roles in controlling viral infection within mucosal surfaces (mordstein et al., 2010; pott et al., 2011; lazear et al., 2015) . we and other groups previously demonstrated that porcine ifn-λdisplays powerful antiviral activity against pedv infection in both vero e6 cells and porcine intestinal epithelia (li et al., 2017 (li et al., , 2019 . pedv has evolved multiple strategies to escape ifn responses, including the degradation of stat1 and the suppression of type i ifn production . although type i and type iii ifns have a large overlap in the spectrum of induced antiviral isg responses, recent studies demonstrated that type iii ifn is a critical non-redundant antiviral mediator of type i ifns in the gi tract and elicits a unique transcriptional profile that does not completely overlap with that induced by ifn-α (wells and coyne, 2018) . it is necessary to clarify how pedv evades type iii ifn following infection. unlike ample studies reporting that pedv escapes type i ifns, limited studies demonstrate that pedv escapes ifn-λ response. pedv suppresses irf1-mediated type iii ifn responses by reducing the number of peroxisomes and counteracting type iii ifn response by pedv nsp15 endoribonuclease deng et al., 2019) . deng et al. showed that type i and type iii ifns exhibit different modulation in response to pedv infection and that the discrepancy of type i and type iii ifn responses is independent of pedv endoribonuclease activity (deng et al., 2019) , suggesting that there are distinct strategies to modify host type i and type iii ifn responses during pedv infection. because cells generally produce both type i and type iii ifns in response to viral infection, it is challenging to elucidate how viruses escape ifn-λ response separately to type i response. in this study, we used vero cells, a cell line with a defective function, to produce endogenous type i ifns. vero cells are widely used as an in vitro model to study the interactions between viruses and hosts including pedv. we and others reported that vero cells respond well to both porcine type i and type iii ifns shen et al., 2016; li et al., 2017) . ifn-λ is rapidly produced after infection and following engagement with its receptor induces ifn-stimulated gene (isg) expression to mediate antiviral activity (kotenko et al., 2003; dellgren et al., 2009; lazear et al., 2015) . binding of ifnλ to its receptor, which consists of two subunits, ifn-λr1 and il-10r2, leads to activation of jak1 and tyk2, which mediates the phosphorylation of stat1 and stat2 proteins (sheppard et al., 2003; palma-ocampo et al., 2015) . the suppressor of cytokine signaling protein 1 (socs1), a negative regulator of janus family kinase (jak) signal transducer, simultaneously binds the receptors and jaks and prevents stats from accessing the receptor kinase complex (de weerd and nguyen, 2012; palma-ocampo et al., 2015) . previous reports demonstrated that socs1 is an inducible negative regulator of ifn-λ-induced gene expression in vivo (blumer et al., 2017) . socs1 was also associated with denv-2 escape from ifn-λ response during infection (palma-ocampo et al., 2015) . however, the role of socs1 during pedv infection remains unclear. micrornas (mirnas), as important post-transcriptional modulators of gene expression, participate in modulating the host innate and adaptive immune responses in response to pathogen invasion (baltimore et al., 2008; gottwein and cullen, 2008; o'neill et al., 2011) . increasing evidence has shown that mirnas of viral and cellular origin can help viruses evade host immune responses by targeting critical components in the host immune system (cullen, 2006; sullivan et al., 2006; kincaid and sullivan, 2012) . for example, mir-30c is a potent negative regulator of type i ifn signaling by targeting jak1, resulting in the enhancement of prrsv infection . the mir-30 family is a well-studied host mirna that plays an important role in viral infection by modulating ifn signaling (zhu et al., 2014; liu et al., 2018; ma et al., 2018) . our previous study revealed that tgev escapes type i ifn response by engaging the ire1-mir-30a-5p/socs1/3 axis (ma et al., 2018) . the potential role of mirnas in coronavirus escape from ifn-λ response remains elusive. in this study, we showed that pedv escaped ifn-λ responses by upregulating socs1 expression in type i ifn-defective vero e6 cells. in addition, we demonstrated that pedv infection increased socs1 expression by decreasing the expression of host mir-30c-5p, which modulates socs1 expression by specifically targeting the 3 ′ utr of socs1. our findings identify a new strategy by pedv to escape ifn-λ-mediated host innate immune defenses. african green monkey kidney cells (vero e6 cells) were stocked by our laboratory and grown in dmem (gibco, gaithersburg, md, usa) supplemented with 10% fbs (gibco) and antibiotics (100 u/ml of penicillin and 100 µg/ml of streptomycin) at 37 • c in a humidified atmosphere of 5% co2. pedv-cv777 (genbank accession no. kt323979) stocked in our laboratory was propagated in vero e6 cells as previously described (hofmann and wyler, 1988; sun et al., 2015) . to evaluate the anti-pedv activity of porcine ifn-λ (prosit sole biotechnology, co., ltd., beijing, china), vero e6 cells were pretreated with designated concentrations of ifn-λ for 12 h and then infected with pedv (moi of 0.1). all of the mirna mimics, mirna inhibitors, and short hairpin rnas (shrnas) were synthesized by gene pharma (shanghai, china). the mirnas and shrnas sequences are listed in table 1 . lipofectamine 2000 (invitrogen, carlsbad, ca, usa) or lipofectamine rnaimax (invitrogen) was used to transfect cells with plasmid dna or synthetic oligonucleotides according to the manufacturer's instructions. the cells were infected with pedv as previously described after transfection for 24 h. the cells were harvested for quantitative real-time pcr (rt-qpcr) or treated with np-40 lysis buffer for western blotting after infection for 36 h. sequences total rna isolation, reverse transcription, and qpcr total cellular rna was isolated using an rneasy mini kit (qiagen sciences, hilden, germany) according to the manufacturer's instructions. total rna was extracted and reverse transcribed as previously described (ma et al., 2018) . for mirna reverse transcription, cdna was prepared with a mirna first strand cdna synthesis kit (sangon biotech, shanghai, china). qpcr was conducted in triplicate with power sybr green pcr master mix reagents (takara) on a lightcycler480 ii system (thermo fisher scientific, waltham, ma, usa) as previously described (ma et al., 2018) . the mirna expression levels were normalized to the internal control of u6. the sequences of rt-qpcr primers for pedv, ifn-λ, socs1, ifit1, isg15, mxa, gapdh, mir-30c, and uni-mir transcription are listed in table 2 . the results are presented as the means ± sem from three separate trials. targetscan release 7.1 (http://www.targetscan.org) was used to predict the targets of mir-30c-5p. socs1 3 ′ utrs as a prospective target was cloned and constructed as previously described (ma et al., 2018) . to construct the monkey socs1 expression vector, the full-length cds region of monkey socs1 was amplified from vero e6 cellular mrna pcr and cloned into pcaggs-ha vector (clontech, mountain view, ca, usa) using ecor i and kpn i restriction sites. the 3 ′ utr of socs1 (genbank:100307052) was amplified and inserted into the pmirglo luciferase reporter vector. the socs1 3 ′ utr mutant vector was produced by mutating five seed nucleotides using a site-directed mutagenic kit (stratagene, la jolla, ca, usa) according to the manufacturer's instructions. the constructed plasmids were verified by sequencing. the luciferase activities were tested using a dual-luciferase reporter assay system (promega, madison, wi, usa) based on the manufacturer's instructions. wild-or mutant-type socs1 3 ′ utr luciferase reporter vectors were co-transfected with mir-30c-5p mimics (mir-30c), mimic nc (nc), mir-30c-5p inhibitor (mir-30c-i), or inhibitor nc (nc-i) into vero e6 cells for 24 h. then prl-tk was co-transfected with either mir-30c, nc, mir-30c-i, or nc-i for 24 h. the cells were collected and the luciferase activity was evaluated with a dual-luciferase reporter assay system (promega). the prl-tk vector expressing the renilla luciferase gene was used as a normalization control. vero e6 cells were fixed with 4% paraformaldehyde for 30 min at 4 • c and permeabilized with 0.2% triton x-100 for 15 min, then blocked with blocking buffer (pbs with 5% fbs) for 2 h at 37 • c. the cells were incubated with an anti-ha monoclonal antibody (sigma-aldrich, munich, germany, 1:5000) at 37 • c for 2 h, followed by labeling with an alexa fluor 546 goat anti-mouse igg antibody (thermo fisher scientific, 1:500) at 37 • c for 1 h. 4 ′ ,6-diamidino-2-phenylindole (dapi, 1:100) was used to stain the cellular nuclei. the stained cells were visualized using an amg evos f1 fluorescence microscope. vero e6 cells were lysed with np-40 lysis buffer (beyotime, china) supplemented with 0.1 mm of phenylmethylsulfonyl fluoride (pmsf) (roche, indianapolis, in, usa). target proteins were separated on sds-page gels then transferred onto nitrocellulose membranes (ge healthcare, chicago, il, usa). after blocking with tbs-t containing 5% non-fat milk at room temperature (rt), the membranes were incubated with primary antibody at 4 • c for 18 h. antibodies included: β-actin (sigma-aldrich, 1:5000) and socs1 (sigma-aldrich, 1:500). the membranes were incubated with secondary antibody goat antimouse-hrp or goat anti-rabbit-hrp, diluted at 1:2000 for 1 h at room temperature, and visualized using an ecl system (thermo fisher). the results were analyzed using imagej software. all of the data are described as the means ± the standard error of the mean (sem). graphpad prism (graphpad software, inc.) was used to analyze the data using student's t-test. each experiment was repeated three times. p-values < 0.05 were considered significant: * p <0.05; * * p < 0.01; * * * p < 0.001; * * * * p < 0.0001, and ns, not significant. kinetic curve of pedv replication in vero e6 cells. vero e6 cells were inoculated with pedv at an moi of 0.1, and the level of pedv infection compared to mock controls at 12, 24, and 48 h was quantified by rt-qpcr and tcid 50 . the results were obtained from three independent experiments. mean ± sem, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, and ns, not significant. previous research showed that the pretreatment of porcine ifn-λ inhibits pedv infection in vero e6 cells and ipec-j2 (li et al., 2017) . it is well-established that pedv replicates efficiently in vero e6 cells. to determine whether pedv elicits an endogenous ifn-λ response in vero e6 cells following infection, we initially infected vero e6 cells with pedv at moi = 0.1 and monitored the ifn-λ expression. compared with a mock uninfected control, pedv did not increase the expression of ifnλ transcripts as observed until 12 hpi, and then gradually induced ifn-λ expression, indicating that pedv infection elicits type iii ifn expression at the late stage of infection instead of the early stage of infection in the vero e6 cells (figure 1a) , which was consistent with the results in porcine enteroids (li et al., 2019) . and pedv propagated efficiently in vero e6 cells by quantifying viral genomes and titers (figures 1b,c) . the virus titer increased up to 10 5 /0.1 ml at 48 hpi ( figure 1c) . interestingly, despite the increased expression of endogenous ifn-λ at the late stage of infection, the pedv virus titer did not decrease. this indicates that there are mechanisms explored by pedv to antagonize the endogenous ifn-λ isg response at the late-stage infection. pedv infection increased the expression of socs1 in vero e6 cells socs1, a typical member of the socs family of proteins, is a well-known negative feedback inhibitor of jak/stat signaling pathway induced by cytokines (ma et al., 2018) . to explore the underlying mechanisms exploited by pedv to escape ifnλ-induced antiviral responses, we initially assessed whether pedv infection induces socs1 expression in vero e6 cells. the mrna levels of socs1 significantly increased following pedv infection and displayed a time-dependent response (figure 2a) . the induction of socs1 by pedv infection was further verified by socs1 western blotting ( figure 2b ). counteracted the anti-pedv activity of ifn-λ socs1 is a potent inhibitor of the type i and type ii ifn signaling pathway (skjesol et al., 2014) . we next investigated whether socs1 suppresses ifn-λ-mediated antiviral activity. first, we silenced endogenous socs1 expression by specific shrnas. socs1 shrnas or a non-targeting shrna (nc) were transfected into vero e6 cells. the efficiency of socs1 knockdown was confirmed by western blotting (figure 3a) . shsocs1 #2 and #3 led to a 55 and 65% decrease in socs1 expression, respectively, compared with nc ( figure 3a) . silencing of endogenous socs1 by shsocs1 #2 or #3 significantly reduced pedv replication in vero e6 cells without the addition of exogenous ifn-λ ( figure 3b) . the decreased levels of pedv infection were in line with the knockdown efficiency of socs1 shrnas, indicating the specific effect of socs1 shrnas. as previously reported, exogenous ifn-λ significantly inhibited pedv infection, whereas silencing of endogenous socs1 by shsocs1 #2 or #3 further enhanced the pedv inhibition by ifn-λ in vero e6 cells compared with untreated ifn-λ mock control ( figure 3b) . the knockdown of endogenous socs1 by shsocs1 #3 resulted in a more than 3.4-fold decrease in pedv titer ( figure 3b ) and degraded to 7.2-fold of pedv titers with ifn-λ treatment in vero e6 cells (figure 3b) , indicating that socs1 knockdown increases the antiviral effects of ifn-λ. inconsistent with the viral results, socs1 knockdown increased the mrna levels of isg15, mxa, and ifit1 ( figure 3c) . we subsequently investigated the role of socs1 overexpression on the anti-pedv effects of ifn-λ. the transient overexpression of socs1 in vero e6 cells was verified by ha ifa (figure 3d ). as expected, socs1 overexpression substantially elevated pedv figure 4 | mimics or inhibitor for 24 h, cells were pretreated with ifn-λ or dmem for 12 h and then infected with pedv (moi = 0.1) and harvested at 36 hpi for viral rna quantification and tcid 50 . (f) the socs1 expression levels in vero e6 cells were measured by rt-qpcr at 36 hpi at different mois. p values represent the difference from the mock-infected control for time kinetics, the socs1, and mir-30c-5p levels. error bars, mean ± sem. (n = 3 independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001, and ns, not significant. infection ( figure 3e ) and blunted the expression of isg15, mxa, and ifit1 ( figure 3f) . thus, these data indicate that socs1 counteracts the anti-pedv activity of ifn-λ. modulating mir-30c-5p the porcine mir-30 family (five members: mir30a-e) has been demonstrated to modulate host type i ifn response during virus infection (zhu et al., 2014; liu et al., 2018; ma et al., 2018) . the targetscan (http://www.targetscan. org) prediction program indicated that socs1 was targeted by mir-30c-5p through a site in the 3 ′ utr conserved in the socs1 of seven representative mammals ( figure 4a ). to investigate whether mir-30c-5p is involved in modulating ifnλ signaling by directly targeting socs1 and downregulating endogenous socs1 expression, we conducted a computational analysis using targetscan release 7.1 (http://www.targetscan. org). the result showed that mir-30c could directly target the site on the 3 ′ utrs of socs1 ( figure 4a) . we cloned the predicted target sites in porcine socs1 3 ′ utr, and constructed the firefly luciferase reporter vector of porcine socs1 3 ′ utr ( figure 4a) . overexpression of mir-30c-5p, the luciferase reporter containing the socs1 wild-type target sequence, decreased to ∼65% relative to nc mimics, whereas the blockage of mir-30c by mir-30c inhibitor increased socs1 3 ′ utr luciferase activity. however, the mutation of the socs1 target 3 ′ utr site of mir-30c-5p disrupted the effects of mir-30c-5p on modifying the luciferase activity in vero e6 cells relative to the ncs ( figure 4b) . these results confirmed that mir-30c-5p directly targets the 3 ′ utr of socs1. consistent with the luciferase results, mir-30c-5p overexpression reduced socs1 expression measured by western blotting (figure 4c) . conversely, blockage of endogenous mir-30c-5p increased the expression of socs1 in vero e6 cells compared with the nc inhibitor. to further validate the modulation of socs1 expression by mir-30c-5p during pedv infection, we examined the expression of socs1 in pedvinfected vero e6 cells with overexpression or inhibition of mir-30c-5p, and found that the expression pattern of socs1 in pedv infected vero e6 cells was similar to that in pedv-uninfected e6 cells ( figure 4c) . taken together, these data demonstrated that mir-30c-5p downregulates the expression of socs1 by directly targeting socs1 3 ′ utr. to verify whether pedv escape the ifn-λ antiviral signaling through mir-30c-5p mediated modification of socs1 expression, we then explored the effect of mir-30c-5p on pedv infection and ifn-λ antiviral signaling. transient mir-30c expression reduced pedv titers and promoted ifn-λ anti-pedv activity compared with the mock control ncs, whereas mir-30c-5p inhibitor significantly increased pedv infection and undermined the anti-pedv activity of ifn-λ (figures 4d,e) . furthermore, the socs1 expression increased starting at 12 hpi and substantially increased at 24 hpi, which was inversely correlated with the kinetic expression profiles of mir-30c-5p ( figure 4f ). in agreement with the kinetics pattern of mir-30c-5p and socs1 in vero e6 cells, pedv infection reduced the levels of mir-30c-5p and increased socs1 expression in ipec-j2 starting at 12 h post-infection (data not shown). collectively, pedv infection upregulates socs1 expression by modulating host mir-30c-5p abundance at the late stage of infection. to further verify whether pedv escape ifn-λ response through the mir-30c-5p/socs1 axis, we co-transfected mir-30c-5p with socs1 and measured the replication of pedv with or without ifn-λ treatment. socs1 overexpression promoted pedv replication with or without ifn-λ treatment. mir-30c-5p largely abolished the role of socs1 in promoting pedv replication, and the effect was more pronounced in the presence of ifn-λ stimulation ( figure 5a) . consistent with this, socs1 inhibited ifn downstream isgs expression such as ifit1 and isg15 expression (figures 5b,c) , whereas overexpression of mir-30c-5p abrogated the isg inhibition of socs1, which was more pronounced in the presence of ifn-λ priming (figures 5b,c) . in summary, these data indicate that pedv escapes the response of ifn-λ through the mir-30a-5p/socs1 axis. ifn-λ is an antiviral innate cytokine induced by virus infection that plays vital roles in controlling mucosal infection (blumer et al., 2017) . we and other groups previously showed that ifnλ substantially inhibits pedv (li et al., 2017; zhang et al., 2018) . however, whether pedv has evolved a mechanism to counteract endogenous ifn-λ just as pedv does the type i ifn response remains unclear. in this study, we found that pedv propagated well despite the significant production of endogenous ifn-λ induced at the late stage of infection in vero e6 cells, indicating that pedv escaped the ifn-λ response at the late stage of infection not through suppressing ifn-λ production. we further defined the mechanism that pedv counteracted ifnλ-elicited antiviral isg responses by exploiting the mir-30c-5p/socs1 axis. pedv has evolved multiple strategies to escape type i ifn response. whether pedv exploits similar mechanisms to counteract type iii ifn remains elusive. one previous study demonstrated that pedv escaped type iii ifn by suppressing irf1-mediated ifn-λ production through pedv viral nsp1 protein . in that study, pedv actually upregulates ifn-λ expression at 3 h post-infection and then decreased to minimal levels of ifn-λ expression until 12 hpi figure 5 | mir-30c-5p inhibited the infection of pedv by regulating the expression of socs1. (a) socs1 overexpression increased pedv infection and undermined the anti-pedv activity of ifn-λ. vero e6 cells were transfected as described with pcaggs-ha, pcaggs-socs1, and mir-30c-5p for 24 h, followed by incubation with porcine ifn-λ (100 ng/ml) or dmem for 12 h. the cells then were infected with pedv at an moi of 0.1; pedv infection was determined at 36 hpi. (b,c) mir-30c-5p abolished the impairment of the overexpression of socs1 to ifn-λ signaling under ifn-λ-stimulated or pedv-infected conditions. e6 cells were treated as described in the legend for panel a. the cells were collected for rt-qpcr analysis of ifit1 and isg15 expression relative to that of gapdh. error bars, mean ± sem (n = 3 independent experiments). *p < 0.05, **p < 0.01, and ns, not significant. . in agreement with this, we did not observe increased ifn-λ expression at 12 hpi ( figure 1a) . they did not show the ifn-λ expression at the late stage of pedv infection. in the current study, we observed that pedv elicited substantially increased ifn-λ expression in vero e6 cells only after 24 hpi (figure 1a) , which is consistent with the results observed in porcine enteroids following pedv infection (li et al., 2019) , indicating that pedv has evolved mechanisms to escape ifn-λ antiviral response instead of ifn-λ production at the late stage of infection. it is possible that pedv exploits varying strategies at different infection stages. this is also observed in other rna viruses such as influenza virus (chung et al., 2018) . to prevent over-activation of the ifn signaling pathways, the host evolves a few negative regulators of ifn signaling, and socs1 is one of the canonical inhibitors of ifn signaling (shao et al., 2013) . socs1 has been reported to be exploited by multiple viruses to abrogate ifn antiviral signaling (shao et al., 2013; wei et al., 2014; ma et al., 2018) . we showed that pedv significantly induced the expression of socs1 at the late stage of infection (figure 2) . as expected, increased socs1 impaired the antiviral isgs expression and impaired the anti-pedv activity of ifn-λ (figure 3) . this is in agreement with the results of tgev, another swine alphacoronavirus (ma et al., 2018) . therefore, unlike previously published studies with the modification of ifn production mediated by viral proteins such as nsp1, our study found that pedv largely evades innate immunity of ifn-λ by modulating the antiviral signal of ifn-λ rather than manipulating the production of ifn-λ at the late stage of infection. mirna plays a vital role in regulating gene expression through post-transcription modification. increasing evidence demonstrates that viruses escape ifn antiviral activity for optimal infection by modifying the cellular abundance of mirna targeting vital components of the ifn response (zhu et al., 2014; liu et al., 2018; ma et al., 2018) . jev infection downregulates the expression of mirna mir-432, which directly targets the suppressor of cytokine signaling protein 5 (socs5) and manipulates the jak-stat1 signaling cascade (sharma et al., 2016) . the mir-30 family has been reported to target socs family members and manipulate the jak/stat signaling pathway (zou et al., 2017; ma et al., 2018; yuan et al., 2019) . in this study, we showed that pedv infection suppressed mir-30c-5p expression, which was conversely related to socs1 expression during pedv infection (figure 4) . just as other members of mir-30, mir-30c-5p specifically targeted the 3 ′ utr of socs1 and inhibited socs1 expression (kobayashi et al., 2012; ma et al., 2018; yuan et al., 2019) (figure 4) . however, the mechanism of pedv decreasing mir-30a-5p remains unclear and deserves further study. in summary, we determined that pedv escaped ifn-λ response at the late stage of infection by downregulating mir-30c-5p, thus increasing socs1 expression. therefore, unlike previously published studies with defined mechanisms such as nsp1, we demonstrated that pedv escapes ifn-λ response through another pathway of the mir-30c-5p/socs1 axis. our results highlight the important role of mir-30c-5p in the regulation of interferon pathways during pedv infection, improve the current knowledge of pedv infection, and expand the role of micro-rna in viral infection. the datasets generated for this study are available on request to the corresponding author. pl, cw, ls, and wx designed the research studies and analyzed and interpreted the data. cw, ls, sq, mx, kw, ff, lw, zw, and lf conducted the experiments and collected the data. pl, cw, and ls drafted the manuscript. all of the authors contributed revisions. micrornas: new regulators of immune cell development and function socs1 is an inducible negative regulator of interferon lambda (ifnlambda)-induced gene expression in vivo construction of a transcriptomedriven network at the early stage of infection with influenza a h1n1 in human lung alveolar epithelial cells viruses and micrornas the interferons and their receptors-distribution and regulation human interferon-lambda3 is a potent member of the type iii interferon family coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type i and type iii interferon responses viral and cellular micrornas as determinants of viral pathogenesis and immunity porcine epidemic diarrhea virus infection inhibits interferon 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signaling microrna-30e * suppresses dengue virus replication by promoting nf-kappab-dependent ifn production microrna-30c-5p ameliorates hypoxia-reoxygenation-induced tubular epithelial cell injury via hif1alpha stabilization by targeting socs3 the authors declare that the research was conducted in the key: cord-259324-g8kv4pvq authors: ko, suk-min; sun, hyeon-jin; oh, myung jin; song, in-ja; kim, min-jae; sin, hyun-sook; goh, chang-hyo; kim, yong-woo; lim, pyung-ok; lee, hyo-yeon; kim, suk weon title: expression of the protective antigen for pedv in transgenic duckweed, lemna minor date: 2011-10-28 journal: hortic environ biotechnol doi: 10.1007/s13580-011-0007-x sha: doc_id: 259324 cord_uid: g8kv4pvq duckweeds are small, floating aquatic plants with a number of useful characteristics, including edibility, fast-growing, and a clonal proliferation. duckweed is also fed to animals as a diet complement because of its high nutritional value. porcine epidemic diarrhea virus (pedv) is a major causative agent of fatal diarrhea in piglets and is a serious problem in the hog-raising industry. in this study, we assessed the feasibility of producing a protective antigen for the pedv spike protein 1 using duckweed, lemna minor. stably transformed lemna were obtained by co-cultivation with a. tumefaciens eha105 harboring the pedv spike protein gene. transgene integration and expression of the pedv spike protein 1 gene were confirmed by genomic pcr and rt-pcr and western blot analysis of transgenic lemna, respectively. this is the first report of the expression of a vaccine antigen against an animal infectious disease in duckweed. plants have many potential advantages in the production of high-value proteins, such as therapeutic proteins, including a low risk of contamination with potential animal pathogens, possessing the proper protein modification machinery, and providing low-cost scalable system for commercial production. it may also be possible to use them as a vehicle for oral delivery of vaccine antigens, resulting in an 'edible plant vaccine' (fischer et al., 2004; koprowski and yusibov, 2001; ma et al., 2005) . duckweeds are small, floating aquatic plants belonging to the lemnaceae, a monocotyledonous family containing four genera: spirodella, lemna, wolffia, and wolfiella (stomp, 2005) . useful characteristics of duckweed include being fastgrowing with clonal proliferation, a small size and a simple growth habit, suggesting its use as a biomanufacturing platform for recombinant proteins (stomp, 2005) . duckweed is also fed to animals and fish to complement their diets because of its high nutritional value (leng, 1995) . duckweed has been used to successfully produce several high-value proteins, including human monoclonal antibody modified to lack plantspecific n-glycans (cox et al., 2006) , a bacterial endoglucanase (sun et al., 2007) , alpha-interferon (gasdaska et al., 2003) and aprotinin, used in cardiac surgery (rival et al., 2008) . porcine epidemic diarrhea (ped) is a contagious, enteric disease clinical symptoms of which include vomiting, diarrhea and dehydration resulting in a loss of appetite and weight in adult pigs and death in neonatal piglets (pensaert and de bouck, 1978) . porcine epidemic diarrhea virus (pedv) is a known causative agent of ped. pedv is an enveloped rna with a single, positive-stranded genome, belonging to the group i coronaviruses (lai and cavanagh, 1997) . pedv infects intestinal epithelial cells and causes loss of enterocytes with villous atrophy, resulting in diarrhea and dehydration (coussement et al., 1982) . development of effective vaccination strategies against pedv infection is important because pedv-induced disease is a serious problem in the hog-raising industry. edible vaccines delivered to mucosal surfaces may be good candidates for a vaccination system for inducing modulated systemic immune responses without injection-related hazards. the antigen protein corresponding to the neutralizing epitope of the pedv spike protein has been successfully expressed in a transgenic tobacco plant (kang et al., 2005 (kang et al., , 2006 . in this study, we report the stable transformation and expression of a protective antigen for pedv in lemna minor with potential for use as an effective complement to the diets of animals. lemna minor was collected from a rice field near daejeon, korea, and surface-sterilized with 1% sodium hypochlorite solution for 10 min. fronds of lemna were cultured on halfstrength ms medium supplemented with 1 mg l -1 ba, 0.4 mg l -1 thiamine hcl, 100 mg l -1 myo-inositol, 15 g l -1 sucrose, and 4 g l -1 gelrite (½ms1ba). the ph of the medium was adjusted to 5.8 using 1 n naoh before autoclaving. all cultures were maintained at 26 under continuous fluorescent light (70 µmol m -2 s -1 , photoperiod 16/8 h). to examine the phytotoxic level of antibiotics, fronds were cultured separately on ½ms1ba medium supplemented with kanamycin at 0, 50, 100, 200, 300, and 500 mg l -1 . each treatment consisted of 20 fronds per dish with three replicates. data for the mean percentage of productive fronds was calculated for each treatment. a single cell colony of the bacteria was grown in 4 ml yep liquid medium containing 50 mg l -1 kanamycin for 12 h, and 2 ml were transferred to 50 ml yep liquid medium containing 50 mg l -1 kanamycin for another 12 h at 28 . bacterial cells were pelleted by centrifugation (4000 rpm, 15 min), and the pellet was dissolved in liquid ½ms1ba containing 100 µm acetosyringone. for transformation, fronds were injured with a pair of forceps and scalpel, and immersed in the bacterial suspension for 30 min. fronds were then blotted onto sterile filter paper, and co-cultivated with agrobacterium tumefaciens strain eha105 harboring the pedv spike protein 1 gene fused to a c-myc tag for 72 h on antibiotic-free ½ms1ba medium. after co-cultivation, the fronds were rinsed with liquid ½ms1ba supplemented with 500 mg l -1 cefotaxime, and cultivated on solid ½ms1ba medium supplemented with 300 mg l -1 cefotaxime and 200 mg l -1 kanamycin. kanamycin-resistant fronds were subcultured at 3 week intervals. total genomic dna from the putative transgenic and untransformed wild-type lemna was extracted according to dellaporta et al. (1983) . pcr was carried out using the specific forward/reverse primers designed for the pedv spike protein 1 gene. the gene-specific primers used for genomic pcr were as follows: 5'-atggaacagccaatttcttt-3' and 5'-a aaagatctatggtacaagc-3'. after denaturation for 5 min at 95 , samples were carried through 30 cycles using the following pcr conditions: 95 for 1 min, 55 for 1 min, and 72 for 1 min. total rna was extracted from the whole lemna plants using the trizol reagent (invitrogen, usa). cdna synthesis was carried out using 2 µg of dnase-digested total rna with oligo (dt) primers using a m-mlv reverse transcriptase kit (promega, madison wi) according to the manufacturer's protocol. the gene-specific primers used for rt-pcr were the same as those used for genomic pcr analysis. as a control, the actin gene was amplified using two degenerate primers (5'-tctggcatcacaccttctacaac-3' and 5' ca gtgtggctgacaccatcacca-3'). the following thermocycling conditions were employed: initial denaturation at 94 for 5 min, 25 cycles of 94 for 30 s, 55 for 30 s and 72 for 30 s, with a final extension at 72 for 7 min. the amplified products were separated on a 1.2% agarose gel and photographed. lemna tissue was ground in liquid nitrogen to a fine powder. the powder (0.1 g) was resuspended in 200 µl of extraction buffer consisting of 50 mm tris-hcl (ph 8.0), 150 mm nacl, 2 mm edta, and 2% pvpp. the extract was centrifuged (12,000 rpm, 30 min, 4 ), and the resulting supernatant was used for western blot analysis. extracted proteins were separated on a 10% gradi-gel ii gradient gel (elpis biotech, korea) and transferred onto a nitrocellulose membrane (ge healthcare, uk). after blocking with 5% non-fat dried milk, the blots were incubated with mouse anti-myc antibody (igtherapy, korea), followed by incubation with goat antimouse igg coupled to horseradish peroxidase (bio-rad, hercules, ca, usa). immunoreactive signals were detected using picoepd western blot detection reagents (elpis biotech, korea), according to the manufacturer's instructions. despite the useful characteristics of duckweed as a biomanufacturing platform for foreign proteins, so far there have only been a few reports on the production of recombinant proteins, as described above. more protein trials and data collection are necessary to expand the industrial availability of duckweed as a biomanufacturing platform. in the present study, we assessed the feasibility of producing a protective antigen for pedv in lemna minor, a member of the duckweed family. first, to determine the appropriate conditions for selecting lemna transformants, the effect of increasing concentrations of kanamycin was assessed on lemna fronds (fig. 1a) . approximately 78% of the fronds were bleached on day 30 in culture medium containing 100 mg l -1 kanamycin. the phytotoxic effect of kanamycin was noticeable at 200 mg l -1 in the medium as fronds started to bleach between day 10 and day 14 of culture, resulting in a total loss of chlorophyll pigmentation (bleaching) by day 30. thus, in subsequent experiments, we supplemented the ½ ms1ba medium with 200 mg l -1 kanamycin to select the putative transformed fronds. yamamoto et al. (2001) established efficient genetic transformation protocols for two duckweed species, l. gibba (geographic isolate g3) and l. minor (geographic isolates 8627 and 8744; strain number is designation in lemnaceae germplasm collection). only selection with 10 mg l -1 kanamycin was used for the transformation of l. gibba g3 and l. minor 8744, while concentrations of up to 100 mg l -1 were used for l. minor 8627 (yamamoto et al., 2001) . in our experiments, kanamycin at a comparatively high concentration (200 mg l -1 ) was used. this difference may be due to different sensitivity to antibiotics of various geographic isolates of lemna. moon et al. (1998) reported that proliferation of lemna fronds varied six-fold across the 25 geographic isolates of lemna collected at various geographical locations around the world. as described above, the lemna used in this study was collected from a rice field in daejeon, korea. the fronds of lemna were transformed by co-cultivation with a. tumefaciens strain eha105 harboring the pedv spike protein1 gene fused to a c-myc tag. the c-myc tag sequence was introduced into the vector to facilitate purification of the target recombinant proteins. kanamycin-resistant fronds were transferred to liquid medium containing kanamycin and subcultured under the high selection pressure. the liquid environment allows the antibiotic to contact the entire surface of the plant, thus ensuring reliable selection (yamamoto et al., 2001) . a liquid medium was used due to the differences in the tissue uptake of kanamycin in different media environments and different availability of kanamycin due to its binding to certain gelling agents (chauvin et al., 1999; yamamoto et al., 2001) . after an additional selection for 3 months under high kanamycin selection pressure, lemna fronds resistant to kanamycin in liquid medium were obtained. the genomic integration of the pedv spike protein 1 gene in the putative transformants was confirmed by genomic pcr analysis (fig. 2) . pcr products of the expected size (330 bp) corresponding to primers designed on the internal pedv spike protein 1 gene were detected from kanamycin-resistant lemna, whereas no dna band corresponding to the target gene was detected in untransformed wild-type lemna. to confirm whether the pedv spike protein 1 gene was transcribed in lemna transformants, the genomic integration of the target gene was confirmed by genomic pcr analysis. a reverse transcription-pcr (rt-pcr) assay was performed with the specific primers used in genomic pcr analysis, corresponding to the pedv spike protein 1 gene (fig. 3) . pcr products of the expected size corresponding to the specific primers were detected in the lemna transformants, whereas no amplified pcr product was detected in untransformed wildtype lemna (fig. 3) . the actin gene and 18s ribosomal dna were used as internal controls and are shown in the lower panel. this result demonstrated that the pedv spike protein 1 gene was correctly transcribed in the transgenic lemna. to further investigate the expression of the pedvsp1myc protein in the lemna transformants in which transcription of pedvsp1 mrna was confirmed by rt-pcr, immunoblot analysis was performed using a monoclonal anti-myc primary antibody. the sds-page profile of total soluble proteins extracted from whole lemna plants, including wild-type and transformants, and stained with coomassie blue and immunoblotted is shown in fig. 4 . no specific band was detected in the coomassie blue-stained gel, but immunoblotting with a monoclonal anti-myc revealed the specific band at a molecular weight of about 28 kda (fig. 4) . this result indicated the pedv spike1-myc fusion protein was produced in the transgenic lemna. in this study, we assessed the feasibility of producing a protective antigen for pedv in lemna minor that could be used as an effective complement in the diets of animals. this is the first report of the expression of a vaccine antigen for an animal infectious disease in duckweed, and may expand the industrial availability and development of effective edible vaccines. the national research foundation (nrf) funded by the ministry of education effects of gelling agent on in vitro regeneration and kanamycin efficiency as a selective agent in plant transformation procedures pathology of experimental cv777 coronavirus enteritis in piglets glycan optimization of a human monoclonal antibody in the aquatic plant lemna minor plant-based production of biopharmaceuticals advantage of therap eutic protein production in aquatic plant lemna expression of the synthetic neutralizing epitope gene of porcine epidemic diarrhea virus in tobacco plants without nicotine expression of synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic b subunit of escherichia coli heat-labile enterotoxin in tobacco plants the green revolution: plants as heterologous expression vectors the molecular biology of coronaviruses duckweed -a potential high-protein feed resource for domestic animals and fish plant-derived pharmaceuticals-the road forward effect of media components and phytohormones on in vitro frond proliferation of lemna gibba g3 and 24 additional lemna gibba strains a new coronavirus-like particle associated with diarrhea in swine the duckweed: a valuable plant for biomanufacturing expression and characterization of acidothermus cellulolyticus e1 endoglucanase in transgenic duckweed lemna minor 8627 spirodela (duckweed) as an alternative production system for pharmaceuticals: a case study, aprotinin genetic transformation of duckweed lemna gibba and lemna minor key: cord-273712-r2akpce8 authors: wang, jingjing; deng, feng; ye, gang; dong, wanyu; zheng, anjun; he, qigai; peng, guiqing title: comparison of lentiviruses pseudotyped with s proteins from coronaviruses and cell tropisms of porcine coronaviruses date: 2016-02-19 journal: virol sin doi: 10.1007/s12250-015-3690-4 sha: doc_id: 273712 cord_uid: r2akpce8 the surface glycoproteins of coronaviruses play an important role in receptor binding and cell entry. different coronaviruses interact with their specific receptors to enter host cells. lentiviruses pseudotyped with their spike proteins (s) were compared to analyze the entry efficiency of various coronaviruses. our results indicated that s proteins from different coronaviruses displayed varied abilities to mediate pseudotyped virus infection. furthermore, the cell tropisms of porcine epidemic diarrhea virus (pedv) and transmissible gastroenteritis virus (tgev) have been characterized by live and pseudotyped viruses. both live and pseudoviruses could infected veroccl-81 (monkey kidney), huh-7 (human liver), and pk-15 (pig kidney) cells efficiently. ccl94 (cat kidney) cells could be infected efficiently by tgev but not pedv. overall, our study provides new insights into the mechanisms of viral entry and forms a basis for antiviral drug screening. [image: see text] coronaviruses (covs) are important infectious pathogens that are associated closely with respiratory and enteric diseases in humans and animals (perlman and netland, 2009; belouzard et al., 2012; li, 2013) . covs have a single-strand positive sense rna genome and consist of four groups: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (table 1) . viral entry into cells is highly dependent on the interaction between viral particles and the host cells. covs use a variety of cellular receptors and co-receptors, including proteins and sugars, to facilitate their entry into cells. infection begins with an interaction between the virus and its specific receptors (li, 2015) . severe acute respiratory syndrome coronavirus (sars-cov) of group beta uses a type i integral membrane protein, angiotensin-converting enzyme 2 (ace2), as a receptor . there is no structural homology between sars-cov and the human coronavirus nl63 (hcov-nl63) receptor-binding domain (rbd), but they recognize the same receptor, ace2 (wu et al., 2011) . although the rbd crystal structures of transmissible gastroenteritis virus (tgev) and hcov-nl63 are similar, they use different receptors (reguera et al., 2012) . aminopeptidase n (apn), also known as cd13, is a type ii transmembrane protein (tusell et al., 2007) . tgev and porcine epidemic diarrhea virus (pedv) infect cells through interaction with porcine (p) apn (li et al., 2007; nam and lee, 2010) . human (h) apn is a receptor for hcov-229e (belouzard et al., 2012) . in the same beta group, the receptors for mouse hepatitis virus (mhv) and bovine coronavirus (bcov) are carcinoembryonic antigen-related cell adhesion molecule 1 (ceacam1) and a sugar, respectively, despite their high sequence homology peng et al., 2012) . ceacam1, the first identified coronavirus receptor (dveksler et al., 1991) , is a type i transmembrane multifunctional protein of the immunoglobulin superfamily (de haan et al., 2005) . cell entry and interspecies transmission of covs are mediated by the spike protein (s). cov s is a class i fusion protein (bosch et al., 2003) . in covs, s is a significant surface protein and plays an important role in mediating infection of virions (schwegmann-wessels et al., 2009) . s is also responsible for receptor binding and the fusion of viral and cellular membranes. all cov s share the same functional component in two domains: an nterminal domain called s1 that is responsible for receptor binding, and a c-terminal s2 domain that is responsible for fusion. s1 contains two subdomains, an n-terminal domain and a c-terminal domain ( figure 1a ). both function as rbds and bind a variety of proteins and sugars (belouzard et al., 2012) . because some covs have strong pathogenicity, the lentiviral pseudotype system is a reliable tool to study the proteins of highly pathogenic viruses under conventional biosafety conditions. hiv-luc is an hiv-1 based lentiviral vector bearing the luciferase reporter gene and has been used in the production of pseudotyped viruses lu et al., 2012; tang et al., 2012) . pseudovirus entry efficiency is characterized based on the luciferase activity. viral particles pseudotyped by various s proteins have been described for several viruses. in our study, we compared the efficiency of pseudotyped viruses with s proteins from different groups of covs. furthermore, the cell tropisms of tgev and pedv were characterized by live and pseudotyped viruses. 293t cells were cultured and maintained in rpmi 1640 medium (invitrogen, carlsbad, ca, usa) supplemented with 10% fetal bovine serum (fbs) at 37 °c in an atmosphere of 5% co 2 . pk-15, huh-7, vero-ccl-81, mdbk, ccl94, bsr, and mdck cells were cultured and maintained in dulbecco's modified eagle's modium (dmem) (invitrogen) supplemented with 10% fetal bovine serum (fbs) at 37 °c in an atmosphere of 5% co 2 . the pedv and tgev strains (genbank accession numbers: kt021232.1 for pedv, hq462571.1 for tgev) were isolated from a suckling piglet. rabbit anti-pedv n protein and rabbit anti-tgev n protein polyclonal antibodies are stored by the lab. the s protein sequences of different groups of covs (genbank accession numbers: abd72982.1 for sars-cov-s, aar92025.1 for mhv-s, np_073551.1 for hcov-229e-s, abg89335.1 for tgev-s, np_598310.1 for pedv-s, aat84362.1 for hcov-oc43-s, abm66810.1 for bcov-s, and np_040831.1 for avian infectious bronchitis virus (ibv) s) were codon-optimized and synthesized (genscript, nanjing, china). the s fragments were cloned into the pcdna3.1 (+) vector with a c-terminal his-tag. on the day before transfection, 293t cells were seeded in six-well plates. the next day, the cells were transfected with s plasmids plus the lentiviral vector hiv-luc lentiviruses pseudotyped with s proteins were solubilized by boiling in sodium dodecyl sulfate sample buffer. the s proteins were fractionated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. the nitrocellulose membrane was incubated with a 1:4000 dilution of a mouse anti-his antibody (proteintech group, chicago, il, usa). goat anti-mouse igg (boster, wuhan, china) was used as the secondary antibody. a plasmid (vector pcdna 3.1(+)) with each viral receptor gene (ace2, ceacam1, hapn, and papn) was transfected into 293t cells with lipofectamine 2000. after incubation for 5-6 h at 37 °c, the supernatant was replaced by dmem containing 2% fbs. after 48 h, the cells were infected by lentiviruses pseudotyped with the s proteins from covs of different groups. for analysis of the lentiviruses pseudotyped with the s proteins from covs of different groups, different cell lines were seeded in 96-well plates. 293t cells expressing receptors were infected by the same lentiviruses and incubated at 37 °c for 2 h. dmem (150 µl) containing 2% fbs was added subsequently to all cells and incub-ated for an additional 48-72 h. the pseudovirus entry efficiency was characterized based on luciferase activity. pedv and tgev were used to infect various cell lines from different species, including pk-15 (pig kidney), huh-7 (human liver), vero-ccl-81 (monkey kidney), mdbk (bovine kidney), ccl94 (cat kidney), bsr (hamster kidney), and mdck (canine kidney) cells at a multiplicity of infection of 0.01. trypsin (10 µg/ml) was included in the cell culture medium to facilitate live virus infections. cells infected with viruses were fixed with 4.0% (v/v) paraformaldehyde at 24 or 48 h post-inoculation. pedv or tgev was detected with fluorescein isothiocyanate-labeled rabbit anti-pedv or anti-tgev n protein antibodies, respectively, and observed under a fluorescence microscope. all experiments were done at least twice (in most cases three or more times). data were analyzed statistically using two-tailed student's t-tests for comparison of pcdna3.1 and cov s pseudovirus. the pseudovirus entry efficiency is reported as the luciferase activity. statistical analyses were performed to compare the treatment groups: ***p < 0.001; **0.001 < p < 0.01. cov diversity is reflected in the variable s proteins (heald-sargent and gallagher, 2012). therefore, we compared the s protein amino acid sequences of covs from different groups, including sars-cov, mhv, hcov-229e, tgev, pedv, hcov-oc43, bcov, and ibv (gen-bank accession numbers abd72982.1, aar92025.1, np_073551.1, abg89335.1, np_598310.1, aat84362.1, abm66810.1, and np_040831.1, respectively) using clustalw ( figure 1b ). because s1 is responsible for receptor binding, the amino acid sequences of the s1 domain were aligned ( figure 1c ). the s proteins from covs of the same group shared sequence similarities of greater than 20%, with some similarities of up to 40%. the s1 sequence similarities of covs from different groups were less than 12%. to determine the success of pseudovirus construction, the s proteins of pseudoviruses were confirmed by western blotting (figure 2a ). the pseudoviruses were quantified with an hiv-1 p24 eia kit and used to infect cells expressing the corresponding receptor. lentiviruses pseudotyped with sars-cov s protein could efficiently infect 293t cells expressing ace2, and the pseudovirus level after entry reached 10 6 relative light units (rlu) ( figure 2b ). the s proteins of hcov-229e, tgev, and pedv exhibit high homology. however, their pseudovirus infection efficiency varies, and they are approximately 50-fold less efficient than the sars-cov-s pseudovirus. the tgev-s and pedv-s pseudoviruses showed similar infection efficiencies. although hcov-oc43, bcov, and ibv utilize a sugar as their receptors, their pseudovirus infection efficiency was not clear. tgev-s and pedv-s pseudoviruses were used to infect pk-15, huh-7, vero-ccl-81, mdbk, ccl94, bsr, and mdck cells ( figure 2c ). the tgev-s and pedv-s pseudoviruses could enter pk-15, huh-7, and vero-ccl-81 cells with a similar efficiency. however, there was an intriguing finding that ccl94 cells could be infected efficiently by tgev-s but not pedv-s pseudoviruses. to further study the cellular entry of covs, we used live pedv and tgev to infect different cell lines. pedv efficiently infected vero-ccl-81 (monkey kidney), huh-7 (human liver), and pk-15 (pig kidney) cells (figure 3) . infection was not evident in mdbk (bovine kidney), ccl94 (cat kidney), bsr (hamster kidney), or mdck (canine kidney) cells. this is consistent with the data in figure 2c . tgev efficiently infected cells from pigs (pk-15), humans (huh-7), monkeys (vero-ccl-81), and felines (ccl94) (figure 4) . overall, our study demonstrates that pedv and tgev can infect cells from different species. covs recognize a variety of cell-surface molecules as their host receptors, including proteins, sugars, and heparan sulfate (li, 2013) . the cell entry mechanisms of these viruses are mediated by the viral s proteins that bind cellular receptors and mediate the fusion of viral and cellular membranes (heald-sargent and gallagher, 2012) . we demonstrated that s proteins from covs of different groups played an important role in mediating viral infection at different efficiencies. sars-cov-s pseudoviruses efficiently entered cells expressing ace2. this result is consistent with previous reports demonstrating that ace2 is a receptor for sars-cov (schwegmann-wessels et al., 2009; wu et al., 2011) . hcov-229e-s and mhv-s pseudoviruses showed strong infectivity, in part because they use proteins as a receptor belouzard et al., 2012) . moreover, pedv-s and tgev-s pseudoviruses had similar infection efficiencies in pk-15, huh-7, or vero-ccl-81 cells, and tgev-s pseudovirus-infected ccl94 cells. the sugar moiety 5-n-acetyl-9-o-acetylneuraminic acid (neu5, 9ac2), found on cell-surface glycoproteins or glycolipids, is recognized by bcov and hcov-oc43. in addition, two other types of sugars, 5-n-glycolylneuraminic acid (neu5gc) and 5-n-acetylneuraminic acid (neu5ac), can serve as receptors or co-receptors for some alpha-and gamma-covs (cavanagh and davis, 1986; krempl et al., 1997; peng et al., 2012; shahwan et al., 2013) . the s-to-sugar binding affinity is lower than the s-protein interaction, which partially explains why the bcov and ibv pseudoviruses have low entry efficiency. although pedv and tgev use the same receptor (papn) and the s proteins present high homology, live pedv and tgev prefer different cell lines; pedv prefers vero-ccl-81, huh-7, and pk-15 cells. this is consistent with a previous report (liu et al., 2015) . however, tgev prefers vero-ccl-81, huh-7, pk-15, and ccl94 cells. this indicates that pedv and tgev may have different species preferences. these results are consistent with the observation that their receptor-binding domains are located in non-homologous regions (belouzard et al., 2012) . however, the mechanism underlying different cell tropisms requires further investigation. the s protein plays a crucial role in entry into host cells by mediating receptor binding and membrane fusion. covs use a variety of receptors and triggers to activate fusion (belouzard et al., 2012) . the first and most important step of virus infection is the interaction between the virus and its cellular receptor. infection of lentiviruses pseudotyped shows that s proteins from different covs have varied abilities to mediate pseudotyped virus infection in different cell types from different tissues. these findings further our understanding of the mechanisms underlying viral invasion and contribute to the development of drugs against covs. this work was supported by the national natural science foundation of china (grant no. 31372440). this article does not contain any studies with human or animal subjects performed by any of the authors. there is no competing interest in this research. g.p. and j.w. designed the experiments and wrote the paper. j.w. performed the experiments. f.d. and q.h. contributed to critical discussions of the data. all authors approved the final manuscript. mechanisms of coronavirus cell entry mediated by the viral spike protein the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex coronavirus ibv: removal of spike glycopolypeptide s1 by urea abolishes infectivity and haemagglutination but not attachment to cells 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 ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence ccr5 antagonist td-0680 uses a novel mechanism for enhanced potency against hiv-1 entry, cell-mediated infection, and a resistant variant point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus porcine aminopeptidase n is a functional receptor for the pedv coronavirus receptor recognition and cross-species infections of sars coronavirus receptor recognition mechanisms of coronaviruses: a decade of structural studies structure of sars coronavirus spike receptor-binding domain complexed with receptor receptor usage and cell entry of porcine epidemic diarrhea coronavirus f18, a novel small-molecule nonnucleoside reverse transcriptase inhibitor, inhibits hiv-1 replication using distinct binding motifs as demonstrated by resistance selection and docking analysis contribution of the porcine aminopeptidase n (cd13) receptor density to porcine epidemic diarrhea virus infection crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor crystal structure of bovine coronavirus spike protein lectin domain coronaviruses post-sars: update on replication and pathogenesis structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies comparison of vesicular stomatitis virus pseudotyped with the s proteins from a porcine and a human coronavirus sialic acid binding properties of soluble coronavirus spike (s1) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus a single residue substitution in the receptor-binding domain of h5n1 hemagglutinin is critical for packaging into pseudotyped lentiviral particles mutational analysis of aminopeptidase n, a receptor for several group 1 coronaviruses, identifies key determinants of viral host range a virus-binding hot spot on human angiotensin-converting enzyme 2 is critical for binding of two different coronaviruses key: cord-033488-du8heorx authors: ho, thuong thi; nguyen, giang thu; pham, ngoc bich; le, van phan; trinh, thi bich ngoc; vu, trang huyen; phan, hoang trong; conrad, udo; chu, ha hoang title: plant-derived trimeric co-26k-equivalent epitope induced neutralizing antibodies against porcine epidemic diarrhea virus date: 2020-09-16 journal: front immunol doi: 10.3389/fimmu.2020.02152 sha: doc_id: 33488 cord_uid: du8heorx porcine epidemic diarrhea virus (pedv) is a causative agent of a highly infectious disease with a high mortality rate, especially in newborn piglets in asian countries resulting in serious economic loss. the development of a rapid, safe, effective and cost-efficient vaccine is crucial to protect pigs against pedv infection. the coe antigen is regarded to be a major target for subunit vaccine development against pedv infection. the naturally assembled coe protein forms a homotrimeric structure. in the present study, we successfully produced a trimeric coe protein as a native structure by fusion with the c-terminal isoleucine zipper trimerization (gcn4pii) motif in nicotiana benthamiana, with a high expression level shown via semi-quantified western blots. trimeric coe protein was purified via immobilized metal affinity chromatography (imac), and its trimeric structure was successfully demonstrated by a cross-linking reaction, and a native page gel. a crude extract containing the coe trimer was used for evaluating immunogenicity in mice. after 1 and 2 booster immunizations, the crude extract containing trimeric coe elicited elevated pedv-specific humoral responses, as demonstrated by elisa and western blot analyses. notably, a virus-neutralizing antibody assay indicated that the neutralization activities of sera of mice vaccinated with the crude extract containing coe-gcn4pii were similar to those of mice vaccinated with a commercial vaccine. these results suggest that crude extract containing trimeric coe is a promising plant-based subunit vaccine candidate for pedv prevention. porcine epidemic diarrhea (ped) is a highly infectious disease identified by dehydration, acute watery diarrhea, and a high mortality rate, especially in newborn piglets (1) (2) (3) . pedv, the disease causative agent of ped, spreads to several countries in the world, and resulting in serious economic loss to the swineproduction (4) (5) (6) . the pedv genome comprises five open reading frames (orfs) encoding four structural proteins [the spike (s), envelope, membrane and nucleocapsid proteins] and three non-structural proteins [the replicases orf1a and 1b, and orf3; (7, 8) ]. among the structural proteins, the s protein locating on the surface of pedv virion, plays a key role in the attachment of pedv to host cell receptors (9) (10) (11) . in addition, the s protein is a target for neutralizing antibody induction because it harbors virusneutralizing epitopes and is the principle antigenic determinant (8) . the s protein is naturally assembled in homotrimeric form with a number of predicted glycosylation sites (12) . the co-26k-equivalent epitope (coe epitope) is one of the various neutralizing epitopes on the s protein of pedv which have been recognized (10) . coe is the antigen epitope motif that was identified by the monoclonal antibody 2c10 at the cterminal end of the s protein (13) and the s1d domain (14) . the coe protein is regarded as a critical target for the subunit vaccine development against pedv infection (15) . the neutralizing epitope region of coe contains 139 amino acids within the s1 domain extending from amino acid 499-638 (10, 15) . coe has been expressed as monomer or pentamer structures in various plants including tobacco, rice, and lettuce (15) (16) (17) (18) (19) . mice fed transgenic plants or immunized transgenic rice calli protein extracts containing the monomeric coe protein were found to have both systemic and mucosal immune responses against the coe antigen (15, 16) . the immunogenictity tests of pentameric coe have not been tested. to date, the expression of trimeric coe as a native coe structure in plants and immunogenicity of trimeric coe in animals have not been reported. gcn4, known as the gcn4 leucine zipper, is a yeast transcription factor that is responsible for the reductive reaction of amino acid deficiency (20) . gcn4 can be switched from native dimer form to multimeric states by mutations in the a-and dpositions (21) . gcn4pii is generated by a core that was formed entirely of beta branched residues. gcn4pii has been used for the successful production of trimeric ha proteins of h5n1 viruses (22, 23) , and trimeric s protein of pedv (12) . in which, the gcn4pii was used to trigger trimerization of the proteins of interest, and increase protein stability and solubility. the development of a rapid, valid, safe, and cost-effective vaccination strategy to protect swine against pedv is urgently needed, especially in developing asian countries producing pigs. plant-based subunit vaccines have been reported with several advantages including low manufacturing cost, effortlessness of scaling, high stability and long shelf life [for a review, see (24) ]. in addition, low profit margins in the industrial vaccine development are provided from plants with beneficial and economical platforms (25) . agro-infiltration methods can offer various advantages in production of substantial amounts of recombinant proteins in short times (a few days) after completing vector construction process, and therefore, this system generally exhibits as a very fast and efficient method to produce subunit vaccines (26) . in this study, we generated a plant vector containing a dna sequence encoding the coe protein of the pedv dr13 strain fused gcn4pii as a vector model to investigate the immunogenicity of a plant-based coe antigen in mice compared to that of a commercial vaccine. interestingly, the neutralization activities of sera of mice vaccinated with the crude extract containing coe trimer were similar to those of mice vaccinated with the commercial vaccine. our first successful initial results are expected to provide an alternative strategy to generate a plantbased trimeric coe vaccine against pedv infection for national rapid response. the coe nucleotide sequence encoding for amino acid 499-638 of the coe in the s protein of the attenuated pedv dr13 strain (ncbi accession number jq023161.1) was synthesized, codon optimized commercially in tobacco (genebank accenssion number bankit2361779 optimized mt761690), and then inserted in a pez cloning vector (poch, life science missouri city, texas 77489). the coe gene was amplified from the vector, and replaced for the h5 sequence present in prtra-camv35s-h5-gcn4pii-cmyc-his-kdel (22) to via the bamhi and bsp120i sites. the resulting expression cassette ( figure 1a ) was inserted into the expression vector pcb301-kan (27) via hindiii cleavage, then transformed into the agrobacterium tumefaciens [pgv2260 in c58c1; (28) ] strain via electroporation at 2.5 kv, 25 µf capacitance, and 400 ohm resistance. to express the recombinant protein in planta, the agroinfiltration protocol was performed as described by pham et al. (29) , with some modifications. briefly, bacteria containing the expression vector pcb301-coe-gcn4pii-cmyc-his-kdel and plant vector including hcpro (23) , that was used as a gene silencing suppressor for enhancing the expression levels of targeting proteins in plants (30, 31) , were mixed and diluted in an infiltration buffer [10 mm 2-(n-morpholino) ethanesulfonic acid (mes), 10 mm mgso 4, ph 5.6]. n. benthamiana plants (5 weeks old) were completely infiltrated in an agrobacterium solution, and maintained in a greenhouse. six days after agro-infiltration, plant leaf samples were collected and stored at −80 • c. recombinant coe-gcn4pii protein was purified via immobilized metal affinity chromatography (imac) as described by pham et al. (29) . the oligomeric form of purified coe-gcn4pii protein was determined by a cross-linking reaction that was described by weldon et al. (32) and a native page. leaf samples were ground in liquid nitrogen, mixed with pbs buffer (137 mm nacl, 2.7 mm kcl, 10 mm na 2 hpo4, 1.8 mm kh 2 po4, ph 7.4). the crude extract was clarified by centrifugation twice at 13,000 rpm for 30 min at 4 • c. the coe-gcn4pii protein in leaf crude extract and a number of known concentrations of anti-tnfα-nanobody-elp standard protein [100, 200, 400, 800 ng, (33) ] were separated in a 4-12% sds-page gel and transferred to a pvdf membrane (millipore). the protein expression was determined via western blot that was performed as described by pham et al. (29) using monoclonal anti-c-myc antibody. the coe-gcn4pii protein expression level in the leaf crude extract was semi-quantified via western blotting by comparison of the western blot signal intensities and the coe sequence encoding the coe protein of pedv was fused with the c-terminal trimeric motif (gcn4pii) for generating trimeric coe protein. the recombinant protein was fused to a c-myc tag and his-tag for downstream detection by western blot and protein purification by imac, respectively. the legumine b4 signal peptide and the kdel motif were used to target protein retention in the er. camv35s pro, cauliflower mosaic virus 35s ubiquitous promoter; camv 35s term, cauliflower mosaic virus 35s terminator; asterisk, the molecular weight of proteins was calculated for unglycosylated monomers. (b) a total of 11.25 µg of total soluble proteins in crude extract containing coe-gcn4pii or wild type crude extract and a series of known concentrations of the anti-nanobody-elp standard (100, 200, 400, and 800 ng) were separated in 4-10% gel polyacrylamide. an anti-cmyc tag antibody was used as a primary antibody. hrp-linked goat anti-mouse igg was used as a secondary antibody. the coe content in the plant crude extract was determined by comparing the blot signal intensities and those of the standard protein using imagej software. (c) sds-page analysis of the purification procedure of coe-gcn4pii from total soluble protein extracts using imac; re, raw extract; ft, flow through; w, wash fraction and p, purified fraction in sds-page. (d) western blot analysis of the purification procedure of coe-gcn4pii from total soluble protein extracts using imac. coe-gcnpii was immunologically detected via an anti-his monoclonal antibody. those of the anti-tnfα-nanobody-elp standard protein using imagej software. the study was approved by the ethical committee of the institute of biotechnology, academic of science and technology vietnam (vast), hanoi, vietnam. the crude extracts after 6 days of the storage were mixed with the emulsigen r -d adjuvant (mvp technologies, 4805 g street, omaha, ne 68117, usa) with a ratio of 4:1 (v/v), respectively. three groups of 6-8-week-old female balb/c mouse (five per group) respectively numbered g1, g2, g3 were subcutaneously vaccinated at days 0, 14 and 28 with 200 µl of emulsigen r -d adjuvant-formulated crude plant extracts of non-transgenic plants as negative control) or 200 µl of the commercial vaccine against the pedv dr13 strain (4 × 10 6 tcid50/dose, ctc vacc ped, korea) as positive control or 200 µl of emulsigen r -d adjuvant-formulated crude plant extracts containing 18.76 µg of coe-gcn4pii protein that was semiquantified by western blotting. the bloods of mice were collected at seven days after the second and the third immunization via the retro-orbital sinus. all mouse sera were collected separately by centrifugation. to inactivate the non-specific complement, all mouse sera were incubated at 56 • c for 30 min before being stored at −20 • c until used. pedv propagation and purification were carried out as described by hofmann et al. (34) , with modifications. the vero e6 cell line (atcc r crl-1587tm) was propagated and incubated at 37 • c in dulbecco's modified eagle medium (dmem) including 10% fetal bovine serum (fbs) and antibiotics (100 µg/ml penicillin/streptomycin). the cells were cultured in a 5% co 2 at 37 • c. then, the pedv-dr13 strain was propagated in vero cells with 10 µg/ml trypsin treated-tosyl phenylalanyl chloromethyl ketone (tpck) (worthington, lakewood, nj, usa). after 36 h of cultivation, when all cells showed 100% cytopathogenic effects with morphological changes using cell morphology evaluation by inverted light microscopy, the infective culture fluid was harvested andfreeze-thawed three cycles. next, cellular debris was pelleted by centrifugation at 10,000 ×g for 30 min. the clarified supernatant was then enriched by ultracentrifugation at 30,000 ×g. sucrose density gradient centrifugation (20, 40, 60%) was then used to purify pedv. to detect pedv-specific igg mouse antibodies, 1 µg of purified pedv dr13 was loaded onto 3-wells of one sds-page gel. two-fold serial dilutions of serum samples after the 2nd immunization were prepared in α-minimum essential medium (mem) including 1% antibiotic-anti-mycotic solution (invitrogen, usa). then, 10 3 tcid50/0.1 ml of pedv dr13 was added with an equal volume of diluted serum, and the virus-serum mixture was maintained at 37 • c for 1 h. next, 100 µl of each virus-serum mixture was introduced onto vero cell monolayers in 96-well plates. the virus-serum mixture was removed after adsorption at 37 • c for 1 h. the plates were then washed for 10 min with pbs. finally, 200 µl of serum-free α-mem medium containing trypsin were placed into each well and maintained at 37 • c for 6 days. for controlling this assay, the virus control, positive serum control, negative serum control and blank control were used. the serum neutralization titres (sn titres) were defined as the highest serum dilution and consequent on inhibition of the cytopathic effect. statistical analyses for the elisa test and virus neutralization assay were carried out in sigma plot software using a t-test. the difference between sample data mean was compared and is showed as the x ± standard deviation (sd). p-values that were <0.05 were determined to be significantly different. the expression of the coe-gcn4pii protein in planta was successful demonstrated via separation of sds-page under reducing conditions, blotted and detected by western blot using an anti-c-myc monoclonal antibody ( figure 1b) . the apparent band shown in figure 1b with coe molecular weight was larger than the expected coe size predicted from the coe polypeptide sequence (26 kda). one of the possible reasons for the increase in the molecular weight of coe protein is that the n-glycosylation sites located within the coe-s protein of pedv at amino acids 511 and 533 may influence the electrophoretic behavior during the page separation (16, 17) . no coe protein was detected in wild type crude extract. the coe-gcn4pii protein expression level in leaf crude extract was semiquantified by western blotting. the coe-gcn4pii protein was quantified with a high expression level of ∼4% of the total soluble protein. the amount of plant-produced coe-gcn4pii protein was found to be 234 mg/kg wet weight. several publications have reported the accumulation of coe in transgenic plants (15, 17, 19) ; however, the expression level was still lower than that in our report. we demonstrated that the accumulation of coe in tobacco leaves could be significantly improved by codon optimization for plant expression by using a strong expression system, such as agro-infiltration. the purification process was validated by collecting samples from each step of the purification procedure to analyse via sds-page and western blot using a monoclonal anti-his antibody (figures 1c,d) . these results indicate the enrichment and successful purification of coe-gcn4pii protein from n. benthamiana leaves. the oligomeric state of the coe-gcn4pii protein was successfully determined by a cross-linking reaction with bs3 and a separation under native condition by native-page. a band with a molecular weight of approximately 100 kda corresponding to molecular weight of trimeric coe form was detected (figures 2a-c) . these results revealed that the trimeric coe protein was successfully generated in planta by the fusion of coe with gcn4pii motif. since animal vaccine development should minimize downstream processing in pig immunizations, the crude plant extract was chosen for testing immunogenicity in mice. interestingly, after storing the crude extract containing trimeric coe at 4 • c for six days, the coe content was still stable, as revealed by western blotting (see supplementary file) . the antibody-mediated humoral immune responses from vaccinated mice were first examined against the purified pedv dr13 strain by western blot (figure 3a) . before vaccination, pedv-specific igg antibody responses were not detected in mice. the western blots in figure 3a showed that there was a band with molecular weight of over 245 kda detected in mice groups g2 (vaccinated with the commercial vaccine against pedv) and g3 (vaccinated with crude extract containing coe-gcn4pii) that was larger than the expected size of s protein pedv (151.38 kda). the larger band size obtained in western blot might be explained that might be due to the influence on electrophoretic behavior by the 29 potential n-glycosylation sites locating within the s protein of pedv during the page separation (36) . the results indicate that pedv-specific igg antibody responses were induced in mice groups g2 and g3 after the 2nd immunization and the 3rd immunization, and the antibody responses were strongly increased in both mice groups g2 and g3 after the 3rd immunization. in contrast, no pedv-specific igg antibody response was detected in mice group g1 vaccinated with crude extracts of non-transgenic plants. different levels of pedv-specific igg, iga and igm antibodies in each mouse sera group were calculated as the reciprocals of the geometric mean titer of the five mice of each group. end-point titer of each mouse sera group was compared by the t-test. the results showed that after the 2nd immunization, crude extract containing coe-gcn4pii (g3) elicited elevated levels of igg, iga and igm antibody responses against pedv (figures 3b-d) , figure 3 | determination of the levels of pedv-specific igg, iga, and igm antibody responses via a western blot and elisas. (a) sera from five mice from each group immunized with a negative control (wild-type crude extract, g1) or a positive control (commercial vaccine, g2) or the crude extract containing coe-gcn4pii (g3) were mixed, diluted 200 times and used as a primary antibody for detecting 1 µg of purified pedv antigen. hrp-linked goat anti-mouse igg was used as a secondary antibody. in total, 500 ng of purified pedv per well was coated on a plate. each serum was measured in triplicate. the sera were serially diluted and analyzed via elisa. different levels of pedv-specific igg (b), iga (c), and igm (d) antibodies in each mouse sera group were calculated as the reciprocal of the geometric mean titer of the five mice of each group vaccinated with the negative control (wild-type crude extract, g1) or the positive control (commercial vaccine, g2) or the crude extract containing coe-gcn4pii (g3). end-point titer of igg, iga, or igm antibodies against pedv of each mouse sera group was compared using the t-test (sigmaplot), and is presented. *p< 0.05 was defined as a statistically significant difference. reaching end-point antibody titres of 1:216 178, 1:45 429, 1:37 801, respectively. the levels of igg, iga and igm antibody responses in mice group g3 were strongly enhanced after the 3rd immunization, having end-point antibody titres of 1:383 711, 1:195 985, 1:65 198, respectively. notably, no statistically significant difference in pedv-specific igg antibody responses was obtained between mice group g3 and those in mice group g2, with p-values of 0.056 after the third immunization (p < 0.05). therefore, plant crude extract containing the trimeric coe protein had the level of igg antibodies similar to that of commercial vaccines against the pedv dr13 strain after the third injection. however, level of pedv-specific iga and igm antibody responses in mice group g3 were lower than those in mice group g2 after the second and the third immunization. levels of igg, iga and igm antibody responses against pedv found in the sera of negative control mice group g1 were very poor. the cytopathic effect caused by the wild-type pedv dr13 virus of all serum samples was determined using a microscope and a representative cytopathic effect result observed under microscope of a single dilution of a serum from each group was presented (figure 4a) . the highest dilutions of sera that caused cytopathic effect inhibition were defined as the serum figure 4 | virus neutralization assay. (a) cytopathic effect caused by wild-type pedv dr13 virus at the dilution of 1:32 of a single serum from a mouse from the groups vaccinated with the negative control (wild-type plant extract, g1) or the positive control (commercial vaccine, g2) or the crude extract containing coe-gcn4pii (g3) was determined by using a microscope. (b) determination of serum neutralization titer. serum samples were firstly incubated to inactivate for 30 min at 56 • c. two-fold serial dilutions (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256) were applied. a pedv dr13 strain (200 µl 10 3 tcid50/0.1 ml) was mixed with an equal volume of diluted sera. the virus control, positive serum control, blank control, negative serum control, and were used for controlling the assay. the serum neutralizing titres (sn titres) were expressed as the highest serum dilution and consequence on the inhibition of cytopathic effect. statistical analyses were performed using the t-test (sigmaplot) and are shown. a single dot indicates the sn value of a single mouse serum. the sd is included on a single dot that corresponds to the sn data variation of a single mouse serum with three replications. the bars indicate the average value of the test groups. *p < 0.05 was defined as a statistically significant difference. neutralizing antibody titres. the serum neutralizing antibody (sn) titer of every single mouse serum was presented in each dot ( figure 4b ). as expected, cytopathic effect inhibition of sera from the negative control group was observed in a low serum dilution, which produced neutralizing geometric mean titres of this group as low as 1. in contrast, sera of mice vaccinated with the crude extract containing coe-gcn4pii and the commercial pedv vaccine (the positive control) had the ability to neutralize pedv with high neutralizing geometric mean titres: 57.6 and 61.86, respectively. these titres were significantly different from those of the negative control group. notably, the serum neutralization titer in the crude extract containing coe-gcn4pii (g3) was not significantly different compared to that of sera from mice vaccinated with the commercial vaccine, with a p-value of 0.187. in this study, the immunogenicity in mice group g3 vaccinated with the coe trimer in crude extract was compared to those of mice positive control group g2 vaccinated with commercial pedv vaccine and mice negative control group g1 vaccinated with crude extracts from non-transgenic plants. as expected, western blot results demonstrated that pedvspecific igg antibodies were presented in mice group g3 and mice group g2 after the second and the third immunization. more interestingly, the elisa analyses illustrated that crude extract containing coe-gcn4pii (g3) elicited strong levels of igg antibody responses against pedv, and especially the level of pedv-specific igg antibody responses found in mice group g3 was similar to that in mice group g2 after the third immunization. mucosal immune responses play an important role in the defense of pedv, and mucosal iga antibody response was found to correlate with the protection against pedv infection (37, 38) , however igg antibody-mediated humoral response is also essential to protect the neonatal pig against pedv infection (37, 39) . early publications showed the important role of igg in protection of gastrointestinaltract (40, 41) . in this study, trimeric coe antigen (g3) was vaccinated in mice via subcutaneous route that is a conventional vaccination route widely used for various human and animal vaccines to elicit igg antibody-mediated humoral responses. interestingly, beside pedv-specific igg, pedv-specific iga and igm antibodies were presented in mice group g3, however they were lower as compared to those found in mice group g2. our results are comparable to several recent evidence studies that subcutaneous route can induce both antigenspecific igg antibody, and antigen-specific iga and igm antibodies in sera (42) (43) (44) (45) (46) . moreover, su and his co-workers proposed that under some circumstances (antigen, adjuvant, delivery vehicle) systemic routes may induce systemic immune responses and muscosal immune responses against infectious diseases (46) . in addition, the ability of the neutralizing antibodies induced in mouse sera to neutralize pedv via binding to coe epitopes related to pedv neutralization was further determined. after virus neutralization, there was an inhibition generated by neutralizing antibodies to the virus's infection cycle, containing surface binding, fusion, entry, endocytosis, and replication (47) . the assessment of pedv-neutralizing activity illustrated that there was no statistically significant difference between the serum neutralization titres of mice vaccinated with the crude extract containing trimeric coe (g3) and that of mice vaccinated with the commercial vaccine (g2), with a p-value of 0.187. therefore, we concluded that plant crude extract containing the trimeric coe protein had a strong immunogenicity and induced a neutralizing antibody titer similar to that of the commercial vaccine against the attenuated pedv dr13 strain. the enhancement of neutralizing antibody responses induced in animals after vaccination is an important requirement for vaccine development due to the powerful correlation of vaccine efficacy with neutralizing antibodies for numerous commercial vaccines (48) . the crude extract containing trimeric coe (g3) showed neutralizing activity, with a geometric mean titer of 1:57.6. a high neutralizing antibody titer indicated that mice subcutaneously administered the crude extract containing trimeric coe possessed a strong ability to neutralize pedv. to increase the immune response, especially mucosal immune responses against pedv, oral mucosal vaccination with coe but not subcutaneous administration in animals has been previously presented to induce anti-pedv mucosal immune responses (15, 49) . in addition, since pedv causes mainly intestinal infections, the coe antigen was fused with dendritic cell-targeting peptide (dcpep) and m celltargeting peptide (col) for targeting intestinal microfold (m) cells and dendritic cells (dcs) (15, 49, 50) . the neutralizing activity mouse sera orally provided genetically engineered lactobacillus but not plant extracts expressing coe targeting m cells or dcs or both has been previously reported (49, 50) . when compared to the publication of ma and coworkers, the neutralizing antibody titres obtained by oral administration in mice with the recombinant lactobacillus casei strains expressing the pedv coe antigen on the cell surfaces by fusion with dcpep or col or both dcpep and col were 1:24, 1:24, and 1:36, respectively (49) , which were lower than the neutralizing antibody titres observed in this study. these results showed that crude extract containing trimeric coe can be a promising vaccine candidate against pedv infections. in summary, the trimeric coe protein was successfully produced in plants with high expression levels. crude extract containing trimeric coe elicited strong humoral immune responses and elevated neutralizing antibody titres against pedv. in particular, the neutralizing activities of mice vaccinated with the crude extract containing coe-gcn4pii were similar to those of mice vaccinated with the commercial vaccine. these results suggest that crude extract containing trimeric coe might be a potential subunit vaccine antigen against pedv infection. further studies will focus on investigating immune efficacy and protection against pedv in piglets. all datasets presented in this study are included in the article/supplementary material. the animal study was reviewed and approved by the principles of the basel declaration and recommendations of arrive guidelines, ethical committee of institute of biotechnology, academic of science and technology vietnam (vast), hanoi, vietnam on the use of animals for research. hc, np, and th designed the research. th and gn constructed vectors and performed transient expression. th purified protein and performed the cross-linking reaction, performed elisa and western blotting analyses, and performed the calculations, all data analysis and wrote the manuscript. vl and tt purified the pedv dr13 strain and carried out the 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immunity through systemic immunization: phantom or reality occupancy and mechanism in antibody-mediated neutralization of animal viruses maternal antibodies, childhood infections, and autoimmune diseases oral recombinant lactobacillus vaccine targeting the intestinal microfold cells and dendritic cells for delivering the core neutralizing epitope of porcine epidemic diarrhea virus oral delivery of probiotics expressing dendritic cell-targeting peptide fused with porcine epidemic diarrhea virus coe antigen: a promising vaccine strategy against pedv all authors contributed to the article and approved the submitted version. this study was financed by vast through the project: study on the expression of s1 oligomer of porcine epidemic diarrhea virus (pedv) in nicotiana benthamiana for the next generation of vaccine development, listed in scientific program: biotechnology, code: vast02.02/18-19. we would like to thank dr. song daesub for supplying the pedv dr13 strain. we also appreciate dr. do thi thao from ibt, hanoi, vietnam for the mouse experiments. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.02152/full#supplementary-material key: cord-282466-r2sjv9ih authors: antas, marta; woźniakowski, grzegorz title: current status of porcine epidemic diarrhoea (ped) in european pigs date: 2019-10-24 journal: j vet res doi: 10.2478/jvetres-2019-0064 sha: doc_id: 282466 cord_uid: r2sjv9ih porcine epidemic diarrhoea (ped) is a highly contagious and devastating enteric disease of pigs caused by porcine epidemic diarrhoea virus (pedv), an enveloped, single-stranded rna virus belonging to the alphacoronavirus genus of the coronaviridae family. the disease is clinically similar to other forms of porcine gastroenteritis. pigs are the only known host of the disease, and the occurrence of ped in wild boars is unknown. the virus causes acute diarrhoea, vomiting, dehydration, and high mortality in suckling piglets reaching 100%. heavy economic losses in the pig-farming industry were sustained in the usa between 2013 and 2015 when pedv spread very quickly and resulted in epidemics. the loss in the us pig industry has been estimated at almost seven million pigs. the purpose of this review is a description of the current status of porcine epidemic diarrhoea in european pigs and the risk presented by the introduction of pedv to poland in comparison to the epidemics in the usa. coronaviruses (covs) cause a large variety of diseases in humans and animals. in pigs, coronaviruses affect various organs, including the gastrointestinal and respiratory tracts. covs have one of the largest genomes of all rna viruses, which in combination with their high genetic diversity causes mutation and recombination, resulting in new virus variants (24) . besides porcine epidemic diarrhoea virus (pedv), the porcine coronaviruses comprise transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), porcine haemagglutinating encephalomyelitis virus (phev), and porcine deltacoronavirus (pdcov) (26) . the clinical symptoms of ped are diarrhoea, vomiting, and dehydration which result in very high mortality among suckling piglets and large economic losses (5, 8, 21) . various factors influence the clinical signs of ped, mainly the age of the animals, the herd's immune status, and the virulence of the strain (18) . multiple pedv strains are circulating on different pig farms around the world and they differ in virulence. ped was observed for the first time in 1971 in the united kingdom. the virus only infected fattening pigs and sows. subsequently, the disease spread to other european countries. emergence of a new pedv strain in china in 2010 caused serious epidemics and this strain rapidly spread worldwide. in the usa, highly virulent strains infected pigs in 31 states between 2013 and 2015, resulting in significant economic losses (11, 12, 30) . the disease was characterised by rapid diarrhoea onset in pigs of all ages and mortality in suckling piglets reaching 95% (30) . in europe, ped re-emerged in 2014 in germany. subsequently, highly similar strains were also found in several european countries (19, 36) . ped is not a notifiable disease in the european union and is not on the list of diseases reported to the world animal health organization (oie), therefore the status of this disease is not fully recognised. in the last 10 years, only a few countries have reported clinical cases of ped and/or seropositive animals (4). coronaviruses belong to the coronavirinae subfamily. currently, this subfamily is classified into the four genera alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, which fall into distinct groups on the phylogenetic tree. (5, 11, 26) . the taxonomy of swine coronaviruses is shown in table 1 . the coronaviruses are pleomorphic and have one of the largest rna viral genomes and a unique replication strategy (28, 33) . virions are spherical with diameter of approximately 125 nm. the most prominent and the defining feature of coronaviruses are the spike structures on the surface of the virion, which give them the appearance of a solar corona (5) . the pedv genome is single-stranded rna (28,000 nucleotides in length) of positive-sense polarity containing 3′ and 5′ untranslated regions (utr) at both ends. two thirds of the genome from the 5′ end encodes proteins necessary for rna replication, while the one third on the 3′ side of the genome comprises at least seven open reading frames (orfs) that encode four structural proteins: n (capsule), s (spike), e (envelope), and m (membrane), and three nonstructural proteins (replicases 1a and 1b and orf3). in the genome, they are arranged in the order 5′-replicase (1a/1b)-s-orf3-e-m-n-3′. the rna genome of the virus is associated with the n protein to form a long, helical ribonucleoprotein (rnp) complex. the virus core is enclosed by a lipoprotein envelope, which contains the s, e, and m proteins ( fig. 1) (1, 17, 28, 37) . the m protein is the most abundant component in the viral envelope, is necessary for the assembly process, and affects production of protective antibodies with virus-neutralising activity. the small e protein plays an important role during coronavirus budding. the n protein has multiple functions in viral replication, interacts with viral genomic rna, and associates with other n proteins to protect the viral genome. it disturbs antiviral responses by antagonising interferon production (17, 31) . the s protein is encoded by a gene sequence which determines the virulence of a pedv strain (35) . this protein is composed of 1,383 amino acids (aa), significant among which are a signal peptide (1-18 aa), neutralizing epitopes (499-638, 748-755, 764-771, and 1,368-1,374 aa), a transmembrane domain (1,334-1,356 aa), and a short cytoplasmic domain. it consists of the s1 (1-789 aa) and s2 (790-1,383 aa) domains, which are responsible for binding and fusion of the virus. the s1 domain is involved in a specific interaction with the cellular receptor and induction of neutralising antibodies (14, 28) . only one serotype of pedv has been reported. the phylogenetic analysis of the s gene indicates that pedv could be genetically divided into two groups, these being the classical genotype 1 (gi) and the field epidemic or pandemic genotype 2 (gii). the early european cv777 strains (genbank accession number af353511), vaccine strains, and strains adapted to cell cultures were classified to genotype 1, while genotype 2 includes strains responsible for the epidemics in the usa and asia. strains identified in the usa have been (17, 18, 25, 31) . all pedv pandemic strains circulating in china after 2014 were also clustered to genotype 2, and they were genetically separated from other global pedv strains and from earlier chinese strains (29) . isolation of pedv from field samples is very difficult. the virus grows in vero cells (african green monkey kidney). however, its growth there requires the presence of trypsin-supplemented cell culture medium, because this protease plays an important role in cell entry and the release of pedv virions in vero cells. trypsin does this by cleaving the s protein into s1 and s2 subunits, and it enables efficient replication and spread of pedv in vitro (18, 29) . pedv adapted to the vero cell line can be successfully propagated in other cell types. ped viruses cause cell death leading to lysis of infected cells, a change observable under the microscope as a cytopathic effect (cpe) which is characterised by cell membrane vacuolisation and syncytium formation (13, 15, 24) . the ped virus can survive for variable periods outside the host, depending on the temperature and relative humidity. the virus is stable in temperatures ranging from 4°c to 50°c but loses its infectivity above 60°c (24) . survival of the ped virus in different samples is presented in table 2 . pedv is easily inactivated by heating to 71°c for 10 min. at lower temperatures, ph is a factor in pedv inactivation. the resistance of ped virus in different ph values is given in table 3 . pedv-inactivating disinfectants are oxidising agents, bleach, 2% phenolic compounds, 2% sodium hydroxide, formaldehyde and glutaraldehyde, 4% sodium carbonate, ionic and nonionic detergents, iodophors in 1% phosphoric acid, and lipid solvents such as chloroform (15, 24) . direct transmission by the faecal-oral route may also occur through ingestion of virus-contaminated vomit. indirect transmission occurs through contaminated feed trucks, service vehicles, vehicles used for the movement of pigs, and people (pig owners and farm visitors). until the introduction of pedv to the usa, feed for animals was not considered to be a vector in the spread of the virus. recent studies have shown, however, that feed can be a potential vector for pedv transmission and contaminated feed can be a source of disease (27) . it is widely acknowledged that the most important risk factors for spreading the disease are contaminated vehicles (5, 28) . oral ingestion causes viral replication in the epithelial cells of the small intestinal villi and to a lesser extent in the colonic villi, which results in degeneration of enterocytes and shortening of the villi (6, 24) . pedv can infect pigs of all ages, causing watery diarrhoea and vomiting with anorexia and dehydration, which is the major cause of death in young piglets. the clinical signs depend on the age of the pigs, immune status of the herds, and virulence of the strain (1, 4, 20) . lesions are observed in the gastrointestinal tract and are characterised by a distended stomach filled with completely undigested milk dots and thin, transparent intestine walls (34) . pedv infection is clinically indistinguishable from other forms of porcine gastroenteritis diseases such as those caused by tgev and pdcov, therefore pedv diagnosis cannot be made only on the basis of clinical signs and has to be confirmed by laboratory tests to make diagnosis final (7, 24) . a variety of pedv detection methods are applied which include immunofluorescence (if) or immunohistochemistry (ihc) tests, in situ hybridisation, virus isolation, enzyme-linked immunosorbent assays (elisa), and various reverse transcription polymerase chain reaction (rt-pcr) techniques. samples which can be used in laboratory diagnosis are fresh faeces, oral fluids, small intestine tissue, and serum (to determinate the presence of antibodies). to detect pedv rna, rt-pcr can be used for diagnosis of acute outbreaks no longer than 14 days after the onset of the disease. for surveillance and monitoring of ped, serological diagnosis is necessary. antibodies persist for more than one year in the serum of infected pigs (1, 13, 24) . the regenerative ability of villi is instrumental in the recovery of the pig. regeneration speed depends on age of the animals; in adult and fattening pigs, villi are restored in three to four days, while in piglets the process is longer at six to seven days (4) . the percentage of morbidity in the course of infection can climb to 100%. mortality is variable and also depends on the age of animals. in suckling piglets, mortality can attain 100 %, in piglets older than 10 days, it is less than 10%, and in adult and fattening pigs, it falls below 5%. there is no specific treatment other than symptomatic treatment of diarrhoea, however, most growing pigs recover without treatment within 7-10 days unless secondary infections occur. reinfection may occur when the immunity wanes (9, 16) . ped was first observed in the united kingdom and belgium in early 1970s (1) . the disease caused mortality of about 3% in fatteners and adult pigs. suckling piglets were not affected and remained symptom-free even when the sows had watery diarrhoea for several days. at the beginning, the disease was incorrectly diagnosed as tge because the symptoms of these two diseases are almost identical. at a later stage, tge was excluded by laboratory diagnosis. in 1976, new cases of the disease were described in the uk. this outbreak was different in that the virus affected pigs of all ages, including neonatal and suckling piglets, inflicting around 30% mortality. pedv was definitively identified for the first time in 1977 in belgium and was classified to the coronaviridae family, recorded as the cv777prototype strain (19, 22) . in the 1980s and 1990s, pedv was identified as the cause of severe epidemics in japan and south korea (17) . in the same period in europe, outbreaks of ped appeared sporadically but the virus continued to spread and persisted in an endemic form in the pig population. outbreaks of ped were observed in the netherlands in 1989-1991, in hungary in 1995, and in the uk in 1998. typical epidemic outbreaks of ped with high mortality in neonatal piglets were also identified in italy in [2005] [2006] and china in 2010-2012 (8, 32) . the first symptoms of the disease in the usa were observed in april 2013 and were confirmed in the laboratory in may 2013. the ped virus had escaped from biosecurity and control systems, spread very quickly to other areas, and infected hundreds of farms. by march 2015, the presence of pedv had been confirmed in 31 us states. pedv epidemics resulted in significant economic loss there, and the domestic pig industry lost almost 10% of its population (seven million pigs) (3, 12) . the ped outbreak was characterised by watery diarrhoea, dehydration, and variable vomiting. all groups of animals were affected by the epidemics, but the highest mortality was 95% and occurred among suckling piglets (10, 15) . the strains isolated in the usa were genetically related to the chinese pedv strains reported in 2012. pedv was probably dispersed in the usa mainly through contaminated trucks, but other factors that assist the spread of the virus cannot be excluded (15) . two strains of pedv have been identified in the usa: highly virulent non-indel (usa/kansas29/2013) and the milder variant s-indel (usa/oh851/2014) (2) . both variants were classified to genotype 2. in experimental infections, s-indel strains showed lower pathogenicity and mortality (from 0% to 70%) than the non-indel strains, where the mortality rate was up to 100% (2, 30, 36) . since the ped outbreak in the usa, detection of highly virulent strains has also been reported in canada, mexico, taiwan, south korea, japan, and ukraine. detection of s-indel strains has been reported in most european countries (2, 12, 17) . ped is not notifiable in the european union and is not on the list of diseases reported to the world animal health organization (oie), therefore, it is not possible to accurately describe the occurrence of this disease in europe. in the last 10 years, only a few countries have reported clinical cases of ped and/or seropositive animals (4) . the ped outbreaks in europe were significantly different from epidemics in the usa or asia. after the epidemics in 2013 in the usa, ped cases were also confirmed in austria, belgium, france, germany, italy, the netherlands, portugal, and slovenia ( fig. 2) (2, 18, 21) . viruses identified in these european countries were very similar to usa/oh851/2014 strains. in germany ped emerged in 2014, inflicting up to 70% mortality and typified by acute symptoms (30) . in slovenia ped was confirmed in the laboratory in 2015 (23) . at almost the same time, highly virulent strains were described in ukraine. these viruses were similar to usa/kansas29/2013. so far, ukraine is the only european country where highly virulent strains have been identified. nevertheless, taking into account the occurrence of a virulent strain in ukraine and its rapid spread, it is likely that epidemics similar to those in the usa may also occur in europe (17, 30) . considering the presence of pedv in countries neighbouring poland (germany, the czech republic, and ukraine) and the rapid spread of the virus, it is highly probable that pedv will also be introduced to poland. ped is a rapidly spreading global disease and causes large economic losses in pig production around the world. frequently appearing mutations cause variability in its viral genome and can change the pathogenicity of the new pedv variants. biosecurity is a central to the prevention or spread of pedv. applying the principles of biosecurity protocols, it is possible to reduce the risk of introducing the virus into swine herds. disinfection of vehicles entering farms is highly effective because contaminated faeces or vomit can be found on the wheels. in poland, the clinical symptoms of ped have been observed, but until 2014 no studies were conducted to confirm the presence of pedv. in 2015-2017, the presence of the virus and/or of specific antibodies was confirmed on several farms in poland. samples of blood, faeces, slurry, and intestines were collected in herds in which the clinical symptoms of the disease had previously been observed. so far, virus isolation in vero cells has not been carried out in poland and nor has genetic material from positive samples been sequenced. it is foreseen that pedv might be transferred in the future to polish swine herds. the severity of ped which occurred in the usa has shown that this disease should be countered with timely preventive measures and early diagnosis. therefore, the role of the national veterinary research institute in puławy, poland, in the diagnosis of ped is of paramount importance in future pig production. the authors declare that there is no conflict of interests regarding the publication of this article. porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus pathogenesis comparison 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10.1007/s13337-019-00513-w sha: doc_id: 272480 cord_uid: 276r1lh7 porcine epidemic diarrhea virus (pedv) is a highly infectious virus infecting pigs with high morbidity, especially for newborn piglets. several pedv strains were isolated from the intestinal tracts of diarrheic piglets from the beijing area, china. sequencing of the whole-genome of the pedv isolates (genbank numbers mg546687-mg546690) yielded sequences of 28033–28038 nt. the phylogenetic tree revealed that these strains from the beijing area belonged to group ii, while the vaccine strain, cv777, belonged to group i. we also determined the genetic correlation between these strains and cv777 strain. however, it showed that these strains in the beijing area had unique mutations. the sequence identity of pedv strains showed that these strains are most similar to these strains lzw, ch/jx-1/2013, usaillinois972013, usakansas1252014, ch/gdzq/2014, shqpym2013, aj1102, chzmdzy11, koreak14jb01, and chyj130330, respectively. the possible recombination events indicate that pedv in this studies were possibly recombinant strain formed by parent strains usaillinois972013, koreak14jb01, chyj130330, and chzmdzy11. these pedv strains has been genetic recombination and mutations. the variant strains characterized in this study help to the evolutionary analysis of pedv. porcine epidemic diarrhea (ped) is a highly infectious disease of swine. it causes severe watery diarrhea, vomiting, dehydration and death in suckling and nursing piglets that are typically 1-2 weeks of age [1, 2] . porcine epidemic diarrhea virus (pedv) is a member of the coronaviridae family [3, 4] . the virus strain was first isolated in belgium in 1973 and designated coronavirus cv777 [1] . there were only a few reports in the following decades [5] [6] [7] . however, a new pedv strain has emerged in asia since 2010 [7] [8] [9] [10] . in china, the positive rate of pedv in some provinces was over 50% in 2010-2011 [7] . in usa, classical pedv first identified in the state of ohio in 2013, and a variant pedv was reported in 2014 [11] . in japan, more than 490,000 pigs died of ped from 2013 to 2015 [12] . in korea, ped outbreaks reoccurred in 2013 [13, 14] . in europe, isolates were reported from the ukraine, belgium, holland, france, germany, portugal and italy from 2013 to 2015 [15] . these isolates were closely related to those strains reported from north america [11, 15] . diarrhea disease broke out in some large-scale pig farms in the beijing area of china from 2015 to 2016. the newborn piglets had high mortality rates, resulting in heavy loss to pig farms. the cv777 vaccines used failed. pedv strains were isolated and identified from the small intestine of diarrhea samples obtained at these pig farms. whole genome sequencing and recombinant analysis was performed, with the aim of examining the genomes for viral mutations and recombination. the molecular epidemiological information of pedv can help develop efficient diagnostic reagent and vaccine. intestine samples of dead piglets with diarrhea were collected from several piglets infected with pedv from 2015 to 2016. these infected piglets were chosen from 4 different large-scale swine farms located in beijing and hebei provinces in china. pedv detection were positive via rt-pcr. for total rna extraction, samples were frozen with liquid nitrogen. all procedures were approved by the animal care and use committee of beijing university of agriculture (beijing, china). the fresh intestine samples were collected under sterile conditions, made then into slurry, filter sterilization. vero e6 cells were washed with pbs twice. after adsorption for 60 min at 37°c, pedv growth medium was added [16] . the vero e6 cell cultures were observed for 4 days for cytopathic effects (cpe). total rna was obtained from intestine samples using trizol (invitrogen, usa), per the manufacturer's instructions (promega, usa), and was used for cdna synthesis. pcr was performed to amplify the target gene using specific primers (reference sequence number: nc003436.1), as shown in table 1 . pcr reaction contained primers, cdna, dntp mix and hifi polymerase (transgen, china) in 19 hifi buffer. pcr products were sequenced from both ends by sangon biotech co. ltd. the coding sequences for pedv were deposited in the gen-bank database under accession numbers mg546687-mg546690. nucleotide and amino acid sequences of pedv were aligned by the clustalw method. sequence alignments of the whole genome and construction of trees were also performed by the neighbor-joining method in mega7 software. information of the other strains that were chosen in this study, including years and locations of isolation and genbank accession numbers proposed, are shown in table 2 . comparison and analysis of the whole pedv genome sequences and the reference pedv strains were conducted using megalign programs (dnastar, madison, wi). the putative recombinant sequence of these strains in this study and its parental strains were conducted using recombination detection program (rdp) version 4.97 with seven recombination detection methods (rdp, gene-conv, chimaera, maxchi, bootscan, siscan, and 3seq). in order to improve the reliability of detection, the recombination breakpoints through at least 5 of these methods was taken as confirmatory for any putative recombination event (p value is 0.01). the phylogenetic tree of recombination and non-recombination were constructed to verify the reliability of these results. vero e6 cells were used for viral multiplication. cytopathic effects (cpe) appeared 48 h after inoculating virus, and more than 70% cpe with cell clustering, detachment, and syncytium formation were observed at 72 h. these results indicated that the isolated pedv could cause pathological changes in vero e6 cells (fig. 1 ). the organization of the genome of pedv was characteristic of the gene order 5 0 -orf1a/1b-s-orf3-e-m-n-3 0 [2, 4, 17, 18] . sequencing of the complete gene of the four isolates (genbank numbers: mg546687-mg546690) yielded sequences of 28033-28038 bp, which compared with 18 typical strains available in genbank ( table 2 ). the phylogenetic tree showed that these 22 pedv strains divided into two groups (fig. 2 ). the sequence identity between pedv strains in this studies and typical strains in terms of their according genes or regions were calculated. the highest percentage of nucleotide identities of 5 0 -utr, orf1a/1b, s, m, n, e, orf3, and 3 0 -utr of pedv compared with other pedv strains were show in table 3 . for orf1a/1b gene, these strains are most similar to these strains, lzw, shqpym2013, aj1102, ch/ynkm-8/2013, ch/jx-1/ 2013, usakansas1252014, and chzmdzy11, respectively. for s gene, these strains are most similar to these strains lzw, koreak14jb01, usaillinois972013, usa-kansas1252014, and ch/gdzq/2014, respectively. the percentage of nucleotide identities of s of pedv strains in this study compared with recombined strains secov is 90.4-90.7%. for m gene, these strains are most similar to these strains usaillinois972013 and usakansas1252014. (table 3) . the names of the strains, years and places of isolation, as well as genbank accession numbers proposed, are shown in table 2 . the strains in this study are indicated by underline isolation and recombinant analysis of variants of porcine epidemic diarrhea virus strains from… 297 frequent mutation and recombination of genes may occur in epidemics of pedv. in order to detect these possible recombination events, 19 reference strains were used as parent strains for rdp, geneconv, chimaera, maxchi, bootscan, siscan, and 3seq program analysis. the results showed that pedv in this study were possibly recombinant strain formed by parent strains usailli-nois972013, koreak14jb01, chyj130330 and chzmdzy11 (fig. 3) . usaillinois972013 is major parent of pedv strain ch/bj9/2015, the recombination region locates on 4669-8176nt; koreak14jb01 is minor parent of pedv strains ch/bj2/2016 and ch/bj11/2016, the recombination region locates on 21744-27721nt; chyj130330 is major parent of pedv strain ch/hb9/ 2016 and chzmdzy11 is minor parent of ch/hb9/2016, their recombination region locates on 2218-15942nt. however, there may be a deviation in the prediction of software. it requires further confirms these recombination events. since 2011, a large number of piglets died as a result of pedv outbreaks, causing much economic loss in china [2-4, 7-10, 18-21] . in this study, the deceased piglets that provided intestine samples had been immunized with the cv777 vaccine previously. this showed that the classic vaccine strain exhibited lower protective efficacy against the variant pedv [22, 23] . the phylogenetic trees showed that, these pedv isolates from the beijing area belonged to group ii, while the traditional vaccine strain, cv777, belonged to group i based on the comparison of several important genes in pedv, such as orf1a/1b, s, m, n, e, and orf3. however, n, as a conserved gene, was inconsistent with the above results, which also suggested that the n sequence or protein is suitable for universal detection. the orf1a/1b gene were two long orfs which encoding the non-structural replicase polyproteins of pedv. the strain from 2015 in the beijing area shared the greatest homology in orf1a/1b with lzw, which was also isolated in beijing; it showed the continuity of heredity. the s sequence of isolates in 2015 in the beijing area showed a closer relationship with a previous isolate, lzw. other isolates in 2016 in the beijing area showed a closer relationship with previous isolates in guangdong. this revealed that s genes accumulated variations faster based on inherent genetic characteristics or recombinant genes from other isolates [10, 20, 24] . the 1-1180 bp section in the s1 region of pedv genes is prone to mutate [24] . sequence analysis revealed that isolated pedv strains and these variant pedv strains were reported had insertion in 167-178 sites (gtgaaaac-cagg) and 421-423 sites (tga), also deletion in 218 (a), 471-474 (atat) and 481-482 (tg) comparing with cv777. these insertion, deletion, or introducing stop codon (tga) of sequence resulted in frameshift mutation and abnormally transcripts of s1 gene. it is a reason for failed vaccination with cv777 vaccine. however, different from other recent isolates, these strains in the beijing area had shared sites with cv777 in the s1 region, such as a5g (this specific nucleotide change led to the amino acid change k-r), c70t, and t1043c (which led to the amino acid change f-s). this determined the genetic correlation between these strains in the beijing area and the cv777 strain. it was remarkable that e sequences of isolates in the beijing area had distinct differences from other isolates. there were unique variations in the e gene in these strains, such as t27g. this showed that these isolates from the beijing area had unique mutations. this might provide a gene reference for other mutations of pedv in the future. viral gene recombination and mutation results in evolution [25] . gene recombination and mutation often occurs in coronavirus, such as pedv, sarscov, and infectious bronchitis virus (ibv) [26] [27] [28] [29] . there is evidence that the s gene of epidemic pedv strains and genome of transmissible gastroenteritis virus (tegv) strains have recombined when both were present in the same host to form a new recombinant porcine coronavirus, secov [30, 31] . this s sequence of secov strain share above 90% homology with s sequence of pedv strains in this study. however, analysis of the recombination of pedv strains showed that, no recombination event was occurred between secov strain and pedv strains in this study. these pedv strains isolated in beijing area shows recombination events with pedv isolated in usa, pedv isolated in korea, and pedv isolated in china in sequence. it suggests the accelerated spread of pedv virus. viral recombination and mutation causes immune failure. the new and effective vaccine is critical to control the occurrence of pedv. the study of the effective vaccine must be based on the characteristic analysis of variant viruses [32] . the new data on the evolutionary analysis of pedv is provided in this study. these data will be helpful for research on pedv vaccine and control. several pedv strains were isolated from the intestinal tracts of diarrheic piglets from the beijing area, china. these pedv strains has been genetic recombination and mutations. the variant strains characterized in this study help to the evolutionary analysis of pedv. a new coronavirus-like particle associated with diarrhea in swine molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field strains in south china sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china whole-genome analysis of porcine epidemic diarrhea virus (pedv) from eastern china an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in 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novel strain with a large deletion in the spike gene molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea isolation, molecular characterization and an artificial infection model for a variant porcine epidemic diarrhea virus strain from jiangsu province, china evolutionary characterization of the emerging porcine epidemic diarrhea virus worldwide and 2014 epidemic in taiwan detection and phylogenetic analysis of porcine epidemic diarrhea virus in central china based on the orf3 gene and the s1 gene characterization of chinese porcine epidemic diarrhea virus with novel insertions and deletions in genome porcine epidemic diarrhea: a review of current epidemiology and available vaccines cross protective immune responses in nursing piglets infected with a us spike-insertion deletion porcine epidemic diarrhea virus strain and challenged with an original us pedv strain s1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen epidemiology, genetic recombination, and pathogenesis of coronaviruses recombination in avian gamma-coronavirus infectious bronchitis virus evolution of infectious bronchitis virus in china over the past two decades evolution and variation of the sars-cov genome viral evolution and the emergence of sars coronavirus porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus characterization of a novel chimeric swine enteric coronavirus from diseased pigs in central eastern europe in 2016 evaluation of a recombination-resistant coronavirus as a broadly applicable, rapidly implementable vaccine platform acknowledgements the authors would like to thank the pig farm owners their collaboration.funding this work was supported by the national natural science foundation of china (no. 31572499). the funders did not play any role in the design, conclusions or interpretation of the study. the authors declare that they have no competing interests.ethics approval and consent to participate experimental animals used in the study were cared for in accordance with the internationally accepted standards for the care and handling of laboratory animals. all procedures were approved by the animal care and use committee of beijing university of agriculture (beijing, china). the animal ethics committee approval number was buaacuc2016-1. all samples were collected after informed written consent was obtained from the owners. key: cord-292734-2g2ym81n authors: jung, kwonil; kang, bo-kyu; lee, chul-seung; song, dae-sub title: impact of porcine group a rotavirus co-infection on porcine epidemic diarrhea virus pathogenicity in piglets date: 2008-06-30 journal: research in veterinary science doi: 10.1016/j.rvsc.2007.07.004 sha: doc_id: 292734 cord_uid: 2g2ym81n abstract porcine epidemic diarrhea virus (pedv) and porcine group a rotavirus (pgar) are the main causative agents of acute diarrhea in piglets. in south korea, pgar is prevalent in piglets naturally infected with pedv. piglets naturally co-infected with pedv and pgar appeared to have severe and prolonged diarrhea that was distinct from that commonly observed. the aim of this study was to determine the impact of pgar co-infection on pedv pathogenicity in piglets. thirty-six colostrum-deprived, one-day old, large white-duroc crossbred pigs were randomly divided into four equal groups: pedv, pedv/pgar, pgar, and control groups. the piglets were euthanized at 1, 2, or 3 days post-inoculation (dpi) to measure the villous height:crypt depth (vh:cd) ratio and to collect fecal samples for rt-pcr and virus isolation. no significant differences in mean vh:cd ratio and clinical symptoms (diarrhea, vomiting, dehydration, and anorexia) were observed between the pedv/pgar-infected and pedv-infected groups of piglets at 1, 2 and 3 dpi; however, at 2 and 3 dpi, pgar was detected in all fecal samples by rt-pcr and virus isolation. these findings failed to detect any interaction between pedv and porcine rotavirus in the small intestines of piglets, suggesting that concurrent infection of pgar may not synergistically enhance intestinal villous atrophy of piglets with pedv disease. we propose that the severe diarrhea exhibited in pedv and pgar co-infected piglets may be more associated with the immunity level of the host rather than to any synergistic effect of pgar on pedv enteritis. porcine epidemic diarrhea virus (pedv), a member of the genus coronavirus (family coronaviridae, order nidovirales), causes highly contagious diarrhea in pigs of all ages. the disease leads to significant morbidity and mortality in neonatal piglets. pedv infections are characterized by acute destruction of intestinal villous enterocytes and villous atrophy within the jejunum and ileum (pensaert and de bouck, 1978) . pedv has become an economic concern, mainly in south korea as well as in japan and china (chae et al., 2000) . porcine group a rotavirus (pgar) is a causative agent of acute diarrhea in neonatal and preweaning piglets. intestinal lesions by pgar resemble those of pedv and another porcine enteric coronavirus, transmissible gastroenteritis virus (tgev), but result in milder diarrhea (paul and stevenson, 1999) . outbreaks of pgar are sporadic, whereas pedv and tgev infections are explosive throughout porcine herds. it is believed that porcine rotavirus diseases may be associated with host immune and environmental factors (paul and stevenson, 1999) . a recent research study reported that the concurrent infection rate of pedv and pgar in korean piglets was high (43.2%), indicating that pgar is prevalent in piglets naturally infected with pedv (song et al., 2006) . in some korean pig herds, pedv-induced diarrhea in neonatal piglets with pgar co-infection seemed to be more severe and prolonged when compared to commonly observed diarrhea. gross and histological findings also showed that the piglets diagnosed with pedv and pgar co-infection had more severe atrophic enteritis than those diagnosed with pedv infection or pgar infection (personal observation, d.s. song). pgar can predispose the host to developing concurrent enterotoxigenic esherichia coli, clostridium perfrigens, and coccidian infections, which can increase the severity of the diarrhea (paul and stevenson, 1999) . it was therefore speculated that concurrent infection of pgar might affect pedv pathogenicity in neonatal piglets. the aim of this study was to determine the impact of pgar co-infection on pedv pathogenicity by subjecting piglets experimentally co-infected by pedv and pgar to evaluation of clinical signs (severity in diarrhea, dehydration, and dehydration) and histological morphometric analysis (villous height: crypt depth) in mid-jejunum where pedv lesions were evident. the pedv strain wild type dr13 (passage level 4 (p4)) was used as inoculum after the fourth passage in tissue culture and the pgar strain gottfried were used after the 3 passage in ma-104 cells (song et al., 2003 (song et al., , 2006 . pedv strain dr13 (p4) was a virulent strain that caused watery diarrhea in one-day old piglets within 1 day after oral inoculation in a preliminary study. for pgar strain gottfried, the virus showed less virulence compared to the pedv strain dr13 by causing mild to moderate diarrhea in one-day old piglets between 2 and 3 day after oral infection. piglets infected with pgar showed mild to moderate villous atrophy in mid-jejunum as well as in distal/ ileal intestines at 2 and 3 day after infection. each virus was titrated by analyzing the growth of vero cells or ma-104 cells inoculated with 10-fold serial dilutions of the pedv and pgar virus stocks, respectively, following the reed and muench method (reed and muench, 1938) . thirty-six colostrum-deprived, one-day old, large white-duroc crossbred pigs were randomly divided into four equal groups: pedv, pedv/pgar, pgar, and control. the piglets were acquired from pregnant sows that had not been vaccinated with any pedv or porcine rotavirus before parturition. of the three treatment groups, 27 piglets were inoculated orally with either 3 ml of virus stock of the pedv strain dr13 (10 5 tissue culture infective doses 50% [tcid 50 ]/ml), 3 ml of virus stock of the pgar strain gottfried (10 4 tcid 50 /ml), or with 3 ml of both pedv and gar at a 1:1 ratio. for the pedv/pgar group, the piglets were infected orally with pedv at 12 h after inoculation with pgar so that pedv would replicate in intestinal epithelial cells with an established pgar infection and at the time, pgar group piglets were inoculated with pgar. the experiment was designed in this way to evaluate the potential effect of a secondary pgar infection on the intestinal villous atrophy induced by a primary pedv infection. nine control piglets were similarly treated with uninfected cell culture medium. all piglets were maintained in stainless steel isolates (three per isolator), and fed a commercial sterile milk substitute. animals were examined three times daily for clinical signs. three piglets from each group were euthanized by electrocution at 1, 2, and 3 days post-inoculation (dpi) for tissue collection and analysis. all piglets were sedated with an intravenous injection of sodium pentobarbital, and euthanized by electrocution, as described previously (anonymous, 2001) . complete necropsies were performed on all the pigs, and all organs were grossly and histologically examined. three intestinal samples (from a 15 cm sample of the intermediate jejunal zone) were collected and fixed in 10% neutral buffered formalin for histological morphometric analysis. rt-pcr was performed as described previously, with slight modifications (song et al., 2006) . viral rna was extracted from fecal samples using trizol ls reagent (gibco brl) according to the manufacturer's instructions, and then precipitated with ethanol to collect rna. the forward and reverse primers used to detect pedv were 5 0 -ttctgagtcacgaacagcca-3 0 (1466-1485) and 5 0 -catatgcagcctgctctgaa-3 0 (2097-2116), respectively, which amplified a 651-base pair (bp) region from the spike gene of pedv. the forward and reverse primers used to detect pgar were 5 0 -aaagatg-ctagggacaaaattg-3 0 (57-78) and 5 0 -ttca-gattgtggagctattcca-3 0 (344-365), respectively, which amplified a 309-bp region from the segment 6 region of group a rotavirus. first-strand cdna synthesis was performed as follows: 1 ll of viral rna (5 ng/ll) was supplemented in a total reaction volume of 20 ll with 1 · rt buffer (50 mm tris-hcl, 8 mm mgcl 2 , 30 mm kcl, 1 mm dithiothreitol (ph 8.3)), 0.5 mm of each deoxynucleotide triphosphate (dntp), 2.5 lm random hexanucleotide mixture, 20 u of rnase inhibitor, and 50 u of moloney murine leukemia virus reverse transcriptase. after incubation for 45 min at 42°c, the mixture was heated for 5 min at 99°c to denature the products, and then stabilized at 4°c. the composition of the pcr mixture (150 ll) was 30 ll of cdna (5 ng/ll), 2 ll of each primer (250 nm), 15 ll of 10 · pcr buffer (10 mm tris-hcl, 40 mm kcl, 1.5 mm mgcl 2 (ph 8.3)), 1.2 ll of each dntp (0.2 mm), 29 ll of 2.5 units of taq polymerase, and 67.2 ll of distilled water. the pcr reaction for both pedv and pgar was carried out under the following conditions in a thermal cycler (applied-biosystem): 1 cycle of 5 min at 94°c; 35 cycles of denaturation at 94°c for 30 s, annealing at 53°c for 1 min, and elongation at 72°c for 1 min. the pcr products were analyzed by electrophoresis. three sections of formalin-fixed jejunum were obtained from each virus-infected and control piglet for morphometric analysis. only well-oriented sections were measured, and care was taken to ensure that only transverse sections, cut perpendicularly from villous enterocytes to the muscularis mucosa, were included. villous height and crypt depth were estimated by measuring 10 villi and crypts throughout the section. villous height and crypt depth (vh:cd) ratios were calculated according to a previous report (jung et al., 2006b) . differences in vh:cd ratio between infected and control piglets were assessed by the kruskal-wallis test using statistical analysis systems software (sas institute inc., cary, nc). a value of p < 0.05 was considered to be statistically significant. clinical symptoms in pedv or pgar-infected piglets ranged from diarrhea to vomiting, anorexia and dehydration. in general, piglets from the pedv and pedv/pgar groups showed more severe diarrhea and dehydration than piglets from the pgar group. eighteen piglets (18/18, 100%) from the pedv and pedv/pgar groups exhibited severe watery diarrhea and intermittent vomiting from 1 dpi. anorexia was generally observed at 1, 2, and 3 dpi, although not in all cases. moreover, severe dehydration was prevalent in most piglets at 2 and 3 dpi. no differences in the severity of clinical signs were observed between the pedv and pedv/pgar piglets. pgarinfected piglets, however, showed moderate diarrhea at 2 and 3 dpi, and displayed better health compared to the pedv and pedv/pgar piglets. no clinical signs were observed in the control piglets at each dpi. the results are summarized in table 1 . all fecal samples collected from pedv-infected piglets at 1, 2, and 3 dpi were positive for pedv by both rt-pcr and virus isolation. of the piglets in the pedv/pgar group, 2 of 3 fecal samples collected at dpi 1 were positive for pgar by judged by both rt-pcr and virus isolation. at dpi 2 and 3, however, all fecal samples were positive for pgar by both rt-pcr and virus isolation. fecal samples collected from piglets in the pgar group at dpi 1 were negative for pgar by both rt-pcr and virus isolation. at dpi 2, however, 2 of 3 samples were positive for pgar by both rt-pcr and virus isolation, and at dpi 3, all fecal samples were positive for pgar. pedv/pgar and pedv infected piglets had severe atrophic enteritis at 1, 2, and 3 dpi, whereas pgar infected piglets exhibited mild villous atrophy at 1 dpi, and moderate lesions at 2 and 3 dpi (fig. 1) . no histological lesions were detected in the control piglets. there was no autolysis in the intestines of all pig groups. the mean vh:cd ratios of the pedv/pgar and pedv groups were significantly decreased at 1, 2, and 3 dpi compared to control piglets; however, the mean vh:cd ratios of the pedv/pgar piglets were not significantly different from those of the pedv piglets, but were significantly lower than those of the pgar piglets (p < 0.05) (fig. 1) . at 1 dpi, the mean vh:cd ratio of the pgar group piglets was similar to that of the control, but was significantly decreased at 2 and 3 dpi. the mean vh:cd ratios of the pgar piglets were significantly greater than those of the pedv/pgar and pedv piglets at 2 and 3 dpi (p < 0.05). this study was designed to determine whether there is any interaction between pedv and porcine group a rotavirus (pgar) in the small intestine of neonatal piglets showing classical pedv symptoms such as vomiting and the highest susceptibility to the viruses. the results suggest that concurrent infection of pgar does not synergistically enhance intestinal villous atrophy of piglets with pedvinduced diarrhea. no significant differences in mean vh:cd ratio or clinical symptoms were observed between pedv/pgar and pedv group piglets at 1, 2 and 3 dpi; table 1 diarrhea and detection of porcine epidemic diarrhea virus (pedv) and porcine group a rotavirus (pgar) by rt-pcr and virus isolation in the fecal samples from piglets infected with pedv, pedv/pgar, or pgar, or in control piglets at different days post-inoculation (dpi) group dpi diarrhea rt-pcr virus isolation 1 2 3 1 2 3 1 2 3 pedv 3/3 a 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 pedv, pgar 3/3 3/3 3/3 3, 2 b /3, 3 3, 3 b /3, 3 3, 3 b /3, 3 3, 2 c /3, 3 3, 3 c /3, 3 3, 3 c /3, 3 pgar 0/3 2/3 3/3 0/3 2/3 3/3 0/3 2/3 3/3 control 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 a number of positive pigs/number of pigs tested. b number of pgar-positive pigs by rt-pcr. for the pedv/pgar group, pedv was infected orally 12 h after inoculation with pgar. c number of pgar-positive pigs by virus isolation. for the pedv/pgar group, pedv was infected orally 12 h after inoculation with pgar. was significantly decreased at 1, 2, and 3 days post-inoculation compared to negative control piglets; however, the mean vh:cd ratios of pedv/ pgar piglets were not significantly different from those of pedv piglets. each value represents the mean (standard deviation). however, at 2 and 3 dpi, pgar was detected in fecal samples by rt-pcr and virus isolation. this indicates that pgar concurrently replicated with pedv in the intestinal epithelial cells at the specified time points. we collected the middle jejunal intestine to evaluate the changes of pedv pathogenicity using morphometric analysis (vh:cd). intestinal villous atrophy in pigs with pedv infection was observed more in middle jejunum than in distal jejunum or ileum, whereas less for pgar infection. however, rotaviruses infect different regions of the intestines depending on the difference in the virulence of each virus strain, and the infected regions include the middle jejunum as well as the distal jejunum or ileum (bridger, 1994; hall et al., 1993) . the piglets with pgar gottfried strain infection also showed marked villous atrophy in the middle jejunal intestine at 3 dpi (fig. 1) . we guess that the pgar gottfried strain could affect replication and distribution of pedv in villous epithelial cells of the middle intestines of the pigs co-infected with pedv. the severity of intestinal villous atrophy by pedv may depend on the type of virus strain used to inoculate the piglets. the pedv strain dr13 (p4) was virulent enough to cause watery diarrhea in piglets within 24 h after oral infection. however, the dr13 strain was not as virulent as the one described in previous reports, where the vh:cd ratio of experimentally pedv-infected piglets decreased to less than 3 at 3 dpi (jung et al., 2006b,c) . virulence and inoculum dose of the pedv dr13 strain were considered to be appropriate at evaluation of severity of villous atrophy (vh:cd ratio). in our study, equivalent doses of the two viruses (3 · 10 5 tcid 50 /pig for pedv and 3 · 10 4 tcid 50 /pig for pgar in the study) were not used. a difference in the inoculum dose between these two viruses could affect the outcome. however, the effects were considered to be minor to get the same results, because the inoculum dose (3 · 10 4 tcid 50 ) of pgar showed enough clinical symptoms and villous atrophy in pgar only-infected group. porcine group a rotavirus infection, as well as pedv disease, is prevalent in neonatal piglets in south korea and japan (katsuda et al., 2006; song et al., 2006) . piglets naturally co-infected with pedv and pgar seem to have severe and prolonged diarrhea that is distinct from that commonly observed (personal observation, d.s. song). pgar infection is known to be associated with host immune and environmental factors (paul and stevenson, 1999) . rotavirus diarrhea is more prolonged and severe in immuno-compromised animals and humans (eiden et al., 1985; liakopoulou et al., 2005; paul and stevenson, 1999) . considering that there was no interaction between pedv and pgar in the present study, we speculate that the severe diarrhea observed in pedv and pgar coinfected piglets may be more a consequence of the immunity level of the host rather than the result of any synergistic effect of pgar on pedv-induced enteritis. a previous study using a virulent pedv strain demonstrated that immunosuppression due to the transplacental infection with porcine circovirus type 2 (pcv2) probably potentiates pedv pathogenicity in piglets by causing more severe intestinal villous atrophy compared to pedv only-infected pigs (jung et al., 2006a) . the piglets co-infected with pcv2 and pedv and piglets infected with pedv alone were histologically different with respect to their vh:cd ratios at 2 and 3 pid (jung et al., 2006b) . although most korean pig herds have been vaccinated against pedv and porcine rotavirus, the disease continues to resist eradication in south korea. the present investigation could not demonstrate any interaction between pedv and porcine group a rotavirus in the small intestine of piglets. however, further studies, such as those designed to evaluate cytokine changes and titre the virus contents of the small intestine of piglets, will be needed to confirm any synergistic relationship between these two viruses. in the present study, we propose that concurrent infection with porcine group a rotavirus does not affect pedv pathogenicity in piglets. report of the amva panel on euthanasia a definition of bovine rotavirus virulence prevalence of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus infection in korean pigs rotavirus rna variation during chronic infection of immunocompromised children variation in rotavirus virulence: a comparison of pathogenesis in calves between two rotaviruses of different virulence identification of porcine circovirus type 2 in retrospective cases of pigs naturally infected with porcine epidemic diarrhoea virus the effects of transplacental porcine circovirus type 2 infection on porcine epidemic diarrhea virus-induced enteritis in preweaning piglets decreased activity of brush border membrane-bound digestive enzymes in small intestines from pigs experimentally infected with porcine epidemic diarrhea virus. research in veterinary science frequency of enteropathogen detection in suckling and weaned pigs with diarrhea in japan rotavirus as a significant cause of prolonged diarrhoeal illness and morbidity following allogeneic bone marrow transplantation diseases of swine a new coronavirus-like particle associated with diarrhea in swine a simple method of estimating fifty percent endpoints differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf3 multiplex reverse transcription-pcr for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group a rotavirus key: cord-287913-pe6ot11q authors: jung, kwonil; kang, bo-kyu; kim, jeom-yong; shin, kyoung-sun; lee, chul-seung; song, dae-sub title: effects of epidermal growth factor on atrophic enteritis in piglets induced by experimental porcine epidemic diarrhoea virus date: 2007-06-18 journal: vet j doi: 10.1016/j.tvjl.2007.04.018 sha: doc_id: 287913 cord_uid: pe6ot11q epidermal growth factor (egf) promotes gastrointestinal mucosal recovery by stimulating the mitogenic activity of intestinal crypt epithelial cells. the aim of this study was to determine the effects of egf on atrophic enteritis induced in piglets by experimental infection with porcine epidemic diarrhoea virus (pedv) strain dr13. two groups of 12 conventional, colostrum-deprived, 1-day-old, large white-duroc cross breed piglets were inoculated orally with pedv (3 × 10(5) 50% tissue culture infective doses), with or without egf (10 μg/kg/day, intraperitoneally once daily for 4 days after infection) and compared to 12 uninfected, untreated control piglets. pedv + egf piglets had less severe clinical signs than pedv only piglets at 48 and 60 h post-infection (hpi). histologically, the ratio of villous height:crypt depth of pedv + egf piglets was significantly higher than pedv only piglets at 36 and 48 hpi. immunohistochemistry for ki67 demonstrated increased proliferation in intestinal crypt epithelial cells of pedv + egf piglets compared to pedv only piglets at 36, 48 and 60 hpi. egf stimulates proliferation of intestinal crypt epithelial cells and promotes recovery from atrophic enteritis in pedv-infected piglets. porcine epidemic diarrhoea virus (pedv), a member of the genus coronavirus (family coronaviridae, order nidovirales) causes highly contagious diarrhoea in pigs of all ages and is particularly fatal to piglets less than 2 weeks of age (pensaert and de bouck, 1978; chae et al., 2000) . pedv is an economically important cause of disease in pigs in korea, resulting in significant morbidity and mortality in neonatal piglets and increased costs due to vaccination and disinfection (chae et al., 2000; jung et al., 2006a; song et al., 2006) . although vaccines can be used to prevent pedv, there are no effective strategies for treatment of affected pigs during pedv outbreaks. pedv infections are characterised by acute destruction of intestinal villous enterocytes and villous atrophy in the jejunum and ileum, producing severe malabsorptive and maldigestive diarrhoea (pensaert and de bouck, 1978; jung et al., 2006b) . severe intestinal villous atrophy, with 50-60% loss of villi, develops within 3 days in piglets experimentally infected with pedv (jung et al., 2006a,b) . the role of growth factors in intestinal mucosal repair has been studied extensively (lemoine et al., 1992; stephen murphy, 1998; playford, 1995; playford and wright, 1996) . five egf-like peptides, including egf, transforming growth factor-a, heparin-binding egf-like growth factor (hb-egf), amphiregulin and betacellulin, have been identified in the intestinal lumen (marti et al., 1989; lemoine et al., 1992; barnard et al., 1995) . among these, egf acts primarily as a gastrointestinal surveillance peptide involved in mucosal repair (playford, 1995; playford and wright, 1996) . epidermal growth factor (egf) is secreted in the gastrointestinal tract by salivary glands, duodenal brunner's glands and ulcer-associated cell lineage (uacl) cells (barnard et al., 1995) . egf binds to a specific receptor expressed on the basolateral surface of enterocytes during mucosal damage and stimulates mitogenic activity (playford, 1995; playford and wright, 1996) . egf has positive effects on the recovery of damaged intestinal villi by promoting the proliferation of intestinal crypt epithelial cells (skov olsen et al., 1986; chao et al., 2003) . the aim of this study was to determine the effects of egf on atrophic enteritis induced in piglets by pedv and to assess whether egf has therapeutic potential for treatment of viral enteritis in piglets. recombinant human egf was produced as described previously (lee et al., 2003) and stored lyophilised at à20°c. egf administered to piglets was diluted with sterile saline. inoculations were performed with fourth passage tissue culture propagated pedv strain dr13, a virulent isolate that causes yellowish watery diarrhoea in piglets within 24 h of oral infection (song et al., 2003) . the virus was titrated in vero cells and viral antigen was detected by immunofluorescence using a monoclonal antibody (jung and chae, 2004) . five conventional, colostrum-deprived, 1-day-old, large white-duroc crossbreed piglets were used from a pedv-seronegative sow from a herd with no history of pedv to determine the optimal dose and time schedule for treatment with egf. the intraperitoneal route was selected to minimise the unpredictable toxicological effects of recombinant proteins on piglets. no adverse effects were detected following daily administration of 10 lg/kg/day egf for 2-6 days. thirty-six conventional, colostrum-deprived, 1-day-old, large white-duroc crossbreed piglets were obtained from 10 pedv-seronegative sows from a herd with no history of pedv and randomly divided into three groups. twenty-four piglets were inoculated orally with 3 ml of 1 · 10 5 50% tissue culture infective doses/ml of pedv strain dr13. twelve pedv-infected piglets (pedv + egf) were treated with 10 lg/kg/day egf intraperitoneally once daily from 0-4 days after infection. twelve control piglets were inoculated with uninfected cell culture medium. experimental piglets were held in groups of three in isolators at 28.5°c and fed a commercial sterile milk substitute. animals were examined three times daily for clinical signs. three piglets from each group were sacrificed at 24, 36, 48 and 60 h post-infection (hpi) by electrocution after sedation with iv sodium pentobarbital. three segments of jejunum from each piglet were fixed in 10% neutral buffered formalin, dehydrated in graded ethanol solutions and embedded in paraffin wax for histopathology. ten villi and 10 crypts were measured in well orientated histological sections. the ratio of villous height:crypt depth (vh:cd) was calculated and values were expressed as means ± standard deviation (jung et al., 2006a,b) . significant differences in vh:cd between groups of piglets were assessed using wilcoxon matched pairs test. p < 0.05 was considered statistically significant. proliferation of intestinal crypt epithelial cells was evaluated by immunohistochemistry for ki67 (danilenko et al., 1995) . antigens were retrieved from formalin-fixed paraffin-embedded tissues by pressure cooking, then were incubated with normal goat serum (sigma) in phosphate-buffered saline (pbs) for 30 min at room temperature to block nonspecific reactivity. sections were incubated with mouse anti-human ki67 monoclonal antibody (dako, 1:200) overnight at 4°c in a humid chamber. after three washes with pbs, sections were incubated for 1 h at 36°c with goat anti-mouse igg (dako, 1:200) conjugated to alkaline phosphatase. after colorimetric detection with red substrate (roche applied science) for 10 min at room temperature, sections were counterstained with mayer's haematoxylin. the ki67 score (number of positive cells per field under 200· magnification) was evaluated using an image analyser (image measurement standard v4.01, bersoft). values were expressed as means ± standard deviation and statistical analysis was performed by analysis of variance (anova) and student's t test. pedv only piglets exhibited severe watery diarrhoea and intermittent vomiting from 24 to 60 hpi, some exhibited anorexia from 36 to 60 hpi and most had severe dehydration from 48 to 60 hpi. most pedv + egf piglets had moderate diarrhoea from 24 to 36 hpi and severe diarrhoea table 1 mean ratio of villous height to crypt depth (vh:cd) in piglets infected with porcine epidemic diarrhoea virus with (pedv + egf) or without (pedv only) epidermal growth factor relative to uninfected piglets (control) hours post-inoculation ( values are given as mean ± standard deviation. * significant difference (p < 0.05) in ki67 score between pedv + egf and pedv only piglets. from 48 to 60 hpi, although some piglets had severe diarrhoea at 24 hpi. anorexia and dehydration were observed in pedv + egf piglets from 36 to 60 hpi. subjectively, clinical signs were less severe in pedv + egf piglets than pedv only piglets. at 24 hpi, the mean vh:cd of pedv-infected piglets was similar to that of control piglets, but decreased signif-icantly from 36 to 60 hpi (table 1 ). the mean vh:cd of pedv + egf piglets was significantly higher than that of pedv only piglets from 36 to 48 hpi (p < 0.05). ki67 scores peaked at 60 hpi in all piglets infected with pedv (table 2) . at 24 hpi, the ki67 scores of ped-v + egf piglets were not significantly different from that of pedv only piglets. however, from 36 to 60 hpi, the ki67 scores of pedv + egf piglets were significantly higher than pedv only piglets (p < 0.05) ( table 2 ; fig. 1 ). egf exerts positive effects on the recovery of damaged intestinal villi by accelerating the proliferation of crypt epithelial cells (skov olsen et al., 1986; chao et al., 2003) . in our study, immunohistochemistry for ki67 revealed that mucosal proliferation in the small intestine of ped-v + egf piglets was significantly enhanced in comparison with pedv only piglets from 36 to 60 hpi. similarly, vh:cd of pedv + egf piglets were higher than those of pedv only piglets from 36 to 48 hpi. these findings suggest that egf stimulates the proliferation of intestinal crypt cells and promotes the recovery of atrophied villi in the small intestine of pedv-infected piglets. a variety of growth factors have been used in clinical therapeutics in humans. egf and fibroblast growth factor are used for skin wound healing, such as in diabetic foot ulcer (ito et al., 2005; hong et al., 2006) . keratinocyte growth factor (kgf) has been used to assist recovery of damaged mucosa in the alimentary tract following radiation and chemotherapy for cancer (farrell et al., 1998 (farrell et al., , 2002 . kgf increases the thickness and cellularity of the intestinal mucosa by accelerating mitotic activity in crypt epithelial cells (housley et al., 1994; farrell et al., 1998; playford et al., 1998) . egf may have a similar mechanism of action to kgf in the small intestine. severe intestinal villous atrophy develops within 3 days after experimental infection of piglets with pedv (jung et al., 2006a,b) , whereas intestinal mucosal damage following radiation or chemotherapy is manifested after 5-10 days (dorr et al., 2005) . in our study, egf was effective in promoting recovery from acute atrophic enteritis within three days after treatment. this study has shown that egf enhances proliferation of intestinal crypt epithelial cells and promotes the recovery of damaged villi in piglets infected with pedv. while treatment with recombinant egf is currently expensive and requires further toxicological and field studies, we propose egf as a potential novel therapy to promote intestinal villous recovery in piglets with pedv infection and possibly other species with viral atrophic enteritis. epidermal growth factor-related peptides and their relevance to gastrointestinal pathophysiology prevalence of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus infection in korean pigs effect of oral epidermal growth factor on mucosal healing in rats with duodenal ulcer differing targets and effects of keratinocyte growth factor, platelet-derived growth factor-bb, epidermal growth factor, and neu differentiation factor reduction of radiochemotherapy-induced early oral mucositis by recombinant human keratinocyte growth factor (palifermin): experimental studies in mice keratinocyte growth factor protects mice from chemotherapy and radiationinduced gastrointestinal injury and mortality the effects of keratinocyte growth factor in preclinical models of mucositis recombinant human epidermal growth factor (egf) to enhance healing for diabetic foot ulcers keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract reconstruction of the soft tissue of a deep diabetic foot wound with artificial dermis and recombinant basic fibroblast growth factor effect of temperature on the detection of porcine epidemic diarrhea virus and transmissible gastroenteritis virus in fecal samples by reverse transcription-polymerase chain reaction the effects of transplacental porcine circovirus type 2 infection on porcine epidemic diarrhea virus-induced enteritis in preweaning piglets decreased activity of brush border membrane-bound digestive enzymes in small intestines from pigs experimentally infected with porcine epidemic diarrhea virus purification of soluble human epidermal growth factor (hegf) from recombinant escherichia coli culture broth by using expanded-bed adsorption chromatography biological effects of epidermal growth factor, with emphasis on the gastrointestinal tract and liver: an update growth factors in the gastrointestinal tract a new coronavirus-like particle associated with diarrhea in swine peptides and gastrointestinal mucosal integrity why is epidermal growth factor present in the gut lumen? effects of keratinocyte growth factor (kgf) on gut growth and repair oral administration of synthetic human urogastrone promotes healing of chronic duodenal ulcers in rats differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf3 multiplex reverse transcription-pcr for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group a rotavirus growth factors and the gastrointestinal tract key: cord-255862-84u3c33m authors: kim, ji won; ha, thi-kim-quy; cho, hyomoon; kim, eunhee; shim, sang hee; yang, jun-li; oh, won keun title: antiviral escin derivatives from the seeds of aesculus turbinata blume (japanese horse chestnut) date: 2017-07-01 journal: bioorganic & medicinal chemistry letters doi: 10.1016/j.bmcl.2017.05.022 sha: doc_id: 255862 cord_uid: 84u3c33m abstract porcine epidemic diarrhea virus (pedv) causes severe diarrhea and high fatality of piglets, influencing the swine industry. japanese horse chestnut (seed of aesculus turbinata) contains many saponin mixtures, called escins, and has been used for a long time as a traditional medicinal plant. structure-activity relationship (sar) studies on escins have revealed that acylations at c-21 and c-22 with angeloyl or tigloyl groups were important for their cytotoxic effects. however, the strong cytotoxicity of escins makes them hard to utilize for other diseases and to develop as nutraceuticals. in this research, we investigated whether escin derivatives 1–7 (including new compounds 2, 3, 5 and 6), without the angeloyl or tigloyl groups and with modified glycosidic linkages by hydrolysis, have pedv inhibitory effects with less cytotoxicity. compounds 1–7 had no cytotoxicity at 20μm on vero cells, while compounds 8–10 showed strong cytotoxicity at similar concentrations on pedv. our results suggest that escin derivatives showed strong inhibitory activities on pedv replication with lowered cytotoxicity. these studies propose a method to utilize japanese horse chestnut for treating pedv and to increase the diversity of its bioactive compounds. from the middle east. until the patient was diagnosed as infected with mers-cov, 29 secondary infections occurred by visiting different clinics, resulting in 186 confirmed cases. 5 coronaviruses at a molecular level have important features such as high rates of rna recombination, extraordinarily large rna genomes and rapid stability after transmission to other species, and leading to genetic diversity, unlike other enveloped rna viruses. 6 pedv of family coronaviridae shares phylogenetically common features with other coronaviruses. pedv causes severe diarrhea, dehydration, vomiting in pigs of all ages, and high mortality of piglets, resulting in tremendous financial loss. 7 thus, these results imply the necessity of studying the characteristics of coronaviruses and discovering active drugs to prevent the fast and extensive spread of coronaviruses. aesculus l (hippocastanaceae) contains 12 species of deciduous trees and has been cultivated as pharmaceutical crops for the production of standardized therapeutics extracts of escins in china. the common name ''horse chestnut" came from the uses of seeds for horses to treat overexertion or coughs, and it has been used as therapeutics purposes for anti-fever. 8 japanese horse chestnut (aesculus turbinata) is a medicinal plant widely distributed in japan and also has a small amount of cultivation in korea and china. 9 the seeds, which a large amount of escins were reported as its constituents, 9 have been used for diverse biological activities including anti-inflammatory, anti-obesity, hypoglycemic, and anti-cancer effects. [10] [11] [12] [13] [14] escins were also reported to possess strong antiviral effects against sars-cov with an ec 50 of 6.0 lm (si value of 2.5) 15 and against anti-hiv-1 protease. 9 however, the industrial utilization of escins for application to diseases and development as nutraceuticals has been limited to date due to their strong nonspecific cytotoxic effects. these reports prompted us towards the development of safer escin derivatives with anti-cov activities. previous studies on structure-activity relationship with escins suggested that acylation at c-21 and c-22 was necessary for the cytotoxic effects. [16] [17] [18] [19] [20] the cytotoxicity can be enhanced with methylation at c-24 and a free hydroxyl at c-16 at oleanane-type structure and altered by the site of glycosides. 14, 21 thus, alkaline and acid hydrolysis of escins was applied to detach acyl moieties at c-21 and c-22, and provide varieties of sugar moieties at c-3. in this research, we reported ten compounds (1-10), including four new compounds 2, 3, 5 and 6, from the extract of a. turbinate after the two-step hydrolysis. we also measured their antiviral activities using the pedv assay with isolated compounds and each fraction for safer utilization of japanese horse chestnut. the air-dried seeds of a. turbinata were extracted and separated through column chromatography using silica gel, rp-c 18 and preparative hplc to afford ten compounds, including four new (2, 3 and 5, 6) and six known (1, 4 and 7-10). 22 11 . the ir spectrum showed absorption due to hydroxyl table 1 1 h nmr and 13 c nmr spectroscopic data of compounds 2, 3, 5, and 6 in pyridine-d 5 . table 1) . the 1 h and 13 c nmr data of 2 were consistent with those of escinidin (1), except for the chemical shift of c-3 and the presence of one b-d-glucopyranosiduronic acid moiety in 2 (fig. 1) . these results indicated the attachment of b-d-glucopyranosiduronic acid to c-3 (d c 89.0). the linkage position of this b-dglucopyranosiduronic acid was confirmed by the hmbc experiment from the correlation from h-1 0 (d h 5.03) to c-3 (d c 89.1) ( fig. 2a) . therefore, the structure of 2 was elucidated as (3b,16a,21b,22a)16 . the 13 c nmr spectrum of 3 was very similar to that of 2, apart from the presence of one b-d-glycopyranosyl moiety in 3 (table 1) table 1 ). the hmbc correlation between h-3 (d h 3.63) and c-1 0 (d c 106.7) confirmed the position of b-d-glucopyranosiduronic acid. the relative configuration of 5 was investigated by analysis of its roesy spectrum (fig. 2b) . correlations 4 .64) were observed in the roesy data, implying that all these protons were on the same side of the molecule. the relative configuration of compound 5 remained unaltered even after the two-step reaction except for the deacylation and the cleavage of the glucose linkage. therefore, the structure of 5 was identified as (3b,16a,21b,22a)-16,21,22,24,28-pentahydroxyolean-12-en-3-o-b-d-glucopyranosiduronic acid. 23 compound 6 by analysing the roesy data of compound 5, we also confirmed that the overall skeleton and relative configurations of new compounds 2, 3, 5, and 6 were identical with the escin series, after a two-step hydrolysis. six known compounds 1, 4, and 7-10 were determined as protoaescigenin (1), 24 escinidin (4), 25 aesculuside b (7), escin ia (8), escin ib (9), 13 and isoescin ia (10) 26 by comparison with literature data. the cytotoxicity assay 27 was done at a concentration of 10 lg/ ml to compare the cytotoxic effects of the total extract and partitioned fractions before and after a two-step hydrolysis (fig. 3a) . the n-buoh fraction, containing a large amount of escins, showed strong cytotoxicity compared to fractions obtained after the twostep hydrolysis. interestingly, compounds 1-7 isolated from the fraction with the two-step hydrolysis were evaluated to have much lower cytotoxic effects than compounds 8-10 from the n-buoh part at concentration of 20 lm (fig. s22) . additionally, dosedependent cytotoxic effects of compounds 8-10 were ascertained at concentrations of 2, 5 and 10 lm (fig. s23) . the n-buoh and the other fractions from a two-step hydrolysis were evaluated for their pedv inhibitory activities with 6-azauridine as positive control at 1, 2, 5, and 10 lg/ml (fig. 3b) . 28 up to 2 lg/ml, both fractions showed similar and mild inhibitory effects on pedv replication, proving the original horse chestnut's antiviral activities. the fraction after a two-step hydrolysis inhibited pedv replication in a dose-dependent manner without cytotoxicity. the n-buoh fractions above 5 lg/ml, which are expected to contain many escins, exhibited poor cell viability because of strong cytotoxic effects, even if it could show better pedv inhibitory effects than the fraction from a two-step hydrolysis. based on these data, the ten purified oleanane triterpenoids (1-10) were evaluated for their pedv inhibitory effects with the same methods (fig. s24) . as compounds 8-10 showed strong cytotoxic effects on vero cells at 20 lm, their pedv inhibitory activities were evaluated at a concentration of 2 lm. compounds 1-7 were tested at a concentration of 20 lm to compare their inhibitory effects on pedv replication, providing less cytotoxicity in relatively high concentrations. compound 4 showed the strongest inhibitory activity among the ten compounds 1-10. additionally, compounds 4-6 exhibited concentration-dependent inhibition of pedv replication at concentrations of 10, 20 and 40 lm, indicating improved cell viability from the two-step hydrolysis (fig. s25 ). based on cytotoxicity and cpe assays, structure-activity relationships (sars) were studied. isolated compounds 1-10 after the two-step hydrolysis suggested the presence of three important groups: (1) acylation at c-21, c-22 or c-28 (1-7 and 8-10), (2) methylation at c-24 (1-3 and 4-6), group 1 (1-7 and 8-10) indicated that deacylation at c-21 could improve the cell viability (figs.3a and s22) . the pedv inhibitory effects of group 2 (1-3 and 4-6) demonstrated that methylation at c-24 could reduce antiviral activity. and (4 and 5-7) ] showed that the absence of glycosidic linkage also improved the antiviral effects (fig. s24) . during the pedv replication, two key structural proteins, spike and nucleocapsid proteins, take part in important roles. 29 the spike protein regulates the entry stage of the virus 30 and binding of nucleocapsid protein to viral rna is crucial for viral transcription. 31 following the data of the cytotoxicity and cpe assays (figs. s22-24), the five compounds 1 and 4-7 were selected for further evaluation. the inhibitory effects of compounds 1 and 4-7 on nucleocapsid protein synthesis at 20 lm were measured using western blot (fig. 4a ). 32 the five compounds showed moderate inhibitory effects on nucleocapsid protein synthesis, and compound 4 significantly inhibited nucleocapsid protein synthesis. thus, compound 4 was further analyzed for its effects in nucleocapsid and spike protein synthesis with western blot at concentrations of 10, 20 and 40 lm, and it was found to inhibit pedv replication in a concentration-dependent manner (fig. 4b) . on the basis of the above findings, compounds 4 and 6 were also measured with key genes and proteins crucial for pedv replication by real time qpcr (qpcr). 33 to measure the expression level of viral rna encoding nucleocapsid and spike proteins, compounds 4 and 6 were treated in vero cells at a concentration of 40 lm and total rna was extracted for reverse transcription followed by polymerase chain reaction using the primers for pedv (stable 1 ). fig. 5a shows the rna expression levels of two kinds of proteins with compounds 4, 6 and positive control. when the inhibitory effect of compound 4 was analyzed in detail at the concentrations (b) cpe inhibition assay of the n-buoh fraction and the reaction fraction at concentrations of 1, 2, 5, and 10 lg/ml. up to 2 lg/ml, the n-buoh fraction and the reaction fraction from a two-step hydrolysis showed similar activities, but at high concentrations, the n-buoh fraction showed cytotoxic effects and the reaction fraction had pedv inhibitory effects in dose-dependent manner. of 10, 20 and 40 lm, compound 4 inhibited the rna expression of nucleocapsid and spike proteins in a dose-dependent manner (fig. 5b) . on the basis of inhibition of pedv rna expression, compound 4 was further studied for its inhibitory effects on pedv replication, by performing an immunocytochemistry assay (fig. 5c ). 34 we observed green fluorescence in virus-infected cells but no signals in mock-treated cells. this result revealed that compound 4 had noticeable inhibitory effects on pedv replication in a dose-dependent manner at concentrations of 10, 20 and 40 lm. 3c-chymotrypsin-like protease (3cl protease) is vital for proteolytic processing of viral replication in coronaviruses. as escin was reported as a sars-cov 3cl protease inhibitor, 15 we performed docking modelling of compound 4 into the active site of sars-cov 3cl pro (pdb id code 3v3m). 35 the binding site was predicted by the 2d program of ds 4.0. as shown in fig. 5d , the hydroxyl group of c22 and c16 of 4 formed hydrogen bonds with the oxygen atom of the carbonyl group of glu166. additionally, the methyl group of c23 and the b ring of 4 showed hydrophobic interactions with cys145 and leu27 through their side chains. the cdocker interaction energy was calculated to be à38.63 kcal/mol. the 3cl pro binding energy value of compound 4 was unstable and weaker than that of the reference ligand 0em. however, clear key amino acid interactions of compound 4 with 3cl pro , proposed the mode of action as inhibition of 3cl protease and explained inhibitory possibility of the sars-cov of escin derivatives. this research demonstrated that including the four new compounds (2, 3, 5, and 6), ten oleanane-type triterpenoids (1-10) were isolated from the seeds of aesculus turbinata (japanese horse chestnut). the cytotoxicity of the n-buoh fraction was decreased with compounds 1-7 isolated from two-step hydrolysis. especially, two compounds 4 and 6 showed strong inhibitory activities against pedv in a dose-dependent manner. the present study proposed a way to utilize japanese horse chestnut for treating pedv with lowered cytotoxic effects and to increase the diversity of bioactive compounds. which is funded by the korean government. references 1 since the n-buoh-soluble fraction (70.8 g) contains a large amount of mixed triterpenoidal saponins called escins, this fraction was directly applied to hydrolysis reactions of two steps. acyl group hydrolysis was done at 90°c with 0.5 n naoh in 50% etoh aqueous solution for 2 h. further partial hydrolysis of the glucose moieties was also followed with 1.0 n hcl in 50% etoh aqueous solution at 90°c for 2 h. this reaction mixture was directly placed on an hp-20 cc (10 â 60 cm) to discard salt, washed with 10% etoh (3 l), and finally eluted with etoh (3 l) for the saponin fraction. the partial saponin fraction (5.59 g) was then chromatographed over an rp-c 18 cc (40-63 lm particle size) and eluted with a gradient solvent system of meoh:h 2 o (from 4:6 to 1:0), to yield five fractions (f1-f5). fraction f1 was further applied to semi-preparative hplc :40) over 25 min) resulted in the isolation of compound 4. fraction f5 was purified by preparative hplc (mobile phase mecn/h 2 o (30:70-60:40) over 25 min) to provide compound 1. compounds 8-10 were isolated from the n-buoh fraction of the dried seeds of a. turbinata extract by semi-preparative hplc using an isocratic solvent of 40% mecn 1023 cm à1 ; see table 1 for 1 h (500 mhz) and 13 c nmr 3901 (calcd for c 36 h 57 o 11 [màh] à , 665.3906). (3b,16a,21b,22a)-16,21 1028 cm à1 ; see table 1 for 1 h (500 mhz) and 13 c nmr 4434 (calcd for c 42 h 67 o 16 [màh] à , 827.4435). (3b,16a,21b,22a)-16,21 3852 (calcd for c 36 h 57 o 12 [màh] à , 681.3856). (3b,16a,21b,22a)-16,21,22,24,28-pentahydroxyolean-12-en-3-yl-o ir (kbr) mmax 3405, 2942, 1604, 1051, 1033 cm à1 ; see table 1 for 1 h (800 mhz) and 13 c nmr hresims m/z 843.4384 (calcd for c 42 h 67 o 17 vero cells (african green monkey kidney cell line cells were grown in dulbecco's modified eagle's medium (dmem) supplemented with virus stock was maintained at à80°c before use. to assess the cell viability, a mtt (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide) assay was carried out. vero cells were seeded for 24 h in 96-well plates at 1 â 10 5 cells per well. then, the cells were exposed to different concentrations of fractions and compounds for 48 h. the final concentration of dmso was maintained at 0.05% (v/v) to avoid solvent toxicity. twenty microliters of mtt solution (2 mg/ml) was then added to cultures and incubated for 4 h pedv (0.01 moi) were inoculated onto confluent monolayers of vero cells for 2 h. the media were replaced by dmem with different concentrations of compounds aliquots of lysates were separated by 10-12% sds-page and electrophoretically transferred to pvdf membranes (pvdf 0.45 lm. immobilon-p, usa) after 24 h, total rna from the cells was isolated by the trizol method and reverse transcribed using random primer (intron biotechnology, seongman, korea) according to the manufacturer' protocol. amplifications were carried out using selective primers for pedv, which are listed in s-table 1 (supporting information), using 2 ll of cdna and maxima sybr green qpcr master mix 2x ) for 1 h. after washing three times with pbs (ph 7.4), the slides were stained with 500 nm dapi solution for 10 min at room temperature and washed with pbs (ph 8.0) three times. mounting reagent (vectashield this work was supported in part by grants from the marine biotechnology program of the ministry of oceans and fisheries (pjt200669) and the korea bioactive natural material bank supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.05. 022. key: cord-277194-mj85mpo2 authors: perri, amanda m.; poljak, zvonimir; dewey, cate; harding, john c. s.; o'sullivan, terri l. title: factors associated with time to elimination of porcine epidemic diarrhea virus in individual ontario swine herds based on surveillance data date: 2019-05-08 journal: front vet sci doi: 10.3389/fvets.2019.00139 sha: doc_id: 277194 cord_uid: mj85mpo2 porcine epidemic diarrhea virus (pedv) emerged into canada in january of 2014. the virus was considered to be of high importance and the number of new cases were tracked using different mechanisms by stakeholders such as veterinary services from the provincial government and the swine industry. in addition to the initial date of infection, veterinary organizations in the swine industry maintained a disease control program (dcp) database that contained the date of declaration of freedom from pedv in individual herds. such data allowed for the determination of the duration of pedv infection in individual herds based on herd type, year and season of diagnosis. therefore, the objective of this study was to determine time to pedv elimination in ontario swine herds infected between 2014 and 2017, on the basis of records from the dcp database; and to identify factors associated with the likelihood of elimination. duration of time to eliminate pedv was estimated using kaplan-meier survival curves. the final cox's proportional hazard model included herd type, season and year of diagnosis. the hazard of pedv elimination for premises that were farrow-to-wean was 3.36 times larger (p-value: 0.044, 95% ci: 1.03, 10.93) than for farrow-to-feeder herds. herds diagnosed in the summer and fall had hazard ratios of 1.40 (p-value: 0.044, 95% ci: 1.03, 10.93) and 7.32 (p-value: <0.001, 95% ci: 3.12, 17.18), respectively compared to herds diagnosed in the winter months. the hazard ratio for herds diagnosed in 2015 was 0.54 (p-value: 0.015, 95% ci: 0.33, 0.89) compared to herds diagnosed in 2014. factors associated with time to elimination are likely reflective of the complexity of infection control practices applied in herds with different demographics and population structures, seasonal variability in the pathogen transmissibility, and the availability of resources to manage an emerging production-limiting disease. the median times to elimination were relatively long, which could be due to how it was measured, decisions made at the level of individual herds or delays related to reporting pedv elimination. design of control measures for production-limiting diseases at the regional level should take these factors into consideration. porcine epidemic diarrhea virus (pedv) emerged into canada in january 2014, soon after the initial detection in the united states (1) . the virus is highly contagious and is associated with mortality ranging between 80 and 100% in suckling pigs (2) (3) (4) . incursion of porcine epidemic diarrhea (ped) has a large impact on animal health and profitability of individual farms; which can result in high loses for the entire swine-producing sector when a large outbreak occurs (4, 5) . despite this, ped is not considered a reportable disease at the federal level in canada, similarly to other jurisdictions. nonetheless, it is considered as provincially reportable in several canadian provinces including ontario (6) . both, legislative framework in the province of ontario and concerns about the impact of this disease in the swine-producing sector supported establishment of several mechanisms of pedv surveillance with different surveillance coverage (7) . one of the surveillance mechanisms is based on the disease control program (dcp) database, which is known as the ped ontario area regional control and elimination program (arc&e). the dcp is based on swine producer volunteer participation and was implemented to monitor disease trends over time. the uniqueness of the dcp database is that it tracks the dates of the initial pedv incursion, as well as the dates the herds declare freedom from infection from pedv on the basis of established criteria. this allowed detailed estimation of incidence and prevalence over time in this source population (7) . briefly, the estimated prevalence and 95% confidence intervals (ci) of the virus at the end of 2014, 2015, and 2016 were 4.36 (3.07, 5.99), 2.25 (1.49, 3.26), and 1.35 (0.79, 2.16), respectively (7) . a decrease in prevalence, despite occurrence of new cases, has been achieved through implementation of targeted elimination programs at the individual herd level. soon after pedv emerged, veterinary practitioners developed approaches that allowed planned elimination of pedv from swine herds. however, the time to elimination of the virus was premises-dependent and depended on the elimination strategy employed. for planning purposes, the time to pedv elimination for specific herds could be projected on the basis of the herd type, its demographics, and infection control practices that are planned to be implemented. however, under field conditions, additional factors such as the demographics of the entire production system, the number of animal movements, availability of resources and the herd owners' overall willingness to eliminate a production-limiting disease could affect time to pedv elimination for specific herds. since the dates of disease incursion and elimination in individual herds are available, the dcp database could be an appropriate resource for evaluating the time to pedv elimination under field conditions in the entire population (source population) participating in the dcp program. therefore, the objective of this study was to determine time to pedv elimination in ontario swine herds infected between 2014 and 2017, on the basis of records from the dcp database; and to identify factors associated with the likelihood of elimination. the source population for this study was the oshab ped ontario area regional control and elimination program (arc&e) database. this dcp and database was initially created for controlling porcine reproductive and respiratory syndrome virus (prrsv) (8) and then was adapted to include pedv when it emerged into canada in 2014. the dcp is a voluntary program that collects diagnostic data including pedv herd status of ontario swine herds as outbreaks are reported, or as herds are classified as having eliminated pedv from premises. the data collected from the participating herds include the premises identification number, herd type, herd size, date of enrollment into the database, pedv status of premises on date of enrollment and the date(s) in which the premises changed their pedv status to "free-from-pedv." for a premises to be included in the current study the following inclusion criteria were fulfilled: (1) the premises participated in the dcp from january 2014 to october 2017, (2) the premises was located in ontario, and (3) the pedv infection status of the premises was available. the dcp monitors infection status of the volunteer premises over time. thus, the database contains herd (premises) infection status information i.e., whether a herd has eliminated the virus, whether any subsequent infection has occurred or any other changes in infection status, and the dates when the changes in infection status occurred. in the database, there are 4 types of premises infection status classifications: (1) confirmed positive, (2) presumed positive, (3) presumed negative and (4) confirmed negative. premises that were classified as ped confirmed positive were premises that had confirmed positive real-time reverse, transcriptase polymerase chain reaction (rt-pcr) test for pedv at the animal health laboratory (ahl) at the university of guelph. a presumed positive status was declared based on pig flow and movement as identified by the premises' veterinarian and did not require any diagnostic testing. thus, premises that housed animals that were sourced from a ped-positive premises were classified as presumed positive due to movement of presumed infected pigs. presumed negative premises were previously positive premises (i.e., either previously confirmed or presumed positive), where the producer implemented measures to eliminate pedv from the herd and confirmed the virus to be eliminated through animal or environmental testing. sampling methods for classifying premises as presumed negative were based on herd type and pig flow, and considered different types of samples (i.e., individual swabs, swiffer samples, oral fluids, etc). the basic considerations for all sampling types were: 98% individual test sensitivity, 100% individual test specificity, maximum design prevalence of 10%, and 95% confidence in detection of disease at the design prevalence level (9) . lastly, premises that were classified as confirmed ped negative were premises in which there were no clinical or diagnostic evidence of ped for at least 6 months after the presumed negative status update. data was entered into microsoft excel version 16.14.1 (microsoft, redmond, washington, usa) and then imported into stata version 13.1 (statacorp, college station, texas, usa). the proportion of premises that were confirmed pedpositive, presumed ped-positive and presumed ped-negative by herd type were documented. also, the proportion of herds to eliminate pedv by herd type, season and the year of pedv diagnosis were recorded. the median time to elimination and the 25th percentile, along with 95% confidence intervals (ci) were estimated by herd type, season and the year of pedv diagnosis. the dcp database consisted of 144 confirmed or presumed pedpositive case herds. four herds reported subsequent infections, which were excluded from further analysis. in addition, one herd was excluded because the herd type was unknown and another herd was excluded because it was categorized as an isolation/acclimatization unit. therefore, 138 confirmed or presumed ped-positive case herds were included in the study. a binary variable was created to indicate whether the case herds eliminated pedv (censored = 1) off-site during the study duration and if the herds did not eliminate pedv (censored = 0) during the study duration or due to loss-to-follow-up (censored = 0). for the case herds that did not report a change in the virus status over the study period of interest (n = 8), the herds were considered to be censored at times when their observation period ended. similarly, there were cases (n = 14) that reported a change in infection status change that was >100 weeks (∼2 years) after the initial date of infection. these herds were censored at 100 weeks. consequently, a total of 22 herds had their time censored and 116 herds had the event of interest (i.e., reported to have eliminated the virus at least 10% level with 95% confidence). the time taken to eliminate pedv from participating premises were estimated using kaplan-meier survival curves by herd type, season of diagnosis and year of diagnosis. the variable season was computed and based on northern meteorological seasons. winter was defined as any confirmed or presumed pedv diagnosis between december 1st and february 28th, as well as february 29th for the year of 2016 to account for the leap year (10). any confirmed or presumed pedv diagnosis between march 1st and may 31st, june 1st and august 31st and, september 1st and november 30th were classified into the variable season as spring, summer, and fall, respectively (10). log-ranked tests were computed for the 3 categories of kaplan-meier survival curves (herd type, year of diagnosis and season of diagnosis). a cox's proportional hazard model was constructed to investigate the effect of explanatory variables including herd type, season of diagnosis and year of diagnosis on the time to eliminate pedv from the premises. the time to event (i.e., elimination) was identified as the time in weeks for a premises to change from confirmed or presumed ped-positive to presumed pednegative. a failure occurred if the premises eliminated pedv. univariable analysis was done using the 3 predictor variables mentioned above, separately. the multivariable model was built using a manual forward selection procedure, with a p < 0.10, based on a partial likelihood ratio test as an inclusion criterion. the assumption of the cox's proportional hazard model was evaluated graphically showing the logarithm of the estimated cumulative hazard function. goodness-of-fit was evaluated using a hosmer-lemeshow test and a harrell's c concordance statistic. deviance and score residuals were evaluated. from january 2014 to october 2017, a total of 138 ped cases were reported in the dcp database. from the participating premises in the dcp database, 60.1% were finisher sites (n = 83), 11.6% were nursery sites (n = 16), 10.2% were farrow-to-finish (n = 14), 10.2% were farrow-to-wean (n = 14), 4.3% were wean-to-finish (n = 6) and 3.6% were farrow-to-feeder (n = 5), respectively. ninety-four cases (65.2%, 90/138) reported that they were confirmed ped-positive. of these 90 cases, 92.2% (n = 83) reported that they eliminated pedv and therefore gained a presumed-negative status. forty-eight cases (34.8%, 48/138) reported that they were initially presumed ped-positive, at their initial date of infection. of these 48 cases, 97.8% (n = 47) reported that they eliminated pedv during the study period and achieved a presumed-negative status. kaplan-meir estimates of the median and 25th percentile time in weeks to eliminate pedv are displayed in table 1 . nursery herds had the shortest median (23 weeks, 95% ci: 14, 31) and 25th percentile (16 weeks, 95% ci: 1, 23) for the duration of time it took in weeks to eliminate pedv. farrow-to-feeder herds had the longest median time (43 weeks 95% ci: 18, na) and second longest 25th percentile (27 weeks, 95% ci: 18, 79) for the amount of time it took in weeks to eliminate pedv. cases that were diagnosed in the spring and winter seasons had higher medians and 25th percentiles for the amount of time it took in weeks to eliminate pedv compared to cases that were diagnosed in fall and summer seasons ( kaplan-meier survival functions based on herd type, season and year of diagnosis are presented in figures 1-3 . the log-rank test statistic evaluating the equality of survival functions between herd types was statistically significant (p = 0.0029). similarly, the season a premises was declared as pedv-positive (p < 0.001) and the year of initial pedv confirmation (p = 0.0105) were both statistically significant. the results of the univariable analyses conducted through cox's proportional hazard model are reported in table 2 . briefly, herd type (p = 0.011), season (p < 0.001), and year of initial diagnosis (p = 0.019) were all associated with the likelihood of elimination in univariable analyses. the final multivariable model also included herd type, season of diagnosis and year of diagnosis and is presented in table 3 . farrow-to-wean premises were 3.36 times more likely than farrow-to-feeder herds (referent category) to eliminate the virus throughout the study period ( table 3) . the hazard ratio for premises diagnosed in the summer and fall months was 1.40 (p < 0.001, 95% ci: 2.74, 9.27) and 7.32 (p <0.001, 95% ci: 3.12, 17.18), respectively. thus, premises that were diagnosed in the summer and fall months were more likely than herds diagnosed in winter months (referent category) to eliminate pedv. premises that were diagnosed with pedv in 2015, had a hazard of eliminating pedv that was 0.54 times the hazard of eliminating pedv in herds diagnosed with pedv in 2014 (p = 0.015, 95% ci: 0.33, 0.89). this suggests that herds that were diagnosed with pedv in 2015 were less likely to eliminate the virus compared to premises that were diagnosed in 2014 (referent category). in contrast, premises that were diagnosed in 2016 were 1.62 times more likely to eliminate the virus compared to herds diagnosed in 2014 (p = 0.10, 95% ci: 0.91, 2.89). the assumption of the cox's proportional hazard model was examined graphically showing the logarithm of the estimated cumulative hazard function. there was no indication that the season of diagnosis and herd type variables had a time varying effect and therefore, the assumption of proportional hazards was met. the hosmer-lemeshow test indicated that the model fits the data (p = 0.46). also, harrell's c concordance statistic computed (0.72) found that the model had good overall predictive ability. there were no outliers or influential observations found. following the emergence of pedv into the united states in 2013, many actions were taken in ontario in anticipation of the emergence of the virus into ontario. newsletters, producer meetings and advertisements were communication tools that were used to inform producers of the risk of ped entry and to elaborate on prevention strategies (11) . following the initial emergence, the outbreak in the province of ontario was well controlled, which was achieved through quick identification of the suspected source of outbreak and implementation of biosecurity practices aimed to prevent further spread of infection. this resulted in a relatively low prevalence of infected herds (7), which could have contributed to willingness to eliminate pedv infection. veterinarians have implemented site-specific elimination strategies in ontario, however the duration of time for a premises to eliminate the virus is variable based on a multitude of factors (i.e., the initial start time for the elimination process may depend on the pedv status of the sow herd, or the season). the starting time for the time to elimination in this study was not the start date of control measures aimed at elimination, but the date of original infection. in part, due to this reason, the median time to elimination was relatively long. however, we believe that this time to elimination gives veterinary authorities reasonable overview of time to elimination for a newly emerging disease in the area, for which previous experience in elimination did not exist. an important finding in this study is that with the exception of 2015, the estimated hazard of eliminating pedv increased over the years examined. although exact reasons are difficult to determine, it is possible that a combination of factors played a role. veterinary practitioners were initially dealing with a new emerging disease into canada, and it is possible that they developed more expertise in procedures to eliminate ped from herds as time went on. additionally, most cases occurred during the first 2 years (n = 92 in 2014 and n = 27 in 2015) of the outbreak and it is possible that resources needed to be prioritized between actions needed to prevent further spread and actions to eliminate infection from already infected sites, particularly if such sites required substantial planning. in contrast, the number of new cases in 2016 (n = 16) and 2017 (n = 3) was substantially lower. another important finding in this study was that herds diagnosed in winter and spring months required more time to eliminate the virus. this was likely due to pedv's survivability and ability to remain infectious. typically, coronaviruses can survive temperatures from 56 • c for 10-15 mins, 37 • c for several days, 4 • c for several months, and while frozen at −60 • c many years without losing infectivity (12) . thus, it was likely difficult to eliminate the virus due to its survivability in ontario's temperatures in the spring and winter months. it is also possible that due to the lack of external pressures, producers who had positive herds waited until warmer months to start with the pedv elimination protocol. farrow-to-wean herds were found to eliminate the virus in a shorter amount of time compared to farrow-to-feeder herds. this was an expected finding, since in a farrow-to-wean operation; the system is generally less complex than a farrow-to-feeder or farrow-to-finish operation. for instance, farrow-to-wean herds have fewer types of production classes than farrow-to-finish herds. the presence of nursery pigs on the same site as suckling pigs complicates infection control practices since a separate set of control measures and operating procedures need to be designed and implemented for the nursery stage of production. this requires resources, strict adherence to internal biosecurity protocols and often demographic measures, such as creation of an interruption, or gap, in pig flow. pig flow through a production system, and more specifically, the creation of a gap in pig flow, is now recognized as an essential aspect of achieving earlier farrowing site elimination by allowing more effective cleaning and disinfection protocols required for successful elimination (13) . pig flow through a production system is the frequency of introducing new pigs into a population and the amount of opportunity these pigs have to come in contact with other pigs. a gap in pig flow however is often a one-time event to prevent the entrance of new animals to control the spread of the virus. a partial depopulation could present a gap in pig flow, where infected animals are removed from the herd, followed by cleaning and decontaminating the site. the database did not include details about specific infection control practices, such as the details of pig flow or attempts to generate a gap in pig flow. nonetheless, it is also worth pointing out that the variability in the time to elimination was markedly higher in farrow-to-feeder than in other herd types. it is possible that this time to elimination is not only driven by herd demographics and pig flow, but also with other factors such as willingness to eliminate, which was not directly measured in this study. an important concept for this study is that the data collected was from a large-scale industry-based surveillance program. this study does present limitations. firstly, the dcp is based on voluntary participation. the animal health act in ontario required that all ped-positive herds report to the ontario ministry of agriculture, food and rural affairs (omafra), by law, when the hazard was deemed emerging. the omafra surveillance program only accounts for primary case herds, which are case herds with a positive diagnostic test (rt-pcr) for pedv (14) . thus, secondary cases due to animal movement were not included in the omafra surveillance program. unlike the surveillance program managed by omafra, where 100% coverage of primary pedv-infected cases were included, the dcp used in the study only includes primary case herds that volunteered to participate in the program, and secondary cases resulting from animal movement from such cases (7) . it is also possible that some producers or veterinarians did not follow up to report that the case indeed eliminated pedv from the premises, in which case the estimated time to elimination would be longer than in reality. there were 14 premises for which the records indicated that the time between initial infection and a change in status to presumed negative was longer than 100 weeks. the survival time of these premises was censored at 100 weeks. since participation in this large-scale disease monitoring program is not mandatory, it is possible that some of these premises were not working toward eliminating the virus, since there was no external pressure to do so. alternatively, it is likely that owners that had a low prevalence of pedv on-site, may have not tested pigs to confirm pedv status (i.e., the absence from infection). however, both of these scenarios could occur with a production-limited disease. if large-scale disease control programs are initiated at the level that is different than a premises, or production-system level; veterinary authorities should be aware of the situations where time to negativity could take a long time. in addition, populations with high replacement and/or birth rates such as swine herds could have considerable number of susceptible animals introduced into a population that is partially immune due to recent exposure. this situation could provide opportunity for infectious agents to continue circulating at low levels. consequently, declaring freedom from infection at 10% may not be sufficient. however, making a decision about the design prevalence should be weighed against the disease epidemiology and cost to producers. another limitation is the database was missing variables for herd size. due to this, the authors decided not to consider this variable in the analysis. however, despite these limitations, this study provided novel insight in regards to pedv elimination times in ontario. in conclusion, this study allowed estimation of time to pedv elimination based on a large-scale disease control program database, which considered time between initial infection and confirmation of pedv freedom at a minimum level of 10%. under such assumptions, the median time to elimination of pedv from ontario swine herds varied between 23 weeks in nursery herds (standard error =1 week), and 43 weeks (standard error =17.5 weeks) in farrow-to-feeder herds. herd type, season, and year of original diagnosis were all associated with the time to negativity (p < 0.05) in the multivariable model. among the sow herds, farrow-to-wean herds had the highest hazard of pedv elimination. these results are reflective of the complexity of the infection control practices applied in herds with different demographics and population structures. the hazard of elimination was also higher in herds that had the initial infection during summer and fall than in herds that had the initial infection during winter. this could be a reflection of seasonal variability in the pathogen transmissibility or decisions made at the level of individual herds to proceed with infection control measures when the likelihood of success is the highest. with the exception of the second year after initial emergence, the hazard of elimination increased over years, which could reflect the availability of resources to manage an emerging productionlimiting disease. the median time to elimination was relatively long in all herd types. however, this could be a consequence of the way it was measured, the decisions about implementation of infection control measures which could be made at the level of individual herds, multi-site production systems or possibly delays related to reporting pedv elimination. nonetheless, the design of control measures for production-limiting diseases at the regional level should consider these factors. research ethics board of the university of guelph approved the study. data for this study were obtained from swine health ontario under an appropriate data sharing agreement based on secondary data usage. investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in canada update on porcine epidemic diarrhea status report on porcine epidemic diarrhea virus in the united states determination of the infectious titer and virulence of an original us porcine epidemic diarrhea virus pc22aa strain an evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of naïve pigs following consumption via natural feeding behaviour: proof of concept porcine epidemic diarrhea current global situation and possible threat for europe herd-level prevalence and incidence of porcine epidemic diarrhoea virus (pedv) and porcine deltacoronavirus (pdcov) in swineherds in ontario descriptive analysis and spatial epidemiology of porcine reproductive and respiratory syndrome (prrs) for swine sites participating in area regional control and elimination programs from 3 regions of ontario oshab guidelines for declaring presumed negative ped site status what are the seasons? porcine epidemic diarrhea update effects of air temperature and relative humidity on coronavirus survival on surfaces risk factors for sow farms becoming chronically infected with porcine epidemic diarrhea virus (pedv) outbreak investigation of porcine epidemic diarrhea in swine in ontario ap conducted data management, analysis, and interpretation of results under guidance of zp and to. manuscript was written by ap, with input from zp, to, cd, and jh. this study relied on collaborative efforts with the ontario swine health advisory (oshab) ped ontario area regional control and elimination program (arc&e) and animal health laboratory, the university of guelph. this work was funded by omafra (#030042), ontario pork (#052556), nserc-collaborative research development (crd) (#401204) grants and ovc fellowship. key: cord-276989-441aclcc authors: liu, jianbo; gao, ran; shi, hongyan; cong, guangyi; chen, jianfei; zhang, xin; shi, da; cao, liyan; wang, xiaobo; zhang, jialin; ji, zhaoyang; jing, zhaoyang; feng, li title: development of a rapid immunochromatographic strip test for the detection of porcine epidemic diarrhea virus specific siga in colostrum date: 2020-03-12 journal: j virol methods doi: 10.1016/j.jviromet.2020.113855 sha: doc_id: 276989 cord_uid: 441aclcc porcine epidemic diarrhea virus (pedv) causes very high mortality in newborn piglets. the mucosal immune system in the gut must eliminate potential pathogens while maintaining a mutually beneficial relationship with the commensal microbiota. antibodies derived from the secretory immunoglobulin a (siga) class, act as the first line of antigen-specific immunity in the gut by recognizing both pathogens and commensals. therefore, the measurement of siga levels is an important index in evaluating pedv infections and immune status. a simple and rapid method for the detection of pedv-specific siga using an immunochromatographic test strip has been developed; incorporating a colloidal gold-labeled anti-siga secretory component (sc) mab probe for the detection of anti-pedv-specific siga in swine. on the strip, a gold-labeled anti-siga sc mab was applied to a conjugate pad; purified pedv particles and goat anti-mouse antibodies were blotted onto a nitrocellulose membrane to form the test and control lines, respectively. results showed that the immunochromatographic test strip had high sensitivity and specificity. when compared with enzyme-linked immunosorbent assay, kappa value suggesting that the strip could be used to detect pedv specific siga in colostrum samples. furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. we found that the immunochromatographic test strip was a rapid, sensitive, and reliable method for the identification of pedv specific siga, indicating its suitability for epidemiological surveillance as well as vaccine immunity when studying pedv. porcine epidemic diarrhea virus (pedv) belongs to the family coronaviridae, first recorded in pigs in england in 1971 (pensaert and de bouck, 1978) , and subsequently spread to other european and asian countries (song and park, 2012) and north america (huang et al., 2013) . porcine epidemic diarrhea is a global infectious disease and is characterized by high morbidity and mortality in pre-weaned piglets, and causes serious economic losses to the swine industry in china (gao et al., 2013; li et al., 2014) . pedv is an enveloped coronavirus with a 28 kb positive-stranded rna genome, containing at least seven open reading frames (kocherhans et al., 2001) . these mainly encode four major structural proteins: the spike (s), nucleocapsid, membrane, and envelope proteins. the s protein is a glycoprotein and can form homotrimers to mediate membrane fusion and gain entry into host cells (bosch et al., 2003) . it is known that the s protein of coronavirus plays a crucial role in the induction of neutralizing antibodies, and it has been used to prepare effective vaccines (brian and baric, 2005; tuboly and nagy, 2001) . the amino-terminal portion of the s protein of several coronaviruses has been shown to contain key antigenic sites that are responsible for eliciting humoral and cellular immune responses (gebauer et al., 1991) . the mucosal immune system in the gut faces the formidable task of eliminating potential pathogens while maintaining a mutually beneficial relationship with the commensal microbiota. antibodies of the secretory immunoglobulin a (siga) class act as the first line of antigenspecific immunity in the gut, and can recognize both pathogens and commensals (song and park, 2012) . in contrast to serum iga, which is derived from plasma cells in the bone marrow, siga is generated locally by plasma cells in the lamina propria, which lies beneath the intestinal epithelium (kaetzel, 2005) . the protective action of siga in the infant gut is a result of many processes, including intracellular neutralization t and viral particle excretion, immune exclusion, whereby siga agglutinates bacteria and viruses, as well as prevents the binding of pathogens to mucosal surfaces, and interference with bacterial motility (van egmond et al., 2001v) . at high endogenous concentrations of siga, infants are less likely to have experienced illness in the preceding and subsequent months (breakey et al., 2015) . pedv can affect the intestinal tract, and based on the above results, we can infer that the siga concentration in the colostrum and rectal swab is a better marker than iga of protection and survival rate after virulent pedv challenge. tools to monitor pedv mucosal immunity and colostral immunity could be useful for the development of preventative programs on affected farms. therefore, the aim of this study was to develop a simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled anti-siga sc mab probe for the detection of anti-pedv-specific siga in swine, and to compare its performance with an indirect siga elisa based on the whole pedv virus (cong et al., 2019) . vero e6 cells (american type culture collection no. crl-1586) were used to propagate pedv (genbank accession no. kt323980). virus was propagated according the method described by liu et al. (2017) with minor modifications. briefly, growth medium was removed (containing 5 % fbs) when the cells formed a confluent monolayer and were then washed with phosphate-buffered saline (pbs, ph 7.2). then, pedv containing 1 × 10 5 plaque forming units (pfu) were diluted in 10 ml of dmem (dmem, gibco brl life technologies, usa) (containing 100 μg trypsin), and added to each culture bottle and incubated at 37°c. virus was harvested and purified according to the methods described by liu et al. (2017) . the purified viruses were negatively stained with 3 % phosphotungstic acid (ph 7.0) for 30 s according to previously described procedures (chen et al., 2014; jung et al., 2014) . and then identified by h-7650 electron microscopy (hitachi, japan). spf swine were used to produce colostrum. a total of four specific pathogen free swine (supplied by experimental animal base, harbin veterinary research institute of the chinese academy of agricultural sciences) were used in this study. three swine were immunized with pedv (lnct2 strain), transmissible gastroenteritis virus (tgev) strain h16 (genbank accession no. fj755618), and porcine rotavirus (porv) a (genbank accession no. jf781161), respectively. all the viruses were inactivated by 0.5 % methanol solution (pbs, ph 7.2) at 37°c for 24 h, and then emulsified with freund's complete adjuvant (sigma-aldrich, st. louis, mo, usa). and they were performed by neck intramuscular injection. the fourth animal was immunized with dmem as control and each swine was immunized twice. the first immunization was 40 days before delivery whereas the second immunization was 20 days before delivery. after delivery, colostrum from the four swine was collected and store at −20°c. the protocols involving animal immunity in this study were approved by our institute's animal care and use committee and performed in accordance with standard ethical guidelines. the hybridoma cell line 2f9, stably secreting mab against siga sc protein (gao et al., 2018) was cultured in the peritoneal cavities with pristane (sigma-aldrich, st. louis, mo, usa) primed balb/c mice to obtain ascites fluid. the ascites fluid was purified by hitrap protein g hp (ge healthcare) according the manufacturer's instruction and the purified antibody was identified by sds-page. the gel was stained by coomassie blue. colloidal gold with an average particle diameter of approximately 20 nm was purchased from shanghai kinbio tech. co. ltd. (china). the colloidal gold-labeled mab was prepared according to the methods described by li et al. (2017) with some modifications. briefly, 0.9 ml (1 mg/ml) of purified mab was incubated with 100 ml of colloidal gold solution (ph 8.2) for 20 min at 20°c. after addition of 1 ml of a 3% casein solution, the mixture was incubated at 20°c for 10 min. then, the mixture was centrifuged at 40,000 × g for 10 min at 4°c. the supernatant was discarded and the pellet of the colloidal gold-labeled mab was suspended in 4 ml of 0.02 m sodium borate buffer (containing 0.1 % nan3, 2 % bovine serum albumin (bsa), 1 % sucrose) and stored at 4°c. the colloidal gold-based lateral flow test strips were generated according to the procedures described by liang et al. (2015) , with some modifications. briefly, 25 × 300-mm pieces of glass fiber was immersed in phosphate-buffered saline (0.2 m, ph 8.0, containing 10 mm edta, 1 % tween-20, and 10 % bsa) for 2 h at 20°c. next, the pieces were dried for 2 h at 37°c and stored for use as the sample pad. glass fiber was also immersed in phosphate-buffered saline (0.1 m, ph 7.4, containing 2.5 % mycose, 1 % bsa, 1 % tween-20) for 2 h at 20°c and then dried for 2 h at 37°c and stored for use as the conjugate pad. the conjugate pad was covered with an appropriate volume of colloidal gold-labeled anti-siga sc mabs (2f9) using a xyz3060 dispense platform (biodot, inc., irvine, ca, usa), and then dried at 37°c for 2 h and stored dry. purified pedv particles were dispensed onto the nitrocellulose (nc) membrane to serve as the test line, and goat antimouse igg polyclonal antibody (pab) was dispensed onto the nc membrane, (being separated by a distance of 7 mm) to serve as the control line. the pedv particles and goat anti-mouse igg pab were diluted with coating buffer to a final concentration of 0.02 and 1.0 mg/ ml, respectively. then, the nc membrane was dried at 37°c for 2 h and stored in the dry at room temperature until use. the test strips were assembled and used as described by liang et al. (2015) . the sample pad, conjugate pad, nc membrane, absorbent pad and backing plate were assembled in sequence. the sample pad and the conjugate pad were overlapped by 2 mm with one end of the conjugate pad; the other end of the conjugate pad was overlapped (2 mm) with one end of the nc membrane underneath the conjugate pad; the absorbent pad was stuck to the other side of the nc membrane. after this, the whole plate was cut into 3 mm-wide strips, which were assembled in the strip cassettes with desiccant. to test for colostrum, we diluted the samples 1: 20 in sample buffer and mixed thoroughly then, 100 μl of the solution was dispensed onto the sample pad well of the strip apparatus to determine the presence of pedv specific siga, as described above. with this system, the liquid migrates towards the absorption pad by capillary action. the siga in the colostrum samples will form a "gold-labeled antibody-siga" complex with the gold-labeled antibody on the conjugate pad. if the colostrum sample has pedv specific siga present, the pedv specific "gold-labeled antibody-siga" complex, while migrating to the nc membrane, will bind to pedv, thereby displaying a red line in the t line area. additional complex migrates further to react with the goat antimouse igg pab, showing another red line in the c line area. negative samples do not produce a red line in the t area, whereas the c line will always show a red line. the results can be seen within eight minutes at room temperature. 2.7. validation of specificity and sensitivity of the immunochromatographic strip test pedv siga positive colostrum, tgev siga positive colostrum, porv siga positive colostrum, and pedv siga negative colostrum were used to test the specificity of the strip. the sensitivity of the strip was evaluated by comparing the detection of the same samples with pedv siga elisa kit. the detection limit of the strip was evaluated by using pedv siga positive-colostrum and colostrum titers determined by using an elisa kit (cong et al., 2019) as the reference standard with minor modifications. in brief, sample buffer was used to dilute colostrum from 1:10 to 1:100, and 100 μl of the solution was added into microtiter plates (costar, corning, ny, usa) incubated at 37°c for 1 h. after a washing step, a horseradish peroxidase mouse anti-pig siga sc antibody 1: 3000 dilution was added and incubated at 37°c for 45 min. after three times wash with pbst, the peroxidase reaction was visualized using 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (bbi, shanghai, china) as the substrate for 15 min at 37°c , and the reaction was stopped by adding 50 μl of 2.5 m sulfuric acid to each well. optical densities (ods) were measured at 405 nm using an elisa plate reader (biotek instruments, winooski, vt, usa). for the sensitivity of the elisa kit, one pedv specific siga positive colostrum was still positive when diluted at 1: 6400. for the specificity, the elisa kit has no cross-reaction with tgev as well as porv specific siga positive colostrum (cong et al., 2019) . sixty three colostrum field samples from hebei, shandong and heilongjaing province were assayed by the strip and the pedv specific siga elisa (cong et al., 2019) . by comparing results obtained by the two methods, kappa statistic value was used to evaluate the effectiveness of the test strip. after removing sucrose, the bands between the 20 % and 40 % and 40 % and 60 % sucrose solutions were identified by electron microscopy. in the 40 % and 60 % sucrose solution band, pedv particles were observed and their diameter was approximately 130 nm (fig. 1) . ascites fluid containing mab 2f9 was purified by hitrap protein g hp and the purified antibody was identified by sds-page. as the results show (fig. 2) , the purified antibody had two brands, a light chain and heavy chain. the purified antibody was stored until further use. the assay specificity was determined using anti-pedv specific siga positive colostrum, anti-tgev specific siga positive colostrum, anti-porv specific siga positive colostrum, and anti-pedv specific siga negative colostrum. as shown by the strips listed in fig. 3 , the anti-pedv specific siga positive colostrum was positive at the test line; the other colostrum samples were negative, suggesting that the immunochromatographic strip had good specificity. anti-pedv specific siga positive colostrum was diluted to determine the sensitivity of the strip. according to the results shown in fig. 4 , the strip had a sensitivity of up to 1: 50, while the detection limits of the elisa kit reached 1:160 dilution (table 1) . all of the 63 colostrum samples were detected by the strip and elisa mentioned above. as shown in table 2 , 53 colostrum samples were anti-pedv specific siga positive as determined by the strip assay, whereas 55 colostrum samples were anti-pedv specific siga positive as detected by the elisa. the kappa statistic value for the comparison of the strip versus the elisa kit, was 0.612. for the interpretation of agreement of the kappa statistic, a value of 0.2 to 0.4 is considered fair, fig. 1 . identification of purified pedv by electron microscopy. the band between the 40 % and 60 % sucrose solutions was collected with a syringe, and after removing the sucrose the purified viruses were identified by electron microscopy. scale bar =100 nm. 0.4 to 0.6 is moderate, 0.6 to 0.8 is good, and 0.8 to 0.9 is very good (altman, 1990) . the kappa statistic value was 0.612, suggesting that the strip could be used to detect pedv specific siga in colostrum samples. antiviral antibody detection is key for vaccine immunity surveillance and in determining pig exposure to pedv. currently, indirect elisas used to assay igg, iga, siga, have been established to monitor antibodies against pedv li et al., 2015; cong et al., 2019) . also, reverse transcription-polymerase chain reaction (rt-pcr) (ishikawa et al., 1997) , duplex rt-pcr (kim et al., 2001) , and other methods have been used to monitor pedv. although the presence of antibodies in serum is not directly related to protection of sows or piglets, serological examinations facilitate the assessment of the humoral responses to pedv, elicited either through vaccination or natural infection. however, the above-mentioned methods are time-consuming and require professional/technical personnel and are mainly limited to laboratory use. the immunochromatographic test strip used to detect anti-pedv specific siga described here is easy to operate, and sensitive. it is known that pedv is mainly transmitted by the fecal-oral route and disease is initiated following interaction with the mucosal surface lining the digestive tract. the primary defense used by this tissue is the mucosal immune system. antibodies from the siga class act as the first line of antigen-specific immunity in the gut. they prevent pathogens from binding to mucosal surfaces, and agglutinate bacteria and viruses (van egmond et al., 2001v) . therefore, siga levels are an important index by which to evaluate pedv infection and immune states. in this study, siga, as opposed to igg, detection was developed to assess pedv infections and vaccination. here a pedv siga-specific immunochromatographic test strip was developed. the purified pedv-lnct2 particles, which belongs to the g2 genotype, were used as capture antigen. the shared amino acid identity of the pedv lnct2 s protein and the pedv cv777 (g1 genotype) s protein (genbank accession number kt323979) is 93.2 %, whereas it is only 46.1 % for the pedv lnct2 s protein and the tgev s protein (purdue strain, genbank accession number dq811789). reactivity using the polyclonal antibodies (pabs) revealed significant cross-reactivity between the two pedv subtypes, although there was a two-fold difference in the antigenic responses based on pab titers in the elisa and ifa (wang et al., 2016) . therefore, the elisa antigen based on the pedv-lnct2 particles could detect both g1 and g2 genotype strains. additionally, the pedv particles used in the present study had no cross-reactivity with anti-tgev positive specific colostrum or anti-porv positive specific colostrum (fig. 3) . furthermore, the strip had a sensitivity of up to 1:50, whereas the elisa kit limits for the same sample was 1:160 (table 1) . based on these results, the anti-pedv specific siga immunochromatographic test strip could be used to effectively monitor siga levels. previously, researchers have used elisas to study the effect of pedv on mucosal immunity; an indirect elisa based on the s1 protein of pedv was used to detect iga levels in colostrum and fecal samples to evaluate pedv colostral immunity (gerber et al., 2014 and . iga plays an important role in providing protection at mucosal surfaces, and it is effective in neutralizing bacterial toxins. furthermore, polymeric iga has been shown to be more effective at neutralizing exotoxins from clostridium difficile when compared to either monomeric iga or igg with the same variable regions (stubbe et al., 2000) . siga consists of the sc, two iga molecules, and a linking chain. sc protects siga from proteolytic degradation (kaetzel, 2005) , making the siga more stable. it has been estimated that approximately 3 g of siga is transported daily fig. 3 . specificity of the immunochromatographic strip. lanes 1 to 6: strip detection of the anti-pedv specific siga positive colostrum, anti-tgev specific siga positive colostrum, anti-porv specific siga positive colostrum, anti-pedv specific siga negative colostrum, water, milk. the samples were diluted at 1: 20 in sample buffer and mixed thoroughly. then, 100 μl of the solution was dispensed onto the sample pad. c stands for control line, t stands for test line. into the intestines of the average adult (mestecky et al., 1986; conley and delacroix, 1987) , where siga forms the first line of antigen-specific immune protection against ingested, inhaled, or sexually transmitted pathogens and antigens at mucosal surfaces (kaetzel, 2005) , making it more important than iga. in this study, we investigated the sc portion to detect siga in colostrum samples, as this can eliminate the effect of monomeric iga. after infection with coronaviruses, such as pedv or tgev, or after vaccination, siga is generated locally by plasma cells located in the lamina propria, which underlies the epithelium (kaetzel, 2005) . therefore, the anti-pedv specific siga immunochromatographic test strip established in this study could be used to detect infections and immune states. by determining the siga level, we could assess whether pig herds (unvaccinated) have been infected by pedv, as well as pigs (vaccinated) could been protected against pedv, highlighting the versatility of our method. therefore, monitoring siga titers in colostrum and milk samples could be performed to ensure that piglets receive adequate passive immunity. the anti-pedv specific siga immunochromatographic test strip for the detection of siga antibodies developed in our study provides a simple, sensitive and specific tool for monitoring passive immunity and pedv infection. null stands for not done. the cut off value of od 405 value was 0.305. the colostrum samples with an od405 value ≥ 0.305 were considered to be positive while < 0.305 were considered to be negative. practical statistics for medical research the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex illness in breastfeeding infants relates to concentration of lactoferrin and secretory immunoglobulin a in mother's milk coronavirus genome structure and replication isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states establishment and application of indirect elisa for detection of porcine epidemic diarrhea virus specific siga intravascular and mucosal immunoglobulin a: two separate but related systems of immune defense? development of an enzyme-linked immunosorbent assay for themonitoring and surveillance of antibodies to porcine epidemicdiarrhea virus based on a recombinant membrane protein phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china preparation and identification of monoclonal antibodies against porcine siga sc fragment residues involved in the antigenic sites of transmissible gastroenteritis coronavirus s glycoprotein detection of immunoglobulin (ig) a antibodies against porcine epidemic diarrhea virus (pedv) in fecal and serum samples detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states direct and rapid detection of porcine epidemic diarrhea virus by rt-pcr pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs the polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex rt-pcr completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field strains in central china based on the orf3 gene and the main neutralization epitopes development of an indirect elisa based on a truncated s protein of the porcine epidemic diarrhea virus development of an immunochromatographic strip for detection of antibodies against porcine reproductive and respiratory syndrome virus rapid and sensitive lateral flow immunoassay method for determining alpha fetoprotein in serum using europium (iii) chelate microparticles-based lateral flow test strips neutralization of genotype 2 porcine epidemic diarrhea virus strains by a novel monoclonal antibody the human iga system: a reassessment a new coronavirus-like particles associated with diarrhea in swine porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines polymeric iga is superior to monomeric iga and igg carrying the same variable domain in preventing clostridium difficile toxin a damaging of t84 monolayers construction and characterization of recombinant porcine adenovirus serotype 5 expressing the transmissible gastroenteritis virus spike gene iga and the iga fc receptor immunogenicity and antigenic relationships among spike proteins of porcine epidemic diarrhea virus subtypes g1 and g2 this work was supported by grants from the national key r & d plan for the 13th five year plan (2017yfd0501603), national natural science foundation of china (no. 31502066), the natural science foundation of heilongjiang (no. qc2016043). all authors have carefully revised the manuscript point-by-point according to the reviewers' comments. none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper. none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper. key: cord-261993-u2a26brw authors: kim, yonghyan; krishna, venkatramana d.; torremorell, montserrat; goyal, sagar m.; cheeran, maxim c.-j. title: stability of porcine epidemic diarrhea virus on fomite materials at different temperatures date: 2018-02-13 journal: vet sci doi: 10.3390/vetsci5010021 sha: doc_id: 261993 cord_uid: u2a26brw indirect transmission of porcine epidemic diarrhea virus (pedv) ensues when susceptible animals contact pedv-contaminated fomite materials. although the survival of pedv under various phs and temperatures has been studied, virus stability on different fomite surfaces under varying temperature conditions has not been explored. hence, we evaluated the survival of pedv on inanimate objects routinely used on swine farms such as styrofoam, rubber, plastic, coveralls, and other equipment. the titer of infectious pedv at 4 °c decreased by only 1 to 2 log during the first 5 days, and the virus was recoverable for up to 15 days on styrofoam, aluminum, tyvek(®) coverall, cloth, and plastic. however, viral titers decreased precipitously when stored at room temperature; no virus was detectable after one day on all materials tested. a more sensitive immunoplaque assay was able to detect virus from styrofoam, metal, and plastic at 20 days post application, representing a 3-log loss of input virus on fomite materials. recovery of infectious pedv from tyvek(®) coverall and rubber was above detection limit at 20 days. our findings indicate that the type of fomite material and temperatures impact pedv stability, which is important in understanding the nuances of indirect transmission and epidemiology of pedv. porcine epidemic diarrhea virus (pedv) is an alphacoronavirus in the family coronaviridae. this enveloped rna virus causes highly contagious viral enteritis in swine [1, 2] . the virus was first identified in england in 1971 and then in other european countries [3] . from 1982, pedv outbreaks were observed in asian countries including japan, korea, philippines, vietnam, and china. until 2009, pedv strains in asia were genotypically similar to those in europe. since 2010, however, pedv has caused outbreaks in asia with severe clinical signs and high mortality in suckling pigs, resulting in heavy economic losses to the pig farmers [3] [4] [5] [6] [7] . in may 2013, a pedv strain, genetically related to a chinese strain, was introduced in the us, possibly by importation of contaminated feed or contaminated feed tote bags [8, 9] . the virus spread rapidly across the country causing high mortality in piglets [10] . over eight million pigs were killed during this outbreak, leading to an estimated loss of 1.8 billion us dollars [11] [12] [13] [14] . transmission of pedv primarily occurs by the fecal-oral route, but indirect transmission can occur when an animal comes in contact with inanimate objects (fomites) contaminated with the feces of pedv-infected animals. disease outbreaks primarily occur in early fall and winter, but the rationale for seasonality remains unclear [15] . previous studies indicate that pedv is stable at 50 • c and at ph between 7.2 and 10.2 [16] [17] [18] [19] . a recent study found that the virus can survive for up to nine months at the center of manure lagoons [20] . we designed this study to evaluate the survival of pedv on various fomite surfaces at both room temperature (rt) and 4 • c. inanimate objects such as rubber boots, gloves, coveralls, and other equipment are routinely used on swine farms and have the potential of being contaminated with manure from pedv infected animals, thereby helping the indirect mode of virus transmission. a strain of pedv (pedv usa/colorado/2013; genbank accession number kf272920) was propagated on vero 76 cells (atcc crl-1587, manassas, va, usa) using dulbecco's modified eagle medium (dmem) supplemented with 0.5 µg/ml tpck-trypsin (worthington biochemical corporation, lakewood, nj, usa) and 0.3% tryptose phosphate broth (sigma, st. louis, mo, usa). the virus was harvested three days post infection (d.p.i.) using one freeze-thaw cycle. after centrifugation at 500× g for 10 min, the supernatant was aliquoted and stored at −80 • c. virus titration was performed on vero 76 cells' monolayers contained in 96-well microtiter plates using serial 5-fold dilutions of samples. virus titer was calculated by the spearman-kärber method [21] and expressed as 50% tissue culture infective dose (tcid 50 )/ml. circular pieces (approximately 1 cm 2 ) of various fomite materials were cut to fit into individual wells of costar ® 24-well cell culture plates (corning inc., corning, ny, usa). materials used were styrofoam, nitrile gloves, cardboard, aluminum foil, tyvek ® coveralls, cloth, metal, rubber, and plastic. cell culture-grown stock virus was applied on each type of material contained in triplicate wells (200 µl of virus containing 2.1 × 10 6 tcid 50 /ml). virus applied on the bottom of 24-well plate without any fomite material served as control. the applied virus was air dried for 2 h in a biosafety cabinet (bsc). sets of virus-contaminated fomites were stored at either at room temperature (~25 • c) or at 4 • c. at various times thereafter (0, 1, 2, 5, 10, 15, 20, and 30 days post application), the surviving virus was eluted from each material in 1 ml of 3% beef extract −0.05 m glycine buffer placed in 50 ml centrifuge tubes. after thorough mixing for 20 s on a vortexer (scientific industries inc., bohemia, ny, usa), the eluate was filtered through a 0.22 µm membrane filters (millipore, billerica, ma, usa) followed by virus titration on vero cell monolayers. virus stability on each fomite material was assessed as three independent replicates for all the time points tested. separate fomite samples were used at every time point, and eluates were processed independently for quantification of infectious virus and viral rna. to compare the survival of cell culture-adapted virus with that of virus contained in fecal material, a separate experiment was done. fecal material obtained from pedv negative pigs was spiked with 200 µl of cell culture propagated pedv (2.1 × 10 6 tcid 50 /ml). after thorough mixing, contaminated fecal matter was applied in 200 µl amounts on fomite materials in triplicate. virus was eluted and titrated as described above for cell culture derived virus. stability of viral rna on fomites was also tested by real-time rt-pcr. briefly, rna was extracted from the eluates using magmax™ 96 viral rna isolation kit (thermo fisher scientific, waltham, ma, usa). primer pair was designed to amplify a portion of the pedv s gene with the following sequences: forward 1910: acgtccctttactttcaattcaca and reverse 2012: tatacttggtacacacatccagagtca. pcr amplification was quantified using a fam labeled probe 1939: fam-tgagttgattactggcacgcctaaaccac-bhq. the primer and probe were added to agpath-id™ one-step rt-pcr reagents (thermo fisher scientific, waltham, ma, usa) along with 5 µl of extracted total rna followed by amplification with 7500 fast real-time pcr system (thermo fisher scientific, waltham, ma, usa) using the following conditions: reverse transcription at 48 • c for 10 min; denaturation at 95 • c for 10 min; 40 cycles of denaturation at 95 • c for 15 s; and annealing at 60 • c for 45 s. immunoplaque assays were performed monolayers of vero cells contained in 24-well tissue culture plates. duplicate wells were infected with 200 µl of 10-fold serially diluted sample eluate or stock pedv (positive control). plates were incubated for 1 h at 37 • c, and the inoculum was removed and replaced with infection medium with or without 1% agarose. at 24 h post infection (without agarose overlay) or 72 h post infection (with agarose overlay), the medium was removed from the wells and the cells were fixed with 4% paraformaldehyde for 20 min at 4 • c. all subsequent washes and incubations were done in pbs, containing 5% normal goat serum and 0.3% triton x-100. after three washes and blocking for an hour in buffer, plates were incubated overnight at 4 • c with one of the following primary antibodies: (i) mouse monoclonal antibody for pedv spike protein (diluted 1:500; clone 3f12, median diagnostics, chuncheon, korea); or (ii) mouse monoclonal antibody for pedv spike protein (diluted 1:500; clone s1d12, vmrd, pullman, wa, usa). wells were then washed three times with wash buffer and incubated for 1 h at room temperature with alkaline phosphatase conjugated anti-mouse igg secondary antibody (diluted 1:200; thermo fisher scientific, waltham, ma, usa). after three washes in buffer, plates were incubated for approximately 20 min with 1-step™ nbt/bcip substrate solution (thermo fisher scientific, waltham, ma, usa). immunostained cells were observed under a light microscope (nikon, tokyo, japan). results are presented as mean ± sem of the three independent replicates of commonly used fomite materials on a farm. virus sample eluted from each fomite replicate was processed independently by tcid 50 assay, immunoplaque assay, and real-time quantitative rt-pcr. an anova was used to determine statistical significance between data sets using graphpad prism 7 software (graphpad software inc., san diego, ca, usa). infectious pedv was recovered from fomite materials for up to 15 days post application at 4 • c; only 1 to 2 logs of virus were inactivated during the first 5 days post application ( figure 1 ). varying levels of virus loss were observed in different fomite materials used. after 10 days at 4 • c, rubber, nitrile gloves, and metal showed residual virus at or below the detection limit of the assay (≤2 × 10 2 tcid 50 /ml), while all other materials tested had ≥10 3 tcid 50 /ml of residual virus. infectious pedv was recovered after 15 days at 4 • c on styrofoam, aluminum foil, tyvek ® coverall, cloth, and plastic, with titers between 10 2 to 10 3 tcid 50 /ml. however, virus recovery from nitrile gloves, cardboard, metal, and rubber was below the detection limit of the assay (2 × 10 2 tcid 50 /ml) after 15 days at 4 • c. on the other hand, pedv survival decreased precipitously at room temperature within 1 to 2-days post application, losing 2 to 4 log titers within 24 h (figure 1 ). infectious pedv was not recovered from any fomite material after 2 days at rt. virus recovery from surfaces of styrofoam, nitrile gloves, aluminum foil, tyvek ® coverall, metal, rubber, plastic, cardboard, and cloth showed no significant differences between the materials at rt, suggesting that storage temperature had a substantial influence on virus survival. virus survival on nine fomites at room temperature (rt) and at 4 °c. pedv was applied on fomite materials or in control wells followed by storage at rt (▲) or at 4 °c (■). the surviving virus was eluted after various time periods and titrated. decay of infectious virus was rapid at rt but delayed when stored at 4 °c. black dotted line represents detection limit of tcid50 assay, which is 2 × 10 2 tcid50/ml. data presented are average of three values (± sem) obtained from independent assessments at each time point indicated. we used a highly sensitive immunodetection method to identify pedv positive plaques on vero cells. the lower limit of detection by this method is 24 focus forming units (ffu)/ml. positive plaques stain purple after immunostaining with an antibody to pedv spike protein, followed by nbt/bcip substrate ( figure 2 ). this assay was performed at 24 h p.i. without using an agarose overlay, because the results obtained with or without agarose were similar. using the immunoplaque assay, we re-tested virus stability on certain fomite materials after 20 days of storage at 4 °c. varying levels of viable pedv were detected ( figure 3 ). titers of approximately 1 × 10 3 ffu/ml were observed in eluates from styrofoam, metal, and plastic, representing a 3-log virus inactivation after 20 days. the surviving virus on tyvek ® coverall and rubber surfaces was moderately above detection limit (24 ffu/ml). in contrast, no plaques were detected from fomite materials stored at rt for 48 h post application (data not shown). we used a highly sensitive immunodetection method to identify pedv positive plaques on vero cells. the lower limit of detection by this method is 24 focus forming units (ffu)/ml. positive plaques stain purple after immunostaining with an antibody to pedv spike protein, followed by nbt/bcip substrate ( figure 2 ). this assay was performed at 24 h p.i. without using an agarose overlay, because the results obtained with or without agarose were similar. using the immunoplaque assay, we re-tested virus stability on certain fomite materials after 20 days of storage at 4 • c. varying levels of viable pedv were detected (figure 3 ). titers of approximately 1 × 10 3 ffu/ml were observed in eluates from styrofoam, metal, and plastic, representing a 3-log virus inactivation after 20 days. the surviving virus on tyvek ® coverall and rubber surfaces was moderately above detection limit (24 ffu/ml). in contrast, no plaques were detected from fomite materials stored at rt for 48 h post application (data not shown). to determine the amount of viral rna remaining on fomites under different storage conditions, eluates were tested by quantitative rt-pcr. viral rna was detected after 2 days at rt and 20 days at 4 • c, although no infectious virus was detected at rt after 2 days ( figure 4a ). in fact, all materials tested had cycle threshold (ct) values similar to those of input virus (~16-17), with the exception of eluates from cardboard that showed a ct value of 21 (table 1) . quantifiable viral rna was detected in styrofoam, tyvek, and cardboard materials, although infectious pedv titer decreased by 3 to 4 logs at 4 • c after 20 days. in fecal material spiked with cell culture grown virus, no infectious virus was detected by either tcid 50 or immunoplaque assay. in fact, eluates from fecal samples induced remarkable cell toxicity, making the infectious virus assays difficult to interpret. however, viral rna was detected in these samples after 2 days at rt and 20 days at 4 • c; the rna levels were similar to those of input virus indicating no significant changes in viral genome levels in feces ( figure 4b and table 1 ). to determine the amount of viral rna remaining on fomites under different storage conditions, eluates were tested by quantitative rt-pcr. viral rna was detected after 2 days at rt and 20 days at 4 °c, although no infectious virus was detected at rt after 2 days ( figure 4a ). in fact, all materials tested had cycle threshold (ct) values similar to those of input virus (~16-17), with the exception of eluates from cardboard that showed a ct value of 21 (table 1) . quantifiable viral rna was detected in styrofoam, tyvek, and cardboard materials, although infectious pedv titer decreased by 3 to 4 logs at 4 °c after 20 days. in fecal material spiked with cell culture grown virus, no infectious virus was detected by either tcid50 or immunoplaque assay. in fact, eluates from fecal samples induced remarkable cell toxicity, making the infectious virus assays difficult to interpret. however, viral rna was detected in these samples after 2 days at rt and 20 days at 4 °c; the rna levels were similar to those of input virus indicating no significant changes in viral genome levels in feces ( figure 4b and table 1 ). our study demonstrates that cell culture-grown pedv remains viable for extended periods when dried and maintained in a cold environment. the virus remained viable at 4 • c for up to 20-days on styrofoam, metal, and plastic, although viral titers decreased by 3 logs in 20 days. when stored at rt, pedv decreased by 4 to 5 logs within 48 h, rendering it undetectable using infectious virus assays. this observation suggests that the storage temperature of the fomite material has a major impact on virus stability. it appears that low storage temperature delays virus degradation on fomite material. generally, enveloped viruses are more vulnerable to environmental conditions [22] . however, pedv was found to have higher stability when stored in spray dried bovine plasma, being stable for up to 3 weeks at 4 • c, 2 weeks at 12 • c, and 1 week at 22 • c [18] . in addition, other coronaviruses, like transmissible gastroenteritis virus (tgev) and mouse hepatitis virus (mhv), were also found to be more stable at 4 • c, surviving for as long as 28 days at 4 • c and up to 5 days at rt [23] . we demonstrated in this study that the infectious virus decay rate on all fomites increased rapidly at rt. this temperature-sensitive feature of pedv may be applied to routine procedures in the farms to help eradicate pedv in the environment and prevent transmission via fomites [16, 24, 25] . the type of fomite material also has implications on viral stability. for example, the survival of prrsv (porcine reproductive and respiratory syndrome virus) differed in different types of materials including solid, porous, and liquid substances [26] . we found that infectious pedv survived at 4 • c on styrofoam, metal, and plastic, retaining 3 logs of infectious virus (from~10 6 ffu/ml) at 20 days. the rate of loss in infectivity was uniform on styrofoam, aluminum, plastic, and tyvek ® coverall, whereas viral infectivity on metal, nitrile gloves, and rubber dropped rapidly after 5 days at 4 • c. furthermore, at 0 days post application (i.e., post 2 h of air drying), there was decreased virus recovery from cardboard and cloth, indicating that the composition of the fomite and the porosity of the pedv-contaminated materials may influence virus survival. our results are consistent with previous studies on virus survival on glass, stainless steel, and plastic for up to 10 days [26, 27] . specifically, styrofoam, metal, and plastic provided extensive pedv stability. feed totes made of polypropylene (commonly used in plastic material) allowed pedv survival for 10 weeks [9] . in addition to storage temperature, porosity of fomite may also influence virus survival and hence virus transmission. our data suggest that long-term persistence of pedv on contaminated surfaces could have an epidemiological impact on disease outbreaks, given that >200 infectious virus units were recovered after 20 days at 4 • c on fomite material and that an infectious dose as low as 56 tcid 50 units infects 100% of animals [28] . pedv transmission appears to be relatively effective via transportation vehicles [29, 30] and feed [18, 20, 31, 32] . our data showed that pedv can survive on metal for up to 20 days at 4 • c, which may play a significant role in promoting the spread of pedv. the longer survival of the virus at cold temperatures may also explain the increase in outbreaks during the winter season [15] . furthermore, enveloped viruses generally survive better in the presence of organic material [33] . in fact, a large amount of lagoon manure possibly contributes to increased survivability of pedv, in which the virus can remain infective for up to nine months after being shed by pigs [20] . in this study, viral rna copy numbers did not correlate with the cell-based assays. it is possible that pedv infectivity is destroyed by factors that affect the integrity of the viral envelope without affecting rna degradation at rt. in a recent study, it was shown that pedv viral rna steadily degrades over time at temperatures above 37 • c in cell culture medium, but the rapid decline of viral rna that is detected by pcr occurs only at temperatures higher than 60 • c [34] . although viral rna degradation was not assessed in the present study, it is clear that the detection of the viral genome does not affect the infectious nature of the sample. besides temperature, the stability of pedv on fomite is also dependent on presence or absence of other organic material and ph. although we used both cell culture propagated pedv and pedv spiked fecal material on fomites to mimic actual situation in the field, we were unable to determine the survival of the virus in pedv-spiked fecal material, since the eluates from fomites with feces were not suitable for infectious virus titer due to the cytotoxic effect of the fecal content. in conclusion, our findings provide a new perspective on how fomite material and temperature impact viral stability over time, indicating the significance of understanding the nuances of indirect transmission in the epidemiology of pedv. a new coronavirus-like particle associated with diarrhea in swine diseases of swine isolation of porcine epidemic diarrhea 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investigating the introduction of porcine epidemic diarrhea virus into an ohio swine operation effect of surfactants on the survival and sorption of viruses effectiveness of composting as a biosecure disposal method for porcine epidemic diarrhea virus (pedv)-infected pig carcasses this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license this study was funded in part by the state of minnesota, the university of minnesota college key: cord-284322-synuzaxm authors: borel, nicole; dumrese, claudia; ziegler, urs; schifferli, andrea; kaiser, carmen; pospischil, andreas title: mixed infections with chlamydia and porcine epidemic diarrhea virus a new in vitro model of chlamydial persistence date: 2010-07-27 journal: bmc microbiol doi: 10.1186/1471-2180-10-201 sha: doc_id: 284322 cord_uid: synuzaxm background: chlamydiae induce persistent infections, which have been associated with a wide range of chronic diseases in humans and animals. mixed infections with chlamydia and porcine epidemic diarrhea virus (pedv) may result in generation of persistent chlamydial infections. to test this hypothesis, an in vitro model of dual infection with cell culture-adapted pedv and chlamydia abortus or chlamydia pecorum in vero cells was established. results: infected cultures were investigated by immunofluorescence (if), transmission electron microscopy (tem) and re-infection experiments. by if, chlamydia-infected cells showed normal inclusions after 39 hpi. dual infections with chlamydia abortus revealed a heterogenous mix of inclusion types including small inclusions consisting of aberrant bodies (abs), medium-sized inclusions consisting of abs and reticulate bodies and normal inclusions. only aberrant inclusions were observable in dual infection experiments with chlamydia pecorum and pedv. tem examinations of mixed infections with chlamydia abortus and chlamydia pecorum revealed aberrant chlamydial inclusions containing reticulate-like, pleomorphic abs, which were up to 2 μm in diameter. no re-differentiation into elementary bodies (ebs) was detected. in re-infection experiments, co-infected cells produced fewer ebs than monoinfected cells. conclusions: in the present study we confirm that pedv co-infection alters the developmental cycle of member species of the family chlamydiaceae, in a similar manner to other well-described persistence induction methods. interestingly, this effect appears to be partially species-specific as chlamydia pecorum appears more sensitive to pedv co-infection than chlamydia abortus, as evidenced by tem and if observations of a homogenous population of aberrant inclusions in pedv chlamydia pecorum co-infections. chlamydiae are implicated in a wide variety of diseases in both animals and humans. although acute infections in animal chlamydioses are the most commonly reported, chronic chlamydial infections are also associated with a variety of diseases in humans and animals. these latter infections are characterized by inflammation and scarring resulting in significant damage of the host. a causative role in chronic diseases requires that chlamydiae persist within infected tissue for extended periods of time. current theories, based primarily on in vitro data, suggest that chlamydial persistence, and the resulting chronic inflammation, is linked to morphological and metabolic conversion of the actively replicating and intracellular reticulate body (rb) into an alternative, non-replicative form known as an aberrant body (ab) [1] . in vitro, alterations of the normal developmental cycle of chlamydia trachomatis and chlamydia pneumoniae can be induced by interferon-γ (ifn-γ), tumor necrosis factor-α (tnf-α) and penicillin g exposure as well as amino acid or iron deprivation and monocyte infection [2, 3] . to date, in vitro models for animal pathogens, chlamydia abortus and chlamydia pecorum have not been described although both organisms are associated with chronic disease in koalas and small ruminants [1] . in pigs, several chlamydial species, including chlamydia abortus, chlamydia psittaci, chlamydia pecorum and chlamydia suis, have been implicated in a variety of disease conditions including conjunctivitis, pneumonia, pericarditis, polyserositis, arthritis, abortion and infertility [4] . in the gastrointestinal tract, chlamydiae appear to be highly prevalent but only occasionally cause enteritis. they have been found in the intestine of diarrheic and healthy pigs and could be demonstrated in mixed enteric infections [5] [6] [7] . pospischil and wood [7] first described an association between chlamydiaceae and lesions in the intestinal tract of pigs and assumed a synergistic effect in co-existence with salmonella typhimurium. further, mixed infections with eimeria scabra, cryptosporidia, and porcine epidemic diarrhea virus (pedv) have been described in the past. pedv, a member of the family coronaviridae, is a well-known cause of diarrhea in pigs. after the identification of pedv in 1978 by pensaert and debouck [8] , more than a decade passed before the virus could be adapted for propagation in cell cultures. examination of infected vero cell cultures by direct immunofluorescence revealed single cells with granular cytoplasmic fluorescence as well as formation of syncytia with up to 50-100 nuclei or more. typical features of syncytial cells were growth, fusion and detachment from cell layers after they had reached a certain size [9] . biomolecular studies revealed major genomic differences between cell culture-adapted (ca)-pedv and wild type virus [10, 11] . cell culture model of co-infection with ca-pedv and chlamydia has been established recently [12] to investigate the interaction of ca-pedv and chlamydiaceae in mixed infections and to detect possible synergistic or additive effects of possible significance in clinical enteric disease in pigs. in that study, abnormally large chlamydial forms were observed in dually infected cell layers by immunofluorescence suggesting that ca-pedv co-infection might alter the chlamydial developmental cycle in a manner similar to that observed during persistent infections. to confirm these initial observations, we established a cell culture model of mixed infections with chlamydia and a cell culture-adapted porcine epidemic diarrhea virus (ca-pedv) and hypothesized that this would result in the generation of persistent chlamydial forms. this data demonstrates that ca-pedv co-infection, indeed, alters the developmental cycle of chlamydia pecorum and chlamydia abortus in a similar manner to other inducers of chlamydial persistence. vero cells can be co-infected with chlamydia and ca-pedv immunofluorescence (if) labeling was used to investigate the morphologic differences of chlamydia between monoinfected and dually infected monolayers using chlamydia and ca-pedv specific antibodies. control and mock-infected cells did not stain with either antibody. ca-pedv monoinfected cells showed brilliant and distinct, red cytoplasmic fluorescence. syncytia were characterized by accumulation of nuclei in the center or the periphery of the multi-nucleated cells and moderate to bright, fine-granular, cytoplasmic ca-pedv labeling (figure 1b) . syncytia were categorized into small (2-15 nuclei), medium (16-30 nuclei) and large (more than 30 nuclei). in single infection experiments, syncytia at 24 h post infection were mostly large with fewer medium sized syncytia observed (data not shown). numbers of syncytia in ca-pedv single and dual infections were counted on the whole coverslip and mean values were determined. no difference of viral syncytia numbers for ca-pedv monoinfection and dual infection with chlamydia abortus were seen (data not shown). in contrast, numbers of viral synyctia in dual infections with chlamydia pecorum were diminished compared to the respective ca-pedv single infections (table 1) . if microscopy of chlamydial single infections revealed intracytoplasmic, mainly round to ovoid, sharply outlined inclusions with brilliant, green fluorescence. chlamydia abortus and chlamydia pecorum infected cells had one to five, finely granular (consisting mainly of ebs) inclusion(s) per cell at 39 h post infection ( figure figure 1 morphology of chlamydia pecorum mono-and coinfection with pedv. a) vero cells were infected with chlamydia pecorum 1 moi for 39 h, with subsequent pedv inoculation and labelled with an anti-chlamydia antibody (green); b) double infected monolayer were labelled for ca-pedv in red, chlamydia in green and dna in blue; c) chlamydia pecorum mono-infected vero cells labelled with an anti-chlamydia antibody (green) and dna staining (blue); d) inclusion size was measured as described and the frequency of chlamydial inclusions assembled into sizes of 50 μm 2 area groups depicted. the difference between mono and double infected monolayers was statistically analyzed using students t-test. the groups were significantly different with p = 0.0044. 1c &2a). in general, chlamydial inclusions were smaller and had more variable forms in chlamydia pecorum than in chlamydia abortus single infections. infectivity was almost 100% and a moderate number of free ebs could be observed. compared to single infections, the size and shape of chlamydial inclusions in pedv co-infections was highly variable. in chlamydia abortus co-infection experiments, three types of inclusions were observed: (i) small inclusions consisting of 1-10 aberrant bodies (abs), (ii) medium-sized inclusions consisting of abs and reticulate bodies (rbs), and (iii) large (normal) inclusions consisting of ebs as seen in the single infection experiments (figure 2b ). in contrast, dual infections with ca-pedv and chlamydia pecorum resulted in the exclusive production of aberrant inclusions containing between 2-50 abs. chlamydial inclusions in viral syncytia grew even larger than in non-viral infected vero cells. overall, no normal chlamydial inclusions were observed (figure 1a &1b) . image analysis was used to compare inclusion size in single chlamydiae-infected vero cells with the inclusion size in vero monolayers that subsequently underwent ca-pedv virus infection. to this end, inclusion size was determined in μm 2 and all inclusions were assembled into groups covering 50 μm 2 and multiples of this area. the average frequency of chlamydia pecorum inclusions between 100 μm 2 and 400 μm 2 was significantly reduced when cells were subsequently infected with ca-pedv. in other words, chlamydia pecorum inclusions vero cells were infected with chlamydia abortus with subsequent pedv inoculation and stained as with an anti-chlamydia antibody and dapi; c) frequency of inclusions with various sizes was calculated and mono and double infected cells were compared according to the inclusion size. the difference between mono and double infected monolayers was statistically analyzed using students t-test. the groups were significantly different with p = 0.0132. a numbers of syncytia for ca-pedv monoinfection and dual infection with chlamydia pecorum were counted on the whole coverslip. b vero cells were mock infected (mock), c. pecorum infected, ca-pedv infected (ca-pedv) and chlamydia pecorum/ca-pedv co-infected as described. were highly significant smaller in ca-pedv dual infections than in those infections without the addition of virus ( figure 1d ) as analyzed by t-test (p = 0.0044). the additional changes observed in the shape of all inclusions growing in virus-infected monolayers indicated the induction of chlamydia pecorum persistence, since the finely dispersed staining reverted to grape-like structures (figure 1a &1b ). the changes of chlamydial inclusion size by subsequent virus addition to chlamydia abortus are different to those we observed in the chlamydia pecorum dual infection experiments. the frequency of inclusions observed between a size range of 0-200 μm 2 was significantly (p = 0.0132) reduced under virus infection but the amount of medium sized and big inclusions 300 -700 μm 2 was increased ( figure 2c ). the morphology of chlamydia abortus inclusions was also found to differ in the population when co-infected with ca-pedv. smaller inclusions were generally observed in aberrant shapes compared to larger inclusions, which appeared similar to normal actively growing inclusions showing finely dispersed staining (figure 2b ). this effect might be due to an incomplete induction of persistence of chlamydia abortus when cells were ca-pedv coinfected. co-infection with ca-pedv induced ultrastructural morphological changes in chlamydia abortus and chlamydia pecorum persistent forms of chlamydia trachomatis and chlamydia pneumoniae are well described by their characteristic electron microscopic appearance [2, 13, 14] . thus, chlamydial ultrastructure in single and co-infected cells was compared by transmission electron microscopy (tem). at 24 h after viral infection, viral-induced syncytia containing vacuoles filled with viral particles were present in ca-pedv-monoinfected and dual infections. the viral particles showed the typical coronavirus morphology with a diameter between 50 to 130 nm (data not shown). at 39 h after chlamydial infection, large intracytoplasmic chlamydial inclusions in single infected cells could be observed in vero cells infected with chlamydia abortus or chlamydia pecorum. the inclusions observed contained variable numbers of morphologically normal rbs and ebs and were generally located near the host cell nucleus, often surrounded by mitochondria (figure 3a and 3b) . tem examinations of mixed infections (ca-pedv and chlamydia abortus or chlamydia pecorum) revealed aberrant chlamydial inclusions containing fewer bacteria than typical inclusions and were located in viral syncytia or single cells without viral infection. aberrant inclusions consisted of reticulate-like, pleomorphic, aberrant bodies (abs), which were in general larger in diameter (up to 2 μm) than typical reticulate bodies (rbs), with a sparse densitometric appearance and no re-differentiation into elementary bodies (ebs). as already observed in if investigations, three types of inclusions were present in dual infections with ca-pedv and chlamydia abortus (figure 3c ), whereas dual infections with ca-pedv and chlamydia pecorum resulted in the exclusive production of aberrant inclusions consisting of 2-50 abs (figure 3d ). neither chlamydial inclusions nor ca-pedv virions were visible in mock-infected cells. previous studies have demonstrated that chlamydial persistent forms are non-infectious [2] . reduced number or even a lack of ebs in co-infected cells in tem suggested arrested chlamydial developmental cycle with halted maturation from rb to eb. to ascertain the effect of ca-pedv inhibition of chlamydial eb production, the yield of infective chlamydial progeny was determined after 40 h of re-infection in three independent experiments for chlamydia abortus (figure 4a ) and for chlamydia pecorum (figure 4b ). neither mock nor ca-pedv monoinfected cells produced detectable infectious ebs, whereas chlamydia abortus and chlamydia pecorum single infections cells produced abundant ebs. coinfected cells produced fewer infectious ebs than nonviral infected cells, demonstrating that production of infectious chlamydial progeny was essentially diminished by ca-pedv-co-infection. eradication of infectious eb production was almost complete in chlamydia pecorum double infection, analyzed by reinfection experiments and found to be statistically different as analyzed by ttest (p = 0.0145) (figure 4b ). in chlamydia abortus reinfection analysis, several ebs could still be observed in spite of the co-infection with ca-pedv (figure 4a this data is consistent with the observations from our if and ultrastructural analysis. chlamydial co-infection does alter ca-pedv infection depending on the chlamydial species but does not alter viral ultrastructure to determine whether chlamydial pre-infection altered subsequent viral infection, numbers of syncytia and ca-pedv-infected cells from single and co-infected monolayers of three unrelated experiments were counted. mock-infected and chlamydia only infected cells produced no virions. the difference between virus-infected cells and co-infection with chlamydia abortus was minimal. the number of syncytia detected were within the same range (data not shown) indicating that chlamydial co-infection with chlamydia abortus does not alter ca-pedv infection or the development of syncytia. in contrast, numbers of syncytia in co-infection with chlamydia pecorum were reduced compared to single ca-pedv infection (table 1) . overall numbers of single viral infected cells were low in both single and co-infection experiments, and no significant difference between the two chlamydial species was obvious (data not shown). viral morphology was also studied by tem. in ca-pedv single and co-infected cells, viral particles were unaltered indicating that chlamydial co-infection did not induce any changes in viral ultrastructural morphology. while a previous study [12] primarily investigated the interaction of ca-pedv and chlamydiaceae in mixed infections to detect possible synergistic or additive effects of these two pathogens, questions remained about whether viral infection could potentially induce the persistent chlamydial phenotype. enlarged chlamydial inclusions were described in that study in the ca-pedv co-infection model with chlamydia abortus and chlamydia pecorum but no further ultrastructural analysis has been subsequently performed. in this study, in vitro models of chlamydia abortus and chlamydia pecorum persistence were established using co-infection with ca-pedv. several experimental methods were used to demonstrate the characteristic features of chlamydial persistence, including altered ultrastructural morphology and decreased production of infectious ebs. our results demonstrated that ca-pedv-co-infection alters the chlamydial developmental cycle similarly to other inducers of chlamydial persistence. a similar co-infection model has been recently described by deka et al. (2006) [15] . in that study, it was shown that chlamydia trachomatis enters a viable but non-cultivable, persistent state with herpes simplex virus type 2 (hsv-2) co-infected host cells. in contrast, a similar study investigating a coinfection model with chlamydia trachomatis and genital mycoplasmas, mycoplasma genitalium and mycoplasma hominis, did not change the morphology of chlamydial rbs, indicating that co-infection of these two microorganisms is likely to be independent and not related to the onset of chlamydial persistence [16] . in the study by deka et al. (2006) [15] , hela monolayers were first infected with chlamydia trachomatis and 24 h later with hsv-2. in our study, the optimal experimental protocol for co-infection procedure was developed, based on our own earlier study [12] , and optimization experiments performed as a part of the current study (data not shown). to this end, vero monolayers were first infected with chlamydia and later with ca-pedv, thus the suspected inducer of persistence would be introduced after chlamydial infection and differentiation into rbs. simultaneous infection of chlamydia and ca-pedv has been performed earlier [12] , but did not result in persistent infection in our preliminary experiments (data not shown) and was not considered further as interference of chlamydial infection and concurrent viral uptake could have influenced the results. viral infection and subsequent development of syncytia was not affected by co-infection with chlamydia abortus as demonstrated by unaltered numbers of syncytia observed in the coinfection experiments. in contrast, viral syncytia formation was dramatically decreased in vero cells double infected with ca-pedv and chlamydia pecorum. if chlamydia pecorum infection might induce a down regulation of the host pedv receptor needed for syncytium formation at 14-15 hours post-chlamydial infection, this could produce a reduction in syncytium formation without reducing viral entry or replication -the possible persistence inducer mechanism. interestingly, chlamydial persistence was more prominent in co-infection with chlamydia pecorum than with chlamydia abortus, indicating possible species-specific differences. limited reports are available for in vitro models of chlamydial persistence from non-chlamydia trachomatis and chlamydia pneumoniae strains. kaltenboeck and storz (1992) [17] suggested that strain 1710s of chlamydia pecorum is highly nutrient dependent and this could elicit aberrant forms. indeed, aberrant forms of this strain were significantly present in our study. previously, only limited data have been published on persistent infection of l cells with an ovine abortion strain of chlamydia psittaci (current classification: chlamydia abortus) [18] . it should be noted, that in the latter study, chlamydial persistence was not demonstrated using the characteristic features now associated with the morphology of persistent chlamydial infections. detailed description of electron microscopic observations on the effects of penicillin on the morphology of chlamydia psittaci cal10 in l cells showing aberrant chlamydial stages were published by matsumoto and manire [13] . the different occurence of persistent forms in coinfection with chlamydia abortus and chlamydia pecorum, respectively, has not been described before. differences between persistence behaviour are already known (reviewed by hogan et al., 2004) [1] not only between different chlamydial species but also between different serovars and strains of chlamydia pneumoniae and chlamydia trachomatis, respectively. the fact that chlamydia pecorum strain 1710s is an original swine isolate whereas chlamydia abortus strain s26/3 originates from a sheep abortion and, thus, from another animal species could have an impact but needs further investigation. the mechanism by which ca-pedv interferes with chlamydial developmental cycle and chlamydial persistence is still unclear. it is known that vero cells, a monkey kidney epithelial cell line, is deficient for interferon production [19] ; thus, this cytokine group well known to be capable of inducing in vitro persistence in chlamydia pneumoniae [1] , cannot be relevant for our co-infection persistence model. co-infection experiments with ca-pedv are best performed with vero cells, as they have been shown to be permissive for viral replication in contrast to other cell lines such as pd5, pk 15, and hrt18 cell lines [9] . specific measurements of primate cytokines in our co-infection model are planned in the future to elucidate the mechanism leading to chlamydial persistence. the herpes simplex virus (hsv) co-induced chlamydia trachomatis persistence model [15] has been recently been shown not to be mediated by any known persistence inducer or anti-chlamydial pathway recently [20, 21] . instead, it was hypothesized by the authors that hsv-2 attachment and/or entry into the host cell is sufficient for stimulating chlamydial persistence, suggesting a potential novel host signaling pathway could be responsible for inducing chlamydial persistence. a very recent publication by the same group showed that hsv replication is not necessary for persistence induction and that chlamydial activity could be recovered after coinfection with uv-inactivated hsv-2. finally, it was concluded that the interaction of hsv glycoprotein d with the host cell surface is crucial to trigger chlamydial persistence [22] . female genital tract infection often has a complex etiology, where chlamydia trachomatis is present together with one or more genital agents. epidemiological and clinical studies have shown that double infection with hsv-2 and chlamydia trachomatis occurs in vivo; thus, the in vitro model described by deka et al. (2006) [15] represents a realistic situation in human medicine. similarities exist to the in vitro model established in this study as simultaneous intestinal infection with different pathogens is possible in swine in vivo. a recent study [23] documented the occurrence of aberrant chlamydial bodies in vivo in intestinal tissues of pigs. in this study, aberrant bodies of chlamydia suis were demonstrated and characterized in the gut of pigs experimentally infected with salmonella typhimurium by transmission electron microscopy. it was concluded by pospischil et al. [23] that aberrant bodies occur in vivo in pigs and that the gnotobiotic pig model might be suitable for the study of chlamydial persistence in vivo. available intestinal tissues from experimentally infected gnotobiotic piglets (single infection and coinfection with chlamydia and ca-pedv, respectively) will be investigated in the future with the aim of further characterization of abs in vivo. although chronic chlamydial diseases in animals are of economic impact, the pig model may also reveal the important link between persistence in vitro and in vivo and, thus, help to elucidate mechanisms of chronic human chlamydial infections in the future. the present study reports a new persistence model of chlamydia in co-infection with porcine epidemic diarrhea virus (pedv). pedv-co-infection altered the chlamydial developmental cycle similarly to other known inducers of chlamydial persistence. this new animal model could provide the important link between persistence in vitro and in vivo and, thus, would help to elucidate mechanisms of chronic human chlamydial infections in the future. growth medium (gm) for normal cell propagation was minimal essential medium (mem) with earle's salts, 25 mm hepes, without l-glutamine (gibco, invitrogen, carlsbad, ca) and supplemented with 10% fetal calf serum (fcs) (bioconcept, allschwil, switzerland), 4 mm glutamax-i (200 mm, gibco) and 0.2 mg/ml gentamycin (50 mg/ml, gibco). gm without gentamycin was used for the propagation of cells for infection experiments. infection medium was prepared as gm but without gentamycin and fcs, and was used for the infection and for the 24 h incubation period after the infection with ca-pedv, respectively. incubation medium was prepared as gm without gentamycin, freshly supplemented with 1 μg/ml cycloheximide (sigma, buchs sg, switzerland), and used after an infection for estimation of the chlamydial titer (ifu determination). vero 76 cells (african green monkey kidney cells, crl 1587 american type culture collection) were seeded on round plastic coverslips (13 mm diameter, bibby sterilin, stone, uk) and cultured in gm without gentamycin at 37°c until they reached confluence. before inoculation, the cells were washed once with phosphate buffered saline (pbs). two different chlamydial strains of chlamydiaceae were used in this study: chlamydia abortus s26/3 (ovine abortion strain, kindly donated by dr. g.e. jones, moredun research institute, edinburgh, gb) and chlamydia pecorum 1710s (intestinal swine isolate, kindly provided by prof. j. storz, baton rouge, louisiana, la, usa). for initial culturing, chlamydial strains were cultured in embryonated chicken eggs, and yolk sac material was harvested, diluted 1:2 in sucrose-phosphate-glutamate (spg) medium and stored at -80°c. yolk sac-derived chlamydiae were then propagated in hep-2 cell (atcc ccl-23) monolayers and elementary bodies (ebs) were harvested and purified by disruption of hep-2 cell monolayers with a cell scraper, sonication and centrifugation over a renografin density gradient as described elsewhere [24] . eb suspensions were stored in sucrosephosphate-glutamic acid buffer at -80°c, after which viable titers were established using standard methods. moi of 1 was used for chlamydial monoinfection and mixed infection, respectively. ca-pedv strain cv777 (kindly provided by prof. dr. m. ackermann, institute of virology, university of zurich) was propagated as previously described [9] . the virus (10 5,5 tcid 50 /ml) was used undiluted (1 ml 10 5,5 tcid 50 /ml). vero cells, an african green monkey kidney cell line (atcc crl 1587), were used for all infection experiments. they were propagated in gm without gentamycin at 37°c in an atmosphere of 5% co 2 . vero cells were divided into four groups: for mock infection, chlamydial infection, ca-pedv infection, and both chlamydia and ca-pedv double infection. host cells were infected with a moi of 1 for chlamydia and an infective dose of 1 × 10 5,5 tcid 50 /ml for ca-pedv, respectively. for ca-pedv monoinfections and negative controls, infection medium was used. all co-infection experiments were done three times and monoinfections with chlamydia and ca-pedv were performed simultaneously. the optimal experimental protocol (adding the virus several hours after chlamydial infection) for co-infection procedure was developed before (data not shown). for dual infections, cell monolayers were first infected with chlamydia at a moi of 1. all coverslips were centrifuged at 1000 × g for 1 h at 25°c. timepoint 0 (t 0 ) was defined after centrifugation and supernatant was replaced subsequently by incubation medium. infected monolayers were then incubated for 14 h at 37°c (t 0 -t 14 ). all cell layers for dual infections or ca-pedv monoinfection were exposed to a ca-pedv suspension (1 × 10 5,5 tcid 50 ), the samples were centrifuged again for 1000 × g for 1 h at 25°c and incubated for 24 h at 37°c. after this incubation period, all monolayers were fixed and further investigated by indirect immunofluorescence and transmission electron microscopy. re-infection experiments were performed to compare the production of infectious chlamydial elementary bodies (ebs) between monoinfections and mixed infections. for indirect immunofluorescence analyses, infected cells were fixed in absolute methanol (-20°c) for 10 min. and if labeling of cell cultures was performed immediately after fixation. for viral antigen detection, a mouse monoclonal antibody against the m protein of pedv (mcab 204, kindly provided by prof. dr. m. ackermann, institute of virology, university of zurich), diluted 1:4 in pbs supplemented with bsa, and an alexa fluor 594-conjugated secondary antibody (goat anti-mouse, 1:500, molecular probes, eugene, usa) were used. chlamydial inclusions were labeled with a chlamydiaceae family-specific mouse monoclonal antibody directed against the chlamydial lipopolysaccharide (mlps; clone aci-p, progen, heidelberg, germany) and a secondary alexa fluor 488-conjugated secondary antibody (goat anti-mouse, 1:500, molecular probes). dna was labeled with 1 μg/ml 4', 6-diamidin-2'-phenylindoldihydrochlorid (dapi, molecular probes). all staining procedures were conducted at room temperature. antibody incubations were carried out for 1 h (primary antibodies) or 45 min (secondary antibodies), respectively, with three washes of pbs following fixation, between and after applications of the different antibodies. dually infected cell layers were stained using sequential double immunofluorescence labeling. uninfected vero cells were used as a negative control. coverslips were mounted with immumount (shandon, pittsburgh, usa) on glass slides and investigated using a leica fluorescence microscope. coverslips from all experimental conditions were fixed in 2.5% glutaraldehyde (electron microscopy sciences, ft. washington, usa) for 1-2 h, and processed by routine methods for embedding in epoxy resin (fluka). appropriate areas for ultrastructural investigation were selected using semithin sections (1 μm) stained with toluidine blue (fluka, buchs sg, switzerland). ultrathin sections (80 nm) were mounted on gold grids (merck eurolab ag, dietlikon, switzerland), contrasted with uranyl acetate dihydrate (fluka) and lead citrate (lead nitrate and tri-natrium dihydrate; merck eurolab ag) and investigated in a philips cm10 electron microscope. at 39 h after chlamydial infection, monolayers were scraped into 1 ml of cold infection medium, pelleted and resuspended in 1 ml of fresh medium. infected host cells were lysed by sonication and centrifuged (500 g for 5 min) to remove pellet cell debris. supernatants were centrifuged once (4,000 g for 60 min). final eb pellets were resuspended in 200 μl of spg and used to infect vero cells plated on glass coverslips in duplicate in dilution series. all coverslips were centrifuged at 1000 × g for 1 h at 25°c. after centrifugation, the vero cells were refed with medium containing 1 μg/ml cycloheximide and subsequently incubated for 40 h at 37°c. fixation and staining of chlamydia, ca-pedv and dna was performed as described above. the number of inclusions in 20 random microscopic fields per sample was determined using a leica fluorescence microscope at a magnification of 200 ×. duplicate coverslips were counted and the counts were averaged. the number of inclusion-forming units (ifu) in the indiluted inoculum was then calculated and expressed as ifu per 10 6 cells as described by deka et al., 2006 [15] . from duplicate samples of three independent experiments uniform random sampled images were acquired using a widefield microscope (leica lx, leica microsystems mannheim, germany). cells and inclusions were automatically detected according to size, shape and intensity and counted using imaris (bitplane ag, zürich switzerland). chlamydial persistence: beyond the biphasic paradigm persistent chlamydiae: from cell culture to a paradigm for chlamydial pathogenesis morphologic and antigenic characterization of interferon gamma-mediated persistent chlamydia trachomatis infection in vitro prevalence of intestinal chlamydial infection in pigs in the midwest, as determined by immunoperoxidase staining intestinal chlamydia in finishing pigs intestinal chlamydia in pigs a new coronavirus-like particle associated with diarrhea in swine propagation of the virus of porcine epidemic diarrhea in cell culture sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf pedv leader sequence and junction sites mixed infections in vitro with different chlamydiaceae strains and a cell culture adapted porcine epidemic diarrhea virus electron microscopic observations on the effects of penicillin on the morphology of chlamydia psittaci chlamydia pneumoniae expresses genes required for dna replication but not cytokinesis during persistent infection of hep-2 cells chlamydia trachomatis enters a viable but non-cultivable (persistent) state within herpes simplex virus type 2 (hsv-2) co-infected host cells chlamydia trachomatis and genital mycoplasmas in the co-infection model -in vitro study biological properties and genetic analysis of the omp a locus in chlamydiae isolated from swine persistent infection of l cells with an ovine abortion strain of chlamydia psittaci characterization of the interferon regulatory factor 3-mediated antiviral response in a cell line deficient for ifn production an early event in the herpes simplex virus type-2 replication cycle is sufficient to induce chlamydia trachomatis persistence herpes simplex virus co-infection-induced chlamydia trachomatis persistence is not mediated by any known persistence inducer or anti-chlamydial pathway interaction of hsv-2 glycoprotein d with the host cell surface is sufficient to induce chlamydia trachomatis persistence aberrant chlamydial developmental stages in the gastrointestinal tract of pigs spontaneously and experimentally infected with chlamydia suis purification on renografin density gradients of chlamydia trachomatis grown in the yolk sac of eggs submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors would like to thank lisbeth nufer of the laboratory staff at the institute of veterinary pathology, zurich, for her excellent technical assistance. we would also like to thank dr. monika engels and eva loepfe, institute of virology (head: prof. m. ackermann), vetsuisse faculty, university of zurich for providing the porcine epidemic diarrhea virus. we thank dr. adam polkinghorne, queensland university of technology, brisbane, australia, for manuscript editing. this work was supported by a grant from the university of zurich (forschungskredit).author details 1 institute of veterinary pathology, vetsuisse faculty, university of zurich, zurich, switzerland. 2 center for microscopy and image analysis, university of zurich, zurich, switzerland. authors' contributions nb conceived of the study, planned the experiments, and drafted the manuscript. cd and uz performed the imaging and statistical analyses. as and ck carried out the cell culture experiments including immunofluorescence and transmission electron microscopy. ap participated in the design and coordination of the study and helped to draft the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-301655-6nxhvvm4 authors: lei, xi-mei; yang, yong-le; he, yong-qiang; peng, lei; zhao, pengwei; xu, shu-ya; cao, hongwei; fang, pengfei; qiu, wenying; qin, pan; wang, bin; huang, yao-wei title: specific recombinant proteins of porcine epidemic diarrhea virus are immunogenic, revealing their potential use as diagnostic markers date: 2019-08-10 journal: vet microbiol doi: 10.1016/j.vetmic.2019.108387 sha: doc_id: 301655 cord_uid: 6nxhvvm4 given the highly contagious and acute nature of porcine epidemic diarrhea (ped), especially in piglets, there is an urgent need for the development of rapid and sensitive diagnostic assays. the diagnostic potentials of specific porcine epidemic diarrhea virus (pedv) accessory and nonstructural proteins, if any, have not yet been investigated. in order to determine and compare which of the viral proteins may be useful as diagnostic antigens, whole virus (wv) particles and a panel of structural and nonstructural pedv proteins [spike subunit 1 (s1), the c-terminal part of orf3 (orf3c), envelope (e), nonstructural protein 1 (nsp1), nsp2, ac (acidic domain of nsp3), and adrp (adp-ribose-1-monophosphatase domain of nsp3), expressed individually in bacterial and/or mammalian cells] were tested for reactivity with sera from pedv-infected pigs by elisa and/or western blot analysis. according to western blots, serum antibody interactions with the s1 protein were relatively more sensitive and specific than orf3c, e and ac. furthermore, a total of 851 serum samples from diarrheal pigs of different ages were analyzed by elisa, with most showing immune-reactivity towards the wv, s1, orf3c, and e proteins. the earliest igg antibody response was observed in the one-week-old piglets, with similar antibody ontogeny and patterns of seroconversion for s1, orf3c, e, and wv antigens. in addition, the pattern of neutralizing antibody was more similar to that of iga in weaning piglets after pedv infection. collectively, these data provide more reliable information on the host immune response to different viral proteins, which will be useful for development of novel serological assays and for design of vaccines that better stimulate protective immunity. porcine epidemic diarrhea (ped) is characterized by severe diarrhea, vomiting, and dehydration followed by high mortality in suckling piglets . the causative agent, porcine epidemic diarrhea virus (pedv), was initially identified in europe in 1978, and its genome (˜28 kb in size) consists of seven open reading frames (orfs) (kocherhans et al., 2001) . the 5' two-thirds of pedv genome encodes orf1 (consisting of overlapping orf1a and orf1b), and the 3' onethird harbors orfs encoding four structural proteins, the spike (s), envelope (e), membrane (m) and nucleocapsid (n), and an accessory orf3 between s and e kocherhans et al., 2001; tian et al., 2014) . rna synthesis in pedv is carried out by a replicase-transcriptase composed of 16 nonstructural proteins (nsp1-16) encoded by orf1a and orf1b. among them, nsp3 comprises multiple structural domains, including a highly acidic domain at the amino terminus (ac), and a highly conserved adp-ribose-1-phosphatase (adrp) macrodomain. the ac domain of nsp3 is essential for virion assembly and plays a critical role in interaction with the viral nucleocapsid during early infection, whereas the adrp provides activities necessary for synthesis of genomic and subgenomic rnas (hurst-hess et al., 2015) . as pigs of all ages are susceptible to pedv (alvarez et al., 2015; annamalai et al., 2015) , there is an urgent need for the development of highly sensitive and specific diagnostic assays for use in the field (diel et al., 2016) . since the identification of pedv, several diagnostic tests based on pcr detection of viral rna have been described in the literature (diel et al., 2016) . another common diagnostic method is serological testing for the presence of specific antibodies against viral proteins, which is also fast and convenient for epidemiologic investigations. many tools have been developed for the detection of anti-pedv antibodies based upon the major structural proteins (such as s, m or n proteins) in serum, colostrum, milk, feces and oral fluid, including indirect immunofluorescence assays (ifa), virus neutralization assays (sn), enzyme-linked immunosorbent assays (elisa), and fluorescent microsphere immunoassays (fmia) (diel et al., 2016; gerber et al., 2014; gerber and opriessnig, 2015; gimenez-lirola et al., 2017; okda et al., 2015) . but comparative studies of the above assays using different pedv structural and nonstructural proteins as antigens are rarely conducted. meanwhile, the diagnostic potentials of specific pedv accessory and nonstructural proteins, if any, have not yet been investigated in details. in this study, a panel of recombinant pedv orfs encoding structural and nonstructural proteins were expressed in mammalian and/or bacterial cells and screened for reactivity with porcine sera from seven provinces of china by elisa and/or western blot analysis, in order to determine which antigen is most suitable as a diagnostic marker for pedv infection. several rabbit polyclonal antibodies against these recombinant proteins were also generated and validated for use as diagnostic tools upon pedv infection in vitro. vero cells (atcc® ccl-81™) were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 8% fetal bovine serum (fbs). the pedv virulent strain zju/g2/2013 (genbank accession no. ku558701) was used in the study qin et al., 2017) . a total of 851 serum samples were collected in 2015-2016 from diarrheic pigs at 24 commercial farms in the shandong, henan, jiangxi, hunan, jiangsu, heilongjiang and zhejiang provinces of eastern and northern china. there were 295 samples from sows (76 primiparous sows, and 219 multiparous), 131 from 1-week-old piglets, 85 from 3 week-olds, 11 from 5 week-olds, 70 from 7 week-olds, 116 from 11 week-olds, 97 from 17 week-olds, and 46 from 20 week-olds. another 88 serum and fecal samples were obtained from weaning piglets experimentally challenged with pedv-zju/g2/2013 in three pedv challenged experiments. the procedure of the pedv challenge has been described previously . the briefly, the 3-dayold conventional piglets were inoculated with about 5 ml 10 6 infectious titer (tcid 50 ) pedv in 1 × pbs (ph 7.4). blood and fecal samples were collected prior to inoculation and at 1, 3, 5, 7, 10, 14, 17 days postinoculation (dpi). samples from a total of 11 survival piglets but not from dead pigs at each time point were collected. after centrifugation at 1500 ×g for 10 min, serum was harvested, aliquoted and stored at −80°c until use. fecal swab samples were individually mixed with 1 ml of 1 × pbs (ph 7.4) immediately after collection, placed in a 2 ml cryogenic tube (bd falcon™), and stored at −80°c until use. the animal experiments were approved by the experimental animal ethics committee of zhejiang university (approval no. zju20170026). the pedv-zju/g2/2013 strain was propagated in vero cells in dmem supplemented with 5 μg/ml trypsin according to the standard method . briefly, a confluent cell monolayer was washed with minimum essential media (mem) twice before infecting with the virus [moi (multiplicity of infection) = 1] for 2 h at 37°c, after which additional culture medium was added without removing the inoculum. observed cytopathic effects (cpe) reached approximately 90% in 2-3 days, and the virus culture supernatant was collected after three freezethaw cycles, then clarified by high speed centrifugation (4000×g for 30 min) and further purified by ultracentrifugation through a 20% (wt/ vol) sucrose cushion (140,000 ×g for 3 h). the purified virus was further exposed by a negative staining technique, examined using electron microscopy, then stored at −80°c until use. concentration of viral proteins was measured by bca protein assay kit (beyotime, shanghai, china). full-length pedv cdna was used as a template for amplification and cloning of various pedv genes, including those encoding the c terminus of pedv orf3 (orf3c) and the complete sequences of e, nsp1, nsp2, ac, and adrp. the constructs used for this study are listed in table 1 . for transient expression in mammalian cells, the pcdna-3.1 vector was used, and the expression plasmid pcdna-pedv-s1-fc has been described in the previous study (gerber et al., 2014) . for expression of his-tag fusion proteins in bacteria, pedv target genes were cloned in-frame with n-terminal 6×his tags in the pet-28a (novagen; for ac, adrp, nsp1 and nsp2) or the pet-28a-derived psmart-i vector with an n-terminal sumo (small ubiquitin-related modifier) tag (smart-lifesciences, changzhou, china; for orf3c and e). the oligonucleotide primer sequences and approaches used are available upon request. the sequences of all constructs were confirmed by dna sequencing (huada gene technology co., ltd). the above recombinant plasmids were transformed into bl21 (de3) competent cells, respectively, and grown in 1 l of luria-bertani (lb) medium (invitrogen) containing 100 μg/ml kanamycin at 37°c with shaking at 220 rpm. when an od 600 of 0.6 was reached, 1 m isopropyl β-d-1-thiogalactopyranoside (iptg) was added to the final concentration of 0.5 mm in the 1 l of lb medium, and bacteria were grown for an additional 14 h at 20°c. cells were chilled at 4°c and harvested by centrifugation at 5000 ×g for 5 min, resuspended in 30 ml lysis buffer (20 mm tris−hcl, ph 8.0), and disrupted by ultrasonication. crude extracts were centrifuged at 10,000 ×g for 10 min at 4°c, and soluble expression of the his-tagged fusion peptides was confirmed by sds-page analysis prior to purification by the ni-nta his•bind® resin system (transgen tech, dp101, beijing, china) according to the manufacturer's instructions. for the polypeptides that were expressed in inclusion bodies, they were first solubilized in a denaturing buffer (20 mm tris−hcl, with 8 m urea, ph 8.0), and purified by ni-chelating chromatography (ge healthcare). elutions were pooled, dialyzed at 4°c against 20 mm pbs (ph 7.4) with 150 mm nacl and 4 m urea, and analyzed by sds-page and western blot. five purified, recombinant pedv peptides (orf3c, ac, adrp, nsp1, and nsp2) were selected and separately used to immunize two new zealand white rabbits, using a custom antibody production service at hangzhou belta-biotechnology co., ltd. (hangzhou, china). rabbits were not immunized with recombinant e protein. the purified recombinant pedv s1, orf3c, e, adrp, nsp1, nsp2, and ac peptides were resolved on a 5-10% bis-tris polyacrylamide gel (invitrogen) by electrophoresis and subsequently transferred onto a polyvinylidene difluoride (pvdf) membrane. proteins were then detected using an anti-6×his mab (1:1000 dilution; protein-tech, wuhan, china) or rabbit polyclonal antisera at 4°c, followed by incubation with hrp-conjugated goat anti-mouse or anti-rabbit igg (1:10,000 dilution; thermo fisher scientific, usa), as appropriate, at room temperature. after four washing steps using tris-buffered saline with 0.05% tween 20 (tbst), membranes were analyzed using an enhanced chemiluminescence kit (beyotime ecl plus kit, china). for serum western blot analysis, purified s1, orf3c, e, and ac peptides were incubated after sds-page and membrane transfer with individual porcine sera diluted 1:1000, or with anti-his mab or polyclonal rabbit serum as positive controls. hrp-conjugated rabbit antiswine igg and goat anti-mouse igg (1:10,000 dilution; abcam, united states) were used, as appropriate, as secondary antibodies. antigen concentration and dilutions of sera and hrp-conjugated antibodies were optimized by checkerboard titrations. the optimal amount of pedv wv, s1, orf3c or e antigen used for coating was 7.8, 0.44, 1.56 or 0.78 ng/well/100 μl. microtiter plates were blocked with 300 μl/well blocking buffer (thermo fisher scientific, usa) for 1.5 h at 37°c. after coating, 100 μl of serum samples (1:100 dilution) were transferred in triplicate and incubated at 37°c for 2 h. afterwards, 100 μl diluted hrp-conjugated goat anti-swine igg or iga (1:10,000 dilution; thermo fisher scientific, usa) was added to each well and incubated at 37°c for 1 h. wells were washed between incubation steps three times with 300 μl pbs (10 mm, ph 7.4) with 0.05% tween-20 (pbs-t washing buffer). finally, 100 μl tmb color liquid (solarbio, beijing, china) was added to each well and incubated for 10 min at room temperature, after which the reaction was stopped by addition of 50 μl/well of 2 m sulfuric acid, and the plates were read at 450 nm using a spectrophotometer. initial pedv-negative sera were obtained from the united states (a gift from dr. tanja opriessnig) that was subsequently used for screening negative porcine sera in china as reported previously (gerber et al., 2014) . the elisa positive cutoff values were calculated as the mean od of negative controls (n = 4) plus three standard deviations. the positive and negative sera from experimentally-infected piglets were also confirmed by western blot on purified pedv wv and s1 protein antigens as previously described (huang et al., 2011) . positive and negative controls were run in duplicate on each elisa plate. antibody response in all tested samples was represented as a corrected sample-to-positive (s/p) ratio, calculated as follows: s/p ratio = (sample odmean od, negative controls) / (mean od, positive controlsmean od, negative controls). vero cells were seeded in confluent monolayers in 96-well plates (co star™, corning) for 24 h, then infected with 100 μl/well of pedv (10 5 tcid 50 /ml) for 2 h at 37°c, followed by removal of the inoculum. mem supplemented with 0.5% (w/v) trypsin (mmt) was added to each well and incubated at 37°c for 48 h, then cells were fixed with 4% paraformaldehyde. for specific detection of pedv proteins, different rabbit anti-pedv polyclonal antibodies were used as appropriate, with a mouse anti-pedv s1 mab (cat no: 9191, jbt, korea) used as a positive control. secondary alexa fluor 488-conjugated goat anti-mouse igg or goat anti-rabbit igg (invitrogen) were used at a 1:1000 dilution, incubated for 1 h at room temperature. plates were washed three times between antibody incubations with 300 μl/well of pbs-t. nuclei were stained with 4', 6-diamidino-2-phenylindole (dapi; kpl, inc.) at a 1:1000 dilution, and visualized under a fluorescence microscope. sera from challenged piglets were tested for neutralizing antibodies (na), according to the protocol with slight modification (kusannagi et al., 1992) . briefly, serum samples were inactivated at 56°c for 30 min and then 2-fold serial dilutions (1:4˜1:512) were prepared in 96well plates. after mixing 50 μl of each dilution with 50 μl pedv (10 5 tcid 50 /ml), samples were incubated for 1 h at 37°c and used to infect monolayers of vero cells in 96-well plates. after adsorption for 2 h at 37°c, the inoculum was discarded, plates were washed three times with mem, and maintenance medium (containing 5 μg/ml trypsin) was added to each well. after incubation at 37°c for 48 h, cells were observed on an inverted microscope for cpe such as cell fusion and nuclear atrophy. sn titers were calculated using the reed and muench method and expressed as the reciprocal of the highest serum dilution resulting in 50% inhibition of pedv infection, relative to controls. all data were processed using spss (version 20.0) software and the graphpad prism program as described previously (gimenez-lirola et al., 2017; huang et al., 2012) . the cutoff value and diagnostic performance of each pedv antigen was determined by receiver operating characteristic (roc) analysis (sas version 9.4, sas institute, inc., cary, nc, usa) based upon the elisa results. pedv-zju/g2/2013 strain was propagated in vero cells and purified by ultracentrifugation on a sucrose-gradient. electron microscopy revealed that the purified virus was comprised of a great number of vesicles with morphological heterogeneity and envelope fragments carrying spikes (fig. 1a) . previously, the virions of several coronaviruses such as transmissible gastroenteritis virus (tgev), turkey and bovine enteric coronaviruses have been observed with a diameter ranging between 60-220 nm (dea and garzon, 1991) . to our knowledge, the pleomorphic property of pedv virions containing not only the small or defective particles, but also the giant spherical particles ranging up to 350 nm in diameter, is reported here for the first time. although a few of the pedv particles had lost partial spikes, they were relatively intact. as spike glycoproteins are known to be the most immunogenic proteins of coronaviruses , purification by ultracentrifugation through sucrose cushions in this study proved to be reliable. also, the level of pedv protein was relatively high as detected by bca protein kits, thus confirming that the quality, integrity and quantity of the purified virions were sufficient for use as the antigen in subsequent elisa assays. initially, we failed to detect the expression of the complete orf3 using the pet-28a, the psmart or the other bacterial expression vectors under different conditions by western blot analysis (data not shown). therefore, the 127-aa of the c terminal part of orf3 (orf3c) showing the predicted hydrophilicity profile was chosen as the target antigen for orf3. the ac, e and orf3c recombinant peptides were expressed in the inclusion bodies, whereas the nsp1, adrp and nsp2 proteins displayed soluble expression in the cultured supernatants. expression yields of ac, e, and orf3c were very low, hardly visible, when examined by coomassie blue staining after sds-page (data not shown); but confirmation of these three peptides with predicted sizes (ac:˜23 kda; e fused with a sumo tag:˜28 kda; orf3c fused with a sumo tag:˜34 kda) could be done using an anti-his-tag antibody by western blot (fig. 1b) . on the other hand, sds-page and western blot analyses of purified nsp1, adrp and nsp2 soluble proteins showed bands that were consistent with the predicted sizes of 13, 18 and 87 kda, respectively (fig. 1c) . the purified s1 protein expressed in mammalian cells was also identified as a single band by sds-page (fig. 1d ) and by western blot using an anti-s1 monoclonal antibody as described previously (gerber et al., 2014) . due to glycosylation of the s1 protein, the size of the band in the gel was larger than its predicted size (˜86 kda). 3.3. antibodies generated against recombinant pedv ac, orf3c, and nsp2 proteins resulted in specific fluorescence in vitro purified recombinant pedv peptides (orf3c, ac, adrp, nsp1, and nsp2) were used to immunize rabbits, generating polyclonal sera that were used to detect viral proteins on pedv-infected vero cells by ifa. the e protein was not used to immunize rabbits in this study. staining of anti-ac polyclonal serum resulted in specific fluorescence at 48 h post-infection ( fig. 2a, b ) similar to the signal observed using the anti-s1 mab as the positive control (fig. 2g, h) . specific fluorescence was also detected with the anti-orf3c (fig. 2c, d) and the anti-nsp2 polyclonal antibodies (fig. 2e, f) . in contrast, no specific fluorescence was observed when using the anti-adrp and anti-nsp1 polyclonal antibodies. ifa with pre-immune rabbit sera displayed no fluorescent signal in vero cells infected with pedv (fig. 2i, j) . the viral antigens were all detected in the cytoplasm of the infected cells. the anti-s1 mab reacted more strongly than the three positive rabbit antisera based on a comparison of the positive cell numbers and fluorescence intensities. infection of vero cells or vero cells expressing the entry receptor porcine apn with the other swine enteric coronaviruses , such as swine enteric alphacoronavirus , porcine deltacoronavirus (pdcov) and tgev, had no detectable fluorescence after ifa with the anti-pedv polyclonal antibodies described above (data not shown). therefore, anti-pedv-ac, anti-pedv-orf3c and anti-pedv-nsp2 are pedv-specific and do not cross-react with these known porcine coronaviruses. previously, we have developed and validated indirect elisa based on the s1 protein to monitor serum anti-pedv igg and serum and fecal anti-pedv iga antibodies in postweaning pigs (gerber et al., 2014; gerber and opriessnig, 2015) . in this study, in order to determine the pattern of antibody response of weaning piglets in a 17-day weaning period after pedv infection, serum or fecal samples from experimentally-infected 3-day-old piglets were examined by elisa based on the pedv wv or the s1 protein, and by serum neutralization test (fig. 3) . the results indicated that igg and iga responses against both antigens were detected in serum at different time points after pedv infection ( fig. 3a and b ). despite challenge with pedv in these piglets, levels of serum igg and iga decreased from 1 dpi, reaching a minimum after 7 dpi as detected by both wv and s1 antigens (fig. 3a, b) , and the pattern or trend of neutralizing antibody (na) was more similar to that of iga (fig. 3c) . a good linear relationship between the s1-based iga elisa titers and na titers was observed (spearman's rank correlation coefficient of 0.98; p < 0.001), demonstrating the correlation between them (fig. 3d ). there were some differences in the sensitivity of the antigens to detect antibodies, as levels of serum iga were slightly higher when the s1 protein was used as the detection antigen. levels of fecal iga were also highest before challenge (0 dpi), and continuously declined after challenge (fig. 3e) . the specificity and sensitivity of detection of s1 and wv antigens were similar for serum iga. the high igg and iga antibodies and na detected at the early stages of the weaning piglets are presumably maternal antibodies received from sows that were not pedv negative. in addition, piglets during weaning have not developed their own immunity to the virus. these results also demonstrated that the s1-based elisa is an alternative (to wv-based) and ideal serological assay for detection of anti-pedv antibodies. 3.5. antibodies against specific pedv peptides were detectable in sera from naturally infected pigs recombinant pedv s1, orf3c, e, ac and nsp2 peptides were used as antigens in western blots to detect antibodies in porcine sera from commercial farms since they produced antibodies in rabbits reacted to pedv antigens in infected cells except for the e protein that was not tested (fig. 2) . the criterion for determining the seropositivity to a particular antigen of a sample was whether the expected protein band a mouse anti-s1 monoclonal antibody (g, h) and pre-immunization rabbit serum (i, j) were used as positive and negative controls, respectively. alexafluor 488-conjugated goat anti-rabbit igg and goat anti-mouse igg (green) were used as secondary antibodies, as appropriate. antibody staining merged with dapi nuclear staining (blue) is shown; magnification = 20× (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). was presented on the membrane. according to this, serum western blot analysis of the anti-nsp2 antibody showed high-background results and thus further investigation of nsp2 was not performed. a main specific band appeared for s1, ac, e or orf3c protein antigen tested (arrows indicated in representative samples in fig. 4) , though additional "fuzzy" bands were present at low or high molecular weights that were likely from nonspecific reactivity of serum components. compared with the other proteins, the s1 band appeared to be more specific, with a cleaner background (fig. 4a) . pedv-positive sera were screened and verified viathis method, which were used to compare with the results of the elisa (fig. 5; see below) , whereas negative sera from s1-based elisa were also analyzed as negative controls, and none of them showed reactivity (fig. 4, the second lanes in all panels) . however, in the case of ac, the specific 23-kda bands also appeared in some sera that were negative by elisa (data not shown). furthermore, the specific igg antibody responses in sera from a total of 851 diarrheic pigs from farms in china were analyzed using elisa assays based on pedv wv, s1, orf3c or e proteins (fig. 5a-d) . the results indicated good correlations among these antigens. overall, the levels of antibodies against pedv wv and the three proteins antigens were generally higher in primiparous and multiparous sows than those in 3-5 weeks-old pigs (p < 0.01). the trend of serum igg response to each pedv antigen from the 1-week-old to the 3-week-old pigs was similar to that of the experimentally-infected weaning piglets (fig. 3a) . antibodies were highest in the 1 week-old piglets (p < 0.01) except for wv antigen, and then declined to a minimum by 5 weeks-of-age significantly (p < 0.01) before increasing again to a relatively stable level from 7 to 20 weeks-of-age (fig. 5b ,c; p < 0.01). this pattern was in agreement with a recent report about the prevalence of pedv antibodies in swine farms (bertasio et al., 2016) , which might reflect that newborn piglets receive maternal igg antibodies from sows, though the level of the antibodies decline quickly, and the weaned piglets must begin to develop their own immunity to pedv. however, there were some differences in reactivity to the antigens used in the elisas. igg detected by wv-based elisa had a similar pattern to that detected by the other antigens, as the antibodies were in a relatively dispersed distribution, although its cutoff value (0.281) was closest to the orf3c elisa (0.224). antibody levels against the pedv e protein were higher with a relatively concentrated distribution, especially in pigs older than 7 weeks. however, the e protein-based elisa had the highest cutoff value (0.511), and the distribution of antibodies from 1 to 20 weeks-of-age was obviously different from the s1-based elisa, which had the lowest cutoff (0.184). collectively, the data indicated that the pedv s1 protein was more specific and accurate as a detection antigen in elisa tests. with the introduction of pedv into the north american herd in 2013-2014 tian et al., 2014) , the need for a suitable diagnostic marker for the accurate, rapid, and early diagnose of pedv infection has become much more urgent. pedv herd-infection status is very important for biosecurity and the control of ped. compared to rt-pcr and other nuclei acid detection assays, serological tests are advantageous to study immune responses related to vaccination, wild-type virus infection, and to determine whether sow immunity is adequate in individual litters after pedv exposure (diel et al., 2016) . pedv infection is not always obvious in finishing pigs, which increases the risk of widespread disease in pigs of all ages (bertasio et al., 2016) . thus, sensitive serological tests would allow detection of recent infections, to avoid the introduction of these animals into naïve herds. however, there are many different structural and non-structural proteins to choose from when selecting an antigen to use in novel diagnostic assays. previously, we have developed and validated indirect elisa based on the s1 protein to detect anti-pedv igg and iga antibodies in postweaning pigs (gerber et al., 2014) . the present study was set up to investigate diagnostic potentials of specific pedv accessory and nonstructural proteins, which have not yet been reported systematically. fig. 4 . detection of antibodies against pedv peptides in porcine sera by western blot. representative serum samples from diarrheic pigs were used in western blots to detect pedv s1 (a), ac (b), orf3c (c), and e proteins (d); 5 μg of recombinant peptides were loaded in each lane. hrp-conjugated goat anti-porcine igg was used as the secondary antibody, and pedv-negative serum was used as negative control. fig. 5 . distribution of cumulative igg elisa sample-to-positive (s/p) ratios in serum samples collected from diarrheic pigs at commercial pig farms. various indirect elisa assays based on pedv whole virus (wv) (a), s1 (b), orf3c (c), and e proteins (d) were used to test serum samples from commercial sows and pigs with diarrhea at different ages with unknown pedv infectious status. sera from naïve piglets were used as negative controls; samples above the determined s/p cutoff (dashed line) were considered positive. one-way analysis of variance (anova) was used for multiple comparisons between different ages among the individual antigens with an alpha value of 0.01. the "ns" denoted no statistical differences. the sera and feces from the experimental infected piglets at weaning were first used for detection of antibodies using the pedv wv and s1 protein-based indirect elisas. the pattern of change in anti-pedv igg/iga from serum samples and in anti-pedv iga fecal samples (immediate decline post-infection) indicates that the piglets had obtained maternal antibodies at birth, and did not produced antibodies even after pedv challenge until their immune system matured. additionally, levels of na were more closely correlated with iga than igg (fig. 3b-d) , as seen in other studies (paudel et al., 2014) . abundant anti-pedv na have been demonstrated in colostrum on the day of birth, decreasing rapidly in milk by day 3, and gradually declining further from days 3-19 post-farrowing, which may contribute to variable protective capacity . the current study showed a similar decline, further confirming the reliability of the s1-based elisa assays applicable to weaning pigs in addition to postweaning pigs (gerber et al., 2014; gerber and opriessnig, 2015) , and also highlighting the importance of accurate diagnosis in a short window for proper immunization of sows and piglets. compared to the pedv wv, s1-based assays showed good reactivity and high sensitivity/specificity (figs. 3, 4a and 5 b) . it is worth noting that the recombinant s1 protein was expressed in a eukaryotic expression system and should display a natural conformation with high glycosylation, as shown in fig. 1d , which may be one reason for its higher detection sensitivity. on the other hand, wv was mainly purified by sucrose density gradient centrifugation, differential centrifugation or polyethylene glycol (peg) precipitation (hoffmann and robert, 1990) . these methods would damage the integrity of the virus, especially the surface spike glycoprotein. therefore, the eukaryotic-expressed s1 protein is likely more advantageous than the wv as the antigen for pedv serological assays. for another two major structural proteins m and n, due to common epitopes shared by pedv, tgev and pdcov, several studies have previously demonstrated that pedv m or n presented some cross-reactivity to tgev or pdcov (gimenez-lirola et al., 2017; lin et al., 2015; ma et al., 2016) . in contrast, recombinant pedv-s1 had no cross-reactivity with sera from these porcine coronaviruses, showing the best diagnostic sensitivity (gimenez-lirola et al., 2017) . therefore, we did not pursue the development of serological assays based on m or n proteins in this study. the accessory orf3 protein is thought to have high potassium channel activity and may be associated with the virulence of pedv (wang et al., 2012) . the small structural e protein has important roles in the assembly of coronavirus virions, virus egress and in the host stress response (ruch and machamer, 2012) . besides the structural proteins, pedv has several non-structural proteins (nsp1, nsp2, nsp3, among others) that express in the early stage of virus infection and have important functions in the viral replication cycle. the coronavirus nsp3 is a conserved component of the viral protein processing machinery, and may be incorporated in the virion viaits intimate association with viral rna (neuman et al., 2008) . the nsp3 is known as the largest replicase subunit, consisting of numerous distinct structural domains separated by disordered linkers. some of these, such as the ac and adrp (macrodomain), are well conserved across all genera of coronaviruses, though there have been no reports about serological assays based on pedv ac and adrp domains. considering their potential use in the study of host immune response, these proteins were specifically included in the current study. recombinant orf3c, e, ac, adrp, nsp1 and nsp2 peptides were expressed and purified (fig. 1) ; however, only orf3c, ac and nsp2 produced functional rabbit antibodies recognizing pedv antigens in infected cells (fig. 2 ; the anti-e was not generated and tested). subsequently, they were used individually as detection antigens in western blot and/or indirect elisas to detect anti-pedv igg antibodies in sera from diarrheic pigs (fig. 5) . the pedv wv and the recombinant s1 expressed from mammalian cells was used for comparison. the s1, orf3c, and e peptides each reacted strongly with the sera, reflecting expected distributions of pedv-specific antibodies. the reactivity of ac, adrp, nsp1 and nsp2 peptides was less pronounced (data not shown), thus they were discarded from consideration as novel diagnostic antigens. the generally high level of igg against s1, orf3c, and e proteins in old-age pigs was consistent with recent reports that anti-pedv igg in infected pigs persisted for over than 17 weeks after the onset of diarrhea symptoms (lin et al., 2016) . all antibody levels declined to their lowest levels at 3 weeks-of-age, which was consistent with the result from experimentally-infected piglets at weaning (fig. 3) . western blot was implemented to confirm the elisas, with additional "fuzzy" bands appeared when the orf3c, ac and e were used as detection antigens, indicating non-specific recognition (fig. 4) . this may be related to differences in antigens used and/or the sensitivity of the assays for detecting anti-pedv antibodies. the orf3c antigen has moderate immune reactivity as evidenced by staining of anti-orf3c in pedv-infected cells displaying specific fluorescence in ifa ( fig. 2c and d) , by serum western blot (fig. 4d) , and by indirect elisa (fig. 5c) . in future studies, we plan to employ the pedv mutants with the orf3 deletion generated by reverse genetics zhao et al., 2017) in comparison with the wild type pedv for more detailed evaluation of the protein for use as a marker in diagnostic assays. the accessory protein e showed intermediate sensitivity in western blot and extremely high sensitivity in elisa assays compared with the other antigens ( fig. 4c and 5 d) . the elisa sensitivity was too strong, as nearly all of the sera from sows and 0-1 weekold piglets were strongly positive, thus it could not properly reflect the trend of pedv-specific antibodies. to our knowledge, this is the first report about the use of an orf3-or e-based elisa on such a large scale. of the non-structural proteins, ac displayed strong immune reactivity ( figs. 2a and 4 b) , suggesting that it may be released into circulation or is picked up by antigen-presenting cells (hurst-hess et al., 2015) . therefore, the study of anti-ac antibodies may contribute to a better understanding of the detailed function of nsp3. our results also complement previous mass spec identification of nsp3 within purified virions (neuman et al., 2008) . in summary, this study is the first to dissect the range of antibody responses against pedv during infection, using different assays (elisa, western blot, sn) to comprehensively analyze pedv antibodies in porcine sera from china. the results confirmed high pedv prevalence in china (sun et al., 2016) . the antibody profiles provided by the study offer more reliable information on the host immune response to different viral proteins, and will be useful for design of vaccines that better stimulate protective immunity. above all, our data identified that besides s1, the recombinant orf3c and e proteins can also be used as diagnostic markers; but s1 represents greater sensitivity for a wide range of pedv-specific antibodies. the authors declare no conflict of interest. impact of porcine epidemic diarrhea on performance of growing pigs age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs porcine epidemic diarrhea virus shedding and antibody response in swine farms: a longitudinal study evaluation of serological cross-reactivity and cross-neutralization between the united states porcine epidemic diarrhea virus prototype and s-indel-variant strains identification of coronaviruses by the use of indirect protein agold immunoelectron microscopy porcine epidemic diarrhea virus: an overview of current virological and serological diagnostic methods detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa detection of immunoglobulin (ig) a antibodies against porcine epidemic diarrhea virus (pedv) in fecal and serum samples reactivity of porcine epidemic diarrhea virus structural proteins to antibodies against porcine enteric coronaviruses: diagnostic implications enzyme-linked immunosorbent assay for the detection of porcine epidemic diarrhea coronavirus antibodies in swine sera origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states expression of the putative orf1 capsid protein of torque teno sus virus 2 (ttsuv2) and development of western blot and elisa serodiagnostic assays: correlation between ttsuv2 viral load and igg antibody level in pigs serological profile of torque teno sus virus species 1 (ttsuv1) in pigs and antigenic relationships between two ttsuv1 genotypes (1a and 1b), between two species (ttsuv1 and -2), and between porcine and human anelloviruses dissection of amino-terminal functional domains of murine coronavirus nonstructural protein 3 aminopeptidase-n-independent entry of porcine epidemic diarrhea virus into vero or porcine small intestine epithelial cells completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate.pdf antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains two-way antigenic cross-reactivity between porcine epidemic diarrhea virus and porcine deltacoronavirus proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3 development of an indirect elisa, blocking elisa, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to north american strains of porcine epidemic diarrhea virus discovery of a novel swine enteric alphacoronavirus (seacov) in southern china comparison of serum neutralization and enzyme-linked immunosorbent assay on sera from porcine epidemic diarrhea virus vaccinated pigs genetic and pathogenic characterization of a novel reassortant mammalian orthoreovirus 3 (mrv3) from a diarrheic piglet and seroepidemiological survey of mrv3 in diarrheic pigs from east china the coronavirus e protein: assembly and beyond characterization of anti-porcine epidemic diarrhea virus neutralizing activity in mammary secretions epidemiology and vaccine of porcine epidemic diarrhea virus in china: a mini-review evidence of recombinant strains of porcine epidemic diarrhea virus porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase n for infectious cellular entry pedv orf3 encodes an ion channel protein and regulates virus production identification of a peptide derived from the heptad repeat 2 region of the porcine epidemic diarrhea virus (pedv) spike glycoprotein that is capable of suppressing pedv entry and inducing neutralizing antibodies this work was supported by the national key research and development program of china (2016yfd0500102), the key research and development program of zhejiang province (2015c02021), and the national natural science foundation of china (31872488). we thank the professional editing service nb revisions for technical preparation of the text prior to submission. key: cord-286831-ni7qfjk9 authors: choi, hwa-jung; kim, jin-hee; lee, choong-hwan; ahn, young-joon; song, jae-hyoung; baek, seung-hwa; kwon, dur-han title: antiviral activity of quercetin 7-rhamnoside against porcine epidemic diarrhea virus date: 2008-11-06 journal: antiviral res doi: 10.1016/j.antiviral.2008.10.002 sha: doc_id: 286831 cord_uid: ni7qfjk9 porcine epidemic diarrhea virus (pedv) is the predominant cause of severe entero-pathogenic diarrhea in swine. the lack of effective therapeutical treatment underlines the importance of research for new antivirals. in this study, we identified q7r, which actively inhibited pedv replication with a 50% inhibitory concentration (ic(50)) of 0.014 μg/ml. the 50% cytotoxicity concentration (cc(50)) of q7r was over 100 μg/ml and the derived therapeutic index was 7142. several structural analogues of q7r, quercetin, apigenin, luteolin and catechin, also showed moderate anti-pedv activity. antiviral drugs and natural compounds revealed ribavirin, interferon-α, coumarin and tannic acid have relative weaker efficacy compared to q7r. q7r did not directly interact with or inactivate pedv particles and affect the initial stage of pedv infection by interfering of pedv replication. also, the effectiveness of q7r against the other two viruses (tgev, prcv) was lower compared to pedv. q7r could be considered as a lead compound for development of anti-pedv drugs to may be used to during the early stage of pedv replication and the structure-activity data of q7r may usefully guideline to design other related antiviral agents. porcine epidemic diarrhea virus (pedv), family coronaviridae, causes porcine epidemic diarrhea, an enteric disease characterized by acute watery diarrhea, dehydration, vomiting, and high mortality in nursery piglets (debouck and pensaert, 1980) . infection with this virus has become a serious issue in the swine industry and outbreaks have lead to serious economic losses in many countries (hofmann and wyler, 1987) . unfortunately, now there are no effective commercial vaccines or specific treatments available and the only measures to control the disease are those directed to preventing the entrance of the virus on the farm. medicinal plants are increasingly being pursued as suitable alternative sources for discovery of antiviral agents (briskin, 2000; cowan, 1999; jassim and naji, 2003; vlietinck and vanden berghe, 1991; williams, 2001) . recent studies have demonstrated that houttuynia cordata thunb. (saururaceae) is effective in treating anaphylaxis and cancer (kwon et al., 2003; li et al., 2005; lu et al., 2006) . it has also been shown that the essential oil prepared * corresponding author. tel.: +82 42 860 4189; fax: +82 42 860 4309. e-mail address: dhkwon@kribb.re.kr (d.-h. kwon) . from fresh plants of h. cordata possessed direct inhibitory activity against herpes simplex virus type 1 (hsv-1), influenza virus and human immunodeficiency virus type 1 (hiv-1), without showing cytotoxicity (lu et al., 2006) . in particular, quercetin, a flavonoid present in h. cordata, has been reported to have inhibitory effects on several viruses (mucsi and pragai, 1985) . although a variety of pharmacological activities associated with chemicals in h. cordata has been demonstrated, antiviral activities against pedv have not been reported. in this study we examine the positional effects of the hydroxyl group within basic flavonoid structure, and the type and position of attached sugars against pedv replication. furthermore, to elucidate the action of q7r on pedv multiplication in more detail, we investigated the effect of q7r on the infection cycle of pedv through time-of-addition study, rt-pcr analysis, effects of q7r on the infectivity of pedv particles. tamiflu (f. hofmann-la roche ltd., switzerland), relenza (laboratoire glaxosmithkline, france) and lamivudine (glaxosmithkline australia pty ltd., australia) were purchased from a pharmacy in korea as prescribed by a medical doctor. acriflavine, acycloguanosine, azidovudine caffeine, cumarin, glycyrrhizin, interferon-␣, riboflavin, tannic acid, quercetin, apigenin, luteolin, catechin, quercetrin, genistin, hesperidin, rutin and sulforhodamine b (srb) were purchased from sigma-aldrich (st. louis, mo, usa). all chemicals were a reagent grade. vero (african green monkey kidney cell line; atcc ccr-81), st (pig testis cell line; atcc crl-1746), tgev (transmissible gastroenteritis virus; atcc vr-763) and prcv (porcine respiratory coronavirus; atcc vr-2384) were kindly provided by atcc (american type culture collection, manassas, va, usa). pedv cv 777 (porcine epidemic diarrhea virus) was obtained from national veterinary research & quarantine service in korea. vero and st cell lines were maintained in minimal essential medium (mem) supplemented with 10% fetal bovine serum (fbs) and 0.01% antibiotic-antimycotic. antibiotic-antimycotic, trypsin-edta, fbs and mem were supplied by gibco brl (grand island, ny, usa). the tissue culture plates were purchased from falcon (bd biosciences, nj, usa). the aerial parts of h. cordata were collected at gyeongsangnamdo agricultural research & extension services, korea, in august 2004. the dried and ground materials (1 kg) were extracted three times with meoh (3 × 10 l) at room temperatures for 3 days, and this extract was performed to srb-based cytotoxicity and antiviral activity assay ( table 1 ). the meoh extract (247 g; ic 50 , 82.2 g/ml; cc 50 , 1036.0 g/ml) was separated into two fractions using etoac as the non-aqueous phase. the concentrated etoac fraction (16.0 g; ic 50 , 4.8 g/ml; cc 50 , 62.1 g/ml) was loaded on a silica gel (merck, 230-400 mesh, 300 g) column, and eluted with a step-gradient of chcl 3 :meoh (100: 0, 98.5:1.5, 97:3, 93.75:6.25, 87.5:12.5, 25:75, 50 :50 and 0:100; each 1 l) to afford eight fractions. the active fraction (chcl 3 :meoh, 50:50 fraction; 7.97 g; ic 50 , <1g/ml; cc 50 , >100g/ml) was separated using sephadex lh-20 (amersham pharmacia biotech ab, sweden, 18-111 mesh, 200 g), eluting successively with 80% methanol/water (v/v = 80/20; each 0.02 l), and 70 fractions were obtained. for further purification, the active fraction (combination of fractions from 41 to 43, 1.22 g; ic 50 , <1g/ml; cc 50 , >100 g/ml) was subjected to analytical hplc (ymc-pack ods-ma; i.d. 250 mm × 10 mm; eluent, 25% aq. acetonitrile; flow rate, 1.5 ml/min; uv, 220 nm), and gave the pure compounds 1 (840 mg; ic 50 , 0.014 g/ml; cc 50 , >100 g/ml). based on electron spray ionization-mass spectrometry, 1 h nmr and 13 c nmr spectral data, the purified compound was verified as quercetin 7-rhamnoside (awaad et al., 2006) . one day before infection, vero cells were seeded onto a 96well culture plate at a concentration of 2 × 10 4 cells per well. next day, medium was removed and then washed with 1 × phosphate buffered saline (pbs). infectivity of virus stock was determined by the srb method using cytopathic effect (cpe) reduction and was determined as infectivity of the virus by srb id 50 (50% infective dose). following this, 0.09 ml of diluted virus suspension of pedv containing ccid 50 (50% cell culture infective dose) of the virus stock to produce a appropriate cytopathic effects within 2 days after infection and 0.01 ml of medium supplemented with typsin-edta containing an appropriate concentration of the compounds were added. the antiviral activity of each test material was determined with a 10-fold diluted concentration ranging from 0.1 to 100 g/ml. three wells were used as virus controls (virus-infected non-drug-treated cells) while three wells were used as cell controls (non-infected non-drug-treated cells). the culture plates were incubated at 37 • c in 5% co 2 for 2 days. after washing 1 times with 1 × pbs, 100 l of cold (−20 • c) 70% acetone were added to each well and left for 30 min at −20 • c. 70% acetone was removed and 96-well plates were left at dry oven for 30 min. 100 l of 0.4% (w/v) srb in 1% acetic acid solution were added to each well and left at room temperature for 30 min. unbound srb was removed and the plates were washed 5 times with 1% acetic acid before oven drying and were then left in a dry oven for 1 day. bound srb was solubilized with 100 l of 10 mm unbuffered tris-base solution and plates were left on a table for 30 min. the absorbance was read at 540 nm by using a versamax microplate reader (molecular devices, palo alto, ca, usa) with a reference absorbance at 620 nm. to calculate the ic 50 values, the results were transformed to percentage of controls and the ic 50 values were graphically obtained from the dose-response curves. the percent protection achieved by the test compound in pedv-infected cells was calculated by the following formula: where (od t ) pedv is the optical density measured with a given concentration of the test compound in pedv-infected cells; (od c ) pedv is the optical density measured for the control untreated pedvinfected cells; and (od c ) mock is the optical density measured for control untreated mock-infected cells. the concentration achieving 50% protection according to the formula above was defined as the 50% inhibitory concentration (ic 50 ). the therapeutic index was defined as cc 50 /ic 50 . to test the effect of q7r on the infectivity of pedv particles, pedv was pre-incubated with q7r of 10 g/ml for 1 h at 4 • c and vero cells were infected with pretreated or untreated pedv for 1 h at 37 • c. unbound virus was removed and washed 2 times with pbs, ant then cells were incubated in infection medium supplemented with or without q7r of 10 g/ml at 37 • c. antiviral activity was determined by srb assay after 2 days. the time-of-addition effect of q7r was examined according to previously described procedures with minor modifications (chiang et al., 2003a,b) . vero cells were seeded onto 96-well culture plates at density of 2 × 10 4 cells per well and incubated for 1 day. after washing with 1 × pbs, each 10 g/ml of the q7r and ribavirin was then added onto the cells at either before (−1 h), during (0 h) or after (1, 2, 4, 6, 8, 12 and 24 h) the period of pedv infection. after 2 days, antiviral activity was carried out as above described. ribavirin was used as positive control. the cytotoxicity was evaluated by modified the srb assay previously described (lin et al., 1999) . vero cells were seeded onto a 96-well culture plate at a concentration of 2 × 10 4 cells per well. next day, medium was removed and the 96-well plates were replaced with media containing the serially diluted compounds and the cells were further incubated for 48 h. the culture medium was removed and washed with 1 × pbs. the next step was conducted by antiviral activity assay obove described. to calculate the cc 50 values, the results were transformed to percentage of controls and the cc 50 values were graphically obtained from the dose-response curves. vero cells were seeded onto a 96-well culture plate at a concentration of 2 × 10 4 cells per well. the next day, medium was removed and the cells were washed with 1 × pbs. then, 0.09 ml of diluted virus suspension and 0.01 ml of medium supplemented with typsin-edta containing q7r or ribavirin of 10 g/ml were added. after incubation at 37 • c in 5% co 2 for 24 or 48 h, the next step was performed. total rna was extracted from the cultured cells by the acid guanidinium thiocyanatephenol-chloroform extraction method (chomczynski and mackey, 1995) . total rna was dissolved in 0.1% diethylpyrocarbonate (sigma)treated water. the amount of rna was determined by measuring spectrometric absorbance at 260 nm. to check purity of the 18s ribosomal rna, rna of 2 g/ml were loaded on a 0.9% rna gel in 1umops buffer, and the 18s ribosomal rna bands were compared. first-strand cdna was synthesized from total rna in an rnasefree condition. the reaction was performed with 5 g of total rna using a prostar1 first-strand rt-pcr kit (stratagene, la jolla, ca, usa), according to the manufacturer's instructions. pcr was performed in a geneamp pcr system 2700 (perkinelmer/cetus, norwalk, ct, usa) using the first-strand cdna and taq polymerase (takara shuzo, kyoto, japan) as follows for each primer: membrane (m) gene of pedv, 5 -cggttctattcccgttgatg-3 and 5 -ccacaaccgaatgctattga-3 ; ␤-actin, 5 -gccatgtacg-ttgctatccaggctg-3 and 3 -agccgtggccatctcttgctcgaag-5 . pcr-amplified products were separated on 1.5% agarose gels containing 0.1 g/ml ethidium bromide and visualized under uv light. current antiviral drugs, natural compounds and flavonoids were further studied for their inhibitory effects on replication of the pedv and cytotoxicity in vero cells, among which ribavirin, tannic acid, coumarin and interferon-␣ exhibited the activities with ic 50 of 4.1, 47.4, 9 g/ml and 0.52 unit, respectively. their cc 50 were 423.3, 229.3, 83.3 g/ml and >100 unit, and the therapeutic indices were 103.2, 4.8, 9.2 and >192.3, respectively. quercetin, apigenin, luteolin and catechin showed anti-pedv activity with ic 50 of less than 12 g/ml and cc 50 of 365.2, >50, 6.7 and >100 g/ml, respectively. furthermore, q7r showed the highest ti (>7142.86) for pedv tested with ic 50 of 0.014 g/ml and cc 50 of >100 g/ml (table 2) . on the basis of the above results, the relationship between flavonoid structure and antiviral activity against pedv was further evaluated. among the flavonoids tested, flavones (i.e. apigenin and luteolin), flavonol (i.e. q7r) and flavan (i.e. catechin), which all have o-dihydroxy functional groups at c-3 and c-4 , showed significant anti-pedv activity. in addition, flavonols q7r possessed one rhamnoside at position 7 with a 3 ,4 -dihydroxyl group was also found to have an antiviral activity than higher that of the 3 ,4dihydroxylated flavonoids. but, the anti-pedv activity of quercetin with five hydroxyl groups decreased (ic 50 value of less than 50%). this indicates that sugars groups at c-7 of a-ring are an important feature for the anti-pedv activity of flavonoids. to observe the effects of q7r on the infectivity of pedv particles, this exam was performed. antiviral activity of pre-incubation with q7r and ribavirin resulted in 14.9%, 0.9%, respectively. in contrast, continuous presence of q7r during infection led to a significant increase in antiviral activity. a similar result was obtained in control infections with treated ribavirin (fig. 1 ), but antiviral activity was shown to be lower than that of q7r. this indicates that q7r does not interact with the particles of pedv, as pre-exposure of the virus to q7r did not alter the infectivity of pedv particles. the q7r was added at different periods (before, during, and after) of pedv infection. results showed that the q7r suppressed pedv infection, when added just after the virus inoculation (0 h) results are presented as the mean ic50 values obtained from three independent experiments carried out in triplicate ± s.d. a concentration required to inhibit virus-induced cpe by 50% (g/ml). b concentration required to reduce cell growth by 50% (g/ml). c therapeutic index = cc50/ic50. fig. 1 . the effects of q7r on the infectivity of pedv particles. pedv particles were incubated with q7r of 10 g/ml for 1 h at 4 • c. afterwards, vero cells were incubated with q7r-treated or untreated virus for 1 h at 37 • c. unbound virus was removed by extensive washing and infection was continued by cultivating cells in infection medium with or without q7r of 10 g/ml at 37 • c. antiviral activity was determined by titration using srb assays 2 days post-infection. preinc, pre-incubation was expressed incubation without q7r of 10 g/ml after washing; inc., incubation was expressed incubation with q7r of 10 g/ml after washing. and also early after the virus inoculation (1, 2 and 4 h). ribavirin also showed a weak effect on pedv infection. the inhibitory either amount of level of q7r was higher than 85% ( fig. 2) but that of ribavirin was lower than 50%. however, the inhibitory rate of two compounds declined to 40% or less when added at either prior (−1 h) or post (6, 8, 12 and 24 h) infection. this observation indicated that q7r affects the initial stage of pedv infection. further evidence of the inhibitory effects of q7r on infection pedv and viral replication in vero cells was provided by pcr analysis. the rna extraction was performed at 24 and 48 h after pedv infection. q7r (10 g/ml) decreased the intensity of the product fig. 3 . rt-pcr analysis. replication of pedv from vero cells before and at 24 and 48 h after infection by pedv in the presence of q7r (10 g/ml) or ribavirin (10 g/ml) or vehicle alone (control, 0.1% dmso), as detected by rt-pcr. band of pedv at 24 and 48 h after infection, but ribavirin (10 g/ml) did not decrease the intensity of the product band of pedv at 48 h after infection (fig. 3) . the two viruses selected for these experiments included tgev and prcv in st cells. when the antiviral activity and cytotoxicity of q7r or ribavirin was examined by srb assay, the two compounds did not exhibit any cytotoxicity at the highest concentrations tested ( table 3 ). the ic 50 value of q7r and ribavirin in tgev was 63.31 and 55.09 g/ml, respectively, while that in prcv was 59.76 and 61.6 g/ml, respectively. furthermore, ti value was no great different between the two viruses. several veterinary coronavirus vaccines are currently available, but their efficacy is variable. the vaccine for prevention of infectious bronchitis virus (ibv), which infects chickens, is effective (ladman et al., 2002) , while the canine and porcine vaccines are only partially effective (pratelli et al., 2004) . ribavirin has been used as treat against various dna and rna virus infections, although virusacquired resistance to it was isolated from various virus populations and observed in some patients (jason and craig, 2006) . antiviral studies have shown that essential oil prepared from fresh plants of h. cordata has an inhibitory effect on herpes simplex virus type 1 (hsv-1), influenza virus and human immunodeficiency virus type 1 (hiv-1), without showing cytotoxicity (lu et al., 2006) . quercetin has been reported to have inhibitory effects on several viruses (mucsi and pragai, 1985) . these studies indicate that h. cordata possesses compound exhibited antiviral effects. in this study, q7r is an effective antiviral compound against pedv. q7r did not have a marked inhibitory effect on the growth of tgev or prcvinfected cells. the antiviral activity of this compound appeared to be strongly influenced by the strain of the coronaviruse tested. ribavirin, which is a broad-spectrum nucleoside analogue, exhibited results are presented as the mean ic50 values obtained from three independent experiments carried out in triplicate ± s.d. a concentration required to inhibit virus-induced cpe by 50% (g/ml). b concentration required to reduce cell growth by 50% (g/ml). c therapeutic index = cc50 / ic50. expected antiviral activity. this broad-spectrum antiviral drug was previously shown to be inhibitory to 16 dna and rna viruses in cell cultures (sidwell et al., 1972) . trials of ribavirin in this study showed that the drug had favorable effects on antiviral activity in vero cells infected with pedv. in tgev or prcv, ribavirin had a weaker influence on survival of in vero cells after infection with tgev or prcv. we were however able to ascertain that ribivirin does possess some antiviral activities. to elucidate the action of q7r on pedv multiplication in more detail, we investigated the effect of q7r on single steps during the infection cycle of pedv. as a result, it can be concluded that q7r does not interact with the particles of pedv, as pre-exposure of the virus to q7r did not alter the infectivity of pedv particles (fig. 1) . based on the results of the time-course study, pre-incubation of the vero cells with q7r did not protect the cells from pedv infection (fig. 2) . furthermore, q7r can inhibit pedv infection only when it was added on, during, and early stages after the virus inoculation (1, 2, 4 h), but not after 6 h or later (fig. 2) . this suggests that the mode of action is not deriven from inhibiting the absorption of virus but results from inhibition at an early stage of viral replication after infection ( figs. 1 and 2 ). among the flavonoids tested, only q7r possessed significant activity against pedv. in a previous report, rutin (quercetin-3rutinoside) did not express antiviral activity whereas quercitrin (quercetin 3-rhamnoside) possessed similar activity to quercetin against human herpesviruses and adenoviruses (chiang et al., 2003a,b) . they found that the antiviral activity among the flavonoid glycosides containing the quercetin moiety might be correlated with the species of sugar group at the 3 position. our results suggest that the antiviral activity of flavonoids against pedv is correlated with the species of sugar group at the 7 position. in conclusion, the present study described that q7r possesses strong anti-pedv activity among the flavonoids. it will be interesting to further investigate the antiviral activity of the q7r in preventing various pedv-mediated injuries in in vivo pathological situations. further studies will be required to explore the detailed antiviral mechanism of q7r. hepatoprotective activity of schouwia thebica web medicinal plants and phytomedicines linking plant biochemistry and physiology to human health in vitro antiviral activities of caesalpinia pulcherrima and its related flavonoids in vitro cytotoxic, antiviral and immunomodulatory effects of plantago major and plantago asiatica substitution of chloroform by bomochloropropane in the single-step method of rna isolation plant products as antimicrobial agents experimental infection of pigs with a new porcine enteric coronavirus cv 777 study of the occurrence of epizootic viral diarrhea in swine in switzerland mechanisms of action of ribavirin against distinct viruses novel antiviral agents: a medicinal plant perspective herba houttuyniae extract induces apoptotic death of human promyelocytic leukemia cells via caspase activation accompanied by dissipation of mitochondrial membrane potential and cytochrome c release protection of chickens after live and inactivated virus vaccination against challenge with nephropathologenic infectious bronchitis virus pa/wolgemuth/98 inhibitory effects of houttuynia cordata water extracts on anaphylactic reaction and mast cell activation sulforhodamine b assay for measuring proliferation of a pigmented melanocyte cell line and its application to the evolution of crude drugs used in the treatment of vitiligo anti-inflammatory effect of houttuynia cordata injection inhibition of virus multiplication and alteration of cyclic amp level in cell cultures by flavonoids safety and efficacy of a modified-live canine coronavirus vaccine in dogs broad-spectrum antiviral activity of virazole: 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide can ethnopharmacology contribute to the development of antiviral drugs review of antiviral and immunomodulating properties of plants of the peruvian rainforest with a particular emphasis on una de gato and sangre de grado this work was supported by a grant from research institutes of bioscience & biotechnology (kribb), republic of korea. this research was also partially supported by a grant from biogreen 21 program rural development administration, by the ministry for food, agriculture, foresty and fisheries, republic of korea. key: cord-274765-3wzht843 authors: kweon, chang-hee; kwon, byung-joon; lee, jae-gil; kwon, geon-oh; kang, yung-bai title: derivation of attenuated porcine epidemic diarrhea virus (pedv) as vaccine candidate date: 1999-06-04 journal: vaccine doi: 10.1016/s0264-410x(99)00059-6 sha: doc_id: 274765 cord_uid: 3wzht843 the field isolate of porcine epidemic diarrhea virus (pedv) was serially passaged in vero cells. the cell passaged pedv, designated kpedv-9, was tested for its pathogenicity in the neonatal pigs, immunogenicity and safety in the pregnant sows. the result indicated that kpedv-9 at the 93rd passage revealed reduced pathogenicity in the neonatal pigs. pregnant sows inoculated with the attenuated virus showed increased immune responses by elisa. in addition, delivered piglets were protected from challenge of wild type pedv. the safety test in pregnant sows indicated that all inoculated animals farrowed the average numbers of litters of piglets. the results of this study supported that the attenuated virus derived from serial passage could be applied as vaccine for protecting suckling piglets against pedv infection. porcine epidemic diarrhea virus (pedv), a member of coronaviridae, is the etiological agent of enteropathogenic diarrhea in swine [1±3] . although the clinical symptoms of pedv infection are similar to transmissible gastroenteritis virus (tgev) infection, pedv has a wider variety of clinical signs in pigs [4] . propagation of pedv in vitro was rather limited until vero cells were found to support the growth of virus in the presence of trypsin [5] . in this study, we described the derivation of an attenuated strain of pedv, as a potential vaccine candidate, through cell adaptation. vero cells obtained from atcc (vero c1008) were regularly maintained in alpha-mem supplemented with 5% fetal bovine serum, penicillin (100 unit/ml), streptomycin (100 unit/ml) and amphotericin (0.25 g/ ml). the isolate of pedv was originated from a neonatal pig and plaque puri®ed in vero cells. the isolate, designated kpedv-9, was passaged in 80±90% monolayers of vero cells in alpha-mem with 0.02% yeast extract, 0.3% tryptose phosphate broth (tpb) and 1±2 mg of trypsin as described [5, 6] . sequential passages of the virus were normally conducted in roller culture. each passage level of virus was stored at à708c or freeze-dried with equal volume of stabilizer (0.217 m vaccine 17 (1999) 2546±2553 0264-410x/99/$ -see front matter # 1999 elsevier science ltd. all rights reserved. pii: s 0 2 6 4 -4 1 0 x ( 9 9 ) 0 0 0 5 9 -6 lactose, 0.0038 m khpo 4 , 0.0072 m k 2 hpo 4 , 0.0049 m monosodium glutamate, 1% gelatin). the ®nal stock of the pedv was characterized in two ways. firstly, the culture supernatant was subjected to direct centrifugation at 60,000 â g for 2 h and the pellet was resuspended to 1/200 of initial volume for morphological identi®cation by transmission electron microscopy. secondly, the virus infected cells were subjected to reverse transcription polymerase chain reaction (rt-pcr) to detect speci®c pedv sequences. three primers for rt-pcr were selected from the sequences information of membrane protein (m) gene of duarte et al. [7] . p1 (27mer); 5 '±ccccagtactgttat-tgacgtataaac±3 ' (position 974±1000), p2 (24mer); 5 '±gtttagactaaatgaagcactttc± 3 ' (position 1665±1688) for pcr and p3 (25mer); 5 '± gccataaagtttctgtttagactaa±3' (1702± 1678) as the primer for synthesis of complementary dna, respectively. the extraction of rna and rt-pcr were conducted according to the instructions of commercially available kit (stratagene). pcr reactions were performed in the conditions as described previously [8] . after ampli®cation, the pcr products were cloned into puc19 vector for sequencing using sequenase version 2.0 (usb, usa). in addition, the presence of adventitious virus such as porcine parvovirus (ppv), japanese encephalitis virus (jev), hog cholera virus (hcv) and other cythopathogenic viruses were examined as described previously [9±11]. for the detection of attenuation, viral stocks at 90 passages were tested in 4 days old piglets. from 10 6.0 tcid 50 /ml to 10 8.0 tcid 50 /ml of cell adapted pedv was inoculated into suckling piglets intramuscularly or through the oral route. in order to compare the pathogenicity, pedv isolate before cell adaptation was prepared from the small intestines of neonatal piglet. the intestine was ground in phosphate buered saline (pbs, ph 7.4). the 10% suspension was then ®ltered using a 0.2-mm membrane ®lter (acrodisk, gelman) and further diluted to 5-, 10-and 20-fold in pbs. four groups of ®ve piglets were orally fed with the suspensions of diluted stock of intestine. the animals were observed for clinical symptoms of diarrhea and mortality in the inoculated animals was observed for 10 days. three pregnant sows 4±5 weeks prior to farrowing were tested for the detection of immune responses. pigs were inoculated intramuscularly with the attenuated viruses at the titer of at 10 7.0 tcid 50 /ml. two pregnant sows remained as uninoculated control. after two weeks, second inoculations of same titer were followed. each paired serum before and after inoculation was collected at two-week interval. the collected sera and colostrum at delivery were tested for the presence of antibodies by elisa. after delivery, suckling piglets of 2 days old were orally challenged with 10 or 5 ld 50 of wild pedv. the clinical signs of diarrhea and mortality of challenged piglets were observed for two weeks. for the preparation of antigen, the cell adapted pedv was concentrated with polyethylene glycol (peg, mw 6000) as described [12] . the peg treated viral solution was then precipitated and resuspended at 1/10th of original volume with ten buer (0.01 m tris, 0.001 m edta, 0.1 m nacl, ph 7.4). the puri-®ed virus was then used for the elisa. the procedures for elisa were basically the same with a previous study [13] . brie¯y, the dilution of antigen and second antibodies were adjusted to the optical density (od) around 0.2 (a 490) using the negative porcine sera. usually each well in 96-well microplate (costar) was coated with 1±2 mg of protein in 50 mm carbonate buer (ph 9.6) at 58c overnight, followed by the blocking with 1% skim milk at 378c. the 1/400 diluted porcine sera in pbs with 0.01% bovine serum albumin (bsa) and 0.05% tween 20 (pbst) were reacted at 378c for 30 min and then washed extensively with pbst three times at 5-min intervals. the reacted plate was washed again at the same condition and incubated with 2000-fold diluted horseradish peroxidase (hrp) labelled anti-porcine igg (kpl) for 1 h at 378c. the plate was developed in o-phenylenediamine (opd) at room temperature for 20 min. the reaction was stopped with 2 m h 2 so 4 before measuring od at 490 nm. a total of 63 pregnant sows were inoculated intramuscularly with 1 ml of the virus containing 10 7.0 tcid 50 /ml. twenty-three pregnant sows received one injection 3±4 weeks prior to farrowing. in another farm, 40 pregnant sows received two injections at 2±3week intervals before farrowing. the average number of the litters were compared with the data of uninoculated pregnant sows at the corresponding farm during same period of time. the sequence accession number for m protein of attenuated kpedv-9 is genebank accession number afo15888. the pedv was continuously passaged in vero cells. sequential passage of virus regularly conducted every 4±5 days postinfection in cells. the supernatant was harvested and used for next inoculation in vero cells up to 93 passages. however, cytopathogenic eect (cpe) in vero cells was not so clear that the culture supernatant of virus infected cell was subjected to morphological and genetic characterization. when the culture supernatant of virus infected cell was examined by the transmission electron microscopy, characteristic shape of coronavirus with diameter of 100±150 nm was possible to identify (fig. 1 ). in addition, the com-parision of m gene of cell passaged virus showed the 98.97% in nucleotide and 98.24% in amino acid identity with previously reported pedv strain (fig. 2) . no adventitious viral contaminations were detected in the ®nal stock of pedv. the pathogenicity of attenuated virus was tested in the 1-day-old piglets before taking colostrum. six separate litters of 53 piglets and 15 litters of 111 piglets were inoculated intramuscularly with virus of 10 7.0 and 10 6.0 tcid 50 /ml, respectively. in this experiment, all the inoculated piglets failed to show signs of diarrhea and symptoms related to pedv infection. in order to avoid any possible eects from the maternal immunity through the colostrum and detect the potential pathogenicity of the attenuated virus, eight piglets of 4 days old were infected orally with 10 ml of virus stock, which contained virus of 10 8.0 tcid 50 /ml, and were arti®cially fed with dairy milk. although three piglets showed signs of anorexia and mild signs of diarrhea in two or three days after inoculation, nevertheless, the signs seemed to be transient. in fact, all piglets recovered in the next 2±3 days. however, in the groups of piglets fed with wild virus before cell passages, all the piglets developed symp-toms of watery diarrhea in 2±3 days, and the mortality reached up to 10±100%, depending on the dilution of virus within one week as shown in table 1 . when the collected sera were tested for the presence of the antibodies by elisa, all inoculated sows showed the rising elisa titers (fig. 3) . on the other hands, the antibody titers of control pigs decreased at the time of delivery. in addition, colostrum at delivery showed higher or similar level of antibodies of corresponding sows. after challenge exposures mortality of piglets were compared with uninoculated control. although the mortality of piglets after challenge with 10 ld 50 of wild pedv was reduced to 20% compared to 100% in control litter, all piglets survived in the litters after challenge experiment with 5 ld 50 of virus compared to 60% in control (table 2) . however, mild signs of diarrhea were also detected in one litter of piglets 2 days after challenge, but they recovered the next day. the safety test of the attenuated virus in pregnant sow was conducted in two separate farms. one farm has not had any history of epidemic diarrhea in last few years and another farm had the experience of pedv outbreak in the previous year. a total of 63 pregnant sows were inoculated once or twice before farrowing. as shown in table 3 , all the inoculated sows farrowed the same average numbers of litters of uninoculated control group without any clinical problems. in this study, we investigated the attenuation of pedv through serial passages in vero cell cultures and its prophylactic eect in pregnant sows. after serial passages in vero cells, the growth of virus was rather trypsin-independent and the detection of cytopathic eect (cpe) was rather variable, depending on clones of vero cell lines (data not presented here). since the spf or gnotobiotic piglets were not used in this experiment, it might not reasonable to ®gure out the exact dierences in pathogenicity between the wild and the attenuated virus. nevertheless, when compared with the wild pedv, the animals inoculated with the high passage level of virus did not show any severe signs of diarrhea or death in piglets, supporting attenuation. it is known that pedv replicates mainly in the villi of small intestines [14] . like attenuated tgev, the replication of attenuated pedv may be limited to the small portion of intestine with short duration of secretion compared to virulent virus [15] . in fact, the detection of signs of diarrhea from piglets inoculated orally with attenuated virus delayed at least by two days and lasted one day compared with the signs from piglets inoculated with wild virus. when we tested the immunoprophylactic eect in pregnant sows, it was demonstrated that the vaccinated swine resulted in reduced piglet mortality after challenge experiment (20±100% in vaccinates compared with 0±40% in controls), indicating that the attenuated pedv could induce the status of immunity in pregnant pigs, providing protection in piglets like other enteric disease in swine. previously, we found that pedv infections were related to more than 20% of diarrheal cases in the neonatal pigs, thus causing a considerable losses in pig industry [13] . in fact, ®eld application of attenuated virus as vaccine resulted in the overall reduction of mortality of neonatal pigs (2± fig. 3 . immune responses of pregnant sows inoculated with cell attenuated kpedv-9 strain. animals were inoculated twice at 2-week intervals and serum samples were tested by elisa. table 2 survival of piglets from attenuated kpedv-9 vaccinated sows (v) and unvaccinated control (c) after challenge exposure 52% compared with before vaccination) in the farms having pedv outbreak [16] . nevertheless, it is worthy to note that the ecacy of protection was rather complicated and con¯icting according to the challenge dose like other enteric diseases. although the pregnant pigs inoculated with attenuated virus showed the increased immune status by elisa as compared to uninoculated control, it is dicult to explain the exact relation to mucosal immunity for protection. since it was well con®rmed that the mechanisms of the passive immunity are extensively related to the presence of iga and igm antibodies [17] , further experiments, including detection of antibody secreting cells of iga and igm, may give the practical information for immunoprophylaxis against pedv. an apparently new syndrome of porcine epidemic diarrhea some characteristics of a new porcine coronavirus and detection of antigen and antibody by elisa experimental infection of pigs with a new porcine enteric coronavirus cv 777 porcine epidemic diarrhea virus as a cause of persistent diarrhea in a herd of breeding and ®nishing pigs propagation of the virus of porcine epidemic diarrhea in cell culture isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf rapid diagnosis of porcine epidemic diarrhea virus infection by polymerase chain reaction porcine parvovirus: properties and prevalence of a strain isolated in the us procedures for identi®cation of arthropod borne viruses a new in vitro method (end) for detection and measurement of hog cholera virus and its antibody by means of eect of hc virus on newcastle disease virus in swine tissue culture. i. establishment of standard procedure morphology of bovine viral diarrhea virus cell adaptation and serological survey on porcine epidemic diarrhea virus (pedv) infection in korea use of two enzyme-linked immunosorbent assays to monitor antibody responses in swine with experimentally induced infection with porcine epidemic diarrhea virus comparison of two methods for detection of transmissible gastroenteritis virus in feces of pigs with experimentally induced infection field trial of attenuated porcine epidemic diarrhea virus (kpedv-9) as vaccine development of an elispot for the detection of antibody secreting cells against the porcine epidemic diarrhea virus (pedv) in dierent tissues we thank joong-won park's technical assistance for the electron microscopy. we also appreciate the help of dr. t.w. molitor of university of minnesota, usa for his review and comments on this manuscript. this study was supported by the research grant from rda, ministry of agriculture, republic of korea. key: cord-272728-inndwa61 authors: jung, kwonil; miyazaki, ayako; saif, linda j. title: immunohistochemical detection of the vomiting-inducing monoamine neurotransmitter serotonin and enterochromaffin cells in the intestines of conventional or gnotobiotic (gn) pigs infected with porcine epidemic diarrhea virus (pedv) and serum cytokine responses of gn pigs to acute pedv infection date: 2018-08-31 journal: research in veterinary science doi: 10.1016/j.rvsc.2018.06.009 sha: doc_id: 272728 cord_uid: inndwa61 abstract serotonin is a critical monoamine neurotransmitter molecule stored and released from enterochromaffin (ec) cells into the gut submucosa, transmitting the vomiting signal to the brain. we studied one mechanism by which vomiting is induced in pigs infected with porcine epidemic diarrhea virus (pedv) by characterization of swine ec cells by immunohistochemistry. conventional or gnotobiotic (gn) 9-day-old pigs [pedv-inoculated (n = 12); mock (n = 14)] were inoculated orally (8.9–9.2 log10 genomic equivalents/pig) with pedv pc21a strain or mock. this is the first identification of serotonin-positive ec cells in swine by immunohistochemistry and mainly in intestinal crypts, regardless of infection status. they were morphologically triangular-shaped or round cells with or without apical cytoplasmic extensions, respectively. at post-inoculation hour (pih) 16 or 24, when vomiting was first or frequently observed, respectively, pedv infection resulted in significantly reduced numbers of serotonin-positive ec cells in duodenum, mid-jejunum, ileum, or colon. however, two of three pedv-inoculated gn pigs that did not yet show vomiting at pih 16 had numbers of serotonin-positive ec cells in duodenum, ileum and colon similar to those in the negative controls. these findings suggest that serotonin release from ec cells (increased serotonin levels) into the gut submucosa might occur early pedv post-infection to stimulate the vagal afferent neurons, followed by vomiting. serotonin might be involved in the mechanisms related to vomiting in pedv-infected piglets. we also found that mid-jejunum was the primary site of acute pedv infection, and that systemic innate and pro-inflammatory cytokine responses were induced during the acute stage of pedv infection. vomiting is a defensive reaction of the body to rapidly remove ingested toxins from the gastrointestinal tract. vomiting is generally induced when either of the two medullary centers in the brain, integrative vomiting center and chemoreceptor trigger zone (crtz), is activated via a variety of their surface chemoreceptors, such as 5-ht 3 (endo et al., 2000; sikander et al., 2009; spiller, 2008) . the integrative vomiting center and crtz are triggered by activation of vagal afferent neurons in the gastrointestinal tract and circulating toxins in blood, respectively. there are vagal or enteric afferent neurons in the gut submucosa. vagal afferent neurons are stimulated by a monoamine neurotransmitter molecule, serotonin (5-hydroxytryptamine, 5-ht), released from enterochromaffin (ec) cells in the gastrointestinal tract. subsequently, vomiting signals are transmitted to the integrative vomiting center and then to the central nervous system (cns). emetic efferent signals from the cns reach the gastrointestinal and abdominal muscles, leading to their expulsive actions by which vomiting is induced (endo et al., 2000; sikander et al., 2009; spiller, 2008) . in humans, approximately 95% and 5% of serotonin is stored in the gut and brain, respectively. in the gut, 90% of serotonin is deposited in the secretory granules of ec cells mainly located in intestinal crypts, and 10% is in neurons in the submucosa (endo et al., 2000; spiller, 2008) . in humans or rats, by immunohistochemistry (ihc) using antibodies against serotonin, ec cells were serotonin-positive and triangular-shaped with apical cytoplasmic extensions (gustafsson et al., 2006; spiller, 2008) . the ec cells likely originate from intestinal crypt stem cells. a mechanical, biological, or chemical stimulus causes ec cells to secret serotonin into the lamina propria or lumen that acts on serotonin receptors (5-ht 3 ) at the terminals of the vagal afferent neurons (endo et al., 2000; gustafsson et al., 2006; spiller, 2008) . serotonin also functions to promote immune activation through the receptors expressed on macrophages, dendritic cells and t and b cells (li et al., 2011; shajib and khan, 2015) . porcine epidemic diarrhea virus (pedv) (family coronaviridae, genus alphacoronavirus) causes acute diarrhea, vomiting, decreased or loss of appetite, dehydration and high mortality in neonatal piglets . diarrhea is frequently accompanied by vomiting in pedv-infected nursing piglets during the acute stage of infection, exacerbating dehydration . however, the mechanisms by which vomiting is induced in pedv infection are poorly understood. we hypothesized that: i) serotonin is involved in the mechanisms related to vomiting; ii) serotonin release from ec cells into the intestinal lamina propria or lumen occurs early pedv post-infection to stimulate the vagal afferent neurons, followed by vomiting; and iii) expression levels of serotonin in intestinal ec cells or numbers of serotonin-positive ec cells in the small or large intestine differ between pedv-infected and uninfected pigs. the in situ distribution and characterization of ec cells in swine intestine are also unknown. in our study, therefore, we aimed to develop an ihc to detect and characterize ec cells in swine intestine and to determine whether pedv infection alters the number of serotonin-positive ec cells in the small and large intestines (primary sites of pedv infection) of infected gnotobiotic (gn) or conventional, 9-day-old piglets during disease progression. we also aimed to detail the pathogenesis of acute pedv infection, including intestinal distribution of pedv antigen and serum innate and pro-inflammatory cytokine profiles in infected gn pigs to understand the relationship of acute pedv infection with the frequency of serotoninpositive ec cells in the small and large intestines. the us virulent (wild-type) pedv strain pc21a was obtained from intestinal contents of a diarrheic 1-day-old piglet in an ohio farm in june 2013 (jung et al., 2014) . the original sample was serially passaged 2 times in gn pigs, and the intestinal contents were negative for other enteric viruses [transmissible gastroenteritis virus, porcine deltacoronavirus (pdcov), porcine rotavirus groups a-c, etc.] by pcr/rt-pcr and electron microscopic examination. the titer of gn pig 2ndpassaged pc21a was 11.8 log 10 ge/ml and was used as virus inoculum after dilution in minimal essential medium (mem) (invitrogen, carlsbad, ca, usa), as described previously . six large white × duroc crossbred gn pigs were acquired by hysterectomy from a pedv-seronegative pregnant sow obtained from a pedv-free, specific-pathogen-free (spf) (confirmed by history and seronegative sows; lack of qrt-pcr-positive fecal samples) swine herd of the ohio state university. the spf herd was seronegative for antibodies to porcine reproductive and respiratory syndrome virus, porcine respiratory coronavirus, transmissible gastroenteritis virus and porcine circovirus type 2. six 9-day-old gn piglets were randomly assigned to one of two groups: pedv-infected (n = 3; pigs 1-3) and mock (n = 3; pigs 4-6). pigs were inoculated orally with 2 ml of pedv strain pc21a [8.9 log 10 genomic equivalents (ge)/ml] [9.2 log 10 ge (≈3.2 log 10 pfu) per pig], a dose similar to that (8.9 log 10 ge/pig) used in the previous study , or mock inoculated with mem. after pedv inoculation, the pigs were monitored frequently for clinical signs, such as diarrhea, appetite, activity, etc., especially vomiting. inoculated and negative control pigs (n = 3/group at each time-point) were euthanized for virological and pathological examination at an acute-stage of infection when or shortly after vomiting was first detected, i.e. approximately post-inoculation hour (pih) 16 in this study. diarrhea was assessed by scoring fecal consistency. fecal consistency was scored as follows: 0 = solid; 1 = pasty; 2 = semi-liquid; 3 = liquid, with scores of 2 or more considered diarrheic. the institutional animal care and use committee (iacuc) of the ohio state university approved all protocols related to the animal experiments in this study. the other tissue samples tested were archival formalin-fixed, paraffin-embedded tissues acquired from twenty 9-day-old [pedv-infected (n = 9) and mock (n = 11)] conventional pigs inoculated orally with 8.9 log 10 ge of pedv strain pc21a or mock (mem) . the clinical disease, fecal virus shedding, and gross and histopathology were described in a previous paper . however, the previous report included only limited information to understand intestinal distribution of serotonin-positive ec cells at the different locations of the intestine and the relationship of the frequency of serotonin-positive cells with vomiting. therefore, more detailed clinical and histopathological observations relevant to the aims of the current study were described in the results section. pigs (n = 3-4/time-point) were euthanized for pathologic examination at post-inoculation days (pids) 1, 3, and 5. rectal swabs and serum samples were collected from gn pigs 1-6 at pih 16. two rectal swabs were suspended in 4 ml mem. the rna was extracted from 200 μl of centrifuged (2000 ×g for 30 min at 4°c) fecal suspensions using the mag-max viral rna isolation kit (applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. pedv rna titers in rectal swabs and serum samples were determined as described previously (jung et al., 2014) . there were increases in serum innate (ifnα) and pro-inflammatory (tnfα and il-12) cytokine levels in pedv-infected conventional pigs (10-day-old) at pid 1 and increased mrna levels of pro-inflammatory cytokines (il-1β, il-6, il-8 and tnfα) in ipec-j2 cells infected with pedv (lin et al., 2017) . therefore, serum innate (ifnα and il-22) cytokines, which are known to play a role in antiviral immune responses (gimeno brias et al., 2016; , and pro-inflammatory (tnfα, il-6, and il-12) cytokines were evaluated in gn pigs 1-6 at pih 16 to confirm the previous findings as well as to investigate whether acute pedv infection induces systemic innate and pro-inflammatory immune responses. serum ifnα, il-6, il-12, il-22, and tnfα cytokine levels were quantitated by elisa in the serum samples collected from gn pigs 1-6 at pih 16, as described previously azevedo et al., 2006; chattha et al., 2013) . briefly, nunc maxisorp 96-well plates were coated with anti-porcine il-6 (0.75 μg/ml, goat polyclonal antibody), anti-porcine il-12 (0.75 μg/ml, goat polyclonal antibody), antiporcine ifnα (2.5 μg/ml, clone k9) (r&d systems, minneapolis, mn), anti-porcine il-22 (2.0 μg/ml, rabbit polyclonal antibody), and antiporcine tnfα (1.5 μg/ml, goat polyclonal antibody) (kingfisher biotech, saint paul, mn) overnight at 4°c or 37°c (for ifn-α only). biotinylated anti-porcine il-6 (0.1 μg/ml, goat polyclonal antibody), antiporcine il-12 (0.2 μg/ml, goat polyclonal antibody), anti-porcine ifnα (3.75 μg/ml, clone f17) (r&d systems, minneapolis, mn), anti-porcine il-22 (0.5 μg/ml, rabbit polyclonal antibody), and anti-porcine tnfα (0.4 μg/ml, goat polyclonal antibody) (kingfisher biotech, saint paul, mn) were used as detection antibodies. porcine ifn-α detection antibody was biotinylated using a commercial kit, as described previously (chattha et al., 2013) . plates were developed and cytokine concentrations were calculated, as previously described azevedo et al., 2006) . the samples were tested in duplicate, and cytokine levels were expressed as the mean values. detection limits of our elisa were 1 pg/ml for ifnα and il-12, 4 pg/ml for tnfα and il-22, and 16 pg/ml for il-6, respectively. small intestinal tissues [duodenum, proximal (distant by 15-20 cm from the pylorus), middle (mid-location of the small intestine (duodenum to ileum) dissociated from the mesentery) and distal jejunum (distant by 15-20 cm from the ileocecal junction), and ileum] and large (cecum/colon) intestinal tissues were examined grossly and histologically. ihc results from only the mid-jejunum and cecum/colon of the conventional pigs were reported in the previous paper . therefore, duodenal, mid-jejunal, ileal, and colonic tissues of conventional or gn pigs were also conducted or re-tested by ihc, as described previously (jung et al., 2007; jung et al., 2014) , for the detection of pedv antigen (using monoclonal antibody 6c8-1 specific to pedv provided by dr. daesub song, korean research institute of bioscience and biotechnology, korea) (jung et al., 2014) . for gn pigs 1-6 euthanized at pih 16, proximal and distal jejunum were additionally tested by ihc for the detection of pedv antigen to clarify the tissue tropism of the virus during the early stage of infection. duodenal, midjejunal, ileal, and colonic tissues of conventional or gn pigs were conducted by ihc, as described previously (jung et al., 2007; jung et al., 2014) , for the detection of serotonin [using monoclonal antibody 5ht-h209 against human serotonin (novus biologicals, littleton, co, usa)]. monoclonal antibodies 6c8-1 and 5ht-h209 were diluted 1 in 200 and 1 in 50, respectively, in phosphate-buffered saline. mean ratios of jejunal or ileal villous height to crypt depth (vh:cd) were calculated in gn pigs, as described previously . only well-orientated, hematoxylin and eosin-stained jejunal or ileal sections were measured and care was taken to ensure that only transverse sections cut perpendicularly from the villus tip to the muscularis mucosa were included. villous height and crypt depth were estimated by measuring 4-5 villi and crypts throughout the section from each gn pig. pedv antigen-positive scores were computed by estimating the number of ihc-positive cells in the intestinal section, as described previously (jung et al., 2014) , as follows: +/− (few but clearly positive), < 1% of villous epithelial cells showed staining; + (low), 1%-29% of villous epithelial cells showed staining; ++ (moderate), 30%-59% of villous epithelial cells showed staining; +++ (high), 60%-100% villous epithelial cells showed staining; and -(negative), no cells showed staining. mean numbers of serotonin-positive ec cells were evaluated per microscopic area, at ×250 magnification by a leitz light microscope, where approximately 10-18 intestinal crypts and 4-7 entire or lower half of villi were included. the variable numbers were dependent on the location of intestinal tissues. in pedv-inoculated pigs at pids 1-5, unlike mock-inoculated pigs, entire jejunal and ileal villi were infrequently observed even at ×250 magnification, because the villi were severely atrophied. only single crypt layer-included areas in well-orientated intestinal tissue sections were evaluated to count the number of serotonin-positive ec cells. mean numbers of serotonin-positive ec cells were estimated by measuring at least 4 different tissue areas for each pid and from pedv-inoculated or mock-inoculated pigs. all values were expressed as the means ± standard deviation of the means (sdm). mean numbers of serotonin-positive ec cells per microscopic area in duodenum, mid-jejunum, ileum, and colon between pedv-and mock-inoculated pigs at the same time-points were analyzed and compared by a student's t-test using graphpad prism software (graphpad prism inc.). a value of p < .05 was considered statistically significant. 3.1. clinical observations and histologic lesions in conventional 9-day-old nursing pigs inoculated orally with pedv watery diarrhea (9/9 pigs) and vomiting (7/9 pigs) were first detected at pid 1 in pedv-inoculated piglets. at pids 3-5, however, all the inoculated piglets exhibited watery diarrhea, lethargy, and dehydration, but not all showed vomiting, whereas none of the mock-inoculated pigs showed clinical signs. in pedv-inoculated piglets, histologic lesions were limited to the jejunum and ileum, and included diffuse, severe atrophic enteritis at pids 1-5. the duodenum showed only mild villous atrophy at pids 1-5. no histologic lesions were evident in the large intestine and other organs of the inoculated nursing pigs and negative controls. pedv antigens were mostly found in the villous epithelial cells. of 3 pedv-inoculated pigs at pid 1, 2 pigs showed moderate numbers of pedv antigen-positive cells in the mid-jejunum and ileum only, and 1 pig showed low numbers of pedv antigen-positive cells in all intestinal segments (duodenum to colon). at pid 3, all 3 pedv-inoculated pigs showed high numbers of pedv antigen-positive cells in the mid-jejunum and ileum and few to low numbers of positive cells in the colon. however, there were no pedv antigen-positive cells in the duodenum. at pid 5, all 3 pedv-inoculated pigs tested showed high numbers of pedv antigen-positive cells in the mid-jejunum and ileum only. no pedv antigen-positive cells were detected in the intestines of the negative control pigs. 3.3. identification, characterization, and distribution of serotonin-positive enterochromaffin cells in duodenum, mid-jejunum, ileum, and colon of conventional 9-day-old nursing pigs inoculated orally with pedv or mock in duodenum, mid-jejunum, ileum, and colon of mock-inoculated nursing pigs at pids 1-5 (10-14 days of age), serotonin-positive ec cells were found mainly in the crypts (fig. 1a -e) and occasionally, in the lamina propria or epithelium of the lower half of the villi (fig. 1b) (except for colon, where ec cells were detected in the entire colonic epithelium). in intestinal crypts, the majority of serotonin-positive ec cells were triangular-shaped or round cells with or without apical cytoplasmic extensions, respectively, that project into the lumen ( fig. 1b and c). occasionally, serotonin-positive ec cells were closely associated with the basement membrane of crypt cells. they were spindle cells with extensions surrounding mucus in crypt cells. in the lamina propria or epithelium of villi, serotonin-positive ec cells mostly appeared to be round cells, whereas no serotonin-positive cells were detected in the submucosa. on the other hand, in the duodenum, mid-jejunum, ileum, and colon of pedv-inoculated nursing pigs at pids 1-5 (10-14 days of age), the morphology and intestinal distribution of serotonin-positive ec cells were similar to those found in the mock-inoculated control pigs. however, a few serotonin-positive ec cells were also detected in the entire epithelium of the atrophied jejunal or ileal villi (fig. 1f) . 3.4. serotonin-positive ec cells in duodenum, mid-jejunum, ileum, and colon of conventional 9-day-old nursing pigs inoculated orally with pedv or mock during disease progression at pid 1 when vomiting was observed in pedv-inoculated pigs, mean numbers of serotonin-positive ec cells per microscopic area (×250) were significantly (p < .05) reduced in the duodenum, midjejunum, ileum, and colon, compared with the corresponding negative controls (table 1 ; fig. 1b , d, e, and f). at pid 3 when vomiting had ceased, mean numbers of serotonin-positive ec cells per microscopic area (×250) were significantly (p < .05) increased in duodenum but reduced in ileum of the pedv-inoculated pigs, compared with the corresponding negative controls, but they did not differ in mid-jejunum and colon (table 1) . at pid 5, mean numbers of serotonin-positive ec cells per microscopic area (×250) in duodenum, mid-jejunum, ileum, and colon did not differ between the pedv-and mock-inoculated nursing pigs (table 1) . 3.5. clinical observations and pedv rna titers in fecal and serum samples in gnotobiotic 9-day-old pigs inoculated orally with pedv during the subclinical incubation stage of infection (prior to pid 1-5 when conventional 9-day-old pigs showed clinical signs) the significantly reduced numbers of serotonin-positive ec cells in the small and large intestines of pedv-inoculated conventional piglets at pid 1, compared with the corresponding negative controls, might be a result of the early release of serotonin from ec cells into the intestinal lamina propria or lumen to stimulate the vagal afferent neurons, followed by vomiting. in an attempt to explore this effect, a further study using six 9-day-old gn pigs inoculated orally with 9.2 log 10 ge/pig (pigs 1-3) or mock (pigs 4-6) was conducted to investigate alterations in the number of serotonin-positive ec cells in the small and large intestines at an earlier time-point concurrent with or shortly after vomiting was first detected, as compared with pid 1 when most of the conventional 9-day-old pigs tested already exhibited vomiting. after virus inoculation, vomiting and other clinical signs such as diarrhea were monitored frequently to determine an earlier time-point appropriate for euthanasia. in our experiments, at pih 16, pig 3 began to exhibit vomiting (but no diarrhea), whereas pigs 1 and 2 did not show any clinical signs. thus, inoculated pigs 1-3 and negative control pigs 4-6 were euthanized at pih 16 for virological and pathological examination to determine their infection status and to understand the relationship of acute pedv infection and frequency of serotonin-positive ec cells in the small and large intestines. the results are summarized in table 2 . at pih 16, by qrt-pcr, no pedv-inoculated gn pigs 1-3 or negative control pigs 4-6 had detectable viral rna (< 4.8 log 10 ge/ml) in the feces. however, pigs 1-3 had moderate viral rna titers in serum at pih 16, ranging from 8.1 to 8.5 log 10 ge/ml, whereas no negative control pigs 4-6 had detectable viral rna (< 3.8 log 10 ge/ ml) in the serum. 3.6. trend toward increased innate (ifnα and il-22) and proinflammatory (tnfα, il-6, and il-12) cytokine levels in the sera of gnotobiotic 9-day-old pigs inoculated orally with pedv during the subclinical incubation stage of infection as compared with uninfected gn pigs 4-6 at pih 16, mean serum ifnα, il-22, tnfα, il-6, and il-12 levels in the infected gn pigs 1-3 at pih 16 were increased by 45-fold, 56-fold, 6-fold, 3-fold, and 3-fold, respectively (table 3) . since there were large standard deviations of mean values of most of the cytokines in infected pigs as well as too few pigs (n = 3) in each group, statistical analysis was not done. interestingly, the infected gn pig 3 with vomiting at pih 16 showed increased or higher serum ifnα, il-22, tnfα, il-6, and il-12 cytokine levels compared with the other infected gn pigs 1 and 2 with no vomiting, or uninfected gn pigs 4-6. as compared with uninfected gn pigs 4-6 at pih 16, serum ifnα, il-22, tnfα, il-6, and il-12 cytokine levels in the infected gn pig 3 at pih 16 were increased by 119-fold, 123-fold, 12fold, 6-fold, and 6-fold, respectively (table 3) . the data clearly indicate that infected gn pigs exhibited systemic innate and pro-inflammatory cytokine responses to acute pedv infection concurrent with the detection of pedv rna in serum. 3.7. gross and histologic lesions and intestinal distribution of pedv antigen in gnotobiotic 9-day-old pigs inoculated orally with pedv during the subclinical incubation stage of infection (prior to pid 1-5 when conventional 9-day-old pigs showed clinical signs) at pih 16, neither pedv-inoculated gn pigs 1 or 2 exhibited gross lesions in the small and large intestines, whereas pedv-inoculated gn pig 3 had accumulation of moderate amounts of watery liquid in the small intestinal lumen, but not large intestine. no gross lesions were evident in the other organs of the pedv-inoculated gn pigs 1-3 and negative controls 4-6. histologic lesions and the distribution based on table 1 mean numbers ( ± sdm) of serotonin-positive enterochromaffin cells by immunohistochemistry in the crypt layers and entire or lower half of villi of duodenum, mid-jejunum, ileum, and colon per microscopic area, at ×250 magnification, of conventional 9-day-old nursing pigs inoculated with virulent us pedv strain pc21a or mock at post-inoculation days (pids) 1, 3, and 5. in pedv-inoculated pigs at pids 1-5, unlike mock-inoculated pigs, entire jejunal and ileal villi were infrequently observed even at ×250 magnification, because their villi were severely atrophied during the period. c bold numbers, p < .05 (statistically significant differences between the pedv-inoculated and mock-inoculated pigs by student's t-test). intestinal locations are summarized in table 2 . duodenum and proximal jejunum of pedv-inoculated gn pigs 1 and 2 showed normal mucosa, submucosa, and serosa. however, their mid-jejunum and ileum showed acute, diffuse, mild (pig 2) to moderate (pig 1) swelling and coagulative necrosis of villous epithelial cells lining up to 100% of the villous epithelium, accompanied by diffuse, mild subepithelial edema and multifocal, mild exfoliation of enterocytes located on the villous tip. also, for pig 1, multifocal, moderate suppurative inflammation was evident among the affected villi in the lumen. the length of the villi appeared normal. distal jejunum showed diffuse, lipid accumulationtable 2 design and results of a pathology study of the original us pedv strain pc21a in gnotobiotic 9-day-old pigs at post-inoculation hour (pih) 16 when or shortly after vomiting was first detected. pig no. 1 2 3 4, 5 and 6 oral inoculum, log 10 ge/ml 2 ml, 8.9 (9.2 log 10 ge/pig) 2 ml, 8.9 (9.2 log 10 ge/pig) 2 ml, 8.9 (9.2 log 10 ge/pig) 2 ml, mock mean (sd) c 1 (0) 8 (6) 4 (0) 142 (72) 18 (9) a the samples were tested in duplicate, and cytokine levels were expressed as the mean values. detection limits of our elisa were 1 pg/ml for ifnα and il-12, 4 pg/ml for tnfα and il-22, and 16 pg/ml for il-6, respectively. b bold fonts and numbers, pig 3 with vomiting at pih 16 showed higher serum ifnα, tnfα, il-6, il-12, and il-22 cytokine levels compared with the other infected gn pigs 1 and 2 with no vomiting, or uninfected gn pigs 4-6. c since there were no or little standard deviations of mean values of some cytokines in mock-inoculated pigs and large standard deviations of mean values of most of the cytokines in infected pigs as well as too few animals in each group, statistical analysis was not conducted. related, cytoplasmic vacuolation of enterocytes lining up to 100% of villous epithelium, with normal length of the villi. on the other hand, pedv-inoculated gn pig 3 had more progressed lesions compared with pigs 1 and 2. duodenum and proximal jejunum mostly had normal mucosa, submucosa, and serosa; however, mild, multifocal subepithelial edema was observed in villi of the proximal jejunum. midjejunum showed acute, diffuse, severe coagulative necrosis of villous epithelial cells, and the affected villi was shortened, with villous height: crypt depth (vh:cd) ratios ranging from 3 to 4 (vs. 6.8 to 7.9 in negative controls). distal jejunum showed diffuse, lipid accumulationrelated, cytoplasmic vacuolation of enterocytes lining up to 100% of the villous epithelium, with normal length of the villi. ileum showed acute, mild, diffuse swelling and coagulative necrosis of villous epithelial cells, accompanied by diffuse, moderate subepithelial edema and diffuse, mild exfoliation of enterocytes located on villous tip. however, the length of the villi appeared to be within the normal range, as the vh:cd ratios ranged from 5 to 6 (vs. 5.0 to 5.6 in negative controls). no histologic lesions were evident in the large intestine and other organs of the pedv-inoculated gn pigs 1-3 and negative controls 4-6. 3.8. intestinal distribution of pedv antigen in gnotobiotic 9-day-old pigs inoculated orally with pedv during the subclinical incubation stage of infection (prior to pid 1-5 when conventional 9-day-old pigs showed clinical signs) like pedv-inoculated conventional pigs tested earlier, pedv antigens were mostly found in the villous epithelial cells, not the crypt epithelial cells ( fig. 2a-f) . at pih 16, pedv-inoculated gn pig 1 showed moderate to high numbers of pedv antigen-positive cells in up to 100% of the mid-jejunal and ileal epithelium ( fig. 2b and c) , low to moderate numbers of pedv antigen-positive cells in the villus-crypt interface of the proximal and distal jejunum ( fig. 2a) , few positive cells in the villus-crypt interface of the duodenum, and no positive cells in the colon. pedv-inoculated gn pig 2 showed high numbers of pedv antigen-positive cells in up to 100% of the mid-jejunal epithelium, low numbers of pedv antigen-positive cells in the villus-crypt interface of the proximal and distal jejunum and ileum, and no positive cells in the duodenum and colon. pedv-inoculated gn pig 3 showed high numbers of pedv antigen-positive cells in up to 100% of the epithelium of atrophied mid-jejunal villi (fig. 2e ), low to moderate numbers of pedv antigen-positive cells in the villus-crypt interface of the proximal and distal jejunum and ileum (fig. 2f) , low numbers of positive cells in the villus-crypt interface of the duodenum (fig. 2d) , and no positive cells in the colon. no pedv antigen-positive cells were detected in the intestines of the negative control pigs. 3.9. serotonin-positive ec cells in duodenum, mid-jejunum, ileum, and colon of gnotobiotic 9-day-old pigs inoculated orally with pedv or mock during the subclinical incubation stage of infection (prior to pid 1-5 when conventional 9-day-old pigs showed clinical signs) the morphology and intestinal distribution of serotonin-positive ec cells identified in pedv or mock-inoculated gn pigs 1-6 were similar to those observed in the pedv-or mock-inoculated conventional pigs. at pih 16, mean numbers of serotonin-positive ec cells per microscopic area (×250) were significantly (p < .05) reduced in duodenum, midjejunum, and colon, but not ileum, of pedv-inoculated gn pigs 1-3, compared with the corresponding negative controls (table 4) . however, further analyses were done based on presence or absence of onset of vomiting in the pedv-inoculated gn pigs tested. in pedv-inoculated gn pigs 1 and 2 with no vomiting, mean numbers of serotonin-positive ec cells per microscopic area (×250) in duodenum, ileum, and colon, but not mid-jejunum, were comparable to those in the corresponding negative controls (table 4 ). like pedv-inoculated conventional pigs tested at pid 1, however, pedv-inoculated gn pig 3 with vomiting exhibited a trend toward reduced numbers of serotonin-positive ec cells per microscopic area (×250) in the duodenum, mid-jejunum, ileum, and colon, compared with the corresponding negative controls (table 4) . our study demonstrates that the monoclonal antibody 5ht-h209 is useful for the detection of serotonin in ec cells in swine intestinal tissues. similar to serotonin-positive intestinal ec cells identified by ihc in humans or rats (gustafsson et al., 2006; spiller, 2008) , swine ec cells were also mainly localized in intestinal crypts, and they were morphologically triangular-shaped or round cells with or without apical cytoplasmic extensions, respectively. like ec cells in humans, swine intestinal ec cells were the most frequent in duodenum compared with the other intestinal segments (tables 1 and 4) (endo et al., 2000; spiller, 2008) . at pih 16 or pid 1, when vomiting was first or frequently observed in pedv-inoculated gn or conventional pigs, respectively, the number of serotonin-positive ec cells was significantly reduced in the small and large intestines, compared with the negative controls. however, two of three pedv-inoculated gn pigs (pigs 1 and 2) that did not exhibit vomiting at pih 16 concomitantly showed numbers of serotonin-positive ec cells in the small (but not mid-jejunum) and large intestines similar to those in the negative controls. these findings suggest that serotonin release from ec cells into the intestinal lamina propria or lumen (increased serotonin levels in the gut submucosa) occurred early post-infection to stimulate the vagal afferent neurons, followed by vomiting. the procedure may be essential for inducing vomiting in pedv-infected piglets. at pid 1, reduced numbers of serotonin-positive ec cells were observed from the duodenum to the colon of conventional pigs, although extensive villous atrophy with a large amount of pedv antigen was confined to the jejunum and ileum. in contrast, the duodenum and colon showed no or mild villous atrophy with little pedv antigen at pid 1. we speculate that regardless of the tissue tropism of the virus, ec cells in all intestinal segments (duodenum to colon) might be involved in the induction of vomiting in pedv-infected piglets. in our study, the detailed pathogenesis of pedv in infected gn pigs was examined to understand the relationship of acute pedv infection and frequency of serotonin-positive ec cells in the small and large intestines. also, there was little information on the pathogenesis of acute pedv infection in young piglets prior to onset of clinical signs. at pih 16, pedv rna was not detected in feces but was detected in serum (8.1-8.5 log 10 ge/ml) of pedv-inoculated gn pigs. our observations are similar to a previous study, showing that only 1 of 20 (5%) 1-dayold, caesarean-derived, colostrum-deprived (cdcd) pigs inoculated orally with 10 3 pedv plaque-forming units/pig were positive for pedv rna in the feces at pih 12 (madson et al., 2016) . in the same study, however, 4 of 5 pigs (80%) were positive for pedv rna in serum at the same time-point. based on these and our observations, viremia (viral rna) may be detected earlier than fecal virus rna shedding in pedvinfected piglets. as our ihc study also revealed large amounts of pedv antigens in the small intestine of pedv-inoculated gn pigs 1-3 at pih 16, viremia might be a result of diffusion or spread of replicated pedv from the infected intestine to the blood, although the detailed mechanisms need to be studied further. based on our histopathologic and ihc observations in pedv-inoculated gn pigs 1-3 at pih 16, pedv initially and mainly infected the mild-jejunum and to a lesser extent, ileum; however, pedv infection of the duodenum, proximal jejunum, and distal jejunum was less compared with the mid-jejunum and ileum. in addition, our ihc study did not find any pedv antigen in the pylorus of the pedv-inoculated pigs 1-3. our pilot study also included another location of proximal jejunum distant by approximately 30 cm from the pylorus. we found that infectivity of pedv in this intestinal segment was more similar to that found in the mid-jejunum compared with the other proximal jejunum segment (distant by 15-20 cm from the pylorus) closer to the pylorus. these observations underscore that the mid-jejunum and ileum may be the primary sites of acute pedv infection , although the exact cause of the distinct tissue tropism remains obscure in terms of virus-host cell interactions at the molecular level. it is debatable whether pedv induces innate immune or pro-inflammatory responses in infected pigs, because most of the research has been conducted in cell cultures in vitro . however, our previous study showed increased serum ifnα and il-12 at pid 1 in conventional 9-day-old nursing pigs infected with pedv . our current study also clearly showed that infected gn pigs exhibited systemic innate (ifnα and il-22) and pro-inflammatory (il-6, tnfα, and il-12) cytokine responses after acute pedv infection, similar to the reported increased mrna levels of pro-inflammatory cytokines (il-1β, il-6, il-8 and tnfα) in ipec-j2 cells infected with pedv (lin et al., 2017) . in particular, 3-to 6-fold increases in the pro-inflammatory cytokines observed in infected gn pigs 1-3 coincided with the decreased or loss of appetite following acute pedv infection in young suckling piglets (langhans, 2000) . ec cells secrete serotonin in response to mechanical stimulation as well as recognition of pathogens via toll-like receptors (worthington, 2015) . several studies showed that serotonin functions to promote immune activation through the receptors expressed on macrophages, dendritic cells, and t and b cells (li et al., 2011; shajib and khan, 2015) . in our pilot study, increased serum levels of pro-inflammatory and innate cytokines coincided with reduced numbers in serotonin-positive intestinal ec cells, i.e. serotonin release from ec cells in the gut submucosa, during the early stage of infection (pih 16 to pid 1) (tables 3 and 4 ). this suggests a possible involvement of ec cells and serotonin, not only in triggering an emetic response but also in development of innate or pro-inflammatory immune responses to acute pedv infection. however, the potentially beneficial or detrimental effects or roles of those innate and pro-inflammatory cytokines and serotonin in infected piglets need to be studied further. as tested in pedv-inoculated gn pigs 1-3 at pih 16, our ihc observations revealed that early localization of pedv antigen is evident in the villous-crypt interface of the duodenum, jejunum, and ileum during the acute or incubation stage of infection ( fig. 2a-f) , similar to previous observations (madson et al., 2016) . a similar observation was also detected by ihc in 1-day-old, cdcd pigs inoculated with a porcine deltacoronaivrus at pih 12-24 (jung and saif, unpublished) , implying a similar tissue tropism between these two viruses during the early stages of infection. the pedv antigen-positive regions appeared first in the villus-crypt interface and subsequently expanded to the upper and then entire villous epithelium of the jejunum to ileum. our data indicate that enzymatically immature (young) enterocytes in the villous-crypt interface of the small intestine, relative to the enzymatically mature (older) enterocytes located at the villous tips, might be the major infection site of pedv. during the incubation period, similar to vibrio cholera (cholera toxin) or rotavirus (non-structural viral protein 4) (spiller, 2008) , pedv itself or undefined secondary mediators from the infected host might act to trigger serotonin release from ec cells. because the infection site of pedv is close to the crypt layer where the majority of serotonin-positive ec cells are located, it might be beneficial for pedv itself or secondary mediators to access ec cells in the crypt layers. in our study, although pedv antigen-positive cells were confined to villous epithelium (not crypt epithelium where the majority of serotonin-positive ec cells were localized), whether pedv can also infect ec cells needs to be studied further. at pid 5 when vomiting disappeared completely, the numbers of serotonin-positive ec cells in the small and large intestines of the conventional pigs recovered and were comparable to those in the negative controls. the rapid recovery could imply a rapid turnover of ec cells to compensate for the reduced numbers. however, the turnover time of ec cells (15-150 days) is much slower than for enterocytes (2-4 days) in rats (de bruine et al., 1992) . thus, the rapid recovery of the number of serotonin-positive ec cells might be a result of increased synthesis and deposition of serotonin in ec cells. collectively, serotonin might be involved in the mechanisms related to vomiting in pedv-infected nursing pigs. serotonin receptor (5-ht 3 ) antagonists may be useful targets as a therapeutic intervention to suppress acute vomiting in pedv-infected pigs, especially in cases when the clinical signs are severe. therefore, further studies are needed to test whether 5-ht 3 antagonists are effective to inhibit or suppress acute vomiting in pedv-infected pigs. to evaluate the disease mechanisms and comparative pathogenesis of pedv strains in young piglets during the acute or incubation stage of pedv infection, the mid-jejunum and ileum are the most critical and useful intestinal locations for histological analysis. our study also revealed that acute pedv infection with evidence of pedv rna in serum, induces systemic innate (ifnα and il-22) and pro-inflammatory (il-6, tnfα, and il-12) cytokine responses in young gn pigs. none of the authors of this paper have a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. table 4 mean numbers ( ± sdm) of serotonin-positive enterochromaffin cells by immunohistochemistry in the crypt layers and lower half of villi of duodenum, mid-jejunum, ileum, and colon per microscopic area, at ×250 magnification, of gnotobiotic 9-day-old pigs inoculated with virulent us pedv strain pc21a or mock at postinoculation hour (pih) 16 when or shortly after vomiting was first detected. pedv-inoculated gn pigs at pih 16 (10 days of age) mock-inoculated gn pigs 4-6 (10 days of age) (n = 3) based on the time-point, pih 16 based on presence or absence of vomiting at pih 16 pigs 1-3 (n = 3) pigs 1 and 2 with no vomiting (n = 2) pig 3 with onset of vomiting (n = 1) age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs cytokine responses in gnotobiotic pigs after infection with virulent or attenuated human rotavirus divergent immunomodulating effects of probiotics on t cell responses to oral attenuated human rotavirus vaccine and virulent human rotavirus infection in a neonatal gnotobiotic piglet disease model renewal of enterochromaffin cells in the rat caecum neurochemistry and neuropharmacology of emesis -the role of serotonin the role of il-22 in viral infections: paradigms and paradoxes a new method for visualization of gut mucosal cells, describing the enterochromaffin cell in the rat gastrointestinal tract porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs comparative pathogenesis of us porcine epidemic diarrhea virus (pedv) strain pc21a in conventional 9-day-old nursing piglets vs. 26-day-old weaned pigs anorexia of infection: current prospects serotonin activates dendritic cell function in the context of gut inflammation differential protein analysis of ipec-j2 cells infected with porcine epidemic diarrhea virus pandemic and classical strains elucidates the pathogenesis of infection characterization of porcine epidemic diarrhea virus isolate us/ iowa/18984/2013 infection in 1-day-old cesarean-derived colostrum-deprived piglets the role of serotonin and its receptors in activation of immune responses and inflammation role of serotonin in gastrointestinal motility and irritable bowel syndrome serotonin and gi clinical disorders the intestinal immunoendocrine axis: novel cross-talk between enteroendocrine cells and the immune system during infection and inflammatory disease immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp1 we thank dr. juliette hanson, andrew wright, megan strother, and ronna wood for assistance with animal care; and xiaohong wang, zhongyan lu, john blakenship, and thavamathi annamalai for technical assistance. salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development center, the ohio state university. key: cord-261043-f9w310tp authors: ajayi, toluwalope; dara, rozita; poljak, zvonimir title: forecasting herd-level porcine epidemic diarrhea (ped) frequency trends in ontario (canada) date: 2019-03-01 journal: prev vet med doi: 10.1016/j.prevetmed.2019.01.005 sha: doc_id: 261043 cord_uid: f9w310tp porcine epidemic diarrhea virus (pedv) emerged in north america in 2013. the first case of pedv in canada was identified on an ontario farm in january 2014. surveillance was instrumental in identifying the initial case and in minimizing the spread of the virus to other farms. with recent advances in predictive analytics showing promise for health and disease forecasting, the primary objective of this study was to apply machine learning predictive methods (random forest, artificial neural networks, and classification and regression trees) to provincial pedv incidence data, and in so doing determine their accuracy for predicting future pedv trends. trend was defined as the cumulative number of new cases over a four-week interval, and consisted of four levels (zero, low, medium and high). provincial pedv incidence and prevalence estimates from an industry database, as well as temperature, humidity, and precipitation data, were combined to create the forecast dataset. with 10-fold cross validation performed on the entire dataset, the overall accuracy was 0.68 (95% ci: 0.60 – 0.75), 0.57 (95% ci: 0.49 – 0.64), and 0.55 (0.47 – 0.63) for the random forest, artificial neural network, and classification and regression tree models, respectively. based on the cross-validation approach to evaluating predictive accuracy, the random forest model provided the best prediction. porcine epidemic diarrhea (ped) is a highly infectious swine disease which can cause high morbidity and mortality in swine populations (carvajal et al., 2015) . its causative agent, porcine epidemic diarrhea virus (pedv), recently emerged in north america, with the first reported case occurring in may 2013 in the united states (hill et al., 2014) . in canada, the first case was identified in january 2014 on an ontario swine farm, with additional cases reported in several provinces, from manitoba on the west to prince edward island on the east (kochhar, 2014) . the combined efforts of federal and provincial authorities, as well as industry organizations, were instrumental in controlling the spread of the disease in ontario (kochhar, 2014) . furthermore, the successful collaboration has led to a low ped prevalence situation in ontario, with provincial disease elimination now viewed as a real possibility. the ontario swine health advisory board (oshab) is an industry organization which administers a ped surveillance program and database under its area regional control and elimination (arc&e) project. the ped database allowed for tracing the ped status of individual herds in discrete locations, from time of initial infection until time of declaration of freedom from pedv infection based on established criteria. as such, it was successfully utilized to obtain ped incidence and prevalence estimates for the province (ajayi et al., 2018) . with limited surveillance and disease control resources, accurate predictions for ped trends would be invaluable in allocating such resources, and fortunately, the ped database provides data which can be used for predictive purposes. recent advances in forecasting and predictive methods have shown great promise for both human and animal medicine (tu, 1996; leung and tran, 2000; er et al., 2010; mancia et al., 2012) , and it seemed logical to explore these methods to determine the most appropriate one for pedv prediction. predictive accuracy of the methods could then be considered as one criterion for selecting the most suitable method for ongoing surveillance, which would contain short-term prediction of disease trends and possibly translate into prediction of resources needed to control the outbreak. therefore, the primary objective of this study was to determine the most accurate machine-learning approach for forecasting future pedv trends in ontario swine herds. specifically in this study, random forest, classification and regression trees, and artificial neural networks were utilized. the secondary objective was to determine variables highly ranked as determinants for pedv trends. the methods considered in t this study were selected for various reasons. random forest, based on different metrics, showed slightly better performance in comparison to other methods when applied to similar datasets (kane et al., 2014; petukhova et al., 2018) . among the data-driven prediction methods, classification and regression trees is the most transparent method with results that are easy to interpret (shmueli et al., 2017) , a feature which may be preferred by end users. in addition, classification and regression trees form the basis for random forests. by contrast, results of analysis conducted via artificial neural networks cannot be directly interpreted through coefficients or through other means, however they have been successfully applied to the analysis of complex relationships between predictors and an outcome, and have often resulted in high prediction accuracy (shmueli et al., 2017) . data about weekly incidence and prevalence measures were obtained from an industry database, which tracks the pedv infection status of individual premises participating in an industry-driven voluntary disease control program. the program is known as the ontario area regional control and elimination (arc&e) project. detailed explanation of variables representing herd-level pedv infection status is provided elsewhere (ajayi et al., 2018) . briefly, (i) confirmed positive premises had to have at least one rt-pcr positive test for the ped virus, (ii) presumed positive premises did not have diagnostic testing conducted but housed animals that were moved from known positive sites (e.g. nurseries supplied from known positive sow sites), (iii) presumed negative premises were previously confirmed or presumed positive sites that underwent diagnostic testing based on industry standards (at least 10% design prevalence with 95% herd sensitivity), which resulted in all negative tests, and (iv) confirmed negative premises were premises with no clinical signs or positive tests for pedv for at least 6 months after the presumed negative status had been achieved. the source population for the outcome measurement consisted of commercial swine herds which included: 14 farrow-wean herds (9.3%), 5 farrow-feeder (3.3%), 16 farrow-finish (10.6%), 17 nurseries (11.3%), 7 wean-finish facilities (4.6%), 91 finisher sites (60.3%) and 1 isolation/acclimation unit (0.7%). the median number of sows on study sites was 750 sows (interquartile range = 925). furthermore, in calculating herd-level ped prevalence, all swine herds available in the database in a given week were used, with their pedv status set as "undetermined". these were herds that existed in the disease control database but were not tested for pedv. however, due to the emerging nature of pedv in ontario during the study period, as well as reporting obligations in this phase of the epidemic, this "undetermined" status can be equated to negative pedv status. these statuses were then processed to obtain weekly measures of disease frequency, including number of new cases, herd-level prevalence (expressed as a percentage), and number of infectious sows (expressed as raw counts). the number of new cases was of primary interest and was used to form the final outcome for the analysis, whereas herd-level prevalence and number of infectious sows were a-priori considered as potentially important predictors for the number of new cases and severity of the outbreak. province-level temperature and humidity values representative of swine locations in ontario were obtained by aggregating corresponding county-level weather data, first to daily and then to weekly level. this was done by: 1) identifying ontario counties with the highest pig counts as documented by the ontario ministry of agriculture food and rural affairs (omafra, 2014), namely, the perth, huron, middlesex, and wellington counties; 2) locating a weather station in each of those counties using the weather underground website (www. wunderground.com), and 3) for each weather station, obtaining the following daily values for november 24, 2013 -may 6, 2017: (i) high, average, and low temperature; (ii) high, average, and low humidity; (iii) precipitation. if daily weather-related values were not reported for a weather station, daily values from another weather station in the same county, either from the weather underground website or environment canada's historical weather data, were substituted. temperature, humidity and precipitation were selected for inclusion for two principal reasons. first, these measurements were readily available from the majority of weather stations. second, temperature and humidity are reported to influence survival of different viruses (lowen et al., 2007; casanova et al., 2010) including pedv (thomas et al., 2015) . the daily weather data for each county were imported into r (r core team, 2017) and a singular dataset of temperature and humidity values for all 4 counties created. the representative daily high, average, and low temperature and humidity values for ontario were obtained by averaging the corresponding values across the counties (i.e. perth values + huron values + middlesex values + wellington values divided by 4). a similar averaging was done for county precipitation values. once daily county values were aggregated to the provincial level using the methods noted above, there were seven variables in all: (i) ontario_hightemphighest temperature reading in ontario for the day, (ii) ontario_avgtempaverage temperature reading in ontario for the day (iii) ontario_lowtemplowest temperature reading in ontario for the day; (iv) ontario_highhumidhighest humidity reading in ontario for the day; (v) ontario_avghumidaverage humidity reading in ontario for the day; (vi) ontario_lowhumidlowest humidity reading in ontario for the day; (vii) ontario_precipprecipitation reading in ontario for the day. for aggregation to the weekly level, a week was defined as beginning on sunday and ending on saturday. as such, for each week beginning november 24, 2013, a corresponding weekly value for each variable was generated by simply averaging the daily values (adding all the daily values in the week and dividing by 7), with the only exception being precipitation where a weekly sum (rather than an average) of precipitation values was generated. the weekly temperature and humidity values were then combined with weekly pedv data (incident cases, infectious sows, and prevalence) to produce the forecast dataset with 10 variables. as pedv emerged in canada in 2014, the starting week in the forecast dataset was set to january 5, 2014, with the ending week set to april 30, 2017. each variable was then lagged five times (i.e. the corresponding values for prior weeksup to five weeks in the pastwere aligned with current values), with each weekly lag resulting in an additional variable. with the lags completed, the final incident cases dataset had a total of 174 observations and 60 variables. a 4-week moving sum of incident cases (i.e. total number of new ped case herds at the province-level over a four-week period, with week beginning january 5, 2014) was calculated with the zoo rollapply function in r (zeileis and grothendieck, 2005) . a moving 4-week sum started with the current week of interest and included 3 additional prospective weeks in the future. then, a 4-week moving sum equal to '0′ incident cases (i.e. zero new positive ped herds in the entire province of ontario) was classified as trend "zero", a moving sum equal to '1′ or '2′ incident cases was classified as trend "low", a moving sum between '3′ and '6′ incident cases was classified as trend "medium", while a moving sum greater than '6′ incident cases was classified as trend "high". such classification was made with a rationale that the capacity needed to manage a production-limiting disease in the entire source population -swine herds in the province of ontario -relies heavily on industry resources (i.e. different industry organizations and a limited number of veterinary practitioners with other daily responsibilities). the weekly count of zero is a preferred condition and qualitatively different from other outcomes; trend "low" within a 4-week window would not be considered as unusual, particularly during periods when the disease peaks seasonally due to environmental conditions (e.g. winter); trend "medium" would be considered as manageable, but would also be reason for further investigation. finally, greater than 6 new cases in a 4-week period (i.e. trend "high") would be considered as an alert and a potentially re-emerging scenario. it follows then that the authors were particularly interested in accurate classification of trends "zero" and "high". the weekly count of incident cases was then removed from the dataset, however, lags 1-5 of the weekly count of incident cases was retained. the final trend forecast dataset had a total of 171 observations and 60 variables. for this dataset, the response variable was set to the 4-week trend (with possible values "high", "medium", "low", and "zero"), and the explanatory variables set as the remaining 59 variables. descriptive statistics were generated and visually assessed by separating the time-series of incident cases and explanatory variables into long-term trend, seasonality and error components via the stl function (r core team, 2017). in this study, we aimed to predict the general trend in the number of new cases over a future 4-week window, and to subsequently evaluate the accuracy of such predictions with three different classification approaches. the random forest, artificial neural nets, and classification tree algorithms were selected for forecasting pedv trends. the following sections provide a brief overview of the methods. the random forest algorithm (breiman, 2001 ) is a non-parametric predictive modeling method which works by: 1) constructing multiple classification or regression trees, and 2) aggregating results from these trees to generate a prediction ('y' or response variable) for a specified set of input values ('x' or explanatory variables). regression trees are constructed for a continuous response variable with the final prediction 'y' determined by averaging results across all trees. for a categorical response variable, classification trees are constructed and the final prediction 'y' is determined by a majority class vote across all trees. furthermore, rather than determine the best node split by looking at all explanatory variables at a given node (as is the case with standard classification and regression tree algorithms), the random forest algorithm randomly selects a subset of explanatory variables at each nodewhich reduces the correlation between subtrees (ho, 2002) -and then determines the best (or homogenous) binary split at the node. the random forest implementation in r -randomforest (liaw and wiener, 2002) -was used in this study. it provides, amongst other features, tuning functions for ascertaining the number of explanatory variables which should be randomly sampled at each node, as well as the optimal number of variables for predicting 'y'. in addition, the random forest implementation provides a variable importance measure, which ranks each explanatory variable per the mean decrease in prediction accuracy when the variable is randomly permuted and other explanatory variables left unchanged. neural nets (or artificial neural networks) are predictive algorithms developed to mimic biological activity in the human brain, specifically the learning patterns for neurons. neural networks have an input layer, hidden layer(s), and an output layer made up of interconnected neurons and an activation function. predictors are supplied to the input layer, which transfers these values to one or more hidden layer(s) for processing via a system of weighted connections. these hidden layer(s) in turn link to an output layer which provides the final prediction result. tuning parameters, such as the maximum number of learning iterations, learning rate, and number of hidden layers and weights, can be set for artificial neural networks (shmueli et al., 2010) . the current neural network implementations in r are nnet (venables and ripley, 2002) and neuralnet (fritsch and guenther, 2016) . the nnet implementation permits one hidden layer, and has several tuning parameters, such as size (for specifying the number of neurons in the hidden layer), decay (a weight decay value to aid in the model optimization process and avoid overfitting), and maxit (the maximum number of permitted iterations). classification trees are predictive algorithms which utilize recursive partitioning, a step-by-step process which splits a node into sub-nodes by evaluating a boolean condition at each node. observations which meet the boolean condition are placed in one node, while the remaining observations are placed in another node. the process is repeated until a terminal node (which can no longer be split) is reached and a class label is assigned. sub-trees are built with each recursive split, and each split (or partition) is constructed such that the resulting nodes are homogenous in nature (izenman, 2008) . the classification tree implementation in r -rpart (therneau et al., 2017) was used in this study. the rpart implementation has several tuning parameters, such as the minimum number of observations which must be present in a node for a split to be attempted, the number of cross-validations performed on observations to determine the best split, as well as complexity parameter cp, where any split that does not decrease the overall lack of fit by a factor of cp is not attempted. the forecast dataset was split into 70% training and 30% testing using functionality available in the caret package (kuhn, 2008) . class distribution was taken into account for the dataset splits, which ensured a balance of "high", "medium", "low", and "zero" trend observations for the training and test set. initially, the random forest model was fitted on the training dataset with all 59 explanatory variables -the number of variables sampled at each split was obtained from the tuning function and a variable importance plot was also constructed. going forward, this model will be known as the "full" model. furthermore, the random forest tuning function, which provides the optimal number of variables for predicting 'y', indicated the lowest prediction error rate at 30 variables. as such, all subsequent models, including the random forest model, were constructed with the top 30 variables from the full model (as obtained from the variable importance plot). for each of the three methods used herein, different parameters for each modelling approach, were then fine-tuned by conducting repeated 10-fold crossvalidation. for the subsequent random forest model, the number of trees grown was set to 500, the number of variables randomly sampled at each split was 3, and a variable importance plot was also generated. for the subsequent neural nets model, the dataset was centered, and scaled, with the number of nodes in the hidden layer, decay function and number of iterations set to maximize the classification accuracy on the testing dataset. the number of nodes in the final neural nets model (i.e. size) was set to 11, decay was set to 0.2, and the maximum number of iterations was set to 1000. the neural nets algorithm used was based on the feed-forward method. for the subsequent classification trees model, the minimum number of observations for a split attempt was set to 14, and the complexity parameter was set to 0.01. for the classification approach with 70% training and 30% test sets, model performance was assessed via confusion matrices, overall classification accuracy, as well as model sensitivity and specificity values by trend (i.e. "high", "medium, "low", and "zero"). due to concerns about limited observations (i.e. small dataset) and to further validate the model's performance for future observations, an additional approach was used to evaluate the predictive accuracy of the models. this was based on 10-fold cross validation, which was performed once on the entire dataset using functionality available in the caret package. specifically, the entire dataset was randomly split into 10 equal-sized groups (or folds), with one fold allocated as the test set and the remaining nine as the training set. the test and training sets were then fed to random forest, neural nets, and classification tree models, with explanatory variables and tuning parameters set to the values used previously (i.e. for the corresponding models with a 70-30 split of the data). the process of allocating test and training sets was repeated until each fold had been used exactly once as a test set. class distribution for the outcome of interest was not considered for each fold, and as such, the randomly selected training and test sets did not have balanced "high", "medium", "low", and "zero" trend observations representative of the entire dataset. for the 10-fold cross-validation approach, model performance was assessed via a summary confusion matrix with overall classification accuracy (across the 10 folds), model sensitivity and specificity values for each trend (i.e. "high", "medium, "low", and "zero"), as well as a boxplot of model sensitivity and specificity values by trend across all 10 folds. model estimates of accuracy based on the cross-validation approach were compared using a paired t-test (kuhn and johnson, 2016) , in which each fold represented the unit of observation. in the full dataset, 19 observations were classified as having "high" trend, 42 as "medium" trend, 55 as trend "low", and 55 as "zero" (tables 1 and 2) . for the forecasting of pedv trends, the confusion matrix in table 1 provides accuracy measures on the training set for a static 70-30 split of the data. table 2 provides accuracy measures on the test dataset for the same split, with random forest, artificial neural nets, and classification trees reporting an overall accuracy of 71%, 75%, and 45% respectively. if non-tolerable errors are considered as misclassification into non-adjacent categories (e.g. high as low, high as zero, medium as zero, and vice versa), and tolerable errors considered as misclassification into adjacent categories (e.g. high as medium, medium as low, low as zero, and vice versa), then random forest had 3 non-tolerable errors and 11 tolerable ones, neural nets had 4 non-tolerable errors and 8 tolerable ones, while classification trees had 10 nontolerable errors and 20 tolerable ones. for the additional models constructed with random training and test sets (using 10-fold cross validation on the entire dataset), the summary confusion matrix in table 3 indicates overall accuracy values of 68%, 57%, and 55% for random forest, neural nets, and classification trees respectively. paired t-test results confirmed accuracy estimates for random forests as being different from neural nets (p = 0.02) and classification trees (p < 0.01), whereas there was no difference in accuracy estimates between neural nets and classification trees (p = 0.68). with non-tolerable errors and tolerable errors defined as before, random forest had 5 non-tolerable errors and 50 tolerable ones, neural nets had 16 non-tolerable errors and 58 tolerable ones, while classification trees had 18 non-tolerable errors and 59 tolerable ones. the sensitivity and specificity for all models are presented in table 4 , while the boxplot of sensitivity and specificity values across 10 folds on the entire dataset is presented in fig. 1 . the boxplot of sensitivity and specificity values indicate higher median values for the random forest model across all 10-folds, with the only exception being trends "zero" and "low" for sensitivity and specificity respectively, where neural nets had a higher median value. the variable importance plot for the random forest model is presented in fig. 2 , while a description of variable names for the 30 explanatory variables in all classification models is provided in stable 1. for random forest classification, prevalence-related variables, whether current or lagged, were the driving force for the 4-week incidence trend (fig. 2) , however, the current week's mean low temperature (i.e. ont_meanlowtemp) was also highly ranked. it appears the current week's low temperature, alongside prior and current prevalence values, determine the pedv trend 4 weeks into the future. time series decomposition plots for weekly pedv incident cases and prevalence, as well as weekly low temperature, average temperature, and average humidity are available in sfigures 2, 3, 4 , 5, and 6 respectively. each plot shows a strong seasonality component, however, there are notable differences where the trend is concerned. for example, incident cases (sfigure 2 ) started with a sharp trend decrease at the beginning of 2014 but then followed with a gradual decrease, while prevalence (sfigure 3 ) had a gradual trend increase at the beginning of 2014 followed by a decrease. both low and average temperature (sfigures 4 and 5) show a trend increase over the study period, while average humidity (sfigure 6 ) started with a trend decrease but then switched to a trend increase in mid-2016. determination of long-term trend for weather variables was, however, based on little informative data (s figures 4-6) . porcine epidemic diarrhea virus (pedv) continues to be a costly disease for swine producers, and is still cause for concern in the united the pedv incursion into canada was less severe, and although ped is not listed as a reportable or notifiable disease by the canadian food inspection agency (cfia, 2017), it is provincially regulated and remains a reportable disease in several provinces, including alberta (alberta ministry of agriculture and forestry, 2014) , saskatchewan (government of saskatchewan, 2017), and manitoba (manitoba agriculture, 2017) . pedv is no longer provincially reportable in ontario (omafra, 2017), and while quebec is free of the virus, pedv remains a reportable disease in the province (quebec ministry of agriculture, fisheries and food, 2017) . while it appears pedv is under control in most regions in canada, the recent outbreaks in manitoba (spanning may -august 2017) are a reminder that pedv is still present, and underscores the importance of monitoring, surveillance, and intervention strategies in controlling and possibly eliminating the disease. to this end, forecasting methods can be added to the surveillance toolkit as an early warning system for ongoing outbreaks. for the above-noted pedv forecast models with a static 70-30 split of the dataset, the artificial neural nets model was the best-performing model with the highest overall accuracy of 0.76 on the test dataset, a result which confirms one of its key strengths, which is its ability to capture complex non-linear relationships between explanatory and response variables (shmueli et al., 2017) . however, judging by an overall accuracy of 0.71 on the test dataset and the misclassification error, the random forest model is a close second. in addition, the random forest model generated just one additional misclassification error on the test dataset when compared to corresponding errors generated by neural nets, and the error was a tolerable one. in this sense, random forest performance is comparable to neural nets. classification trees, on the other hand, was the worst-performing model, a result which confirms a key weakness of the method, which is its inability to accurately capture complex relationships between explanatory and response variables. for the random training and test sets based on 10-fold cross validation, random forest is the best-performing model, a finding which confirms its robustness against majority-class overfitting due to an imbalanced dataset (breiman, 2001) , which in this case would be a model's tendency to classify most, if not all, observations as trend "zero" (the majority class). given the nature of the data, there are few "high" trend observations and many "zero" trend observations. a more pronounced decrease in accuracy during cross-validation than during validation based on a split into training and validation datasets could be a consequence of the sampling approach. it is possible that randomization during cross-validation resulted in imbalanced distribution of observations across outcome categories for training or validation datasets. alternatively, the cross-validation approach may have been a more robust approach to evaluating accuracy on new data, rather than evaluating accuracy on a single draw of randomly selected data. in addition, while the evaluation of classification trees based on the cross-validation approach reported higher accuracy than the evaluation on a single static test dataset, it was still the worst performing modelfurther confirmation that this predictive method is not well-suited to the dataset. the cross-validation approach also allowed for accuracy comparisons among the three different methods via the paired t-test procedure. the results of this test further supported random forests as having different (i.e. higher) accuracy estimates than neural nets and classification and regression trees. based on different metrics, results from random forests have been shown to be more accurate than several other methods on surveillance datasets of roughly comparable size (kane et al., 2014; petukhova et al., 2018) . nonetheless, this should not be over-interpreted since the majority of the datasets used have been moderate in size. however, what is clear from this study is that random forest, as an ensemble of classification trees, had considerable higher accuracy on the test dataset than the method that was based on a single classification tree. in terms of model sensitivity and specificity, especially as they pertain to trends "high" and "zero", neural nets outperform all other models with a static 70-30 split of the data. however, with random training and test sets (10-fold cross-validation), it appears random forest outperforms all other models, the only exception being trends "zero" and "low" for sensitivity and specificity respectively, where neural nets had a higher median value. since neural networks require sufficient records to "learn" the patterns for a minority class (in this case trend "high"), it is perhaps not surprising that the neural nets model focused on majority class "zero" and "low", and as such provided better sensitivity and specificity values. overall, the pedv trend classification models have much higher specificity values than they do sensitivity, indicating that they're much better at identifying when a specific trend is not present, as opposed to when the trend is present. however, in this instance and for the purposes of surveillance for an emerging disease, the cost of a false negative (i.e. false "non-zero" trend being high), outweighs the cost of a false positive, and as such, sensitivity values are paramount. although the random forest classification model outperforms other models, its sensitivity value across all trends is mediocre, indicating that it may not be the best option for avoiding false negatives. it is also likely that the nature of the data puts the random forest method at a disadvantage. while random forest has shown promise for time series data related to endemic animal diseases (kane et al., 2014) , studies highlighting its use for time series data related to emerging animal diseases, especially non-zoonotic ones, are limited (xie et al., 2016) . unlike endemic diseases, for which there are frequent infection peaks and lows, emerging diseases typically have an initial sustained peak (usually at the start of the epidemic), after which the epidemic is typically brought under control, preventive measures initiated, and case counts become low or zero. in summary, further work is needed to develop suitable models for emerging infectious disease data, as the number of observations in the dataset are likely inadequate for the training needs of machine learning models (e.g., random forest, neural nets, and classification trees). with respect to variable importance as assessed by random forests, fig. 2 . variable importance plot for the random forest classification model with 30 co-variates (pedv long-term prediction), as applied to the ontario pedv and weather dataset for january 2014 -april 2017. the x-axis represents the decrease in predictive accuracy once this variable has been omitted from the random forest model, with longer bars representing a larger loss in accuracy, therefore indicating the variable is of higher importance in predicting trends in the number of new cases. variables are further colored based on whether they are related to environmental factors or level of pedv infection. among the top ten important variables, eight of them were related to existing frequency of cases and two were related to environmental conditions. this in itself is not surprising since the biggest risk factor for the number of new cases of a communicable disease is the prevalence of infection (krämer et al., 2010) . nonetheless, prevalence and other measures of disease frequency for newly emerging diseases are often changing rapidly, as was the case for this disease (ajayi et al., 2018) , and are frequently not known with reasonable certainty. therefore, building surveillance systems which are capable of providing up-to-date prevalence estimates for ped and similar production diseases in the source population would be valuable for making predictions about short-term disease frequency trends. this would require notification at the start of infection, as well as accurate data about declaration of freedom from infection for individual establishments. for pedv prediction (i.e. pedv trends), the highly-ranked prevalence, lagged prevalence and temperature variables (from the random forest model) could be a consequence of the nature of swine production in the source population, which to a large degree is organized through segregated phases of production at various locations. for example, an outbreak in a large sow herd will eventually lead to spread of infection from a sow herd to one or more nursery sites, and eventually finisher herds. this requires varying lengths of time, depending on the organization of pig flow in affected farms. alternatively, for swine herds not connected through pig flow to the existing cases, the length of time for pedv transmission between existing cases and naïve herds could vary. yet another possible contributor to the lags are reporting delays due to a variety of reasons (e.g. low clinical impact in growing pigs). it is worth mentioning that the random forest importance measures do not necessarily indicate that the underlying associations are positive (i.e. that an increase in values of a predictor variable leads to an increase in the values of one or more outcome classes). in addition, while the above-noted situations are possible reasons lagged measures of infection and prevalence are identified as important, the reader should be reminded that in the study population, the status of nursery and finisher herds were tracked together with the status of sow herds. in fact, the majority of sites in the study and in the source population of ped positive herds consisted of sites that were housing nursery and finisher pigs (a total of 76.2% of herds). therefore, the results may not be extrapolated directly to target populations consisting of sow herds only. however, this source population mirrors the target population of ontario commercial herds with respect to major demographic characteristics, and both are reflective of the type of segregated production which has been dominant in commercial swine production. among the environmental variables, mean low temperature and mean average temperature were identified among the top ten important variables. it is paramount to note that since pedv is more stable at low rather than high temperatures (thomas et al., 2015) , pedv biosecurity measures are more difficult to implement in cold weather. furthermore, it has been shown that transport vehicles play a critical role in pedv transmission (lowe et al., 2014) , and as such, transport biosecurity guidelines are readily available (national pork board, 2014). however, some guidelines are harder to implement in cold weather since certain disinfectants have reduced efficacy at low temperatures (bowman et al., 2015; eqsp, 2014) , and washed transport vehicles are less likely to dry completely as freezing is more likely. the impact of low temperatures on pedv transport biosecurity has led to the publication of cold weather disinfection guidelines (oshab, 2014), application of disinfectants which maintain their efficacy at low temperatures (ferry and benjamin, 2015) , and temporary funding for costs associated with enhanced biosecurity for transport vehicles (oscia, 2014) . the most noteworthy limitation of the current study is the small dataset, which made evaluation of predictive accuracy challenging. in addition, classifying the number of new ped cases over a 4-week period into four classes was probably too detailed. nonetheless, such a decision was made early in the modeling phase with the rationale that it was worthwhile exploring how the models would perform if such fragmented classification was required. in retrospect, classification into two groups would probably be more robust and permit the use of additional measures of classification accuracy. with such approaches, the use of model ensembles could have been explored in a straightforward manner. furthermore, our decision to reduce the number of variables through the use of the random forest variable importance plot may have impacted the results, as it pre-selected variables that were potentially important for this algorithm but perhaps not as important for the other two. nonetheless, comparison of the accuracy measures between the training and test datasets did not suggest overfitting, at least not for this specific method. in conclusion, this study investigated the use of predictive analytics for the prediction of pedv trends. the results show that the random forest classification model with 30 explanatory variables is the best model for forecasting future pedv trends for this target population. furthermore, variable importance measures from the random forest models confirm prevalence and temperature as important contributors to future pedv trends. herd-level prevalence and incidence of porcine epidemic diarrhea virus (pedv) and porcine deltacoronavirus (pdcov) in swine herds in ontario swine delta coronavirus (sdcv) [online]. available at effects of disinfection on the molecular detection of porcine epidemic diarrhea virus random forests terrestrial animal diseases porcine epidemic diarrhoea: new insights into an old disease effects of air temperature and relative humidity on coronavirus survival on surfaces useful information on disinfectants after contamination with novel swine enteric coronavirus diseases (secd) chest diseases diagnosis using artificial neural networks michigan state university extension, pork producers have another option for disinfecting against pedv neuralnet: training of neural networks. r package version 1 emergence of porcine epidemic diarrhoea in north america a data complexity analysis of comparative advantages of decision forest constructors modern multivariate statistical techniques: regression, classification, and manifold learning, 1st edn comparison of arima and random forest time series models for prediction of avian influenza h5n1 outbreaks porcine epidemic diarrhea in canada: an emerging disease case study chapter 5. principles of infectious disease epidemiology building predictive models in r using the caret package applied predictive modelling predicting shrimp disease occurrence: artificial neural networks vs. logistic regression classification and regression by random forest role of transportation in spread of porcine epidemic diarrhea virus infection, united states influenza virus transmission is dependent on relative humidity and temperature health status, infection and disease in california sea lions (zalophus californianus) studied using a canine microarray platform and machine-learning approaches national pork board livestock disease control and prevention -ontario-specific immediately notifiable diseases number of pigs, by county ped program deadline nears and pork industry tightens biosecurity cold weather trailer disinfection procedure assessment of autoregressive integrated moving average (arima), generalized linear autoregressive moving average (glarma), and random forest (rf) time series regression models for predicting influenza a virus frequency in swine in ontario porcine epidemic diarrhea (dvs) and porcine deltacoronavirus (dcvp) [online]. available at r: a language and environment for statistical computing data mining for business analytics: concepts, techniques, and applications in r rpart: recursive partitioning and regression trees. r package version 4 evaluation of time and temperature sufficient to inactivate porcine epidemic diarrhea virus in swine feces on metal surfaces advantages and disadvantages of using artificial neural networks versus logistic regression for predicting medical outcomes disease bioportal, bioportal | dashboard -trends in us pedv and pdcov diagnostic data swine enteric coronavirus disease (secd) weekly situation report projecting the global distribution of the emerging amphibian fungal pathogen, batrachochytrium dendrobatidis, based on ipcc climate futures zoo: s3 infrastructure for regular and irregular time series the authors are thankful to ontario swine health advisory board for sharing the data and to producers who contributed to the surveillance system. source of funding for this work was national science and engineering research council. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.prevetmed.2019.01. 005. key: cord-278641-8lh5y7j0 authors: chen, jianing; cui, yaru; wang, zemei; liu, guangliang title: identification and characterization of pedv infection in rat crypt epithelial cells date: 2020-09-17 journal: vet microbiol doi: 10.1016/j.vetmic.2020.108848 sha: doc_id: 278641 cord_uid: 8lh5y7j0 porcine epidemic diarrhea (ped) is a devastating enteric disease to the world's swine production. porcine epidemic diarrhea virus (pedv), as the ped causative agent, has been commonly propagated and investigated in vero cells, as well as in ipec-j2, a porcine epithelial cell-jejunum 2. however, vero cells, which are defective in interferon production, cannot represent the host response in enteric cells while pedv replicates poorly in ipec-j2 cells. in this study, we observed that rat crypt epithelial cells (iec-6) were highly susceptible to different subtypes of pedv. the replication kinetics of pedv in iec-6 cells is similar to that in vero cells, but it is much higher than in ipec-j2 cells. besides that, pedv infection in iec-6 cells can induce the production of inflammatory cytokines and interferon, especially the type iii ifns. collectively, our findings suggest that iec-6 is an ideal cell line for pedv replication and immune response studies. the swine enteric coronaviruses, a family of the most significant problematic pathogens to the world's swine industry, include porcine epidemic diarrhea virus (pedv), porcine transmissible gastroenteritis virus (tgev), porcine deltacoronavirus (pdcov), and swine acute diarrhea syndrome coronavirus (sads-cov) (qiuhong et al., 2019) . among these four viruses, pedv has been recognized as the most devastating pathogen, causing up to 100% mortality in piglets younger than one week old. since its first discovery in the 1970s, pedv has then subsequently identified nearly worldwide (wood, 1977) . later in the 2010s, variant pedv strains were emerged and killed hundreds of millions of suckling piglets (jung and saif, 2015; wang et al., 2016) . pedv is an enveloped, single-stranded, positive-sense rna virus, which targets the small intestine, especially the ileum (lee, 2015) . pedv infection in enterocytes can exhibit acute necrosis, leading to marked villous atrophy in the small intestine (ducatelle et al., 1982) . consequently, malabsorption, caused by the massive loss of absorptive enterocytes, contributes to diarrhea and even death of newborn piglets. the cell line, which is permissive for pedv replication and possesses the intestine function and responses, may facilitate the studies about pedv. vero cells are commonly used for pedv propagation. however, this cell line is characterized by lacking the production of interferon (desmyter et al., 1968) . consequently, it is valuable in vaccine production but defective in immune studies. ipec-j2 is a non-transformed porcine columnar epithelial cell line and used in the investigations of pedv lin et al., 2017) . however, it is semi-permissive to pedv infection and defective in the production of several cytokines (brosnahan and j o u r n a l p r e -p r o o f brown, 2012) . these drawbacks of the two cell lines restrict further understandings of pedv. therefore, it is of great importance to screen proper cell lines for pedv investigations. in this study, we identified rat crypt epithelial cells (iec-6) was highly susceptible to a different subtype of pedv, which could be an ideal cell model for pedv studies. the rat small intestine epithelium iec-6 cells were purchased from american type culture collection (atcc) and cultured in dulbecco's modified eagle's medium (dmem) (sigma, germany) supplemented with 0.1 unit/ml bovine insulin and 10% fetal bovine serum (gemini, usa). african green monkey epithelial vero cells were cultured in dmem medium supplemented with 10% fetal bovine serum. the intestinal porcine epithelial cell line j2 (ipec-j2) cells were cultured in dmem/f12 (sigma, germany) supplemented with 10% fetal bovine serum, 5 μg/ml insulin/selenium/transferrin (life, usa) and 5 ng/ml epidermal growth factors (egf) (life, usa). the pedv ljx01/gs/2014 strain was isolated and conserved in our laboratory. the pedv cv777 strain were isolated from attenuated vaccine and maintained in our laboratory. iec-6 cells were seeded in 48-well plates. when grown to 80% confluency, cells were infected with pedv at a moi=0.01. after 48 hours, the cells were fixed with 4% j o u r n a l p r e -p r o o f paraformaldehyde for 30 min and then permeabilized with 0.5% triton x-100 for 15 min at room temperature. after blocking with 5% skim milk for 1 h, cells were incubated with mouse anti-pedv-nucleocapsid monoclonal antibody (1:5000 dilution) for 1 h, followed by incubating with the alexa fluor 488 goat anti-mouse igg antibody (1:1000 dilution) (thermo, usa) for 1 h. the cell nuclei were stained by dapi (beyotime, china). the cells were then examined under a fluorescence microscope (te2000u; nikon) with a video documentation system. to assess the host response against pedv, the three cell lines were infected with pedv at a moi=0.01. the supernatant was collected at 12, 24, 36, 48, 60 and 72 hours postinfection and used for viral titration. at 24 hours post-infection, the cells were also lysed with trizol (takara, japan) for rna extraction. confluent monolayers of iec-6 cells in 6-well plates were inoculated with pedv and incubated for 1 h at room temperature on rocker. after three washes, the cells were overlaid with 0.75% low melting point agarose (sigma, germany) in dmem containing 5% fbs and then incubated at 37℃ for around 72 h. to visualize plaques, cells were stained with 1% crystal violet in ethanol. vero cells were seeded into 96-well plates and cultured for 80-90% confluence. the cell monolayers were washed three times with pbs. serial 10-fold dilutions of the collected j o u r n a l p r e -p r o o f supernatant were made, and 100 l of each dilution were inoculated to five wells of the 96well plate. the plates were incubated at 37 • c and supplied with 5% co2. and then, the cultures were checked under a light microscope for cytopathology every day for 3 days. the dilution of the supernatant was titrated by tcid50 assay and calculated based on the reed-muench method. for the detection of host gene expression level, the real-time qpcr was performed with unique aptamer qpcr sybr green master mix (novogen, china) in the bio-rad cfx96 system. the reactions were incubated at 94°c for 30 s, followed by 45 cycles at 94°c for 5 s and 60°c for 30 s. all reactions were run in triplicate using the primer sets listed in table 1 . the 2 -∆∆ct method was employed to measure the expression level of target genes. the student's t-test was used for the examination of statistical significance between matched groups. an unadjusted p value of less than 0.05 was considered significant; a p value of less than 0.01 was considered highly significant. iec-6 cell line, originally derived from the rat small intestinal crypt, was commonly used in the nutritional investigations (quaroni et al., 1979) . to investigate if it is susceptible to pedv, the iec-6 cells were infected with two different subtypes of pedv at a moi=0.01 and observed for cytopathic effect (cpe) under a microscope. the pedv j o u r n a l p r e -p r o o f vaccine strain cv777 belongs to subtype g1, while the field strain ljx isolated by our group belongs to subtype g2 (guo et al., 2018) . as shown in fig. 1a , typical cpe became visible at 24 hours post-infection. the morphology of pedv-infected cells became enlarged fusiform at early stage. lately, they were wrinkled and detached. however, the mock-infected cells remained normal during the whole process. ifa was then performed to confirm its infection by using a mab against pedv nucleocapsid protein (yang et al., 2019) . the results showed that strong pedv-n positive signals were observed in iec-6 cells infected with either ljx strain or cv777 strain (fig. 1b) , indicating iec-6 cells were successfully infected by pedv. then the total rna was extracted and analyzed by rt-pcr. the results demonstrated that the pedv-specific bands were amplified from virusinoculated cells (fig. 1c) . finally, the western blotting analysis showed that the pedv nucleocapsid protein was detected in cells infected with pedv (fig. 1d) . we also attempted to identify whether the iec-6 were susceptible to tgev but no positive signal were obtained (data not shown). taken together, the above data illustrated that iec-6 cells were susceptible to pedv but not tgev. vero and ipec-j2 cell lines were commonly used for pedv investigation. to test and compare the replicative capacity of pedv in different cell lines, the pedv replication kinetics were measured in these three cell lines. these cells were seeded in the 24-well plate and inoculated with pedv ljx strain or cv777 strain at a moi=0.01. the supernatant was collected every 12 hours and used for viral titration. the results showed that the pedv ljx strain replicated poorly in ipec-j2 cells with viral titers lower than 10 4 j o u r n a l p r e -p r o o f tcid50/ml. pedv ljx strain exhibited a robust and similar growth curve in vero and iec-6 cells, showing higher than 10 7 tcid50/ml viral titers. pedv cv777 strain reached its most elevated viral titers in both vero and iec-6 cells but poorly replicated in ipec-j2 cells (fig. 1e) . the plaque assay is usually employed for virus purification and titration. it is of great importance to virus related studies. we also tested whether iec-6 could be used to conduct plaque assay. the data showed that the infection of both ljx and cv777 strains resulted to the formation of plaque (fig. 1f) . these results further showed that iec-6 cells supported pedv infection and replication. pedv infection in the porcine small intestine is characterized by inflammatory cytokines and interferon (jung and saif, 2015) . the type iii interferon is considered to play a critical role in enteric immunity. therefore, we examined whether pedv infection could induce the typical enteric immunity in iec-6 cells compared to its infection in ipec-j2 and vero cells. pedv ljx was a field strain and was used to infect the three cell lines. after 24 hours post-infection, the rna was extracted for rt-qpcr analysis. the results showed that both type i and type iii interferon responses were significantly activated in pedv infected iec-6 and ipec-j2 cells while the type i interferon were absent in vero cells ( fig. 2a, 2b, 2d, 2e ). due to the lack of ifn-λ3 gene of green monkey in the ncbi or ensembl database, we failed to detect its gene expression level. although the ifn-λ3 gene of green monkey was strongly upregulated in pedv infected vero cells, its ct value was about 30, while the ct value to gapdh was about 18 in pedv infected cells. in mock j o u r n a l p r e -p r o o f cells, the ct values of about 39 for ifn-λ3 and 18 for gapdh. for type ii interferon, pedv infection led to a significant upregulation in iec-6 cells but not in ipec-j2 and vero cells (fig. 2c) . pedv infection also enhanced the expression of il-1, il-6, il-8, and tnfα in iec-6 cells except for il-17a (fig. 2f, 2g, 2h, 2i, 2j ). all these results demonstrated that iec-6 cells were able to generate a robust immune response against pedv infection. although vero and ipec-j2 were commonly used in pedv studies, they were defective in immune response and viral replication. there were also other cell lines identified permissive for pedv infection recently. pedv can also infect hek293t, ip2-2i cells, and primary bovine mesenchymal cells (jung et al., 2020; wang et al., 2019; zhang et al., 2017) . however, hek293t is not an intestinal epithelium cell line, while ipi-2i is semi-permissive to pedv infection. the primary bovine mesenchymal cells cannot culture for a long time. the rat small intestinal crypt epithelium cell line iec-6 is non-transformed. and it was often used as a necrotizing enterocolitis model system and nutritional studies (braga-neto et al., 2012; ruthig and meckling-gill, 1999; yan et al., 2018) . the uptake, metabolism, and transport processes are observed in iec-6 cells (said et al., 1997; sanderson and he, 1994) . therefore, iec-6 is recognized as an ideal model for intestine relating studies. however, it is rarely used in viral studies. only modified vaccinia virus ankara was reported to multiply in iec-6 cells (okeke et al., 2006) . in this study, we tested the susceptibility of iec-6 cells to pedv infection and found it was highly susceptible. the growth curve of pedv in iec-6 was similar to it in vero cells with the viral titers higher than 10 7 tcid50/ml. it was also found that iec-6 could mimic the antiviral immune response in the small intestine. pedv infection in the small intestine is characterized by the induction of proinflammatory responses (huan et al., 2017) . in our study, il-1b, il-6, il-8, and tnfα were upregulated by pedv infection in iec-6 cells. only tnfα, il-17a and il-8 were found increasingly expressed in ipec-j2 cells. il-6 was only activated by pedv infection in iec-6 cells. besides that, interferon is recognized to play critical roles in the innate immune response. the type iii interferon is significantly essential in maintaining intestinal homeostasis, especially to pedv (ingle et al., 2018; zhang et al., 2018) . in pedv infected iec-6 cells, the expression of type i and type iii interferon was drastically upregulated, while type ii interferon was also increasingly expressed. vero cells are defective in interferon production. the ct values of all genes were higher than 29 while it was about 20 for gapdh. although ifnλ1 was highly upregulated in vero cells, its ct value was also higher than 29. in ipec-j2 cells, the production of ifnα, ifnλ1, and ifn-λ3 was increased but not as high as in iec-6 cells. all these data suggested that pedv infection in iec-6 cells could induce strong antiviral responses. in summary, our studies identified the rat small intestinal crypt epithelium iec-6 cells were highly susceptible to pedv. pedv could efficiently replicate in iec-6 cells as it does in vero cells. besides that, pedv infection activated potent inflammatory cytokines and j o u r n a l p r e -p r o o f interferon, primarily type iii interferon in iec-6 cells, which highly simulated its infection in the small intestine. all these data above suggested that iec-6 cells can be used as an excellent cell model for pedv studies. protective effects of alanyl-glutamine supplementation against nelfinavir-induced epithelial impairment in iec-6 cells and in mouse intestinal mucosa porcine ipec-j2 intestinal epithelial cells in microbiological investigations porcine endemic diarrhea virus infection regulates long noncoding rna expression defectiveness of interferon production and of rubella virus interference in a line of african green monkey kidney cells (vero) evolutionary and genotypic analyses of global porcine epidemic diarrhea virus strains glycyrrhizin inhibits porcine epidemic diarrhea virus infection and attenuates the proinflammatory responses by inhibition of high mobility group box-1 protein distinct effects of type i and iii interferons on enteric viruses porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis replicative capacity of porcine deltacoronavirus and porcine epidemic diarrhea virus in primary bovine mesenchymal cells porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus differential protein analysis of ipec-j2 cells infected with porcine epidemic diarrhea virus pandemic and classical strains elucidates the pathogenesis of infection modified vaccinia virus ankara multiplies in rat iec-6 cells and limited production of mature virions occurs in other mammalian cell lines emerging and re-emerging coronaviruses in pigs. current opinion in virology epithelioid cell cultures from rat small intestine. characterization by morphologic and immunologic criteria both (n-3) and (n-6) fatty acids stimulate wound healing in the rat intestinal epithelial cell line, iec-6 intracellular regulation of intestinal folate uptake: studies with cultured iec-6 epithelial cells nucleotide uptake and metabolism by intestinal epithelial cells porcine epidemic diarrhea in china susceptibility of porcine ipi-2i intestinal epithelial cells to infection with swine enteric coronaviruses an apparently new syndrome of porcine epidemic diarrhoea. the veterinary record fish oil-derived lipid emulsion induces rip1-dependent and caspase 8-licensed necroptosis in iec-6 cells through overproduction of reactive oxygen species generation, identification, and functional analysis of monoclonal antibodies against porcine epidemic diarrhea virus nucleocapsid characterization of porcine epidemic diarrhea virus infectivity in human embryonic kidney cells type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp1 in irf1 signaling. 92 key: cord-299988-jaekryq5 authors: karte, claudia; platje, nadine; bullermann, johannes; beer, martin; höper, dirk; blome, sandra title: re-emergence of porcine epidemic diarrhea virus in a piglet-producing farm in northwestern germany in 2019 date: 2020-09-10 journal: bmc vet res doi: 10.1186/s12917-020-02548-4 sha: doc_id: 299988 cord_uid: jaekryq5 background: porcine epidemic diarrhea (ped) is a viral enteric disease of pigs. it affects all age classes of animals but lethality is mainly seen in suckling piglets. after its first appearance in england in 1971, porcine epidemic diarrhea virus (pedv) has spread worldwide. while sporadic outbreaks prevailed in europe, the disease had high impact in asia. following particularly severe outbreaks in 2011, high impact cases were also reported in the united states and neighboring countries in 2013. subsequently, outbreaks were also reported in several european countries including germany. these outbreaks were less severe. this case report describes a recent case of ped re-emergence in germany and the sequence analyses of the causative pedv. case presentation: in spring 2019 5 years after re-introduction of ped into central europe, a piglet-producer in northwestern germany experienced an outbreak that affected sows, their suckling piglets, and weaners. after initial confirmation of pedv by real-time rt-pcr, fecal material and small intestine samples from affected pigs were subjected to metagenomic analyses employing next-generation sequencing. phylogenetic analyses showed high identities among the pedv sequences obtained from samples of different animals and a close relation to recent strains from hungary and france. compared to the pedv strains analyzed in 2014, genetic drift could be confirmed. changes were mainly observed in the spike protein encoding s gene segment. in addition, metagenomic analyses showed multiple picobirnavirus reads in all investigated samples. conclusion: this case report shows that pedv is still circulating in europe. the causative strains are moderately virulent and are still closely related to the so-called indel strains reported previously in europe, including germany. however, a genetic drift has taken place that can be seen in a novel cluster comprising strains from germany, hungary and france in 2019. relevance and impact of the detected picobirna sequences need further investigations. porcine epidemic diarrhea (ped) is an acute and highly contagious enteric disease of swine that results in severe enteritis, diarrhea, vomiting, and dehydration. especially in suckling pigs, lethality can be very high [1] [2] [3] . the causative agent, porcine epidemic diarrhea virus (pedv), is an enveloped positive single-stranded rna virus that belongs to the family coronaviridae, genus alphacoronavirus [4] . the complex coronavirus particles are pleomorphic and possess club-shaped surface projectors [5] . the length of the genome ranges from 27 to 31 kilobases [6] . coronaviruses have a low tenacity [7] but are shed in high amounts and are thus easily transmitted by the fecal-oral route [8] . after its first recognition in the 1970s in europe [7, 9] , the disease caused considerable economic losses especially in asia, where the disease remains endemic [10, 11] . in europe, the disease disappeared quickly, and from most countries, only very sporadic cases were reported over the last three decades. after reports from asia, that a new pedv variant caused considerable losses [12, 13] , that highly virulent pedv variant emerged also in the united states (us) in 2013, with swine farms experiencing explosive epidemics affecting all age classes of animals, with up to 95% mortality in suckling pigs [2, 14] . in 2014, several cases of ped were also reported from southern and western germany. in most cases, fattening pigs were affected showing high morbidity with almost non-existent mortality [15] . however, some breeding herds reported high mortality rates with up to 85% losses in suckling piglets [1] . similar outbreaks were observed in several other central european countries including france, the netherlands, italy, slovenia, belgium, romania, portugal, spain, and austria [16] [17] [18] [19] [20] [21] [22] [23] . the characterization of the involved virus strains revealed that so-called s-indel variants of the virus were involved in central europe, which, in contrast to the highly virulent non-indel strains from asia and the usa, are characterized by deletions and insertions in the spike protein encoding s gene [23] . in the majority of cases, the s-indel variants are associated with milder ped courses. in the absence of reporting obligations, and following the confirmation that the pedv strains in the eu did not belong to the highly virulent non-inde l type, notification and broader follow-up of cases decreased. however, sporadic outbreaks, sometimes with severe problems to get rid of the disease, were still reported from all production systems from different regions of germany and other countries (personal communications and unpublished data). from this time, pedv sequence information is largely missing. when a new wave of ped struck a piglet producer in northwestern germany in 2019, questions were raised to what extent the virus might have changed and if a new emerging variant was causing the clinical case in sows, piglets and weaners. here, we report on the clinical presentation and the whole-genome sequencing of the causative 2019 pedv strain. the affected piglet producer is located in northwestern germany. the farm keeps approximately 350 sows in seven groups (50 sows each), and has a total of 2200 piglet rearing places (1000 on site and 1200 in a leased farm). on the premise, sows and piglets are kept in the same building complex. the farm also includes a fattening unit with 1500 fattening slots. this unit is in close proximity to the above-mentioned units but has its own building with a hygiene lock. three other pig farms are in the radius of 500 m around the holding. prior to the disease event, the farm recorded 33 weaned piglets per sow and year with suckling losses below 10%. loss in piglet rearing and fattening was < 2%. the animals were routinely screened for enteric pathogens and only rotavirus types a and c were detected every now and again (rotavirus type c only very sporadically). the farm was unsuspicious for dysentery and was tested negative for tgev and pdcov prior, during and after the disease event. the routinely applied immunization scheme included maternal vaccinations against colibacillosis, oedema disease, and necrotic enteritis. depending on the infection pressure, rotavirus a vaccines were used in gilts. piglets received vaccinations against porcine circovirus type 2 (pcv-2), mycoplasma, and shiga toxin producing e. coli. sows in integration and reproduction received additional vaccination e.g. against porcine respiratory and reproductive syndrome virus, influenza virus, and parvovirus. in spring 2019, massive diarrhea occurred in sows and suckling pigs. at first, nursing sows (60% of the sows in the unit) in the farrowing unit showed inappetence and shortly afterwards mushy diarrhoea. fever or increased temperature were not detected. the sows recovered completely after three to 4 days. the suckling piglets, which were about 14 days old, showed the first signs of diarrhea two to 3 days after the mothers. about 70% of the litters of this first affected farrowing group showed diarrhea and losses rose to 10% (see table 1 ). immediate investigations confirmed pedv (rt-qpcr from fecal samples and organs). sick piglets were treated with commercial electrolyte solution and additional water supply was given to sows. to increase maternal immunity, infection was enforced in the waiting area. weaned piglets with secondary infections received antibiotic treatment. in total, three farrowing groups (sows and suckling pigs) showed clinical signs of ped and increased loss rates in suckling pigs (10 to 30%). morbidity reached 60 to 90% in sows and 70 to 100% in piglets (see table 1 ). some of the weaned piglets also showed diarrhea, wasting, and growth retardation. the overall losses in piglet rearing rose to 5 to 10% (details see table 1 ). in the fourth farrowing group (approx. eight weeks after the first clinical signs) no clinical signs indicative for ped were recorded and up to now no further ped suspicions arose. in autumn, five suckling piglets were randomly selected and subjected to necropsy and ped screening. all samples were negative for pedv. in the connected fattening unit, no ped signs were recorded at any time. serological checks in the sow rearing unit (separate building) gave negative results. during the disease event, intensive cleaning and disinfection was carried out in the farrowing unit, on driveways, in the waiting areas, and all related stables. disinfectants were chosen in accordance with the list recommended by the germany veterinary society (dvg) for enveloped viruses. purchase of gilts was stopped and replaced by self-remounting. follow-up investigations showed that neighboring farms were also affected by ped shortly before the onset in the described farm. upon initial confirmation of ped by a private laboratory, fecal samples from five sows and feces and intestines from two affected piglets were sent to the friedrich-loeffler-institut (fli) for further analyses and nextgeneration sequencing. ribonucleic acids were extracted from fecal samples or supernatants from homogenized intestines using trizol reagent (lifetechnologies, darmstadt, germany) in combination with the rneasy mini kit (qiagen, hilden, germany) and dnase digestion on the spin column. all rnas were confirmed to be pedv positive by rt-qpcr [24, 25] . subsequently, all samples were subjected to whole genome sequencing and metagenomic analyses using the illumina miseq platform as previously described [26] . in brief, nucleic acids were processed into shotgun dna libraries and then deep-sequenced. the resulting raw data was taxonomically classified using the software pipeline riems [27] . the obtained sequence reads were assembled to determine the genomes in full length. all sequences are available from the insdc databases under study accession prjeb38314. with the help of the geneious prime software suite (v. 2019.2.3; biomatters ltd., auckland, new zealand), phylogenetic analyses were performed (for details see legend fig. 1 ). the genomes originating from the reported german case form a new and distinct cluster within the s-indel strains (see fig. 1 ). close relatives are three virus strains reported in 2019, two from hungary [16] (accessions mh593900 and kx289955) and one from france (accession mn056942). identity among the new german pedv strains was almost 100% (> 99.9%) whereas identities of > 98.8% were found with regard to the hungarian and french sequences. comparing the german strains from 2014 with the german strains detected in 2019, identities are higher than 99.5%. comparisons between german prototype strains from 2014 (the first reported strain, bh76/14-01_l00719_ farm a) and 2019 (894_3_l03204_ger) show high similarities in the nucleotide sequence (see supplementary figure 1 ). in total, 135 nucleotides exchanges are the rna-shotgun sequencing approach allowed metagenomic analyses using riems. in this analysis, several reads were classified taxonomically as picobirnaviridae sequences. multiple reads of the rna-dependent rnapolymerase gene as well as the gene segment encoding the capsid were found in the fecal but not the intestine samples. porcine epidemic diarrhea can have a tremendous impact on the pig industry as was seen in the us following the introduction of pedv in 2013 [2, 29] . critical losses occurred especially in piglet rearing companies and the losses impacted the whole pork industry [15, 29] . following the devastating outbreaks on the american continent, re-emergence of pedv was also reported from europe after intensified surveillance [23] . however, here, strains of lower virulence were circulating and the reporting and follow-up of cases abated quickly despite ongoing cases. one reason for the subsiding of official follow-up is that ped is neither notifiable nor reportable but still has impact on trade and reputation. against this background, most farmers had no interest to make their cases public. thus, official and published information on the german ped situation in general and viral evolution in particular is missing roughly from 2016 onwards. when pedv was introduced in a piglet-producing farm in northwestern germany in 2019, clinical disease and losses were rather disturbing and the farmer and responsible veterinarian initiated a closer follow-up. one hypothesis for the observed impact was a change in virulence and thus, next-generation sequencing was employed to test this hypothesis. our data show that the causative virus strains are still s-indel variants with close relationship to those found in 2014 and the following years. however, viral evolution has taken place and the drift gave rise to a new cluster that comprises recent fig. 1 phylogenetic tree of current pedv strains. phylogenetic tree of 2019 pedv strains from germany, hungary, and france as well as 2014 pedv strains from germany, usa, and china. the complete genome sequences were aligned using mafft and a phylogenetic analysis was performed using phyml, with a gtr substitution model and tree reconstruction supported by 1000 bootstrapping replicas [28, 29] . green branches show the 2019 pedv isolates from germany, blue branches highlight the isolates from hungary and france and red branches are the highly virulent non-indel strains from the usa and china strains from germany, hungary, and france. given the accordant drift, one can speculate that pedv is still circulating in europe. there is no indication that these variants have a higher virulence per se. the previously observed variation seems still present. the affected farm described in this report was finally able to control the outbreak by forced infection in the waiting unit of sows, biosecurity and strict cleaning and disinfection. yet, the history of ped in neighboring fattening farms also shows that the virus was able to enter the farm and room for improvement was given in veterinary hygiene and biosafety. the exact route of introduction remained unclear. supplementary studies into the metagenomic data set showed picobirnaviral sequences in the fecal material. picobirnaviruses are non-enveloped double-stranded rna viruses. they are bisegmented with segment one consisting of 2.3 to 2.6 kilobases and segment two of 1.5 to 1.9 kilobases [30] . picobirnaviruses are often associated with cases of gastroenteritis or infections in the respiratory tract [31] . the role in diarrhea diseases in piglets is unclear, since picobirnaviruses were found in piglets with and without diarrhea [32] . the transmission is fecal-oral [33] and these viruses have so far been detected mainly in feces in various species [30, 33] . impact and relevance of these findings remains to be clarified by future studies. in conclusion, ped re-emerged in northwestern germany in 2019 leading to high morbidity and substantial impact in a piglet-producing farm. the causative virus strains are still s-indel variants but a genetic drift occurred since 2014. this drift is accordant with the evolution in other european countries. the relevance of picobirnavirus detections in fecal samples from pedvpositive animals remains unclear. supplementary information accompanies this paper at https://doi.org/10. 1186/s12917-020-02548-4. emergence of porcine epidemic diarrhea virus in southern germany emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences genetic properties of endemic chinese porcine epidemic diarrhea virus strains isolated since 2010 the springer index of viruses a new coronavirus-like partiele assoeiated with diarrhea in swine diagnostic notes: update on porcine epidemic diarrhea verlaufsuntersuchung über die ausscheidung von porcine epidemic diarrhea virus (pedv) und die serokonversion nach feldinfektion bei saugferkeln und mastschweinen porcine epidemic diarrhoea (ped) -neuausbrüche in deutschen mastschweinebeständen chinese-like strain of porcine epidemic diarrhea virus pig farming. letter to the editor new variant of porcine epidemic diarrhea virus the prevalence of intestinal trichomonads in chinese pigs isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states comparison of porcine epidemic diarrhea viruses from germany and the united states isolation and characterisation of porcine epidemic diarrhoea virus in hungary -short communication complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france in complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium porcine epidemic diarrhea virus (pedv) introduction into a naive dutch pig population in 2014 outbreak of porcine epidemic diarrhea virus in portugal first detection, clinical presentation and phylogenetic characterization of porcine epidemic diarrhea virus in austria porcine epidemic diarrhoea virus in italy: disease spread and the role of transportation porcine epidemic diarrhea in europe: in-detail analyses of disease dynamics and molecular epidemiology genomnachweis des porzinen epidemischen diarrhoe virus (pedv) mittels real-time rt-pcr. avid-methodensammlung: avid-methode vir03 evidence of infectivity of airborne porcine epidemic diarrhea virus and detection of airborne viral rna at long distances from infected herds a versatile sample processing workflow for metagenomic pathogen detection riems: a software pipeline for sensitive and comprehensive taxonomic classification of reads from metagenomics datasets mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform factors associated with time to elimination of porcine epidemic diarrhea virus in individual ontario swine herds based on surveillance data molecular detection and characterization of picobirnaviruses in piglets with diarrhea in thailand detection and molecular characterization of porcine picobirnavirus in feces of domestic pigs from kolkata, india publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors thank patrick zitzow, robin brandt and ulrike kleinert for technical assistance. authors' contributions ck performed next-generation sequencing and was a major contributor in writing the manuscript, np and jb collected and analyzed clinical data, mb interpreted the overall dataset and critically revised the manuscript, sb conceived the study, assisted in interpreting the data and contributed in writing the manuscript, dh analyzed and interpreted whole-genome data and contributed to the manuscript. all authors read and approved the final manuscript. this study was funded by federal excellence initiative of mecklenburg western pomerania and european social fund (esf) grant koinfekt (esf_14-bm-a55-00xx_16). the excellence initiative covered consumables for next-generation sequencing and the personnel costs of the major contributor (ck). open access funding provided by projekt deal.availability of data and materials sequence information was deposited at the european nucleotide archive (ena) under study id prjeb38314 (accessions lr812928; lr812932; lr812927; lr812929, lr812930, lr812926, and lr812931). additional metadata are available from the authors upon reasonable request. the sample material was submitted to the friedrich-loeffler-institut for enhanced diagnostics and was taken by the responsible farm veterinarian in the context of the health monitoring program of the respective farm (in accordance with the regulation on hygiene requirements for the keeping of pigs; online available at http://www.gesetze-im-internet.de/schhalthygv/). in germany, every keeper of pigs must have his herd supervised by a veterinarian as part of the on-farm inspections, this includes clinical and laboratory checks to maintain and improve the health status of the herd. no permissions were necessary to collect the mainly non-invasive specimens. the farmer approved in-detail analyses of the ped case and shipment of samples (verbal agreement with the responsible veterinarian). all procedures were carried out in accordance with the relevant regulations. the owner of the case farm approved submission of the report. all authors have approved submission of the manuscript. the authors declare that they have no competing interests. key: cord-296791-h8ftslps authors: junwei, ge; baoxian, li; lijie, tang; yijing, li title: cloning and sequence analysis of the n gene of porcine epidemic diarrhea virus ljb/03 date: 2006-10-01 journal: virus genes doi: 10.1007/s11262-005-0059-z sha: doc_id: 296791 cord_uid: h8ftslps the nucleocapsid (n) gene of the porcine epidemic diarrhea virus (pedv) strain ljb/03 which was previously isolated in heilongjiang province, china, was cloned, sequenced and compared with published sequences of other avian and mammalian coronavirus. the nucleotide sequence encoding the entire n gene open reading frame (orf) of ljb/03 was 1326 bases long and encoded a protein of 441 amino acids with predicted mr of 49 kda. it consisted of 405 adenines (30.5%), 294 cytosines (22.1%), 329 guanines (24.8%) and 298 thymines (22.5%) residues. sequence comparison with other pedv strains selected from genbank revealed that the ljb/03 n gene has a high sequence homology to those of other pedv isolates, 97.4% with js2004, 95.6% with chinju99, 96.6% with br1/87, and 96.8% with cv777. the encoded protein shared 96.4% amino acid identities compared with cv777, 96.1% with brl/87, 98% with js2004, 96.90% with chinju99, respectively. the amino acid sequence contained seven potential protein kinase c phosphorylation sites, nine casein kinase ii phosphorylation sites, one tyrosine kinase phosphorylation site, two campand cgmp-dependent protein kinase phosphorylation sites. porcine epidemic diarrhea virus (pedv) is classified as a member of the coronaviridae and causes acute enteritis in pigs [1] , which was first reported in england in 1971 and has been reported in many countries such as germany, canada, japanese, korea, france, belgium, switzerland, etc. [2] [3] [4] [5] . the disease was first reported in china in 1976 [6] , caused serious economic losses due to the death of neonatal piglets and weight loss of the infected pigs. clinical signs of ped include anorexia, vomiting, diarrhea, and dehydration. morbidity and mortality in infected neonatal piglets less than 5 days old approach 100% because of severe diarrhea and dehydration. however, mortality in infected piglets older than 10 days is less than 10% [1] . the coronavirus genome consists of a positive-sense, single-stranded rna molecule that is 20-30 kb in size [7] . virions are enveloped, pleomorphic, and 80-220 nm in diameter, and they have club-shaped peplomers approximately 20 nm in length. coronavirus possesses four major structural proteins including a phosphorylated nucleocapsid (n) protein and three envelope proteins, membrane protein (m), spike protein (s), and envelope protein (e); the first two envelope proteins are major envelope proteins, while the amount of e protein in virion is low [8] [9] [10] , s glycoprotein makes up the large surface projections of the virion and the m and e proteins are essential for viral envelope formation and release [11, 12] . studies indicate that the n proteins of coronaviruses are extensively phosphorylated, highly basic, and binds, to the viral genomic rna forming a helical ribonucleoprotein (rnp) [13] . a variety of functional activities have been ascribed to the n proteins of previously known coronaviruses, including participation in transcription of the viral genome, the formation of viral core, and packaging viral rna [14] . the n protein is highly immunogenic, more then, the cellular immune response against n protein of some animal coronaviruses can enhance the recovery from the virus infection [15, 16] . n protein can accumulates intra-cellularly even before it is packed in the mature virus [17] and is the most abundant virus derived-protein throughout the infection, probably because its template mrna is the most abundant subgenomic rna [18] . these features make it a suitable candidate for the accurate and early diagnosis and develop genetically engineered vaccines [19] . the aim of present study was to determine the complement nucleotide sequence of the pedv n gene and get more information about pedv isolates comes from different region. in this study, the rna of pedv was extracted directly from the feces samples of piglets naturally infected with pedv ljb/03. the n gene has been cloned, sequenced and compared with other pedv strains. these data are useful for further the study of molecular biology of pedv strains that are prevalent in china. virus strain pedv ljb/03 was collected from the feces of piglets suffering from severe diarrhea in heilongjiang, china. the feces sample was operated following the methods of fan jinghui and li yijing [20] . the feces sample was diluted 1-10 in a disruption buffer (500 mm tris-hci [ph 8.3], 2% (w/v) pvp-40, 1% (w/v) peg6000, 140 mm nacl, 0.05% (v/v) tween 20), vortexed, incubated at room temperature for 10 min, and centrifuged using a beckman f3602 rotor at 2000 · g at 4°c for 5 min. the supernatant was removed and used for the extraction of the viral rna using the trizol reagent (invitrogen usa) according to the manufacturer's protocol and dissolved in diethyl procarbonatetreated distilled water. a pair of sense and antisense primer was designed and aligned based on nucleotide sequences of the n gene of cv777 and brl/87 available in genbank. the sense primer 5¢-ttatggcttctgtcagcttt-3¢ and antisense primer 5¢-acattgtttaatttcctgtatc-3¢ were used to amplify the n gene coding for the n protein of pedv strain ljb/03. synthesis of the first-strand cdna for n gene was carried out by reverse transcription using promega reverse transcription reagent. the viral rna (50 ll) was mixed with 2.5 ll of 10 pm of the antisense primer, incubated at 65°c for 5 min, and then placed on ice for 2 min. after that, 4 ll of 5· rt buffer, 4 ll of 2.5 mm dntp mixture, 1 ll of rnase inhibitor (40 u/ll), 1 ll of reverse transcriptase (200 u/ll), 2.5 ll h 2 o was added and mixed gently. the reaction mixture was incubated for 50 min at 42°c, and was terminated by heating for 10 min at 65°c. rnase h (1 ll) was added to degrade rna template for 20 min at 37°c prior to pcr amplification. pcr was carried out in a 50 ll volume by mixing the cdna above with 2.5 ll of each 10 pm sense and antisense, 1 mm each of datp, dgtp, dttp, dctp, 5 ll of 10· pcr buffer (100 mm tris-hcl, 1.5 mm mgc1 2 , 50 mm kc1, ph 8.3), and 2.5 u taq dna polymerase (takara biotechnology (dalian) co. ltd.). cycles were as follows: 94°c for 15 s, followed by 30 cycles of 94°c for 40 s, 49°c annealing for 30 s, 72°c extension for 1 min and a final extension of 72°c for 5 min. the pcr product was analyzed by electrophoresis through an agarose gel (fig. 1) , and visualized by staining with ethidium bromide, the target cdna band was extracted from the gel using the qiagen ò gel extraction kit according to the manufacturer's instructions. the purified pcr products were cloned into the pgem-t easy vector (promega, madison, usa) with t4 dna ligase. the plasmids were transformed into e. coli dh5a using standard molecular technique. plasmid dna was extracted by alkaline-lysis from e. coli dh5a culture and verified by using restriction enzyme digestion, pcr and electrophoresis in 1% agarose (fig. 2) . colonies with correct sizes was named pgem-t-n and at least three independent plasmid clones were analyzed, confirmed and sequenced. the nucleotide sequence of the n gene of ljb/03 was, determined by takara biotechnology (dalian) co. ltd. amino acid sequences were aligned using the clus-tal w method, and phylogenetic trees were constructed using the neighbor-joining method. analyses were done using the megalign application of the lasergene software package. the identification of sequence motifs was done with the psi-blast program using the swiss-prot database through the myhits web server (http://myhits.isb-sib.ch). by using rt-pcr method, we successfully amplified the nucleocapsid gene. the pcr products were approximately 1.3 kb in size and cloned into the pgem-t easy vector. the complete nucleotide sequence of nucleocapsid gene has been deposited in genbank, accession number is dq072726. sequence analysis indicate that the compete open reading frame (orf) for the nucleocapsid gene of pedv ljb/03 consists of 1326 bases and codes for a basic protein of 441 amino acid. it consisted of 405 adenines (30.5%), 294 cytosines (22.2%), 329 guanines (24.8%) and 298 thymines (22.5%) and a g+c content of 47.0%. the result of motif blast indicated the ljb/03 n protein had seven potential protein kinase c phosphorylation sites, nine casein kinase ii phosphorylation sites, one tyrosine kinase phosphorylation site, two camp-and cgmp-dependent protein kinase phosphorylation sites. the gene had 43 nucleotide mismatches compared to cv777, a substantial portion (72%, 31/43) of the substitutions was transversions, about 60% of the substitutions were non-synonymous mutations. table 1 shows that the percent similarity of the n nucleotide sequences varied from 95.6% to 97.4% between ljb/03 and the other four strains of pedv, and a high degree of identity (94.9-99.8%) was observed between the nucleotide sequences of pedv strains. the alignment of the nucleotide sequences shows that no deletion or insertion event was detected, and there is a large region of absolute identity such as in the region from nucleotide 517 to nucleotide 614 (517-614 bases). the entire nucleocapsid protein of pedv ljb/03 aligned with the published sequences of cv777, brl/87, chinju99 and js2004. this alignment indicates that overall the sequences are, highly conserved with some regions showing no variation at all, and the 15 nucleotide acid substitutions in the 5¢ region (1-249 bases) did not arouse amino acid changes, which may suggestion the n-terminal of the protein had more homologous than the c-terminal. two-way comparisons among the nucleocapsid proteins of these five strains of pedv indicate that the identities range from 95.9% to 98.0%, with cv777 and brl/87 having the most identity, and ljb/03 and chinju99 the least. a phylogenetic tree was prepared to further examine relationships between pedv and other coronaviruses based on a comparison of n protein amino acid sequences (fig. 3) . phylogenetic analysis showed that pedv was more closely related to group 1 (tgev, hcv 229e and fipv) than to members of group 2 korea are more closely related to each other than they are to those two isolates european cv777 and brl/87. in the present study, the n gene of ljb/03 was cloned and sequenced. the result sequence revealed the n gene has a orf of 1326 nucleotides coding for a 441 amino acids protein. sequence comparison with other pedv strains selected from genbank indicated that the n gene of pedv was highly conserved even though comes from different geographic region, and the alignment result showed there is some region of absolute identity in the sequences. previous studies showed the chinju99 n protein had 7 potential t-or s-linked phosphorylation sites and seven potential casein kinase ii phosphorylation sites, the result in this study indicated the ljb/03 n protein had seven potential protein kinase c phosphorylation sites, nine casein kinase ii phosphorylation sites, one tyrosine kinase phosphorylation site, two camp-and cgmp-dependent protein kinase phosphorylation sites. the entire nucleocapsid protein of pedv ljb/03 aligned with the published sequences of cv777, brl/87, chinju99 and js2004. this alignment of nucleocapsid protein sequences indicates that overall the sequences are highly conserved with some regions showing no variation at all. this can be the feasible information for the development of genetically engineered n protein for vaccine to prevent pedv infections. shuichi et al. developed a method of detection of pedv using polymerase chain reaction based on part of nucleocapsid nucleotide, and then compare the nucleocapsid nucleotide among strains of the virus, the result of restriction analysis the pcr products were that cv777 and all the korean strain can be digested with dra i, ecor i, but the korean strain was not digested with pst i. we found the n gene of ljb/03 and js2004 (another china isolate) have the same restriction patterns with the korean strains [21] . coronaviruses have been subdivided into three major antigenic groups based on antigenic differences identified by serological analyses and nucleotide sequence analyses [22, 23] . group i members are the porcine transmissible gastroenteritis virus (tgev) and epidemic diarrhea virus (pedv), feline and caamino acid sequences were aligned using the clustal method, and phylogenetic . trees were constructed using the neighbor-joining method. analyses were done using the megalign application of the lasergene software genbank accession numbers of sequences in the phylogenetic tree are: ljb/03 dq072726; chinju99 af237764; cv777 nc003436; brl/ 87 z24733 (britain isolate); js2004 ay653206 (china field isolate) nine coronavirus (fcov and ccov), and human coronavirus 229e (hcov-229e). group ii includes porcine hemagglutinating encephalomyelitis virus (hev), murine hepatitis virus (mhv), bovine, equine, and rat coronavirus (bcov, ecov, and rtcov), and human coronavirus oc43 (hcov-oc43). group iii is specific for avian species including turkey coronavirus (tcov), pheasant coronavirus and avian infectious bronchitis virus (ibv). the coronavirus n protein has been shown to be highly variable in size as well as in amino acid composition between the viruses that comprise the three coronavirus antigenic groups but highly conserved within these groups. group i viral genomes have the smallest nucleocapsid protein with 378-389 residues, group ii genomes have the largest with 449-455 residues and group iii 409 residues. all 5 pedv strains had 441 amino acid residues, and have a longer peptide than other group i members, which illuminate pedv, a particular case is an exception to the rule of the coronavirus n protein has been shown to be highly conserved within these groups. in the study, we acquired the nucleotide sequence of the n gene pedv ljb/03 and did the nucleotide sequence analysis to establish the phylogenetic relationships between several strains of pedv. this work showed that the nucleotide sequence can form a base for further study on the epidemiological study of pedv infections. diease of swine pig farming acknowledgement the financial support of this work was provided by grants from ''project of the tenth-five'' of heilongjiang provincial scientific and technique committee, china. key: cord-268010-1m5h3krw authors: jung, kwonil; hu, hui; saif, linda j. title: porcine deltacoronavirus infection: etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis date: 2016-12-02 journal: virus res doi: 10.1016/j.virusres.2016.04.009 sha: doc_id: 268010 cord_uid: 1m5h3krw porcine deltacoronavirus (pdcov) (family coronaviridae, genus deltacoronavirus) is a novel swine enteropathogenic coronavirus that causes acute diarrhea/vomiting, dehydration and mortality in seronegative neonatal piglets. pdcov diarrhea was first reported in the us in early 2014, concurrently with co-circulation of porcine epidemic diarrhea virus (pedv) (family coronaviridae, genus alphacoronavirus). the origin of pdcov in pigs and also its sudden emergence or route of introduction into the us still remains unclear. in the us, since 2013–2014, the newly emerged pdcov and pedv have spread nationwide, causing a high number of pig deaths and significant economic impacts. the current us pdcov strains are enteropathogenic and infect villous epithelial cells of the entire small and large intestines although the jejunum and ileum are the primary sites of infection. similar to pedv infections, pdcov infections also cause acute, severe atrophic enteritis accompanied by transient viremia (viral rna) that leads to severe diarrhea and/or vomiting, followed by dehydration as the potential cause of death in nursing piglets. at present, differential diagnosis of pdcov, pedv, and transmissible gastroenteritis virus (tgev) is essential to control viral diarrheas in us swine. cell culture-adapted us pdcov (tc-pdcov) strains have been isolated and propagated by us and in several other laboratories. tc-pdcov strains will be useful to develop serologic assays and to evaluate if serial cell-culture passage attenuates tc-pdcov as a potential vaccine candidate strain. a comprehensive understanding of the pathogenesis and epidemiology of epidemic pdcov strains is currently needed to prevent and control the disease in affected regions and to develop an effective vaccine. this review focuses on the etiology, cell culture isolation and propagation, molecular epidemiology, disease mechanisms and pathogenesis of pdcov infection. porcine deltacoronavirus (pdcov), a member of the genus deltacoronavirus in the family coronaviridae of the order nidovirales, causes acute diarrhea, vomiting, dehydration and mortality in neonatal piglets (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . pdcov was first reported in pigs in hong kong in 2012 (woo et al., 2012) . among a total of 7140 rectal, tracheal, or cloacal swab samples from wild asian leopard cats, bats, wild birds, domestic cats, cattle, chickens, dogs, pigs, rodents, monkeys, and humans in china and hong kong in 2007-2011, dcovs were detected only in pigs and wild birds, with a 10.1% positive rate for pdcov in the 169 swine rectal swab samples tested (woo et al., 2012) . however, it appears that dcovs were present in rectal swabs of small mammals, such as asian leopard cats and chinese ferret badgers, at live-animal markets in china since 2005 -2006 (dong et al., 2007 . pdcov diarrhea was first reported in the us in early 2014 (li et al., 2014; marthaler et al., 2014b; wang et al., 2014a, b) , concurrently with porcine epidemic diarrhea virus (pedv) (family coronaviridae, genus alphacoronavirus), which causes similar, but more severe clinical disease in neonatal piglets (cima, 2013; stevenson et al., 2013) . recent studies reported that pdcov chn-ah-2004 and ch/sichuan/s27/2012 strains, genetically related to the current us pdcov strains, were previously present in diarrheic piglets in china in 2004 and 2012, respectively, prior to the first outbreak of pdcov infection in the us in early 2014 (dong et al., 2015; wang et al., 2015b) . the family coronaviridae can be genetically divided into the four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (woo et al., 2012) . bats are the projected host for the gene source of alphacoronaviruses and betacoronaviruses, while birds are the suspect host for gammacoronaviruses and deltacoronaviruses (woo et al., 2012) . the other swine enteropathogenic coronavirus, transmissible gastroenteritis virus (tgev), also belongs to alphacoronavirus. similar epidemiological, clinical, and pathological features among pdcov, pedv and tgev have complicated their diagnosis, requiring differential laboratory tests . in the us, since 2013-2014, the newly emerged pedv and pdcov have spread nationwide and affected pigs alone or jointly, causing a high number of pig deaths and significant economic impacts and hindering the control of both viral infections in farms dually affected by these two viruses (anon., 2014; jung and saif, 2015) . at present, differential diagnosis of pedv, pdcov, and tgev is critical to control the coronaviral epidemic diarrheas in us pig farms. cultivable, cell culture-adapted pdcov (tc-pdcov) strains include the two isolates, usa/il/2014 and michigan/8977/2014, isolated at the usda national veterinary services laboratories (chen et al., 2015b; ma et al., 2015) and tc-pdcov strain oh-fd22 isolated in our lab (hu et al., 2015) . the tc-pdcov oh-fd22 has been serially passaged >90 times on llc-pk cells, with high sustained infectious titers (hu et al., 2015) . the tc-pdcov strains are useful for pdcov pathogenesis studies, virological and serological assays, and vaccine development. this review focuses on the current understanding of the etiology, molecular epidemiology, disease mechanisms and pathogenesis of pdcov, and cell culture isolation and propagation of the virus. pdcov is enveloped and pleomorphic with diameter of 60-180 nm, excluding the projections (ma et al., 2015) . pdcov has a single-stranded positive-sense rna genome of approximately 25.4 kb in size (excluding the poly a-tail) that encodes four structural proteins, namely, spike (s), envelope (e), membrane (m), and nucleocapsid (n), and four nonstructural proteins (lee and lee, 2014; li et al., 2014; ma et al., 2015; marthaler et al., 2014a; wang et al., 2015b) . the pdcov genome organization and arrangement consist of: 5 untranslated region, open reading frame 1a/1b (orf1a/1b), s, e, m, nonstructural protein 6 (ns6), n, nonstructural protein 7 (ns7), and 3 utr. although general characteristics of the structural and nonstructural proteins of coronaviruses and their roles in viral replication have been identified saif et al., 2012) , the detailed functions and roles of structural and nonstructural proteins of pdcov in host cells are unknown. a recent study reported multifunctional roles of pdcov n protein similar to those of other coronaviruses. the n protein was identified in both the nucleus and cytoplasm of stably n-expressing porcine kidney cells, suggesting rna synthesis or ribosome biogenesis through its interactions with ribosomal subunits or nucleolar proteins . there was no cross-reactivity of pdcov usa/il/2014 strain with antibodies to either pedv or tgev, as determined by indirect immunofluorescence assays of virus-infected cells using a rabbit antiserum against synthetic m protein of pdcov, a pedv monoclonal antibody (sd6-29) targeting the n protein, and a tgev polyclonal antiserum (chen et al., 2015b) . this finding was also supported by other reports (jung et al., 2015c; ma et al., 2015) , showing no cross-reactivity of pig hyperimmune or convalescent antiserum to pdcov oh-fd22 or cvm1 strains with pedv, as determined by immunohistochemical staining of frozen or formalin-fixed intestinal tissues of pedv-infected pigs. however, a recent study reported evidence of antigenic cross-reactivity between pdcov michigan/8977/2014 strain and pedv, possibly sharing at least one conserved or similar epitope on their n proteins, as determined by enzyme-linked immunosorbent assay (elisa) and western blot using monoclonal pedv and pdcov n-specific antibodies, whereas no cross-reactivity was detected when virus neutralization, indirect immunofluorescence, and immunostaining assays were conducted on either virus-infected cells or intestinal tissues using pig hyperimmune antisera to pedv or pdcov (ma et al., 2016) . llc porcine kidney (llc-pk) and swine testicular (st) cells support the isolation and serial propagation of pdcov in cell cultures supplemented with exogenous protease trypsin or pancreatin (hu et al., 2015) . trypsin contributed to a significant increase in pdcov growth after several passages in llc-pk cells. however, even without supplemental trypsin, pdcov replicated in llc-pk cells, but without cytopathic effects (cpe) and at relatively lower virus titers compared to the trypsin supplemented cell cultures (hu et al., 2015) . thus, trypsin is not essential, but is beneficial for the propagation of pdcov in llc-pk cells. the cell-culture conditions required for the propagation of pdcov in st cells differ from those in llc-pk cells. trypsin did not support pdcov propagation in st cells. in contrast, pdcov could be serially propagated in st cells supplemented with pancreatin or small intestinal contents from gnotobiotic (gn) piglets (hu et al., 2015) . the addition of trypsin and pancreatin in pdcov-inoculated llc-pk and st cells, respectively, resulted in similar cpe that consisted of enlarged, rounded, and densely granular cells that occurred singly or in clusters and then, cell shrinkage and detachment as a result of apoptotic cell death hu et al., 2015) . the roles of trypsin or pancreatin in cell entry and release of pdcov virions in llc-pk or st cells remain unknown. coronaviruses in the genus deltacoronavirus have been identified recently in a variety of wild small mammals and avian species (dong et al., 2007; woo et al., 2009a; woo et al., 2009b; woo et al., 2012) . in 2006, novel coronaviruses were detected in feces of wild asian leopard cats (prionailurus bengalensis) and chinese ferret badgers (melogale moschata) during a virological surveillance for severe acute respiratory syndrome (sars) coronavirus in southern china. the isolates were genetically similar to each other and commonly had low amino acid similarity and unique phylogeny distinct from other coronavirus groups (dong et al., 2007) . the two isolates were subsequently shown genetically to belong to deltacoronavirus (woo et al., 2012) . another molecular epidemiology study was conducted in dead wild birds for a 7-month period (december 2006 to june 2007 in hong kong (woo et al., 2009b) . three novel avian coronaviruses (bucov hku11, thcov hku12, and mucov hku13) were identified and proposed as a novel coronavirus subgroup 3c under the group 3 coronaviruses (woo et al., 2009b) . recently, the three isolates were further identified genetically to belong to deltacoronavirus (king et al., 2012) . a coronavirus surveillance study was conducted in a variety of domestic and wild animal species from 2007 to 2011, and the presence of dcovs in the feces of domestic pigs was first reported in 2012 (woo et al., 2012) . seven dcovs in pigs and wild birds were identified, which were designated porcine coronavirus hku15 and the avian coronavirueses, white-eye coronavirus hku16, sparrow coronavirus hku17, magpie robin coronavirus hku18, night heron coronavirus hku19, wigeon coronavirus hku20, and common moorhen coronavirus hku21 (woo et al., 2012) . this study first verified that pdcov was present in the feces of pigs in mainland china and hong kong in 2007-2011, with a positive rate of 10.1%. two chinese pdcov complete genome sequences were generated from two different isolates, hku15-44 and hku15-155 (genbank accession no. jq065042 and jq065043, respectively) (woo et al., 2012) . the helicase, s and n genes of pdcov hku15-44 and hku15-155 were closely related to those of the coronaviruses isolated from the asian leopard cats and chinese ferret badgers [nucleotide (nt) similarity ≥99.8%] (woo et al., 2012) (fig. 1a) , implying the potential interspecies transmission of a dcov between these wild small mammals and pigs. further molecular surveillance studies are needed to define the potential role of the small mammals as an intermediate host of pdcov and the mechanisms of interspecies transmission of dcovs between the small mammals and domestic pigs or wild birds, and also between domestic pigs and wild birds. on february 11, 2014, the ohio department of agriculture first announced the presence of pdcov in us swine. in early february 2014, the animal disease diagnostic laboratory at the ohio department of agriculture received 42 fecal or intestinal samples from diarrheic sows and piglets on 5 ohio farms, and 39 (92.9%) were positive for pdcov by rt-pcr (wang et al., 2014a) . the pdcov hku15-oh1987 strain identified (genbank accession no. kj462462) had a 99% nt identity to pdcov hku15-44 and hku15-155 (wang et al., 2014a) (fig. 1b) , implying a possible importation of a chinese pdcov into us swine. during a similar period, the university of minnesota veterinary diagnostic laboratory also received fecal swabs from diarrheic pigs. the causative agent of the diarrhea was identified as the pdcov sdcv/usa/illinois121/2014 strain (genbank accession no: kj481931.1) that also had approximately 99% nt identity to the two hong kong pdcov strains (marthaler et al., 2014a) . the iowa state university veterinary diagnostic laboratory also identified another us pdcov strain usa/ia/2014/8734 (genbank accession no: kj567050) that had 98.9% nt identity to the hku15-44 strain and 99.2% nt identity to the hku15-155 strain (li et al., 2014) . pdcovs were further detected in other us states (minnesota, south dakota, nebraska, illinois, indiana, michigan, kentucky, pennsylvania, and ohio) (wang et al., 2014b) . of the 435 samples tested, 109 (25%) were positive for pdcov by rt-pcr, and they shared high nt similarity (≥99.8%) with each other and also 98.9-99.2% nt similarity with pdcov hku15-44 and hku15-155 (wang et al., 2014b) . as of march 2016, pdcov has been detected in 18 us states according to a report of the usda animal and plant health inspection service (www.aphis.usda.gov/ animal-health/secd). several retrospective studies were conducted to determine the earliest date when pdcov existed in us swine. a retrospective study using real-time rt-pcr was performed on swine samples that were submitted to the iowa state university veterinary diagnostic laboratory for investigation of enteric diseases between october 2012 and december 2013 (sinha et al., 2015) . a total of 5 samples were positive for pdcov rna among the 1734 clinical samples from 18 states tested. pdcov rna was detected in fecal samples collected on august 19th 2013 from minnesota, august 20th and august 27th from iowa, and august 29th 2013 from illinois (sinha et al., 2015) . based on these data, pdcov has been present in us swine since at least august 2013. another retrospective study using an indirect elisa for the detection of pdcov igg antibodies demonstrated their prevalence in us pigs (thachil et al., 2015) . a total of 968 serum samples collected between 2006 and 2014 were tested. the pdcov igg antibodies were first detected in the archival serum samples collected in 2010 (thachil et al., 2015) . however, the origin of us pdcov in us swine remains unclear. to date, pdcov also has been reported in canada, korea, china, and thailand. epidemic diarrhea related to pdcov was first detected in 6 ontario farms in canada in march 2014 (marthaler et al., 2014b) . in april 2014, pdcov (kun14-04 strain, genbank accession no. km820765) was first identified in feces of diarrheic piglets in south korea. the korean strain had nt identities of 98.8-99.0% to hku15-44 and hku15-155 and 99.6-99.8% to us pdcov strains (lee and lee, 2014) . a recent survey conducted in south korea reported that of 691 diarrheic fecal samples collected on 59 pig farms in january 2014 to march 2015, only 2 samples from 1 pig farm (march 2015) were pdcov-positive by rt-pcr (lee et al., 2016) . two korean pdcov strains sl2 and sl5 isolated in the study were also closely related to us pdcov strains, but they appeared to genetically differ from the previous korean strain knu14-04 isolated in 2014 (lee et al., 2016) . the first identification of pdcov hku15-44 and hku15-155 strains in swine in hong kong, china for the period 2007-2011 were documented in 2012 (woo et al., 2012) . since then, pdcov has been identified in diarrheic pigs in mainland china (dong et al., 2015; song et al., 2015; wang et al., 2015b) . a study also found pdcov rna in diarrheic pig samples collected in 2004 in china (dong et al., 2015) . the prevalence of pdcov in mainland china was high (>30%), and co-infections with pdcov and pedv (51%) were also common (dong et al., 2015; song et al., 2015) . all pdcov strains reported from china shared high nt identities (≥98.9%) with each other and with the other pdcov strains found globally (dong et al., 2015; song et al., 2015) (fig. 1b) , implying a possible global circulation of a single genotype. pdcov strains with multiple mutation or deletion sites in their s, nonstructural, or 3 utr genes have been also found in china, whereas the mutations were not found in the genomes of us pdcov strains (wang et al., 2015a; wang et al., 2015b) . in june 2015, a pdcov-related diarrhea outbreak was identified on a pig farm in thailand (janetanakit et al., 2016) . pdcov strains detected in thailand shared the highest nt identities (≥98.4%) with the chinese pdcov strain chn-ah-2004 (janetanakit et al., 2016) . like pedv and tgev saif et al., 2012) , the fecal-oral route may be the main means of pdcov transmission. since aerosolized pedv remains infectious (alonso et al., 2014) , whether aerosolized pdcov is also infectious should be investigated. like pedv transmission, diarrheal feces and/or vomitus and other contaminated fomites, such as transport trailers (lowe et al., 2014) and feed (dee et al., 2014) , may be major transmission sources of the virus. another possible reservoir for pdcov includes carriers, such as older pigs with subclinical infection, similar to pedv infection in weaned pigs (jung et al., 2015a) . like pedv and tgev saif et al., 2012) , pdcov is cytolytic, and infected enterocytes rapidly undergo acute necrosis, leading to marked villous atrophy in the small but not in the large intestine ( fig. 2a) (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . like pedv and tgev (kim et al., 2000) , pdcov may not induce apoptotic death of enterocytes in the small intestine of infected pigs . pdcov antigens ( fig. 2b and c) and nucleic acids (fig. 2d ) are observed mainly in villous enterocytes of the small (duodenum to ileum) and large intestines (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . as tested at post-inoculation days (pid) 3-7, pdcov antigens were detected mainly in the villous epithelium of the atrophied mid-jejunum to ileum of experimentally infected pigs and to a lesser extent, in duodenum, proximal jejunum and cecum/colon (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . occasionally, a few pdcov antigenpositive cells are detected in the intestinal crypts . porcine small intestinal villous enterocytes express large amounts of aminopeptidase n (apn), a 150-kda glycosylated transmembrane protein, identified as the cellular receptor for pedv and tgev (delmas et al., 1992; li et al., 2007) . whether apn also functions as the cellular receptor for pdcov or if other cellular receptors are involved in cell entry of this virus is unknown. in addition, a few pdcov antigen-positive cells were also detected in the intestinal lamina propria, peyer's patches, and mesenteric lymph nodes (mln) during the early stages of infection jung et al., 2016) . as tested at pid 23-24 after experimentally infected pigs had recovered from clinical disease and ceased fecal virus shedding, larger numbers of pdcov antigen-positive cells were detected in these tissues . the pdcov antigen-positive cells might include antigen-presenting cells, such as macrophages. pdcov antigens were not detected in other organs, such as stomach, lung, heart, tonsil, spleen, liver and kidney (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . however, by qrt-pcr, pdcov rna could be detected in low to moderate quantities in multiple organs, possibly due to the effect of viremia (chen et al., 2015b; ma et al., 2015) . lung tissues of orally infected pigs were negative for pdcov antigen (chen et al., 2015b; hu et al., 2016; jung et al., 2015c) . in our preliminary study, four 10-to 14-day-old gn pigs orally and nasally inoculated with tc-pdcov oh-fd22 at llc-pk cell-culture passages 5 (p5) or 80 (p80) were negative for pdcov rna in nasal swabs and bronchoalveolar lavage fluids at pid 3-10, although the inoculated pigs were positive for both clinical disease and fecal virus shedding for the period, suggesting lack of pdcov replication in the respiratory tract. however, further studies are still needed to investigate whether extra-intestinal replication of pdcov occurs. based on clinical observations in experimentally infected gn or conventional nursing pigs, like pedv and tgev saif et al., 2012) , pdcov also induces acute, watery diarrhea, frequently accompanied by acute, mild to moderate vomiting, ultimately leading to dehydration, loss of body weight, lethargy, and death (jung et al., 2015c; ma et al., 2015) . similar to pedv infection (jung et al., 2014) , in a controlled experimental setting, clinical pigs exhibited dehydration, loss of bodyweight, and lethargy, but frequently maintained their appetite until sudden death (ma et al., 2015) . among the different clinical signs identified in gn or conventional pigs, only diarrhea could be observed in experimentally infected conventional nursing pigs (chen et al., 2015b) , indicating that a variety of disease outcomes are expected based on different experimental conditions. however, conventional pigs appeared to be more susceptible to pdcov infection, compared to gn pigs, under similar experimental conditions, such as when the same virus and inoculation dose and age of pigs were used (ma et al., 2015) . based on field observations in us swine (anon., 2014) , pdcov caused a number of deaths (up to a 40% mortality rate) among suckling pigs, a lower rate than that found in pedv infections. the clinical impact and disease severity of pdcov in the field may be less than that of pedv (anon., 2014) . on the other hand, a pdcov-related diarrhea outbreak in a breeding farm in china resulted in a higher mortality rate (more than 80%) among suckling piglets (song et al., 2015) . like pedv and tgev saif et al., 2012) , diarrhea induced by pdcov may be a consequence of malabsorption due to massive loss of absorptive enterocytes. functional disorders of infected enterocytes may also contribute to the malabsorptive diarrhea. like pedv , mild vacuolation observed in the infected colonic epithelial cells may interfere with the vital reabsorption of water and electrolytes (jung et al., 2015c) . as a consequence of extensive loss of enterocytes, brush border membrane-bound digestive enzymes such as disaccharidases (lactase, sucrase, and maltase), leucine apn, and alkaline phosphatase were significantly decreased in the small intestine of diarrheic piglets after pedv infection jung et al., 2006) , resulting in maldigestive diarrhea. because of the similar pathological features between pdcov and pedv, pdcov infection may also result in maldigestive diarrhea. dehydration is exacerbated by vomiting, but the mechanisms by which vomiting is induced in pdcov infection are poorly understood. whether pdcov infection induces metabolic acidosis, similar to hyperkalemia and acidosis in acute tgev and pedv infections saif et al., 2012) , needs to be studied. based on experimental findings (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) , the onset of diarrhea after pdcov infection appears to be acute, which coincided mostly with or was detected 1-2 days later than the detection of viral rna in feces. fecal viral rna titers peaked 1-3 days later. the findings of these studies are summarized in table 1 . five 14-day-old gn pigs inoculated with 8.8 or 11.0 log 10 ge of two field pdcov strains oh-fd22 and oh-fd100 all showed severe diarrhea and/or vomiting at post-inoculation hours (pih) 21-24, which coincided with the detection of viral rna in feces (jung et al., 2015c) . six 14-day-old gn pigs inoculated with 10.1-10.8 log 10 ge [≈11 log 10 50% tissue culture infectious dose (tcid 50 ) or 9 log 10 plaque forming units (pfu) per pig (hu et al., 2015) ] of the tc-pdcov oh-fd22 p5, p20, and p40 all showed severe diarrhea and/or vomiting at pid 2, which also coincided with the detection of viral rna in feces . in this study, fecal viral rna titers peaked, ranging from 7.8 to 8.9 log 10 ge/ml, at pid 2-5 and then decreased gradually thereafter . another study using conventional 5-day-old pigs and a cell culture-adapted pdcov usa/il/2014 strain (p11) reported the onset of diarrhea at pid 5 in 5 of 5 pigs orally inoculated with 3 × 10 4 tcid 50 /pig of the virus, which was 1 day later or coincided with the detection of viral rna in feces at pid 4 (3/5 pigs tested) or 5 (2/5 pigs tested) (chen et al., 2015b) . fecal viral rna peaked at approximately pid 5-7 (chen et al., 2015b) . in another study using another strain genetically similar to the usa/il/2014 strain, four 10-dayold conventional and three 10-day-old gn pigs orally inoculated with 10 6 pfu (≈10 9 genomic rna copies) of a cell culture-adapted pdcov michigan/8977/2014 strain (p15) all exhibited moderate to severe diarrhea at pid 1 (conventional pig) and pid 3 (gn pig), respectively (ma et al., 2015) . fecal shedding occurred at pid 1 (1/4 pigs tested) or pid 2 (3/4 pigs tested) in conventional pigs, whereas fecal shedding was detected at pid 1 (1/3 pigs tested) or pid 2 (2/3 pigs tested) in gn pigs prior to the onset of diarrhea (ma et al., 2015) . in the infected conventional pigs, fecal viral rna titers peaked, ranging from 6 to 7 log 10 rna copies/gram, on pid 7 and then decreased gradually thereafter (ma et al., 2015) . based on experimental findings jung et al., 2015c; ma et al., 2015) , persisting diarrhea in infected nursing pigs is observed for approximately 5-10 days. prolonged fecal virus rna shedding was also evident in pdcov-infected nursing pigs. after recovery from disease, pigs continued to shed pdcov rna in the feces. the findings of these studies are summarized in table 1 . the gn pig inoculated with 8.8 log 10 ge of the field pdcov strain oh-fd22 developed diarrhea at pih 21-24, which was consistently observed until pid 7 (jung et al., 2015c) . four 10-day-old conventional pigs orally inoculated with 10 6 pfu of the michigan/8977/2014 strain all exhibited diarrhea at pid 1, which then persisted for another 6-9 days (ma et al., 2015) . the gn pigs inoculated with 10.1-10.8 log 10 ge of the tc-pdcov oh-fd22 p5, p20, and p40 all showed diarrhea at pid 2, which then persisted for another 4-6 days . hu et al. (2016) reported that similar to prolonged fecal virus rna shedding (until pid 20) observed in the wild-type pdcov oh-fd22-inoculated pigs, fecal virus shedding in the tc-pdcov oh-fd22 p5 or p20-inoculated pigs was detectable until pid 18 or 20, respectively . the tc-pdcov oh-fd22 p40-inoculated pig also had pdcov rna-positive feces until pid 15 . one of the four 10-day-old conventional pigs (25%) inoculated with the michigan/8977/2014 strain had detectable pdcov rna in the feces until pid 21 (ma et al., 2015) . there is little information on intestinal replication of pdcov during disease progression in pigs of varying ages. in previous experimental infection studies, most of the infected gn or conventional suckling pigs were euthanized at pid 3-7 (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . thus, the earlier, pathological characteristics related to pdcov infection at pid 1-3 are unknown. in experimental pedv infections in seronegative suckling pigs, during the incubation period, i.e. at approximately pih 12-24, pedv antigen-positive cells were seen throughout the small intestine and as many as 30-100% of the absorptive epithelial cells were positive (debouck et al., 1981; madson et al., 2015) , consistent with fecal virus shedding in subclinical pigs. after that, during the clinical period, moderate to large numbers of pedv antigen-positive cells were observed throughout the small intestine (debouck et al., 1981; jung et al., 2015a; madson et al., 2015) . whether pdcov also replicates similarly in the small intestine of nursing pigs, needs to be studied in well-controlled experimental settings with large numbers of pigs and multiple time-points. more information on the pathogenesis of pdcov in pigs at earlier timepoints (pids 1-2) is needed to fully understand disease progression. relative to experimental pedv infections (jung et al., 2015a; jung et al., 2014; madson et al., 2015) , pdcov-infected pigs also appeared to shed less pdcov rna and had lower peak shedding titers in the feces (chen et al., 2015b; jung et al., 2015c) , implying a potentially lower replication rate of pdcov in the gastrointestinal tract of pigs. studies are also needed to investigate if this might contribute to the lower mortality of pdcov-infected nursing pigs, as compared to pedv infections. based on field observations (anon., 2014; song et al., 2015) , pdcov caused a number of deaths (up to a 40% mortality rate in us and >80% in china) among suckling pigs. similarly, ped is the most devastating in nursing piglets causing 100% morbidity and 50-100% mortality saif et al., 2012) . several mechanisms by which pedv infection induces greater disease severity in nursing versus weaned pigs, have been defined (annamalai et al., 2015; jung et al., 2015a) . compared to 9-day-old nursing pigs that began to show severe clinical disease, villous atrophy and fecal virus shedding at pid 1, a longer incubation period of pedv was evident before 26-day-old weaned pigs shed fecal virus rna (by 1 more day) or developed lesions and clinical disease (by 2 more days) (annamalai et al., 2015; jung et al., 2015a) . there were significantly decreased functional natural killer cells in the ileum and blood of nursing piglets, compared to weaned pigs, which may have contributed to the greater susceptibility of nursing pigs to pedv infection (annamalai et al., 2015) . studies are needed to confirm if nursing pigs also have higher susceptibility to pdcov infection. anatomical and physiological factors that may contribute to the longer recovery of nursing pigs from ped include: 1) the slower turnover of enterocytes (5-7 days) in neonatal piglets compared to 2-3 days in 3-week-old weaned pigs (moon et al., 1973) ; and 2) the anatomically underdeveloped large intestine of nursing piglets that may increase their vulnerability to dehydration, compared to weaned pigs (jung et al., 2015a) . there was also a lack of crypt stem cells and lower numbers of proliferating crypt cells in the small intestine of nursing pigs, compared to weaned pigs (jung et al., 2015a) . this could lead to the slower turnover of enterocytes in nursing vs weaned pigs, contributing to a slower recovery from ped in nursing piglets and the greater susceptibility of nursing pigs to ped (diarrhea and dehydration). because of the similar pathological features between pdcov and pedv, studies are needed to confirm if pdcov infection may also induce greater disease severity and deaths of nursing piglets vs. weaned pigs by mechanisms similar to those contributing factors to pedv infection. viremia where viral rna in serum ranged from 10 0.5 -10 3 tcid 50 /ml was detected in conventional 5-day-old pigs (15/15; 100%) inoculated with the usa/il/2014 strain at pid 4 (incubation period) and pid 5-7 (clinical period) (chen et al., 2015b) . simi-larly, viral rna in serum ranged from 3.4-4.1 log 10 copies of pdcov rna/ml in 19-day-old gn pigs inoculated with the ohio wild-type field cvm1 strain at pid 1-3 (clinical period) (ma et al., 2015) . in another study, viral rna in serum ranged from 4.8-5.2 log 10 ge/ml in gn piglets (6/6; 100%) inoculated with the us tc-pdcov oh-fd22 during the clinical period at pid 3 . the early, severe diarrhea/vomiting might be accompanied by acute, transient viremia, but no one has yet confirmed the presence of infectious virus in the serum. similar to pedv infection of nursing pigs that was accompanied by early viremia (viral rna, up to 100%), viremia in pdcov-infected pigs might also be related to the severity of atrophic enteritis and structural alteration of tight and adherens junctions in the jejunal and ileal villous epithelium in the small intestine (jung et al., 2015b; jung et al., 2014) . there is a dearth of information on the innate and adaptive immune responses to pdcov. after pdcov infection, infiltration of macrophages, lymphocytes, eosinophils, and neutrophils were observed in the lamina propria of the small intestine (chen et al., 2015b; ma et al., 2015; wang et al., 2015a) . our study reported the development of pdcov-specific antibodies in serum of pdcovinfected pigs . the 14-day-old gn pigs orally inoculated with the original oh-fd22 and tc-pdcov oh-fd22 (p5, p20, and p40) had detectable serum igg, iga and virus neutralization (vn) antibodies at pid 14. thereafter, pdcov-specific igg, iga and vn antibody titers increased and remained high at the end of experiment, pid 23/24, when the pigs were fully recovered from clinical disease and fecal virus rna shedding. similarly, in pedv cv777-infected pigs, serum pedv-specific igg antibody was first detected at approximately pid 7-12 (de arriba et al., 2002) . 6.9. virulence of high cell culture-passaged pdcov (p80) the tc-pdcov oh-fd22 has been serially passaged >p90 on llc-pk cells. our study confirmed the enteropathogenicity of tc-pdcov oh-fd22 p5, p20, and p40 in gn pigs . the virulence appeared to be similar to that of the parent wild-type virus, as evident by severe diarrhea/vomiting, atrophic enteritis, and high levels of serum igg, iga and vn antibodies in inoculated gn pigs. our pilot study also revealed that two 10-day-old gn pigs inoculated orally with 10.2 log 10 ge of the tc-pdcov oh-fd22 p80 (passage 80) developed delayed (by 1-3 days), but severe clinical signs with high viral rna shedding titers in the feces, ranging from 7.7 to 8.8 log 10 ge/ml at pids 5 and 6, which is similar to those in gn pigs inoculated with the original field strain oh-fd22 or tc-pdcov oh-fd22 (cell passages 5-40). therefore, the tc-pdcov oh-fd22 needs to be further passaged to evaluate if higher cell-culture passaged virus becomes attenuated, because attenuation of the virulence of pedv strains has been induced through high cell-culture passages (93rd-144th) (chen et al., 2015a; kweon et al., 1999; sato et al., 2011; song et al., 2003) . the severity of clinical disease caused by pdcov in nursing piglets may be affected by co-infections with other viruses including rotavirus (rv) and pedv. however, the impact of dual pdcov and pedv or rotavirus infection on the disease outcome in pigs needs to be delineated. a survey conducted in the us reported that of 293 samples collected from diarrheic pigs in the us and canada in early 2014, 89 (30%) were pdcov-positive by rt-pcr (marthaler et al., 2014b) . of the 89 pdcov-positive samples, 69 (78%) were positive for pedv, group a rv (rva), rvb, or rvc. co-infections with pdcov and rvc were the most common [52 (58%) samples], and 29 (29/89; 33%) were also positive for pedv. our lab also identified that seven (41.2%) of 42 pdcov-positive samples, collected on different farms with diarrhea outbreaks in ohio and indiana, were also positive for pedv (3 samples) or rvc (4 samples) by rt-pcr (hu et al., 2015) . in mainland china, a surveillance study based on nested rt-pcr results reported that of 356 porcine diarrheic samples collected in jiangxi during 2012-2015, 120 (33.7%) were pdcov-positive; 231 (64.9%) were pedv-positive; and 281(78.9%) were positive for either pedv or pdcov (song et al., 2015) . coinfections with pdcov and pedv (19.7%) were common in diarrheic pigs in jiangxi, china. another study tested by rt-pcr reported that of 215 diarrheic samples collected in anhui, guangxi, hubei, and jiangsu during 2004-2014, 14 (6.51%) were positive for pdcov, 110 (51.2%) were positive for pedv, and 5 (2.3%) were positive for tgev. of the 14 pdcov-positive samples, 7 (50%) were also positive for pedv; and 2 were co-infected with pedv, tgev, and pdcov. in infected nursing pigs, similar to pedv and tgev infections saif et al., 2012) , gross lesions are limited to the gastrointestinal tract and are characterized by thin and transparent intestinal walls (proximal jejunum to colon) with accumulation of large amounts of yellow fluid in the intestinal lumen (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . the stomach is filled with curdled milk. the transparency and fragility of affected intestines appeared milder, as compared to pedv infections. in infected nursing pigs, similar to pedv and tgev infections saif et al., 2012) , histological lesions are characterized by acute, multifocal to diffuse, mild to severe atrophic enteritis in the proximal jejunum to ileum (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015; wang et al., 2015a) , occasionally accompanied by mild vacuolation of superficial epithelial cells in the cecum and colon that coincided with a few pdcov antigen-positive cecal and colonic epithelial cells (jung et al., 2015c) . no villous atrophy or histologic lesions were evident in the remainder of the small intestine, duodenum (chen et al., 2015b; hu et al., 2016; jung et al., 2015c) , which coincided with few pdcov antigen-positive duodenal epithelial cells. during acute infection, vacuolated enterocytes or massive cell exfoliation were seen on the tips or the entire villi in the jejunum and ileum. atrophied villi are frequently fused and covered with a degenerated or regenerated flattened epithelium. infiltration of inflammatory cells, such as macrophages, lymphocytes, neutrophils, and eosinophils, was evident in the lamina propria (chen et al., 2015b; ma et al., 2015; wang et al., 2015a) . no lesions were seen in other organs, such as stomach, lung, spleen, liver, kidney, and mln of infected pigs, all of which were also negative for pdcov antigen (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . however, one study found an acute, focal, mild degeneration or necrosis of gastric epithelial cells in the gastric pits of stomach (ma et al., 2015) . pdcov is a novel enteropathogenic coronavirus in pigs. the disease related to pdcov was first reported in the us in early 2014, when pedv infections were also epidemic. however, since 2005-2006, dcovs already had been identified in rectal swabs of wild small mammals (asian leopard cats and chinese ferret badgers), domestic pigs, and wild birds. moreover, pdcov (chn-ah-2004 and ch/sichuan/s27/2012 strains), genetically related to the current us pdcov strains, has been present in chinese diarrheic pigs since 2004, prior to the first outbreak of pdcov infection in us swine. although birds are thought to be the suspect host for deltacoronaviruses, the detailed genetic relatedness of pdcov with other dcov isolates from wild birds and other mammal species should be studied further to clarify the origin of pdcov in pigs and also the source of the sudden emergence of us pdcov in us swine. us pdcov strains are enteropathogenic and infect villous epithelial cells of the entire small and large intestines, although the jejunum and ileum are the primary sites of infection. similar to pedv (alphacoronavirus) infections, pdcov (deltacoronavirus) infections also cause acute, severe atrophic enteritis accompanied by viremia (viral rna) that leads to severe diarrhea and/or vomiting, followed by dehydration as the potential cause of death in nursing piglets. differential diagnosis of pedv, pdcov and tgev is essential to control viral diarrheas in us pig farms. currently, cultivable, us tc-pdcov strains have been isolated and propagated in several laboratories. tc-pdcov strains will be useful to evaluate if high level cell-culture passaged viruses become attenuated and can be potential vaccine candidate strains. relative to pedv infections, causing similar, but more severe clinical disease and lesions, the lack of comprehensive understanding of the pathogenic characteristics of pdcov and effective preventive (pdcov vaccine) and therapeutic interventions has impeded biosecurity and development of methods for prevention and control of pdcov in us swine. it is also critical to understand the pathogenic aspects of pdcov infection distinct from those of pedv, possibly clarifying why pdcov infection results in distinctively lower mortality rates 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appropriated to the ohio agricultural research and development center, the ohio state university. this paper was in part supported by a grant from the oardc seeds grants program, grant ohoa1536 (jung k, pi). none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. the authors have no conflict of interest to declare. key: cord-302286-wu6csxve authors: song, d. s.; oh, j. s.; kang, b. k.; yang, j. s.; moon, h. j.; yoo, h. s.; jang, y. s.; park, b. k. title: oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr13 strain date: 2007-02-28 journal: research in veterinary science doi: 10.1016/j.rvsc.2006.03.007 sha: doc_id: 302286 cord_uid: wu6csxve abstract a vero cell attenuated porcine epidemic diarrhea virus (pedv) strain, dr13, was distinguished from wild-type pedv using restriction enzyme fragment length polymorphism (rflp). cell attenuated dr13 was orally or intramuscularly (im) administered to late-term pregnant sows, and mortality resulting from the highly virulent pedv challenge was investigated in passively immunized suckling piglets of the two different groups. the mortality rate of the oral group (13%) was lower than that of the im group (60%). in particular, the concentration of iga against pedv was higher in piglets of sows in the oral group, compared to the im group. the attenuated dr13 virus remained safe, even after three backpassages in piglets. the findings of this study support the theory that the vero cell attenuated dr13 virus may be applied as an oral vaccine for inducing specific immunity in late-term pregnant sows with a high margin of protection against pedv infection. porcine epidemic diarrhea virus (pedv), a coronavirus, is an etiological enteropathogenic agent in swine (debouck and pensaert, 1980; ducatelle et al., 1981; wood, 1977) . the virus replicates in differentiated enterocytes covering the villi of the small intestine, leading to villous atrophy and maladsorption (debouck and pensaert, 1980; moon, 1978) . ped occurs in most swine-raising countries in europe, as well as china, korea, and japan. thus, economic losses from the disease are serious every winter in these countries (pensaert, 1999; saif and wesley, 1999) . infection is associated with >20% diarrheal cases in neonatal pigs in korea (kweon et al., 1999) . live vaccines for ped are used in korea (kpedv-9) and japan (p-5v), due to the endemic nature of the disease (kweon et al., 1999; kadoi et al., 2002) . the efficacy of commercially available vaccines is limited in field conditions, and the protective immunity induced is insufficient. this is directly attributed to the attenuated strain employed, and the intramuscular (im) route. in a previous study, a serially vero cell passaged pedv, designated ''dr13 0 ', with a restriction fragment length polymorphism (rflp) pattern distinct from wild-type pedvs was tested for pathogenicity in piglets and safety in pregnant sows after oral inoculation (song et al., 2003) . in this investigation, we report the results of a field trial comparing oral versus im inoculation of the dr13 virus in pregnant sows for protecting piglets against pedv challenge. the continuous vero cell line (atcc, ccl-81) was maintained in a-minimum essential medium (a-mem) supplemented with 5% fetal bovine serum, penicillin (100 units/ ml), streptomycin (100 lg/ml), and amphotericin b (0.25 lg/ ml). vero cell attenuated pedv dr13 was propagated, as described previously (hofmann and wyler, 1988; kweon et al., 1999) . briefly, prior to virus inoculation, the growth medium of confluent cells in 25 cm 2 flasks (falcon, usa) was removed, and cells washed three times with phosphate-buffered saline (pbs, ph 7.4). next, cells in each flask were inoculated with 1 ml virus. after adsorption at 37°c for 1 h, cells were incubated in a-mem supplemented with 0.02% yeast extract, 0.3% tryptose phosphate broth, and 2 lg trypsin. challenge ped virus was prepared from small intestines of suckling pigs inoculated orally with the field isolate, dr13, before cell attenuation. intestines were homogenized to a 10% (v/v) suspension with phosphate-buffered saline (pbs; 0.1 m, ph 7.2). suspensions were mixed by vortexing, and clarified by centrifugation for 10 min at 4800g. supernatant fractions passed through a 0.2 lm syringe filter (acrodisk, gelman) were used for challenge experiments. five commercial farms were used for the pedv dr13 virus field trials. three farms had no outbreaks or vaccination of ped, while the other two farms had a history of ped. a mixed breed of pigs was employed in this study (yorkshire · landrace · duroc). all sows from farms a, b, and c, located in the kyungsang and chuchung provinces, were serologically negative for ped virus neutralizing antibodies. a number of pregnant sows in the three ped-free commercial farms (500 sows in farm a; 300 in b; 200 in c) were inoculated orally (o group) or intramuscularly (im group) with 1 ml dr13 (passage level 100) at a titer of 10 6.0 tcid 50 /0.1 ml, 4 weeks prior to farrowing. a second inoculation with an equivalent titer of virus was administered after 2 weeks. sows that were not vaccinated with the virus comprised the control group. paired serum samples before and after inoculation were collected at two-week intervals. paired sera (2 and 4 weeks before farrowing, and at farrowing) and colostrum at delivery were tested for the presence of antibodies against pedv using elisa, as reported previously (kweon et al., 1999) . piglets of all groups were housed with their mothers with no artificial supply of colostrum or milk. thirty 3-day-old piglets were selected randomly from farrowing sows in vaccinated (o and im) and control groups for challenge exposure with virulent pedv. piglets from all groups were challenged orally with 5 ml wild-type pedv (the virus titer was not determined). before challenge, sera from piglets were collected for elisa and serum neutralization (sn) analysis of antibodies against pedv. nursing 3-day-old pigs were removed from the sow 1 h prior to challenge with 5 ml virulent pedv (which previously caused 100% mortality in 3day old pigs) . piglets from each group were housed in a 1.3 m · 1.3 m (1.69 m 2 ) pen of an environmentally controlled building, and fed substitute milk. clinical signs of diarrhea and mortality in challenged piglets were observed for 10 days, and antibody response against pedv was examined during the experimental period. the duration of pedv shedding after challenge with wild-type pedv was monitored using rt-pcr. twenty-four pregnant sows from the two other farms (d and e) were inoculated orally with 1 ml virus (passage level 100) containing 10 6.0 tcid 50 /0.1 ml at 2 and 4 weeks prior to farrowing. the 24 colostrum samples were collected at delivery from each farrowing sow. moreover, two colostral samples were collected from control sows of each farm within 24 h after parturition. serum samples were obtained from the cranial vena cava, and colostrum samples obtained manually from 2 or 3 glands and pooled after im injection of 1 iu oxytocin. whey was obtained from colostral samples for use in a sn test. eight 3-day-old piglets were selected randomly from farrowing sows of farms d and e, and challenged using a similar method. non-vaccinated sows provided the source of control pigs in farms a, b, and c. control piglets (yorkshire · landrace · duroc) of farms d and e (seronegative against pedv, 1: 6 2 in sn test), which aged at 3days, were provided by yangsung laboratory animal inc (yongin, korea). all animal experiments complied with the current laws of korea. animal care and treatment were conducted in accordance with guidelines established by the seoul national university institutional animal care and use committee. antigen was prepared, as described previously, with minor modifications (kweon et al., 1999; oh et al., 2005) . briefly, dilutions of antigen and secondary antibody were adjusted to an optical density (od) of around 0.1 (a 405 ), using negative porcine sera. each well in a 96-well microplate (costar) was coated with 0.1 lg protein in 50 mm carbonate buffer (ph 9.6) at 5°c overnight, followed by blocking with 5% rabbit serum and 3% gelatin at 37°c. the 1/100 diluted sample porcine sera, colostrum and negative porcine sera (used for adjusting the antigen concentration) in pbs with 0.05% tween 20 (pbst) were reacted at 37°c for 1 h, and washed 5 times with pbst. the collection of positive and negative porcine sera was described previously . the reacted plate was re-washed under the same conditions, and incubated with 10,000-fold (igg) and 2000-fold (iga) diluted horseradish peroxidase (hrp)-labeled anti-porcine igg or iga (kpl) for 1 h at 37°c. the plate was developed in abts substrate (kpl) at room temperature for 20 min. the reaction was terminated with 2 m h 2 so 4 before od measurement at 405 nm. all samples were tested at the same time at the same plate in triplicate for minimizing intraassay variation. the sn test was performed using a previously published method, with some modifications (kusanagi et al., 1992) . the hyperimmune pedv reference serum was prepared from an antibody free and pedv-free 2-week-old pig inoculated with 1 ml of pedv, kpedv-9 strain (105.5 tcid50/0.1 ml) and then the serum was collected at 4 weeks post-inoculation. negative reference serum was also obtained from a mock-infected pig of the same age. briefly, swine sera were inactivated at 56°c for 30 min, and stored at à20°c until use. after twofold dilution, serum was mixed with pedv (200 tcid 50 /0.1 ml) at an equal volume, and incubated for 1 h at 37°c. subsequently, 0.1 ml of each virus-serum mixture was transferred to vero cell monolayers of a 96-well tissue culture plate washed twice with pbs. after adsorption for 1 h at 37°c, inocula were discarded, and washed twice with pbs. next, maintenance medium containing trypsin (2 lg/ml) was added to each well, and the plate incubated for 5 days at 37°c. sn titers were expressed as reciprocals of the highest serum dilution, resulting in cytopathic effect (cpe) inhibition. fecal samples from all groups were collected every day with cotton swabs (3 cotton tipped sticks, 15 cm long per pig) upto 10 days post-challenge. pedv was identified by the presence of the s gene in rt-pcr experiments (kim et al., 2001; song et al., 2006) . the following primers were employed for amplification of pedv: 5 0 -ttctgagtca-cgaacagcca-3 0 (forward), and 5 0 -catatgcag-cctgctctgaa-3 0 (reverse). the size of the amplified product was 651 bp. for the positive control, we utilized cell-attenuated pedv (kped-9 strain) provided by the virology lab, national veterinary research and quarantine service of korea. reverse primer was used for the synthesis of complementary dna. three-step procedures were performed in a thermal cycler (perkin-elmer, applied biosystems, inc., foster city, ca). samples were amplified using a program that consisted of: incubation at 94°c for 5 min; followed by five cycles of denaturation at 94°c for 30 s, annealing at 55°c for 30 s, and extension at 72°c for 30 s; and 30 cycles of denaturation at 94°c for 30 s, annealing at 53°c for 30 s, and extension at 72°c for 30 s, adding 1 s every cycle. at the completion of cycling, samples were kept at 72°c for 7 min and then cooled. specificity of pcr was evaluated using several causative agents of diarrhea, tgev, porcine rotavirus, and bovine viral diarrhea virus. 2.6. passage of dr13 (passage level 100) in colostrumdeprived 3-day-old pigs dr13 in piglets was passaged as reported previously, with some modifications (welter, 1998) . the rflp marker stability was assessed in vivo at passage level 100 of dr13. colostrum-deprived 3-day-old pigs were employed in three consecutive passages (3, 3 and 2 pigs in order), allowing one pig as a control for each passage. at 3 days pi, a pig was sacrificed, and duodenum, jejunum and ileum were collected for gross histopathology and rt-pcr detection of virus (kim et al., 2001) . to prepare an inoculum for the next passage, a 10% (w/v) tissue suspension of small intestine was additionally prepared. the 5 ml suspension was fed orally to the next pig passage. the hindiii and xhoii-specific restriction pattern of the rflp marker of shedding virus was analyzed, as described above. results were expressed as means ± sd of at least two independent experiments. all data of immune response (sn test) were converted into log 2 and used in statistical analysis. a one-way anova using sas version 8.0 software was employed for multiple comparisons, and a value of p < 0.05 was considered statistically significant. igg-specific elisa results revealed no significant differences between the od values of o-and im-inoculated groups from farms a and b. however, a difference in od values between vaccinated and control groups was evident. interestingly, in iga-specific elisa, significant od differences were observed between the sera of o-and iminoculated groups at delivery and colostrums (figs. 1a and b) (p < 0.05). in colostrums samples from farm a and b, a difference in od values between o and im group was evident (o group: 0.515 ± 0.13, im group: 0.226 ± 0.07). moreover, od values of all groups increased considerably after a second vaccination (fig. 1b ). an igg-specific elisa comparison of the od values obtained from serum with those of colostrum of corresponding sows at delivery disclosed slightly higher values in colostrum, regardless of the inoculation route. interestingly, in iga-specific elisa, the values of the o group were significantly increased in colostrum, compared to sera. in contrast, no major differences were observed between the two sample types (colostrum and sera) in the im group. furthermore, the antibody responses of sows in the sn test were distinct between samples of o and im groups in the b farm (figs. 2a and 2b). optical density (od) values of colostrum samples from pregnant sows (farm d and e) orally inoculated with cell-adapted pedv dr13 ranged from 0.409 to 0.733 (0.53 ± 0.11 from 24 colostrum samples), while those of the control group were from 0.129 to 0.155 (0.151 ± 0.02 from 4 colostrum samples). the mortality of piglets after challenge with wild-type pedv was 13% in the o-inoculated group (3/23), compared to 100% in the control group (14/14). in contrast, piglet mortality in the im-inoculated group was 60% (9/ 15). antibody responses of 3-day-old piglets before challenge are presented in figs. 3 and 4a. piglets delivered from oinoculated sows displayed a higher concentration of iga and sn titer than those from im-delivered sows (p < 0.05). however, no significant differences in the igg level were observed between piglets from the o-and iminoculated groups. there were no significant differences in virus shedding between the o-and im-inoculated groups. virus shedding in the ogroup lasted for 3-4.3 days after challenge. in the im group, shedding lasted for 4-5.3 days after challenge. however, the endpoint of virus shedding in the control group was not detected, due to mortality 3-6 days postchallenge. at the first passage, virus shedding lasted for 6 days, similar to data shown in table 1 . from the second passage, the duration of virus shedding was decreased to 4 days, and only lasted for 3 days in the third passage (table 1 ). the rflp marker of the dr13 inoculum was maintained during passage, as identified by restriction enzyme patterns (hindiii and xhoii). in three consecutive passages, inoculated pigs remained clinically and histopathologically normal with no villous atrophy. moreover, no reversion of virulence was observed during passage. examination of the immunoprophylactic effect in pregnant sows after oral inoculation disclosed decreased mortality of piglets from vaccinated animals, indicating that cell-adapted dr13 induces immunity status. previously, protective immunity was induced by an attenuated strain of pedv inoculated intramuscularly (kweon et al., 1999) . pedv dr13 was isolated through serial passage in vero cell cultures, and differentiated from the wild-type strainusing rt-pcr rflp with hindiii and xhoii enzymes (song et al., 2003) . moreover, attenuation and safety of pedv dr13 (passage level 100) after oral inoculation have been reported (song et al., 2003) . since lactogenic immunity protects suckling pigs from pedv infection, induction of mucosal immune responses in lactating pigs may be an effective way of protection. efficacy studies on similar enteric diseases, such as bovine coronavirus, revealed that the modified live oral vaccine significantly protected calves from highly virulent challenges with newborn calf diarrheal coronavirus isolates (welter, 1998) . highly attenuated pedv (orally inoculated) conferred partial protection against challenge with the virulent virus in conventional pigs. this protection is related to the inoculation dose, and is enhanced with increasing concentrations (de arriba et al., 2002) . in this study, we compare the efficacy of cell-adapted dr13 after inoculation via oral (o) and intramuscular (im) routes, using the sn test and elisa. discrepancies between data obtained with elisa and sn tests under our experimental conditions are possibly due to the addition of trypsin required for pedv propagation, which degrades antibodies (bae et al., 2003) . the correlation of sn titer and elisa was described previously (oh fig. 3 . antibody responses from serum samples against porcine epidemic diarrhea virus in 3-day-old piglets using igg and iga-specific elisa before challenge exposure ( * p < 0.05). fig. 4 . antibody responses from serum samples against porcine epidemic diarrhea virus in 3-day-old piglets using the sn test before challenge exposure ( * p < 0.05). 1 1-a à +/+ a +/+ +/+ +/+ +/+ +/+ à 1-b à +/+ +/+ +/+ b control à à à à à à à à 2 2-a à +/+ +/+ +/+ +/+ à à à 2-b à +/+ +/+ +/+ b control à à à à à à à à 3 3-a à +/+ +/+ +/+ à à à à control à à à à à à à à a pedv detection in feces using rt-pcr/rflp patterns (hindiii andxhoii) of the virus. b pigs 1b and 2b were sacrificed at day 3 after oral inoculation, and small intestines collected for preparing the next oral inoculum. et al., 2005) . briefly, the maximum agreement was obtained from the serum samples of 1: p 32 of sn titer, however, the least was from the samples of 1:4 of sn titer. the elisa has been shown to detect igg, including maternal antibodies at a very low level. in contrast, the sn test detects both igm and igg antibodies. the sn test can detect maternal antibodies once the titers drop to level (1:<16) that are more difficult to interpret because of possible nonspecific reactions that can interfere with the test. the protective effects observed with high titers of iga and sn from maternal colostrum are consistent with the findings of other reports . high titers of passive serum antibodies in colostrumfed conventional piglets are associated with reduced mortality, following inoculation of virulent viruses. the differences in mortality between the o and im inoculated groups may be due to the iga content in colostrum. in fact, the iga concentration before challenge exposure in piglets delivered from the o-inoculated group was higher than that in the im-inoculated group. the results indicate that iga plays a more central role in preventing ped than igg. moreover, there is a strong correlation between the iga concentration detected in blood and gut associated lymphoid tissue (de arriba et al., 2002) . similarly, yuan et al. (1996) reported that in pigs orally inoculated with rotavirus, the iga antibody secretion response in blood was analogous to that in the gut. in piglets, the extent of protection against pedv is based on the presence of specific iga antibodies in the milk of immune sows (de arriba et al., 1995) . after antigenic sensitization in the gut, iga immunocytes migrate to the mammary gland, where they localize and secrete iga antibodies into colostrum and milk. this ''gut mammary'' immunologic axis is an important concept in designing optimal vaccines to provide effective lactogenic immunity (saif et al., 1972) . sows that are immune after infection with pedv protect the suckling pigs through lactogenic immunity (pensaert et al., 1994) . virus specific antigen stimulation of lymphocytes occurs during intestinal infection in the sow. the stimulated cells subsequently circulate in the body, mature to plasma cells, and migrate to the gut and settle in other organs, such as mammary gland. secretory iga production occurs at these sites. lactogenic iga-producing cells are thus stimulated in the intestine but produce iga in the mammary gland itself. pigs that regularly suckle the immune mother are constantly provided with milk-bound iga antibodies in the lumen. this passive immunity is related to the presence of specific virus neutralizing antibodies in the gastrointestinal tract of suckling pigs. igg accounts for more than 60% of the ig in colostrum. however, iga is more effective to neutralize the orally infected pathogens than igg and igm because iga is more resistant to proteolytic degradation in the intestinal tract and has more virus neutralizing ability than igg or igm (offit and clark, 1985) . accordingly, we performed iga-specific elisa to elucidate the relationship between protection and iga content in colostrum. notably, variable piglet immunity is observed, according to the degree of colostrum uptake, litter size, antibody uptake, and quality of colostrum (song et al., 2003) . to determine the specific roles of iga and igg in preventing ped after vaccination, the changes in antibody concentrations in milk during lactation require investigation. the duration of virus shedding after virulent challenge is an indication of the protective level (yuan et al., 1998; ward et al., 1996) . three of the farms selected for the investigation were pedv-free and the piglets from those farms did not shed pedv before challenge. therefore, the virus shed after challenge exposure was possibly the same as inoculum with no rflp marker. earlier studies report that complete protection from pedv infection prevents virus shedding after challenge exposure (de arriba et al., 2002) . high titers of serum antibodies in piglets are related to reduced severity and duration of diarrhea, and shorter period of virus shedding . accordingly, virus shedding in piglets was assessed after challenge, using rt-pcr. no significant differences in duration of virus shedding were observed between o and im groups. pedv shedding may be variable, depending on the sensitivity of the detection tool. moreover, shedding may be influenced by the viral strain employed. in the case of cell attenuated dr13, virus shedding lasted for 8 days, and that in sows for 3 days . a comparison of the duration of virus shedding between vaccinated and control groups was not performed due to the death of control piglets after 3-6 days post-challenge. these results indicate that passive immunity by pedv dr13 does not prevent virus shedding after challenge. additionally, a study on reversion to virulence was conducted through serial pig passage. cell-adapted dr13 pedv remained safe, even after three passages in colostrumdeprived pigs. accordingly, we propose that cell-adapted dr13 may be applied as a vaccine candidate under commercial farm conditions, although there is rising concern over the possibility of confounding factors introduced by genetic variation or housing variables (hoblet et al., 1986) . in conclusion, cell-adapted pedv dr13 prevents mortality after challenge, and induces higher iga concentrations in colostrum. this elevated iga concentration may explain the reduced mortality in the o-inoculated group, compared to im-inoculated and control groups. further experiments, including lymphoproliferative response and antibody-secreting cells in lymphoid tissue of pigs after challenge, are required to improve pedv immunity in pigs. induction of antigen specific systemic and mucosal immune responses by feeding animals transgenic plants expressing antigen development of an elisa for the detection of antibody isotypes against porcine epidemic diarrhea virus (pedv) in sow's milk mucosal and systemic isotype-specific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhea virus experimental infection of pigs with a new porcine enteric coronavirus cv777 in vivo morphogenesis of a new porcine enteric coronavirus, cv777 efficacy of an orally administrated modified-live porcine origin rotavirus vaccine against postweaning diarrhea in pigs propagation of the virus of porcine epidemic diarrhea in cell culture the propagation of a porcine epidemic diarrhea virus in swine cell lines differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex rt-pcr isolation and serial propagation of porcine epidemic diarrhea virus infection in cell cultures and partial characterization of the isolate derivation of attenuated porcine epidemic diarrhea virus (pedv) as vaccine candidate mechanism in the pathogenesis of diarrhea: a review protection against rotavirus induced gastroenteritis in a murine model by passively acquired gastrointestinal but not circulating antibodies comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection porcine epidemic diarrhea enteric coronaviruses of animals isolation of porcine immunoglobulins and determination of the immunoglobulin classes of transmissible gastroenteritis viral antibodies transmissible gastroenteritis and porcine respiratory coronavirus differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf 3 fecal shedding of a highly cell adapted porcine epidemic diarrhea virus after oral inoculation in pigs use of an internal control in a quantitative rt-pcr assay for quantitation of porcine epidemic diarrhea virus shedding in pigs development of mucosal and systemic lymphoproliferative response and protective immunity to human group a rotavirus in a gnotobiotic pig model adaptation and serial passage of bovine coronavirus in an established diploid swine testicular cell line and subsequent development of a modified live vaccine an apparent new syndrome of porcine epidemic diarrhea systemic and intestinal antibody secreting cell responses and correlations of protective immunity to human rotavirus in a gnotobiotic pig model of disease antibodysecreting cell response and protective immunity assessed in gnotobiotic pigs inoculated orally or intramuscularly with inactivated human rotavirus key: cord-003503-t6cnjwpd authors: sung, ming-hua; lin, chao-nan; chiou, ming-tang; cheng, i-ju; thanh, quang-hien; chao, day-yu; lan, yu-ching title: phylogeographic investigation of 2014 porcine epidemic diarrhea virus (pedv) transmission in taiwan date: 2019-03-06 journal: plos one doi: 10.1371/journal.pone.0213153 sha: doc_id: 3503 cord_uid: t6cnjwpd the porcine epidemic diarrhea virus (pedv) that emerged and spread throughout taiwan in 2014 triggered significant concern in the country’s swine industry. acknowledging the absence of a thorough investigation at the geographic level, we used 2014 outbreak sequence information from the taiwan government’s open access databases plus genbank records to analyze pedv dissemination among taiwanese pig farms. genetic sequences, locations, and dates of identified pedv-positive cases were used to assess spatial, temporal, clustering, gis, and phylogeographic factors affecting pedv dissemination. our conclusion is that s gene sequences from 2014 pedv-positive clinical samples collected in taiwan were part of the same genogroup 2 identified in the us in 2013. according to phylogenetic and phylogeographic data, viral strains collected in different areas were generally independent of each other, with certain clusters identified across different communities. data from gis and multiple potential infection factors were used to pinpoint cluster dissemination in areas with large numbers of swine farms in southern taiwan. the data indicate that the 2014 taiwan pedv epidemic resulted from the spread of multiple strains, with strong correlations identified with pig farm numbers and sizes (measured as animal concentrations), feed mill numbers, and the number of slaughterhouses in a specifically defined geographic area. porcine epidemic diarrhea virus (pedv) causes acute diarrhea, vomiting, and dehydration, resulting in high mortality rates for suckling piglets [1] . since 2010, a new pedv variant belonging to genogroup 2 has spread throughout the united states and across multiple asian countries, including china and taiwan [2, 3] . an initial identification in the us was made in april of 2013 [4] . a 40.5% premises-level incidence of pedv caused the deaths of more than 8 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 million newborn piglets in a single year-an event that significantly affected the american swine industry [5] [6] [7] [8] [9] . subsequent genogroup 2 epidemics have been reported in major swineproducing countries such as canada, mexico, taiwan, korea and japan [8, [10] [11] [12] [13] . one of the most notable pedv outbreaks occurred in south korea in late 2013 [14] , more than a half million pigs died from pedv infections in japan between 2013 and 2016 [15] , and a significant increase in pedv outbreaks occurred in taiwan around the same time [16] . pedv is now considered the world's most catastrophic swine disease, with major financial impacts noted throughout the global pork industry. the pedv genome is comprised of at least seven open reading frames (orf1a, orf1b and orf2-6) encoding four structural (s), envelope (e), membrane (m), and nucleocapsid (n) proteins [17] . a high degree of genetic diversity has been observed in the s glycoprotein gene [18] [19] [20] . partial spike (s) polyprotein genes located in the virus envelope are central to pedv biological properties such as interactions with cellular receptors during virus entry, the neutralizing of antibody induction in natural hosts, growth adaptation in vitro, and virulence attenuation in vivo [19] . the pedv spike (s) protein is a type 1 transmembrane envelope glycoprotein with a 4,158 nucleotide sequence divided into s1 and s2 domains. the s1 region (aa 26-734) is responsible for viral binding, while the s2 domain (aa 735-1383) serves as an anchor for viral membrane and fusion activity [21, 22] . thus, the s glycoprotein is considered a primary target for pedv vaccine development. as the major envelope glycoprotein found in virion, s serves as an important viral component for studying genetic relationships among pedv isolates, and for determining pedv epidemiological status [23, 24] . pedv is believed to infect pigs by both direct and indirect fecal-oral routes. due to the scales and complexities of modern swine production systems, pedv is likely transmitted between farms via diarrheic feces or vomitus; contaminated environmental sources involving clinically or sub-clinically infected pigs; trailers used to transport livestock, manure, or food sources; farmers or visitors wearing contaminated clothes; or wild animals and birds [5, 25] . other potential sources include contaminated fomites (e.g., raw food, feed, sow milk), food ingredients or additives, and environmental features such as wind direction, farm altitude, terrain slope, and tree coverage [26] . after an initial outbreak, pedv may spread at an increasingly rapid rate due to inadequate farm hygiene management procedures such as improper disinfection and poor biosecurity. the virus can remain dormant in weaning pigs or growth finishing units, eventually triggering mild symptoms and resulting in low mortality rates [6] . although researchers believe that pedv infections primarily result via fecal-oral routes, the rapid regional spread of the disease raises the possibility of airborne transmission [4] . support for this hypothesis includes an identified correlation between disease-spread direction and prevailing wind direction [7] , with environmental features such as land coverage, altitude, and slope possibly influencing airborne disease dissemination [26] . to determine specific temporal and geographic relationships associated with pedv strain transmission, we used phylogenetic, phylodynamic and phylogeographic methods to systematically evaluate potential temporal and spatial transmission routes among taiwanese swine farms during the 2014 outbreak. epidemiological and geographic data were collected from 92 animal lab reports of pedv viral infections involving 8,557 pig farms, 37 pig feed mills, 57 slaughterhouses, and 5,806,237 animals. these reports are available from an open database maintained by the taiwan government. additional epidemiological and genetic information was gathered for purposes of determining details of the disease spread. a total of 48 global pedv whole genome sequences (s1 table) and 49 taiwan partial s1 gene sequences (648 nucleotide of pedv s1 gene position 1468-2115) (s2 table) were downloaded from genbank, and information for the tw4 whole genome sequence was collected from a previous study [27] . information datasets focused on pig feeding and disease were collected from the taiwan open data website (http:// data.gov.tw/), the animal health research institute of the taiwan executive yuan's agricultural council (http://eng.nvri.gov.tw/fmodule/default.aspx), and genbank (https://www.ncbi. nlm.nih.gov/genbank/). in the first stage of this study, pedv phylogenetic and phylogeographic data analyses were performed for purposes of organizing viral transmission evidence and tracking possible transmission routes. in the second, data for variables of interest associated with the pig feed industry were collected and combined with geographic information system (gis) data for investigation using open source quantum gis (qgis v3.2.3) software [28] . an sas mixed procedure was used to create linear regression models, with livestock breeding variables employed to predict pedv infections. gene sequences for the 49 global pedv whole genomes and 49 taiwan pedv partial s1 gene sequences were downloaded from genbank and aligned with a single taiwan pedv complete genome from a previous study [27] using the clustalw multiple alignment feature in bioedit [29] . the best-fit model, as determined using jmodeltest 2.1.7 [30] , had a gamma distribution (gtr+g+i). to ensure topological consistency, phylogenetic trees were constructed using maximum likelihood (ml) and bayesian methods (mega version 6.0 and beast v1.8.2, respectively). branch support was evaluated using bootstrap analyses based on 1,000 ml tree replications. bootstrap values >75% were considered as belonging to the same monophyletic group. to identify the specific locations of migration events, we grouped the 49 pedv isolates into different counties and used various pedv viral strains as references. spatial location reconstruction and viral migration activity were estimated using discrete coalescent tree and bayesian phylogeographic methods. bayesian markov chain monte carlo (mcmc) sampling using beast v1.8.2 [31] was employed to infer the time-scaled phylogenies of partial pedv s genes. hky+g and relaxed clock exponential models were used prior to setting coalescent population constants in the mcmc simulations. estimated convergence and effective sampling sizes were visually assessed using tracer v1.6. multiple chains were combined based on a 10% burnin using the version of logcombiner (v1.8.2) included in the beast package. maximum clade credibility trees with temporal and spatial annotations were summarized with the 10% burn-in removed using treeannotator (v1.8.2, also in the beast package). figtree (v1.4.2) was used to generate presentation figures. bayes factor (bf) tests were conducted to build statistical support for transmission routes among geographic locations using spread3 (v0.9.6; bf cutoff = 3) [32] . bf values were used to indicate differences between posterior and prior probabilities so that rates between any two locations were non-zero. routes with high bf values were considered as having greater potential for viral strain migration. to create animations of viral dispersion over time, annotated mcc trees were converted into a keyhole markup language (kml) file using spread3 (v0.9.6). the kml file can be visualized with an open-access earth map downloaded from natural earth (http://www. naturalearthdata.com) as a qgis software base layer. the maximum-likelihood phylogenetic tree (fig 1) was constructed from 49 global pedv whole genome sequences. sequences found in taiwan clustered with 100% bootstraps containing viruses from other asian countries and the us between 2011 and 2015. a correlation was determined between this cluster and the genogroup 2 originally identified in the us. among the taiwan sequences, some of the viruses collected and identified in 2014 were strongly correlated with one another (bootstrap values >75%), while others were mixed with viral strains collected in other countries at different times, suggesting multiple transmission events (fig 2 & table 1 ). to further evaluate transmission periods and to verify viral strain origins, all 49 partial s gene sequences determined from the taiwan pedv samples were used for phylodynamic data analyses (fig 3) . four clusters (a, b, c and d) exhibited statistically significant posterior probabilities (>0.8), including 9 viral strains on 4 monophyletic branches. the most recent common ancestor (mrca) for the 9 viral strains was traced to july 2013. all other coalescent tree sequences were determined as independent. viral branches had mrcas in 2013 or the spring of 2014, with none identified as statistically significant. thus, the phylodynamic coalescent tree established for this study indicates the involvement of multiple virus strains in the 2014 pedv outbreak. results from our phylogeographic analysis were matched to a map of the country to specify the geographic boundaries of the taiwan outbreak (fig 4) . next, molecular sequence data were combined with isolation time data and geographic coordinates to determine the spatiotemporal distribution of taiwan pedv strains. lineages were identified in several agricultural communities in the far south, with additional virus strains found in central taiwan at approximately the same time. the southern infections were spatially closer to each other. the data indicate an absence of natural and artificial barriers restricting the spread of the virus. the pig feed industry risk map shown was created to assist in the identification of significant risk factors associated with infections ( fig 5) . results from our gis system analysis of positive cases indicate correlations between pedv infection transmission and both pig farm size (number of pigs, not physical size) (0.000232, 95% ci [0.000102, 0.000362]) and slaughterhouse distribution (0.8043, 95% ci [0.4351, 1.1735]) ( table 2 ). our data also indicate higher infection rates in counties with fewer pig feed mills (-0.7857, 95% ci [-1.2298, -0.3415]). phylogeographic inferences are a potential tool for identifying the transmission and dissemination routes of pedv and other potentially much deadlier infectious diseases. however, to date very few research efforts in asia have utilized full genome sequencing for determining geographic structures due to the high costs and enormous amounts of computational time phylogeographic investigation of 2014 porcine epidemic diarrhea virus transmission in taiwan required for analyses [33, 34] . several researchers have suggested using partial s genes for phylogenetic tree construction and for phylodynamic analyses specifically aimed at studying the genetic relatedness of pedv strains [3, 10, 19] . in this study, we investigated temporal and geographic relationships among pedv strains identified as having been transmitted among farms in taiwan in 2014, using partial sequences from s genes extracted from porcine fetus samples and genbank sequences as reference panels. according to the phylogenetic tree we constructed based on these partial s gene sequences, the primary pedv strain in taiwan is related to the genogroup 2 strain identified in samples collected in the us in 2013 [35] . as previously suggested, the most recently identified taiwan pedv strains have greater similarity with us strains than with chinese or earlier taiwanese strains [3, 36] . transmission may have occurred as early as december 2013 [36] , but the crossborder route to taiwan remains unknown. results from our phylogenetic analysis of pedv viruses associated with the taiwan outbreak confirm independence in multiple counties. although these viruses share a common ancestry with the us genogroup 2 pedv, our coalescent tree data indicate that only 9 of the taiwanese viruses were significantly clustered (fig 3) . further, no recombination involving taiwanese strains was observed in the present study. five independent pedv strains were identified in taiwan on or before september 16, 2013 in wanda (km246707 and km246708) and jhutian (km246673, km246674 and km246675) townships. these are considered starting points for pedv complex dissemination throughout sections of taiwan up to may 15, 2014. according to a mix of phylogenetic and coalescent data ( table 2) , clustering was limited to 3 counties in southern taiwan (wanluan, jiouru and daliao) (fig 3) , with most of the identified viruses existing independently. combined, data for pedv divergence during the taiwan outbreak suggest a common ancestry shared by multiple virus lineages. since that time period, no evidence has been found indicating outbreaks involving multiple virus strains in taiwan. according to our phylogenetic analysis, most of the identified pedv strains from the 2014 outbreak were independent, despite sharing a common ancestor. multiple pedv invasions from abroad were also identified in japan during the 2014 outbreak [15, 37] . the widespread dissemination that followed presumably resulted from the movement of pigs, agricultural vehicles, farmers, farm visitors, commercial feed products, and other materials [15, 37] . reproducing novel approaches used in the molecular and spatial surveillance of the porcine reproductive and respiratory syndrome virus (prrsv) in the us and in pedv studies in japan [26, 38] , we utilized a combination of phylogeographic and gis approaches in our effort to profile the taiwan pedv outbreak. gis has been used to investigate correlations between diseases and factors that include pig farm size, number of feed mills, and number of pig slaughterhouses in a specified geographic zone (fig 5) . we used a mixed linear regression to identify factors associated with the number of pedv cases in taiwan. results indicate positive correlations between the number of cases and both slaughterhouse number and pig farm size, and a negative correlation with number of feed mills ( table 1 ). the highest concentration of pedv cases was identified in a multi-county section of southern taiwan characterized by a large number of pig farms with high animal densities. our results are in agreement with findings from previous studies suggesting that excessive farm capacity (measured as the total number of pigs on a farm) is a risk factor for the spread of pedv [5] . further, aerosol transmission is considered a viable dissemination route in environments marked by high pig densities and close animal proximities [4] . regarding the negative correlation between pedv cases and number of feed mills, our data indicate that pig farms at the end of feed routes likely have higher probabilities of infections. some reports suggest that vehicles used for the dual purposes of transporting swine to slaughterhouses and delivering feed to farms may increase the potential for pedv due to feedbag contamination [5, 25, 39, 40] . our finding of a high correlation between the number of pedv cases and the number of pig slaughterhouses suggests that transport trucks may have been a factor in the 2014 taiwan outbreak. other risk factors requiring further research include spray-dried porcine plasma (sdpp, an important blood-based component of nursery pig diets) and improper disposal procedures when pig corpses are collected and sent to rendering plants. there is a clear need to collect more data on feed truck routes, sdpp supplement distribution, rendering plant procedures (especially delivery), and slaughterhouse processes when trying to identify the sources of various strains in taiwan. due to the potential for significant financial losses, there is a strong need to act on these and other possible factors before detailed studies can be designed, funded, and completed. farms, slaughterhouses, and feed suppliers in counties with high pig densities need to immediately enhance their biosecurity measures to prevent future pedv outbreaks, and greater effort is required to monitor potential transmission routes. table. https://doi.org/10.1371/journal.pone.0213153.g004 supporting information s1 conceptualization: yu-ching lan. porcine epidemic diarrhea, diagnosis, and elimination 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pedv strains possessing an intact s gene in domestic pigs in japan: a new disease situation. plos one heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea detection and molecular diversity of spike gene of porcine epidemic diarrhea virus in china the s2 glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes cellular entry of the porcine epidemic diarrhea virus sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea coronaviruses: structure and genome expression role of transportation in spread of porcine epidemic diarrhea virus infection, united states. emerging infectious diseases land altitude, slope, and coverage as risk factors for porcine reproductive and respiratory syndrome (prrs) outbreaks in the united states molecular characterization of the porcine epidemic diarrhea virus tw4/2014 in taiwan. austin virology and retro virology qgis geographic information system. open source geospatial foundation bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt jmodeltest 2: more models, new heuristics and parallel computing bayesian phylogeography finds its roots spread: spatial phylogenetic reconstruction of evolutionary dynamics origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis distinct characteristics and complex evolution of pedv strains us-like strain of porcine epidemic diarrhea virus outbreaks in taiwan molecular characterization of pig epidemic diarrhoea viruses isolated in japan from 2013 to 2014. infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases spatial dynamics of porcine epidemic diarrhea (ped) spread in the southern kyushu, japan. preventive veterinary medicine an evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of naive pigs following consumption via natural feeding behavior: proof of concept an evaluation of a liquid antimicrobial (sal curb(r)) for reducing the risk of porcine epidemic diarrhea virus infection of naive pigs during consumption of contaminated feed key: cord-292690-1p1gnpgf authors: zang, yue; tian, ye; li, yungang; xue, ruixue; hu, liping; zhang, dong; sun, shengfu; wang, guisheng; chen, jing; lan, zouran; lin, shaoli; jiang, shijin title: recombinant lactobacillus acidophilus expressing s(1) and s(2) domains of porcine epidemic diarrhea virus could improve the humoral and mucosal immune levels in mice and sows inoculated orally date: 2020-08-16 journal: vet microbiol doi: 10.1016/j.vetmic.2020.108827 sha: doc_id: 292690 cord_uid: 1p1gnpgf porcine epidemic diarrhea (ped) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (pedv), which is characterized by a high mortality rate in piglets. since 2012, a remarkable growth in ped outbreaks occurred in many pig farms in china, landing a heavy blow on the pig industry. in order to develop a new effective vaccine for the current pedv, oral vaccines were generated by transferring eukaryotic expression recombinant plasmids carrying the s(1) and s(2) (antigenic sites of the s protein) epitopes of pedv into a swine-origin lactobacillus acidophilus (l. acidophilus). after oral immunization of the balb/c mice, higher levels of anti-pedv specific igg and siga antibodies and cellular immune responses were detected in mice orally administered with the recombinant l. acidophilus-s(1) compared to the l. acidophilus-s(2). furthermore, l. acidophilus-s(1) was used to inoculate the pregnant sows orally and the results showed that the recombinant l. acidophilus-s(1) could elicit a specific systemic and mucosal immune response. in summary, our study demonstrated that oral immunization with l. acidophilus-s(1) could improve the humoral and mucosal immune levels in sows and would be a promising candidate vaccine against pedv infection in piglets. porcine epidemic diarrhea (ped), caused by ped virus (pedv), is an acute enteric infectious disease characterized by severe vomiting, diarrhea, and dehydration (debouck and pensaert, 1980; pensaert and de bouck, 1978) . the spike (s) protein of pedv is a type 1 transmembrane envelope glycoprotein, which is responsible for the virus invading host cells through membrane fusion and mediating the production of neutralizing antibodies in infected hosts (lee et al., 2010) . based on the homology of the coronavirus s protein, it can be divided into two domains: s1 (1-789aa) and s2 (790-1383aa) (sun et al., 2006; follis et al., 2006) . there are four neutralizing epitope domains in the pedv s protein: coe (499-638 aa), ss2 (748-755 aa), and ss6 (764-771 aa) are located in the s1 domain, and 2c10 (1368-1374 aa) is in the s2 domain (chang et al., 2002; cruz et al., 2008; j o u r n a l p r e -p r o o f et al., 2008) . s1 is responsible for binding receptors and s2 is responsible for membrane fusion (gallagher et al., 2001; song et al., 2012; oh et al., 2014) . since 2012, outbreaks of ped have significantly increased in china, and the mortality rate of suckling piglets is as high as 90%-100%, which seriously hinders the healthy development of the pig industry (zhang et al., 2017) . due to immature immune system development in newborn piglets, vaccination with traditional vaccines does not produce protection in time, resulting in immune failure zhao et al., 2012) . to prevent intestinal infectious diseases in newborn piglets, an ideal strategy is for maternal antibodies to be produced in immunized pregnant sows and passively transferred to suckling piglets via colostrum . gut-associated lymphoid tissues (galt) are distributed in the pig intestine. the immunity induced at mucosal sites in the pregnant sow and passively transferred to suckling piglets via colostrum and milk (lactogenic immunity) is crucial for immediate protection of neonates against enteric infections. as recognized in previous studies of passive immunity to transmissible gastroenteritis virus (tgev), sows that recovered from tgev infection protected their exposed litters against tgev. this protection was associated with high levels of antibodies in the milk (lactogenic immunity), but not in the serum of the sows bohl and saif, 1975; saif et al., 1972; saif, 1999) . oral immunization can induce more iga-plasmablasts and t cells in the intestine. in addition to localized intestinal tracts, some of these cells migrate to the breast via the "intestinal-mammary-siga axis" to produce more siga and cytokinesin milk (song et al., 2007) . the presence of gastric acids and proteases in the gastrointestinal tract of animals affects the effectiveness of oral vaccines. in recent years, many scholars have studied how to improve the effect of oral immunization, and the application of antigen-presenting carriers is an effective strategy. as an important predominant bacterium in intestine, lactobacillus acidophilus (l. acidophilus) can not only effectively antagonize pathogenic bacteria in the digestive tract and maintain the intestinal microecological balance, but also be used as an antigen-presenting carrier in j o u r n a l p r e -p r o o f practical production. l. acidophilus is the candidate with the greatest potential for oral vaccines with a series of advantages (yu et al., 2013) . in a previous report, tetanus toxin was successfully constructed on a lactobacillus expression vector to be expressed as an immune antigen to induce the immune system to produce specific igg and siga antibodies in mice inoculated by nasal cavity (grangette et al., 2001) . in china, scientists have compared the effectiveness of lactobacillus carrier vaccines on piglets by using both oral and injection immunization routes. the results showed that oral administration of the vp1 gene of foot-and-mouth disease virus using lactobacillus as a carrier could produce a higher antibody titer than injection, and induce humoral and cellular immunity (li et al., 2007) . in this study, eukaryotic recombinant expression plasmids prc/cmv2-s1-rep.8014 and prc/cmv2-s2-rep.8014 carrying the s1 and s2 epitopes of pedv and replication gene rep.8014 of l. acidophilus were constructed and transformed into swine-origin l. acidophilus. subsequently, balb/c mice and pregnant sows were orally immunized with the recombinant l. acidophilus. the results showed that the recombinant l. acidophilus vaccine prc/cmv2-s1-rep.8014 could induce higher levels of humoral and mucosal immune response. the plasmid prc/cmv2 (sigma, germany) is a 5.5 kb vector designed for high-level stable (zhang et al., 2017) . the pcr products were identified and purified by gel electrophoresis. purified s1 and s2 genes and plasmid prc/cmv2 were subjected to a hind iii and not i double digestion separately and ligated by t4 dna ligase (tiangen, beijing, china). the recombinants prc/cmv2-s1 and prc/cmv2-s2 were sequenced and analyzed. the plasmid of pgem-t-rep.8014 (shandong provincial center for animal disease control and prevention, china) carrying replication gene rep.8014 of l. acidophilus (li et al., 2007) and two recombinants were then subjected to a not i and apa i double digestion separately and ligated by t4 dna ligase. the resulting recombinants prc/cmv2-s1-rep.8014 and prc/cmv2-s2-rep.8014 were then sequenced and analyzed. for detection of the displayed s1 and s2 of recombinant plasmids, ifa was used as described previously (xue et al., 2019) . in brief, the bhk-21 cells were transfected with the recombinant plasmids prc/cmv2-s1-rep.8014 and prc/cmv2-s2-rep.8014, as well as the negative control plasmid prc/cmv2 with vigofect transfection reagent (vigorous biotechnology, beijing, china), respectively. after incubation for 48 h, the ifa was conducted with the swine pedv-positive serum al., 2014) . in brief, l. acidophilus sw1 colony was inoculated into 3 ml of mrs broth (hopebiol, qingdao, china) for static cultivation at 37 °c with 5% co2 for 16 h and then further cultured for 2-3 h until the same situation was obtained (od600 0.1-0.2). cells (od600 0.2-0.3, incubation for 1-1.5 h) were chilled on ice for 10 min and washed twice with ice-cold epwb (5 mm sodium, 1 mm mgcl2, ph 7.4). a total of 10 μl of plasmid dna (prc/cmv2-s1-rep.8014 or prc/cmv2-s2-rep.8014, 200 ng/μl) was mixed with 100 μl of the ice-cold cell suspension in a 0.2 cm cuvette on ice for 10 min. bio-rad gene pulser (bio-rad laboratories, richmond, ca) was used for electroporation. meanwhile, the non-electroporated bacterial cells were set as a negative control. positive clones were confirmed with primers ls1 (f and r) and ls2 (f and r) by nucleotide sequencing. to detect the stability of the two recombinant plasmids in l. acidophilus sw1, the recombinant l. acidophilus-prc/cmv2-s1-rep.8014 (l. acidophilus-s1) and l. acidophilus-prc/cmv2-s2-rep.8014 (l. acidophilus-s2) were passaged five times in mrs broth without any antibiotics, and all the generations were detected by pcr with primers ls1 and ls2 respectively. spedv fragment concluding the coe (499-638 aa) and 2c10 (1368-1374 aa) epitopes, based on the nucleotide sequence of spike protein of the strain (genbank acc. no. jq282909), was synthesized after codon-optimization by genscript (piscataway, nj, usa). spedv was cloned into the pet-30a (qiagen, germany) plasmid dna vector, and then positive recombinant plasmids were transformed into escherichia coli (e. coli) bl21 (tiangen, beijing, china) for protein expression. briefly, the positive bl21 colony containing recombinant expression vector pet-30a-spedv was cultured in lb broth containing 5 μg/ml of kanamycin for 8-10 h at 37 °c until the od600 reached 0.6. iptg was then added to a final concentration of 1 mmol/l to induce the expression at 37 °c for 6 h. recombinant protein was purified as described previously (xue et al., 2019) . after sonication, the lysate was centrifuged at 8000 rpm for 10 min. the supernatant and precipitate were collected and j o u r n a l p r e -p r o o f submitted to sds-page and western blot as described previously (zhang et al., 2015) . sds-page was conducted on pre-made 12% polyacrylamide mini-gel run in a mini-protean electrophoresis system (bio-rad, ca, usa). the expressed soluble protein was purified according to the procedure of the his·bind® purification kit (novagen, usa) and determined according to the manufacturer's instructions of the bca protein assay kit (beyotime, shanghai, china). western blotting was conducted with swine pedv-positive serum as the first antibody and hrp-conjugated goat anti-swine igg antibody (biodee, beijing, china) as the second antibody. to detect the igg and siga antibodies specific to spedv epitope, indirect elisa methods were developed referring to published protocols with minor modifications (liu et al., 2010) . briefly, the optimal concentrations of serum and coating antigen were determined by a checkerboard titration; 96-well microtiter plates coated with two-fold diluted spedv protein antigen (from 12 μg/well to 0.325 μg/well, 100 μl/well) were incubated at 37 °c for 1 h and then 4 °c overnight in carbonate buffer (35 mm nahco3, 15 mm na2co3, ph 9.6). after washing, the wells were incubated with two-fold diluted mouse/pig pedv positive sera and negative sera (from 1:25 to 1:400, 100 μl/well) at 37 °c for 1 h. a total of 100 μl hrp-conjugated goat anti-mouse (pig) igg (iga)-specific antibodies (biodee, beijing, china) was added and incubated at 37 °c for 1 h after washing with pbst. o-phenylenediamine dihydrochloride substrate (100 μl/well) (biodee, beijing, china) was added and further incubated for 15 min. the reaction was terminated with stop solution (2 m h2so4, 50 μl/well) and the optical density (od) was read at 450 nm. the optimum sera titer and concentration of coating antigen were established with the chessboard test. a total of 24 six-week-old female balb/c mice (experimental animal center of shandong university, china) were randomly separated into 4 groups, and 6 ones in every group. the first group was orally immunized with 10 9 cfu recombinant l. acidophilus-prc/cmv2-s1-rep.8014 (l. acidophilus-s1) (100 μl of the suspension). the second group was orally immunized with 10 9 cfu j o u r n a l p r e -p r o o f recombinant l. acidophilus-prc/cmv2-s2-rep.8014 (l. acidophilus-s2) (100 μl of the suspension). the third group were subcutaneously immunized with 100 μl pedv commercial inactivated vaccine (lanzhou pharmaceutical factory of biology, china). the last group was orally administered with 100 μl sterile phosphate buffer saline (pbs). the induction of antigen specific serum igg and siga levels were measured by elisa. the assay procedures were the same as described in the indirect elisa section. all mice were boosted at 2 weeks post first immunization by the same strategy. sera for the detection of pedv specific antibody were collected via tail-bleeding at 7, 14, 21, 28, 35, and 42 days post immunization (dpi) and stored at −20 °c until used. fecal samples used for detecting siga antibodies were collected in the same periods and treated according to methods described previously with slight modification (liu et al., 2011) . cytokines ifn-γ and il-4 levels were measured by elisa according to the manufacturer's instructions (r&d systems, shanghai, china) . data were acquired on an automated elisa plate reader at od 450 nm immediately. the most effective vaccination group was used to test protective efficacy in the pig vaccination. pedv-seronegative, crossbred, pregnant sows were obtained from a local farm and all the sows were confirmed to be negative for pedv, from both pathogenic and serological tests by pcr (vipotion, guangzhou, china) and elisa (boyang, guangzhou, china), respectively. nine sows were randomly divided into three groups (3 ones in every group) and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. the first group was orally dosed with 2 ml of l. acidophilus-s1 (1×10 9 cfu/ml). the control groups of sows were orally dosed with 2 ml of pbs and subcutaneously immunized with an equal volume of pedv commercial inactivated vaccine. all pigs were first immunized at 0 dpi and received booster immunization at 21 dpi under the same conditions. sera samples were collected at 0, 7, 14, 21, 28, and 35 dpi. farrowing was induced at 16 days after the last vaccination. colostrum samples were collected on the day of farrowing and treated as described previously (adams and marteau, 1995) . the levels of anti-pedv specific igg antibody in the serum and siga antibody in the colostrum were determined by indirect elisa. piglets were allowed to suckle their dams and their sera samples collected on the 4th day after birth were tested by anti-pedv igg elisa. all of the data were analyzed a paired samples t-test in spss software. comparison of antibody titers and cytokine levels at each time point were conducted by a paired t-test and the values are presented as mean ± standard deviation (sd), with p < 0.05 and p < 0.01 considered as statistically significant and highly significant, respectively. all animal experiments were carried out in accordance with guidelines issued by the shandong agricultural university animal care and use committee (approval number, sdaua-2018-054). the prc/cmv2-s1 and prc/cmv2-s2 plasmids were obtained by amplifying and sub-cloning the s1 and s2 fragments into the prc/cmv2 vector, and the replication gene rep.8014 of l. acidophilus from pgem-t-rep.8014 was then subcloned into prc/cmv2-s1 and prc/cmv2-s2, resulting in prc/cmv2-s1-rep.8014 and prc/cmv2-s2-rep.8014. the expression levels of recombinants were assessed in vitro by ifa. the results showed that positive fluorescence signals were observed in the cytoplasm under confocal microscope, while no fluorescence was observed in the mock cells, indicating that the s1 and s2 epitopes were expressed successfully in bhk-21 cells ( figure 1a ). recombinant plasmids prc/cmv2-s1-rep.8014 and prc/cmv2-s2-rep.8014 were transformed into swine-origin l. acidophilus sw1 by electroporation assay. to determine whether the bacterial j o u r n a l p r e -p r o o f strains were carrying the recombinant plasmids after electroporation assay, the specific primers of s1 and s2 genes were used for colony identification. in order to test if prc/cmv2-s1-rep.8014 and prc/cmv2-s2-rep.8014 could be carried in recombinant l. acidophilus steadily, the positive l. acidophilus were cultured for five generations and analyzed through pcr, and the genes were detected as expected (data not shown). the 2300 bp and 1300 bp bands were observed by agarose gel electrophoresis and the results showed that recombinant plasmids from all five passages were carried in recombinant l. acidophilus steadily without any antibiotics in the media. to establish spedv-based indirect elisa, the protein was expressed and purified in bl21 (de3) figure 3 , the expression of the spedv epitope protein was assessed via sds-page; a band with the expected molecular mass of 16.9 kda was observed upon staining with coomassie brilliant blue (figure 1e ). the spedv protein was purified successfully with the his·bind purification kit (figure 1f) , and the concentration of the purified protein was 0.2 mg/ml referring to bca protein assay. the purified spedv protein was further identified by western blot using pedv positive swine serum (figure 1g) . a checkerboard titration was used to determine the optimal dilutions of antigen and serum. the optimal antigen concentration and serum sample dilution were set at 0.75 μg/well and 1:100, respectively. the levels of anti-pedv igg antibodies in mice induced by the recombinant l. acidophilus were determined by indirect elisa. compared to the pbs mock group, from the 14th day, significant levels of anti-pedv igg antibody (p < 0.01) were induced in mice that were orally administered l. acidophilus-s1 and l. acidophilus-s2 groups, and subcutaneously immunized with commercial inactivated vaccine group (figure 2a) . moreover, the igg antibody levels of the commercial inactivated vaccine group were obviously higher (p < 0.01) than those of oral vaccine groups between 14 dpi to 42 dpi, and the igg antibody level of the l. acidophilus-s1 group were higher (p < 0.05) than that of the l. acidophilus-s2 group. notably, compared to the pbs mock group and commercial inactivated vaccine group, the mucosal siga levels increased significantly (p < 0.01) after first immunization with pedv l. acidophilus-s1 and l. acidophilus-s2 vaccines ( figure 2b ). the pedv specific siga level of the l. acidophilus-s1 group was higher (p < 0.05) than that of the l. acidophilus-s2 group after 14 dpi, suggesting the l. acidophilus-s1 vaccine could efficiently induce mucosal immunity in mice. these results indicated that the oral recombinant l. acidophilus vaccines induced both humoral and mucosal immunity, and the oral recombinant vaccines induced much greater mucosal immunity and significantly less humoral immunity than that of the commercial inactivated vaccine (figure 2 ). the serum ifn-γ and il-4 levels of the three vaccine immunized groups began to increase after immunization and reached the peak at 35 dpi (table 1 and table 2 ). the serum ifn-γ level of the three vaccine groups was obviously higher than that of the pbs mock group (p < 0.01) between 14 dpi and 42 dpi (table 1, figure s1 ), while the il-4 level of the two oral vaccine groups was obviously higher than that of the pbs mock group (p < 0.01) between 28 dpi and 35 dpi (table 2) . though the serum ifn-γ and il-4 levels of the l. acidophilus-s1 group were higher than those of the l. acidophilus-s2 group during the whole test period, the differences between the two orally immunized groups were not significant (p > 0.05). the serum ifn-γ and il-4 levels of the commercial inactivated vaccine group was obviously higher than those of the oral vaccine groups between 35 dpi to 42 dpi (p < 0.01), but the differences between the two oral vaccine groups and the inactivated vaccine group were not significant (p > 0.05) at 7 dpi to14 dpi. to determine the immunogenicity of the recombinant l. acidophilus-s1, 9 pregnant sows were used for the program and the humoral immune responses were examined by indirect elisa. the l. acidophilus-s1 and commercial inactivated vaccine groups both showed growth in specific serum igg after the first and second vaccination (figure 3) , whereas no increasing trend was noticed in the control sow (p < 0.01). furthermore, in contrast to the l. acidophilus-s1 group, the commercial inactivated vaccine group demonstrated obviously higher serum igg levels at 35 dpi (p < 0.05). piglets that regularly suckle the immune mother receive colostrum/milk antibody, a process that transfers passive immunity to the piglets (makadiya et al., 2016) . therefore, we tested colostrum samples collected from the sows on the day of farrowing for the presence of pedv specific siga antibodies. the levels of siga antibodies were obviously higher in colostrum of the recombinant l. acidophilus-s1 group compared to the control and commercial inactivated vaccine groups (p < 0.01) ( figure 4a ). moreover, serum of piglets collected on day 4 after birth had also high pedv specific igg in the recombinant l. acidophilus-s1 group, while no specific antibodies were found in the control and commercial inactivated vaccine groups (p < 0.01) ( figure 4b ). to date, the most effective measure to control viral infectious diseases still depends on vaccination in china. ped is a highly infectious disease with intestinal tissue tropism. immunization with non-oral vaccines mainly stimulates the body to produce igg. igg in serum cannot neutralize the free pedv particles in the intestine and cannot prevent the invasion of pedv in the intestine (song and park, 2012) . oral vaccines cannot only stimulate the body to produce igg, but also stimulate the intestinal mucosa to produce siga, protecting pigs from infection (liu et al., 2012) . however, if a live-attenuated pedv vaccine is directly orally immunized, it will be damaged after entering the gastrointestinal tract. therefore, there are broad prospects for development of a live vector oral vaccine which can effectively stimulate mucosal immunity by simulating the natural infection route of pedv. lactobacillus is a normal intestinal bacterium and has adjuvant properties. it can combine immune adjuvant and target antigen to produce antigens and regulate the body's immune system for a long time to continuously stimulate the body's mucosal surface lymphocytes to produce specific antibodies (yu et al., 2013) . as is well known, the most important features of lactobacillus are its non-toxic side effects and safety in clinical use, and the entire surface of most lactobacillus is covered with a single crystal protein material. this structure can secrete proteins, so can be used as a secretory expression vector. lactobacillus-based vaccines will receive increasing attention. the mechanism by which the lactobacillus vaccine induces an immune response is unknown. in short, based on the outstanding advantages of lactobacillus itself, it will become an attractive engineering bacterium and represents a safe, emerging, and promising oral vaccine carrier (song et al., 2014; shi et al., 2014; yu et al., 2017; song et al., 2019) . in previous studies, a genetically engineered lactobacillus casei (l. casei) oral vaccine in china, outbreaks of ped have caused great economic losses to the swine industry in recent years zhang et al., 2017) . it is speculated that genetic mutations lead to less protective effect of the traditional vaccine. g3 group of pedv is the main predominant in china, and ss6 and coe domains in s protein displayed 2-aa or 3-aa mutations in most field strains of g3 group compared with traditional strain cv777 (zhang et al., 2017) . in this study, s1 and s2 genes were amplified from pedv ch-sdbz-1-2015 strain, which was located in g3 group. we developed two oral vaccines encoding s1 and s2 epitope domains of pedv spike protein, delivered by live l. acidophilus. the mice immunization results suggested that oral l. acidophilus vaccines j o u r n a l p r e -p r o o f were able to induce pedv specific humoral antibodies. moreover, pedv specific siga levels of oral l. acidophilus vaccine groups were significantly higher than the commercial inactivated vaccine group after 14 dpi, and the l. acidophilus-s1 vaccine efficiently induced mucosal immunity in mice compared to the l. acidophilus-s2 group (figure 2) . the serum levels of ifn-γ and il-4 indicated that our two oral l. acidophilus vaccines might mainly enhance a th1-type immune response to stimulate the cellular immune responses. although mice are not susceptible to pedv infection, good immune responses of the oral l. acidophilus vaccine in pigs were also observed in this study. sows vaccinated two times with l. acidophilus-s1 had higher igg antibody levels in the serum as compared to the control sows ( figure 3) . also, obviously higher levels of siga antibodies were found in the colostrum of the orally vaccinated sows. furthermore, maternal transferring of antibody was demonstrated, as only the serum of suckling piglets had higher levels of pedv specific igg. all data indicated that the oral recombinant l. acidophilus-s1 delivering the pedv specific antigens could act as a novel mucosal vaccine formulation and provide a useful strategy to induce efficient immune responses against pedv infection. in this study, the oral vaccines were designed as only s1 or s2 protein was expressed in recombinant l. acidophilus. in previous reseach, lc-expressed n protein as molecular adjuvant or immunoenhancer was able to effectively facilitate the induction of mucosal and systemic immune responses by lc-expressing s1 region (liu et al., 2012 ). an oral vaccine using l. acidophilus to co-express the s and n proteins of pedv should be developed to yield better immune efficacy. in summary, an oral vaccine strategy using l. acidophilus to deliver s1 and s2 epitopes of pedv spike protein was explored to develop an anti-pedv vaccine for oral administration in this study. we demonstrated that the genetically engineered prc/cmv2-s1-rep.8014 could efficiently induce mucosal, humoral, and cellular immune responses against pedv, suggesting a promising vaccine strategy. the authors declare that they have no competing interests. antibody responses in serum, colostrum, and milk of swine after infection or vaccination with transmissible gastroenteritis virus passive immunity in 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bacteria: a comparative survey heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea immune responses generated by lactobacillus as a carrier in dna immunization against foot-and-mouth disease virus molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field strains in south china development of an indirect elisa for the detection of duck circovirus infection in duck flocks comparison of the immune responses induced by oral immunization of mice with lactobacillus casei-expressing porcine parvovirus vp2 and vp2 fused to escherichia coli heat-labile enterotoxin b subunit protein high-level mucosal and systemic immune responses induced by oral administration with lactobacillus-expressed porcine j o u r n a l p r e -p r o o f epidemic diarrhea virus (pedv) oral recombinant lactobacillus vaccine targeting the intestinal microfold cells and dendritic cells for delivering the core neutralizing epitope of porcine 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lactisexpressing the vp1 protein of duck hepatitis a virus type 3 induces mucosal and systemic immune responses adjuvant effects of l. acidophilus lw1 on immune responses to the foot-and-mouth disease virus dna vaccine in mice spike protein region (aa 636789) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein oral delivery of probiotics expressing dendritic cell-targeting peptide fused with porcine epidemic diarrhea virus coe antigen: a promising vaccine strategy against pedv efficacy and immunogenicity of a live l. acidophilus expressing sad epitope of transmissible gastroenteritis virus as an oral vaccine faeg in conjunction with a heat-labile enterotoxin a (ltak63) and heat-labile enterotoxin b (ltb) of enterotoxigenic escherichia coli as an oral adjuvant in mice mucosal lactobacillus vectored vaccines identification of a conserved neutralizing linear b-cell epitope in the vp1 proteins of duck hepatitis a virus type 1 and 3 two distinct genotypes of porcine epidemic diarrhoea virus in vaccinated pig flocks in shandong province of china complete genome sequence of a vero cell-adapted isolate of porcine epidemic diarrhea virus in eastern china this study was funded by the provincial natural science foundation of shandong, china (zr2017bc104). different lowercase letters denote differences among groups (p<0.05) between the assays for the same immune time. different uppercase letters denote significant differences among groups (p<0.01) between the assays for the same immune time.j o u r n a l p r e -p r o o f different lowercase letters denote differences among groups (p<0.05) between the assays for the same immune time. different uppercase letters denote significant differences among groups (p<0.01) between the assays for the same immune time. key: cord-260835-ck9z5xsd authors: kamau, anthony ndirangu; park, jung-eun; park, eui-soon; yu, jung-eun; rho, jaerang; shin, hyun-jin title: porcine amino peptidase n domain vii has critical role in binding and entry of porcine epidemic diarrhea virus date: 2017-01-02 journal: virus res doi: 10.1016/j.virusres.2016.10.004 sha: doc_id: 260835 cord_uid: ck9z5xsd porcine epidemic diarrhea virus (pedv) infects swine intestinal cells causing enteric disease. research has shown that the entry into these cells is through porcine aminopeptidase n (papn) receptor. to gain insights into mechanisms of pedv-papn interactions, the present study aimed at identifying the domain that is critical for pedv binding. to this end, nih3t3 cell lines constitutively expressing papn or papn mutants were generated. the mutants were; domain vii deletion mutant and domains iv–vi deletion mutant. in the latter, domain vii was linked to the transmembrane segment through domain iii. results showed pedv infection was restricted to papn and papn domain vii expressing nih3t3 cells. further, reducing pedv titre 10 fold resulted in 37.8% decrease in foci indicating positive correlation. a time course test at 12, 24, 36, 48 and 60 h showed that foci increased 6 fold in the overall time range. also, pedv harvested from papn or domain vii expressing nih3t3 cells was induced indirect plaques in vero cells confirming successful entry and replication. collectively, our results demonstrate that pedv recognizes papn and that the main interactive point is lodged within domain vii of the papn. these findings are important for therapeutic development as well as creating a platform for future studies on pedv. coronaviruses (covs) are enveloped, positive-stranded rna viruses potentially pathogenic in respiratory and intestinal tracts of humans, animals and birds (david and kathryn, 2007; susan and navas-martin, 2005) . analyses by serological cross-reactivity and genome phylogeny distinguish these viruses into four genera which include alpha, beta, gamma and delta-cov (de groot et al., 2011) . studies have speculated alpha and beta-covs to have originated from bats whereas gamma and delta-covs gene source is the birds (woo et al., 2012) . pedv belongs to the genus alpha-cov (de groot et al., 2011) . host specificity, tissue tropism and virulence of covs are dictated by interactions between the spike glycoproteins of the viruses and cellular glycoproteins on surface receptors. receptor binding and cell entry is essential steps for virus entry into host cell, which leads to internalization of the viruses (park et al., 2014; liu et al., 2015) . an envelope-anchored spike protein mediates coronavirus entry into cells (liu et al., 2015) . the spike ectodomain consists of a receptor-binding subunit, s1, and a membrane fusion subunit, s2. two domains in s1, calls s1-ntd and s1-ctd, can potentially function as receptor-binding domains (li, 2012 (li, , 2015 in some covs, entry mechanisms progress via sialic acid interactions with spike and/or hemagglutinin esterase glycoproteins (haijema et al., 2003; kuo et al., 2000; phillips et al., 1999; sanchez et al., 1999) . the spike protein of murine hepatitis virus (mhv) binds carcinoembryonic cell adhesion molecule 1a for cell penetration while human (hcov-nl63) and severe acute respiratory syndrome (sars)-cov recognize human angiotensin-converting enzyme 2 (hace-2) on host cells (dveksler et al., 1991 (dveksler et al., , 1993 . aminopeptidase n (apn) serves as receptor for feline (fcov), canine (ccov), transmissible gastroenteritis virus (tgev), pedv as well as human (hcov -229e) (delmas et al., 1992; tresnan et al., 1996; yeager et al., 1992; kolb et al., 1998; li, 2015; liu et al., 2015) . apn/cd13 is a 150 kda, zinc dependent metalloprotease consisting of 967 amino acids (rawlings and barrett, 1995) . it is ubiquitously present in diverse organs, tissues and cell types (luan and xu, 2007) . it is expressed as a glycosylated homodimer on the surface of epithelial cells of liver, intestine, kidney and respiratory tract fibroblasts and leukocytes (kenny and maroux, 1982; lendeckel et al., 2000; nam and lee, 2010) . additionally, apn is found on endothelial cells such as cerebral astrocytes as well as pericytes and synaptic membrane in the central nervous system (kay et al., 1994; soichiro et al., 1983; mina-osorio et al., 2008) . apn cleaves the n-terminal amino acids from peptides. in addition, apn plays role as a receptor and signaling molecule (mina-osorio et al., 2008) . a seven domain organization has been described for apn structure. the first domain constitutes a short cytoplasmic tail on the n-terminal end (amino acids [aa] 1-7). domain ii (aa 8-39) consists of the transmembrane segment and links to a stalk forming domain iii (aa 40-70). domain iv spans from aa 71 to 252. the peptidase activity is housed by domains v and vi (aa 253-580). located on the c terminus is domain vii consisting of aa 581-967 (sjostrom et al., 2000) . previous investigations by virus overlay protein binding assay showed that pedv bound papn in permissive cells and it was also shown that anti-papn blocked this binding (oh et al., 2003) . further, expression of papn in non-permissive madin-darby canine kidney cells resulted in susceptibity of these cells to pedv (li et al., 2009) . however, pedv fails to infect native porcine apn expressing swine testis cells which are highly susceptible to tgev (hofmann and wyler, 1988 ). in the current study, by constitutive expression of papn in non-susceptible nih3t3 cells, we have sought to annotate the previous findings stipulating papn receptor function for pedv. in addition, this study aimed at identification of using papn protein domain(s) that are critical for its binding and entry. vero cells were cultured in alpha minimum essential medium ([␣-mem] gibco). mouse embryonic fibroblasts (nih3t3) and human embryonic kidney (hek 293t) cells were cultured in dulbecco's modified eagle medium ([dmem] gibco). all growth media were supplemented with 10% fetal bovine serum ([fbs] gibco). the pedv strain used in this study was korean isolate. the clones used in this study are illustrated (fig. 1b) . three clones were designed and denoted as follows; (1) wild type papn (papn wt ), (2) domain vii deletion mutant of papn (papn di-vi ) and (3) domains iv-vi deletion papn mutant (papn dvii ). these were amplified by pcr (horton, 1993) using pfu polymerase (solgent). primers were designed using genbank sequences (accession number hq824547) and all antisense primer sequences were fused in frame to flag epitope sequence. the wild type papn was amplified using sense 5 -ccc aag ctt acc atg gcc aag gga ttc tac-3 and anti-sense 5 -ccc ctc gag tca ctt gtc gtc atc gtc ttt gta gtc gct gtg ctc tat gaa cca-3 primers. mutants papn di-vi pcr primers were sense 5 -ccc aag ctt acc atg gcc aag gga ttc tac-3 and anti-sense 5 -ccc ctc gag tca ctt gtc gtc atc gtc ttt gta gtc gct gtg ctc tat gaa cca-3 while papn dvii was amplified using 5 -cgg tac cgc gcc tcg gtt atc agg atg ctc-3 and 5 -ccc ctc gag tca ctt gtc gtc atc gtc ttt gta gtc gct gtg ctc tat gaa cca-3 sense and anti-sense primers respectively. the cdnas encoding papn wt and mutants were cloned into the hind iii and xho i sites of pcdna 3.0 (invitrogen). hek 293t cells were transfected using plasmid constructs containing papn clone or mutants cdna using fugene hd (roche) according to the manufacturer. at 24 h after transfection, cells were assayed for papn expression by immunoblotting using mouse anti-papn polysera and anti-flag antibody (santa-cruz). extracellular expression of papn was determined by detection of flag epitope. cells were harvested using cells dissociation solution (sigma). these were washed two times in 2 ml of 1 x phosphate buffered saline (pbs), centrifuged at 300 × g for 5 min and supernatants were discarded. cells were resuspended in 100 l of staining buffer (1 x pbs containing 2% fbs) and surface antigens were stained with anti-flag antibodies. after 2 times wash with 2 ml of 1 x pbs and re-suspension in 100 l staining buffer, cells were incubated with phycoerythrin (pe) conjugated secondary antibody (bd biosciences) and analyzed by flow cytometry (facs calibur, becton-dickinson). nih3t3 cells were transfected with pcdna3.0 with or without papn wt or mutants using fugene hd (roche) as suggested by the manufacturer and 24 h after transfection, cells were subjected to 400 g/ml g418 selection pressure. the cell lines generated were designated as follows; nih3t3 stably harboring only vector (nih3t3 pcdna3.0 ), expressing papn wt (nih3t3-papn wt ), expressing papn di-vi (nih3t3-papn di-vi ) and papn dvii mutant (nih3t3-papn dvii ), respectively . after antibiotic selection, the cells were analyzed for expression by western blot. the kpedv-9 was propagated in vero cells using serum free ␣-mem in the presence of 10 g/ml trypsin (sigma) as described (oh et al., 2003) . for the quantitation of virus, plaque assay was performed as described (dulbecco, 1952) . briefly, vero cells (2.5 × 10 5 cells/ml) were seeded in a 6-well plate 24 h prior to inoculation. cells were washed with 1 ml of 1x pbs and 200 l of inoculum was added. after 1 h adsorption, inoculum was removed and cells were overlaid with serum free ␣-mem containing 0.9% methylcellulose and 10 g/ml trypsin. these were incubated at 37 • c with 5% co 2 until plaques formed. the overlay medium was discarded and cells washed with 1 ml 1x pbs then fixed with 2 ml of fixation solution (5% methanol and 10% acetic acid) for 30 min at room temperature. cells were washed with 1 ml of 1x pbs and incubated with 1 ml of 1% crystal violet for 20 min at room temperature. the dye was then discarded and cells washed were gently with distilled water then air-dried. a plaque count was conducted to deduce the number of plaque forming units (pfu) per ml of virus suspension. this virus culture technique was adapted for inoculation of nih3t3 cells with pedv at a multiplicity of infection (m.o.i.) of 0.06-0.6. the inocula were removed after 1 h adsorption and replaced with fresh medium. after various time periods after inoculation, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% triton x-100. cells were blocked with 4% skim milk for 30 min at room temperature and detection of viral antigens was performed using mouse anti-pedv polysera for 1 h at 37 • c. after 3 times wash with 200 l of 1 x pbs, cells were incubated with horseradish peroxidase (hrp) -conjugated anti-mouse igg (santa-cruz) for 1 h, 37 • c. the cells were washed 3 times with 1 x pbs. reaction was visualized by addition of 3,3 -diaminobenzidine chromogenic substrate (vector lab). pedv titer attained in nih3t3 cell types was analyzed indirectly in vero cells as previously described (yu et al., 2010) . nih3t3 cell lines expressing or not expressing surface papn wt or mutants were seeded at a density of 2.5 × 10 5 per ml. at 24 h post inoculation (p.i.) with pedv at m.o.i. 0.6, virus was harvested after by 3 cycles of freezing-thawing. the supernatants were re-inoculated into vero cells along with pedv control inoculation. since dead or defective virus particle are incapable of forming plaques, virus viability in nih3t3 cell lines was verified by plaque assay. western blot assays using anti-flag antibody and mouse anti-papn polysera revealed papn wt was expressed at approximately 150 kda in both 293t and nih3t3 cells ( fig. 2a and b) . additionally, surface expression was confirmed by flow cytometry using anti-flag and p.e. labeling. the results confirmed 47.88% (293t) and 62.23% (nih3t3) surface expression ( fig. 2c and d) . to investigate susceptibility of nih3t3-papn wt , pedv was inoculated into nih3t3-papn wt , wild type nih3t3 and nih3t3 pcdna3.0 . although the typical cytopathic effects (cpe) such as cell rounding and syncytia formation were not observed in nih3t3-papn wt cells, detection of pedv antigens in fixed cells showed stained foci, but not in wild type nih3t3 cells nor in nih3t3 pcdna3.0 (fig. 3a) . this result confirmed that papn wt is important for pedv infection. further, pedv was serially diluted by a factor of 10 and inoculated to nih3t3-papn wt . comparison of pedv at m.o.i. 0.6 and 0.06 showed that number of foci was positively correlated to concentration (fig. 3b ). the number of foci formed in nih3t3-papn wt at an m.o.i of 0.6 reduced by 37.8% at an m.o.i. of 0.06. although nih3t3-papn wt was susceptible to pedv, the number of progeny virions produced was much lower than those in vero cells. to identify papn wt protein domain(s) important for pedv binding, deletion mutants were generated and their stable expression in nih3t3 was verified by western blotting. as shown in fig. 4 , papn dvii and papn di-vi expression was detected at 61 kda and 55 kda, respectively. susceptibility assays showed infection in papn domain-vii expressing cells but virus entry was abrogated in absence of seventh domain (papn di-vi ) as shown in (fig. 5a) . also, there was no observable infection of both nih3t3 wild type and nih3t3 pcdna3.0 controls. analysis of infection between 12 and 60 h p.i. showed that the average number of foci increased more than 6 times in nih3t3-papn dvii . similarly, pedv induced foci increased more than 6 fold over time (12 h-60 h) in papn wt expressing cells. also notable was the increase in foci spread through time especially in nih3t3-papn wt (fig. 5b) . lysates of pedv or mock-infected nih3t3 cell lines were re-inoculated in vero cells for the indirect plaque assay. nih3t3-papn wt and nih3t3-papn dvii lysates induced plaques but nih3t3, nih3t3 pcdna3.0 and nih3t3-papn di-vi lysates did not (fig. 6a) . on quantification, nih3t3-papn wt had more plaque forming particles (992 pfu/ml) on average while nih3t3-papn dvii lysate had 725 pfu/ml (fig. 6b) . the hallmark of coronaviruses is the spike glycoprotein which is their functional attachment molecule to the surface of host cells. as of fact and characteristic of all viruses, the attachment involves definite interactions with precise receptors which determine host specificity and tissue tropism (sandrine et al., 2012) . the extensively studied coronavirus prototype, mhv is able to penetrate the hepatocytes through the biliary glycoprotein on the surface of the liver and cause hepatitis (godfraind and coutelier, 1998) . in alphacoronaviruses, papn on the microvillar membrane of the small intestine is the known gateway for tgev infections in swine (delmas et al., 1992 (delmas et al., , 1994 while human apn (cd13) is the attachment receptor for hcov-229e which targets infections to lungs where it has been associated with common cold or severe pneumonia in immunocompromised persons (lachance et al., 1998; pene et al., 2003) . apn/cd13 is a monomeric or homodimeric type ii membrane-bound zinc-dependent metalloproteinase, which cleaves neutral or basic amino acids from the n-terminus of oligopeptides and can release neutral and basic amino acid from the n-terminal of peptides (mou et al., 2009) . it is widely expressed on various cells such as epithelial cells of the intestine and kidney, hepatocytes, osteoclasts, endometrial cells, fibroblasts, endothelial cells, bone marrow stromal cells and on neuronal synaptic membranes. apn is involved in many physiology and pathology processes such as hydrolysis of nutrients, inactivation of bioactive peptides, inflammatory bowel diseases, rheumatoid arthritis and cancer (bank et al., 2008; yamashita et al., 2007; shimizu et al., 2002) . in 2006, the 3d structure of apn has been studied according to the co-crystal complex of apn and bestatin by ito (ito et al., 2006) . based on this report on overall structure and subunits of apn, it is composed of consists of 26 ␣helices and 26 ␤-strands and can be divided into four domains, an n-terminal ␤-domain (met 1 -asp 193 ), a catalytic domain (phe 194 -gly 444 ), a middle ␤-domain (thr 445 -trp 546 ), and a c-terminal ␣-domain (ser 547 -ala 870 ). they also reported the c-terminal ␣domain might be important as the active site. more recently, shan et al. reported the protein structure on papn by swiss-model web server (shan et al., 2015) . they reported papn contains small intra-cellular domain and large extra cellular domain divided into 3 subdomains named spa, spb and spc. in virology, it has been reported that apn is involved in binding of corona viruses and mediating cytomegalovirus infection and antigen presentation (shahwan et al., 2013; regan et al., 2012; li et al., 2007; kasman, 2005) . it has been established that tgev binds the papn receptor on the surface of swine enterocytes (delmas et al., 1992 (delmas et al., , 1994 . additionally, tgev along with ccov, tgev and hcov-229e exhibit cross-binding activities to feline apn (fapn), which is the major receptor for feline infectious peritonitis virus and it has been demonstrated that these viruses can utilize this receptor to infect fapn-expressing bhk21 and nih3t3 cells (tresnan et al., 1996; tusell and holmes, 2006; tusell et al., 2007) . pedv was also reported to interact with papn in porcine enterocytes (li et al., 2009) , and is capable of infecting mdck and st cells expressing the papn receptor (oh et al., 2003; nam and lee, 2010) . more recently, li et al. reported papn is important receptor for pedv infection in swine small intestine (iecs). they found higher expression increased the susceptibility by pedv, and this was confirmed by sirna inhibition results. they also found pedv enters via apical plasma membrane and released from there in polarized epithelial cells (cong et al., 2015) . other group reported that porcine and human apn serve as efficient receptor for pedv infection (li, 2015) . they found although tgev spike protein bound porcine apn much more tightly than human apn, pedv spike protein efficiently bound both porcine and human apn. they also reported that neu5ac as the pedv coreceptor. very recently, shirato et al. reported papn is not functional receptor for pedv but promotes the its infection through protease activity (shirato et al., 2016) . taken together it is acceptable that papn as the pedv receptor still little bit controversial. because pedv infects porcine enterocytes causing intestinal lesions that are indistinguishable from those caused by tgev (ducatelle et al., 1982; hooper and haelterman, 1969) , it is conceivable that the two viruses may recognize the same receptor. curiously, tgev readily infects porcine primary cells whereas attempts to isolate pedv in both primary and secondary fetal porcine cells have not been successful (hofmann and wyler, 1988; kusanagi et al., 1992) . this ambiguity is further compounded by the fact that vero cells, with no surface papn, are susceptible to pedv (hofmann and wyler, 1988) . to clarify these doubts, the current study employed the nih3t3 cells which are non-susceptible to pedv infection. by constitutive expression of papn wt on these cells, the receptor function would be established by the conversion of the cells into a susceptible cell line (nih3t3-papn wt ). our results demonstrated that there was extracellular expression of papn wt with a surface presence of 62.23% by flow cytometry. whether this surface receptor would allow pedv entry was investigated by infection of the cells along with experimental controls, nih3t3 wt and empty vector harboring cells (nih3t3 pcdna3.0 ). results of our analyses showed that although cytopathic effects such as cell rounding and or syncytia formation were not vividly evident, pedv antigens were detectable immunocytochemistry. in addition, the number of foci formation was positively correlated with the virus titer and the time allowed for virus replication. this development could only be attributed to the papn wt because the wild type nih3t3 and nih3t3 pcdna3.0 cells were not permissive to pedv infection. since nih3t3 cells being fibroblasts, have a morphology that may not allow close association between cells, spaces in between cells may have resulted in the lack of syncytia or plaque formation in nih3t3. in addition, pedv has already been adapted to vero cells and it is assumable that nih3t3 may be an unfamiliar environment for pedv replication. indeed, even new isolates of pedv are able to show distinct signs of cpe in vero cells only after several passages (pan et al., 2010) , which could probably be an adaptation period. as the functional domain study on papn, shan et al. reported that spc region on papn might be putative functional domain for pedv infection (shan et al., 2015) . they found mdck cells expressing only spc region successfully infected by pedv and even shown cytopathic effects (cpe). they also reported that pedv titer in mdck cell expressing only spc region generated same number of virus in papn expressing mdck and vero cells. in our study, to determine the specific interactive point of papn wt with pedv, domain mutants were expressed stably in nih3t3 cells. the cloning strategy of the mutants followed cues that coronaviruses identify certain intermittent motifs on fapn which are amino acids (aa) 288-290, aa 732-746 (called r1), and aa 764-788 (called r2) which determine their tropism (tusell et al., 2007) . thus, two mutants; (david and kathryn, 2007) expressing domains i-vi of papn wt and (susan and navas-martin, 2005) expressing domain vii of papn wt were constructed and designated papn d1-vi and papn dvii, respectively. nih3t3 stable cell lines expressing these mutants were assayed for their susceptibility to pedv and similar to tgev (delmas et al., 1994) , pedv successfully infected papn dvii expressing cells but that was abrogated in the absence of the seventh domain (papn di-vi ). also noteworthy, pedv harvested from nih3t3-papn wt and nih3t3-papn dvii induced plaques on vero cell monolayers indicating virus entry and propagation in those cells. taken together, all our data confirmed that pedv replicated in both nih3t3-papn wt and nih3t3-papn dvii suggesting successful entry into the cell lines. we therefore concluded that domain vii of papn is critical for pedv entry. although structural domain analysis on papn used in this study is little bit different from those from shan et al. study, but functional binding domain from both studies are identical. especially, our results promising because we studied stable expression of wild type and deletion mutants from stable cell lines expressing those proteins. even if our studies confirmed with stable cell lines but still don't know how those mutations on papn affected on its stability, 3-d structure and especially function. still more studies required to answer on those questions. inflammatory bowel diseases: multiple benefits from therapy with dipeptidyl-and alanyl-aminopeptidase inhibitors porcine aminopeptidase n mediated polarized infection by porcine epidemic diarrhea virus in target cells coronavirus binding and entry virus taxonomy: ninth report of the international committee on taxonomy of viruses aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-n that is distinct from the enzymatic site pathology of experimental cv777 coronavirus enteritis in piglets. ii. electron microscopic study production of plaques in monolayer tissue cultures by single particles of an animal virus 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 morphological analysis of mouse hepatitis virus a59-induced pathology with regard to viral receptor expression switching species tropism: an effective way to manipulate the feline coronavirus genome propagation of the virus of porcine epidemic diarrhea in cell culture lesions of the gastrointestinal tract of pigs infected with transmissible gastroenteritis soeing together tailor-made genes crystal structure of aminopeptidase n (proteobacteria alanyl aminopeptidase) from escherichia coli and conformational change of methionine 260 involved in substrate recognition cd13/aminopeptidase n and murine cytomegalovirus infection localization of aminopeptidase n and dipeptidyl peptidase iv in pig striatum and in neuronal and glial cell cultures topology of microvillar membrance hydrolases of kidney and intestine molecular analysis of the coronavirus-receptor function of aminopeptidase n retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate involvement of aminopeptidase n n (cd13) in infection of human neural cells by human coronavirus 229e the role of membrane peptidases in immune functions porcine aminopeptidase n is a functional receptor for the pedv coronavirus porcine aminopeptidase n is a functional receptor for the pedv coronavirus evidence for a common evolutionary origin of coronavirus spike protein receptor-binding subunits receptor recognition mechanisms of coronaviruses: a decade of structural studies receptor usage and cell entry of porcine epidemic diarrhea coronavirus the structure and main functions of aminopeptidase n cd13 is a novel mediator of monocytic/endothelial cell adhesion design, synthesis and primary activity evaluation of l-arginine derivatives as amino-peptidase n/cd13 inhibitors contribution of the porcine aminopeptidase n (cd13) receptor density to porcine epidemic diarrhea virus infection identification of a putative cellular receptor 150 kda polypeptide for porcine epidemic diarrhea virus in porcine enterocytes isolation and characterization of a variant porcine epidemic diarrhea virus in china clathrin-and serine proteases-dependent uptake of porcine epidemic diarrhea virus into vero cells coronavirus 229e-related pneumonia in immunocompromised patients pathogenesis of chimeric mhv-4/mhv-a59 recombinant viruses: the murine coronavirus spike protein is a major determinant of neurovirulence families of aspartic peptidases, and those of unknown catalytic mechanism characterization of a recombinant canine coronavirus with a distinct receptor-binding (s1) domain targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence mechanisms of coronavirus cell entry mediated by the viral spike protein sialic acid binding properties of soluble coronavirus spike (s1) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus identification of the functional domain of the porcine epidemic diarrhoea virus receptor cd13/aminopeptidase n-induced lymphocyte involvement in inflamed joints of patients with rheumatoid arthritis porcine aminopeptidase n is not a cellular receptor of porcine epidemic diarrhoea virus, but promotes its infectivity via aminopeptidase activity structure and function of aminopeptidase n distribution and biosynthesis of aminopeptidase n and dipeptidyl aminopeptidase iv in rat small intestine coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i molecular interactions of group 1 coronaviruses with feline apn mutational analysis of aminopeptidase n, a receptor for several group 1 coronaviruses, identifies key determinants of viral host range 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 involvement of aminopeptidase n in enhanced chemosensitivity to paclitaxel in ovarian carcinoma in vitro and in vivo human aminopeptidase n is a receptor for human coronavirus 229e protein arginine methyltransferase 1 regulates herpes simplex virus replication through icp27 rgg-box methylation this research was supported by the basic science research program through the national research foundation of korea (nrf) funded by the ministry of science, ict and future planning (grant number 2015r1a2a2a01003785). key: cord-272973-kzaowysv authors: joshi, lok r.; okda, faten a.; singrey, aaron; maggioli, mayara f.; faccin, tatiane c.; fernandes, maureen h. v.; hain, kyle s.; dee, scott; bauermann, fernando v.; nelson, eric a.; diel, diego g. title: passive immunity to porcine epidemic diarrhea virus following immunization of pregnant gilts with a recombinant orf virus vector expressing the spike protein date: 2018-05-03 journal: arch virol doi: 10.1007/s00705-018-3855-1 sha: doc_id: 272973 cord_uid: kzaowysv passive immunity is critical for protection of neonatal piglets against porcine epidemic diarrhea virus (pedv). here, we investigated the immunogenicity of an orf virus (orfv) vector expressing the full-length spike (s) protein of pedv (orfv-pedv-s) in pregnant gilts and its ability to confer passive immunity and protection in piglets. three doses of orfv-pedv-s were given to two groups of pedv-negative pregnant gilts, with the last dose being administered two weeks prior to farrowing. one of the two groups immunized with the orfv-pedv-s recombinant virus was also exposed to live pedv orally on day 31 post-immunization (pi). antibody responses were assessed in serum, colostrum and milk of immunized gilts, and passive transfer of antibodies was evaluated in piglet sera. the protective efficacy of orfv-pedv-s was evaluated after challenge of the piglets with pedv. pedv-specific igg, iga and neutralizing antibody (na) responses were detected in orfv-pedv-s-immunized and orfv-pedv-s-immunized/pedv-exposed gilts. pedv na, igg and iga were detected in the serum of piglets born to immunized gilts, demonstrating the transfer of antibodies through colostrum and milk. piglets born to immunized gilts showed reduced morbidity and a marked reduction in mortality after pedv challenge in comparison to control piglets. piglets born to gilts that received orfv-pedv-s and were exposed to live pedv showed stronger na responses and lower clinical scores when compared to piglets born to gilts immunized with orfv-pedv-s alone. these results demonstrate the potential of orfv as a vaccine delivery platform capable of eliciting passive immunity against pedv. porcine epidemic diarrhea virus (pedv), an alphacoronavirus in the family coronaviridae, is a single-stranded, positive-sense rna virus that causes porcine epidemic diarrhea (ped) in pigs [1] . pedv infects pigs of all age groups resulting in an enteric disease with high morbidity. high mortality rates (50%-100%) are observed in neonatal piglets; however, mortality is usually low in older animals [2] . ped is characterized by vomiting, watery diarrhea and dehydration, which are usually followed by death in suckling piglets [3] . after its introduction in the usa in 2013, pedv caused the deaths of over 7 million piglets, resulting in significant economic losses to the us swine industry [4] . since then, significant investments and efforts to develop pedv vaccines have been made, with a few vaccines receiving a conditional license from the usda. the efficacy of these vaccines in protecting newborn piglets, however, is still unknown. piglets are born agammaglobulinemic due to the impermeable nature of the epitheliocorial swine placenta, and their immune system is immature, which makes them highly susceptible to pedv in the first weeks (1-3) of life. therefore, passive transfer of antibodies through colostrum and milk is critical for protection of neonatal piglets against pedv [5, 6] . previous studies with transmissible gastroenteritits virus (tgev), a coronavirus that is closely related to pedv, have shown a high correlation between milk antibody levels and protection in piglets [7, 8] . hence, a requirement for an effective pedv vaccine is the ability to induce high levels of antibodies in colostrum and milk with their subsequent transfer to piglets born to immunized sows. although several attenuated, killed, or subunit pedv vaccines have been developed, most of them fail to induce sufficient levels of lactogenic immunity and protection in newborn piglets [9, 10] . thus, it is critical to develop improved alternatives to currently available pedv vaccines. recently, we demonstrated that a recombinant orf virus (orfv) expressing the full-length spike (s) protein of pedv (orfv-pedv-s) is capable of eliciting protective immune responses in immunized pigs [11] . three-week-old piglets immunized intramuscularly (im) with the orfv-pedv-s recombinant virus were protected from clinical signs of ped after oral challenge with pedv and showed reduced virus shedding in feces [11] . in the present study, we investigated the immunogenicity of orfv-pedv-s recombinant virus in pregnant gilts and its ability to induce passive immunity and protection in piglets born to immunized animals. the recombinant orfv-pedv-s was previously generated and characterized in our laboratory [11] , propagated and titrated in primary ovine fetal turbinate cells (oftu, provided by d.l. rock, university of illinois) [11] . pedv strain usa/co/2013 (co13) was obtained from the national veterinary services laboratory (nvsl) and propagated in vero-76 cells (atcc ® crl-1587™) in the presence of 1.5 µg of tpck-treated trypsin per ml (sigma aldrich, st. louis, mo). six primiparous gilts were randomly assigned to three experimental groups as follows: group 1 (g1), control (mem; n = 2); group 2 (g2), orfv-pedv-s-immunized (n = 2); group 3 (g3), orfv-pedv-s-immunized/live pedv-exposed (n = 2) ( table 1) . animals from g2 and g3 were immunized intramuscularly (im) with 2 ml of the recombinant orfv-pedv-s containing 10 7.38 tissue culture infectious dose 50 (tcid 50 )/ml in mem. all animals received the first immunization on day 0 and two booster immunizations on days 21 and 42 post-immunization (pi). the second booster was administered two weeks prior to the anticipated parturition date. in addition to the immunization regimen with orfv-pedv-s described above, gilts in g3 were exposed to live pedv orally (1 × 10 5 tcid 50 in 1 ml) on day 31 pi. animals from g1 were sham-immunized with 2 ml mem as described above. animals were housed in bsl-2 animal rooms, and two weeks prior to farrowing, each gilt was transferred to individual farrowing crates (two crates per room). all gilts received prostaglandin-f 2α (10 mg) on day 52 pi by im injection to induce parturition. animals from g1 and g3 farrowed on day 53 pi, while animals from g2 farrowed on day 56 pi. twelve piglets from each sow were randomly selected, kept with their mothers (n = 24 per group) for 17 days and allowed to suckle colostrum and milk ad libitum. excess piglets were euthanized to keep all sows with an equal number of piglets (n = 12). all piglets (g1, g2 and g3) were challenged orally on day 7 post-birth with a virus suspension containing 2.5 × 10 2 tcid 50 of pedv strain co13 (1 ml/piglet). animals were monitored daily for clinical signs and mortality, and the experiment was terminated on day 74 pi or day 11 post-challenge (pc). clinical scores were evaluated based in four criteria, which were modified from a scoring method described by lohse and collaborators [12] as follows: a) well-being: normal = 0, slightly depressed = 1, depressed and lethargic = 2; b) defecation: normal feces = 0, semi-solid and pasty = 1, watery feces = 2; c) vomiting: no = 0, yes = 1; d) body condition: normal = 0, 2 × 10 7.38 24 1 × 10 5 thin = 1, emaciated = 2. mean daily group scores and mortality rates were calculated and compared between different groups. piglets showing severe dehydration and emaciation were euthanized based on the independent evaluation of sdsu's veterinarian. serum was collected from sows on days 0, 21, 28, 35, 42, 49, 54, 60, 63, 67, 70 and 74 pi. additionally, colostrum and milk were collected from sows on days 1 (day of farrowing) 3, 7, 10, 14, 17 post-farrowing. serum and rectal swabs were collected from piglets on days 1 (pre-colostrum), 7 (pre-challenge), 10, 14, and 17 post-birth. all animal studies were conducted at the sdsu animal resource wing (arw), following the guidelines and protocols approved by the sdsu institutional animal care and use committee (iacuc approval no. 16-003a). indirect elisas using a truncated s protein (aa 630-800) were used to assess igg and iga antibody responses in animals immunized with the recombinant orfv-pedv-s virus as described previously [11] . neutralizing antibody responses elicited by immunization with the recombinant orfv-pedv-s were assessed by fluorescent focus neutralization assay (ffn) as described previously [13] . pedv s-specific igg and iga antibody responses were assessed in colostrum and milk by fmia. optimal assay conditions (amount of antigen, colostrum/milk and secondary antibody dilutions) were determined by a checkerboard titration [13] . virus shedding was assessed in rectal swabs by rt-qpcr. viral rna was extracted from rectal swabs using a zymo viral rna extraction kit (zymo research, ca, usa) according to the manufacturer's instructions. primers and a probe targeting the pedv nucleocapsid protein were designed using primerquest tool (integrated dna technologies inc., usa). rt-qpcr was performed using a sen-sifast™ probe lo-rox one-step kit (bioline, ma, usa) following the manufacturer's instructions. genome copy numbers per ml were determined using a relative standard curve method. the amount of viral rna detected in feces was expressed as log 10 genome copies/ml. gilts from both immunized groups (g2 and g3) developed pedv s-specific igg and iga responses ( fig. 1a and b). igg and iga antibodies were first detected on day 21 pi in animals in g2, and the highest level of antibodies was detected on day 35 pi. similarly, in g3, igg and iga antibodies were first detected on day 21 pi, and their levels increased after the booster immunization on day 21. an anamnestic antibody response was observed in g3 gilts after they were exposed to live pedv on day 31 pi (fig. 1a and b) . no spike-specific igg or iga antibodies were detected in the serum of control sows (g1). the ability of orfv-pedv-s to induce neutralizing antibody (na) responses against pedv was assessed using a ffn assay. na were first detected in serum a week after the first booster immunization (day 28 pi; fig. 1c ). an increase in na levels was observed in both immunized groups (g2 and g3) after day 28 pi (fig. 1c) . gilts in g2, which were immunized only with orfv-pedv-s, had the highest neutralizing antibody titers on day 49 pi, a week after the second booster immunization (day 42 pi; fig. 1c ), whereas gilts in g3, which were immunized with orfv-pedv-s and exposed to live pedv (day 31 pi), had the highest antibody titers on day 35 pi, with titers remaining constant thereafter until the end of the experiment (fig. 1c) . similar to the igg and iga responses, higher neutralizing antibody responses were observed in gilts in g3 when compared to g2 animals. no neutralizing antibodies against pedv were detected in control gilts (g1) in serum samples collected pre-farrowing/pre-challenge. animals in g1 seroconverted to pedv, presenting detectable levels of igg, iga and na a week after challenge of the piglets (day 7 post-birth; fig. 1 ). notably, a strong correlation between na and igg and iga levels was observed in sow serum (na vs. igg: r = 0.876, p < 0.0001; na vs. iga: r = 0.822, p < 0.0001; fig. 1d and e). these results confirmed our previous findings demonstrating the immunogenicity of orfv-pedv-s in pigs [11] . high levels of s-specific igg antibodies were detected on days 1 and 3 post-farrowing, decreasing thereafter in animals from g2 and g3 ( fig. 2a) . colostrum collected from sows in g3, exposed to live pedv, had higher levels of igg when compared to g2 on day 1 post-farrowing ( fig. 2a) . high levels of s-specific iga antibodies were also detected in colostrum of animals from g2 and g3 on day 1 post-farrowing (fig. 2b) . notably, the levels of s-specific iga antibodies remained elevated in milk up to day 17 post-farrowing (fig. 2b) . iga antibody levels were higher in milk from animals in g3 when compared to animals in g2 after day 7 post-farrowing (fig. 2b) . no spike-specific igg or iga antibodies were detected in colostrum/milk collected from control sows (g1) ( fig. 2a and b ). these results demonstrate the ability of orfv-pedv-s to induce s-specific igg and iga antibodies in colostrum and milk. passive transfer of antibodies from immunized sows to their offspring was assessed by elisa and ffn assays performed on serum samples collected from piglets pre-and post-ingestion of colostrum/milk. no s-specific antibodies were detected in serum samples collected on day one prior to ingestion of colostrum (fig. 3) . notably, high levels of s-specific igg and iga antibodies were detected in the serum of piglets from g2 and g3 on day 3 post-birth. piglets born to immunized sows had igg and iga antibodies in their serum until the end of the experiment on day 17 post-birth (fig. 3) , with higher levels of igg antibodies being detected until the end of the experiment. both igg and iga levels were higher in piglets born to sows in g3 when compared to piglets born to sows in g2. no spike-specific igg or iga was detected in serum of piglets born to control sows (g1; fig. 3 ). in addition to igg and iga, nas were detected in piglet serum on day 3 post-birth. high levels of na were detected in piglets born to orfv-pedv-s-immunized sows until the end of the experiment on day 17 pi (fig. 3c) . no neutralizing antibodies were detected in the piglets born to control sows (g1, fig. 3c ). notably, a significant correlation between na titers in sow serum and piglet serum was observed (r = 0.5157, p = 0.0285). additionally, a strong correlation between na and igg and iga antibody levels was observed in piglet serum (na vs. igg: r = 0.866, p < 0.0001; r = 0.641; fig. 3e and f). together, these results demonstrate passive transfer of pedv-specific igg, iga and nas from immunized sows to piglets through ingestion of colostrum and/or milk. all piglets born to immunized or control gilts were challenged orally with virulent pedv strain co13 on day 7 post-birth. piglets were monitored daily for characteristic clinical signs of ped and mortality. daily average clinical scores were calculated for each group, and the mean daily scores are presented in fig. 4a . all piglets born to sows in g1 and g2 showed clinical signs of ped starting on day 1 pc which lasted until day 9-10 pc. the highest clinical scores were observed in g1 piglets between days 2 and 5 pc. piglets in g3 started showing clinical signs on day 3 pc, when moderate diarrhea was observed and continued until day 11 pc. as shown in fig. 4a , more-severe clinical signs were observed in piglets from g1 (average daily scores ranging from 4 to 7), followed by g2 (average daily scores ranging from 4 to 6) and then g3 (average daily scores ranging from 2 to 3.5). it is important to note that, while piglets in g1 and g2 experienced vomiting and diarrhea, none of the piglets in group 3 experienced vomiting during experiment. virus shedding in feces was assessed by rt-qpcr using rectal swabs. piglets in g3 had a significantly lower level of viral rna in their feces (p < 0.0001) in comparison to piglets in g1 and g2 on day 3 pc (fig. 4b) . however, no significant differences in virus shedding in feces were observed between the three groups thereafter. the daily mortality recorded for piglets in the three groups is presented in fig. 4c . notably, twelve out of 24 results are presented as group mean s/p ratios or na titers. the error bars represent +/-standard error of mean (sem). statistical significance was determined using two-way anova, and multiple comparisons were ran using tukey's test. a, b, and c represent statistical significance for g1 vs. g2, g1 vs. g3, and g2 vs. g3, respectively. the significance level is < 0.01. s/p, sample-to-positive ratio; the arrowhead represents the day of challenge with pedv. (d) correlation of piglet serum na levels with sow mean na levels. (e) correlation of group mean igg antibodies in piglet serum with piglet serum na levels. (f) correlation of group mean iga antibodies in piglet serum with piglet serum na levels. correlations were calculated using the spearman method with a 95% confidence interval using graphpad prism 7 rt-qpcr in piglet feces expressed as log 10 genome copy number per milliliter. data are presented as group means. error bars represent +/-sem. statistical significance was determined using two-way anova, and multiple comparisons were ran using tukey's test. a, b, and c represent statistical significance for g1 vs. g2, g1 vs. g3, and g2 vs. g3, respectively. the significance level is < 0.01. (c) survival curve demonstrating piglet mortality. the survival curve was gener-ated using the kaplan-meier method. statistical comparison between groups was performed using the log-rank test in graphpad prism 7. the p-value was adjusted using the bonferroni method. (d) correlation of piglet mortality with piglet group mean na levels. correlation of piglet mortality with group mean iga antibodies in sow colostrum/ milk. (f) correlation of piglet mortality with group mean igg antibodies in sow colostrum/milk. correlations were calculated using the spearman method with 95% confidence interval using graphpad prism 7 (12/24; 50%) piglets in control g1 died by day 7 pc, while only one piglet (1/24; 5%) in g2 died on day 5 pc, and none of the piglets (0/24; 0%) in g3 died after the pedv challenge. interestingly, when the correlation between antibody levels in piglets and sows was compared to survival rates in piglets, moderate to strong correlations between na levels in piglet serum (r = 0.4819, p =0.04288) and iga and igg antibody levels in the sow colostrum/milk (r = 0.8759, p < 0.0001; r = 0.6706, p = 0.002322, respectively) were observed (fig. 4d, e and f) . together, these results demonstrate decreased morbidity and mortality in piglets born to immunized gilts and a strong correlation between survival and antibody levels in colostrum and milk. in the present study, we assessed the ability of orfv-pedv-s to induce passive immunity against pedv following immunization of pregnant gilts. one of the goals of the study was to assess whether immunization with orfv-pedv-s alone would be sufficient or if live exposure to pedv would be required for protection of piglets born to immunized gilts. this underlies our experimental design, in which pregnant gilts were either immunized with orfv-pedv-s (g2) or with orfv-pedv-s followed by oral exposure to live pedv (g3). similar to our findings in 3-week-old weaned pigs [11] , im immunization of gilts with orfv-pedv-s elicited pedv-specific igg, iga and nas. as expected, the booster provided by live pedv exposure in gilts from g3 led to higher antibody responses in serum, colostrum and milk of these animals. notably, passive transfer of antibodies from gilts to piglets was observed in both g2 and g3, as pedv-specific igg, iga and nas were detected in serum of piglets born to immunized gilts following ingestion of colostrum and milk. na antibodies detected in piglet serum showed a strong correlation with igg and iga levels ( fig. 3e and f). although the antibody responses elicited by immunization with orfv-pedv-s alone were lower in comparison with those induced by immunization with orfv-pedv-s followed by live pedv exposure, the levels of antibodies induced by im immunization with orfv-pedv-s were sufficient to reduce neonatal mortality after oral challenge with pedv. antibodies present in sow colostrum and milk are derived either from serum or produced locally in the mammary tissue [14] , and transfer of pathogen-specific antibodies from the sow to the piglet via colostrum and milk is critical for protection against the pathogens during the neonatal phase of the piglet's life. in this context, iga plays a key role in protection against enteric pathogens such as pedv, mainly because it is stable to proteolytic degradation in the intestine. by remaining in the gut lumen, pathogen-specific iga can effectively inhibit/ decrease virus infection/replication in the gut epithelium [8, [15] [16] [17] . the results of this study show that increasing levels of iga in milk of immunized animals paralleled lower disease morbidity and mortality in piglets born to immunized gilts after challenge with pedv. while severe ped and high mortality (50%) were observed in piglets born to control gilts in g1 (no antibody responses detected), piglets born to gilts in g2 and g3 (high levels of iga in g2 and even higher in g3) showed less-severe clinical signs and reduced mortality rates (5 and 0%, respectively). these results suggest that pedv-specific iga detected in piglets in g2 and g3 may have contributed to reducing disease severity and mortality in piglets. the possibility that other antibody isotypes or t cell-mediated immunity may have played a role in protection, however, cannot be formally excluded. one of the most important observations of our study was the reduced mortality in piglets from g2 (5% versus 50% in the control group, g1), which were born to gilts immunized im with orfv-pedv-s. notably, the lower piglet mortality in this group paralleled s-specific igg, iga and na responses detected in the gilts and passively transferred to piglets. together, these observations show that parenteral immunization with orfv-pedv-s is sufficient to induce protective levels of passive immunity against pedv. these results corroborate the findings of previous studies in which pedv specific-antibodies have been detected in serum and colostrum/milk and passively transferred to piglets after parenteral immunization with live, inactivated and/or subunit vaccine candidates [18] . although the mechanism(s) underlying this phenomenon remain unknown, it is possible that systemic antibodies could be transferred from the sow's serum to colostrum and milk and then to the piglets. alternatively, the immunomodulatory properties of the orfv vector used here could potentially lead to migration of antibody-secreting plasma cells to the mammary gland, resulting in local antibody production. future studies assessing local immune cells in the mammary gland and antibody isotypes in the intestinal lumen of piglets born to immunized sows will be critical for dissecting the precise mechanism(s) underlying the protective immune responses elicited by im immunization with orfv-pedv-s observed here. in summary, this study demonstrates the ability of orfv-pedv-s to confer passive immunity against pedv following immunization of pregnant gilts. similar to our previous findings in 3-week-old weaned pigs [11] , im immunization of gilts with orfv-pedv-s elicited pedv-specific igg, iga and nas responses. additionally, passive transfer of antibodies from gilts to piglets was observed, as pedv-specific igg, iga and nas were detected in serum of piglets born to immunized gilts following ingestion of colostrum and milk. porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and 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and other swine coronaviruses immunogenicity of a recombinant parapoxvirus expressing the spike protein of porcine epidemic diarrhea virus experimental infection of young pigs with an early european strain of porcine epidemic diarrhoea virus and a recent us strain development of an indirect elisa, blocking elisa, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to north american strains of porcine epidemic diarrhea virus the transfer of immunoglobulins igg, iga and igm from serum to colostrum and milk in the sow mucosal and systemic isotype-specific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus does circulating antibody play a role in the protection of piglets against porcine epidemic diarrhea virus? strategies for design and application of enteric viral vaccines evaluation of antibody response of killed and live vaccines against porcine epidemic diarrhea virus in a field study we thank the staff of the sdsu animal resource wing (arw) for excellent care and handling of animals and for their invaluable help with the animal experiments. funding this study was funded by the national pork board grant no. 15-034 and in part by the national institute of food and agriculture hatch project no. sd00517-14. the authors declare that there is a patent pending related to this work (us patent pending, p11703us00).ethical approval all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. all animal experiments were reviewed and approved by the sdsu iacuc (approval no. 16-003a). this article does not contain any studies with human participants performed by any of the authors. key: cord-292101-lnap47kp authors: jung, kwonil; eyerly, bryan; annamalai, thavamathi; lu, zhongyan; saif, linda j. title: structural alteration of tight and adherens junctions in villous and crypt epithelium of the small and large intestine of conventional nursing piglets infected with porcine epidemic diarrhea virus date: 2015-06-12 journal: vet microbiol doi: 10.1016/j.vetmic.2015.03.022 sha: doc_id: 292101 cord_uid: lnap47kp integrity of the intestinal epithelium is critical for proper functioning of the barrier that regulates absorption of water and restricts uptake of luminal bacteria. it is maintained mainly by tight junctions (tjs) and adherens junctions (ajs). we conducted immunofluorescence (if) staining for in situ identification of zonula occludin (zo)-1 proteins for tj and e-cadherin proteins for aj in the small and large intestinal villous and crypt epithelium of nursing pigs infected with porcine epidemic diarrhea virus (pedv). twenty 9-day-old piglets [pedv-infected (n = 9) and mock (n = 11)] from pedv seronegative sows, were orally inoculated [8.9 log(10) genomic equivalents/pig] with pedv pc21a strain or mock. at post-inoculation days (pids) 1–5, infected pigs showed severe watery diarrhea and/or vomiting and severe atrophic enteritis. by immunohistochemistry, pedv antigens were evident in enterocytes lining the villous epithelium. at pids 1–5, pedv-infected pigs exhibited mildly to extensively disorganized, irregular distribution and reduced expression of zo-1 or e-cadherin in villous, but not crypt epithelial cells of the jejunum and ileum, but not in the large intestine, when compared to the negative controls. the structural destruction and disorganization of tj and aj were extensive in pedv-infected pigs at pids 1–3, but then appeared to reversibly recover at pid 5, as evident by increased numbers of zo-1-positive epithelial cells and markedly improved appearance of e-cadherin-positive villous epithelium. our results suggest a possible involvement of structurally impaired tj and aj in the pathogenesis of pedv, potentially leading to secondary bacterial infections. integrity of the intestinal epithelium is critical for proper functioning of the barrier that regulates absorption of water and restricts uptake of luminal bacteria. it is maintained mainly by tight junctions (tjs) and adherens junctions (ajs). we conducted immunofluorescence (if) staining for in situ identification of zonula occludin (zo)-1 proteins for tj and e-cadherin proteins for aj in the small and large intestinal villous and crypt epithelium of nursing pigs infected with porcine epidemic diarrhea virus (pedv). twenty 9-day-old piglets and mock (n = 11)] from pedv seronegative sows, were orally inoculated [8.9 log 10 genomic equivalents/pig] with pedv pc21a strain or mock. at post-inoculation days (pids) 1-5, infected pigs showed severe watery diarrhea and/or vomiting and severe atrophic enteritis. by immunohistochemistry, pedv antigens were evident in enterocytes lining the villous epithelium. at pids 1-5, pedv-infected pigs exhibited mildly to extensively disorganized, irregular distribution and reduced expression of zo-1 or e-cadherin in villous, but not crypt epithelial cells of the jejunum and ileum, but not in the large intestine, when compared to the negative controls. the structural destruction and disorganization of tj and aj were extensive in pedv-infected pigs at pids 1-3, but then appeared to reversibly recover at pid 5, as evident by increased numbers of zo-1-positive epithelial cells and markedly improved appearance of e-cadherin-positive villous epithelium. our results suggest a possible involvement of structurally impaired tj and aj in the pathogenesis of pedv, potentially leading to secondary bacterial infections. ß 2015 elsevier b.v. all rights reserved. the intestinal epithelium provides a robust barrier that regulates absorption of nutrients and water and also restricts uptake of luminal bacteria (su et al., 2011) . the integrity of the intestinal epithelium is maintained by tight junctions (tjs), adherens junctions (ajs), desmosomes, and gap junctions. the tj is the major paracellular barrier and functions to separate apical and basolateral compartments or membranes. the aj forms a continuous belt between cells and is crucial for the maintenance of intercellular adhesion (hartsock and nelson, 2008; su et al., 2011) . the tj and aj are regulated by transmembrane tj (occludin and claudin) and aj (e-cadherin and catenins) proteins on the opposing cells' plasma membranes and interactions mediated by actin binding proteins such as the zonula occludin (zo) family, which link the aj complex to actin microfilaments (hartsock and nelson, 2008; su et al., 2011) . porcine epidemic diarrhea virus (pedv), which belongs to the genus alphacoronavirus in the family coronaviridae, causes high mortality of suckling pigs and substantial economic losses (saif et al., 2012) . epidemic pedv strains are highly enteropathogenic and acutely infect villous epithelial cells of the entire small and large intestines, but the jejunum and ileum are the primary sites of infection (jung and saif, 2015; jung et al., 2014) . during the early stages of pedv infection, necrosis and exfoliation of infected villous epithelial cells is pronounced, resulting in acute, severe villous atrophy. to what extent the integrity and function of the pedv-infected villous epithelium is restored by intestinal stem cells located at crypt cell layers is currently unclear. the aim of the present study was to determine whether pedv infection causes structurally altered tj and aj in the villous and crypt epithelium of the small and large intestine by immunofluorescence (if) staining for in situ identification of the related proteins, zo-1 and e-cadherin for tj and aj, respectively. the wild-type us pedv strain pc21a was obtained from the intestinal contents of a diarrheic 1-day-old piglet on an ohio farm in june 2013 (jung et al., 2014) . the original sample was serially passaged 2 times in gn pigs. the original sample and gn pig-passaged pc21a strain were confirmed to contain only pedv, as reported in our previous study (jung et al., 2014) . the titer of gn pig second-passaged pc21a strain was 11.8 log 10 ge/ml and was used as virus inoculum after dilution in minimal essential medium (mem). two seronegative, large white â duroc crossbred, pregnant sows to acquire 20 nursing piglets, were obtained from a pedv-free spf (confirmed by history and seronegative sows; lack of qrt-pcr positive-fecal samples) swine herd of the ohio state university. the spf herd was seronegative for antibodies to prrsv, prcv, tgev and porcine circovirus type 2. twenty 9-day-old nursing piglets were randomly assigned to one of two groups: pedv inoculated (n = 9) and mock (n = 11). each experimental group of pigs was housed in a separate room in a high-security isolation facility (biosafety level 2). nursing pigs were inoculated orally [8.9 log 10 ge (%2.9 log 10 plaque forming units)/pig] (jung et al., 2014) with 1 ml of pc21a or mock inoculated with mem. inoculated and negative control pigs (n = 3-4/group at each time-point) were euthanized for pathological examination at an acute-stage (pid 1), at a mid-stage (pid 3), and at a later-stage (pid 5) of infection. after pedv inoculation, the pigs were monitored for clinical signs 2-3 times daily until necropsy. the institutional animal care and use committee (iacuc) of the ohio state university approved all protocols related to the animal experiments and care in this study. small (duodenum, proximal, middle and distal jejunum, and ileum) and large (cecum/colon) intestinal tissues and other major organs (lung, liver, heart, kidney, spleen, and lymph node) were examined grossly and histologically at pids 1, 3, and 5. tissues were placed in 10% phosphate buffered formaldehyde (ph 7.0), dehydrated in graded alcohol, embedded in paraffin, and cut in 3-mm sections onto microscope slides, fixed and stained with hematoxylin and eosin (h&e) then analyzed for histopathological changes. villous height and crypt depth were estimated by measuring at least 10 villi and crypts throughout the section. mean ratios of jejunal villous height to crypt depth (vh:cd) were calculated as previously described (jung et al., 2006) . the formalin-fixed, paraffin-embedded tissues were prepared and tested by immunohistochemistry (ihc) for the detection of pedv antigens, using monoclonal antibody 6c8-1 against the spike protein of pedv strain dr13 (provided by dr. daesub song, korea research institute of bioscience and biotechnology, daejeon, korea). the antibody was diluted 1:200 in pbst [phosphate-buffered saline (pbs) containing tween 20, 0.1%]. ihc was conducted, as described previously (jung et al., 2009 ). the frozen jejunal/ileal and cecal/colonic tissues were prepared in tissue-tek oct compound (sakura, torrance, ca, usa) and tested by if staining for the detection of zo-1 and e-cadherin using monoclonal antibodies against human recombinant zo-1 and human e-cadherin (invitrogen, ca, usa). the anti-zo-1 antibody was diluted 1:100 in pbst and incubated on the tissues at 4 8c overnight. the anti-e-cadherin antibody was diluted 1:100 in pbst and incubated on the tissues at 4 8c overnight, and an anti-mouse antibody conjugated with alexa fluor 1 488 (invitrogen) was used as the detection antibody and incubated on the tissues at 37 8c for 1 h. the stained tissues were examined by fluorescence microscopy. zo-1or e-cadherin-positive scores were computed by estimating the number of if-positive cells in the intestinal section per microscopic area, at â400 magnification based on the following criteria: 0, no positive cells; 1, 1-29% of zo1-or e-cadherin-positive villous or crypt epithelium showed staining; 2, 30-69% of zo1-or e-cadherin-positive villous or crypt epithelium showed staining; and 3, 70-100% of zo1-or e-cadherin-positive villous or crypt epithelium showed staining. because the entire intestinal villous and crypt epithelium of uninfected and pedv-infected pigs (e-cadherin-positive scores, all 3) were positive for e-cadherin, and e-cadherin-stained villous epithelium (but not crypt epithelium) showed evident disorganization, e-cadherinstained tissues were additionally scored by the following system: 0, no positive cells; 1, 1-29% of e-cadherin-positive villous epithelium showed disorganization; 2, 30-69% of e-cadherin-positive villous epithelium showed disorganization; and 3, 70-100% of e-cadherin-positive villous epithelium showed disorganization. disorganization was characterized by irregular distribution and mildly to moderately reduced expression of e-cadherin in villous epithelial cells of the small intestine. all values are expressed as the means ae standard deviation of the means (sdm). zo-1-or e-cadherin-positive scores and disorganization scores of e-cadherin-positive villous epithelium between pedv-infected and uninfected nursing pigs, or between different time-points in the same group, were analyzed and compared by a student's t-test using graphpad prism software (graphpad prism inc.). a value of p < 0.05 was considered statistically significant. clinical signs were first detected at pid 1 in pedvinoculated nursing piglets. all inoculated nursing piglets at pids 1-5 exhibited acute, severe watery diarrhea and/or vomiting, followed by lethargy and dehydration. no negative control nursing pigs showed diarrhea or other clinical signs throughout the experiment. by macroscopic examination, all inoculated nursing pigs tested at pids 1-5 exhibited extensively thin and transparent intestinal walls and accumulation of large amounts of yellowish fluid in the small and large intestinal lumen. the other internal organs appeared normal. in inoculated nursing pigs, histologic lesions were limited to the small intestine, mainly jejunum and ileum, and included acute diffuse, severe atrophic enteritis. no histologic lesions were evident in the large intestine or other organs of the inoculated nursing pigs and negative controls. mean (aesdm) jejunal vh:cd ratios of uninoculated nursing piglets were 8.7 (ae1.4) at pid 1, 7.6 (ae1.2) at pid 3, and 7.1 (ae1.1) at pid 5. on the other hand, mean (aesdm) jejunal vh:cd ratios of inoculated nursing piglets were 1.4 (ae0.5) at pid 1, 1.2 (ae0.5) at pid 3, and 1.1 (ae0.6) at pid 5. all inoculated pigs exhibited pedv antigen-positive cells in the small and large intestine tested at pids 1-5. under our ihc conditions, mean (aesdm) pedv antigen-positive cells per jejunal villus of inoculated nursing pigs were 9.3 (ae7.3) at pid 1, 13.9 (ae5.9) at pid 3, and 9.2 (ae4.3) at pid 5. more detailed clinical disease, pathology, fecal shedding, and viremia (viral rna in serum) results are in a manuscript in preparation by jung et al., 2015 (unpublished data) . no other internal organs of the inoculated pigs showed ihc-positive staining. no pedv antigen-positive cells were detected in the negative control pigs. in frozen jejunal (or ileal) and colonic (or cecal) tissues of the uninfected nursing pigs at pids 1-5, the major amount of zo-1 protein were regularly and discontinuously expressed on the apical surface of villous and crypt epithelial cells by if staining (fig. 1a-c) . transversely sectioned cells frequently exhibited rectangular, pentagonal, or hexagonal if staining for zo-1 (fig. 1a-f ). in the jejunum and colon of all uninfected nursing pigs tested at pids 1-5, the entire villous (zo-1-positive scores, all 3) or crypt (zo-1-positive scores, all 3) epithelium was mostly positive for zo-1 protein ( fig. 1a -c, g and h). no differences in staining results and patterns were evident between jejunal and ileal tissues, or between cecal and colonic tissues. on the other hand, pedv-infected pigs at pids 1-5 had moderately to extensively disorganized, irregular distribution and reduced expression of zo-1 in villous epithelial cells of the small intestine, but not crypt epithelial cells ( fig. 1d and e) , compared to the negative controls ( fig. 1a and b). regardless of the infection stage, most of the crypt epithelium had mildly reduced expression of zo-1 protein or numbers of zo-1-positive cells (mean zo-1 positive scores: 2.7 at pid 1, 2.7 at pid 3, and 2.8 at pid 5) (fig. 1d , e, and h), compared to the negative controls. pedv-inoculated nursing pigs exhibited small numbers of zo-1-positive cells at pids 1 and 3 (mean zo-1-positive scores, all 1.2) ( fig. 1d and g) and moderate numbers of zo-1-positive cells at pid 5 (mean zo-1-positive score, 2.2) ( fig. 1e and g) in the villous epithelium of the small intestine. mean zo-1-positive scores at pid 5 were significantly higher than those at pid 1 or pid3. at pids 1-5, mean zo-1-positive scores in the jejunal villous epithelium of infected nursing pigs were significantly lower than those in the uninfected pigs (p < 0.01 at pids 1 and 3; and p < 0.05 at pid 5). however, there were no significant differences in mean zo-1-positive scores in the colonic villous and crypt epithelium between uninfected and pedv-infected nursing pigs at pids 1-5 ( fig. 1c and f) . apart from zo-1 that was detected mainly on the apical surface of intestinal epithelial cells under the conditions tested, the large amount of e-cadherin protein were strongly expressed on the apical and basolateral surface and also mildly in the cytoplasm of the negative control pigs ( fig. 2a and c) . similar to zo-1 staining, the transversely sectioned cells frequently exhibited rectangular, pentagonal, or hexagonal if staining for e-cadherin ( fig. 2a-f) . in the jejunum (or ileum) and colon (or cecum) of all uninfected and pedv-infected nursing pigs tested at pids 1-5, the entire villous (e-cadherin-positive scores, all 3) and crypt (e-cadherin-positive scores, all 3) epithelium was positive for e-cadherin (fig. 1a-f) . as a result, there were no differences in mean e-cadherin-positive scores between uninfected and pedv-infected pigs. also, no differences in staining results and patterns were detected between jejunal and ileal tissues, or between cecal and colonic tissues. pedv-infected pigs at pids 1-5 showed disorganized, irregular distribution and mildly to moderately reduced expression of e-cadherin in only villous epithelial cells of the small intestine ( fig. 2d and e) , compared to the negative controls ( fig. 2a and b ). e-cadherin-stained tissues were additionally scored based on the extent of disorganization of e-cadherin-positive villous epithelium. mean disorganization scores (aesdm) of e-cadherin-positive villous epithelium of the infected pigs were 2.3 (ae0.8) at pid 1 and 1.8 (ae0.3) at pid 3, which were significantly higher than those (mean disorganization scores, all 0) in the uninfected pigs at the same time-point (p < 0.01 at each note that transversely sectioned cells show rectangular, pentagonal, or hexagonal if staining for zo-1 (arrowheads) (b, e, and f). original magnification, all â400. mean zo-1-positive scores in the jejunal villous (g) or crypt epithelium (h) of pedv-infected nursing piglets compared to uninfected pigs. zo-1positive scores were computed by estimating the number of if-positive cells in the intestinal section per microscopic area, at â400 magnification based on the following criteria: 0, no positive cells; 1, 1-29% of zo1-positive villous or crypt epithelium showed staining; 2, 30-69% of zo1-positive villous or crypt epithelium showed staining; and 3, 70-100% of zo1-positive villous or crypt epithelium showed staining. each bar represents the mean ae sdm. **p < 0.01; *p < 0.05 (statistically significant differences between the pedv-infected and uninfected nursing pigs by student's t-test). time-point) (fig. 2g) . at pid 5, however, there were no significant differences in mean disorganization scores of e-cadherin-positive villous epithelium between uninfected and pedv-infected nursing pigs (fig. 2g) . based on our data, pedv infection resulted in structurally altered tj and aj in the villous (not crypt) epithelium of the primary sites of infection, jejunum and ileum, but not the large intestine (cecum and colon). similar to pedv infection of intestinal epithelium, tj and aj disruption in pulmonary epithelium, which might contribute to the desquamation of the alveolar wall, was observed in lung biopsies from the betacoronavirus sars coronavirus (sars-cov)-infected macaques and patients (nicholls et al., 2003) . a previous study suggested that alteration of tj and aj may create a breach in the epithelial barrier allowing sars-cov to reach the basal matrix and eventually the systemic circulation, i.e. viremia (teoh et al., 2010) . by similar mechanisms, after pedv infection, viremia might be caused during acute infection stages, as reported previously (jung et al., 2014) . in this study, by qrt-pcr, all inoculated nursing pigs tested at pids 1-5 also had low to moderate viral rna titers in serum, ranging from 5.6 to 8.6 log 10 ge/ml. another study showed that impaired tj in intestinal epithelium of patients with human immunodeficiency virus likely contributes to intestinal barrier dysfunction, resulting in note that transversely sectioned cells show rectangular, pentagonal, or hexagonal if staining for e-cadherin (arrowheads) (a, b, e, and f). original magnification, all â400. (g) mean disorganization scores of e-cadherin-positive jejunal villous epithelium of pedv-infected nursing piglets compared to uninfected pigs. e-cadherin-stained tissues were scored by the following system: 0, no positive cells; 1, 1-29% of e-cadherin-positive villous epithelium showed disorganization; 2, 30-69% of e-cadherin-positive villous epithelium showed disorganization; and 3, 70-100% of e-cadherin-positive villous epithelium showed disorganization. each bar represents the mean ae sdm. **p < 0.01 (statistically significant differences between the pedv-infected and uninfected nursing pigs by student's t-test). increased permeability and microbial translocation that cause severe, chronic diarrhea (chung et al., 2014) . the impaired tj and aj caused by pedv might be associated with high co-infection rates of infected nursing piglets with other enteropathogens, such as escherichia coli (turgeon et al., 1980; wang et al., 2013 ). an in vitro study using the porcine in testinal epithelial cell line, ipec-j2, reported a significantly decreased expression (by western blotting) of tj and aj proteins at 40-60 mins after inoculation with another alphacoronavirus, transmissible gastroenteritis virus, or along with pedv, but not pedv alone (zhao et al., 2014) . whether pedv can alter tj or aj in vivo in the intestinal epithelium of pedv-infected pigs was uncertain. our study clearly demonstrated that at pids 1-5, pedv-infected pigs exhibited mildly to extensively disorganized, irregular distribution and reduced expression of zo-1 or e-cadherin in villous, but not crypt epithelial cells of the jejunum and ileum, but not in the large intestine, when compared to the negative controls. structural alterations of tj and aj were extensive in the small intestine of pedv-infected pigs at pids 1-3, but then appeared to recover at pid 5, as evident by increased numbers of zo-1-positive epithelial cells and markedly improved appearance of e-cadherin-positive villous epithelium of the infected pigs. structural destructions and disorganizations of tj and aj in the intestinal villous epithelium with pedv infection might be reversibly recovered during the late stage of infection. our present study revealed evidence of a possible involvement of structurally impaired tj and aj and reduced expression of the related proteins (zo-1 and e-cadherin) in the pathogenesis of pedv. the pedv-infected intestine may have an impaired gut integrity, possibly leading to uptake of luminal bacteria and loss of water into the intestinal lumen with high osmotic pressure caused by pedv infection; however, further studies are needed to delineate the functional mechanisms involved. neither of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. progressive proximal-to-distal reduction in expression of the tight junction complex in colonic epithelium of 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diarrhea in baby pigs in quebec porcine epidemic diarrhea virus variants with high pathogenicity transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j2 cells we thank dr. juliette hanson, andrew wright, megan strother, and ronna wood for assistance with animal care; and xiaohong wang and john blakenship for technical assistance. salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development center, the ohio state university. this work was supported by a grant from the oardc seeds, grant # oaoh1536 (jung k, pi). key: cord-252121-s1zxu5vo authors: lowe, james; gauger, phillip; harmon, karen; zhang, jianqiang; connor, joseph; yeske, paul; loula, timothy; levis, ian; dufresne, luc; main, rodger title: role of transportation in spread of porcine epidemic diarrhea virus infection, united states date: 2014-05-17 journal: emerg infect dis doi: 10.3201/eid2005.131628 sha: doc_id: 252121 cord_uid: s1zxu5vo after porcine epidemic diarrhea virus (pedv) was detected in the united states in 2013, we tested environmental samples from trailers in which pigs had been transported. pedv was found in 5.2% of trailers not contaminated at arrival, , suggesting that the transport process is a source of transmission if adequate hygiene measures are not implemented. after porcine epidemic diarrhea virus (pedv) was detected in the united states in 2013, we tested environmental samples from trailers in which pigs had been transported. pedv was found in 5.2% of trailers not contaminated at arrival, , suggesting that the transport process is a source of transmission if adequate hygiene measures are not implemented. p orcine epidemic diarrhea virus (pedv) was detected in herds of pigs in the united states during april 2013 (1) . pedv is a member of the cornaviridae family that produces a malabsorptive diarrhea secondary to atrophy of the small intestinal villi (2) . initial clinical cases were detected in herds in indiana and iowa during may 2013. the virus spread rapidly across large geographic regions; 218 cases of infection were identified in 16 states during the first 9 weeks of the outbreak (3) . subsequent testing of historical samples collected during the week of april 15, 2013 identified the index herd in ohio (3) . veterinarians became concerned about the role that facilities where pigs are harvested for processing into food and the transportation equipment used to move pigs from farms to those facilities were playing in the spread of pedv. these concerns were based on evidence that equipment used to transport live pigs transmits another enteric coronavirus, transmissible gastroenteritis virus, between sites in the united states (j.f. lowe, unpub. data). pigs are commonly transported to harvest facilities in vehicles that have not been cleaned and disinfected between loads. implementation of "all in-all out" sites, which are sites in which pigs are grown and all pigs in a group are removed before arrival of the next group, limits the spread of disease introduced by transport vehicles. in many cases, the risks and associated costs of disease introduced late in the growing period are thought to be less than the cost of cleaning and disinfecting vehicles. transport vehicles are often shared by different pig owners, enabling the spread of disease across large regions. the objective of this study was to assess the risks that harvest facilities and transport vehicles engendered in promoting the initial outbreak of a novel disease organism by estimating the incidence of trailer contamination with pedv during the unloading process at harvest facilities. environmental samples were collected from 575 livestock trailers before and after pigs were unloaded into holding pens, or lairages, at 6 harvest facilities (83-102 trailers per facility) located in the central united states. samples were collected during a period of 2-3 days at each facility during june 14-20, 2013. for each trailer, the following information was collected: transport company and trailer identification, time of unloading, dock used, whether the truck driver stepped on the dock, and whether facility personnel entered the trailer. sample collection consisted of rubbing a phosphate-buffered saline-moistened pad (swiffer, procter & gamble, cincinnati, oh, usa) over an ≈900 cm 2 area of the trailer floor, 15 cm from the rear door. the pad was placed in a sterile bag (whirl-pac, nasco, fort atkinson, wi, usa) and the liquid was collected by applying manual pressure. the liquid was transferred to a sterile tube (14ml falcon tube, fisher scientific, chicago, il, usa), immediately placed on ice, and maintained at 4°c during transport to the iowa state university veterinary diagnostic laboratory. new latex gloves were worn for each sample collection to minimize the risk for cross-contamination. rna extraction was performed with 100 ml of each environmental sample by using the magmax viral rna isolation kit (life technologies, carlsbad, ca, usa) and a kingfisher 96 instrument (thermo scientific, waltham, ma, usa) and kingfisher program am_1836_ dw_hv_v3 provided by the manufacturer of the viral extraction kits. viral rna was eluted into 90 μl of buffer. real-time reverse transcription pcr (rrt-pcr) was performed on nucleic acid extracts by using the path-id multiplex one-step rt-pcr kit (life technologies) according to the manufacturer's recommendations. primers and probe targeting conserved regions of the pedv nucleocapsid protein gene were as described (4) with modifications specific to the sequence isolated in north america deposited in genbank (accession no. kf272920). the forward primer sequence was 5′-cgcaaagact-gaacccactaacct-3′, the reverse primer sequence was 5′-ttgcctctgttgttacttggagat-3′, and the probe sequence was 5′-tgttgccattaccac-gactcctgc-3′. sequences were labeled by using the fam/zen/3′ iowa black detector (integrated dna technologies, coralville, ia, usa). all rrt-pcr reactions were conducted on an abi 7500 fast (applied biosystems, foster city, ca, usa) and results analyzed by system software. samples were tested separately from routine diagnostic samples in the laboratory to minimize risks for cross-contamination. before unloading, 38 (6.6%) of the 575 trailers were contaminated with pedv. the proportion of contaminated trailers ranged from 2% to 14.6% among the 6 harvest facilities; the facility level median was 5.0%. of the remaining 537, 28 (5.2%) that were not contaminated at arrival were contaminated in the unloading process (table) . of the 38 trailers that were contaminated on arrival, environmental samples from 13 (34.2%) were negative for pedv after unloading. environmental samples from these 13 trailers tended to have higher cycle threshold values than those from the 25 trailers that were positive before and after unloading: 32.3 versus 30.6, respectively. this result suggests that the pigs transported to the harvest facility on the 13 trailers may not have been shedding pedv, but instead, the trailers had been contaminated by previous loads of pigs, so viral quantities in the trailer were low or at the limit of detection. contamination during unloading occurred at a higher rate if harvest facility staff entered the trailer (or 4.15, 95% ci 1.27-13.54) or if unloading occurred immediately after unloading another trailer that was found to be contaminated (or 3.35, 95% ci 1.22-9.18). facilities in which more pedv was identified in truck trailers on arrival had a higher overall incidence of contamination. this was measured by multiplying the prevalence of contamination at arrival by the inverse of the cycle threshold value from trailers contaminated at arrival (r 2 = 0.32, p = 0.01). all drivers stepped into the harvest facility at least once, leading to a high rate of contact between drivers, the trailers, and the harvest facility. harvest facilities serve as a source of contact between many swine farms with different health statuses. this study suggests that collection points, such as harvest facilities and livestock auction markets, can be an efficient source of contamination of transport vehicles that return to pig farms and likely played a role in rapidly disseminating pedv across vast geographic regions shortly after pedv was first identified in the united states. these data also suggest that the contamination of transport vehicles leaving the harvest facilities increased as the prevalence of pedvpositive transport vehicles and virus load coming into the facility increased. the results of this study suggest that proactive disease control measures should include improved sanitation, hygiene, and segregation practices at collection points to limit the spread of the agent early in the outbreak. current data suggest that novel agents, such as pedv, may be present in a country but remain undetected for an extended period. thus, control measures may be implemented too late to limit the spread of the disease through fomites that are identified, such as, in this instance, contaminated vehicles returning from swine collection points. simple measures such as limiting contact between drivers and the collection point and requiring drivers to remain on trucks and out of the collection point during the unloading process may have a dramatic effect on limiting the transmission of novel agents. these biosecurity measures are simple but require a coordinated effort between producers, transporters, harvest facility owners, and regulators to achieve effective implementation. this study of pedv transmission by fomites should serve as an example of the risks that a modern, highly technical animal protein industry may encounter during a novel disease introduction. pedv's introduction and subsequent spread in the united states should spur action to minimize these risks before a disease that can affect international trade or food safety is introduced. emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences diseases of swine animal and plant health inspection service, national veterinary services laboratories. porcine epidemic diarrhea virus (pedv) testing data from nahln laboratories multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus key: cord-289026-v09m2fzw authors: sun, yan-gang; li, rui; jiang, longguang; qiao, songlin; zhi, yubao; chen, xin-xin; xie, sha; wu, jiawei; li, xuewu; deng, ruiguang; zhang, gaiping title: characterization of the interaction between recombinant porcine aminopeptidase n and spike glycoprotein of porcine epidemic diarrhea virus date: 2018-10-01 journal: int j biol macromol doi: 10.1016/j.ijbiomac.2018.05.167 sha: doc_id: 289026 cord_uid: v09m2fzw porcine epidemic diarrhea (ped) has caused huge economic losses to the global pork industry. infection by its causative agent ped virus (pedv), an alpha-coronavirus, was previously proven to be mediated by its spike (s) glycoprotein and a cellular receptor porcine aminopeptidase n (papn). interestingly, some recent studies have indicated that papn is not a functional receptor for pedv. to date, there is a lack of a direct evidence for the interaction between papn and pedv s protein in vitro. here, we prepared papn ectodomain and the truncated variants of pedv s protein in drosophila s2 cells. these recombinant proteins were homogeneous after purification by metal-affinity and size-exclusion chromatography. we then assayed the purified target proteins through immunogenicity tests, pedv binding interference assays, circular dichroism (cd) measurements, papn activity assay and structural determination, demonstrating that they were biologically functional. finally, we characterized their interactions by gel filtration chromatography, native-polyacrylamide gel electrophoresis (page) and surface plasmon resonance (spr) analyses. the results showed that their affinities were too low to form complexes, which suggest that papn may be controversial as the genuine receptor for pedv. therefore, further research needs to be carried out to elucidate the interaction between pedv and its genuine receptor. coronaviruses are enveloped single-stranded positive-sense rna viruses, comprising alpha-, beta-, gamma-, and delta-coronaviruses [1] . they are critical causative agents, whose infections are often associated with respiratory, digestive, and neurological diseases in humans and animals. among these viruses, two beta-coronaviruses severe acute respiratory syndrome coronavirus (sars-cov) [2] [3] [4] [5] [6] and middle east respiratory syndrome coronavirus (mers-cov) [7] [8] [9] [10] are lethal to humans, and an alpha-coronavirus transmissible gastroenteritis virus (tgev) has caused severe intestinal diseases to pigs [11] . the infection of a coronavirus is mediated by its spike (s) glycoprotein and the s protein is further cleaved into s1 and s2 subunits by endogenous and/or exogenous proteases [12] . the s1 subunit recognizes and binds to the corresponding host receptor, whereas the s2 subunit mediates membrane fusion between the virus and host cells [13] [14] [15] . since intervention of s1 subunit binding to host receptor shows a great antiviral potential, there are increasing studies to elucidate their interaction mechanisms. angiotensin-converting enzyme 2 (ace2) [16, 17] and dipeptidyl peptidase 4 (dpp4) [18] [19] [20] have been identified as the receptor for sars-cov and mers-cov, respectively. the viral receptor-binding domains (rbd) are located in the c-terminal domains (ctds) of the s1 subunits (s1-ctds) [17, 19, 20] . human coronavirus 229e (hcov-229e) [21, 22] and tgev [23, 24] utilize aminopeptidase n (apn) as the host receptor and their rbds are also located in the s1-ctds [22, 24] . porcine epidemic diarrhea (ped), characterized by vomiting, diarrhea and dehydration, has caused huge economic losses to the global pork industry [25] [26] [27] [28] [29] [30] . its causative agent, ped virus (pedv), was an alpha-coronavirus. previous studies showed that a porcine receptor apn (papn) mediated pedv infection [31] [32] [33] [34] and its density appeared to be an important factor in contributing to the virus efficient infection [35] . pedv s1-ctd (residues 505-629) was further demonstrated to interact with papn ectodomain (residues 63-963) [36] . interestingly, a few recent studies show that papn is not the functional receptor for pedv infection [37, 38] . however, there is a lack of direct demonstration of the interaction between papn and pedv s protein to clarify this discrepancy. in this study, we prepared the extracellular domain of papn, pedv s1 and its truncated variant (s1t) in drosophila schneider 2 (s2) cells. after purification, we assayed the target proteins for their biological functions. finally, we characterized their interactions by three canonical assays. ipec-j2 cell line (from porcine small intestines) was kindly provided by zhanyong wei of henan agricultural university, china. vero cell line (african green monkey kidney epithelial cell line), drosophila s2 cell line, pmt/bip/v5-his a vector, pcoblast vector [39, 40] and positive serum against pedv were kept in our laboratory. peasy-blunt-papn plasmid was constructed and kept in our laboratory. cellfectin ii reagent, blasticidin s, sf-900 ii serum-free medium (sfm) and trizol reagent were purchased from invitrogen (carlsbad, usa). escherichia coli strain trans5α competent cells were purchased from transgen biotech (beijng, china). fetal bovine serum (fbs), dulbecco's modified eagle's medium (dmem), roswell park memorial institute-1640 medium (rpmi-1640), and antibiotics were purchased from gibco (grand island, usa). schneider's insect medium, tryptose phosphate broth (tpb), tpck-trypsin, and l-alanine 4-nitroanilide hydrochloride were purchased from sigma-aldrich (st. louis, usa). the mouse anti-his epitope tag antibody was purchased from proteintech (wuhan, china). horseradish peroxidase (hrp)-conjugated anti-mouse immunoglobulin g (igg) was purchased from santa cruz biotechnology (delaware ave, usa). pre-stained protein ladder (10 to 180 kda) was purchased from thermo fisher scientific (waltham, usa). mlu i, bgl ii and peptide-n-glycosidase f (pngase f) enzymes were purchased from new england biolabs (massachusetts, usa). enhanced chemiluminescence (ecl) plus reagent was purchased from solarbio (beijing, china). the cdnas encoding pedv s1 (residues 21-793) and its truncated variants (residues 21-249, 253-638 and 505-629) were amplified by pcr using the codon-optimized s gene of the pedv ch/hnxc strain as template. the cdna encoding papn ectodomain (residues 63-963) was amplified by pcr from the peasy-blunt-papn plasmid. all pcr primers were listed in table 1 . the pcr products were isolated and inserted into the pmt/bip/v5-his a expression vector between the bgl ii and mlu i sites. all constructs were transformed into escherichia coli strain trans5α competent cells and the recombinant expression vectors were verified by sequencing at shanghai sangon biotech co. ltd. (shanghai, china). the drosophila s2 cells were kept in schneider's insect medium supplemented with 10% heat-inactivated fbs at 28°c in a humidified incubator. the vero cells were grown in dmem supplemented with 10% heat-inactivated fbs at 37°c in a humidified incubator with 5% co 2 . ipec-j2 cells were grown in rpmi 1640 medium with 15% heat-inactivated fbs at 37°c with 5% co 2 . all cell cultures were supplemented with antibiotics (100 u/ml penicillin, 100 μg/ml streptomycin). the recombinant expression vectors and the pcoblast vector were co-transfected into drosophila s2 cells by cellfectin ii reagent according to the manufacturer's instructions. the stably transfected s2 cells were selected in schneider's insect medium supplemented with 10% fbs, antibiotics, and 25 μg/ml blasticidin s. the stably transfected cell lines were then grown in sf-900 ii sfm and induced by 0.75 mm cuso 4 for 5 days. the culture supernatants were harvested by centrifugation at 2000 rpm at 4°c for 10 min. expression of recombinant proteins was checked by mouse anti-his epitope tag antibody and hrp-conjugated anti-mouse igg using western blot. the supernatant containing papn ectodomain, pedv s1 or s1t protein was adjusted to ph 8.0 by 1.5 m tris-hcl ph 8.8, followed by centrifugation at 10000 rpm at 4°c for 30 min. after filtration through 0.22 μm filter membranes, the supernatant was applied to histrap excel prepacked column (ge healthcare, fairfield, usa) or/and hitrap q hp prepacked column (ge healthcare) for purification. then, each target protein was subjected to size-exclusion column hiload 16/600 superdex 200 pg (ge healthcare) and superdex 200 increase 10/300 gl (ge healthcare) for further purification with 20 mm tris-hcl ph 7.6 and 150 mm nacl as the elution buffer. the purified proteins were treated with pngase f according to the manufacturer's instructions. the pngase f-treated and -untreated proteins were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) under reducing (with β-mercaptoethanol) or non-reducing condition (without β-mercaptoethanol). 2.6. function analyses of pedv s1 and s1t proteins 2.6.1. immunogenicity tests of pedv s1 and s1t proteins purified pedv s1 and s1t proteins were subjected to sds-page and transferred to pvdf (polyvinylidene fluoride) membranes. the immunogenicity was then tested by positive serum against pedv and ecl plus reagent. the pedv ch/hubei/2016 strain was propagated on vero cells as previously described [41] . briefly, when the confluence reached 80%, vero cells were washed three times and inoculated with 200 tcid 50 (50% tissue culture infective dose) pedv at 37°c for 1 h. after that, the viruses not entering were removed and the cells were cultured with growth medium consisting of 0.3% tpb, 0.02% yeast extract and 3 table 1 primers utilized in this study. sequence a a the boldface letters indicate restriction sites. μg/ml of tpck-trypsin. when obvious cytopathic effects (cpe) appeared, the cells were frozen and thawed three times, and centrifuged at 2000 rpm at 4°c for 10 min. finally, the supernatant was stored at −80°c for further use. 2.6.3. interference of pedv binding by purified s1 and s1t proteins the ipec-j2 cells were pre-incubated with indicated concentrations of the purified pedv s1 or s1t protein for 1 h at 4°c. cells were further inoculated with pedv ch/hubei/2016 strain at an moi (multiplicity of infection) of 0.1 for 1 h at 4°c. then, the virus and protein mixtures were washed away. total rna was isolated using trizol reagent according to the manufacturer's instructions and the pedv binding was represented by the copies of the pedv nucleocapsid (n) gene using absolute quantitative pcr (the primers were listed in table 1 ) [42] . 2.7. circular dichroism (cd) measurements of pedv s1 and s1t proteins cd measurements (180-260 nm) were carried out on aviv 420sf spectrometer (lakewood, usa) at room temperature (rt) using a 1 mm path-length quartz cell. the instrument was calibrated with 10 mm phosphate buffer (pb) ph 7.6, and freshly purified pedv s1 and s1t proteins were adjusted to 0.8 mg/ml and 1.9 mg/ml in 10 mm pb ph 7.6, respectively. five scans were accumulated at a scan speed of 1 nm/step with average time of 0.3 s. 2.8.1. activity assay of papn ectodomain 1 nm purified papn ectodomain was mixed with 100 μm to 10 mm l-alanine 4-nitroanilide hydrochloride as substrate in 100 μl 60 mm kh 2 po 4 buffer ph 7.2 at 37°c for 60 min. then, the product pnitroanilide was measured using a microplate reader (bmg labtech, offenburg, germany) at 405 nm [43] . the purified papn ectodomain was concentrated to 10 mg/ml in 20 mm tris-hcl ph 7.2, 150 mm nacl. crystallization of papn ectodomain was carried out by the sitting-drop vapor diffusion method with an equal volume of the target protein and various crystallization reagents from the crystallization screening kits (hampton, usa) at rt. the crystals were acquired with a reservoir solution containing 25% (wt/vol) peg3350, 0.2 m sodium fluoride and 100 mm hepes ph 7.2. the crystals were flash-frozen in liquid nitrogen using a crystal freezing buffer containing 20% ethylene glycol, 30% peg3350, 0.2 m sodium fluoride and 100 mm hepes ph 7.2. x-ray data sets of the crystals were collected at a wavelength of 0.979 å on the beam line bl19u1 at national center for protein sciences shanghai (ncpss) and shanghai synchrotron radiation facility (ssrf). the crystal structure was solved by molecular replacement [44] using the mammalian apn structure (pdb code: 4hom) [43] as the search model. the structure was refined by ccp4 program package [44] and manually adjusted by the molecular graphics program coot [45] until convergence of the refinement. solvent molecules were added using a fo-fc fourier difference map at 2.5σ in the final refinement step. statistics of data collection and final model refinement were summarized in table 2 . the coordinates of papn ectodomain were deposited in the protein data bank (pdb code 5z65, http://www.rcsb.org). the final structures were analyzed by software pymol [46] . papn ectodomain was mixed with excess pedv s1 or s1t protein for 1 h at rt in 20 mm tris-hcl ph 7.6, 150 mm nacl. then, pedv s1 or s1t protein, papn ectodomain and their mixtures were individually loaded on the superdex 200 increase 10/300 gl column (ge healthcare) in the same volume and superposed for uv absorption peaks. the eluted proteins were analyzed by sds-page. the 10% native-page gel and running buffer were prepared without sds. the papn ectodomain and pedv s1 or s1t protein were mixed in 30 μl 20 mm tris-hcl ph 7.6, 150 mm nacl at a molar ratio of 1:1/1:5. after incubation at rt for 30 min, pedv s1 or s1t protein, papn ectodomain and their mixtures were analyzed by native-page. spr analyses were performed on biacore s200 (ge healthcare) to determine the equilibrium binding constants (k d , m), the association rate constants (k on , m −1 s −1 ) and the dissociation rate constants (k off , s −1 ) of papn ectodomain binding to pedv s1 or s1t protein. pedv s1 or s1t protein was coupled to the cm5 sensor chips (ge healthcare) and papn ectodomain was injected with the indicated concentrations (187.5 nm, 375 nm, 750 nm, 1500 nm and 3000 nm). the running buffer was hbs-ep (10 mm hepes ph 7.4, 150 mm nacl, 3 mm edta, 0.005% surfactant p20). the binding kinetics were analyzed with the software biacore s200 evaluation 1.0, and the k on , k off and k d were calculated using a 1:1 interaction model as previously described [47] . the equations used for calculation are represented below with biacore spr terminology: where dr dt (response unit/s, ru/s) is the slope of the curve during association phase, c (m) is the concentration of injected papn ectodomain, rmax (ru) is the signal response for maximum absorption of pedv s1or s1t protein, and r (ru) is the signal response for papn ectodomain binding to pedv s1 or s1t protein. all experimental data were presented as group means and standard errors of the means (sem). the experimental data were analyzed using the unpaired, 2-tailed student t-test using graphpad prism software (graphpad). differences at the 95% confidence level (p b 0.05) were considered statistically significant. the recombinant papn ectodomain (residues 63-963), pedv s1 protein (residues 21-793) and s1 truncated variants (residues 21-249, 253-638 and 505-629) were produced in drosophila s2 cells. western blot analyses verified that papn ectodomain (fig. 1a) , pedv s1 protein (fig. 1b) and a pedv s1 truncated variant (s1t, residues 505-629, fig. 1c) were highly expressed. the expression of other pedv s1 truncated variants (residues 21-249 and 253-638) was too low to be detected (data not shown), which were consequently not considered in our subsequent experiments. the target proteins were purified through metal-affinity and sizeexclusion chromatography. after purification, these proteins achieved to a high purity (n99%, fig. 2 ). papn ectodomain (fig. 2, top) , pedv s1 (fig. 2 , middle) and s1t proteins (fig. 2, bottom) were eluted at 10.9 ml, 12.2 ml and 17.8 ml on the calibrated size-exclusion column, corresponding to approximately 240 kda, 110 kda, 15 kda, respectively. as shown in fig. 2 , there were differences between the bands of each target protein under reducing (lane 2) and non-reducing (lane 1) conditions, which implied that they contained disulfide bonds. in addition, the bands showed a significant change after de-glycosylation using pngase f (lane 2 and lane 3). therefore, the recombinant proteins were heavily glycosylated compared to their calculated molecular masses (papn ectodomain: 104 kda, pedv s1 protein: 85.5 kda, pedv s1t protein: 15.1 kda). based on the results above, recombinant papn ectodomain was obtained as dimers, and pedv s1 or s1t protein existed as monomers, which showed similar natures of mammalian apn [43] and other coronavirus s proteins [48] [49] [50] as previously reported. 3.3. immunogenicity tests, interference of pedv binding and cd measurements of purified pedv s1 and s1t proteins we carried out immunogenicity tests and pedv binding interference assays to analyze whether purified pedv s1 and s1t proteins were functional. as shown in fig. 3a and b, purified pedv s1 and s1t proteins could be recognized by positive serum against pedv, indicating that these recombinant proteins had a comparable immunogenicity to the native virus. since porcine small intestines are the major target organ for pedv infection, ipec-j2 cells from porcine small intestines were applied in interference of pedv binding by purified pedv s1 and s1t proteins. ipec-j2 cells incubated with pedv s1 or s1t protein showed the viral rna reduction in a concentration dependent manner (fig. 3c ). especially at a concentration of 200 μg/ml, pedv s1 or s1t protein pretreatment caused a significant reduction compared to that incubated with control protein buffer (p b 0.05), demonstrating that purified pedv s1 and s1t proteins were in functional form (fig. 3c) . we have taken efforts to crystallize functional pedv s1 or s1t protein, and determine their crystal structures. unfortunately, crystallization of these proteins was unsuccessful (data not shown). cd measurement is usual for determination of protein secondary structures [51] [52] [53] [54] [55] [56] and we performed cd measurements to study their secondary structures. as shown in fig. 4 , one minimum at about 215 nm was observed for both pedv s1 and s1t proteins, suggesting that the two proteins had β-sheets. these results were consistent with other coronavirus s1 proteins, which have a high content of β-sheets [49, 50, [57] [58] [59] [60] . mammalian apn is a member of zinc-dependent m1 metallopeptidases, which is widely distributed on the intestinal epithelia and the nervous system cells [61] . it plays multifunctional roles in tumor angiogenesis and metastasis, signal pathway, and degradation of enkephalins [62] . based on these functions, we performed activity assay for purified papn ectodomain. the enzymatic kinetics showed pedv s1 protein (residues 21-793). lane 1-2: pedv s1 protein; lane 3: mock. (c): pedv s1 truncated variant (s1t, residues 505-629). lane 1: pedv s1t protein. lane m: thermo pre-stained protein ladder (10 to 180 kda). that purified papn ectodomain could degrade the substrate l-alanine 4nitroanilide hydrochloride into the product p-nitroanilide. the k m and k cat were 0.18 ± 0.13 mm and 75.55 ± 7.9/s, respectively (table 2) . furthermore, we determined the crystal structure of papn ectodomain. purified papn ectodomain was crystallized with one molecule in each asymmetric unit and its crystal structure was determined at 2.65 å in a c121 space group (table 3) . as described previously [22, 24, 43, 63, 64] , papn ectodomain adopts a hook-like conformation or so-called seahorse shape ( fig. 5b and c) . it is consisted of four domains (i-iv) and a zinc ion in domain ii, characteristics of m1 metallopeptidases ( fig. 5a and b ). in addition, it is heavily glycosylated, and cys758 and cys765 in association with cys795 and cys831 bridge into two disulfide linkages in domain iv (fig. 5b) , which are consistent with our results stated above (fig. 2) . a close structural comparison of our current papn ectodomain and that determined by others (pdb code: 4hom) reveals that the root mean square deviation (rmsd) differences are slight (0.361 å for 853 matching cα atoms), demonstrating that they share almost identical structural fold (fig. 5c ). taken together, purified papn ectodomain was shown to be biologically active on the basis of its enzymatic activity and crystal structure. papn was previously demonstrated as a functional receptor for pedv [31] [32] [33] [34] . however, the vero cell lines, possessing no apn fig. 4 . cd spectra of functional pedv s1 (a) and s1t proteins (b). one minimum at about 215 nm was observed for both pedv s1 and s1t proteins. fig. 3 . immunogenicity tests and interference of pedv binding by purified s1 and s1t proteins. purified pedv s1 (a) and s1t proteins (b) were detected by western blot using the positive antiserum against pedv. (c) pedv binding interference with purified pedv s1 or s1t protein. data represent means ± sem of three independent experiments. *significantly reduced viral rna (p b 0.05). [32, 41] , are efficiently infected by pedv, and widely utilized for isolation and series propagation of the virus [41, [65] [66] [67] [68] . recently, no effects were reported on the susceptibility to pedv after knocking out apn in porcine swine testis cells and human cell lines (human hepatoma cells, huh7 cells and human cervical cancer cells, hela cells) by crispr/cas9 genome editing [38] . moreover, soluble papn ectodomain neither interacted with pedv nor inhibited its infection [37] . all these studies infer that papn may be not the receptor for pedv. to date, there are few direct evidences to clarify this discrepancy by characterization of the interaction between papn ectodomain and pedv s protein in vitro. we speculate that the lack of related studies is probably due to the difficulty in preparing the large pedv s protein, which contains many disulfide bonds and glycosylation sites as other coronaviruses [48, 49] . in the current study, three canonical assays were carried out to characterize the interaction between papn ectodomain and pedv s1 or s1t protein since these functional target proteins were successfully prepared. according to previous reports, coronavirus s-rbds and their host receptors have high affinities and yield stable complexes in vitro, which can be obtained by gel filtration chromatography [17, 20, 22, 24, 69, 70] . therefore, we firstly characterized the interaction between papn ectodomain and pedv s1 or s1t protein by gel filtration chromatography. the results showed that there were no complexes formed in these protein mixtures as no complex peaks appeared (fig. 6) . additionally, we performed native-page to detect whether they formed complexes. as shown in fig. 7a , there were no bands of the protein complexes compared to single proteins. interestingly, pedv s1t protein showed other forms in addition to monomers (figs. 6b and 7a) . in order to further corroborate the interaction between the proteins, the kinetics of papn ectodomain binding to pedv s1 or s1t protein were measured by spr using biacore s200. the profiles showed that there were no signal responses during their association and dissociation phases, which demonstrated that papn ectodomain neither interacted with pedv s1 nor s1t protein under physiological condition (fig. 7b ). in contrast, hcov-229e [22] and tgev [24] rbds had high affinities to their receptor apn. these results were similar to sars-cov rbd not binding to dpp4 or mers-cov rbd not binding to ace2, while the k d of dpp4 binding to mers-cov rbd was about 16.7 nm (k on : 1.79 × 10 5 m −1 s −1 , k off : 2.99 × 10 −3 m −1 s −1 ) [20] . in conclusion, our work characterized the interaction between recombinant papn ectodomain and pedv s protein in vitro. the results suggest that papn may be controversial as the genuine receptor for pedv because functional papn ectodomain neither binds to pedv s1 nor s1t protein. our current work sheds some light on the invasion mechanism of the virus, and supports a molecular basis for prevention and control of ped. as s protein recognizing and interacting with the host receptor are crucial for pedv infection, further research needs to be carried out to identify the genuine receptor of pedv and elucidate the specific interaction between them. the authors declare that they have no conflicts of interest. papn ectodomain is colored as described for (a). the zinc ion is represented as red sphere and n-linked oligosaccharides are shown in stick representation. the n and the c termini are also labeled. (c) structural comparison of papn ectodomain in the current study with that determined by others (pdb code 4hom) in cartoon diagrams. the papn ectodomain in the current study is colored in green and the papn ectodomain previously determined is in cyan. their n and c termini are labeled. fig. 7 . characterization of the interaction between papn ectodomain and pedv s1 or s1t protein. (a): native-page analysis. lane 1: pedv s1 protein; lane 2: papn ectodomain and pedv s1 protein mixtures (1:1); lane 3: papn ectodomain; lane 4: papn ectodomain and pedv s1t protein mixtures (1:5); lane 5: pedv s1t protein. 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coronavirus spike glycoprotein trimer cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding cryo-em structure of porcine delta coronavirus spike protein in the pre-fusion state the moonlighting enzyme cd13: old and new functions to target the structure and main functions of aminopeptidase n the x-ray crystal structure of human aminopeptidase n reveals a novel dimer and the basis for peptide processing allosteric inhibition of aminopeptidase n functions related to tumor growth and virus infection isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene isolation and characterization of a korean porcine epidemic diarrhea virus strain knu-141112 the dynamics of chinese variant porcine epidemic diarrhea virus production in vero cells and intestines of 2-day old piglets crystal structure of nl63 respiratory coronavirus receptor-binding domain complexed with its human receptor crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor we thank the staff of bl19u1 beamline at national center for protein sciences shanghai (ncpss) and shanghai synchrotron radiation facility (ssrf), shanghai, china, for assistance during data collection. we also acknowledge qiang wei and hongfang ma from henan academy of agricultural sciences for their assistance on crystallization, and baolin xiao from henan university for his assistance on cd measurements. this work was supported by the key: cord-272729-nbgdmavr authors: kim, youngnam; lee, changhee title: ribavirin efficiently suppresses porcine nidovirus replication date: 2012-10-27 journal: virus res doi: 10.1016/j.virusres.2012.10.018 sha: doc_id: 272729 cord_uid: nbgdmavr porcine reproductive and respiratory syndrome virus (prrsv) and porcine epidemic diarrhea virus (pedv) are porcine nidoviruses that represent emerging viral pathogens causing heavy economic impacts on the swine industry. although ribavirin is a well-known antiviral drug against a broad range of both dna and rna viruses in vitro, its inhibitory effect and mechanism of action on porcine nidovirus replication remains to be elucidated. therefore, the present study was conducted to determine whether ribavirin suppresses porcine nidovirus infection. our results demonstrated that ribavirin treatment dose-dependently inhibited the replication of both nidoviruses. the antiviral activity of ribavirin on porcine nidovirus replication was found to be primarily exerted at early times post-infection. treatment with ribavirin resulted in marked reduction of viral genomic and subgenomic rna synthesis, viral protein expression, and progeny virus production in a dose-dependent manner. investigations into the mechanism of action of ribavirin against prrsv and pedv revealed that the addition of guanosine to the ribavirin treatment significantly reversed the antiviral effects, suggesting that depletion of the intracellular gtp pool by inhibiting imp dehydrogenase may be essential for ribavirin activity. further sequencing analysis showed that the mutation frequency in ribavirin-treated cells was similar to that in untreated cells, indicating that ribavirin did not induce error-prone replication. taken together, our data indicate that ribavirin might not only be a good therapeutic agent against porcine nidovirus, but also a potential candidate to be evaluated against other human and animal coronaviruses. the nidovirales are an order of enveloped single-stranded positive-sense rna viruses with animal hosts that include the families arteriviridae, coronaviridae, and roniviridae (cavanagh, 1997; mayo, 2002; spaan et al., 2005) . despite striking differences in genome size and virion morphology, the genome organization and expression strategy of the two groups belonging to the nidovirales order were found to be comparable. the nidovirus genome contains two large orfs, 1a and 1b, comprising the 5 two-thirds of the viral genome encoding non-structural proteins (nsps) and the remaining orfs located in the 3 terminal region coding for structural proteins (lai et al., 2007; snijder and spaan, 2007) . the initial translation from orf1a and orf1b yields the 1a and lab replicase polyproteins, respectively, which are then proteolytically processed into functional nsps including the viral rna-dependent rna polymerase (rdrp) (bautista et al., 2002; van aken et al., 2006; ziebuhr et al., 2000) . the rdrp-containing replication complex mediates genomic rna replication and subgenomic (sg) mrna transcription, eventually generating a nested set of 3 -coterminal sg mrnas that are individually translated to structural proteins (lai et al., 2007; snijder and spaan, 2007) . porcine reproductive and respiratory syndrome virus (prrsv), a pathogenic macrophage-tropic arterivirus of pigs, is the etiological agent of acute respiratory illness in young piglets and reproductive failure in pregnant sows (albina, 1997) . prrsv primarily replicates in porcine alveolar macrophages (pams) and can establish persistent infection in lymphoid tissues of infected pigs that lasts for several months (albina et al., 1994; christopher-hennings et al., 1995; duan et al., 1997; wills et al., 2003) . as a result, prrsv infection results in suppression of normal macrophage functions and immune responses, which may render pigs susceptible to secondary bacterial or viral infections, leading to more severe disease than either agent alone (allan et al., 2000; feng et al., 2001; harms et al., 2001; wills et al., 2000) . porcine epidemic diarrhea virus (pedv), a pathogenic enterocyte-tropic coronavirus of swine, is the etiological agent of acute enteritis, which is characterized by lethal watery diarrhea followed by dehydration leading to death with a high mortality rate in suckling piglets (debouck and pensaert, 1980) . these two viruses, prrsv and pedv, are devastating porcine nidoviral pathogens that have still continued to plague swineproducing nations, causing tremendous economic losses to the global and asian pork industries (neumann et al., 2005; pensaert and yeo, 2006) . ribavirin (1-␤-d-ribofuranosyl-1,2,4-triazole-3-carboxamide, also known as virazole) is a synthetic guanosine analog that exhibits broad-spectrum antiviral activity in vitro (sidwell et al., 1972) . it has been used experimentally against a wide range of both dna and rna viruses, including gb virus b, hantaan virus, hendra virus, respiratory syncytial virus, lassa fever virus, norwalk virus, and west nile virus (chang and george, 2007; cooper et al., 2003; day et al., 2005; mccormick et al., 1986; lanford et al., 2001; rockx et al., 2010; severson et al., 2003) . most notably, ribavirin is used in combination with interferon-␣ for treatment of chronic hepatitis c virus (hcv) infections (cummings et al., 2001; davis et al., 1998) . however, there is still no report regarding an antiviral effect of ribavirin during the replication cycle of porcine nidoviruses. in the present study, therefore, we tried to investigate the antiviral activity of ribavirin and its mechanism of action in target cells upon porcine nidovirus infection. independent treatment of target cells with ribavirin significantly impaired prrsv and pedv infection. further experiments revealed that suppression of ribavirin affects post-entry steps of the replication cycle of prrsv and pedv, including viral genomic and sg rna synthesis, viral protein expression, and virus production. the addition of guanosine to the ribavirin treatment resulted in moderate reversal of the antiviral effects, suggesting that ribavirin activity is involved in the depression of cellular guanosine triphosphate (gtp) levels. sequencing analysis of the prrsv and pedv genomes in the ribavirin-treated and non-treated groups revealed that the mutation rates were similar and indicated that ribavirin did not induce catastrophic mutations during the replication of porcine nidoviruses. altogether, our results suggest that ribavirin may be an excellent therapeutic option for nidovirus infection in a human or veterinary subject. pam-pcd163 cells were cultured in rpmi 1640 medium (invitrogen, carlsbad, ca) supplemented with 10% fetal bovine serum (fbs, invitrogen), antibiotic-antimycotic solutions (100×; invitrogen), 10 mm hepes (invitrogen), 1 mm sodium pyruvate (invitrogen), and nonessential amino acids (100×; invitrogen) in the presence of 50 g/ml zeocin (invitrogen). vero cells were cultured in alpha minimum essential medium (␣-mem, invitrogen) with 10% fbs and antibiotic-antimycotic solutions. the cells were maintained at 37 • c in a humidified 5% co 2 incubator. prrsv strain vr-2332 was propagated in pam-pcd163 cells as described previously . pedv strain sm98-1 was kindly provided by the korean animal, plant and fisheries quarantine and inspection agency and propagated in vero cells as described previously (hofmann and wyler, 1988) . ribavirin and mycophenolic acid (mpa) were purchased from sigma (st. louis, mo) and dissolved in distilled water (dw) or dimethyl sulfoxide (dmso), respectively. a monoclonal antibody (mab; sdow17) against the prrsv n protein was purchased from rural technologies (brookings, sd). the pedv spike (s) glycoprotein-specific and n protein-specific monoclonal antibodies (mabs) were kind gifts from sang-geon yeo (kyungpook national university, daegu, south korea). the anti-␤-actin antibody and horseradish peroxidase (hrp)-conjugated secondary antibody were purchased from santa cruz biotechnology (santa cruz, ca). the cytotoxic effects of reagents on pam-pcd163 and vero cells were analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (mtt) assay (sigma) detecting cell viability. briefly, pam-pcd163 and vero cells were grown at 1 × 10 4 cells/well in a 96-well tissue culture plate with ribavirin or mpa treatment for 24 h. after one day of incubation, 50 l of mtt solution (1.1 mg/ml) was added to each well and the samples were incubated for an additional 4 h. the supernatant was then removed from each well, after which 150 l of dmso was added to dissolve the color formazan crystal produced from the mtt. the absorbance of the solution was measured at 540 nm using an enzyme-linked immunosorbent assay plate reader. all mtt assays were performed in triplicate. pam-pcd163 and vero cells grown on microscope coverslips placed in 6-well tissue culture plates were pretreated with ribavirin or mpa for 1 h and mock infected or infected with prrsv and pedv at a multiplicity of infection (moi) of 01, respectively. the virusinfected cells were further grown in the presence of ribavirin until 48 hpi, fixed with 4% paraformaldehyde for 10 min at room temperature (rt) and permeabilized with 0.2% triton x-100 in pbs at rt for 10 min. the cells were blocked with 1% bovine serum albumin (bsa) in pbs for 30 min at rt and then incubated with n-specific mab 7 for 2 h. after being washed five times in pbs, the cells were incubated for 1 h at rt with a goat anti-mouse secondary antibody conjugated with alexa fluor 488 (molecular probes, carlsbad, ca), followed by counterstaining with 4 ,6-diamidino-2-phenylindole (dapi; sigma). the coverslips were mounted on microscope glass slides in mounting buffer (60% glycerol and 0.1% sodium azide in pbs) and cell staining was visualized using a fluorescent leica dm il led microscope (leica, wetzlar, germany). pam-pcd163 and vero cells were grown in 6-well tissue culture plates for 1 day and were mock infected or infected with prrsv or pedv at an moi of 0.1. at the indicated times, cells were harvested in 50 l of lysis buffer (0.5% tritonx-100, 60 mm ␤glycerophosphate, 15 mm -nitro phenyl phosphate, 25 mm mops, 15 mm, mgcl 2 , 80 mm nacl, 15 mm egta [ph 7.4], 1 mm sodium orthovanadate, 1 g/ml e64, 2 g/ml aprotinin, 1 g/ml leupeptin, and 1 mm pmsf) and sonicated on ice five times for 1 s each. homogenates were lysed for 30 min on ice, and clarified by centrifugation at 15,800 × g (eppendorf centrifuge 5415r, hamburg, germany) for 30 min at 4 • c. the protein concentrations of the cell lysates were determined by a bca protein assay (pierce, rockford, il). the cell lysates were mixed with 4× nupage sample buffer (invitrogen) and boiled at 70 • c for 10 min. the proteins were separated on nupage 4-12% gradient bis-tris gel (invitrogen) under reducing conditions, and electrotransferred onto immunobilon-p (millipore, billerica, ma). the membranes were subsequently blocked with 3% powdered skim milk (bd biosciences, belford, ma) in tbs (10 mm tris-hcl [ph 8.0], 150 mm nacl) with 0.05% tween-20 (tbst) at 4 • c for 2 h, and reacted at 4 • c overnight with the primary antibody against prrsv n, pedv s or ␤-actin. the blots were then incubated with the secondary horseradish peroxidase (hrp)-labeled antibody (santa cruz biotechnology) at a dilution of 1:5000 for 2 h at 4 • c. proteins were visualized by enhanced chemiluminescence (ecl) reagents (amersham biosciences, piscataway, nj) according to the manufacturer's instructions. to quantify viral protein production, band densities of prrsv n and pedv s proteins were quantitatively analyzed using a computer densitometer with the wright cell imaging facility (wcif) version of the imagej software package (http://www.uhnresearch.ca/facilities/wcif/imagej/) based on the density value relative to ␤-actin gene. pam-pcd163 and vero cells were infected with prrsv or pedv, respectively, at an moi of 0.1 as described above. at −1, 0, 1, 2, 4, 6, 8, 10, 12, or 24 hpi, ribavirin was added to give the indicated final concentration for the remainder of the time course experiment. the virus-infected and ribavirin-treated cells were further maintained and the infection was terminated at 48 hpi by fixing the monolayers with 4% paraformaldehyde for 10 min at rt. the fixed cells were subjected to ifa to assess the presence of prrsv or pedv infection as described above. an internalization assay was performed as described previously (cai et al., 2007) . briefly, pam-pcd163 and vero cells grown in 6-well culture plates were infected with prrsv and pedv, respectively, at an moi of 0.1 at 4 • c for 1 h, respectively. unbound viruses were then washed with pbs and the cells were either incubated at 4 • c or 37 • c in the presence ribavirin for 1 h, followed by treatment with protease k (0.5 mg/ml) at 4 • c for 45 min to remove the bound but uninternalized virus particles. the prrsv-infected cells were then serially diluted in rpmi and added onto fresh pam-pcd163 cell monolayers in 96-well tissue culture plates. at 48 h post-incubation, internalized viruses were titrated through ifa as described above and the 50% tissue culture infectious dose (tcid 50 ) was calculated. for pedv, the serially diluted infected cells were added onto uninfected vero cells and after 48 h, internalized viruses were titrated using a plaque assay as described previously , and the plaques were counted after 1% crystal violet staining. pam-pcd163 and vero cells were incubated with ribavirin for 1 h prior to infection and then inoculated with prrsv or pedv at an moi of 1 for 1 h at 37 • c. the virus inoculum was subsequently removed and the infected cells were maintained in fresh medium containing ribavirin for 48 h. total rna was extracted from lysates of the infected cells at 48 hpi using trizol reagent (invitrogen) and treated with dnase i (takara) according to the manufacturer's protocols. the concentrations of the extracted rna were measured using a nanovue spectrophotometer (ge healthcare, piscataway, nj). quantitative real-time rt-pcr was conducted using a thermal cycler dice real time system (takara) with gene-specific primer sets (table 1) as described previously (sagong and lee, 2011) . the rna levels of viral genes were normalized to that of mrna for the ␤-actin or glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene and relative quantities (rq) of mrna accumulation were evaluated using the 2 − ct method. to detect alteration of genomic rna and sg mrna levels in the presence of ribavirin during porcine nidovirus infection, the results obtained using ribavirin-treated samples were compared with vehicle-treated results. pam-pcd163 and vero cells were prrsv or pedv infected with treatment of ribavirin as described above. the culture supernatant was collected at different time points (6, 12, 24, 36, and 48 hpi) and stored at −80 • c. the titer of prrsv was measured by limiting dilution on pam-pcd163 cells through ifa as described above and then quantified as the tcid 50 per ml. the pedv titer was determined by a plaque assay using vero cells and quantified as plaque-forming units (pfu) per ml. pam-pcd163 and vero cells were preincubated with 100 m guanosine for 6 h before ribavirin was added to the medium at various final concentrations and then inoculated with prrsv or pedv as described above. the virus inoculum was removed and the infected cells were maintained in fresh medium containing ribavirin and guanosine. at 48 h dpi, the virus-infected cells were fixed and subjected to ifa to evaluate the presence of prrsv or pedv infection as described above. the n gene coding regions for prrsv and pedv were sequenced to monitor mutation frequency. cells were prrsv or pedv infected and treated with ribavirin as described above. at 48 hpi, total rna was extracted by trizol, and rt-pcr was performed to amplify the region encoding the orf5 to orf7 genes of prrsv or the n gene of pedv using gene-specific primer sets (table 1) . the corresponding pcr product was then cloned into the pgem-t vector (promega, madison, wi). for sequencing of the gene in the recombinant vector, we selected 20 independent bacterial colonies per group and analyzed mutations in the region. a student's t test was used for all statistical analyses and pvalues of less than 0.05 were considered statistically significant. virus-specific cpes were observed daily and were photographed at 48 hpi using an inverted microscope at a magnification of 100× (first panels). for immunostaining, infected cells were fixed at 48 hpi and incubated with mab against the n protein of prrsv or pedv followed by alexa green-conjugated goat anti-mouse secondary antibody (second panels). the cells were then counterstained with dapi (third panels) and examined using a fluorescent microscope at 200× magnification. viral productions in the presence of ribavirin were measured by quantifying the number of cells expressing n proteins through ifa. five fields at 200× magnification were counted per each condition and the total number of cells per field as determined by dapi staining was similar in all fields. values are representative of the mean of three independent experiments and error bars represent standard deviations. *p = 0.001-0.05; † p < 0.001. to examine the effect of ribavirin on porcine nidovirus replication, prrsv and pedv were selected because they are important viral pathogens that economically affect the swine industry. based on mtt assay, none of the doses of ribavirin tested in the present study caused detectable cell death of pam-pcd163 and vero cells (data not shown). pam-pcd163 and vero cells were pretreated with ribavirin at concentrations of 10-50 m or 10-200 m, respectively, or with dw as a vehicle control for 1 h prior to infection. ribavirin was present during the entire period of infection. viral production was initially measured by monitoring the cytopathic effect (cpe) after infection and confirmed by immunofluorescence using n protein-specific mab at 48 hpi (fig. 1) . in vehicle-treated control cells, visible cpe appeared at 24 hpi and became predominant by 48 hpi, and n-specific staining was clearly evident in many cell clusters, indicating infection and the spread of the virus to neighboring cells. in contrast, ribavirin had an obvious inhibitory effect on virus propagation. as shown in fig. 1a and b, ribavirin significantly decreased virus-induced cpe and viral gene expression of prrsv and pedv at concentrations of ∼10 m and ∼50 m, respectively. n protein staining revealed that the number of cells expressing viral antigen, as quantified by n protein staining results, was also reduced during ribavirin treatment, resulting in a maximum of ∼80% inhibition in response to 50 m and 200 m for prrsv and pedv, respectively ( fig. 1a and b) . moreover, the effective doses for inhibiting 50% (ed 50 ) of the replication of prrsv and pedv were determined to be about 21 m and 73 m, respectively. taken together, these data demonstrate that ribavirin efficiently suppresses the replication of porcine nidoviruses. to further determine at which point ribavirin acted during the porcine nidovirus infection period, pam-pcd163 and vero cells were treated independently with ribavirin at various time points post-infection. at 48 hpi, the levels of prrsv or pedv replication were measured indirectly as viral antigen production by quantifying cells expressing the n protein through ifa (fig. 2) . treatment of cells with 50 m ribavirin for up to 4 hpi resulted in more than 80-90% decrease in prrsv production when compared to the untreated control. addition of ribavirin between 6 and 12 hpi led to 75-38% inhibition of prrsv infectivity. similarly, treatment with 200 m ribavirin at −1, 0, and 1 hpi was found to lead to 83-61% suppression of pedv production, while its treatment at 2-12 hpi resulted in 49-28% reduction in pedv infectivity. in contrast, no significant impairment of porcine nidovirus propagation was observed when ribavirin was added at 24 hpi. these results demonstrate that the inhibitory effect of ribavirin against the replication of prrsv and pedv was exerted primarily during the initial period of infection, suggesting that its action occurs mainly at early time points after porcine nidovirus infection. to further assess the antiviral activity of ribavirin against porcine nidovirus replication, virus yield was determined during treatment of ribavirin. upon infection, viral supernatants were collected at 48 hpi and viral titers were measured. as fig. 3a shows, the presence of ribavirin reduced the release of viral progeny in a dose-dependent manner. the peak viral titer was determined to be 10 6 tcid 50 /ml and 10 6.34 pfu/ml in the vehicle-treated control for prrsv and pedv, respectively. however, the addition of 50 m or 200 m ribavirin decreased titers of prrsv and pedv to 10 3.53 tcid 50 /ml and 10 3.52 pfu/ml, respectively (almost 3 log reduction compared with the control). the growth kinetics study further demonstrated that the overall process of porcine nidovirus replication was significantly delayed when cells were treated with ribavirin (fig. 3b) . consequently, these findings revealed that ribavirin inhibits optimal progeny virus release within the natural host cells. to evaluate which steps of the replication cycle of porcine nidoviruses were targeted by ribavirin, we started focusing on the earliest step, virus entry upon the ribavirin treatment. to address this issue, an internalization assay was performed as described previously (cai et al., 2007) . pam-pcd163 and vero cells were inoculated with prrsv and pedv, respectively, at 4 • c for 1 h to allow virus attachment and further maintained either at 4 • c or 37 • c to permit virus internalization in the presence of ribavirin. the samples were then treated with protease k to remove the remaining viruses from the cell surface. the serially diluted infected cells were subsequently subjected to an infectious center assay on uninfected pam-pcd163 and vero cell monolayers and virus titers were measured 2 days later through ifa or by plaque assay. as shown in fig. 3c , the titers of prrsv and pedv were virtually the same in cells treated with ribavirin or without ribavirin at 37 • c and determined to be 10 1.7 -10 2.2 tcid 50 /ml and 2.2 × 10 2 -3.2 × 10 2 pfu/ml, respectively, indicating no difference between those cells. in contrast, only minimal viral productions of about 10 1.3 tcid 50 /ml (prrsv) and 4.5 × 10 1 pfu/ml (pedv) were observed in cells maintained at 4 • c, which was likely due to inefficient removal of the bound virus. these results indicated that ribavirin has no inhibitory effect on the virus entry process. like all positive-sense rna viruses, following the uncoating process, the nidovirus genome is released into the cytoplasm and immediately serves as a template for viral translation by the same cellular cap-dependent mechanism. early nidovirus translation produces replicase polyproteins that are posttranslationally cleaved into nsps by viral proteases. subsequently, the nonstructural replicase proteins mediate de novo synthesis of viral rna, including genomic rna replication and sg mrna transcription. the nidovirus structural proteins are translated lately from respective sg mrna transcripts. thus, to determine the inhibitory mechanism of ribavirin on the postentry steps of the nidovirus life cycle, we first investigated whether viral protein translation was affected by ribavirin. to accomplish this, pam-pcd163 and vero cells were treated with ribavirin for 1 h prior to infection, and the drug was allowed to remain during infection and subsequent incubation. the expression levels of the prrsv n and pedv s proteins in the presence or absence of ribavirin were evaluated at 48 hpi by western blot analysis. the production of both proteins productions was drastically diminished during ribavirin treatment (fig. 4) . densitometric analysis of the western blots revealed that the intracellular expression of both n and s proteins was reduced by ribavirin, with a maximum of about 90% inhibition at the concentration of 50 m and 200 m, respectively (fig. 4) . this marked reduction was probably not the result of a nonspecific decrease in the translation mechanism, since ponceau s staining results indirectly indicated that the expression levels of the overall cellular proteins remained similar following treatment (data not shown). taken together, these results demonstrated the inhibitory effects of ribavirin specifically against viral translation during porcine nidovirus replication. for positive-strand rna viruses, synthesis of the viral nonstructural proteins is required for viral rna replication and transcription. thus, it is conceivable that impaired viral protein expression in would be caused by inhibition of viral rna synthesis. since nidovirus infection produces two rna entities including genomic versus subgenomic, we therefore determined if ribavirin specifically affected genome replication and sg mrna transcription. to answer this question, the relative levels of both genomic rna and sg mrna were assessed by quantitative real-time strand-specific rt-pcr in the presence or absence of ribavirin upon porcine nidovirus infection. as shown in fig. 5a , ribavirin exhibited a maximal 70% and 80% reduction in the synthesis of prrsv genomic rna and sg after washing with cold pbs, infected cells were maintained in the presence or absence of ribavirin either at 4 • c or 37 • c for an additional hour. bound but uninternalized virus particles were removed by treatment with protease k. the infected cells were then serially diluted and plated onto fresh target cells. at 2 days post-incubation, internalized viruses were titrated by ifa and plaque assay for prrsv (left) and pedv (right), respectively. results are expressed as the mean values from triplicate wells and error bars represent standard deviations. *p = 0.001-0.05; † p < 0.001. mrna at a concentration of 50 m, respectively, when compared with untreated infected cells. furthermore, a similar inhibitory effect of ribavirin on genome replication and sg mrna transcription of pedv was observed. the amounts of pedv genomic rna and sg mrna detected in cells treated with 200 m ribavirin were only about 13% and 11% of the untreated level (fig. 5b) . the decreases in viral rna levels after the addition of ribavirin were not due to nonspecific inhibition of transcription, as mrna levels of the internal control (␤-actin or gapdh) remained unchanged in all samples (data not shown). altogether, these results indicated that ribavirin suppresses the synthesis of the nidoviral genomic rna and sg mrna. several mechanisms of action for the antiviral activity of ribavirin have been suggested, including a reduction in cellular gtp pools via inosine monophosphate dehydrogenase (impdh) inhibition and increased mutation frequency on the virus genome leading to error catastrophe (graci and cameron, 2006) . to assess these known mechanisms, we first examined whether replenishment of guanosine affected the antiviral effect of ribavirin against porcine nidovirus infection. the addition of 100 m guanosine to the ribavirin-treated and virus-infected cells was found to moderately reverse the antiviral activity of ribavirin. the incubation of ribavirin alone reduced prrsv production to 29% and 17% at 10 m and 50 m, respectively, whereas supplementation of guanosine to ribavirin inhibited virus production to 40% and 26% at the same concentrations (fig. 6a, left panel) . likewise, while pedv production was declined to 71%, 37%, and 12% in the presence of ribavirin alone at 50 m, 100 m, and 200 m, respectively, it decreased to 100%, 48%, and 31% at the same concentrations when guanosine was added to the ribavirin treatment (fig. 6a, right panel) . to verify these results, we also tested the effects of mpa, a potent inhibitor of cellular impdh, on the replication of prrsv and pedv. as shown in fig. 6b , mpa efficiently reduced porcine nidovirus propagation fig. 4 . inhibition of viral protein translation by ribavirin. ribavirin-treated pam-pcd163 and vero cells were mock-infected or infected with prrsv (a) or pedv (b) for 1 h and were further cultivated in the presence or absence of ribavirin. at 48 hpi, cellular lysates were collected, resolved by sds-page, transferred to a nitrocellulose membrane, and immunoblotted by using the antibody that recognizes the prrsv n protein or the pedv s protein. the blot was also reacted with mouse mab against ␤-actin to verify equal protein loading. each viral protein expression was quantitatively analyzed by densitometry in terms of the relative density value to the ␤-actin gene and ribavirintreated sample results were compared to vehicle-control results. values are representative of the mean from three independent experiments and error bars denote standard deviations. *p = 0.001-0.05; † p < 0.001. inhibition of viral rna transcription by ribavirin. pam-pcd163 and vero cells pretreated with ribavirin were mock-infected or infected with prrsv (a) or pedv (b) for 1 h and were incubated in the presence of ribavirin. total cellular rna was extracted at 48 hpi, and strand-specific viral genomic rnas (black bars) and sg mrnas (white bars) of prrsv and pedv were amplified by quantitative real-time rt-pcr. viral positive-sense genomic rna and sg mrna were normalized to mrna for porcine ␤-actin or monkey gapdh and relative quantities (rq) of mrna accumulation were evaluated. ribavirin-treated sample results were compared with untreated results. values are representative of the mean from three independent experiments and error bars denote standard deviations. *p = 0.001-0.05; † p < 0.001. at concentrations greater than 0.5 m and 0.1 m in a dosedependent manner for prrsv and pedv, respectively. sequence analysis of the porcine nidovirus genome in the presence or absence of ribavirin was conducted to investigate whether it triggers catastrophic mutations. we analyzed 20 recombinant sequences of the orf5 to orf7-coding region corresponding to nucleotide numbers 13,559-15,090 (prrsv) and the n gene region corresponding to nucleotide numbers 26,335-27,660 (pedv) from each virus grown in the presence of ribavirin. as controls, 20 colonies were also obtained individually from untreated prrsv-and pedv-infected a the prrsv orf5-orf7 coding region (1532 bp) and the pedv n (1326 bp) gene were rt-pcr amplified and the amplicon product was then cloned into the pgem-t vector. for sequencing of the gene in the recombinant vector, 20 clones per group were selected and analyzed the number (rate) of mutations. b a total of 30,640 nt and 26,520 nt for prrsv and pedv were sequenced and a mutation frequency was calculated per 1000 nt, respectively. cells. in the ribavirin-treated groups, point mutations occurred in 17 (85%) plasmids for both prrsv and pedv, which corresponded to a mutation frequency of 1.49 and 1.66 per 1000 nt, respectively. interestingly, mutations were also found in 17 (85%) plasmids in the untreated groups for prrsv and pedv, which were calculated as a mutation frequency of 1.69 and 1.24 per 1000 nt, respectively (table 2) . these sequencing results indicated that there were no significant differences in the mutation rates of ribavirin-treated and non-treated groups upon porcine nidovirus infection. the nidovirales are an order of enveloped, positive-strand rna viruses with animal hosts, which synthesize a nested set of multiple sg mrnas. this order includes three families arteriviridae, coronaviridae, and roniviridae, which have similar genome organization and replication strategy, but different virion morphology and genome length (mayo, 2002; lai et al., 2007; snijder and spaan, 2007) . porcine nidoviruses are not only devastating viral pathogens to the pig population, but can also be applied as an animal virus model to study human or veterinary-important nidoviruses. among these, despite tremendous efforts to control the diseases, prrsv and pedv have continuously plagued pigproducing countries, leading to significant economic impacts on the global or asian swine industry, respectively. this is partially due to a lack of efficient vaccines capable of conferring full protection against viral infections and antiviral agents for treating porcine nidoviruses. although ribavirin has an antiviral effect on a variety of dna and rna viruses (sidwell et al., 1972) , its efficacy and mode of action on porcine nidovirus replication are currently unknown. the present study demonstrated that ribavirin also exerts very effective antiviral activity against prrsv and pedv in vitro, as indicated by ed 50 values of approximately 21 m (5.1 g/ml) and 73 m (17.8 g/ml), respectively. ribavirin can potentially act via inhibition of various steps of the virus life cycle, including viral translation, genome or transcript capping, rna synthesis, and progeny virus spread (graci and cameron, 2006) . treatment of cells with ribavirin resulted in significant attenuation of postentry steps during the replication of porcine nidovirus, as determined by lower progeny production, diminished viral protein expression, and decreased synthesis of genomic rna and sg mrna. it was previously reported that the tier of murine norovirus 1 (mnv-1) in the presence of ribavirin dropped about 10-fold after virus infection, but remained similar after 48 h of virus infection (chang and george, 2007) . in this study, growth kinetics experiments also indicated that the reduced rate of nidoviral titers in the presence of ribavirin was found to be comparable between 24 and 48 hpi. since ribavirin should be converted to its monophosphate active form (rmp) to exert antiviral activity, it appears that virus infection may disrupt the normal metabolic processes, leading to interference with the conversion of ribavirin (parker, 2005) . taken together, our data indicate that ribavirin potently elicits antiviral activity against prrsv and pedv in target cells. an important aspect of the antiviral activity of ribavirin may be attributed to the ability to act via multiple mechanisms simultaneously. although the mechanisms of action of ribavirin still remain controversial, five primary mechanisms have been proposed depending on the particular virus. these include reduction in cellular gtp pools by inhibiting impdh, enhanced mutation frequency via its incorporation into the viral genome leading to catastrophic error, modulation of host immune responses, inhibition of 5 mrna capping, and interference with viral rna polymerase. therefore, the mechanisms of action of ribavirin may differ among viruses, and its antiviral activity may be operated predominantly via one or two mechanisms (graci and cameron, 2006; parker, 2005) . to elucidate potential mechanisms responsible for the antiviral effect of ribavirin on porcine nidoviruses, we first tested whether the antinidoviral activity of ribavirin is involved in inhibition of impdh and subsequent depression of cellular gtp levels. in previous studies, the addition of guanosine to the culture medium efficiently reversed the antiviral effects of ribavirin against norovirus, yellow fever virus, and human parainfluenza virus 3 (chang and george, 2007; leyssen et al., 2005) . the present study so investigated the effects replenishing gtp by adding guanosine to ribavirin-treated cells upon virus infection. consistent with previous reports, the addition of guanosine to the ribavirin treatment significantly reversed the antiviral activity of ribavirin in porcine nidoviruses. furthermore, a noncompetitive impdh inhibitor, mpa, was found to strongly inhibit the replication of prrsv and pedv at concentrations above 0.5 m and 0.1 m, respectively. in contrast to ribavirin, which has to be converted to the active rmp form to elicit the antiviral activity, mpa does not require metabolic activation to exert its function. thus, the efficient antinidoviral activity of mpa suggests that ribavirin may directly lead to the inhibition of intracellular impdh levels. since rna viruses replicate with high genetic variation, they exist as viral quasispecies that are complex and dynamic mutant distributions that share a dominant nucleotide sequence and a mutant spectrum (ruiz-jarabo et al., 2000) . therefore, rna viruses reside on the edge of mutation crisis, and the increased average error frequency can cause defective genetic information and reduced viability termed error catastrophe (crotty et al., 2000; day et al., 2005) . ribavirin has been shown to trigger catastrophic mutations including lethal mutations in a variety of viruses and these may accumulate as a virus goes through multiple rounds of replication, resulting in extinction of the viral population (crotty et al., 2001; contreras et al., 2002; day et al., 2005; lanford et al., 2001; severson et al., 2003) . however, sequence analysis of the conserved n gene regions of prrsv and pedv in the ribavirintreated or untreated groups revealed that the two groups produced similar mutation frequencies (1.89/1.66 versus 2.42/1.24). these results were not surprising since rna genomes mutate at average rates of 10 −3 -10 −5 per nucleotide copied during replication of rna viruses by the virus-encoded rna-dependent rna polymerase (rdrp) lacking proofreading functions (drake and holland, 1999) . our data indicated that the antiviral activity of ribavirin against porcine nidoviruses may not be associated with error-prone replication by increasing the probability of ribavirin incorporation into the viral genome. in conclusion, our findings described here demonstrated that ribavirin effectively prevents the replication of porcine nidoviruses in target cells at subcytotoxic doses. additionally, the highest doses of ribavirin used in this study did not increase the frequency of mutations in the nidoviral genome, and instead affected intracellular gtp levels via impdh inhibition. thus, we propose that one of the modes of action of ribavirin to elicit the antiviral effects against porcine nidoviruses occurs via gtp depletion, which may not work in concert with catastrophic mutations. although further studies based on in vivo assessment are needed to evaluate the efficacy and safety of ribavirin, the results presented here indicate that it is a good candidate of choice for antiviral approach against porcine nidoviral diseases. immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview experimental infection of colostrum deprived piglets with porcine circovirus 2 (pcv2) and porcine reproductive and respiratory syndrome virus (prrsv) potentiates pcv2 replication functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus suppression of coronavirus 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and the effectiveness of ribavirin treatment memory in viral quasispecies porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-␤ production by inhibiting irf3 activation in immortalized porcine alveolar macrophages ribavirin causes error catastrophe during hantaan virus replication broad-spectrum antiviral activity of virazole: 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide arteriviridae family coronaviridae proteolytic maturation of replicase polyprotein pp1a by the nsp4 main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp7 synergism between porcine reproductive and respiratory syndrome virus (prrsv) and salmonella choleraesuis in swine duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus virus-encoded proteinases and proteolytic processing in the nidovirales this research was supported by basic science research program through the national research foundation of korea (nrf) funded by the ministry of education, science and technology (2010-0002318). key: cord-311561-eiys4mbf authors: song, deping; chen, yanjun; peng, qi; huang, dongyan; zhang, tiansheng; huang, tao; zhang, fanfan; zhou, xinrong; tang, yuxin title: full-length genome sequence of a variant porcine epidemic diarrhea virus strain, ch/gdzq/2014, responsible for a severe outbreak of diarrhea in piglets in guangdong, china, 2014 date: 2014-12-04 journal: genome announc doi: 10.1128/genomea.01239-14 sha: doc_id: 311561 cord_uid: eiys4mbf the full-length genome sequence of a variant porcine epidemic diarrhea virus (pedv) strain, ch/gdzq/2014, was determined. the isolate was a variant strain with a relatively far relationship with the pedv strains previously identified in the same area between 2011 and 2012 and was genetically distinct from the cv777-based vaccine strain currently being used in china. stranded rna virus, is a member of the genus alphacoronavirus (1, 2) . field strains of pedv, which are genetically distinct from the cv777-based vaccine strain, have caused severe epidemics of diarrhea in swine herds in china since 2010. the mortality rate in neonatal piglets affected with variant field strains of pedv can reach as high as 100% (3) (4) (5) (6) (7) . here, we report the full-length genome sequence of a variant pedv isolate, ch/gdzq/2014, which is responsible for a severe outbreak of diarrhea in guangdong, southern china, in march 2014. fecal samples were collected from piglets infected with pedv. afterward, viral rna was extracted, and then the full-length genome sequence of ch/gdzq/2014 was amplified by 33 pairs of primers specific to pedv. the amplicons obtained were cloned into the pmd18-t vector (takara, japan) and sequenced. the complete genome sequence was assembled and annotated by using dnastar lasergene version 7.10 (dnastar, inc., madison, wi). nucleotide and amino acid sequences along with 32 reference pedv sequences retrieved from genbank were comparatively analyzed. the complete genome sequence of ch/gdzq/2014 is 28,038 nucleotides (nt) in length, excluding the 3= poly(a) tail. (8) (9) (10) (11) , indicating that ch/gdzq/2014 was genetically distinct from these strains. sequence homology analysis showed that ch/gdzq/2014 had 96.7% nt identity with the prototype cv777 strain (12) but shared 97.8 to 99.0% nt identity with the variant pedv strains that have emerged since 2010 in china (13, 14) and 98.8% nt identity with two newly identified u.s. strains (15, 16) . the s protein of ch/ gdzq/2014 was 4,161 nt long, encoding a protein of 1,386 amino acids, and had 93.1 to 98.6% nt identity with the reference strains, showing the lowest identity with the cv777-based vaccine strain (nc_003436) (17) and the highest identity with two chinese strains (bj-2011-1 and js-hz/2012). compared to the deduced amino acid (aa) sequences of the s gene of cv777, ch/gdzq/ 2014 showed 5 aa insertions (59qgvn62, 139n140), 2 aa deletions (161gk162), and 94 aa mutations. of these variations, most were located in the n-terminal of the s protein (18, 19) . ch/gdzq/2014 was a very virulent field pedv strain isolated from guangdong province in southern china. the genome data provided here will help better understand molecular characteristics of variant field strains of pedv currently affecting swine in guangdong province. nucleotide sequence accession number. the complete genome sequence of pedv strain ch/gdzq/2014 was deposited in genbank under the accession number km242131. a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines complete genome sequence of porcine epidemic diarrhea virus strain aj1102 isolated from a suckling piglet with acute diarrhea in china molecular epidemiology of porcine epidemic diarrhea virus in china complete genome sequence of a chinese virulent porcine epidemic diarrhea virus strain new variants of porcine epidemic diarrhea virus, china emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences complete genome sequence of a highly prevalent isolate of porcine epidemic diarrhea virus in south china complete genome sequence of a very virulent porcine epidemic diarrhea virus strain, ch/ gdgz/2012, isolated in southern china complete genome sequence of a novel porcine epidemic diarrhea virus in south china complete genome sequence of novel porcine epidemic diarrhea virus strain gd-1 in china origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states whole-genome analysis of porcine epidemic diarrhea virus (pedv) from eastern china phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china complete genome sequence of porcine epidemic diarrhea virus strain usa/colorado/2013 from the united states new variant of porcine epidemic diarrhea virus completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo cloning and sequence analysis of the spike gene of porcine epidemic diarrhea virus chinju99 key: cord-287947-7kjxbd6t authors: gao, ruyi; zhang, yongshuai; kang, yuhui; xu, weiyin; jiang, luyao; guo, tingting; huan, changchao title: glycyrrhizin inhibits pedv infection and proinflammatory cytokine secretion via the hmgb1/tlr4-mapk p38 pathway date: 2020-04-23 journal: int j mol sci doi: 10.3390/ijms21082961 sha: doc_id: 287947 cord_uid: 7kjxbd6t our previous study showed that glycyrrhizin (gly) inhibited porcine epidemic diarrhea virus (pedv) infection, but the mechanisms of gly anti-pedv action remain unclear. in this study, we focused on the anti-pedv and anti-proinflammatory cytokine secretion mechanisms of gly. we found that pedv infection had no effect on toll-like receptor 4 (tlr4) protein and mrna levels, but that tlr4 regulated pedv infection and the mrna levels of proinflammatory cytokines. in addition, we demonstrated that tlr4 regulated p38 phosphorylation but not extracellular regulated protein kinases1/2 (erk1/2) and c-jun n-terminal kinases (jnk) phosphorylation, and that gly inhibited p38 phosphorylation but not erk1/2 and jnk phosphorylation. therefore, we further explored the relationship between high mobility group box-1 (hmgb1) and p38. we demonstrated that inhibition of hmgb1 using an antibody, mutation, or knockdown decreased p38 phosphorylation. thus, hmgb1 participated in activation of p38 through tlr4. collectively, our data indicated that gly inhibited pedv infection and decreased proinflammatory cytokine secretion via the hmgb1/tlr4-mitogen-activated protein kinase (mapk) p38 pathway. porcine epidemic diarrhea (ped) is characterized by vomiting, dehydration, and watery diarrhea. the causative agent of ped is ped virus (pedv), which emerged in asia, america, and europe and causes high mortality rates in suckling piglets [1] [2] [3] . ped was initially reported in belgium and the united kingdom in the late 1970s. recently, outbreaks in swine-raising countries worldwide have led to tremendous economic losses [1, [3] [4] [5] [6] [7] . ped is a significant public health concern because pedv is a potential threat to humans [8] . pedv, an enveloped rna virus with a single-stranded, positive-sense genome, rapidly becomes epidemic in nearby pig farms [9] [10] [11] . glycyrrhizin (gly) is a well-characterized component of gancao and has been used as traditional medicine. gly is extracted from licorice, which has many pharmacological and biological activities, such as anti-tumor, anti-inflammatory, anti-oxidative, and anti-viral effects [12] [13] [14] [15] . gly contains one molecule of glycyrretinic acid and two molecules of glucuronic acid (823 da) [16, 17] . gly has inhibitory effects on hepatitis c virus (hcv) [18, 19] , herpes simplex type 1 [20] , influenza virus [21] , and kaposi's sarcoma-associated herpes virus [22] . gly is also a high mobility group box-1 (hmgb1) inhibitor. extracellular hmgb1 involves a damage-associated molecular pattern to activate proinflammatory signaling pathways through toll-like receptor 4 (tlr4) [23, 24] . hmgb1 is a unique mediator of inflammation-associated events and innate immune responses [25] . we previously showed that gly inhibited pedv [26] , but the anti-pedv mechanisms of gly remain unclear. the mitogen-activated protein kinase (mapk) cascade pathways are activated by viral infection. the mapk pathways play a critical role in transmitting signals to the intracellular environment and exquisitely controlling numerous cellular activities. three distinct mapks have been identified on the basis of the respective terminal mapk components, namely, extracellular signal-regulated kinases (erk), mapk p38, and c-jun n-terminal kinases (jnk) [27] [28] [29] . the mapk pathways play vital role in viral infection including foot-and-mouth disease and influenza [30, 31] . severe acute respiratory syndrome coronavirus(sars-cov) 3b protein induces activator protein-1 (ap-1) transcriptional activity through activation of jnk and erk pathways, and sars-cov 3b protein has an important role in the stimulation of ap-1-dependent genes, especially proinflammatory cytokines causing the cytokine storm during sars virus infection [32] . the mapk p38 pathway is a key regulator of inflammatory responses [33] [34] [35] [36] . human coronavirus 229e and transmissible gastroenteritis virus wuhan 1 (tgev wh-1) replication requires mapk p38 activation [37, 38] . lipopolysaccharide (lps)-induced inflammation depends on p38 mapk signaling [39, 40] . infection and inflammation of influenza a virus are inhibited on the basis of tlr4-mapk p38 pathways [41] . hmgb1 promotes the synthesis of proinflammatory interleukin (pro-il)-1β and proinflammatory interleukin (pro-il)-18 by activation of mapk p38 [42] , and acts in synergy with lps to augment il-6 production, which depends on mapk p38 and nuclear transcription factor-kappa b (nf-κb) activation [43] . in the present study, we explored the anti-pedv mechanisms of gly. we demonstrated that the tlr4 inhibitor, tak-242 (tak), and mitogen-activated protein kinase p38 (mapk p38) inhibitor, sb202190 (sb), both inhibited pedv infection. in addition, we confirmed that hmgb1 and tlr4 functioned upstream of mapk p38, but that this was not the case for extracellular regulated protein kinases1/2 (erk1/2) and c-jun n-terminal kinase (jnk). the effects of gly against pedv infection depended on the hmgb1/tlr4-mapk p38 pathway. gly is a competitive inhibitor of hmgb1 cytokine activity. our previous study demonstrated that gly inhibited pedv infection, and that extracellular hmgb1 binding to tlr4 promoted inflammatory responses [44] . however, there have been no studies of the effects of pedv on tlr4. thus, we explored the effect of pedv infection on tlr4. vero cells were infected with pedv (0.1 multiplicity of infection (moi)) for different lengths of time (4, 8, 12, 24, and 36 h) . the vero cells were collected for western blotting and qrt-pcr. western blotting showed no change in tlr4 protein levels following infection of vero cells with pedv ( figure 1a ,b). in addition, qrt-pcr showed that pedv infection had no effect on tlr4 mrna levels ( figure 1c ). these results revealed that pedv infection in vero cells had no effect on tlr4 activation. antiviral responses are associated with tlr4, and viral rna and dna can interact with tlr4. previous studies showed that tlr4 can interact with the lipoprotein (ns1) of dengue virus and with ns5a of hcv [3, 18] . pedv infection had no effect on tlr4 expression, but we further explored whether tlr4 regulated pedv infection. we pretreated vero cells with the tlr4 inhibitor tak for 2 h and then infected the cells with pedv (0.1 moi) in the presence of various concentrations of tak for 24 h. vero cells and supernatants were collected for western blotting, plaque formation assays, and qrt-pcr. western blotting showed that tak induced moderately decreased porcine epidemic diarrhea virus nucleocapsid (pedv-n) protein expression in a dose-dependent manner, and about 73% pedv-n protein decreased when treating with 10 µm concentration of tak (figure 2a) , demonstrating the fact that tlr4 might regulate pedv infection. immunofluorescence assays (ifas) confirmed that tak reduced pedv infection rate about 79% at 10 µm concentration ( figure 2b ). in addition, tak caused reductions in viral open reading frame (orf3) mrna levels at a concentration of 1 µm ( figure 2c ). the plaque formation assay showed viral titer in the supernatant of cells was reduced by about 95% at 10 µm concentration ( figure 2d ). these results suggested that tak inhibited pedv infection in a dose-dependent manner, and furthermore that tlr4 potentially regulated this infection. in addition, tak reduced levels of proinflammatory cytokine mrnas, namely, il-1β (88%), il-6 (93%), il-8 (98%), and tumor necrosis factor-a (tnf-a) (97%) during pedv infection ( figure 2e ). however, tak did not have cytotoxic effects in vero cells at concentrations up to 10 µm after 24 h [45] . antiviral responses are associated with tlr4, and viral rna and dna can interact with tlr4. previous studies showed that tlr4 can interact with the lipoprotein (ns1) of dengue virus and with ns5a of hcv [3, 18] . pedv infection had no effect on tlr4 expression, but we further explored whether tlr4 regulated pedv infection. we pretreated vero cells with the tlr4 inhibitor tak for 2 h and then infected the cells with pedv (0.1 moi) in the presence of various concentrations of tak for 24 h. vero cells and supernatants were collected for western blotting, plaque formation assays, and qrt-pcr. western blotting showed that tak induced moderately decreased porcine epidemic diarrhea virus nucleocapsid (pedv-n) protein expression in a dose-dependent manner, and about 73% pedv-n protein decreased when treating with 10 μm concentration of tak (figure 2a) , demonstrating the fact that tlr4 might regulate pedv infection. immunofluorescence assays (ifas) confirmed that tak reduced pedv infection rate about 79% at 10 μm concentration ( figure 2b ). in addition, tak caused reductions in viral open reading frame (orf3) mrna levels at a concentration of 1 μm ( figure 2c ). the plaque formation assay showed viral titer in the supernatant of cells was reduced by about 95% at 10 μm concentration ( figure 2d ). these results suggested that tak inhibited pedv infection in a dose-dependent manner, and furthermore that tlr4 potentially regulated this infection. in addition, tak reduced levels of proinflammatory cytokine mrnas, namely, il-1β (88%), il-6 (93%), il-8 (98%), and tumor necrosis factor-a (tnf-a) (97%) during pedv infection ( figure 2e ). however, tak did not have cytotoxic effects in vero cells at concentrations up to 10 μm after 24 h [45] . we demonstrated the fact that pedv infection was associated with tlr4, but we wanted to further explore which pathways depended on tlr4 during pedv infection. as the mapk pathways play a vital role in viral infection, such as foot-and-mouth disease virus and influenza a virus infection, we therefore assessed the roles of the mapk p38, erk1/2, and jnk pathways during pedv infection. phosphorylation of p38, erk1/2, and jnk was assessed by western blotting in vero cells infected with pedv (0.1 moi) at 4, 8, 12, 24, and 36 h post-infection (h.p.i.). as shown in figure 3a ,b, pedv infection stimulated robust phosphorylation of p38 at 8, 12, 24, and 36 h.p.i. these effects were especially apparent at 24 (4.4 times) and 36 (5.3 times) h.p.i. (figure 3a ,b). however, jnk and erk1/2 phosphorylation were only increased at 36 h.p.i. compared with mock-infection vero cells ( figure 3a ,c,d). levels of p38 phosphorylation were monitored during early pedv infection and during persistent pedv infection. however, jnk and erk1/2 phosphorylation were only monitored at 36 h.p.i. in addition, we revealed that mapk p38, jnk, and erk1/2 phosphorylation were induced at 48 h.p.i., and that phosphorylation was higher at 48 h.p.i. than 36 h.p.i. [45] . phosphorylation of p38 was induced at 24 h.p.i., whereas jnk and erk1/2 phosphorylation were not induced until 24 h.p.i. this result suggested that p38 might play a vital role in pedv infection from 24 h.p.i. onwards. we demonstrated the fact that pedv infection was associated with tlr4, but we wanted to further explore which pathways depended on tlr4 during pedv infection. as the mapk pathways play a vital role in viral infection, such as foot-and-mouth disease virus and influenza a virus infection, we therefore assessed the roles of the mapk p38, erk1/2, and jnk pathways during pedv infection. phosphorylation of p38, erk1/2, and jnk was assessed by western blotting in vero cells infected with pedv (0.1 moi) at 4, 8, 12, 24, and 36 h post-infection (h.p.i.). as shown in figure 3a ,b, pedv infection stimulated robust phosphorylation of p38 at 8, 12, 24, and 36 h.p.i. these effects were especially apparent at 24 (4.4 times) and 36 (5.3 times) h.p.i. (figure 3a ,b). however, jnk and erk1/2 phosphorylation were only increased at 36 h.p.i. compared with mock-infection vero cells ( figure 3a ,c,d). levels of p38 phosphorylation were monitored during early pedv infection and during persistent pedv infection. however, jnk and erk1/2 phosphorylation were only monitored at 36 h.p.i. in addition, we revealed that mapk p38, jnk, and erk1/2 phosphorylation were induced at 48 h.p.i., and that phosphorylation was higher at 48 h.p.i. than 36 h.p.i. [45] . phosphorylation of p38 was induced at 24 h.p.i., whereas jnk and erk1/2 phosphorylation were not induced until 24 h.p.i. this result suggested that p38 might play a vital role in pedv infection from 24 h.p.i. onwards. to explore the roles of mapk p38 during pedv infection, we pretreated vero cells with different concentrations of sb for 2 h before infecting the cells with pedv (0.1 moi). cells and supernatants were collected for western blotting, plaque formation assays, and qrt-pcr 24 h after pedv infection. we assessed the levels of pedv-n protein by western blotting and ifa, and found that sb inhibited . the cells were collected after different lengths of time for western blotting. an equal amount of protein was subjected to western blotting analysis. (a) levels of phosphorylated and total mapk p38, erk1/2, or jnk were analyzed by western blotting. beta-actin was used as a loading control. (b) levels of phospho-p38/total p38 were plotted using imagej. (c) levels of phospho-jnk/total jnk were plotted using imagej. (d) fold changes in the phospho-erk/total erk ratio were plotted using imagej. p-values less than 0.05 were considered statistically significant (** p < 0.01). bars represent standard deviations. to explore the roles of mapk p38 during pedv infection, we pretreated vero cells with different concentrations of sb for 2 h before infecting the cells with pedv (0.1 moi). cells and supernatants were collected for western blotting, plaque formation assays, and qrt-pcr 24 h after pedv infection. we assessed the levels of pedv-n protein by western blotting and ifa, and found that sb inhibited pedv-n expression in a dose-dependent manner ( figure 4a,b) . western blotting revealed that pedv-n expression was reduced about 82% by sb at 5 µm concentration ( figure 4a) , and ifa showed that pedv infection rate was decreased about 84% by sb at the same concentration ( figure 4b ). qrt-pcr showed that sb decreased the level of pedv orf3 mrna about 56% at 1 µm concentration ( figure 4c ). we found that pedv titer in the supernatant was decreased about 81% at 5 µm concentration using a plaque formation assay ( figure 4d ). thus, the mapk p38 inhibitor sb inhibited pedv infection. in addition, levels of proinflammatory cytokine mrna during pedv infection were reduced about 58% (il-1β), 61% (il-6), 64% (il-8), and 68% (tnf-a) by treatment with sb ( figure 4e ). sb did not cause cytotoxic effects in vero cells at concentrations up to 5 µm after 24 h [45] . our results revealed that tlr4 and mapk p38 regulated pedv infection. thus, we assessed whether tlr4 was an upstream modulator of p38 mapk during pedv infection. we pretreated vero cells with tak for 2 h and infected them with pedv (0.1 moi) in the presence of different concentrations of tak for 24 h. the cells were collected to assess levels of p38, jnk, and erk1/2 phosphorylation. tak reduced the levels of p38 phosphorylation about 59% at 10 μm concentration during pedv infection ( figure 5a,b) , but did not inhibit jnk and erk1/2 phosphorylation ( figure 5a,c,d) . these results demonstrated tlr4 as an upstream modulator of mapk p38 during pedv infection, as well as the fact that pedv infection depended on the tlr4-mapk p38 pathway. our results revealed that tlr4 and mapk p38 regulated pedv infection. thus, we assessed whether tlr4 was an upstream modulator of p38 mapk during pedv infection. we pretreated vero cells with tak for 2 h and infected them with pedv (0.1 moi) in the presence of different concentrations of tak for 24 h. the cells were collected to assess levels of p38, jnk, and erk1/2 phosphorylation. tak reduced the levels of p38 phosphorylation about 59% at 10 µm concentration during pedv infection ( figure 5a,b) , but did not inhibit jnk and erk1/2 phosphorylation ( figure 5a,c,d) . these results demonstrated tlr4 as an upstream modulator of mapk p38 during pedv infection, as well as the fact that pedv infection depended on the tlr4-mapk p38 pathway. levels of phospho-p38/total p38 were plotted using imagej. (c) levels of phospho-jnk/total jnk were plotted using imagej. (d) fold changes in phospho-erk/total erk ratio were plotted using imagej. p-values less than 0.05 were considered statistically significant (** p < 0.01). bars represent standard deviations. to explore the effects of hmgb1 on mapk p38, jnk, and erk1/2 during pedv infection, we pretreated vero cells with a hmgb1 competitive inhibitor (gly) for 2 h and infected the cells with pedv (0.1 moi) in the presence of different concentrations of gly for 24 h. we collected vero cells for western blotting analysis. gly inhibited p38 phosphorylation about 85% at 0.8 mm concentration induced by pedv infection (figure 6a,b) . however, gly did not inhibit jnk and erk1/2 phosphorylation. these results showed that gly inhibited pedv infection through effects on mapk p38. however, gly caused little increase in jnk phosphorylation at 0.4 and 0.8 mm and little increase in erk1/2 phosphorylation at 0.8 mm ( figure 6a,c,d) . the explanations of these effects need to be explored in future studies. we performed an hmgb1 antibody neutralization experiment to investigate whether hmgb1 induced activation of mapk p38. vero cells were incubated with an anti-hmgb1 antibody for 24 h, and then the cells were infected with pedv. western blotting demonstrated that p38 phosphorylation was deceased by hmgb1 antibody treatment ( figure 6e,f) . hmgb1 binding to tlr4 requires the presence of a reduced cys106 and a disulfide bond between cys23 and cys45 in hmgb1 [46] . we found that mutant forms of hmgb1 (hmgb1-c45s, hmgb1-c106s, and hmgb1-c45s/c106s) decreased the activation of mapk p38 ( figure 6g,h) . in addition, small interfering rna (sirna) knockdown of hmgb1 inhibited the levels of p38 phosphorylation by about 66% (figure 6i,j) . thus, hmgb1 can lead to activation of mapk p38. to explore the effects of hmgb1 on mapk p38, jnk, and erk1/2 during pedv infection, we pretreated vero cells with a hmgb1 competitive inhibitor (gly) for 2 h and infected the cells with pedv (0.1 moi) in the presence of different concentrations of gly for 24 h. we collected vero cells for western blotting analysis. gly inhibited p38 phosphorylation about 85% at 0.8 mm concentration induced by pedv infection (figure 6a,b) . however, gly did not inhibit jnk and erk1/2 phosphorylation. these results showed that gly inhibited pedv infection through effects on mapk p38. however, gly caused little increase in jnk phosphorylation at 0.4 and 0.8 mm and little increase in erk1/2 phosphorylation at 0.8 mm ( figure 6a,c,d) . the explanations of these effects need to be explored in future studies. we performed an hmgb1 antibody neutralization experiment to investigate whether hmgb1 induced activation of mapk p38. vero cells were incubated with an anti-hmgb1 antibody for 24 h, and then the cells were infected with pedv. western blotting demonstrated that p38 phosphorylation was deceased by hmgb1 antibody treatment ( figure 6e,f) . hmgb1 binding to tlr4 requires the presence of a reduced cys106 and a disulfide bond between cys23 and cys45 in hmgb1 [46] . we found that mutant forms of hmgb1 (hmgb1-c45s, hmgb1-c106s, and hmgb1-c45s/c106s) decreased the activation of mapk p38 ( figure 6g,h) . in addition, small interfering rna (sirna) knockdown of hmgb1 inhibited the levels of p38 phosphorylation by about 66% (figure 6i,j) . thus, hmgb1 can lead to activation of mapk p38. (g) levels of phosphorylated mapk p38, total mapk p38, and wild-type or mutant hmgb1 were analyzed by western blotting. (h) fold change in phospho-p38/total p38 ratio were plotted using imagej. (i,j) vero cells were transfected with small interfering hmgb1 (sihmgb1) to silence hmgb1 expression for 24 h. sinc (an irrelevant small interfering rna (sirna)) was used as a negative control. the cells were infected with pedv for 24 h. (i) levels of total and phosphorylated mapk p38 and of hmgb1 were analyzed by western blotting. (j) levels of phospho-p38/total p38 were plotted using imagej. p-values less than 0.05 were considered statistically significant (* p < 0.05 and ** p < 0.01). bars represent standard deviations. hmgb1 is a ubiquitous and conserved dna-binding protein that was discovered over 30 years ago and participates in dna replication and maintenance of nucleosome structure [47, 48] . extracellular hmgb1 can activate proinflammatory signaling pathways through tlr4 [23, 24] . hmgb1 is a unique mediator of innate immune responses and inflammation-associated events [25] . our previous study indicated that pedv infection caused release of hmgb1, and that the hmgb1 competitive inhibitor gly inhibited pedv infection and attenuated proinflammatory responses by inhibition of hmgb1 [44] . therefore, we explored the relationships between hmgb1, pedv infection, and proinflammatory cytokine production during pedv infection. extracellular hmgb1 can bind to tlr4. thus, we assessed the expression of tlr4 during pedv infection. we found that pedv had no effect on levels of tlr4 protein or mrna. these results were inconsistent with our hypothesis. tlr4 and mapk p38 play an important role in influenza a virus replication and inflammation responses. tlr4 has an antiviral effect on mouse hepatitis virus [49] . the mapk p38 pathway is a key regulator of inflammatory responses [33] [34] [35] [36] . hmgb1 binding to tlr4 triggers cytokine release under the prerequisite of both a disulfide bond between c23 and c45 and a reduced c106 of hmgb1 [46] , and our previous study corroborates the fact that extracellular hmgb1 binding to tlr4 promotes inflammatory responses [26] . therefore, we speculated that tlr4 might regulate pedv infection and that inflammatory responses depended on activation of signaling cascades. we explored the effect of tlr4 on pedv infection and proinflammatory cytokine. we revealed that the tlr4 inhibitor tak significantly attenuated viral infection, judging by the observed downregulation of viral protein, viral rna, and viral titer, as well as decreased proinflammatory cytokine production. these results implied that although pedv infection had no effect on tlr4, tlr4 regulated pedv infection through activation of signaling cascades. our previous study showed that pedv infection caused acetylation and the release of hmgb1, which binds tlr4 [44] . the mapk pathways are involved in cell death [50] , and play a vital role in infection of coronaviruses, such as mouse sars-cov and hepatitis virus [51, 52] . therefore, we assessed whether tlr4 induced signaling cascades depended on the mapk pathways in this study. we found that early pedv infection activated p38, and that activation of p38 is continuous during pedv infection. this phenomenon is consistent with a previous study [53] . maximal activation of p38 was observed at 12 h.p.i. in a previous study [53] , which was inconsistent with our results. in addition, activation of jnk and erk1/2 was only observed at 36 h.p.i.; this is inconsistent with a previous study [54] . lee et al. and kim et al. showed that activation of jnk or erk1/2 occurred at 12 and 24 h.p.i., but that activation of jnk or erk1/2 was not required for productive pedv infection at 36 and 48 h.p.i. [53, 54] . the reasons underlying these discrepancies may be relate to differences between pedv strains, and thus require further exploration. our results showed that only p38 phosphorylation was induced at 4, 8, 12, and 24 h.p.i., suggesting that activation of p38 might have a vital role during pedv early infection. we thus focused our efforts on p38. we explored the effect of the p38 inhibitor sb on pedv infection and proinflammatory cytokine production. we confirmed that the p38 inhibitor sb inhibited pedv infection and decreased proinflammatory cytokine production. our results revealed that tlr4 and p38 have an important role during pedv infection. mapk p38 is a downstream molecule of tlr4 [55, 56] . however, the dependency of the effects of tlr4 on pedv infection and expression of proinflammatory cytokines on the p38 pathway was unknown. we revealed that the tlr4 inhibitor tak decreased phosphorylation of p38, but slightly increased levels of phospho-jnk and erk1/2. these results revealed that tlr4 inhibition of pedv infection and proinflammatory cytokine production depended on p38 but not jnk and erk1/2. treatment of vero cells infected with pedv with gly showed that gly decreased the activation of p38. gly is a competitive inhibitor of hmgb1 cytokine activity. our previous study demonstrated that gly could block of binding hmgb1 to tlr4 and impair the activity of tlr4 [26] . our results showed that gly decreased the activity of p38 dependent on tlr4 activation. however, gly caused slight increases of phospho-jnk and erk1/2, which was consistent with our results using the tlr4 inhibitor tak. this phenomenon was surprising and requires further study. in addition, inhibition of hmgb1 using an antibody, site-specific mutations (hmgb1-c45s, hmgb1-c106s, and hmgb1-c45s/c106s), and sirna knock-down decreased p38 activation. extracellular hmgb1 can bind to tlr4, and pedv infection leads to release of hmgb1. our study showed that extracellular hmgb1 interaction with tlr4 leads to activation of p38, but not jnk and erk1/2, which facilitates pedv infection and increases proinflammatory cytokine production. gly prevented binding hmgb1 to tlr4 to inhibit pedv infection, and this effect depended on the hmgb1/tlr4-mapk p38 pathway (figure 7 ). c45s/c106s), and sirna knock-down decreased p38 activation. extracellular hmgb1 can bind to tlr4, and pedv infection leads to release of hmgb1. our study showed that extracellular hmgb1 interaction with tlr4 leads to activation of p38, but not jnk and erk1/2, which facilitates pedv infection and increases proinflammatory cytokine production. gly prevented binding hmgb1 to tlr4 to inhibit pedv infection, and this effect depended on the hmgb1/tlr4-mapk p38 pathway (figure 7 ). in this study, we explored gly-inhibited pedv infection and decreased proinflammatory cytokine production through effects on the hmgb1/tlr4-p38 pathway. our results suggest targets for pedv prevention and control, and provide a basis for research and development toward novel anti-pedv agents. african green monkey kidney cells (vero cells) were cultured in high-glucose dulbecco's modified eagle's medium (dmem; invitrogen, carlsbad, ca, usa) supplemented with 10% (v/v) newborn calf serum (gibco, carlsbad, ca, usa). pedv (hljby, genbank: kp403802.1) was propagated in vero cells in dmem containing 2% newborn calf serum. gly and tak were purchased from sigma (st. louis, mo, usa). sb was purchased from santa cruz. anti-mapk p38, phospho-p38 mapk (thr180/tyr182), erk1/2, and phospho-erk1/2 (thr202/tyr204) antibodies were purchased from cst (danvers, ma, usa). anti-jnk and phosphor-jnk (thr183/tyr185) antibodies were purchased from santa cruz (santa cruz, ca, usa). the anti-pedv-n antibody was prepared in xiang mao's lab. the anti-pedv-n polyclonal antibody was prepared in rabbits using pedv-n protein as antigen. the work dilutions of this antiserum for western blot and ifa were 1:5000 and 1:500, respectively. in this study, we explored gly-inhibited pedv infection and decreased proinflammatory cytokine production through effects on the hmgb1/tlr4-p38 pathway. our results suggest targets for pedv prevention and control, and provide a basis for research and development toward novel anti-pedv agents. african green monkey kidney cells (vero cells) were cultured in high-glucose dulbecco's modified eagle's medium (dmem; invitrogen, carlsbad, ca, usa) supplemented with 10% (v/v) newborn calf serum (gibco, carlsbad, ca, usa). pedv (hljby, genbank: kp403802.1) was propagated in vero cells in dmem containing 2% newborn calf serum. gly and tak were purchased from sigma (st. louis, mo, usa). sb was purchased from santa cruz. anti-mapk p38, phospho-p38 mapk (thr180/tyr182), erk1/2, and phospho-erk1/2 (thr202/tyr204) antibodies were purchased from cst (danvers, ma, usa). anti-jnk and phosphor-jnk (thr183/tyr185) antibodies were purchased from santa cruz (santa cruz, ca, usa). the anti-pedv-n antibody was prepared in xiang mao's lab. the anti-pedv-n polyclonal antibody was prepared in rabbits using pedv-n protein as antigen. the work dilutions of this antiserum for western blot and ifa were 1:5000 and 1:500, respectively. the cytotoxic effects of tak and sb on vero cells were assessed using the enhanced cell counting kit-8 (cck-8, beyotime, shanghai, china). briefly, 1 × 10 4 vero cells per well were cultured in wells of 96-well plates containing tak or sb. after 24 h, 10 µl of cck-8 was added to each well, and the samples were incubated for 1 h. the absorbance of each well was measured at 450 nm. all assays were performed in triplicate. western blotting analysis was performed as described previously [44] , with slight modifications. proteins in gels were transferred to a 0.22 µm nitrocellulose membrane (nc, pall, new york, ny, usa). the membranes were blocked with 3% (w/v) nonfat milk and treated with the primary antibody hybridized with the secondary antibody, and the target protein bands were visualized via an enhanced chemiluminescence (ecl) exposure according to the producer's instructions (vazyme, nanjing, china). densitometry of tlr4, phospho-p38, phospho-erk, and phospho-jnk expression was performed using imagej. vero cells grown on coverslips were treated with tak or sb and infected with pedv at a multiplicity of infection (moi) of 0.1 for 24 h. to assess the effect of tlr4 and mapk p38 inhibitors on pedv infection, vero cells were pretreated with tak or sb for 2 h and infected with pedv. twenty-four hours following pedv infection in the presence of tak or sb, vero cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% triton x-100 at room temperature for 10 min. vero cells were blocked with 3% bovine serum albumin for 1 h and incubated with anti-pedv-n antibody for 2 h at 37 • c the cells were washed with phosphate-buffered saline (pbs) three times and incubated with an alexa fluor 488-conjugated goat anti-rabbit secondary antibody (invitrogen, carlsbad, ca, usa) and 4 ,6-diamidino-2-phenylindole (beyotime, shanghai, china). fluorescence was visualized using a leica inverted fluorescence microscope after washing three times with pbs. qrt-pcr was performed as described previously [53] . total rna was extracted from vero cells, and gene expression was assessed by qrt-pcr using specific primers (pedv-orf3-f: tttgcactgtttaaagcgtct, pedv-orf3-r: agtaaaagcagactaaacaaagcct). the samples were assessed in triplicate using sybr (vazyme, nanjing, china). the data were analyzed using the 2-∆∆ct method, and expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (gapdh) mrna in the same sample. the plaque formation assay was carried out as previously described [53] . briefly, vero cells were cultured in 6-well plates containing serially-diluted virus for 1 h. overlay medium (2% low melting-point agarose in dmem medium containing 2% fetal bovine serum) was added to the wells for 72 h. the cells were stained with 0.5% crystal violet. all statistical data were expressed as means ± standard deviations of triplicate experiments, and the significance of differences between groups was analyzed by one-way anova using the spss 17.0 software package (version 17.0, spss inc., chicago, il, usa). p-values less than 0.05 were considered statistically significant (* p < 0.05 and ** p < 0.01). in this study, we explored gly-inhibited pedv infection and decreased proinflammatory cytokine production through effects on the hmgb1/tlr4-p38 pathway. our results suggest targets for pedv prevention and control, and provide a basis for research and development toward novel anti-pedv agents. the authors declare no conflict of interest. origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states porcine epidemic diarrhea in europe: in-detail analyses of disease dynamics and molecular epidemiology outbreak of porcine epidemic diarrhea in suckling piglets 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end-products in macrophages. asian pac hmgb1 acts in synergy with lipopolysaccharide in activating rheumatoid synovial fibroblasts via p38 mapk and nf-kappab signaling pathways porcine epidemic diarrhea virus nucleoprotein contributes to hmgb1 transcription and release by interacting with c/ebp-beta the role of mapk pathways in porcine epidemic diarrhea virus infection mutually exclusive redox forms of hmgb1 promote cell recruitment or proinflammatory cytokine release a new group of chromatin-associated proteins with a high content of acidic and basic amino acids nucleosome dynamics: hmgb1 relaxes canonical nucleosome structure to facilitate estrogen receptor binding toll-like receptor 4 deficiency increases disease and mortality after mouse hepatitis virus type 1 infection of susceptible c3h mice opposing effects of erk and jnk-p38 map kinases on apoptosis murine coronavirus replication-induced p38 mitogen-activated protein kinase activation promotes interleukin-6 production and virus replication in cultured cells palese, p. 7a protein of severe acute respiratory syndrome coronavirus inhibits cellular protein synthesis and activates p38 mitogen-activated protein kinase porcine epidemic diarrhea virus uses cell-surface heparan sulfate as an attachment factor extracellular signal-regulated kinase (erk) activation is required for porcine epidemic diarrhea virus replication human endophthalmitis caused by pseudorabies virus infection oridonin protects against the inflammatory response in diabetic nephropathy by inhibiting the tlr4/p38-mapk and tlr4/nf-kappab signaling pathways key: cord-295534-bwa4wz94 authors: jung, kwonil; saif, linda j. title: porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis date: 2015-02-26 journal: vet j doi: 10.1016/j.tvjl.2015.02.017 sha: doc_id: 295534 cord_uid: bwa4wz94 porcine epidemic diarrhea virus (pedv), a member of the genera alphacoronavirus in the family coronaviridae, causes acute diarrhea/vomiting, dehydration and high mortality in seronegative neonatal piglets. for the last three decades, pedv infection has resulted in significant economic losses in the european and asian pig industries, but in 2013–2014 the disease was also reported in the us, canada and mexico. the ped epidemic in the us, from april 2013 to the present, has led to the loss of more than 10% of the us pig population. the disappearance and re-emergence of epidemic ped indicates that the virus is able to escape from current vaccination protocols, biosecurity and control systems. endemic ped is a significant problem, which is exacerbated by the emergence (or potential importation) of multiple pedv variants. epidemic pedv strains spread rapidly and cause a high number of pig deaths. these strains are highly enteropathogenic and acutely infect villous epithelial cells of the entire small and large intestines although the jejunum and ileum are the primary sites. pedv infections cause acute, severe atrophic enteritis accompanied by viremia that leads to profound diarrhea and vomiting, followed by extensive dehydration, which is the major cause of death in nursing piglets. a comprehensive understanding of the pathogenic characteristics of epidemic or endemic pedv strains is needed to prevent and control the disease in affected regions and to develop an effective vaccine. this review focuses on the etiology, epidemiology, disease mechanisms and pathogenesis as well as immunoprophylaxis against pedv infection. porcine epidemic diarrhea virus (pedv), a member of the genera alphacoronavirus in the family coronaviridae of the order nidovirales, causes acute diarrhea, vomiting, dehydration and high mortality in neonatal piglets, resulting in significant economic losses. the disease was initially reported in european and asian pig industries over the last 30 years, with the virus first appearing in england (wood, 1977) and belgium (pensaert and de bouck, 1978) in the early 1970s. recently, pedv has also been reported in the us (stevenson et al., 2013) . since then, the virus has rapidly spread nationwide throughout the usa (cima, 2013) and to other countries in north america, including canada and mexico. as a result of the significant impact of pedv, the us pig industry has lost almost 10% of its domestic pig population after only a 1 year-epidemic period, amounting to approximately 7 million piglets. similar epidemiological and clinical features between pedv and another alphacoronavirus, transmissible gastroenteritis virus (tgev), have led to complications in diagnosis, requiring differential laboratory tests (saif et al., 2012) . since the emergence of a natural spike gene deletion mutant of tgev, porcine respiratory coronavirus (prcv) in 1984, the spread of tgev has been reduced in prcv-seropositive herds due to cross-protective immunity with tgev (saif et al., 2012) . in contrast, pedv continues to spread and cause economic problems worldwide. based on genetic analysis, the family coronaviridae can be divided into the four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. bats are the projected host for the gene source of alphacoronaviruses and betacoronaviruses, while birds are the suspect host for gammacoronaviruses and deltacoronaviruses (woo et al., 2012) . in different us regions where pedv is epidemic, a new coronavirus genetically distinct from pedv, porcine deltacoronavirus (pdcov), has been simultaneously (and frequently) detected in diarrheic fecal samples from pigs (wang et al., 2014a) . the clinical impact and disease severity of pdcov in the field is reportedly less than that of pedv. a recent study confirmed that pdcov is enteropathogenic in pigs and acutely infects the small intestine, causing severe diarrhea and/or vomiting and atrophic enteritis, similar to the clinical signs of pedv and tgev infections (jung et al., 2015) . at present, differential diagnosis of pedv, pdcov, and tgev is critical to control viral epidemic diarrheas in us pig farms. this review focuses on current understanding of the etiology, epidemiology, disease mechanisms and pathogenesis of pedv and the control measures that may be used to prevent pedv infection. pedv is enveloped and pleomorphic with a range in diameter of 95-190 nm, including the projections, which are approximately 18 nm in length (pensaert and de bouck, 1978) . details of the pedv structure and genome can be found elsewhere (song and park, 2012) . pedv has a single-stranded positive-sense rna genome of approximately 28 kb in size (excluding the poly a-tail) that encodes four structural proteins, namely, spike (s), envelope (e), membrane (m), and nucleocapsid (n) protein, and four nonstructural proteins: 1a, 1b, 3a, and 3b (kocherhans et al., 2001) . among the viral proteins, the s protein is critical for regulating interactions with specific host cell receptor glycoproteins to mediate viral entry and for inducing neutralizing antibodies (bosch et al., 2003) . the s protein is also associated with growth adaptation in vitro and attenuation of pedv virulence in vivo (sato et al., 2011) . the m protein is the most abundant component among viral proteins in the envelope and plays an important role in virus assembly by interacting with the s and n proteins (klumperman et al., 1994; vennema et al., 1996) . the n protein of coronavirus binds rna and packages viral genomic rna into the nucleocapsid of virus particles (spaan et al., 1983) . a previous study using the cell-adapted german isolate v215/ 78 documented the biological and physicochemical properties of pedv (hofmann and wyler, 1989) . pedv showed a buoyant density of 1.18. pedv was easily inactivated by ether or chloroform, and it was relatively stable at 4-50°c compared to higher temperatures. after incubation in cell culture media at 4°c with a ph range (3-10) for 6 h, pedv exhibited low to moderate residual infectivity, whereas at 37°c for 6 h, it retained its infectivity only between the ph range 5 and 8.5, but the virus was completely inactivated at ph < 4 and > ph 9. these data indicate that pedv will be inactivated by acidic or alkaline disinfectants if they are applied for a certain period at a higher temperature (>37°c). the pedv strain v215/78 was not neutralized by an antiserum to tgev (hofmann and wyler, 1989) . this finding was supported by another report , which showed no crossreactivity of pedv cv777 strain with either a belgian strain of tgev or feline infectious peritonitis virus (fipv), as determined by immune-electron microscopy and immunofluorescence (if). however, a subsequent study found a detectable, two-way crossreactivity between pedv and fipv by more sensitive assays, such as enzyme linked immune-sorbent assay, immunoblotting and immune-precipitation (zhou et al., 1988) . these discrepancies indicate that cross-reactivity between pedv and other coronaviruses probably varies depending on the sensitivity of the techniques and the viral strains tested. a recent study reported evidence of antigenic cross-activity between the prototype cv777 and recent us pedv strains and tgev (miller strain) by sharing at least one conserved epitope on the n-terminal region of their n proteins (lin et al., 2015) . pospischil et al. (2002) demonstrated that pedv is inactivated by disinfectants, namely, oxidizing agents (virkon s), bleach, phenolic compounds (one-stroke environ; tek-trol), 2% sodium hydroxide, formaldehyde and glutaraldehyde, sodium carbonate (4% anhydrous or 10% crystalline, with 0.1% detergent), ionic and nonionic detergents, 1% strong iodophors in phosphoric acid, and lipid solvents such as chloroform. vero (african green monkey kidney) cells support the isolation and serial propagation of pedv in cell cultures supplemented with the exogenous protease trypsin. another african green monkey kidney cell line, marc-145, also supported a subsequent cell passage of pedv (lawrence et al., 2014) . trypsin plays an important role in cell entry and release of pedv virions in vero cells, contributing to efficient replication and spread of the virus to neighboring cells in vitro (shirato et al., 2011; wicht et al., 2014) . trypsin resulted in the cleavage of the s protein into s1 and s2 subunits, which most likely accounts for cell-to-cell fusion and the release of virions from infected vero cells (shirato et al., 2011) . cytopathic effects consist of vacuolation and formation of syncytia as a result of apoptotic cell death (hofmann and wyler, 1988; kim and lee, 2014) . the hemagglutinating activity of pedv was demonstrated with rabbit erythrocytes only after trypsin treatment (park et al., 2010) . only one serotype of pedv has been reported from different countries (saif et al., 2012) . pedv first appeared in the united kingdom (wood, 1977) and belgium (pensaert and de bouck, 1978) in the early 1970s. the virus was first isolated in 1977 in belgium and was classified in the family coronaviridae (pensaert and de bouck, 1978) . subsequently, in the 1980s and 1990s, pedv was identified as a cause of severe epidemics in japan and south korea (takahashi et al., 1983; kweon et al., 1993) . despite extensive application of pedv vaccines, ped has remained endemic in south korea (park et al., 2013) . during the 1980s and 1990s in europe, outbreaks of ped appeared infrequently, but the virus continued to spread and persisted in an endemic form in the pig population. subsequent serological surveys showed a low to moderate prevalence of pedv in european pigs (van reeth and pensaert, 1994; carvajal et al., 1995) . the prevalence of pedv in european pigs then declined greatly although the reasons are unclear. outbreaks of ped were observed only sporadically in europe: in the netherlands in 1989-1991 (pijpers et al., 1993) ; in hungary in 1995 (nagy et al., 1996) , and in england in 1998 (pritchard et al., 1999) . however, a typical epidemic outbreak of ped was identified in italy in 2005 -2006 (martelli et al., 2008 . in thailand in 2007-2008, several outbreaks of severe ped were reported with thai pedv isolates in the same clade phylogenetically as the chinese strain js-2004-2 (puranaveja et al., 2009) . this new genotype of pedv continues to cause sporadic outbreaks in thailand. in china in 2010-2012, severe ped outbreaks in seropositive pigs were reported in different regions (li et al., 2012; sun et al., 2012; wang et al., 2013) . for almost two decades since pedv first emerged in china, many pig herds have been vaccinated with the prototype strain cv777-inactivated or related vaccines. however, the moderate to high mortality of suckling piglets in vaccinated herds indicates a low effectiveness of the cv777 vaccines (li et al., 2012) . the ped outbreaks in china, in 2010-2012, were caused by both classical and new pedv variant strains that differ genetically from the prototype cv777 (wang et al., 2013) . the us pedv strains identified during the initial outbreak in 2013 were closely related genetically to the chinese strains (china/2012/ ah2012) reported in 2011-2012 chen et al., 2014) , indicating emergence of ah2012-like chinese pedv strains in the us. the us-like pedv strains were also found in diarrheic piglets in south korea and taiwan during late 2013 and early 2014 (cho et al., 2014; lin et al., 2014) , although whether chinese or us pedv strains could have been transmitted to pigs in south korea and taiwan is unknown. further investigations are needed to clarify if chinese or us pedv strains were already present in south korea and taiwan before the related outbreaks were first identified. for <1 year since the first outbreak, other novel us pedv strains (oh/oh851) with multiple deletions and insertions in their s gene, which clustered closely with chinese strain hbqx-2010 or ch/ zmzdy/11, rather than ah2012, were found to possess low nucleotide identity in their 5′-end s1 region (first 1170 nucleotides) and high nucleotide identity in the remaining s gene, compared to the major us pedv strains wang et al., 2014b) . possible recombination events involving strain(s) from china may have contributed to a rapid evolution of us pedv and the emergence of multiple variants, complicating the molecular epidemiology of us pedv strains (tian et al., 2014) . remarkably, another pedv variant, which has a large 197 amino acid (aa) deletion in the n-terminal portion of the s protein of major cell-cultured us pedv strains, such as pedv strain tc-pc22a (genbank accession number km392224), has emerged only 1 year after the first outbreak (oka et al., 2014) . another pedv variant with a large (204 aa) deletion at positions 713-916 of the s protein was identified among korean pedv strains . the fecal-oral route is the main means of pedv transmission, although aerosolized pedv remains infectious (alonso et al., 2014) . diarrheal feces and/or vomitus and other contaminated fomites, such as transport trailers (lowe et al., 2014) and feed (dee et al., 2014) , can be major transmission sources of the virus. another possible reservoir for pedv includes carriers, such as older pigs with a-symptomatic infection, in which the virus spreads subclinically. previous studies showed a low to moderate detection rate (23-41%) of pedv rna in milk samples of affected, lactating sows (li et al., 2012; sun et al., 2012) , suggesting that sow milk might be a potential route for the vertical transmission of pedv. our study demonstrated a significant detection rate of pedv rna in acute serum samples (55-100%) of experimentally infected piglets or naturally infected grower pigs . whether pork plasma used as a feed additive could be a transmission source of pedv remains questionable, since discrepant results were reported in two different infection studies that investigated whether spray-dried porcine plasma that had tested positive for pedv rna was infectious in seronegative pigs (opriessnig et al., 2014; pasick et al., 2014) . porcine small intestinal villous enterocytes express large amounts of aminopeptidase n (apn), a 150-kda glycosylated transmembrane protein, identified as the cellular receptor for pedv (li et al., 2007) . high density of the receptor on enterocytes allows pedv to enter and replicate through virus-receptor interactions (li et al., 2007) . pedv is cytolytic, and infected enterocytes rapidly undergo acute necrosis, leading to marked villous atrophy in the small but not in the large intestine ( fig. 1a ) . pedv antigens are observed mainly in villous enterocytes of the small (duodenum to ileum) ( fig. 1b ) and large intestines (except the rectum) stevenson et al., 2013; jung et al., 2014; madson et al., 2014) . like tgev (kim et al., 2000) , pedv may not induce apoptotic death of enterocytes in the small intestine of infected pigs (figs. 1c, d). occasionally, a few pedv-positive cells were detected in the intestinal crypt cells or peyer's patches during the late-stages of infection sueyoshi et al., 1995; stevenson et al., 2013; jung et al., 2014) . in our preliminary study, mean numbers of goblet cells per intestinal villi of infected gnotobiotic pigs (<2/villus) at postinoculation hours (pih) 30-72 were fewer than those (6-18/ villus) of the negative counterparts (fig. 2) . as with tgev (schwegmann-wessels et al., 2003) , pedv might infect goblet cells, leading to a dramatic decrease in this cell type during the early phase of diarrhea. goblet cells secrete mucins and provide the first line of defense against microbes in the intestine (kim and ho, 2010) . lung tissues of oronasally infected pigs were negative for pedv antigen, indicating no evidence of pedv replication in the lower respiratory tract sueyoshi et al., 1995; stevenson et al., 2013; jung et al., 2014) . pedv antigens were not detected in other major organs, such as pylorus, tonsils, spleen, liver and kidneys. however, a recent study reported the replication of pedv in porcine pulmonary macrophages in vitro and in vivo (park and shin, 2014) . whether extra-intestinal replication of pedv occurs still remains uncertain. pedv binds and infects enterocytes expressing apn. assembly of the virus in infected enterocytes occurs rapidly by budding through intracytoplasmic membranes, such as the endoplasmic reticulum and golgi apparatus (ducatelle et al., 1981) . during the incubation period, pedv antigen-positive cells were seen throughout the small intestine and as many as 30-50% of the absorptive epithelial cells were positive , consistent with fecal shedding of asymptomatic pigs during the acute stage of infection. from the acute stage to mid-stage (24-60 h after onset of clinical signs) of infection, moderate to large numbers of pedv antigenpositive cells were observed throughout the small and large intestine, frequently affecting the entire villous epithelium . during the later-stage of infection (>72 h after onset of clinical signs), large numbers of pedv-infected epithelial cells were still observed, suggesting pedv re-infection of regenerating enterocytes . diarrhea induced by pedv is a consequence of malabsorption due to massive loss of absorptive enterocytes. functional disorders of infected enterocytes also contribute to the malabsorptive diarrhea. in the infected enterocytes examined by electron microscopy, loss of electron density of the cellular cytoplasm and rapid degeneration of mitochondria result in a lack of transport energy needed for absorption . ultrastructural changes and mild vacuolation observed in the infected colonic epithelial cells may interfere with the vital reabsorption of water and electrolytes . dehydration is exacerbated by vomiting but the mechanisms by which vomiting is induced in pedv infection are poorly understood. similar to hyperkalemia and acidosis in acute tgev infections (saif et al., 2012) , our preliminary study showed that pedvinoculated piglets at 1 day after onset of severe watery diarrhea showed hypernatremia, hyperkalemia, and hyperchloremia, but with low calcium and bicarbonate levels. brush border membranebound digestive enzymes such as disaccharidases (lactase, sucrase, and maltase), leucine apn, and alkaline phosphatase are significantly decreased in the small intestine of diarrheic piglets jung et al., 2006a) . reduced enzymatic activity in the small intestine results in maldigestive diarrhea. in our preliminary study, disorganized, irregular distribution and decreased expression of the tight junction protein, zonula occludin (zo)-1, and adherens junction protein, e-cadherin, was observed in the small intestine of infected gnotobiotic pigs at pih 30-120 (fig. 3) . the impaired gut integrity might lead to loss of water into the intestinal lumen with high osmotic pressure caused by pedv infection as well as uptake of luminal bacteria causing co-infections. the mechanisms by which pedv infection induces greater disease severity and deaths in nursing versus weaned pigs have not been clearly defined (shibata et al., 2000; madson et al., 2014) . several anatomical and physiological factors that may influence the higher susceptibility of suckling piglets to pedv infection and a longer recovery from disease include the slower turnover of enterocytes (5-7 days) in neonatal piglets compared to 2-3 days in 3-week-old weaned pigs (moon et al., 1973) . the high turnover rate of the intestinal epithelium depends on the stem cells found in the intestinal crypt. intestinal stem cells consist mainly of three cell types, namely, lgr5 (leucine-rich repeatcontaining g protein-coupled receptor 5)-positive crypt base columnar cells (lgr5+ cells), +4 cells, and paneth cells (sato and clevers, 2013) . however, the presence of paneth cells in the intestine of pigs is debatable (burkey et al., 2009) . our preliminary study revealed localization of large numbers of lgr5+ cells in the crypt cell layers of pedv-infected pigs (fig. 4) , indicating the presence of stem cells that are critical to the epithelial cell renewal during the acute-stage of pedv infection (k. jung et al., unpublished data) . that study also revealed a lack of lgr5+ cells and low proliferation of crypt cells (small expression of ki67 protein in crypt cells) in the small intestine of nursing piglets (9-day-old) without pedv infection, possibly causing the slow turnover of enterocytes. at 3-5 days after pedv infection, however, the number of lgr5+ cells and proliferation of crypt cells were remarkably increased, leading to the replacement of necrotic enterocytes shed from infected villi. on the other hand, weaned pigs (3-week-old) without pedv infection exhibited high proliferation of intestinal crypt cells and large numbers of lgr5+ cells in the crypts, relating to the rapid turnover rate of enterocytes. large numbers of lgr5+ cells and high proliferation of crypt cells were maintained at 1-5 days after pedv infection, possibly resulting in a rapid recovery from ped in weaned pigs. viremia where viral rna in serum ranged from 4.8 to 7.6 log10 genomic equivalents (ge)/ml was detected in gnotobiotic piglets inoculated with a us pedv strain (pc21a) at acute-to later-stages of infection . similar findings were observed in field samples, showing that 11/20 acute serum samples (55%) collected from diarrheic 13-20-week-old pigs had viral rna titers (4.0-6.3 log10 ge/ml) . the early, severe diarrhea/ vomiting and high pedv fecal shedding titers might be accompanied by viremia, but no one has yet confirmed the presence of infectious virus in the serum. there is a dearth of information on the innate and adaptive immune responses to pedv. after pedv infection, infiltration of lymphocytes (cd4+ and cd8+ t cells at pih 30-120) (fig. 5) , mononuclear cells, eosinophils and neutrophil was observed in the lamina propria of the small intestine coussement et al., 1982; sueyoshi et al., 1995) . isotype-specific antibody-secreting cells in systemic and mucosal associated lymphoid tissues and serum antibody responses were studied in conventional pigs inoculated with the cv777 strain (de arriba et al., 2002) . cultured intestinal epithelial cells expressing the e protein of pedv up-regulated interleukin (il)-8 expression in vitro (xu et al., 2013) . the pathogenicity of epidemic pedv strains is commonly severe, as evidenced by the high mortality of infected nursing piglets. however, attenuation of the virulence of pedv strains has been induced through high cell-culture passages (93rd-100th) (kweon et al., 1999; song et al., 2003; sato et al., 2011) . the attenuated pedv strains have multiple nucleotide changes in their s and open reading frame 3 (orf3) genes compared to those of their parent wild-type strains (song et al., 2003; sato et al., 2011) . among the 652 nucleotides of orf3, two deletions and seven changes were identified between the parent wild-type dr13 pedv and the cell-adapted pedv (100th) that was confirmed to be attenuated (song et al., 2003; sato et al., 2011) . notably, the s genes of the two attenuated pedv strains, korean dr13 (100th) and japanese 83p-5 (100th), had a remarkable similarity with comparable nucleotide mutations and aa substitutions relative to their parental viruses. the attenuated 83p-5 had 18 nucleotide mutations and 13 predicted aa substitutions in the s gene. similarly, the sequence analysis of a us pedv strain and in vitro passaged virus (10th in marc-145 cells) showed that the cell culture adaptation specifically modifies pedv s protein (six aa substitutions) whereas the open reading frame 1a/b (orf1a/b)-encoded polyprotein, orf3, e, m, and n proteins remained unchanged (lawrence et al., 2014) . multiple nucleotide mutations and aa substitutions in the s gene of pedv might contribute to attenuation of its in vivo pathogenicity, but the entire pedv genomes should be sequenced to verify other changes after attenuation. detailed clinical disease and complications as a result of typical epidemic ped were documented on seronegative pig breeding farms in the uk in 1976 -1977 (wood, 1977 , belgium in 1977 (pensaert and de bouck, 1978 ), japan in 1982 (sueyoshi et al., 1995 ), italy in 2005 -2006 (martelli et al., 2008 ), thailand in 2007 (puranaveja et al., 2009 , and the us in 2013 (stevenson et al., 2013) . the clinical outbreaks on seronegative farms were characterized by a sudden epidemic of severe diarrhea and/or vomiting, accompanied by anorexia and significantly reduced appetite, in pigs of all ages. the severity of clinical signs and mortality appeared to be inversely related to the age of the pigs (shibata et al., 2000) . in weaner to finisher pigs, including pregnant sows, clinical signs are selflimiting within 5-10 days after onset of disease and are not as severe as those of nursing piglets under 2 weeks of age (martelli et al., 2008; puranaveja et al., 2009) . when pregnant sows become immune after virus exposure, they protect their offspring by lactogenic immunity. the interval between onset and cessation of the disease is generally 3-4 weeks (puranaveja et al., 2009) , however clinical signs mainly develop in the seronegative lactating sows and their suckling piglets. in farrowing herds, morbidity can approach 100% in piglets, but varies in sows. mortality of piglets <2 weeks of age can exceed 95% (50% on average) at 3-5 days after onset of severe watery diarrhea and/or vomiting. field observations on epidemic ped in the uk in 1976 -1977 (wood, 1977 and in the us in 2013 (stevenson et al., 2013) as well as experimental findings (pensaert and de bouck, 1978) suggest that the incubation period of pedv before clinical signs are detected varied, ranging from 1 to 7 days (us pedv) or 5-8 days (uk pedv). experimental studies using the prototype cv777 showed that 3-15day-old, caesarean-derived, colostrum-deprived (cdcd) pigs developed diarrhea within pih 22-36 (pensaert and de bouck, 1978; debouck et al., 1981) . another us pedv infection study using 10-35-day-old gnotobiotic pigs with 6.3-9.0 log10 ge showed that severe diarrhea and/or vomiting were detected commonly within pih 24-48 original magnification, ×600. nuclei were stained with blue-fluorescent 4′, 6-diamidino-2-phenylindole dihydrochloride. immunofluorescence staining using monoclonal antibodies against human recombinant zo-1 and e-cadherin (invitrogen). . unlike cdcd or gnotobiotic pigs, conventional nursing piglets inoculated with 3.2-3.3 log10 50% tissue culture infectious dose (tcid50) of a chinese pedv strain had a longer incubation period (3-6 days after inoculation) before clinical signs were detected (wang et al., 2013) . detailed clinical disease and problems caused by endemic ped were documented in a farrow-to-finish farm in the netherlands in 1989 -1991 (pijpers et al., 1993 . during the outbreak in 1989, diarrhea was most severe in fattening pigs and pregnant sows, and was mild or absent in nursing and weaning pigs with no mortality. for at least 18 months after the onset of the first outbreak, pedv became endemic on this farm and the infection persisted in seronegative gilts or 6-10-week-old pigs newly introduced to the farm. another typical endemic ped has been manifested in korean pig farms. korean farms have employed live or inactivated pedv vaccines using three korean strains dr13, kpedv-9 and sm98-1 or a japanese strain p-5v. studies reported that recent prevalent korean pedv field isolates are closely related to chinese strains and differ genetically from the four vaccine strains used in korea and the prototype cv777 (park et al., 2013) . this divergence of historic vaccine and recent field pedv strains may contribute to the reduced efficacy of the vaccines, causing difficulty with eradication of pedv from pig farms with endemic ped in the korean pig population. like endemic tge, pedv-related mortality and morbidity of nursing piglets passively immunized is lower than is seen in seronegative pigs (bohl et al., 1978) . endemic ped is manifested mainly in weaned pigs (pijpers et al., 1993) , and the severity of clinical disease in nursing piglets may be exacerbated by co-infections of other enteropathogens (escherichia coli, 3% for chinese piglets or 9% for canadian piglets) (turgeon et al., 1980; wang et al., 2013) , or viruses including porcine circovirus type 2 (pcv2) (30-33% for korean piglets), tgev (8% for chinese piglets), and rotavirus (4% for chinese piglets) (hirai et al., 2001; jung et al., 2006b; wang et al., 2013) . gross lesions are limited to the gastrointestinal tract and are characterized by thin and transparent intestinal walls (duodenum to colon) with accumulation of large amounts of yellow fluid in the intestinal lumen sueyoshi et al., 1995; stevenson et al., 2013) . the stomach is filled with curdled milk, possibly due to reduced intestinal peristalsis. congestion of the mesenteric vessels is frequently detected, and mesenteric lymph nodes (mln) are edematous. lack of intestinal lacteals, as an indicator of malabsorption, is frequently seen (puranaveja et al., 2009) . despite persistent severe diarrhea, infected pigs had low to moderate appetite at 3-5 days after onset of diarrhea after which they became moribund . histological lesions consist of acute diffuse, severe atrophic enteritis and mild vacuolation of superficial epithelial cells and subepithelial edema in the cecum and colon coussement et al., 1982; sueyoshi et al., 1995; jung et al., 2014) . based on electron microscopy, one of four piglets infected with cv777 had ultrastructural changes in the colonic epithelial cells, but with a lack of histological lesions . during acute infection, vacuolated enterocytes or massive cell exfoliation were seen on the tips or the entire villi in the jejunum. atrophied villi are frequently fused and covered with a degenerate or regenerated flattened epithelium. infiltration of inflammatory cells is evident in the lamina propria. the crypts of lieberkuhn in the duodenum appeared normal . no lesions were seen in the spleen, liver, lung, kidney, and mln of orally and/or intranasally infected piglets . during the incubation period, i.e. prior to onset of clinical signs, infected pigs exhibited normal villous lengths but with vacuolated enterocytes undergoing necrosis . for 1-3 days after the onset of diarrhea, infected pigs exhibited severe villous shortening . piglets euthanased at a later stage of infection (84-120 h after onset of clinical signs) had moderate to severe villous atrophy jung et al., 2014) , indicative of continued cellular necrosis. after pedv infection, intestinal crypt layers included lgr5+ cells (fig. 4) and crypt cells positive for ki67 protein that is a marker for proliferating cells (jung et al., 2008) . the time of onset and severity of malabsorptive diarrhea induced by pedv may depend on the extent of villous atrophy in the jejunum and the rapidity of replacement by the crypt stem cells. when ped occurs in a seronegative breeding farm, immunization or vaccination of pregnant sows is important in the control of epidemic ped and to reduce the number of deaths of suckling piglets. if the sows are due to farrow within 2 weeks or more, immunization can be undertaken by exposure to virulent autogenous virus, such as fecal slurry or minced intestines from infected neonatal piglets. however, there is a potential risk of incidental widespread infection of other pathogenic viruses, such as pcv2, contained in the pedv-infected piglets' feces or intestines among sows or their suckling piglets via vertical transmission routes (jung et al., 2006c; park et al., 2009; ha et al., 2010) . the importance and mechanisms of passive lactogenic immunity to provide newborn piglets with immediate protection against tgev infection have been reviewed by saif et al. (2012) . all strains of epidemic pedv in europe, asia and the us are highly enteropathogenic, as evidenced by the high mortality of infected nursing piglets. however, attenuation of the virulence of korean (kpedv-9 and dr13) or japanese (83p-5) pedv strains could be induced through high cell-culture passages (93rd-100th) (kweon et al., 1999; song et al., 2007; sato et al., 2011) . in addition, the attenuated cell-adapted pedv strains have been used as oral (korean strain dr13 only) or intramuscular (im) live virus vaccines. the im administration of live attenuated kpedv-9 pedv vaccine (1 ml of 10 7 tcid50/ml; twice at 2 or 4 weeks before farrowing) reduced the 40% mortality rate of piglets challenged with five 50% lethal dose (ld50) of the parent wild-type strain and the 100% mortality rate of piglets challenged with 10 ld50 to 0% and 80%, respectively (kweon et al., 1999) . the efficacy might be associated with high pedv specific igg levels in the serum and colostrum of vaccinated sows (song et al., 2007) . a study using im live attenuated dr13 pedv vaccine (1 ml of 10 7 tcid50/ml; twice at 2 or 4 weeks before farrowing) reduced the 100% mortality of piglets challenged with a high-dose of the parent dr13 to 60% (song et al., 2007) . based on these observations, pregnant sows can be vaccinated using live attenuated pedv strains via an im route, but induction of complete protection was not observed in the nursing piglets. active immunization of nursing or feeder pigs is important for the control of endemic pedv infections (saif et al., 2012) . a field study (song et al., 2007) showed that compared to vaccination via im route, oral administration with live attenuated pedv (dr13 strain) vaccine twice 2 or 4 weeks before farrowing was more effective in boosting or initiating immunity in pregnant sows and their suckling piglets (3-day-old). the vaccinated sows and their piglets exhibited higher iga (mucosal immunity) and virus neutralization antibody (humoral immunity) levels in the colostrum or sera compared to those of the counterparts administered the im vaccine with the same dose. however, the presence of maternal antibodies in vaccinated pigs can interfere with active antibody production after pedv infection, as observed in tgev infection (sestak et al., 1996; saif et al., 2012) . whether the oral live vaccine strain is genetically stable and remains non-infectious in the fields needs to be further studied. disappearance and re-emergence of epidemic ped indicates that pedv is effectively able to escape from the current vaccination protocols, biosecurity and control systems. endemic ped is a significant problem, which is exacerbated by the emergence or potential importation of multiple pedv variants into countries. epidemic pedv strains spread rapidly and cause a high number of pig deaths and substantial economic losses. these strains are highly enteropathogenic and acutely infect villous epithelial cells of the entire small and large intestines although the jejunum and ileum are the primary sites of infection. pedv infections cause acute, severe atrophic enteritis accompanied by viremia (viral rna) that leads to severe diarrhea and vomiting, followed by extensive dehydration and imbalanced blood electrolytes as the major cause of death in nursing piglets. a better understanding of the pathogenic characteristics of epidemic or endemic pedv strains is needed to prevent and control the disease in affected regions and in the development of effective vaccine. high mortality of pedv-infected, seronegative nursing piglets is most likely associated with extensive dehydration as a result of severe villous atrophy. in infected nursing piglets, there is an increased proliferation of crypt cells as well as numbers of lgr5+ crypt stem cells in the intestine, reorganization of the damaged intestinal epithelium, and migration of mature enterocytes to the tips of villi which may be not sufficient to prevent severe dehydration in nursing piglets. the time taken until dehydration of pedv-infected nursing piglets in the field appears to be too short to enable the animals to recover from the disease through naturally occurring epithelial cell renewal by crypt stem cells. further studies are needed to define the extent to which intestinal stem cells in nursing versus weaned pigs organize and migrate to replace pedv-infected villous epithelial cells. pharmacological or biological mediators such as epidermal growth factor that promote stem cell regeneration or maturation would be interesting targets to try to shorten the time for epithelial cell renewal and to reduce pedv death losses from dehydration. neither of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. whose contributions to pedv research may have been inadvertently and unintentionally missed. salaries and research support were provided 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pedv strains porcine epidemic diarrhea virus variants with high pathogenicity detection and genetic characterization of deltacoronavirus in pigs new variant of porcine epidemic diarrhea virus proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture 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 an apparently new syndrome of porcine epidemic diarrhoea porcine epidemic diarrhea virus e protein causes endoplasmic reticulum stress and up-regulates interleukin-8 expression porcine epidemic diarrhea virus (cv 777) and feline infectious peritonitis virus (fipv) are antigenically related we thank bryan eyerly and xiaohong wang for technical assistance in immunofluorescence staining. we apologize to authors key: cord-305156-w6iqeayr authors: gallien, sarah; moro, angélique; lediguerher, gérald; catinot, virginie; paboeuf, frédéric; bigault, lionel; gauger, phillip c.; pozzi, nathalie; berri, mustapha; authié, edith; rose, nicolas; grasland, béatrice title: limited shedding of an s-indel strain of porcine epidemic diarrhea virus (pedv) in semen and questions regarding the infectivity of the detected virus date: 2018-10-11 journal: vet microbiol doi: 10.1016/j.vetmic.2018.09.025 sha: doc_id: 305156 cord_uid: w6iqeayr pedv is mainly transmitted by the oro-fecal route although pedv shedding in semen has already been shown for an s-non-indel pedv strain infection. the aim of this study was to determine if pedv can be shed in semen from spf (specific pathogens free) boars infected by a french s-indel pedv strain (pedv/fr/001/2014) and in case of positive semen to determine the infectivity of that semen. both infected boars had diarrhea after inoculation and shed virus in feces. pedv genome was also detected by rt-qpcr in the sperm-rich fraction of semen (6.94 × 10(3) and 4.73 × 10(3) genomic copies/ml) from the two boars infected with the s-indel pedv strain but only once at 7dpi. in addition, pedv rna in peyer’s patches and in mesenteric lymph nodes was also present for the two inoculated boars. the pedv positive semen (s-non-indel and s-indel) sampled during a previous trial and in this boar trial were inoculated to six spf weaned pigs. the inoculated piglets did not seroconvert and did not shed virus throughout the duration of the study except for one pig at 18 dpi. but, pedv could be detected in intestinal tissues such as duodenum, jejunum and jejunum peyer’s patches by rt-qpcr except for one pig. even if pedv genome has been detected in semen, experimental infection of piglets with positive semen failed to conclude to the infectivity of the detected pedv. porcine epidemic diarrhea (ped) was described for the first time in europe in the 1970s (jung and saif, 2015; song and park, 2012) . this disease is characterized by a severe, profuse watery diarrhea with or without vomiting and dehydration and is caused by an etiologic agent called porcine epidemic diarrhea virus (pedv), a positive-sense, singlestranded rna enveloped coronavirus of 28-kb (jung and saif, 2015) . after several outbreaks in the 70's, ped has been persisting in europe with sporadic cases until the late 1990's (jung and saif, 2015) . outbreaks of ped have also been described in asia in the 1980s and from 2010 (jung and saif, 2015; wang et al., 2013) . currently, two genotypes are circulating in several regions in the world, namely the s-non-indel strains and the s-indel strains, showing insertions-deletions in the s1 segment of the s gene (jung and saif, 2015; vlasova et al., 2014) . from clinical reports, these two types of strains seemed to be different in terms of morbidity and mortality with the s-non-indel pedv strains associated with more severe clinical cases and higher case-fatality rate compared to the others (vlasova et al., 2014; wang et al., 2014) . in contrast to s-non-indel pedv strains, exclusively reported from america and asia, s-indel pedv strains have also been identified in europe since 2014 (efsa, 2016; stadler et al., 2015; vlasova et al., 2014) . pedv is transmitted mainly by the oro-fecal route, but also contact with contaminated equipment, vehicles used for pig transport or the staff (bowman et al., 2015; jung and saif, 2015; lowe et al., 2014) . airborne transmission of the virus was also shown for s-non-indel strains (alonso et al., 2014) . pedv transmission through contaminated milk from dam to piglets can also occur (sun et al., 2012) but vertical transmission of pedv through semen has never been shown. s-non-indel pedv strain shedding has recently been evidenced in the different fractions of semen (seminal and sperm-rich fractions) and in gelatin plug of specific pathogen free (spf) boars experimentally inoculated (gallien et al., 2018b) . however, to the best of our knowledge, the presence of pedv rna in semen of boars infected by an s-indel pedv strain has not been studied so far. thus, the aim of this study was to determine if pedv can be shed in semen from spf boars infected by an s-indel french pedv strain (pedv/fr/001/2014). subsequently, the infectivity of quantitative rt-pcr (rt-qpcr) positive semen was evaluated in an experimental infection of spf weaned piglets. two experimental trials were carried out in the air-filtered level 3 biosecurity facilities of the french agency for food, environmental and occupational health & safety (anses) in accordance with the european and the french regulations on animal welfare. protocols used were approved by the ethics committee registered under number #16 by the french ministry of . the first trial (trial 1) was performed to evaluate pedv shedding in semen from large white spf inoculated boars. two boars (boars i1 and i2 aged 2 ½ and 1 ½ years-old) were housed in the same room but in two separate pens. the two other boars (boars c1 and c2), of similar age to boars i1 and i2, were used as negative controls and housed within the biosafety level-3, air filtrated anses spf herd. this experiment lasted for 51 days post-inoculation (dpi). at 51 dpi, the inoculated boars were euthanized including anesthesia (zoletil®, virbac, carros, france, 15 mg/kg) followed by bleeding before necropsy. the boars c1 and c2 were not necropsied. the second trial (trial 2) was carried out for 18 days to assess the infectivity of rt-qpcr pedv-positive semen. the positive semen fractions from two experiments were used and were collected from trial 1 with the s-indel pedv strain and from a previously described trial involving an s-non-indel strain (gallien et al., 2018b) . six spf weaned pigs of three week-old were housed in the same room in two different pens separated by a plastic partition. two pigs of the same age housed in another room were used as controls. at 18 dpi, pigs were euthanized after anesthesia as already described and necropsied. inoculation procedures the two boars of the trial 1 received orally 5 ml of a homogenate of the pedv french s-indel strain pedv/fr/001/2014 (genbank number: kr011756) titrating 10 8 viral genome copies/ml (equivalent to ≈10 5 tcid 50 /ml). the pedv french s-indel strain was amplified in a threeweek-old spf weaned pig inoculated with a homogenate of jejunum collected from ped-affected pigs belonging to a french farm in 2014. the inoculum was prepared from the jejunum of this spf pedv-inoculated pig by homogenization in dulbecco's phosphate-buffered saline (sigma-aldrich, saint louis, mo, usa) (20% w/v). the homogenate was centrifuged at 10,000×g for 10 min at 4°c and the supernatant filtered through a 0.45 μm filter. next-generation sequencing (ngs) was performed on the inoculum to obtain the pedv complete genome sequence and to ensure the absence of other viral rna sequences. the absence of porcine circovirus type 2 (pcv2) and porcine reproductive and respiratory syndrome virus (prssv) in the inoculum was assessed by the absence of seroconversion against pcv2 and prssv in the inoculated boars at the end of the trial. the six spf weaned pigs of trial 2 were orally inoculated with 10 ml of contaminated semen: pigs #1, #2, #3, #4 with the s-non-indel and pigs #5, #6 with the s-indel (table 1 ). the semen samples inoculated were stored at −80°c until inoculation of the piglet. the semen were defrosted on ice just before the inoculation and reconstituted in cold dulbecco's phosphate-buffered saline to avoid thermal shock. for the two trials, clinical signs (lethargy, outward appearance, behavior, breathing, diarrhea and vomiting) were recorded daily. pedv shedding in fecal samples was evaluated daily the first week after inoculation, and then three times a week until 51 dpi for trial 1 and for trial 2, at 0.5 dpi, twice a day from 1 to 4 dpi, once at 5 and 6 dpi and then three times a week until the end of the trial. pedv shedding was also assessed in semen for trial 1. semen was collected before inoculation and every day the first week post-inoculation and then twice a week until the end of the trial. the belly and the sheath of the boars were cleaned with cleaning wipes before sampling in order to avoid any contaminations of the semen with positive pedv-feces/aerosols. a swab of the prepuce was also done before each collection to rule out the possibility of external contamination of semen. semen was collected manually without sexual stimulation with support of a collection dummy. the gelatin plug was also collected at the end of the semen ejaculate. blood samples were also collected before inoculation and at the end of trials for the two trials, and at 21 dpi for trial 1 and once a week until the end of the trial for trial 2 to assess seroconversion and viremia by rt-qpcr ( fig. 1) . during necropsy, organs of the digestive tract (duodenum, jejunum, ileum, colon, peyer's patches (jejunum and ileum)), spleen, liver, mesenteric and inguinal lymph nodes, lungs, were collected and stored in rna later tissue storage reagent (sigma-aldrich, saint louis, mo, usa) for the two trials. organs of the reproductive tract were also collected for the trial 1 (vas deferens (right and left), testicles (right and left: apical pole, distal pole, median axis), epididymis (right and left: head, body, tail), prostate, cowper's glands (right and left), seminal vesicles (right and left: apical and distal pole) and spermatic cords (right and left)) and stored in the same tissue storage reagent. macroscopic lesions were also evaluated on necropsy for the two trials. all samples collected during the two trials were stored at −80°c. fresh semen samples were centrifuged at 8000×g for 20 min at 4°c (pal et al., 2008) . this centrifugation step allowed separating the seminal fraction from the sperm-rich fraction of the semen. these two fractions and the gelatin plug were stored at −80°c. blood samples were centrifuged at 1200×g for 10 min at 4°c and then sera were stored at −80°c until use. one gram of feces (or 1 ml of feces in case of liquid feces) was homogenized in 9 ml of dulbecco's phosphate-buffered saline (sigma-aldrich, saint louis, mo, usa). the fecal homogenates were then centrifuged at 15,000×g for 10 min at 4°c. tissues were homogenized in dulbecco's phosphate-buffered saline (sigma-aldrich, saint louis, mo, usa) at 20% v/w using a bead mill (retsc, haan, germany). these homogenates were centrifuged at 10,000×g for 10 min at 4°c and the supernatants were stored at −80°c. total rnas were extracted from the fecal and tissue homogenates, from the preputial swabs and from sera using the qiagen rneasy mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. five μl of eluted rna were used as templates for pedv rt-qpcr. rna extraction controls were performed for every five samples to check for any pedv contamination by replacing sample with rnase free water. rna extracted from feces with the rneasy mini kit was diluted 1:10 to avoid any pcr inhibition. total rnas were extracted from the two semen fractions (seminal and sperm-rich fractions) and the gelatin plug using trizol® assay (thermo fisher scientific, waltham, ma, usa) (gallien et al., 2018b) . the number of pedv genome copies was assessed by real-time rt-pcr using power sybr® green rna-to-ct ™ 1-step kit (thermo fisher scientific, waltham, united states of america) on an applied biosystems® 7500 real time pcr system (thermo fisher scientific, waltham, united states of america) as already described (gallien et al., 2018b; kim et al., 2007) . the primers used were designed from the conserved regions of the pedv nucleocapsid gene for universal detection of both strains (forward, 5′-cgcaaagactgaacccactaa-3′; reverse, 5′-ttgcctctgttgttacttggagat-3′). for each pcr run, a positive control containing pedv rna extract from a pedv cell culture supernatant was included. two negative controls were also included on the plate, the rna samples were replaced by rnase free water. one negative control was placed close to the positive control and the second one at the end of the plate. all samples were processed in duplicate. sera were tested for pedv antibodies using a commercial elisa test, id screen® pedv indirect (id vet, grabels, france). the elisa test is validated if the mean value of the positive control optical density (od) is greater than 0.350 and if the ratio of the mean values of the positive and negative controls is greater than 3. for each sample, the s/p (sample-to-positive) ratio was calculated. samples with s/p ratios equal to or higher than 60% were considered positive for pedv antibodies (fig. 1) . both inoculated boars demonstrated clinical signs. they were lethargic as soon as 2 dpi and until 6 dpi. a reduction of food intake was also observed for the two inoculated boars at 3 dpi and until 9 dpi. diarrhea was also observed at 3 dpi. none of the control boars demonstrated clinical signs during the trial. the inoculated boars were pedv seronegative before inoculation and became seropositive at 21 ± 1 dpi (s/p% i1 = 62.68% and s/p% i2 = 66.83%). only boar i2 was still positive for pedv antibodies at the end of the trial (s/p% i2 = 74.29%). the two control boars were seronegative before and at the middle of the trial. all boars were rt-qpcr negative for pedv in feces prior to inoculation. the two control boars remained pedv negative in feces until the end of the trial. however, pedv rna was first detected in the feces of boar i1 at 2 dpi (4.67 × 10 5 genome copies/ml of feces). at 3 dpi, boars i1 and i2 showed 6.02 × 10 8 and 3.47 × 10 7 genome copies/ml of feces, respectively. the viral genome was continuously detected until 14 dpi. maximum shedding was detected between 4.5 and 6 dpi for the two boars (2.82 × 10 9 genome copies/ml for boar i2 at 4.5 dpi and 3.49 × 10 9 genome copies/ml for boar i1 at 6 dpi). pedv rna was then sporadically detected for the two boars (at 24 dpi for boar i1 and at 18 and 42 dpi for boar i2) (fig. 2) . no pedv nucleic acid was detected in the seminal or the sperm-rich fractions of semen and in the gelatin plug collected from boars i1, i2, c1 and c2 before inoculation. the two control boars remained negative for pedv rna in all fractions of semen and in the gelatin plug throughout the trial. pedv rna was detected for both inoculated boars at 7 dpi in the sperm-rich fraction of semen with 6.94 × 10 3 and 4.73 × 10 3 genome copies/ml for boars i1 and i2, respectively. no pedv rna was detected by rt-qpcr thereafter in this fraction. the inoculated boars remained rt-qpcr negative for the seminal fraction and the gelatin plug throughout the trial. all swabs collected from the sheath tested negative for pedv rna, even when the virus was detected in the spermrich fraction (7 dpi). pedv rnas were detected in the jejunum peyer's patches (2.49 × 10 6 and 2.82 × 10 6 genome copies/ml for boars i1 and i2 respectively), in the ileum peyer's patches (2.85 × 10 6 and 4.58 × 10 7 genome copies/ml for boars i1 and i2 respectively) and in the mesenteric lymph nodes (2.74 × 10 6 and 1.23 × 10 6 genome copies/ml for boars i1 and i2 respectively). pedv rna was not detected from the organs of the reproductive tract. no lesions were observed in the gastrointestinal or reproductive tract. none of the inoculated piglets showed clinical signs throughout the trial. no seroconversion of these pigs was observed at 18 dpi. no pedv nucleic acid was detected in feces of pigs #1, #2, #3, #4, #5 and #6 during the trial except for pig #5 at 18 dpi (8.94 × 10 4 genome copies/ ml). some digestive tissue samples were found positive for pedv in rt-qpcr for pigs #2, #3, #4, #5 and #6 at 18 dpi (table 2 ) but no lesion was observed in the gastrointestinal tract. oral inoculation of mature boars by an s-indel french pedv strain demonstrated less severe clinical signs compared to those observed in boars inoculated with a s-non-indel us pedv strain (gallien et al., 2018b) . however, the infection induced profuse diarrhea and affectd growth performances similarly to weaned pigs inoculated with this s-indel french pedv strain (gallien et al., 2018a) . the duration of virus fecal shedding determined in our study (11 days) was shorter compared to weaned pigs inoculated with an s-indel pedv strain (18-20 days) (leidenberger et al., 2017; lohse et al., 2017) or for boars inoculated with an s-non-indel pedv strain (16-19 days) (gallien et al., 2018b) . in this experimental challenge, the presence of pedv rna in the fig. 2 . fecal pedv shedding detected from boar i1 and i2 (log(pedv-genome copies/ml)) in trial 1. pedv genome loads (genome copies/g) detected in the tissues collected at necropsy from spf weaned pigs inoculated with pedv rt-qpcr positive semen in trial 2. sperm-rich fraction of semen of both inoculated boars was detected at 7 dpi only, which is much more limited than in the sperm-rich fraction of semen from boars inoculated with an s-non-indel pedv strain (gallien et al., 2018b) . in the previous study, pedv rna could be detected for a longer period in the sperm-rich fraction of semen: i.e. transient detections of pedv rna during three distinct periods comprised between 3 and 16 days. pedv rna was also detected in the seminal fraction of semen and in gelatin plug in s-non-indel pedv inoculated boars contrasting to the observations from the present study. this difference regarding pedv rna detection in semen could be linked to the viral strain used for inoculation and its virulence. clinical signs induced with s-non-indel pedv were more severe compared to clinical signs observed in s-indel pedv challenged boars (gallien et al., 2018b) . viral shedding in the sperm-rich fraction of semen has already been reported for prrsv which belongs to the same order as pedv, the nidovirales order, and differences in terms of shedding in semen between genotype 1 and highly virulent genotype 2 prrsv strains have already been shown (christopher-hennings et al., 1995; prieto and castro, 2005) . prrsv genotype 2 strains could be detected during longer periods in semen compared to genotype 1 strains. hence, the duration of detection of highly virulent genotype 2 prrsv strains in semen was reported between 25 to 92 days (christopher-hennings et al., 2001) while the presence of genotype 1 prrsv strains in semen could be observed more sporadically: only at one point after infection, at 7 dpi (prieto et al., 1996) or during shorter periods comprised between 10 and 40 days (swenson et al., 1994) . detection of s-indel pedv rna in semen appeared sporadically after the occurrence of clinical signs and detection in feces in contrast to what we observed for an s-non-indel pedv strain. moreover, no s-indel pedv rna was detected in semen after cessation of clinical signs and fecal shedding in contrast to what was observed with the s-non-indel strain (gallien et al., 2018b) . these data suggest that the risk of introduction of pedv shedding boars via imports should be more predictable from clinical exams or fecal shedding assessment for s-indel strains than for s-non-indel. absence of detection of pedv rna in the genital tract has been shown in boars inoculated with s-indel pedv strain as well as in boars inoculated with an s-non-indel strain at 51 dpi (gallien et al., 2018b) . mc carty and al., showed that pedv rna could be detected in penis and testicle at 5 days post-infection (mccarty et al., 2015) . that presence was noticed at infection peak when a viremia could be noticed too. that could explain the absence in reproductive tract of pedv we noticed at 51 days post infection, a very late date compare to the infection peak. during the second trial, no shedding was detected in feces of spf weaned pigs inoculated with s-indel and s-non-indel rt-qpcr pedv positive semen except for pig #5 at 18 dpi. the presence of low pedv genomic loads could be noticed in different intestinal tissues. however, clinical signs and seroconversion were absent in all inoculted piglets. the results of the present study neither confirms nor denies if the rt-qpcr pedv positive semen contained infectious virus. additional trials with longer periods of observation, sequential slaughters or the use of a more sensitive model such as neonatal piglets should be conducted in order to determine if the pedv rna detected in the tissues were caused by pedv infection. the use of neonatal piglets could have been in fact a most sensitive model to evaluate pedv infectivity but to realize that kind of trial in our experimental facility, we should have infected two sows with their suckling piglets and for material reasons (place, cost, animal availability…), this option could not be selected. moreover, with these results, it is impossible to speculate whether pedv-positive semen could infect sows via natural insemination. in fact, it has already been shown for other porcine viral pathogens that contaminated semen could be infectious, but could not infect sows or gilts through artificial insemination. weaned pigs inoculated with pcv2 positive semen, for example, presented a viremia and anti-pcv2 antibodies but the same pcv2 positive semen could not infect sows through artificial insemination (madson et al., 2009) . the amount of virus contained in semen could explain the impossibility of viral transmission through artificial insemination (grasland et al., 2013; madson et al., 2009 ). an impact of the amount of virus contained in semen on the capacity of the transmission of viral pathogens has also been shown for prrsv. the prrsv can be transmitted through semen but only when it is present in this matrix at a given concentration. under experimental conditions, prrsv transmission was successful when semen contained 2 × 10 5 tcid 50 /ml of virus i.e. on average 10 7 genome copies/ml. however, when the amount of virus contained in semen was approximately 2 × 10 3 tcid 50 /ml (10 6 genome copies/ml on average), transmission through semen was limited. when the amounts were even lower (2 × 10 2 tcid 50 /ml, 10 5 genome copies/ml on average), transmission was no longer effective (prieto and castro, 2005) . a possible impact of the amount of genomic load of pedv contained in semen on the capacity of the transmission of pedv might be suspected in the present case. to conclude, a very transient shedding of pedv in semen was observed in case of infection by an s-indel pedv strain conversely to what was observed with s-non-indel pedv strain. however the infectivity of the virus present in pedv s-indel or s-non-indel positive semen was not evidenced. evidence of infectivity of airborne porcine epidemic diarrhea virus and detection of airborne viral rna at long distances from infected herds investigating the introduction of porcine epidemic diarrhea virus into an ohio swine operation persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from yorkshire, hampshire, and landrace boars collection and review of updated scientific epidemiological data on porcine epidemic diarrhoea better horizontal transmission of a us non-indel strain compared with a french indel strain of porcine epidemic diarrhoea virus evidence of porcine epidemic diarrhea virus (pedv) shedding in semen from infected specific pathogenfree boars evaluation of the transmission of porcine circovirus type 2 (pcv-2) genogroups a and b with semen from infected specific-pathogen-free boars porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis multiplex realtime rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus virulence of current german pedv strains in suckling pigs and investigation of protective effects of maternally derived antibodies experimental infection of young pigs with an early european strain of porcine epidemic diarrhoea virus and a recent us strain role of transportation in spread of porcine epidemic diarrhea virus infection, united states infectivity of porcine circovirus type 2 dna in semen from experimentallyinfected boars case report describing the clinical course of porcine epidemic diarrhea in a commercial boar stud and return of the stud to service after whole-herd inoculation with porcine epidemic diarrhea development and validation of a duplex real-time pcr assay for the simultaneous detection and quantification of porcine circovirus type 2 and an internal control on porcine semen samples porcine reproductive and respiratory syndrome virus infection in the boar: a review semen changes in boars after experimental infection with porcine reproductive and respiratory syndrome (prrs) virus porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines emergence of porcine epidemic diarrhea virus in southern germany outbreak of porcine epidemic diarrhea in suckling piglets excretion of porcine reproductive and respiratory syndrome virus in semen after experimentally induced infection in boars distinct characteristics and complex evolution of pedv strains porcine epidemic diarrhea virus variants with high pathogenicity new variant of porcine epidemic diarrhea virus the authors are grateful to anses, inra and lncr for their financial support. we also would like to thank phillip gauger from iowa state university who provided the us non-indel strain and all the team members at the experimental laboratory for their contribution. all the authors designed and carried out the experiments. sg analyzed the data and wrote the manuscript. bg, nr and mb supervised the project. all the co-authors revised the manuscript. all authors read and approved the final manuscript. the authors declare that they have no conflict of interests. key: cord-259296-qsaewje2 authors: wang, pengcheng; bai, juan; liu, xuewei; wang, mi; wang, xianwei; jiang, ping title: tomatidine inhibits porcine epidemic diarrhea virus replication by targeting 3cl protease date: 2020-11-11 journal: vet res doi: 10.1186/s13567-020-00865-y sha: doc_id: 259296 cord_uid: qsaewje2 porcine epidemic diarrhea virus (pedv) causes lethal diarrhea in suckling piglets, leading to severe economic losses worldwide. there is an urgent need to find new therapeutic methods to prevent and control pedv. not only is there a shortage of commercial anti-pedv drugs, but available commercial vaccines fail to protect against highly virulent pedv variants. we screened an fda-approved library of 911 natural products and found that tomatidine, a steroidal alkaloid extracted from the skin and leaves of tomatoes, demonstrates significant inhibition of pedv replication in vero and ipec-j2 cells in vitro. molecular docking and molecular dynamics analysis predicted interactions between tomatidine and the active pocket of pedv 3cl protease, which were confirmed by fluorescence spectroscopy and isothermal titration calorimetry (itc). the inhibiting effect of tomatidine on 3cl protease was determined using cleavage visualization and fret assay. tomatidine-mediated blocking of 3cl protease activity in pedv-infected cells was examined by western blot detection of the viral polyprotein in pedv-infected cells. it indicates that tomatidine inhibits pedv replication mainly by targeting 3cl protease. in addition, tomatidine also has antiviral activity against transmissible gastroenteritis virus (tgev), porcine reproductive and respiratory syndrome virus (prrsv), encephalo myocarditis virus (emcv) and seneca virus a (sva) in vitro. these results may be helpful in developing a new prophylactic and therapeutic strategy against pedv and other swine disease infections. pedv, an enveloped, positive-sense, single-stranded rna virus, is a member of the coronavirinae subfamily [1, 2] , which comprises viruses that cause a variety of diseases in mammals and birds, ranging from enteritis in cows and pigs, to upper respiratory disease in chickens, and potentially lethal human respiratory infections, such as severe acute respiratory syndrome (sars) [3] , middle east respiratory syndrome (mers) [4] , and the novel coronavirus disease 2019 (covid-19) [5] . fecal-oral transmission is believed to be the main mode of pedv transmission [6] . the latest research indicates that airborne transmission may also contribute to a pedv outbreak [7] , similar to sars-cov-2 and mers-cov. owing to antigenic, genetic (> 10% amino acid variation between respective s-proteins) and phylogenetic (g1 vs g2) differences between vaccine and field epidemic strains, current pedv vaccines appear to have low to moderate efficacy [8] . there is a need for alternative approaches to control this disease, such as effective antiviral drugs for pedv treatment. the interaction between pedv and pigs has been well studied. several host antiviral factors, including the bone marrow stromal cell antigen 2 (bst2) [9] , interleukin-11 (il-11) [10] , interferon-lambda (ifn-lambda) [11] , gtpase-activating protein-binding protein 1 (g3bp1) [12] , and cholesterol 25-hydroxylase (ch25h) [13] , have been reported to show antiviral activity against pedv infection. some natural compounds and compositions have also been reported to show anti-pedv activity in vitro, such as griffithsin [14] , coumarin [15] , and prenylated phenolic compounds [16] . however, the mechanism of antiviral activity is not well understood, and there are no commercial anti-pedv drugs available to the pig breeding industry. in this study, we screened a library of 911 natural products and found that tomatidine, a steroidal alkaloid that can be extracted from the skin and leaves of tomatoes [17] , significantly inhibited the replication of pedv by directly inhibiting 3clpro activity, and showed antiviral activity against other swine disease viruses as well, demonstrating excellent potential as a natural broad-spectrum antiviral product. vero cells, st cells, marc-145 cells, bhk-21 cells, and ipec-j2 cells were maintained in dulbecco's modified eagle's medium (dmem) (gibco, usa) with 10% fetal bovine serum (lonsera, uruguay), penicillin (250 u/ml), and streptomycin (250 ug/ml). the cells were incubated at 37 °c in a humidified incubator with 5% co 2 . pedv ms (genbank accession no. mt683617), yz (genbank accession no. mk841495.1), sh (genbank accession no. mk841494.1) [18] , and cv777 (genbank accession no. af353511.1) strains were maintained in our laboratory and passaged in vero cells with 2.5% trypsin. the ms, yz, and sh strains belong to variant strains; cv777 belongs to a classical strain. the ms strain was used for all experiments and is represented by "pedv" in this article. tgev js2012 (genbank accession no. kt696544.1) passaged in st cells, the highly pathogenic prrsv strain bb0907 (genbank accession no. hq315835.1) passaged in marc-145 cells, emcv nj08 (genbank accession no. hm641897), and sva ch-sd (genbank accession no. mh779611.1) passaged in bhk-21 cells, were maintained in our laboratory. tomatidine with purity > 99% was used for in vitro experiments (selleck chemicals, usa). a library (fda approved) of 911 natural products was purchased from selleck chemicals. these compounds were stored as 10 mm stock solutions in dmso at − 80 °c until use. the workflow for high-throughput screening (hts) of the library was carried out as shown in figure 1a and b. vero cells were seeded in 96-well plates at 5 × 10 4 cells per well. when approximately 90% confluent, the cells were treated with 10 μm compound or dmso (1μl, 0.2% v/v) for 1 h and then infected with pedv (0.01 moi) or mock infected for 1 h. the cells were then washed with pbs, then culture medium containing 10 μm compound was added back to each well. at 16 hpi, cpe and ifa were observed under a microscope. the fluorescence intensity of ifa was measured by imagej software. the percentage of inhibition of fluorescence intensity is calculated by drug treatment relative to dmso treatment. during primary screening, compounds were ruled out if they resulted in any visible cytotoxicity or demonstrated less than 50% reduction of cpe compared with the dmso control group. for the second round of screening, cell viability had to be 80% or greater, and the inhibition of pedv had to be 80% or greater as determined by ifa. the ic 50 (concentration of compounds was 0.5, 2.0, 4.0, 6.0, 8.0, and 10.0 μm) and cc 50 (concentration of compounds was 1.0, 5.0, 10.0, 25.0, 30.0, 50.0, and 100.0 μm) of each remaining candidate compound were calculated using log (inhibitor) vs. response-variable slope (four parameters) method by graphpad prism 6.0 software (graphpad software, ca, usa), and those that displayed dose-dependent inhibition of pedv and selectivity index (si, si = cc 50 /ic 50 ) over 10 were considered for further study. in addition, cell viability was tested using an enhanced cell counting kit-8 (cck-8) (beyotime, china) following the manufacturer's instructions. the cc 50 was calculated using graphpad prism 6.0 software. dmso was used as the negative control. figure 1 screening protocol for pedv inhibitors. a screening procedure. vero cells were treated with 10 μm compound for 1 h, then infected with pedv (0.01 moi) for 1 h. the cells were washed with pbs, then incubated in medium containing 10 μm compound for another 15 h. b screening process flowchart. the criteria for passing the primary screening were that the compound must have no apparent cytotoxicity and must reduce cpe by at least 50% compared with the dmso treatment. the criteria for passing the secondary screening were that the compound must leave cells at least 80% viable and inhibit pedv by more than 80%. compounds that passed the third screen inhibited pedv in a dose-dependent manner and had a selective index (si) higher than 10. c each dot represents the percent inhibition of each compound. the dots located above the dotted line indicate 80% or greater inhibition. d ifa of infected cells treated with one of the four designated compounds. pedv n-protein is colored green; a bright field shows cpe. e ic 50 and cc 50 curves of the four designated compounds. cell viability is calculated as a percentage of the viability in the cells treated with the compounds divided by that in the dmso-treated cells. the structure of each compound is inset. f sis of the four designated compounds. (see figure on next page.) wang et al. vet res (2020) the cells were lysed with 100 μl of ripa lysis buffer (beyotime, china) on ice for 15 min, then resolved by sds-page and transferred to a nitrocellulose membrane. after transfer, the membrane was incubated in blocking buffer (5% non-fat milk in pbst w/v) for 2 h at room temperature, washed three times with pbst, then probed with the following antibodies: anti-pedv n-protein ( the detection of mrna levels of tgev, prrsv, and sva was performed as described previously [13, 19, 20] . quantitative rt-pcr was performed using aceq ® qpcr sybr ® green master mix (vazyme, china). each reaction was performed in triplicate and results are expressed as mean ± standard deviation (sd). vero cells grown in 96-well plates were infected with tenfold serial dilutions of pedv samples in four replicates. after 1 h at 37 °c, the culture medium was replaced with fresh dmem. the plates were incubated for 48-72 h at 37 °c. st cells grown in 96-well plates were infected with tenfold serial dilutions of tgev samples in four replicates. after 1 h at 37 °c, the culture medium was replaced with fresh dmem. the plates were incubated for 48-72 h at 37 °c. marc-145 cells grown in 96-well plates were infected with tenfold serial dilutions of prrsv samples in four replicates. after 1 h at 37 °c, the culture medium was replaced with fresh dmem. the plates were incubated for 72-96 h at 37 °c. bhk cells grown in 96-well plates were infected with tenfold serial dilutions of emcv/sva samples in four replicates. after 1 h at 37 °c, the culture medium was replaced with fresh dmem. the plates were incubated for 48-72 h at 37 °c. virus titers are expressed as tcid 50 , calculated using the reed-muench method. tomatidine (10 μm) or dmso was incubated with pedv (0.01 moi) at 37 °c for 3 h and 5 h. a mixture of tomatidine (10 μm) or dmso and pedv was placed into vero cells seeded in 24-well plates. after incubation at 37 °c for an additional 1 h, the culture supernatants were replaced with fresh dmem and incubated for an additional 12 h ( figure 3a ). the cells were then washed with pbs, and the mrna levels of pedv n and gapdh in the cells were measured using qrt-pcr. vero cells were pretreated with tomatidine (10 μm) or dmso for 1 h at 37 °c, and then infected with pedv (0.01 moi) for the time indicated (15 min, 30 min, 1 h) at 4 °c ( figure 3b ). the cells were then washed with icecold pbs, and the mrna levels of pedv n and gapdh in the cells were measured using qrt-pcr. vero cells were infected with pedv (0.01 moi) at 4 °c for 1 h. the supernatant was replaced with dmem containing tomatidine (10 μm) or dmso, and then incubated at 37 °c for the time indicated (30 min, 1 h, 2 h) ( figure 3c ). the cells were washed with citrate buffer (ph 3) to remove non-internalized virus. the mrna levels of pedv n and gapdh in the cells were then measured using qrt-pcr. vero cells were incubated with pedv (0.01 moi) at 37 °c for 1 h and washed three times with pbs to remove free virus. at 4 hpi, the culture medium was replaced with fresh dmem containing tomatidine (10 μm) or dmso, and the cultures were incubated at 37 °c ( figure 3d ). the mrna levels of pedv n and gapdh in the samples collected at 6, 8, 10 hpi were measured using qrt-pcr. vero cells were infected with pedv (0.01 moi) for 1 h at 37 °c. the culture medium was then replaced with fresh dmem. at 10 hpi, the cells were washed three times with pbs and the culture medium was replaced with fresh dmem containing tomatidine (10 μm) or dmso. the cultures were incubated at 37 °c for 0.5, 1, and 2 h, at which time the supernatants were harvested ( figure 3e ). the mrna levels of pedv n in the released virus was quantified by absolute fluorescence quantification. the crystal structure of pedv nsp5 was obtained from the protein data bank (3clpro, pdb:4xfq). due to lack of crystal structure, the protein sequences of nsp3 (plp2), nsp12 (rdrp), nsp13 (ntp), nsp14 (exon), nsp15 (nendou), and nsp16 (2′-o-methyltransferase) were subjected to comparative homology modeling using swiss model, which has been widely used and cited by more than 470 articles, to generate a putative 3d model. swiss model performs the sequence alignments and searches the putative template protein to generate a 3d model for a query sequence. all the modeling parameters were set to default. the three-dimensional structure of tomatidine was obtained from pubchem (compound cid: 65576). the autodock 4.2 program was used for the docking of tomatidine to the active pocket of potential replication-relevant proteins. we generated a grid map around selected active site residues with grid point spacing of 0.375 å. the potential tomatidine-protein complex was generated by genetic algorithm using the default parameters. the estimated free energy of binding was ranked, and the top two complexes were employed for further md analysis. the docking results were visualized using pymol 2.3.2. the ligand bound to the gromos87/gromos96 force field generated by prodrg 2.5. simulations were conducted with the gromacs package using the gro-mos96 43a1 force field. the simple point charge 216 (spc 216) model of water was used to solvate the protein in a periodic dodecahedron box extending 10 å from the nearest protein atom. the solvated system was then neutralized with na+ and cl− ions, minimized by the steepest descent method (50 000 steps), and equilibrated with a 100-ps constant volume (nvt) simulation. the production runs were conducted in a constant pressure ensemble (npt). the temperature was set to 300 k and controlled with v-rescale. long-range electrostatic interactions were treated with the particle-mesh ewald (pme) method. the pressure was coupled to 1 bar using the parrinello-rahman method. all bond lengths were constrained with the linear constraint solver (lincs) algorithm. a cut-off of 14 å was used to calculate short-range van der waals and electrostatic interactions. the time step was 2 fs and the total simulation time was 10 ns. rmsd, distance, and the number of hydrogen bonds between tomatidine and active pockets of the proteins were analyzed to judge binding stability and convergence. the pet-32a-3clpro was transformed into e. coli strain bl21, and the cells were cultured at 37 °c in lb medium. when optical density at 600 nm (od600) reached 0.8, the culture was cooled to 27 °c and supplemented with 1 mm iptg. the cells were harvested after incubation at 27 °c for 7 h, resuspended in pbs and disrupted by ultrasonication. the supernatant was filtered and loaded onto ni-sepharose (ge healthcare, usa). finally, the histagged protein was eluted using a linear gradient between the binding buffer and elution buffer a (20 mm tris, ph 7.4, 500 mm nacl, and 250 mm imidazole). low concentration imidazole (50 mm) was used to wash impurities, and high concentration imidazole (250 mm) was used to elute targeted protein. the target protein was condensed and desalinated using amicon ultra-4 (30 kda, ge healthcare, usa). the proteins were analyzed by sds-page. all of the purification procedures were performed at 4 °c to avoid unexpected degradation. the fluorescence quenching assay was measured by a perkinelmer enspire multimode plate reader. the reaction medium (200 μl) contained 190 μl of 3clpro solution at the concentration of 1 μm and 10 μl of tomatidine with different final concentrations (0, 30, 60, 90, 120, and 150 μm). following incubation at room temperature for 15 min, the fluorescence spectra of 3clpro with the different concentrations of tomatidine were recorded in the wavelength range of 300-500 nm upon excitation at 280 nm. the stern-volmer equation was used to describe fluorescence quenching as follows: . in this equation, f 0 and f represent the fluorescence intensities in the absence and presence of tomatidine. τ 0 (10 −8 s) indicates the lifetime of the fluorophore without quencher. k q is the bimolecular quenching constant. [q] refers to the concentration of the quencher, and k sv is the stern-volmer quenching constant. hence, the above equation may be applied to determine k sv by linear regression of a plot of f 0 /f versus [q] . each measurement was repeated three times. all measurements were performed using the microcal itc200 calorimeter in an itc buffer (20 mm tris, ph 7.4, 500 mm nacl, ph 8.2) while stirring at 700 rpm. the stock solution of compound and 3clpro protein were diluted with the itc buffer to a compound concentration of 300 μm and a protein concentration of 10 μm before titrations. the final concentration of dmso in the reaction buffer was less than 2% of the total volume. protein solution was titrated using the compound. all titrations were performed using an initial injection of 0.4 μl followed by 19 identical injections of 2 μl with a duration of 4 s and an interval of 120 s between injections. the data were analyzed by microcal itc200 software. the last three data points were averaged and subtracted from each titration to account for the heat of dilution. each measurement was repeated three times. for pedv 3clpro eukaryotic and prokaryotic expression plasmids, the coding sequence for 3clpro was reverse transcribed and amplified from pedv ms strains. the resulting pcr products were assembled into pcaggs-flag and pet-32a. mutagenesis of pedv 3clpro constructs were carried out by overlap extension pcr using specific mutagenic primers. the plasmids were verified by sequencing and double enzyme digestion. for functional substrates of pedv 3clpro, a nucleotide sequence encoding for an 8-amino-acid stretch corresponding to the cleavage site of pedv 3clpro derived from the junction of the nsp5 and nsp6 genes (ygvnlq^sg) in the pedv genome was inserted into the coding sequence of gfp between amino acids g190 and d191. the first pcr product (n-gfp) was amplified with a reverse primer harboring the n-terminal of the cleavage site coding sequence (ygvnlq). the second pcr product (c-gfp) was amplified with a forward primer harboring the c-terminal of the cleavage site coding sequence (vnlqsg). the first and second pcr products were assembled using overlap pcr and cloned into pcaggs. vero cells cultured in 24-well plates were co-transfected with 250 ng/well of gfp nsp5/6 encoding plasmid, 250 ng of 3clpro expressing plasmid, or an empty vector. at 12 hpi, the culture supernatants were replaced with fresh dmem containing the indicated concentrations (10, 20, 30 μm) of tomatidine or dmso. at 24 hpi, the cells were harvested and the cleaved fragment of gfp nsp5/6 was visualized using western blot. the polypeptide substrate dabsyl-ynstlq↓aglrkm-e-edans was chemically synthesized by the genscript corporation. pedv 3cl protease was used at final concentrations of 0.5, 1, and 1.5 μm; and fret peptide was added to the protein in a black 96-well plate at final concentrations of 10 μm. prrsv gp5 protein expressed and purified at the same time with 3cl protease was used as a negative control at final concentrations of 10 μm. the mixtures were then further incubated at 37 °c for 20 min, and fluorescence was monitored at 340 nm excitation and 485 nm emissions every minute. obacunone was used as a control drug. it is an fdaapproved compound with molecular weight similar to tomatidine, but it has no antiviral activity on pedv (according the data from hts). tomatidine (25, 50 μm), obacunone (50 μm, negative control) or dmso were pre-incubated with 1 μm 3clpro for 20 min at 37 °c, and 10 μm fret peptide was added to the mixture in a black 96-well plate [21] . the mixtures were then further incubated at 37 °c for 20 min, and fluorescence was monitored at 340 nm excitation and 485 nm emissions every minute. the mock represents fret peptides with no protease. the relative fluorescence units (rfu) of the experimental group were obtained after subtracting fluorescence readings of the mock. the percentage of inhibition was calculated as follows: percentage of inhibition (%) = 100 × [1 − rlu of the tomatidine group (20-0 min)/rlu of the negative control group (20-0 min)] (20) . all procedures involving animals for this study were approved by and performed in accordance with the animal ethics committee and nanjing agricultural university animal experiment central guidelines, respectively. female 6-8 week-old balb/c mice were immunized with 50 μg of purified 3clpro protein in 0.2 ml, emulsified in the same amount of freund's complete adjuvant. two booster injections were administered with equal amounts of immunogen and freund's incomplete adjuvant at 3-week intervals. ten days after the last injection, antiserum was collected. pedv was cultured and passaged by 10 passages in ipec-j2 cells in the presence of tomatidine at an increasing concentration of 2-4-6 μm or in dmso. the total rna was extracted from the virus cultures, tomatidine-f10, dmso-f10, and f0. the 3clpro gene was amplified and sequenced by using the primers located at ~ 150 bp upstream and downstream of 3clpro. to assess the sensitivity of f10 viruses to tomatidine, ipec-j2 cells were inoculated with the virus (tomatidine-f10, dmso-f10, and f0) at 0.1 moi, and the culture medium was replaced with fresh dmem containing tomatidine (10 μm) or dmso at 4 hpi. the mrna levels of pedv n and gapdh in the samples collected at 16 hpi were measured using qrt-pcr. western blot, qpcr, and tcid 50 were used to examine tomatidine's inhibition of other swine disease viruses. three designated compound concentrations were added to the culture medium (final concentrations were 2.5, 5, and 10 μm). dmso was used as the negative control. statistical analysis was performed using graphpad prism6 software. results are expressed as mean ± standard deviation (sd). differences between groups were examined for statistical significance using one-way or two-way analysis of variance (anova). the asterisks in the figures indicate significant differences (*p < 0.05; **p < 0.01; ***p < 0.001; ns = not significant). vero cells were treated with 10 µm natural product compounds, and infected with pedv as detailed in the timeline ( figure 1a) . the results indicated that 57 (6.26%) compounds showed no apparent cytotoxicity, and reduced cytopathic effect (cpe) by 50% compared with dmso alone. these compounds were then subjected to a second round of screening ( figure 1b) . four of the compounds produced negligible cytotoxicity and inhibited pedv infection by over 80% as determined by indirect immunofluorescence assay (ifa) ( figure 1c and d). in addition, they inhibited pedv in a dose-dependent manner and had a selectivity index (si) greater than 10 (figure 1e and f) . tomatidine was chosen for further study as it had the highest si and the lowest price. to determine the dose range of tomatidine having anti-pedv activity, vero cells were treated with 2.5, 5, and 10 μm tomatidine for 1 h and then infected with pedv. median tissue culture infectious dose (tcid 50 ), western blot and qrt-pcr analysis showed that the virus titers, n-protein, and mrna levels decreased in a dosedependent manner (figure 2a-c) . at 16 h post-infection (hpi), ifa showed that the number of infected cells in the tomatidine-treated groups were obviously lower than those in the negative control ( figure 2d ). in addition, tomatidine had similar inhibitory effects on the different pedv variant strains tested (yz, ms, and sh), as well as on the classic cv777 strain ( figure 2e ). to understand the anti-viral activity of tomatidine in porcine cells, ipec-j2 cells were treated with 2.5, 5, and 10 μm tomatidine for 1 h and then infected with pedv (0.1 moi). tcid 50 and qrt-pcr analysis showed that the virus titers and orf7 mrna levels significantly decreased in a dose-dependent manner ( figures 2f, g) . meanwhile, the ipec-j2 cells indicated that the tomatidine displayed no cytotoxicity ( figure 2h ). to further explore the mechanism by which tomatidine inhibits pedv infection, we first tested whether tomatidine is directly virucidal and kills pedv particles. as shown in figure 3a , tomatidine treatment failed to directly inactivate pedv. next, the relative amount of pedv n mrna to gapdh was detected by qrt-pcr to determine the effect of tomatidine on pedv attachment, internalization, and replication. the results showed that tomatidine treatment prior to pedv infection did not significantly block virus attachment to vero cells, indicating that tomatidine does not inhibit pedv attachment to cells ( figure 3b ). in evaluating the effect of tomatidine on pedv internalization, as shown in figure 3c , we determined that tomatidine treatment accompanied by virus internalization did not significantly impede virus replication compared to treatment with dmso. we then examined the effect of tomatidine on pedv replication by adding tomatidine during the replication stage. as shown in figure 3d , tomatidine treatment decreased pedv n mrna levels by approximately tenfold compared to treatment with dmso, suggesting that tomatidine inhibits pedv replication. in addition, the virus release assay showed that there was no significant difference in pedv rna levels in the supernatants between the treatment with tomatidine and dmso ( figure 3e ). taken together, these results indicate that tomatidine inhibits pedv infection primarily by affecting viral replication. several replicative enzymes regulate virus replication [22] . considering that tomatidine significantly inhibited infection at the virus replication stage, we speculated that tomatidine may work directly with important viral replicative enzymes. to ascertain which replicative enzymes were targeted by tomatidine, potential binding sites were analyzed in detail using autodock to dock tomatidine into the pedv replicative enzyme structures ( figure 4a ). the estimated binding free energies were ranked as shown in figure 4b , and the top two complexes (binding energy = − 9.14 kj/mol, − 8.96 kj/mol) nsp5 and nsp16 were selected for further molecular dynamics (md) analysis. md utilizes newtonian physics to simulate atomic movements in a solvated system and is an accurate computational method for simulating protein-drug interactions [23] . to judge the findings of our docking model, a 10 ns molecular dynamic simulation was carried out for the assessment of receptor and ligand stability via root mean square deviation (rmsd), the distance and the number of hydrogen bonds between tomatidine and the active pockets of the proteins. in tomatidine-nsp5 (3clpro) simulation, the rmsd values of the backbone converged at less than 0.25 nm and remained stable starting at 7 ns. the higher and more volatility rmsd in tomatidine-nsp16 implied structure instability (figure 4c(i) ). distances between tomatidine and the active pocket of nsp5 are highly conserved and tight during simulation calculations, whereas those between tomatidine and nsp16 fluctuated wildly ( figure 4c(ii) ). we measured the number of hydrogen bonds during the md simulations to better capture the intermolecular polar interactions. no hydrogen bonds were detected between tomatidine and nsp16. however, tomatidine did form hydrogen bonds with the active pocket of 3clpro, which may contribute to the stability of the tomatidine-3clpro complex ( figure 4c(iii) ). this means that 3clpro is a more likely target of tomatidine than nsp16. in addition, the low binding energy of tomatidine to inactive 3cl (see figure on next page.) figure 4 in silico tomatidine targeted the active pocket of pedv 3cl protease. a docked conformations of tomatidine with pedv nsp3 plp2, nsp5 3clpro, nsp12 rdrp, nsp13 ntp, nsp14 exon, nsp15 nendo u, and nsp16 2′-o-methyltransferase. the compounds and proteins are represented as sticks and cartoons, respectively. the compounds are colored green. the proteins are colored according to their secondary structures (helix = blue, sheet = purple, loop = pink). the active sites of enzyme pockets are shown as a mesh. b the binding energy of the tomatidine-protein complex, calculated using autodock, is listed. c overall dynamic behaviors in the md simulations. (i) rmsd of backbones of nsp5 (red) and nsp16 (blue); (ii) distance between tomatidine and active pocket of nsp5 (red) and nsp16 (blue); (iii) number of hydrogen bond interactions of tomatidine with nsp5 (red) and nsp16 (blue). d docked conformations of tomatidine with pedv 3clpro or inactive 3clpro. the compounds and proteins are represented as sticks and cartoons, respectively. the compounds are colored green. the proteins are colored according to their secondary structures (helix = blue, sheet = purple, loop = pink). the active sites of enzyme pockets are shown as a mesh. e the binding energy of the tomatidine-protein complex, calculated using autodock, is listed. wang et al. vet res (2020) 51:136 protease in silico strengthens the possibility that 3clpro is indeed the target for tomatidine ( figure 4d and e) . to verify the binding of 3clpro and tomatidine, pedv 3clpro fused with his tag was expressed in soluble form using an e. coli expression system ( figure 5a(i) ). after ni-sepharose purification, a single clear target band indicated that the 3clpro obtained could be used for further experiments ( figure 5a (ii), (iii)). the purified recombinant 3clpro was then used for a fluorescence quenching assay, itc and fret. a fluorescence quenching assay was carried out to determine the interaction between 3clpro and tomatidine. the results showed that the fluorescence emission intensity of 3clpro decreased distinctly in a dose-dependent manner with increasing concentration of tomatidine ( figure 5b upper) . the stern-volmer plot is shown in figure 5b lower; the kq value was calculated to be 2.7 × 10 11 l·mol −1 s −1 . the maximum scatter collision quenching constant of the various quenchers is 2 × 10 10 l·mol −1 s −1 . this indicates that the quenching mechanism was static and the interaction between 3clpro and tomatidine was strong. the binding affinities of tomatidine with pedv 3clpro were measured by itc, which is widely used to determine thermodynamic parameters of protein-ligand interactions such as the dissociation constant (kd) and binding stoichiometry (n). the results showed that tomatidine directly interacted with pedv 3clpro with a kd of 2.78 μm and n of 0.723 sites. this result further supports the premise that pedv 3clpro is the target protein of tomatidine. these results allowed us to speculate that tomatidine may block the activity of the 3clpro, thereby inhibiting pedv replication. to explore the impact of tomatidine on 3clpro activity, we constructed plasmids expressing nsp5 (3clpro), a series of inactive 3clpro mutants (h41a, c144a, h41/ c144a), and gfp nsp5/6 containing the nsp5/nsp6 junction (ygvnlq^sg) of pedv. after the vero cells were co-transfected with these plasmids, western blot assay showed that cleaved fragments of gfp nsp5/6 were detected in the presence of 3clpro, but not in the presence of 3clpro mutants h41a, c144a, and h41/c144a, indicating that pedv 3clpro was active ( figure 6a ). when vero cells were treated with increasing concentrations of tomatidine, the cleaved fragments clearly decreased in a dose-dependent manner ( figure 6b ), indicating that tomatidine inhibited the activity of pedv 3cl protease. we also used fret to confirm the effect of tomatidine on 3clpro activity. the dose-dependent increase of fluorescence intensity shows that the 3clpro obtained possessed catalytic activity ( figure 6c ). moreover, compared with obacunone, tomatidine significantly inhibited the activity of pedv 3clpro ( figure 6d ). the percentage of inhibition of 50 μm and 25 μm tomatidine was 75.5% ± 2.3% and 47.1% ± 3.8% respectively, at 20 min incubation. each reaction was performed in triplicate and the results are expressed as mean ± standard deviation (sd). in order to explore the inhibition of viral 3clpro activity of tomatidine in pedv-infected cells, anti-3clpro serum antibody was prepared by vaccinating mice with the purified recombinant 3clpro ( figure 6e, lines 1-2) . vero cells were inoculated with pedv (0.01 and 0.02 moi) and 6 μm tomatidine. western blot showed that the partial virus polyprotein (nsp5-6 complex, rather than 3clpro) was detected with the anti-3clpro serum antibody at 16 hpi, as previously described [24] . the ratios of nsp5-6 complex/n in pedv not treated with tomatidine were similar to each other between the two doses of 0.01 and 0.02 moi. but the ratios of nsp5-6 complex/n in pedv treated with tomatidine were significantly higher than those treated with dmso ( figure 6e , line 3-7), indicating that tomatidine may block the 3cl protease activity in a natural situation. tomatidine strongly inhibits virus particle production of dengue virus and chikungunya virus [25, 26] . to investigate whether tomatidine has an antiviral effect against other swine disease viruses, we selected several other viruses including a coronavirus, tgev, [27], an arterivirus, prrsv [28] , and picornaviruses emcv and sva [20, 29, 30] . st cells were treated with 2.5, 5, and 10 μm tomatidine for 1 h and then infected with tgev for 24 h. the results indicated that tgev proliferation was significantly suppressed by tomatidine and almost completely cut off at the concentration of 10 μm ( figure 7a-c) . marc-145 cells were treated with the indicated concentrations of tomatidine for 1 h and infected with prrsv for 48 h. these results indicated that tomatidine exhibits antiviral activity against prrsv ( figure 7d -f). bhk-21 cells were treated with the indicated concentrations of tomatidine for 1 h and then infected with emcv or sva for 18 h. western blot, qpcr, and tcid 50 analysis revealed substantial inhibition activity of tomatidine against emcv and sva (figures 7g-i, and j-l). none of the various cells treated with the indicated concentrations of tomatidine showed any cytotoxicity ( figure 7m ). outbreaks of porcine epidemic diarrhea cause significant lethality rates in neonatal piglets, creating a heavy economic burden in the global swine industry. unfortunately, available commercial vaccines fail to protect against the high virulence of pedv variants [31, 32] . because there is no antiviral drug available to treat this disease, we need to develop new therapeutic methods to prevent and control pedv. natural compounds and compositions have been a rich source of drugs against many viral infections. for example, griffithsin, a highmannose-specific lectin, has been shown to reduce pedv infection in vero cells by preventing viral attachment and disrupting cell-to-cell transmission [14] . prenylated phenolic compounds from the leaves of sabia limoniacea figure 6 tomatidine directly inhibited the activity of pedv 3cl protease. a vero cells cultured in 24-well plates were co-transfected with 250 ng of gfp nsp5/6 encoding plasmid, 250 ng of 3clpro or inactive mutants expressing plasmid or empty vector. at 24 hpi, the cells were harvested and the cleaved fragment of gfp nsp5/6 was visualized using western blot. b vero cells cultured in 24-well plates were co-transfected with 250 ng of gfp nsp5/6 encoding plasmid, 250 ng of 3clpro expressing plasmid or empty vector. at 12 hpi, the culture supernatants were replaced with fresh dmem containing the indicated concentrations (10, 20, and 30 μm) of tomatidine or dmso. at 24 hpi, the cells were harvested and the cleaved fragment of gfp nsp5/6 was visualized using western blot. c pedv 3clpro was used at final concentrations of 0.5, 1, and 1.5 μm; and 10 μm (3clpro substrate) was added to the protein in a black 96-well plate. prrsv gp5 protein was used as a negative control. the mixtures were then further incubated at 37 °c for 20 min, and fluorescence was monitored at 340 nm excitation and 485 nm emissions every minute. the rfu were calculated by subtracting the mock from the fluorescence readings to eliminate the effect of background signals. d tomatidine (25, 50 μm) or obacunone (50 μm, negative control) or dmso was pre-incubated with 1 μm protease for 20 min at 37 °c, and 10 μm (3clpro substrate) was added to the mixture in a black 96-well plate. the mixtures were then further incubated at 37 °c for 20 min, and fluorescence intensity was monitored at 340 nm excitation and 485 nm emissions every minute. rfu were calculated as above. e vero cells were inoculated with pedv (0.01 and 0.02 moi) and 6 μm tomatidine. cells were transfected with 250 ng pcaggs-3clpro as 3clpro positive control. after incubation for 16 h, the cellular proteins were collected and the virus polyprotein was detected with western blot using anti-3clpro mouse antibody, as previously described (20) . meanwhile, pedv n-protein, gapdh, and 3clpro-flag were detected with western blot using molecular antibodies against pedv n-protein, gapdh, and flag. the ratio of nsp5-6 complex/n protein levels was depicted by integrated density analysis. the arrow indicates the location of nsp5-6 complex. all results are mean ± sd from three independent experiments performed in triplicate. exhibit promising antiviral activities against pedv replication [16] . but the mechanisms involved in these studies are not well understood. in this study, tomatidine, screened from a library of 911 natural products, was shown to inhibit pedv proliferation in vitro. tomatidine displayed remarkable inhibition of pedv replication by targeting 3cl protease, which we investigated using molecular docking and md analysis, fluorescence spectroscopy, itc, 3cl protease activity, and fret assays. the pedv life cycle is composed of four stages: attachment, entry, replication, and release [33] . our results indicate that tomatidine significantly inhibits viral infection at the replication stage. several nonstructure proteins are key to viral replication, hence we speculated that tomatidine, which is shown to inhibit viral replication, may target a non-structural protein. making use of molecular docking and molecular dynamics, we speculated that tomatidine may inhibit pedv proliferation by directly targeting 3cl protease. fluorescence quenching is an important technique for measuring binding affinity between ligands and proteins. itc is a technique used in a wide variety of quantitative studies of biomolecular interactions. itc works by directly measuring the heat that is either released or absorbed during a biomolecular binding event. in our study, 3clpro-tomatidine binding was indicated by fluorescence quenching and itc. in order to confirm the effect of tomatidine on 3clpro activity, we constructed a plasmid expressing gfp nsp5/6 protein containing the nsp5/nsp6 junction (ygvnlq^sg) of pedv. our results show that gfp nsp5/6 was cleaved into two fragments by 3clpro, indicating that pedv 3clpro was active. tomatidine inhibited the activity of pedv 3cl protease in vero cells in a dose-dependent manner. the inhibition ratio of tomatidine was also shown in a fret assay. the 485 nm fluorescence intensity remained stable until the labeled substrate was fractured by the protease, which indicates that the purified protein possesses 3cl protease activity. compared with the control drug obacunone, tomatidine significantly reduced the fluorescence intensity. the ratio of nsp5-6 complex/n in virus treated with tomatidine was significantly higher than in the dmso treated virus, indicating that tomatidine blocked the 3cl protease activity in a natural situation. however, anti-3clpro serum raised in mice detected nsp5-6 complex rather than 3clpro (nsp5) only. this phenomenon underlines the possibility that there is a quantitative difference between nsp5-6 complex and nsp5 of products in pedv-infected cells at 16 hpi. the 3c-like protease, which is responsible for processing polyproteins of nidoviruses and picornaviruses, is an attractive target for drug therapy. it has been reported that a series of compounds such as flavonoids, chalcones, and benzothiazolium display significant antivirus activity by inhibiting 3clpro activity. we found that tomatidine strongly inhibits pedv replication by targeting 3cl protease. tomatidine has many cell biological properties. it enhances expression of nuclear factor erythroid 2-related factor 2 (nrf2) [34] , which when knocked out benefits viral propagation [19] . 5′-amp-activated protein kinase catalytic subunit alpha-2 (ampk) promotes innate immunity and antiviral defense through modulation of sting signaling [35] . tomatidine also stimulated ampk phosphorylation by activating the camkkβ pathway [36] . tomatidine inhibits dengue virus particle production partly by inhibiting cyclic amp-dependent transcription factor atf-4 (atf4) [26] . in this study, we noted that the ic 50 (50% inhibitory concentration) of tomatidine against pedv in cells was low micromolar (3.4 μm); the cc 50 (50% cytotoxic concentration) was about 45.68 μm; while the inhibition of pedv 3clpro by tomatidine in the fret assay at 50 μm was only 75%. in addition, the binding energy of tomatidine with the viral nsp16 was close to that of the 3clpro. these results suggest that there may be other antiviral mechanisms involved. 3clpro of pedv, as well as of other coronaviruses (porcine deltacoronavirus), picornaviruses (hepatitis a virus, foot and mouth disease virus), and arterivirus (prrsv), has been shown to inhibit production of infβ, hijacking the host's innate antiviral immune response [37] [38] [39] [40] . this effect has been attributed to the direct cleavage of nf-kappa-b essential modulator (nemo) figure 7 tomatidine shows broad-spectrum antiviral activity against other swine disease viruses. tcid 50 , western blot, and qpcr were used to examine the inhibition activity of tomatidine against other swine disease viruses. three designated concentrations of compounds were added to the culture medium (final concentrations were 2.5, 5, 10 μm, with no-observable cytotoxicity). dmso was used as the negative control. tgev (0.01 moi), prrsv (e), and emcv (h)/sva (k) were then used to infect st, marc-145, and bhk-21 cells, respectively, and samples were harvested at 24 h, 48 h, and 18 h. the tcid 50 of tgev (a), prrsv (d), emcv (g), and sva (j) treated with 10 μm tomatidine or dmso were calculated using the reed-muench method. the n-protein level of tgev (b), prrsv (e), emcv (h), and sva (k) were determined by western blot. relative tgev n (c), prrsv n (f), emcv vp1 (i), and sva vp1 (l) mrna levels, was determined by qrt-pcr, and expressed relative to that in dmso-treated cells. m viability of st, marc-145, and bhk-21 cells pretreated with the indicated concentrations of tomatidine and incubated for 24 h, 48 h, and 18 h, respectively in medium containing tomatidine. the results are from one of three independent experiments. the internal loading control was β-actin. error bars represent the sd. the asterisks in the figures indicate significant differences (*p < 0.05; **p < 0.01; ***p < 0.001; ns = not significant). (see figure on next page.) (an important substrate in the rlr cascade) [41, 42] . by blocking 3clpro, tomatidine may also prevent 3clpro cleaving nemo, thereby enhancing the innate antiviral immunity, which could in turn help to inhibit virus replication. tgev, prrsv, emcv, and sva contain 3c or 3cl protease. our results showed that tomatidine effectively inhibits tgev, prrsv, emcv, and sva replication in vitro. but the antiviral activity of tomatidine against picornaviridae looks more significant than tgev and prrsv. it may be related to the different characterization of the virus replication. the molecular docking analysis demonstrated that the binding energy between the 3c or 3cl protease of the viruses with tomatidine were weak and no hydrogen bonds were detected, which being different from that of pedv 3clpro (additional file 1). it indicates that the antiviral activity of tomatidine is likely to rely on other pathways rather than only targeting 3c proteases, which is worthy being further explored. tomatidine has been proved to be safe within a certain dosage in mice. unfortunately, as a dietary supplement approved by the fda, tomatidine lacks detailed safety and pharmacokinetics evaluation in pigs. the safety, pharmacokinetics, and efficacy of tomatidine for prevention, treatment, and control of pedv and perhaps other porcine nidoviruses need to be further evaluated in pigs. it would be interesting to test whether its 3clpro blocking activity also holds for nidoviruses infecting humans. we conclude that tomatidine effectively inhibits pedv proliferation via inhibition of 3clpro activity. in addition, tomatidine displays antiviral activity against tgev, prrsv, emcv, and sva. these findings offer novel and promising therapeutic possibilities for fighting infections caused by these viruses. supplementary information accompanies this paper at https ://doi. org/10.1186/s1356 7-020-00865 -y. additional file 1. the binding energy of tomatidine to 3cl or 3c protease of tgev, prrsv, emcv, and sva in silico. a docked conformations of tomatidine with 3cl or 3c protease of tgev, prrsv, emcv, and sva in silico. the compounds and proteins are represented as sticks and cartoons, respectively. the compounds are colored green. the proteins are colored according to their secondary structures (helix = blue, sheet = purple, loop = pink). the active sites of enzyme pockets are shown as a mesh. b the binding energy of the tomatidine-protein complex, calculated using autodock, is listed. porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains characterization of a novel coronavirus associated with severe acute respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study structural basis for the recognition of 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processing of the arterivirus replicase orf1a polyprotein: evidence that nsp2 acts as a cofactor for the nsp4 serine protease tomatidine, a natural steroidal alkaloid shows antiviral activity towards chikungunya virus in vitro efffects of electrostatic and hydrophobic interaction on the stability of the tgev main proteinase dimer structure and cleavage specificity of the chymotrypsinlike serine protease (3clsp/nsp4) of porcine reproductive and respiratory syndrome virus (prrsv) encephalomyocarditis virus 3c protease attenuates type i interferon production through disrupting the tank-tbk1-ikkepsilon-irf3 complex seneca valley virus 3c(pro) abrogates the irf3-and irf7-mediated innate immune response by degrading irf3 and irf7 two novel porcine epidemic diarrhea virus (pedv) recombinants from a natural recombinant and distinct subtypes of pedv variants genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from henan, china coronaviruses: an overview of their replication and pathogenesis tomatidine enhances lifespan and healthspan in c elegans through mitophagy induction via the skn-1/nrf2 pathway amp-activated kinase (ampk) promotes innate immunity and antiviral defense through modulation of stimulator of interferon genes (sting) signaling tomatidine reduces palmitate-induced lipid accumulation by activating ampk via vitamin d receptor-mediated signaling in human hepg2 hepatocytes aspartic acid at residue 185 modulates the capacity of hp-prrsv nsp4 to antagonize ifn-i expression porcine deltacoronavirus nsp5 antagonizes type i interferon signaling by cleaving stat2 hepatitis a virus 3c protease cleaves nemo to impair induction of beta interferon foot-andmouth disease virus antagonizes nod2-mediated antiviral effects by inhibiting nod2 protein expression arterivirus nsp4 antagonizes interferon beta production by proteolytically cleaving nemo at multiple sites porcine epidemic diarrhea virus 3c-like protease regulates its interferon antagonism by cleaving nemo publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors wish to thank miss. huixin zhu for her generous help with the animal experiment. in addition, the critical and helpful comments from the reviewers are highly appreciated. conception of the work: pj and pw; cellular and animal experiment: pw, jb, xl and mw; analysis and interpretation of data: pj, xw and pw; preparation of the manuscript: pj and pw. all authors read and approved the final manuscript. not applicable. all animals were housed in the animal facility of nanjing agricultural university (nau), nanjing, jiangsu, china. all experimental protocols for animals were conducted following the national guidelines for housing and care of laboratory animals (china) and performed in accordance with nau institutional regulations after approval by the institutional animal care and ethics committee of nau (syxk(su)2017-0007). not applicable. none of the authors have any possible conflicts of interest. key: cord-305859-vt8vwo3y authors: jung, kwonil; hu, hui; saif, linda j. title: calves are susceptible to infection with the newly emerged porcine deltacoronavirus, but not with the swine enteric alphacoronavirus, porcine epidemic diarrhea virus date: 2017-04-03 journal: arch virol doi: 10.1007/s00705-017-3351-z sha: doc_id: 305859 cord_uid: vt8vwo3y fecal virus shedding, seroconversion and histopathology were evaluated in 3-7-year-old gnotobiotic calves orally inoculated with porcine deltacoronavirus (pdcov) (9.0-9.6 log(10) genomic equivalents [ge] of oh-fd22-p5; n=4) or porcine epidemic diarrhea virus (pedv) (10.2-12.5 log(10) ge of pc21a; n=3). in pdcov-inoculated calves, an acute but persisting fecal viral rna shedding and pdcov-specific serum igg antibody responses were observed, but without lesions or clinical disease. however, no fecal shedding, seroconversion, histological lesions, and clinical disease were detected in pedv-inoculated calves. our data indicate that calves are susceptible to infection by the newly emerged pdcov, but not by the swine coronavirus, pedv. coronaviruses (covs) are enveloped, single-stranded rna viruses of positive-sense polarity. their genomes range from approximately 26 to 32 kb in size [16] . the family coronaviridae of the order nidovirales is divided into the four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. bats are the projected host reservoir for alphacoronaviruses and betacoronaviruses, while birds are thought to be the host for gammacoronaviruses and deltacoronaviruses [18] . the two betacoronaviruses, severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov), were transmitted by civet cats and camels, respectively, to humans, but they have shown limited capacity for adaptation to humans [16] . some covs can be transmitted to different animal species, and subsequently adapt to and be maintained in the new host, because they can exploit or share a variety of host cell surface molecules or other undefined factors [16] . the species porcine epidemic diarrhea virus belongs to the genus alphacoronavirus. porcine epidemic diarrhea virus (pedv) causes acute diarrhea, dehydration and high mortality in neonatal piglets [9] . for the last four decades, since the first appearance of pedv in 1977, pedv infection has resulted in significant economic losses in the european, asian and us swine industries. pedv has been found only within the pig population, indicating that the virus might have adapted only to pigs [9] . the species porcine deltacoronavirus belongs to the genus deltacoronavirus. porcine deltavoronavirus (pdcov) is a novel enteropathogenic cov infecting pigs and was previously reported in birds [10] . pdcov was first identified in pigs in hong kong in 2012 [18] and the associated enteric disease was first reported in us swine only in early 2014 [17] . however, the origin of pdcov infection in pigs and also the sudden emergence and route of introduction of this virus in the us remains unclear [10] . pdcov may have only partially adapted to pigs and still retain some potential to infect different animal species, particularly poultry or other livestock, which can often have frequent contact with pigs in small-scale, backyard farms in the us. therefore, our aim was to determine whether calves are susceptible to infection with either the newly emerged pdcov or the swine enteric cov, pedv. fecal virus shedding, seroconversion and histopathology were evaluated in gnotobiotic (gn) calves orally inoculated with pdcov or pedv. the pdcov oh-fd22 virus was isolated and then serially passaged five times (p5) in llc porcine kidney (llc-pk) cells (atcc cl-101) [2] . the virus was orally inoculated and propagated in a 9-day-old gn pig. the viral rna titer of oh-fd22-p5 used as inoculum in the intestinal contents (ics) was 9.0 log 10 genomic equivalents (ge)/ml. the wild-type us pedv strain pc21a, propagated in a gn pig [7] , was also used in this study. all ics were negative for other enteric viruses, such as rotavirus groups a-c, by pcr/rt-pcr [6] . near-term angus 9 jersey crossbred gn calves were delivered aseptically by caesarean section [5] . eight 3-to 7-day-old calves were randomly assigned to three groups: pdcov infection (n=4; calves #1-4), pedv infection (n=3; calves #5-7), and mock (minimum essential medium [mem]; n=1; calf #8, 3 days of age) ( table 1 ). calves #1-4 were inoculated orally with 9.0-9.6 log 10 ge of the oh-fd22-p5, and calves #5-7 were inoculated orally with 10.2-12.5 log 10 ge of the pc21a (table 1) . after viral inoculation, we monitored clinical signs daily. diarrhea was assessed by scoring fecal consistency as follows: 0=solid; 1=pasty; 2=semi-liquid; 3=liquid, with scores of 2 or more considered diarrheic. calves #1 (pdcov) and #5 (pedv) were monitored for long-term clinical signs and virus shedding until post-inoculation day (pid) 16-17. the other inoculated or mock-infected calves were kept for short-term studies and were euthanized for histopathological examination at acute to mild stages (pids 3, 8 or 9) of viral infection (table 1) . rectal and nasal swabs or serum samples were collected and prepared as described previously [4, 6] . rectal and nasal swabs were diluted 1:10 and 1:50, respectively, in mem. virus rna was extracted using the mag-max viral rna isolation kit (applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. titers of virus shed in feces were determined by qrt-pcr using the onestep rt-pcr kit (qiagen, valencia, ca, usa) [6, 8] . the detection limit of qrt-pcr for pdcov was 10 ge per reaction, corresponding to 5.3, 4.6, and 3.6 log 10 ge/ml of pdcov in nasal, rectal swab, and serum samples, respectively [8] . the detection limit of qrt-pcr for pedv was 10 ge per reaction, corresponding to 4.8 log 10 ge/ml of pedv in rectal swab samples [6] . pdcov oh-fd22-p5 (passage five) and pedv pc22-p40 (passage 40) viruses, grown on llc-pk or vero cells, respectively, were used to infect llc-pk or vero cells, respectively, in 96-well plates. the viral antigens expressed were detected by indirect immunofluorescence assay (ifa) [2, 3, 12] . a multiplicity of infection of 0.1 was used for each viral inoculation in llc-pk and vero cells treated with 5 and 10 lg/ml of trypsin, respectively [2, 3, 12] . the cell culture conditions used to infect llc-pk or vero cells with each virus were described previously [2, 3, 12] . characteristic cytopathic effects (cpe) [2, 3, 12] were monitored regularly in inoculated llc-pk or vero cells. when cpe was pronounced, pdcov-inoculated llc-pk (pid 1) and pedv-inoculated vero cells (pid 2) were fixed with 100% ethanol at 4°c overnight for ifa. the fixed cells were washed with 0.01 m phosphate buffered saline (pbs) (ph 7.4). blocking was performed with 19 buffered solution of casein (109 power block tm universal blocking reagent, biogenx, ca, usa) in distilled water for 1 hr at room temperature. four-fold serial dilutions, starting at 1:4, of the paired serum samples of calves #1 and #5, obtained at pid 0 and pid 16-17 were added to the wells and the plates were incubated overnight. swine hyperimmune antiserum against pdcov (1:100) or monoclonal antibody against the spike (s) protein of pedv (6c8-1) (1:200), with the appropriate detection antibodies, were added to the wells as ifa positive controls [2, 3, 12] . goat polyclonal antibodies specific for bovine, swine or mouse igg (whole igg) conjugated to fluorescein (kpl, gaithersburg, md, usa) were diluted 1:100 in 0.01m pbs. the plates were incubated for 1 hr at 37°c. the stained plates were evaluated using a fluorescence microscope. the antibody titers were expressed as the reciprocal of the highest serum dilution in the wells that were positive for pdcov or pedv antigen detection. formalin-fixed or frozen small (duodenum, jejunum and ileum) and large (cecum/colon) intestinal tissues and other major organs were examined macroscopically and histologically and tested by if for pdcov or pedv antigens [5, 6, 8] . tissues from the negative control calf (#8) were tested for histological comparisons and as an if negative control. tissues from pdcov-or pedv-infected gn pigs at pid 3 were also tested as positive controls [6, 8] . clinical observations revealed that none of the gn calves inoculated with pdcov (calves #1-4), pedv (calves #5-7), or mock (calf #8) exhibited diarrhea or other clinical signs throughout the course of the experiment (fig. 1) . pdcov rna was first detected in fecal samples at pid 2 (calves #1 and 2) or pid 3 (calves #3 and 4) by qrt-pcr (table 1) . fecal pdcov rna titers, ranging from 7.2 to 8.4 log 10 ge/ml, peaked at pid 2 (calf #2), pid 3 (calf #1), or pid 4-6 (calf #3) ( table 1) . calf #1, was followedup long-term and had prolonged viral rna shedding until pid 16 (fig. 1) . the highest fecal pdcov rna titer of calf #1 was at pid 3 (8.4 log 10 ge/ml), and the titers decreased progressively thereafter (fig. 1) . on the other hand, none of the gn calves inoculated with pedv shed detectable pedv rna in the feces at pids 1-9 (calves #6 and #7) and pids 1-17 (calf #5). the negative control (calf #8) did not shed detectable pdcov or pedv rna in the feces during the experiment. nasal swab or serum samples collected from pdcov-inoculated calf #1 were also tested by qrt-pcr. serum samples were collected at pids 3 and 15, and nasal swab samples were collected at pids 2, 6, 9, 13, and 16. no pdcov rna was detected in the serum (\3.6 log 10 ge/ml) and nasal swab (\5.3 log 10 ge/ml) samples tested at the time-points indicated. ifa showed that calf #1 had serum igg antibodies (titer of 1,024) against pdcov oh-fd22 at pid 16, indicating seroconversion. large numbers of if-stained cells were consistently observed when pdcov-infected llc-pk cells were incubated with the serum diluted 1:4 to 1:1,024 (fig. 2) . no pdcov-specific igg antibodies were detected in the prebled serum samples of calf #1 and negative control calf #8 (fig. 2) . in pedv-inoculated calf #5, there were no detectable pedv-specific igg antibodies in the serum samples at pid 0 and pid 17 (fig. 2) . light microscopy analysis revealed that none of the pdcov-or pedv-inoculated calves had major histological changes in the intestine. additionally, by if, no pdcov or pedv antigen-positive cells were observed in the small and large intestinal tissues of pdcov or pedv-inoculated gn calves, while the intestinal tissues (mainly villous enterocytes) of pdcov or pedvinfected gn pigs were found to be positive for pdcov or pedv antigen in earlier studies [6, 8] . our study demonstrated that gn calves orally inoculated with the pdcov strain oh-fd22 (ics of cell-culture grown pdcov from infected gn pigs) develop an acute infection with persistent fecal pdcov rna shedding and pdcovspecific serum igg antibody responses, but show no signs of significant intestinal lesions or clinical disease [8] . these observations are similar to a previous report showing that, human sars-cov strain urbani, prior to its adaptation to mice (ma105 strain), replicated in the lungs of young mice, as indicated by their lung homogenates testing positive by qrt-pcr. however, the urbani strain-inoculated mice did not show any clinical signs, but only mild histological lesions with low expression of viral antigens [13] . in our study, there were no detectable fecal viral rna shedding, virus-specific serum igg antibody responses, histological lesions, and clinical disease in gn calves orally inoculated with the pedv strain pc21a (ics of wild-type pedv-infected gn pigs) [6] . for the past 40 years, pedv has been found only within pigs, indicating that pedv may have only adapted to pigs [9] . in contrast, pdcov may not yet be completely adapted to pigs, and the disease caused by pdcov has emerged only 2-3 years ago. pdcov-infected pigs appeared to shed less pdcov rna and had a lower peak of viral titer in the feces, compared with experimental pedv infections [7, 11] , suggesting that there is a lower replication of pdcov in the intestine of pigs, possibly due to its incomplete adaptation to pigs. in a molecular surveillance study in china and hong kong conducted in 2007-2011, deltacoronaviruses (dcovs) were detected only in pigs and wild birds [18] . however, dcovs were detected previously in rectal swabs of small mammals, such as asian leopard cats and chinese ferret badgers, in live-animal markets in china, in 2005-2006 [1] . the helicase and s genes of the covs isolated from the wild small mammals were closely related to those of pdcov [18] , suggesting that a potential interspecies transmission event may have occurred leading to dcov transmission between wild small mammals and pigs. however, further studies are needed to define the potential role of small mammals as an intermediate host of pdcov and the mechanisms of interspecies transmission of dcovs between small mammals and domestic pigs or wild birds. our data also support the fig. 1 persisting fecal viral rna shedding of calf #1 inoculated with pdcov strain oh-fd22-p5, but without diarrhea. calf #1 was inoculated orally with 9.6 log 10 ge of the gnotobiotic pig-passaged oh-fd22-p5. it was monitored for long-term clinical signs and virus shedding at pids 1 to 16. rectal swabs were collected daily throughout the experiment. the pdcov fecal shedding titers were determined by qrt-pcr. the dotted line indicates the detection limit (4.6 log 10 ge/ml) of the qrt-pcr potential ability of the newly emerged pdcov to infect another animal species, such as cattle [16] . however, our study did not demonstrate pdcov's ability of direct interspecies transmission between pigs and cattle. the s protein of covs is critical for regulating interactions with specific host cell receptor glycoproteins to mediate viral entry [15] . based on our data, it is possible that interactions between pdcov s protein and the host cell receptor or co-receptor binding may occur in the bovine intestine, whereas this may not be the case for pedv s protein. the major cellular receptors of pedv and pdcov in pigs are unknown [10, 14] . the cov s proteins can be divided into two functional subunits, s1 and s2. the s1 is involved in the receptor recognition and binding via the n-terminal or c-terminal virus receptor binding domains. therefore, proteolysis between s1 and s2 is a critical determinant for cov tropism and pathogenesis. the cov s proteins can be cleaved by different animal proteases [15] . the extent of cleavage of pedv and pdcov s proteins might differ in the bovine intestine, possibly depending on their adaptation to the bovine intestinal environment; for example, exogenous proteolytic enzymes of bovine origin may influence pedv or pdcov s proteinhost cell receptor binding. however, these assumptions should be verified by further studies. in our study, pdcov-inoculated calves showed acute fecal viral rna shedding, followed by progressively decreased titers thereafter. there were also no pdcov rnapositive nasal or serum samples from pdcov-inoculated calf #1, indicating that pdcov infection may be limited to the intestine, similar to pdcov infections in pigs [10] , although pdcov-infected pigs also showed viremia (viral rna in serum) [10] . our study did not identify which type of cells in the intestines of inoculated calves could be the target for pdcov replication, resulting in fecal shedding. our if staining experiments identified pdcov antigen-positive cells in intestinal tissues from pdcov-infected gn pigs used as positive controls. similar to the giii.2 bovine norovirus (cv186-oh strain) that was shown to cause fecal virus rna shedding in infected gn calves, but in the absence of histological lesions or viral antigen/rna in the intestine [5] , pdcov did not induce major histological changes in the intestine of gn calves, such as necrosis of intestinal epithelium or villous atrophy. this result is also in line with data showing no pdcov-positive cells in the intestinal epithelium. based on these observations, pdcov may have infected the intestine of calves as an attenuated virus that does not cause clinical disease and histological lesions, with low levels of viral antigen detected in enterocytes in inoculated animals. nevertheless, our study did not clearly rule out an abortive infection with limited pdcov replication in calves. in addition, interestingly, no pedv-specific igg antibodies were detected by ifa in the serum of pedv-inoculated calf #5 at pid 16. however, a virus neutralization (vn) assay using a pedv strain iowa106 [3, 12] , showed low pedv neutralizing activity in the sera of calf #5 at pid 16 and prebled prior to pedv inoculation, with vn titers of 4 2.8 (pid 16) and 4 2.5 (prebled). additional studies using larger numbers of serum samples are needed to confirm whether bovine sera have pedv antibodies or neutralizing substances. collectively, our data indicate that calves are susceptible to infection with the newly emerged pdcov, but not with the swine enteric cov, pedv. however, the infectivity of pdcov in calves was limited, as manifested by no clinical disease and histological lesions, possibly due to its incomplete adaptation to the bovine intestine. other animal species with frequent contacts with pigs, such as poultry, also need to be monitored for potential infectivity by pdcov. detection of a novel and highly divergent coronavirus from asian leopard cats and chinese ferret badgers in southern china isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the united states experimental infection of gnotobiotic pigs with the cell-culture-adapted porcine deltacoronavirus strain oh-fd22 altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus pathogenesis of giii.2 bovine norovirus, cv186-oh/00/us strain in gnotobiotic calves pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs comparative pathogenesis of us porcine epidemic diarrhea virus (pedv) strain pc21a in conventional 9-day-old nursing piglets vs. 26-day-old weaned pigs pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis porcine deltacoronavirus infection: etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis characterization of porcine epidemic diarrhea virus isolate us/iowa/18984/2013 infection in 1-day-old cesarean-derived colostrum-deprived piglets cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice porcine aminopeptidase n is not a cellular receptor of porcine epidemic diarrhoea virus, but promotes its infectivity via aminopeptidase activity proteolytic activation of the sars-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research epidemiology, genetic recombination, and pathogenesis of coronaviruses detection and genetic characterization of deltacoronavirus in pigs 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 ethical approval all applicable international, national, and/or institutional guidelines for the care and use of animals were followed.conflict of interest neither of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. all authors have seen and approved the manuscript. key: cord-288253-wqrhiq08 authors: park, jung-eun; park, eui-soon; yu, jung-eun; rho, jaerang; paudel, sarita; hyun, bang-hun; yang, dong-kun; shin, hyun-jin title: development of transgenic mouse model expressing porcine aminopeptidase n and its susceptibility to porcine epidemic diarrhea virus date: 2015-02-02 journal: virus res doi: 10.1016/j.virusres.2014.12.024 sha: doc_id: 288253 cord_uid: wqrhiq08 porcine coronavirus infections have known as they are specific to pigs with predominantly enteric or respiratory diseases. no laboratory animal model is yet been developed in porcine coronaviruses study. here, we report that development of a transgenic mouse model expressing porcine apn which is susceptible to porcine coronavirus infection. the porcine apn transgene was constructed by fusing with mouse proximal apn promoter at 5′ terminus and bovine growth hormone polyadenylation site at its 3′ terminus. after screen on pubs from the microinjected mice, we confirmed two transgenic lines expressing porcine apn in various organs. we confirmed the susceptibility to porcine epidemic diarrhea virus, one of the porcine coronaviruses. these transgenic mice will be an important tool for research into the porcine coronaviruses. the coronaviruses belong to the family coronaviridae within the order nidovirales. they are classified into four genera, alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, based on genetic similarities (adams et al., 2014) . the primary replication of the coronaviruses is often confined to respiratory-or gastrointestinal-tract epithelial cells, so they usually induce respiratory or enteric diseases, but also hepatic, renal and neuronal infections (lednicky et al., 2013; masters, 2006; weiss and navas-martin, 2005) . the pathogenesis of several porcine coronaviruses, including transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcov), has been broadly studied (enjuanes et al., 1995; saif, 2004a,b; saif, 2004a,b; weiss and navas-martin, 2005) . tgev is a major cause of viral enteritis and fetal diarrhea in swine, most severely in neonates and with a high mortality rate, which causes significant economic losses worldwide (enjuanes et al., 1995) . prcov is reported to be an attenuated variant of tgev. prcov infects lung epithelial cells, and prcov antigen has been found in type i and type ii pneumocytes and alveolar macrophages, and infection is followed by interstitial pneumonia (halbur et al., 1993; saif, 2004a,b) . porcine epidemic diarrhea virus (pedv) is the causative agent of porcine epidemic diarrhea, which is characterized by watery diarrhea, as is tgev infection (chasey and cartwright, 1978; pensaert and de bouck, 1978; turgeon et al., 1980) . pedv-infected piglets usually show typical enteric signs, including profuse watery diarrhea, weight loss, and loss of milk uptake, entailing high mortality. pedv has been reported in europe and asia, and also recently in the united states (hess et al., 1980; huang et al., 2013; pan et al., 2012; park et al., 2013; wang et al., 2014) . all these porcine coronaviruses belong to the genus alphacoronavirus. a hemagglutinating enteric coronavirus, a member of the genus betacoronavirus, is antigenically unrelated to the other porcine coronaviruses and uses a 5-nacetyl-9-o-acetylneuraminic-acid-containing moiety as its cellular receptor. coronavirus infections are mediated by the spike (s) glycoprotein, a large surface glycoprotein on the viral envelope (delmas and laude, 1990; masters, 2006) . coronavirus s glycoproteins recognize cellular receptors and mediate virus-cell fusion (masters, 2006; peng et al., 2011) . most alphacoronavirus, including tgev, pedv, and prcov, use aminopeptidase n (apn/cd13) as their cellular receptor (delmas et al., 1992; li et al., 2007; ren et al., 2010; tresnan and holmes, 1998; yeager et al., 1992) . apn/cd13 is a 150-kda, zinc-dependent metalloprotease consisting of 967 amino acids (rawlings and barrett, 1995) . mammalian apn is ubiquitously expressed as a glycosylated homodimer on the surfaces of epithelial cells in the liver, intestine, kidney, and respiratory tract, and in fibroblasts and leukocytes, and plays multiple roles in many physiological processes, including coronavirus entry (barnes et al., 1994; luan and xu, 2007; mina-osorio et al., 2008; miura et al., 1983) . the natural hosts of porcine coronaviruses are young piglets, and clinical illness has only been observed in sucking piglets. however, in vivo studies using suckling piglets have many disadvantages, including their high cost, difficulty in handling the piglets, limited reagents, etc. because the use of laboratory animal models (e.g., mouse models) can circumvent these limitations, several transgenic animal models have been generated to study porcine viruses (benbacer et al., 1998; ono et al., 2006) . here, we generated transgenic mice expressing porcine apn under the control of the mouse proximal apn promoter. we tested their susceptibility to pedv with reverse transcription-polymerase chain reaction (rt-pcr) and immunochemical analyses, and found that the porcine apn transgenic mice were susceptible to pedv infection. vero monkey kidney cells were maintained in minimal essential medium containing 10% fetal bovine serum (fbs), 100 u/ml penicillin g, 100 g/ml streptomycin, and 250 g/ml amphotericin b in a 5% co 2 atmosphere at 37 • c. 293t human embryonic kidney cells were maintained in dulbecco's modified eagle's medium containing 10% fbs, 100 u/ml penicillin g, 100 g/ml streptomycin, and 250 g/ml amphotericin b. all tissue culture reagents were purchased from gibco (carlsbad, ca, usa). kpedv-9, a cell-adapted vaccine strain of pedv, was grown and titrated in the vero cells, as described previously, and stored at −80 • c until use (cruz and shin, 2007; hofmann and wyler, 1988) . the polyclonal antibodies specific for pedv and porcine apn were generated in balb/c mice immunized with kpedv-9 and purified porcine apn, respectively, as described previously (cruz et al., 2008) . the antibody specificities were confirmed with enzyme-linked immunosorbent assays. the anti-flag m2 monoclonal antibody (anti-flag) and anti-flag m2 affinity gels (gel beads) were purchased from sigma-aldrich (st. louis, mo, usa). fluorescein isothiocyanate (fitc)-conjugated anti-mouse igg antibody was purchased from santa cruz biotechnology (santa cruz, ca, usa). the mouse proximal apn promoter region (starting at nucleotide-1044) was amplified from c57bl/6j genomic dna by pcr with primers 5 -cccgcggccgcaagatttgaaacagtgga-3 and 5 -cccaagcttgatgccggtggacaggga-3 , containing flanking noti and hindiii restriction endonuclease sites, respectively. the pcr product was cloned into the pbluescript ks (+) vector and the pgl3-basic vector (promega, madison, wi, usa), which contains a promoterless luciferase reporter gene. the porcine apn gene was amplified from the total rna isolated from porcine enterocytes with rt-pcr using specific primers and cloned into the pbluescript ks (+) vector. a sequence encoding the flag epitope (dykddddk) was fused to the 3 terminus of porcine apn with pcr with primers 5 -cccaagcttaccatggccaagggattctac-3 and 5 -cccctcgagtcacttgtcgtcatcgtctttgtagtcgctgtgctctat-gaacca-3 , which contain flanking hindiii and xhoi restriction endonuclease sites, respectively. the bgh-polya sequence was amplified from the pcdna3.1 vector (invitrogen, carlsbad, ca, usa) with pcr using primers 5 -cccctcgagcgactgtgccttctagtt-3 and 5 -cccggtaccccatagagcccaccgcat-3 , which contain flanking xhoi and kpni restriction endonuclease sites, respectively. the pcr product was cloned into the pbluescript ks (+) vector. all pcr products were confirmed with automated sequencing. to generate the porcine apn transgene, porcine apn-flag and the mouse proximal apn promoter were ligated into pbluescript ks-bgh-polya after they were digested with hindiii/xhoi and noti/hindiii, respectively. 293t cells (2.5 × 10 5 cells/ml) were plated in 24-well plates. after 24 h, the cells were transfected with pgl3-basic-mouse proximal apn promoter (mapn-luc) (0.2 g or 1 g). the cells were transfected in triplicate using lipofectamine 2000 (invitrogen), according to the manufacturer's instructions. after 24 h, the cells were lysed for assay with the luciferase assay system (promega), according to the manufacture's protocol. all luciferase activities were normalized to ␤-galactosidase activity. the porcine apn transgene was linearized by restriction with noti and purified with gel extraction (qiagen, valencia, ca, usa). gain-of-function gene transfer was performed by microinjecting the purified dna into the pronuclei of icr mouse zygotes, which were then transferred into the oviducts of female recipient mice. the transgenic mice were identified with pcr analysis of tail genomic dna with primers: pcr1-f: 5 -cccaagcttaccatggccaagggattctac-3 and pcr1-r: 5 -gaagttggagagcatcct-3 ; and pcr2-f: 5 -ggcgtcctacttgcatgc-3 and pcr2-r: 5 -cccctc-gagtcacttgtcgtcatcgtctttgtagtcgctgtgctctatgaacca-3 . the founder mice were backcrossed to the c57bl/6j background for five generations. all the mice used in this study were maintained in a specific-pathogen-free facility at the biomedical research center at the korea advanced institute of science and technology, daejeon, korea. all animals were cared for and the experiments were performed at the animal facility at chungnam national university (cnu), korea, with the permission of and according to protocols approved by the institutional animal care and ethics committee of cnu (permission number 20110825). the porcine apn transgenic and nontransgenic wild-type mice were orally inoculated with 5 × 10 6 tcid 50 of kpedv-9 or phosphate-buffered saline (pbs, ph 7.2) as the negative control. their clinical signs were monitored and their feces collected for 5 days. two mice from each group were killed on the indicated days after viral infection. the tissues were aseptically collected and prepared for rt-pcr and immunohistopathological analysis. total rnas were extracted from the feces and tissue samples using trizol ® reagent (invitrogen) and transcribed to cdna using power cdna synthesis kit (intron biotechnology, korea), according to the manufacturer's protocol. the porcine apn-flag cdna was pcr amplified with the specific primers used for the genotyping pcr. as a control, specific primers were used to amplify ␤-actin (mouse ␤-actin-f: 5 -cggttccgatgccctgaggctctt-3 and mouse ␤-actin-r: 5 -cgtcacacttcatgatggaattga-3 ). the cycling parameters were initial denaturation at 94 • c for 2 min, followed by 30 cycles of denaturation at 94 • c for 15 s, annealing at 60 • c for 40 s, and extension at 72 • c for 90 s, with a final extension step at 72 • c for 5 min. to detect the viral genome, viral rna was extracted from tissue homogenates using the viral gene-spin viral dna/rna extraction kit (intron biotechnology), according to the manufacturer's instructions. m-mlv reverse transcriptase (intron biotechnology) was used for first-strand cdna synthesis, together with 10 l of extracted viral rna in a 50 l randomly primed reaction. the specific primers used for the detection of the n gene were pedv-n forward (5 -ggtaccatggcatctgtcagcttt-3 ) and pedv-n reverse (5 -ggatccttaatttcctgt tcgaa-3 ). rt-pcr was performed at 94 • c for 2 min, followed by 30 cycles of 94 • c for 20 s, 54 • c for 10 s, and 72 • c for 2 min, with a final extension at 72 • c for 10 min. the pcr products were analyzed by gel electrophoresis and visualized with ethidium bromide staining and uv transillumination. 293t cells were lysed with cell lysis buffer (25 mm tris-cl [ph 7.5], 150 mm nacl, 1 mm edta, 1 mm naf, 1 mm sodium orthovanadate, 1 mm pmsf, 5% glycerol, 0.5% triton x-100, and protease inhibitors [roche, indianapolis, in, usa]). the small intestines were rinsed with pbs and homogenized in lysis buffer (1% np40, 150 mm tris-cl, 50 mm nacl, 1 mm edta). the cell or tissue lysates were separated with 8-15% gradient sds-page and transferred into pvdf membrane (amersham-pharmacia biotech, piscataway, nj, usa). the proteins were probed with an anti-flag monoclonal antibody diluted 1:4000 and an anti-␤-actin antibody diluted 1:10,000. the immune complexes were detected with peroxidase-conjugated goat anti-mouse igg antibody (santa cruz biotechnology) and visualized with amersham ecl western blotting detection reagent (amersham-pharmacia biotech). 293t cells were transfected with the porcine apn transgene. after 24 h, the cells were rinsed with pbs and detached with non-enzymatic cell dissociation solution (sigma-aldrich) for 2 min. the detached cells were harvested and resuspended in fluorescenceactivated cell sorting (facs) staining buffer (pbs containing 2% fbs). the cells were stained with the anti-flag m2 monoclonal antibody (1:200) for 15 min at 4 • c and then with fitc-conjugated anti-mouse igg secondary antibody (1:500) for 15 min at 4 • c. the antibody-stained cells were analyzed with flow cytometry (fac-scalibur, becton-dickinson biosciences, franklin lakes, nj, usa). kidney and liver tissues collected from mice were rinsed with pbs, and the renal or hepatic cells were isolated from the tissues with a 70 m mesh filter (becton-dickinson biosciences), resuspended in facs staining buffer, and analyzed as described above. small intestine samples were fixed in 10% buffered formalin and embedded in paraffin. serial 5 m sections were mounted on silane-coated slides. the sections were deparaffinized for immunohistochemical analysis, and the endogenous peroxidases were blocked with 0.6% h 2 o 2 in methanol for 30 min. antigens were retrieved with microwave heating in citrate buffer for 10 min, and the slides were incubated with 1.5% horse serum for 30 min at room temperature. polyclonal anti-porcine apn antiserum, monoclonal anti-flag antibody, or polyclonal anti-pedv antiserum were applied overnight at 4 • c. the biotinylated anti-mouse igg secondary antibody was detected with an avidin-biotin-peroxidase kit (vectastain abc kit, vector laboratories, burlingame, ca, usa). antibody binding was detected with the chromogen diaminobenzidine (vector laboratories), and the cells were counterstained with hematoxylin and eosin. 2.11. pedv replication in the small intestines of porcine apn transgenic mice both wild type and porcine apn transgenic mice were infected with pedv orally on day 0. viral titer in inoculum was 5x tcid 50 10 6 . two mice from each group were killed daily up to 5 days, and small intestine samples from all mice were collected in mem and homogenized. samples were centrifuged and supernatants were kept in −80 • c until processed for titration. titration was performed using vero cells. the pedv titer was measured and described in log value. the transcription of mouse apn is regulated by two different promoters, as shown in fig. 1a (bhagwat et al., 2001) . the distal promoter is located 8 kb upstream from the transcription -1 (3 and 4) , and cnupedm-3 mice (5 and 6). porcine apn rnas were amplified using specific primers targeting porcine apn, as described in section 2. ␤-actin was detected as the loading control. (b) expression of porcine apn protein in the small intestines. small intestine samples were collected from cnupedm-1 and cnupedm-3 mice. porcine apn protein was detected in small intestine homogenates with immunoblotting using an anti-flag monoclonal antibody (2 and 4). no recombinant protein was detected in the small intestine samples derived from nontransgenic wild-type mice (1 and 3). ␤-actin was used as the loading control. (c) immunohistochemical analysis of porcine apn transgenic mice. small intestine sections were prepared from porcine apn transgenic mice (a-c: cnupedm-1; d-f: cnupedm-3) and incubated with anti-flag monoclonal antibody (anti-flag) (a and d), mouse anti-porcine apn polyclonal antiserum (anti-papn) (b and e), or normal mouse serum (c and f). the immune complexes were visualized with avidin-biotin-peroxidase and the cells were counterstained with hematoxylin and eosin. magnification, ×40. start site and controls apn expression in myeloid and fibroblast cells, whereas the proximal promoter regulates apn expression in epithelial cells (olsen et al., 1991; shapiro et al., 1991) . following references and preliminary studies, we choose the mouse proximal apn promoter to express porcine apn in mouse epithelial cells. to test the promoter's activity, the sequence encoding the promoter region was amplified and cloned into the pgl3-basic promoterless vector (fig. 1b) , and the promoter activity was measured by luciferase assay. as shown in fig. 1b , the luciferase activity was about eight-fold higher in 293t human embryonic kidney cells transfected with the vector containing the mouse proximal apn promoter (mapn-luc) than in cells transfected with the empty vector. we constructed a vector encoding porcine apn, which was regulated by the mouse proximal apn promoter (porcine apn transgene) (fig. 2a) . the porcine apn cdna was amplified from porcine enterocytes and tagged at the c-terminus with the flag epitope to distinguish exogenously expressed porcine apn from endogenously expressed murine apn. the 2.9-kb flag-epitopetagged porcine apn cdna was cloned into the pbluescript ks (+) vector under the control of a 1.1-kb genomic sequence containing the mouse proximal apn promoter. the splice sites and polyadenylation signal for the cdna were provided by a 0.2-kb bovine growth hormone polyadenylation signal (bgh-polya). the expression of the porcine apn protein was detected with immunoprecipitation and immunoblotting in 293t cells transfected with the porcine apn transgene. the flag-tagged recombinant protein, with a molecularweight of about 150 kda (the expected molecular mass for porcine apn), was confirmed with immunoprecipitation (fig. 2b) . we also measured the surface expression of porcine apn with flow cytometry using an anti-flag antibody, and found that 21.3% of cells were flag-positive (fig. 2c ). our data demonstrate that the mouse proximal apn promoter efficiently induced porcine apn expression from our construct. the porcine apn transgene was linearized with noti restriction and the dna microinjected into mouse zygotes. to screen for porcine apn expression, we designed two genomic pcr primers, as indicated in fig. 3a (arrowheads) . the presence of the porcine apn transgene in mouse litters was monitored with pcr analysis on tail genomic dna (fig. 3b) . microinjection of the porcine apn transgene produced two transgenic founders, designated cnupedm-1 and cnupedm-3. the transgenic mice were healthy and showed no negative effects of transgene expression. because the major pathological changes of the porcine coronaviruses (e.g., tgev and pedv) involves enteric diseases, we measured porcine apn expression in the small intestine by rt-pcr, immunoblotting, and ihc. all the tested litters descended from cnupedm-1 or cnupedm-3 expressed porcine apn mrna in their small intestines (fig. 4a ). the small intestines expressed a recombinant protein with the molecular mass expected for porcine apn (fig. 4b ). an immunohistochemical analysis, with both anti-flag and anti-porcine apn antibodies, clearly confirmed porcine apn expression in the brush borders of the absorptive cells in the small intestines of the mouse model (fig. 4c) . these results demonstrate that the porcine apn transgenic mice expressed porcine apn in their small intestines. we also screened for porcine apn expression in various other tissues with rt-pcr. porcine apn mrna was strongly expressed in the lung, kidney, and the intestine, and was slightly expressed in the liver (fig. 5a) . the extracellular expression of porcine apn was examined in the kidney and liver by facs analysis (fig. 5b) . consistent with the rt-pcr results, the number of flag-positive renal cells was significantly higher (2-4.3-fold) in the porcine apn transgenic mice than in the nontransgenic wild-type mice, whereas the number of flag-positive hepatic cells was also slightly higher (1.6-3-fold) in summary, we confirmed the expression of porcine apn in the small intestines, lungs, livers, and kidneys of the porcine apn transgenic mice. we examined the susceptibility of the porcine apn transgenic mice to pedv, one of the enteropathogenic porcine coronaviruses. porcine apn transgenic or nontransgenic wild-type mice were inoculated orally with kpedv-9, a vero-cell-adapted korean strain of pedv. in infected transgenic mice, other than slightly watery feces observed at 3 days after inoculation (fig. 6a) , whereas no clinical signs (e.g., watery diarrhea, vomiting, fever, weight loss, or death) were observed for 5 days. viral replication was detected in various tissue extracts with rt-pcr. viral rna was confirmed in the small intestines (for at least 5 days), kidneys (for at least 2 days), and spleen (for at least 3 days) only in the porcine apn transgenic mice (table 1) , and not in the nontransgenic mice. an immunohistochemical analysis detected pedv antigen in the brush borders of the small intestines, at the same location at which porcine apn is expressed (fig. 6b) , which has been demonstrated in pedvinfected piglets (debouck and pensaert, 1980; pensaert et al., 1981) . however, no histopathological changes were observed in the small intestines that shown in pedv infected piglets. overall, these data indicate that porcine apn transgenic mice are susceptible to pedv infection, although they showed none of the enteric disease symptoms typical of pedv-infected piglets (fig. 7) . 3.6. pedv replication in the small intestines of porcine apn transgenic mice pedv replication in both wild type and porcine apn transgenic mice were confirmed and compared by viral load in the small intestines. there were no detectable pedv in any of wild type mice samples. in porcine apn transgenic mice, the mean value of pedv was tcid 50-10 3.1 on day 1. it increased to tcid 50-10 3.8 , tcid 50-10 4.8 , tcid 50-10 5.0 , tcid 50-10 5.1 , on day 2, 3, 4, and 5, respectively. there was no big change in titer between 3 and 5 days pi. we could confirm clear pedv replication in the small intestines of porcine apn transgenic mice. laboratory animal models are crucial tools for the study of viral pathogenesis in vivo, especially for highly pathogenic human viruses or viruses with restricted host ranges (calvert et al., 2014; chiu et al., 2014; deruaz and luster, 2013) . unlike in vitro cell culture systems, laboratory animal models offer researchers invaluable opportunities to study the biological, pathological, and histological characteristics of human and animal diseases. for these purposes, many transgenic mouse models have been developed to study viral pathogenesis, immune responses, and vaccines (darling et both wild type and porcine apn transgenic mice were infected with pedv (5x tcid5010 6 ) orally on day 0. two mice from each group were killed daily up to 5 days, and intestine samples were collected and prepared for titration. titration was done using vero cells. y value is pedv titer in tcid50 in log value. reynaud et al., 2014) . in the field of coronavirus research, studies of severe acute respiratory syndrome coronavirus (sars-cov) and human coronavirus 229e (hcov-229e) have been conducted with transgenic mouse models (lassnig et al., 2005; tseng et al., 2007) . the host range of the porcine coronaviruses is strongly limited to pigs, so pigs are the only animals available in which to study viral pathogenesis. however, in vivo experiments using pigs are relatively challenging because they require special treatments, reagents, and facilities. because most porcine coronaviruses use porcine apn as their receptor, we generated porcine apn transgenic mice, which are susceptible to porcine coronaviruses. we constructed the porcine apn transgene by attaching the mouse proximal apn promoter to the 5 terminus of the porcine apn gene and the bgh-polya signal at its 3 terminus. we thus generated porcine apn transgenic mice expressing porcine apn in the brush borders of their small intestines and various tissues (lungs and kidneys). previous attempts to generate apn transgenic mice susceptible to tgev or hcov-229e have been documented (benbacer et al., 1998; lassnig et al., 2005) . benbacer et al. generated an rt-pcrpositive lineage, but failed to confirm either the expression of the porcine apn protein in the intestine or tgev replication in their porcine apn transgenic mouse model (benbacer et al., 1998) . a susceptible mouse model for hcov-229e infection was successfully developed using comprehensive apn regulatory elements to generate human apn +/+ stat1 −/− double transgenic mice, and a virus was adapted to grow in primary embryonic fibroblasts from these mice (lassnig et al., 2005) . a transgenic mouse model of sars was generated by expressing human angiotensin-converting enzyme 2, a functional receptor for the virus, under the regulation of a global promoter (tseng et al., 2007) . the significance of our study is that we successfully generated transgenic mice expressing porcine apn with minimal modifications, by using the mouse apn proximal promoter and the bgh-polya signal to ensure the strong expression and correct splicing of the gene (pfarr et al., 1986) . therefore, we could express porcine apn in the brush borders of the mouse small intestines and on the surfaces of their renal cells (figs. 4 and 5) . among the porcine coronaviruses, pedv is relatively poorly studied, and many questions and controversies remain regarding its outbreaks and pathogenesis. outbreaks of porcine epidemic diarrhea have been limited to few countries in east asia in the cold season, whereas it has spread to most swine farms in the asian region (mole, 2013; pan et al., 2012; park et al., 2013; song and park, 2012; stevenson et al., 2013) . recently, pedv has also spread rapidly among swine farms in the united states, causing high piglet mortality in more than 17 states (huang et al., 2013; mole, 2013; stevenson et al., 2013; wang et al., 2014) . more importantly, although vaccination is practiced on most swine farms in korea, reported outbreaks are still increasing. moreover, a method for isolating wild-type pedv is not well established. for these reasons, we tested the susceptibility of porcine apn transgenic mice to pedv. although significant clinical illness was not observed when the transgenic mice were infected with pedv, their susceptibility to the virus was confirmed by the detection of viral rna in various organs with rt-pcr and viral proteins in the small intestines with ihc. more clearly, pedv replication was confirmed by increase in viral titer up to 5 days pi compared to those in wild type mice. we could found clear increase in viral titer in the small intestines of papn transgenic mice but not from wild type mice. these results also confirmed that porcine apn plays a role as the cellular receptor for pedv. however, similar experiments using other porcine coronaviruses are required. although we have presented evidence of pedv infection in the small intestines of porcine apn transgenic mice, no clinical signs were observed, apart from moderately soft feces. it has previously been shown that pedv can infect cells lacking extracellular trypsin, although trypsin is essential for the generation of a cytopathic effect (cpe) in infected cells (park et al., 2011) . therefore, we assume that cellular cofactors (e.g., trypsin) other than the primary receptor might be involved in and required for pedv pathogenesis in vivo. however, these factors are probably present at insufficient levels in mice. proteases are generally required for efficient viral entry and the cpes of coronaviruses (matsuyama et al., 2005; simmons et al., 2004 simmons et al., , 2013 . although the viral receptors are usually expressed in various organs, the diseases caused by coronavirus infections are limited to the small intestines and/or lungs, which are enriched cellular proteases. this suggests that proteases play a role in the pathogenesis of the coronaviruses in vivo, as is already well established for influenza virus infections (hatesuer et al., 2013; sakai et al., 2014; tarnow et al., 2014) . the porcine apn transgenic mouse model should extend our understanding of the pathogenesis of and other studies to porcine coronavirus infections. this laboratory animal model should also circumvent all the disadvantages and difficulties related to the study of porcine 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work was supported by a grant from animal and plant quarantine agency of korea (grant no. z-ad-14-2009-10-02), and a grant from the technology development program for agriculture and forestry, ministry for food, agriculture, forestry and fisheries, the korean research foundation grants (grants no. 20120008358, 2011grants (grants no. 20120008358, -0023942, 211-2006. key: cord-302890-eenijt7f authors: shi, da; wang, xiaobo; shi, hongyan; zhang, jiyu; han, yuru; chen, jianfei; zhang, xin; liu, jianbo; zhang, jialin; ji, zhaoyang; jing, zhaoyang; feng, li title: significant interference with porcine epidemic diarrhea virus pandemic and classical strain replication in small-intestine epithelial cells using an shrna expression vector date: 2019-11-02 journal: vaccines (basel) doi: 10.3390/vaccines7040173 sha: doc_id: 302890 cord_uid: eenijt7f porcine epidemic diarrhea (ped) re-emerged in china in 2010 and is now widespread. evidence indicates that highly virulent porcine epidemic diarrhea virus (pedv) strains belonging to genotype g2 caused a large-scale outbreak of diarrhea. currently, vaccines derived from pedv classical strains do not effectively prevent infection by virulent pedv strains, and no specific drug is available to treat the disease. rna interference (rnai) is a novel and effective way to cure a wide range of viruses. we constructed three short hairpin rna (shrna)-expressing plasmids (shr-n307, shr-n463, and shr-n1071) directed against nucleocapsid (n) and determined their antiviral activities in intestine epithelial cells infected with a classical cv777 strain and lnct2. we verified that shr-n307, shr-n463, and shr-n1071 effectively inhibited the expression of the transfected n gene in vitro, comparable to the control shrna. we further demonstrated the shrnas markedly reduced pedv cv777 and lnct2 replication upon downregulation of n production. therefore, this study provides a new strategy for the design of antiviral methods against coronaviruses by targeting their processivity factors. porcine epidemic diarrhea (ped), caused by porcine epidemic diarrhea virus (pedv), is a highly contagious intestinal infectious disease. ped is an important disease in swine-producing countries. ped causes the death of newborn piglets and weight loss in pigs of all ages from pedv-induced severe symptoms, such as serious diarrhea, vomiting, and dehydration, which seriously damage the swine industry [1] . following reports in 1978 [1] , ped had an outbreak in swine-farming countries in asia, north america, south america, and europe [2] [3] [4] [5] . starting from the end of 2010, highly virulent pedv variants that differed from the classic european strain cv777 were widespread in china, resulting in high mortality of newborn piglets and huge economic losses [6] [7] [8] [9] . pedv can be divided into genotypes g1 and g2 based on phylogenetic analysis of full-length s gene sequences [10] . pedv strains detected in china since 2010 mostly belonged to genotype g2, which differed genetically from the pedv g1 cv777 vaccine strain (genbank accession: af353511.1) was preserved at harbin veterinary research institute (harbin, china). pedv g2 strain lnct2 (genbank accession: kt323980.1) was isolated in vero e6 cells in our laboratory. vero e6 cells were cultured and used to amplify pedv as previously described [9] . after 70% of virus-infected cells showed cytopathic effects (cpes), cultures were collected for three freeze-thaw cycles. viral titration used 96-well microplates with vero e6 cells. viral cultures were 10-fold serially diluted with virus replication medium containing trypsin (10 µg/ml). confluent vero e6 cells from microplates were washed three times with phosphate-buffered saline (pbs) and inoculated at 0.1 ml per well into eight wells. following adsorption for 1 h at 37 • c, the inocula were removed, and cells were washed three times with pbs. subsequently, 0.1 ml of fresh virus replication medium was transferred into each well, and cells were incubated 4-5 days at 37 • c. the 50% tissue culture infective dose (tcid 50 ) was expressed as the reciprocal of the highest dilution showing cpe by the reed and muench method. assays were performed in triplicate in three independent experiments. plasmids expressing gfp-tagged n and myc-tagged n were described previously [20] . the design of shrnas targeting the pedv strain lnct2 genome n gene ( figure 1a ) used methods from the literature [21] and the web-based block-it tm rnai designer program [22] . a basic local alignment search tool (blast) search [23] was performed to exclude possible homologous sequences. three individual targeting sites were selected and chemically synthesized (sangon biotech, shanghai, china) ( table 1) . control shrna was designed at the same time to have no homology with pedv or the iec cell genome. all sequences were arranged as bbsi + sense + loop + antisense + termination signal + bamhi and cloned into the pgpu6-hygro vector to make the shrna-expressing plasmids shr-n307, shr-n463, shr-n1071, and shr-nc ( figure 1b ,c). expression of sirnas was driven by the u6 promoter. vaccines 2019, 7, x 3 of 12 amplify pedv as previously described [9] . after 70% of virus-infected cells showed cytopathic effects (cpes), cultures were collected for three freeze-thaw cycles. viral titration used 96-well microplates with vero e6 cells. viral cultures were 10-fold serially diluted with virus replication medium containing trypsin (10 μg/ml). confluent vero e6 cells from microplates were washed three times with phosphate-buffered saline (pbs) and inoculated at 0.1 ml per well into eight wells. following adsorption for 1 h at 37 °c, the inocula were removed, and cells were washed three times with pbs. subsequently, 0.1 ml of fresh virus replication medium was transferred into each well, and cells were incubated 4-5 days at 37 °c. the 50% tissue culture infective dose (tcid50) was expressed as the reciprocal of the highest dilution showing cpe by the reed and muench method. assays were performed in triplicate in three independent experiments. plasmids expressing gfp-tagged n and myc-tagged n were described previously [20] . the design of shrnas targeting the pedv strain lnct2 genome n gene ( figure 1a ) used methods from the literature [21] and the web-based block-it tm rnai designer program [22] . a basic local alignment search tool (blast) search [23] was performed to exclude possible homologous sequences. three individual targeting sites were selected and chemically synthesized (sangon biotech, shanghai, china) ( table 1) . control shrna was designed at the same time to have no homology with pedv or the iec cell genome. all sequences were arranged as bbsi + sense + loop + antisense + termination signal + bamhi and cloned into the pgpu6-hygro vector to make the shrna-expressing plasmids shr-n307, shr-n463, shr-n1071, and shr-nc ( figures 1b,c) . expression of sirnas was driven by the u6 promoter. plasmid dna was transformed into electrocompetent dh5α escherichia coli and purified with endofree plasmid maxi kits (qiagen, hilden, germany). we validated the inhibitory effects of the shrnas against n by target gene expression in an in vitro transfection system. one day before transfection, iec cells were seeded into 12-well plates at 5 × 10 4 cells per well without antibiotics and grown at 37 • c overnight with 5% co 2 . the 60-80% confluent cells were transiently transfected with the indicated plasmid using lipofectamine 3000 (invitrogen, carlsbad, ca, usa), according to the manufacturer's instructions. at 48 h after transfection, n expression was analyzed by western blot or inverted fluorescence microscope. to examine the inhibitory effects of shrnas against n on target gene expression during pedv replication, iec cells were transfected with or without 1 µg, 2 µg, or 4 of µg shr-n307, shr-n463, and shr-n1071 or 4 µg of shr-nc for 24 h and infected with 100 tcid 50 /ml pedv strain cv777 or lnct2. at 48 h post-infection (hpi), antigen slides were prepared to detect pedv n protein expression by western blot. in parallel experiments, cells from individual wells were collected and frozen and thawed twice, followed by centrifugation at low speed (1000× g), and supernatants were serially diluted, inoculated with vero e6 cells, and titrated by the reed and muench method. iec cells grown in 12-well plates were transfected and infected as described. cells were harvested at the indicated time points after transfection or virus infection, washed once with cold pbs, and lysed in radioimmunoprecipitation assay (ripa) buffer (sigma-aldrich, st. louis, mo, usa) to determine protein concentrations. equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) followed by blotting onto nitrocellulose membranes. membranes were washed twice in tris-buffered saline (tbs) and incubated 2 h in superblock blocking buffer (thermofisher, waltham, ma, usa). membranes were incubated with anti-n monoclonal antibody (mab) 3g2 (prepared by our laboratory, diluted 1:1000), anti-myc monoclonal (sigma, diluted 1:1000) or anti-glyceraldehyde 3-phosphate dehydrogenase (gapdh) monoclonal antibody (sigma, diluted 1:10,000). proteins were revealed by irdye 800cw goat anti-mouse immunoglobulin g (igg) (h + l) (1:10,000) (licor biosciences, lincoln, ne, usa), and blots were visualized using an odyssey infrared imaging system (licor biosciences). cell viability assays were performed using cell counting kit-8 (cck-8) (ck04; dojindo, shanghai, china), according to the manufacturer's protocol. in brief, iec cells were seeded in 96-well plates at 10,000 per well and incubated at 37 • c for 24 h. cells were transfected or not with shrnas, and plates were incubated for 48 h before 10 µl of cck-8 was added to wells for incubation for 2 h. optical density at 450 nm was measured. viability of treated cells was expressed as a percentage relative to untreated cells. iec cells were seeded in 12-well plates, and confluent cell monolayers were transfected with shr-n307, shr-n463 and shr-n1071, or shr-nc (4 µg) with lipofectamine 3000 (invitrogen) before pedv infection. cells were infected with pedv strain cv777 or lnct2 at 100 tcid 50 /ml. pedv infection was analyzed using immunofluorescence assays (ifas) at 48 hpi. cells were fixed with 4% paraformaldehyde at 4 • c for 30 min and washed with pbs. fixed cells were permeabilized with 0.2% triton x-100 for 15 min at room temperature and blocked with blocking buffer (pbs with 5% bovine serum albumin (bsa)) for 2 h. preparations were labeled with the mouse anti-pedv n mab (1:100 dilution) at 37 • c for 2 h followed by labeling with alexa fluor 488 goat anti-mouse igg antibody (1:200 dilution) (thermofisher) for 1 h at 37 • c. cell nuclei were stained with 4 ,6-diamidino-2-phenylindole (dapi) (0.05 µg/ml) (d9542; sigma) for 15 min and analyzed using an amg evos f1 florescence microscope. all statistical data are expressed as means ± standard deviation (sd) of three independent experiments and analyzed using the student's t-test. a p-value <0.05 was considered statistically significant. phylogenetic analysis of cv777 and lnct2 was based on complete genomic sequences ( figure 2a ). cv777 clustered with pedv g1 genotype (classic strains), whereas lnct2 clustered with pedv g2 genotype (epidemic strains), with >98% nucleotide identity to these strains. to determine pedv propagation in iec cells, cpes and ifas were used to monitor after infection ( figure 2b ). at 48 hpi, cv777 and lnct2 caused similar cpes in iec cells, characterized by rounding, aggregation, and rupturing. the morphology of negative control cells remained unchanged and did not exhibit any signs of cpes. inoculation of cv777 and lnct2 with iec cells resulted in positive immunofluorescent staining with mab that recognized the virus n protein. controls were negative for immunostaining. vaccines 2019, 7, x 5 of 12 4′,6-diamidino-2-phenylindole (dapi) (0.05 μg/ml) (d9542; sigma) for 15 min and analyzed using an amg evos f1 florescence microscope. all statistical data are expressed as means ± standard deviation (sd) of three independent experiments and analyzed using the student's t-test. a p-value <0.05 was considered statistically significant. phylogenetic analysis of cv777 and lnct2 was based on complete genomic sequences ( figure 2a ). cv777 clustered with pedv g1 genotype (classic strains), whereas lnct2 clustered with pedv g2 genotype (epidemic strains), with >98% nucleotide identity to these strains. to determine pedv propagation in iec cells, cpes and ifas were used to monitor after infection ( figure 2b ). at 48 hpi, cv777 and lnct2 caused similar cpes in iec cells, characterized by rounding, aggregation, and rupturing. the morphology of negative control cells remained unchanged and did not exhibit any signs of cpes. inoculation of cv777 and lnct2 with iec cells resulted in positive immunofluorescent staining with mab that recognized the virus n protein. controls were negative for immunostaining. the prevalent strains that caused the outbreak of pedv in china in 2010 and in north america in recent years belonged to the g2 genotype. pedv is a positive-stranded rna virus with higher mutation rates than dna viruses [24] . specific shrnas were designed to target the conserved gene. we used the n gene of lnct2 as the target sequence. using nucleotide substitutions in the n gene and previous reports [25] , we chose three unused regions that were well conserved in n genes among 25 isolates of pedv g1 and g2 genogroups. the sites were at +307 to +327 nt, +463 to +483 nt, and +1071 to +1091 nt relative to the 5 atg initiation codon (table 1) . shrnas induce large amounts of cell death when transfected at large volume, resulting in interference with experimental results. to detect if the three shrnas we used led to cytotoxicity at 4 µg, iec cells were seeded in 96-well microplates and transfected with shr-n307, shr-n463, shr-n1071, or shr-nc (4 µg) using lipofectamine 3000 (invitrogen), or transfected with transfection reagent alone as mock. after transfecting for 48 h, cck-8 solution (10 µl) was added to wells, and plates were incubated at 37 • c for 2 h. absorbance was measured at 450 nm using a microtiter plate reader (bio-rad, hercules, ca, usa). viable cells that were mock transfected with transfection reagent alone were the reference of 100% cell viability. shr-n307, shr-n463, and shr-n1071 exhibited no obvious cytotoxicity in transfected vero e6 and iec cells at 4 µg ( figure 3 ). china; red circle, g1 cv777 strain from this study; red rhombus, g2 lnct2 strain from this study. (b) production and growth properties of pedv cv777 and lnct2 in iec cells. cpe 48 h post-infection (hpi) (upper panels, bar: 400 μm) and immunofluorescence assay (ifa) 48 hpi (lower panels, bar: 200 μm) of cv777 and lnct2 in iec cells. iec cells were infected with pedv cv777 or lnct2 at 100 50% tissue culture infective dose (tcid50)/ml. cpe and ifa were examined at 48 hpi, and cell images were captured. the prevalent strains that caused the outbreak of pedv in china in 2010 and in north america in recent years belonged to the g2 genotype. pedv is a positive-stranded rna virus with higher mutation rates than dna viruses [24] . specific shrnas were designed to target the conserved gene. we used the n gene of lnct2 as the target sequence. using nucleotide substitutions in the n gene and previous reports [25] , we chose three unused regions that were well conserved in n genes among 25 isolates of pedv g1 and g2 genogroups. the sites were at +307 to +327 nt, +463 to +483 nt, and +1071 to +1091 nt relative to the 5′ atg initiation codon (table 1) . shrnas induce large amounts of cell death when transfected at large volume, resulting in interference with experimental results. to detect if the three shrnas we used led to cytotoxicity at 4 μg, iec cells were seeded in 96-well microplates and transfected with shr-n307, shr-n463, shr-n1071, or shr-nc (4 μg) using lipofectamine 3000 (invitrogen), or transfected with transfection reagent alone as mock. after transfecting for 48 h, cck-8 solution (10 μl) was added to wells, and plates were incubated at 37 °c for 2 h. absorbance was measured at 450 nm using a microtiter plate reader (bio-rad, hercules, ca, usa). viable cells that were mock transfected with transfection reagent alone were the reference of 100% cell viability. shr-n307, shr-n463, and shr-n1071 exhibited no obvious cytotoxicity in transfected vero e6 and iec cells at 4 μg ( figure 3 ). we designed three shrnas specifically targeting regions shown in figure 1 , named shr-n307, shr-n463, and shr-n1071. western blots determined the inhibitory effects of the shrnas on n we designed three shrnas specifically targeting regions shown in figure 1 , named shr-n307, shr-n463, and shr-n1071. western blots determined the inhibitory effects of the shrnas on n gene expression. pmyc-n was co-transfected into iec cells with increasing doses of shr-n307, shr-n463, or shr-n1071 using lipofectamine 3000. at 48 h after transfection, n protein expression was analyzed by western blots using mouse anti-myc mab. western blots demonstrated that shr-n307, shr-n463, and shr-n1071 inhibited myc-tagged n protein expression in a dose-dependent manner ( figure 4a ). cassettes. at 48 h post-transfection, the effects of shrna on enhanced gfp (egfp) expression were monitored by fluorescence microscopy. the shr-n307, shr-n463, and shr-n1071 expression cassettes targeting n gene inhibited the expression of acgfp to some extent compared with cells transfected with plasmid pacgfp-n alone. the shr-n307, shr-n463, and shr-n1071 expression cassettes targeting the n sequence were similar, with weak fluorescence observed from cells transfected with 4 μg of shrnas ( figure 4b ). western blots for effect of shr-n307, shr-n463, and shr-n1071 on n gene expression. pmyc-n was co-transfected with increasing doses of shr-n307, shr-n463, or shr-n1071 into iec cells. after 48 h, transfected cells were lysed. equal amounts of cell lysates were resolved by 12.5% sds-page. reaction products were probed with anti-myc or anti-glyceraldehyde 3-phosphate dehydrogenase (gapdh). densitometric data for n/gapdh from three independent experiments are shown as means ± sd. * , p < 0.05. the p value was calculated using student's t-test; (b) shr-n307, shr-n463, and shr-n1071 influence on pacgfp-n expression in cultured iec cells. pacgfp-n was co-transfected with increasing doses of shr-n307, shr-n463, or shr-n1071 into iec cells. pacgfp-n expression plasmid was the unrelated control. images show enhanced gfp (egfp) expression at 48 h post-transfection. to examine the inhibitory effects of shrnas against n on target gene expression during cv777 and lnct2 replication, iec cells were transfected with indicated plasmids for 24 h and then infected with pedv cv777 orlnct2 strain (100 tcid50/ml). at 48 hpi, pedv-infected cells were lysed with ripa buffer containing 1 mm phenylmethylsulfonyl fluoride (pmsf), and western blots the n gene was fused with the acgfp gene to make pacgfp-n. the effect of shrnas on n gene expression was monitored by acgfp expression. plasmid pacgfp-n was transfected into iec cells alone or with the indicated concentrations of shr-n307, shr-n463, or shr-n1071 expression cassettes. at 48 h post-transfection, the effects of shrna on enhanced gfp (egfp) expression were monitored by fluorescence microscopy. the shr-n307, shr-n463, and shr-n1071 expression cassettes targeting n gene inhibited the expression of acgfp to some extent compared with cells transfected with plasmid pacgfp-n alone. the shr-n307, shr-n463, and shr-n1071 expression cassettes targeting the n sequence were similar, with weak fluorescence observed from cells transfected with 4 µg of shrnas ( figure 4b ). to examine the inhibitory effects of shrnas against n on target gene expression during cv777 and lnct2 replication, iec cells were transfected with indicated plasmids for 24 h and then infected with pedv cv777 orlnct2 strain (100 tcid 50 /ml). at 48 hpi, pedv-infected cells were lysed with ripa buffer containing 1 mm phenylmethylsulfonyl fluoride (pmsf), and western blots were used to analyze n expression using mab 3g2. treatment with shr-n307, shr-n463, and shr-n1071 significantly reduced n expression compared to shr-nc-or mock-transfected iec cells. the inhibitory effects of the three shrnas on n expression were elevated with increased shrna concentration (figures 5a and 6a) . these results show that n-targeted shrna efficiently inhibited target gene expression during pedv replication. with cv777 or lnct2 (100 tcid50/ml). cell cultures were collected at 48 hpi, and viral titers were determined. transfection with shr-n307, shr-n463, or shr-n1071 significantly reduced pedv cv777 and lnct2 replication compared to shr-nc-or mock-transfected iec cells (p < 0.05) ( figure 5b and figure 6b ). although differences in inhibition of pedv cv777 and lnct2 strain replication induced by 1 μg, 2 μg, or 4 μg of shr-n307, shr-n463, and shr-n1071 were seen, pedv replication gradually reduced with increased shrna concentrations. anti-n protein monoclonal antibody (mab). densitometric data for n/gapdh from three independent experiments, shown as means ± sd. * p < 0.05. the p value was calculated using student's t-test; (b) pedv cv777 titers in shrna-transfected iec cells. shrna transfection and viral infection were as in panel a. viral titers in supernatants collected at 48 hpi were determined using the reed-muench method. error bars represent standard errors of the mean from three independent experiments. * p < 0.05. the p value was calculated using student's t-test. to determine if downregulation of n expression by shrnas decreased pedv cv777 and lnct2 strain replication, iec cells were transfected with indicated plasmids for 24 h and infected with cv777 or lnct2 (100 tcid 50 /ml). cell cultures were collected at 48 hpi, and viral titers were determined. transfection with shr-n307, shr-n463, or shr-n1071 significantly reduced pedv cv777 and lnct2 replication compared to shr-nc-or mock-transfected iec cells (p < 0.05) (figures 5b and 6b ). although differences in inhibition of pedv cv777 and lnct2 strain replication induced by 1 µg, 2 µg, or 4 µg of shr-n307, shr-n463, and shr-n1071 were seen, pedv replication gradually reduced with increased shrna concentrations. in this study, we demonstrated that shrnas against pedv n protein broadly inhibited pedv g1 and g2 strains. this report found that shrnas inhibit swine coronavirus replication in epithelia. most coronaviruses such as severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), and pedv infect epithelial cells in the respiratory and/or enteric tracts. the control of coronavirus diseases is important for human public health security. further developing rnai as a potential therapeutic agent against coronavirus infection is worthwhile. the pedv n protein is predominantly produced in susceptible cells, which makes it a major target for early and accurate diagnosis [26] . the n protein forms complexes with coronavirus genomic rna and enhances the viral transcription and assembly. because of the importance of n protein in viral replication and the coronavirus n gene long used as a major target for shrna design, we focused on the inhibition of pedv infection using rnai targeting the n gene of pedv and systematically evaluated suppression efficiency. alterations of viral genomes such as nucleotide substitution, insertion, and deletion have the potential to decrease the inhibitory effect of shrna. we analyzed 25 n gene sequences derived from pedv genotypes g1 and g2 and only found single-nucleotide substitutions. considering the nucleotide substitutions in the n gene, we chose three conserved and unexploited regions for the design of shrnas. pedv is primarily transmitted through the fecal-oral route and infects intestinal villous epithelial cells in vivo [27] . current in vitro cell cultures of pedv include vero e6 cells, marc-145 cells (another monkey kidney cell line), and hek293 cells [28] . most are non-porcine intestinal epithelial cells and, thus, not ideal in vitro cellular models for studying the interaction between pedv infection and the host response due to interspecific variation. the iec cell line represented a better model of normal porcine intestinal epithelium than transformed cell lines, and provided a unique opportunity to explore host-pathogen interactions in an in vitro system [29] . using this cell model, we showed that shrna expression did not affect the viability of iec cells. co-transfection of recombinant plasmid pmyc-n and different concentrations of shrnas highlighted the success of the gene knockdown at the protein level. gfp was an important reporter for n gene expression. for example, if the n gene was silenced by the shrnas, the translation of gfp protein in frame with the n gene in the recombinant plasmid would also be inhibited. this hypothesis was consistent with our findings. our shrnas shr-n307, shr-n463, and shr-n1071 strongly reduced gfp expression, and the inhibition was dose-dependent. in addition, the model system was exploited to further study the pathological functions of pedv genes. we assessed the capacity of shr-n307, shr-n463, and shr-n1071 for inhibiting the gene and protein expressions of pedv cv777 and lnct2 strains in vitro. we observed that shr-n307, shr-n463, and shr-n1071 were able to knock down target gene expression of pedv cv777 and lnct2 strains at 48 hpi. we also showed that shr-n307, shr-n463, and shr-n1071 against the n gene suppressed pedv cv777 and lnct2 strain replication. currently, sirna import into cells requires vectors such as plasmids and recombinant lentiviruses that express shrnas efficiently and stably. as recombinant lentiviruses have a higher adaptability and replication ability in host cells, and as their genes can be integrated into cellular genomes, the silencing effect of lentiviruses is more efficient than plasmids [30] . however, this kind of gene integration may induce host genome mutations and cause cellular injury. in addition, excessive expression of shrnas competitively inhibits cell endogenous mirnas and causes cytotoxicity [31] . therefore, rnai mediated by a plasmid is currently safer for high gene silencing efficiency. however, off-target effects from using plasmid-based sirna are still a shortfall [32] . to solve this problem, designing improved sirna sequences, as well as developing novel and progressive vectors, is necessary. in conclusion, plasmids expressing three shrnas targeting different sites of the n gene of pedv were constructed and transfected into iec cells. infection of pedv cv777 (g1) or lnct2 (g2) occurred post transfection. detection by western blot and viral titer assays was used to measure levels of viral replication in cells. the results demonstrated that pedv g1 and g2 strains were susceptible to rnai pathways targeting the n gene. all shrna tests led to the silencing of n gene expression and suppressed proliferation of pedv g1 and g2 strains in iec cells. in sum, our data showed the potential for the shrna expression vectors to precisely and effectively interfere with the replication of pedv g1 and g2 strains in vitro. to our knowledge, this is the first report of the inhibition of pedv g1 and g2 strain infection with shrnas in iec cells. therefore, this report enriches the antiviral spectrum of rnai treatment. although iec cells are a non-transformed porcine intestinal epithelial cell line, these cells lack the complexity of the cell types found in the architecture of the intestinal epithelium and, thus, do not satisfactorily mimic the natural infection process. this method merits further investigation in animal studies to define its therapeutic potential. determining if the technology could be used in vivo for anti-pedv therapy is still under investigation. molecular characterizations of subcellular localization signals in the nucleocapsid protein of porcine epidemic diarrhea virus a previously undescribed coronavirus associated with respiratory disease in humans new variants of porcine epidemic diarrhea virus, china fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin the molecular biology of coronaviruses repeated administration of hepatitis c virus core-encoding plasmid to mice does not necessarily increase the immune 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rnai efficacy, toxicity, and persistence in the adult mouse liver dicing and slicing: the core machinery of the rna interference pathway inactivation of expression of gene 4 of mouse hepatitis virus strain jhm does not affect virulence in the murine cns porcine epidemic diarrhea in china nucleocapsid interacts with npm1 and protects it from proteolytic cleavage a 2 ,5 -oligoadenylate analog inhibits murine hepatitis-virus strain-3 (mhv-3) replication in-vitro but does not reduce mhv-3-related mortality or induction of procoagulant activity in susceptible mice basic local alignment search tool do viral proteins possess unique biophysical features? effective inhibition of porcine epidemic diarrhea virus by rna interference in vitro analyses of coronavirus assembly interactions with interspecies membrane and nucleocapsid protein chimeras evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains characterization of porcine epidemic diarrhea virus infectivity in human embryonic kidney cells porcine small intestinal epithelial cell line (ipec-j2) of rotavirus infection as a new model for the study of innate immune responses to rotaviruses and probiotics transfection of small rnas globally perturbs gene regulation by endogenous micrornas recent advances in understanding of the immunological off-target effects of sirna the authors declare no conflicts of interest. key: cord-315885-iu5wg5ik authors: hoang, hai; killian, mary l.; madson, darin m.; arruda, paulo h. e.; sun, dong; schwartz, kent j.; yoon, kyoungjin j. title: full-length genome sequence of a plaque-cloned virulent porcine epidemic diarrhea virus isolate (usa/iowa/18984/2013) from a midwestern u.s. swine herd date: 2013-12-19 journal: genome announc doi: 10.1128/genomea.01049-13 sha: doc_id: 315885 cord_uid: iu5wg5ik porcine epidemic diarrhea (ped) was recognized in u.s. swine for the first time in early 2013. a plaque-purified ped virus (pedv) isolate (usa/iowa/18984/2013) was obtained from a diarrheic piglet. the isolate is genetically close to other previously reported u.s. pedvs and recent chinese pedvs and was virulent when inoculated into neonatal pigs. genus alphacoronavirus (1) (2) (3) , was first identified in england in 1971 and later in other countries, such as belgium, china, hungary, italy, japan, korea, and thailand (4) (5) (6) (7) (8) (9) (10) (11) . in april of 2013, ped emerged in u.s. swine (12) and was detected in swine herds in 18 u.s. states by the end of october (http://www.aasv.org/pedv /pedv_weekly_report_103013.pdf), causing considerable economic losses. an isolate of ped virus (pedv) was sought for use in studies to address concerns regarding diagnosis, transmission, pathogenesis, and vaccination. virus isolation was attempted from field samples submitted to the iowa state university veterinary diagnostic laboratory that were positive for pedv by a real-time reverse transcriptase pcr (rt-pcr) assay (13) using vero cells (atcc ccl-81), as previously described (14) . a cytopathic virus with coronavirus morphology was isolated from intestinal tissues collected from a 1.5week-old piglet from a swine farm in iowa. in the cells that were inoculated with the virus, the virus induced syncytia and eventually cell death. the presence of pedv in the inoculated cells was confirmed by immunofluorescence microscopy using a pedvspecific monoclonal antibody, 6c8 (12) . after 3 cell culture passages, the primary isolate was subjected to plaque purification. a plaque-cloned virus isolate was further propagated in the cells for 2 more passages, for a total of 6 cell passages (p6). the plaque-cloned pedv p6 isolate (usa/iowa/18984/2013) was able to cause cytopathic effects at 12 to 18 h postinoculation (hpi) and reach a titer of 10 5.5 pfu/ml within 48 hpi. caesareanborn colostrum-deprived piglets orally inoculated with the isolate at a rate of 10 3 pfu/ml developed severe watery diarrhea and dehydration within 24 hpi and eventually died. microscopically, intensive immunohistochemical staining of almost all enterocytes for pedv was observed at 18 hpi, leading to severe villous atrophy at a later time. the entire genome of the isolate usa/iowa/18984/2013 was sequenced using next-generation sequencing technology on the ion torrent platform (life technologies, austin, tx) as per the manufacturer's instructions, and the data were assembled using dnastar ngen based on known pedv sequences. the genomic rna of the isolate is 28,039 nucleotides long, excluding the 3= poly(a) tail. the genomic organization of the isolate is similar to what was previously described (2, 15, 16) and includes a 5= untranslated region (5= utr), open reading frame 1a (orf1a)/ orf1b, s, orf3, e, m, n, and a 3= utr with a slippery sequence ( 12610 tttaaac 12616 ) in orf1. there is an insertion between nucleotides 20204 and 20205 in orf1 that causes a reading frameshift, shortening the replicase polyprotein 1ab (6,649 amino acids long). phylogenetically, the isolate is 99.8 to 99.9% similar to other u.s. pedvs reported earlier (12, 15, 17) , 97.2 to 99.6% similar to recent chinese pedvs (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) , with ah2012 (genbank accession no. kc210145) being the closest, and 96.9% similar to the prototype pedv strain cv777 (1). in conclusion, the pedv isolate usa/iowa/18984/2013 is a virulent strain with a genetic profile similar to those of other u.s. pedvs reported to date. such a representative purified virulent pedv isolate can be a valuable reagent for studying the pathogenesis and immunobiology of pedv and developing diagnostic reagents and kits, as well as effective vaccines. nucleotide sequence accession number. the complete genome sequence of pedv strain usa/iowa/18984/2013 was submitted to genbank under the accession no. kf804028. we are grateful to practicing swine veterinarians for submitting samples from suspect cases to the iowa state university veterinary diagnostic laboratory and procuring additional samples. we also thank drew magstadt (veterinary diagnostic laboratory, iowa state university, ames, ia) and john neil (ruminant diseases and immunology research unit, national animal disease center, usda/ars, ames, ia) for assistance in animal work and laboratory support, respectively. the study was supported in part by funding from the iowa pork producers association (npb no. . sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhoea virus confirms that this virus is a coronavirus related to human coronavirus 229e and porcine transmissible gastroenteritis virus genome organization of porcine epidemic diarrhoea virus sequence analysis of the nucleocapsid protein gene of porcine epidemic diarrhoea virus letter to the editor a new coronavirus-like particle associated with diarrhea in swine molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china enterotoxigenic escherichia coli, rotavirus, porcine epidemic diarrhoea virus, adenovirus and calicilike virus in porcine postweaning diarrhoea in hungary epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan prevalence of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus infection in korean pigs chinese-like strain of porcine epidemic diarrhea virus emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus propagation of the virus of porcine epidemic diarrhea in cell culture complete genome sequence of porcine epidemic diarrhea virus strain usa/colorado/2013 from the united states porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states complete genome sequence of porcine epidemic diarrhea virus strain aj1102 isolated from a suckling piglet with acute diarrhea in china complete genome sequence of a chinese virulent porcine epidemic diarrhea virus strain complete genome sequence of a porcine epidemic diarrhea virus variant complete genome sequence of a novel porcine epidemic diarrhea virus in south china phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china complete genome sequence of a recombinant porcine epidemic diarrhea virus strain from eastern china complete genome sequence of a highly prevalent isolate of porcine epidemic diarrhea virus in south china complete genome sequence of a variant porcine epidemic diarrhea virus strain isolated in central china complete genome sequence of novel porcine epidemic diarrhea virus strain gd-1 in china complete genome sequence of a vero cell-adapted isolate of porcine epidemic diarrhea virus in eastern key: cord-294559-u0r7oh9z authors: bian, hongfen; xu, fei; jia, yumin; wang, lei; deng, shengchao; jia, aiqing; tang, yong title: a new immunochromatographic assay for on-site detection of porcine epidemic diarrhea virus based on monoclonal antibodies prepared by using cell surface fluorescence immunosorbent assay date: 2019-01-18 journal: bmc vet res doi: 10.1186/s12917-019-1773-4 sha: doc_id: 294559 cord_uid: u0r7oh9z background: porcine epidemic diarrhea virus (pedv) is a highly effective pathogen that can cause death of new-born piglet, resulting in big economical loss in pig farming industry. for rapid detection of pedv, a new immunochromatographic assay (ica) based on monoclonal antibodies (mabs) was developed in this study. results: the mabs were prepared by using pedv positive hybridoma cells that were selected by using cell surface fluorescence immunosorbent assay (csfia). fourteen mabs against pedv strain isolated from south of china were prepared. the optimal mab 4a11 was coated on nc membrane as the capturing reagent and the mab a11h7 was coupled to gold nanoparticles (aunps) as detection reagent for the new ica. the new ica was used to measure pedv in phosphate buffer containing tween-20. results indicated that the limit of detection (lod) of the new ica was 0.47 μg/ml (5.9 × 10(3) tcid(50)/ml) and the liner detection range of the ica was 0.625–10 μg/ml (7.8 × 10(3)–10(5) tcid(50)/ml). the specificity analysis results showed that this new ica had no cross reaction in the presence of other porcine viruses. the ica was also validated for the detection of pedv in swine stool samples with little interference from swine stool. to compare its accuracy to other traditional detection methods, 27 swine stool samples from south of china were investigated with the new developed ica, commercial strip and rt-pcr. results showed that the new ica was more comparable to rt-pcr than commercial test strip. conclusions: a new ica based on mabs prepared by csfia was developed in this study. it was a sensitive, specific and rapid method that could be used for on-site detection of pedv and therefore was useful for the diagnosis and prevention of ped. electronic supplementary material: the online version of this article (10.1186/s12917-019-1773-4) contains supplementary material, which is available to authorized users. porcine epidemic diarrhea virus (pedv), which belongs to the genus alphacoronavirus and the family coronaviridae, is an enveloped single-stranded positive-sense rna virus [1, 2] . pedv is a highly effective pathogen responsible for causing porcine epidemic diarrhea (ped), a disease characterized by severe and acute watery diarrhea, dehydration and vomiting that results in high mortality rate of one-week-old piglets [3, 4] . since the first appearance in england in the early 1970s, outbreaks of ped have been reported in several european and asian countries [5, 6] . ped was first reported in china in 1973, followed by a large-scale outbreak of ped in december 2010 that led to heavy economic loss [7, 8] . therefore, it is of great importance to properly and routinely monitor pedv. unfortunately, due to the fact that pedv and a different alphacoronavirus, transmissible gastroenteritis virus (tgev), have the same epidemiological and clinical features. these two viruses have led to complications in clinical diagnosis. therefore, it is of great importance to develop differential laboratory tests [9, 10] . to date, several methods including rt-pcr [11] , separation identification [12] , serological method [13] , enzyme-linked immunosorbent assay (elisa) [14] and colloidal gold method [15] [16] [17] have been used to detect the pedv antigen. although rt-pcr serves as a good standard due to its high sensitivity and accuracy, this method relies heavily on sophisticated equipment and expensive apparatus. several other common methods are also not suitable for on-site diagnosis due to the following reasons. separation identification means to isolate pedv in vero cell cultures from the small intestine of a pedv infected piglet. it is a classical method for pedv detection, but it is time-consuming for it needs at least one week. the serological method can only detect pedv antibody. however, whether the pedv antibody is from vaccine infection or wild infection is still unknown. elisa is a cheap and simple approach for pedv detection, however, it involves long incubation period, multiple washing steps, and the assistance of a microplate reader. in contrast, recently developed immunochromatographic assay (ica) is much more applicable for on-site diagnosis of pedv for it is simple, sensitive, specific and can be operated without training. therefore, this method could be useful for monitoring the infection of ped [18] [19] [20] . currently, the most widely used commercial pedv test strip in china (bionote test strip), which is made by utilizing ica, is based on the pedv-dr13 strain. the strain was harvested from a suspension of minced small intestine from infected neonatal pigs in south of korea [21] . considering regional differences in strains, it might not be the best choice for pedv detection in south of china. a pedv strain chyj130330 was isolated from the fecal specimen of a 3-day-old dead piglet in march 2013 on a commercial swine farm in guangdong province in the south of china [22] . in this study, we prepared monoclonal antibodies (mabs) using cell surface fluorescence immunosorbent assay (csfia) [10] , a simple and rapid method for selecting positive hybridoma cells. fourteen mabs against this pedv strain were prepared. relying on signals emitted from gold nanoparticles labeled mab (aunps-mab), a new ica was developed for sensitive, specific and on-site detection of pedv in swine stool in china. a comparison between rt-pcr, bionote test strip and the ica was performed to confirm the applicability of the newly developed ica for on-site pedv detection. vero cell, porcine epidemic diarrhea virus (pedv) strain named chyj130330, pseudorabies virus (prv), classical swine fever virus (csfv), transmissible gastroenteritis virus (tgev) and swine stool were obtained from guangdong haid institute of animal husbandry & veterinary (guangdong, china). porcine reproductive and respiratory syndrome virus (prrsv) and porcine circovirus-2 (pcv-2) were obtained from winsun bio (guangdong, china). balb/c mice were purchased from southern medical university. myeloma cells (sp2/0) were purchased from shanghai cell biology (shanghai, china). gibicorpmi-1640 was purchased from asegene (guangzhou, china). ninety-six-well polystyrene plates were purchased from jet biofil (guangzhou, china). goat anti-mouse igg h&l (hrp) was purchased from abcam (cambridge, ma). mab subtype classification kit was purchased from sigma (usa). the hrp-labeling kit was purchased from galaxy-bio (beijing, china). bca kit was purchased from thermo (usa). nitrocellulose (nc) membrane (hf18002s25), conjugate pad, sample pad, plastic backing and absorbent pad were purchased from millipore (shanghai, china). pedv ag test kit was obtained from bionote (korea). ultrapure water produced by a milli-q ultra pure system (millipore, usa) was used throughout this study. all chemicals used were of analytical grade or higher. absorbance of the hrp-based elisa was measured using a synergy h1 hybrid multi-mode microplate reader (bio--tek instruments, inc., winooski, vt). the following equipments were also utilized in this study: centrifuge 5810 r (eppendort, germany), xw-80 avortex mixers (shanghai, china), magnetic stirrer (beike, beijing), dynabeads mx mixer (invitrogen, america), programmable strip cutter hgs201 (autokun, china), xyz3060 platform (bio-dot scientific equipment, china), and electrothermal forced air convection drying oven (taisite instrument, china). pedv was inoculated onto a vero cell monolayer for 45 min at 37°c. cell infection at a moi of 0.005. then maintenance medium was added and cultured for 2 or 3 days. when the cytopathic changes reached above 85%, the cells were collected and repeatedly frozen and thawed for 3 times. after ultrasonic cracking and centrifugation at 10000 rpm for 1 h, the supernatant was further centrifuged at 45000 rpm for 3 h and the precipitate was resuspended in pbs (0.01 m, ph 7.4). the crude extraction virus was successively added into 30%~60% sucrose-pbs and centrifuged at 33000 rpm for 3 h. then it was resuspended in pbs and centrifuged at 40000 rpm for 3 h to remove sucrose content. the purified virus was resuspended in pbs. the uninoculated vero cells were subjected to the same treatment and used as negative control. the pre-purification protein concentration was measured by nanodrop2000 micro nucleic acid tester. virus titer was expressed as tcid 50 , 10 6 tcid 50 /ml. viral protein concentration was measured using bca, 80 μg/ml. 1.25 ml of the purified pedv antigen and an equal volume of freund's complete adjuvant, was injected into each mouse (6 weeks, female, balb/c) for the first immunization. mice were immunized every 2 weeks. tail blood was taken on the tenth day after the third immunization. mice with high titer were selected for fusion 3 days after they were boosted with immune antigen pedv. to prepare the hybridomas, the mice were euthanized by cervical dislocation. all the mice were sent to huaqiao hospital for centralized treatment after the study. csfia, a simple and rapid method for selecting positive hybridoma cells, was used to screen for positive hybridoma cell clones. in this study, pedv antigens were first anchored to the hybridoma cell surface through a dual functioning molecular oleyl-peg4000-nhs. specific antibodies secreted from hybridoma cells were then captured by the antigens on the cell surface. positive hybridoma cells were stained using a fluorescently labeled anti-mouse igg-fc antibody (fig. 1) . after the addition of a methylcellulose semisolid medium, positive clones were picked using a pipet. these positive cell clones were used to produce monoclonal antibodies after direct expansion [10] . mabs were prepared by inducing ascites in mice. they were then purified by ammonium sulphate, desalt column, and protein g affinity chromatography column. mabs were preserved at − 20°c after freeze-drying. titer of mabs was determined by indirect elisa with pedv as coating antigen and hrp-goat anti-mouse igg as secondary antibody. the sensitivity of mabs was determined by indirect competition elisa with pedv as coating antigen and pedv specific hrp-monoclonal antibody as the competitors. the mab classification kit was used to identify the category and subclass of immunoglubulin. the mab pair for pedv detection was selected by double antibody sandwich elisa matrix fig. 1 the mechanism of csfia based on the specific binding of antigens anchored on the positive hybridoma cell surface and capturing of secreted antibodies from the same cells method with hrp labeled mabs as secondary antibodies and mabs as coating substrates. the preparation of gold nanoparticles (aunps) and gold nanoparticles labeled mab (aunps-mab) to prepare aunps, trisodium citrate dehydrate was used to reduce gold chloride (haucl 4 ). in brief, 50 ml ultrapure water was added to a clean triangle flask and heated and stirred on a magnetic stirrer until boil. then, 1 ml of 1% haucl 4 was quickly added to the triangle flask and 1.2 ml trisodium citrate dihydrate (10 mg/ml) was added after a few seconds. the solution was heated for 10 min, and then moved to an unheated plate for continued stirring. the prepared aunps were then diluted with ultrapure water to 50 ml. aunps-mab was prepared by labeling mab to aunps. first, 1 ml of aunps (0.02 mg/ml) was added to a clean centrifuge tube, and 16 μl of 0.25 m k 2 co 3 were added; the solution was mixed on an avortex mixers. then, 2 μl mab was quickly added to the centrifuge tube with mixing. the mixture was rotated for 15 min and then kept still for 15 min at room temperature. subsequently, 100 μl of 10% bsa were added to cover the unconjugated site. the mixture was rotated for another 15 min and then kept still for 15 min at room temperature. finally, the mixture was centrifuged at 11000 rcf for 15 min and the precipitate was resuspended in 60 μl pbs (0.015 m, ph 7.4, containing 20% (w/v) sucrose, 20% (w/v) trehalose, 0.1% (w/v) pvp k40, 0.1% (w/v) s-9, 1% (w/v) bsa, and 0.5% (w/v) tween-20). this new ica was sandwich type, in which two monoclonal antibodies were used separately as capture and detecting reagents to detect pedv (fig. 2) . gray value corresponding to signal from the red t-line was obtained using image j [23] [24] [25] . the assembly of the new ica was as follows: the new ica was made of pvc plate, nc membrane, sample pad, conjugate pad and absorbent pad. pbs (0.015 m, ph 7.4) diluted mab (0.875 mg/ml) and goat anti-mouse igg (1 mg/ml) were dispensed on specific areas of nc membrane called the test line (t-line) and calibration line (c-line) using an automatic dispenser at a volume of 1 μl/cm. the nc membrane was dried at 37°c and saved for later use. the aunps-mab was loaded onto the conjugate pad using an automatic dispense system set to 5.0 μl/cm and then dried at 37°c. the pretreatment of sample pad involved soaking in pbs (0.015 m, ph 7.4, containing 2.5% (w/v) sucrose, 2% (w/v) bsa, and 1% (w/v) tween-20) and drying at room temperature and then at 37°c. the pvc plate acted as the holder of the new ica, onto which sample pad, conjugate pad, nc membrane and absorbent pad were pasted with an overlap of 2 mm between each part. after the assembly was completed, the new ica was cut into 3.8-mm-wide and 60-mm-long strips using a programmable hgs201 strip cutter and kept in a suitable plastic cassette for further use. a fully assembled new ica is shown in fig. 2a . to optimize the quality of the new ica, some important single factors were optimized. they were capture and detection mab, the size of gold nanoparticles, the type of sample pad, the type of conjugate pad, the type of nitrocellulose membrane, the type of absorbent pad, the amount of tween-20 addition and the spray volume of aunps-mab. the optimization methods are shown in the supplemental materials. to verify the specificity of the new ica, pedv, prv, pcv-2, prrsv, csfv, and tgev at the same concentrations were prepared and tested following similar procedures. the stability of the new ica was tested by comparing results from the experiments performed now to those from experiments performed a month later using the same icas. for the detection of pedv in spiked samples, negative samples with different pedv concentrations were prepared. first, 800 μl pb (0.2 m, ph 7.4, containing 1% (w/v) tween-20) was added to a 1.5 ml centrifuge tube. then, 200 μl negative swine stool a7 was added to the solution and mixed for 5 min. after centrifugation at 11000 rcf for 15 min, the solution was kept still for 15 min. the supernatant was collected and the precipitate was discarded. pedv was incorporated into the sample at 0.1, 1, 5, 10 and 20 μg/ml. finally, 80 μl of the spiked solutions were added to the sample holes of the new icas respectively. photos were taken to record the results after 15 min of reaction. for detection of pedv in swine stool samples, 50 μl of swine stool samples and 50 μl of pb (0.2 m, ph 7.4, containing 1% (w/v) tween-20) were mixed and incubated for 5 min. after centrifugation at 11000 rcf for 15 min, the solution was kept still for 15 min. the supernatant was collected and the precipitate was discarded. the swine stool samples were analyzed using rt-pcr, bio-note test strips and the new ica, respectively. preparation and characterization of antibody to pedv cell lesion was above 85% after pedv was inoculated onto vero cell monolayer for 36 h. after repeated freezing and thawing, cracking and sucrose-pbs gradient centrifugation, the virus bands between the 40 and 60% interface were collected. after identification, protein concentration was determined to be 4 mg/ml. the prepared pedv was used to immunize mice to generate mabs against pedv [24] . from the hybridoma cells generated against pedv, 14 clones that could produce mabs with strong pedv binding ability were selected. immunoglobulin isotyping suggested these clones were igg2a. to select pedv mab pairs with high sensitivity and specificity, a double antibody sandwich elisa was used. the amount of capture antibody for coating and detecting antibody in both experimental and control group was 10 μg/ml. 1 μg/ml pedv was added to each well of experimental group while an equal volume of pbs was added to the control group. only the mab pairs which have strong signal (od ≥ 1.0) in pedv group and no obvious signal (od ≤ 0.2) in pbs group were marked as positive. all the mab pairs which have no strong signal in pedv group or have obvious signal in pbs group were marked as negative. as shown in tables 1, 5 of the 14 mabs were successfully paired using double antibody sandwich elisa matrix method. these successfully paired mabs (4a11, 5h9, 5a9, a11h7 and 4h7) were used in the new ica for their outstanding binding ability. first, the new ica was optimized by selecting the best pair from the 5 selected mabs. the optimization results showed that 4a11 was the best capturing mab and a11h7 was the best detection mab (see additiona file 1: figure s1 . in the supporting information, number 10 represent 4a11 as capturing mab and a11h7 as detection mab). in addition, since gold nanoparticles of suitable size is essential to a successful ica, an assay was performed to optimize the size of gold nanoparticles. in brief, 16 nm, 24 nm, 30 nm and 40 nm aunps were used to label mab a11h7. with increasing size of gold nanoparticles, the red signal of the t-line also increased, while the red signal of the c-line remained the same (see additional file 2: figure s2 . in the supporting information). in addition, the reaction was not blocked at any time during the process despite the increasing size of the gold nanoparticles. we found that aunps of 40 nm was associated with the strongest red signal at the t-line with to no impairment to the reaction process. therefore, 40 nm was chosen as the optimized size for aunps for subsequent experiments (see additional file 2: figure s2 . in the supporting information). interestingly, the type of sample pad, conjugate pad, nc membrane and absorbent pad also appeared to have great impact on reaction rate of the new ica. to optimize these factors of the ica, different types of sample pad, conjugate pad, nc membrane and absorbent pad were tested in the optimization experiment. as shown in additional file 3: figure s3 . in the supporting materials, the new ica with gl-b04 as sample pad showed the most uniform red c line without any break. therefore, gl-b04 was chosen as the optimized sample pad. it can be seen from additional file 4: figure s4 . in the supporting materials, the new ica with polyester film as conjugate pad showed the most uniform red c line without any break. thus, polyester film was selected as the optimized conjugate pad. what can be learned from additional file 5: figure s5 . was that the new ica with millipore 135 as nc membrane showed the most uniform red c line without any break. therefore, millipore 135 was chosen as the optimized nc membrane. as for the optimization of absorbent pad, the new ica with h2 as absorbent pad (see additional file 6: figure s6 . in the supporting materials) showed strong color signal at the t-line. therefore, h2 was chosen as the optimal absorbent pad for the new ica. as tween-20 can remove unbound aunps-mab on the t-line and reduce nonspecific binding. sample solutions with different volumes of tween-20 (5, 10, 15, 20 and 40 μl/ml) were added to icas. results confirmed that no red line on the t-line was observed in the negative sample. at 15 μl/ml upward, stronger red color at the t line of negative sample was observed with increasing volume of tween-20 added (see additional file 7: figure s7 . in supporting information). results for positive sample remained almost the same with increasing volume of tween-20. therefore, 10 μl of tween-20 to 1 ml of sample solution was chosen as the optimized ratio for subsequent experiments. furthermore, the spray volume of aunps-mab was also important to the new ica. as shown in additional file 8: figure s8 . in supporting information, no red line at the t-line of the negative sample ica was observed until the spray volume of aunps-mab was at 6 μl/cm and the red color on the t-line of the negative sample ica was more intense with increasing spray volume. however, the red line at the t-line of positive sample test strip remained almost the same. taking nonspecific adsorption and sensitivity into consideration, 5 μl/cm was chosen as the optimal spray volume for subsequent experiments. the new ica was designed for efficient detection of pedv. the performance of the new ica for detecting pedv at various concentrations was tested using a serial dilution of pedv in pbt (0, 0.625, 1.25, 2.5, 5 and 10 μg/ml) under the optimized condition (fig. 3a) . the limit of detection (lod) was found to be 0.47 μg/ml (5.9 × 10 3 tcid 50 /ml). to quantitatively analyze the performance of the new ica, gray values obtained from image j were used to draw a standard curve in origin 7.0 (fig. 3b) . the liner detection table 1 the selection results of paired antibodies in double antibody sandwich elisa capture antibody a11h7 5h12 4d5 4a11 5e2 2c11 2h2 4h7 3g10 5h9 5a9 3g9 3c11f5 3c11f9 detection antibody hrp-a11h7 ---++ --------- range was 0.625 μg/ml to 10 μg/ml (7.8 × 10 3 to 10 5 tcid 50 /ml) and the coefficient (r 2 ) was 0.991. the exact value of pedv concentration could be read directly by comparing results and the photos corresponding to the standard curve. these results indicated that this new ica could determine pedv in a precise and quantitative manner. to confirm the specificity of the new ica, 5 kinds of swine viruses were tested and pedv was used as positive control. these swine viruses were assayed by the new ica at the same concentrations as pedv. the red line at the t-line was obvious in pedv sample, while it was invisible in samples containing other swine viruses (fig. 4a) . these results indicated high specificity of the new ica. results were further analyzed using gradation histogram made by origin 7.0 based on gray values obtained from image j. no significant cross-reaction of other swine viruses was observed in the developed new ica (fig. 4b) , which further confirmed its high specificity. to verify the stability of the new ica, experiments were carried out at different time points using the same ica and pedv. in both experiments, the positive sample solution showed a red line at t-line while the negative sample solution did not (see additional file 9: figure s9 . in the supporting information). these results indicated that the new ica was stable over time. negative swine stool was mixed with pedv to prepare the spiked samples in this study. spiked samples that included 0.1, 1, 5, 10, or 20 μl pedv were tested. the red color at the t-line became more intense with increasing pedv concentration. each spiked sample was analyzed in six repeated experiments and the gray values were calculated using the standard equation corresponding to the standard curve. as shown in table 2 , the recoveries of the new ica ranged from 90 to 110.27% with the highest relative standard deviation (rsd) calculated to be 12.63%. the result indicated that the new ica was ideal for detection of pedv in spiked samples. to analyze the performance of the new ica in detecting pedv in swine stool samples, 29 swine stool samples were collected from different swine farms from south of china. rt-pcr, commercial bionote test strip and the new ica were used to test these swine stool samples, respectively. as shown in table 3 , the total coincidence of bio-note test strip to rt-pcr was 62.69% while that to the new ica was 74.07%, indicating that the new ica was more accurate than bionote test strip. in the present study, an immunochromatographic assay for pedv detection was developed and tested with spiked samples and swim stool samples from field infected pigs. the assay was based on mabs to chyj130330, which is a new strain of pedv found in south of china and therefore is an appropriate target antigen for pedv detection. first, to prepare pedv specific monoclonal antibody, immune antigen pedv was cultured and purified in the normal way to ensure the quality of immune effect. t he well prepared 14 pedv mabs were used to do a double antibody sandwich elisa matrix, in which these 14 mabs were used as capture antibody and the same 14 mabs labeled with hrp were used as detection antibody. the aim was to find which mab as capture antibody and which hrp-mab as detection antibody can produce the best detection result. that means, which mab pair had the best binding ability to pedv. results showed that 4a11, 5h9, 5a9, a11h7 and 4h7 were these mabs that can pair successfully. second, due to the fact that the paired antibodies selected by double antibody sandwich elisa matrix may not be suitable for the new ica due to the differences in materials and environment, the new ica was optimized by selecting the best pair from the 5 selected mabs. 4a11 was proved to be the best capturing mab while a11h7 was the best detection mab. this optimization experiment greatly improved the sensitivity of the new ica. third, gold nanoparticles were important for a successful ica. if they were too big, they would block nc membrane and weaken red signal of t and c line. on the other hand, if too small, the red signal of t and c line was weak and it was not easy to observe it. considering both, the aunps of 40 nm were found to be the optimal aunps in this study. the spray volume of aunps-mab was also optimized. if the spray volume was too high, it was easy to produce nonspecific adsorption. however, if it was too low, the result would have low sensitivity. 5 μl/cm was selected considering both sides. forth, different types of sample pad, conjugate pad, nc membrane and absorbent pad had different influences on running speed, releasing amount and nonspecific absorption. so they were optimized. results showed that the optimized condition was gl-b04 as sample pad, polyester film as conjugate pad, millipore 135 as nc membrane and h2 as absorbent pad. fifth, tween-20 in sample solution acted as surface active agent, which can accelerate running speed. a high running speed may be not good because it can reduce sensitivity. therefore, the tween-20 concentration was optimized and results showed that 10 μl of tween-20 added to 1 ml of sample solution was the best choice. specific experiment showed that there was no cross reaction between this ica and other swine viruses, indicating that this ica can distinguish pedv from other swine viruses. when detecting pedv in pbt, the lod was 0.47 μg/ml (5.9 × 10 3 tcid 50 /ml) and the liner detection range was 0.625-10 μg/ml (7.8 × 10 3 -10 5 tcid 50 /ml). the pedv concentration in cultured pedv was pretty high. therefore, it can be used to detect cultured pedv. the data obtained by the new ica were compared with those obtained by both rt-pcr and commercial strip. it has been described that rt-pcr is the most sensitive and trustworthy method for pedv detection. in this study, the commercial strip was included for comparison purpose. the data analyzed in the present work showed that the total coincidence of bionote test strip to rt-pcr was 62.3% while that to the new ica was 74.07%, indicating that the new ica was more comparable to rt-pcr than commercial test strip. these results suggested that the new ica was suitable for monitoring pedv. the new ics shows pretty good detection ability of detecting pedv in swine stools, which will help pig farms in diagnosis of pedv. based on the success of making this new ics, double pathogen detection is going to be done. it can satisfy the need of pig farm. in addition, pedv fluorescent test strips will be done for precise quantitative detection. we believe that these new icas will bring great help to agriculture. coronavirus genome structure and replication further analysis of the genome of porcine epidemic diarrhoea virus porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs an apparently new syndrome of porcine epidemic diarrhoea porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis complete genome sequence of porcine epidemic diarrhea virus strain aj1102 isolated from a suckling piglet with acute diarrhea in china new variants of porcine epidemic diarrhea virus. china emerging infectious diseases multiplex reverse transcription-pcr for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group a rotavirus a rapid method for antigenspecific hybridoma clone isolation direct and rapid detection of porcine epidemic diarrhea virus by rt-pcr isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate porcine epidemic diarrhea virus: an overview of current virological and serological diagnostic methods an elisa optimized for porcine epidemic diarrhoea virus detection in faeces development of rapid immunochromatographic strip test for the detection of porcine epidemic diarrhoea virus reverse transcription crosspriming amplification-nucleic acid test strip for rapid detection of porcine epidemic diarrhea virus a novel diagnostic approach to detecting porcine epidemic diarrhea virus: the lateral immunochromatography assay triple lines gold nanoparticle-based lateral flow assay for enhanced and simultaneous detection of leishmania dna and endogenous control microchip based and immunochromatographic strip assays for the visual detection of interleukin-6 and of tumor necrosis factor alpha using gold nanoparticles as labels multicolor immunochromatographic strip test based on gold nanoparticles for the determination of aflatoxin b1 and fumonisins fecal shedding of a highly cell-cultureadapted porcine epidemic diarrhea virus after oral inoculation in pigs complete genome sequence of chyj130330, a highly virulent strain of porcine epidemic diarrhea virus in south china a new lateral-flow immunochromatographic strip combined with quantum dot nanobeads and gold nanoflowers for rapid detection of tetrodotoxin a turn-on competitive immunochromatographic strips integrated with quantum dots and gold nano-stars for cadmium ion detection identification and quantification of eight listeria monocytogene serotypes from listeria spp. using a gold nanoparticle-based lateral flow assay this work was conducted in the guangdong province key laboratory of molecular immunology and antibody engineering and guangdong haid institute of animal husbandry & veterinary. this manuscript has been thoroughly edited by a native english speaker from an editing company. editing certificate will be provided upon request. this work was supported by the national key research and development program of china (2016yfd0500600). the funders had no role in study design, or the collection, analysis, and interpretation of data. in addition, they were not involved in the writing of the report or the decision to submit the article for publication. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. molecular immunology and antibody engineering, jinan university, guangzhou 510632, people's republic of china. 2 in conclusion, a new ica based on mabs prepared by csfia was developed in this study. the gold nanoparticle signal of the test strip was of superior strength with very low background noise. the lod of the new ica was 0.47 μg/ml (5.9 × 10 3 tcid 50 /ml) and the liner detection range was 0.625 to 10 μg/ml (7.8 × 10 3 to 10 5 tcid 50 /ml). in addition, the new ica exhibited high specificity with little interference from swine stool, and could achieve recoveries ranging from 90 to 110.27% in spiked samples. compared to rt-pcr and elisa, the new ica does not require sophisticated instruments and training. relative to commercial test strips, the new ica provides more accurate detection of pedv in swine stool samples from south of china. our study highlighted the potential of the on-site application of this new ica for diagnosis and prevention of ped. additional file 1: figure s1 . authors' contributions hb, fx and yj performed the laboratory tests and drafted the manuscript. yt and aj conceived the study and participated in its design. lw and sd participated in the data analysis and results interpretation. all authors critically read and contributed to the manuscript, approving its final version.ethics approval and consent to participate all samples were obtained from healthy or naturally infected animals in the field by qualifiers. therefore, no aggressive operation was conducted against pigs for sampling purpose and no pigs were sacrificed in this study. the study used mice for generation of mabs and those mice had to be sacrificed. the mice were euthanized by cervical dislocation. thumb and forefinger were used to press down on the mice's head. the other hand grabbed the mice's tail and pulled it slightly back to the top to make the cervical spine disengage. the spinal cord was separated from the brain and the animal died immediately. there was no destruction of brain or section of major blood vessels in the neck during the process. all animal experiments were performed using protocols approved by laboratory animal ethics committee of jinan university. not applicable. 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-305315-0qt7eth0 authors: cao, liyan; ge, xuying; gao, yu; herrler, georg; ren, yudong; ren, xiaofeng; li, guangxing title: porcine epidemic diarrhea virus inhibits dsrna-induced interferon-β production in porcine intestinal epithelial cells by blockade of the rig-i-mediated pathway date: 2015-08-18 journal: virol j doi: 10.1186/s12985-015-0345-x sha: doc_id: 305315 cord_uid: 0qt7eth0 background: the lack of optimal porcine cell lines has severely impeded the study and progress in elucidation of porcine epidemic diarrhea virus (pedv) pathogenesis. vero cell, an african green monkey kidney cell line, was often used to isolate and propagate pedv. nonetheless, the target cells of pedv in vivo are intestinal epithelial cells, during infection, intestinal epithelia would be damaged and resulted in digestive disorders. the immune functions of porcine epithelial cells and interactions with other immune cell populations display a number of differences compared to other species. type i interferon (ifn) plays an important role in antiviral immune response. limited reports showed that pedv could inhibit type i interferon production. in this study, porcine small intestinal epithelial cells (iecs), the target cells of pedv, were used as the infection model in vitro to identify the possible molecular mechanisms of pedv-inhibition ifn-β production. results: pedv not only failed to induce ifn-β expression, but also inhibited dsrna-mediated ifn-β production in iecs. as the key ifn-β transcription factors, we found that dsrna-induced activation of ifn regulatory factor 3 (irf-3) was inhibited after pedv infection, but not nuclear factor-kappab (nf-κb). to identify the mechanism of pedv intervention with dsrna-mediated ifn-β expression more accurately, the role of individual molecules of rig-i signaling pathway were investigated. in the upstream of irf-3, tank-binding kinase 1 (tbk1)-or inhibitor of κb kinase-ε (ikkε)-mediated ifn-β production was not blocked by pedv, while rig-i-and its adapter molecule ifn-β promoter stimulator 1 (ips-1)-mediated ifn-β production were completely inhibited after pedv infection. conclusion: taken together, our data demonstrated for the first time that pedv infection of its target cell line, iecs, inhibited dsrna-mediated ifn-β production by blocking the activation of ips-1 in rig-i-mediated pathway. our studies offered new visions in understanding of the interaction between pedv and host innate immune system. porcine epidemic diarrhea virus (pedv) is an enveloped, single-stranded, rna virus of coronaviridae family, which is the main etiological agent of severe diarrhea in pigs of all ages and fatality in neonates [1] . outbreaks of porcine epidemic diarrhea (ped) have received extensive attention for the considerable economic losses to the swine industry worldwide. great advances have been made in elucidation of the molecular epidemiology, diagnosis, prevention, and treatment of ped [2] . recently, coronavirus interaction with host innate immune system has been a hot research field. previous studies indicated that transmissible gastroenteritis virus (tgev) infection enhanced type i interferon expression and its protein 7 modulated type i ifn expression [3, 4] . for mouse hepatitis virus (mhv), ifn production among different cell populations varied due to their diverse susceptibility to this virus [5] [6] [7] [8] [9] . furthermore, both severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) do not induce type i ifn (ifn-α/ β) activation [10] [11] [12] . so far, limited reports showed that pedv could inhibit type i interferon production [13, 14] . during viral infection and replication, the host innate immune response is the first line of defense; therefore, the ability of viruses to suppress or avoid this response is crucial for their pathogenic potential. ifn-α/β is an essential element of the host innate immune response against viral infections. double-stranded rna (dsrna), the replicative intermediate of most viruses, is a potent inducer of ifn-β, which is recognized as a pathogen-associated molecular pattern (pamp) by host pattern recognition receptors (prrs). two of major prrs, retinoic acid-inducible gene i (rig-i) and melanoma differentiation-associated gene 5 (mda5) detect dsrna in the cytoplasm [15] . following dsrna binding, rig-i and mda5 recruit corresponding adapter protein ifn-β promoter stimulator 1 (ips-1) that, in turn, activate downstream signaling of tank-binding kinase 1 (tbk1) and inhibitor of κb kinase-ε (ikkε) transduction, leading to the activation of transcription factor ifn regulatory factor 3 (irf-3) and nuclear factor-kappab (nf-κb). activated irf-3, and nf-κb bind to ifn-β enhancer and initiate ifn-β transcription [16] . vero cell, an african green monkey kidney cell line, was often used to isolate and propagate pedv [17] . however, it was often considered that vero cells might lack genetic component necessary for ifn production [18] [19] [20] . porcine intestinal epithelial cells (iecs) are thought to the target cells of pedv, which play an important role in the activation of host immune responses by induction of key signaling molecules, including cytokines, surface molecules, and chemokines during microoganism invasion [21, 22] . in the present study, to determine if pedv infection suppresses ifn-β activation, we chose iecs as an infection model to research the molecular mechanisms of pedv infection and the host antiviral innate immune response. our results clearly suggested that pedv prevented dsrna-induced ifn-β synthesis by blocking rig-i-mediated pathways. pedv failed to induce ifn-β expression and inhibited poly (i:c)-mediated ifn-β production in iecs type i ifns (ifn-α/β) are critical to the host antiviral innate immune response. however, there is no evidence suggesting that iecs produce type i ifns in response to pedv infection. previous studies have showed that pedv could be propagated in iecs [ 23, 24,] . to confirm whether pedv infection could induce ifn-β production in iecs or not, we transiently cotransfected the ifn-β/ luciferase reporter plasmid (ifn-β-luc) and the renilla luciferase construct phrl-tk and then infected with pedv (at an moi of 1 or 0.1, respectively) or mockinfected for 24 h. the cells were retransfected with 1 μg of poly (i:c) as a positive inducer. as shown in fig. 1a , ifn-β luciferase activity enhanced markedly in positive controls, while it was almost not detected in pedv-infected iecs. in addition, ifn-β mrna expression was hardly detected in pedv-infected iecs similar to mock-infected group, however, it had significant expression in poly (i:c)-transfected group at the indicated times (12 h and 24 h, p < 0.01) (fig. 1b) . this result was consistent with the luciferase reporter assay. taken together, pedv infection of iecs did not induce ifn-β activation. increasing evidence showed that viruses not only inhibit the induction of type i ifns, but also block dsrnainduced production of type i ifns to escape the innate immune surveillance of the host [25] [26] [27] . to identify whether pedv was able to inhibit dsrna-induced ifn-β production, ifn-β-luc was transfected into pedv-infected and uninfected cells, respectively. the cells were retransfected with or without poly (i:c) 24 h later. as a result, activation of the ifn-β promoter decreased significantly in poly (i:c)-transfected, pedvinfected cells compared with mock-infected cells transfected with poly (i:c) (fig. 1c) . it showed pedv also inhibited poly (i:c)-mediated ifn-β induction. pedv impeded poly (i:c)-mediated activation of irf-3, but not nf-κb irf-3 and nf-κb are two essential ifn-β transcription factors. in unstimulated cells, irf-3 is ubiquitously present in the cytoplasm as an inactive monomer, whereas nf-κb is present as a homodimer or heterodimer bound to the inhibitory proteins iκb in the cytoplasm [28] . phosphorylation, which is a key step during irf-3 and nf-κb activation, in turn, leads to nuclear translocation. therefore, we evaluated whether pedv infection induced irf-3 and p65 activation by western blot analysis. following pedv infection, the whole cell extracts were prepared for the indicative times, in fig. 2a , irf-3 still existed in the cytoplasm, and the levels of irf-3 protein was almost equal with mock-infected cells, while the phosphorylation of irf-3 (p-irf-3) did not detect in the pedv infected cells in comparision to a obviously signal in poly (i:c)-transfected cells. on the contrary, compared with the amount of nf-κb subunit p65 in the cytoplasm, the phosphorylation of p65 (p-p65) increased with progression of pedv infection in the nucleus. meanwhile, the concentration of poly (i:c)induced p65 nuclear translocation was clearly increased. pedv n protein was also detected in pedv-infected cells. these data suggested that pedv did not induce activation of irf-3, but nf-κb. we then used luciferase reporter assay system to determine whether irf-3 and nf-κb are linked with the inhibition of ifn-β production after pedv infection. as shown in fig. 2b , irf-3 luciferase activity was sharply decreased in pedv-infected cells, and poly (i:c)-induced irf-3 activation was also inhibited by pedv in comparison to a remarkably signal in poly (i:c)-transfected cells. however, in fig. 2c , compared with mock-infected to detect the activation of irf-3 and p65 after pedv infection, the cell extracts were prepared at the indicated times and subjected to western blot analysis with antibodies specific for irf-3, p65, p-irf-3, p-p65 and pedv n mcab. anti-β-actin was included as a control for sample loading. these experiments were performed in duplicate. b and c iecs were infected or mock-infected with pedv at an moi of 1 for 12 h, and then cells were cotransfected with (prdiii-i) 4-luc (b) or pnf-κb-luc (c) and phrl-tk for additional 24 h. cells were retransfected with poly (i:c) for 12 h, harvested, and then subjected to a dual-luciferase assay. all data are expressed as means ± sd of 3 independent experiments. **p < 0.01 as compared with poly (i:c). d irf3-gfp fusion protein transfected with iecs and then infected with pedv at an moi of 1 and mock-infected cells served as negative controls. 24 h later, cells were retransfected with poly (i:c) (positive control) (c and d) or untransfected (a and b) for 12 h. cells were fixed with 4 % paraformaldehyde, permeabilized with 0.1 % triton x-100, and stained by dapi (blue). cells were incubated with anti-pedv rabbit polyclonal antibody (red) and tritc-labeled goat anti-rabbit secondary antibody, then analyzed for fluorescence by confocal microscopy. magnification, ×20 (leica, wetzlar, germany) (see figure on previous page.) fig. 1 pedv does not induce ifn-β production and inhibits poly (i:c)-mediated ifn-β induction. a iecs were cotransfected with ifn-β-luc and phrl-tk, then infected with pedv at an moi of 1 and 0.1 for 24 h. cells were retransfected with poly (i:c) as a positive control. after 12 h, the cells were harvested and subjected to a dual-luciferase assay. b iecs were infected with pedv at an moi of 1, mock-infected as a negative control, or transfected with poly (i:c) as a positive control. at the indicated time points, total rna was extracted and ifn-β and β-actin mrna were subjected to real-time pcr. rna expression levels were normalized to β-actin. c in contrast to a, iecs were first mock-infected or infected with pedv at an moi of 1 for 12 h and then cotransfected with ifn-β-luc and phrl-tk for 24 h. cells were retransfected with or without poly (i:c) for an addition 12 h, harvested, and then subjected to a dual-luciferase assay. all data are expressed as means ± sd of 3 independent experiments. *p < 0.05; **p < 0.01 as compared with poly (i:c) cells, nf-κb luciferase activity significantly enhanced both in pedv-infected and poly (i:c)-transfected cells. in addition, we found that poly (i:c)-induced activation of nf-κb was not blocked by pedv. to further identify pedv-inhibited poly (i:c)-mediated activation of irf-3, confocal microscopy assay was used. as a result, irf3-gfp remained in the cytoplasm of both mockinfected ( fig. 2d. a) and pedv-infected (fig. 2d . b) iecs compared with poly (i:c) controls, in which clear translocation to the nucleus was observed (fig. 2d. d) . furthermore, pedv could block poly (i:c)-mediated irf-3 nucleus migration (fig. 2d. c) . taken together, our date clearly implied that pedv impeded dsrna-mediated ifn-β transcription primary by interfering with irf-3 activation, but not nf-κb. pedv failed to block tbk1/ikkε activity tbk1 and ikkε are essential kinases for the irf-3 activation [29] . in order to ascertain whether pedv inhibited poly (i:c)-induced irf-3 activation by impeding tbk1/ikkε kinase activity, we cotransfected plasmids expressing tbk1/ikkε kinase and a plasmid encoding the ifn-β promoter of the luciferase reporter into infected or mock-infected iecs and retransfected the cells with or without poly (i:c) at 36 h.p.i. as show in fig. 3 , tbk1/ikkε overexpression increased ifn-β promoter activity in both infected and mock-infected iecs, and obviously upregulation was detected in iecs transfected with poly (i:c), suggesting that pedv failed to block tbk1/ikkε activity. however, poly (i:c)-induced ifn-β promoter activity in iecs overexpression of tbk1/ ikkε plasmids was significantly inhibited by pedv. it showed that pedv interrupting dsrna-induced ifn-β production should localize upstream from tbk1/ikkε. it is possible that pedv blocks poly (i:c)-mediated ifnβ production by suppression of the individual molecules upstream of tbk1/ikkε in rig-i signaling pathway. to explore this possibility, mock-and pedv-infected iecs were cotransfected with ips-1 expression plasmid and ifn-β promoter luciferase reporter plasmid, respectively. as shown in fig. 4a , overexpression of ips-1 in iecs could enhance ifn-β luciferase activity in mock-infected cells, but it was completely inhibited in pedv-infected cells. for the poly (i:c) transfection experiments, there appeared significant restriction of ifn-β luciferase expression in pedv-infected cells compared with that of mock-infected cells. these date indicated that pedv interacted with ips-1 to block poly (i:c)-mediated ifn-β transcription. ips-1 is an adapter molecule of rig-i, the data showed that pedv blocked ips-1-induced ifn-β production in dsrna signaling pathway, thus, we speculated that rig-i-induced ifn-β production in this signaling pathway was also inhibited. to verify it, mock-and pedvinfected iecs were transfected with rig-i expression and ifn-β reporter plasmids. the results showed that ifn-β luciferase activity was markedly increased in iecs overexpressing rig-i, but was completely inhibited by pedv infection. and the ifn-β reporter signal could be observed in iecs stimulated with poly (i:c), while this signal could be sharply reduced in rig-itransfected, poly (i:c)-stimulated and pedv-infected iecs (fig. 4b) . in summary, the findings of the present study suggested that pedv-infection in iecs inhibits dsrna-induced ifn-β induction by interfering with irf-3 activity associated with rig-i-mediated signaling pathway. the target interaction molecules of pedv intervention of dsrnainduced ifn-β production primarily was ips-1. however, as a limitation to this study, host cells may inhibit or activate multiple signaling pathways simultaneously in response to exogenous stimulus, and some other transcription factors may have been blocked or activated in this process. here, we only addressed the mechanisms of pedv-induced inhibition of ifn-β production in relation to the molecules of rig-i signaling pathways in vitro. further studies are needed. overall, elucidation of the cells were harvested and subjected to a dual-luciferase assay. data were analyzed and the ratio of firefly luciferase expression to renilla luciferase activity was shown. all data are expressed as means ± sd of 3 independent experiments. **p < 0.01 compared with pedv-infected, expression plasmids or vector-transfected control influence of pedv evasion of the host innate immune response will aid in the development of antiviral agents to prevent the spread of pedv during the early infection phase. the african green monkey kidney cell line veroe6 and swine small intestine epithelial cells (iecs) [30, 31] were respectively cultured in dulbecco's modified eagle's medium (dmem) and dulbecco's modified eagle's f12 ham medium (dmem-f12) supplemented with 10 % fetal bovine serum at 37°c in a humidified atmosphere of 5 % co 2 . pedv strain cv777 was propagated in veroe6 cells in dmem containing 2.5 μg/ml of trypsin. poly (i:c) was purchased as a sodium salt (sigma-alorch, saint louis, mo, usa) and dissolved in water to obtain a stock solution of 10 mg/ml. the dual-luciferase® reporter assay system was purchased from promega corporation (madison, wi, usa) and monoclonal anti-β-actin antibody was purchased from sigma-aldrich (st. louis, mo, usa). anti-irf-3, anti-p65 anti-p-irf-3 and anti-p-p65 rabbit polyclonal antibodies and secondary horseradish peroxidase (hrp)-conjugated anti-rabbit igg were purchased from cell signaling technology, inc. (beverly, ma, usa). rhodamine isothiocyanate (tritc)-labeled goat anti-rabbit igg were purchased from the zhongshan company (beijing, china). anti-pedv rabbit polyclonal antibodies and anti-pedv n protein monoclonal antibody (mcab) were prepared in our laboratory, which could specifically react with pedv. the plasmids ifn-β-luc for ifn-β, prd (iii-i) 4-luc for irf-3, and pnf-κb-luc for nf-κb were kindly donated by dr. shaobo xiao (huazhong agricultural university, wuhan, hubei province, china) [25] . the pef-bos empty vector and pef-flag-rig-i recombinant expression plasmid were kindly provided by t. fujita (tokyo metropolitan institute of medical science, tokyo, japan) [32] . the pef-bos-flag-trif, pcdna3-flag-ikkε and pcdna3-flag-tbk1 recombinant expression plasmids, and the pcdna3 empty vector, and the conjugate irf3-green fluorescence protein (gfp) expression construct were kindly provided by k. fitzgerald (university of massachusetts medical school, worcester, ma, usa) [27] . the porcine ips-1 (ncbi accession no: eu082069.1) gene was cloned from porcine kidney cells by reverse transcription polymerase chain reaction (rt-pcr) using the specific primer pair. the ips-1 primers were 5′-ccgggtaccaccatgacgtttgccgaggacaa-3′ and 5′-tttctcgagtcactggggcaggcgccgcc-3′. porcine ips-1 was inserted into pcdna3.1 (+) using the restriction enzymes kpni and xhoi. cells were harvested and luciferase activity was analyzed using a dual-luciferase assay. all data are expressed as means ± sd of 3 independent experiments. **p < 0.01 compared with pedv-infected, expression plasmids or vector-transfected control when the cells reached 70 %-80 % confluence. the cells were then infected or mock-infected with pedv for 24 h. cells were retransfected with or without poly (i:c) (1.0 μg) for an additional 12 h. or iecs were infected or mock-infected with pedv for 12 h prior to transfection the luciferase reporter plasmids alone or cotransfection the indicated expression plasmids (0.5 μg). the cell lysates were harvested and luciferase activity was analyzed using a dual-luciferase assay system and a luminometer (turner biosystems, inc. sunnyvale, ca, usa) according to the manufacturer's instructions. data represent relative firefly luciferase activity normalized to renilla luciferase activity. the resulting ratios were used to compare the expression of the firefly luciferase gene in pedvinfected cells to that present in mock-infected cells. total rna was extracted from the transfected cells using triquick reagent (beijing solarbio science & technology co., ltd., beijing, china) according to the manufacturer's instructions and then reverse-transcribed into complementary dna (cdna) using murine leukemia virus reverse transcriptase (gbi labs/golden bridge international, inc., mukilteo, wa, usa) with oligo dt random hexamers (haigene technology, harbin, china). the cdna was then subjected to real-time pcr with specific primer pairs targeting ifn-β (f: 5′-gctaac aagtgcatcctccaaa-3′ and r: 5′-ccaggagc ttctgacatgcca-3′) and β-actin (f: 5′-ggctcag agcaagagaggtatcc-3′, and r: 5′-ggtctcaaa catgatctgagtcatct-3′. β-actin mrna was used as an endogenous control. iecs were seeded in 24-well plates and then transfected with 1 μg of irf3-gfp fusion expression constructs per well using lipofectin transfection reagent (invitrogen corp.) when cells reached confluence. cells were then mock-infected or infected with pedv at a multiplicity of infection (moi) of 1. at 24 h postinfection, cells were transfected with 1 μg of poly (i:c) or left untransfected. after 12 h, cells were removed from the culture medium and washed three times in cold phosphatebuffered saline (pbs). next, cells were fixed in 4 % paraformaldehyde for 15 min at room temperature, quenched with 0.1 m glycine for 5 min, and then permeabilized with 0.1 % triton x-100 for 10 min. afterward, the cells were incubated with anti-pedv antibody (dilution, 1:500) for 1 h followed by tritclabeled goat anti-rabbit secondary antibody (dilution, 1:200) for 30 min at 37°c. the nuclei were stained with 4′,6-diamidino-2-phenylindole-dihydrochloride (dapi) (invitrogen corp.). cells were examined using a tcs sp2 aobs confocal microscope (leica camera ag, wetzlar, germany). iecs were infected with pedv at an moi of 1 or treated with poly (i:c) for the indicative times, lysed in 2× sodium dodecyl sulfate (sds) sample buffer and boiled for 10 min. whole cells extracts were separated by 12 % sds-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane, which was blocked with 5 % (w/v) bovine serum albumin (bsa) in tris-buffered saline (10 mm tris-cl at ph 7.5 and 150 mm nacl) containing 0.05 % tween 20 (tbst) at room temperature for 1 h. the membranes were then incubated with a primary antibody (dilution, 1:1000) at 4°c overnight and a secondary hrp-conjugated antibody (dilution, 1:2500) for 1 h at room temperature. protein blots were developed using an enhanced chemiluminescence (ecl) detection system and exposed to x-ray film (clinx science instruments co., ltd., shanghai, china). all data were expressed as means ± standard deviations (sd) of 3 independent experiments. the statistical significance was tested by student 's t-test and p-values less than 0.05 were considered statistically significant. porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines induction of alpha interferon by transmissible gastroenteritis coronavirus: role of transmembrane glycoprotein e1 alphacoronavirus protein 7 modulates host innate immune response the biological relationship of mouse hepatitis virus (mhv) strains and interferon: in vitro induction and sensitivities murine coronavirus induces type i interferon in oligodendrocytes through recognition by rig-i and mda5 inhibition of the alpha/beta 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paramyxoviruses bind the ifn-inducible rna helicase, mda-5, and inhibit its activation of the ifn-beta promoter virus induction of human ifn beta gene expression requires the assembly of an enhanceosome propagation of the virus of porcine epidemic diarrhea in cell culture defectiveness of interferon production and of rubella virus interference in a line of african green monkey kidney cells (vero) regulation of the interferon system: evidence that vero cells have a genetic defect in interferon production sars coronavirus and innate immunity first line of defense: the role of the intestinal epithelium as an active component of the mucosal immune system characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine porcine epidemic diarrhea virus infection induces nf-kappab activation through the tlr2, tlr3, and tlr9 pathways in porcine intestinal epithelial cells porcine aminopeptidase n mediated polarized infection by porcine epidemic diarrhea virus in target cells porcine reproductive and respiratory syndrome virus (prrsv) suppresses interferon-beta production by interfering with the rig-i signaling pathway hepatitis a virus suppresses rigi-mediated irf-3 activation to block induction of beta interferon human rhinovirus attenuates the type i interferon response by disrupting activation of interferon regulatory factor 3 induction of irf-3/-7 kinase and nf-kappab in response to double-stranded rna and virus infection: common and unique pathways ikkepsilon and tbk1 are essential components of the irf3 signaling pathway porcine epidemic diarrhea virus e protein causes endoplasmic reticulum stress and upregulates interleukin-8 expression porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin-8 expression submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank shaobo xiao, takashi fujita, kate fitzgerald for kindly providing important constructs. this work was supported by the programme for new century excellent talents at the heilongjiang provincial university all authors declare that there are no financial or other relationships that might lead to a conflict of interests.authors' contributions lyc response for carrying out the experiments, date analysis and drafting the manuscript. xyg and yg construct the recombinant expression plasmids. ydr participated in date analysis. gh, xfr and gxl designed the experiments and reviewed manuscript. all authors have seen and approved the manuscript and have contributed significantly to the work. key: cord-301175-6alsigxk authors: okda, faten; liu, xiaodong; singrey, aaron; clement, travis; nelson, julie; christopher-hennings, jane; nelson, eric a.; lawson, steven title: development of an indirect elisa, blocking elisa, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to north american strains of porcine epidemic diarrhea virus date: 2015-08-01 journal: bmc vet res doi: 10.1186/s12917-015-0500-z sha: doc_id: 301175 cord_uid: 6alsigxk background: recent, severe outbreaks of porcine epidemic diarrhea virus (pedv) in asia and north america highlight the need for well-validated diagnostic tests for the identification of pedv infected animals and evaluation of their immune status to this virus. pedv was first detected in the u.s. in may 2013 and spread rapidly across the country. some serological assays for pedv have been previously described, but few were readily available in the u.s. several u.s. laboratories quickly developed indirect fluorescent antibody (ifa) assays for the detection of antibodies to pedv in swine serum, indicating prior exposure. however, the ifa has several disadvantages, including low throughput and relatively subjective interpretation. different serologic test formats have advantages and disadvantages, depending on the questions being asked, so a full repertoire of tests is useful. therefore, the objective of this study was to develop and validate multiple improved serological assays for pedv, including an indirect elisa (ielisa); a highly specific monoclonal antibody-based blocking elisa (belisa); fluorescent microsphere immunoassays (fmia) that can be multiplexed to monitor exposure to multiple antigens and pathogens simultaneously; and a fluorescent focus neutralization assay (ffn) to measure functional virus neutralizing antibodies. results: a recombinant north american nucleoprotein (np) based ielisa was developed and validated along with a belisa using newly developed pedv-np specific biotinylated monoclonal antibodies (mabs) and an fmia using magnetic beads coupled with expressed na pedv-np. receiver operating characteristic (roc) analysis was performed using swine serum samples (ielisa n = 1486, belisa n = 1186, fmia n = 1420). the roc analysis for the fmia showed estimated sensitivity and specificity of 98.2 and 99.2 %, respectively. the ielisa and belisa showed a sensitivity and specificity of 97.9 and 97.6 %; and 98.2 and 98.9 %, respectively. inter-rater (kappa) agreement was calculated to be 0.941 between ielisa and ifa, 0.945 between belisa and ifa and 0.932 between fmia and ifa. similar comparative kappa values were observed between the ielisa, belisa and fmia, which demonstrated a significant level of testing agreement among the three assays. no cross-reactivity with the closely related coronaviruses, transmissible gastroenteritis virus (tgev) or porcine respiratory coronavirus (prcv) was noted with these assays. all three assays detected seroconversion of naïve animals within 6–9 days post exposure. the ffn assay allows relative quantitation of functional neutralizing antibodies in serum, milk or colostrum samples. conclusion: well-validated ielisa, belisa and fmia assays for the detection of pedv antibodies were developed and showed good correlation with ifa and each other. each assay format has advantages that dictate how they will be used in the field. newly developed mabs to the pedv-np were used in the belisa and for expediting ffn testing in the detection and quantitation of neutralizing antibodies. in addition, these pedv mabs are useful for immunohistochemistry, fluorescent antibody staining and other antigen-based tests. measurement of neutralizing antibody responses using the ffn assay may provide a valuable tool for assessment of vaccine candidates or protective immunity. porcine epidemic diarrhea virus (pedv) was first described in europe in the 1970s with more recent and severe outbreaks in asia [1, 2] . the virus was identified in the united states in may 2013, causing severe diarrhea and vomiting in pigs across age groups, with high mortality of up to 90 −95 % in suckling pigs [3] . pedv is an enveloped, single stranded rna virus belonging to the coronaviridae family. the coronaviruses taxonomically form a subfamily (coronavirinae) within the order nidovirales. recently, the international committee on taxonomy of viruses (ictv) recognized four genera within the coronavirinae subfamily: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus [4] . pedv belongs to the genus alphacoronavirus along with other swine viruses including transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv). the genome is composed of a large~28 kb molecule consisting of a 5′ untranslated region (utr), a 3′ utr, and at least seven open reading frames (orfs) encoding three nonstructural proteins: orf1ab (pp1a and pp1ab) and orf3, an accessory protein. the four major structural proteins of the mature virion include the spike (s) glycoprotein (mr 150-220 kda), the nucleoprotein (np) (mr 45-57 kda) that is associated with the positive stranded rna providing integral support for its helical structure, the glycosylated membrane protein (m) (mr 20-30 kda), and the glycosylated envelope protein (e) (mr 7 kda) [5] [6] [7] . coronaviruses are taxonomically assigned to different genera based on their rooted phylogeny and calculated evolutionary distance for seven highly conserved genomic domains within orf 1ab [8] . the genetic diversity of coronaviruses may be due to their high frequency of recombination [9] . the heterogeneity among coronavirus subfamilies is well documented [7] , and the factors that contribute to pedv's ability to gain or lose parts of its transcriptome are believed to have contributed to quasispecies with novel traits that are able to adapt to new hosts, ecological niches and zoonotic events. the exact origin of pedv in north america is not entirely clear, but there is evidence of genetic similarities to chinese pedv strains [10] . recently, a novel na pedv recombinant strain was identified (s indel) containing both insertions and deletions within the n-terminal domain of the orf 3 and s1 genes. specifically, sequence alignment indicated spike gene nucleotide deletions at positions 164-169 that correspond to amino acid deletions at positions 55 and 56 in addition to substitutions at positions 23 (i), 31 (h), 57 (k), and 59 (e) as compared to the cv777strain [10, 11] . the relatedness of several pedv strains circulating in china was evaluated by li et al. [5] using phylogenic analysis of the np gene and no insertions or deletions were noted. sequence comparison with other european and korean pedv strains obtained from genbank indicated that the np genes were highly conserved (94.7−97.7 %) even though these strains originated from different geographic regions [5] . in addition to being highly conserved among pedv variants, the np is the most abundant viral protein expressed in pedv infected cells [12, 13] . in contrast, the spike protein is presented on the viral surface and subject to various host immune pressures, which predisposes it to a greater range of genetic heterogeneity including insertions and deletions. because the np protein is highly abundant in virus infected cells, it provides an attractive target for the development of antigen-based serological assays. taken together, this evidence provided rationale for using it as our antigen of choice for the ielisa, belisa and fmia. in response to the recent outbreaks of highly virulent pedv in north america (na), pcr assays were quickly developed to detect the presence of pedv rna in intestine or fecal material. these assays provide an important tool in control of the virus; however, well-validated, high-throughput assays to detect antibodies following infection would provide additional valuable diagnostic tools for the swine industry. the ability to detect and evaluate antibody responses using serologic tests is important in efforts to answer basic production related questions. these questions may include whether a production site is naïve or has historically experienced a pedv exposure, even though a producer has not seen obvious clinical signs; the level of immune response sows may have in relation to vaccination, initial wildtype virus infection or intentional feedback exposure; and whether sow immunity is inadequate when clinical infection occurs in individual litters after initial pedv exposure in a herd. one of the most pressing issues of pedv disease is maintaining herd site biosecurity through exclusion measures to prevent viral entrance into swine units. however, pedv infection may not always be obvious in finishing pigs so the widespread transport of these animals may represent additional risks. thus, sensitive serological tests provide a valuable tool in the detection of recent infection to avoid the introduction of these animals into naïve herds. since pedv was widespread in europe in the 1970s and 1990s and more recently in asia, various serologic tests have been developed and subjected to varying degrees of validation [14] [15] [16] [17] [18] [19] [20] . however, few assays have been developed using antigens associated with contemporary strains currently circulating across na. the need to develop more sensitive serological assays has become paramount in order to address questions regarding pedv infections and epidemiological transmission patterns, as well as to analyze disease progression. currently, serum virus neutralization (svn) tests are the most widely employed serological assays used to detect pedv antibodies. it is a test that is highly specific and useful for screening of antibody titer post vaccination [1, 16] . however, the test is expensive and labor intensive, requiring manual reading and interpretation of virus induced cytopathic effect (cpe) endpoints. moreover, serum cytotoxicity can be mistaken for viral cpe, giving rise to false interpretations at lower serum dilutions. several laboratories have generated in-house indirect elisas using either virus derived antigen or recombinant structural proteins. early indirect elisas were developed using vero cell derived, whole virus preparations [15, 21] or vero cell expressed viral proteins [16] . these methods may be problematic because serum from animals vaccinated with cell culture derived pedv may cross-react with cellular components of elisa antigen, causing low specificity and high background. other groups have used recombinantly expressed, purified, structural s and np proteins for ielisa serodiagnosis, but because low numbers of experimentally derived samples were used to evaluate the performance of the assay, full validation of the diagnostic sensitivities and specificities could not be assessed [19, 20] . both the ielisa and belisa formats have proven useful for the serodiagnosis of experimental and natural infections. blocking or competitive elisas have been shown to be especially useful where a higher level of specificity is required. the increased specificity has been shown to be dependent on both the isotype and on the target specificity of the monoclonal or polyclonal antibodies [6, 17, 22] . various laboratories have developed sensitive blocking elisas, and carvajal et al. [17] demonstrated their belisa was able to detect an antibody response 3 to 5 days earlier than ifa, which suggested higher sensitivity of the belisa. in addition, the belisa is valuable as a confirmatory test where unexpected positive results appear in presumably negative herds. the fluorescent microsphere immunoassay is based on fluidic, particle array technology (luminex corp., austin, tx) and has become increasingly standardized and accepted in applications involving the serologic diagnosis of autoimmune and animal infectious diseases [23, 24] . there are distinct advantages of the fmia over the elisa, which include higher sensitivity, higher sample throughput analysis, and the ability to multiplex and monitor exposure to multiple pathogens simultaneously in a single sample. in addition, multiple bead sets in the fmia could be added to a standardized assay against newer virus subtypes that continue to emerge in the field or to assess antigenic/phylogenetic differences between genera of coronaviruses. in this study, we report the adaptation of a recombinant, highly purified, na pedv-np antigen to the development of ielisa, belisa and fmia platforms for the detection of pedv antibodies in serum. these assays provide high throughput serological tests designed to address pedv disease diagnostics. they were fully validated using a large number of serum samples of known status, and validation of the tests was detailed using methods for the validation of serological assays for the diagnosis of infectious diseases previously described by jacobson for the office international des epizooties [25] . in addition, a fluorescent focus neutralization (ffn) assay was developed for the rapid evaluation of neutralizing antibody responses. procedures involving animals were approved by the south dakota state university institutional animal care and use committee (iacuc) under approval numbers 13-054a and 04-a034. time course swine serum samples provided by kansas state university were part of a separate pedv challenge study conducted at the biosecurity research institute approved by the kansas state university institutional animal care and use committee. all other serum samples were obtained as routine diagnostic sample submissions at the south dakota adrdl. for time course studies, serum samples from experimentally infected animals were obtained courtesy of dr. richard hesse (kansas state university veterinary diagnostic laboratory, national pork board grant #13-228). thirty-three pedv naïve 3-week-old feeder pigs were obtained from a private, high-health status swine production farm. . of the 33 pigs, 23 were inoculated with pedv at 4 weeks of age via intranasal and oral routes with a pool of gut derived intestinal contents that had been used as "feedback" inocula for controlled exposure of a sow herd. serum was collected prior to challenge and at days 0, 6, 9, 14, 21, 28, 35 and 43 days post-infection (dpi). multiple aliquots of all samples collected were shared with requesting laboratories to expand diagnostic testing and vaccine development capabilities. to accurately assess the diagnostic sensitivity and specificity of the assays, samples of known serostatus for pedv were used. this included sera from multiple animal populations including experimentally infected animals and serum samples from animals with known historical exposure to pedv that were submitted to the south dakota animal disease research and diagnostic laboratory (adrdl). pedv negative sample sets included samples from pedv negative control pigs used in experimental studies and selected high biosecurity herds with no history of pedv. in addition, archived serum samples collected prior to the emergence of pedv in the u.s., including samples testing positive for the related swine coronaviruses tgev and prcv (n= > 50), were used. the exact number of positive and negative sera used for sensitivity and specificity calculations per assay with statistical testing agreement calculations based on serum numbers is listed in table 1 . the majority of these sera were identical among assays, but limited serum volume did not allow for use of all sera samples among all assays. the development and validation of the ielisa and belisa made use of a recombinantly expressed full length na pedv-np. the np open reading frame (orf) of pedv was amplified from rna extracted directly from intestinal contents by rt-pcr from a case submitted to the south dakota adrdl. it was subsequently directionally cloned into the e. coli, pet 28a(+), plasmid expression vector (novagen, madison, wi), then transformed into bl21-codon plus (de3)-rp competent cells (stratagene, la jolla, ca) for protein expression. primers used for the amplification of the full length (1323 bp) nucleoprotein were: pedv-np-fwd (5′-cg cggatccatggcttctgtcagttttcag-3′); ped v-np-rev (5′-cacactcgagatttcctgtgtcgaa gatctc-3′). next, 20 μl of transformed cells were plated onto luria-bertani agar plates containing 50 μg of kanamycin/ml and incubated overnight. the following morning, colonies from the agar plates were added to 1 l of pre-warmed 2x yeast extract tryptone (yt) culture medium containing 50 μg kanamycin/ml and allowed to grow to an od 600 of 0.5 at 37°c. pedv-np expression was induced using isopropyl β-d-1-thiogalactopyranoside (iptg) at a final concentration of 1.0 mm to induce transcription of the lac operon, and the e. coli was allowed to incubate for an additional 8 h at 37°c with shaking at 200 rpm. the agar was strained out and bacteria pelleted by centrifugation at 12,000 g for 10 min at 4°c. the pellet was resuspended in 40 ml of lysis buffer solution (b-per, pierce, rockford, il), incubated for 15 min at 20-22°c, then centrifuged at 12,000 g to separate the soluble from insoluble proteins. the pedv-np recombinant protein was expressed as insoluble periplasmic inclusion bodies. the resulting 441 amino acid recombinant protein was denatured using 8 m urea, subsequently purified three times using nickel-nta affinity column chromatography and refolded back to its native conformational state. individual affinity column elutions were collected, pooled and confirmed by sds-page, then aliquoted/frozen at −80°c. the correct nucleotide sequence was confirmed by sequence and restriction endonuclease analysis. the average protein yield produced by the pet28a-pedv-np plasmid construct was calculated to be 11 mg pedv-np per liter of 2xyt under the aforementioned conditions. the recombinant protein was detected and a predicted molecular weight of 51 kda was confirmed via western blotting using convalescent sera, a 6x histidinespecific mab (novagen, madison, wi) and a pedv-np specific mab (figs. 1 and 2). two separate mabs were developed in our laboratory (sd6-29 and sd17-103) that recognize both the native conformation of the pedv-np and the full length, linear, recombinant protein used in all antibody capture assays. hybridomas were produced as previously described [26, 27] . immunoglobulin isotyping of the resulting mabs was performed using a commercial lateral flow assay (serotec, raleigh, nc). subsequently, mouse ascites fluid was produced in pristane-primed mice, and the antibodies were purified and biotinylated for use as the detection moiety for the belisa [23] . the conjugated antibody solution was quantified via the lowry protein method, and carrier bsa was added to a final concentration of 10 mg/ml, then aliquoted and stored at −20°c. after 1 h of incubation at 37°c, plates were rinsed 3x with pbs and examined using fluorescent microscopy. for each individual test, each pedv infected well was compared to its respective uninfected partner well, and a positive sample was indicated if a pedv specific fluorescent signal was observed at a serum dilution of 1:40 or greater. all samples were tested in duplicate, and the antibody titer was expressed as the mean of all replicates. ielisa the serological pedv-np indirect elisa was performed by coating alternate wells of immulon 1b, 96-well, microtiter plates (thermo labsystems, franklin, ma) with 250 ng/well of purified, recombinantly expressed pedv-np antigen. the optimal dilution of the recombinant protein and secondary detection antibody was determined by a checkerboard titration that gave the highest signal to noise ratio. in addition, a single lot of pooled convalescent serum from pedv infected pigs was used to generate quality control standards that gave high and low optical density (high od = 2.0 to 2.5; low od = 0.5 to 1.0; and negative od < 0.2). pedv-np recombinant protein was diluted to 2.5 μg/ml in 15 mm sodium carbonate-35 mm sodium bicarbonate-antigen coating buffer (acb) ph 9.6. odd-numbered columns were coated with 100 μl of acb plus antigen, while the evennumbered columns were coated with acb without antigen, serving as background control. the plates were incubated for one hour at 37°c, then washed 3x with pbs plus 0.05 % tween 20 (pbst). each well was then blocked with 200 μl of sample milk diluent (pbst plus 5 % nonfat dry milk, (smd)) and allowed to incubate overnight at 4°c. the following day, the plates were washed 3x with 300 μl of pbst. test and control sera were diluted 1/50 in smd, mixed, and 100 μl of the solution was added to each well. the plates were incubated for 1 h at 20-22°c. next, 100 μl of biotinylated, goat anti-swine detection antibody (bethyl laboratories, tx) was added at a concentration of 200 ng/ml of pbst and allowed to incubate at 20-22°c for 1 h. the plate was washed 3x with 300 μl of pbst, then 100 μl of streptavidin-hrp conjugate (pierce, rockford, il) was added and incubated for another hour at 20-22°c, then washed and developed with 3,3′,5,5′-tetramethylbenzidine, peroxidase substrate (tmb) (surmodics, eden prairie, mn). color development progressed until the positive control attained a standard od and was stopped using 2 n h 2 so 4 . colorimetric development was quantified spectrophotometrically at 450 nm with a elx800 microplate reader (biotek instruments inc., winooski, vt) controlled by xcheck software (idexx laboratories, westbrook, me). the raw data was normalized and transformed into an excel spreadsheet. sample to positive (s/p) ratios were calculated using the following formula: s/p = optical density (od) of sample -od of buffer/od of positive control -od of buffer. belisa the serological belisa was performed using immulon 1b, 96-well microtiter plates (thermo labsystems, franklin, ma). alternate wells of each plate were coated with 500 ng per well of expressed pedv-np antigen. the optimal dilution of the recombinant protein and mab antibody was determined by a checkerboard titration that gave the highest signal to noise ratio with an od reading of approximately 2.0, in the absence of swine serum/competitor antibody. first, pedv-np recombinant protein was diluted to 2.5 μg/ml of acb. odd-numbered columns were coated with 100 μl of acb plus antigen, while the even-numbered columns were coated with acb without antigen serving as background control. the plates were incubated for 1 h at 37°c, washed 3 times with pbst, then placed at 4°c overnight. the following day, each well was blocked with 300 μl of smd and incubated one hour at 37°c. plates were washed 3 times with pbst, and 100 μl of test and control sera were diluted 1/3 with pbst + 0.1 % nonfat dry milk and added to each of the duplicate wells. plates were incubated 1 h at 37°c. during sample incubation, pedv-np specific biotinylated, mabs (sd6-29 and sd17-103) were adjusted to equal titers and mixed together in a 1:1 ratio. next, 100 μl of a 1:40,000 dilution of the antibody detection mixture was added to the microtiter plate containing the competitive swine antibody, then swirled and incubated for an additional 30 min at 37°c. the plates were washed 3 times, and 100 μl of high sensitivity, streptavidin-horseradish peroxidase conjugate (pierce, rockford, il) was added to all wells of the microtiter plate for 1 h at 37°c. plates were washed 4 times with pbst, and 100 μl of tmb was added to all wells and gently swirled. after ap-proximately15 min, color development progressed until the negative control attained a standard od of approximately 2.0 and was subsequently stopped using 2 n h 2 so 4 . colorimetric development was quantified spectrophotometrically at 450 nm with an elx800 microplate reader (biotek instruments inc., winooski, vt) controlled by xcheck software (idexx laboratories, westbrook, me). the raw data was normalized and transformed into a excel spreadsheet, and the percent inhibition (pi) ratio was calculated using the following formula: pi = 1-{(od of sample -od of buffer)/(od of negative control -od of buffer)} x 100. a two-step carbodiimide coupling procedure was used to couple na pedv-np protein to luminex™ microspheres. briefly, the coupling of fluorescent microsphere was performed by washing 3.125 × 10 6 microspheres twice with 250 μl activation buffer (0.1mnah 2 po 4 , ph6.2) and sonicating them for 60 s after each wash. microspheres were activated for 20 min at 20-22°c in 500 μl activation buffer containing 2.5 mg n-hydroxysulfosuccinimide (sulfo-nhs) and 2.5 mg n-(3dimethylaminopropyl)-n-ethylcarbodiimide (edc) (pierce chemical, rockford, il). activated microspheres were washed twice with pbs and sonicated. coupling was initiated by the addition of 12.5 μg of recombinant na pedv-np protein, brought to a final volume of 500 μl with pbs and incubated in the dark for 3 h at 20-22°c with rotation. coupled microspheres were washed once with 1 ml of pbs plus 0.05 % nan 3 and 1.0 % bovine serum albumin (pbs-nb). next, the microspheres were blocked with 1 ml of pbs-nb for 30 min to reduce nonspecific binding. microspheres were then washed twice with pbs-nb and resuspended in pbs-nb to a final concentration of 2.0 × 10 6 antigen-coupled microspheres/ml. a 96-well hydrophilic membrane filter plate was blocked for 2 min with 150 μl of pbs-nb, and then the liquid was aspirated via vacuum manifold. the plates were wetted with 20 μl of pbs-nb buffer to prevent drying. next, 50 μl of serum (diluted 1:50 in pbs-nb) was added to duplicate wells of the filter plate along with 50 μl of pbs-nb containing 2.5 × 10 3 antigen-coupled microspheres. since the microspheres and reporter moieties are light sensitive, all incubations were performed in the dark by sealing the plate with foil. subsequently, the fmia plate was incubated at 20-22°c for 1 h on a plate shaker rotating at 600 rpm. the plate was washed 3 times with 200 μl of pbst. next, 50 μl of anti-swine, biotinylated iga (heavy & light chain, diluted in pbs-nb; bethyl laboratories) or igg-fc specific polyclonal antibodies (diluted 1:2,000 dilution in pbs-nb; bethyl laboratories) were added to the filter plate and incubated at 20-22°c for 1 h. np igm and igg isotypespecific antibody levels were detected using pedv-np coated microspheres, but speciated by means of individual and separate igm and igg-specific secondary antibodies. since validation was performed using serum, and because iga is present in very low amounts, iga specific secondary antibodies were not used at this step. after incubation with the secondary antibodies, 50 μl of streptavidin phycoerythrin (2.5 μg/ml in pbs-nb, molecular probes) was added to each well and incubated for 30 min at 20-22°c with shaking. the supernatant was aspirated, and the plate was washed 3 times with pbst. finally, the microspheres were resuspended in 125 μl of pbst per well and transferred to a clear 96-well polystyrene optical plate. coupled microspheres were analyzed through a dual-laser bio-rad bio-plex 200 instrument. the median fluorescent intensity (mfi) for 100 microspheres corresponding to each individual bead analyte was recorded for each well. all reported mfi measurements were normalized via f -f 0 , where f 0 was the background signal determined from the fluorescence measurement of a test sample in uncoated beads and f was the mfi for a serological test sample using antigencoated beads. four serological reference serum sets were constructed as standards termed high, medium, low and negative to serve as internal quality control standards and to mathematically normalize individual samples for objective comparisons between testing platforms. the high-labeled standard was designed to generate an od above 2.0 for the ielisa and belisa and an mfi of approximately 25,000 for the fmia. the high standard was used exclusively for the mathematical determination of the serological response (s/p ratio) of samples used for test validation. the medium standard generated a response of between 1.5 and 2.0 od for the two elisas and approximately 10,000 mfi for the fmia. the low standard was designed to deliver a signal slightly above threshold level for all 3 tests, and the negative serum generated an od or mfi to a background level of less than 0.2 od for the elisas and 600 mfi for the fmia. validation methods for the determination of diagnostic sensitivity, specificity, repeatability and threshold cutoff level to accurately assess the diagnostic sensitivity and specificity of the assays, samples of known serostatus for pedv were used. this included sera from multiple animal populations including experimentally infected animals and serum samples submitted to the south dakota adrdl. pedv negative sample sets included samples from selected high biosecurity herds with no history of pedv and archived serum samples collected prior to the emergence of pedv in the u.s., including samples testing positive for the related swine coronaviruses tgev and prcv. known positive samples were collected from pigs that were naturally infected at least 3 weeks prior to collection and were previously positive by pcr. the negative-testing sample population (uninfected animals) consisted of maximally 980 pedv negative serum samples, while the positive-testing (infected) population was composed of 516 serum samples. receiver operating characteristic (roc) analysis was calculated for each assay to assess diagnostic performance, which included determination of sensitivity, specificity and threshold cutoff using medcalc version 11.1.1.0 (medcalc software, mariakerke, belgium). the repeatability of each assay was assessed by running the same internal quality control serum standards in multiple replicates within the same run or between runs. for the ielisa and the belisa, the intra-assay repeatability was calculated for 48 replicates on 3 separate plates, then repeated over a 3-day period for inter-assay repeatability assessment. the values for each assay were expressed as a mean, standard deviation and percent coefficient of variation (cv%) for repeated measure. multiple comparison, inter-rater agreement (kappa measure of association) was calculated among all four tests (belisa, ielisa, fmia and ifa) using ibm, spss version 20 software (spss inc., chicago, il). the sample cohort used was a well-characterized set of serum samples collected from "positive testing" experimentally infected pigs over time courtesy of dr. richard hesse (n = 158) and from archived experimental control uninfected pedv "negative testing" animals. the interpretation of kappa can be rated as follows: kappa less than 0.0, "poor" agreement; between 0.0 and 0.20, "slight" agreement; between 0.21 and 0.40, "fair" agreement; between 0.41 and 0.60, "moderate" agreement; between 0.61 and 0.80, "substantial" agreement; and between 0.81 and 1.0, "almost perfect" agreement [28, 29] . a pedv virus neutralization assay using a ffn format was developed for rapid detection of neutralizing antibodies produced in response to pedv infection. the ffn was evaluated using serum samples or rennet treated milk and colostrum samples. heat-inactivated samples were diluted in a 2-fold dilution series starting at 1:10 in mem plus 1.5 μg/ml tpck-treated trypsin in 96-well plates. an equal amount of cell culture adapted pedv stock at a concentration of 100 foci forming units/100 μl was added to each well and plates incubated for 1 h at 37°c. the virus/sample mixture was then added to washed confluent monolayers of vero-76 cells and incubated for 2 h at 37°c. plates were washed again with mem/tpck-trypsin medium and incubated 20-24 h to allow for replication of non-neutralized virus. plates were then fixed with 80 % acetone and stained with fitc conjugated mab sd6-29 to allow visualization of infected cells. endpoint neutralization titers were determined as the highest serum, milk or colostrum dilution resulting in a 90 % or greater reduction in fluorescent foci relative to controls. as shown in fig. 1 , the purity of the recombinant protein was assessed via sds-page and gave a band that migrated corresponding to the expected molecular mass of 51 kda upon staining with coomassie brilliant blue r250. the protein yield of the iptg induced e. coli culture was calculated to be approximately 11 mg pedv-np/liter of 2xyt medium with a purity of greater than 95 %. the identity of the protein was further characterized by western blot using convalescent swine serum, an anti-his mab and an anti-pedv-np mab (fig. 2) . to optimize the serologic assays, various antigen and serum dilutions were used to determine optimum concentrations. all 3 tests were optimized in a checkerboard fashion to maximize signal-to-noise ratios. it was determined by antigen titration that the optimal coating of luminex™/fmia microspheres was achieved at a concentration of 12.5 μg protein per 3.125 × 10 6 microspheres. similarly, the optimum coating of both the ielisa and belisa plates was achieved at a concentration of 250 ng/well. in addition, to determine the optimum serum dilution for each of the testing platforms, a well-characterized pedv "high" positive serum standard was serially diluted in a log 2 titration against antigen coated microspheres (fmia) or antigen coated elisa wells at a fixed concentration. figure 3 shows concentration-dependent od or mfi signals of various serum standards. overall, sample absorbance increased inversely proportional to the serum dilution. however, based upon the highest signalto-noise ratio, it was determined that the optimal serum dilution for the belisa was 1/3, while the ielisa and fmia each demonstrated an optimum dilution of 1/50 as indicated by arrows (fig. 3) . roc analysis to determine sensitivity, specificity and threshold cut-off levels was performed using large numbers of swine serum samples and demonstrated excellent agreement (>0.91 kappa scores) between assays with good intra and inter assay repeatability ( table 1) . none of the known positive tgev or prcv samples tested was shown to cross-react. the optimal cutoff values and corresponding sensitivity and specificity of each individual test are presented in fig. 4 . specifically, roc analysis for the ielisa and belisa showed similar sensitivity and specificity of 97.9 and 97.6 %; and 98.2 and 98.9 %, respectively. the roc analysis for the fmia showed estimated sensitivity and specificity of 98.2 and 99.2 %, respectively. although the fmia showed an identical sensitivity as the belisa, it demonstrated the highest degree of specificity of all three assays at 99.2 %. this observation was not surprising given that fmia technology inherently imparts greater sensitivity and a larger dynamic range than the elisa platform [30] . in addition to determining cutoff values, sensitivities and specificities, multiple comparison tests were performed to calculate the degree of agreement among the elisa, fmia and ifa tests. specifically, the kappa test demonstrated all diagnostic platforms had kappa values greater than 0.91, which demonstrates that all 4 tests are in "almost perfect" agreement with each other. the ielisa and belisa demonstrated slightly lower %cvs than the fmia with 3.7 %, 6.8 %, 10.7 % intra-assay variability for belisa, ielisa and fmia respectively. inter-assay %cvs were 5.0, 5.6 and 7.7 % for the belisa, ielisa and fmia respectively. nonetheless, all the cvs were 10.7 % or less, which demonstrated that the tests were highly repeatable in a diagnostic application. as shown in fig. 5 , a mean antibody response to pedv-np could be detected as early as 9 dpi for both the ielisa and belisa. the fmia detected pedv-np antibodies slightly earlier at 6 dpi. all 3 tests detected the duration of antibody out to the 43 dpi time-point in this study but demonstrated a decline in detectable antibody after 21 dpi. high levels of pedv-np specific igm antibodies were observed at 7 dpi compared to igg (fig. 6) . however, the igm antibodies decreased to barely detectable levels by 20 dpi. igg continued to increase linearly to 20 dpi. there is a concomitant appearance of neutralizing antibodies by 14 dpi. the ffn assay was initially evaluated using sequential serum samples from experimentally inoculated piglets. additional evaluation was conducted using 250 serum samples from known pedv naïve herds and 250 samples from herds with documented pedv exposure, collected at least 3 weeks after initial pcr diagnosis and whole herd feedback. experimentally inoculated piglets demonstrated detectable seroconversion by 14 dpi (fig. 6 ). essentially all samples from pedv naïve animals had serum ffn endpoint titers of <1:20 while most samples from the pedv positive set had endpoint titers ranging from 1:40 to 1:1280 (data not shown). further evaluation of the ffn included serum, milk and colostrum samples from 27 sows from a herd that had experienced an acute pedv outbreak 6 to 7 weeks prior to farrowing. all animals were exposed to live virus twice within the first week of the outbreak, followed by one dose of harrisvaccines porcine epidemic diarrhea vaccine, rna (harrisvaccines, inc., ames, ia) at 1 week pre-farrow. serum and colostrum samples were tested at the time of farrowing, followed by serum and milk samples at 1 week and 2 weeks later. as shown in fig. 7 , mean colostrum titers were approximately 4-fold higher than serum titers at the time of farrowing. at later time-points, serum and milk titers were similar in magnitude, although substantial animal to animal variation was apparent. overall, this repertoire of assays is useful for initial identification and efficient, high throughput quantitation of pedv antibodies. we evaluated all three diagnostic platforms against a well characterized ifa and compared the individual serum igm and igg kinetic antibody responses in an fmia to the appearance of neutralizing antibody as detected by the ffn assay. each of the antibody-capture assays was validated using a large number of serum samples (n >1100) based upon the assay validation methods of jacobson, which is supported by the office international des epizooties [25] . since pedv was first identified in the u.s. in may 2013, it has spread rapidly to at least 33 states (www.aasv.org) and has been reported in mexico and canada [31] . the virus causes severe gastroenteritis, destroying villus enterocytes in pigs of all ages, and is characterized by vomiting and diarrhea, leading to subsequent dehydration, high mortality rates and economic losses, particularly in nursery piglets [3, 32] . a variety of serological tests have been developed against pedv, but they vary by antigen used and in the degree of validation. in addition, few have used north american np based antigens or compared the array of serologic assays described here. in the current study, four tests (ifa, belisa, ielisa, fmia) showed strong correlation. each has advantages, which dictate how they will be used in the field. in addition, newly developed np mabs were used in the belisa and for expediting ffn testing in the detection of neutralizing antibodies. in the development of the elisas and fmia, the full length na pedv-np gene was amplified directly from rna extracted from pedv-infected ileal tissue. multiple sequence alignment analysis showed that the amplified np gene shared a 100 % nucleotide homology with that of the us colorado strain isolated in 2013 (genbank accession no. 13-019349). several authors confirm that the np carries multiple antigenic determinants that are conserved among the coronaviridae [33, 34] . however, we performed one-way cross-reactivity testing using serum from tgev and prcv, and no antibody crossreactivity was detected within any of our assays. in addition to being highly conserved among various pedv variants, the np is the most abundant viral protein expressed in pedv infected cells, making it an attractive target antigen [12, 13] . using western blotting experiments, we confirmed the finding of hou et al. [19] , in which they observed the level of expression of np protein to be significantly higher than the level of the spike protein. our study demonstrated that it is possible to achieve a protein yield of over 10 mg per liter of culture with a purity of greater than 95 %. the recombinant np has previously been identified as a useful antigen in other elisas developed to detect antibodies in pigs located in china and korea [19] . in a study by hou et al. [19] , the authors showed similar sensitivities and specificities of their ielisa compared to the ielisa described in this study. however, smaller numbers of known positive and negative samples were evaluated than in the current study. since no test has 100 % specificity, a belisa was developed that is useful for confirmatory testing due to its higher inherent specificity than the ielisa [35] . blocking or competitive elisas have been constructed using monoclonal antibodies in pedv serodiagnosis, and the specific methodology can affect the overall specificity and performance of the assay. our method was based upon coating plates with highly purified na pedv-np, then using a combination of two separate na, anti-pedv-np specific, biotinylated, monoclonal antibodies as the blocking/competitive detection step. this allows the capture of anti-np antibodies at higher quantities and those with a greater range of antigen specificities. the analytical specificity of the np-based belisa is also dependent on the affinity of the mabs used. the antibodies used in this study are directed against conserved epitopes on the nucleocapsid protein without any evidence of cross-reactivity to any other genera of alphacoronavirus tested. a previous assessment of antigenic cross-reactivity was performed using these same mabs against different strains of pedv and tgev [7] . in that study, the authors reported that both mabs used in the belisa reacted with all pedv strains tested, namely the homologous us isolate pc22a and the heterologous strains s indel iowa 106, s 197del pc177 and cv777, at similar titers. neither of the pedv-np mabs cross-reacted with either the tgev miller or purdue strains. not only were the belisa mabs tested for heterologous cross-reactivity, but all three diagnostic platforms were evaluated in their ability to capture antibody against tgev and prcv, and there was no cross-reactivity to either heterologous virus. serology testing with ifa, ielisa, belisa or fmia is useful in determining whether pigs were previously infected with pedv, or if piglets have acquired antibodies through colostrum (eg. passive antibody transfer). however, tests that evaluate the functionality of the antibodies such as the ffn are needed to determine if the detected immune response could be helpful in providing protection to nursing piglets. neutralizing antibodies may be protective through actions including blocking uptake of the virus into cells, preventing virus binding to receptors on cells, preventing uncoating of the virus genomes in endosomes and/or causing aggregation of virus particles. for enveloped viruses, such as pedv, lysis of the virus may also occur when antiviral antibodies and serum complement disrupt the viral membrane. for these reasons, an ffn-based virus neutralization assay was developed to assess levels of pedv neutralizing antibodies in serum, milk or colostrum samples. the ffn provides a more rapid determination of neutralizing antibody levels than is possible with traditional virus neutralization assays that rely on visualization of virusinduced cpe after three or more days incubation to allow for full development of pedv cpe. the direct observation of fluorescent stained infected cells, or lack of stained infected cells in the case of virus neutralization, allows for simple endpoint determination. this feature is particularly valuable when dealing with a fastidious, trypsin-dependent virus such as pedv where cpe-based endpoints may not be obvious or may be confused with trypsin-induced cpe in the cell monolayer. although neutralizing antibodies present in the serum would not be expected to provide direct protection from a strictly enteric infection such as pedv, our data suggest a correlation between detectable neutralizing antibody levels in the serum and those present in milk and colostrum of previously exposed or vaccinated sows. some correlation between pedv neutralization results and elisa results exists as described in the literature. one study performed a comparative analysis between a whole-virus antigen elisa and a serum neutralization test for the serodiagnosis of pedv [21] . the presence of antibodies was confirmed by each test, and an overall testing agreement of 84.2 % was demonstrated using 1024 field serum samples. furthermore, a pairwise correlation was performed that showed corrected cutoff values between the elisa od and sn titers having an r value of 0.837, indicating that the cpe-based neutralization test had roughly the same reliability as the elisa test [21] . newer technologies such as the fmia are useful for the detection of antibodies against multiple antigens simultaneously for surveillance purposes. fmia are bead based assays for simultaneous high throughput detection of antibodies to multiple antigens. the fmia differs from the elisa since it involves a fluid incubation step with "beads suspended in solution, which allows for higher surface area exposure in 3 dimensions" [30] . therefore, there is a shorter diffusion path to antibody binding sites on the antigen coated beads resulting in rapid reaction times. instead of a method using an enzymatic reaction such as with the elisa, the fmia detection is with laser technology, which results in a shorter detection time. this pedv antigen specific bead set can be "mixed" with additional coated beads to other antigens, such as siv, pcv2, prrsv or other pathogens, for simultaneous detection of antibodies to these antigens. in addition, an fmia could be developed for differentiation of wild-type infected vs vaccinated animals (diva) if proteins used in the vaccine were different from those produced in a wild-type infection. individual kinetic serum igg and igm levels were measured by fmia in experimentally infected animals over time. the appearance of the igm subclass is considered an immunological parameter of early infection and generally appears prior to the appearance of igg, and this was confirmed in our study. this was in contrast to the data of woo et al. [36] , which was unable to detect igm antibodies using their np-based indirect elisa. igg antibodies may be more easily detected as they are characterized by higher antigen affinity but lower avidity than igm [37] . further understanding of various antibody profiles will provide important information on the ability of vaccines to stimulate a protective immune response. these well-validated na pedv ielisa, belisa, fmia and ffn assays are useful for a range of serological investigations. they can serve as a complement to nucleic acid detection and determine the pedv status of asymptomatic individuals for cost-effective tools in management strategies and monitoring virus exposure within the herd. the fmia will be useful for isotyping the antibody responses and in multiplexing for determining exposure to multiple pathogens simultaneously. in addition, the ffn is useful for determining whether the antibodies measured are providing a biological function of blocking virus infectivity. work is ongoing to further validate these assays on other sample matrices such as milk and colostrum for measuring passive transfer of antibodies and oral fluids for pen-based surveillance. porcine epidemic diarrhea virus; ifa: immunofluorescent assay; ielisa: indirect enzyme linked immunosorbent assay; belisa: blocking enzyme linked immunosorbent assay; fmia: fluorescent microsphere immunoassay; ffn: fluorescent focus neutralization; orf: open reading frame; utr: untranslated region tgev: transmissible gastroenteritis virus; prcv: porcine respiratory coronavirus; na: north american; cpe: cytopathic effect; dpi: days post-infection; adrdl: animal disease research and diagnostic laboratory sodium dodecyl sulfate-polyacrylamide gel electrophoresis iacuc: institutional animal care and use committee; hat medium: hypoxanthine-aminopterin-thymidine medium; dmso: dimethyl sulfoxide; bsa: bovine serum albumin fbs: fetal bovine serum; nvsl: national veterinary services laboratories moi: multiplicity of infection; tpck: l-1-tosylamide-2-phenylethyl chloromethyl ketone; fitc: fluorescein isothiocyanate; acb: antigen coating buffer; smd: sample milk diluent; hrp: horseradish peroxidase 5′-tetramethylbenzidine; pi: percent inhibition; mfi: median fluorescent intensity; siv: swine influenza virus; pcv-2: porcine circovirus type 2; prrsv: porcine reproductive and respiratory syndrome virus evaluation of antibody response of killed and live vaccines against porcine epidemic diarrhea virus in a field study experimental infection of pigs with a new porcine enteric coronavirus, cv 777 emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences ratification vote on taxonomic proposals to the international committee on taxonomy of viruses sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china an elisa optimized for porcine epidemic diarrhoea virus detection in faeces antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans establishing a genetic recombination map for murine coronavirus strain a59 complementation groups origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states third strain of porcine epidemic diarrhea virus, united states coronavirus immunogens the molecular biology of coronaviruses a new coronavirus-like particle associated with diarrhea in swine enzyme-linked immunosorbent assay for the detection of porcine epidemic diarrhea coronavirus antibodies in swine sera an elisa for detection of antibodies against porcine epidemic diarrhoea virus (pedv) based on the specific solubility of the viral surface glycoprotein evaluation of a blocking elisa using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies development of a porcine epidemic diarrhea virus m protein-based elisa for virus detection development and evaluation of enzyme-linked immunosorbent assay based on recombinant nucleocapsid protein for detection of porcine epidemic diarrhea (pedv) antibodies detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection porcine epidemic diarrhea: kinetics of actively and passively acquired serum antibodies and the effect of reinfection development of an 8-plex luminex assay to detect swine cytokines for vaccine development: assessment of immunity after porcine reproductive and respiratory syndrome virus (prrsv) vaccination development of a fluorescent microsphere immunoassay for detection of antibodies against prrsv using oral fluid samples as an alternative to serum-based assays validation of serological assays for diagnosis of infectious diseases differentiation of u.s. and european isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies antibodies to major histocompatibility antigens produced by hybrid cell lines the measurement of observer agreement for categorical data development and laboratory evaluation of a lateral flow device (lfd) for the serodiagnosis of theileria annulata infection opportunities for bead-based multiplex assays in veterinary diagnostic laboratories distinct characteristics and complex evolution of pedv strains pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs antigenic relationships among homologous structural nucleotide sequences of porcine, feline and canine coronaviruses porcine epidemic diarrhea virus (cv777) and feline infectious peritonitis virus (fipv) are antigenically related critical factors affecting the diagnostic reliability of enzyme-linked immunosorbent assay formats longitudinal profile of immunoglobulin g (igg), igm, and iga antibodies against the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in patients with pneumonia due to the sars coronavirus the distribution and functions of immunoglobulin classes the authors declare that they have no competing interests.authors' contributions fo: conducted elisa and fmia development/validation, statistical analysis and co-wrote paper. xl: conducted elisa and fmia development/validation. as: assisted with sample acquisition, assay development and study design. tc: assisted with sample acquisition and study design. jn: conducted virus neutralization assay development and testing. jch: assisted with study design and co-wrote paper. ean: developed study concept and design, edited paper. sl: directed assay development and validation, co-wrote paper. all authors read and approved the final manuscript. key: cord-308170-uqezwbzn authors: dee, scott; neill, casey; clement, travis; christopher-hennings, jane; nelson, eric title: an evaluation of a liquid antimicrobial (sal curb®) for reducing the risk of porcine epidemic diarrhea virus infection of naïve pigs during consumption of contaminated feed date: 2014-09-25 journal: bmc vet res doi: 10.1186/s12917-014-0220-9 sha: doc_id: 308170 cord_uid: uqezwbzn background: since its initial detection in may 2013, porcine epidemic diarrhea virus (pedv) has spread rapidly throughout the us swine industry. recently, contaminated feed was confirmed as a vehicle for pedv infection of naïve piglets. this research provides in vivo data supporting the ability of a liquid antimicrobial product to reduce this risk. results: sal curb® (kemin industries, des moines, ia, usa) is a fda-approved liquid antimicrobial used to control salmonella contamination in poultry and swine diets. to test its effect against pedv, sal curb®-treated feed was spiked with a stock isolate of pedv (ct = 25.22), which pedv-naïve piglets were allowed to ingest via natural feeding behavior (ad libitum) for a 14-day period. for the purpose of a positive control, a separate group of piglets was allowed to ingest non-treated (sal curb®-free) feed also spiked with stock pedv (ct = 25.22). a negative control group received pedv-free feed. clinical signs of pedv infection (vomiting and diarrhea) and viral shedding in feces were observed in the positive control group 2–3 days post-consumption of non-treated feed. in contrast, no evidence of infection was observed in pigs fed sal curb®-treated feed or in the negative controls throughout the 14-day study period. in addition, the sal curb®-treated feed samples had higher (p < 0.0001) mean pedv ct values than samples from the positive control group. conclusions: these data provide proof of concept that feed treated with sal curb® can serve as a means to reduce the risk of pedv infection through contaminated feed. furthermore, the results from the positive control group provide additional proof of concept regarding the ability of contaminated feed to serve as a risk factor for pedv infection of naïve piglets. porcine epidemic diarrhea virus (pedv) is an enveloped single-stranded positive sense rna virus belonging to the order nidovirales, the family coronaviridae and the genus alphacoronavirus [1] . following detection in the us swine population during may, 2013, the virus spread rapidly across the country with 6659 cases of porcine epidemic diarrhea (ped) confirmed across 30 states as of may 17, 2014 [2, 3] . recently, proof of concept that contaminated feedstuffs can serve as a route of pedv transmission to naïve pigs was reported [4] . this study evaluated the risk of pedv-contaminated complete feed through a novel on-farm sampling method for detection of virus in feed along with an in vivo experiment (swine bioassay) using at-risk feed material and normal feeding behavior [4] . as this new information confirmed feed as a risk factor, it became imperative to seek solutions. therefore, a follow up study was conducted to evaluate whether a liquid antimicrobial product containing formaldehyde and organic acid could mitigate said risk. the rationale for this approach was based on previous publications indicating that products containing formaldehyde and organic acids have a positive effect on salmonella reduction in feed [5] [6] [7] . furthermore, additional studies have demonstrated that formaldehyde treatment of organ inoculums containing turkey coronavirus (tcov) rendered this material non-pathogenic, whereas other treatments failed to ameliorate its negative effects [8, 9] . as pedv and tcov are both coronaviruses, it was hypothesized that formaldehyde treatment of pedvcontaminated feed may induce an anti-viral effect and prevent infection of susceptible pigs. this study was conducted in biosafety level 2+ rooms at the animal resource wing (arw) at south dakota state university (sdsu). all procedures involving animals throughout the study were performed under the guidance and approval of the sdsu institutional animal care and use committee. animals (n = 12, six-week old piglets) were sourced from a pedv-naïve herd and were tested on arrival to the arw via blood sampling and collection of rectal swabs from each pig. prior to animal arrival, all rooms (walls, ceilings, floors and drains) were monitored for the presence of pedv by pcr using sampling procedures previously described [10] . using a validated swine bioassay method [4] , the 12 piglets were divided into 3 groups and each group was housed in an individual room as follows: treatment group: five piglets consumed feed treated with sal curb® and spiked with stock pedv [11] . positive control group: five piglets consumed saline-treated feed spiked with stock pedv. negative control group: two piglets consumed feed + saline without pedv. feed was sourced from a pedv-naïve farm and screened by pcr prior to use. for the purpose of the study, a 45.5 kg allotment of feed was treated with 147.42 ml of sal curb®, (kemin industries, des moines, ia usa), based on an inclusion rate (per label) of 3 kg/ton of complete feed. sal curb® is a premix of aqueous formaldehyde solution 37% (for maintenance of complete animal feeds or feed ingredients salmonella-negative for up to 21 days) and propionic acid (as a chemical preservative for control of mold in feed or feed ingredients). while sal curb® provides effective salmonella control for up to 21 days, it is not approved for use by the u.s. food & drug administration or the u.s. department of agriculture as a treatment for pedv. the liquid antimicrobial was added to the feed using a syringe, injecting approximately 30 ml in 5 different locations within the 45.5 kg of feed. to promote proper mixing, the feed was stirred manually for 10 minutes using wooden spoons and strainers. upon completion of the 10 minute mixing period, treated feed was spiked with 100 ml of a stock isolate of pedv at a cycle threshold (ct) value of 25.22. twenty ml aliquots of virus were injected into 5 different locations within the feed. this level of pedv contamination was selected based on data from actual field cases of pedv-contaminated feed as well as levels of challenge used in the proof of concept study [4] . for the purpose of a positive control, a 45.5 kg quantity of feed was also spiked with 100 ml of stock pedv (ct = 25.22) along with 147.42 ml of sterile saline. finally, feed for the negative control group was treated with 147.42 ml of sterile saline (no pedv and no sal curb®). the total time required for preparation of feed batches was 60 minutes, followed by placement into the feeders of the respective rooms, allowing immediate adlibitum access to feed for a 14-day period [4] . separate mixing instruments were used to prepare the feed for all 3 groups of pigs. following access to treated feed, the pedv status of all 3 groups was monitored. on a daily basis, arw personnel inspected animals for clinical signs of ped and collected rectal swabs (dacron swabs, fisher scientific, franklin lakes, nj, usa) from each pig. personnel moved from the negative control group, to the treatment group and then to the positive control group every day. showers were taken between rooms and room-specific coveralls, footwear, hairnets, gloves and p95 masks (3 m, st. paul, mn usa) were worn. in addition, each room was ventilated individually and hepa filtration for both incoming and outgoing air was employed per room. if clinically affected animals were observed, swabs of diarrhea and/or vomiting, in conjunction with the daily rectal swab, were collected. swabs were submitted to the sdsu adrdl and tested by pcr. on day 15 of the study, animals were humanely euthanized with intravenous sodium pentobarbital and small intestinal tracts submitted for pcr and immunohistochemistry (ihc) testing and microscopic evaluation. on 9 designated days during the study period, (days 0, 1, 3, 5, 7, 9, 11, 13 and 15), feed samples were collected from the 3 respective groups. the purpose of sampling was to document the presence of pedv in feed and to determine whether a change in viral load occurred over time. samples were collected from the material hopper from the feeder in each room. as these hoppers were rectangular in shape (91.44 cm deep × 61 cm long × 30.5 cm wide), protocols used to sample feed from flatbottom trucks were referenced per the usda grain inspection, packers and stockyards administration [12] . for collecting feed samples, a model of a grain probe was constructed [12] . this model consisted of 2 pvc tubes (heritage plastics inc., carrolton, oh, usa), one placed inside the other per standard probe design. the outer tube was 64 cm in length with a diameter of 4.45 cm and the inner tube was 73 cm in length with a diameter of 3.18 cm. to facilitate feed entry into the lumen of the probe, seven 1.91 cm slots were drilled into each tube. rotation of the outer tube aligned the slots across both tubes, resulting in the entry of feed into the probe via gravity flow. once sampling was complete, the outer tube was rotated in the opposite direction, thereby closing the slots. to maintain feed in the probe lumen during sampling, as well as facilitate sample removal post-collection, both ends of the model were covered by a 3.18 cm plastic cap. three models were constructed, one for each room. using the flat-bottom truck protocol, 5 samples of feed were taken from each hopper, one from each corner (n = 4) and one from the center. the goal was to collect approximately 50 grams of feed per sampling time. at each point, the probe was inserted at an angle of 0 0 to a depth of 50 cm into the hopper, thereby "burying" the probe. during placement, the outer tube was rotated clockwise to close the slots and prevent feed entry. for sample collection, the outer tube was rotated counter-clockwise; opening the slots in both tubes and the model was moved in an up-and-down manner 2 times [12] . the outer tube was rotated to close the slots and the probe was removed. one end cap was removed and the sample was deposited into a 50 gram plastic specimen container. once approximately 50 grams of feed were collected, a 10 ml aliquot of sterile saline was added to the specimen container. a sterile dacron swab was inserted into sample and rotated 5 times clockwise and 5 times counter-clockwise to contact any pedv present. the swab was removed, placed into a 3 ml plastic tube (falcon, franklin lakes, nj, usa) containing 2 ml of sterile saline and submitted for testing. all diagnostic testing was conducted using protocols developed and validated by the south dakota state university animal disease research and diagnostic laboratory. the magmax™ 96 viral isolation kit (life technologies, waltham ma, usa) was used to obtain viral rna from the samples, as described in the instructions provided (1836 m revision f). a 175-μl volume of sample was used for the extraction. the magnetic bead extractions were completed on a kingfisher96 instrument (thermo scientific, waltham ma, usa). a commercially available real-time, single tube rt-pcr multiplex assay for the detection of pedv and transmissible gastroenteritis virus (tgev) was used in this study per kit instruction (tetracore, rockville, md, usa). briefly, 7 μl of the extracted rna was added to 18 μl of the master mix. the one-step real-time rt-pcr amplification conditions started with 15 minutes at 48°c, followed by 2 minutes at 95°c. the final cycles consisted of 5 seconds at 95°c and then 40 seconds at 60°c (data collection step). the program was run for 40 cycles (cycle time) and the fam detector was used for pedv and the tamra detector was used for tgev. positive and negative controls were included on each run. all amplification was completed on the abi7500 instrumentation (austin, tx, usa). for pedv propagation, vero 76 cells (atcc crl-1587) were maintained in mem plus 10% fetal bovine serum and antibiotics. three-day old confluent monolayers of vero 76 cells in 150 cm 2 flasks were washed 3 times with serum free minimum essential media (mem) prior to inoculation. monolayers were infected at~0.1 moi of pedv in mem containing 2.5ug/ml tpck-treated trypsin, incubated at 37°c for approximately 48 hours until obvious cpe was apparent. flasks were frozen at −80°c until needed. immunohistochemistry slides of porcine gi tracts were prepared using the standard sdsu adrdl ihc procedure, with the following modification being the use of pedv monoclonal antibody sd-6-29, of mouse ascites origin, courtesy of steve lawson, sdsu, at a 1:1000 dilution. the in vivo phase of the study was conducted from march 20 to april 5, 2014 (table 1 ). prior to initiation of the bioassay, all samples from incoming piglets, arw facilities and feed were pcr negative. throughout the 14 day study period, pedv rna in rectal swabs and clinical signs of ped were not observed in the treatment group or the negative control group. in addition, small intestinal tract samples from all 5 pigs in the treatment group and the 2 pigs in the negative control group were negative by pcr and ihc and no evidence of microscopic lesions of ped was observed. in contrast, in the positive control group pedv rna was detected in a rectal swab from the index piglet on day 2 post-ingestion (table 1) . on day 3, clinical signs of diarrhea were observed in the index piglet and another piglet was pcr positive on rectal swab. from day 4-7 post-ingestion, pedv rna was detected in diarrhea and rectal swabs from 3 piglets, with lethargy, vomiting and diarrhea noted. for the remainder of the 14 day study period, sporadic shedding of pedv in feces was detected with poor condition in piglets. small intestinal tract samples from all 5 pigs were positive by pcr and ihc. in addition, microscopic evaluation of small intestinal tissues indicated lesions indicative of ped, including re-epithelialization with diffuse villous blunting and fusion. the results of the feed sampling are summarized in figure 1 with raw data provided in table 2 . all 9 feed samples from the positive control group were positive for the presence of pedv rna by pcr with a mean ct of 25.15 (range = 24.15-26.74). in contrast, the mean ct value of the feed from the treatment group was 35.79 (range 25.89-40). when analyzed by t-test, the difference in the mean ct levels between the treatment group and the positive control group was significant at p < 0.0001. all samples were pcr-negative from the negative control group. based on the results of the bioassay, sal curb® treated feed prevented infection and clinical disease in naïve piglets. in contrast, pigs allowed to ingest non-treated feed spiked with pedv became infected. while both the treatment and the positive control feed contained a similar level of pedv immediately post-processing (day 0), there was a significant difference in mean ct at the end of the sampling period (day 15) across the 2 groups. while ct values in treated feed changed over time, values detected in the positive control feed samples remained relatively constant. one interpretation of this observation, in conjunction with the swine bioassay data, is that the sal curb® product had an adverse effect of viral load and viability, while in the absence of sal curb® the quantity of pedv remained constant and that virus survived over time. an acknowledged limitation was that the results are based on very small populations of pigs housed under experimental conditions and cannot be extrapolated to today's large-scale commercial farm conditions until further testing can be conducted. in addition, the study was not designed to answer questions which still remain regarding the liquid antimicrobial product, such as the duration of activity against pedv, its effects on other viral pathogens, its effect on dietary nutrients and the logistics of application and daily use. in closing, this is the first publication providing evidence that a means to "biosecure" feed against a globally significant virus may be possible. future studies should investigate whether application of the liquid antimicrobial product may have broader application at the international level and could possibly reduce the risk of the introduction of emerging and re-emerging pathogens through feed and feed ingredients that cross borders. finally, as "feed biosecurity" is a new paradigm for the swine industry, veterinarians, producers and representatives from the feed industry will need to work together and pursue novel means to implement such a strategy. the results of this study provide initial proof of concept that the application of a liquid antimicrobial product (sal curb®) reduced the risk of pedv infection through contaminated feed. furthermore, data from the positive control group once again provide proof of concept regarding the ability of contaminated feed to serve as a risk factor for pedv infection of naïve piglets. the data set(s) supporting the results of this article is included within the article. diseases of swine isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states usda aphis vs nvsl nahln umn swine health monitoring report: porcine epidemic diarrhea virus reporting an evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of naïve pigs following consumption via natural feeding behavior: proof of concept assessment of anti-salmonella activity of commercial formulations of organic acid products nutritional strategies to combat salmonella in mono-gastric food animal production chemical treatment of animal feeds and water for the control of salmonella spiking mortality of turkey poults: 2. effect of six different in vitro disinfection techniques on organ homogenates capable of reproducing smt spiking mortality of turkey poults: 1. experimental reproduction in isolation facilities role of transportation in spread of porcine epidemic diarrhea virus infection, united states environmental stability of a cell culture adapted u.s. isolate of pedv usda grain inspection, packers and stockyards administration: chapter 2: probing and sampling an evaluation of a liquid antimicrobial (sal curb®) for reducing the risk of porcine epidemic diarrhea virus infection of naïve pigs during consumption of contaminated feed the authors would like to recognize dr. michele mucciante and the animal resource wing team for their significant contributions to the success of this study. the authors would also like to thank dr. mark bienhoff and kemin industries for providing the technical expertise, funding and in-kind resources necessary to complete this project. the authors declare that they have no competing interest.authors' contributions sd: developed study, co-wrote paper. cn: identified sal curb product. tc: conducted molecular diagnostics, co-wrote paper. jch: provided critical review and revising of paper. en: provided virological expertise at the laboratory level and co-wrote paper. all authors read and approved the final manuscript. key: cord-293512-rcwwx7qw authors: steinbach, falko; dastjerdi, akbar; peake, julie; la rocca, s. anna; tobin, frank p.; frossard, jean-pierre; williamson, susanna title: a retrospective study detects a novel variant of porcine epidemic diarrhea virus in england in archived material from the year 2000 date: 2016-10-27 journal: peerj doi: 10.7717/peerj.2564 sha: doc_id: 293512 cord_uid: rcwwx7qw outbreaks of porcine epidemic diarrhea (ped) were first recorded in england in the 1970s and continued to be confirmed until 2002. retrospective analysis of archived material from one of the last confirmed cases in england in the year 2000 demonstrates the previous existence of a very diverse ped virus strain. following the outbreaks of ped in north america in 2013, there has been renewed interest in phylogenetic analysis of sequences from pedv strains worldwide. there is a gap in the available sequence data between the mid 1980s and the mid 2000s. this work is an example of how this gap can be at least partially filled by the examination of archived material. porcine epidemic diarrhea virus (pedv), an alphacoronavirus in the order nidovirales, was first described in england in the 1970s and subsequently emerged across most of europe (wood, 1977; pensaert & de bouck, 1978) . while the virus reached southeast asia in the 1980s, reports of ped declined in europe and no cases have been reported from the field in england since 2002 (williamson et al., 2013) . in recent years, however, variant strains of pedv have emerged in china causing high mortality in sucking piglets and spreading rapidly between farms (sun et al., 2012) . in 2013 these virulent pedv strains were reported in pig herds in the united states (stevenson et al., 2013) . this was followed by a report from ohio of another pedv strain characterised by insertions/deletions in the s-gene, known as the indel strain, oh851 being the prototype (wang, byrum & zhang, 2014) , associated with reportedly milder disease in the field. most recently indel pedv strains have been detected in outbreaks of diarrhoea in western europe, including germany, the netherlands, france and italy (hanke et al., 2015; efsa, 2016) . in parallel, the pedv strain associated with higher virulence in asia and north america was detected in the ukraine (dastjerdi et al., 2015) . the molecular characterization of pedv has been established (huang et al., 2013) . the virus is an enveloped, positive-sense, single-stranded rna coronavirus possessing a genome of ≈28 kilobases. to classify strains involved in the previous and recent outbreaks, a phylogenetic nomenclature using genogroups or clades has been suggested (huang et al., 2013) . markers for pathogenicity are poorly defined for coronaviruses, thus any focused sequencing of the s1 region only, for example, might introduce unreasonable bias into the analysis. this paper reports the results of analysis of the pedv from a historic field case in england, including comparisons with currently circulating north american/asian viruses, indel strains and the european prototype strain cv777 (kocherhans et al., 2001) . between 1999 and 2002, the national veterinary diagnostic database for gb (vida) recorded 13 diagnoses of ped on nine pig units in five counties of england, with the last diagnosis being made in 2002 and none since. unfortunately, the archived data does not allow us to draw epidemiological links between these cases. archived material was only available from one outbreak from october 2000; a finishing unit in yorkshire reporting an outbreak of diarrhoea. three fecal samples were submitted at that time, and a diagnosis of ped was made by testing the samples using a duplex rt-pcr (primer sequences tttattctgtcacgccatgt and ccagatttacaaacacctatgtta spanning a 199 bp fragment) designed to detect and discriminate the s gene from both pedv and transmissible gastroenteritis virus (tgev). all three samples tested positive for pedv. differential diagnostic testing for salmonella and brachyspira species by selective culture identified salmonella enterica ser. typhimurium phage type 193 only after enrichment culture in one of the three fecal samples, negating a diagnosis of salmonellosis or swine dysentery. one of the samples, from a 14-week-old pig, was retained in the cryostore archive, and was retrieved for further analysis in 2014. the original positive pedv pcr result was re-confirmed on the archived faeces, and also using a further in-house pcr targeting the n gene. the initial pcr amplicon was subjected to conventional sanger sequencing to confirm the detection of pedv. upon recognition of the significant differences to other pedv strains, next generation full genome sequencing was carried out to avoid the necessity of designing novel primers in multiple rounds of sanger sequencing for such a large rna genome. in brief, the extracted pedv rna was subjected to dnase digestion and used as template for cdna generation using a cdna synthesis system (roche) for preparation of sequencing libraries using a nextera xt kit (illumina, san diego, ca, usa). paired end sequencing was performed on an illumina miseq. finally, the consensus sequence was obtained by de novo assembly using velvet 1.2.10 as previously described (miller, koren & sutton, 2010 ) and re-evaluated using the templated genome assembly function of the seqman ngen v13 software (dnastar inc. madison, wi, usa). the consensus sequence was generated from 321425 sequence reads; considering length of each read the average coverage for each base pair is 1341.15, which is well above the optimum coverage. for phylogenetic analysis, comparisons were made with published spike protein amino acid sequences of 32 other pedv strains and two other coronaviruses, and similarly for the orf1a/b nucleotide sequence. sequence alignments were performed using the clustal w algorithm, and phylogeny was performed using the maximum likelihood method with 1,000 bootstrap replications, both using mega version 6 (tamura et al., 2013) . pcr testing of the faecal sample from 2000 confirmed the presence of pedv nucleic acid and the absence of tgev, indicating involvement of pedv in the outbreak of diarrhoea in the finishing pigs. as shown in fig. 1 , phylogenetic analysis of the consensus whole genome sequence obtained directly from the archived faecal sample indicates that the virus, while clearly belonging to the pedv species, lies distinct from any of the genogroups described by huang et al. (2013) . at the amino acid level, as shown in table 1 , proteins from this england-1-2000 strain vary in similarity from 91.5% to 96.3% compared to those of the cv777 strain. at the nucleotide level, similarities for the orf1a/b region vary from 95.1% to 96.3% when compared to the other pedv sequences analysed. while this strain is significantly different to other pedv strains known so far, it is in no part more closely related to other known coronaviruses, thus does not resemble the result of inter-species recombination such as described for the italian swine enteric coronavirus (boniotti et al., 2016) . the complete genome sequence of pedv england-1-2000 has been deposited at genbank under the accession number ku836638. while pedv england-1-2000 was significantly different from the european prototype cv777 from the early 1980s (ducatelle et al., 1981) , current and established pcr tests in 2000 were able to detect the virus without modification, suggesting that similar strains were not simply missed due to lack of diagnostic potential. the virus also differs from the virulent north american/asian and the indel variants such as oh851 currently circulating in western europe. the lack of other published pedv sequences from historic outbreaks of ped to the 2000s prevents further comparisons, but this clearly represents a genotype not described previously. no detailed morbidity or mortality data are available from records of the ped outbreak from which this virus was identified, but a previous diagnosis of ped was made on the farm in an earlier batch of finishing pigs in november 1999. the isolation of salmonella typhimurium in just one of the three fecal samples by enrichment culture only, and lack of isolation of brachyspira species from the samples, indicates no significant involvement of salmonellosis or brachyspira-related colitis in this diarrhea outbreak. in general, ped outbreaks at that time caused watery diarrhea without mortality in growing pigs and sows the evolutionary history was inferred by using the maximum likelihood method. the tree with the highest log likelihood (−10946.4157) is shown. initial trees for the heuristic search were obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using a jtt model. a discrete gamma distribution was used to model evolutionary rate differences among sites (5 categories (+g, parameter = 1.4131)). the rate variation model allowed for some sites to be evolutionarily invariable ([+i], 0% sites). (b) molecular phylogenetic analysis of 33 pedv orf1 nucleotide sequences. the evolutionary history was inferred by using the maximum likelihood method based on the tamura-nei model. the tree with the highest log likelihood (−85920.7638) is shown. initial tree(s) for the heuristic search were obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using the maximum composite likelihood (mcl) approach. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. the trees are drawn to scale, with branch lengths measured in the number of substitutions per site. the percentage of trees in which the associated taxa clustered together is shown next to the branches. evolutionary analyses were conducted in mega6 (tamura et al., 2013) . the genbank accession numbers, country and year of isolation are indicated, along with the genogroups as described by huang et al. (2013) . porcine respiratory coronavirus (prcv) and transmissible gastroenteritis (tgev) virus sequences are shown for reference. the prototype pedv strains cv777, oh851, colorado 2013 and england-1-2000 strains are underlined. rather than piglets, and tended to spread rapidly through the herd. this clinical presentation may, in part, reflect immunity through natural infection in the national herd at that time. unfortunately, no viable virus could be recovered from the limited amount of material available after the prolonged storage period to assess the relative virulence of this strain in experimental infections. this finding shows the presence of a diverse field pedv strain in england at the turn of the century, the pathogenicity of which requires further investigation that will only be possible upon rescuing the strain via reverse genetics. outbreaks of ped were diagnosed in gb until 2002 although the annual incidence of laboratory confirmed outbreaks was quite low, and there is no evidence that this strain is still present as a cause of disease in gb (efsa, 2016) . this report highlights the merit of further investigation of archived material from europe and elsewhere to establish the degree of heterogeneity of historic pedv strains and determine whether this england-1-2000 strain was typical of those circulating at the time in europe. assembly algorithms for next-generation sequencing data a new coronavirus-like particle associated with diarrhea in swine emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences outbreak of porcine epidemic diarrhea in suckling piglets mega6: molecular evolutionary genetics analysis version 6.0 new variant of porcine epidemic diarrhea virus, united states emergence of porcine epidemic diarrhoea in pigs in the usa an apparently new syndrome of porcine epidemic diarrhea we are grateful to sonia zuñiga and luis enjuanes, madrid for assisting attempts to isolate this strain, and to richard j. ellis for ngs sequencing. the authors declare there are no competing interests.frank tobin is an employee of holmefield farm services ltd, murton, yorkshire, united kingdom. • falko steinbach and susanna williamson conceived and designed the experiments, wrote the paper, reviewed drafts of the paper.• akbar dastjerdi conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.• julie peake and s. anna la rocca performed the experiments, reviewed drafts of the paper.• frank p. tobin contributed reagents/materials/analysis tools, reviewed drafts of the paper.• jean-pierre frossard analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. the following information was supplied regarding data availability: genbank accession number ku836638. key: cord-261372-xjbs09gi authors: sozzi, enrica; luppi, andrea; lelli, davide; martin, ana moreno; canelli, elena; brocchi, emiliana; lavazza, antonio; cordioli, paolo title: comparison of enzyme-linked immunosorbent assay and rt-pcr for the detection of porcine epidemic diarrhoea virus date: 2010-02-28 journal: research in veterinary science doi: 10.1016/j.rvsc.2009.05.009 sha: doc_id: 261372 cord_uid: xjbs09gi abstract porcine epidemic diarrhoea (ped) is a contagious enteric disease of pigs caused by a coronavirus. a double antibody sandwich enzyme-linked immunosorbent assay (das-elisa) based on the use of monoclonal antibodies was developed for the detection of porcine epidemic diarrhoea virus (pedv). the das-elisa was compared with rt-pcr in the examination of 506 specimens collected during 2006–2007 from pigs originating from different farms located in the po valley. both faecal samples obtained directly from the rectum of live animals showing clinical signs and intestinal samples collected from the caecum of deceased pigs were included in the study. the correlation between the two methods was higher when testing faecal samples (k =0.97, 95% ci: 0.94–1.00) than testing intestinal samples (k =0.62, 95% ci: 0.35–0.89). the use of elisa technology provided an efficient and effective mean of evaluating the presence of coronavirus ped antigen in field samples and indicates that this procedure is a very useful tool in epidemiological studies. porcine epidemic diarrhoea virus (pedv) is a member of the order nidovirales, family coronaviridae, genus coronavirus and is part of group 1 species. pedv was first reported in belgium and united kingdom in 1978 (pensaert and debouck, 1978) . since then, several outbreaks of the disease have been reported in many swineraising countries, most notably in europe and east asia (pensaert and sang-geon, 2006) . pedv induces an enteric disease similar to that caused by transmissible gastroenteritis virus (tgev) in pigs of all ages with high morbidity and mortality related to the age. mortality rate averages 50% but may be as high as 90% in pigs less than 7 days of age and very low or nil in pigs older than 2 weeks (pensaert and debouck, 1978; pensaert and sang-geon, 2006; pritchard et al., 1999) . since the 1990s pedv has caused severe epidemics in some asian countries such as japan, korea and china (kweon et al., 1993; takahashi et al., 1983) . in these countries massive losses of piglets have been reported and mortality usually reached high percentages (30-80%) in pigs younger than 1 week (shibata et al., 2001) . between may 2005 and june 2006, an epidemic of watery diarrhoea in pigs of all ages, including piglets, occurred in italy in the po valley, a densely pig populated area. in previous years, similar but sporadic outbreaks had been observed in growers and finishers but that was the first time that ped ''re-emerged" in epidemic form in a european country in many years (martelli et al., 2008) . the objective of the present study was to develop a double antibody sandwich (das) elisa, based on the use of monoclonal antibodies for pedv detection in swine intestinal and faecal samples useful for routine examinations of field samples. the diagnostic performances of the das-elisa and the correlation with rt-pcr were evaluated by testing 506 samples collected from pig herds in the po valley during the 2006-2007 period. two hundred and fifteen faecal samples were obtained directly from the rectum of live animals showing clinical signs and 291 intestinal samples were collected from the caecum of deceased pigs. these specimens were diluted with phosphate-buffered saline (pbs; 0.1 m, ph 7.2) to obtain a 10% suspension (v/v), clarified by centrifugation at 2000g for 10 min and then stored at à80°c. the pedv reference strain (cv-777) was propagated in vero cells grown in minimum essential medium eagle (mem) in the presence of trypsin (hofmann and wyler, 1988) . as soon as the cytopathic effect was fully developed, the virus was released from the cells by repeated freezing/thawing. after centrifugation (2000g for 15 min), the supernatant of the infectious culture medium was kept at à80°c before being used as positive control in the elisa reaction. negative control antigen was similarly prepared from non-infected cell lines. monoclonal antibodies (mabs) were prepared using standard methods (galfre and milstein, 1981) . mice were immunised with partially purified cv-777 strain. forty hybridomas were screened for secretion of desired antibodies by indirect immunoflorescence (ifi) and indirect elisa, set up using pedv infected and non-infected cells. only six mabs specific for the pedv were selected and further characterised. purified mabs from hybridomas culture supernatants or ascitic fluids conjugated with horseradish peroxidase (hrpo) using a modification of the method described by tjissen and kurstak (1984) . the different combinations of the six selected mabs were tested in das-elisa using as antigen both vero cells infected or not with pedv and cells infected with other coronaviruses or other pig viruses. the intensity and specificity of the reaction using each mab as catcher or tracer were considered as criteria for selection of the best combination, i.e. mab 4c3 as antigen catching antibody and 1f12 as conjugated mab. in the standard procedure, nunc maxisorb immunoplates were coated overnight at 4°c with purified mab 4c3 optimally diluted in 0.05 m carbonate-bicarbonate buffer, ph 9.6 (2 lg/well). samples were dispensed to duplicate wells and, after incubation a 37°c for 1 h, the hrpo-conjugated mab 1f12 was added at a pre-determined optimal dilution. positive and negative controls for pedv were included in each plate. the diluting buffer consisted of pbs (ph 7.4) with 0.05% tween 20 and 1% yeast extract. following incubation at 37°c for 1 h, the substrate solution (orthophenylenediamine dihydrochloride 0.5 mg/ml and 0.02% h 2 o 2 in 50 mm phosphate citrate buffer, ph 5.0) was then added. the colorimetric reaction was stopped after 10 min by addition of 2 n sulphuric acid and the absorbance values were read at 492 nm using a spectrophotometer. results were expressed as an optical density (od). fifty microliters per well of reagent were dispensed; three washings with pbs-tween 20 were performed after each incubation. a panel of four faecal samples, that had a different degree of viral load (from high positive to negative) when examined in dilution in rt-pcr (data not shown), were analyzed by das-elisa in serial two fold dilutions (from 1/1 to 1/512) in order to select the optimal dilution for test samples. the titration curves based on the od values provided evidence of a clear separation between the positive and negative samples (fig. 1) . the dilutions 1/1 and 1/2 provided the widest window between them and were selected as screening dilutions combined with an od cut-off value 0.2. all the 506 field samples were examined in parallel using das-elisa and a rt-pcr performed using primers specific for the spike protein (s-protein) as previously described (kim et al., 2001) . the agreement between pcr and elisa techniques was measured with the kappa statistic value (landis and koch, 1977) . the results obtained by examining, respectively, faecal and intestinal samples with both methods are shown in table 1 . when examining faeces, only two samples gave discordant results, i.e. they were negative by elisa, but were identified as pedv-positive by rt-pcr. the kappa value was high (0.97), suggesting an almost perfect agreement between the two methods. the elisa test may fail to detect antigens in faecal samples with very low viral titres specially when clinical specimens are collected in the recovery phase of the disease. other reasons could be the presence in faeces of specific antibodies that form immunocomplexes, or an excessive delay between collection and examination resulting in sample degradation and loss of antigenic sites. a lower number of intestinal samples resulted pedv positive in both arrays (6 vs 42 faecal samples) and, in addition, the two methods gave more discordant results, indicating just a substantial agreement (k = 0.62). in particular, 6 of the 291 intestinal samples were positive by both methods, whereas 7 samples resulted elisa positive, but rt-pcr negative. such disagreement could be due to the presence of pcr inhibitors and dna damaging substances in stool samples, but can also result from poor-quality specimens, e.g. extremely autolysed tissues or those stored at room temperature for prolonged periods. in addition, false-positive reactions in the elisa assay may be due to the non-specific binding of antibodies used as reagents to intestinal bacteria or their products (brandt et al., 1981) . however, this was probably not the case since the results did not change, even after incubation of elisa pos/rt-pcr neg samples with a negative mouse serum in order to block a potential binding between murine antibody (mabs) and pig specimens. it is already known that elisa results depend on the clinical and pathological data: in intestinal contents and faecal specimens obtained from experimentally infected pedv piglets, the virus shedding is detected with high consistency during the acute phase of disease, but much less frequently during the incubation period and the recovery phase (callebaut et al., 1982) . our data confirm that for a successful and correct diagnosis of pedv it is advisable to collect faecal materials at the onset of illness rather than taking intestinal contents from dead or suppressed animals with prolonged clinical signs. using das-elisa the presence of pedv was found in 55 of 506 (10.8%) field samples of pigs with diarrhoea originating from different farms located in the po valley. a high proportion of positive results was detected in samples taken between january and june 2006 (20 of 30 examined, 66.6%), during an epidemic of diarrhoea in the same area of italy (martelli et al., 2008) , from pigs of all ages. it is therefore possible that virus persisted after the acute phase in some groups of animals, for example after weaning, but this aspect was not specifically investigated by serology. during the latter part of 2006 and 2007, the pedv was detected in field samples at a lower rate, i.e. 35 of 476 (7.3%). the rapid decrease of the frequency of detection is probably also related to the quick development of a sufficient level of immunity in the population that caused a reduction of virus circulation. these findings are similar to those found in other countries with developed swine production (pensaert and sang-geon, 2006) . on the whole the results obtained by testing samples of naturally infected swine indicate that the das-elisa here described could be considered as a reliable and accurate assay for the diagnosis of pedv in clinical specimens. such data agree with previous studies in which the pedv-elisas were employed for the screening for pig herds during epizootic outbreaks (carvajal et al., 1995; rodak et al., 2005; van nieuwstadt and zetstra, 1991) . the advantages of the das-elisa compared with rt-pcr assay derive from the simple and rapid procedure, suitable for the screening of a large number of specimens, and from the use of mabs, that ensure standardisation and reproducibility. when taking into consideration the epidemiological characteristics of pedv, the rapid identification of the etiological agent would facilitate the implementation of effective control measures. comparison of direct electron microscopy and rotavirus enzyme-linked immunosorbent assay for detection of gastroenteritis viruses in children enzyme-linked immunosorbent assay for the detection of the coronavirus-like agent and its antibodies in pigs with porcine epidemic diarrhea evaluation of a blocking elisa using monoclonal antibodies for the detection of porcine epidemic diarrhoea virus and its antibodies preparation of monoclonal antibodies, strategies and procedure propagation of the virus of porcine epidemic diarrhoea in cell culture differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by rt-pcr isolation of porcine epidemic diarrhea virus (pedv) infection in korea the measurement of observer agreement for categorical data an epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhea transmissible gastroenteritis and porcine epidemic diarrhoea in britain an elisa optimized for porcine epidemic diarrhoea virus detection in faeces passive protection against porcine epidemic diarrhoea (ped) virus in piglets by colostrum from immunized cows an outbreak of swine diarrhoea of a new type associated with coronavirus-like particles in japan highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibodies conjugates for enzyme immunoassays use of two enzyme-linked immunosorbent assays to monitor antibody responses in swine with experimentally induced infection with porcine epidemic diarrhoea virus key: cord-267446-rpv19oy6 authors: park, jung-eun; cruz, deu john m.; shin, hyun-jin title: receptor-bound porcine epidemic diarrhea virus spike protein cleaved by trypsin induces membrane fusion date: 2011-06-12 journal: arch virol doi: 10.1007/s00705-011-1044-6 sha: doc_id: 267446 cord_uid: rpv19oy6 porcine epidemic diarrhea virus (pedv) infection in vero cells is facilitated by trypsin through an undefined mechanism. the present study describes the mode of action of trypsin in enhancing pedv infection in vero cells during different stage of the virus life cycle. during the viral entry stage, trypsin increased the penetration of vero-cell-attached pedv by approximately twofold. however, trypsin treatment of viruses before receptor binding did not enhance infectivity, indicating that receptor binding is essentially required for trypsin-mediated entry upon pedv infection. trypsin treatment during the budding stage of virus infection induces an obvious cytopathic effect in infected cells. furthermore, we also show that the pedv spike (s) glycoprotein is cleaved by trypsin in virions that are bound to the receptor, but not in free virions. these findings indicate that trypsin affects only cell-attached pedv and increases infectivity and syncytium formation in pedv-infected vero cells by cleavage of the pedv s protein. these findings strongly suggest that the pedv s protein may undergo a conformational change after receptor binding and cleavage by exogenous trypsin, which induces membrane fusion. porcine epidemic diarrhea virus (pedv), a member of the family coronaviridae, is an economically important pathogen of swine. pedv causes acute watery diarrhea, resulting in approximately 50% mortality among suckling piglets and reduced weight among fattening pigs [10] . although the structural and pathological properties of pedv are similar to those of other group 1 coronavirus, including human coronavirus 229e (hcov-229e), transmissible gastroenteritis virus (tgev) and feline infectious peritonitis virus, many biological issues, such as the role of trypsin in infection, remain unresolved [7, 12, 36] . the first successful propagation of pedv in cell culture was done by supplementing the vero cell culture medium with trypsin [15] . the addition of trypsin was shown to induce fusion of the infected vero cells, resulting in the formation of multiple syncytia, and produced a significant increase in virus titer after several passages. soon afterwards, several other groups performed pedv infection in vitro using the same conditions and reported similar findings [20, 21] . on the other hand, another study reported the successful propagation of the p-5 v strain in porcine enterocyte cell lines without trypsin supplementation of the medium, suggesting that the proteolytic processing of pedv in enterocytes may have occurred during maturation or prior to virus release [17] . the spike (s) glycoprotein is the dominant surface protein in coronaviruses. the protein is responsible for virus attachment and fusion. the requirement of proteinase-cleaved s glycoprotein has been reported for almost all group 2 and 3 coronavirus. for example, infection by severe acute respiratory syndrome coronavirus (sars-cov) and murine hepatitis virus strain 2 (mhv-2) requires proteolytic cleavage in their target cells, which is mediated by trypsin-like proteases [24, 29, 32] . the situation for group 1 coronavirus is unclear. recently, the s protein of the group 1 coronavirus hcov-229e was reported to be cleaved by treatment with cathepsin l and trypsin, which prompts the fusion of the viral envelope and the cell membrane, similar to sars-cov [18] . the present study reports the putative role of trypsin in cell-adapted pedv infection of vero cells. trypsin treatment was performed in the early and late stages of viral infection, and its influence on viral titer and syncytium formation was examined. furthermore, the effect of trypsin on the s protein was compared in free and receptor-bound virions. the results suggest that trypsin activity is involved mainly with receptor-bound s protein of pedv, leading to the conclusion that the effect of trypsin on pedv is similar to that of the group 2 coronaviruses, sars-cov and mhv. african green monkey kidney cells (vero, ccl-81) were prepared in minimum essential medium (mem, gibco) supplemented with 5% fetal bovine serum (fbs, gibco). the cell-adapted strain of the korean pedv isolate, kpedv-9, was propagated as described elsewhere [15] , with some modifications. briefly, vero cells were inoculated with kpedv-9 at a multiplicity of infection c1 and cultured in serum-free mem at 37°c, 5% co 2 for 48-60 h. the supernatant was harvested and then clarified by centrifugation at 12,000 g for 10 min at 4°c. concentration and partial purification were performed by ultracentrifugation under a 20% sucrose cushion at 26,000 rpm for 3.5 h. the pellet was resuspended in 10 mm phosphate-buffered saline (pbs, ph 7.4) and stored at -70°c. mouse polyclonal antibodies against pedv were generated by immunizing 6-week-old female balb/c mice (samtako) intraperitoneally with 1 9 10 5 focus-forming units (ffu) of purified kpedv-9 emulsified in an equal volume of complete freund's adjuvant (sigma-aldrich) on day 1 and incomplete freund's adjuvant (sigma-aldrich) on days 14, 21 and 28. whole blood was collected from the retro-orbital sinus on days 0 and 35 and centrifuged at 1500 g for 10 min to separate the sera. cultured vero cells were inoculated with kpedv-9 as described above and allowed to adsorb for 2 h at 37°c. the vero cells were washed twice with pbs and cultured in serum-and trypsin-free mem or mem containing trypsin (10 lg/ml, sigma-aldrich). at 8, 12, 24, and 48 h postinoculation (hpi), culture supernatants were collected for titration in a focus-formation assay, and cells were fixed with 4% formaldehyde in pbs for 30 min and permeabilized with 1% np-40 (sigma-aldrich) in pbs, followed by immunocytochemistry using mouse anti-pedv polyclonal sera [8] . clusters of infected cells staining dark gray were counted under an inverted microscope and reported as ffu. trypsin treatment at various stages of virus infection cells or viruses were treated with trypsin at various stages of virus infection as described in fig. 1 . to investigate the effects of proteolytic cleavage of the surface protein of vero cells and free virions by trypsin, vero cells or kpedv-9 were pre-treated with trypsin prior to infection. trypsin treatment was performed for 30 min at 37°c prior to inoculation, and enzyme activity was neutralized with 2 lg/ml aprotinin (sigma-aldrich). trypsin-pretreated vero cells were inoculated with kpedv-9, and untreated vero cells were inoculated with trypsin-pretreated kpedv-9. after a 2-h incubation to allow adsorption, cells were washed three times with pbs and then cultured in serum-free mem without trypsin for 24 h. in another experiment, trypsin treatment was carried out during the virus adsorption stage to determine whether trypsin is involved in the entry of kpedv-9. vero cells were inoculated with kpedv-9 in the presence of various concentrations of trypsin (5, 10, 20, 40 and 80 lg/ml) during the adsorption period and were cultured in serum-free mem at 37°c for 24 h. to investigate the effect of trypsin on the budding stage of pedv infection, kpedv-9-infected vero cells were prepared by inoculating them with purified kpedv-9 and then cultured in mem for 20 h. prior to trypsin treatment, the cell monolayer was washed three times with pbs to remove residual fbs and released virions, prior to treatment with trypsin for 10 min. after neutralization of trypsin by the addition of aprotinin, cells were cultured for an additional 4 h. the culture supernatants were harvested for virus titration, and cells were fixed for immunocytochemistry at the indicated times. virus-infected cells were detected by probing with mouse anti-pedv polyclonal antisera and biotinylated rabbit antimouse igg and visualized by treatment with streptavidinbiotinylated horseradish peroxidase (vector labs) followed by 3,3'-diaminobenzidine tetrahydrochloride dihydrate (dab, vector labs). all specimens were observed under an inverted microscope. ultrapurified kpedv-9 was treated with various concentrations of trypsin at room temperature (rt) for 10 min and then analyzed by western blotting. vero cells were infected with kpedv-9 in the absence of trypsin for 24 h, and kpedv9-infected vero cells then were harvested and treated with trypsin for 10 min at rt. mock-infected trypsin-treated vero cells were used as a negative control. samples for western blot analysis were treated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) sample buffer and electrophoresed in an 8% sds-page gel system. the separated proteins were electrically transferred onto a polyvinyl difluoride membrane (amersham bioscience). the antibodies used in this study were mouse anti-pedv polyclonal antibodies against pedv s protein, and monoclonal anti-b-actin-peroxidase (sigma-aldrich). the bands were visualized using supersignal west dura (pierce) with las-1000plus (fujifilm). statistical analysis was performed using spss, version 7.5, for windows. correlation coefficients were calculated using pearson's correlation coefficient. error bars represent the standard deviations from at least three replicates. trypsin is not essential for pedv infection pedv propagation in vero cells results in low infection rates, even after subsequent passages in the absence of trypsin supplementation [15] . however, with the addition of trypsin, virus adaptation to vero cells increased, and a prominent cytopathic effect (cpe) marked by formation of syncytia was observed in subsequent passages. following this observation, the growth rate of kpedv-9 in vero cells in the presence or absence of trypsin supplementation was compared. as shown in fig. 2 , detectable levels of progeny virions were observed from 8 hpi in both trypsin-and nontrypsin-supplemented media. at 12 hpi, the titer was higher (3.09 9 10 3 ffu/ml) in trypsin-supplemented samples than in non-trypsin-supplemented samples (1.08 9 10 3 ffu/ml). the rate of virus production in trypsin-supplemented cultures was also significantly higher than in trypsin-free cultures (virus titer of 1.83 9 10 5 and 1.65 9 10 4 ffu/ml at 24 hpi, respectively). even at 48 hpi, the titer in the trypsinfree cultures only reached peak titer levels of 4.52 9 10 4 ffu/ml, which was significantly lower than the peak titer attained at 24 hpi in trypsin-supplemented cultures. these results are consistent with the suggestion that trypsin is not absolutely essential for vero-cell-adapted pedv infection, as reported for other group 1 coronaviruses, but the titer increases during infection with trypsin-treated virus. trypsin mediates the penetration of cell-attached pedv to investigate how trypsin enhances pedv infectivity of vero cells, trypsin was added during various stages of infection. trypsin treatment of kpedv-9 prior to inoculation did not significantly differ from non-trypsin-treated virus after 20 hpi (1.24 9 10 4 and 1.32 9 10 4 ffu/ml, respectively) (fig. 3) . this suggests that proteolytic processing of the surface glycoprotein by trypsin prior to receptor binding does not have a significant effect on enhancing infectivity. to determine whether trypsin interaction with vero-cell-surface proteins contributes to enhanced pedv infectivity, vero cells were pre-treated with 10 lg/ml trypsin for 30 min before inoculation. this treatment did not significantly alter virus titer when compared to the untreated cells. interestingly, addition of trypsin immediately after inoculation during the absorption to investigate the mechanism of trypsin in more detail, vero-cell-bound pedv was treated with trypsin. vero cells were inoculated with kpedv-9 in serum-and trypsin-free media for 2 h and then washed twice to remove un-bound kpedv-9. the cell-bound kpedv-9 was treated with different concentrations of trypsin for 10 min, and the titers of penetrating and produced virus were determined. when the concentration of trypsin in the medium was increased from 5 lg/ml to 80 lg/ml, the number of pedv penetrating the vero cells also increased from 4 9 10 2 ffu/ml to 9 9 10 2 ffu/ml. the enhanced trypsin-mediated penetration during initial infection resulted in an increase in virus titer at 24 hpi, from 3.0 9 10 4 ffu/ml to 1.0 9 10 5 ffu/ml (fig. 4) . these findings are consistent with the notion that trypsin activity during the initial stage of virus infection enhances the efficiency of virus penetration into vero cells, thereby increasing viral infectivity. although the penetration of cell-attached virions was facilitated by trypsin treatment, virions treated with trypsin before cell attachment did not show any difference when compared to the results obtained in the absence of trypsin. based on these findings, it is appropriate to suggest that trypsin might only affect the receptor-bound spike, inducing fusion between the cell membrane and the virus envelope, leading to increased virus penetration. to investigate the role of trypsin on the late stage of infection and syncytium formation, kpedv-9-infected vero cells were prepared by inoculation for 20 h. the cells were washed extensively and treated with various concentrations of trypsin for 10 min prior to continuing cell cultivation in fresh serum-and trypsin-free medium. kpedv-9-infected vero cells did not show visible signs of syncytium formation in the absence of trypsin, while kpedv-9-infected vero cells treated with 5, 10 and 20 lg/ml trypsin at 20 hpi contained multiple syncytia (fig. 5a) . without trypsin treatment, the virus titer at 4 hpi was 1.4 9 10 3 ffu/ml while kpedv-9-infected vero cells treated with 5, 10, 20, 40 and 80 lg/ml trypsin showed virus titers of 2.1 9 10 3 , 3.0 9 10 3 , 3.5 9 10 3 , 4.1 9 10 3 and 6.7 9 10 3 ffu/ml, respectively (fig. 5b) . in virus budding stage, trypsin also activated syncytium formation of infected vero cells and consequently increased virus infectivity. newly packaged virions budding from infected vero cells could be activated by trypsin, which caused the infected vero cells to form syncytia. this finding was consistent with the previous results shown in figs. 3 and 4 . the collective results supported the idea that trypsin acts on cell-attached virions, both during virus attachment and during virus release and induces membrane fusion between the host-cell membrane and the virus envelope, and also between host-cell membranes. cleavage of receptor-bound s protein by trypsin the s protein from ultrapurified virions and receptor-bound virions was treated with trypsin and analyzed by western blotting. s protein from both ultrapurified virions and kpedv-9-infected vero cells that had not been treated with trypsin was apparent as a species of about 220 kda, which represented the glycosylated native s protein (fig. 6) . in ultrapurified virus, only this protein species was detected, even after trypsin treatment, while 140-kda and 125-kda proteins, likely trypsin-cleaved s protein, were detected in trypsin-treated kpedv-9-infected vero cells (fig. 6) . the findings supported the suggestion that pedv s protein has a site that is highly sensitive to trypsin cleavage to produce two fragments, as has been reported for other coronaviruses [23, 24, 32] . however, this fig. 4 enhancement of cellattached kpedv-9 penetration by trypsin. vero cells were inoculated with kpedv-9 for 2 h and then washed to remove unbound kpedv-9. only cellattached kpedv-9 was treated with trypsin for 30 min, and penetrated virus (j) and progeny virus were titrated after 24 h (h). increasing the amount of trypsin added during virus adsorption resulted in increased virus penetration into vero cells during initial entry and higher virus titers after 24 hpi cleavage only occurred when the virus was associated with receptor protein. several enterotropic or pneumotropic viruses, such as those belonging to the families orthomyxoviridae and paramyxoviridae, undergo proteolytic cleavage of their surface glycoprotein prior to entry into host cells to facilitate virus penetration by activating the fusion domain of the surface glycoprotein [6, 26] . the activated fusion protein undergoes conformational changes that induce fusion of the viral envelope and host membranes [3, 27, 30] . this process usually occurs during the period between virus maturation and virus attachment to the host receptors [25, 33] . after the fusion process, the viral core, including the viral genome, is transported into the cytoplasm where uncoating and replication ensue [22] . in natural infections, the viral surface glycoproteins that are not cleaved during maturation are subsequently cleaved by exogenous proteases secreted from host pancreas, liver and bronchiolar epithelia [19] . in cell culture, the protease cleavage that is required for virus propagation is carried out by exogenous proteases such as trypsin or pancreatin [1, 15, 28] . these exogenous proteases induce syncytium formation by activating the fusion domain of the viral glycoprotein expressed on the surface of infected cells [2, 16] . several studies of different coronaviruses have shown that proteolytic cleavage of the s protein enhances viral infectivity. for mhv, separation of the s1 and s2 subunits enhances the fusion activity of the s2 subunit and increases viral infectivity [5, 32] . mutations that alter the furin protease recognition sequence (rxr/kr) located at the junction of the s1 and s2 subunits as well as treatment with a peptide furin inhibitor prevent the proteolytic cleavage of the s protein, resulting in reduced cell-cell fusion activity, although viral entry is not significantly affected [4, 9, 14] . conversely, the addition of trypsin to the culture medium can enhance the fusion of mhv-infected cells [13] . in contrast to mhv, the s protein of sars-cov does not show any evidence of proteolytic maturation to cleaved s1 and s2 subunits in mature virions [35] . instead, proteolytic cleavage of the s protein on the surface of infected cells occurs by exogenous proteases, mediating cell-cell fusion [25, 29] . similar to sars-cov, the s protein in most group 1 coronaviruses also does not exhibit cleaved s1 and s2 subunits during virus maturation and biogenesis [34] . several studies on tgev and pedv have used trypsinsupplemented culture media to induce cpe by syncytium formation in st cells and vero cells, respectively [15, 31] . furthermore, in the case of pedv, trypsin facilitates successful propagation in vero cells as well as other primate cell lines [15, 20] . however, the role of cellular and exogenous proteases on the cell entry of group 1 coronaviruses, particularly pedv, as well as the putative proteolytic cleavage site on the s protein, remains unclear. based on the present results summarized in figs. 3, 4 and 5, enhancement of virus penetration and cellcell fusion induced by the addition of trypsin suggests that the pedv s protein may also be cleaved into s1 and s2 subunits during the course of infection. electrophoretic examination of purified virions resulted in the detection of the pedv s protein as a monomer of about 220 kda in the absence of trypsin, while the protein was detected as two fragments of 140 kda and 125 kda in the presence of trypsin (fig. 6) . it may be that the pedv s protein on native virions adopts a conformation that protects it from various exogenous proteases, but the s protein attached to the host receptor protein may undergo a conformational change that exposes a trypsin cleavage site. previous reports have described the formation of syncytia by pedvinfected cells only upon addition of trypsin in the culture medium, and sequence analysis of the pedv s protein has revealed the absence of the rrx(r/h)r motif, which is associated with cleavage into the s1 and s2 subunits [11] . this suggests that the pedv surface glycoprotein does not undergo proteolytic processing upon maturation and release. conversely, the observation that trypsin can induce temperature for 10 min. pedv s was detected using anti-pedv polyclonal antibodies raised in mice. uncleaved s protein and cleaved s protein are indicated by black and white arrows, respectively cell-cell fusion in pedv-infected cells suggests that proteolytic processing of the s protein by exogenous trypsin may augment viral entry by facilitating fusion of the viral membrane with the host membranes [11, 15] . it seems that the timing of the cleavage of the s protein by trypsin is critical for the activation of fusion activity. as shown in fig. 3 , early activation of the s protein before binding to cellular receptors did not enhance viral entry into the host cell, while addition of trypsin shortly after receptor binding increased the efficiency of virus entry. while the s protein in infected cell lysates and receptorbound virions was cleaved into two fragments, the s protein cleavage in pedv was different from that of other coronaviruses, as pedv s protein was only cleaved when associated with its host cell. this implies that cleavage of the s protein by trypsin occurs only when it is bound at the surface of host cells to the host receptor protein, which presumably induces a conformational change in the bound s protein. this conformational change might expose a trypsin cleavage site. cleavage of the s protein could result in membrane fusion. it would be of interest to determine the nature of the conformational change that is involved and the location of the s protein cleavage site. in summary, the present results reveal the role and importance of trypsin in pedv infection of vero cells. trypsin is not essential for pedv infection but enhances its infectivity and cpe formation. trypsin cleaves pedv s protein only when it bound its cell receptor and in the later stages of infection. the association of the s protein with the host receptor could induce conformational changes that expose a trypsin cleavage site(s). the resulting cleavage might expose or activate the fusion peptide and activate pedv entry and pathogenesis. plaque formation by influenza viruses in the presence of trypsin characterization of the infectious salmon anemia virus fusion protein structural basis for paramyxovirus-mediated membrane fusion mutational analysis of the murine coronavirus spike protein: effect on cellto-cell fusion the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between ha1 and ha2 determines proteolytic cleavability 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proteolytic cleavage of the glycoproteins and its significance for the virulence of newcastle disease virus structural polypeptides of mumps virus analysis of the relationship between cleavability of a paramyxovirus fusion protein and length of a connecting peptide characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoproteinmediated viral entry changes in the conformation of influenza virus hemagglutinin at the ph optimum of virus-mediated membrane fusion transmissible gastroenteritis (tge) of swine: effect of age of swine testes cell culture monolayers on plaque assays of tge virus proteolytic cleavage of the e2 glycoprotein of murine coronavirus: activation of cellfusing activity of virions by trypsin and separation of two different 90k cleavage fragments evolution and ecology of influenza a viruses nucleotide sequence and expression of the spike (s) gene of canine coronavirus and comparison with the s proteins of feline and porcine coronaviruses the sars-cov s glycoprotein: expression and functional characterization porcine epidemic diarrhea virus (cv777) and feline infectious peritonitis virus (fipv) are antigenically related key: cord-320559-up1q3k6q authors: dortmans, j.c.f.m.; li, w.; van der wolf, p.j.; buter, g.j.; franssen, p.j.m.; van schaik, g.; houben, m.; bosch, b.j. title: porcine epidemic diarrhea virus (pedv) introduction into a naive dutch pig population in 2014 date: 2018-05-24 journal: vet microbiol doi: 10.1016/j.vetmic.2018.05.014 sha: doc_id: 320559 cord_uid: up1q3k6q porcine epidemic diarrhea virus (pedv) is the highly contagious, causative agent of an economically important acute enteric disease in pigs of all ages. the disease is characterized by diarrhea and dehydration causing mortality and growth retardation. in the last few decades, only classical pedv was reported sporadically in europe, but in 2014 outbreaks of pedv were described in germany. phylogenetic analysis showed a very high nucleotide similarity with a variant of pedv that was isolated in the us in january 2014. the epidemiological situation of pedv infections in the netherlands in 2014 was unknown and a seroprevalence study in swine was performed. in total, 838 blood samples from sows from 267 farms and 101 samples from wild boars were collected from may till november 2014 and tested for antibodies against pedv by elisa. the apparent herd prevalence of 0.75% suggests that pedv was not circulating on a large scale in the netherlands at this time. however, in november 2014 a clinical outbreak of pedv was diagnosed in a fattener farm by pcr testing. this was the first confirmed pedv outbreak since the early nineties. sequence analyses showed that the viruses isolated in 2014 and 2015 in the netherlands cluster with recently found european g1b strains. this suggests a one event introduction of pedv g1b strains in europe in 2014, which made the netherlands and other european countries endemic for this type of strains since then. porcine epidemic diarrhea (ped) is an economically important acute enteric disease in pigs of all ages. the disease is characterized by diarrhea and dehydration causing mortality -particularly in neonatal pigletsand growth retardation. the causative agent is porcine epidemic diarrhea virus (pedv), which is an enveloped, positive singlestranded rna virus, belonging to the family of coronaviridae (pensaert and de bouck, 1978) . the genome of pedv is approximately 28 kb long and about two-third encodes for non-structural proteins and one-third of structural proteins (kocherhans et al., 2001) . among these proteins, the main research interest is focused on the spike (s) gene and its glycoprotein product s, mediating receptor binding and membrane fusion (li et al., 2016) . although only one serotype has been described, phylogenetic studies of the s gene showed that pedv can be genetically separated into two groups: genogroup 1 (g1) and genogroup 2 (g2). each genogroup can be further divided into subgroups 1a and 1b, and 2a and 2b, respectively (lee, 2015) . classical ped, now grouped g1a, was first recognized as a severe swine enteric disease separate from transmissible gastro enteritis (tge) in the united kingdom in 1971 and first described in belgium in 1978 (pensaert and de bouck, 1978) . in the eighties and nineties, the virus was detected in many countries in europe including the netherlands and from europe pedv spread to asia, where it caused large outbreaks with considerable losses in the pig industry (song and park, 2012) . until 2013, north america was considered to be free of pedv infections (cima, 2013) , but in that same year highly virulent strains of pedv emerged in the united states of america (us), causing diarrhea, vomiting and loss of appetite in pigs of all age groups and up to 100% of mortality in suckling piglets (chen et al., 2014; huang et al., 2013; stevenson et al., 2013) . this strain, typed as g2b, rapidly spread across the us, canada, mexico and several countries in south america . in the last few decades, only classical pedv (or g1a) was reported sporadically in europe (alborali et al., 2014; martelli et al., 2008) . in 2014, outbreaks of pedv were described in germany and phylogenetic t analysis showed a very high nucleotide similarity with a variant of pedv (oh851) containing nucleotide insertions and deletions in the s gene (s-indel) that was isolated in the us in january 2014 stadler et al., 2015) . this variant, typed as g1b, caused mild clinical signs and lower mortality rates in suckling piglets compared to other circulating pedv g2b strains in the us . since the reports of outbreaks in germany, more reports about outbreaks of this particular s-indel virus in several european countries have been published, among which france, belgium, spain, portugal and austria (efsa, 2016; grasland et al., 2015; mesquita et al., 2015; steinrigl et al., 2015) . this suggests that this mild pedv variant is circulating in europe since the beginning of 2014. the aim of this study was to determine the status of pedv in the netherlands with a serological survey and to investigate the first pedv outbreak in the netherlands since the early nineties. the number of required blood samples from animals and farms to estimate the seroprevalence of pedv in dutch sow herds was calculated based on the following assumptions: pedv is highly contagious and no vaccination against this virus was carried out in the netherlands. as a result, it was expected that, if pedv was present in a sow herd, the within herd prevalence would be very high (bertasio et al., 2016; goede et al., 2015) . the required number of blood samples per herd was calculated using winepiscope 2.0 (thrusfield et al., 2001) . to detect infection in a herd with 95% probability, an estimated within herd prevalence of 70%, and an average herd size of 464 sows per farm (wur, 2016), three blood samples per farm were required. in 2014, there were approximately 2061 sow farms in the netherlands (wur, 2016) . in order to show with a high probability (95%), that less than 1% of the dutch farms (n = 20) were infected, 286 farms would need to be tested (thrusfield et al., 2001) . herds were randomly selected stratified by pig density per province to represent the total dutch sow herd population ( fig. 1a and b). statistical analyses were performed using stata/se version 14.1 software (stata corporation, 2017). for the serological survey, 410 blood samples were selected from samples collected for the obligatory monitoring of aujeszky's disease (pseudorabies), swine vesicular disease (svd) and classical swine fever (csfv). additionally, 428 blood samples were collected from sows at slaughter (vion, groenlo, the netherlands). herds were identified based on their unique herd number (ubn). samples were randomly selected given the availability of enough serum to perform all assays. samples were collected from may till up to and including november 2014. commissioned by the dutch government, gd animal health was also monitoring wild boars for aujeszky's disease, svd, foot and mouth disease, csfv, trichinellosis and african swine fever. because it is suggested that wild boars may play an important role as a pedv reservoir (lee et al., 2016) , blood samples of wild boar collected as part of this monitor were also tested. these blood samples were collected from regions close to the borders with germany and belgium, from april till august 2014. blood samples collected were stored at −70°c, before dispatched to the virology division of the faculty of veterinary medicine, at utrecht university (uu). for the detection of pedv antibodies in serum samples an in-house indirect elisa based on the viral spike (s) protein s1-part of the g2b strain gdu (non-s-indel, genbank ku985230.1) was used, similar as the elisa previously described (gerber et al., 2014) . the s1 antigen used in this study is produced in hek293 t cells, a mammalian expression system. to facilitate the purification from the cell culture supernatant, the protein is associated with the fc part of murine igg. per well 4 ng of protein was used to coat the plates. the in-house elisa was optimized by uu with sera from pedvinfected (g2b) pigs from the us and with hyperimmune sera of animals vaccinated with the pedv s1 protein. sera were tested in duplicate at a 1:100 dilution, the absorbance was measured with an elisa plate reader at 450 nm. the mean od value of pedv-negative sera was 0.11. sera with od values of > 0.33 (3× od value pedv negative sera) were considered positive. the aforementioned pedv-positive sera from infected animals were high positive (> 0.8 od) in this elisa ( supplementary fig. 1 ). the calculated sensitivity and specificity of the elisa was 100% (ci 95%: 90-100%; n = 43) and 100% (ci 95%: 92-100%; n = 57), respectively. for the detection of virus-specific antibodies in serum samples the virus neutralization test (vnt) was used as an alternative sero-diagnostic assay. a previously described vnt was used to test all samples positive by elisa. the validation of this test is shown in fecal swabs or eswabs (copan diagnostics incorporated) of individual animals were taken by the local practitioner and sent in the same day for pcr testing. also three pigs of the index farm with severe diarrhea were euthanized and submitted for post mortem examination. rna was isolated from fecal samples and the detection pcr was performed as previously described (kim et al., 2007; lowe et al., 2014) . in order to type the viruses the pedv s gene was sequenced with primers previously described (chen et al., 2014; oka et al., 2014) and designed in this study (table 1) , and compared with sequences available in genbank. a phylogenetic tree was constructed using mega6 software by neighbor-joining method maximum likelihood method. branch lengths represent the predicted number of substitutions and are proportional to the differences between the isolates (tamura et al., 2013) . after confirmation of the first g1b outbreak in the netherlands it was unquestionable to try to prevent the further spread of the outbreak. therefore, it was decided to monitoring and follow up of specific management advices to the farmers. five fattening herds, five sow herds and one nursery herd were selected after pedv infection was confirmed by pcr on feces. for control and eradication of pedv, a tailor made advice per farm, mainly based on biosecurity measures, was given. advise was mainly focused on the separation between pedvinfected and non-infected healthy animals. this separation was applied in stables, animal categories and compartments. it was made using the following measures: dress code (change between compartments), additional disinfection cleaning and disinfection of compartments and corridors and improving pest control. furthermore, all professional herd visitors were informed and required to take measures, in particular those aimed at cleaning and disinfection, which would prevent the transfer of the disease to another pig farm. regular testing of pooled fecal samples was done to monitor the effect of interventions. in sow herds, nursery piglets and replacement gilts were sampled; in fattening herds a random sampling in all age groups was performed. after introduction of pedv, the virus could be detected in a compartment for 4-6 weeks. at farm level the virus was detectable much longer due to transmission to new susceptible animals. farms were presumed negative for pedv if three sampling rounds with at least 2-weeks interval, of thirty randomly taken individual fecal samples each, proved to be pcr negative. in total, sera from 838 sows originating from 267 farms, and 101 sera from wild boar were collected. the blood samples came from all provinces in the netherlands and are fairly representative for the distribution of farms across provinces (fig. 1b ). all sera were tested in the indirect pedv s1-based elisa (group 1, table 2 ). nine samples, originating from nine different farms located in four different provinces, tested seropositive. the od values were low in all seropositive samples (od: 0.3-0.8) relative to positive control sera (convalescent or hyperimmune sera) with values od: 0.8-3.2. two of the nine elisa-positive sera from the serological study also tested positive in the vnt. with two confirmed positive samples of the 838 samples the animal prevalence was 0.24% (ci 95%: 0 -0.9%). the herd prevalence with 2 out of 267 farms was 0.75% (ci 95%: 0-2.7%). for all herds, three samples were tested. the additional samples of seven herds with a seropositive sample of which one vnt positive, were examined. these samples, 36 in total, were all negative in the pedv elisa. the proportion of seropositive samples (7/838) falls within the expected proportion of false positives given the specificity of the elisa which is estimated at > 99%. so we either detected pedv outbreaks that had not spread yet or the seven original samples were false positives. for two herds no additional samples from the monitoring programs were available (including one sow farm with a vnt positive sample). all tested sera obtained from wild boar were negative for pedv antibodies in the used indirect elisa (group 2, table 2 ). in the first week of november 2014, gd animal health received a report of pigs showing lethargy and anorexia for up to 24 h after which profuse diarrhea occurred in almost all pigs in nine compartments within the fattening barn. diarrhea was watery, light greyish, sometimes yellow or green colored. body temperature in the clinical phase reached 39.8°c. after the first days of disease lethargy subsided and lack of appetite diminished, a profuse diarrhea became more prominent. in later stages of the infection the consistency of the diarrhea changed to slightly more solid. pigs did not seem to suffer much and no animals died, although some pigs did not grow for a week and within the group body weights started to differ. after an extra week of feeding, pigs did recover and had a normal weight at slaughter. the fattening barn, located in a pig dense area of the netherlands, consisted of 18 compartments with 104 pigs each, divided over 8 pens. first symptoms occurred on october 26 th 2014 in one compartment followed by symptoms in consecutive compartments in the following days. initial diagnostic tests for the presence of e. coli, salmonella, lawsonia intracellularis and brachyspira pilosicoli were, except for low numbers of pathogenic e. coli, negative. based on the low numbers of pathogenic e. coli and the age of the pigs involved, e. coli was ruled out as causative agent in this case. subsequently, it was decided to test for porcine deltacoronavirus (pdcov) and pedv. six fecal samples were found to be negative for pdcov, but positive for pedv rna on november 14 th . on the same day, three pigs with severe diarrhea were euthanized and were submitted for post mortem examination. pathological examination showed severe villus atrophy in the small intestine, and pcr tested positive for pedv. during the outbreak, in total 67% of the animals (12 compartments) were affected by pedv as confirmed by pcr on fecal samples. based on the results of the serological survey, over 99% of the dutch farms were pedv negative in 2014; it was decided to try to prevent the further spread of the outbreak. to control and eradicate pedv the biosecurity measures taken, as described in the materials and methods section, seemed to be of great importance. also pet control was applied since some farms suffered from mice and rats. furthermore, it became clear that in compartments in which the infection was present, after thorough cleaning and disinfection, pedv free piglets could be introduced and that those stayed free from pedv infection. three fattening and three sow herds were presumed free of pedv within 6 months after the diagnosis of ped was confirmed. the nursery herd was depopulated, and, after double cleaning and disinfection of all compartments, repopulated. despite the immediate action of all parties in the dutch pig production industry to optimize their hygiene measures to ensure that infection by pedv would be avoided as much as possible, it could not be prevented that pedv spread to other farms. most infected farms were located on the east side of the netherlands at the border of germany (fig. 1c) . at the end of 2015, in total 75 farms were confirmed pedv positive by gd animal health. in fig. 2 the number of new pedv pcr positive farms per month since the beginning of the outbreak is shown. in order to characterize the virus originating from outbreaks in 2014 and 2015, the s gene of three isolates of different farms was sequenced and the sequences ned/gd001/2014, ned/gd002/2014 and ned/ gd001/2015 were deposited in the genbank database and received accession numbers kr011121 (index farm), kr011122 and mf974246, respectively. together with sequences available in genbank, the isolates were compared in a phylogenetic tree (fig. 3) . sequence analyses showed that the isolates had a 99.5% homology with the usa/oh851 strain and a 99.8% homology with the 2014 german g1b strains. furthermore, the european strains available in genbank (2014-2016) cluster together with this oh851 strain, or the so called s-indel strain (fig. 3) . this study showed that most likely pedv and particularly the genogroup 1b (s-indel) strains did not circulate in the netherlands on a large scale till the end of 2014. only a very small part of the dutch sow farms tested positive on antibodies against pedv (0.75% (ci 95%: 0-2.7%)) and no pedv antibodies were detected in wild boars. the used elisa based on the s1 protein of a g2b strain showed a high sensitivity and specificity against antibodies raised against g1a, g1b en g2b strains. however, the elisa was validated with a limited amount of samples ( supplementary fig. 1 ) and field samples may react differently, just like in a similar elisa recently described (gerber et al., 2014) . the viral spike (s) protein is prominent on the virus surface and is very immunogenic. all animals that have been infected with pedv have antibodies against the s protein and particularly against the s1part. the s1-part of the spike protein is the most variable part between related coronaviruses and there is no cross reactivity with other coronaviruses such as transmissible gastroenteritis coronavirus (tgev), porcine respiratory coronavirus (prcov) and porcine delta coronavirus (pdcov) as previously described (gimenez-lirola et al., 2017; lin et al., 2015b) . because results of immunoassays based on the s protein correlate well with viral neutralization (paudel et al., 2014) , as shown in supplementary table 1 , we decided to use the vnt as an alternative sero-diagnostic assay. based on case reports from germany stadler et al., 2015) , austria (steinrigl et al., 2015) and the netherlands (gd animal health) a pedv infection with the european circulating strain (g1b) will spread very rapidly within a herd and a single pedv positive animal on a farm is not likely to occur. the number of elisa positive animals (67%, table 2 , group 3) on the index farm after the clinical signs started, seemed to justify the assumption of the high within herd prevalence of 70% for sample size calculation for the serological survey. eventually, there were fewer herds sampled than planned (267 instead of 286) since it was decided to stop the serological survey, because the first case of pedv was diagnosed in the netherlands on november 14 th 2014. sequence analyses of the viruses isolated in 2014-2015 showed a 99% homology with oh851, a less virulent pedv strain found in the us and in germany in 2014 . although the g1b virus strain present in europe is less virulent (efsa, 2016; grasland et al., 2015; hanke et al., 2015; mesquita et al., 2015; steinrigl et al., 2015) compared to the strain that caused the us outbreaks in 2013 , this european strain showed to be very contagious and still could cause severe economic damage in pedv naive herds. after infection on sow farms loss off piglets could range up to 100% in the sucking piglets ( (lin et al., 2015a) and individual case reports, gd animal health). in the foreseeable future, vaccination will not be possible. therefore, the authorities in the netherlands advised all parties in the pig production industry to optimize their hygiene measures to ensure that infection by this virus would be avoided as much as possible. the strict preventive biosecurity measures taken in these herds demonstrated that most herds that were monitored could prevent the transfer to pedv naive compartments within the herd and some were able to obtain a presumed pedv free status for the whole farm. additionally, a higher biosecurity level helps preventing the introduction of other pathogens and controls the spread of infection within that herd (fao, 2010) . however, an increasing number of herds became infected (fig. 2) and pedv spread across the country after the initial outbreak (fig. 1c) . the transport trucks with positive animals between herds seemed to have the highest transmission risk (data not shown). the number of new pedv pcr positive farms per month since the beginning of the outbreak is most likely an underestimation (fig. 2) . since ped is not a notifiable disease, nor have all veterinarians performed diagnostic testing in all cases, as the clinical signs of an outbreak are very typical. it seems that pedv g1b became endemic in the netherlands since its initial outbreak in november 2014, just like in most countries in europe. that g1b viruses isolated in europe in 2014-2016 phylogenetically cluster together with a high similarity (fig. 3) suggests a onetime introduction event. however, pedv is endemic and many other coronaviruses are circulating that may result in coronavirus variants through mutation or recombination (fehr and perlman, 2015) . therefore, it is of upmost importance that the presence of circulating pedv is being monitored and genetically analyzed, in order to update diagnostic tools where necessary. we declare no conflict of interest. surveillance and control of ped coronavirus in pigs in italy porcine epidemic diarrhea virus shedding and antibody response in swine farms: a longitudinal study isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states viral disease affects u.s. pigs: porcine epidemic diarrhea found in at least 11 states updated epidemiological data on ped good practices for biosecurity in the pig sector -issues and options in developing and transition countries coronaviruses: an overview of their replication and pathogenesis detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa reactivity of porcine epidemic diarrhea virus structural proteins to antibodies against porcine enteric coronaviruses: diagnostic implications previous infection of sows with a "mild" strain of porcine epidemic diarrhea virus confers protection against infection with a "severe complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france comparison of porcine epidemic diarrhea viruses from germany and the united states origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states multiplex realtime rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus wild boars harboring porcine epidemic diarrhea virus (pedv) may play an important role as a pedv reservoir manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination cellular entry of the porcine epidemic diarrhea virus experimental infection of a us spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original us pedv infection antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains role of transportation in spread of porcine epidemic diarrhea virus infection epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy outbreak of porcine epidemic diarrhea virus in portugal cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene comparison of serum neutralization and enzyme-linked immunosorbent assay on sera from porcine epidemic diarrhea virus vaccinated pigs a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines emergence of porcine epidemic diarrhea virus in southern germany first detection, clinical presentation and phylogenetic characterization of porcine epidemic diarrhea virus in austria emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences mega6: molecular evolutionary genetics analysis version 6.0 win episcope 2.0: improved epidemiological software for veterinary medicine distinct characteristics and complex evolution of pedv strains new variant of porcine epidemic diarrhea virus pig farming the authors would like to thank d. oorburg en c. sima of vion, groenlo, the netherlands, for providing blood samples from slaughter sows, g. spierts and laboratory staff for collecting and archiving blood samples and n. schuurman and j. de jong for performing the elisa. furthermore, r. dijkman for implementing the pcr at the gd animal health lab, a. van lenthe for advice during the survey, m. meijerink for assisting in sample collection during herd visits and a. veldhuis and h. brouwer-middelesch for making the maps of the netherlands (fig. 1) . the authors thank the owner of the index farm and the practitioner involved in this farm for their information and their cooperation in collecting the samples.the authors would like to thank the dutch ministry of economic affairs and the product board for livestock and meat for funding the monitoring system for pig health in the netherlands. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetmic.2018.05.014. key: cord-306517-tls0849i authors: leidenberger, s.; schröder, ch.; zani, l.; auste, a.; pinette, m.; ambagala, a.; nikolin, v.; de smit, h.; beer, m.; blome, s. title: virulence of current german pedv strains in suckling pigs and investigation of protective effects of maternally derived antibodies date: 2017-09-07 journal: sci rep doi: 10.1038/s41598-017-11160-w sha: doc_id: 306517 cord_uid: tls0849i porcine epidemic diarrhea (ped) has caused tremendous losses to the united states pig industry since 2013. from 2014, outbreaks were also reported from central europe. to characterize the central european pedv strains regarding their virulence in suckling piglets, and to assess the protective effect of maternally derived antibodies (mda), four trial groups were randomly assigned, each consisting of two pregnant sows and their litter. to induce mda in a subset of piglets, two sows received a cell culture-adapted pedv strain, and another two sows were inoculated with field material from german ped outbreaks. four sows stayed naïve. subsequently, all piglets were inoculated with the corresponding pedv strains at an age of 3 to 6 days, and virus shedding, clinical signs and occurrence of specific antibodies were assessed. piglets without mda showed a morbidity of 100% and low lethality, while almost all mda-positive piglets stayed clinically healthy and showed considerably lower virus shedding. taken together, the central european pedv strains showed rather low virulence under experimental conditions, and pre-inoculation of sows led to a solid protection of their offspring. the latter is the prerequisite for a sow vaccination concept that could help to prevent ped induced losses in the piglet sector. clinical and pathological observations. clinical signs upon inoculation of sows. two sows, which were inoculated with a cell culture-adapted pedv strain (pedv eu, group a1) showed slight depression over a period of four days, accompanied by reduced feed intake. in contrast, the two sows of group b1 (inoculated with german field pedv, de) showed diarrhea and vomiting upon inoculation. moreover, both sows were anorectic for one day. thereafter, one of the sows (ear tag 6150) was still showing slight depression over four days whereas the other was free of clinical signs again. all four sows of groups a2 and b2, which stayed naïve prior to farrowing, remained healthy during the pre-farrowing time. clinical signs upon inoculation of piglets. sows which were naïve before farrowing and got infected via their inoculated piglets during the trial, showed more severe clinical signs according to our standardized score system for sows (table 1) than sows, which were directly inoculated four weeks before farrowing (see above). almost complete anorexia, watery diarrhea, and decrease in milk production was observed from 60 h after the inoculation of the piglets in sows of groups a2 and b2 (see supplementary fig. f1 ). the lack of milk production led to a more severe situation for their unprotected infected piglets (malnutrition). sows, which were inoculated before farrowing (sows of groups a1 and b1), did not show any clinical signs indicative for ped upon challenge of their piglets. one sow of group a1 died from septicaemia two days after farrowing (necropsy revealed severe peritonitis after uterine rupture and intestinal invagination). the respective piglets (which had all taken colostrum) were reared by the remaining sow of group a1. as can be seen in fig. 1 , none of the mda-positive piglets showed any severe clinical signs indicative for ped upon challenge inoculation. very mild signs were discontinuously observed in some animals (pasty feces). one litter (group b1, sow 4423) showed general weakness and remittent shivering leading to clinical scores prior to inoculation. a viral genesis could not be detected by routine pcrs (porcine enteroviruses (pev), suid alphaherpesvirus-1, classical and african swine fever virus, atypical porcine pestivirus). piglets without mda and inoculated with the cell culture-adapted virus (pedv eu, group a2) started vomiting 24 hours post inoculation (hpi), and showed diarrhea from 36 hpi. the clinical signs remained over 6 to 7 days with diarrhea as the leading sign (see fig. 1 , upper graph). mda-negative piglets challenged with the german field material (de), started to show signs indicative of ped at 24 hpi, also beginning with vomiting followed by diarrhea. clinical signs persisted over 10 to 11 days (fig. 1, lower graph) . as the respective sows showed an almost complete cessation of milk production, piglets showed signs of malnutrition. two piglets had to be euthanized with an endpoint clinical score because of dehydration and general weakness. necropsy of the euthanized piglets showed gas-filled intestines. all other piglets were without specific results indicative for ped at the time point of necropsy (end of the trial). clinical score values differed significantly between groups with and without mda from 2 to 10 days post inoculation (dpi). in addition, a statistically significant difference was observed at 1 dpi between groups a1 and a2, and 12 to 14 dpi between groups b1 and b2. the above mentioned condition in one litter of group b1 led to a significant difference at the day of challenge that was however not ped related. interested in feed without clear feed intake watery feces, reddened anal region, vomiting 3 strong depression, almost entirely resting, decreased milk production, abortion total anorexia watery feces with blood or fibrin added, highly reddened anal region, vomiting table 1 . clinical score system for sows in ped infection studies. the humane endpoint was defined as a cumulative score >6. moreover, euthanasia was carried out after abortion or other signs indicative for inacceptable suffering. shed virus from 4 or 5 days post inoculation (dpi) for 7 to 10 days, while the group inoculated with the field virus (b1) was pcr-positive from 4 dpi for 9 to 10 days. all naïve sows were negative prior to the inoculation of piglets. pedv shedding of piglets. detection of pedv genome occurred in all groups but to a much lesser extent in the mda-positive animals (see fig. 2 , pedv eu in the upper graph, field material de in the lower). in general, the amount of detectable pedv genome was influenced by the faecal load on the swab. however, as this phenomenon occurred in all groups, it was regarded as systematic error. all below mentioned genome copy numbers refer to 1 µl rna extracted from 100 µl faecal suspension (all swabs were submerged in 1 ml medium, irrespective of their load). piglets of groups a1 and a2 showed viral rna in rectal swabs beginning 1 dpi until 20 dpi, and the genome load was markedly lower in piglets of group a1 with mda. significant differences were found among groups a1 and a2 on 1, 2, 13, and 17 dpi with higher genome loads in the animals without mda (a2). on the other days, the genome loads were still higher in group a2, but not significantly different. it was quite the same in group b. in this case, genome loads were significantly higher on 1 to 5 dpi and 13 to 18 dpi in piglets without mda (b2). in more detail, all animals of group a2 were found positive at 1 dpi with genome loads between 1100 and 147000 genome copies per µl, whereas only low virus shedding could be detected in group a1 with genome copy numbers ranging from 0.2 to 64 per µl (see fig. 2 , upper chart). at 2 dpi, a comparable picture was seen at lower level. however, single results varied considerably (genome copies ranging from 0 to 47000 in group a2 and 0 to 26 in group a1). only low copy numbers were detected for all animals at 3 dpi (no significant differences). up to 7 dpi, mainly low genome loads were detected with single animals showing considerable shedding (up to 213500 copies per µl for a mda negative piglet at 5 dpi). at 7 dpi all but one piglets of group a2 still showed genome loads of 0.4 to 33000 genome copies per µl. animals of group a1 showed generally lower genome loads with 0.2 to 850 genome copies per µl. only one piglet of this groups shed higher genome loads with 9600 genome copies per µl. thereafter, shedding decreased, with faster decline in the mda positive groups (see fig. 2 , upper graph). compared to group a2, mda negative animals of group b2 showed higher initial virus shedding (up to 18000000 copies per µl) at 1 and 2 dpi. only a few animals of the mda positive group were detected positive for a few days with genome loads ranging from 0.15 and 37 genome copies. from 3 dpi, virus shedding decreased gradually with scattered positive findings and high variability. from 17 dpi, all mda positive animals (b1) were negative while shedding still occurred in mda negative animals of group b2 (see fig. 2 , lower graph). detection of other relevant pathogens. the inoculum of the sows was tested negative for transmissible gastroenteritis virus and porcine delta coronavirus. positive results were obtained for porcine circovirus 2, rotavirus a, and porcine enteroviruses (pev). the piglet inoculum was tested positive for pev genome (cq value: 30) and showed negative results for all other pathogens. however, no pev-shedding could be detected in piglets, which showed signs that might have been indicative for pev infection (group b2; litter of sows 4423 and 6150). the cell culture adapted virus (pedv eu, groups a1 and a2) was free of all tested pathogens. prior to the study, sows were tested for pedv-specific antibodies by three commercial indirect igg isotype antibody elisa. all but one animal showed clear negative results. the serum sample of the remaining sow which had shown inconclusive results was retested by indirect immunofluorescence with clear negative results. all sows, which were inoculated with pedv prior to farrowing (groups a1 and b1), showed igg isotype antibodies in blood and colostrum samples at the day of farrowing using the elisa assays mentioned above. in general, the used elisa kits seemed to vary regarding specificity and sensitivity. nevertheless, the overall tendency of antibody levels in the different groups was comparable (see fig. 3a -c). colostrum samples were also tested positive for pedv s protein specific iga isotype antibodies using an in-house elisa. in those pre-inoculated animals, antibody levels in blood decreased till the end of the trial. sows in groups a2 and b2 had no detectable pedv specific igg isotype antibodies in blood or colostrum at the day of farrowing but developed rising levels till the end of the trial once they got exposed to pedv from their offspring. blood samples of all but two piglets received from pre-inoculated sows (both in group a1) were shown to contain pedv antibodies prior to inoculation (see fig. 3a -c, upper right chart for pedv eu and lower right chart for de). at the end of the trial 72.0% (b1/b2) to 81.8% (a1/a2) of the piglets and all of the sera from the sows were positive in the igg-elisa (see fig. 3a -c). all milk samples collected at the day of slaughter or death were negative for pedv-specific igg, but showed varying amounts of iga isotype antibodies. this was also found in samples of sows which were naïve prior to farrowing (groups a2 and b2) (see supplementary fig. f2 ). as can be seen in fig. 3a -c, a clear increase of serum antibodies was observed over the course of the trial in mda negative piglets of groups a2 and b2, whereas antibody levels markedly decreased in the mda positive groups a1 and b1. bacteriology. the fecal swabs taken at 0 dpi and 2 dpi did not show any growths of pathogenic bacteria. the bacterial flora was similar in the samples at 0 and 2 dpi. following the disastrous ped outbreaks in the us, re-introduction was also confirmed in central europe 25, 27, [29] [30] [31] [32] 34 . with the exception of the highly virulent strains occurring in ukraine 28 , all recent european pedv strains were so-called s-indel variants with an expected lower virulence than the us-type non-indel pedv variants. overall, the ped impact on pig industry seemed to be low, but sporadic cases with high mortality and long persistence of the disease in continuously producing enterprises was observed 37 . follow-up investigations showed that the reported virulence of the german pedv strains varied considerably among breeding herds (suckling pigs) while only mild diarrhoea was observed in fatteners. interestingly, the causative strains were highly identical, even on the full-genome level 33 . detailed molecular analyses did not reveal any significant impact of viral and bacterial co-infections or viral variants 38 . in order to assess strain-related virulence and disease dynamics among suckling pigs in the presence and absence of maternally derived antibodies, the presented study was carried out under controlled laboratory conditions. in a nutshell, the pedv strains obtained from recent german outbreaks (both cell culture and field material) showed indeed a low virulence with only two mda-negative piglets that had to be euthanized at the humane endpoint. all remaining piglets recovered completely and were able to compensate the weight loss by the weaning age. however, piglets with mda were almost completely protected from clinical disease. in direct comparison, the field material induced more severe signs in naïve piglets than the cell culture isolate. possible reasons include attenuation by cell culture adaptation but also differences in inoculation dose and secondary pathogens. regarding the inoculation dose, it can be stated that the genome load was higher in the cell culture material than in the field material. provided that this gives at least an indication for the virus titer, we can assume that the virus titer of the field material was at least not considerably higher than that of the cell culture material. testing of the field materials for secondary pathogens showed low rotavirus a and enterovirus loads. the secondary materials used for the inoculation of the piglets in groups b1 and b2 contained pedv and low pev genome loads. the latter was however not found in any of the piglets. despite the observed differences, 100% morbidity and long-term shedding of viral rna was still observed for both variants in mda-negative piglets. in contrast to the study with a cell culture adapted us-type pedv, published by poonsuk et al. in 2016 39 , all sows that were naïve prior to farrowing showed diarrhea after challenge of their piglets. disease progression in these sows was worse than the disease course in the sows following direct inoculation prior to farrowing (groups a1 and b1). diarrhea and depression even led to a severe decrease of milk production and thus inadequate nutrition of piglets. this underlines the importance of the sow's health status for the fate of ped-diseased piglets. the increased severity could probably be explained by the more fragile immune system during the farrowing and lactating period, but also with the higher virus loads in the stable and ongoing contact to pedv shed by the infected piglets 39 . shedding of viral rna in both groups was detected over three weeks and beyond, which is in line with former studies 30, 33, 40 and field observations. the variability of individual results was probably due to the different content of fecal material on the swab. however, also the desquamation of intestinal cells and hereby the amount of virus at rectal swabs might vary. in order to gain individual samples, pooled faecal samples were not considered appropriate. the long-term shedding of virus could be a most important issue for disease control and elimination. with shedding over such a long time, it is possible that clinically healthy but still shedding piglets are sold and brought to another holding for subsequent production steps where they pose a risk for naïve stable/pen mates. the risk of disease transmission by those piglets would however need further investigation. most importantly, our study clearly shows that maternally derived immunity against pedv is able to protect piglets in the most vulnerable phase after birth. this is prerequisite for a sow vaccination strategy which should be explored in light of the tremendous impact of ped in the us and also farms in the ukraine. with neonatal piglets being born agammaglobulinemic and possessing limited, undeveloped lymphoid tissues and no effector and memory t-lymphocytes 41 , it is absolutely necessary that they receive maternal cell-mediated and humoral immune components through the ingestion of colostrum and milk 42 . it is already shown for tgev that natural oral infection of sows leads to a more effective maternal derived lactogenic protection for suckling pigs than systemic immunization 43, 44 . this effect is mainly based on a larger amount of secretory iga in colostrum and milk because of stimulation of the gut-mammary-axis of the sow. systemic immunization usually induces high levels of igg in both the blood and colostrum, but iga titers in blood might stay on a low level offering no reliable lactogenic protection against infection of piglets 45 . this is in line with the results of the provided study, in which high amounts of igg could be detected in colostrum but not in milk samples, whereas iga could be found in both -colostrum of pre-infected sows and milk of all sows. thus, also the sows that stayed naïve prior to farrowing seroconverted upon challenge of their piglets and low levels of iga could be detected in milk samples at the end of the trial. when revisiting ped vaccination, this route-dependent issue should be kept in mind as it could explain the unsatisfactory protection against virus shedding and morbidity of piglets from sows that were only parenterally immunized using different types of vaccines 46 . therefore, a sow vaccination concept against pedv should probably prefer oral or other mucosal immunization routes for proper protection of suckling pigs if possible 9, 47, 48 . regarding the duration of immunity in both sows and piglets, we could confirm that the antibodies are rather short-lived. sows pre-exposed prior to farrowing showed already decreasing levels of igg at the end of the trial (with elisa results still being positive). with a high antibody titer in the blood being a requirement for solid and protective antibody titers -especially secretory iga -in colostrum and milk, it seems that sows would have to be re-vaccinated/re-infected prior to each farrowing to ensure piglet protection. moreover, immunization is needed close to the farrowing date. from literature it is known that antibody levels of igg and iga in serum of primiparous and multiparous sows can remain stable up to 6 months post field infection as measured by high titers in elisa and virus neutralization assay 49 . the respective sows were obtained from commercial breeding farms that had reported previous ped outbreaks. thus, booster/reinfection seems to play a role for the duration of immunity. with regard to piglets, field studies observed antibody levels in piglets that lasted up to 100 days 49, 50 . for the piglets/young weaners, the source of antibodies clearly influences the duration of detection. while a decay of mda is visible already after a few weeks, antibodies induced by active immunization or infection might last much longer. in our study, mda-positive animals had only low antibody levels by the end of the trial (at the age of weaning). in these animals, protection in later stages of pig production would have been questionable and this could explain field observations that showed reinfection of finishing pigs that had been exposed to pedv as suckling pigs. these aspects also need consideration when designing a vaccination concept. another important fact is the reliability of commercial elisa assays. in this study three commercial elisa assays were compared and variations in specificity and sensitivity were found as already shown in other studies 51 . therefore, the combined use of different diagnostic tools to investigate the serological status of an animal should be taken into account. in conclusion, the tested pedv strains were of moderate to low virulence and natural infection of sows during gestation led to an effective lactogenic immunity in piglets. this is a promising outcome when vaccination concepts are discussed. as a spin-off of our trial, a simple and standardized score system was implemented that worked well even with limited clinical signs. the presented score system (see table 2 ) is less detailed than others that were previously described 52 . nevertheless, it seems sufficiently reliable and will be used for subsequent immunization-challenge experiments. study design. in total, eight sows and their offspring were used in this study. the multiparous sows were purchased in late pregnancy from a commercial breeding farm with a high veterinary hygiene standard (bundes hybrid zucht programm, bhzp, dahlenburg-ellringen, germany). they were transported to the high containment facilities at the friedrich-loeffler-institute greifswald, insel riems, germany, five weeks before farrowing. according to eu legislation, the animals were kept in open pens in sets of two before they were moved to commercial farrowing pens one week before farrowing. two farrowing pens were integrated in one stable room so that each experimental group could be kept together. all animals had access to water ad libitum and were fed with commercial feed for breeding sows and after farrowing for lactating sows. all applicable animal welfare regulations, including eu-directive 2010/63/ec and institutional guidelines, were taken into consideration. the animal experiment was approved by the competent authority, landesamt für landwirtschaft, lebensmittelsicherheit und all animals were tested prior to arrival with negative pcr-results for pedv and antibodies against pedv. after arrival, the animals were randomly assigned to four treatment groups. two sows (ear tags 1871 and 4249) were orally inoculated with cell culture adapted pedv (pedv eu) four weeks before farrowing to induce mda-positive piglets (group a1). another two sows (ear tags 4423 and 6150) were treated the same way orally with german field material (organ suspension and fecal homogenate; de) containing pedv to also obtain mda-positive piglets (group b1). the remaining four sows stayed untreated to obtain mda-negative piglets (group a2: ear tags 4343 and 4454; group b2: ear tags 1866 and 4365). all piglets born alive were individually ear tagged prior to challenge inoculation. piglets born to sows of groups a1 (9 piglets of sow 4249 and 12 piglets of sow 1871) and a2 (7 piglets of sow 4454 and 7 piglets of sow 4343) were orally challenged with pedv eu at an age of three to six days whereas piglets born to sows of groups b1 (7 piglets of sow 6150 and 5 piglets of sow 4423) and b2 (8 piglets of sow 4365 and 8 piglets of sow 1866) received the german field material (de). during the whole trial, daily rectal swabs (copan plain cotton swabs without medium) were taken of all animals for real-time reverse transcription polymerase chain reaction (rt-qpcr) analyses. samples of days 0 to 7 post inoculation (pi) as well as days 10 pi and 13 pi were tested. moreover, samples were tested from day 21 pi till the end of the trial. additional rectal swabs were taken of four randomly chosen piglets of each sow prior to inoculation and two days afterwards for bacteriological examination. moreover, clinical signs indicative for ped were recorded using a standardized score system (see table 2 ). blood samples were taken at the day of inoculation and the day of slaughter or euthanasia. additional blood and colostrum samples were collected from all sows during farrowing. furthermore, milk samples were collected from all but two sows at the day of slaughter (samples missing from sow 4249 which died from septicemia, and sow 1871 which milk production had already ceased). at the end of the trial, all remaining piglets were euthanized and seven sows were slaughtered (electro-stunning and subsequent exsanguination). all animals were necropsied and samples of the small intestine were taken. for inoculation of animals of groups a1 and a2, cell culture adapted pedv was provided by the boehringer ingelheim veterinary research center (pedv eu). this cell culture isolate was obtained from a ped outbreak in northern germany with high mortality rates in suckling piglets. it is sharing over 99% nucleotide identity with the u.s. strain oh851 and recent central european pedv strains. the initial isolate was obtained using the following (standard) protocol: 5 × 10 5 vero cells were seeded into 6-wells plates. after 24 h of incubation at 37°c, confluent monolayers were obtained and the cell culture medium was removed. all wells were washed three times with 3 ml phosphate-buffered saline (pbs). thereafter, cells were inoculated with 200 µl of filtrated (0.22 µm) homogenate from piglet guts. after the addition of 2 ml of pedv media containing 15 µg/ml trypsin, cultures were incubated for 24-48 h at 37°c. the cultures were checked daily for the presence of characteristic fusion formation, and positive materials were further passaged. for the purpose of our study, the titer was defined by end point titration and recorded as tissue culture infection dose 50 (tcid 50 )/ ml. the stock titer was 3.16 × 10 5 tcid 50 /ml (rt-qpcr: cq value 15). each sow of group a1 received 6 ml of this stock diluted with 14 ml pbs. the solution was orally fed using a 20 ml syringe. piglets of groups a1 and a2 were also orally inoculated. in this case, each piglet received 2 ml of a 1:10 diluted viral stock (titer 3.16 × 10 4 tcid 50 / ml) using 2 ml syringes. to inoculate animals of groups b1 and b2, pedv-pcr-positive field material (de) from recent clinical cases in south-western germany was used. in these cases mainly fattening pigs were affected by rather mild clinical signs. the material was a pool of different fecal samples and intestines. to obtain the inoculum, intestines were homogenized with sterile sea sand using mortar and pestle. faecal samples were mixed and also homogenized. each sow of group b1 received 20 ml of the organ suspension (rt-qpcr: cq value 23) and 20 ml of the faecal homogenate (rt-qpcr: cq value 15) orally using 20 ml syringes. all piglets of groups b1 and b2 received a faecal homogenate obtained from the sows of group b1 at 5 dpi and 6 dpi (rt-qpcr: cq value 19). the piglets were inoculated orally with 2 ml of this faecal homogenate using 2 ml syringes. sample preparation and nucleic acid extraction. rectal swabs were submerged in 1 ml dulbecco's modified eagle medium with standard antibiotics and antimycotics (antibiotic-antimycotic 100×, gibco) and incubated for 1 hour at room temperature. viral rna was extracted using either the manual qiamp viralrna mini kit (qiagen) or the nucleomagvet-kit (macherey-nagel) in combination with the kingfisher extraction platform (thermo scientific). the rna was stored at −20 °c until further use. blood samples were centrifuged at 2000 × g for 20 minutes at room temperature to obtain serum. the resulting serum was aliquoted and stored at −20 °c. colostrum and milk samples were aliquoted and stored at −20 °c until further use. to detect pedv genome in fecal swabs, a rt-qpcr system targeting the s-gene of pedv was used as described previously 33 . cq values above 40 were considered negative and the amount of pedv genome copies was calculated by using a standard curve. in addition, the inoculum for sows and piglets was tested for porcine circovirus 2, porcine enteroviruses (including enteroviruses, teschoviruses, and sapeloviruses), rotavirus a, transmissible gastroenteritis virus, and porcine delta coronavirus using specific in-house rt-pcr assays (primer sequences and reaction conditions are available from the authors upon request). swabs of animals which showed central nervous signs that could have been indicative for infection with teschoviruses were also tested in the pev pcr assay (concerns the litters of sows 4423 and 6150, group b1). plots and statistics. creation of different plots and charts was performed using sigmaplot for windows version 11.0 (systat software). shapiro-wilk test was used for normality testing and a mann-whitney rank sum test was conducted as implemented in the software package. statistical significance (p <= 0.05 was considered significant) was tested using sigmaplot software. veterinary treatments unrelated to ped. six sows (all except animals 4249 and 1871) received cloprostenol (estrumate, intervet) to induce farrowing. moreover, all sows were treated with antibiotics (riketron n, ani-medica) and anti-inflammatory drugs (metacam, boehringer) for a mild metritis-mastitis-agalactia-syndrome over 3 days. piglets of sow no. 6150 (group b1) received baytril orally over 4 days because of mild enteritis prior to inoculation. where needed, piglets received anti-inflammatory drugs (metacam, boehringer) for lameness (arthritis, panaritia). antibody detection. three commercial indirect elisa (swinecheck ped indirect, biovet, gc kerkrade, the netherlands; ingezim pedv, ingenasa, madrid, spain; id screen pedv indirect, grabels, france) were performed with all sera according to the producer's manual. colostrum and milk samples were tested in the same manner after initial validation. while elisa kits of biovet and idvet are using recombinant nucleoprotein, the ingenasa elisa plates are coated with recombinant spike protein. in cases of ambiguous results, the respective samples were tested in indirect immunofluorescence assays using commercial pedv fa substrate slides (vmrd, pullman, washington) following the manufacturer's instructions. the slides were screened for specific fluorescence with a standard fluorescence microscope (zeiss axio vert.a1, oberkochen, germany). in addition, colostrum and milk samples were tested for pedv specific iga antibodies using an in-house indirect elisa (assay specifications are available from the authors upon request). data availability statement. the datasets generated and analysed during the current study are available 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of their suckling pigs against virulent tgev challenge exposure evaluation of the effects of pedv vaccine on pedv naïve and previously pedv-exposed sows in a challenge model comparing immune response and preweaning mortality porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr13 strain immunogenicity and protective efficacy of recombinant s1 domain of the porcine epidemic diarrhea virus spike protein evaluation of humoral immune status in porcine epidemic diarrhea virus (pedv) infected sows under field conditions porcine epidemic diarrhea virus shedding and antibody response in swine farms: a longitudinal study inter-laboratory study to characterize the detection of serum antibodies against porcine epidemic diarrhoea virus experimental infection of young pigs with an early european strain of porcine epidemic diarrhoea virus and a recent us strain this research was supported by boehringer ingelheim. we thank our colleagues from boehringer for sharing knowledge and discussion of research results. our special thanks goes to svenja mamerow who helped us throughout the trial. we are also grateful to all of our colleagues in the laboratory and stable. n.v., des. h., b.m. and b.s. designed the experiment. l.s., s. ch., au. a., b.s., and z.l. carried out the animal trial and collected clinical samples. sample analyses were performed by l.s., z.l., au. a., l.s., m.p. and a.a. analysed data. the manuscript was written by l.s., b.s. and b.m. all authors took part in discussion and interpretation of results. all authors read, advised and approved the final manuscript. supplementary information accompanies this paper at doi:10.1038/s41598-017-11160-w competing interests: the authors were part of an industrial funded research project from boehringer ingelheim. the pedv cell culture isolate was kindly provided by boehringer ingelheim. no other conflicts of interest exist.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-310579-tnxokfwu authors: kim, sung-jae; nguyen, van-giap; huynh, thi-my-le; park, yong-ho; park, bong-kyun; chung, hee-chun title: molecular characterization of porcine epidemic diarrhea virus and its new genetic classification based on the nucleocapsid gene date: 2020-07-23 journal: viruses doi: 10.3390/v12080790 sha: doc_id: 310579 cord_uid: tnxokfwu porcine epidemic diarrhea virus (pedv) causes continuous, significant damage to the swine industry worldwide. by rt-pcr-based methods, this study demonstrated the ongoing presence of pedv in pigs of all ages in korea at the average detection rate of 9.92%. by the application of bayesian phylogenetic analysis, it was found that the nucleocapsid (n) gene of pedv could evolve at similar rates to the spike (s) gene at the order of 10(−4) substitutions/site/year. based on branching patterns of pedv strains, three main n gene-base genogroups (n1, n2, and n3) and two sub-genogroups (n3a, n3b) were proposed in this study. by analyzing the antigenic index, possible antigenic differences also emerged in both the spike and nucleocapsid proteins between the three genogroups. the antigenic indexes of genogroup n3 strains were significantly lower compared with those of genogroups n1 and n2 strains in the b-cell epitope of the nucleocapsid protein. similarly, significantly lower antigenic indexes in some parts of the b-cell epitope sequences of the spike protein (coe, s1d, and 2c10) were also identified. pedv mutants derived from genetic mutations of the s and n genes may cause severe damage to swine farms by evading established host immunities. porcine epidemic diarrhea virus (pedv) is an enveloped, single-stranded rna virus belonging to the family coronaviridae, subfamily coronavirinae, genus alphacoronavirus, and subgenus pedacovirus. pedv is one of the major pathogens causing acute enteritis disease, which is characterized by vomiting and watery diarrhea and commonly leads to high rates of mortality and morbidity in suckling piglets [1] . the disease was first reported in the uk in 1971, and the prototype virus-designated as pedv cv777-was subsequently identified in belgium [2] . since the 1980s, pedv has been widespread throughout asia, where it has been regarded as an endemic disease for many years [3, 4] . in the late 2010s, new and highly pathogenic strains were reported in china. these new strains were pathologically more critical than the classic strains, resulting in morbidities of 80-100% and mortality rates of 50-100% in infected suckling piglets [5] . in may 2013, these new highly pathogenic strains moved from china to the usa and rapidly spread across the country, massively impacting the swine industry; they affected more than 4000 farms, accounting for the death of more than 7 million piglets within the year [6, 7] . subsequently, these strains have become pandemic [8] . pedv has an approximately 28 kb long genome and consists of seven open reading frames (orf), encoding non-structural or structural proteins [9] . orf1ab and orf3 genes encode non-structural proteins. orf1a codes for the large polyprotein pp1a, while orf1b is always expressed with pp1a as the fusion protein pp1a/b through ribosomal frameshifting. pp1a and pp1a/b are further processed into 16 non-structural proteins (nsp1 to nsp16). orf3 codes an accessory protein that is likely to be an additional non-structural protein [10] . the envelope (e), membrane (m), spike (s), and nucleocapsid (n) genes encode four major structural proteins [11] . the n protein, which is the most abundant protein in the virus particle, provides the structural basis for the helical nucleocapsid surrounding the virus genome [12, 13] . the m and e proteins form a viral envelope by assembly. the m protein is the most abundant component, and the e protein is less abundant in the viral envelope. [14] . the s protein is very exposed and forms large petal-shaped spikes on the surface of the virus [15] . among these structural proteins, the s protein plays important roles in virus infection and the induction of neutralizing antibodies [16] and shows substantial genetic diversity [11] . as a result of these features, genetic analyses based on the s gene have been commonly used to investigate pedv evolution [7, 8, 17] . the pedv has diverged into several subgroups based on the genetic diversification of the s gene. so far, two genotypes (g1 and g2) have been identified by s gene phylogenetic analysis. each genogroup can be further divided into two subtypes, g1a/b and g2a/b, respectively [18, 19] . recently, a third subtype (g2c) was identified within the g2 genogroup [8] . the n protein is involved in several biological and immunological activities of the virus, including viral nucleolar localization, host cycle er stress, s-phase prolongation, the inhibition of interferon-β production [20] [21] [22] , and the induction of abundant antibodies [12, 13] . despite the significant features of the n protein, there has been less investigation on the n protein compared to the s protein. in order to better understand the evolutionary aspects of pedv, this study applied multiple bioinformatic tools to investigate the genetic diversity of pedv. six hundred and seventy-two fecal samples were collected from 83 farms in 2017, and 235 fecal samples were collected from 33 farms in 2018. rectal swabs were randomly collected from pigs of all stages (suckling, weaned, growing, finishing, gilt, sow) in swine farms dispersed throughout 9 provinces of south korea. rna extraction from the samples was performed using an rna/dna extraction kit (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions, and the extracted rna samples were stored at −70 • c. the rna was converted into cdna with a commercial kit (rna to cdna ecodry premix, clontech, otsu, japan), following the manufacturer's protocol. pedv in the collected fecal samples was detected by pedv-pcr [4] . commercial pcr kits (median diagnostics, chuncheon-si" korea) were used to detect porcine reproductive and respiratory syndrome virus (cat. ns-prr-11), porcine parvovirus (cat. ns-abo-11), and japanese encephalitis virus (cat. ns-abo-12). the other three enteric pathogens were detected by specific primers and pcr conditions reported previously, such as porcine deltacoronavirus [23] , transmissible gastroenteritis virus, and porcine group a rotavirus [24] . full-length genome sequencing was conducted with 26 overlapping primer pairs using positive samples from swine farms that were severely damaged by porcine epidemic diarrhea [25] , and 7 strains (y178, s6, s10, s12, s14, s97 and s100) were fully sequenced. information relating to the 7 strains is provided in table 1 . the full-length genome sequences of the 7 strains were registered in genbank (accession numbers mh891584-mh891590). for the genetic analyses, 72 other previously registered complete genome sequences were retrieved from genbank, and the sequences originated from asia (china, korea, japan), america (usa), and europe (germany, belgium) from 1978 to 2018. rdp v4.51 program [26] with 5 default algorithms (rdp, geneconv, maxchi, siscan, and bootscan) was applied to identify recombinants in the alignment of the s and n genes. the general options were "sequences are linear", "highest acceptable p-value = 0.05", and "bonferroni correction" = true. upon detection, new recombination-free alignments were created by the option of "save alignment with recombinant regions removed". four datasets with identical number of sequences (n = 79, seven obtained in this study) were generated for subsequent analyses: complete s gene (d1), s gene without recombinant regions (d2), complete n gene (d3), and n gene without recombinant regions (d4). beast package v2.6.1 [27] , which is available at the cipres science gateway [28] , was used to infer the phylogenetic relationships between sequences and co-estimate the substitution rates from the above-mentioned d1-d4 datasets. the genetic classification of pedv based on the s gene followed previous publication [8] . for the model of nucleotide substitution, bmodeltest tool [29] implemented in beast 2 was selected, which helps infer the most appropriate substitution model. for the molecular clock model, four models were specified: strict clock, uncorrelated lognormal and exponential relaxed-clock [30] , and random local clock [31] . for tree prior, three coalescent models implemented in beast 2 were tested, including coalescent constant population, coalescent exponential population, and coalescent bayesian skyline plot [32] . each analysis was run for 100 million chains, sampling every 10,000 generations. the output log files were analyzed in tracer v1.7.1 [33] to assess the convergence (effective sample size > 100). path sampling analyses [34] were also performed to select the best fitting molecular clock and tree prior models for each dataset. for that analysis, the number of path steps was 100, and the length of each chain was one million iterations. the nucleotide substitution rates and phylogenetic tree of each d1-d4 dataset were inferred from the data best-fit combining models (supplementary material tables s1 and s2). the phylogenic trees were summarized with treeannotator v2.6.1 to produce the maximum clade credibility tree, which was displayed using figtree v1.4.3. pairwise genetic distances (p-distances) within each dataset were calculated using mega v. 7 software [35] . the option for gaps/missing data treatment was specified as "partial deletion". the obtained results were displayed in a frequency distribution histogram of p-distance. basically, a lower genetic relationship between two sequences indicated a higher p-distance, and well-bounded areas with peaks in the histogram indicated the presence of different genetic clusters [36] . the baseml program implemented in package paml 4.9j [37] was used to reconstruct amino acid changes on the evolutionary path of pedv based on the n gene. the input tree topology for that analysis was the maximum clade credibility tree inferred by beast 2 under the data best-fit combining models. nonsynonymous substitutions that occurred on the given branches of a phylogeny were annotated by the treesub program [38] . amino acid sequences deduced from nucleotide sequences of the n gene were aligned and comparatively analyzed to investigate the nonsynonymous changes according to genetically distinct clusters. subsequently, antigenic index analysis of the complete amino acid sequences of the n gene was performed to evaluate the possible antigenic variation of n proteins. the antigenic index of each amino acid was calculated by the jameson-wolf method [39] , and the calculated indexes of each strain were compared. antigenic index analyses were performed to evaluate the antigenic variation of s proteins within the korean pedv strains. the antigenic index of each amino acid was calculated by the jameson-wolf method for 3 previously identified s protein-neutralizing epitopes, coe (within the s1 b region) [40] , s1d [41] , and 2c10 [42] . subsequently, the calculated indexes of the korean strains were compared. the detection rate of pedv from 2017 to 2018 was 9.92% (90/907). specifically, the positive rates in 2017 and 2018 were 8.63% (58/672) and 13.62% (32/235), respectively. the positive rate in 2018 had somewhat increased compared with that in 2017. from the detection rates of each growth stage, the highest rate was seen in the suckling stage (table 2) . geographically, the province with a higher concentration of swine farms showed higher positive samples of pedv (figure 1 ). in the phylogenetic trees inferred from the d1-d2 datasets of the s gene ( figure 2 , supplementary figure s1-s2), the pedv strains were classified into five sub-genogroups (g1a, g1b, g2a, g2b, and g2c), which were previously designated [8] . however, the relationships between sub-genogroups differed. the d1 dataset contains the complete s gene supported for the clusters of (g1a, g1b) and (g2c, (g2a, g2b)). the d2 dataset contains the s gene without recombinant regions supported for the different clusters of (g1b, g2c) and (g2b, g2a). in both datasets, histograms of pairwise p-distances exhibited a discrete distribution betwen sub-genogroups. that was in agreement with the tree topologies and posterior support values at the nodes to each sub-genogroup ( figure 2 ). in both the d1 and d2 datasets, the seven korean strains identified in this study (s6, s10, s12, s14, s97, s100, and y178) were clustered within sub-genogroup g2a. in the phylogenetic trees inferred from the d1-d2 datasets of the s gene ( figure 2 , supplementary figures s1 and s2) , the pedv strains were classified into five sub-genogroups (g1a, g1b, g2a, g2b, and g2c), which were previously designated [8] . however, the relationships between sub-genogroups differed. the d1 dataset contains the complete s gene supported for the clusters of (g1a, g1b) and (g2c, (g2a, g2b) ). the d2 dataset contains the s gene without recombinant regions supported for the different clusters of (g1b, g2c) and (g2b, g2a). in both datasets, histograms of pairwise p-distances exhibited a discrete distribution betwen sub-genogroups. that was in agreement with the tree topologies and posterior support values at the nodes to each sub-genogroup ( figure 2 ). in both the d1 and d2 datasets, the seven korean strains identified in this study (s6, s10, s12, s14, s97, s100, and y178) were clustered within sub-genogroup g2a. regions (d1) and with the recombinant regions removed (d2). the sub-genogroups were designated as g1a, g1b, g2a, g2b, and g2c. each sub-genogroup was colored consistently between the d1 and d2 datasets. the inserted histograms of pairwise p-distances were between sub-genogroups g1a-g1b, g1a-g2a, g1a-g2b, and g1a-g2c. the p-distance is calculated by dividing the number of nucleotide differences by the total number of nucleotides compared. after removing recombinant regions, some sequences might contain large deletions. in other words, the total number of nucleotides became smaller. thus, the p-distance on the right panel was larger than that on the left panel. the korean strains identified in this study are marked by arrows. supported by high posterior probability values (0.90-1), the phylogenetic trees inferred from the d3-d4 datasets of the n gene (figure 3, supplementary figures s3 and s4) suggested that the classification of pedv strains into four s gene-based sub-genogroups g1a, g1b, g2b, g2a/g2c was more reliable. in both datasets, it was observed that the s gene-based g2a and g2c were not monophyletic (pink branches). differing from the s gene-based phylogenies (figure 2) , the ngene-based trees with or without the elimination of recombinant regions had identical topologies of (g1a, (g1b, (g2b, g2a/g2c))). as the result, this study proposed an n-based genotyping of pedv as n1, n2, n3b and n3a, which were equivalent to the s-based genotyping of g1a, g1b, g2b, and g2a/g2c, respectively. that classification was also supported by a clear bimodal distribution of genetic distance between the proposed genogroups n1-n2 and n1-n3a/n3b (inserted histograms, figure 3 ). according to that scheme, the seven korean strains identified in this study (s6, s10, s12, s14, s97, s100, and y178) were within sub-genogroup n3a. the estimated mean nucleotide substitutions of the s and n genes were at the order of 10 −4 nucleotide substitutions/site/year ( table 3 ). the substitution rates of the complete s and n genes were not significantly different, as the 95% highest posterior density (hpd) overlapped (table 3) . inferring from two datasets where the recombinant regions were removed, the s gene showed substantially higher substitution rates than the n gene because of non-overlapping 95% hpd (6.18 × 10 −4 -10.02 × 10 −4 versus 2.12 × 10 −4 -4.52 × 10 −4 , respectively). the estimated mean nucleotide substitutions of the s and n genes were at the order of 10 −4 nucleotide substitutions/site/year ( table 3 ). the substitution rates of the complete s and n genes were not significantly different, as the 95% highest posterior density (hpd) overlapped (table 3) . inferring from two datasets where the recombinant regions were removed, the s gene showed substantially higher substitution rates than the n gene because of non-overlapping 95% hpd (6.18 × 10 −4 -10.02 × 10 −4 versus 2.12 × 10 −4 -4.52 × 10 −4 , respectively). * random local clock model (rlc). ** the geometric mean nucleotide substitution rate (substitutions/site/year) was inferred from the data that best fit the molecular clock and coalescent tree prior of constant population size, exponential population size, and bayesian skyline plot (bsp). *** highest posterior density (hpd). several consistent changes allowed the differentiation of the genogroups and sub-genogroups based on the n gene (figure 4, supplementary figure s5 ). the complete list of nonsynonymous changes was given in the supplementary figure s5 . genogroup n1 differed from genogroups n2 and n3 by 7 nonsynonymous substitutions (a84g, k205n, m216v, p381l, q395l, n398h, and v408a). unique changes leading to branches (n2, (n3a, n3b)) were a142t, h242l, q397l, and e400d. genogroup n2 was further characterized by two nonsynonymous substitutions (a145t, k380i). finally, the main branch leading to sub-genogroup n3a displayed five changes (k123n, m216v, r241k, k252r, and n255s). in the antigenic index analysis of n gene sequences, significant reductions were found in amino acid positions 122-126 according to the genogroups ( figure 5 ). genogroup n3 exhibited lower antigenic indexes, below 0.5 (the cut-off value), compared with genogroups n1 and n2. these amino acid sequences were located in a b-cell epitope sequence of the n protein, which is one of the pedv-specific epitopes (amino acids 18-133 and 252-262) previously identified [43] . in the antigenic index analysis of n gene sequences, significant reductions were found in amino acid positions 122-126 according to the genogroups ( figure 5 ). genogroup n3 exhibited lower antigenic indexes, below 0.5 (the cut-off value), compared with genogroups n1 and n2. these amino acid sequences were located in a b-cell epitope sequence of the n protein, which is one of the pedv-specific epitopes (amino acids 18-133 and 252-262) previously identified [43] . nonsynonymous substitutions were mapped to the branches of the phylogeny. for clarity, posterior values were shown for main separating nodes. in the antigenic index analysis of n gene sequences, significant reductions were found in amino acid positions 122-126 according to the genogroups ( figure 5 ). genogroup n3 exhibited lower antigenic indexes, below 0.5 (the cut-off value), compared with genogroups n1 and n2. these amino acid sequences were located in a b-cell epitope sequence of the n protein, which is one of the pedv-specific epitopes (amino acids 18-133 and 252-262) previously identified [43] . when analyzing the korean pedv strains-newly identified in this study-based on the three major b-cell epitope sequences of the s protein (coe, s1d, and 2c10), the six strains exhibited significantly lower antigenic indexes in some parts of the b-cell epitope sequences ( figure 6 ). the strains s6, s10, s12, and s100 exhibited lower antigenic indexes, below the cut-off value of 0.5, in amino acid positions 623-627 of the coe region, which contained the nonsynonymous change k623n. the s97 strain exhibited lower antigenic indexes, below the cut-off value of 0.5, in amino acid positions 634-638 in the coe region, and the nonsynonymous change e635v was observed in this region. the y178 strain also showed antigenic indexes below 0.5 at amino acids 764-771 of the s1d region, which contained the nonsynonymous change s768f. it is likely that each of the mentioned nonsynonymous changes affected the antigenic indexes of the adjacent regions. viruses 2020, 12, x for peer review 9 of 13 when analyzing the korean pedv strains-newly identified in this study-based on the three major b-cell epitope sequences of the s protein (coe, s1d, and 2c10), the six strains exhibited significantly lower antigenic indexes in some parts of the b-cell epitope sequences ( figure 6 ). the strains s6, s10, s12, and s100 exhibited lower antigenic indexes, below the cut-off value of 0.5, in amino acid positions 623-627 of the coe region, which contained the nonsynonymous change k623n. the s97 strain exhibited lower antigenic indexes, below the cut-off value of 0.5, in amino acid positions 634-638 in the coe region, and the nonsynonymous change e635v was observed in this region. the y178 strain also showed antigenic indexes below 0.5 at amino acids 764-771 of the s1d region, which contained the nonsynonymous change s768f. it is likely that each of the mentioned nonsynonymous changes affected the antigenic indexes of the adjacent regions. figure 6 . antigenic index analysis of s protein b-cell epitopes in korean pedv strains. strains s6, s10, s12, and s100 exhibited lower antigenic indexes, below the cut-off value of 0.5, at amino acids figure 6 . antigenic index analysis of s protein b-cell epitopes in korean pedv strains. strains s6, s10, viruses 2020, 12, 790 9 of 12 s12, and s100 exhibited lower antigenic indexes, below the cut-off value of 0.5, at amino acids 623-627 of the coe region. strain s97 exhibited reduced antigenic indexes, below 0.5, at amino acids 634-638 of the coe region. strain y178 exhibited reduced antigenic indexes, below 0.5, at amino acids 764-771 in the s1d region. first of all, this study reflected the ongoing circulation of pedv in korea at about 10% (table 1) and its wide distribution (figure 1 ). at the same time, obtaining genomic sequences of pedv from field samples provided good opportunity for studying the genetic evolution of the virus. as a result, this study applied multiple bioinformatics tools to investigate the genetic diversity of pedv. based on phylogenetic analysis of the complete s gene or s gene excluding recombinant regions, global pedv strains ( figure 2 ) can be classified into two genogroups, and each genogroup may be further subdivided into two (g1a and g1b) and three (g2a, g2b, and g2c) sub-genogroups. this result was consistent with that of guo et al. [8] . additionally, guo et al. reported that all these different subgroups existed in korea, with the most prevalent subgroup being g2a, when they analyzed the korean pedv strains identified prior to 2016. similar to the previous data, all the korean field strains identified from 2017 to 2018 in this study were included in subgroup g2a, indicating that the most prevalent korean subgroup has not changed since 2016. genogroups g1 and g2 are known to have different s protein neutralization activities [16] . however, differences within the genogroups have not been investigated. the six korean strains (s6, s10, s12, s97, s100, and y178) had significantly reduced antigenic indexes compared with other korean strains in some parts of the coe [40] and s1d [41] , which encode the b-cell epitopes of the s protein. these antigenic index reductions may induce somewhat different s protein antigenicities, even within the same genogroup. in fact, strains s10, s12, s97, and s100 originated from swine farms that had suckling piglet mortalities of almost 100%. these swine farms had been regularly using killed vaccines containing the new genogroup (g2) pedv strain but were seriously damaged by porcine epidemic diarrhea. in the literature, the n gene had been used to infer the phylogenetic relationships of pedv strains [44, 45] . it was noteworthy that the n gene showed a similar evolutionary rate to the s gene in this study. this high evolutionary rate implies that the n gene, as well as the s gene, is likely to have high genetic diversity; accordingly, several sub-genogroups could have diverged. comparing to the s-based phylogenetic topology, the classical strains (g1 strains) presented the same topology in the n-based and s-based analysis. however, there were some differences in the classification of subgroups on the new genotype strains (g2 strains). the n3 genogroup consisting of the g2 strains was divided into only 2 subgroups (n3a and n3b) not following the s-based subgroups (g2a, g2b and g2c). specifically, the n3b strains were consistent with the g2b strains, but the g2a and g2c strains were grouped into the same subgroup, n3a, in the n-based topology. this classification of the three genogroups (n1, n2, and n3) was also supported by the consistent variation in the antigenic indexes depending on the genogroups. the number of antigenic indexes of the n3 strains significantly decreased compared to those of the n1 and n2 strains within amino acids 122-126, which code for the b-cell epitope of the n protein [43] . the pedv n protein has an important immunological aspect. abundant antibodies against the n protein are induced at the early stages of pedv infection [12, 13] . furthermore, the n protein is considered to play an important role in inducing cell-mediated immunity [13] . as a result of these features, the n protein is commonly used as a target for diagnosis and vaccine development [10, 46] . as mentioned above, several geno-and sub-genogroups based on the n gene were identified in this study. this genetic diversity may change their antigenicities according to their geno-or sub-genogroup ( figure 5 ). in the immunological diagnosis of pedv, commercial pedv elisa kits have been showing poor performance, and the results of neutralizing assays using cell culture sometimes mismatch with those of the elisa assays. in fact, chang et al. recently reported that the antibodies induced by the g2b pedv strain poorly reacted with a commercial n-based elisa kit that showed a sensitivity of 37% [47] , which may be the result of antigenicity differences between the genogroups. however, further study is required to validate this hypothesis. indeed, if there are antigenic differences between the genogroups, a combination of n proteins derived from both genogroups would be required for the accurate immunological diagnosis of pedv. overall, this study revealed that pedv displayed genetic diversity in both the s and n genes, which resulted in the divergence into different sub-genogroups and altered antigenic indexes. such pedv mutants derived from genetic mutations of the s and n genes may cause severe damage to swine farms because of their ability to evade the unprepared host immune systems. table s1 : supplementary-material-table-s1-model-comparison-1. table s2 : supplementary-materialtable experimental infection of pigs with a new porcine enteric coronavirus, cv 777 a new coronavirus-like particle associated with diarrhea in swine isolation of porcine epidemic diarrhea virus (pedv) in korea isolation of porcine epidemic diarrhea virus during outbreaks in south korea new variants of porcine epidemic diarrhea virus, china ped virus reinfecting u.s. herds. virus estimated to have killed 7 million-plus pigs distinct characteristics and complex evolution of pedv strains evolutionary and genotypic analyses of global porcine epidemic diarrhea virus strains emerging and re-emerging coronaviruses in pigs porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis the molecular biology of coronaviruses coronavirus immunogens identification of the membrane protein of porcine epidemic diarrhea virus cellular entry of the porcine epidemic diarrhea virus neutralization of genotype 2 porcine epidemic diarrhea virus strains by a novel monoclonal antibody molecular characterization of a korean porcine epidemic diarrhea virus strain nb1 molecular characterization of us-like and asian non-s indel strains of porcine epidemic diarrhea virus (pedv) that circulated in japan during 2013-2016 and pedvs collected from recurrent outbreaks detection and phylogenetic analysis of porcine epidemic diarrhea virus in central china based on the orf3 gene and the s1 gene porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf3 and tbk1 molecular characterizations of subcellular localization signals in the nucleocapsid protein of porcine epidemic diarrhea virus porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin-8 expression detection and phylogenetic analysis of porcine deltacoronavirus in korean swine farms multiplex reverse transcription-pcr for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group a rotavirus phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china rdp4: detection and analysis of recombination patterns in virus genomes 5: an advanced software platform for bayesian evolutionary analysis creating the cipres science gateway for inference of large phylogenetic trees bayesian phylogenetic site model averaging and model comparison relaxed phylogenetics and dating with confidence bayesian random local clocks, or one rate to rule them all bayesian coalescent inference of past population dynamics from molecular sequences posterior summarization in bayesian phylogenetics using tracer 1.7 improving the accuracy of demographic and molecular clock model comparison while accommodating phylogenetic uncertainty mega2: molecular evolutionary genetics analysis software new phylogenetic groups of torque teno virus identified in eastern taiwan indigenes phylogenetic analysis by maximum likelihood annotating ancestral substitution on a tree the antigenic index: a novel algorithm for predicting antigenic determinants heterogeneity in membrane protein genes of porcine epidemic diarrhea viruses isolated in china spike protein region (aa 636789) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies the gprlqpy motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus the identification and characterization of two novel epitopes on the nucleocapsid protein of the porcine epidemic diarrhea virus sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china genetic variation of nucleocapsid genes of porcine epidemic diarrhea virus field strains in china development and evaluation of enzyme-linked immunosorbent assay based on recombinant nucleocapsid protein for detection of porcine epidemic diarrhea (pedv) antibodies development and comparison of enzyme-linked immunosorbent assays based on recombinant trimeric full-length and truncated spike proteins for detecting antibodies against porcine epidemic diarrhea virus 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 eun ok kim and jung ah kim for their excellent technical assistance. the authors declare that there is no conflict of interest. key: cord-298401-4szmu1dh authors: lyoo, kwang-soo; yeom, minjoo; kim, jungho; kim, donghyuk; ha, gunwoo; na, woonsung; le, van phan; song, daesub title: development of rapid immunochromatographic strip test for the detection of porcine epidemic diarrhoea virus date: 2017-12-02 journal: vet rec doi: 10.1136/vr.103959 sha: doc_id: 298401 cord_uid: 4szmu1dh porcine epidemic diarrhoea virus (pedv) causes acute and severe watery diarrhoea and dehydration, as well as 50–100 per cent mortality in piglets. for the pedv diagnosis, a rapid test kit that is specific and sensitive to pedv is critical to monitor this disease at pig farms. the present study aimed to develop an immunochromatographic assay (ica) strip test for detecting pedv in faecal swabs. the newly developed diagnostic test showed a detection limit of 10(4.0) tcid(50)/ml of pedv. using faecal swab samples, the relative sensitivity and specificity of the ica kit were 95.0 per cent and 98.6 per cent, respectively, compared with those of real-time rt-pcr. in samples from piglets experimentally infected with pedv, the results showed 100 per cent agreement with those found by real-time rt-pcr. our developed test strip will be useful for rapid diagnosis and can be used for epidemiological surveillance of pedv infection. porcine epidemic diarrhoea virus (pedv) is a highly contagious pathogen that causes acute and severe watery diarrhoea, vomiting and dehydration, and shows 50-100 per cent mortality in up to one-week-old piglets and less severe disease in older pigs. 1 pedv belongs to genus alphacoronavirus, family coronaviridae, and is an enveloped single-stranded positive sense rna virus. 2 3 outbreaks of porcine epidemic diarrhoea (ped) have been reported in several countries in europe, as well as in asian countries including china, thailand and south korea, since the disease was first identified in england in the early 1970s. 1 3 recently, pedv was diagnosed in the midwestern region of the usa in 2013. 4 5 since then, the virus has spread rapidly and was confirmed throughout 30 states in the usa. pedv has significant economic impacts on the pig industry, causing the loss of approximately seven million piglets within the one-year epidemic period in the usa. 3 moreover, the emergence of pedv has been reported in western, central and eastern europe, and the genome sequences were found to be closely related to pedv strains from the usa, showing a sequence identity of more than 99 per cent. [6] [7] [8] the genome of pedv is approximately 28 kb in size and encodes four structural proteins, including spike (s), membrane (m), envelope (e) and nucleocapsid (n), and four non-structural proteins, including 1a, 1b, 3a and 3b. 2 the s protein is critical to regulating interactions between receptor glycoproteins and virus ligands and for mediating viral entry into host cells, while the n protein is a basic phosphor-protein important for maintaining the nucleocapsid structure. epitopes of the n protein may play a role in inducing cell-mediated immunity. 2 additionally, the n protein may be an appropriate target because it is the predominant antigen expressed in coronavirus-infected cells. 9 to diagnose pedv, laboratory-based techniques are necessary, because ped is clinically indistinguishable from other diarrhoea diseases such as transmissible gastritis-enteritis virus infection. 10 to detect the pedv antigen, virus isolation, immunofluorescence testing, immunohistochemical techniques, elisa, and molecular techniques including rt-pcr and real-time rt-pcr are currently used in most laboratories. 1 however, to obtain accurate results, these methods require welltrained technicians and specific equipment, and are time-consuming. moreover, issues such as transportation delays or temperature changes during shipping from outbreak locations to diagnostic laboratories may affect the diagnosis results. 1 10 therefore, a rapid test kit that is specific and sensitive to pedv is critical to monitoring this disease on pig farms. here, we aimed to develop and validate an immunochromatographic assay (ica) for the rapid and qualitative detection of pedv antigen from diseased animals. the assay detects n protein of pedv using highly selective monoclonal antibodies. to express the pedv-n protein, cdna was generated by rt-pcr using rna extracted from the pedv-dr13 strain (accession no. jq023161) provided by green cross veterinary product (suwon, korea). the n protein gene was amplified using primers 5-gga tcc atg gct tct gtc agc ttt-3 and 5-gtc gac tta att tcc tgt gtc aaa-3. the pcr products were cloned into the bamh i/sal i restriction site (underlined) of the pfastbac vector (invitrogen, carlsbad, ca, usa), and this recombinant plasmid was transformed into escherichia coli dh10bac containing a baculovirus shuttle vector (bacmid, invitrogen). the recombinant bacmid dna carrying the pedv-n protein gene was transfected using cellfectin reagent (invitrogen) into spodoptera frugiperda (sf-9) cells. expression of the n protein gene of pedv was confirmed by immunoblot analysis using sds-page. for immunoblotting, sf-9 cells expressing n protein of pedv were lysed in radioimmunoprecipitation assay buffer (ripa) lysis buffer (1 per cent triton x-100, 1 per cent deoxycholate and 0.1 per cent sds). the proteins were then separated on 10 per cent sds polyacrylamide gels, and then transferred onto a nitrocellulose membrane. the membrane was blocked in 5 per cent skimmed milk buffer and incubated with polyclonal anti-pedv from mice immunised with pedv at 4°c overnight. for protein detection, the membrane was incubated with antimouse igg horseradish peroxidase (hrp)-conjugated secondary antibody and visualised using the atto ez-capture ii system (atto, tokyo, japan). recombinant baculoviruses expressing pedv-n protein were propagated in sf-9 cells. the recombinant n protein was purified by affinity chromatography using ni-nta resin (qiagen, hilden, germany) according to the manufacturer's protocol. to produce monoclonal antibodies, balb/c mice (six to eight weeks old) were intraperitoneally immunised with the purified n protein antigen emulsified in complete freund's adjuvant. three identical boosters were administrated with the n protein emulsified with incomplete freund's adjuvant one-week intervals. the immunised mice were sacrificed to isolate spleen cells, which were washed in serum-free cell culture medium. splenocytes (1×10 8 ) were mixed with myeloma cells line sp2/0-ag-14 (atcc crl 1581) (2×10 7 ) in a ratio of 5:1 in the presence of polyethylene glycol for cell fusion. the fused cells were seeded into 96-well tissue culture plates with hypoxanthine-aminopterin-thymidine (hat) selection medium. culture medium was exchanged with fresh hat selection medium eight days after fusion. on day 11, the supernatants were screened by elisa, and then mab anti-ped 3b12-1a6 and mab anti-ped 1h12-1c6 hybridomas, which strongly reacted with the pedv protein, were selected. each hybridoma cell was intraperitoneally injected into naïve balb/c mice to generate ascetic fluid. the ascetic fluid was then purified on protein g sepharose 4 fast flow (ge healthcare, uppsala, sweden) according to the manufacturer's protocol. the antibody concentrations were measured using the bradford protein assay (bio-rad laboratories, hercules, ca, usa). the binding affinity of the mabs was calculated following surface plasmon resonance (spr) analysis using proteon xpr36 system (bio-rad laboratories). purified mab 3b12-1a6 or mab 1h12-1c6 was immobilised to a glc chip (bio-rad laboratories) using a standard ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ed-c)/n-hydroxysuccinimide (nhs) cross-linking reaction, and affinity values (ka, kd, kd) of each mab for pedv were determined by proteon manager rm 2.1 software. calculation of the binding affinity was performed in gyeonggi bio-center (suwon, republic of korea). to further characterise the mabs for epitope recognition, we used a competition elisa to test whether the mabs recognise different epitopes of pedv as in a previously described method. 11 briefly, the recombinant n protein was coated onto a microtitre plate, and the plate was blocked with casein buffer. biotinylated mab and/or non-biotinylated mab were added to the wells, and the plate was reacted with hrp-conjugated avidin (thermo fisher scientific, waltham, ma, usa). the colour reaction was stopped and read as optical density at 450 nm using an automated plate reader. characterisation of the monoclonal antibody was conducted by indirect immunofluorescence assay (ifa). for ifa, vero cells inoculated with pedv, st cells inoculated with transmissible gastroenteritis virus (tgev) or porcine respiratory coronavirus (prcv), and ma-104 cells inoculated with porcine rotavirus were grown in cell culture chamber slides. the slides fixed with cold acetone were incubated with the pedv mabs at 37°c for 45 minutes. after washing, the slides were reacted with goat antimouse igg conjugated with fitc at 37°c for 45 minutes and examined under a fluorescence microscope. establishment and assembly of the ica kit for establishment of ica kit, the monoclonal antibody, anti-ped 3b12-1a6, purified from ascetic fluid using protein g resin (ge healthcare) was coated onto a specific area (test line) of a nitrocellulose membrane, while goat antimouse igg was coated onto another specific area (control line) on the same membrane. to produce the test conjugate, a different monoclonal antibody, anti-ped 1h12-1c6, was mixed with colloidal gold prepared by trisodium citrate reduction method as previously described, 12 and then the antibody mixture was treated by a previously described method. 12 the assay strips were prepared by assembly with the colloidal gold-conjugated glass fibre, the nitrocellulose membrane and an absorbent paper using polyvinyl chloride self-adhesive floor. the specificity of the ica kit for other pedv strains was tested with pedv-knu1309 (accession no. kj451044), pedv-knu1303 (accession no. kj451038) and pedv-knu1402 (accession no. kj451048), which have 99 per cent identity with the viruses identified in the us pig populations. additionally, the specificity of the ica kit against pedv was evaluated for other porcine viruses including tgev, prcv and porcine rotavirus obtained from green cross veterinary product. for the ica kit test, the culture supernatant (100 µl) of each virus or cell culture medium as a negative control was transferred into a test tube containing 100 µl of assay diluent (50 mm tris (ph 8.5), 10 mm nacl, 0.1 per cent tween 20, 1 parts per million proclin 300). the mixture (100 µl) was applied to the sample well of the test kit and the test signal for each test was visualised after 15 minutes. the sensitivity of ica was evaluated by comparing the detection limit using real-time rt-pcr as previously described with some modifications. 13 briefly, real-time rt-pcr was carried out in a total volume of 20 µl containing viral rna, pedv forward (5'-cgc aaag actg aacc cactaattt-3') and reverse (5'-ttg cctc tgtt gtta cttggagat-3') primers, probe (6-carboxyfluorescein [fam]-5'-tgt tgcc attg ccac gactcctgc-3'-bhq1), 4x taqman fast virus 1-step master mix (thermo fisher scientific) and water. the reaction was performed on a lightcycler 480 system (roche diagnostics, basel, switzerland) beginning with reverse transcription at 58°c for 30 minutes, followed by heat denaturation at 95°c for 5 minutes, 40 cycles of denaturation at 95°c for 30 seconds, and annealing and extension at 60°c for 1 minute. the data were analysed using lc480 software (roche diagnostics). serial tenfold dilutions of the pedv-dr13 strain including tegv, prcv and porcine rotavirus corresponding 1×10 6.0 to 1×10 1.0 tcid 50 /ml were analysed with the ica kit and real-time rt-pcr. evaluation of ica kit using clinical samples a total of 130 rectal swab samples consisting of faeces were collected from pig farms in eight provinces in south korea between january 2012 and may 2014. these herds were suspected to have enteric viral infection showing typical clinical signs such as diarrhoea, vomiting, high mortality and no response to antibiotic treatment. all samples were tested to detect pedv infection by real-time rt-pcr and evaluated using the ica kit to determine the relative specificity and sensitivity. for the experimental infection study, a total of eight piglets (three-day-old, seronegative for pedv) were obtained from sows with no disease history. these piglets were orally inoculated with 3 ml of pedv-dr13 strain (10 2.0 ld 50 /ml) in minimum essential medium cell culture medium, and clinical signs in all piglets were monitored for 10 consecutive days postinoculation (dpi). the rectal cavity of piglets was gently rubbed with a sterile moistened cotton swab, which were taken daily for monitoring. to evaluate the applicability of the ica kit for diagnosing pedv infection in pigs, 100 µl rectal swab transport medium was transferred into a test tube containing 100 µl of the assay diluents, and then a total of 100 µl of the supernatant was added to the test well of the ica kit using a disposable dropper. all animal experiments complied with the current laws in the republic of korea. expression of the pedv n protein was confirmed by immunoblot analysis (fig 1a) . the binding affinity calculated by spr analysis of mab 3b12-1a6 and mab 1h12-1c6 were kd: 4.91e-09 m (ka: 1.97e+05 1/ms; kd: 9.67e-04 1/s) and kd: 5.09e-09 m (ka: 6.00e+04 1/ ms; kd: 3.05e-04 1/s), respectively. the kinetics of the mabs to bind pedv were similar in the examination. a competition elisa was performed to test whether the mabs recognise different epitopes of pedv. as shown table 1 , recognition level of mab 3b12-1a6 to its epitope was not blocked either alone or in combination with different mab, and binding of mab 1h12-1c6 to its epitope was also not blocked. these results indicate that both mabs recognise different epitopes on pedv. cells infected with pedv and stained with mab 3b12-1a6 or mab 1h12-1c6 showed typical cytoplasmic fluorescence of pedv on the ifa, while the other cells infected with tgev, prcv and porcine rotavirus did not show fluorescence for both pedv mabs (fig 1b) . the best result for detecting the pedv detection threshold was obtained using the optimised concentration of the monoclonal antibodies in the ica kit. we applied 0.60±0.12 µg mab anti-ped 3b12-1a6 to a nitrocellulose membrane. the maximum intensity of test line colouring was detected when the mab anti-ped 1h12-1c6 conjugated with 0.1±0.02 µg colloidal gold of coated on a glass fibre membrane for immobilisation. further increasing the mab concentration did not increase test line intensity. positive results for pedv-dr13, pedv-knu1309, pedv-knu1303 and pedv-knu1402 strains were demonstrated by the visualisation of two red-purple bands at the test line and control line on the ica kit. however, tgev, prcv and porcine rotavirus were considered negative when only one band appeared in the control line, such as the cell culture medium. in the determination of the sensitivity of the ica kit, real-time rt-pcr showed a sensitivity of up to 10 2.0 tcid 50 /ml of pedv, while the detection limit of the ica kit was 10 4.0 tcid 50 /ml (table 2) . of the 130 swab samples tested, 58 were positive for pedv and 72 were negative according to the ica kit, whereas 60 were positive and 70 were negative according to real-time rt-pcr (table 3) . thus, three samples were positive (threshold cycle value of 29.5, 30.2 and 31.7) by real-time rt-pcr, but not detected in the ica kit test. the relative sensitivity and specificity of the ica kit compared with those of real-time rt-pcr were 95.0 per cent (95 per cent confidence interval: 86.08-98.96) and 98.6 per cent (95 per cent confidence interval: 92.30-99.96), respectively. piglets inoculated with the pedv strain experienced moderate to severe diarrhoea starting on 1 dpi. most infected piglets showing clinical signs died between 2 and 9 dpi except for one piglet, which recovered from clinical signs at 9 dpi and survived during the experimental challenge (table 4 ). in a comparison of the diagnosis methods, examination with the ica kit using swab samples showed consistent results with real-time rt-pcr in identifying pedv. since the pedv emergency in the usa in 2013, ped outbreaks have occurred worldwide. the prototype of pedv is the cv777 strain, which was identified in 1977 in belgium. next, pedv spread throughout europe and asia during the 1980s and 1990s and recently emerged in the usa. based on extensive phylogenetic analysis using obtained sequences, pedv strains are classified into two major categories: the classical pedv strain showing lower pathogenicity and highly virulent pedv strain that emerged in 2010. however, most studies aimed at developing techniques for diagnosing pedv infection have attempted to differentiate various diarrhoea-causing antigens rather than differentiating between classical and novel pedv strains. in the present study, we established an ica kit that can be universally applied for pedv infection screening without differentiation. the first advantage of this diagnostic kit is that it can be used for any diarrhoea case in which viral infection is suspected, but for which there is no available information regarding pedv strain involvement. second, ica is useful upon emergence of novel pedv strains, for which a strain-specific diagnostic method may be ineffective. thus, another optimised diagnostic method should be considered to differentiate classical and novel pedv strains if additional detection becomes essential for epidemiological studies or disease control. the detection limit of the ica kit was10 4.0 tcid 50 / ml, while its sensitivity was less than that of real-time rt-pcr (10 2.0 tcid 50 /ml). it is generally accepted that rapid screening test kits may not be as sensitive as gold standard methods for virus diagnosis such as real-time pcr. however, high relative specificity and sensitivity results for pedv were observed using field samples. considering the performance of the ica kit, we additionally assessed the sensitivity of the test in samples from piglets experimentally infected with pedv; the results showed 100 per cent agreement with real-time rt-pcr results. these results of the challenge study clearly indicate the value of well-defined serial stool samples from experimentally infected animals, as the results of these samples are not influenced by previous infections with pedv or other gastroenteric pathogens. furthermore, the ica kit targets the nucleocapsid protein of pedv, which is a conserved region that may be applicable for assessing not only prototype strains, but also novel strains of pedv from different geographical regions. 14 15 therefore, the presence of new pedv variant strains should be monitored, and the test kit may be useful for rapid diagnosis of pedv in the usa and other countries. the ica kit has the advantages of being easy, user-friendly and deliverable without specific equipment available in field conditions. examination of clinical samples showed that the sensitivity and specificity of the assay approached 95.0 per cent and 98.6 per cent, respectively, compared with those of the real-time rt-pcr, and 100 per cent agreement of the results were observed in faecal samples from experimentally infected piglets. thus, the ica kit developed in this study can be used for rapid diagnosis and can assist in epidemiological surveillance of pedv infection. the development of a rapid and easily confirmed diagnosis for pedv antigen detection is important for controlling ped outbreaks and limiting their spread; however, the ica kit cannot differentiate novel variant strains. therefore, an ica kit that can differentiate pedv variant strains using a multiplex strip should be developed in future studies. porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines coronavirus genome structure and replication porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences porcine epidemic diarrhea virus among farmed pigs, ukraine outbreak of porcine epidemic diarrhea virus in portugal emergence of porcine epidemic diarrhea virus in southern germany development and evaluation of enzyme-linked immunosorbent assay based on recombinant nucleocapsid protein for detection of porcine epidemic diarrhea (pedv) antibodies multiplex reverse transcription-pcr for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group a rotavirus characterization of neutralization epitopes of simian immunodeficiency virus (siv) recognized by rhesus monoclonal antibodies derived from monkeys infected with an attenuated siv strain development and validation of a rapid immunochromatographic assay for detection of middle east respiratory syndrome coronavirus antigen in dromedary camels multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china sequence analysis of the nucleocapsid protein gene of porcine epidemic diarrhoea virus the authors would like to thank dr hyoungjoon moon (green cross veterinary product) for providing porcine viruses: pedv-dr13 strain, tgev, prcv and porcine rotavirus. open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by-nc/ 4. 0/ key: cord-302083-9q1i20o6 authors: jung, kwonil; saif, linda j.; wang, qiuhong title: porcine epidemic diarrhea virus (pedv): an update on etiology, transmission, pathogenesis, and prevention and control date: 2020-06-02 journal: virus res doi: 10.1016/j.virusres.2020.198045 sha: doc_id: 302083 cord_uid: 9q1i20o6 porcine epidemic diarrhea virus (pedv), a member of the genus alphacoronavirus in the family coronaviridae, causes acute diarrhea and/or vomiting, dehydration and high mortality in neonatal piglets. two different genogroups of pedv, s indel [pedv variant containing multiple deletions and insertions in the s1 subunit of the spike (s) protein, g1b] and non-s indel (g2b) strains were detected during the diarrheal disease outbreak in us swine in 2013-2014. similar viruses are also circulating globally. continuous improvement and update of biosecurity and vaccine strains and protocols are still needed to control and prevent pedv infections worldwide. although the non-s indel pedv was highly virulent and the s indel pedv caused milder disease, the latter has the capacity to cause illness in a high number of piglets on farms with low biosecurity and herd immunity. the main pedv transmission route is fecal–oral, but airborne transmission via the fecal–nasal route may play a role in pig-to-pig and farm-to-farm spread. pedv infection of neonatal pigs causes fecal virus shedding (alongside frequent detection of pedv rna in the nasal cavity), acute viremia, severe atrophic enteritis (mainly jejunum and ileum), and increased pro-inflammatory and innate immune responses. pedv-specific iga effector and memory b cells in orally primed sows play a critical role in sow lactogenic immunity and passive protection of piglets. this review focuses on the etiology, transmission, pathogenesis, and prevention and control of pedv infection. porcine epidemic diarrhea virus (pedv), a member of the genus alphacoronavirus in the family coronaviridae of the order nidovirales, causes acute diarrhea, vomiting, dehydration and high mortality in neonatal piglets. the disease was reported in the european and asian pig industries over the last 30 years, with the virus first appearing in england (wood, 1977) and j o u r n a l p r e -p r o o f one study reported that four sows exposed via feedback to a g1b s indel pedv at approximately seven months pre-farrowing and then re-exposed to a g2b non-s indel pedv at day 109 of gestation provided long-term (7 months), passive immune protection of piglets against challenge with the same g2b non-s indel pedv (goede et al., 2015) . there was 0% mortality rate of piglets (with 57% reduced incidence of diarrhea), compared with mean 33% mortality and 100% morbidity rates of piglets born to non-immunized sows. however, another study revealed that more than 80% of the piglets inoculated orally with a g1b s indel pedv (iowa106 strain) at 3-4 days of age had diarrhea after challenging with a g2b non-s indel pedv at 24 days of age, whereas none of the piglets inoculated previously and then challenged with the same non-s indel pedv showed diarrhea (annamalai et al., 2017; lin et al., 2015a) . antigenic cross-reactivity or cross-neutralization between pedv and feline infectious peritonitis virus (fipv) was detected by enzyme linked immunosorbent assay (elisa), immunoblotting and immune-precipitation (zhou et al., 1988) , or vn tests (zhao et al., 2019) . despite no serological cross-neutralization detected between pedv and tgev (hofmann and wyler, 1989; pensaert and de bouck, 1978) , investigators found some antigenic cross-activity between pedv and tgev (miller, but not purdue strain) based on at least two conserved epitopes on the n-terminal region of their n proteins, as well as via their m proteins or whole virus particles (gimenez-lirola et al., 2017; lin et al., 2015b; xie et al., 2019) . truncation of the n-terminal region of the n protein or avoidance of m-or purified whole virus-based serologic tests may eliminate pedv and tgev shared epitope(s) or reduce the possible cross-reactivity for serologic j o u r n a l p r e -p r o o f assays (gimenez-lirola et al., 2017; xie et al., 2019) . polyclonal hyperimmune antisera against pdcov did not cross-react with pedv . among n, m and e proteins and whole pedv particles, only the m protein of pedv showed cross-reactivity in 1/12 convalescent sera from pdcov-infected pigs (gimenez-lirola et al., 2017) . however, one study reported antigenic cross-reactivity between us pdcov and pedv strains, possibly due to at least one shared epitope in the n-terminal region of their n proteins . for sads-cov, monoclonal antibody against the n protein did not cross-react with pedv, tgev or pdcov (pan et al., 2017) . the fecal-oral route is the main means of direct transmission of pedv via the feces and/or vomitus of infected pigs (jung and saif, 2015) . indirect contact transmission of pedv is also frequent within and between farms, particularly, with a low biosecurity, via other contaminated fomites including (jung and saif, 2015; kim et al., 2017b) : transport trailers (lowe et al., 2014) , farm workers' hands, boots and clothes (kim et al., 2017b) , feed (bowman et al., 2015a; dee et al., 2014; schumacher et al., 2017) , feed ingredients and additives, such as spray-dried porcine plasma (pasick et al., 2014; perri et al., 2018) , and feed totes used for transporting bulk feed or feed ingredients (anon., 2015; scott et al., 2016) . pedv remained infectious on tote material for 35 days at room temperature (scott et al., 2016) . pedv cross-contamination also occurred during feed manufacturing (schumacher et al., 2018) . j o u r n a l p r e -p r o o f 8 the fecal-nasal route is another route of pig-to-pig, or farm-to-farm (up to 10 miles away) airborne transmission of pedv via aerosolized pedv particles that are infectious in nursing pigs (alonso et al., 2014; alonso et al., 2015; beam et al., 2015; gallien et al., 2018a; . airborne pedv transmission occurs within farrowing herds where newborn piglets, highly susceptible to the virus, are raised (alonso et al., 2015; niederwerder et al., 2016) . the nasal cavity of naïve pigs housed at a distance from clinical pigs was frequently positive for pedv rna (niederwerder et al., 2016) . aerosolized pedv does not infect only the intestine of pigs (alonso et al., 2015) , but it also infects the epithelium lining the nasal cavity . reported that dendritic cells in the lamina propria of the nasal mucosa or lymphoid tissue carry and transfer pedv to cd3 + t cells. pedv-loaded t cells may reach the intestine via unknown specialized endothelial venules during blood circulation. the virus loaded onto t cells had the capacity to infect intestinal epithelial cells via cell-to-cell contact and transfer infection . compared with neonatal piglets, however, higher doses of aerosolized pedv may be required to infect weaned and older pigs (niederwerder et al., 2016) . in the study, none of aerosol contact, 28-day-old pigs (0/5 pigs) was infected, although their nasal cavities (5/5 pigs) were positive for pedv rna. the severity of pedv infection and the disease, and the transmissibility of pedv, depends on the overall immunity and health status of the pig population and the levels of biosecurity on farms (pensaert and martelli, 2016) . nevertheless, transmissibility of pedv via direct or aerosol j o u r n a l p r e -p r o o f contact also varies dependent on the pedv genogroup. direct contact or aerosol transmission rates were significantly higher in pigs infected with non-s indel pedv compared with s indelinfected pigs (gallien et al., 2018a) . gallien et al. (2018) revealed that despite the presence of aerosolized s indel pedv, none of aerosol contact pigs (0/10 pigs) was infected, whereas 10/10 aerosol contact pigs were infected by non-s indel pedv (gallien et al., 2018a) . porcine small intestinal villous enterocytes express aminopeptidase n (apn), a 150-kda glycosylated transmembrane protein, which was tentatively identified as the cellular receptor for pedv (li et al., 2007; liu et al., 2015a; nam and lee, 2010) . however, porcine apn may not be the major cell surface receptor for pedv (ji et al., 2018; shirato et al., 2016) . concomitantly, apn knockout pigs were susceptible to infection with pedv, but not with tgev that uses apn as the cellular receptor (whitworth et al., 2019; zhang et al., 2019) . cell membrane cholesterol or two cell surface molecules, such as sialic acids and occludin expressed on the apical surface of secretary (goblet) or absorptive enterocytes, respectively, were involved partially in binding and entry of pedv into enterocytes (deng et al., 2016; jeon and lee, 2017; luo et al., 2017) . the strains of pedv, such as cell culture-adapted vs. wild-type, may be a factor involved in the cellular entry via sialic acids (deng et al., 2016; . intracytoplasmic localization of pedv antigen in goblet cells of infected pigs and porcine intestinal enteroids in vitro (jung and saif, 2017; raises questions on whether or how goblet cells are utilized for pedv replication, or if they only transfer pedv to absorptive enterocytes. j o u r n a l p r e -p r o o f we demonstrated the tissue tropism of pedv to certain intestinal locations (jung et al., 2018) . during acute pedv infection [post-inoculation hour (pih) 12-24] in nursing pigs, pedv initially infected mainly the mid-jejunum and ileum and to a lesser extent, the proximal and distal jejunum and duodenum (jung et al., 2018) (fig. 1) . the pylorus was not the site of acute pedv infection (jung et al., 2018) , whereas the villous enterocytes of the large intestine were frequently infected; however, infected colonic enterocytes did not undergo necrosis (jung et al., 2015a; jung et al., 2014) . unlike colonic enterocytes, pedv-infected small intestinal villous enterocytes undergo acute necrosis and exfoliation from the lamina propria, leading to marked villous atrophy or fusion in the small intestine (jung et al., 2018; jung et al., 2014) . like tgev (kim et al., 2000) and pdcov (jung et al., 2016) , pedv may not induce apoptotic death of intestinal villous enterocytes in vivo (jung and saif, 2015) . however, pedv-infected vero cells in vitro underwent apoptosis (kim and lee, 2014) . during acute pedv infection (pih 12-24) of nursing pigs, early localization of pedv antigen was evident in the villous-crypt interface of the small intestine, rather than the villous tips (jung et al., 2018) (fig. 1) . iimmature enterocytes were the major site of initial pedv infection, although the exact reason remains obscure (jung et al., 2018) . the pedv antigen-positive regions subsequently expanded to the upper and then the entire villous epithelium of the jejunum to ileum (<24 hours after oral inoculation) (jung et al., 2018) . the villous-crypt interface is close to blood vessels in the submucosa. although pedv-related viremia might be a result of diffusion of replicated pedv from the acutely infected intestine to blood (jung et al., 2018) , further studies are j o u r n a l p r e -p r o o f needed to investigate whether pedv or pedv-loaded cd3+ t cells circulating in blood reach and infect the villous-crypt interface or other villous regions, and other types of cells of extra-intestinal origin (jung et al., 2018; . pedv antigens were also frequently detected in the intestinal crypt cells or antigen presenting cells, such as macrophages, in the lamina propria or peyer's patches (debouck et al., 1981; jung et al., 2014; lin et al., 2015a; madson et al., 2016; stevenson et al., 2013; sueyoshi et al., 1995) . pedv remains infectious in dendritic cells for <24 hours in vitro (gao et al., 2015) , but dendritic cells may not be a site of pedv replication . instead, pedv appeared to employ dendritic cells to cross the epithelial barrier of the nasal cavity . lung tissues of oronasally infected pigs were negative for pedv antigen (debouck et al., 1981; jung et al., 2014; stevenson et al., 2013; sueyoshi et al., 1995) . however, the upper respiratory tract may be the site of pedv infection, because the epithelium lining the nasal cavity was positive for pedv antigens . although acute viremia was frequently noted in infected pigs during the acute or incubation stage of infection (jung et al., 2018; jung et al., 2014; , pedv antigens were not detected in other major organs, such as liver and kidneys (debouck et al., 1981; jung et al., 2014; stevenson et al., 2013; sueyoshi et al., 1995) . the reproductive organs of experimentally infected boars, such as cowpers's glands, were also negative for pedv rna, but their semen was transiently positive for pedv rna (gallien et al., 2018b; gallien et al., 2019) . nevertheless, whether the pedv rna-positive semen contained infectious viral particles is unclear. j o u r n a l p r e -p r o o f during the incubation period (<pih 16) in nursing pigs, a few to 50% of the villous epithelial cells positive for pedv antigen were seen in the villous-crypt interface and upper villous epithelium of the small intestine (debouck et al., 1981; (jung et al., 2018) . during this period, infected enterocytes underwent necrosis, but complete villous atrophy was not observed (jung et al., 2018) . when clinical signs were first seen (pih 16-24), moderate to large numbers of infected villous epithelial cells were observed throughout the small intestine (debouck et al., 1981) . during this period, up to 100% of the epithelial cells were positive for pedv antigen in the mid-jejunum to ileum where moderate to severe villous atrophy was observed (jung et al., 2018) . during the middle [post-inoculation day (pid) 1-3] to late (pid 4-5) stages of pedv infection of nursing pigs (5-9-day-old), regardless of the locations (except for the duodenum with milder villous atrophy), all small intestinal segments exhibited similar, extensive villous atrophy (mean 1-2 intestinal villous height to crypt depth ratios, compared with 5-9 of uninfected pigs) (chen et al., 2016a; jung et al., 2015a) . during this period, large numbers of pedv-infected epithelial cells could still be observed (debouck et al., 1981) . diarrhea induced by pedv is a consequence of malabsorption and maldigestion due to massive loss and functional disorders of infected enterocytes, followed by decreased brush border membrane-bound digestive enzymes (coussement et al., 1982; jung et al., 2006) . dehydration is exacerbated by vomiting and decreased appetite (jung and saif, 2015) . serotonin is involved in induction of vomiting, and increased pro-inflammatory cytokine responses in part led to decreased appetite (jung et al., 2018) . pedv-infected piglets also showed hyperkalemia and acidosis due to j o u r n a l p r e -p r o o f the loss of bicarbonate (jung and saif, 2015) . the cardiac contractility is impaired by acidosis and dehydration. in the small intestine of clinically infected pigs, the tight or adherens junctions of the villous enterocytes were destroyed (jung et al., 2015b) . expression of the junction proteins, such as zonula occludin-1, was also decreased or altered, followed by decreased transepithelial resistance (curry et al., 2017; jung et al., 2015b; luo et al., 2017; zong et al., 2019) . during the clinical period, there were remarkably reduced numbers of goblet cells in the small intestine (jung and saif, 2017) , followed by decreased amounts of mucins (curry et al., 2017; jung and saif, 2017) . the damaged gut integrity may lead to uptake of luminal food and bacteria causing allergic reactions and co-infections. the impaired gut integrity or digestive function usually recovered by the second week after infection in survived pigs (curry et al., 2017) . in infected gnotobiotic or conventional nursing piglets (1-10-day-old), a few (<5%) to all of the inoculated pigs began to show diarrhea at pid 1, which mostly coincided with or was detected several hours later than the first detection of viral rna in serum or feces (chen et al., 2016a; gerber et al., 2016; jung et al., 2015a; jung et al., 2018; liu et al., 2015c; madson et al., 2016; thomas et al., 2015a) . fecal viral rna titers shed peaked by pid 1, or occasionally, 1-3 days later, and the titers decreased remarkably during the middle to late stages of infection and then titers remained low (with at least one more recurrent peak, possibly due to ped re-infection of regenerating enterocytes) during the recovery stage of infection (chen et al., 2016a; jung et al., 2015a; lin et al., 2015a; madson et al., 2016) . a one-three-day delay in onset of diarrhea was observed in nursing pigs inoculated with s indel pedv compared with the non-s indel counterparts (gallien et al., 2018a; lin et al., 2015a) , indicating a longer incubation time for s j o u r n a l p r e -p r o o f indel pedv. in infected conventional weaned to feeder pigs (21-56-day-old), a few (<5%) to all of the inoculated pigs began to show diarrhea at pid 1-4, which coincided with or was detected 1-2 days later than the first detection of viral rna in feces (gallien et al., 2018a; gerber et al., 2016; jung et al., 2015a; lohse et al., 2017; madson et al., 2014; niederwerder et al., 2016; thomas et al., 2015a) (table 1) . fecal viral rna titers shed peaked by 1-3 days after diarrhea was first detected (gallien et al., 2018a; jung et al., 2015a; lohse et al., 2017; madson et al., 2014; niederwerder et al., 2016; thomas et al., 2015a) . detailed data from boars or sows are summarized in table 1 . duration of diarrhea in infected nursing pigs varied from 2-8 days, with a higher variability for the us s indel strain (2-7 days) compared with the us non-s indel (7-8 days) (lin et al., 2015a) . prolonged fecal viral rna shedding (at least pid 14) was evident in 20/33 (61%) us non-s indel pedv-infected nursing (10-day-old) pigs (gerber et al., 2016) (table 1) . diarrhea in weaned to feeder pigs (21-56-day-old) inoculated orally with us non-s indel pedv was observed for approximately 6 days niederwerder et al., 2016; thomas et al., 2015a) . prolonged fecal viral rna shedding was evident in non-s indel or s indel pedvinfected weaned or feeder pigs for 15-26 days (gallien et al., 2018a; lohse et al., 2017; madson et al., 2014; niederwerder et al., 2016; thomas et al., 2015a) . duration of fecal shedding was similar between non-s indel-(15-26 days) (gallien et al., 2018a; lohse et al., 2017; niederwerder et al., 2016; thomas et al., 2015a) and s indel-infected weaned or feeder pigs (15-18 days) (gallien et al., 2018a; lohse et al., 2017) . longer fecal viral rna shedding was observed in weaned pigs infected via direct contact (42-46 days) or aerosol contact (48 days) compared with the orally infected counterparts (18-21 days) (crawford et al., 2015; gallien et al., 2018a) . however, infectious pedv was shed in feces only for the initial 2 weeks tested using sentinel naïve pigs (crawford et al., 2015) . the minimum infectious dose [0.56 median tissue culture infectious dose (tcid50)] of non-s indel pedv required to infect 5-day-old nursing pigs was lower than for 3-week-old weaned pigs (56 tcid50) (thomas et al., 2015a) . similarly, 3 plaque-forming unit (pfu)/pig of the non-s indel pc22a strain caused diarrhea in 100% of 4-day-old cesarean-derived, colostrum-deprived (cdcd) piglets (liu et al., 2015c) . compared with nursing pigs that began to show diarrhea, villous atrophy, and fecal virus shedding at pid 1, a longer incubation time of non-s indel pedv was required for weaned pigs to show fecal virus shedding (1 more day), or diarrhea and lesions (2 more days) (jung et al., 2015a) . one factor contributing to the higher susceptibility of nursing pigs to non-s indel pedv infection is immaturity of gut innate immunity, as evident by a functional defect in natural killer cells in the ileum and blood (annamalai et al., 2015) . the lack of diversity and decreased firmicutes to bacteroidetes ratio in the gut microbiota of neonatal piglets contributed to the immature gut innate immunity . compared with grower pigs (gastric ph 2-3), neonatal pigs have a higher gastric ph (4-6) that allows pedv to survive in the stomach (mavronmichalis, 2016). compared with nursing pigs, several factors that influence a shorter recovery of weaned pigs from ped include: 1) the faster regeneration of enterocytes in weaned pigs compared with j o u r n a l p r e -p r o o f nursing pigs (moon et al., 1973) ; and 2) the anatomically and physiologically fully developed large intestine of weaned pigs where water reabsorption is greater (jung et al., 2015a) . there was a lack of lgr5 (leucine-rich repeat-containing g protein-coupled receptor 5, marker for crypt stem cells)-positive crypt base columnar cells (lgr5+ cells) and low proliferation of crypt cells in the small intestine of naïve nursing piglets (9-day-old), causing the slower turnover of enterocytes (jung et al., 2015a) . unlike nursing pigs, naïve weaned pigs (3-week-old) exhibited high proliferation of intestinal crypt cells and large numbers of lgr5+ cells in the crypts, leading to the higher turnover rate of enterocytes (jung et al., 2015a) . after pedv infection, large numbers of lgr5+ cells and high proliferation of crypt cells were maintained, possibly relating to the rapid recovery from ped in weaned pigs. detection of viremia where viral rna in serum ranged from 4.5-8.6 log10 genomic equivalents (ge)/ml was identified in gnotobiotic neonatal (5/5; 100%), or conventional 9-dayold nursing (16/16; 100%) and 26-day-old weaned pigs (11/20; 55%) infected with a us non-s indel pedv strain at pid 1-5 (jung et al., 2015a; jung et al., 2014) . pedv rna was detected in all sera from 20 nursing pigs infected with either one of three different us non-s indel strains or a us s indel strain at pid 3 (chen et al., 2016a) . the classical pedv br1/87 also induced viremia in 5/13 inoculated weaned pigs (38.5%) at pid 2-7 (lohse et al., 2017) . in experimentally infected nursing pigs, the frequency of viremia and serum viral rna titers peaked at pid 1, then decreased progressively, and ceased at pid 14-21 (chen et al., 2016a; jung et al., 2015a; opriessnig et al., 2014) . a significantly higher frequency of viremia in weaned pigs infected via direct or aerosol contact, or orally with non-s indel pedv was identified, compared with the s j o u r n a l p r e -p r o o f indel counterparts (gallien et al., 2018a) . the role of viremia in the pathogenesis of pedv is still obscure, although pedv identified in the serum could be infectious pasick et al., 2014; perri et al., 2018) . during the clinical period, alteration in the abundance or proportion of gut microbiota (dysbiosis) occurred in nursing pigs and their dams koh et al., 2015; liu et al., 2015b; song et al., 2017; tan et al., 2019) . the transfer of a healthy gut microbiota from infected sows to their offspring was impaired . the dysbiosis was characterized by reduced numbers of beneficial bacteria (verrucomicrobia) and increased numbers of harmful bacteria (fusobacterium and proteobacteria) (liu et al., 2015b; tan et al., 2019) , followed by decreased cellular transport and catabolism . a study showed that feeding bacillus subtilis to 2-week-old pigs infected with pedv reduced intestinal pathology (but not clinical disease) (canning et al., 2017) . similarly, live or cell-free supernatants of some lactobacillus plantarum strains had antiviral effects against pedv infection in vero cells (sirichokchatchawan et al., 2017) . however, this feed additive method (1/77 herds surveyed; 1.2 %) was rarely used to mitigate ped on us pig farms during the 2013-2017 epidemic (niederwerder and hesse, 2018) . early studies verified high enteropathogenicity of the g1a cv777 in nursing pigs (coussement et al., 1982; pensaert and martelli, 2016) . the s1 of the s protein and the rest of the genome of the s indel pedv (g1b) was genetically closest to that of g1a and the highly virulent j o u r n a l p r e -p r o o f g2 pedv, respectively (lee, 2015; pensaert and martelli, 2016; vlasova et al., 2014) , the strain was initially thought to be virulent. however, during the initial outbreaks by s indel pedv in the us and europe, the low to zero mortality rates of pigs in the field were distinct from those of us non-s indel infections with up to 100% mortality rates in nursing pigs (boniotti et al., 2016; goede et al., 2015; stevenson et al., 2013; wang et al., 2014b) . nevertheless, difference in virulence of the two types of strains in the field was not often observed (grasland et al., 2015; mesquita et al., 2015; stadler et al., 2015; steinrigl et al., 2015) . a study using a wild-type strain of s indel pedv reproduced as severe ped in infected sows and their piglets as that observed in the field (leidenberger et al., 2017) . other studies showed generally milder pathogenicity of s indel pedv in 3-7-day-old conventional pigs, compared with non-s indel pedv (chen et al., 2016a; lin et al., 2015a; yamamoto et al., 2015) . however, the virulence of s indel pedv was increased in the pedv challenged suckling piglets when the mothers became sick, corresponding to poor immune, nutritional or health conditions (lin et al., 2015a) . the mortality rates of piglets increased from 0% to 75%, and the peak fecal pedv rna titers shed were also as high as those of non-s indel strain. the virulence of pedv was attenuated by: 1) high cell-culture passage (93 rd -160 th ) in the cell line of non-swine origin, vero cells (with trypsin in the cell culture medium) (chen et al., 2015; kweon et al., 1999; lin et al., 2017a; sato et al., 2011; song et al., 2003) ; 2) modified cell . in addition, pedv variants of mild virulence, such as the s1 n-terminal domain (ntd)-del variant, also occurred naturally (su et al., 2018; suzuki et al., 2016; zhang et al., 2018a) . pedv strains fully adapted to vero cells and serially passaged at least 70 times (with trypsin or gcdca in the cell culture medium) tended to replicate less efficiently in the intestine of pigs (chen et al., 2015; kim et al., 2017a; lin et al., 2017a; sato et al., 2011; wu et al., 2019) (table 2 ) and elicited low serum pedv-specific antibody titers (lin et al., 2017a) . twelve-week-old pigs orally inoculated with 8.2 log10 tcid50 of g1a pedv 83p-5 (100 th passage in vero cells) exhibited only intermittent fecal viral rna shedding in 3 of 4 pigs (sato et al., 2011) . none of the 10-dayold pigs orally inoculated with 6 log10 tcid50 of g2b pedv yn (144 th passage in vero cells) showed fecal viral rna shedding; however, pedv antigen was detected in the small intestine (chen et al., 2015) . compared with g2b pedv yn (144 th ) and pc22a (140-160 th passage in vero cells) grown with trypsin, g2b pedv 8aa (70 th -105 th passage in vero cells) grown with gcdca and attenuated at lower cell passage numbers, caused no or little fecal viral rna shedding in 1day-old pigs (6 log10 tcid50/pig) (kim et al., 2017a) . four-day-old cdcd pigs orally inoculated with 2 log10 pfu of pc22a (140 th -160 th ) showed low to moderate fecal viral rna shedding titers, with mild villous atrophy (lin et al., 2017a) (table 2) . six-day-old conventional pigs orally inoculated with 6 log10 tcid50 of g2b pedv ct (120 th passage in vero cells) showed mild diarrhea and moderate fecal viral rna shedding titers, with mild villous atrophy . on the other hand, pregnant sows orally inoculated with g1a pedv dr13 (100 th passage in vero cells) showed no clinical signs but high vn antibody levels in the colostrum (song et al., 2007) . il-17) responses in acutely infected neonatal pigs (annamalai et al., 2015; jung et al., 2018) . this coincided with increased numbers of pedv antigen-positive cells (gao et al., 2015; madson et al., 2016) or inflammatory cells (lymphocytes or neutrophils) in the gut-associated lymphoid tissue (galt) (coussement et al., 1982; de arriba et al., 2002b; debouck et al., 1981; jung and saif, 2015) , and the onset of necrosis and exfoliation of infected enterocytes (jung et al., 2018) . necrosis of enterocytes is in part involved in the innate or pro-inflammatory cytokine responses in infected pigs, as also observed in vitro (koonpaew et al., 2019; lin et al., 2017b) . the increased systemic innate and pro-inflammatory cytokine responses that occur during acute pedv infection are beneficial for differentiation of naïve t cells into cytotoxic or helper t cells, leading to removal of infected cells or production of vn antibodies, respectively (siegrist, 2013) . however, highly increased, prolonged pro-inflammatory cytokines that can cause decreased appetite, etc. are also detrimental (jung et al., 2018; langhans, 2000) . increased systemic ifnα and ifnγ cytokine responses were also observed in pedv-infected gilts at pid 13-17 and pid 6-8, respectively (langel et al., 2019b) . clinically infected pigs responded to a primary pedv infection and seroconverted at pid 7-14 (mean pid 14) (pid 14-21 for serum pedv-specific iga antibodies), with peak serum j o u r n a l p r e -p r o o f pedv-specific igg or iga antibody titers at pid 21 (annamalai et al., 2017; chen et al., 2016b; de arriba et al., 2002a; de arriba et al., 2002c; gallien et al., 2018a; gerber et al., 2016; niederwerder et al., 2016; opriessnig et al., 2014; thomas et al., 2015a) . there were lower serum igg or vn antibodies in s indel-infected pigs compared with the non-s indel counterpart (annamalai et al., 2017; chen et al., 2016b) . serum vn antibodies were detected 7 days earlier than serum pedv-specific igg antibodies (chen et al., 2016b; thomas et al., 2015a) . there were low titers of pedv-specific secretory iga (siga) in the feces of pedv-infected pigs (10-day or 8-week-old) by pid 14 (gerber et al., 2016) . serum pedv-specific iga and igg antibodies in naturally infected sows remained high for 6 months, whereas fecal pedv-specific siga antibodies disappeared 1-2 months after infection (ouyang et al., 2015) . pedv-specific iga or vn antibody titers of sows infected at 3 months pre-farrowing peaked in the colostrum at post-farrowing day (pfd) 1, decreased rapidly in milk by pfd 3, and then declined only gradually during pfd 4-19 . memory b cell responses were first identified by in vitro secondary stimulation in mononuclear cells from pedv cv777-infected nursing pigs at pid 21 (de arriba et al., 2002a) . the number of pedv-specific iga antibody-positive pigs also rose from 30% at the day of reinfection to 70% 3 days later (gerber et al., 2016) . similarly, the number of pedv-specific iga antibody secreting cells of sows infected at 3-4 months pre-farrowing and then after re-exposure to pedv at pfd 3-5, increased in the milk at pfd 8-14, compared with pfd 0 (colostrum) or pfd enhanced biosecurity, such as decontamination and disinfection of transport trailers and other potential contaminated fomites, supply of secure feed or feed additives (gordon et al., 2019; niederwerder and hesse, 2018) , and maintenance of strong hygiene measures on swine farms and farm workers (kim et al., 2017b) , is critical to prevent direct or indirect contact transmission of pedv. transport trailers (19/72 herds surveyed; 26.4%) or feed (21/72 herds surveyed; 29.2%) were thought to be the main contaminated fomites (55.6% in total) in us swine farms during the 2013-2017 epidemic (niederwerder and hesse, 2018) . spillover of pedv from pedv-infected farms to others or wildlife should also be controlled through high quarantine protocols that should include no movement of pigs until the diarrhea is terminated and the disease is controlled (niederwerder and hesse, 2018) . indeed, 28 of 287 fecal samples ( use of 0.5% virkon s and 2.06% clorox that reduced the quantity of pedv rna were the best disinfectants for pedv inactivation in feces (bowman et al., 2015b) . super-oxidized water j o u r n a l p r e -p r o o f (ph 6.0) also effectively inactivated pedv at room temperature after 10-90 min contact . washing trailers with a high pressure power washer, followed by disinfection and drying, was effective for their sanitation and decontamination (baker et al., 2018; thomas et al., 2015b) . accelerated hydrogen peroxide or peroxygen-based disinfectants effectively inactivated pedv on contaminated metal (trailer) surfaces at 20 °c after 30 min contact (holtkamp et al., 2017) and at -10 °c after 40-60 min contact (baker et al., 2017) , or at -10 to 4 °c after 10-30 min contact (baker et al., 2018) , respectively. pedv rna was still detectable after disinfection, but no infectious virus was detected. the levels of pedv-specific siga antibody in the colostrum/milk of dams are key to determine the extent of passive immunity of their piglets against pedv infection (langel et al., 2019a; langel et al., 2019b; langel et al., 2016; langel et al., 2020; saif et al., 2012) . the siga antibody is resistant to proteolytic enzymes and is highly efficient at neutralizing pedv in the intestinal mucosa (langel et al., 2016) . in the mammary glands of female swine, however, there is an absence of lymphoid follicles for antigen processing and iga or igg antibody production ( fig. 2a and b) . the pedv-specific siga antibodies in the colostrum and milk are produced by effector b cells or plasmablasts that migrate from the galt of the pedv-infected intestine to the mammary gland (langel et al., 2016; langel et al., 2020; saif et al., 2012) . this is defined as the gut-mammary-siga axis, that was first described by saif and bohl in studies of immunity to tgev [reviewed in (saif et al., 2012) ]. the details were reviewed previously (langel et al., 2016; langel et al., 2020; saif et al., 2012) . the endothelium in the mammary glands contains specialized j o u r n a l p r e -p r o o f endothelial venules that attract and bind to the specific integrins on the surface of the igaplasmablasts, such as α4β7 and chemokine receptor 9 (ccr9) (bourges et al., 2008; jiang et al., 2016; salmon, 2000) . mucosal addressin cellular adhesion molecule 1 (madcam-1) and chemokine ligand 25 expressed in the specialized venules ( fig. 2d and e) interact with α4β7 and ccr9, respectively. extravasated siga antibody-secreting cells reside in the alveoli-supporting fibrous stroma of the mammary glands ( fig. 2a-c) . the number of pedv-specific siga antibodysecreting cells may positively correlate with the amount and density of these endothelial surface molecules in the mammary glands (salmon, 2000) , although the specific role of each surface molecule in the gut-mammary-siga axis needs to be better characterized. immunization of pregnant sows is important in the control of epidemic ped and to reduce the number of deaths of suckling piglets (jung and saif, 2015; langel et al., 2020) . the most common practice used to initiate herd immunity in us pig farms during the 2013-2017 epidemic when no pedv vaccines were available, was use of whole-herd feedback using a load-closeexpose protocol (59/77 herds surveyed; 76.6%), in which a controlled exposure for gilt acclimation (23/77 herds surveyed; 29.9%) was included (niederwerder and hesse, 2018) , whereas 19/77 herds surveyed (24.7%) used vaccination. there is no standard feedback protocol for any type of pig production systems to have the most consistent and highest efficacy (niederwerder and hesse, 2018) . immunization of pregnant sows, or gilts expected to be bred (gilt acclimation), is undertaken by exposure to virulent autogenous virus, such as minced intestines from infected neonatal piglets negative for other infectious agents (jung and saif, 2015; niederwerder and hesse, 2018) . feedback stimulated lactogenic immunity via the gut-mammary-siga axis in sows (saif et j o u r n a l p r e -p r o o f al., 2012), as well as prolonged, passive immune protection of piglets against pedv infection (goede et al., 2015) . in our study, strong protective immunity (100% survival rate of the piglets challenged with pc22a) was induced in gilts inoculated orally once with virulent non-s indel pedv strain pc22a (10 5 pfu/gilt) during mid-gestation (day 57-59) (langel et al., 2019a ). gilts exposed to the virus earlier (day 19-22) or later (day 96-97) in the gestation period showed less protective immunity, as evident by 87.2% or 55.9% survival rates of their piglets, respectively. sows given feedback or orally inoculated with virulent pedv during mid-gestation also discontinued fecal pedv shedding prior to farrowing (langel et al., 2019a; leidenberger et al., 2017) . whether prolonged pedv shedding post-exposure and during farrowing and lactation may cause infection of piglets, remains unclear. however, the most efficient and safest timing for feedback should be mid-gestation. because of the frequent inefficiency or lack of safety, feedback should be replaced by oral immunization with high, consistent doses of live attenuated pedv that is minimally replicative, but immunogenic in the intestine of pigs (song et al., 2007) . pregnant sows orally inoculated with live attenuated pedv (dr13 strain) vaccine twice at 2 and 4 weeks pre-farrowing exhibited higher pedv-specific iga and vn antibody levels in the colostrum compared with the counterpart sows administered the same vaccine im (song et al., 2007) . although the im route has also been used as an administration option using live pedv vaccine (kweon et al., 1999) , to what extent pedvspecific iga memory b cells are induced in sows initially primed via an im route with live pedv vaccine, has not been confirmed. the efficacy of pedv im boost vaccination is dependent on induction of iga memory b cells in initially field exposed or orally primed sows (gillespie et al., j o u r n a l p r e -p r o o f 2018), similar to the success of tgev oral prime/parenteral boost vaccine strategies (saif et al., 2012) . live attenuated strains of pedv have been isolated from the feces of pigs, suggesting that recombinant or mutated strains of pedv might have emerged (gerdts and zakhartchouk, 2017; guo et al., 2016) . for maintaining or boosting herd immunity initiated through feedback or oral live vaccines in farrowing herds, im administration with either live pedv (sato et al., 2018) or inactivated, adjuvanted whole-virus pedv vaccine (gillespie et al., 2018) once or twice at 4-5 or 2 weeks prefarrowing was effective in boosting lactogenic immunity in sows positive for pedv-specific iga memory b cells in galt. many vaccine candidates or products have been tested and developed in the us and worldwide. the details were reviewed previously gerdts and zakhartchouk, 2017) . im live vaccine booster plus feedback was more effective in reducing the number of deaths of suckling piglets, compared with either im live vaccine alone or feedback alone (sato et al., 2018) . prime and boost im vaccination with inactivated pedv provided incomplete protection of piglets against pedv infection (gerdts and zakhartchouk, 2017; niederwerder and hesse, 2018) , because of poor lactogenic immunity induced in their vaccinated dams (gillespie et al., 2018) . despite the safety issue, therefore, feedback alone, or prime and boost vaccination via oral and/or im routes with live pedv have been frequently used for maternal immunization against pedv infection. j o u r n a l p r e -p r o o f pedv remains a threat to the swine industry in the us and worldwide because no effective vaccines are currently available. regardless of the genogroup, pedv can be deadly in pig populations with no immunity or low immunity or health status. the extent of pedv transmission and ped on farms has a reverse relationship with the levels of biosecurity. high biosecurity to control ped should be practiced prior to seasonal or expected epidemics. concurrently, pregnant sows are immunized by either feedback or vaccination. however, the current feedback or vaccination protocols are frequently inefficient or unsafe, due to: 1) no standardized protocol for feedback; 2) poor capacities of current im vaccines (live or killed) to induce lactogenic immunity; 3) antigenic differences of vaccine vs. epidemic strains; and 4) possibility of continuous reinfection by pedv used for feedback within herds. in addition, current live vaccines, which were generated by conventional cell culture-adaptation methods, may revert to virulent pedv or recombine with field pedv strains to generate new strains after they are applied in the field. therefore, better vaccination protocols and vaccine strains (attenuated oral vaccines) are required for prevention and control of ped. pedv-specific iga memory b cells in orally 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epidemic diarrhea virus infection on tight junction protein gene expression and morphology of the intestinal mucosa in pigs histologic (a and b) and immunohistochemical (c) localization of mammary igasecreting cells, and expression of mucosal addressin cellular adhesion molecule hematoxylin and eosin-stained mammary gland tissue of a gilt at 4 days post-farrowing, showing fully expanded alveoli, with mostly empty lumens. (b) higher magnification of panel a (inset), showing clusters of plasma cells characterized by the typical peripheral nuclei in the abundant cytoplasm in the interlobular stroma. original magnification, 200 (a) and 400 (b). (c) immunohistochemistry (ihc)-stained mammary gland tissue of a gilt at 5 days post-farrowing, showing a moderate number of iga-positive cells we sincerely thank all the authors cited in our review for their efforts and contributions to understand pedv. we apologize to authors whose contributions to pedv research may have been up to pid 21 9.7-10.2 log10 rna copies/ml at pid 4-6 direct contact pid 4 pid 4-14 up to pid 49 6.6-9.3 log10 rna copies/ml at pid 6-21 indirect aerosol contactno clinical signs in 10 pigs (0/10) loose stool in 2/5 pigs (40%) at pid 1-10 or 15low (with mean peak viral rna shedding titer of 5.00 log10 ge/ml), as tested at pid 1-10 or 15mild villous atrophy at pid 9 (mean vh:cd ratio of 5.2 vs. 6.8 in the control)no detection of pedv antigen at pid 9ct (120 th ) 2b (non-s indel) 6 log10 tcid50 6-day 5-6 0 at pid loose stool in some pigs at pid 4-5, at monitored at pid 1-10moderate (with mean peak viral rna shedding titer of approximate 6-7 log10 ge/ml at pid 4-5), as tested at pid 1-10 mild villous atrophy in a pig at pid 3no or little detection of pedv antigen in a pig at pid 3wu et al., 2019 key: cord-308344-ao9z00t7 authors: diep, nguyen van; norimine, junzo; sueyoshi, masuo; lan, nguyen thi; yamaguchi, ryoji title: novel porcine epidemic diarrhea virus (pedv) variants with large deletions in the spike (s) gene coexist with pedv strains possessing an intact s gene in domestic pigs in japan: a new disease situation date: 2017-01-17 journal: plos one doi: 10.1371/journal.pone.0170126 sha: doc_id: 308344 cord_uid: ao9z00t7 since late 2013, after an absence of seven years, outbreaks of porcine epidemic diarrhea virus (pedv) infection have reemerged and swept rapidly across japan, resulting in significant economic losses. in this study, we report the emergence, mixed infection, and genetic characterization of 15 novel field pedv variants with large genomic deletions. the sizes of deletion varied between 582 nt (194 aa) and 648 nt (216 aa) at positions 28–714 (10–238) on the s gene (protein). among 17 pedv samples isolated from individual pigs, all of them contained at least two distinct genotypes with large genomic deletions, and 94.1% of them were found to consist of strains with an intact s gene. these variants were found in eight primary and nine recurrent outbreaks, and they might be associated with persistent pedv infection in the farms. full-length s and orf3 genes of eight variants derived from 2 samples were characterized. this is the first report of mixed infections caused by various genotypes of pedv and would be important for the studies of viral isolation, pathogenesis, and molecular epidemiology of the disease. porcine epidemic diarrhea (ped) is a highly contagious enteric disease characterized by vomiting, acute watery diarrhea, and in particular, a high mortality rate in suckling piglets, resulting in substantial economic losses [1] . the etiologic agent of this disease is the ped virus (pedv), which is an enveloped, positive-sense, single-stranded rna virus that belongs to the order nidovirales, family coronaviridae, and genus alphacoronavirus. the disease was initially reported in england in 1971 [2] and belgium in 1978 [3] . since then, it has been identified in many swine-producing countries such as those in europe and asia, notably belgium, czech a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the samples were obtained from a naturally infected animal in the field by qualified veterinarians as a part of normal veterinary care and diagnostic testing procedures. samples of intestine used were collected from dead piglets and the fecal samples were non-invasively collected immediately after excretion. therefore, no aggressive operation had been conducted against pigs for sampling purpose. no piglets or other animals were sacrificed for the purposes of this study. the university of miyazaki dna recombinant committee approved the protocol for our cloning work of pedv (protocol number 2014-464). amplification of the partial s gene, full-length s gene, and orf3 gene reverse transcription was performed using random primers and oligo-dt primers from reverse transcription system kits (promega, madison, wi, usa). to determine the complete s gene sequence, five primer sets (cs1-cs5, table 1 ) were used to amplify five dna fragments spanning the entire s gene. these primers were designed on the basis of the published sequences of reference pedv strains [5, 9, 33] . emeraldamp max pcr master mix kit (takara bio, japan) containing high fidelity dna polymerases was used for performing the pcr amplifications under the following conditions: denaturation at 94˚c for 2 min; 35 cycles of denaturation at 94˚c for 30 s, annealing at 54˚c for 30 s and extension at 72˚c for 1 min; a final extension step at 72˚c for 10 min. when a partial s gene was amplified using the primer pair cs1-f/cs1-r from generated cdna, one or two distinct bands of unexpected shorter sizes were observed in each pcr product in addition to the predicted band (fig 1) . the same result was observed when one-step rt-pcr tests (accessquick rt-pcr system kit, promega corp, wi, usa) were performed from extracted rna and pcr tests using other kits containing high fidelity dna polymerases (kod fx, kod fx neo, and blend-tag plus kits, toyobo co., japan) were performed from cdna templates to amplify the cs1 fragment. the unexpected and expected dna bands were also observed and distinguished upon performing pcr amplifications with longer fragments containing the first portion of the s gene (primer pairs cs1-f/cs2-r, cs1-f/cs3-r, and cs1-f/cs4-r). the pcr products containing the cs1 fragment were individually gel-extracted and cloned into pcr2.1-topo vectors (thermo fisher scientific, us). the cloning reaction was then chemically transformed into one shot top10 to obtain the sequence of the full-length s gene and orf3 gene of pedv strains coexisting in a single pedv sample, a fragment containing these two contiguous genes was amplified using the primer set fs-f/orf3-r and the emeraldamp max pcr master mix kit (takara bio, japan). the pcr conditions were as follows: denaturation at 94˚c for 2 min; 35 cycles of denaturation at 94˚c for 30 s, annealing at 55˚c for 30 s and extension at 72˚c for 5 min; a final extension step at 72˚c for 10 min. the expected size of this amplified product was 4955 bp. subsequently, the pcr products were gel-extracted and cloned into pcr-xl-topo plasmid vectors (topo xl pcr cloning kit, invitrogen, us). after transformation into competent cells, plasmid dna isolation was performed as described previously. the purified plasmid dna was sequenced using the primer sets cs1-cs5 and the primer set orf3-f/orf3-r [15] . however, when the fragment containing entire s and orf3 genes was amplified, pcr products derived from only two samples jka-295 and jmi-277 were visualized distinctly in agarose gel 0.8%. therefore, these two samples were selected for cloning and sequencing of the fulllength s and orf3 genes. all sequences were determined in both directions, and sequencing reactions were performed using bigdye terminator v3.1 cycle sequencing kits and an abi prism 3130xl genetic analyzer (applied biosystems, us). nucleotide and deduced amino acid sequences were edited, assembled, and analyzed using geneious v9.0.4 software (http://www.geneious.com) and molecular evolutionary genetics analysis (mega) software version 6.0 [34] . the resultant nucleotide sequences were deposited in genbank under the following accession numbers: ku363044-ku363130. phylogenetic trees based on the nucleotide sequences of the full-length s gene and orf3 gene were constructed by the maximum likelihood method using the tamura-nei substitution model with a discrete gamma distribution; then gaps/missing data treatment was done by complete deletion, and bootstrap tests of 1000 replicates in the mega program. a total of 248 field samples (70.7%) collected from animals at 145 farms (82.4%) experiencing acute diarrhea in six prefectures were positive for pedv. the result indicated that pedv has a high prevalence rate in japanese pig herds. notably, ped outbreaks were commonly observed in ped-vaccinated farms, and there was a significant difference in the mortality rate (0-100%) for piglets and durations of diarrhea among pedv-infected farms. this prompted critical questions regarding the molecular characteristics and virulence of circulating pedv as well as efficacy of pedv vaccines that have been used in japan. to monitor heterogeneity among pedv strains, 52 pedv-positive samples were selected for further sequence analyses. however, when cs1-f/cs1-r primers were used for pcr to amplify the first portion of the s gene, one or two distinct bands of unexpected size (approximately 0.2-0.3 kbp) were observed in each pcr product of 17 pedv samples (32.7%) in addition to the predicted band (872 bp). the pcr products were individually gel-extracted, cloned, and sequenced. sequencing results illustrated that the unexpected bands were fragments with large deletions in the s gene, and a total of 15 novel pedv genotypes with large deletions were identified ( table 2 although two genotypes feature 606-nt deletions, the deletions were located at two distinct positions (94-699 and 46-651) in the s gene. furthermore, the genotype with a 203-aa deletion at residues 10-217 featured a 5-aa insertion (yamff) at the position of the deletion. interestingly, the identical nucleotide sequence corresponding to the insertion (tacgccatgttcttt) existed in the middle of the s gene (nt 2091-2105). additionally, all pedv variants of the s protein identified in this study were predicted to have signal peptide cleavage sites using the signalp 4.1 server (http://www.cbs.dtu.dk/services/signalp-4.1). this indicates that the s proteins of the variants are most likely expressed as structural proteins. surprisingly, mixed infection with multiple variants was found in all pedv samples (17/17) collected from individual pigs in primary or recurred ped outbreaks. particularly, in sample jka-295, we found nine coexisting genotypes including a genotype with an intact s gene and eight genotypes with deletions of 194, 197, 201, 204, 205, 211, 215, and 216 aa (table 3) . identical genotypes were also isolated from different farms of various prefectures that are geographically distant from each other. in addition to the variants, pedv strains with an intact s gene were detected in 94.1% of the samples (16/17), with no intact gene detected in sample jao-56. these results revealed that complex mixed-genotype infection of pedv had occurred in individual pigs and farms. full-length s and orf3 genes of pedv strains derived from samples jka-295 and jmi-277 were cloned and sequenced. identical nucleotide sequences of both s and orf3 genes were distinguished and excluded, resulting in the identification of 11 individual strains (table 3) . three strains that were obtained had an intact s gene (4161 nt, 1386 aa), whereas eight other variants possessed deletions of 582 (194 aa), 591 (197 aa), and 645 (215 aa) nt. notably, the variants with a 197-aa deletion were identical to that of us strain pc177-p2, which was proposed to be obtained during adaptation of the virus in cell culture [17] . in addition, the 194-aa deletion from the variants in this study was similar to that of the tottori2 strain that has been recently isolated from domestic pigs in japan [19] . however, apart from tottori2, all variants with a 194-aa deletion in our study were found to coexist with other variants as well as strains with an intact s gene. the pedv s protein is a type i glycoprotein [35] . its identified epitope regions include the coe domain (499-638 aa) and epitopes ss2 (748-755 aa), ss6 (764-771 aa), and 2c10 (1368-1374 aa) [36] [37] [38] . it was demonstrated that the large deletions found in the s gene of the variants did not occur in these four regions, and there was no significant difference in the epitope regions between the pedv variants and pedv strains with an intact s gene (s1 fig) . sequencing analysis indicated that strains found in a single pedv sample displayed genetic diversity in their s genes. in sample jmi-277, three strains with an intact s gene had 99.64-99.78% nucleotide sequence identity with each other and 99.64-99.89% nucleotide sequence identity with three variants possessing the 197-aa deletion when pairwise distances were measured using mega v6.05 software. within jka-295, three variants with the 197-aa deletion shared 99.72-99.92% nucleotide sequence identity with each other but had 99.38-99.55% nucleotide sequence identity with jka-295fsde204co25, which possessed the 204-aa deletion (s1 table) . on the contrary, all strains found in jka-295 and jmi-277 had the greatest [39] . furthermore, phylogenetic analysis based on the full-length s gene classified these japanese strains into the north american (highly virulent) type as shown in fig 2a. it is suggested that the variants originated from north american type of pedv. the sequencing results illustrated that the orf3 gene of the 11 strains in jka-295 and jmi-277 had the same length of 675 nt, and it did not possess any deletion found in known pedv vaccine strains [15, 27, 40] . all of the variants with large deletions have the same orf3 nucleotide sequence excluding jka-295fsde194co25. nucleotide identity of the orf3 gene between the variants and intact s strains was 98.81-99.41% which was lower than the 28 field isolates (99.6-100%) collected from 5 prefectures in japan during 2103-2104 [15] . eight distinct snps between the variants and the strains with an intact s gene were also identified at positions 136, 154, 178, 228, 302, 561, 645, and 657 in the orf3 gene (s2 fig) . phylogenetic analysis revealed that the strains with an intact s gene and the variants fell into two distinct clades (fig 2b) . the result indicated that the variants were not vaccine-related, and might have evolved independently as compared to the intact s strains which coexisted in the individual samples. in this study, variants with large deletions in the s gene were found in eight primary and nine recurrent outbreaks from 16 pig farms, and they mostly (94.1%) coexisted with pedv strains with an intact s gene. in four farms (te, sa, is, and at), the variants were not found in the primary outbreaks, but they appeared in subsequent pandemics. this indicated that pedv variants could be transmitted from farm to farm and/or that the proportion of the variants in samples collected at the primary outbreaks was insufficient to permit detection of the variants by pcr. additionally, ped outbreaks in japan since late 2013 were proposed to be caused by the invasion of recent overseas strains [15, 31] . taken together, it is possible that in the recent pandemic, both the pedv variants and the strains with an intact s gene were introduced together from other countries, after which they spread across japan. however, the earliest ped outbreak of the variants possessing the large s deletions occurred on kagoshima on december 2013 (sample jka-292) while the first ped case in japan was reported earlier in okinawa on october 2013. presently, no pedvs possessing the large deletions in the n-terminus of the s gene analogous to the japanese variants was reported in other countries except pc177-p2 which was concluded to derive from cell adaptation in vitro. therefore, the variants might have arisen spontaneously during the pedv spreading in japan. further studies are required for the confirmation. in this study, due to the coexistence of the variants and pedv strains with an intact s gene in all of the samples excluding jao-56, it might not be accurate to evaluate the virulence of individual strains based solely on the information from clinical signs and mortality rates recorded from the pig farms. in jao-56, we could not identify any pedv strain with an intact s gene. in the farm from which this sample was collected, the sows had never been vaccinated with modified live pedv. piglets in the farm manifested various severities of diarrhea from mild to severe, and the mortality rate reached 72.4% (670/926). the mortality rate was much higher than the mortality rate recorded in the epidemic during which tottori2 was isolated (0%) [19] . therefore, the large deletion in the variants observed in this study might not necessarily lower the virulence of this virus. further investigations are needed to elucidate the virulence of variants in ped outbreaks. after the primary outbreaks occurred in 2013 and the first half of 2014, there was a sharp decrease of ped cases that were reported during the second half of 2014 and 2015 in japan. among the few recurrent ped outbreaks reported in 2014 and 2015, all the samples collected from pig farms in miyazaki (6 farms) and aichi (2 farms) were found to contain the pedv variants. in those farms, diarrhea in piglets was observed for a long time (up to three months), and outbreaks of ped were reported to frequently recur in intervals from three weeks to several months (s2 table) . although many strict measures for establishing a high level of security were implemented such as quarantine of reposition, strict visitor policies, banning of unwashed vehicles, controlling of carcasses, movement of the caretakers on the farms and using virucidal disinfectants; ped still recurred in the farms. therefore, it is suggested that mixed infection of pedv variants with large genomic deletions may play an important role in the persistence of pedv in pig farms. defective interfering (di) particles are a common observation in many virus families [41] . di particles are defective for replication in the absence of the product of the deleted gene, and its replication requires the presence of the complete functional virus particle to co-infect a cell with it, in order to provide the lost factors [42] . the pedv variants wherein large genomic deletions were observed may be di particles. however, we speculated otherwise because the reported pedv strains pc177-p2 and tottori2 possessing the same deletions found in the variants of this study could replicate well in cell culture and cause cytopathic effect [17, 19] . besides, the mutant strain pc177-p2 can propagate well and induce diarrhea in piglets [43] . a deletion of 215 aa (position in the n-terminal domain of the s protein analogous to the deletion observed in the variants revealed a not impaired propagation of pedv in vivo [44] . moreover, only variants with large s deletions were found in the field sample jao-56, which indicated that the variants were able to independently propagate well in pigs. additionally, all the deletions found in the variants are in-frame and the s proteins were predicted to express as a functional structure protein. taken together, it is suggested that the variants with the large s deletions in this study were not di particles. functions of the spike protein n-terminal domain of pedv and other alphacoronaviruses were previously elucidated [44] . the loss of this domain occurred in a number of alphacoronaviruses correlates with a loss or reduction of enteric tropism. the most obvious example of this phenomenon is transmissible gastroenteritis virus (tgev) that has a dual tropism targeting the respiratory and gastrointestinal tract of the pig. meanwhile, porcine respiratory coronavirus (prcv) is a natural variant of tgev which lacks the s protein n-domain. it has reduced considerably its enteric tropism and primarily replicated in the respiratory tract then, produced an attenuated disease in comparison with parental tgev strains. another typical example is feline coronaviruses that have 2 pathotypes: feline enteric coronavirus (fecv) causing a clinical mild or asymptomatic disease and feline infectious peritonitis (fipv) inducing a highly pathologic disease. fipv is thought to be derived from fecv by natural mutation. however, compared to fecv, pipv has a large deletion in the s protein n-domain which results in the loss of tropism for enterocytes [44, 45] . these examples revealed an important role of alphacoronavirus spike n-domain for viral replication in the enteric tract. the n-terminus (residues 1-249) of the s protein was demonstrated to be responsible for the sialic acid binding activity of pedv [44] . the loss of enteric tropism observed in tgev was specifically associated with the loss of the sialic binding activity that located in the n-domain. however, how the sialic acid binding activity affects the tropism of these alphacoronaviruses remains unclear. the s protein n-domain of some pedv strains were thought to be dispensable for replication of pedv in vitro [44] [45] [46] . the large deletions in the s protein of the pedv variants in this study indicated that this domain might also be dispensable in vivo. in the present study, the characteristics of large deletions found in the japanese variants were highly similar to those observed in the prcv that have a 224 or 227-aa deletion in the n-terminal domain of the s protein [47] . prcv can persist in pig herds throughout the entire year, or it can disappear temporarily from a herd for several months and reappear subsequently. the emergence and widespread prevalence of prcv lessened the clinical impact of tge in the united states and europe due to cross-protective immunity with tgev [48] . thus, the pedv variants identified in this study might have biological characteristics analogous to prcv and may persist in the pig farms of japan. unfortunately, we did not collect lung tissues from pedv-infected pigs in these outbreaks, and virus isolation for the individual variants has not yet been completed. therefore, it remains unclear whether the pedv variants found in this study exhibited altered virulence, pathogenicity, and tissue tropism from the strains with an intact s gene. in future studies, we will continuously seek to answer this question. this is the first report describing ped outbreaks with mixed infections of various genotypes. our study identified 15 field pedv genotypes with large deletions in the s gene that have been circulating in japan from 2013 to 2015. the full-length s gene and orf3 gene of eight variants and three strains with an intact s gene were characterized. the variants coexisted with intact s strains in high frequency and they might be associated with persistent pedv infection in pig farms in japan. such a reemerging and mixed infection of the variants in this study have put ped in a new situation indicating this disease has become more complex in terms of viral isolation, pathogenesis, and epidemiology. the mechanism by which the pedv variants were generated and evolved remains unclear. hence, further studies are required to elucidate biological properties such as changes in tissue tropism and the virulence patterns of the new variants. table. nucleotide sequence identity based on the full-length spike genes of 11 japanese pedv field strains identified in this study and pedv reference strains ã . (docx) s2 table. status of pig farms where the pedv samples using in this study were collected. (docx) porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines an apparently new syndrome of porcine epidemic diarrhoea a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea porcine epidemic diarrhea virus variants with high pathogenicity new variants of porcine epidemic diarrhea virus, china emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences distinct characteristics and complex evolution of pedv strains comparison of porcine epidemic diarrhea viruses from germany and the united states complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france in complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium pubmed central outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea us-like strain of porcine epidemic diarrhea virus outbreaks in taiwan us-like isolates of porcine epidemic diarrhea virus from japanese outbreaks between new variant of porcine epidemic diarrhea virus, united states cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene novel porcine epidemic diarrhea virus variant with large genomic deletion new porcine epidemic diarrhoea virus variant with a large deletion in the spike gene identified in domestic pigs detection and molecular diversity of spike gene of porcine epidemic diarrhea virus in china cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus dr13 porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus genetic diversity of orf3 and spike genes of porcine epidemic diarrhea virus in thailand molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) samples from field cases in fujian differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf 3 pedv orf3 encodes an ion channel protein and regulates virus production molecular epidemiology of porcine epidemic diarrhea virus in china isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate an immunohistochemical investigation of porcine epidemic diarrhoea porcine epidemic diarrhea: its diagnosis and control molecular characterization of pig epidemic diarrhoea viruses isolated in japan from differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex rt-pcr sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhoea virus confirms that this virus is a coronavirus related to human coronavirus 229e and porcine transmissible gastroenteritis virus mega6: molecular evolutionary genetics analysis version 6.0 coronavirus spike proteins in viral entry and pathogenesis identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein the gprlqpy motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo genetic variation analysis of reemerging porcine epidemic diarrhea virus prevailing in central china from defective viral particles and viral disease processes defective interfering viruses and their potential as antiviral agents evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains cellular entry of the porcine epidemic diarrhea virus establishment of feline intestinal epithelial cell cultures for the propagation and study of feline enteric coronaviruses manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination genetic evolution and tropism of transmissible gastroenteritis coronaviruses diseases of swine, tenth ed the work was conducted with funding from the university of miyazaki (grant number 2014-70). key: cord-331509-p19dg1jw authors: bigault, lionel; brown, paul; bernard, cécilia; blanchard, yannick; grasland, béatrice title: porcine epidemic diarrhea virus: viral rna detection and quantification using a validated one-step real time rt-pcr date: 2020-05-31 journal: j virol methods doi: 10.1016/j.jviromet.2020.113906 sha: doc_id: 331509 cord_uid: p19dg1jw since 2014, porcine epidemic diarrhea virus (pedv) has reemerged in europe. rt-pcr methods have been described for the detection of pedv, but none have been validated according to a norm. in this study we described the development and validation of a sybr™ green one-step rt-qpcr according to the french norm nf u47-600, for the detection and quantification of pedv viral rna. the method was validated from sample preparation (feces or jejunum) through to nucleic acid extraction and rt-qpcr detection. specificity and sensitivity, limit of detection (lod), limit of quantification (lq), linearity, intra and inter assay variability were evaluated using transcribed rna and fecal and jejunum matrices spiked with virus. the analytical and diagnostic specificities and sensitivities of this rt-qpcr were 100% in this study. a lod of 50 genome copies/5 µl of extract from fecal matrices spiked with virus or rna transcript and 100 genome copies/5 µl of extract from jejunum matrices spiked with virus were obtained. the lower lq (llq) was 100 genome copies/5 µl and the upper lq (ulq) 10(8) copies/5 µl. this method is the first, validated according a norm for pedv and may serve as a global reference method to harmonize detection and quantification of pedv viral rna in both field and experimental settings. porcine epidemic diarrhea (ped) was first described in europe in 1971. it is characterized by watery diarrhea, vomiting, dehydration, and is most notable in young piglets. the etiologic agent, porcine epidemic diarrhea virus (pedv) which was first identified by electron microscopy (em) in 1977 (chasey and cartwright, 1978; debouck and pensaert, 1980) is now characterized as an enveloped virus with a single stranded positive sense rna genome, member of the order nidovirales, suborder cornidovirinae, family coronaviridae, subfamily orthocoronavirinae, genus alphacoronavirus, subgenus pedacovirus (walker et al., 2019) . in the 1980's, pedv was detected for the first time in asia whilst in europe it was endemic. during the 90's only few sporadic cases were reported in europe and most of these were reported in italy were it remains endemic (martelli et al., 2008) . during the last two decades new pedv strains have appeared in china and some of these strains have caused extremely severe outbreaks characterized by a morbidity of 100% and a mortality of 80-100% on suckling piglets (sun et al., 2012) . this has led to the naming of pedv as either s-non-indel or s-indel genotypes. in general the more virulent viruses belong to the s-non-indel group. in the last decade both s-non-indel and s-indel viruses have emerged in the usa with serious consequences for the industry. throughout europe, the predominant types are now closely related to the viruses circulating in asia and north and central america (boniotti et al., 2016) . furthermore, all viruses reported in europe since 2014 belong to the s-indel group (grasland et al., 2015; stadler et al., 2015; steinrigl et al., 2015; theuns et al., 2015) except for one in the ukraine (dastjerdi et al., 2015) . this data highlights the importance of pedv diversity across several continents. in france, since 2014, ped caused by s-non-indel is a notifiable disease. for territory monitoring purpose, all pedv suspicions have to be notified to french ministry of agriculture and the pedv genotype has to be confirmed by the national reference laboratory at the french agency for food, environmental and occupational health safety (anses). until today, no official method has been validated for the detection and quantification of the pedv viral rna. since the 2000s, real-time pcr emerged as a tool of choice for the detection and quantification of viral rna and has multiple benefits: i) these tests are highly specific ii) are easily standardized compared to "classical" virology procedures, iii) are much less time consuming, and iv) are highly reproducible. several rt-pcrs have been described for the detection of pedv rna (kim et al., 2007; miller et al., 2016) . for a rapid, accurate and reliable diagnosis of ped in the veterinary laboratory, a method for the detection of pedv viral rna has been developed and more importantly validated according to the "association francaise de normalisation" (afnor) french nf u47-600 norm entitled "requirement and recommendation for the implementation, development and validation of pcr in animal health" (afnor, 2015a; afnor, 2015b) . this validated sybr tm green one-step rt-qpcr was based on a previously published taqman® probe real time rt-qpcr (kim et al., 2007) and targeted the same zones of sequence in the conserved n open reading frame (orf) as this had previously allowed for broad range detection and the capability to differentiate between the closely related virus transmissible gastro-enteric virus (tgev). the method developed in the current study under nf u47-600, unlike other molecular tests developed for pedv, evaluates the whole process from sample preparation through to the detection and quantification by rt-qpcr. this method should help harmonize detection and quantification of viral rna from pedv belonging to both s-non-indel and s-indel strains in both field and experimental settings. all commercial methods were performed according to the manufacturers' recommendations unless otherwise stated. an alignment of 192 pedv n orf sequences that were available on the data base at the time of the study (2014) was made using mafft (katoh and standley, 2013) and the probabilities of the nucleotides at the priming zones defined by kim et al. (2007) (pednf : 5'-cgcaaagactgaacccactaattt-3', and pednr : 5'-ttgcctctgttgttactt-ggagat-3') were calculated using r (wagih, 2017) (fig. 1 ). based on these probabilities forward primer mpednf (5'-cgcaaagactgaacccactaa-3') and reverse primer pednr were chosen (fig. 1) . these primers were subsequently checked against n orfs of the s-indel and s-non-indel pedv strains circulating in europe (dastjerdi et al., 2015; grasland et al., 2015; hanke et al., 2017; martelli et al., 2008; stadler et al., 2015; steinrigl et al., 2015; theuns et al., 2015) . original cv777, the pedv reference strain isolated in 1977, was collected from perfused jejunum performed in 1981 and kept at -80°c. this stock was named wtcv777. wtcv777 was propagated in cell culture as previously described (hofmann and wyler, 1988) and was named cccv777. a stock of cccv777 was produced as follows: 20 x 175 mm 2 confluent monolayer of vero cells (atcc® ccl-81) were infected each with 500 µl of 6.8x10 4 tcid50 of cccv777 in infection media; emem (thermofisher scientific, france) supplemented with 0.3% tryptone phosphate broth, 0.02% yeast extract, 1% penicillin/streptomycin and 10µg/ml trypsin. after 24 hours of infection, cells were subjected to three freeze thaw cycles and the culture medium was clarified by centrifugation at 10000g for 10 minutes. a total volume of 1l of supernatant was then centrifuged for four hours at 20000g to pellet the virus. the pellet was then resuspended in 100ml of pbs. the infectious viral titer of cccv777 was determined by immuneperoxidase monolayer assay according kärber's method (kärber, 1931) . the virus stock solution was titrated by immuno-peroxidase monolayer assay to 1.2x10 7 tcid50/ml. four other pedv strains were used: three french field strains (pedv/fr/001/2014 genbank accession number (gb acc) kr011756, pedv/fr/001/2017 and pedv/fr/001/2019 gb acc mn056942), and one american strain (pedv/usa/2014/iowa gb acc mf373643, kindly provided by dr p.gauger from iowa state university). nine other 'non-pedv' rna viruses were also used: one pig alpha-coronavirus (porcine respiratory coronavirus, prcv), and two gamma-coronaviruses (infectious bronchitis virus (ibv) gb acc fj904713), turkey coronavirus (tcov) gb acc kr822424) as well as other pig viruses: a pig artevirus (porcine reproductive and respiratory syndrome virus (prrsv), gb acc ky366411), a pestivirus (classical swine fever virus (csfv)), three pig ortomyxoviruses (swine influenza viruses h1ni, h1n2, h3n2), and two swine dna virus, one circovirus (porcine circovirus type 2 (pcv2) gb acc af201311), and an asfavirus (african swine fever virus (asfv) bankit1774827 anses-mada68322). jejunum and fecal samples were collected from both specific pathogen free (spf) pigs confirmed negative for coronavirus rna by deep sequencing and from pedv infected pigs positive for pedv rna. the pedv positive samples had been collected during previous experimental studies (gallien et al., 2018a; gallien et al., 2018b; gallien et al., 2019) . spf samples were used as negative controls or were spiked with pedv produced in vitro as described in section 2.2. spiked spf samples were used for the validation of the method and are later referred to as 'infectious reference materials'. for each jejunum sample, 200mg were homogenized in 1ml of phosphate buffered saline (pbs) (merck, france) with 4mm stainless steel beads in a tissuelyserii (qiagen, france). samples were then clarified by centrifugation at 10000g for 10 minutes. for each fecal sample, 1ml was diluted in 9ml of pbs and vortexed for 5 minutes before clarification by centrifugation as describe above. to determine the limit of quantification (lq) of the pcr and produce standard for quantification, a rna transcript was produced by in vitro transcription of the pedv wtcv777 n orf sequence. wtcv777 rna was extracted using trizol (thermofisher scientific, france). viral rna extract was subjected to reverse transcription using hexanucleotide primers and superscript iii reverse transcriptase (thermofisher scientific, france). reverse transcription was performed at 55°c for 1 hour followed by enzyme inactivation at 70°c for 15 minutes. to amplify the n orf, 5µl of rt were subjected to pcr amplification in 50µl reaction containing 400nm of primers ogvb160-f (gtcggatccactttatggcttct) and ogvb160-r (gtcctcgagatt gtttaatttccterror! reference source not found.), 2.5 units of platinum taq hifi (invitrogen, france), 5µl of 10x high fidelity pcr buffer, and mgso4 at a final concentration of 2mm. the pcr was performed as follows: 95°c for 2 minutes for initial denaturation, 5 cycles of 95°c for 15 seconds, 30 seconds at 55°c decreasing by 2.5°c per cycle and then 68°c for 2 minutes, follow by 40 cycles of 95°c for 15 seconds, 60°c for 30 seconds and 68°c for 2 minutes. amplified pedv n cdna was separated on 2% agarose gel and extracted using montage gel extraction kit (millipore, france). 100ng of extracted product were cloned in pcr4-topo vector (invitrogen, france). plasmid dna was prepared using nucleospin® plasmid kit (macherey nagel, france). in vitro transcription was performed with maxiscript tm t7 transcription kit (thermofisher scientific, france) using 1µg of precipitated spei linearized n orfs plasmid. rna was purified with agencourt® rnaclean xp kit (beckmancoulter, france), and quantified using qubit® fluorometer (life technology, france, saint aubin). stock of in vitro transcribed rna was stored at -80°c. number of molecular copies was calculated according the following formula: ) × 6.023 × 10 23 rna transcript was diluted to 10 9 molecules/5µl, aliquoted in 100µl, supplemented with 20µl of rnastable® (m, france) and dried in speedvac® vacuum concentrator (thermoelectron, france). the standard transcript was resuspended in 1ml in deionized nuclease-free water and then log10 serially diluted from 10 8 to 10 2 copies/5µl and stored at -80°c. all rna extractions were performed using rneasy® mini kit (qiagen, france) with the following modifications. 120µl of sample mixture containing 100µl of sample, 10µl of an external exogenous control (eec) and 10µl of proteinase k were used as opposed to 100µl of sample alone as recommended by the kit. rna was eluted with 50µl of nuclease-free water and stored at -80°c until use. eec used in this study was viral rna genome (mengovirus). reactions were carried out in an applied biosystems 7500 real-time pcr system, with power sybr tm green rna-to-ct tm 1-step kit (applied biosystems, saint aubin, france). the final pcr mix volume was composed of 12.5µl of master mix (2x), 0.2µl of enzyme mix, 5µl of rna template, primers mpednf and pednr at 300nm or 600 nm, h2o to final volume of 25µl. rt-pcr cycles were as follows: reverse transcription at 48°c for 30 minutes, followed by 95°c for 10 minutes, then 40 cycles of 95°c for 15 seconds, 60°c for 1 minute, and a final melting curve analysis step as defined by the applied 7500 software v2.3. all sample amplifications with a melting temperature corresponding to the standard with a viral rna concentration equal to, or above to the limit of detection (lod) were considered positive. all of the following tests were performed using primers at 300nm. j o u r n a l p r e -p r o o f the analytical sensitivity and specificity were determined as described in the nf u47-600 norm. all nucleic acid extractions from viruses listed in 2.2 were tested. five strains of pedv were tested for inclusivity, and eleven other virus for exclusivity, among which, four coronaviruses, five other rna viruses, and two dna virus, all known as pathogens in pigs. the diagnostic sensitivity and specificity were determined as described in the nf u47-600 according to nf u47-600, lod is the last dilution of reference material that allows a detection of the target with a confidence level of 95%. n rna transcript dilutions were tested for the lod of the pcr. six points of a two-fold dilution series ranging from 400 to 12.5 genome copies/5µl were analyzed in eight replicates. three independent assays were performed for rna transcripts (lodpcr). to determine the lod of the method, spf jejunum and fecal samples spiked with cccv777 from 10 6 to 10 -2 tcid50/ml, were tested in two independent assays on a hundredfold serial dilution ranging from 10 8 to 10 2 and 50 n transcripts equivalent/5µl, as infectious reference materials (lodjejunum or lodfeces). lod's were determined by probit calculation (finney and stevens, 1948) . according to nf u47-600, lq is defined as the lowest (lower lq, llq) and highest level (upper lq, ulq) between which, for each dilution, the statistical bias is under or equal to 0.25log10. the bias is the difference between the measured value and the theoretical value calculated by linear regression on all dilutions. uncertainty is calculated as the variance of calculated point plus the medium bias value. the statistical bias is defined as the medium of uncertainty. for the lq, seven points of a ten-fold serial dilution of n rna transcript were tested (10 8 to10 2 ). ten independent assays were performed on four independent serial dilutions. the lq for organic matrices were calculated on results obtained for the lod assessment (hundredfold dilution from 10 8 to 10 2 ). pcr efficiency was evaluated by plotting the ct against an expected rna copy number in respect to the tcid50/ml (data not shown) for infectious reference material or by qubit j o u r n a l p r e -p r o o f quantification for rna transcript. in agreement with the nf u47-600 norm, an efficiency of 75 -125% was accepted. the forward primer of kim et al. (kim et al., 2007) (pednf) had perfect base pairing with 7 of the 192 (3.6%) n orfs sequences. the forward primer designed in the current study (mpednf) which did not contain the last three bases of kim et al. (2007) had perfect base pairing with 188 of 192 (97,9%) and of those that did not match at 100 % only one had a mismatch at the last 3' position ( fig. 1 a) . sequence of the reverse primer (pednr) had perfect base pairing with 123 of 192 sequence (64.1%) and those that did not match at 100 % did not have any mismatches in the last three nucleotides of the 3' end ( fig. 1 b) . concerning the alignments with the european strains available after may 2014, pednf had perfect base pairing with 7 of 56 n orfs sequences (fig. 1 c) . mpednf had perfect base pairing with 54 of 56 sequences, those sequences that did not match at 100 % only contained one mismatch and these were localized close to the 5' end ( fig. 1 c) . pednr had perfect base pairing with 55 of 56 sequences and only one single miss-match with the remaining sequence at the 5' end. amongst the different viruses strains listed in 2.2, only the pedv strains (cv777, american field strain, and three french field strains) were positive. wtcv777 (ct = 20 ), cccv777 (ct = 12), all with a tm of 79.5 ± 0.5°c which is the expected tm for the pedv sequence amplicon according to the in vitro transcription control. all the other viruses were negative. the analytical specificity and sensibility were both 100%. efficiency of the method, calculated by linear regression, was 91.04% ± 1.31(0.01) for rna transcripts, 93.51% ± 3.97(0.04) for spiked jejunum and 99.36% ± 5.12(0.05) for spiked feces. different concentrations of primers had no effect on the efficiency of the method (data not shown), however melting curve analysis showed the presence of primer dimers at 600nm and not at 300nm (figure 2 ). the lod was determined at 50 copies/5µl for the rna transcript, 50 copies/5µl (0.5x10 0.01 tcid50/ml for the spiked feces and 100 copies/5µl (10 0.01 tcid50/ml) for spiked jejunum (table 1) . for every selected rna dilution tested, from 10 8 to 10 2 copies/5µl, bias enlarged of uncertainty were included in the norm limits (-0.5 to 0.5) and statistical bias (mean of uncertainty) were < 0.25 log10 ( table 2 ). the ulqs and llq were 10 8 and 10 2 copies/5µl respectively for all matrices. calculations were done when a minimum of 23 out of 24 results were positive for the lod and for all replicates for lq. all coefficients of variation (cv) were below the 0.1 limit given by the norm nf u47-600 with 0.004 -0.032, 0.002 -0.035, 0.0004 -0.018, for rna transcript, jejunum and feces intra-assay cvs respectively and 0.022 -0.064, 0.008 -0.031, 0.007 -0.031 for rna transcript, jejunum and feces inter-assay cvs respectively (table 1) . the diagnostic sensitivity was 100% at two and fourteen dpi, pedv viral rna were detected in all true positive pigs. the diagnostic specificity was 100% as all non pedv infected pigs were found negative all along all experiments. pedv is of global importance to the pig industry with many different strains and genotypes existing in different continents. after 2013 and the introduction of both s-indel and s-non-indel strains to north america and the resulting huge economic losses, the french ministry for agriculture classified ped caused by the s-non-indel virulent strains as a notifiable disease. thus there was a need for a reliable method for rapid, accurate and specific detection and quantification of a broad range of pedv strains and one that was completely validated according to french norm nf u47-600. many methods have been developed and used for pedv detection and quantification as previously reviewed (diel et al., 2016) such as direct viral isolation, but it is laborious, time consuming, and requires a reliable model for all possible strains. furthermore, many pedv strains cannot be isolated in vitro. many immuno-assay tests have been developed to detect viral proteins (ifa, blotting, elisa) but all these methods are time consuming, have a low sensitivity and reaction, and are subject to cross reactivity decreasing the specificity. for these reasons the current study focused on developing and validating a specific and rapid diagnostic test for the detection of pedv viral rna. basing this test on a taqman® multiplex rt-qpcr, published by kim et al. (2007) , we developed and validated a sybr tm green one-step rt-qpcr method. the development and validation of the complete method, including the steps of sample preparation, rna extraction, and rt-qpcr, were done according to the french standard nf u47-600. this norm is an adaptation to the french context of the manual of diagnostic tests and vaccines for terrestrial animals (international office of epizootics, 2018) and respects the criteria stated by the world organization for animal health (oie). these standards describe the validation criteria for a pcr method in animal health and allows the characteristics not only of rt-qpcr to be determined, but also of the complete method, including sample preparation and extraction. for this, fecal and jejunum samples were used as this material has previously been described as the best matrices for detection of pedv rna in animals (gallien et al., 2018a) . validating the complete method in this way means that the method is applicable for both experimental and diagnostic purposes. in the current study the primers used by kim et al. in 2007 were refined by in silico analysis. n orf alignments of the priming site showed that the pednf forward primer of kim et al. (2007) had mismatches with several different pedv n orfs and that the last three nucleotides at the 3' end only matched with 3.6% of the sequences. removing these three nucleotides in primer mpednf allowed a 100% match with 97.9% of international sequences and with 96.4% of european strains. the method using the new coupled primers demonstrated sufficient sensitivity to detect all tested pedv strains (historical, s-indel and s-non-indel strains). although sybr tm green pcrs are characteristically less specific than probe based pcrs, the specificity of the method was 100% against all viral types tested. primer dimer formation, which are problematic for fluorescent dye based methods as they interfere dramatically with quantification, were eliminated by optimizing the primer concentration to 300nm. during validation, the sample preparation and rna extraction step were optimized by the addition of a proteinase k treatment step which allowed the statistical bias to be maintained in acceptable limits (<0.25log10). the statistical bias obtain with the proteinase k treatment confirms a correct reproducibility at all quantification points, and guarantees a near or equivalent lod (50 and 100 copies/5µl for feces and jejunum) for the different matrices than for the transcribed rna (50 copies/5µl). in addition, the detection limit determined in this study (10 0.01 tcid50/ml) is very similar to other rt-qpcrs (10 0.03 tcid50/ml) (miller et al., 2016) . in conclusion, many pcrs have been developed to detect and monitor the presence of pedv, but, as yet to the authors' knowledge none have been developed with a complete validation according to a norm such as the french nf u47-600. this fully validated method is the first of its kind for pedv and should help harmonize detection and quantification of pedv viral rna in both field and experimental settings. nucleotide probabilities at each position are shown as coloured text above the alignments. red text in the alignment sequences represent a mismatch. sequences of primers are shown above the alignment (pednf, mpednf or pednr). pednr is shown as reverse complement. each line represents a hybridization sequence, the number of strains presenting this sequence is indicated to the left of the sequence. j o u r n a l p r e -p r o o f afnor nf u47-600-1 méthodes d'analyse en santé animale -pcr (réaction de polymérisation en chaîne) -partie 1 : exigences et recommandations pour la mise en oeuvre de la pcr en santé animale afnor nf u47-600-2 méthodes d'analyse en santé animale -pcr (réaction de polymérisation en chaîne) -partie 2 : exigences et recommandations pour le développement et la validation de la pcr en santé animale porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus virus-like particles associated with porcine epidemic diarrhoea porcine epidemic diarrhea virus among farmed pigs experimental infection of pigs with a new porcine enteric coronavirus, cv 777 porcine epidemic diarrhea virus: an overview of current virological and serological diagnostic methods a table for the calculation of working probits and weights in probit analysis better horizontal transmission of a us non-indel strain compared with a french indel strain of porcine epidemic diarrhoea virus evidence of porcine epidemic diarrhea virus (pedv) shedding in semen from infected specific pathogen-free boars limited shedding of an s-indel strain of porcine epidemic diarrhea virus (pedv) in semen and questions regarding the infectivity of the detected virus complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france porcine epidemic diarrhea in europe: in-detail analyses of disease dynamics and molecular epidemiology propagation of the virus of porcine epidemic diarrhea in cell culture manual of diagnostic tests and vaccines for terrestrial animals : (mammals, birds and bees) beitrag zur kollektiven behandlung pharmakologisher reihenversuche mafft multiple sequence alignment software version 7: improvements in performance and usability multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy evaluation of two real-time polymerase chain reaction assays for porcine epidemic diarrhea virus (pedv) to assess pedv transmission in growing pigs first detection, clinical presentation and phylogenetic characterization of porcine epidemic diarrhea virus in austria outbreak of porcine epidemic diarrhea in suckling piglets complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium ggseqlogo: a versatile r package for drawing sequence logos changes to virus taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses the authors wish to thanks ms. cherbonnel-pansart for her help with afnor validation methodology, phd. le guyader for the furniture of the mengovirus and phd p.gauger for the s-indel strain furniture. this work was partially funded by "direction générale de l'alimentation" of the french ministry of agriculture (project n°2014-145). key: cord-330825-apfcql4m authors: paraguison-alili, rubigilda; domingo, clarissa yvonne j. title: phylogenetic tracking of current porcine epidemic diarrhea virus (pedv) strains in the philippines date: 2016-06-28 journal: arch virol doi: 10.1007/s00705-016-2938-0 sha: doc_id: 330825 cord_uid: apfcql4m to trace the possible route of introduction of porcine epidemic diarrhea virus (pedv), the phylogenetic relationships of pedv strains in the regions of epidemicity in the philippines to pedv strains that are endemic in other countries were investigated. partial nucleotide sequences of the s1 spike gene was determined from the pedv-positive samples and compared with s1 sequences from other countries. phylogenetic analysis indicated that pedv strains in the philippines segregate into two groups. members of group 1 are related to strains from the usa, taiwan, japan and canada, while those in group 2 are related to strains from china and vietnam. porcine epidemic diarrhea (ped) is an infectious enteric disease that poses a threat to the swine industry and is characterized by acute enteritis and diarrhea, with the infection causing more-severe disease in piglets. the causative agent of ped is porcine epidemic diarrhea virus (pedv), which belongs to group 1a of the genus coronavirus, family coronaviridae, order nidovirales. pedv is an enveloped rna virus with a single-stranded positivesense rna genome approximately 28 kb in size that contains six genes: replicase (rep), spike (s), orf3, envelope (e), membrane (m), and nucleoprotein (n). the full-length s gene is about 4.1 kb and consists of the s1 and s2 domains of approximately 2.2 and 1.9 kb in length, respectively. since the receptor-binding sites and majority of the neutralization epitopes are located in the s1 portion, this region has been subjected to sequencing and molecular analysis to determine the genetic relatedness of different pedv viruses [1] [2] [3] . the current study characterizes the spike s1 portion of the virus. the s protein has a crucial role in viral and cell fusion activity and in inducing a host immune response [4] [5] [6] . the spike protein makes up the surface projections of the virus and plays a major function in interaction with cell receptors [4] . the sequence of the full-length s gene, the s1 portion, or the s2 portion reflects the genetic diversity of the virus. the s gene, particularly the s1 portion, is a highly variable region and is appropriate for sequencing to study the genetic relatedness and molecular epidemiology of pedv [1] . ped may occur year round, with peak incidence during the cold season, and it occurs in herds that have been exposed to the virus and have developed partial immunity. pigs from 1 week of age are usually affected, and those that survive ped can be carriers for up to 104 days. infected sows may be febrile, with occasional abortion, but they usually recover within 5-10 days. in the philippines, the first case was reported in 1975 and was identified as a tge-like virus on a farm of 800 sows in pampanga. recorded outbreaks occurred in january 2005 in pampanga, tarlac, pangasinan, batangas, cavite, laguna, quezon and negros occidental. sporadic and isolated outbreaks were recorded every year thereafter. through 2016, outbreaks have been reported particularly in the provinces of bulacan, rizal, pampanga, tarlac, pangasinan, batangas, cavite, laguna, bacolod and general santos, with severe cases in adult animals. the mortality rates due to ped were as follows: birth to 7 days old, 80 % to 100 %; 8 to 14 days old, 40 % to 50 %; 15 to 21 days old, 20 % to 25 %; 22 days or older, rare. the mortality decreases as age increases. morbidity is high in growers and finishers, but mortality is low, and affected pigs may be stunted. in may to august 2010, an outbreak of ped occurred in the province of batangas, killing 67 % (11,414 head) of 17,115 sick pigs, resulting in huge economic loss [7] . linkages with swine farms in the provinces of pampanga, tarlac, batangas and agusan were established. using data from swine farms in these provinces and diagnosis data from the college of veterinary science and medicine in central luzon state university, the incidence is 67 % in pampanga, 79 % in tarlac, 61 % in batangas, and 90 to 100 % in agusan. the overall prevalence of ped is 65 %, 73 % on smallholding farms and 57.5 % on commercial farms. during the wet season of 2015, from july to october, swine farms from the provinces of agusan, batangas, pampanga and tarlac, which are known to experience ped outbreaks, were requested to provide clinical samples such as intestines, fecal samples, fecal swabs and environment swabs whenever there were reported cases. selection of commercial and small swine farms was done in coordination with provincial veterinarians. reports on cases of gastrointestinal infections with symptoms of diarrhea were obtained from veterinary offices. a list of affected farms was obtained and used in constructing the sampling frame. rna was isolated using trizol reagent (invitrogen usa) according to manufacturer's recommendations. reverse transcription pcr was performed using a onestep rt-pcr kit (qiagen germany). a pair of primers was designed to specifically amplify a portion of the s1 region: fs1m2 (gtc atg gca ctg acg atg atg ttt c) and peds1r (cag atg tgt aat aaa cac ctg cca a). thermocyling conditions were as follows: 45°c for 30 minutes and 95°c for 10 minutes for the rt reaction, followed by 35 cycles of amplification at 94°c for 30 seconds, 60°c for 30 seconds, and 72°c for 1 minute, with a final extension at 72°c for 7 minutes. good-quality rt-pcr products from samples that were positive for pedv were selected and processed for dna sequencing: batangas, 1 sample; tarlac, 5; pampanga, 8 and agusan, 2. a total of 16 pedv-positive samples were processed for dna sequencing of the 753-bp portion of the s1 gene. genbank accession numbers for pedv strains or clones from other countries are listed in table 1 . the partial s1 gene sequences of pedv were aligned with those of strains from other countries, using clustalw. a phylogenetic tree for the 753-bp gene segment was constructed using the maximum-likelihood method with the general time-reversible nucleotide substitution model. the confidence level for each branch was tested by the bootstrap method with 1,000 replicates. phylogenetic and molecular evolutionary analysis was conducted using mega version 7.0.14 software [8] . this is the first report of pedv s1 gene sequences from the provincial pedv hotspots of the philippines, which include batangas, pampanga, tarlac and agusan. a phylogenetic tree was created by the maximum-likelihood method with 1,000 bootstrap replicates, using the mega7.0.14 software [8] . interestingly, sequence analysis of the s1 gene from current pedv strains from the philippines revealed that these current strains cluster into two groups. viruses in group 1 share common phylogenetic roots with strains from the usa, taiwan, japan and canada, while those in group 2 were more closely related to strains from china and vietnam. pedv identified on swine farms in pampanga, tarlac (central luzon), batangas (southern luzon), and agusan (mindanao) were clustered within group 1. interestingly, other samples from central luzon, particularly pampanga and tarlac, clustered separately within group 2 (fig. 1) . it has been reported that all us pedv strains form a tight cluster and are most closely related to a strain isolated in 2012 in china (ah2012) [9] , while philippine strains are closely related to vietnamese pedvs in an orf3 genetic tree [10] . in another study, comparative analysis of fulllength sequences of the whole genome revealed that isolates from germany show very high nucleotide sequence similarity to strain oh851, which was found in the united states in 2014 [11] . phylogenetic analysis of the complete pedv genome also indicated that 38 japanese strains, table 1 reference sequences from ncbi genbank, described by strain or clone names and accession numbers including two novel pedv variants, were closely related to strains that were widespread in the united states and korea in 2013-2014 [12] . moreover, sequencing and phylogenetic analysis of the spike gene and orf3 of pedv revealed that the prevailing pedv isolates in japan had the greatest genetic similarity to us isolates [13] . in taiwan, phylogenetic analysis of the s gene showed that all pedv strains from taiwan were closely related to the non-s indel strains from the usa, canada and china [14] . taken together, recent reports are consistent with our findings that the philippine pedv strains share common ancestors with those from the usa, taiwan, japan and canada (group 1) as well as those from china and vietnam (group 2). pedv is present in many countries in asia and has been present in europe since the 1970s. it was first discovered in europe, but it has become increasingly problematic in asian countries such as korea, china, japan, the philippines, and thailand. it has also spread to north america and was discovered in 2013 in indiana, usa [14] , and in canada in the winter of 2014. in january 2014, a new variant strain of pedv with three deletions, one insertion, and several mutations in the spike 1 region was identified in ohio by the animal disease diagnostic lab of the ohio department of agriculture [15] . findings presented in this paper provide important information regarding the route of introduction of pedv in the philippines and provide linkages to other countries. the emergence of different pedv strains can occur via introduction of the virus from overseas, since this virus is versatile and can be easily spread. our study also detected pedv contaminants from inanimate objects such as swabs of floor pens and farrowing crates, fan blades of tunnel vent farms, raw feed ingredients, and inner surfaces of empty feed sacks. hence, proactive disease control measures should include the identification of the source of imported goods for pigs, which may require thorough biosecurity procedures to limit the spread of the agent and the outbreak. acknowledgments this project was funded by the philippine council for agriculture, aquatic, and natural resources research and development (pcaarrd). grateful acknowledgments to our colleagues at the college of veterinary science and medicine, central luzon state university for helpful advice. we thank the veterinarians for submitting the clinical samples from the different provinces for pedv screening. we also thank the division of bioresources hokkaido university for dna sequencing of the rt-pcr products. above all, to god for the wisdom and knowledge. conflict of interest the authors whose names are listed below certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria, educational grants, participation in speakers' bureaus, membership, phylogeny was based on partial spike s1 gene sequences. evolutionary history was inferred using the maximumlikelihood method based on the kimura 2-parameter model [16] . the initial tree for the heuristic search was generated automatically by applying neighbor-join and bionj algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (mcl) approach. evolutionary analysis was conducted using mega7 [8] employment, consultancies, stock ownership, or other equity interest, and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. we have no conflict of interest. statement of human rights all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. statement on the welfare of animals all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. informed consent informed consent was obtained from all individual participants included in the study. pathogenesis comparison between the united states porcine epidemic diarrhea virus prototype and s-indel-variant strains in conventional neonatal piglets veterinary virology: the third edition characterization of the structural proteins of porcine epizootic diarrhea virus, strain cv777 the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus lipa city veterinary office mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets phylogenetic analysis of the spike (s) gene of the new variants of porcine epidemic diarrhoea virus in taiwan molecular characterization of the spike and orf3 genes of porcine epidemic diarrhea virus in the philippines comparison of porcine epidemic diarrhea viruses from germany and the united states molecular characterization of pig epidemic diarrhoea viruses isolated in japan from us-like isolates of porcine epidemic diarrhea virus from japanese outbreaks between origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states american association of swine veterinarians presentation to the 2014 pork management conference a simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences key: cord-301355-9lswjro2 authors: fan, jing-hui; zuo, yu-zhu; shen, xiao-qiang; gu, wen-yuan; di, jing-mei title: development of an enzyme-linked immunosorbent assay for the monitoring and surveillance of antibodies to porcine epidemic diarrhea virus based on a recombinant membrane protein date: 2015-12-01 journal: j virol methods doi: 10.1016/j.jviromet.2015.07.021 sha: doc_id: 301355 cord_uid: 9lswjro2 the recent dramatic increase in reported cases of porcine epidemic diarrhea (ped) in pig farms is a potential threat to the global swine industry. therefore, the accurate diagnosis, serological monitoring, and surveillance of specific antibodies in pigs resulting from porcine epidemic diarrhea virus (pedv) infection or vaccination would be essential in helping to control the spread of ped. we developed and validated an indirect enzyme-linked immunosorbent assay (elisa) based on the recombinant membrane (m) protein of pedv. to detect pedv antibodies in eight herds, 382 serum samples were collected from sows that had been immunized with a ped vaccine, and screened using the developed elisa in parallel with a serum neutralization (sn) assay. of the tested samples, 276 were positive for the presence of pedv antibodies according to both assays, while 98 were negative. an excellent agreement between the elisa and the sn assay was observed (kappa = 0.947; 95% confidence interval = 0.910–0.984; mcnemar's test, p = 0.727). no cross-reaction was detected for the developed elisa with other coronaviruses or other common pig pathogens. the developed elisa could be used for serological evaluation and indirect diagnosis of ped infection. porcine epidemic diarrhea (ped) is a devastating swine disease and its etiological agent is ped virus (pedv). porcine epidemic diarrhea is characterized by watery diarrhea, dehydration, and a high death rate among suckling pigs (pensaert and debouck, 1978) . the disease was first documented in the united kingdom in 1971 as a swine disease that resembled transmissible gastroenteritis (oldham, 1972) . outbreaks of ped have since been reported in europe, asia, and north america (nagy et al., 1996; takahashi et al., 1983; sun et al., 2011; fan et al., 2012; stevenson et al., 2013) . porcine epidemic diarrhea virus has spread rapidly across the united states, resulting in the high mortality of piglets, and substantial economic losses (cima, 2013) . its recent emergence in north america suggests that this virus is a threat to the swine industry worldwide. accurate diagnosis and serological detection of specific antibodies in pigs as a result of pedv infection or vaccination are required to control the spread of ped. porcine epidemic diarrhea virus is a coronavirus within the coronaviridae family. the genome of pedv contains genes encoding spike (s), membrane (m), small membrane (sm), open reading frame 3, and nucleocapsid (n) proteins (park et al., 2011) . the m protein is a structural membrane glycoprotein, and the most abundant of all the envelope proteins. it has a short amino-terminal domain that exists outside of the virion, with the long carboxyterminal domain of the protein present inside the virion (utiger et al., 1995) . immune reactions to the m protein of coronaviruses play an important role in the induction of protection, and in mediating the course of the disease (fleming et al., 1989; vennema et al., 1991) . the nucleotide sequence of the pedv m gene exhibits low homology (around 50%) with the m gene of other coronaviruses; however, its sequence is highly conserved among different pedv strains arndt et al., 2010) . therefore, the m protein could be a suitable candidate for the detection of pedvspecific antibodies and in diagnosing pedv infections. many enzyme-linked immunosorbent assays (elisas) have been developed for the detection of antibodies against pedv. however, the preparation of an appropriate antigen for most of these methods requires cultivation of pedv, which is time consuming and expensive. in the current study, we expressed and purified the m protein of pedv. was used as a coating antigen in an indirect elisa that we developed. we then screened serum samples from pigs to determine the presence of antibodies specific for the pedv m protein. we collected 36 blood samples from healthy unvaccinated pedv-free pigs of various ages. the sera derived from these samples were negative for the presence of antibodies against pedv according to serum neutralization (sn) assays (sn titers <1:2). the sera from 20 blood samples, taken from pedv-infected pigs, were used as reference positive sera (sn titers ≥1:16). the pedv infection status of pigs was confirmed by reverse transcription polymerase chain reaction (rt-pcr) assays targeting the pedv m gene, with viral rna extracted from fecal samples. porcine sera containing antibodies against transmissible gastroenteritis virus (tgev; sn titer 1:413), porcine rotavirus (prv; 1:305), porcine reproductive and respiratory syndrome virus (prrsv; 1:376), porcine circovirus 2 (pcv-2; 1:317), and classical swine fever virus (csfv; 1:289) were obtained from the hebei center for disease prevention and control (shijiazhuang, people's republic of china). a total of 382 serum samples were collected from eight pig herds, in which the animals had been administered a pedv vaccine. all samples were tested by the developed indirect elisa. porcine epidemic diarrhea virus strain hb/bd (genbank accession no. jf690777.1) was isolated from the feces of a pig from hebei province, china, and adapted to cell culture for 32 passages in vero cells. viral rna was extracted from pedv using trizol reagent (invitrogen, carlsbad, ca, usa), following the manufacturer's instructions. the sequence corresponding to the m gene was amplified from viral rna by rt-pcr using oligonucleotide primers mp1 (5 -gga tcc atg tct aac ggt-3 ) and mp2 (5 -aag ctt tct gtt tag act aaa t-3 ). the resulting pcr products were separated by agarose gel electrophoresis and purified using a gel extraction mini kit (tiangen biotech co. ltd., beijing, china) according to the manufacturer's recommendations. purified amplicons were cloned into the pmd18-t vector (takara biotech co. ltd., dalian, china) to yield the recombinant plasmid pmd-m, which was then transformed into competent escherichia coli jm109. positive clones were selected according to their lacz phenotype, and verified by restriction enzyme digestion, pcr screening, and dna sequencing. the m gene contained within pmd-m was subcloned into the prokaryotic expression vector pgex-6p-1 (sunbiotech inc., beijing, china) to yield pgex-6p-m. the pgex-6p-1 vector also contained a sequence for a gst tag, which was designed so as to be added at the amino terminal of the expressed protein. the pgex-6p-m vector was verified by dna sequencing. the recombinant m protein was transformed into e. coli bl21 (tiangen biotech co. ltd., beijing, china). e. coli bl21 cells were cultured and transformed with pgex-6p-m, in luria-bertani broth supplemented with 100 g/ml ampicillin until the optical density at 600 nm (od 600 ) was 0.6-1.0, at which point protein expression was induced via the addition of 1.0 mm isopropyl-␤-d-thio-galactopyranoside. at 5 h post-induction, bacterial cells were collected by centrifugation. the recombinant protein was purified from the bacterial lysate using a gst fusion protein purification kit (transgen biotech co. ltd., beijing, china), according to the manufacturer's recommendations. fractions of purified recombinant m protein were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), with polyacrylamide gels stained with coomassie brilliant blue r-250. expression of the recombinant m protein was confirmed by immunoblotting, using porcine pedv antisera. the prokaryotic expression vector pgex-6p-1 was also transformed into e. coli bl21 as a negative control. fractions of purified recombinant proteins were separated by sds-page and electrophoretically transferred to nitrocellulose membranes (gibco brl, gaithersburg, md, usa). to prevent nonspecific reactions, membranes were blocked with 5% (w/v) non-fat milk powder in tris-buffered saline containing 0.05% (v/v) tween 20 (tbst) at 37 • c for 2 h. membranes were incubated with pig pedv antisera (diluted 1:200) at 37 • c for 1 h. after three washes with tbst, membranes were incubated with an anti-pig igg conjugated to horseradish peroxidase (hrp; sigma-aldrich, st louis, mo, usa) at 37 • c for 1 h. we used 3,3 -diaminobenzidine (sigma-aldrich) for the development of color reactions to visualize protein bands. a checkerboard titration involving each combination of antigen (purified recombinant pedv m protein) and sera was used to determine optimal dilutions for use in the indirect elisa we developed. the antigen and serum were diluted from 30 to 0.938 g/ml and 1:10 to 1:320 (two-fold dilution), respectively. ninety six well plates (nunc maxisorp, denmark) were coated with the recombinant m protein, which was diluted in phosphate-buffered saline (pbs), and allowed the plates to incubate at 4 • c overnight. pbs was used as a blank control. wells were blocked with 5% fetal bovine serum in pbs (ph 7.4) for 1 h at 37 • c. wells were then washed three times with pbs containing 0.05% tween 20 (pbst) and diluted serum samples were added, in duplicate, to the antigen-coated wells. following incubation at 37 • c for 1 h, plates were washed three times with pbst and 100 l of diluted goat anti-pig igg conjugated to hrp (kpl inc., gaithersburg, md, usa) was added to each well. after incubation at 37 • c for 45 min, wells were washed with pbst, and 100 l of 3,3 ,5,5 ,-tetramethyl-benzidine (tmb) was added to each well and the plates were incubated for 15 min at room temperature. color development reactions were stopped with 100 l of 2 m h 2 so 4 . the od 450 was determined for each well using a microplate reader. dilutions that resulted in the highest od 450 ratio between the positive and negative serum samples (p/n value), along with an od 450 value for positive serum samples close to 1.0, were considered optimal working conditions. to establish negative and positive cutoff values for this assay, 36 pedv-negative serum samples, as determined by sn assays, were tested in triplicate using the elisa we developed. a cutoff value was defined as the mean absorbance value plus three standard deviations (sd). repeatability assays were conducted by comparing the ratios of od 450 values for triplicate results from each serum sample tested on the same plate (intra-assay repeatability) or on different plates (inter-assay repeatability). the coefficient of variation (cv) was calculated according to the following formula: where x was the average od 450 for the field serum samples. a cv value less than 10% was considered an acceptable level of variation. to determine the specificity of the developed elisa, crossreactivity was assessed by determining the reactivity of the purified antigen with serum samples containing antibodies against tgev, prv, prrsv, pcv2, csfv and pedv. reference serum samples were diluted 1:40, with three replicates of each dilution tested using the indirect elisa. serum samples from pedv-infected pigs (n = 10) were selected at random to determine the sensitivity of the indirect elisa. samples were serially diluted two-fold from 1:2 to 1:4096. eight replicates of each diluted serum sample were tested to determine the titers of antibodies in samples. the titers were also determined by sn. the indirect elisa that we developed was used to evaluate the presence of pedv antibodies in 382 serum samples from sows with varying immune status. these serum samples were also subjected to sn assays. the level of agreement between the sn and elisa assay was determined using a cohen's kappa, and a mcnemar's test was performed to investigate the differences between both assays. a p-value < 0.05 was considered significant. all analyses were performed in spss version 22.0. the m gene (approximately 680 bp) of pedv was successfully amplified and cloned it into a ta vector. positive clones were identified by sequencing. sequence analysis indicated that the pedv m gene was 681 bp, and encoded 227 amino acids. the complete nucleotide sequence of the m gene has been deposited in genbank (accession number jf690777). recombinant pedv m gene was expressed in e. coli bl21. analysis of the bacterial cell lysate by sds-page and western blotting revealed a prominent band of around 53 kda. the recombinant protein we identified was approximately 26 kda heavier than the predicted molecular weight of 27 kda, which was due to the gst tag (fig. 1) . the recombinant protein was predominantly in the insoluble fraction of the bacterial cell extract, and purified using a gst fusion protein purification kit. western blotting analysis showed that the purified protein was recognized by pedv antiserum (fig. 2) . the optimum antigen concentration and serum sample dilution were determined to be 3.75 g/ml and 1:40, respectively. the optimum dilution of the anti-pig igg conjugated to hrp was found to be 1:4000. the incubation conditions for primary and secondary antibodies were 1 h at 37 • c and 45 min at 37 • c, respectively. the optimum blocking buffer was found to be 5% fetal bovine serum in pbs, while pbst was the most appropriate washing buffer. the detection threshold of our indirect elisa was determined using 36 serum samples that were negative for the presence of pedv antibodies. the cutoff point was specific for each individual assay and was based on the od 450 of negative samples included in each assay. the mean od 450 value for the elisa was 0.192, with an sd of 0.054. the cutoff value was determined to be 0.354 (mean + 3sd); therefore the negative-positive threshold was set at 0.35. serum samples returning an elisa od 450 value below table 1 cross-reaction analysis of the m-based i-elisa with anti-sera against other pig viruses: od value (mean ± 3sd). pedv, tgev, prv, pcv-2, csfv, prrsv represent pig sera that were positive for pedv, tgev, prv, pcv-2, csfv, prrsv, respectively. 'noninfected' represents the serum from a pig that was negative for pedv. 0.354 were regarded as negative; samples that returned an od 450 value higher than this were considered to be seropositive for pedv antibodies. reproducibility within and between elisa plates was evaluated by testing ten sn-negative serum samples and ten sn-positive serum samples in triplicate. the inter-assay cv of the indirect elisa ranged from 4.7% to 7.3% with a median value of 5.8%. the intraassay cv ranged from 5.4% to 8.9%, with a median value of 6.3%. these results indicate that the indirect elisa we developed to detect antibodies against the pedv m protein was repeatable, with low and acceptable levels of variation. the specificity of our elisa was evaluated by testing the reactivity of the pedv m protein antigen with antibodies against pedv, tgev, prv, prrsv, pcv-2, and csfv. we found that pedv antisera significantly reacted with the pedv m antigen, as expected. in contrast, the od 450 values for all other serum sample containing antibodies against other porcine viruses were significantly lower than the established negative cutoff value (table 1) . our results suggest that little or no cross-reactivity occurs between the pedv m protein and antibodies against other porcine viruses, and that the m protein antigen was specific for antibodies against pedv. we evaluated the sensitivity of our indirect elisa using ten pedv-positive serum samples, and found that we could detect pedv antibodies in a serum sample that had been diluted out to 1:2048, while only 1:532.5 in sn. this result indicated that our indirect elisa was more sensitive than the sn assay for pedv antibody detection (table 2) . of the 382 serum samples collected from animals with a known immunization history across eight pig farms, 281 (73.6%) were positive for pedv antibodies according to our elisa, while 101 (26.4%) were pedv antibody-negative. in comparison, according to the sn assay we used in parallel with the indirect elisa, 73% (279/382) of samples were pedv antibody-positive and 27.0% (103/382) of samples were antibody-negative (table 3) . according to both assays, 276 and 98 of the screened serum samples were pedv antibody-positive and -negative, respectively. three of the serum samples that were positive according to the sn assay, were porcine epidemic diarrhea virus is a member of the coronaviridae, and is known to cause fatal diarrhea in newborn piglets. the virus was first identified in 1978 (pensaert and debouck, 1978; chasey and cartwright, 1978) and has since been found to be prevalent in many countries. infection with pedv has resulted in significant economic losses, mainly in europe and asia, and recently the usa (fan et al., 2012; chae et al., 2000; martelli et al., 2008; stevenson et al., 2013) . although there are commercial vaccines available to prevent and control ped in china, the damage caused by pedv is significant and the threat continuous. to effectively control and prevent this disease, detection methods that can assess the current epidemic situation in herds are required. in addition, for subsequent immunoprophylaxis, assays that can monitor serum antibody levels of immunized or infected pigs need to be developed. before local immunity is actively established, the piglet intestine is protected against pedv infection by maternal antibodies. although the presence of antibodies in serum is not directly related to the protection for sows or for piglets, serological examination facilitates the assessment of humoral response to pedv elicited either through vaccination or natural infection. porcine epidemic diarrhea virus was isolated for the first time in 1988 using the vero cell line and trypsin-supplemented medium (hofmann and wyler, 1988) . various diagnostic methods have been described for the detection of pedv antigen and pedv antibodies (carvajal et al., 1995; knuchel et al., 1992; kweon et al., 1997; song et al., 2006) . at present, serological diagnostic methods for the diagnosis of pedv infection are more common. given that elisas are simple, sensitive, and convenient serological detection methods, a number of commercial and in-house elisas have been developed to detect pedv antigens, or antibodies against pedv (song and park, 2012) . the majority of in-house assays for the detection of antibodies against pedv are based on using the whole virus as an antigen (carvajal et al., 1995; oh et al., 2005) , or preparations of viral antigen (knuchel et al., 1992) . the cultivation of pedv is laborious and time consuming, especially considering the bio-security measures that must be taken in handling this virus; therefore it is difficult to upscale the production of these serological assays. the use of a recombinant viral protein as an antigen would allow researchers to avoid some of the problems associated with the large-scale preparation of native pedv antigen. recently, an elisa based on a recombinant n protein was validated in china (hou et al., 2007) . however, these researchers did not assess the cross-reactivity of the elisa with other coronaviruses. the m protein of pedv protrudes from the viral envelope, and is considered a superior diagnostic antigen compared with other pedv proteins (shenyang et al., 2007) . in the current study, we chose the pedv m protein for use as an elisa antigen because it is highly conserved among pedv strains, and because it can elicit the formation of protective antibodies (zhang et al., 2012) . we expressed the m protein in e. coli and confirmed its antigenicity using pedv-specific antibodies. our results indicated that the expressed recombinant m protein was indeed antigenic, and could feasibly be used as a coating antigen in an indirect elisa. the indirect elisa that we developed exhibited low levels of variability among replicates, according to intra-and inter-assay tests. these minor variations in results indicated that the indirect elisa was reproducible. furthermore, this elisa was able to detect pedv antibodies in serum samples that had been diluted out to 1:2048, and exhibited no cross-reactivity to antibodies against other common pig pathogens. these findings indicated that the developed indirect elisa could be used widely in the future. vaccination is one of the most effective techniques in controlling ped in china. however, since late 2010, ped has been reemerging in immunized swine herds with devastating impact. to confirm the effects of targeted immunoprophylactic measures, we used our indirect elisa to screen serum samples from vaccinated pigs. we found that 73.6% and 73% of samples were seropositive according to elisa and sn assay, respectively. this indicated that vaccination elicited neutralizing antibodies against pedv in sows to some degree. however, 26.4% and 27% of samples from vaccinated pigs were seronegative by the elisa and sn assays, respectively. although these antibodies could not be used to assess protection against pedv, these vaccinated, but seronegative sows are not able to vertically transmit effective antibodies to their neonates. it indicating that the vaccine used requires some improvement in antigenicity. a comparison of the elisa and sn assay results revealed eight differences between positive and negative samples. there were five elisa-positive samples that were negative according to the sn assay we used, which could be explained by the higher sensitivity of the elisa. the reason for the elisa-negative results for the three sn-positive serum samples might be due to errors inherent in the tests that we applied. in conclusion, this is the first report of an indirect elisa using the recombinant pedv m protein as a coating antigen for the detection of antibodies against pedv in china. the developed assay is quick, convenient, and not labor-intensive, and could facilitate the development of a reliable tool or kit for the large-scale detection of antibodies against pedv. the authors have no 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supported by the research foundation of the education bureau of hebei province, china (no. 2011180). key: cord-280183-fxhkfjl6 authors: have, p.; moving, v.; svansson, v.; uttenthal, å.; bloch, b. title: coronavirus infection in mink (mustela vision). serological evidence of infection with a coronavirus related to transmissible gastroenteritis virus and porcine epidemic diarrhea virus date: 1992-04-30 journal: veterinary microbiology doi: 10.1016/0378-1135(92)90135-g sha: doc_id: 280183 cord_uid: fxhkfjl6 abstract antibodies to a transmissible gastroenteritis virus (tgev)-related coronavirus have been demonstrated in mink sera by indirect immunofluorescence, peroxidase-linked antibody assays and immunoblotting. this is the first serological evidence of a specific coronavirus infection in mink. the putative mink coronavirus (mcv) seems to be widespread in the danish mink population with a prevalence approaching 100%. analysis by immunoblotting has shown that mcv is closely related to tgev by the spike (s), matrix (m) and nucleoprotein (n) polypeptides. furthermore, antibodies to mcv also cross-reacted with n and m polypeptides of porcine epidemic diarrhea virus (pedv). thus mcv may occupy an intermediate position between the tgev group of coronaviruses and pedv. the possibility that mcv may be associated with syndromes of acute enteritis in preweaning mink is discussed. coronaviruses infect a number of mammalian and avian species and the mammalian viruses are usually placed in two major serological groups represented by mouse hepatitis virus (mhv) and transmissible gastroenteritis virus (tgev) (sturman and holmes, 1983, siddell et al., 1983) . the mhv group includes bovine coronavirus (bcv), porcine hemagglutinating encephalomyelitis virus (hev) and human coronavirus (hcv) oc-43. the tgev group includes porcine respiratory coronavirus (prcv) (pensaert et al., 1986 (pensaert et al., , s~inchez et al., 1990 , canine coronavirus (ccv), feline infectious peritonitis virus (fipv) and feline enteric coronavirus (fecv) . mature virions of coronaviridae contain three major structural proteins (cavanagh et al., 1990) of which the spike (s) glycoprotein and transmembrane matrix (m) glycoprotein are glycosylated. the nucleoprotein (n) is associated with the positive-stranded rna genome. coronaviruses are responsible for various disease manifestations in their host species, depending on virus tropism (e.g. pneumotropic, enterotropic or neurotropic viruses ). porcine epidemic diarrhea coronavirus (pedv) (pensaert et al., 1981 ) , causing a gastroenteritis similar to tge in pigs, is as yet unclassified, although a slight serological relationship with fipv has been demonstrated (yaling et al., 1988 ) . until recently, no known cell culture system was available for propagation of pedv in vitro but this has now been accomplished using vero cells and medium including proteases such as trypsin (hofmann and wyler, 1988 ) . the apparent mr values of pedv structural proteins have been found to be 85 000-135 000k for the s protein, 58 000 for the n protein and 20 000-32000 for the m protein . this serological study on mink coronavirus was initiated by our observations in electron microscopy of coronavirus-like particles in stool samples from neonatal mink kits suffering from diarrhea. specific coronavirus infections have so far not been described in mink but coronavirus-like particles in feces have recently been associated with epizootic catarrhal gastroenteritis in mink (gorham et al., 1990) . the present paper provides the first serological evidence for infection in mink with a coronavirus (here designated mcv) serologically related to tgev and pedv. tgev (purdue strain) and prcv (danish isolate dk1/86, have, 1991 ) were grown in a swine testicle (st) cell line (mcclurkin and norman, 1966 ) . ccv (strain 1-71 ) was grown in a-72 cells (atcc crl 1542 ). fipv (strain df2, atcc vr-2004) and fecv (strain wsu 79-1683, atcc vr 989) were grown in crandell feline kidney (crfk) cells. pedv (strain 1/87 ) was kindly provided by dr. s. cartwright, weybridge, as an intestinal passage of pedv-infected colostrum-deprived newborn piglets. after a further two passages in piglets the virus was adapted to grow in vero cells by the method of hofmann and wyler (hofmann and wyler, 1988 ) . four groups of mink sera were selected for evaluation of mcv antibodies: a, 71 mink sera were obtained during 1990 from the danish fur breeders association as part of routine sampling for plasmacytosis testing (aleutian disease, adv). b, 23 sera were obtained in 1989 from a mink farm showing a high level of neonatal mortality. c, 17 sera were obtained from mink kept for experimental purposes. d, 41 mink sera collected during 1981-1988 were examined in order to trace the history of coronavirus infection in mink. hyperimmune or convalescent porcine, canine and feline sera against tgev, prcv, ccv, fipv and pedv were included as controls. porcine sera were produced by experimental oronasal inoculation of spf pigs with tgev, prcv or pedv, respectively. canine antiserum to ccv was obtained from a recovered dog in a kennel having an acute outbreak of ccv gastroenteritis. fipv antiserum was from a cat (field case) with clinically diagnosed fip. monolayers for ifat were prepared by infecting secondary porcine kidney cells with tgev and acetone fixation after 24 h. sera were examined at a dilution of 1/2 in pbs using acetone-fixed monolayers of virus-infected cells on multitest slides. following incubation in a moist chamber for 1 h at 37 ° c, the slides were rinsed three times in pbs and incubated with fitc-conjugated protein-a (pharmacia) at a dilution of 1/50 in pbs. after incubation for 30 min at 37 °c slides were rinsed and mounted for microscopy. virus-infected cells were prepared in 96-well tissue culture microplates by mixing cell suspensions with an appropriate dilution of virus. following incubation for 24 h cells were fixed in 30% acetone in pbs for 10 min, air dried at 37°c for 1 h and stored at -20°c or used immediately (jensen, 1981 ) . monolayers were incubated with dilutions of test sera in pbs-0.1% tween 20 (pbst) for 1 h at 37°c. following washing in pbst, peroxidase-conjugated protein a (zymed) or peroxidase-conjugated anti-swine igg (dako), both diluted 1/200 in pbst, were added and incubated for 30 min at 37 °c. finally, the substrate 3-amino-9-ethylcarbazole was added and left for 30 min. serum titres were recorded as the highest dilution causing specific cytoplasmic staining. neutralization tests were carried out in 96-well microplates. sera were heatinactivated and two-fold dilutions were mixed with 100 tcidso of virus and incubated for 1 h at 37 ° c. following incubation, the appropriate cell suspension was added, plates were sealed and incubated at 37°c in a 7% co2-air mixture. neutralizing titres were evaluated after 3-4 days using the method of reed and muench ( 1938 ) . tgev, ccv or pedv culture supernatants were clarified at 3500 g in a ja 14 rotor for 20 min followed by pelleting at 20 000 g for 4 h in the same rotor. pelleted virus was separated by sds-page on a 10% gel after solubilisation at 100°c for 2 min in sample buffer (0.06 m tris-hc1, 9% glycerol, 3% sds, ph 6.8) under non-reducing conditions. electrophoresis was performed using laemmli buffers (laemmli, 1970) . proteins were transferred electrophoretically (200 ma, 90 min ) to nitrocellulose or polyvinylidene difluoride sheets by semi-dry blotting using tris-glycine-methanol buffer (60 mm glycine, 50 mm tris base, 0.03% (w/v) sds, 20% methanol) ph 8.9. strips were blocked by incubation for 1 h with 5% (w/v) skimmed milk powder in pbst. sera were diluted 1 / 100 in the same buffer and applied for 1 h on a tilt shaker. peroxidase-conjugated protein a (zymed) was diluted 1/500 in pbst and applied for 30 min. specific bands were revealed using the substrate o-dianisidine dihydrochloride (sigma, 0.3 mg/ml in pbs). initially, mink sera were examined in ifat using acetone fixed monolayers of secondary swine kidney cells infected with tgev as substrate and fitcconjugated protein-a to detect specific binding of antibodies to tgev. of 71 sera collected during 1990 for routine testing of adv antibodies, 68 were found to contain tgev cross-reacting antibodies in ifat at a dilution of 1 / 2. the pattern of fluorescence was most often diffusely cytoplasmic and very similar to that seen with sera from fipv-infected cats. the serological data on mink sera have been summarized in table 1 . all of 23 sera from a farm showing high neonatal mortality contained tgev cross-reacting antibodies in ifat at a dilution of 1/2. these sera represented adult females as well as 6 months old male and female kits. also, all of 17 sera from experimental animals were positive in ifat for tgev cross-reacting antibodies. sera obtained during 1981-1988 were titrated in tgev pla and 31 of 41 samples were positive with titres ranging from 5 to 320. positive samples were evenly distributed over the years with no obvious time-clustering. due to cytotoxicity some of the sera included in this study could not be examined in neutralizatioti test. all of the 17 sera originating from experimental animals were tested in neutralization tests against tgev and prcv (initial dilution 1 / 10), ccv, fipv and fecv (initial dilution 1/5 ) and none of the sera neutralized any of these viruses. furthermore, 20 sera ( 13/20 positive by pla) tested against tgev and prcv and 16 sera ( 16/16 positive by pla or ifat) tested against fipv and fecv were also negative for neutralizing antibodies. a high-titered mink antiserum and representative high-titered antisera against tgev, prcv, ccv, fipv and pedv were selected for further studies of the serological relationship of the putative mink coronavirus to other coronaviruses. these sera were used to develop western blots of tgev, ccv and pedv. it should be noted, that antisera to ccv and fipv are field sera and not homologous to the strains applied in this study. it is apparent, that antisera against tgev, prcv, ccv and mcv all react strongly with all three major structural proteins s (200k), m (27k) and n (47k) of tgev (fig. 1) . a fourth polypeptide (14-15k) is revealed by antiserum to tgev only. in contrast, fipv antiserum reacts primarily with n and m of tgev. antiserum against pedv reacts weakly with s and n of tgev. a similar pattern was found when using ccv instead of tgev as antigen (fig. 2) except that staining of the s protein was only evident with ccv antiserum. in parallel experiments weak staining of the s protein of ccv has been observed with tgev, prcv and mcv antisera (not shown). antiserum against pedv reacts with s, m (28k) and n (55k) of pedv whereas antisera against tgev, prcv, ccv and fipv barely reacts with n of pedv (fig. 3 ) . mink antiserum occupies an intermediate position in that there is a distinct reaction with both m and n of pedv. by cross-neutralization tgev, prcv, ccv, fipv and fecv were found to be very similar, all being neutralized by antisera to tgev, prcv and ccv (table 2) . on the other hand antisera to fipv, pedv and mcv did not neutralize any of the viruses. in particular, antiserum to fipv did not neutralize the strains of fipv and fecv applied in this study. neutralization of pedv was not attempted, due to the extreme dependance of replication on trypsin. the reactivity of these sera was also tested in pla against tgev, fecv/ fipv and pedv (table 3 ). the titres of antisera against tgev, prcv and ccv are higher against tgev and fecv/fipv than against pedv. antiserum against fipv and mcv are broadly reacting with tgev, fecv/fipv and pedv whereas antiserum against pedv reacts strongly with the homologous virus and only weakly with fecv. examination of mink sera by ifat has shown a high prevalence of antibodies reacting with tgev. these results indicate that one or more coronaviruses related to tgev are widespread in the danish mink population and that such viruses have been present at least since 1981. the high prevalence of seropositive animals over several years points towards an enzootic coronavirus infection in balance with its host population. at present, it is unknown whether this coronavirus infection causes any disease but if so, it may be expected that the virus would manifest itself primarily in young animals either as a consequence of waning maternal antibodies or inadequate lactogenic immunity. a number of clinical syndromes which might be associated with coronavirus infection have been described in mink. epizootic catarrhal gastroenteritis (ecg) (larsen and gorham 1975 ) occurs primarily in mink four months and older exhibiting a mucoid diarrhea over 2-6 days. coronavirus-like particles have recently been identified by negative contrast electron microscopy in feces of animals with ecg (gorham et al., 1990) . a similar observation has been made by one of us (bloch, unpublished results), who found coronavirus-like particles in stool samples from neonatal mink kits. these mink kits originated from farms with a high neonatal mortality. the mink kits were suffering from diarrhea, referred to by farmers as "wet mink kits". the epizootiology of this syndrom is still unclear (henriksen 1989 ) . fip-like symptoms (pyogranulomas, vasculitis and immune-complex disease ) have not been described in mink except for adv-associated immune-complex disease (porter et al., 1980) but such symptoms should be kept in mind when looking for possible effects of coronavirus infection. from the data presented here it is clear that there is a close serological relationship between the putative mcv and both tgev and ccv. the results of western blots show that antibodies to mcv react with all three major structural proteins of tgev and ccv. however, none of the mink sera neutralized any of these viruses, indicating that the antigenic domains responsible for neutralization in the spike (s) protein of tgev and ccv are probably absent from mcv. in this respect mcv may resemble field strains of fipv/ fecv as it has been found that feline field sera having ifat and pla titres of 20 480 or higher against tgev generally do not neutralize tgev or the laboratory strains of fipv and fecv used in this study (have, unpublished data) . it would thus be interesting to have more recent isolates of feline coronavirus and analyze their relationship to mcv. no clear relationship has been found between pedv and any other known coronavirus (pensaert et al., 1981 ) except for a weak cross-reaction with the n protein of fipv (yaling et al., 1988 ) . the present results have confirmed this cross-reaction and further shown that it extends to tgev, prcv, ccv and mcv. in addition, the m proteins of pedv and mcv are clearly related as seen by the reaction of mink antiserum with the m protein of pedv. the present study has shown that infection with a coronavirus serologically related to tgev and pedv is common in the danish mink population. at present, nothing is known of the pathogenic potential of mink coronavirus, if any, nor the preferred site of replication in the host. studies aimed at answering these questions as well as attempts to isolate the virus have been initiated. recommendations of the coronavirus study group for the nomenclature of the structural proteins, messenger rnas, and genes of coronaviruses characterization of the structural proteins of porcine epizootic diarrhea virus, strain cv777 detection of coronavirus-like particles from mink with epizootic catarrhal gastroenteritis serologic differentiation from transmissible gastroenteritis virus using monoclonal antibodies so-called "wet mink kits" -acute enteritis in pre-weaning mink propagation of the virus of porcine epidemic diarrhea in cell culture detection of antibodies against hog cholera virus and bovine viral diarrhea virus in porcine serum cleavage of structural proteins during the assembly of the head of bacteriophage t4 a new mink enteritis: an initial report studies on transmissible gastroenteritis of swine. ii. selected characteristics of a cytopathogenic virus common to five isolates of transmissible gastroenteritis an immunelectron microscopic and immunofluorescent study on the antigenic relationship between the coronavirus-like agent, cv 777, and several coronaviruses isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis aleutian disease of mink a simple method of estimating fifty percent endpoints antigenic homology among coronaviruses related to transmissible gastroenteritis virus the biology ofcoronaviruses the molecular biology of coronaviruses porcine epidemic diarrhea virus (cv 777) and feline infectious peritonitis virus (fipv) are antigenically related key: cord-319712-3dikelw6 authors: pujols, joan; segalés, joaquim title: survivability of porcine epidemic diarrhea virus (pedv) in bovine plasma submitted to spray drying processing and held at different time by temperature storage conditions date: 2014-12-05 journal: vet microbiol doi: 10.1016/j.vetmic.2014.10.021 sha: doc_id: 319712 cord_uid: 3dikelw6 bovine plasma was inoculated with porcine epidemic diarrhea virus (pedv) at an average final titer of 4.2 log10 tcid(50)/ml to determine the effect of spray drying on viral inactivation. using a laboratory scale drier, inoculated plasma was spray dried at 200 °c inlet temperature and either 70 or 80 °c throughout substance. both liquid and dried samples were subjected to three passages on vero cell monolayers to determine pedv infectivity. results indicated liquid samples contained infective virus, but none of the spray dried samples were infectious. also, survivability of pedv inoculated on spray dried bovine plasma (sdbp) and stored at 4, 12 or 22 °c was determined for 7, 14 and 21 days. commercial sdbp powder was inoculated with pedv to an average final titer of 2.8 log10 tcid(50)/g. five samples per time and temperature conditions were subjected to three passages on vero cell monolayers to determine pedv infectivity. the virus was non-infectious for all samples stored at 22 °c at 7, 14 and 21 days. pedv was infective in 1 out of 5 samples stored at 12 °c at 7 days, but none of the samples stored for 14 and 21 days were infectious in cell culture. for samples stored at 4 °c, 4 out of 5 samples were infectious at 7 days, 1 out of 5 samples were infectious at 14 days, but none were infectious at 21 days. in summary, pedv was not infectious on cell culture within 7 days when stored at room temperature and within 21 days when stored at refrigerated temperature. porcine epidemic diarrhea (ped) virus (pedv) is an rna enveloped virus ranging in diameter from 90 to 190 nm, which is taxonomically classified into the coronaviridae family (song and park, 2012) . pedv is antigenically unrelated to other porcine coronaviruses like gastroenteritis transmissible virus and hemaglutinating encephalomyelitis virus (kusanagi et al., 1992) . pedv is highly infectious and transmission is primarily through pig-to-pig contact or indirect contact with fecal material from infected pigs. ped was linked to diarrhea in nursery and fattening pigs in europe in 1971, and pedv was initially recognized in 1978 in belgium (pensaert and debouck, 1978) . ped has also been documented in south-east asia since the 1980s (song and park, 2012) . however, variant strains of pedv causing severe outbreaks of disease in suckling pigs with high morbidity and mortality appeared in 2010 in china (li et al., 2012) . furthermore, and since april 2013, very similar pedv strains to the chinese variants have been veterinary microbiology 174 (2014) [427] [428] [429] [430] [431] [432] bovine plasma was inoculated with porcine epidemic diarrhea virus (pedv) at an average final titer of 4.2 log10 tcid 50 /ml to determine the effect of spray drying on viral inactivation. using a laboratory scale drier, inoculated plasma was spray dried at 200 8c inlet temperature and either 70 or 80 8c throughout substance. both liquid and dried samples were subjected to three passages on vero cell monolayers to determine pedv infectivity. results indicated liquid samples contained infective virus, but none of the spray dried samples were infectious. also, survivability of pedv inoculated on spray dried bovine plasma (sdbp) and stored at 4, 12 or 22 8c was determined for 7, 14 and 21 days. commercial sdbp powder was inoculated with pedv to an average final titer of 2.8 log10 tcid 50 /g. five samples per time and temperature conditions were subjected to three passages on vero cell monolayers to determine pedv infectivity. the virus was noninfectious for all samples stored at 22 8c at 7, 14 and 21 days. pedv was infective in 1 out of 5 samples stored at 12 8c at 7 days, but none of the samples stored for 14 and 21 days were infectious in cell culture. for samples stored at 4 8c, 4 out of 5 samples were infectious at 7 days, 1 out of 5 samples were infectious at 14 days, but none were infectious at 21 days. in summary, pedv was not infectious on cell culture within 7 days when stored at room temperature and within 21 days when stored at refrigerated temperature. ß 2014 elsevier b.v. all rights reserved. detected in north america resulting in significant pig mortality and economic loss (huang et al., 2013) . phylogenetic analyses suggested that the pedv in the usa likely originated from china (huang et al., 2013) . taking into account that the exact source of origin of the us strains has not been identified, the potential continued spread of the virus worldwide is a critical concern. under this scenario, feed or feed ingredients, including spray-dried porcine plasma (sdpp), have recently been suggested as potential sources for transmission of pedv (pasick et al., 2014) . spray-dried plasma (sdp) proteins are used extensively in nursery pig feed to enhance feed intake, growth, and feed efficiency during the post-weaning period (coffey and cromwell, 2001; van dijk et al., 2001) and represents an excellent alternative to antibiotics (torrallardona, 2010) . the spray drying process used to manufacture sdp involves sudden changes in temperature and pressure that causes rapid evaporation of water which is detrimental to a variety of pathogens including bacteria and viruses (polo et al., 2005 (polo et al., , 2013 . a series of studies have demonstrated that manufacturing conditions to produce commercial spray-dried porcine plasma (sdpp) inactivate several viruses of importance to the swine industry such as porcine reproductive and respiratory syndrome virus (prrsv), pseudorabies virus (prv) (polo et al., 2005) or swine vesicular disease virus (svd) (pujols et al., 2007) . in addition, several studies have reported that orally consumed commercial sdpp does not transmit either porcine parvovirus (ppv) or porcine circovirus type 2 (pcv2) (polo et al., 2005; pujols et al., 2008 pujols et al., , 2011 shen et al., 2011) , which are two of the most heat and chemically resistant swine viruses. the first objective of this study was to determine the survival of pedv inoculated in liquid bovine plasma that was then submitted to two different spray drying conditions (70 and 80 8c throughout its substance). a second objective was to determine survival time of pedv inoculated on spray-dried bovine plasma when stored under different temperatures (4, 12 and 22 8c) for different periods of time (0, 7, 14 and 21 days). the cv777 strain (pensaert and debouck, 1978; debouck and pensaert, 1980) , prototype pedv strain from belgium, belongs to group g1-1 and is similar to isolates br-1/87 (britain), js-2004 (china), p-5v (japan) and kped-9, sm98-1 and dr-13 (south korea) (park et al., 2007) . this strain of pedv is well known to propagate on vero cells and was selected as a model swine coronavirus to study inactivation of pedv by spray drying. pedv was propagated in vero cell line, grown in eagle's minimum essential medium (mem), supplemented with 0.3% tryptose phosphate broth (sigma), 10% fetal bovine serum and an antibiotic combination (penicillin 100 iu/ml, streptomycin 100 mg/ml). virus stock was produced in vero cells seeded in 175 cm 2 flask to confluent monolayers, cells were rinsed 3 times with phosphate buffered saline and inoculated with 10 ml of pedv 3.5 log10 tcid 50 /ml (moi of 0.002) to each bottle. the virus-cells mixture was incubated 1 h at 37 8c, and then the inoculum was replaced with post-infection media (pm), which consisted of mem supplemented with 0.3% tryptose phosphate broth (sigma), 12.5 mg/ml trypsin (sigma) and the above mentioned antibiotic combination (hofmann and wyler, 1989) . cultures were checked at 18 and 24 h after infection to assess syncytia formation and the evolution of cytopathogenic effect (cpe) until reaching nearly 100% cpe. liquid supernatants and scraped cells were collected. the pooled suspensions were centrifuged at 1000 g for 20 min at 4 8c and supernatants were stored at 4 8c. the resulting pellet was suspended in pm and subjected to freeze-thaw, centrifuged and supernatants were harvested and added to bulk supernatants. stock virus was stored at à80 8c or used immediately after its production (hofmann and wyler, 1989) . a pedv virus stock was produced to a virus titer ranging between 4.0 log10 and 5.0 log10 tcid 50 /ml (hofmann and wyler, 1989; song et al., 2003; oh et al., 2005) . total pedv rna genome copies of virus production was estimated by quantitative reverse-transcriptase pcr (qrt-pcr) with a swine enteric panel assay for pedv and other enteric viruses (thermo fisher scientific inc., waltham, ma). correlation between pedv loads by tcid50/ml on cell culture and ct values by qrt-pcr was calculated. for this, 10-fold dilutions of 4.6 ae 0.2 log10 tcid 50 of strain pedv cv777 were extracted two times and amplified 3 times each one. the reason for using bovine plasma was mainly to avoid the presence of potential antibodies against pedv european strains or cross-reaction with antibodies against other porcine coronavirus that may be present in swine plasma. bovine plasma was collected from an eu inspected slaughter facility. animals were inspected and approved for slaughter for human consumption. blood was collected into stainless steel pans containing sodium phosphate as anticoagulant. blood was transported refrigerated to the laboratory and plasma was separated by centrifugation and three 0.135-l bottles were prepared and frozen at à20 8c. a 10-ml sample of each of the 0.135-l batches was stored at à80 8c and used as controls to determine absence of virus neutralization antibodies and viral contamination. each batch of plasma was inoculated with stock pedv at 1:1 dilution. pedv was added to each 0.125 l of bovine plasma to get an approximate virus titer of 4.0 log10-4.5 log10 tcid 50 /ml. each of the three 0.25-l batches was sampled in three 5 ml tubes just after the virus inoculation and immediately frozen at à80 8c. the three 0.235-l batches were dried using a bü chi 190 mini spray dryer (bü chi labortechnik ag, flawil, switzerland). all the bottles were dried with an inlet temperature of 200 ae 5 8c and outlet temperature of 80 ae 1 8c. before spray-drying plasma and virus mixture, the spray-drier was drying control bovine plasma at the target temperature for 45 min to ensure that the system was processing at the target parameters when the virus inoculated plasma was dried. the spray drying parameters used were similar to those previously established (polo et al., 2005) . airflow (at 20 8c) through the column was set at 45 m 3 h à1 and the suspension flow to the nozzle was set at 0.2 m 3 h à1 . the airflow through the feeding nozzle was set at 0.7 m 3 h à1 (at 20 8c). estimated dwell time was 0.41 s. the dwell time of industrial size driers used in manufacturing plants to produce commercial sdp ranges between 20 and 90 s, depending on size of the spray drier and configuration (fluid bed, etc.), whereas the dwell time of the lab drier was <1 s. in an attempt to more closely simulate industrial size driers and conditions, immediately after the spray-drying process, 6 sub-samples (0.5 g each) of sdp were placed in 0.5 cm (inner diameter) glass tubes and held at 89-91 8c in a water bath for a dwell time of 30 s or 60 s to achieve an interior plasma powder temperature of 70.4 8c and 80.7 8c, respectively. the temperature was determined with thermocouple probe (crison instruments sa, alella, spain) inserted in the glass tube containing the sdbp. dried samples were immediately frozen in dry ice and stored at à80 8c prior to analysis. liquid and dried frozen samples were analyzed for infectivity in vero cell cultures, using the microtiter assay procedure. dried samples were reconstituted by adding 0.5 g of plasma to 5.5 ml of distilled water to each sample. to determine that no minute quantities of virus had survived, 5 ml of each of the reconstituted spray-dried samples were inoculated on a 75 cm 2 flask of vero cell monolayers. after 1 h of incubation at 37 8c the inoculum was discarded and cultures were rinsed 3 times with pbs and then pm media was added. after four days of incubation at 37 8c and 5% co 2 , cell cultures were frozen-thawed, harvested, gently vortex to full homogenization and 5 ml supernatants seeded onto new 75 cm 2 bottles with vero cell monolayers. three serial passages were made. if cpe was observed, then the initial sample was titrated on vero monolayers grown in 96-well tissue culture plates. a virus neutralization (vn) test was performed on the three liquid samples to verify that the bovine plasma used had no neutralizing antibodies against pedv. briefly, 2 log dilutions of bovine plasma were incubated 1 h at 37 8c with 200 tcid 50 /ml of pedv. then 0.1 ml of this mixture was added to a vero cells monolayer grown in 96-well plates that previously had been rinsed 3 times with pbs, and were incubated for 1 h at 37 8c. after adsorption, inoculum was removed and plates were rinsed 3 times with pbs. pm was added and plates were incubated for 3 days at 37 8c. virus neutralization was expressed as the reciprocal of highest serum dilution that abrogates the cpe (oh et al., 2005) . 2.3. experiment 2: survival of pedv inoculated on spray dried bovine plasma stored over time at different temperatures spray dried bovine plasma (sdbp) was selected from commercial production for use in the experiment. a 10-g sample of the sdbp was stored at à80 8c and used as a control to determine absence of pedv neutralization antibodies and pedv contamination. sixty grams of sdbp contained in a large petri dish were inoculated with 9.0 ml of pedv cell culture by spraying the liquid virus culture onto the sdbp using a spray bottle. the petri dish was sealed with plastic paraffin film and shaken vigorously for 15 min to ensure a good homogenization of the virus with the sdbp. one gram of inoculated sdbp was added to 50 different glass tubes (0.5 cm inner diameter). one set of 15 tubes was stored at 4 ae 2 8c in the lab refrigerator, another set of 15 tubes was stored at 12 ae 2 8c in a calibrated water bath and the last set of 15 tubes was stored at room lab temperature of 22 ae 2 8c. for each temperature storage condition, 5 tubes were selected at 7, 14 and 21 days and stored at à80 8c until their analysis. at the time of analysis, the à80 8c stored samples (the initial samples and the samples stored at different time by temperature conditions) were analyzed to detect the survival of pedv in cell culture. each dried sample was reconstituted by adding 1 g of sdbp to 10 ml of distilled water. to determine that no minute quantities of virus survived, 5 ml of each of the reconstituted sdbp samples were inoculated on a 75 cm 2 flask of vero cell monolayers and, after 1 h incubation at 37 8c, the inoculum was discarded and cultures were rinsed 3 times with pbs and then pm media was added. after four days, cell cultures were thawed, harvested, clarified by centrifugation and passaged to new vero cell cultures. three serial passages were made. if cpe was observed, then virus was titrated on vero cell monolayers grown in 96-well tissue culture plates. a tissue culture vn test was performed on the control sdbp used to ensure lack of neutralizing antibodies against pedv in the original sample. briefly, 2 log dilutions of sdbp re-suspended in distilled water (0.5 g sdbp to 5 ml distilled water) were incubated 1 h at 37 8c with 200 tcid 50 /ml of pedv. then, the same procedure explained before for liquid bovine plasma was followed. statistical analysis were performed on qrt-pcr results by glm procedure and turkey-kramer multiple comparison test (ncss 2004, statistical systems, ncss llc, kaysville, ut). correlation between pedv titers expressed as tcid 50 /ml and pedv loads expressed in qrt-pcr ct values are displayed in fig. 1 . under the range of detection on cell culture, the qrt-pcr results displayed an increased range of standard deviation, which was attributed to non-encapsidated virus material, still present at higher dilutions. the regression results showed a close correlation (r = 0.9745; y = à3.30x + 28.60). the non-inoculated liquid bovine plasma stored at à80 8c was determined negative for presence of pedv and negative for neutralizing antibodies against the virus. total concentration of pedv in the pellet used to inoculate the liquid bovine plasma was determined to be 4.6 log10 tcid 50 /ml (ct = 12.1 ae 0.2 by means of qrt-pcr). results of the liquid and the sdbp samples are presented in table 1 . analyses of the initial 3 batches of inoculated liquid bovine plasma stored at à80 8c gave an average titer of 4.2 ae 0.2 log10 tcid 50 /ml (calculated for dry product: 5.15 ae 0.2 log10 tcid 50 /g). the samples processed at both temperatures (70 and 80 8c) throughout its substance were negative for pedv infectivity on cell culture. the calculated inactivation rate at these temperatures was at least greater than the initial titer of 5.15 log10 tcid tcid 50 /g. the qrt-pcr ct results for the virus inoculated plasma were 13.9 ae 0.3, and for the spray dried plasma at 70 and 80 8c throughout its substance were 23.3 ae 0.6 and 23.8 ae 0.8, respectively. the non-inoculated sdbp stored at à80 8c was determined negative for pedv in vero cell culture and qrt-pcr. total concentration of pedv in the pellet used for sdbp inoculation was titrated to be 4.6 log10 tcid 50 /ml (ct = 12.1 ae 0.2). the expected titer after virus inoculation was 3.8 log10 tcid 50 /g. results of the sdbp pedv inoculated samples are presented in table 2 . analyses of the initial 5 contaminated sdbp samples that were immediately stored at à80 8c averaged 2.8 log10 tcid 50 /g (average ct = 22.0 ae 0.8). for samples stored at 4 ae 2 8c, 4 out of the 5 samples contained infectious pedv at day 7 of storage, but at day 14 only 1 out of the 5 samples contained infectious virus and none of the samples contained infectious pedv by day 21 of storage. the ct value for samples stored at 4 ae 2 8c averaged 21.6 ae 0.7. for samples stored at 12 ae 2 8c, only 1 out of the 5 samples stored for 7 days contained infectious virus and by days 14 and 21 none of the samples retained infectivity. the ct value for samples stored at 12 ae 2 8c averaged 22.2 ae 1.1. for samples stored at room temperature (22 ae 2 8c), none of them remained infectious at 7, 14 and 21 days of storage. the ct value for samples stored at 22 8c averaged 21.1 ae 0.6. the primary route of infection of pedv is direct contact with infected pigs or from the manure of infected pigs (oie, 2014) . other routes of infection responsible for spreading the virus have not been clearly identified, but transport vehicles for swine and feed or feed ingredients have been shown to contain pedv genome by rt-pcr (dee et al., 2014; oie, 2014) . the role of feed or feed ingredients in the spread of pedv was suggested after the first report of ped in ontario (canada). the subsequent investigation conducted by the canadian food inspection agency (cfia) concluded that nursery feed containing sdpp might have been a source of pedv transmission. although the cfia reported that infective virus was detected in samples of sdpp, infective virus could not be detected in the feed containing the corresponding sdpp (pasick et al., 2014) . in spite of this conflicting data, the opinion that sdpp may contain infective pedv as a reason to explain the spread of pedv across north america has been adopted by some swine industry professionals. the results obtained in the present study indicated that pedv inoculated in bovine plasma was apparently inactivated when spray-dried at both simulated commercial processing conditions of 70 and 80 8c throughout its substance. these results are in agreement with previous publications indicating that pedv is a low thermal stable virus (song and park, 2012) . in addition, these results agree with previous reports with different enveloped the spray dryer concentrated liquid plasma 10 times; the expected virus titer in the dry product was 5.15 ae 0.2 log10 tcid 50 /g. c the theoretical limit of detection of the method used was estimated to be able to detect as low as 0.7 viral particles/g. viruses like prrsv and prv (polo et al., 2005) and a nonenveloped virus like svd (pujols et al., 2007) . these studies demonstrated that the commercial spray drying conditions used for the manufacturing process of commercial sdp should be sufficient to eliminate low-medium heat resistant viruses like pedv. the normal processing temperature condition used by commercial sdp manufacturing plants is >80 8c throughout its substance. the present data indicated that drying at 70 8c throughout its substance was already adequate to inactivate at least 5.15 log10 tcid50/g of this virus. therefore, the heat treatment used during the commercial spray-drying process is theoretically more than adequate to inactivate pedv if present in the raw material. recent data would support such conclusion opriessnig et al., 2014) . in the first study, researchers used raw plasma from a non-infected pig spiked with wild type us strain of pedv and either fed directly as raw liquid spiked plasma or spray-dried it (in a laboratory drier at 83 8c outlet temperature) and fed to naïve pigs. their results indicated that the raw liquid spiked plasma was infective but the spray dried spiked plasma was not infective, indicating that spray drying at outlet temperature around 80 8c should be enough to inactivate the pedv . in the second study, piglets received a diet with 5% sdpp containing 5.1 ae 0.1 log10 pedv rna copies/g and showed no evidence of infectivity by the existing pedv rna present in the sdpp lot utilized . pedv viremia has been reported only once in gnotobiotic pigs experimentally infected with pedv genogroup 2 (jung et al., 2014) . it is usually accepted that pedv rna detected in serum is possible at the peak of disease, but viremia is reported to be short and of low magnitude (pensaert, 1999) . therefore, it appears unlikely that pedv viremia and utilization of blood from viremic pigs is a main source of pedv contamination of sdpp. probably, the presence of this virus in raw blood is more likely attributed to carcass contamination at time of slaughter opriessnig et al., 2014) . the present data studying the survival of pedv inoculated on sdbp appeared to be dependent upon storage temperature and time, with virus survivability associated with lower temperature storage conditions. however, virus concentration diminished considerably for the samples stored at 4 8c or 12 8c for 7 days compared to samples immediately stored frozen at à80 8c. importantly, samples stored at room temperature did not contain infective pedv by 7 days of storage, which is consistent with previous data reported by other researchers. they demonstrated that pedv-positive feces spread evenly on the bottom surface aluminum tray and treated at 20 8c for 7 days were non infective when feed-back to naïve pigs by oral-gastric tube (thomas et al., 2014) . also, it is important to highlight that, even at 4 8c, the virus was not infectious in cell culture after 21 days of storage. the fact that spray dried blood products are powders with low moisture (below 8%) and water activity (<0.6) are important, since pedv does not survive very long under dry conditions (oie, 2014) . the ct values for samples stored at 4, 12 and 22 8c for 7 days were 21.59 ae 0.73, 22.18 ae 1.08 and 21.12 ae 0.62, respectively. these results indicated that qrt-pcr ct values were not different between samples (p > 0.05), independently of the storage conditions. however, differences between days (p < 0.05) at each temperature condition should be indicative of progressive genome degradation. moreover, the qrt-pcr positive results compared to those generated by cell culture of the virus indicated that the former technique is not a good indicator to establish pedv infectivity. however, it is also true that virus isolation might not be highly sensitive; therefore, a swine bioassay would be the most definitive way to demonstrate the putative infectivity of those qrt-pcr positive samples which were negative after inoculation of vero cells. in summary, results of the present work demonstrated that pedv (at least the european prototype strain, cv777) was apparently inactivated by spray drying conditions mimicking those used by the spray-dried blood products industry. moreover, if sdp would become contaminated use of spray-dried animal plasma in diets for weanling pigs experimental infection of pigs with a new porcine enteric coronavirus cv777 an evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of naive pigs following consumption via natural feeding behavior: proof of concept the spray-drying process is sufficient to inactivate infectious porcine epidemic diarrhea virus in plasma origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (pedv) pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate new variants of porcine epidemic diarrhea virus, china comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection infection with porcine epidemic diarrhoea virus porcine epidemic diarrhea virus rna present in commercial spray-dried porcine plasma is not infectious to naive pigs sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in canada porcine epidemic diarrhea a new coronavirus-like particle associated with diarrhea in swine efficacy of spray-drying to reduce infectivity of pseudo rabies and porcine reproductive and respiratory syndrome (prrs) viruses and seroconversion in pigs fed diets containing spray-dried animal plasma neutralizing antibodies against porcine circovirus type 2 in liquid pooled plasma contribute to the biosafety of commercially manufactured spray-dried porcine plasma inactivation of swine vesicular disease virus in porcine plasma by spray-drying lack of transmission of porcine circovirus type 2 to weanling pigs by feeding them spray dried porcine plasma commercial spray-dried porcine plasma does not transmit porcine circovirus type 2 in weaned pigs challenged with porcine reproductive and respiratory syndrome virus commercially produced spray dried porcine plasma contains high levels of porcine circovirus type 2 (pcv2) dna but did not transmit pcv2 when fed to naïve pigs porcine epidemic diarrhea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf 3 evaluation of time and temperature sufficient to inactivate porcine epidemic diarrhea virus in swine feces on metal surfaces spray dried animal plasma as an alternative to antibiotics in weanling pigs -a review growth performance of weanling pigs fed spray-dried animal plasma: a review funding for this study was provided by apc europe, s.a., granollers, barcelona, spain. the authors thank dr. hans nauwynck, laboratory of virology, faculty of veterinary medicine (ghent university) for supplying the pedv cv777 strain and the vero cell line. we also thank nuria navarro, nuria pujol, marta valle and lorena có rdoba from cresa and carmen rodríguez and neus saborido from apc europe, s.a. for their assistance providing the raw material and with the laboratory work. the authors declare no conflict of interest. key: cord-314751-i9rxesrg authors: oh, jongsuk; lee, kyung-won; choi, hwan-won; lee, changhee title: immunogenicity and protective efficacy of recombinant s1 domain of the porcine epidemic diarrhea virus spike protein date: 2014-07-10 journal: arch virol doi: 10.1007/s00705-014-2163-7 sha: doc_id: 314751 cord_uid: i9rxesrg porcine epidemic diarrhea virus (pedv) is a highly contagious enteric pathogen of swine. acute pedv outbreaks have continually emerged in most swine-producing asian countries and, recently, in the united states, causing significant economic losses in the pig industry. the spike (s) protein of pedv is a type 1 transmembrane envelope glycoprotein and consists of the s1 and s2 domains, which are responsible for virus binding and fusion, respectively. since the s1 domain is involved in a specific high-affinity interaction with the cellular receptor and induction of neutralizing antibody in the natural host, it is a primary target for the development of effective vaccines against pedv. in this study, a codon-optimized pedv s1 gene containing amino acid residues 25–738 was synthesized based on a multiple alignment of the s amino acid sequences of pedv field isolates and used to establish a stable porcine cell line constitutively expressing the pedv s1 protein. the purified recombinant s1 protein was found to mediate highly potent antibody responses in immunized rabbits. the antibodies strongly recognized the recombinant s1 protein from cell lysates and supernatants of s1-expressing cells, whereas they bound weakly to the authentic s protein of pedv vaccine strain sm98-1. furthermore, a serum neutralization test revealed that the rabbit antisera completely inhibit infection of the pedv vaccine strain at a serum dilution of 1:16. we then tested the ability of vaccination with the recombinant s1 protein to protect piglets against pedv. late-term pregnant sows were inoculated intramuscularly with the purified s1 protein, and the outcome was investigated in passively immunized suckling piglets after a virulent pedv challenge. the results showed that vaccination with s1 protein efficiently protected neonatal piglets against pedv. our data suggest that the recombinant s1 protein shows potential as an effective and safe subunit vaccine for ped prevention. porcine epidemic diarrhea (ped) is a devastating swine disease that is characterized by acute enteritis and lethal watery diarrhea followed by severe dehydration leading to death, with a high mortality rate in piglets [6, 26, 28] . the disease was initially recognized in england in 1971 [23] , but the causative agent of this disease, ped virus (pedv), was later identified in 1978 [25] . ped epidemics were first reported in asia in 1982, and since then, ped has continued to threaten swine health, causing substantial economic losses in the asian swine industry [4, 19, 27, 33] . in 2013, ped outbreaks suddenly occurred in the united states and have swept through the pork industry across the country, raising concerns about control measures for ped prevention [21, 30] . in korea, pedv appeared in 1992 [14] ; however, a retrospective study indicated that the virus had been present as early as 1987 [24] . although periodic vaccination strategies have been implemented nationwide to control ped in korean swine herds, pedv has continually emerged, causing tremendous harm to the productivity of korean pig farms. pedv, a member of the genus alphacoronavirus within the family coronaviridae of the order nidovirales, is a large, enveloped virus possessing a single-stranded, positive-sense rna genome of approximately 28 kb with a 5 0 cap and a 3 0 polyadenylated tail [25, 28] . the pedv genome is composed of the 5 0 untranslated region (utr), at least seven open reading frames (orf1a, orf1b, and orf2 through 6), and the 3 0 utr [13] . the two large orfs 1a and 1b make up the 5 0 two-thirds of the genome and encode the non-structural replicase genes. the remaining orfs in the 3 0 terminal region code for four major structural proteins: the 150-220-kda glycosylated spike (s) protein, the 20-30-kda membrane (m) protein, the 7-kda envelope (e) protein, and the 58-kda nucleocapsid (n) protein [8, 28] . the s protein of pedv is a type i membrane glycoprotein composed of 1,383 to 1,386 amino acids (aa), depending on the strain. it contains a putative signal peptide (aa 1-24), a large extracellular region, a single transmembrane domain (aa 1,334-1,356), and a short cytoplasmic tail. although pedv has an uncleaved s protein because it lacks a furin cleavage site, the s protein can be divided into s1 (aa 1-735) and s2 (736-the last aa) domains based on homology with s proteins of other coronaviruses [7, 11, 16, 31] . like other coronavirus s proteins, the pedv s protein is known to play a pivotal role, interacting with the cellular receptor to mediate viral entry and inducing neutralizing antibodies in the natural host [2, 3] . more precisely, previous studies have shown that the s1 domain includes the main neutralizing epitopes and the receptor-binding region [17, 32] . furthermore, along with the full-length s gene, the s1 portion is known to be a suitable region for determining genetic relatedness among the different pedv isolates and for developing differential diagnostic assays [5, 16] . considering these molecular and biological features of the s1 domain, it would be an appropriate target for developing effective vaccines against pedv. in the present study, therefore, we first synthesized a full-length, codon-optimized pedv s1 gene and then generated a stable porcine-origin cell line constitutively expressing the recombinant s1 protein. the protective efficiency of a recombinant-s1-protein-based vaccine against pedv was then evaluated in the natural host. the recombinant s1 protein was capable of inducing an efficient antibody response in immunized rabbits. moreover, immunization with the pedv s1 protein was found to elicit specific neutralizing antibodies in sows and to protect passively immunized suckling piglets against pedv challenge. cells, virus, antibodies, and plasmid hek-293t cells (crl-1573) were purchased from the american type culture collection (atcc, manassas, va) and cultured in dulbecco's modified eagle medium (dmem) with high glucose (invitrogen, carlsbad, ca) with 10 % fetal bovine serum (fbs, invitrogen) and antibiotic-antimycotic solutions (1009; invitrogen). pk-15 cells were grown in rpmi 1640 medium (invitrogen) supplemented with 10 % fbs and antibiotic-antimycotic solutions. vero cells were cultured in alpha minimum essential medium (a-mem, invitrogen) with 10 % fbs and antibiotic-antimycotic solutions. the cells were maintained at 37°c in a humidified 5 % co 2 atmosphere. the pedv vaccine strain sm98-1 was obtained from the korean animal and plant quarantine agency and propagated in vero cells as described previously [10] . challenge pedv was prepared from the small intestine of a 4-day-old suckling piglet orally inoculated with small intestine homogenate containing the field virus. small intestine tissues were collected and homogenized in a 10 % suspension with a-mem using a magna lyser (roche diagnostics, mannheim, germany) with three repetitions of 15 s at a speed of 7,000 rpm, and suspensions were clarified by centrifugation at 4,500 9 g (hanil centrifuge fleta5, incheon, korea). the clarified supernatant was filtered through a 0.22-lm-pore-size syringe filter (millipore, billerica, ma), aliquoted, and stored at -80°c until use as crude challenge virus. all of the horseradish peroxidase (hrp)-conjugated secondary antibodies were purchased from santa cruz biotechnology (santa cruz, ca). the pedv s-protein-specific monoclonal antibody (mab) was a kind gift from sang-geon yeo (kyungpook national university, daegu, korea). a plasmid encoding the s1 fragment of severe acute respiratory syndrome coronavirus (sars-cov), pcdm8-sars-cov-s1-ig, was kindly provided by hyeryun choe (harvard medical school, boston, ma). dna manipulation and cloning were performed according to standard procedures [29] . the e. coli strain dh5a (rbc bioscience, taiwan) was used as the host for general cloning. the plasmid encoding the full-length s1 gene of the pedv field strain knu-0801, pcdm8-pedv-s1-ig, was described previously [17] . to construct the plasmids expressing s1 and its variants, the consensus sequence of pedv s1 was identified based on a multiple alignment of the s aa sequences of pedv field isolates [16] and utilized to synthesize a full-length, codon-optimized pedv s1 gene (encoding aa 24-735) according to a method described previously [1] . the codon-optimized pedv s1 gene was cloned into a modified expression vector, pcdm8-fc, which contains the cd5 signal sequence and the fc domain of human igg1 [9] , thereby producing a human fc-tagged fusion protein, rs1-ig. all of the pedv s1-truncated variants used in this study were generated using this template with the previously described primer sets [17] . an fctagged pedv rs1-ig fragment obtained from pcdm8-fc-rs1-ig was then subcloned into a pfb-neo retroviral vector (stratagene, la jolla, ca) using the sali and xhoi restriction sites to construct a pedv rs1-ig gene expression plasmid, pfb-neo-pedv-rs1-ig. all of the constructed plasmids were verified by nucleotide sequencing. generation of a stable pk-15 cell line expressing pedv rs1-ig the retrovirus gene transfer system (stratagene) was applied to generate a cell line constitutively expressing the recombinant pedv rs1-ig gene or an empty retroviral vector as described elsewhere [16, 22] . antibiotic-resistant continuous cell clones were examined by rt-pcr to verify the presence of the full-length rs1-ig gene, and the positive clones (pk-rs1-ig) were then amplified for subsequent analyses. immunofluorescence assay (ifa) pk-rs1-ig cells were grown on microscope coverslips placed in 6-well tissue culture plates. at 48 h post-seeding, the cells were fixed with 4 % paraformaldehyde for 10 min at room temperature (rt) and permeabilized with 0.2 % triton x-100 in pbs at rt for 10 min. the cells were subsequently blocked with 1 % bovine serum albumin (bsa) in pbs for 30 min at rt and then incubated with a goat anti-human igg antibody conjugated with fluorescein isothiocyanate (fitc) (santa cruz biotechnology). finally, the cells were counterstained with 4 0 ,6-diamidino-2-phenylindole (dapi; sigma, st. louis, mo), and cell staining was visualized using a fluorescent leica dm il led microscope (leica, wetzlar, germany). fluorescence-activated cell sorting (facs) analysis rs1 expression in pk-rs1-ig cells was analyzed by flow cytometry. briefly, cells were trypsinized at 48 h postseeding and centrifuged at 250 9 g (hanil centrifuge fleta 5) for 5 min. the cell pellet was washed with cold washing buffer (1 % bsa and 0.1 % sodium azide in pbs) and 1 9 10 6 cells were resuspended in 1 % formaldehyde solution in cold wash buffer and fixed at 4°c in the dark for 30 min, followed by incubation in 0.2 % triton x-100 in pbs for permeabilization at 37°c for 15 min. following centrifugation, the cell pellet was resuspended in normal mouse igg1 antibody (santa cruz biotechnology) and incubated at 4°c for 30 min. the cells were washed and reacted with fitc-conjugated anti-human or anti-mouse igg secondary antibody at 4°c for 30 min in the dark. the stained cells were washed again and analyzed by facscan flow cytometry. western blot analysis hek 293t cells were transfected with each plasmid using lipofectamine 2000 (invitrogen) according to the manufacturer's protocol and solubilized in lysis buffer at 48 h post-transfection as described previously [22] . cell lysates were also prepared from vero cells infected with pedv sm98-1 at a multiplicity of infection (moi) of 0.1 at the indicated time points using lysis buffer. the protein concentrations of the cell lysates were determined using a bca protein assay (pierce, rockford, il). pk-rs1-ig cells were grown at 5 9 10 5 cells/well in a 6-well tissue culture plate, and the protein-containing culture supernatants were harvested on days 1, 2, and 3. soluble proteins were immunoprecipitated with protein a sepharose cl-4b beads (ge healthcare, piscataway, nj) in the presence of protease inhibitors at 4°c for 16 h. the beads were collected by centrifugation at 5,000 9 g (eppendorf centrifuge 5415r, hamburg, germany) for 5 min at 4°c and washed three times with 0.5 m nacl in pbs. the cell lysates or the beads were mixed with 49 nupage sample buffer (invitrogen) and boiled at 70°c for 10 min. the proteins were separated on a nupage 4-12 % gradient bis-tris gel (invitrogen) under reducing conditions, and electrotransferred onto immunobilon-p (millipore). the membranes were then blocked with 3 % powdered skim milk (bd biosciences, belford, ma) in tbs (10 mm tris-hcl [ph 8.0], 150 mm nacl) with 0.05 % tween-20 (tbst) at 4°c for 2 h and reacted directly with the goat anti-human igg hrp-conjugated secondary antibody, the anti-s1 rabbit serum or the anti-pedv s mab, followed by the corresponding hrp-labeled secondary antibody at a dilution of 1:5,000 for 2 h at 4°c. finally, the proteins were visualized using enhanced chemiluminescence (ecl) reagents (ge healthcare) according to the manufacturer's instructions. protein purification pk-rs1-ig cells were grown at 5 9 10 5 cells/well in a 6-well tissue culture plate in serum-free medium (optipro sfm; invitrogen). at 72 h post-seeding, the protein-containing culture supernatants were harvested and soluble proteins were immunoprecipitated with protein a sepharose cl-4b beads in the presence of protease inhibitors at 4°c for 16 h. the beads were collected and washed as described above. the samples were subsequently eluted with 50 mm sodium citrate/50 mm glycine (ph 2.0) and neutralized with 1 m tris-hcl (ph 8.0). the purified proteins were concentrated with amicon ultra centrifugal filters 100k (millipore). protein concentration was measured using a bca protein assay (pierce, rockford, il) and the final products were analyzed by western blotting to confirm target protein purification. two new zealand white rabbits were immunized intradermally with 250 lg of purified rs1-ig resuspended in pbs in the presence of freund's complete adjuvant and boosted four times with a freshly prepared emulsion of 250 lg immunogen and freund's incomplete adjuvant at 2-week intervals. pre-immune sera were collected before starting the immunization, and antisera were collected at each boost. the in vivo swine experiments described here were performed at the choongang vaccine laboratory animal facility under the guidelines established by its institutional animal care and use committee. a total of eight pregnant sows were obtained from a pig farm with no outbreaks or vaccination with pedv and randomly divided into four groups of two sows. all animals were determined to be free of antibodies to pedv. the design for the present immunogenicity study involving eight pregnant sows is outlined in table 1 . the sows in group 1 were immunized intramuscularly with attenuated pedv live vaccine and inactivated pedv vaccine obtained from choongang vaccine laboratory, in order at 2-week intervals prior to farrowing. the pigs in group 2 were immunized intramuscularly with pedv live vaccine and 400 lg of purified rs1-ig resuspended in pbs in the presence of an oil-in-water adjuvant, in order at 2-week intervals before farrowing. both the pedv live and inactivated vaccines were administrated according to the manufacturers' manuals. the sows in group 3 and group 4 were inoculated intramuscularly three times at 2-week intervals with 400 lg of purified rs1-ig mixed with the oil adjuvant or with cell culture medium as a negative control. paired blood samples and colostrum were collected at 3-week intervals before farrowing and at farrowing, at delivery, respectively. one 4-to 5-day-old piglet (8 piglets in total) was selected randomly from each farrowing sow in the immunized and control groups for challenge exposure with virulent pedv. piglets from all groups were challenged orally with 1 ml of small intestine homogenate containing 10 5 tcid 50 /ml of pedv field virus, equivalent to 10 9.8 viral genome copies per ml, determined using real-time rt-pcr as described previously [12] . clinical signs of diarrhea and death in challenged piglets were monitored daily throughout the study. stool samples from all groups were collected every day with 16-inch cotton-tipped swabs and subjected to rt-pcr using a tge/ped detection kit (intron biotechnology, seongnam, korea) according to the manufacturer's protocol. all piglets from the vaccinated and control groups were euthanized at 5 days after challenge for post-mortem examination. small-intestinal tissue specimens collected from each piglet (\3 mm thick) were fixed with 10 % formalin for 24 h at rt and embedded in paraffin according to standard laboratory procedures. the formalinfixed paraffin-embedded tissues were cut into 5-to 8-lmthick sections on a microtome, floated on a 40°c water bath containing distilled water, and transferred onto glass slides. the tissues were then deparaffinized in xylene for 5 min and washed in decreasing concentrations of ethanol (100, 95, 85, 70, and 50 %) for 3 min each. the deparaffinized intestinal tissues sections were subjected to immunofluorescence assay using an n-specific mab and alexa fluor 594-conjugated goat anti-mouse antibody as described above. the presence of pedv-specific neutralizing antibodies in serum and colostrum samples collected from sows in all groups was determined using a serum neutralization (sn) test in 96-well microtiter plates using pedv vaccine strain sm98-1 as described previously [15] . briefly, individual virus stocks were diluted in serum-free a-mem to make 200 pfu in a 50 ll volume. the diluted virus was then mixed with 50 ll of twofold dilutions of individual pedv s1 immunogen a pedv s1 immunogen pedv s1 immunogen 4 placebo placebo placebo a placebo (cell culture medium) and protein (pedv s1) immunizations were given intramuscularly with the oil-in-water adjuvant b commercial pedv live and inactivated vaccines were administrated intramuscularly according to the manufacturers' instructions with the oil-in-water adjuvant inactivated sera in 96-well plates and incubated at 37°c for 1 h. next, the mixture was incubated at 37°c for 1 h, approximately 1 9 10 4 vero cells in 100 ll of a-mem were added to each well, and the mixture was maintained at 37°c in a 5 % co 2 incubator for 3 to 4 days. the neutralization titer was calculated as the reciprocal of the highest dilution of serum that inhibited the virus-specific cytopathic effect in both of the duplicate wells. results were also visualized by staining the wells with a crystal violet-formaldehyde staining reagent (0.013 % crystal violet, 2.5 % ethanol, and 10 % formaldehyde in 0.01 m pbs) for 1 h at rt. the student's t test was used for all statistical analyses, and p-values of less than 0.05 were considered statistically significant. generation of stable porcine-origin cell lines expressing the full-length, codon-optimized s1 protein we have previously constructed a series of expression plasmids encoding the full-length s1-ig (s1 ). although expression of this gene could be detected in transiently transfected cells by western blot analysis, its expression level was relatively low and insufficient for further protein purification study (fig. 1, lane 2) . thus, to enhance s1 protein expression, we first synthesized the full-length, codon-optimized s1 gene and constructed a panel of expression plasmids encoding the codon-optimized rs1-ig (s1 ) and two c-terminally truncated s1 variants: rs1 25-543 -ig and rs1 25-432 -ig. these constructs were independently transfected into hek-293t cells, and expression of each construct was robustly detectable by western blot analysis using an anti-human igg secondary antibody (fig. 1, lanes 3 to 5) , compared with a normal s1 protein (lane 2). this result indicates that codon optimization greatly increases the expression level of s1 upon transient transfection. subsequently, to easily produce preparative amounts of the s1 protein, sublines of pk-15 cells were established to stably express the recombinant codon-optimized s1 under the control of a retroviral ltr promoter. ten generated cell clones were initially collected and subjected to rt-pcr and western blotting to identify s1 gene expression at the mrna and protein level, respectively (data not shown). based on the results of western blot analysis, one pk-rs1-ig cell clone that consistently expressed the highest level of s1 was chosen for subsequent studies. to characterize pk-rs1-ig cells, intracellular and extracellular expression levels of s1 were examined by immunofluorescence, facs analysis, and western blotting. as shown in fig. 2a , specific cell staining was clearly evident when pk-rs1-ig cells were reacted with the antihuman igg antibody, confirming a consistent high expression level of the s1 protein. the majority of the cells consistently exhibited specific fluorescent signals, indicating a homogenous population of cells expressing s1 (fig. 2b) . time-course western blot analysis using culture supernatants revealed that the pk-rs1-ig cells stably express and cumulatively secrete high levels of approximately 160-kda s1 (fig. 2c ). in addition, the overall growth kinetics of s1-gene-expressing pk-15 cells were found to be similar to those of the parental pk-15 cells, indicating that s1 expression has no effect on cell growth (data not shown). further, the recombinant s1 protein tagged with the human igg fc domain expressed in the supernatants of stable pk-rs1-ig cells was purified using protein a sepharose beads. the purified s1 protein was detectable at a high level by ponceau s staining, and this was confirmed by immunoblotting with anti-human igg antibody (fig. 2d ). using our purification and concentration procedures, we were able to obtain approximately 15 lg of the s1 protein from 1 ml culture supernatant of pk-rs1-ig cells cultivated in one well of a 6-well plate for 72 h. antibody response of the recombinant s1 immunogen in rabbits rabbit antisera were collected before immunization (preimmune) and at each boost at 2-week intervals. the serum samples at 1:10,000 to 1:100,000 dilutions were examined for binding to the recombinant fusion protein rs1-ig or the authentic s protein by western blot analysis. the antiserum collected at the second boost reacted with the purified rs1 protein in a concentration-dependent manner (fig. 3a, top panel) , similar to the binding capacity of the anti-igg secondary antibody, which can directly recognize the protein fused to the fc region of human igg1 (middle panel). the antisera were found to retain nearly equivalent reactivity at the third and fourth boosts (data not shown). in contrast, the rabbit antiserum bound weakly to the authentic s protein prepared from cells infected with a pedv vaccine strain (top panel), whereas an mab raised against the vaccine strain reacted strongly with the authentic s protein (bottom panel). the rabbit antisera were further tested for their neutralizing activity against the pedv sm98-1 vaccine strain, which can be grown in vero cells in the absence of trypsin. as shown in fig. 3b , the antisera collected at the final boost at 1:16 dilution fully protect vero cells from sm98-1 infection (left panel), whereas pig hyperimmune sera raised against the vaccine virus were highly effective in inhibiting virus infection with neutralizing antibody titers of [1:256 (right panel). the rabbit sera at the second and third boosts possessed comparable neutralizing activities against the vaccine strain (data not shown). taken together, our data indicated that the recombinant s1 immunogen elicits potent antibody responses in immunized rabbits. however, the rabbit antisera strongly recognized the homologous s1 protein representing the s protein of field isolates but recognized the heterologous s protein of the pedv vaccine strain inefficiently, suggesting that there are antigenic differences between the vaccine strain and field pedvs. to determine the immunogenicity of the s1-based subunit vaccine, pregnant sows assigned to four groups were immunized intramuscularly, as outlined in table 1 . serum samples were collected at 6 weeks and 3 weeks prior to farrowing and at delivery and were subjected to a serum neutralization test against the pedv vaccine strain. nonimmunized sows showed only minimal neutralizing antibody titers, whereas immunized sows exhibited gradually increased neutralizing antibody titers, which increased considerably after the final vaccination (fig. 4) . briefly, sows immunized with two doses of pedv live and inactivated vaccines at 2-week intervals (group 1) had neutralizing titers of [1:256 at delivery. in contrast, sows immunized with two doses of pedv live and s1 protein vaccines (group 2) or three doses of s1 protein vaccine (group 3) at 2-week intervals produced relatively lower neutralizing antibody titers of 1:16 to 1:32 compared to those in group 1. in addition, colostrum samples from each group were found to have neutralizing antibody titers comparable to those in the corresponding serum samples (fig. 4b) . these results were coincident with the rabbit study described above, since the rabbit antisera generated by immunization with the recombinant s1 protein also contained low levels of neutralizing antibodies to the heterologous pedv vaccine strain. lastly, to assess the efficacy of immunization with the s1 protein, eight 4-to 5-day-old neonatal piglets from each sow were arbitrarily selected and challenged orally with wild-type pedv. clinical observations of death, diarrhea, and virus shedding in challenged piglets are summarized in table 2 . in the control group, one piglet died and the other piglet experienced severe watery diarrhea post-challenge. although none of the piglets from any of the groups of immunized sows died during the challenge experiment, the number of piglets exhibiting diarrhea after challenge varied depending on the group. all piglets delivered from group 1 sows showed mild-to-severe diarrhea lasting for the entire experiment at 2 days post-challenge. in contrast, piglets from sows in groups 2 and 3 experienced only mild diarrhea for 1 or 2 days after challenge or throughout the challenge experiment. likewise, all piglets from immunized sows (groups 1, 2, and 3) exhibited mild intestinal lesions, and viral antigens were detected only in their small intestines, whereas the majority of enterocytes over the entire villi in the control piglets (group 4) were affected by pedv, showing moderate-to-severe villous atrophy (fig. 5) . altogether, all immunization methods used in this study were capable of protecting passively immunized neonatal piglets against mortality and severe disease after challenge. however, immunizations involving at least one dose of pedv s1 protein vaccine (groups 2 and 3) were more efficacious than immunization of sows with two doses of pedv live and killed vaccines (group 1) in reducing the overall degree of diarrhea, in terms of the duration and severity, in the suckling piglets. pedv continues to have a severe economic impact in swine-raising countries in asia and, more recently, in the united states. vaccination against pedv is an important and effective prevention measure. pedv entry into host cells is mediated by the s glycoprotein on the viral surface, which interacts with the cellular receptor and induces direct membrane fusion. the pedv s protein is also responsible for inducing neutralizing antibodies in the natural host and hence is a logical target in the development of effective vaccines. furthermore, the s1 domain is a key functional portion of the s protein, which is associated with viral binding to host-cell receptors and contains neutralizing epitopes [17, 32] . therefore, the s1 protein of pedv is considered to be a potential candidate antigen for vaccination attempts. in the present study, the first aim was to stably express the full-length, codon-optimized s1 gene of pedv in porcine-origin cells and to evaluate the immunogenicity and efficacy of the recombinant s1 protein. our codon-optimization approach dramatically enhanced the expression level of the gene of interest upon transient transfection using a mammalian expression system. subsequently, we were able to successfully generate a stable pk cell line continuously producing large amounts of the codon-optimized s1 protein. following the purification and concentration processes, approximately 15-20 lg of the recombinant s1 protein could be consistently harvested from pk-rs1-ig cells in each well of a 6-well plate. since the antibody response is a critical indicator to assess the effect of a vaccine, we immunized rabbits with the s1-based immunogen prepared from culture supernatants of pk-rs1-ig cells and determined whether or not they developed humoral immunity. pedv-specific antibodies were strongly detectable in rabbit sera collected from the second boost, even at the highest dilution (1:100,000), when reacted with the recombinant pedv-s1 protein purified from s1-expressing pk-rs1-ig cells. in contrast, the binding capacity of the antiserum was dramatically reduced when the authentic s protein in wholecell lysates prepared from cells infected with sm98-1 was used as the antigen for western blotting. moreover, the rabbit antisera could completely block infection of sm98-1 only at a serum dilution of 1:16, whereas the pig sera raised against sm98-1 contained high levels of neutralizing antibody against the homogeneous virus ([1:256). since the pedv field isolate propagated in the current culture system is unavailable to us, we were unable to test the neutralizing capacity of those antisera against the field virus in this study. however, it is conceivable that rabbit antisera directed against the s1 protein may be more effective in neutralizing infection with the field virus than with sm98-1. on the other hand, the weak interaction and neutralizing activity of the anti-s1 rabbit serum against the sm98-1 s protein may be attributed to genetic variations between the s proteins of the vaccine strain and field isolates. indeed, the korean field isolates, including the challenge strain used in this study, were found to display a high degree ([10 %) of genetic heterogeneity, especially in the s1 domain, compared with the pedv vaccine strain sm98-1 [16, 18] . nevertheless, western blot and virus neutralizing assays showed that the recombinant s1 protein efficiently elicits humoral immune responses against pedv. in korea, several management strategies, including vaccination, have been employed to control pedv in pig farms. the most highly recommended immunization schedule involves two doses of attenuated live and inactivated killed vaccines in gilts at 2-to 3-week intervals before mating and in pregnant sows at 12 and 14 weeks of gestation. in the present study, we compared the efficacy of this common vaccination protocol and s1 protein-based vaccination. although all of the immunized sows developed neutralizing antibodies against sm98-1, only the sows immunized with live and killed vaccines (group 1) had high neutralizing antibody titers, ranging from 1:32 to 1:1024. however, consistent with the rabbit immunization study, sows in groups 2 and 3, immunized with at least one dose of the s1-protein-based subunit vaccine, developed weak neutralizing responses to the vaccine virus, with titers ranging from 1:8 to 1:32. successful protection against pedv is based on the presence of specific neutralizing antibodies in immune sows that are passively transferred to their piglets through colostrum and milk. our data showed that, regardless of vaccination group, all neonatal piglets from immunized sows survived after challenge with virulent pedv, suggesting that the s1 subunit vaccine provides effective lactogenic immunity to prevent mortality comparable to whole-virus-based vaccines. however, the s1based vaccination strategies, which produced relatively low neutralizing antibody responses, exhibited more-efficient protection, with respective to the duration and severity of diarrhea, than the common live and killed vaccination procedure. it is therefore plausible that the actual levels of neutralizing antibodies raised against the s1 subunit vaccine are underestimated by the vaccine-virus-based neutralization test. thus, improved diagnostic tools are needed to differentiate vaccinal antibodies from those resulting from natural infection with the field virus. for this purpose, the recombinant s1 protein purified from pk-rs1-ig cells could be further used as the diagnostic antigen in an enzyme-linked immunosorbent assay (elisa), and this aspect is currently under investigation. additionally, it is possible that the full-length s protein may induce stronger immune responses than s1 alone because it contains multiple functional domains and neutralizing epitopes. in fact, meng et al. [20] have recently reported that the full-length pedv s gene induces a better immune response than the n-terminal half alone, using the recombinant dna plasmid in a mouse model. however, production of the full-length s protein may be unachievable in our system because its expression may cause cytotoxicity due to the presence of potential fusion activity in the c-terminal portion of the s protein. in conclusion, to the best of our knowledge, this is the first evaluation of the immunological and protective effects triggered by recombinant s1 protein in rabbit and pig models. the results presented here indicate that the recombinant s1 protein can elicit a specific antibody response and induce neutralizing antibodies, suggesting its excellent immunogenicity in the natural host. furthermore, challenge experiments revealed that the s1protein-based vaccine protected passively immunized suckling piglets against field pedv. despite the nationwide use of commercial live and killed pedv vaccines, swine herds in korea have continued to experience repeated outbreaks, leading swine practitioners and researchers to question their protective efficacy. based on the present and previous studies, we hypothesize that antigenic and genetic variation between the vaccine virus and field pedvs responsible for periodic outbreaks in herds may be the cause of the low efficacy or failure of vaccination. accordingly, current pedv vaccines manufactured from cell-adapted viruses should be reassessed to determine their efficacy and improved if necessary. although further studies with a larger number of animals will be needed to better evaluate the efficacy of the s1 protein vaccine and to optimize immunization procedures, the recombinant s1 protein has potential for use in improving or developing effective and safe vaccines for ped prevention. amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor the coronavirus spike 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diarrhea virus deadly pig virus slips through us borders contribution of the porcine aminopeptidase n (cd13) receptor density to porcine epidemic diarrhea virus infection letter to the editor retrospective study of porcine epidemic diarrhea virus (pedv) in korea by in situ hybridization a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs chinese-like strain of porcine epidemic diarrhea virus coronaviruses molecular cloning: a laboratory manual emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences proteolytic cleavage of peplomeric glycoprotein e2 of mhv yields two 90k subunits and activates cell fusion spike protein region (aa 636789) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan acknowledgments we gratefully thank hyeryun choe from harvard medical school for providing reagents. this research was supported by basic science research program through the national research foundation of korea (nrf) funded by the ministry of education, science and technology (2010-0002318). key: cord-298922-k568hlf4 authors: sun, dongbo; shi, hongyan; guo, donghua; chen, jianfei; shi, da; zhu, qinghe; zhang, xin; feng, li title: analysis of protein expression changes of the vero e6 cells infected with classic pedv strain cv777 by using quantitative proteomic technique date: 2015-06-15 journal: j virol methods doi: 10.1016/j.jviromet.2015.03.002 sha: doc_id: 298922 cord_uid: k568hlf4 recent outbreaks of porcine epidemic diarrhea virus (pedv) have caused widespread concern. the identification of proteins associated with pedv infection might provide insight into pedv pathogenesis and facilitate the development of novel antiviral strategies. we analyzed the differential protein profile of pedv-infected vero e6 cells using mass spectrometry and an isobaric tag for relative and absolute quantification. a total of 126 proteins were identified that were differentially expressed between the pedv-infected and mock-infected groups (p < 0.05, quantitative ratio ≥1.2), among which the expression of 58 proteins was up-regulated and that of 68 proteins was down-regulated in the pedv-infected vero e6 cells, involving in integrin β2/β3, cystatin-c. the gene ontology analysis indicated that the molecular function of the differentially expressed proteins (deps) was primarily related to binding and catalytic activity, and that the biological functions in which the deps are involved included metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases. among the disease-related functions, certain anti-viral pathways and proteins, such as the rig-i-like receptor, rap1, autophagy, mitogen-activated protein kinase, pi3k-akt and jak-stat signaling pathways, and integrin β2/β3 and cystatin-c proteins, represented potential factors in pedv infection. our findings provide valuable insight into pedv-vero e6 cell interactions. the porcine epidemic diarrhea virus (pedv) is an enveloped, single-stranded positive-sense rna virus that causes porcine epidemic diarrhea (ped), an acute and highly contagious enteric disease in pigs. ped is characterized by severe diarrhea, vomiting, dehydration, and a mortality rate of up to 90% in suckling piglets (pensaert and debouck, 1978) . ped was first reported in belgium and the united kingdom in 1978, and frequent outbreaks have occurred in various asian countries . since 2007, acute ped outbreaks have continually occurred in thailand, china, and the usa, which have resulted in substantial economic losses (puranaveja et al., 2009; li et al., 2012; chen et al., 2013; huang et al., 2013; marthaler et al., 2013; stevenson et al., 2013; yang et al., 2013; chen et al., 2014) . the continued outbreaks of ped, despite control efforts, have caused widespread concern. the pedv belongs to the genus alphacoronavirus, in the family coronaviridae and order nidovirales (belouzard et al., 2012) . previous studies have investigated various control measures to protect against pedv infection, such as vaccines, diagnostic tools, and therapeutic drugs (sun et al., 2008; ren et al., 2011; sun et al., 2012; zhu et al., 2013; guo et al., 2013; kim and lee, 2013) . various aspects of pedv infection remain unclear, for example, swine testis (st) cells expressing porcine aminopeptidase n of pedv receptor were not susceptible to pedv infection. african green monkey kidney (vero) cells are highly susceptible to pedv infection, and are widely used for the primary isolation and cultivation of pedv guo et al., 2014) . therefore, vero lineages are suitable hosts for understanding the mechanisms of pedv infection. proteomics techniques are effective tools for characterizing protein expression profiles, and have been used widely to investigate disease-associated proteins (hondermarck et al., 2008; boja et al., 2011; he et al., 2012; sun et al., 2013) . among current proteomics methods, quantitative high-throughput proteomics approaches are useful for the analysis of infection-associated proteins of pathogens (linde et al., 2013; papachristou et al., 2013; ye et al., 2013; zeng et al., 2015) . in our current study, we used a quantitative proteomics approach based on an itraq tandem mass spectrometry (ms/ms) technique to identify proteins differentially expressed between pedv-infected and mock-infected vero e6 cells. the functions of the differentially expressed proteins (deps) were analyzed to determine whether they might be associated with pedv infection. our findings provide valuable insight into the changes in cellular processes that occur during pedv infection. the cv777 strain of pedv, kindly provided by maurice pensaert at ghent university (merelbeke, belgium), was used in all of our experiments after being adapted to vero e6 cells, as previously described (hofmann and wyler, 1988) . the vero e6 cell-adapted pedv, the vero e6 cells, and the monoclonal antibody against the nucleocapsid protein (np) of pedv were stored at the diarrhea-related viruses section, division of swine infectious diseases, national key laboratory of veterinary biotechnology, harbin veterinary research institute of the chinese academy of agricultural sciences. the vero e6 cells were cultured in dulbecco's modified eagle's medium containing 10% fetal bovine serum (fbs) in 75-cm flasks at 37 • c in a 5% co 2 atmosphere. when the cells reached 70-80% confluence, they were inoculated with the pedv at a multiplicity of infection of 1 in presence of 5 g/ml trypsin. at 48 h postinoculation, the cells began to exhibit cytopathic effects (cpes) of viral infection, but no cells lysis or shedding had occurred. the cells were washed three times with cold phosphate-buffered saline (pbs, ph 7.4). a 1.5-ml aliquot of lysis buffer containing 4% sds, 1 mm dtt, and 150 mm tris-hcl (ph 8.0) was added to each flask, and the flasks were incubated at 37 • c for 5 min. the cell lysates were collected using a cell scraper, and boiled for 5 min. three cell lysate replicates were prepared for the pedv-infected (v1-v3) and mock-infected (c1-c3) vero e6 cells, and stored at −80 • c. western blotting was performed to confirm pedv infection by detecting the presence of the np of pedv in the vero e6 cells. aliquots of the cell lysates were subjected to sds-page on a 12% acrylamide gel, and the protein bands were transferred to a nitrocellulose membrane using a semi-dry transfer device (bio-rad, hercules, ca, usa). the membrane was blocked using 5% (w/v) nonfat dried milk in pbs at 37 • c for 1 h, before incubation in pbs containing the anti-np monoclonal antibody (1:2000 dilution) at 37 • c for 1 h. after washing three times with 5% tween 20 in pbs (pbst), the membrane was incubated in pbst containing a horseradish peroxidase-conjugated goat anti-mouse igg (1:4000 dilution) at 37 • c for 1 h. after washing three times with pbs, the membrane was incubated with enhanced chemiluminescence detection reagents (biotopped, beijing, china) at room temperature for 3 min, and the peroxidase-mediated luminescence was digitally captured using the molecular imager chemidoc xrs+ system (bio-rad) and the image lab software (bio-rad). to verify the differential expression of the selected deps, equivalent volumes of the cell lysate replicates from the pedv-infected (v1-v3) and mock-infected (c1-c3) vero e6 cells were pooled into the v and c samples, respectively, and western blotting was performed as described above, with the following exceptions: a 1:1000 dilution of the polyclonal antibodies anti-␤ tubulin, anti-integrin-␤3, anti-cystatin-c, anti-protein s100-a2, anti-apolipoprotein e4, and anti-centrin from rabbit (beijing biosynthesis biotechnology, beijing, china) was used as the primary antibody, and a 1:5000 dilution of the hrp-conjugated goat anti-rabbit igg (sigma-aldrich, st. louis, usa) was used as the secondary antibody. protein digestion of the samples was performed according to the fasp procedure described by wiśniewski et al. (2009) . an aliquot of each cell lysate containing 200 g of protein was combined with 30 l of std buffer containing 4% sds, 100 mm dtt, and 150 mm tris-hcl (ph 8.0). the detergent, dtt, and other low-molecular-weight components were removed by dilution in ua buffer containing 8 m urea and 150 mm tris-hcl (ph 8.0) and repeated ultrafiltration using microcon (30 kda) ultrafiltration units. the reduction of cysteine residues was blocked by the addition of 100 l of 0.05 m iodoacetamide to the ua buffer. the samples were incubated for 20 min in darkness before ultrafiltration. the microcon filters were washed three times with 100 l of ua buffer, followed by two washes with 100 l ds buffer containing 50 mm triethyl ammonium bicarbonate (ph 8.5). the final protein suspensions were digested using 2 g of trypsin (promega, madison, wi, usa) in 40 l of ds buffer overnight at 37 • c, and the digested peptides were collected as the filtrate. the peptide content was quantified based on absorbance at 280 nm using an extinction coefficient of 1.1 for a 0.1 mg/ml solution. the digested peptide mixture was labeled using the 8-plex itraq reagent (life technologies, carlsbad, ca, usa), according to the manufacturer's instructions. each itraq reagent was dissolved in 70 l of ethanol, and added to the digested peptide mixture. the samples were labeled as c1-113, c2-114, c3-115, v1-116, v2-117, or v3-118 . the samples were multiplexed, and vacuum dried. the itraq labeled peptides were fractionated by scxc using the akta purifier system (ge healthcare, waukesha, wi, usa). the dried peptide mixture was reconstituted, and acidified by the addition of 2 ml of buffer a containing 10 mm kh 2 po 4 in 25% acetonitrile (ph 2.7). the samples were loaded onto a 4.6 mm × 100 mm column packed with polysulfoethyl (5 m, 200å) chromatography resin (polylc, columbia, maryland, usa). the peptides were eluted at a flow rate of 1 ml/min using a gradient of 0-10% buffer b containing 500 mm kcl and 10 mm kh 2 po 4 in 25% acetonitrile (ph 2.7). the gradient elution consisted of 10-20% buffer b for 25 min, 20-45% buffer b for 5 min, and 50-100% buffer b for 5 min. the absorbance of the eluate was monitored at 214 nm, and fractions were collected at 1-min intervals. thirty fractions were combined into ten pools, and desalted using empore standard density spe c18 cartridges (sigma-aldrich, st. louis, mo, usa) with a bed diameter of 7 mm and a volume 3 ml. each fraction was concentrated by centrifugation in a vacuum, and reconstituted in 40 l of 0.1% (v/v) trifluoroacetic acid. all samples were stored at −80 • c until the ms analysis was performed. the lc-ms/ms experiments were performed using a q exactive mass spectrometer coupled to a proxeon biosystem easy nanolc (thermo fisher scientific, waltham, ma, usa). ten microliters of each fraction was injected for nanolc-ms/ms analysis. the peptide mixture (5 g) was loaded onto a c18-reversed phase column (15 cm × 75 m) packed with rp-c18 (5 m) resin in buffer a containing 0.1% formic acid, and eluted with a linear gradient of buffer b (80% acetonitrile and 0.1% formic acid) at a flow rate of 0.25 l/min for 140 min using the intelliflow technology. the eluate underwent electrospray ionization for the ms/ms analysis. the ms/ms instrument was run in the peptide recognition mode, and the spectra were acquired using a data-dependent top-10 method based on the selection of the most abundant precursor ions from the survey scan (300-1800 m/z) for hcd fragmentation. the determination of the target value was based on the predictive automatic gain control, and the dynamic exclusion duration was 60 s. survey scans were acquired at a resolution of 70, 000 at m/z 200, and the resolution for the hcd spectra was set to 17, 500 at m/z 200. the normalized collision energy was 30 ev, and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%. the ms/ms spectra were compared to the uniprot cercopithecidae database (107 051 sequences, downloaded november 25, 2013) and a decoy database using the mascot search engine, version 2.2 (matrix science, london, uk), embedded in the proteome discoverer 1.4 software (thermo electron, san jose, ca). the following parameters were used for protein identification: a peptide mass tolerance of 20 ppm; an ms/ms tolerance of 0.1 da; trypsin digestion; a missed cleavage value of 2; the fixed modifications included carbamidomethyl, itraq8plex(k), and itraq8plex(n-term); the variable modification was oxidation; and an fdr value ≤0.01. protein quantification was performed using the proteome discoverer 1.4 software based on the centroided reporter ion peak intensity. the average quantitative value of each protein in samples c1, c2, and c3 (mock-infection group) was used as the internal reference. the value of the quantitative ratio for each protein relative to the internal reference was calculated, and averaged to obtain the quantitative ratio (v/c) of the proteins identified in the treatment groups (unwin et al., 2010) . a protein was considered to be differentially expressed between the pedv-infected and mock-infected groups based on the following criteria: the protein had to be present in three replicates of both groups, the difference in the level of the protein between the two groups had to be statistically significant (p < 0.05), and the change ratio for the protein had to be ≥1.2 (yuan et al., 2012) . the expression of a protein with a v/c > 1.0 was considered to be up-regulated, and those with a v/c < 1.0 were considered to be down-regulated. the data were analyzed using a two-tailed, paired student's t test. the statistical analysis was performed using the excel 2007 software (microsoft, redmond, wa, usa). the deps were annotated using the blast2go, version 2.7.0, program (ashburner et al., 2000; quevillon et al., 2005; götz et al., 2008) . the deps were blasted against the kegg genes database (human). the gene ontology categories (gocs) were retrieved, and mapped to pathways in the kegg database (kanehisa et al., 2012) . table 1 the proteins identified from pedv-infected and mock-infected groups. the vero e6 cells inoculated with pedv displayed distinct cpes at 48 h postinoculation, including cell shrinkage, cell fusion, and a rounded cell morphology, but no cells lysis or shedding was observed (fig. 1a) . the immunoblotting analysis confirmed that the vero e6 cells were pedv-infected. the band corresponding to the np of pedv was detected in samples v1, v2, and v3, whereas none was detected in samples c1, c2, and c3 (fig. 1b) . the identified peptides, identified proteins, quantified proteins, known/uncharacterized proteins, and the goc annotations are showed in table 1 . a total of 3178 proteins, including 15 564 peptides, were identified in the pedv-infected and mock-infected groups using the itraq-ms/ms approach, among which 3171 (99.78%) were quantified, 1859 (58.50%) were known proteins, and 1319 (41.50%) were uncharacterized/putative proteins. based on the gocs, 2061 (64.85%) of the proteins were annotated as biological process, 2495 (78.51%) were annotated as molecular function, and 1917 (60.32%) were annotated as cellular components. the quantification and significance of the identified proteins are shown in fig. 2 . the changes in the levels of expression between the two groups were analyzed based on statistical significance. of the 3178 proteins identified, 2496 (78.54%) were not differentially expressed (p > 0.05), and 675 (21.24%) were expressed at statistically different levels between the pedv-infected and mockinfected vero e6 cells (p < 0.05), including 357 proteins (11.23%) with a p-value between 0.01 and 0.05, 227 proteins (7.14%) with a p-value between 0.001 and 0.01, and 91 proteins (2.86%) with a p-value <0.001. the proteins with a p-value <0.05 were also filtered based on whether the v/c or c/v was ≥1.2. based on these criteria, a total of 126 (3.96%) of the 3178 identified proteins were determined to have been differentially expressed between the pedv-infected and mock-infected groups (table 2) . among the 126 deps, 46.03% (58/126) were up-regulated, and 53.97% (68/126) down-regulated. the known proteins and uncharacterized/putative proteins accounted for 69.05% (87/126) and 30.95% (39/126) of the deps, respectively. the dep displaying the greatest increase in expression in the pedv-infected vero e6 cells was isoform 2 of the ovarian cancer immunoreactive antigen domaincontaining 1 protein (1:2.5), and the dep displaying the greatest decrease in expression in the pedv-infected vero e6 cells was cystatin-c (1:2.2). the gene ontology (go) database has been widely used for describing protein function in a standardized format. according to their gocs, the 126 deps were annotated as cellular component, biological process, or molecular function. the go annotations are shown in table 2 , and distributions of the go annotations are shown in fig. 3 . seventy-eight deps were distributed among 16 groups of biological processes (fig. 3a) . the metabolic process (go:0008152), cellular process (go:0009987), single-organism process (go:0044699), and biological regulation (go:0065007) groups contained the highest proportions of the biological process deps. there were more up-regulated proteins in the cellular component organization group (go:0071840) than down-regulated proteins. seventy-four deps were distributed among eight cellular component groups (fig. 3b) , among which the organelle (go:0043226) and cell (go:0005623) groups contained the highest proportion of cellular component deps. there were more down-regulated deps in the membrane group (go:0016020) than up-regulated deps, and there were more up-regulated deps in the macromolecular complex group (go:0032991) than downregulated deps. ninety-seven deps were distributed among eight molecular function groups (fig. 3c) , among which the binding (go:0005488) and catalytic activity (go:0003824) groups contained the greatest proportion of molecular function deps. the kyoto encyclopedia of genes and genomes (kegg) pathway is a collection of pathway maps that represent molecular interactions and reaction networks in cells. seventy-five of the 126 deps identified were annotated, and mapped to a total of six kegg pathway categories, which included the metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases pathway categories (fig. 4) . the annotations in the metabolism, organismal systems, and diseases pathway categories represented 32, 25, and 36 pathway groups, respectively (fig. 4a, b and f). the annotations in metabolism pathways category included the carbohydrate, energy, lipid, nucleotide, amino acid, glycan biosynthesis, cofactors and vitamins, biosynthesis of other secondary metabolites, and xenobiotics pathway groups (fig. 4a ). the annotations in the organismal systems category included the tolllike receptor (tlr) signaling (ko04620), rig-i-like receptor (rlr) signaling (ko04622), and natural killer cell mediated cytotoxicity (ko04650) pathway groups (fig. 4b) , which represent pathways related primarily to the immune response to virus infection. the largest number of deps in the cellular process category were mapped to the lysosome (ko04142) pathway group, all ten of which were down-regulated deps (fig. 4c ). the annotations in the genetic information processing category included pathway groups related to dna replication and repair, transcription, translation, and the folding, sorting, and degradation of proteins (fig. 4d ). the annotations in the environmental information processing proteins and one up-regulated protein. overall, more disease pathway groups were assigned to a single down-regulated dep than those assigned to up-regulated deps. the integrin (␤2 and ␤3 subunits) protein was annotated to the largest number of pathway groups (28), which included the organismal systems, environmental information processing, cellular processes, and diseases categories. the ␤ tubulin as loading control, three down-regulated deps cystatin-c, apolipoprotein e4 and centrin-2, two up-regulated deps integrin-␤3 and protein s100-a2, were selected to verify differential expression between the pedv-infected and mock-infected vero e6 cells. the immunoblotting analysis showed that the ratios of these proteins between the pedv-infected and mock-infected groups were consistent with those obtained using the quantitative proteomics analysis (fig. 5) . in our study, pedv infection significantly alters protein expression in vero e6 cells. the differentially expressed proteins (deps) annotated to virus infection-associated signaling pathways, autophagy, and virus entry-associated proteins were analyzed further to assess their potential roles in pedv infection. in mammals, the first line of defense against virus infection is the innate immune system. early antiviral responses are initiated upon the recognition of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs), resulting in the production of interferons for the innate immune response and the maturation of dendritic cells for establishing acquired immunity (yokota et al., 2010) . the prrs are grouped into the tlrs, rlrs, and nucleotide binding-oligomerization domain-like receptors. our results showed that pedv infection induced the deps that participated in six signaling pathways involved in viral infection, including the rlr, rap1, pi3k-akt, mapk, jak-stat, and tlr signaling pathways. the pedv is an enteric virus that infects the intestinal epithelial cells (iec) of swine, causing severe diarrhea. hirata et al. (2007) reported the rig-i signaling pathway plays an important role in antiviral innate immunity mechanisms in iecs. sheikh et al. (2013) reported the rap1a signaling pathway was associated with secretory diarrhea. the jak-stat signaling pathway regulates the adaptive and innate mechanisms related to mucosal immunity (heneghan et al., 2013; wang et al., 2013) . our results showed that deps induced by pedv infection in vero e6 cells involved in the rlr, rap1, and jak-stat signaling pathways. it has been reported that the tlr, mapk, and pi3k-akt signaling pathways play roles in host cell responses to coronaviruses (mizutani et al., 2004; integrins are cell surface ␣/␤ heterodimeric glycoproteins that contribute to a variety of cellular functions (stewart and nemerow, 2007) . combinations of the various isotypes of the ␣ and ␤ subunits of integrins generate more than 20 different integrin proteins. previous studies have shown that various integrin molecules are used as receptors for virus attachment (stewart and nemerow, 2007; sun et al., 2013) . in our current study, the expression of integrin-␤2 and -␤3 was down-regulated and up-regulated, respectively, in response to pedv infection. the upregulation of integrin-␤3 expression is consistent with that observed in response to dengue virus infection (zhang et al., 2007) . our pathway analysis revealed that both integrin-␤2 and -␤3 are involved in 28 pathways that contribute to organismal systems, environmental information processing, cellular processes, and diseases. the integrin ␣v␤3 protein has been shown to serve as an entry receptor for various viruses (guerrero et al., 2000; neff et al., 2000; chu and ng, 2004; parry et al., 2005; wang et al., 2005) , some of which bind the integrin through an rgd sequence in a viral structural protein to initiate infection (stewart and nemerow, 2007) . the s protein of pedv is a glycoprotein peplomer on the viral surface that plays an important role in receptor-mediated binding and cell membrane fusion. in our study, the integrin recognized sequences of pedv s protein was analyzed based on ruoslahti's (1996) report. the results indicated that four conserved integrin-recognized amino acid motifs (asp-gly-glu, lys-gly-glu, arg-leu-asp, and leu-asp-val) were found in the s proteins of various pedv strains (data not shown). these data suggest that integrin proteins may act as an infection associated protein for the attachment and entry of pedv. autophagy is an essential component of host defenses against viral infection (dong and levine, 2013) . maier and britton (2012) reported that ␤-coronaviruses induced autophagy. in our study, more deps were mapped to the autophagy pathway group than any of the other pathway groups. fifteen deps were mapped to the lysosome and phagosome pathways. of the 15 proteins, 12 (80%) were down-regulated deps. although the autophagy pathway plays an antiviral role in virus-infected cells, the autophagy machinery is exploited by certain viruses for viral evasion and propagation. our results showed that pedv infection induced the downregulation of the expression of many autophagy-associated proteins. therefore, pedv infection might inhibit autophagy in vero e6 cells, thus facilitating virus replication. previous studies have shown that the microtubule-associated protein 1b is a useful biomarker protein for autophagy (dong and levine, 2013) . we found that the expression of map1b was up-regulated 1.37-fold in the pedv-infected vero e6 cells. these results suggest that the pedv induces autophagy. cystatin-c has been shown to reduce the replication of certain viruses, including the poliovirus, rhinovirus, and human coronaviruses oc43 and 229e (korant et al., 1986; collins and grubb, 1991) . the cleavage of s protein has been shown to be essential for the induction of cell-to-cell fusion and coronavirus entry into cells (sturman et al., 1985) . shirato et al. (2011) reported the transmembrane type ii serine protease 2 enhanced infection of pedv in vero cells by increasing virus release. in our study, the reduced expression of cystatin-c might facilitate pedv replication and release through the activation of cysteine-associated proteases in vero e6 cells. apolipoprotein e4, galectin, clusterin, and transferrin receptor 1 have also been shown to be associated with virus infection (hishiki et al., 2010; peng et al., 2011; martin and uprichard, 2013; tripathi et al., 2013) , and may therefore function as infectionassociated proteins in pedv-infected vero e6 cells. additionally, the decreased in vitro expression of the adherens junction protein, such as cadherin, might be associated with a reduced integrity of pedv-infected intestinal epithelial cells in vivo. to the best of our knowledge, our study represents the analysis of the interactions between pedv and vero e6 cells using a quantitative proteomics technique. pedv infection-associated pathways and proteins are described and discussed based on the bioinformatics analysis of the differentially expressed proteins. our analysis of vero e6 cell responses to pedv infection identified relevant targets for subsequent in-depth studies of pedv pathogenesis, expand the current knowledge base regarding the interaction between the pedv and the host cell, and provide useful basic information about other coronaviruses. although the vero e6 cells are highly susceptible to pedv infection and facilitate experimental design and performance for proteomics, the vero e6 cell line is an interferondeficient cell line and not a pig cell line. so, the detailed functions of these pathways and proteins in pedv infection require further verification in the actual host cells of pedv. gene ontology: tool for the unification of biology mechanisms of coronavirus cell entry mediated by the viral spike protein evolution of clinical proteomics and its role in medicine detection and molecular diversity 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potential disease-associated factors identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein influenza a virus nucleoprotein induces apoptosis in human airway epithelial cells: implications of a novel interaction between nucleoprotein and host protein clusterin simultaneous analysis of relative protein expression levels across multiple samples using itraq isobaric tags with 2d nano lc-ms/ms porcine reproductive and respiratory syndrome virus nsp1␤ inhibits interferon-activated jak/stat signal transduction by inducing karyopherin-␣1 degradation integrin alphavbeta3 is a coreceptor for human cytomegalovirus universal sample preparation method for proteome analysis genetic variation analysis of reemerging porcine epidemic diarrhea virus prevailing in central china from quantitative proteomics by amino acid labeling in foot-and-mouth disease virus (fmdv)-infected cells the battle between virus and host: modulation of toll-like receptor signaling pathways by virus infection cytotoxicity evaluation of oxidized single-walled carbon nanotubes and graphene oxide on human hepatoma hepg2 cells: an itraq-coupled 2d lc-ms/ms proteome analysis proteome analysis of porcine epidemic diarrhea virus (pedv)-infected vero cells up-regulated expression of beta3 integrin induced by dengue virus serotype 2 infection associated with virus entry into human dermal microvascular endothelial cells expression and purification of the scfv from hybridoma cells secreting a monoclonal antibody against s protein of pedv this work is supported by the national natural science foundation of china (grant no. 31472209), the state national key laboratory of veterinary biotechnology (grant no. sklvbf201506/201302), and the program for new century excellent talents in heilongjiang provincial university (grant no. 1252-ncet-016). key: cord-313126-7hrjzapj authors: chen, fangzhou; knutson, todd p.; rossow, stephanie; saif, linda j.; marthaler, douglas g. title: decline of transmissible gastroenteritis virus and its complex evolutionary relationship with porcine respiratory coronavirus in the united states date: 2019-03-08 journal: sci rep doi: 10.1038/s41598-019-40564-z sha: doc_id: 313126 cord_uid: 7hrjzapj the epidemiology and genetic diversity of transmissible gastroenteritis virus (tgev) in the united states (us) was investigated by testing clinical cases for tgev by real time rt-pcr between january 2008 and november 2016. prevalence of tgev ranged between 3.8–6.8% and peaked during cold months until march 2013, in which prevalence decreased to < 0.1%. nineteen complete tgev genomes and a single strain of porcine respiratory coronavirus (prcv) from the us were generated and compared to historical strains to investigate the evolution of these endemic coronaviruses. sixteen of our tgev strains share 8 unique deletions and 119 distinct amino acid changes, which might greatly affect the biological characteristics of the variant tgev, and resulted in a “variant” genotype of tgev. the “variant” genotype shared similar unique deletions and amino acid changes with the recent prcv strain identified in this study, suggesting a recombination event occurred between the ‘‘variant’’ tgev and prcv. moreover, the results indicate the “variant” genotype is the dominant genotype circulating in the us. therefore, this study provides insight into the occurrence, origin, genetic characteristics, and evolution of tgev and prcv circulating in the us. is also associated with protease and adp-ribose 1"-monophosphatase activities 19 . the functions of other nsps of tgev are unknown. the s glycoprotein attaches to the host cellular receptor porcine aminopeptidase n (papn) or sialic acid, induces cellular fusion, stimulates neutralizing antibodies, and has hemagglutination activity [20] [21] [22] [23] . the papn-binding domain of the s protein has two major antigenic sites, a and b 24 . in addition, deletions in the orf3 gene led to attenuation and reduced pathogenesis of tgev and in vivo 25 . in 1984, porcine respiratory coronavirus (prcv), which is believed to have evolved from tgev since prcv and tgev shared a high nucleotide percent identity, was first identified in belgium. compared to the s gene of tgev strains, the s gene of prcv has 621-681 nt deletions at n-terminal, including in strains from asia 26 . thus, the s gene is used to differentiate prcv from tgev. recently, a novel strain of prcv (oh7269 28 ) was identified in the us, but the origin and evolutionary relationship to current tgev strains in the us is unknown. while the decline of tgev is believed to occur in response to partial immunity from prcv infections [29] [30] [31] [32] , the united states swine industry also made significant changes (increased biosecurity, 3 site production model, etc.) to raise healthier pigs, which may have contributed to the reduction of tgev infections as well. the porcine enteric coronaviruses (including tgev, porcine epidemic diarrhea virus [pedv] , and porcine deltacoronavirus [pdcov]) cause similar clinical presentation, and co-infection of these enteric coronaviruses can occur 33 . in 2010, a highly virulent pedv emerged in china 34 and later spread to the us in april 2013 35 . within less than a year, pdcov was identified in the us 36, 37 . pedv quickly spread throughout the us 38 , and by march 2014, approximately 41% of the sow herds were infected with pedv 39 . while the identification of the pedv lead to an increased biosecurity measure within the us swine industry, prevalence of pedv in the sow herds did not significantly decrease until july 2014 39 . within the past couple of years, a chimeric tgev and pedv virus (consisting of a tgev backbone and the spike of pedv) was identified in multiple countries in europe [40] [41] [42] , illustrating the potential emergence of a chimeric tgev and pedv virus in the us. the occurrence and genetic diversity of tgev was investigated prior to and after the identification of pedv and pdcov in the us from the diagnostic cases submitted to the university of minnesota veterinary diagnostic laboratory since pedv recently emerged in the us, and a chimeric tgev and pedv virus was identified in europe. nineteen tgev strains (us, n = 18 and mexico, n = 1) and a single prcv strain from the us were sequenced, analyzed, and compared with other global tgev and prcv strains to characterize historical and currents strains. this research will further our understanding of the occurrence, genetic variability, and evolution of an endemic coronavirus in the us and will provide guidance for future efforts to prevent, monitor, and control endemic coronaviruses. decline of tgev positive cases from 2008-2016. between january 2008 and november 2016, 29,397 porcine enteric cases, distributed across 41 states in the us and mexico, were tested for tgev by real time rt-pcr, and 2.3% of the cases (n = 667) were positive for tgev ( table 1 ). the percentage of tgev positive cases was 4.0% in 2008, increased to 6.8% in 2010, and decreased to 0.4% in 2014 (table 1) . after the introduction of pedv in the us, the prevalence of tgev decreased further to less than 0.1%. positive tgev cases were detected in the main pig raising regions (midwest, south-central, and southeast) of the us (fig. 1a) between january 2008 and november 2016. most of the cases (n = 13,190) were from minnesota where 2.7% (n = 356) were positive for tgev. the percent of positive tgev cases per state ranged between 0.4-20.6%, with the highest percentage found in tennessee. a seasonality trend occurred with the positive cases between winter and spring (november to april) compared to summer and fall (may to october) (fig. 1b) . genomic characteristic, entropy and recombination analyses. the genomic nucleotide sequence alignments of the 37 tgev and 3 pcrv strains revealed two main genotypes (traditional and variant genotypes) ( fig. 2a) there were 8 major regions of insertion or deletion (indels) between the traditional and variant tgev strains. in the variant group, 3 deletion regions occurred within nsp3, 2 deletion regions occurred between the s and orf3a genes, 1 deletion regions occurred in orf3a genes, 1 deletion region occurred between the orf3a and orf3b genes and a single deletion occurred in the m genes ( table 2 and fig. 2b) . interestingly, the same orf3a and orf3b deletions were present in some of the historical tgev strains. however, these deletions were not present in the tgev strain purdue, which was used to create the attenuated tgev vaccine in the us. the variant tgev strain illinois139/2006 had a 128-nucleotide deletion in orf3a, which resulted in the truncated protein compared with our variant tgev strains. the traditional tgev strains z/1986, hb/1988, and mex/145/2008 shared the same 6 nucleotide deletions in the s gene that were present in the tgev strain, purdue. surprisingly, the pcrv strains contained an assortment of these deletions in their genomes, and compared to the historical tgev strains, the tgev strains from our study (excluding z/1986, hb/1988 and mex/145/2008) contained 8 deletions and 119 amino acid changes similar to the recently reported prcv strain oh7269 and the single prcv strain minnesota155/2016 from our study. www.nature.com/scientificreports www.nature.com/scientificreports/ to determine the level of nucleotide or amino acid variation across the tgev genomes, entropy analysis was conducted with an alignment of the complete genomes and concatenated amino acid sequences (fig. 3) . based on the level of diversity in the dataset and previous work 43 , entropy values greater than 0.7 were considered highly variable for the nucleotide and amino acid sequence alignments. the orf1 and s genes had the highest number of nucleotides with entropy levels above 0.7 (n = 20 and n = 12, respectively) while the orf3b, e and, orf7 genes lacked diversity in nucleotide positions (fig. 3a ). within orf1, the nsp2 and nsp3 had the highest number of nucleotide positions with entropy values greater than 0.7 (n = 4 and n = 6, respectively) while a single position was identified in nsp4, nsp6, nsp8, nsp12, nsp14, and nsp15. within the amino acid alignment, the m, n and orf7 genes lacked positions with entropy levels above 0.7 while orf1 and s proteins (n = 10 and n = 9, respectively) had residues with entropy values higher than 0.7 (fig. 3 ). there were 7 high-entropy positions at orf1b www.nature.com/scientificreports www.nature.com/scientificreports/ gene in the nucleotide sequence entropy value analysis while entropy value higher than 0.7 were lacking in the amino acid sequence entropy value analysis. recombination analysis was performed with the 37 tgev and 3 prcv strains. a single recombination event was detected by chimera, bootscan, maxchi, and siscan and rdp within the dataset between the recently identified variant tgev strains and the novel prcv strain minnesota155/2016 (fig. 4) . the tgev variant oklahoma147/2012 shares a high nucleotide identity of the first 19,941 nucleotides (breakpoint) with prcv strain minnesota155/2016 while the remainder of the genome shares a high nucleotide identity with tgev strain minnesota141/2007, indicating a complex nucleotide relationship between prcv and tgev. phylogenetic analyses of tgev and prcv strains. phylogenetic trees of complete genome sequence (n = 40) and complete s gene (n = 56) revealed two distinct genotypes, representing the traditional and variant tgev strains from the us (fig. 5a,b, respectively) . in the complete genome and s gene phylogenetic trees, the traditional genotype contained historical strains from the us, the recent strain from mexico, and historical and recent strains from china. from our study, the 16 recent tgev strains from the us formed the variant tgev group, which share a common ancestor with the prcv strain ohio-oh7269/2014. also, the variant tgev strains were identified in the three main swine production regions of the us (midwest, south-central, and southeast) indicating substantial geographical distribution. interestingly, whole genome phylogenetic analysis of prcv strain minnesota155/2016 clustered within the variant genotype and not with historical prcv strains, indicating significant genetic diversity within prcv strains circulating in the us. since additional partial s gene sequences (first 1383 nt) were available from geographically different locations, a partial s gene phylogenetic tree was constructed to further investigate the global evolutionary relationship between tgev and prcv strains (fig. 5c) . the traditional tgev group consisted of tgev strains isolated from the us, china, japan, south korea, england and mexico. the variant tgev group consisted our 16 us tgev (fig. 6a) . the different amino acid between traditional and variant tgev strains were highlighted in the predicted crystal structure of rbd ( fig. 6b-d) . four of eight amino acid substitutions within the rbd (l626f, f628l, v642a, and d649e) were exposed on the viral protein while two of the four amino acid changes in papn (d600e and t602a) were exposed on the viral protein ( fig. 6b-d) . porcine enteric coronaviruses (pedv, pdcov, and tgev) are significant, emerging pathogens causing severe enteric diseases in the global swine industry. while global historical strains of tgev caused severe enteritis and prcv is endemic in europe, asia, and the us, the mortality rate due to tgev infections has declined in these countries 3, 44, 45 . however, a virulent tgev or prcv strain could emerge since the epidemiology of tgev and prcv is different in china. in 1992, prcv was first reported in china 46 and was not identified or reported in china afterwards while tgev is constantly reported as a significant viral pathogen for the chinese pig industry 10, 15, 16 . the different clinical status of tgev in china and the western countries could be due to the stringent biosecurity measures in western countries compared to china. tgev was consistently detected in the us between 2008 and 2016, but after the introduction of pedv into the us, the swine industry significantly increased their biosecurity to prevent and control pedv infections and the prevalence of pedv were reduced after march 2014 in us 39,47,48 , which indirectly may have reduced the prevalence of tgev to less than 0.1%. our study illustrates a seasonality pattern with tgev in the us with infection peaking during cold months, which has been identified in our swine pathogens as well 37, 45, 46 . while tgev has been identified throughout europe 1, 8, 49, 50 , asia 11, 15, 16, 45 , and north america 5,6 , only the variant tgev genotype was detected in us pig farms since 2006, suggesting that variant tgev is the dominant genotype currently circulating in the main pig raising midwest, south-central, and southeast regions. in addition, the first tgev genome from mexico was characterized, which helps us to understand the evolutionary relationship of tgev strains from different countries. surprisingly, the mex/148 strain is phylogenetically related to the traditional tgev, and not the variant tgev strain. given the close geographical proximity of mexico and us and that the mexican and us pedv strains are phylogenetically related 51 , the variant tgev strains could be circulating in mexico, but were not represented in our study since our study had only a single tgev strain from mexico. the circulation of tgev and pedv in the us indicates a chimeric virus could emerge in the us similar www.nature.com/scientificreports www.nature.com/scientificreports/ as the chimeric virus emerged in europe. characterizing of the current tgev strains will aid in understanding the emergence of virulent or chimeric tgev strains in the us, if such an event would occur in the future. prcv was first identified in belgium in 1984 44 and has been identified multiple countries including belgium 52 , china 53 , japan 26 , uganda 12 and the us 37 . the evolutionary relationship between the recently identified prcv strain (ohio-oh7269/2014) and our prcv strain (minnesota155/2016) was unclear given the limited number of globally available prcv strains. ideally, additional prcv strains would have been sequenced, but routine screen of prcv does not occur since prcv is not considered as a significant pathogen in swine. the lab accidently isolated the prcv strain, which was added to the study since the genetic and evolutionary relationship of prcv is unknown in the us. the variant tgev strains shared the same nucleotide mutations and amino acid deletions with the novel prcv strain ohio-oh7269/2014, and a recombination event between prcv and tgev was identified, illustrating the complex evolutionary relationship between tgev and prcv strains in the us. www.nature.com/scientificreports www.nature.com/scientificreports/ hopefully, future us studies will assess the genetic and evolutionary of prcv strains to fully elicit the complex relationship with tgev. historically, a partial gene fragment or a single gene of tgev was sequenced 6 to differentiate viral strains due to limitations in technology and economic constraints. in this study, ngs technology generated the whole tgev genome, revealing a total of 8 unique indels located in nsp3, s, orf3a, orf3b and m protein, and these regions, excluding the m protein, had high entropy levels in our study (fig. 3a) . the nsp3, s, orf3a and orf3b proteins of tgev are associated with enteric tropism, immunogenicity, neutralization, sialic acid and other receptor binding activity ability, virulence, protease and adp-ribose 1 ″-monophosphatase activities [18] [19] [20] [21] [22] [23] [24] [25] , and changes in these proteins might reduce indels efficacy to cause clinical disease. mutations in the spike gene in other coronaviruses has impact on initiation of membrane fusion 54 , sialic acid binding activity 22 , confer resistance to virus neutralization 55 , and render trypsin independent for cell entry and fusion 56 , which could be contribution to a reduction of the overall fitness of the indels. the attenuation of murine coronavirus in the natural host occurred due to deletion of the he, 2a/he, and 4/5a genes 57 , epidemics of feline infectious peritonitis were contributed to deletions in the orf7a and orf7b of feline coronavirus 58, 59 , and disruption of functional expression of accessory protein occurred due to deletion of orf8 in human severe acute respiratory syndrome coronaviruses 60 . given the results from the previous studies, we hypothesis the indels are significantly attenuated compared to the traditional strains of tgev. further studies are needed to confirm this hypothesis and investigate the effect of these deletions and mutations on the biological characteristics and fitness of the new tgev genotype. in conclusion, a variant genotype of tgev is the predominant in the us and evolutionary relationship between tgev and prcv is complex. the decline of tgev positive diarrhea cases after may 2013 in the us may be indirectly associated with the outbreak of ped in the us via increased biosecurity. compared with the www.nature.com/scientificreports www.nature.com/scientificreports/ traditional tgev strains, the unique amino acid indels might affect the biological characteristics of the variant tgev, which could lead to changes in pathogenesis or chimeric virus in the future. veterinarians routinely send samples to the university of minnesota veterinary diagnostic laboratory (mnvdl) to determine potential pathogenic agents contributing to disease and to promote the health of swine herds. the samples may represent clinical outbreaks of diarrhea or are for routine monitoring of enteric pathogens in swine herds. upon arrival, ownership of the samples belongs to the mnvdl and client(s) confidentiality is retained by removing identifiers associated with client(s) information. between january 2008 and november 2016, a total of 29,397 samples, including fresh intestines and fecal samples from of clinical cases were submitted from 41 us states and mexico and tested for tgev by real time rt-pcr under standard operation procedures (available upon request) and various bacterial and viral enteric pathogens dependent on the veterinarian's request. request may include beta-hemolytic escherichia coli, non-beta-hemolytic escherichia coli, rotavirus a, b, and c, pedv, and pdcov. randomly selected historical positive tgev samples from the mnvdl (n = 17) were saved from previous enteric studies, two tgev samples from ohio (z and hb) were supplied from dr. linda saif. routine testing for prcv does not occur since it is not considered a major swine pathogen. however, a prcv isolate from mn was obtained from a nasal swab in 2016 during an attempted to isolate other viruses. all samples (n = 20) were selected for whole genome sequencing using next generation sequencing as previously described 36 . tgev prevalence information was exported from the mnvdl database and analyzed at the case-level with r software 61 using the ggplot2 62 and maps 63 . to investigate the differences in tgev strains and phylogenetic relationship with prcv, our 20 sequences and available sequences from genbank (table s1 ) were aligned using clustalw in geneious v9.1.4 64 . nucleotide and amino acid entropy analyses of the concatenation orfs (orf1a/b, s, orf3a/3b, envelope, membrane, nucleocapsid and orf7) was performed using the matlab 65 to determine regions of diversity within the alignment. entropy values higher than 0.7 in the nucleotide and amino acid alignments were identified as high variation positions 43 . recombination analysis was performed using recombination detection program (rdp) 66 v4 with rdp, bootscan, geenecov, siscan and maxchi algorithms (window size is 100 bp). recombination event was represented using similarity plot, with oklahoma147 as the query strain. the similarity plot was implemented in the simplot, v. 3.5.1 package 67 , using the two-parameter (kimura) distance model with a sliding window of 1000 bp and step size of 30 bp. whole genome (n = 40), the whole s gene (n = 56), and the partial s gene (first 1383 nt) (n = 70) (table s1 ) phylogenetic trees were constructed using the maximum likelihood algorithm, with a gtr nucleotide substitution model (bootstrap analysis with 1,000 replicates) by mega v6.06 68 . the s protein receptor binding domains (rbds) of tgev and prcv were modeled using the open-source modeling server swiss-model 69 provided by the swiss institute of bioinformatics. predicted tertiary structure of the rbds of tgev and prcv were modeled using prcv rbd (pdb accession no. 5szs) reported in the previous study. spike monomer and trimer models were performed using human coronavirus nl63 model as a template to theatrically visualize the changes in the residues since tgev and prcv templates are lacking 70 . 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recombination patterns in virus genomes full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination molecular evolutionary genetics analysis version 6.0 swiss-model: modelling protein tertiary and quaternary structure using evolutionary information glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy the pymol molecular graphics system, version 2.0 schrödinger, llc the study was funded by the mnvdl. the authors thank the faculty and personal at the university of minnesota veterinary diagnostic laboratory (mnvdl) for their technical services. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-40564-z. 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-302323-vvo8a4hp authors: wang, xiaobo; chen, jianfei; shi, da; shi, hongyan; zhang, xin; yuan, jing; jiang, shibo; feng, li title: immunogenicity and antigenic relationships among spike proteins of porcine epidemic diarrhea virus subtypes g1 and g2 date: 2015-11-26 journal: arch virol doi: 10.1007/s00705-015-2694-6 sha: doc_id: 302323 cord_uid: vvo8a4hp porcine epidemic diarrhea virus (pedv) is a coronavirus that infects cells lining the small intestine of swine, resulting in vomiting, diarrhea, and dehydration. the amino acid sequence of the spike (s) protein, which is the principal target recognized by host immune cells, has multiple mutations that distinguish the two pedv genotypes, g1 and g2. to determine whether these mutations lead to changes in antigenicity, as suggested by the failure of pedv vaccines in china, we first optimized the codons of typical s genes of the cv777 vaccine strain (g1 subtype) and lnct2 strain (g2 subtype) and expressed the recombinant full-length sequence of the s protein in a eukaryotic expression system. the igg antibody levels of serum from mice immunized with purified s protein were markedly high. antigenicity was compared by detection of polyclonal antibodies (pabs) against the virus and s protein using an enzyme-linked immunosorbent assay (elisa), an indirect immunofluorescence assay (ifa), and a serum cross-neutralization (sn) assay. reactivity with the pabs revealed significant cross-reactivity between the two pedv subtypes, although there was a twofold difference in the antigenic responses based on pab titers in the elisa and ifa. consistent with the variation in the s gene sequences, the sn titer suggested differences in the neutralization activity of the s protein between the two subtypes, which could explain the antigenic variation between the pedv subtypes g1 and g2. porcine epidemic diarrhea virus (pedv) is an acute and highly contagious enteric infectious disease characterized by vomiting, diarrhea, and dehydration in pigs of all ages, but especially in newborn piglets [1, 2] . pedv was first reported in england in 1971 [3] and then detected in japan in 1982 and subsequently confirmed in other southeastern asian countries [4] . in the usa, pedv was first reported in 2013 and has since rapidly spread throughout the country [5, 6] . in china, the incidence of pedv outbreaks has rapidly increased since 2010, especially among newborn piglets aged from a few hours to one week, often resulting in death due to watery diarrhea and dehydration [7, 8] . although the use of inactivated and attenuated vaccines may have helped to reduce the prevalence of disease, pedv has continually emerged, causing tremendous losses to the swine industry in china [9] . pedv, a member of the genus alphacoronavirus in the family coronaviridae, has a single-stranded positive-sense rna genome of approximately 28 kb encoding four structural proteins: the spike (s), nucleocapsid (n), envelope (e), and membrane (m) proteins [10, 11] . the s protein is a glycoprotein consisting of 1383-1387 amino acids (aa) on the viral surface, which is composed of four regions, a signal peptide (aa , an extracellular region, a transmembrane domain (aa 1,334-1,356), and a short x. wang [12] . unlike other coronaviruses (i.e., murine coronavirus and bovine coronavirus), the s protein of pedv cannot be cleaved into s1 and s2 domains after pedv maturation. thus, the s1 domain (residues 1-789) and s2 domain (aa 790-1383) of pedv are artificially defined based on homology to other coronavirus s proteins [13, 14] . the s protein is responsible for binding and fusion of the virus to the host cells. it therefore not only determines cellular tropism but also plays a vital role in inducing production of neutralizing antibodies in the host [15, 16] . the s gene of pedv is often used to evaluate the genetic diversity of coronaviruses [17, 18] . based on phylogenetic analysis of the full-length s sequences isolated in china, pedvs can be divided into two genotypes, g1 and g2 [19] . furthermore, pedv detected in china from 2010 to 2012 were mostly variant strains (most belonging to subtype g2) that differed genetically from the cv777 vaccine strain (belonging to subtype g1) [20] . however, many pig herds vaccinated with inactivated or attenuated cv777 vaccines still experienced high mortality rates among newborn piglets [17, 21] . therefore, further evaluation of the immunogenicity and antigenic relationships of pedv subtypes g1 and g2 is of vital importance. in the present study, we expressed the full-length s gene in a eukaryotic expression system, prepared anti-s polyclonal antibodies (pabs), and performed subsequent experiments to investigate differences in antigenicity between the pedv subtypes to determine if mutations in the s gene are the cause of immunization failure. the findings of this study also lay a foundation for further study of the function of the s protein and to facilitate development of pabs against pedv. the pedv g1 cv777 vaccine strain (genbank accession number: kt323979) was preserved at harbin veterinary research institute (harbin, china). pedv g2 strain lnct2 (genbank accession number: kt323980) was isolated in vero e6 cells in our laboratory. vero e6 cells and hek 293t cell were cultured in gibco ò dulbecco's modified eagle medium (dmem; thermo fisher scientific, inc., waltham, ma, usa) with 10 % fetal bovine serum (thermo fisher scientific, inc.) and maintained at 37°c in a humidified atmosphere of 5 % co 2 . the expression vector paav-ires-hrgfp was kindly provided by professor shibo jiang (fudan university, shanghai, china). x-tremegene hp dna transfection reagent was purchased from roche diagnostics gmbh (mannheim, germany). mouse monoclonal anti-flag ò antibody produced in mouse and fluorescein isothiocyanate (fitc)conjugated goat anti-mouse igg were purchased from sigma-aldrich corporation (st. louis, mo, usa). anti-dykddddk g1 affinity resin was purchased from genscript usa, inc. (piscataway township, nj, usa). a bca protein assay kit was purchased from thermo fisher scientific, inc. horseradish peroxidase (hrp)-conjugated goat anti-mouse igg was purchased from zsgb-bio (beijing, china), and irdye@680rd goat anti-mouse igg was purchased from li-cor biosciences (lincoln, ne, usa). full-length s sequences of the pedv cv777 vaccine strain, the classical chinese strain ch/s, and 20 epidemic chinese strains isolated from 2011 to 2015 were used for sequence alignments and phylogenetic analysis. homology among multiple aa sequences was analyzed using megalign sequence alignment software (dnastar, inc., madison, wi, usa). a phylogenetic tree was constructed from the aligned nucleotide sequences by the maximumlikelihood method using mega 5.05 statistical analysis software (http://www.megasoftware.net/). to improve the expression levels of the s protein, s gene sequences were codon optimized with the strategy of 'one amino acid-one codon', in which the most preferred codon of the host for a given amino acid is used in the target sequence without changing the amino acid sequences [22] . the nucleotide sequences of the cv777 and lnct2 s genes were edited in accordance with the codon table for homo sapiens in the codon usage database (http://www. kazusa.or.jp/codon/) for optimal expression in hek 293t cells and biochemically synthesized for cv777-s and lnct2-s by beijing genomics institute (beijing, china). full-length sequences of the s gene were cloned between the bamhi and xhoi restriction sites in the paav-ires-hrgfp plasmid. the recombinant plasmids paav-opti cv777 s-flag and paav-opti-lnct2 s-flag were extracted using a plasmid maxi kit (qiagen, hilden, germany). hek 293t cells were cultured to 60 % confluency in a sixwell tissue culture plate at 37°c and then transfected with 2 lg of paav-opti cv777 s-flag and paav-opti lnct2 s-flag together with 2 ll of transfection reagent. after 48 h, the hek 293t cells were collected, treated with ip lysis buffer (thermo fisher scientific, inc.), and centrifuged at 12,000 rpm for 30 min. next, proteins in the supernatants were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) on 10 % gels and transferred to a polyvinylidene difluoride (pvdf) membrane for 60 min. the pvdf membrane was blocked with 5 % skim milk for 2 h at 37°c, incubated with mouse anti-flag antibody (dilution, 1:5,000) at 37°c for 1 h, and then further incubated with irdye 680rd goat anti-mouse igg (dilution, 1:4,000) at 37°c for 45 min. the protein bands were visualized using an odyssey ò clx infrared imaging system (li-cor biosciences). hek 293t cells were cultured in 75-cm 2 bottles and transfected with 20 lg of paav-opti pedv-s-flag plasmids. at 48 h post-transfection, the cell lysate was harvested as described above, recombinant s protein was purified using anti-dykddddk g1 affinity resin, the cell lysate was loaded onto a column, and the soluble proteins were immunoprecipitated with anti-flag resin. the purified proteins were concentrated using amicon ultra centrifugal filters (molecular weight cutoff, 100 k; emd millipore, billerica, ma, usa). protein concentrations were measured using the bicinchoninic acid (bca) protein assay, and the final products were analyzed by sds-page to confirm purification of the target protein. all animal procedures were approved by the ethics committee of harbin veterinary research institute. three groups of 6-week-old female pathogen-free balb/c mice were respectively immunized subcutaneously three times, at two-week intervals, with 50 lg/mouse of recombinant cv777 s protein, lnct2 s protein formulated in freund's adjuvant (sigma-aldrich corporation) and phosphate-buffered saline (pbs) which was used as a negative control. blood was collected from the tail vein of three mice from each group, and serum samples were prepared prior to the first immunization and at one-week intervals, then stored frozen until assayed. detection of igg antibodies of immunized mice using an enzyme-linked immunosorbent assay (elisa) igg antibodies of the immunized mouse's serum samples were detected by elisa [23] . ninety-six-well plates (costar tm ; thermo fisher scientific, inc.) were coated with the purified recombinant cv777 and lnct2 s proteins (200 ng/well) in coating buffer (20 mm na 2 co 3 , 20 mm nahco 3 , ph 9.6) at 4°c overnight. the antigencoated plates were washed three times with pbs with tween ò 20 (pbst) and blocked with 200 ll of 5 % skim milk for 1 h at 37°c. the blocked wells were washed three times with pbst. anti-s lnct2 or anti-s cv777 polyclonal antisera (pabs) collected at different time points were diluted from 1:200 to 1:204,800 with pbs and added to the wells. the plates were incubated at 37°c for 1 h, washed three times with pbst, and hrp-conjugated goat antimice igg (1:5000) was added to each well. the plates were incubated at 37°c for 45 min and washed three times with pbst. then, 100 ll of abts [2,2 0 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] peroxidase substrate was added to each well, and the plates were incubated for an additional 20 min at 37°c in the dark. the reaction was stopped by the addition of 1 m naf, and the absorbance was read at a wavelength of 405 nm (od 405 ) using an enzyme-linked immunosorbent assay (elisa) plate reader. confluent monolayers of vero e6 cells in 24-well plates were washed three times with pbs. subsequently, each well was incubated with 10 4 times the median tissue culture infective dose (tcid 50 ) of strain cv777 and/or lnct2 in dmem supplemented with 10 lg of trypsin per ml. after incubation at 37°c for 48 h, monolayers were rinsed with pbs and fixed with 4 % paraformaldehyde at 4°c for 30 min. they were then washed twice with pbs and incubated with 0.2 % tritonx-100 at 37°c for 20 min. after washing with pbs, the wells were blocked with 1 % bovine serum albumin at 37°c for 2 h, washed three times with pbs, and incubated with cv777 or lnct2 anti-s pabs at 37°c for 1 h. to determine titers, serum samples were initially diluted 400-fold and then serially diluted twofold and rinsed three times with pbs. fluorescein-isothiocyanate-conjugated goat anti-mouse igg at a dilution of 1:2,000 in pbs was added to each well. after 45 min of incubation in the dark at 37°c, the plates were washed three times with pbs and incubated with 4 0 ,6diamidino-2-phenylindole at a dilution of 1:1,000 for 15 min in the dark at 37°c. after washing three times with pbs, the samples were examined using a digital inverted microscope (thermo fisher scientific, inc.). the cross-reactivity between two types of virus s protein and anti-s protein pabs were assessed by western blot analysis. briefly, vero e6 cells in 75-cm 2 bottles were infected with either the cv777 or lnct2 virus variation between porcine epidemic diarrhea virus subtypes 539 (multiplicity of infection = 1) for 48 h, treated with mammalian protein extraction reagent (thermo fisher scientific, inc.) for 20 min, and centrifuged at 12,000 g for 30 min. the supernatants containing total virus proteins were subjected to 10 % sds-page followed by western blot analysis as described above with anti-s pabs diluted to 1:1,000. the sn test was performed according to the fixed-virusdilution serum method described by reed and muench [24] . briefly, confluent monolayers of vero e6 cells in 96-well plates were washed three times with dmem. pabs against the s protein were inactivated at 56°c for 30 min and then diluted twofold starting at 1:25. they were then mixed with the same volume (50 ll) of 200 tcid 50 of virus diluted with dmem supplemented with 10 lg of trypsin per ml and incubated at 37°c for 1 h. subsequently, 0.1 ml of each virus-serum mixture was inoculated onto vero e6 cell monolayers in 96-well tissue culture plates. after 5 days, specific cytopathic effects (cpes) of cells were observed under an inverted microscope. sn titers were expressed as the titer of the highest serum dilution resulting in 50 % inhibition of pedv infection. one-way analysis of variance was used to determine statistical differences between groups. all statistical analysis was performed using graphpad prism version 5.0 software (graphpad software, inc., la jolla, ca, usa). probability (p) values of .05 and .01 were considered statistically significant and highly statistically significant, respectively. the maximum-likelihood phylogenetic relationships inferred from the aa sequences of the s protein confirmed that the two genotypes were pedv strains g1 and g2. the cv777 vaccine strain was subtype g1, while the lnct2 strain was subtype g2 (fig. 1) expression and purification of full-length recombinant pedv s proteins hek 293t cells in six-well tissue culture plates were transfected with recombinant plasmids (paav-cv777 s-flag and paav-lnct2 s-flag) containing unoptimized s genes and recombinant plasmids (paav-opti cv777 s-flag and paav-opti-lnct2 s-flag) containing optimized s genes. as the s protein was co-expressed with the hrgep protein, the fluorescence intensity reflected the expression level of the s protein. we found that the expression levels of the s proteins were significantly increased by codon optimization ( fig. 2a) . s proteins were detected by western blotting using anti-flag mouse monoclonal antibody (mab) as the primary antibody. the results showed that the recombinant proteins reacted specifically with the primary antibody, and each had a molecular weight of 250 kda (fig. 2b) . these results were in accordance with those of the sds-page analysis with the purified s protein (fig. 2c) . specific igg antibodies were detected in serum samples collected at different time points using an indirect elisa, as described previously. a shown in fig. 3 , igg antibody levels against the s protein in serum from immunized mice were significantly increased at 14 days post-immunization (p \ 0.05) and then highly significantly increased at 21 days, as compared with mice immunized with pbs (p \ 0.01), while there were no significant differences in titers of igg antibodies induced by cv777 and lnct2 s proteins at different time points (p [ 0.05). the elisa results indicated high cross-reactivity between the pabs and the two types of pedv s protein. the titers of anti-s pabs reacting with same subtype s protein reached 1:204,800, while the titers reacting with different subtype s proteins reached 1:102,400. as shown in fig. 4a and b, the od 405 values of anti-s cv777 protein pabs at different dilutions reacting with the cv777 s protein were much higher than those reacting with the lnct2 s protein. the ifa results indicated that pedv s protein was present in the cytoplasm of vero e6 cells (fig. 5a ). both pedv cv777 and lnct2 could induce obvious cpe: vero e6 cell fused with each other and formed syncytia. the structure of cells infected by lnct2 was still clear, while cv777-infected cells were all fused and no single cellular morphology was generally observed. the syncytia of cv777-infected cells were larger than those of lnct2infected cells (fig. 5a and b) . the ifa results confirmed that the anti-s pabs crossreacted with pedv strains cv777 and lnct2. next, we detected the fluorescence intensity of the antigenic crossreaction between the the anti-s protein pabs and the two pedv subtypes. consistent with the elisa results, which showed twofold differences between different pedv subtypes based on titers of the anti-s pabs, the highest titer of anti-s pabs reacting with pedv within subtypes (observing green fluorescence) reached 1:51,200, while the reaction of anti-s pabs and pedv across subtypes only reached 1:25,600 ( table 2) . anti-s pabs bound with the pedv s protein, which was about 220 kda, slightly smaller than the recombinant s protein, as was expected. the results of the western blot assay demonstrated that the anti-s pabs cross-reacted with strains cv777 and lnct2 (fig. 6a and b) , indicating that pedv s proteins had at least one common antigenic epitope across the g1 and g2 strains. the titers of anti-s pabs neutralizing pedv were calculated by the kärber method. the average titer of anti-s cv777 pab, which protected 50 % of cv777-infected cells, was 1:336, while the titer cross-neutralizing lnct2 was 1:150. the average titer of anti-s lnct2 pabs neutralizing lnct2 reached 1:230, while the titer of neutralizing cv777 was 1:387 (fig. 7) . the sn titer of anti-s cv777 pabs neutralizing cv777 strain was more than twofold higher than those neutralizing the lnct2 strain, while the neutralizing titers of anti-s lnct2 pabs were less than twofold of those reacting with the two strains. the ongoing epidemic of pedv has resulted in significant economic losses to the swine industry in asia as well as europe and north america. in china, mortality due to pedv infection can reach 80 %-100 % in piglets less than 10 days old [25] . in the usa, pedv infection has resulted in a loss of almost 10 % of the domestic pig population after only a 1-year epidemic period [26] . located on the surface of pedv, the s protein plays an pivotal role in recognizing receptors of target cells, thereby inducing production of neutralizing antibodies by activated host immune cells [15, 16] . although there have been relatively few studies using full-length sequences of the pedv s protein, most investigating the immunogenicity of the pedv s protein have focused on the s1 region [27] [28] [29] . as pedv s protein cannot be cleaved into s1 and s2 domains after virus maturation and s2 domain may also have potential neutralizing linear and conformational epitopes, the whole s protein with its native conformation might be a better immunogen than the s1 protein. previously, severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) spike proteins have been shown to produce high-titer antibodies in mice [30] . in the present study, we successfully expressed and purified the full-length s protein using a eukaryotic expression system. western blot analysis confirmed that the s protein was specifically detected by anti-flag antibodies (fig. 1b) . the ifa indicated that the s protein was expressed at higher levels in hek 293t cells through codon optimization (fig. 1a) , as demonstrated with other coronaviruses [31] . the pedv s protein has 27-30 potential glycosylation sites that help to maintain its biological function [32] . therefore, we chose a eukaryotic expression system that was particularly useful for the production of such a large multi-domain protein requiring complex folding machinery and post-translational modifications [33] so that the structure of the s protein was close to the naturally occurring conformation. other advantages of choosing a eukaryotic expression system were that the expressed fusion protein was soluble as the biological function was maintained [34, 35] . in the present study, we established a method to express and purify the pedv s protein, which can be used not only to evaluate immunogenicity but also to study s protein function. the igg levels of mice immunized with the s protein were significantly increased beginning on post-immunization day 14, and antibody titers reached 1:204,800 on post-immunization day 42 according to the elisa results (fig. 2) . the high igg antibody titers of immunized mice indicated that purified s protein may be a good candidate as an immunogen for pab production and diagnostic methods. since 2010, pedv outbreaks in several provinces have caused significant economic losses to the swine industry in china [7, 8] . although a cv777-inactivated vaccine has been used to immunize swine herds against pedv infection, its efficacy has been rather low [8] . therefore, it is of vital importance to develop a cv777 vaccine that can cross-protect against epidemic strains of the g2 subtype. previous studies have found differences in the antigenicity between pedv cv777 and strains isolated in the usa, and some pig antibodies and mabs showed 4-to 16-fold differences between the titers of the homologous and heterologous strains ccif and vn, as at least one epitope on the n-terminal region of pedv/tgev n protein contributed to this cross-reactivity [36] . however, the mabs used in the previous study were against the n protein, which is not the major antigen that induces production of neutralizing antibodies by host cells. in our previous study, we also found twofold differences in pig antibodies based on sn titers between strains cv7777 and lnct2 (data not shown). a comparison of sequences of two types of pedv g2 s protein from 14 recent chinese epidemic strains from 2011 to 2015 showed that they had 90.4 %-93.8 % sequence identity to the cv777 vaccine strain (table 1) , and sequence variations were concentrated in the n-terminal region of the s protein due to a point mutation, a deletion, and an insertion of four amino acids. the lnct2 strain we chose belonged to the g2 subtype, and its s sequence was 92.3 % identical to that of the cv777 vaccine strain (g1 subtype). the neutralizing epitope domain coe (aa 499-638) had eight different amino acids, and the the luminescence intensity was divided into four degrees: ???, strong; ??, moderate; ?, weak; -, negative epitope motif ss6 (764lqdgqvki771) had two aa mutations between the two pedv subtypes [19] . based on these differences in s protein sequences, we investigated whether mutations in the s protein resulted in a change in antigenicity between strains g1 and g2. the elisa and ifa results showed significant antigenic cross-reactivity between the two pedv subtypes. however, there was approximately a twofold difference in the antigenic responses based on the titers of the pabs. especially, the elisa results showed that reactivity of the anti-s cv777 pabs at different dilutions with the cv777 s protein was much greater than that with the lnct2 s protein, indicating that the ability of anti-s cv777 pabs to crossreact with different subtypes was lower than that of the anti-s lnct2 pabs. in addition, previous studies have shown that the pedv cv777 strain induces larger syncytia than korean strain dr13 and three us strains [36] [37] [38] . comparing the ifa results of the cv777 and lnct strains, we also found a similar phenomenon (fig. 7) . these results indicated that cv777 and epidemic strains may have differences in their ability to enter cells and induce fusion and may have different effects on cell morphology. the results of western blot analysis indicated that the pedv s proteins had at least one common antigenic epitope, allowing the s protein pabs to cross-react with different pedv subtypes. likewise, the results of the sn assay confirmed the presence of high titers of pabs against the s protein that were able to cross-neutralize other pedv subtypes. however, consistent with the elisa and ifa results, there were obvious differences in serum neutralizing responses across subtypes. the sn titer of anti-s cv777 pabs neutralizing strain cv777 was more than twofold higher than the pabs neutralizing strain lnct, while the differences in the neutralizing titers of anti-s lnct2 pabs across subtypes was less than twofold. these results indicate that the neutralizing epitopes of the lnct2 s protein had changed or that new neutralizing epitopes had emerged due to mutations in the s protein. in the subsequent experiments, we found that the 5f7 monoclonal antibody against the lnct s protein could react with strain lnct, but not strain cv777 (data not shown). further studies to identify the mab 5f7 epitope are underway. in summary, the full-length sequence of the pedv s gene was codon-optimized and successfully expressed in hek293t cells. the findings of this study confirmed that the recombinant s protein was highly immunogenic in cross serum neutralization (sn) between the s proteins of the two pedv subtypes and anti-s pabs. reciprocals of pedv neutralizing antibody titers were expressed as the dilution inhibiting pedv infection by 50 %, which was calculated as follows: log50 % neutralizing titer = l-d (s-0.5), where l is the log of the lowest dilution factor, d is the difference between the dilution factors, and s is the sum of the ratios of positive wells variation between porcine epidemic diarrhea virus subtypes 545 mice and could effectively induce production of antibodies, especially neutralizing antibodies. furthermore, we found significant antigenic relationships between the pedv s protein of subtypes g1 and g2, which indicated that the cv777 vaccine could cross-protect against g2 epidemic strains, at least to some extent. however, the antigenic and serologic neutralization responses against the s protein also reflected antigenic differences of twofold between the two pedv subtypes, based on anti-s pabs titers. therefore, future development of vaccines against emerging pedv strains 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fusion variation between porcine epidemic diarrhea virus subtypes 547 acknowledgments this work was supported by grants from the national natural science foundation of china (grant number 31172350), innovation capability project for research institutions of heilongjiang province (grant number yc2015d011). key: cord-276542-lxwls664 authors: pan, zhongzhou; lu, jiaxuan; wang, ningning; he, wan-ting; zhang, letian; zhao, wen; su, shuo title: development of a taqman-probe-based multiplex real-time pcr for the simultaneous detection of emerging and reemerging swine coronaviruses date: 2020-06-03 journal: virulence doi: 10.1080/21505594.2020.1771980 sha: doc_id: 276542 cord_uid: lxwls664 with the outbreak of the recent severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in 2019, coronaviruses have become a global research hotspot in the field of virology. coronaviruses mainly cause respiratory and digestive tract diseases, several coronaviruses are responsible for porcine diarrhea, such as porcine epidemic diarrhea virus (pedv), porcine deltacoronavirus (pdcov), and emerging swine acute diarrhea syndrome coronavirus (sads-cov). those viruses have caused huge economic losses and are considered as potential public health threats. porcine torovirus (ptov) and coronaviruses, sharing similar genomic structure and replication strategy, belong to the same order nidovirales. here, we developed a multiplex taqman-probe-based real-time pcr for the simultaneous detection of pedv, pdcov, ptov, and sads-cov for the first time. specific primers and taqman fluorescent probes were designed targeting the orf1a region of pdev, ptov, and sads-cov and the orf1b region of pdcov. the method showed high sensitivity and specificity, with a detection limit of 1 × 10(2) copies/μl for each pathogen. a total of 101 clinical swine samples with signs of diarrhea were analyzed using this method, and the result showed good consistency with conventional reverse transcription pcr (rt-pcr). this method improves the efficiency for surveillance of these emerging and reemerging swine enteric viruses and can help reduce economic losses to the pig industry, which also benefits animal and public health. coronaviruses, belonging to the family coronaviridae, order nidovirales, are single-stranded, positive-sense rna viruses with the largest genome among known rna viruses [1, 2] . according to genetic and antigenic characteristics, coronaviruses can be divided into four genera: α-coronaviruses, β-coronaviruses, γcoronaviruses, and δ-coronaviruses [3] [4] [5] . coronaviruses can cause respiratory and gastrointestinal diseases in animals and humans [6] . generally, αand β-coronaviruses only infect mammals, while γ-and δ-coronaviruses mainly infect birds, but some of them can also infect mammals [4] . of note, coronaviruses exhibit a pronounced propensity for interspecies transmission as illustrated by important emerging viruses in humans such as sars-cov and middle east respiratory syndrome-related coronavirus (mers-cov), as well as the recent sars-cov-2 that is causing a major human pandemic [7, 8, 9] . compared to many other species, pigs are in frequent contact with both humans and other animals such as pets, livestock and wild animals, and theoretically possess a greater chance to promote cross-species viral transmission. there are currently six coronaviruses that can infect pigs: pedv, transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), porcine hemagglutinating encephalomyelitis virus (phev), sads-cov, and pdcov [10] . with the exception of prcv and phev, the remaining four can all cause severe diarrhea, dehydration, and death in pigs [3, 11] . pedv is an αcoronavirus that causes long-lasting and extremely harmful swine diarrhea worldwide. pedv may have originated from bat coronavirus [12] . it has high genome variability and different degrees of virulence among different strains [13] . pedv strains can be divided into genotype g1 and genotype g2 with high genetic diversity. both genotypes can cause catastrophic herd harm, of which the g2 variant has spread rapidly worldwide since 2010 [14] [15] [16] [17] . sads-cov is also an αcoronavirus and is regarded to share a relationship with rhinolophus bat coronavirus hku2 [18] . it was first discovered in january, 2017 in guangdong, china. afterward, there were no other reemerging sads-cov strains detected until it appeared again in guangdong, china causing devastating damage to the local pig industry in 2019 [11] . this suggests possible periodic outbreaks of sads-cov could be observed in years to come. pdcov, a δ-coronavirus, was first detected in 2012 and caused pedv-like signs in pigs [19] [20] [21] 22] . birds can be considered as the natural hosts of δ-coronaviruses. based on their ability to spread across species, δ-coronaviruses may "jump" the species barrier and adapt to mammals [23] . it has been reported that δ-coronaviruses have also been detected in asian leopard cats and chinese ferret-badgers [10, 24] . ptov was first discovered by kroneman et al. in 1998 [2] . ptov was considered as a coronavirus for a long time before the ictv (international committee on taxonomy of viruses) classified it into family tobaniviridae, order nidovirales. ptov has a high positive rate in swine diarrhea samples [25] . however, it remains to be a potential pathogen in pigs [25] [26] [27] . it should be noted that frequent recombination events involving ptov have been discovered, some of which, especially those on the hemagglutinin-esterases (he) or spike (s) genes, which encode proteins relating to attachment and invasion, could lead to changes in pathogenicity or host-specificity [28] [29] [30] [31] [32] . so far, pedv, pdcov, sads-cov, and the coronavirus-like virus ptov have caused huge economic losses to the pig industry worldwide. in addition, their cross-species transmission ability may pose a threat to public health. in order to monitor these four viruses more efficiently, it is extremely important to develop a fast, simple, and accurate method for the detection and differentiation of those viral swine diarrhea pathogens. in clinical testing, multiplex real-time pcr has excellent performance. it has a larger detection capacity with higher speed and lower labor costs. however, the main reason hindering the application of multiplex real-time pcr on pathogen detection is the difficulty in assay design. here, we developed a multiplex taqman-probe-based real-time pcr method for these four emerging and reemerging swine enteric viruses. to ensure the detection performance of primers used in the multiplex real-time pcr method, all available sequences of pedv, pdcov, ptov, and sads-cov from genbank were obtained and analyzed. the conserved region of orf1a was chosen for designing primers and probes for pedv, ptov, and sads-cov, and the orf1b region was chosen for pdcov. four sets of primers and probes were designed using the oligo 7 (version 7.60) software (table 1) . primers and probes were synthesized by sangon biotech (shanghai) co., ltd. primers were also used for the construction of plasmid standards. all primers for conventional rt-pcr in this study referred to former reports [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] . clinical samples collected during 2017-2019 were preserved at −80°c in our laboratory. those samples were mainly from henan, jiangsu, anhui and guangdong provinces in china. pcr templates were dna or cdna, preserved at −20°c. all positive samples were identified by singleplex conventional rt-pcr in our laboratory and confirmed with dna sequencing by sangon biotech (shanghai) co., ltd. intestinal tissues or feces samples were treated with 3 to 5 volumes of pbs, mixed by shaking or vortex and supernatant was collected after centrifuged at 12,000 × g at 4°c for 15 minutes. nucleic acids were extracted using the rnapure virus kit (beijing comwin biotech co., ltd.) following the manufacturer's instructions. reverse transcription was performed using the hiscript iii rt supermix for qpcr (+gdna wiper) kit (nanjing vazyme biotechnology co., ltd.). the target fragments of pedv, pdcov, ptov, and sads-cov were amplified separately via pcr using the cdna obtained in the previous step with the phanta max super-fidelity dna polymerase (nanjing vazyme biotechnology co., ltd.) following the manufacturer's instructions. primers used in the amplification were the same as used in multiplex real-time pcr method. the pcr fragments were then cloned into the pmd18-t vector (takara biomedical technology (beijing) co., ltd.) through ta colony and confirmed by dna sequencing. the plasmid copy number was calculated and the plasmids were diluted from 1 × 10 7 copies/μl to 1 × 10 1 copies/μl. singleplex real-time pcr was performed for each virus using the 10-fold diluted plasmids to generate standard curves, based on which the e value (amplification efficiency), r 2 (correlation coefficient), and the standard equation were calculated. as shown in table s1 , the total volume of the singleplex real-time pcr reaction was 20 μl, consisting of 10 μl of 2× aceq qpcr probe master mix (aceq qpcr probe master mix kit, nanjing vazyme biotechnology co., ltd.), 0.4 μl of each forward and reverse primer (10 μm), 0.2 μl of taqman probe (10 μm), 2 μl of template, and the remaining volume of nuclease-free water. amplification was carried out on a roche lightcycler® 96 instrument (roche life science) using the following program: 95°c for 600 s; 40 cycles of 95°c for 10 s, 55°c for 10 s, and 72°c for 20 s. fluorescence signal was automatically collected at the end of each cycle. the singleplex real-time pcr assays for pedv, pdcov, ptov, and sads-cov described above were multiplexed into one reaction system consisting of 2× aceq qpcr probe master mix (aceq qpcr probe master mix kit, nanjing vazyme biotechnology co., ltd.), primers and probes for all four viruses, and templates. the multiplex reaction system was then optimized using different volumes of primers (10 μm) and probes (10 μm), and the optimal volumes of templates were determined. in the optimization stage, the final concentration of primers and probes in the system ranged from 1200 nm to 2400 nm and 200 nm to 1000 nm, respectively. the plasmid standards containing 1 × 10 3 copies/μl were chosen as templates. the same instrument and real-time pcr program were used as described above. to determine the limit of detection (lod) of the multiplex detection method, we performed real-time pcr reactions for each virus separately, using 10-fold serial dilutions of standard plasmid templates ranging from 1 × 10 7 copies/μl to 1 × 10 1 copies/μl. to confirm the detection limit, a multiplex real-time pcr was performed using plasmid templates of all four viruses at the concentration of the presumable detection limit with 23 replicates for each concentration. the lowest concentration that met the positive detection rate of 95% was considered as the reliable lod. to rule out potential false positives caused by other viruses that may present in the samples, positive samples for pedv, pdcov, ptov, sads-cov, tgev, porcine kobuvirus (pkv), classical swine fever virus (csfv), porcine sapelovirus (psv), porcine teschenvirus (ptv), and porcine rotavirus (porv) were tested using the multiplex real-time pcr detection method. all the cdna samples were previously synthesized and stored in our laboratory. the assay was repeated three times with a 7-days interval, using 10-fold dilutions of the standard plasmid of each pathogen ranging from 1 × 10 7 copies/μl to the lod with three replicates per reaction. each template was a mixture of standard plasmid of four pathogens at the same concentration. the coefficient of variation (cv) of the cq values of the samples at each concentration in the three experiments was calculated to estimate repeatability. plasmid standards of two, three or four target pathogens at the same concentration were randomly chosen and mixed as templates and detected using our new method. three concentrations (1 × 10 7 copies/μl, lod and 10 times the lod) of the plasmid standards were tested. to simulate actual co-infection events, we mixed the plasmid standards of the four target pathogens with one at 1 × 10 7 copies/μl and the other three at the lod and then detected the template mixture using our multiplex detection method. we tested 45 newly-collected samples from pigs showing signs of diarrhea and 56 previouslyconfirmed positive samples (31 with pedv only, 16 with pdcov only, 3 with ptov only, 1 with sads-cov only, 4 with pedv and ptov, and 1 with pedv, pdcov and ptov) using our multiplex detection method. the clinical performance of our established methods was evaluated by comparing the results with those of singleplex conventional rt-pcr. positive samples detected by either method were then confirmed through dna sequencing by sangon biotech (shanghai) co., ltd. as shown in table 1 , the sequences of the primers and probes designed in this study are presented. pedv, pdcov, ptov, and sads-cov probes were labeled with fam, vic, texas red, and cy5 separately. plasmid standards with concentrations ranging from 1 × 10 7 copies/μl to 1 × 10 1 copies/μl of each pathogen were selected to perform a singleplex realtime pcr (figure 1 ). the standard curves showed an acceptable amplification efficiency and correlation coefficient: pedv r 2 = 0.9957, e value = 99%; pdcov r 2 = 0.9990, e value = 100%; ptov r 2 = 0.9943, e value = 94%; and sads-cov r 2 = 0.9982, e value = 99%, indicating that our plasmid standards were qualified, and the primers and probes designed were efficient. among the four fluorophores used in the multiplex detection method, the fluorescence of cy5 was the weakest and very susceptible to interference from other fluorophores due to its own physical properties. therefore, the main purpose of the reaction optimization system was to improve the performance of the cy5 fluorophores without hindering other fluorophores, and to achieve the best amplification efficiency (i.e. the lowest cq value). we performed multiplex realtime pcr with final concentration of probes ranging from 200 nm to 1000 nm, and of primers ranging from 1200 nm to 2400 nm and compared the fluorescence intensity and cq values of each possible combination. we concluded that the best final concentrations for probes and primers are 1000 nm and 2400 nm, respectively (table 2 and figure 2 ). the maximum volume of template that can be loaded in this system is 4.8 μl as shown in table s2 . using the optimized system and the plasmid standards of each pathogen with concentrations ranging from 1 × 10 7 copies/μl to 1 × 10 1 copies/μl, we found that the method could identify positive samples with the concentrations as low as 1 × 10 1 copies/μl (figure 3a and table s3 ). however, follow-up experiments indicated that the detection rate of samples at 1 × 10 1 copies/μl was less than 95% of replicates (table 3) . thus, the reliable lod of this method is 1 × 10 2 copies/μl. in this experiment, the cutoff line of positivity was automatically decided by the lightcycler® 96 instrument. we set the cutoff line of positivity of our method at 35, which means samples with a cq value less than or equal to 32 (≤ 32) are regarded as positive, higher than 32 but less than or equal to 35 (32 < and ≤ 35) are invalid, higher than 35 (>35) are negative. the criteria were set based on two reasons. first, the lod of our detection method was 1 × 10 2 copies/μl, of which the cq value was around 32. second, some samples at 1 × 10 1 copies/μl were detectable, however, the detection rate was unqualified, and the cq values of those detectable samples were around 35. all following experiments complied with these criteria. the optimized method was used to detect pedv, pdcov, ptov, and sads-cov in positive samples and some other samples derived from pigs with diarrhea positive for tgev, pkv, csfv, psv, ptv, and porv. as shown in figure 3b and table s4 , the target pathogens were detected while the other pathogens were negative, indicating good specificity. as is shown in table s5 , most %cv values of the cq values of the plasmid standard were less than 1% (81/ 96) with only a few %cv values ranging from 1% to 5% (15/96), indicating that this multiplex detection method is stable. we selected plasmid standards with concentrations of 1 × 10 7 copies/μl, 1 × 10 3 copies/μl, and 1 × 10 2 copies/μl of different pathogens as templates to perform a co-infection simulation experiment. as shown in figures 4, 5, and 6 , the multiplex detection method could detect duplex, triplex, or quadruplex simulation co-infections of the target pathogens, even pathogens with different concentrations (figure 7) . two batches of clinical samples were tested by our established method to validate its performance in clinical use. the first batch consisted of 45 digestive tract samples recently collected from pig farms in china. the results of our method were 100% consistent with the results of singleplex conventional rt-pcr (table s6 ). the second batch consisted of 56 positive samples stored in our laboratory, of which 31 were positive for pedv alone, 16 were positive for pdcov alone, 1 was positive for sads-cov alone, and 8 were positive for ptov (not necessarily alone). our method was 100% (table s6 ). virus cross-species transmission from wildlife reservoirs poses a remarkable threat to human and domestic animal health [18, 43] . coronaviruses can cross the species barrier and gradually adapt to new hosts [44, 45] . for example, the recently emerged sars-cov-2 was estimated to have originated from bats spreading to wild animals or livestock and then to humans [46] . animals may potentially serve as mixing vessels for the generation of novel recombinant coronaviruses and facilitate the viruses to expand their host tropism to humans [47] . in current swine breeding practices, humans have close contact with pigs, which further increases the possibility of viral transmission to humans, posing potential threat to human health [10] . therefore, the detection of emerging and reemerging coronaviruses is of significance for farming and public health. among the four target pathogens in this study, pedv is a coronavirus with considerably high prevalence and frequent recombination and rapid evolution rate [13] . sads-cov and pdcov are two emerging coronaviruses and have been reported to be spreading and causing economic losses [11, 23] . ptov is regarded as a swine diarrhea virus that has not caused huge economic losses [25] . however, given the high recombination rate and evolutionary characteristics of coronavirus and torovirus, we cannot underestimate their potential threat [28] [29] [30] . it is very likely that a virulent strain could emerge and cause unpredictable damage to the pig industry [13] . thus, monitoring these currently less harmful viruses, such as sads-cov, pdcov, and ptov, is of great significance. pathogen monitoring nowadays largely relies on laboratory detection methods. singleplex conventional rt-pcr has been widely applied for pathogen detection [34] [35] [36] 38] . however, such kind of assays are not convenient for the simultaneous detection of co-infection of multiple pathogens, which hinders further study of viral recombination events [3] . given that co-infections of different coronaviruses and torovirus are common in the field and the high recombination frequency of coronavirus and torovirus, it is necessary to develop a fast, convenient detection method (e.g. multiplex realtime pcr) for the diagnosis of co-infections. pcr is fast, accurate and convenient for clinical sample screening. co-infection of all the four pathogens at different concentrations. a: the concentration of plasmid standard of pedv was 1 × 10 7 copies/μl and the others were 1 × 10 2 copies/μl; b: the concentration of plasmid standard of pdcov was 1 × 10 7 copies/μl and the others were 1 × 10 2 copies/μl; c: the concentration of plasmid standard of ptov was 1 × 10 7 copies/μl and the others were 1 × 10 2 copies/μl; d: the concentration of plasmid standard of sads-cov was 1 × 10 7 copies/μl and the others were 1 × 10 2 copies/ μl. two replicates were set per reaction. x-axis: cycle, y-axis: fluorescence. real-time pcr is better than conventional pcr due to its faster, more sensitive and accurate detection capacity [48] . the use of probes is the most obvious and critical difference [49, 50] . in general, conventional rt-pcr is less sensitive than real-time pcr. the lod of our multiplex real-time pcr detection method can reach as low as 1 × 10 2 copies/μl for each pathogen, while that of the singleplex conventional rt-pcr is generally around 1 × 10 3 copies/μl to 1 × 10 4 copies/μl [51] . likewise, multiplex conventional rt-pcr shows no noticeable advantage in terms of sensitivity [34, 52] . hui et al. developed a multiplex conventional rt-pcr assay for pancoronaviruses, in which the limits of detection were no less than 1 × 10 3 copies/μl [40] . zhao et al. developed a multiplex rt-pcr detection for csfv, porcine reproductive and respiratory syndrome virus (prrsv), pedv, and tgev. the limit of detection of this method was 1 × 10 3 copies/μl [52] . on the other hand, although multiplex real-time pcr combines high sensitivity and high detection efficiency, the design of qualified multiplex real-time pcr assays, especially of quadruplex quantitative real-time pcr assays, is challenging [48, 49, 53] . in multiplex real-time pcr assays, multiple sets of oligonucleotides exist simultaneously in the reaction system, increasing the possibility for nonspecific amplification, which poses high demands on the specificity of primers and probes. improvements in detection method can bring many benefits. accurate detection of virus at lower concentration enables the diagnosis and prevention of porcine diarrhea at an early stage. some virulent strains may cause severe signs at a low titer, and thus a sensitive detection method is indispensable in such situations. due to its lower false negative rate caused by lower lods, our new detection method enables more powerful surveillance over those four swine diarrhea viruses and, hopefully, can benefit the construction of pig farms with higher biosecurity. however, the improvement in sensitivity also increases the possibility of false positive results, which imposes higher requirements on the prevention of contamination during sampling and assay set up. good practice in the laboratory is necessary in order to get credible results. in short, an excellent and efficient detection method should accurately reflect the epidemiological data (e.g. the scale of the disease, the rate of transmission, and the severity of the epidemic), so as to be beneficial to the monitoring and prevention of the disease [53] . multiplex real-time pcr achieves better detection capability in less time and with lower labor cost, but its development is still challenging compared to singleplex conventional rt-pcr due to technical difficulties at the development stage. to the best of our knowledge, this is the first multiplex real-time pcr detection method for pedv, pdcov, ptov, and emerging sads-cov, which often present simultaneously among pigs. the multiplex detection method developed here can detect multiple pathogens in a single reaction, making detection for co-infection more convenient. this method can ensure good specificity and sensitivity, which will undoubtedly save labor and material costs. here, we developed a taqman-probe-based multiplex real-time pcr method for the simultaneous detection of emerging and reemerging pedv, pdcov, ptov and sads-cov of swine. the limit of detection can reach as low as 1 × 10 2 copies/μl for each pathogen with good specificity and repeatability. the application of this method in clinical detection will not only improve the detection capacity, but also reduce workload and cost, benefiting clinicians and epidemiologists. program ss provided the study design and experiment, other authors contributed to the experiment. all authors contributed to writing the manuscript. all authors have approved the final version of the manuscript. coronavirus genome structure and replication identification and characterization of a porcine torovirus a taqman-probe-based multiplex real-time rt-qpcr for simultaneous detection of porcine enteric coronaviruses coronavirus genomics and bioinformatics analysis bats and coronaviruses origin and evolution of pathogenic coronaviruses return of the coronavirus: 2019-ncov covid-19: epidemiology, evolution, and cross-disciplinary perspectives comparison of sars-cov-2 spike protein binding to ace2 receptors from human, pets, farm animals, and putative intermediate hosts bat-origin coronaviruses expand their host range to pigs the re-emerging 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recent advances real time quantitative pcr basic principles of real-time quantitative pcr development of a multiplex taqman probe-based real-time pcr for discrimination of variant and classical porcine epidemic diarrhea virus a multiplex rt-pcr assay for rapid and simultaneous detection of four rna viruses in swine molecular diagnostic techniques we thank professor huang yaowei (college of animal sciences, zhejiang university) and professor zhou jiyong (college of animal sciences, zhejiang university) for their guidance on our coronavirus research and the help they provided on biological materials. we thank haitao qi (new hope liuhe company) and ao liao (han shiwei company) for providing pig samples. we thank jiexiong xie (laboratory of virology, faculty of veterinary medicine, ghent university) for helping us improve the paper. the authors declare no competing financial interest. this work was financially supported by the national natural science foundation of outstanding youth fund in china (nsfc grant no. 31922081), the national key research and development key: cord-306502-jkqg1qal authors: dee, scott; clement, travis; schelkopf, adam; nerem, joel; knudsen, david; christopher-hennings, jane; nelson, eric title: an evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of naïve pigs following consumption via natural feeding behavior: proof of concept date: 2014-08-05 journal: bmc vet res doi: 10.1186/s12917-014-0176-9 sha: doc_id: 306502 cord_uid: jkqg1qal background: since its initial detection in may 2013, porcine epidemic diarrhea virus (pedv) has spread rapidly throughout the us swine industry. initially, contaminated feed was proposed as a risk factor for pedv; however, data were not available to support this theory. here we provide proof of concept of this risk by describing a novel means for recovering pedv-contaminated complete feed material from commercial swine sites and conducting an in vivo experiment to prove its infectivity. results: for on-farm detection of pedv rna in feed, paint rollers were used to collect material from at-risk feed bins from 3 clinically affected breeding herds. this material was tested by pcr and determined to be positive for pedv-rna (ct = 19.50-22.20 range). to test infectivity, this material was pooled (ct = 20.65) and a treatment group of 3-week old pedv-naïve piglets were allowed to consume it via natural feeding behavior. for the purpose of a positive control, piglets were allowed to ingest feed spiked with stock pedv (ct = 18.23) while the negative control group received pedv-free feed. clinical signs of pedv infection (vomiting and diarrhea) and viral shedding were observed in both the positive control and treatment group’ post-consumption with virus and microscopic lesions detected in intestinal samples no evidence of infection was observed in the negative controls. conclusions: these data provide proof of concept that contaminated complete feed can serve as a vehicle for pedv infection of naïve pigs using natural feeding behavior. porcine epidemic diarrhea virus (pedv) is an enveloped single-stranded positive sense rna virus belonging to the order nidovirales, the family coronaviridae and the genus alphacoronavirus (saif et al. [1] ). following detection in the us swine population during may, 2013, the virus spread rapidly across the country and 6317 cases of porcine epidemic diarrhea (ped) have been confirmed in 29 states as of may 3, 2014 [2, 3] . while little information is known regarding the routes of pedv transmission between herds, potential risk factors include infected pigs, contaminated transport and pedv-positive aerosols [4] [5] [6] . recently, contaminated feedstuffs have been proposed as a route of pedv transmission to naïve pigs but its current status is unclear [7] . while an initial report from the canadian food inspection agency indicated that consumption of pedv-positive porcine blood plasma caused disease in pigs, a follow-up study could not demonstrate that the feed pellets (complete feed) containing the blood plasma in question were capable of causing disease [8, 9] . despite this lack of evidence, dietary modifications to enhance the biosecurity of feed have been recommended to reduce this perceived risk [10] . as more data regarding the risk of pedv transmission via complete feed are needed, we conducted a study to test the risk of pedv-contaminated complete feed using a novel on-farm sampling method for virus detection in feed along with an in vivo experiment (swine bioassay) using at-risk feed material. the study was based on the hypothesis that contaminated complete feed can serve as a vehicle for pedv infection of naïve swine. during the period of january 9-13, 2014, clinical porcine epidemic diarrhea was diagnosed in 3 breeding herds following acute outbreaks of anorexia, diarrhea and vomiting in isolated groups of sows. these herds were part of an organized system of commercial pork production; farm a (4973 sows) was located in nw iowa, while farms b and c, 3390 sows and 3016 sows respectively, were located in sw minnesota. all 3 herds emphasized strict biosecurity, using protocols previously validated to reduce the risk of prrsv infection [11, 12] . once a diagnosis of pedv was confirmed, an investigation of each site was conducted to identify possible routes of viral entry. during the investigation, a consistent observation common to all 3 herds was noted. specifically, from january 6-9, 2014, all 3 farms experienced an unexpected feed outage which required an "emergency" delivery. the emergency delivery had been deposited into a designated external storage bin which sourced feed to a distinct subpopulation of the herd. following consumption of said feed, clinical signs became apparent only in the animals that had consumed this feed, i.e. no other signs were noted in other animals consuming other feed from other bins. based on this history, information regarding dates corresponding to recent feed deliveries, the location of the associated storage bin, the period of time between delivery of feed and clinical signs, the location of index cases in each farm, mill source and whether porcine byproducts were present in feed was collected during the investigation. in addition, all transport-related activities, diagnostic data pertaining to recent genetic introductions, and records of personnel and supply entry to each farm were reviewed. finally, as farms a and b were air filtered, an evaluation of filter integrity and inspection for the potential of air bypass (entry of non-filtered air through improperly sealed fans, etc.) was conducted. because of the potential link to feed, it was planned to sample the designated bin on each farm to determine whether pedv could be detected in "at-risk feed", which was defined as feed consumed by the index population. unfortunately, across all 3 sites, the majority of feed defined as "at-risk" had been consumed, leaving the designated bins nearly (or completely) empty. however, upon inspection of the bin lumen it was observed that clusters of feed material (feed particles and feed dust) were adhered to the interior walls. to access this material, a modification of a published method for sampling contaminated transport for pedv rna was devised [5] . specifically, synthetic woven paint roller pads, 23 cm in length, 0.95 cm nap length (sherwin williams, cleveland oh, usa) were attached to 3.6 m extension poles to access the cylindrical surface area of interior bin walls at multiple heights. to minimize environmental contamination of the roller prior to placement within the bin interior, a 4.4 l plastic bag (ziploc, sc johnson & son inc, racine, wi, usa) covered the roller during ascension of the bin ladder. following insertion of the roller into the bin lumen, the bag was removed and the roller was drawn across the inner walls, forcing large quantities of the adhered feed material to attach to the pad. in addition, if stored feed was present in a bin, the pad was drawn across the top layer to collect more material. upon completion of sampling, the bag was replaced over the roller and the entire sampling apparatus was removed from the bin. once on the ground, 200 ml of 7.2% phosphate buffered saline was poured into the bag, immersing the pad and promoting absorption of liquid. using manual pressure, liquid was then forced from the pad into the bag and a 10 ml aliquot was decanted into a 15 ml plastic falcon tube (becton dickenson, franklin lakes, nj, usa) for diagnostic testing. in addition to the sampling of the "at risk feed bin" on each farm, an "on-farm control bin" was also sampled. control bins were located within 10 m of the "at-risk bin" but had not received a recent feed delivery and animals consuming feed from these bins were not clinically affected. finally, to insure that the method did not generate false positive results, 8 feed bins across 4 pedv negative farms were also sampled. all samples were tested for the presence of pedv rna using a rt-pcr at the south dakota state university animal disease research and diagnostic laboratory (sdsu adrdl). a sample with a cycle threshold (ct) of less than 38 was considered pedv positive. the swine bioassay component of this study was conducted in biosafety level 2+ rooms at the animal resource wing (arw) at sdsu. all procedures involving animals throughout the study were performed under the guidance and approval of the sdsu institutional animal care and use committee. animals (n = 11, three-week old piglets) were sourced from a pedv-naïve herd and were tested on arrival to the arw via blood sampling and collection of rectal swabs from each pig. prior to animal arrival, all rooms (walls, ceilings, floors and drains) were monitored for the presence of pedv by pcr using sampling procedures previously described (8) . in addition, feed was sourced from a pedv-naïve farm and screened by pcr prior to use. for the purpose of the swine bioassay, 11 piglets were divided into 3 groups and house each group in a pen within a designated room as follows: treatment group: 5 piglets to be fed challenge material consisting of the pcr-positive feed bin samples from herds a, b and c. positive control group: 4 piglets to be challenged with feed spiked with stock pedv [13] . negative control group: 2 piglets to be fed a placebo (feed + saline). the study encompassed an 8-day period with challenge (consumption of designated feed material) occurring on day 0, followed by 6 days of diagnostic monitoring with necropsies conducted on day 7 post-challenge. piglets were offered free-choice access to challenge material on day 0 of the study, allowing for natural feeding behavior, rather than to administer the challenge via gavage. following iacuc approval, feed was withheld from all piglets for 12 hours prior to challenge. for the preparation of challenge for the treatment group, 30 grams of feed material from the pcr-positive bin samples from farms a, b and c was pooled and diluted in 30 ml of sterile phosphate buffered saline. the solution was vortexed for 2 minutes and then centrifuged at 16,000 g for 2 minutes. the supernatant was used in the pcr extraction and was then mixed with 454 grams of documented pedv-free feed. in the case of the positive control group, 30 ml of stock pedv was added to 454 grams of documented pedv-free feed. finally, for the negative control group, an equivalent quantity of saline was added to 454 grams of pedv-negative feed. following consumption of challenge material, piglets were fed pedv-free feed ad libitum for the remainder of the study. following consumption of their respective challenge material on day 0, the pedv status in piglets across all 3 groups was monitored over time. on a daily basis, arw personnel inspected animals for clinical signs of ped and collected samples as needed. personnel moved from the negative control group, to the treatment group to the positive control group every day. showers were taken between rooms and room-specific coveralls, footwear, hairnets, gloves and p95 masks were worn. in addition, each room was ventilated individually, and hepa filtration for both incoming and outgoing air was employed per room. if clinically affected animals were observed, rectal swabs (dacron swabs, fisher scientific, franklin lakes, nj, usa) were collected, along with swabs of any detectable diarrhea and vomiting. swabs were submitted to the sdsu adrdl and tested by pcr. on day 7 of the study, animals were humanely euthanized with intravenous sodium pentobarbital and intestinal tracts submitted for pcr and immunohistochemistry (ihc) testing and microscopic evaluation. select samples were nucleic acid sequenced. all diagnostic testing was conducted using protocols developed and validated by the south dakota state university animal disease research and diagnostic laboratory. a commercially available real-time, single tube rt-pcr assay for the detection of pedv and tgev was used in this study per kit instruction (tetracore, rockville, md, usa). briefly, 7 μl of the extracted rna was added to table 1 temporal relationship between the delivery of "at-risk" feed and the onset of clinical ped in the index cases across the 3 affected breeding herds 18 μl of the master mix. the one-step real-time rt-pcr amplification conditions started with 15 minutes at 48°c, followed by 2 minutes at 95°c. the final cycles consisted of 5 seconds at 95°c and then 40 seconds at 60°c (data collection step). the program was run for 38 cycles (cycle time) and the fam detector was used for pedv and the tamra detector was used for tgev. positive and negative controls were included on each run. all amplification was completed on the abi7500 instrumentation (austin, tx, usa). a for select samples, it was planned to conduct nucleic acid sequencing of the pedv s1 gene. specifically, fragments of the s1 domain of the spike gene were amplified from extracted rna. product size of 565 bp and primer pair 7 and 8 obtained a pcr product size of 745 bp. fragments were assembled sing the vector nti software (life technologies, waltham, ma, usa) for a complete s1 domain. qiagen one-step master mix (valencia, ca, usa) was used per kit instructions with an annealing temperature of 58°c for 30 seconds. immunohistochemistry slides were prepared using the standard sdsu adrdl ihc procedure, with the following modification being the use of pedv monoclonal antibody, of mouse ascites origin, courtesy of steve lawson, sdsu, at a 1:1000 dilution. during the on-farm inspection, no obvious breaches in any of the biosecurity protocols were detected across all 3 herds. no evidence of viral entry through genetic introduction, personnel error, contaminated transport or supplies were noted upon review of on-farm documentation. finally, no breaches in the air filtration system (filter integrity, air bypass etc.) were noted on farms a and b. in regards to feed, all herds received feed from different mills and diets contained corn, soybean meal, vitamins and trace minerals. no porcine by-products were included in any diet. upon review of the history, a temporal relationship between the delivery of at-risk feed and the onset of clinical signs in index cases was observed (table 1) . across all 3 herds, clinical signs were observed within 2 days post-delivery of at-risk feed. in addition, index cases were isolated to isolated areas of each farm which only received at-risk feed. specifically, farm a cases were located in the exterior row of stalls in the east gestation room and in gilts housed in a single room in the developer facility. for farm b, index cases were located in the west farrowing room while the exterior row of stalls in the north gestation room housed index cases for farms c. assessment of feed material in the at-risk bins across the 3 affected sites indicated the presence of pedv rna with ct values ranging from 19.50-22.20 (table 1 ). in contrast, all samples from on-farm control bins and samples from bins on pedv-negative sites were pcr-negative. the in vivo phase of the study was conducted from january 17-24 and results are summarized in table 2 . prior to initiation of the in vivo phase of the study, all samples from incoming piglets, arw facilities and feed were pcr negative. in regards to the treatment group, the pooling of feed material from farms a, b and c resulted in challenge material having a ct value of 20.65. following challenge, clinical signs of diarrhea were observed in the index piglet in the treatment group on day 4 post-ingestion. pedv-rna was detected in a rectal swab from this animal, along with swabs collected from diarrhea in the pen. this piglet continued to shed through the remainder of the study period and 1 other piglet displayed clinical signs of diarrhea and vomiting on day 6 post-ingestion (figures 1, 2 and 3 ). following euthanasia, rectal swabs and intestinal tract samples from all 5 pigs were positive by pcr and ihc (figure 4 ). in addition, microscopic evaluation of small intestinal tissues indicated re-epithelialization with diffuse villous blunting and fusion was noted ( figure 5 ). in the positive control group, the ct of the stock virus used to spike its respective challenge feed was 18.23. following consumption, shedding and clinical signs were observed in the index piglet on day 2 post-consumption with subsequent evidence of viral shedding to 2 other piglets on days 3 and 4. similar to the treatment group, all rectal swab samples and intestinal tract samples were pcr and ihcpositive at necropsy, along with evidence of microscopic lesions. in contrast, clinical signs, viral shedding or pedv-positive intestinal tract samples were not observed in the negative control group. s1 pedv sequencing was completed on pcr-positive feed challenge material from both the positive control group and the treatment group, as well as an intestinal homogenate from the index piglet in the treatment group. the sequencing results of the intestine and the treatment feed material were similar, but different from the positive control sample, indicating intestinal infection from consumption of the feed material. the purpose of this paper was to provide proof of concept that complete feed that was contaminated with pedv could act as a vehicle for infection of naïve pigs. to accomplish this goal, we developed a novel means of figure 4 presence of pedv antigen in a jejunal section from a treatment group piglet. this photomicrograph (400x) illustrates the presence of pedv antigen in multiple infected enterocytes following application of immunohistochemical staining. sampling at-risk feed material under controlled field conditions and conducted a swine bioassay to demonstrate transmission under controlled experimental conditions. under the conditions of this study, we successfully detected pedv rna in complete feed material across all 3 sites and proved its infectivity using natural feeding behavior, a novel finding not yet reported. these results were strengthened through the inclusion of on-farm control feed bins and bins from naïve farms and the use of a negative control group and natural feeding behavior during the in vivo phase. the sequencing data also suggest that the intestinal infection in the treatment group resulted from ingestion of pooled feed material from the 3 affected herds. an acknowledged limitation of the study was that the in vivo study was not designed to estimate the frequency of feed-related pedv infections. these results were based on very small populations of pigs and cannot be extrapolated to today's commercial farm conditions. however, the ability to complete a successful swine bioassay using a small number of animals cannot be ignored and the fact that our at-risk feed samples were collected from large commercial production sites adds to the credibility of this first attempt to investigate this risk factor. it was interesting to note the pattern of shedding (as detected by rectal swabs) in the treatment and positive control groups. despite the fact we employed small numbers of animals, shedding was first detected in an index case and spread occurred throughout the group of piglets and differences were observed between the 2 groups. this suggests that when small groups are employed and/or field samples used for inoculation, the bioassay period may need to be extended to avoid the risk of false negative results. in closing, this is the first publication providing proof of concept of the risk of pedv-contaminated complete feed to naïve pigs. it was interesting to note that since neither feed source contained animal by-products, suggesting that contamination may have occurred post-processing, however, this was not proven. further studies should focus on understanding the possibility of post-processing contamination as well as evaluating the ability of intervention strategies, i.e. the application of heat and pressure through the pelleting process and/or the inclusion of select feed additives which may have anti-viral effects, for reduction of this risk. finally, it is the authors hope that the results from this study will assist in uniting the north american veterinary profession with the feed and swine industries as we collectively move forward in reducing the threat of this devastating transboundary disease. these data provide the initial proof of concept that contaminated complete feed can serve as a vehicle for pedv infection of naïve pigs. information from this study should be used to justify the need for further research towards the mitigation of said risk. the data set(s) supporting the results of this article is included within the article. diseases of swine isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states usda aphis vs nvsl nahln umn swine health monitoring report: porcine epidemic diarrhea virus reporting prevalence of infections with enzootic respiratory and enteric virus in feeder pigs entering fattening units role of transportation in spread of porcine epidemic diarrhea virus infection, united states detection of porcine epidemic diarrhea virus in air samples at varying distances to epidemic farms in oklahoma american association of swine veterinarians and usda center for epidemiology and animal health: ped epidemiologic survey canadian food inspection agency: statement on porcine epidemic diarrhea virus in feed canadian food inspection agency: investigation into feed as a possible source of porcine epidemic diarrhea (ped) kansas state university applied swine nutrition team provides nursery diet options in response to pedv concerns use of a production region model to assess the airborne spread of porcine reproductive and respiratory syndrome virus use of a production region model to assess the efficacy of various air filtration systems for preventing the airborne transmission of porcine reproductive and respiratory syndrome virus and mycoplasma hyopneumoniae. results of a 2-year study environmental stability of a cell culture adapted u.s. isolate of pedv 23rd ipvs congress submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors would like to recognize dr. michele mucciante and the animal resource wing team for their significant contributions to the success of this study. funding for this study was provided by pipestone applied research the authors declare that they have no competing interests.authors' contributions sd: developed study, co-wrote paper. tc: conducted molecular diagnostics, co-wrote paper. as: participated in on-farm collection. jn: participated in on-farm collection. dk: conducted pathological assessment of tissues and developed figures 4-5. jch: provided critical review and revising of paper. en: provided virological expertise at the laboratory level and co-wrote paper. all authors read and approved the final manuscript. key: cord-273610-cfoq3r3i authors: tian, peng-fei; jin, yu-lan; xing, gang; qv, ling-ling; huang, yao-wei; zhou, ji-yong title: evidence of recombinant strains of porcine epidemic diarrhea virus, united states, 2013 date: 2014-10-17 journal: emerg infect dis doi: 10.3201/eid2010.140338 sha: doc_id: 273610 cord_uid: cfoq3r3i to investigate the evolutionary process by which porcine epidemic diarrhea virus (pedv) in the united states hypothetically descended from strains in china, we analyzed pedv-positive samples collected in china during january 2012–july 2013. recombination in 2 strain sublineages was likely associated with identification of pedv in the united states in 2013. to investigate the evolutionary process by which porcine epidemic diarrhea virus (pedv) in the united states hypothetically descended from strains in china, we analyzed pedv-positive samples collected in china during january 2012-july 2013. recombination in 2 strain sublineages was likely associated with identification of pedv in the united states in 2013. phacoronavirus that causes enteric disease in swine. the disease, ped, is characterized by acute vomiting and watery diarrhea and causes high mortality rates in newborn piglets (1) . ped was first reported in 1971 in the united kingdom and was soon identified in many european and asian countries (1, 2) . variant pedv strains that were fatal to young pigs, initially isolated during late 2010 in china and southeast asia (3, 4) and in may 2013 in the united states (5, 6) , have posed a serious threat to the pork industry. incidence of pedv-associated large-scale outbreaks of diarrheal disease in china was reported at 80%-100% in suckling piglets (3, 7) and outbreaks in the united states had spread to 25 states by february 2014 (http://www.aasv. org/news/story.php?id=7038), causing numerous deaths in neonatal piglets (5, 6, 8, 9) . how the virus entered the united states remains unknown. a phylogenetic analysis based on available fulllength genomic pedv sequences indicated that all pedv strains were classified into 2 distinct genogroups: g1 and g2 (6) . pedv field strains isolated before 2010 and the derived vaccine strains were in the g1 genogroup, whereas all the new pedv strains isolated since 2011 in china and the united states (us pedv) are in g2. the us pedv sequences were >99% identical to strains found in china in the subgroup g2a, suggesting their origin. in particular, the us pedv are most closely related to strain ah2012, which was isolated in eastern china and was proposed to have come from multiple recombination events among g2 lineages of pedv (6) . divergence of pedv is driven by genetic recombination, as in other coronaviruses (10) . details of recombination events in the process are needed to investigate origins. to investigate the evolutionary process by which us pedv strains hypothetically descended from precursors in china, we conducted a molecular epidemiologic analysis using pedv-positive samples collected from eastern china since 2012. a total of 169 fecal and intestinal samples were collected from pigs with typical ped symptoms on 26 farms in 4 provinces of eastern china during january 2012-july 2013. the rate of pedv-positive samples was 56.8% (96/169) as had been determined by using reverse transcription pcr (rt-pcr) specific for the spike (s) gene (11) . from the positive samples, we selected 24 representative samples (table) to examine. using rt-pcr, we determined the sequences of the full-length genomic cdna for the strain ch/zjcx-1/2012, identified the spike (s) gene for strains ch/ zjqz-2w/2012 and ch/zjdx-1/2012, and identified the region encoding structural protein genes by an order of 5′-s-orf3-e-m-n-3′ (5′-spike protein-open reading frame 3-envelope-membrane-nucleoprotein-3′) for the remaining 21 strains. all primers were designed based on the pedv mn strain (genbank accession no. kf468752). we purified and cloned pcr products into a vector using ta cloning. we used vector nti software (http://www. lifetechnologies.com/us/en/home/life-science/cloning/ vector-nti-software.html) to assemble and analyze the sequences. we performed multiple alignments of s-orf3-e-m-n, s, orf3, m, n, and full-length genomes with available sequences from asia and the united states (5, 6, 8, 12) and performed phylogenetic analyses using the mega5.2 program (http://www.megasoftware.net/) with the neighbor-joining method. similar to most of the sequences recently documented in pedv strains in china and the united states, the s genes of the 24 samples have a 4,161-nt sequence that, compared with the prototype cv777 strain, shows 97.9%-100% sequence identities, and contain 2 notable insertions at amino acids (aa) 56-59 (igen) and 139 (n) and a deletion of 2 aa (gk) at aa positions 160 and 161 at the n terminus (6) . a phylogenetic analysis comparing s genes showed that, based on the complete genome (online technical appendix figure 1 , wwwnc.cdc.gov/eid/article/20/10/14-0338-techapp1.pdf), all 24 strains were classified into the same group corresponding to g2. however, it is notable that the chinese sublineage (branch) most closely related to the us pedv strains did not include the ah2012 strain. instead, this sublineage contained the strain ch/zmdzy/11 and 4 other strains determined in this study. analyses of the phylogenetic trees constructed on the basis of the s-orf3-e-m-n genes ( figure) , orf3 or m (online technical appendix figure 1 ) also indicated that the ah2012 strain was not closely related to the us branch, relative to the sublineage represented by strains ch/zmdzy/11, ch/ hubwhyq/2012, ch/jxzs-1223l/2012, and ch/jxzs-3l/2012 (designated the zmdzy sublineage hereafter; figure) . the exception is the n gene-based tree, in which the ah2012 was grouped more closely to the sublineage associated with the united states than the strains in the designated zmdzy sublineage (online technical appendix figure 1 ). the relationship of the ah2012 strain with the 33 pedv strains identified in china, the united states, south korea, and belgium (online technical appendix figure 1 ) in nonstructural protein genes was also determined by generation of 3 phylogenetic trees based on orf1ab, orf1a, and orf1b genes, respectively. in accordance with the results from the n gene and the pedv genotyping based on the full-length genomes (6), the ah2012 strain in these trees was most closely related to the us strains (online technical appendix figure 1 ). therefore, the strains ah2012 and ch/zmdzy/11 displayed different phylogenetic relationships in different genome regions. overall, the ah2012 strain was clustered closely with the us strains in the orf1ab and the n gene region, whereas the zmdzy sublineage was clustered closely with the us strains in the s-orf3-e-m region. to accurately determine how the us strains are related to strains ah2012 and the zmdzy sublineage, we performed a recombination analysis using the recombination detection program and available pedv sequences (13) . we used a multiple-comparison-corrected p-value cutoff of 0.05 in all methods. recombination events were identified by 6 methods (recombination detection program, geneconv, bootscan, maxchi, chimaera, and siscan) when the us pedv sublineage represented by the mn strain was used as a query. by bootstrap analysis, 3 putatively major recombination breakpoints were detected at nucleotides 6699, 21840, and 26882 (online technical appendix figure 2 ), which generated 2 regions: 1 covered the 3′ half of orf1a, complete orf1b, and the n terminus of the s (first 1,207 nt); the other spanned partial s, orf3, e, m, and partial n (first 504 nt) between the strain ah2012 (as the major parent) and the zmdzy sublineage (as the minor parent). although the second region (partial s-orf3-e-m-partial n) of the us strains is associated with the zmdzy sublineage, the source of genetic material in this region is not known, because none of the pedv strains in this sublineage had a highly identical sequence to the consensus sequence of the us strains. it is possible that the other recombination breakpoints exist within the s gene, according to the bootstrap supports in this region, which may be determined by future study with available new sequence data. we showed that the emergent us pedv strains are possibly descendent of 2 major lineages derived from the zmdzy sublineage and ah2012 through recombination. our study provides further information on the origin of the us pedv in 2013. we identified 21 s-orf3-e-m-n genes, 2 s genes, and 1 full-length genomic cdna of pedv from pedv-positive samples collected in eastern china. comparative genomic, phylogenetic, and recombination analyses using new and known sequence data demonstrated that the ah2012 strain is likely not the direct progenitor of emergent us pedv strains during 2013. it is possible that replacement of a region within the partial s-orf3-e-m-partial n region of the ah2012 strain with a corresponding fragment close to the zmdzy sublineage (including several newly identified strains) resulted in a recombinant strain related to emergence of this virus in swine in the united states. other unidentified recombination events and accumulation of adapted mutations within the structural protein genes were also likely involved in this process. porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines a new coronavirus-like particle associated with diarrhea in swine isolation and characterization of a variant porcine epidemic diarrhea virus in china chinese-like strain of porcine epidemic diarrhea virus complete genome sequence of porcine epidemic diarrhea virus strain usa/colorado/2013 from the united states origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states outbreak of porcine epidemic diarrhea in suckling piglets emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission multiplex reverse transcription-pcr for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group a rotavirus isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states recombination detection and analysis using rdp3 key: cord-288058-oilurica authors: cui, tingting; theuns, sebastiaan; xie, jiexiong; den broeck, wim van; nauwynck, hans j. title: role of porcine aminopeptidase n and sialic acids in porcine coronavirus infections in primary porcine enterocytes date: 2020-04-05 journal: viruses doi: 10.3390/v12040402 sha: doc_id: 288058 cord_uid: oilurica porcine epidemic diarrhea virus (pedv) and transmissible gastroenteritis virus (tgev) have been reported to use aminopeptidase n (apn) as a cellular receptor. recently, the role of apn as a receptor for pedv has been questioned. in our study, the role of apn in pedv and tgev infections was studied in primary porcine enterocytes. after seven days of cultivation, 89% of enterocytes presented microvilli and showed a twoto five-fold higher susceptibility to pedv and tgev. a significant increase of pedv and tgev infection was correlated with a higher expression of apn, which was indicative that apn plays an important role in porcine coronavirus infections. however, pedv and tgev infected both apn positive and negative enterocytes. pedv and tgev miller showed a higher infectivity in apn positive cells than in apn negative cells. in contrast, tgev purdue replicated better in apn negative cells. these results show that an additional receptor exists, different from apn for porcine coronaviruses. subsequently, treatment of enterocytes with neuraminidase (na) had no effect on infection efficiency of tgev, implying that terminal cellular sialic acids (sas) are no receptor determinants for tgev. treatment of tgev with na significantly enhanced the infection which shows that tgev is masked by sas. coronaviruses are known as human and animal pathogens that mainly infect the epithelium of the respiratory or intestinal tract. porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), and its variant porcine respiratory coronavirus (prcv) are classified as alphacoronavirus. they are enveloped viruses containing a single-stranded, positive-sense rna genome of approximately 28.5 kb. the positive ssrna serves as mrna for the generation of viral replicative proteins by translation of open reading frame (orf) 1a and orf1b. the genome contains a 5 untranslated region (utr), a 3 utr, and at least seven orfs. orf1a and orf1b make up two-thirds of the viral genome and encode the non-structural replicase polyproteins (replicases 1a and 1b), which further guide the viral replication and translation, regulate cellular processes, and potentially fulfill other unknown functions. the remaining proximal third of the genome encodes four structural proteins (spike (s), envelope (e), membrane (m), and nucleocapsid (n) by orf2, orf4, orf5, and orf6, respectively). the s protein is a type i glycoprotein that projects from the virions surface forming in the present study, the co-culture system of primary porcine enterocytes that was established in our laboratory was used to study the role of apn in pedv and tgev infection in their target cells [19] . further, it was investigated whether sialic acids are cellular receptors for tgev in primary enterocytes. primary porcine enterocytes were isolated from the ileum of three-day-old piglets and co-cultured with porcine myofibroblasts [19] . euthanizing piglets was done in agreement with the european legislation on animal experiments. all experimental procedures were approved by the local ethical committee of the faculty of veterinary medicine, ghent university (ec2013/97), and all methods were carried out in accordance with the approved guidelines. the enterocytes were maintained with dulbecco's modified eagle's-f12 ham medium (dmem-f12). tgev purdue and miller grown on swine testicle (st) cells and pedv cv777 strain grown on vero cells were used in this study. pedv cv777 fecal suspension was collected from a three-day-old infected suckling piglet. a twenty percent fecal suspension was prepared in phosphate buffered saline (pbs) containing 1000 u/ml penicillin (continental pharma, puurs, belgium), 1 mg/ml streptomycin (certa, braine l'alleud, belgium), 1 mg/ml gentamicin (gibco brl, merelbeke, belgium), and 0.01% v/v fungizone (bristol-myers squibb, braine l'alleud, belgium). hydrocortisone, spermidine, and wnt agonist were purchased from sigma-aldrich (sigma, st-louis, mo, usa). porcine insulin was purchased from protein specialists (prospec, rehovot, israel). the small intestinal contents (ic) were collected from the duodenum of a three-day-old suckling piglet. after euthanasia, a 20 cm long segment of duodenum was closed by two surgical clamps and was removed from a piglet. then, one clamp was removed and the intestinal contents were released from the lumen into a 15 ml centrifugation tube. in order to collect all the intestinal contents, the lumen was washed once by filling it with 5 ml dulbecco's modified eagle's medium (dmem) containing 1000 u/ml penicillin, 1 mg/ml streptomycin, 1 mg/ml gentamicin, and 0.01% v/v fungizone. then, the dmem was released from the lumen into the 15 ml tube that already contained the intestinal contents. after centrifugation (1200 rpm, 10 min at 4 • c), the supernatant of the intestinal contents was collected and stored at −20 • c until use. three-and seven-day-old primary porcine enterocytes were fixed in hepes-buffered glutaric aldehyde (sigma, st-louis, mo, usa) for scanning electron microscopy as described previously [20] . after 24 h fixation, the samples were treated with 1% osmium tetroxide for 2 h at room temperature (rt), followed by ascending grades of alcohol dehydration. in order to avoid the water vaporization obstructing the electron beam and interfering with image clarity, the dehydrated samples were transferred to a critical point drier (cpd, bal-tec, balzers, liechtenstein) for complete drying. finally, the dried samples were mounted on a metal stub and were sputter-coated with platinum. the microvilli of all the samples were acquired with a jeol jsm 5600 lv scanning electron microscope (jeol ltd., tokyo, japan). twenty-four h post co-cultivation, monolayers of enterocytes were cultured with medium containing hydrocortisone (1 and 10 µg/ml), spermidine (50 and 500 µm), insulin (1 and 10 µg/ml), wnt agonist (0.1 µm), or intestinal contents (1%) for 24 h. the cells were fixed with 4% paraformaldehyde for 10 min and immunofluorescence staining was conducted for apn expression analysis. cells were incubated with mouse monoclonal anti-porcine apn antibodies (imm013; kindly provided by prof. eric cox, ghent university) containing 10% normal goat serum for 1 h at 37 • c, followed by goat anti-mouse-igg fitc labeled antibodies for 1 h at 37 • c. nuclei were stained with hoechst for 10 min at rt. the percentage of apn positive cells were analyzed by fluorescence microscopy (leica microsystems gmbh). at twenty-four h of co-cultivation, monolayers of enterocytes were treated with 1 µg/ml hydrocortisone, 50 µm spermidine, 1 µg/ml insulin, 0.1 µm wnt agonist, or 1% intestinal contents for another 24 h. then, the susceptibility of treated enterocytes to pedv and tgev was tested. cells were inoculated with 200 µl of tgev miller at a multiplicity of infection (m.o.i.) of 1 and 200 µl of the pedv cv777 vero adapted strain at 10 5.6 tcid 50 /ml or 10 7 viral rna copies/ml of fecal suspension with 10 µg/ml trypsin. after 60 min of incubation at 37 • c, unbound viral particles were removed by three washing steps with dmem. cells were further incubated in medium containing 1 µg/ml hydrocortisone, 50 µm spermidine, 1 µg/ml insulin, 0.1 µm wnt agonist, or 1% intestinal contents for 24 h (37 • c, 5% co 2 ) and fixed with 4% paraformaldehyde for immunofluorescence staining. to determine the co-localization of viral antigens and apn, co-cultured enterocytes were infected with tgev miller and purdue and pedv vero adapted and non-adapted strains. after 24 h incubation, cells were fixed with 4% paraformaldehyde for 10 min and immunofluorescence staining was performed. cells were incubated with mouse monoclonal anti-porcine apn antibodies containing 10% normal goat serum for 1 h at 37 • c, followed by goat anti-mouse-igg1 fitc labeled antibodies for 1 h at 37 • c. then, cells were permeabilized with 0.1% triton x-100 for 5 min at rt and incubated with mouse monoclonal anti-porcine pedv antibodies (kindly provided by prof. luis enjuanes, national center for biotechnology) or swine polyclonal anti-tgev antibodies [19] containing 10% normal goat serum for 1 h at 37 • c, followed by goat anti-mouse igg2a af594 labeled antibodies or goat anti-swine texas red labeled antibodies (molecular probes). nuclei were stained with hoechst for 10 min at rt and the results were analyzed by a leica tcs spe laser scanning spectral confocal system linked to a dm b fluorescence microscope (leica microsystems). to remove sas from enterocytes, monolayers of co-cultured enterocytes were washed three times with warm dmem. then, cells were incubated with 50 mu/ml na from vibrio cholerae (roche diagnostics, risch-rotkreuz, switzerland) at 37 • c for 1 h. cells that were mock-treated were incubated with dmem and underwent the same manipulations as na-treated cells. to remove sas from the virus, virus suspensions were incubated with 50 mu/ml na from vibrio cholerae at 37 • c for 1 h. the mock-treated virus was incubated in dmem and underwent the same manipulations as the na-treated virus. afterwards, cells were inoculated with either na-treated or mock-treated virus (m.o.i of 1 for tgev purdue and miller). after 60 min incubation at 37 • c, cells were washed three times with medium and further incubated in medium for 24 h (37 • c, 5% co 2 ). then, cells were fixed with 4% paraformaldehyde for 10 min at rt. immunofluorescence was performed to measure the percentage of infected cells. the cells were permeabilized with 0.1% triton x-100 for 5 min at rt. then, cells were incubated with swine polyclonal anti-tgev antibodies containing 10% normal goat serum for 1 h at 37 • c, followed by goat anti-swine-igg fitc labelled antibodies for 1 h at 37 • c. nuclei were stained with hoechst for 10 min at rt. the percentages of infected cells were determined by fluorescence microscopy. to determine the co-localization of viral antigens and sas, co-cultured enterocytes were infected with tgev miller and purdue. after 24 h incubation, cells were fixed with 4% paraformaldehyde for 10 min and a double immunofluorescence staining was performed. cells were incubated with biotinylated maackia amurensis lectin ii (vector laboratories) for 1 h at 37 • c. the lectin was subsequently stained with streptavidin-fitc (invitrogen) for 1 h at 37 • c. then, cells were permeabilized with 0.1% triton x-100 for 5 min at rt and incubated with swine polyclonal anti-tgev antibodies containing 10% normal goat serum for 1 h at 37 • c, followed by goat anti-swine-igg texas red labeled antibodies. nuclei were stained with hoechst for 10 min at rt and results were analyzed by a leica tcs spe laser scanning spectral confocal system linked to a dm b fluorescence microscope. to characterize the attachment of tgev to primary enterocytes, direct virus-binding studies were carried out with tgev particles. cells were chilled on ice for 5 min and washed three times with dmem. then, cells were inoculated with tgev miller and purdue particles at a m.o.i. of 10 for 1 h at 4 • c. unbound virus particles were removed by three washings with dmem. cells were fixed with 4% paraformaldehyde for 10 min and a double immunofluorescence staining was performed. cells were incubated with biotinylated maackia amurensis lectin ii or mouse monoclonal anti-porcine apn antibodies for 1 h at 37 • c, followed by streptavidin-fitc or goat anti-mouse-igg1 fitc labeled antibodies for 1 h at 37 • c. then, cells were permeabilized with 0.1% triton x-100 for 5 min at rt and incubated with swine polyclonal anti tgev antibodies containing 10% goat serum for 1 h at 37 • c, followed by goat anti-swine-igg texas red labeled antibodies. nuclei were stained with hoechst for 10 min at rt and the results were analyzed by a leica tcs spe laser scanning spectral confocal system linked to a dm b fluorescence microscope. data were statistically processed by spss (t-test). the data are represented as means with standard deviation (sd) of three independent experiments. results with p-values of <0.05 were considered significant. the percentages of microvilli positive enterocytes were analyzed by scanning electron microscopy. a higher percentage of microvilli positive cells (89%) was observed at seven days cultivation compared to three days cultivation (66%). the expression of apn at seven days cultivation (28.7 ± 2.3%) was significantly higher than at three days cultivation (14.8 ± 3.2%; figure 1a ,b). the data suggest that primary enterocytes underwent a differentiation process in vitro. they terminally differentiate into mature enterocytes during the long cultivation time. next, enterocytes were inoculated with tgev and pedv at three and seven days of cultivation. the results showed that a significantly higher infection was detected in enterocytes at seven days cultivation (miller: 3.6 ± 1.1%; purdue: 7.3 ± 0.7%) than at three days cultivation (miller: 0.7 ± 0.7%; purdue: 3.5 ± 0.5%) for tgev. an increased trend of infection was detected in enterocytes at seven days cultivation (39 infected cells per well) than at three days cultivation (16 infected cells per well) for pedv but without significance (p = 0.06; figure 1c ). data are expressed as mean ± standard deviation (sd) of the results of three separate experiments. statistically significant differences in comparison with data from three days cultivation are presented as *p < 0.05 or **p < 0.01. cells were treated with hydrocortisone, spermidine, porcine insulin, wnt agonist, or small intestinal contents to analyse their effects on enterocyte differentiation. cells were treated with the aforementioned products for 24 h. afterwards, the differentiation marker apn was stained by immunofluorescence and the percentage of apn positive cells was counted ( figure 2a ). the results showed that without treatment, 13.8 ± 2.6% of cells were apn positive. the treatment with 1 and 10 µg/ml hydrocortisone significantly increased the apn expression to 25 ± 5.5% and 25 ± 8.8%, respectively. the treatment with 50 mm and 500 mm spermidine significantly enhanced the apn expression to 27 ± 8.1% and 26 ± 5.8%, respectively. similarly, 1 and 10 µg/ml insulin treatment significantly increased the apn expression to 25 ± 6.4% and 27 ± 10.1%, respectively. since there was no dose-dependent enhancement, the lower concentration of each product (1 µg/ml of hydrocortisone, 50 µm of spermidine and 1 µg/ml of porcine insulin) was used for the next cells were treated with hydrocortisone, spermidine, porcine insulin, wnt agonist, or small intestinal contents to analyse their effects on enterocyte differentiation. cells were treated with the aforementioned products for 24 h. afterwards, the differentiation marker apn was stained by immunofluorescence and the percentage of apn positive cells was counted ( figure 2a ). the results showed that without treatment, 13.8 ± 2.6% of cells were apn positive. the treatment with 1 and 10 µg/ml hydrocortisone significantly increased the apn expression to 25 ± 5.5% and 25 ± 8.8%, respectively. the treatment with 50 mm and 500 mm spermidine significantly enhanced the apn expression to 27 ± 8.1% and 26 ± 5.8%, respectively. similarly, 1 and 10 µg/ml insulin treatment significantly increased the apn expression to 25 ± 6.4% and 27 ± 10.1%, respectively. since there was no dose-dependent enhancement, the lower concentration of each product (1 µg/ml of hydrocortisone, 50 µm of spermidine and 1 µg/ml of porcine insulin) was used for the next experiment. the treatment of wnt agonist and intestinal contents showed a trend of increased apn expression up to 22 ± 7% (p = 0.1) and 22 ± 9.9% (p = 0.3), respectively ( figure 2b ). to determine the effect of apn on coronavirus replication, the enterocytes were precultured with 1µg/ml hydrocortisone, 50 µm spermidine, 1 µg/ml insulin, 0.1 µm wnt agonist, or 1% intestinal contents for 24 h prior to inoculation with pedv cv777 vero adapted strain, cv777 fecal suspension, and tgev miller. for pedv cv777 vero adapted strain, only 30 ± 11 cells were infected per well without pretreatment. the highest infection (169 ± 81 infected cells per well) was observed in cells that were pretreated with intestinal contents. because of the variation between the three replicates, the treatment with intestinal contents was not significantly different from the mock treatment (p = 0.09). when cells were pretreated with wnt agonist, a significant increase of infection (87 ± 23 infected cells per well) was observed (p = 0.018). an increased trend of infection (but not significantly different) was observed after pretreatment with hydrocortisone (42 ± 34 infected cells per well, (p = 0.6), spermidine (50 ± 6 infected cells per well, p = 0.052), and porcine insulin (46 ±27 infected cells per well, p = 0.4). for cv777 fecal suspension, the highest infection was observed when to determine the effect of apn on coronavirus replication, the enterocytes were precultured with 1µg/ml hydrocortisone, 50 µm spermidine, 1 µg/ml insulin, 0.1 µm wnt agonist, or 1% intestinal contents for 24 h prior to inoculation with pedv cv777 vero adapted strain, cv777 fecal suspension, and tgev miller. for pedv cv777 vero adapted strain, only 30 ± 11 cells were infected per well without pretreatment. the highest infection (169 ± 81 infected cells per well) was observed in cells that were pretreated with intestinal contents. because of the variation between the three replicates, the treatment with intestinal contents was not significantly different from the mock treatment (p = 0.09). when cells were pretreated with wnt agonist, a significant increase of infection (87 ± 23 infected cells per well) was observed (p = 0.018). an increased trend of infection (but not significantly different) was observed after pretreatment with hydrocortisone (42 ± 34 infected cells per well, (p = 0.6), spermidine (50 ± 6 infected cells per well, p = 0.052), and porcine insulin (46 ±27 infected cells per well, p = 0.4). for cv777 fecal suspension, the highest infection was observed when cells were pretreated with the wnt agonist (12 ± 7 infected cells per well), showing a four-fold higher infection compared to mock-treated cells (3 ± 4 infected cells per well). when cells were pretreated with intestinal contents, 9 ± 6 cells were infected. for tgev miller, incubation with intestinal contents, porcine insulin, and wnt agonist significantly increased the virus infection from 1.0 ± 0.3% to 3.6 ± 0.9%, 3.2 ± 0.9% and 3.0 ± 0.5%, respectively. hydrocortisone and spermidine increased the virus infection to 2.7 ± 0.5% and 2.5 ± 0.6% (figure 3) . the results show that pretreatment of primary enterocytes with hydrocortisone, spermidine, porcine insulin, wnt agonist, and intestinal contents could stimulate the expression of apn and enhance the infection of pedv cv777 vero adapted and non-adapted strains and the tgev miller in the enterocytes. statistically significant differences in comparison with data from mock treatment are presented as *p < 0.05 or **p < 0.01. to assess the role of apn in the replication of pedv and tgev, a double immunofluorescence staining of apn and virus was performed. for cv777 vero adapted strain, 0.040 ± 0.002% of apn positive cells and 0.020 ± 0.006% of apn negative cells were infected. for cv777 fecal suspension, 0.02 ± 0.02% of apn positive cells and 0.003 ± 0.001% of apn negative cells were infected. for tgev miller, 4.0 ± 2.6% of apn positive cells and 1.1 ± 0 3% of apn negative cells were infected. for tgev purdue, more infection was found in apn negative cells (3.5 ± 0.6%) than in apn positive cells (2.0 ± 1.2%; figure 4 ). the results suggest that for pedv and tgev miller, apn may be the predominant receptor, while tgev purdue mainly uses an additional receptor for virus infection. to assess the role of apn in the replication of pedv and tgev, a double immunofluorescence staining of apn and virus was performed. for cv777 vero adapted strain, 0.040 ± 0.002% of apn positive cells and 0.020 ± 0.006% of apn negative cells were infected. for cv777 fecal suspension, 0.02 ± 0.02% of apn positive cells and 0.003 ± 0.001% of apn negative cells were infected. for tgev miller, 4.0 ± 2.6% of apn positive cells and 1.1 ± 0 3% of apn negative cells were infected. for tgev purdue, more infection was found in apn negative cells (3.5 ± 0.6%) than in apn positive cells (2.0 ± 1.2%; figure 4 ). the results suggest that for pedv and tgev miller, apn may be the predominant receptor, while tgev purdue mainly uses an additional receptor for virus infection. to assess the role of sas as receptor determinants, enterocytes were pretreated with 50 mu/ml na prior to inoculation with tgev miller and purdue. miller infected 1.0 ± 0.5% of the na-treated enterocytes and 1.4 ± 0.4% of mock-treated cells. for purdue, the percentage of infection was 2.3 ± 1.1% and 2.3 ± 0.9% for na-treated and mock-treated cells, respectively. this implies that tgev does not depend on terminal sa residues on the enterocytes surface for infection. since it has been reported that removal of sas on the surface of coronaviruses improves binding and infection [21] , the replication of mock-treated, and na-treated viruses was compared in untreated epithelial cells. na pretreatment of virus significantly enhanced infection from 1.4 ± 0.4% to 2.9 ± 0.5% for miller. for purdue, na pretreatment of virus significantly increased infection from 2.3 ± 1.1% to 7.9 ± 2.1% ( figure 5 ). these data show that removal of sa from tgev promotes binding and replication of tgev in enterocytes. viruses 2020, 12, 402 10 of 18 to assess the role of sas as receptor determinants, enterocytes were pretreated with 50 mu/ml na prior to inoculation with tgev miller and purdue. miller infected 1.0 ± 0.5% of the na-treated enterocytes and 1.4 ± 0.4% of mock-treated cells. for purdue, the percentage of infection was 2.3 ± 1.1% and 2.3 ± 0.9% for na-treated and mock-treated cells, respectively. this implies that tgev does not depend on terminal sa residues on the enterocytes surface for infection. since it has been reported that removal of sas on the surface of coronaviruses improves binding and infection [21] , the replication of mock-treated, and na-treated viruses was compared in untreated epithelial cells. na pretreatment of virus significantly enhanced infection from 1.4 ± 0.4% to 2.9 ± 0.5% for miller. for purdue, na pretreatment of virus significantly increased infection from 2.3 ± 1.1% to 7.9 ± 2.1% ( figure 5 ). these data show that removal of sa from tgev promotes binding and replication of tgev in enterocytes. data are expressed as mean ± sd of the results of three separate experiments. statistically significant differences in comparison with data from mock treatment are presented as *p < 0.05. double immunofluorescence staining was further performed to assess the role of sa on tgev replication. tgev miller could infect both sa positive cells (1.7 ± 0.3%) and sa negative cells (1.7 ± 0.2%). the tgev purdue replicated slightly better in sas negative cells (4.5 ± 0.3%) compared to sas positive cells (3.2 ± 0.8%; figure 6 ). double immunofluorescence staining was further performed to assess the role of sa on tgev replication. tgev miller could infect both sa positive cells (1.7 ± 0.3%) and sa negative cells (1.7 ± 0.2%). the tgev purdue replicated slightly better in sas negative cells (4.5 ± 0.3%) compared to sas positive cells (3.2 ± 0.8%; figure 6 ). primary enterocytes were inoculated with tgev particles (m.o.i. = 10) at 4 °c. the binding of tgev to apn positive/negative and sas positive/negative cells was examined by double immunofluorescence staining. no significant differences were observed between the percentage of apn positive cells with bound tgev miller (2.3 ± 0.7%) and the percentage of apn negative cells with bound tgev miller (1.9 ± 0.2%; p = 0.49). the percentage of apn positive cells with bound tgev purdue (1.5 ± 0.7%) was lower than the percentage of apn negative cells with bound tgev purdue (3.9 ± 0.7%), but was not significantly different (p = 0.07). the percentage of miller particles that colocalized with apn (65 ± 11%) was significantly higher than non-colocalized particles (35 ± 11%). the percentage of purdue particles that colocalized with apn (33 ± 2%) was significantly lower than non-colocalized particles (67 ± 2%; figure 7) . the percentage of sa negative cells with bound tgev miller (4.3 ± 0.3%) and with bound tgev purdue (5.0 ± 0.9%) was significantly higher than the percentage of sa positive cells with bound tgev miller (0.9 ± 0.4%) and with bound tgev purdue (2.1 ± 0.3%). the percentage of tgev miller particles (29 ± 13%) and tgev purdue (32 ± 12%) that colocalized with sa was significantly lower than the particles that did not colocalize, with miller at 71 ± 13% and purdue at 68 ± 12% (figure 8 ). primary enterocytes were inoculated with tgev particles (m.o.i. = 10) at 4 • c. the binding of tgev to apn positive/negative and sas positive/negative cells was examined by double immunofluorescence staining. no significant differences were observed between the percentage of apn positive cells with bound tgev miller (2.3 ± 0.7%) and the percentage of apn negative cells with bound tgev miller (1.9 ± 0.2%; p = 0.49). the percentage of apn positive cells with bound tgev purdue (1.5 ± 0.7%) was lower than the percentage of apn negative cells with bound tgev purdue (3.9 ± 0.7%), but was not significantly different (p = 0.07). the percentage of miller particles that colocalized with apn (65 ± 11%) was significantly higher than non-colocalized particles (35 ± 11%). the percentage of purdue particles that colocalized with apn (33 ± 2%) was significantly lower than non-colocalized particles (67 ± 2%; figure 7 ). the percentage of sa negative cells with bound tgev miller (4.3 ± 0.3%) and with bound tgev purdue (5.0 ± 0.9%) was significantly higher than the percentage of sa positive cells with bound tgev miller (0.9 ± 0.4%) and with bound tgev purdue (2.1 ± 0.3%). the percentage of tgev miller particles (29 ± 13%) and tgev purdue (32 ± 12%) that colocalized with sa was significantly lower than the particles that did not colocalize, with miller at 71 ± 13% and purdue at 68 ± 12% (figure 8 ). porcine epithelial cells of the small intestines are the target cells for pedv and tgev in vivo. these cells show a high surface expression of apn, and apn has been reported to be the cellular receptor for pedv and tgev [7, 9] . however, vero cells with undetectable apn expression were historically used for pedv propagation questioning the role of apn as a cellular receptor for pedv [22] . in addition, overexpression of porcine apn in non-susceptible cells did not robustly support pedv infection, and knock-out of apn in susceptible cells did not abrogate pedv infection [13] . the present study was performed to determine the role of apn in pedv and tgev infection in their target primary porcine enterocytes. we found that a higher infection of pedv and tgev was correlated with a higher apn expression. however, both pedv and tgev did not only infect apn porcine epithelial cells of the small intestines are the target cells for pedv and tgev in vivo. these cells show a high surface expression of apn, and apn has been reported to be the cellular receptor for pedv and tgev [7, 9] . however, vero cells with undetectable apn expression were historically used for pedv propagation questioning the role of apn as a cellular receptor for pedv [22] . in addition, overexpression of porcine apn in non-susceptible cells did not robustly support pedv infection, and knock-out of apn in susceptible cells did not abrogate pedv infection [13] . the present study was performed to determine the role of apn in pedv and tgev infection in their target primary porcine enterocytes. we found that a higher infection of pedv and tgev was correlated with a higher apn expression. however, both pedv and tgev did not only infect apn positive enterocytes, but also apn negative cells. our results demonstrated that pedv and tgev may use another additional unknown receptor for entry in primary enterocytes. the epithelium of the small intestines is continuously and rapidly renewed in a process involving cell generation and migration from the multi-potent stem cells in the crypts to the differentiated cells at the tips of the villi within 2-3 days. in our study, after three days cultivation, the primary enterocytes were positive for sucrase and iso-maltase, which are considered as differentiation markers for epithelial cells [19] . however, the expression of aminopeptidase n was only around 11%, indicating that the primary enterocytes are not fully mature at three days cultivation. therefore, we analyzed apn expression in enterocytes at a later time point (seven days cultivation). a significantly higher expression of apn was detected at seven days cultivation compared to three days cultivation. interestingly, the seven-day-cultured enterocytes with a higher apn expression showed significantly higher infection to tgev than that of enterocytes cultured for three days. this agrees with the fact that the virus mainly infects and destroys mature enterocytes lining the villi of small intestines [23] . however, the infection efficiency of pedv in intestinal epithelial cells was still low, indicating that other factors than apn need to be considered for pedv infection. therefore, several positive enterocyte differentiation factors were tested. hydrocortisone plays an important role in the metabolism of proteins, lipids, and carbohydrates, and is a known promoter for differentiation of cultured cells. hydrocortisone was found to be a critical factor for the differentiation of skeletal muscle, osteoblasts, and endothelial cells [24] . sorrell and colleagues demonstrated that hydrocortisone significantly upregulated the expression of apn in human dermal fibroblasts [25] . besides, wnt signaling is required for the formation of normal crypt-villus units of intestines, and stimulates the differentiation of intestinal secretory epithelial cells [26] . activated wnt signaling has also been shown to promote mesenchymal differentiation [27] . the original rationale for including intestinal contents in our primary cell cultures was trying to mimic the in vivo situation in the intestinal tract. intestinal contents contain a large number of enzymes, such as: amylase, which digests carbohydrates to monosaccharides; pancreatic enzymes, which digest proteins into amino acids; and lipase which digests fats. it has been demonstrated that intestinal contents play an important role in virus infection. the proteases (trypsin) in the intestinal contents activate rotavirus infection by cleaving the outer capsid protein vp4 [28] . the propagation of porcine enteric calicivirus (pec) on a cell line critically relies on the presence of intestinal contents in the culture medium [29] . chang et al. demonstrated that the bile salts in intestinal contents are essential for growth of pec by inducing the protein kinase a (pka) signaling pathway [30] . in our study, the intestinal contents collected from the upper duodenum promoted the expression of apn and enhanced the infection of both tgev and pedv, especially the cv777 vero adapted strain. further investigation is needed to determine which growth-promoting factor in the intestinal contents is responsible for the increase of coronavirus infection. to date, coronaviruses use four different proteins as cellular receptors. mouse hepatitis virus (mhv) initiates the infection by binding to the carcinoembryonic cell adhesion molecule 1 on hepatocyte membranes and intestinal brush border membranes [31] . next, apn was found to act as a receptor for porcine, feline, canine, and human coronaviruses [6] . the receptors for the highly pathogenic human respiratory viruses sars-cov type 1 and type 2 as well as middle east respiratory syndrome coronavirus are angiotensin-converting enzyme 2 (ace2) and dipeptidyl peptidase 4 (dpp 4) [32, 33] . cong and colleagues proved that the porcine small intestine epithelial cell line was more susceptible to pedv when a high expression level of apn was present, and that interference with apn expression in epithelial cells inhibited pedv infection, demonstrating that apn serves as an essential receptor for pedv [12] . in addition, a transgenic mouse model expressing porcine apn was proven to be susceptible to pedv, which confirmed that apn plays a role as the cellular receptor for pedv [34] . in our study, we found that the higher infection of pedv in enterocytes is correlated with higher apn expression. both aged enterocytes and enterocytes treated with differentiation factors expressed more apn and were more susceptible to pedv, which indicates that apn may play role in pedv infection in enterocytes. moreover, we found that the apn was expressed at the apical surface of enterocytes and pedv infected 40 times more enterocytes at the apical surface than at the basolateral surface (supplementary figure s1 ). these results were in agreement with the previous finding that pedv enters polarized cells via the apical membrane [12] . our results indicate that apn facilitates the entry of pedv into primary enterocytes. shirato and colleagues indicated that apn may promote pedv replication in porcine kidney cell line, cpk cells via its protease activity [14] . how apn facilitates pedv infection in enterocytes should be further investigated. however, due to the fact that other molecules besides apn will also be expressed at the apical plasma membrane during differentiation, they may also contribute to the higher susceptibility of differentiated enterocytes. recently, increasingly more data has been published that show that apn is not a cellular receptor for pedv. overexpression of apn in non-susceptible cells did not confer susceptibility of the cells to pedv and knocking out apn in susceptible cells did not abrogate pedv infection, which indicates that apn is not required as a cellular receptor for pedv in vitro [13, 14, 22] . furthermore, apn knockout pigs retained their susceptibility to pedv confirming that pedv may use another additional receptor in pigs [35] . in agreement with these findings, we found that pedv can infect apn negative primary enterocytes, which further confirmed that a cellular receptor different from apn exists in enterocytes for pedv replication. the primary enterocytes isolated and cultured in our study are not 100% positive for apn, as we not only get the villi epithelial cells, but also the crypt epithelial cells during our isolation procedure. by immunofluorescence staining of ileum tissue of a three-day-old piglet, we observed that the villi epithelial cells are apn positive, while the crypt epithelial cells are apn negative (data not shown). as pedv has been shown to infect goblet cells and crypt stem cells in addition to villous mature enterocytes [36] , we believe that pedv may also use another cellular receptor besides apn to infect intestinal cells. taken together, our results indicate that although apn could significantly promote pedv infection in enterocytes, an additional cellular receptor exists in enterocytes for pedv replication. since ace2 is expressed in the gut epithelial cells, it will be tested in the near future if it can function as a pedv receptor. in addition to pedv, apn has been identified as a major cellular receptor for tgev. in the present study, it was shown that a higher expression of apn significantly increased the replication of tgev in enterocytes, indicating that apn plays an important role as a cellular receptor for tgev. whitworth and colleagues demonstrated that apn knockout pigs were resistant to tgev infection, indicating that apn is the sole functional receptor for tgev [35] . however, we found that except apn positive enterocytes, tgev could also infect apn negative enterocytes, suggesting that an additional receptor also exists in enterocytes for tgev. the result obtained in our in vitro experiment may not fully reflect the in vivo experiments, as the in vivo situation is composed of a complex set of cells and tissues. the purdue strain used in our study has been passaged 114 times in primary kidney cells and a nucleotide mutation (t to g at nucleotide position 1753), which causes a serine (s) to alanine (a) at aa 585 [37, 38] . this mutation may be correlated with the cell adaptation and also it may result in a broader cell tropism of the adapted strain, which may explain that the virus is able to grow more efficiently in apn negative cells. purdue has been proven to infect primary colon epithelial cells and porcine myofibroblasts, which are both negative for apn [19] . in vivo, tgev shows a higher cell tropism to villous enterocytes of newborn piglets compared to older pigs and causes only high mortality in the early life of piglets. since apn is present on enterocytes from both newborn and older pigs, the age-dependent susceptibility to tgev infection may be caused by an additional receptor that is specifically present in newborn piglets. taken together, we hypothesize that apn is the determinant cellular receptor for tgev, but an additional receptor exists in young piglets. apart from apn, tgev also uses sa as an attachment mediator on the cells. shahwan and colleagues have shown that na treatment of jejunum epithelial cryosections did not reduce the tgev spike protein binding in vitro and were doubting on the role of sa during infection [39] . in our study, removal of sa from intestinal cells had no effect on tgev infection, showing that terminal sa residues are not receptor determinants for tgev. removal of sa from tgev virions significantly enhanced the viral infectivity in vitro. this indicates that sa on the virion surface masks the binding site of the viral protein to cellular receptors. removal of sa from virions facilitates tgev to bind to its functional receptor on the enterocyte membrane. to confirm the role of apn and sa in tgev infection in primary enterocytes, a binding assay was performed in this study. the results demonstrated that both tgev miller and purdue could bind to apn positive and sa positive cells. meanwhile, miller and purdue could also bind to apn negative and sa negative enterocytes. furthermore, our study showed that apn and sa double-negative enterocytes could be infected by tgev miller and purdue (supplementary figure s2) , which suggests that besides apn and sa, tgev can use another cellular receptor for the replication in enterocytes. further investigation will focus on identifying this unknow receptor. a binding assay for pedv could not be conducted due to the low titer of the virus stock. based on our previously established primary enterocyte co-culture system, which is very relevant for the in vivo situation, it was shown that a higher expression of apn on the enterocytes resulted in a higher infectivity of tgev and pedv. however, tgev and pedv could also infect apn negative enterocytes, indicating that an additional receptor exists in enterocytes besides apn. tgev did not show binding activity on sas on the surface of enterocytes. these new insights stimulate the search for unknown receptors for pedv and tgev, which can assist further research on antiviral intervention. coronavirus spike proteins in viral entry and pathogenesis the coronavirus membrane glycoprotein mapping of the coronavirus membrane protein domains involved in interaction with the spike protein absence of e protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis feline aminopeptidase n serves as 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line, through mesenchymal-to-endothelial transition-like effects cytokines and glucocorticoids differentially regulate apn/cd13 and dppiv/cd26 enzyme activities in cultured human dermal fibroblasts canonical wnt signals are essential for homeostasis of the intestinal epithelium expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells trypsin cleavage stabilizes the rotavirus vp4 spike cell-culture propagation of porcine enteric calicivirus mediated by intestinal contents is dependent on the cyclic amp signaling pathway bile acids are essential for porcine enteric calicivirus replication in association with down-regulation of signal transducer and activator of transcription 1 receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 structure of sars coronavirus spike receptor-binding domain complexed with receptor development of transgenic mouse model expressing porcine aminopeptidase n and its susceptibility to porcine epidemic diarrhea virus resistance to coronavirus infection in amino peptidase n-deficient pigs goblet cell depletion in small intestinal villous and crypt epithelium of conventional nursing and weaned pigs infected with porcine epidemic diarrhea virus a competitive inhibition elisa for the differentiation of serum antibodies from pigs infected with transmissible gastroenteritis virus (tgev) or with the tgev-related porcine respiratory coronavirus complete genomic sequences, a key residue in the spike protein and deletions in nonstructural protein 3b of us strains of the virulent and attenuated coronaviruses, transmissible gastroenteritis virus and porcine respiratory coronavirus sialic acid binding properties of soluble coronavirus spike (s1) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus we are grateful to e. cox for supplying the antibody against apn. we also thanks l. enjuanes for supplying the antibody against pedv. special thanks go to marthe pauwels for the excellent technical assistance. the authors declare no conflict of interest. key: cord-307408-6wfx0wey authors: li, renfeng; qiao, songlin; yang, yanyan; su, yunfang; zhao, pu; zhou, enmin; zhang, gaiping title: phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field strains in central china based on the orf3 gene and the main neutralization epitopes date: 2013-11-30 journal: arch virol doi: 10.1007/s00705-013-1929-7 sha: doc_id: 307408 cord_uid: 6wfx0wey since 2010, porcine epidemic diarrhea has re-emerged with devastating impact on the swine-raising industry in central china. to investigate the epidemic characteristics of pedv, the complete orf3 genes of 14 pedv field strains from central china during 2012 to 2013 were cloned, sequenced and compared with reference strains. phylogenetic analysis based on the complete orf3 gene showed that the pedvs in central china and the reference strains could be divided into three groups: g1, g2, and g3. the 14 pedv isolates were classified as g1 and showed a close relationship to some chinese strains isolated previously in central china and differed genetically from recent isolates from southern china, korean strains (sm98 and db1865, 2012), the chinese lzc strain (2007), and the vaccine strain (cv777) being used in china. our findings suggested that the pedvs circulating between 2012 and 2013 in central china might have evolved from earlier strains in the local region. to determine the reason for recent vaccination failures, we also studied variations in antigenicity of field strains by analyzing the three neutralizing epitope regions in the s gene. the results showed that the neutralizing epitopes at aa 245-252 were highly conserved, but most of the amino acid changes occurred in the epitope regions aa 7-146 and 271-278. we speculate that the amino acid mutations in the neutralizing epitope regions may be associated with changes in the antigenicity of pedv and consequently result in vaccination failure. together, these findings may be useful for understanding the epidemiology of pedv and may be relevant for designing of new and more efficacious vaccines. porcine epidemic diarrhea (ped) is an acute, highly contagious viral enteric disease of swine caused by ped virus (pedv), a member of the genus alphacoronavirus [1] . ped was first reported in belgium and the united kingdom in 1971 [2] . since then, the disease has been recognized in many european countries, such as germany, france and switzerland, and more recently in korea and thailand [3] [4] [5] . in china, pedv was first confirmed in 1984 [6] , and since 2010, it has caused enteric disease with a devastating impact on the swine-raising industry. this disease is characterized by high morbidity and mortality among preweaning piglets, causing serious economic losses to the swine industry in china [7] . pedv is an enveloped virus possessing an approximately 28-kb genome. the pedv genome contains at least seven open reading frames (orfs), encoding four structural proteins (spike [s], envelope [e] , membrane [m], and nucleocapsid [n] ) and three non-structural proteins (replicases 1a and 1b and orf3) [8] . the orf3 gene is an accessory gene of pedv and has been demonstrated to be associated with the virulence of pedv [9] . a region (nt 245 to 295) that is crucial for pedv pathogenicity, is deleted in all live vaccine strains, and this could be a marker of adaptation to cell culture and attenuation of the virus. thus, the orf3 gene could be used as a valuable tool for differentiation of wild-and attenuated-type pedvs and molecular epidemiology studies of pedv [10] . the spike protein (s) is the major structural proteins of pedv and consists of 1383 amino acids. similar to those of other coronaviruses, the s protein of pedv consists of three domains, including a large outer domain, a transmembrane domain and a short cytoplasm domain at the carboxyl end. it has been demonstrated to have four neutralizing epitopes (aa 499-638, 748-755, 764-771, and 1,368-1,374) on the surface of the s protein [11] [12] [13] , which are important for receptor binding and virus entry, the induction of neutralizing antibodies, and host-cell fusion [11, 13, 14] . therefore, the s protein is also essential for understanding the epidemiological status of pedv in the field, diversity of pedv isolates, and the association between genetic mutations and viral antigenicity [5, 15] . recent studies have shown that the ped caused by pedv is becoming increasingly serious in china, and current vaccines are ineffective on most swine-raising farms. molecular epidemiology and virus isolation of pedv have been undertaken in some regions of china [7, [16] [17] [18] [19] . these studies are essential for identifying the epidemic characteristics of pedv and have the potential to provide information about its pathogenesis. nevertheless, little is known about the antigenic variability of pedv prevailing currently in the field. in this study, we aimed to investigate the molecular epidemiology and antigenic variability of pedv field strains based on analysis of orf3 and a portion of the s gene encoding three neutralizing epitopes (aa 499-638, 748-755, and 764-771) of the s protein. this study provides new information about the prevalence of pedv strains currently circulating in china. intestinal and fecal samples were collected from piglets suffering from severe diarrhea from september 2012 to june 2013 in central china. the scraped-off mucosa and the contents of the small intestine were pooled, diluted 1:5 in phosphate-buffered saline (pbs; 0.15 m nacl, 0.01 m phosphate buffer [ph 7.4]), homogenized by ultrasonication, and clarified by centrifugation for 15 min at 4,8009g after freezing and thawing three times. the supernatants were collected for rt-pcr. pedv rna was extracted using trizol reagent (invitrogen), dissolved in 30 ll of 1 % diethylpyrocarbonate-treated water, and stored at -70°c. the complete orf3 gene was amplified using previously published primers [9] , and the size of expected product was 833 bp. the primers for amplifying the partial s gene were designed based on the genome of pedv cv777 (genbank no. af353511), and the size of the final fragments, containing three neutralizing epitopes of pedv, was 894 bp. two sets of primers were synthesized by sangon biotech, china. the rt-pcr primers are listed in table 1 . synthesis of the first-strand cdna for the n gene was carried out by reverse transcription using reverse transcription reagents from promega. the viral rna (50 ll) was mixed with 2.5 ll of 10 pm antisense primer, incubated at 65°c for 5 min, and placed on ice for 2 min. after that, 4 ll of 59 rt buffer, 4 ll of 2.5 mm dntp mixture, 1 ll of rnase inhibitor (40 u/ll), 1 ll of reverse transcriptase m-mlv (200 u/ll), and 2.5 ll h 2 o were added with gentle mixing. the reaction mixture was incubated for 1 h at 42°c, and the reaction was terminated by heating for 10 min at 65°c. the cdna was either stored at -20°c or amplified immediately. for pcr, 2 ll of cdna was mixed with a reaction mixture containing 2.5 ll of 109 taq dna polymerase buffer (promega, madison, wi), 3 mm mgcl 2 , 2.0 ll of dntps (2.5 mm), 0.5 ll of each specific primer (10 pmol), 1 ll of taq dna polymerase (promega, madison, wi) and autoclaved, filtered (0.2 lm) distilled water in a total volume of 25 ll. amplification was performed as follows: one cycle at 95°c for 5 min, followed by 30 cycles at 95°c for 1 min, 50°c for 1 min, and 72°c for 1 min, and a final extension at 72°c for 10 min. the rt-pcr products were visualized by electrophoresis in a 1.5 % agarose gel containing ethidium bromide. purified rt-pcr products were identified by electrophoresis in a 1.5 % agarose gel and cloned into pmd ò 19-t. the recombinant vector was identified by pcr and enzyme digestion. the positive clones were sent to sangon biotech, china, for sequencing. all sequencing reactions were performed in duplicate. the expected sizes of the pcr products were 833 and 894 bp. the former contained the complete orf3 gene and its flanking sequences, while the latter contained the partial s gene. the nucleotide sequences of the pedv isolates were deposited in the genbank database, and the corresponding accession numbers are listed in table 2 . the nucleotide sequences were assembled and proofread using contigexpress software. multiple sequence alignments were generated by the clustal w method using the megalign 4.0 program in dnastar (dnastar inc. usa) (version 7.0). phylogenetic trees were constructed by [20] . in addition, 18 reference strains with complete s gene sequences were chosen for the analysis of the region containing neutralizing epitopes (7-146). b-cell epitope prediction was performed using the bepipred 1.0b server (http://www.cbs.dtu.dk/services/bepipred/) [21] . the reference strains used for phylogenetic analysis and the 14 samples strains are listed in table 2 . fourteen (fig. 1) . fourteen partial s genes amplified from the clinical samples (genbank accession nos. kf484733-kf484746) had a size of 894 nt, encoding a 298-amino-acid polypeptide, corresponding to nt 1477-2370 of the complete s gene of the cv777 strain, containing three neutralizing epitopes (499-638, 748-755, 764-771), with 97.5-100.0 % nucleotide and 95.7-100.0 % deduced amino acid sequence similarity to each other, and 94.7-99.9 % nucleotide and 93.6-99.7 % amino acid sequence identity to the reference strains. a phylogenetic tree based on the partial s nucleotide sequence showed that the isolates could be clustered into three groups. all 14 isolates belonged to g1, together with most chinese strains and six foreign strains, including two american strains (usa/colorado/2013 and 13-019349), three strains from thailand (vn12258, ku04rb08, and th/np-65/12) and one strain from japan (mk). the 14 samples in g1 were clearly separated into two different subgroups: ch/fch-01, ch/lh, ch/ly-09, ch/jch, ch/zhz-04, and ch/heb formed a unique subgroup and differed clearly from the other pedv isolates, and the unique subgroup was adjacent to mk, a strain from korea. in addition, g2 included two korean strains (ad01 and nj02). g3 included one european strains (br1/ 7), four korean strains (dr13, kh, nk, and sm98), two early domestic strains (lzc and ch/s), and the vaccine strain cv777). these strains differed genetically from the 14 sample strains because of their distant location in phylogenetic tree (fig. 2) . in addition, the deduced amino acid sequences from the partial s gene analysis showed that there was no amino acid change in the epitope at aa 245-252 in any of the isolates. however, when compared with the reference strains, some variants were observed in the epitope regions at table 3 . only one deletion was observed at aa 85 in the epitope region aa 7-146 of ch/ xip-03. in addition, b-cell epitope in the partial s protein (aa 7-146) was predicted using the bepipred 1.0b server. the results showed that there were three b-cell epitopes with higher scores between aa 7 and 146, located at positions 22-30, 32-40, and 64-75. in the current study, the amino acid mutations at aa 28, 32, and 74 were within the epitopes predicted above (aa 22-30, 32-40, 64-75, respectively; fig. 3 ; table 3 ), indicating that these changes might result in antigenicity differences. pedv has been detected frequently in many provinces in china, including central china, since it was first identified in 1984, and has become one of the most important viral enteric diseases [7] . despite the current vaccination strategy, the losses caused by pedv infection are continuous and serious. it is necessary to understand the epidemiology of pedv and to explore the antigenic variation of the virus. during the epidemiologic investigation, we confirmed that pedv was the predominant causative pathogen contributing to outbreaks of clinical diarrhea in central china and determined that the positive rate of pedv in all diarrhea samples tested was 78.6 %, followed by rotavirus (11.3 %) co-infections with multiple pathogens in diarrheal disease were very frequent, especially mixed infections with pedv and porcine group a rotavirus (garv). these findings are consistent with those reported by zhang et al. [22] . the 14 samples used in this study were mostly from neonatal piglets (91.3 %), and they were obtained mainly in winter. to further investigate the molecular epidemiology of this virus, we chose the orf3 gene and a partial s gene encoding a region including three neutralization epitopes for studying the evolutionary characteristic and antigenic variation of pedv. orf3 is an important virulence gene that can be used to differentiate highly cell-adapted viruses and field isolates of pedv and is a potential tool for studying the molecular epidemiology of pedv [9, 23] . in the present study, 14 orf3 genes of pedv field strains collected in central china between 2012 and 2013 were amplified by rt-pcr, cloned, and sequenced to determine the genetic characteristics of viruses causing ped outbreaks in central china. the results confirmed that none of the 14 strains had the 51-nt deletion in orf3. sequence comparison with other pedv reference strains selected from the genbank database indicated that the orf3 genes from the sample strains had a high degree of homology to most chinese strains. for example, ch/ly-09, ch/fch-01, ch/heb, and ch/lh were distributed in the same subgroup with ch/zmdzy/ 11; ch/yf-01 and ch/kf-01 were in the same subgroup with ah2012; and both ch/zmdzy/11 and ah2012 were collected previously in central china. these results revealed that the pedv strains prevailing in central china might originate from local regions in central china. in addition, although relatively conserved when compared with the reference strains, variable sites were found in the orf3 nucleotide sequences of these 14 isolates (alignment data not shown), leading to the following single amino acid changes: l ? we speculate that these mutations may cause differences in the virulence of pedv in the field and consequently intensify the epidemic. moreover, the results in this study did not show a 51-bp deletion in the complete orf3 nucleotide sequence, indicating that the pedv prevailing on swine farms of central china were wild-type strains. as a glycoprotein on the viral surface, the s protein of pedv is closely associated with its pathogenicity and immunogenicity [10] . in this study, 14 partial s genes from field pedv strains were amplified by rt-pcr, cloned, sequenced, and analyzed to study their antigenic variation. alignment of the deduced amino acid sequences of the partial s regions showed relatively high sequence identity among the pedv strains. in particular, the 14 samples in this study were highly conserved in the neutralizing epitope region aa 245-252, but most of the changes occurred in the core epitope regions of aa 7-146 and aa 271-278. of these, there were 10 samples with amino acid sequences changes at position 28: from a to v (ch/yf-01, ch/qx-02, ch/ kf-01, ch/lshan) and a to s (ch/ly-09, ch/fch-01, ch/lh, ch/xip-03, ch/zhz-04, ch/heb). the amino acid sequences in four samples (ch/yf-01, ch/qx-02, ch/kf-01, ch/ly-09) changed from h to r at aa 32, and (fig. 3) . these changes might alter the antigenicity of pedv and consequently result in vaccination failure. further study is needed to confirm the relationship between amino acid mutations in epitope regions and antigenicity, which would help to improve our understanding of the prevalence of pedv in china. phylogenetic analysis based on the partial s gene showed that the 14 isolates were separated into two groups ( fig. 2) , with ch/fch-01, ch/lh, ch/ly-09, ch/jch, ch/zhz-04, and ch/heb forming a unique subgroup. due to amino acid changes occurring mostly in epitope regions, these results indicated that the antigenicity of these strains differed from that of other strains, and these might be new pedv variants prevailing in central china. interestingly, although isolated from different geographical areas, two american strains (usa/colorado/2013 and 13-019349), three thai strains (vn12258, ku04rb08, and th/np-65/12) and one korean strain (mk) were clustered within the same subgroup with some recent chinese strains, indicating that these strains from different geographic regions might have similar antigenicity. in addition, one european strain (br1/7), two japanese strains (kh and nk), two korean strains (dr13 and sm98), two early domestic strains (lzc and ch/s), and the vaccine strain (cv777) differed genetically from the other strains and clustered in the group g3 in the phylogenetic tree based on the partial s gene (fig. 2) . these results are consistent with those reported previously [24] [25] [26] . based on the sequence analysis of orf3 and partial s genes of pedv, molecular epidemiology was conducted using 14 field strains from central china. our results revealed that the pedvs prevailing in the central china are still wild-type strains rather than the vaccine strain, and they mainly originated from the earlier strains in local regions. the amino acid mutations that occurred in the s epitope region might be associated with vaccination failure and the emergence of new pedv variant strains. further study should be done to examine the pathogenicity, antigenicity, and epidemiology of pedv. sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhoea virus confirms that this virus is a coronavirus related to human coronavirus 229e and porcine transmissible gastroenteritis virus a new coronavirus-like particles associated with diarrhea in swine genetic characterization of porcine epidemic diarrhea virus pedv isolates from southern vietnam during 2009-2010 outbreaks molecular detection of porcine kobuviruses in pigs in korea and their association with diarrhea chinese-like 19 strain of porcine epidemic diarrhea virus study on the culture of porcine epidemic diarrhea virus adapted to fetal porcine intestine primary cell monolayer phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china porcine epidemic diarrhea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf 3 diagnostic notes: update on porcine epidemic diarrhea identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein major receptor-binding and neutralization epitope are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein spike protein region (aa 636-789) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea molecular epidemiology of porcine epidemic diarrhea virus in china molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) samples from field cases in fujian new variants of porcine epidemic diarrhea virus outbreak of porcine epidemic diarrhea in suckling piglets mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods improved method for predicting linear b-cell epitopes occurrence and investigation of enteric viral infections in pigs with diarrhea in china role of proteases in the release of porcine epidemic diarrhea virus from infected cells molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea genetic variation analysis of reemerging porcine epidemic diarrhea virus prevailing in central china from sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china key: cord-313691-6wq64h2b authors: zhang, yuhan; chen, yanjun; yuan, weifeng; peng, qi; zhang, fanfan; ye, yu; huang, dongyan; ding, zhen; lin, longhua; he, houjun; wu, qiong; song, deping; tang, yuxin title: evaluation of cross-protection between g1aand g2a-genotype porcine epidemic diarrhea viruses in suckling piglets date: 2020-09-17 journal: animals (basel) doi: 10.3390/ani10091674 sha: doc_id: 313691 cord_uid: 6wq64h2b simple summary: porcine epidemic diarrhea (ped), caused by ped virus (pedv), is a devastating enteric disease in pigs worldwide. at least two genotypes (g1 and g2) and five subgenotypes (g1a, g1b, g2a, g2b, andg2c) of pedv strains have been identified. to date, the reports on the antigenicity and immunogenicity of those viruses are limited and the results documented on cross-neutralization among different genotypes and/or subgenotypes of pedv were inconsistent. this study aimed to observe the comparative pathogenicity and cross-protection between g1a and g2a pedvs, and thus find a new insight into the antigenicity and immunogenicity of pedvs. the results of the present study demonstrated that the g2a-based inactivated vaccine could provide sterilizing immunity against both highly virulent homologous and heterologous pedv challenges. in contrast, the g1a-based inactivated vaccine could induce a sterilizing immune response against challenge of homologous strain cv777 and only provide partial protection for the challenge of a heterologous g2a pedv ch/jx/01. the findings of this study might explain the underlying mechanism that severe ped and deaths still occurred among the neonatal piglets of which cv777-based pedv vaccine were administered in china, and imply g2a-based pedv vaccine used in this study might be a good vaccine candidate for pedv which may provide solid protection against circulating highly virulent pedvs. abstract: to date, two genotypes, i.e., genotype 1 (g1) and genotype 2 (g2), of porcine epidemic diarrhea virus (pedv) have been identified in swine, while the cross protection between the g2a and g1a subgenotypes is undetermined. hence, in the present study, we attempted to observe a comparative pathogenicity and cross protection of g1a (cv777) and g2a (ch/jx/01) pedvs. initially pregnant sows were vaccinated twice with the two kinds of inactivated g1aand g2a-based pedv vaccines, respectively and the delivered neonatal piglets were challenged with prototype isolates of g1a and g2a pedvs, and then the pathogenicity and cross-protection in neonatal piglets were observed. the results showed that ch/jx/01, a highly virulent and dominant g2a pedv strain currently circulating in china had more severe pathogenicity in vitro and in vivo, and induced more strong immune responses, including higher titers of siga in maternal milk than that induced by cv777 pedv, a prototype of g1a pedv strain. all piglets from the sows immunized with ch/jx/01 could not only survive when challenged with the homologous pedv, but also be fully protected when challenged with heterogenous g1a pedv. in contrast, the piglets from the sows immunized with cv777 could be protected when challenged with homologous pedv and only partially protected when challenged with heterologous g2a strain of pedv (ch/jx/01). the findings of this study provide new insights into the pathogenicity, antigenicity, and immunogenicity of currently circulating wild type g2a pedv, which might be valuable for the development of novel pedv vaccine candidates with improved efficacy. porcine epidemic diarrhea (ped), caused by porcine epidemic diarrhea virus (pedv), is a devastating enteric disease in pigs, characterized with severe diarrhea, vomiting, and dehydration, especially with high morbidity and mortality (up to 100%) in suckling piglets [1] . pedv is an enveloped, single-stranded, positive-sense rna virus that belongs to the order nidovirales, family coronaviridae and genus alphacoronavirus [2, 3] . ped was first identified in the uk in 1971, and then spread to other european and asian countries. in late 2010, severe outbreaks of diarrhea occurred in both non-vaccinated and cv777 vaccinated swine herds in china, attributing to highly virulent emerging pedvs. since then, the emerging variant pedvs have been predominated in other asian, north american, and european countries [4] [5] [6] [7] [8] . subsequently, prototype pedv variant strains with deletion(s) and/or insertion(s) in the spike (s) gene (so called indel in g1b subgenotype), with less severe clinical cases and lower case-fatality rate, was identified in the united states in 2013, and later reported in asian and european countries [9] [10] [11] [12] [13] [14] . phylogenetic analyses based on the whole genome showed that all pedv strains can be classified into two genotypes: genotype i (g i or g1) and genotype ii (g ii or g2). g1 is represented by classical pedv strains of cv777 and dr13, and the g2 compasses the highly virulent strains of variant pedv [15, 16] . the g1 pedvs could be further divided into two sub-genotypes, i.e., g1a and g1b, respectively. the prototype pedv of cv777 is a representative of g1a. and the g2 pedvs are divided into three sub-genotypes, g2a, g2b, and g2c [17] . genetic analyses revealed that the emerging highly virulent pedvs being currently circulating in the field were different in various degrees from the classical pedvs, including the vaccine strain of cv777 being used in china [4, 18, 19] . until now, there have been considerable reports on the comparative pathogenicity and cross reactivity/protection among/between different sub-genotypes pedv strains, including the observation in pathogenicity and cross-protection between g2a and g2b [20] , g2b and g1b [21] , g2b and g2c [22] , g2b and g1 [23] , and g1a and g1b [24] . liu and colleagues reported a cross-protection between g2a and g2b strains, and found that g2a strain-based inactivated vaccine candidates had better efficacy to against both g2a and g2b strains than that of g2b-based candidates, shedding the lights on the development of an effective vaccine against the highly virulent pedv strains [20] . however, the cross-protection between g2a and g1 in vivo, especially the protype g1a pedv is still not documented. in this study, we isolated a highly virulent g2a pedv strain from a diarrheal piglet, designated ch/jx/01, by which an inactivated cell culture-derived vaccine candidate was generated. afterwards, the pathogenicity and cross-protection experiments of g2a strain ch/jx/01 towards g1a strain cv777 were performed by a passive immune protection approach, i.e., immunizing sows and then challenging suckling piglets fed with their mother milk. the jiangxi agricultural university institutional animal care and use committee approved the animal use protocol for this study (protocol number jxau-ae-2017-11). all the procedures were carried out in accordance with the care and use guidelines of experimental animals established by the ministry of agriculture of china. vero-81 cells (atcc ® ccl-81) were grown and maintained in dulbecco's modified eagle's medium (dmem, gibco, usa) supplemented with 10% fetal bovine serum (fbs, bi, usa), 100 u/ml penicillin, and 10 µg/ml streptomycin at 37 • c in a humidified atmosphere with the addition of 5% co 2 . the highly virulent chinese g2a pedv strain, ch/jx/01 (genbank accession number: kx058031) was isolated in our laboratory, and the g1a pedv strain cv777 was generously provided by dr. zhen li from shanghai academy of agricultural sciences. both ch/jx/01 and cv777 were propagated in vero-81 cells according to the previously described method [25, 26] . briefly, culture medium was removed from 90% confluent vero-81 cell monolayers in t-25 culture flasks (biofil, guangzhou, china), and the cells were washed twice with sterile phosphate-buffered saline (pbs, 0.01m, ph 7.2, solarbio, beijing, china). then, pedv ch/jx/01 p10 stock was inoculated into the flask with a multiplicity of infection (moi) of 0.1, supplied with 10 µg/ml final concentration of trypsin (sigma, usa). after incubation for 1 h at 37 • c with 5% co 2 , the supernatants were removed and 4 ml of virus growth medium containing 10 µg/ml trypsin was added, and was then cultured at 37 • c with 5% co 2 . when more than 80% of the cells showed cytopathic effects (cpes), the supernatants and cells were harvested, and stored at −80 • c until use. confluent vero-81 cells in six-well plates were washed twice and then inoculated with 400 µl of viral supernatants containing ch/jx/01 or cv777 at a moi of 0.1. after 1 h incubation at 37 • c with 5% co 2 , the inoculum with unabsorbed viruses was removed and 2 ml of dmem containing 10 µg/ml trypsin were added in each well, and then incubated at 37 • c with 5% co 2 . the cell culture supernatants were collected at the time intervals of 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 , and 48 h(s), respectively, and titrated by the median tissue culture infectious dose (tcid 50 ) according to the protocol described previously [27] . vero-81 cells were cultured in 12-well plates containing sterile covering slides and incubated with cv777 and/or ch/jx/01 at a moi of 0.1, respectively. after the virus was inoculated, the slides were taken out at 3 h, 6 h, 12 h, 24 h, 36 h, and 48 h, and then fixed with pre-cooled glutaraldehyde for 10 min, followed by rinsing with 0.01 m ph 7.4 pbs. the fixed cells were allowed to dry on air for at least 10 min. then, 0.5% v/v triton x-100 (sigma-aldrich, shanghai, china) was applied to each well for 10 min. after washing three times with pbs, the cells were overlaid with 5% w/v bovine serum albumin (bsa) in pbs and incubated at 37 • c for 30 min. the rabbit anti-pedv antibody made by our lab was used as the first antibody with a 1:100 dilution in pbs. after 1 h incubation at 37 • c, the cell monolayers were rehydrated by rinsing three times with pbs. coverslips from each well were stained using 100 µl 1:2000 diluted goat anti-rabbit immunoglobulin g (igg) conjugated with fluorescein isothiocyanate (fitc; sigma-aldrich, st. louis, mo, usa) and incubated for a 1 h at 37 • c. fitc-stained cells were then rinsed with pbs, and immediately examined under a fluorescence microscope (axio observer a1, carl zeiss, oberkochen, germany) [28] . six large white x duroc crossbred pregnant sows at 70 to 74-day of gestation were purchased from a commercial pig farm, which had neither previous outbreak of ped nor vaccination of pedv vaccines during the recent two years. initially, all sows had been tested for antigen negative of pedv by an established real-time pcr assay [26] , and sero-negative for pedv antibody by a commercial elisa kit (biovet, calgary, ab, canada). each sow was housed in a separate room, farrowed routinely in our animal facility in biosafety level-2 laboratory. the eighteenth passage (p18) of cell culture-adapted pedv strain ch/jx/01 with a titer of 10 7.0 tcid 50 / ml were inactivated using binary ethyleneimine (bei) at a final concentration of 0.2% incubating at 30 • c for 72 h. after inactivation, the remaining bei was neutralized by the addition of 20% sodium thiosulfate. to assure a complete inactivation of the virus, a standard procedure was employed by growing the preparation in vero-81 cell cultures. subsequently, the inactivated virus suspension was 1:1 v/v mixed with a water-in-oil-water adjuvant, montanide isa 201 vg (seppic, shanghai, china) according to manufacturer's instructions, and the prepared vaccines were stored at 4 • c until use. the detailed experiment design is shown in figure 1 . six pedv antigen-and sero-naïve sows were assigned randomly to three groups: (1) group 1, designated as exp-im-2a, sows in this group were intramuscularly vaccinated with a dose of 2 × 10 7.0 tcid 50 ch/jx/01 (in 4 ml volume) inactivated vaccine in the neck; (2) group 2, designated as exp-im-1a, sows in this group received 4 ml (2 × 10 7.0 tcid 50 ) of an inactivated cv777-based vaccine intramuscularly into the neck; and (3) group 3, named as mock-control, sows in this group were sham-vaccinated intramuscularly in the neck with 4 ml of pbs. all sows were firstly immunized at 90th pregnant day and re-immunized at 104th pregnant day. elisa kit (biovet, calgary, ab, canada). each sow was housed in a separate room, farrowed routinely in our animal facility in biosafety level-2 laboratory. the eighteenth passage (p18) of cell culture-adapted pedv strain ch/jx/01 with a titer of 10 7.0 tcid50/ml were inactivated using binary ethyleneimine (bei) at a final concentration of 0.2% incubating at 30 °c for 72 h. after inactivation, the remaining bei was neutralized by the addition of 20% sodium thiosulfate. to assure a complete inactivation of the virus, a standard procedure was employed by growing the preparation in vero-81 cell cultures. subsequently, the inactivated virus suspension was 1:1 v/v mixed with a water-in-oil-water adjuvant, montanide isa 201 vg (seppic, shanghai, china) according to manufacturer's instructions, and the prepared vaccines were stored at 4 °c until use. the detailed experiment design is shown in figure 1 . six pedv antigen-and sero-naïve sows were assigned randomly to three groups: (1) group 1, designated as exp-im-2a, sows in this group were intramuscularly vaccinated with a dose of 2 × 10 7.0 tcid50 ch/jx/01 (in 4 ml volume) inactivated vaccine in the neck; (2) group 2, designated as exp-im-1a, sows in this group received 4 ml (2 × 10 7.0 tcid50) of an inactivated cv777-based vaccine intramuscularly into the neck; and (3) group 3, named as mock-control, sows in this group were sham-vaccinated intramuscularly in the neck with 4 ml of pbs. all sows were firstly immunized at 90th pregnant day and reimmunized at 104th pregnant day. healthy neonatal suckling piglets born with the body weight of >1.2 kg were kept for this experiment, and used for virus challenge at 5 d of age. the piglets from group exp-im-2a were randomly divided into two groups (five piglets/group), and orally challenged with prototype cv777 (1 × 10 5.0 tcid50 per piglet) and ch/jx/01 (1 × 10 4.2 tcid50 per piglet), respectively. similar operations were carried out on piglets from exp-im-1a as indicated in figure 1 . piglets from mock-control group were randomly divided into three groups (five piglets each group), and orally challenged with healthy neonatal suckling piglets born with the body weight of >1.2 kg were kept for this experiment, and used for virus challenge at 5 d of age. the piglets from group exp-im-2a were randomly divided into two groups (five piglets/group), and orally challenged with prototype cv777 (1 × 10 5.0 tcid 50 per piglet) and ch/jx/01 (1 × 10 4.2 tcid 50 per piglet), respectively. similar operations were carried out on piglets from exp-im-1a as indicated in figure 1 . piglets from mock-control group were randomly divided into three groups (five piglets each group), and orally challenged with prototype cv777, ch/jx/01, and pbs, respectively ( figure 1 ). the groups were housed in separate rooms after challenge and were observed three times daily for the first 7 days post challenge (dpc). all piglets were weighted at dpc 0 and dpc 7. fecal score (fs) was recorded twice daily after challenge, and was scored under the criteria of 0 = solid, 1 = pasty, 2 = semi-liquid, 3 = liquid, respectively. rectal swabs were collected twice a day from 0 dpc to 7 dpc for pedv fecal shedding. the rna was extracted of rectal swabs using trizol plus (takara, dalian, china), and the titers/copies of pedv rna were quantified by an established taqman real-time rt-pcr in our lab [29] . samples were considered negative when no signal was observed within 40 amplification cycles. all of the lived piglets at 7 dpc were euthanized for gross and histopathology examination. milk samples were also collected from sows at the 7th day post parturition, and whey was prepared from milk samples following previously described protocols [30] . briefly, components of fat globules, casein micelles, and cells, which are known to interfere with immunological assays, were removed, then whey was used to determine siga responses by a commercial indirect elisa kits (yoyoung, guangzhou, china). the levels of tumor necrosis factor-α (tnf-α) in piglet serum samples in all groups were tested with commercial elisa kit following the manufacturer's instructions (invitrogen, frederick, md, usa). at necropsy, both intestine and other major organs were examined. duodenum (5 cm distal to the pylorus), jejunum (three samples taken at 40-60 cm intervals), and ileum (5 cm anterior to the ileocaecal valve) were collected. after 48 h fixation in 4% paraformaldehyde, tissues were serially dehydrated with 30%, 50%, 70%, 95%, and 100% ethanol, cleared in xylene, embedded in paraffin wax, and sectioned at 4-6 µm thickness. after dewaxing in xylene and serially rehydrating with 100%, 95%, and 70% ethanol, tissue sections were stained with hematoxylin and eosin (he, sigma-aldrich, shanghai, china) and then examined by conventional light microscopy. for each tissue section, at least ten villi and crypts were measured using a computerized image system with villous height and crypt depth (vh:cd) ratios calculated as previously described procedures [31] . a randomized design was applied in the study, and the replicate was defined as the experimental unit. statistical analysis was performed using spss software 25.0 (ibm corporation, armonk, ny, usa). the data of this study were assessed for the normal (gaussian) distribution by using the shapiro-wilk test in spss before performing the statistical analysis. statistical analysis of the growth titers of pedv strains belonging to different subgenotypes and body weight changes in piglets was performed using paired-samples student's t test. a kaplan-meier survival curve with log-rank test was used to compare piglet survival rate among the experimental groups. statistical analysis of the indices of villus height, crypt depth, thickness and the titer of siga was carried out by one-way analysis of variance (anova) and significant differences among group means were determined using the least significant difference (lsd) test. statistical analysis of the relative express level of cytokines was performed by using the tukey test. data are presented as the mean ± standard error of the mean (sem). a p-value of <0.05 was set as the statistically significant level. to compare the infectivity of ch/jx/01 and cv777 in vitro, the growth performance was assessed by growth kinetics and ifa. the titers of both viruses reached the plateau at 24 h post infection (hpi) in cell culture supernatants, while the tcid 50 of ch/jx/01 (10 7.32 tcid 50 / ml ) was over seven times as that of cv777 (10 6.47 tcid 50 / ml ) ( figure 2 ). the ifa results indicated that pedv n protein was present in the cytoplasm of vero-81 cells. both ch/jx/01 and cv777 induced an apparent and typical cpe in vero-81 cells, including cell fusion, cell detachment, and multinucleated giant cell formation. the fluorescence signals of ch/jx/01-infected cells appeared as earlier as 3 h post inoculation (hpi) and the signals were stronger than those of cv777-infected cells, which indicated ch/jx/01 was more aggressive and pathogenic than that of cv777 (figure 3 ). at the same time point of post inoculation, the syncytia of ch/jx/01-infected cells appeared earlier and in greater numbers than those of cv777-infected cells, suggesting that ch/jx/01 proliferated faster and was supposed to be more pathogenic than cv777. the results of ifa were consistent with the results of growth kinetics. animals 2020, 10, x 6 of 17 formation. the fluorescence signals of ch/jx/01-infected cells appeared as earlier as 3 h post inoculation (hpi) and the signals were stronger than those of cv777-infected cells, which indicated ch/jx/01 was more aggressive and pathogenic than that of cv777 (figure 3 ). at the same time point of post inoculation, the syncytia of ch/jx/01-infected cells appeared earlier and in greater numbers than those of cv777-infected cells, suggesting that ch/jx/01 proliferated faster and was supposed to be more pathogenic than cv777. the results of ifa were consistent with the results of growth kinetics. formation. the fluorescence signals of ch/jx/01-infected cells appeared as earlier as 3 h post inoculation (hpi) and the signals were stronger than those of cv777-infected cells, which indicated ch/jx/01 was more aggressive and pathogenic than that of cv777 (figure 3 ). at the same time point of post inoculation, the syncytia of ch/jx/01-infected cells appeared earlier and in greater numbers than those of cv777-infected cells, suggesting that ch/jx/01 proliferated faster and was supposed to be more pathogenic than cv777. the results of ifa were consistent with the results of growth kinetics. before challenge, all piglets were healthy and had no any clinical signs, and were antigen/antibody negative for pedv. after challenge, clinical symptoms, fecal scores (fs), body weight, morbidity, and mortality were observed and recorded (table 1) table 1 and supplementary table s1 ). clinically, the piglets in groups exp-1a-c-2a, pos-1a, and pos-2a were all extremely emaciated and severely dehydrated as evidenced by sunken eyes, inelasticity of the skin, and tacky subcutaneous tissues. for piglets in pos-2a, watery diarrhea observed as early as 12 hpc, severe dehydration showed within 24 h after watery diarrhea, and all of the piglets died within 6 dpi. the surviving piglets were euthanized at 7 dpc for the observation of gross and histopathological lesions. necropsy indicated that the small intestine walls were transparent and thin in all challenged groups, with pos-2a being the most severe. in some piglets, the intestinal lumens were filled with large amounts of liquid intestinal content. no gross and histopathological lesions were observed in the piglets of neg-control group. microscopic lesions were seen in the pedv infected piglets, pigs which had mild to severe atrophic enteritis at 7 dpc. shortening, blunting and fusion of the villi, and occasionally, vacuolization and exfoliation of enterocytes were noted. microscopic lesions observed in the ch/jx/01-infected piglets (pos-2a) were more severe than those observed in cv777-inoculated piglets (pos-1a). the vh:cd ratio in duodenum were 0.99 in piglets in pos-2a while 1.81 in piglets in pos-1a ( figure 4 and table 2 ). in the immunization-challenge groups (exp-2a-c-2a, exp-2a-c-1a, exp-1a-c-2a, exp-1a-c-1a), the microscopic lesions in exp-1a-c-2a were more severe than those observed in exp-2a-c-1a, with more shortened, blunted villi in duodenum and jejunum. the vh:cd ratios ranged between 1.55 and 1.97 in duodenum, jejunum, and ileum. no significant gross and microscopic lesions were noted in neg-control, and the vh:cd ratios were 10.62, 14.27, and 14.55 in duodenum, jejunum, and ileum, respectively ( figure 4 and table 2 ). virus shedding in feces in all experimental piglets was examined daily by pedv-specific taqman real-time rt-pcr. all piglets were negative for pedv rna in fecal swabs on 0 dpc and neg-control piglets remained negative for the duration of the 7 days post challenge. fecal shedding was detected in 5/5 pos-1a and 5/5 pos-2a piglets within 1 dpc, and the titers of pedv rna in piglets in pos-2a were magnitude higher than that in piglets within pos-1a. compared to exp-1a-c-1a, piglets in exp-1a-c-2a had more frequent virus shedding and the titers of the virus were higher. among piglets in exp-2a-c-2a, a piglet showed pasty was found virus shedding from dpc 1 to dpc 5 ( figure 5 and supplementary table s2 ). the colostrum antibody responses induced by vaccinated ch/jx/01 and cv777 were assessed by a commercial indirect-elisa kit. as shown in figure 6 , the ch/jx/01 and cv777 strains were all induced specific siga responses, while the titer of the siga stimulated by ch/jx/01 was significantly higher than that induced by cv777 at 7 days post parturition (p < 0.01). the protective effects of two maternal antibodies against challenge with virulent homologous and heterologous virus (ch/jx/01, cv777) were set by the observation of clinical signs and mortality of the piglets during the 7-day observation period. the results indicated that maternal antibodies induced by ch/jx/01 could protect 100% (5/5) piglets against both ch/jx/01 and cv777 challenge, while the maternal antibodies induced by cv777 protected only 40% (2/5) piglets against ch/jx/01. the survival curves were in accordance with the results of cross-protection and maternal siga antibodies (figure 7) . no piglets in group pos-2a survived under ch/jx/01 challenge. the colostrum antibody responses induced by vaccinated ch/jx/01 and cv777 were assessed by a commercial indirect-elisa kit. as shown in figure 6 , the ch/jx/01 and cv777 strains were all induced specific siga responses, while the titer of the siga stimulated by ch/jx/01 was significantly higher than that induced by cv777 at 7 days post parturition (p < 0.01). the protective effects of two maternal antibodies against challenge with virulent homologous and heterologous virus (ch/jx/01, cv777) were set by the observation of clinical signs and mortality of the piglets during the 7-day observation period. the results indicated that maternal antibodies induced by ch/jx/01 could protect 100% (5/5) piglets against both ch/jx/01 and cv777 challenge, while the maternal antibodies induced by cv777 protected only 40% (2/5) piglets against ch/jx/01. the survival curves were in accordance with the results of cross-protection and maternal siga antibodies (figure 7) . no piglets in group pos-2a survived under ch/jx/01 challenge. the expression of tnf-αwas tested in the sera of all piglets. the concentrations of tnf-α in groups pos-2a and pos-1a were significantly higher than the neg-control group at 48 and 96 hpc (p < 0.05, figure 8 ). no significant change of tnf-α was found in piglets within groups exp-1a-c-1a, exp-1a-c-2a, exp-2a-c-2a, and exp-2a-c-1a. the expression of tnf-αwas tested in the sera of all piglets. the concentrations of tnf-α in groups pos-2a and pos-1a were significantly higher than the neg-control group at 48 and 96 hpc (p < 0.05, figure 8 ). no significant change of tnf-α was found in piglets within groups exp-1a-c-1a, exp-1a-c-2a, exp-2a-c-2a, and exp-2a-c-1a. the expression of tnf-αwas tested in the sera of all piglets. the concentrations of tnf-α in groups pos-2a and pos-1a were significantly higher than the neg-control group at 48 and 96 hpc (p < 0.05, figure 8 ). no significant change of tnf-α was found in piglets within groups exp-1a-c-1a, exp-1a-c-2a, exp-2a-c-2a, and exp-2a-c-1a. the continued epidemic of ped has caused huge economic losses to the pig industry around the world. in china, the mortality of piglets within 7-day age associated with pedv infection could reach 80% to 100% [18] . in usa, pedv infection has reduced domestic pig population by nearly 10% during 2013 to 2014 [32] . epidemiologic survey and phylogenetic analysis indicated that most of the pedv strains currently circulating in the field were variants of pedv, which are highly pathogenic and caused severe morbidity and mortality in piglets [31, 33, 34] . the highly virulent pedv strains were genetically different from the classical pedv strains [17] . as reported, there are many nucleotide variations between g1 and g2 pedv strains, especially in spike gene, which may lead to the failure of traditional attenuated vaccines being used in asia [15] . in our study, biological characteristics between ch/jx/01 and cv777 suggested that ch/jx/01 grew faster and induced more severe cpe in cells and caused more severe diarrhea and death in piglets, indicating that the ch/jx/01 were more virulent than that of classic pedv cv777. several subgenotypes of field pedv-based vaccines are being used in china, but the efficacies are tremendously variable. many pig herds vaccinated with classical pedv vaccines failed to provide solid protection against highly virulent pedvs [19, 32] . so, it is essential to investigate the crossneutralization of different subgenotypes of pedv circulating in the field and find novel vaccine candidates with improved efficacy. since it's costly to use pregnant sows as experimental models, many studies on the cross-protections evaluation use neonatal or weaning piglets. in practice, most infections were contracted to piglets within 7 days old due to the lack of adaptive immunity against pedv. for suckling piglets, the maternal antibodies provide the primary protection against the pedv infection. in this study, to evaluate the homologous and heterologous reactivities of g1a and g2a pedvs, we initially immunized pregnant sows twice with inactivated g1a and g2a pedv vaccines and then challenged piglets with the prototype strains of g1a pedv strain cv777 and field very virulent g2a pedv strain ch/jx/01, respectively. the sows vaccinated with the inactivated ch/jx/01 had a stronger siga responses in milk than that of cv777. it has been shown that siga levels in maternal milk correlated with siga measured in sera of suckling piglets [35] . goede and colleagues found that previous infection of sows with a mild virulence pedv strain could confer protection on piglets against high virulent pedv strain [16] . lin et al. reported that vaccinated inactivated pedv could provide piglets against homologous challenge [36] . so, the piglets fed by sows immunized with ch/jx/01 could obtain more anti-pedv siga in milk, and thus passively protected by those siga originated from their mothers. when challenged with very virulent strain of ch/jx/01, piglets in groups pos-2a had earlier disease onset, higher diarrhea index and earlier death time than those infected with cv777 strain, which indicated ch/jx/01 had higher pathogenicity than that of cv777. the continued epidemic of ped has caused huge economic losses to the pig industry around the world. in china, the mortality of piglets within 7-day age associated with pedv infection could reach 80% to 100% [18] . in usa, pedv infection has reduced domestic pig population by nearly 10% during 2013 to 2014 [32] . epidemiologic survey and phylogenetic analysis indicated that most of the pedv strains currently circulating in the field were variants of pedv, which are highly pathogenic and caused severe morbidity and mortality in piglets [31, 33, 34] . the highly virulent pedv strains were genetically different from the classical pedv strains [17] . as reported, there are many nucleotide variations between g1 and g2 pedv strains, especially in spike gene, which may lead to the failure of traditional attenuated vaccines being used in asia [15] . in our study, biological characteristics between ch/jx/01 and cv777 suggested that ch/jx/01 grew faster and induced more severe cpe in cells and caused more severe diarrhea and death in piglets, indicating that the ch/jx/01 were more virulent than that of classic pedv cv777. several subgenotypes of field pedv-based vaccines are being used in china, but the efficacies are tremendously variable. many pig herds vaccinated with classical pedv vaccines failed to provide solid protection against highly virulent pedvs [19, 32] . so, it is essential to investigate the cross-neutralization of different subgenotypes of pedv circulating in the field and find novel vaccine candidates with improved efficacy. since it's costly to use pregnant sows as experimental models, many studies on the cross-protections evaluation use neonatal or weaning piglets. in practice, most infections were contracted to piglets within 7 days old due to the lack of adaptive immunity against pedv. for suckling piglets, the maternal antibodies provide the primary protection against the pedv infection. in this study, to evaluate the homologous and heterologous reactivities of g1a and g2a pedvs, we initially immunized pregnant sows twice with inactivated g1a and g2a pedv vaccines and then challenged piglets with the prototype strains of g1a pedv strain cv777 and field very virulent g2a pedv strain ch/jx/01, respectively. the sows vaccinated with the inactivated ch/jx/01 had a stronger siga responses in milk than that of cv777. it has been shown that siga levels in maternal milk correlated with siga measured in sera of suckling piglets [35] . goede and colleagues found that previous infection of sows with a mild virulence pedv strain could confer protection on piglets against high virulent pedv strain [16] . lin et al. reported that vaccinated inactivated pedv could provide piglets against homologous challenge [36] . so, the piglets fed by sows immunized with ch/jx/01 could obtain more anti-pedv siga in milk, and thus passively protected by those siga originated from their mothers. when challenged with very virulent strain of ch/jx/01, piglets in groups pos-2a had earlier disease onset, higher diarrhea index and earlier death time than those infected with cv777 strain, which indicated ch/jx/01 had higher pathogenicity than that of cv777. results from studies on cross-reactivity, cross-neutralization, or cross-protection between different subgenotypes of pedv were various. researchers evaluated cross-reactivity between g2b non s-indel and g1b s-indel pedvs using convalescent sera from pigs, and confirmed the serological cross-reactivity between the two subgenotype strains in vitro [11] . a study presented by lin et al. showed that previously infected with s-indel pedv could just protect 81.25% (13/16) protection on challenge by virulent non s-indel pedv [37] . in the present study, piglets from sows vaccinated with g2a and g1a pedvs could sustain the challenges of homologous viruses, and provide cross-protection to g1a strain of pedv. however, sows immunized with g1a pedv could not provide cross-protection to the g2a strain virus challenge. this implicated that g2a pedv might provide immunity to both g2a and g1a pedvs. liu and colleagues reported that the g2a strain-based inactivated vaccine candidates are more promising than g1b-based pedv candidates for the development of an effective vaccine against the current highly virulent pandemic pedv strains [20] . so, the g2a pedv strain might be a better vaccine candidate strain for vaccine development. proinflammatory and anti-inflammatory cytokines (e.g., tnf-α and il-6, il-10, il-12, and il-22) are produced to mediate various inflammatory responses and exert antiviral effects [38] [39] [40] . previous studies have shown that tnf-α can induce a striking increase in the number of intra-alveolar neutrophils and their phagocytic capacity against various viruses. in addition, tnf-α induces apoptosis in cells infected with a virus [41, 42] . the levels of serum tnf-α were in accordance with the symptoms of piglets. piglets in group pos-2a with most severe diarrhea and death had the highest tnf-α level, while the levels in piglets within groups exp-1a-c-1a, exp-1a-c-2a, exp-2a-c-2a, and exp-2a-c-1a were not significantly increased. the reason might due to the maternal antibody neutralized part of the viruses and suppressed the inflammatory responses. however, further study is needed to explore the interactions between pedv and cellular cytokines. the highly virulent g2a pedv strain ch/jx/01 induced stronger cpe, grew faster in vero-81 cells, and induced higher siga level in maternal milk in vivo than those in g1a pedv strain cv777. furthermore, vaccination with ch/jx/01 could not only protect the homologous challenge, but also showed a full cross-protection to heterogenous pedv strain cv777 challenge. the findings of this study provide new insights into the pathogenicity, antigenicity, and immunogenicity of currently circulating wildtype g2a pedv, which might be valuable for the development of novel pedv vaccine candidates with improved efficacy. supplementary materials: the following materials are available online at 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providing the pedv cv777 strain. 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. key: cord-322475-i29t7ce8 authors: chen, xi; yang, jinxian; yu, fusong; ge, junqing; lin, tianlong; song, tieying title: molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) samples from field cases in fujian, china date: 2012-07-29 journal: virus genes doi: 10.1007/s11262-012-0794-x sha: doc_id: 322475 cord_uid: i29t7ce8 the outbreak of porcine epidemic diarrhea virus (pedv) has been a big problem of swine industry in china in recent years. in this study, we investigated molecular diversity, phylogenetic relationships, and protein characterization of fujian field samples with other pedv reference strains. sequence analysis of the s1 and sm genes showed that each sample had unique characteristics, and the sample p55 may be differentiated from the others by the unique deletions and insertions of sm gene. phylogenetic analysis based on s1 or sm gene, which have high levels of variations, indicated that each sample was related to the specific reference strain, and this finding was consistent with the protein characterization prediction analysis. the study is useful to better understand the prevalence of pedv and its prevention and control in fujian. porcine epidemic diarrhea (ped) is a devastating swine disease that is characterized by acute enteritis and lethal watery diarrhea, followed by dehydration, and frequently leading to a high mortality in piglets [1] [2] [3] . most of the incidence farms found the disease first in farrowing barns and subsequently 100 % mortality of newborn piglets. the disease was first reported in england in 1971 [4] , and since then, outbreaks of the disease have been reported frequently in europe and asia [5] [6] [7] . since 1990s, the disease has continuous outbreak in pig farms of 26 major cities and provinces in china, causing tremendous economical losses to the swine industry [8] . the causative agent of ped, the porcine epidemic diarrhea virus (pedv), was first described in 1978 [9] . then, a cell culture system was developed for pedv isolation and propagation [10] . pedv is a member of coronavirus genus and the family coronaviridae. the genome consists of a positive-sense, single-stranded rna, with 27-32 kb in size, which can transcribe into several subgenomic mrnas, and encode structure or non-structure proteins in a conserved order [11] . the polymerase gene, which covering 70 % of the genome, encodes the replicase polyproteins. the genes for major structural proteins including the membrane protein (m), the phosphorylated nucleocapsid protein (n), the small membrane protein (sm), and the spike protein (s) are located downstream of the polymerase gene [11] . the s glycoprotein makes up the large surface projections of the virion and plays an important role in the attachment of viral particles to the receptor of the host cell [12] [13] [14] . thus, the s glycoprotein would be a primary target for the development of vaccines against pedv. it is also the major envelope glycoprotein of the virion, which serves as an important viral component to understand genetic relationships of different pedv strains and the epidemiological status of pedv in the field [6, 15, 16] . the sm gene is the only accessory gene of pedv. accessory genes are generally maintained and their loss mainly results in attenuation of the virus in the natural host [17] . for pedv, virulence of the virus can be reduced by altering the accessory gene region in a manner similar with tgev [18] , and its differentiation could be a marker of virus attenuation [19] and a valuable tool for the study of molecular epidemiology of pedv [8] . in china, pedv was first isolated in 1982 [20] , its prevalence has been a big problem of swine industry in recent years, although a periodic vaccination strategy has been applied nationwide to prevent the disease [21] . thereby, a comprehensive study is necessary to better understand the genetic relationships between different strains, and would be helpful to find out the reason of the continuously outbreak of pedv and develop new strategy to control and prevent pedv infection. in this study, we investigated the molecular epidemiology and analyze phylogenetic relationships of fujian pedv field samples with other pedv reference strains. the study mainly focused on s1 and sm gene due to their vital roles in viral function and higher variation. partial of intestine or stool specimens were taken individually from the acute enteritis and watery diarrhea piglets of 3 different big swine farms in fujian province in 2011, and designated as p55, p68, and f422, respectively. intestinal samples were homogenized with 9 times of phosphatebuffered saline (pbs). the suspensions were then vortexed and centrifuged for 10 min at 1,7009g. the supernatants were stored at -80°c before utilization. in order to determine the sequences of the pedv samples, primers were designed based on the sequence of reference pedv strains ( table 1) . partial of s gene, i.e., s1, was amplified for investigation because of its long length. in brief, viral rna was extracted from the supernatants of the homogenized samples with the rnaiso plus agent (takara, japan) according to the manufacturer's instructions. rt-pcr was conducted individually to amplify each fragment from the isolated rna using primescript ò one step rt-pcr kit ver.2 (takara, japan) according to the manufacturer's protocol under the following conditions: reverse transcription at 50°c for 30 min, denaturation at 94°c for 2 min, 30 cycles of denaturation at 94°c for 30 s, annealing at 55°c for 30 s, and extension at 72°c for 1 min. the rt-pcr products were analyzed by 1.5 % agarose gel electrophoresis and visualized by ultraviolet illumination after ethidium bromide staining. bands of the corresponding size of the gene were excised, and the synthesized dna was purified using a qiaquick gel extraction kit (qiagen, germany) according to the manufacturer's instructions, then sequenced by takaka company. the nucleotide and deduced amino acid sequences of s1 and sm genes of pedv samples were independently used for sequence alignments. the multiple-sequencing alignments were generated with clustalw method by megalign 4.0 [22] . phylogenetic tree were constructed with deduced amino acid sequences by the bootstrap neighbor-joining method. in the study, the characterizations of deduced amino acid sequences, including pi value, antigenic peptides, hydrophobic positions, and transmembrane motif, were analyzed by danman program. sequence analysis of s1 region the nucleotide sequences of the sl region are 2,024 bp for p55, 2,032 bp for p68, and 2,036 bp for f422 in length (accession number: jq723739, jq723740, and jq723741). sl protein of p55 is 620 aa in length with a predicted mr of 68.1 kda, sl protein of p68 and f422 is 522 aa in length with a predicted mr of 57.2 kda. twelve homolog sequences were found in the genbank and shared the similarity of 99 % (table 2) . however, mutations were frequently occurred in s1 gene. the alignment analysis indicated that five sequences including p68, f422, ch/ interestingly, most of the mutations were observed in the n-terminal region. these variations of p68 and f422 were probably due to mutation of the gene with filed strains. p55 and dr13 consists of another group (group 2, fig. 1 ) with 8 specific nucleotide changes, and the mutations occurred in the middle of s1 gene, interestingly, the purine (c/g) and pyrimidine (a/t) was found interchanged (c/g$a/t). the relationships of group 1 and group 2 were testified by their deduced amino acids. the sequences of group 1 were found to have a long deletion at the initial followed by a short deletion. the mutations of group 2 were found to have a deletion at position 157 and a substitution at position 329 (s?f). in terms of potential asparagine (n)linked glycosylation sites, only 11 sites were found in group 1, much less than group 2 (14 for p55 and 15 for dr13). unlike the result by lee et al. [23] , neither gtaaac nor similar sequence was found upstream of the initiator atg of the s gene in all of the chinese and english (cv777) strains. the sm gene of 3 fujian pedv field samples were sequenced (accession number: jq723734 for p55, (fig. 2) . p55 and f422 own 7 and 8 unique point mutations, respectively. however, besides the long deletion in p55, only one amino acid was changed by those mutations (f?l at 124 in f422, fig. 2 ). in addition, p55 have one less asparagine (n)-linked glycosylation sites than the others. all the pedv strains including the 3 fujian samples except the sm98 strain (accession number: gu937797) have a conserved sequence (ctagac) at 46 nucleotides upstream of the initiator atg. in order to analyze the phylogenetic relationships between the 3 fujian samples and other pedv strains isolated in various regions worldwide, we constructed 2 phylogenetic trees using the deduced amino acid sequences of s1 and sm, respectively (fig. 3) . the phylogeny based on the s1 glycoprotein indicated all the strains were clustered into 3 major groups, including one big mixed group (group 1) and 2 chinese groups (group 2 and 3). p68 and f422 formed a subgroup (subgroup 4) to differentiate with other strains. the subgroup comprising dr13 and p55 (subgroup 1) located in group 1. the result was correlated with the finding from sequence analysis. quite different from the results from s1 protein, phylogenetic analysis based on the sm protein fragment divided the strains into 2 groups, one of which included p55 and ch/gsjiii/07 (fig. 3b) . the reason might be the deletions occurred in the p55 and ch/gsjiii/07. f422 had a close relationship with dx and formed a subgroup, while p68 formed another subgroup. the characterization of s1 protein confirmed the results from phylogenetic analysis ( table 3 ). the characterizations of p55 and dr13, except antigenic peptide number, were shown to be greatly different from those of other strains; and the strains f422, p68, ch/fjnd-3/2011, cnu-091222-02, and cv777 shared the similar antigenic peptide, but had one for the sm protein, pi varied from about 6.5 to 11 among the 6 chosen strains (table 4) , indicating the potential variation of the protein. it was noteworthy that high identities between f422 and dx were indicated by same characterizations except one hydrophobic region. the identities between p68 and cv777 were less than dx and f422, differences of which involved in little pi variation, one variation in hydrophobic and transmembrane segments and 3 positions' amino acid mutations (table 4 , underlined). consistent with the phylogenetic analysis, the characterizations of p55 and ch/gsjiii/07 were similar and extremely different from the other strains. since the sm determines the virulence of pedv [24] , our results would benefit the research on the variation of virulence of pedv in china. the diversities in s1 and sm were observed to be significant among different strains. although there were so many mutations in this segment, the first unique characteristic was the deletion in the sm gens of ch/gsjiii/07 and p55. compared to ch/gsjiii/07, p55 was found to be more viable due to the existence of insertion within the c-terminus domain, the unique point mutations and less asparagine (n)linked glycosylation sites. the long deletion of sm gene, which was also found in the field strain dr13 (accession number: jq023161) and its attenuated strain (accession number: jq023162) [25] , led to reduced pathogenicity and induced protective immune response in pigs [24] . remarkably, similar results were found in p55 and there were no significant mutations found in the sequences of other structural protein genes including m, n (data not shown), and s gene, whether the mutated strain reduced its pathogenicity or not needs further study. the loss of sm resulted in attenuation of the virus in the natural host. however, we found that the pedv with long deletion of sm gene also caused typical clinical signs of pedv infection, the pathogenesis mechanism of the virus and how the sm mutant strain comes from also need to be clarified. in general, the variation in sm gene different from the various diversities of sm gene, the s1 region of the 3 fujian samples have unique mutations in common. coronaviruses have transcription regulatory sequences (trss) that include a highly conserved core sequence 5 0 -cuaaac-3 or a related sequence at upstream of encoding genes [26] . though the sequence ataaac, agaaac, and ctagac were found respectively upstream of the initiator of m, n (data not shown), and sm gene, the sequence gtaaac reported in the korean strains [23] was not found upstream of the s1 gene of the fujian pedv samples. however, the neutralizing epitome was conserved in s1 that is responsible for mediating the production of anti-viral neutralizing antibodies. phylogenetic trees based on the protein sequence were constructed to analyse the relationship between the fujian samples and the other strains. phylogenetic analysis based on sm protein indicated that the strain ch/gsjiii/07 was relatively close to p55, but distantly related with group 1. however, park et al [27] found that ch/gsjiii/07 was in group 1, which was different from our research. the reason for these might be due to the nucleotides sequences were used in the previous study, but amino acid sequences were used in this study. the location of p68 and f422 in the tree based on s1 protein suggested high variation of fujian samples. dr13 and p55 were within the same subgroup. as dr13 was used to develop the pedv vaccine in korea [28] , it might be interesting to know whether p55 can be used to develop the pedv vaccine in china. table 2 . a tree based on amino acid sequences of s1 protein. b tree based on amino acid sequences of sm protein the results of protein characterization prediction confirmed the relationship and demonstrated specific differences between the close strains obtained from sequence and phylogenetic analysis, which might be useful in further functional exploration. it was noteworthy that the unique hydrophobic region in the n-terminus of s1 protein of cv777, cnu-091222-02, dr13, and p55 that might related to the variation of protein structure and function. in conclusion, the fujian pedv samples were classified into different group. both of p68 and f422 were found to have close relationship with isolated strains from china, but still have some unique characterizations. the p55 had highest variation and a close phylogenetic relationship with filed strain ch/gsjiii/07. the underlines indicate amino acid mutations between strains experimental infection of pigs with a new porcine enteric coronavirus, cv777 porcine epidemic diarrhea, in diseases of swine porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs letter to the editor an immunoelectron microscopic and immunofluorescent study on the antigenic relationship 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glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea coronaviruses: structure and genome expression the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host efficacy of a transmissible gastroenteritis coronavirus with an altered orf-3 gene cloning and further sequence analysis of the orf3 gene of wild-and attenuated-type porcine epidemic diarrhea viruses porcine epidemic diarrhea molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools heterogeneity in spike protein genes of porcine epidemic diarrhea viruses differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf 3 complete genome sequences of a korean virulent porcine epidemic diarrhea virus and its attenuated counterpart complete genome sequence of transmissible gastroenteritis coronavirus pur46-mad clone and evolution of the purdue virus cluster molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus dr13 acknowledgments this work was supported by project management for agricultural science and technology achievements transformation fund (2010gb2c400209), science and technology major project of fujian (2010nz0002-3), and national spark program (2011ga720005). we also thank hualong feedstuffs technology and development group company in fujian province for sample collection and advices. key: cord-321953-yql6gpd3 authors: barrera, maritza; garrido-haro, ana; vaca, maría s.; granda, danilo; acosta-batallas, alfredo; pérez, lester j. title: tracking the origin and deciphering the phylogenetic relationship of porcine epidemic diarrhea virus in ecuador date: 2017-12-12 journal: biomed res int doi: 10.1155/2017/2978718 sha: doc_id: 321953 cord_uid: yql6gpd3 in 2010, new chinese strains of porcine epidemic diarrhea virus (pedv), clinically more severe than the classical strains, emerged. these strains were spread to united states in 2013 through an intercontinental transmission from china with further spreading across the world, evidencing the emergent nature of these strains. in the present study, an analysis of pedv field sequences from ecuador was conducted by comparing all the pedv s gene sequences available in the genbank database. phylogenetic comparisons and bayesian phylogeographic inference based on complete s gene sequences were also conducted to track the origin and putative route of pedv. the sequence from the ped-outbreak in ecuador was grouped into the clade ii of pedv genogroup 2a together with other sequences of isolates from mexico, canada, and united states. the phylogeographic study revealed the emergence of the chinese pedv strains, followed by spreading to us in 2013, from us to korea, and later the introduction of pedv to canada, mexico, and ecuador directly from the us. the sources of imports of live swine in ecuador in 2014 were mainly from chile and us. thus, this movement of pigs is suggested as the main way for introducing pedv to ecuador. porcine epidemic diarrhea (ped) is an acute and highly contagious disease, which causes severe enteritis, vomiting, watery diarrhea, dehydration, and high mortality rates in pigs [1] . ped was first described in 1971 in united kingdom, affecting fattening pigs [2] , and the etiological agent was identified in belgium as a new coronavirus, which was designated as ped virus (pedv) [3] . pedv belongs to the genus alphacoronavirus of the family coronaviridae, subfamily coronavirinae, and order nidovirales. pedv genome consists of 28 kb long single-stranded rna with positive polarity and includes seven known open reading frames (orfs). two large orfs, 1a and 1b, occupying two-thirds of the genome, encode two nonstructural polyproteins (pp1a and pp1b) that direct genome replication and transcription. the remaining one-third of the genome encodes four structural proteins, spike (s), envelope (e), membrane (m), and nucleocapsid (n), and one hypothetical accessory protein encoded by the orf3 gene [4] . of all viral proteins, the pedv s protein has a pivotal function regulating interactions with specific host-cell receptor glycoproteins to mediate viral entry [5] . therefore, pedv s protein has been often used to understand the genetic relationships between different pedv strains and the epidemiological status of pedv in the field (reviewed in [4] ). other genes including orf3, e, m, or n have been also utilized for phylogenetic inference [6] [7] [8] [9] and some studies have included pedv full genome sequences to get better phylogenetic resolution [10, 11] . however, realistically, sequencing the full genome of pedv is still expensive, from both computational and laboratory perspectives. moreover, for many computationally intensive analyses, utilizing the full genome is unfeasible. it would be, therefore, beneficial to use only those genomic regions that contain the highest phylogenetic signal to reduce cost without losing valuable information [12] . in fact, phylogenetic markers together 2 biomed research international with powerful bayesian phylogenetic approaches have been successfully applied to track the origin of important viral outbreaks [13, 14] and establish molecular epidemiology links among different viral strains including coronavirus members [15] . for pedv it was recently shown that the s and nsp3 genes contain the lowest phylogenetic noise; therefore both are the highest recommended for phylogenetic analysis and molecular characterization studies [11] . in 2010, new chinese strains of pedv clinically more severe than the classical strains emerged [16] . these strains were spread to united states in 2013 through an intercontinental transmission from china [17] with further spreading to canada [18] , mexico, and other countries into the american continent including colombia and dominican republic (reviewed in [1] ). moreover, in europe, the recent reports in germany [19] , france [20] , and belgium [21] of ped-outbreaks caused by strains closely related to the variants identified in china and the united states evidence the emergent nature of these strains. the epidemiological situation regarding pedv turned more complicated since in december 2013 a second pedv strain oh851 (later named as s-indel strain) emerged in the us [22] . this new pedv strain was also reported in spring 2014 in germany [19, 23] , as a consequence of a probable single or simultaneous introduction [24] and with later reports in other european countries including france [20] , belgium [21] , italy [25] , austria [26] , and spain [27] . even though the pedv s-indel strains have shown lower virulence in the field [28] the clinical manifestations of the disease in the european countries affected have been very variable, with ranges of mortality between 0 and 70% (reviewed in [29] ). this recent global reemergence of ped requires urgent attention and deeper understanding of pedv epidemiological links driving the changes in viral pathogenicity. therefore, this report was conducted using the complete sequence of the s gene and powerful bayesian phylogeographic reconstructions to clarify the putative origin of pedv in ecuador, revealing the wide expansion of the emergent pedv strains, which caused the first pedv outbreak in this country. international standards for animal welfare were used for all animal samples collected, following the regulations for animal sampling of the article number 134 of animal welfare law included into the national constitution of the republic of ecuador. , and processing of samples. on july 2014, a clinical outbreak with epidemiological characteristics compatible with pedv was reported in a pig farm located in the province of cotopaxi, ecuador. in detail, the commercial farm contained a total of 10,909 animals from which 1,341 animals were affected, showing clinical signs compatible with ped, 1,043 died as a consequence of the disease, and 1,401 were slaughtered as control measure. to avoid further spreading of the disease a national contingency plan was applied including restrictions on the animal movement, increasing the biosafety measures, and establishment of epidemiological surveillance at national level. from the ped-outbreak a viral isolation was obtained and named pedv/cotopaxi/2014. a total of five fragments of ileum from three different diarrheic pigs were taken together with the viral isolate pedv/ cotopaxi/2014 for total rna isolation from mucosal scrapings and cell supernatant, respectively. rna isolation was performed using trizol reagent (invitrogen) following the manufacturer's directions with modifications to ensure a high rna yield and quality. a total of thirteen primers were designed using the primer 3plus software [32] to amplify five overlapping fragments of the s gene (table 1 ) covering the complete s gene: 218 bases before the start codon and 213 bases after the termination codon of strain tc pc170-p2 virus ped united states (accession number genbank: km392227). all segments were amplified using superscript5 iii one-step rt-pcr high fidelity system with platinum5 taq dna polymerase (invitrogen) following the manufacturer's directions. the amplicons were visualized in agarose gel and purified by pure link kit quick gel purification (invitrogen) following the manufacturer's instructions. the resulting products were submitted to bidirectional dna sequencing using bigdye terminator cycling conditions by an external laboratory (macrogen, korea). consensus sequences were generated using the software sequencher version 6.1, 2014 (code gene corporation). nucleotide blast analysis (https://www.ncbi.nlm.nih.gov/ blast/blast.cgi) was initially used to verify the identity of the sequences obtained. to perform sequence comparison analyses and to establish the phylogenetic relationships of pedv sequence from ecuador, alignments using the consensus sequence of complete s gene available at genbank database (supplementary information table s1 ) were conducted by the algorithm clustalw included in the program bioedit sequence alignment editor [33] . to remove sequences with a possible recombinant event from the alignment datasets, searches for recombinant sequences and crossover regions were performed using geneconv, rdp, maxchi, chimera, bootscan, siscan, 3seq and lard, all implemented in rdp3 beta 4.69, as previously described in alfonso-morales et al. [12] . the software jmodeltest 2.0 [34] was used to estimate the best-fit model using the akaike (aic) and bayesian information criteria (bic). the bestfit models for the complete s gene were selected. phylogenetic analyses were performed by bayesian inference (bi), neighbour-joining (nj), and maximum likelihood (ml) methodologies as described elsewhere in [35] . all topologies obtained were compared as described by alfonso-morales et al. [12] . to visualize the structure of s protein and denote amino acids changes, a model of the s protein was kindly provided by professor douglas marthaler from department of veterinary population medicine, college of veterinary medicine, university of minnesota, st. paul, mn, usa, and recreated using chimera software package (http://www.cgl .ucsf.edu/chimera). table s1 , sequences denoted by asterisk) as described by alfonso-morales et al. [14] . briefly, the bayesian markov chain monte carlo (mcmc) analysis was performed in two independent runs. the resulting maximum clade credibility (mcc) phylogenetic tree was obtained by treeannotator and summarized using the spread software [36] . a keyhole markup language (kml) file was generated to identify the major routes of geographic diffusion. the bayes factor (bf) test was used to select the most probable routes of transmission. the resulting kml files from spread with a nonzero expectancy that showed a bf > 5 were visualized by google earth (available at: https://earth .google.com/). after the pedv outbreak in the united states in 2013, followed by the fast spreading of the virus to canada, mexico, korea, and taiwan, an important turn in ped research has taken place [1] . thus, a relevant increase of studies about the epidemiology, genetic structure, and characteristics of pedv has occurred to get a better understanding of this disease, which is currently the most fatal in pigs and one of the economic concerns for the pig industry [4] . in the present study, an analysis of pedv field sequences from ecuador was conducted by comparison with all the pedv s gene sequences available in the genbank database. in addition, phylogenetic comparisons and bayesian phylogeographic inference based on complete s gene sequences were conducted to track the origin and putative route of pedv. the s gene of pedv has more than 4000 nucleotides and encodes the s protein, which constitutes the spikes of the viral envelope, responsible for its high variability. the pedv s glycoprotein is known to be an appropriate viral gene for determining the genetic relatedness among pedv isolates [4] . the sequences obtained from the animals selected and the viral isolate pedv/cotopaxi/2014 were assembled, yielding a final fragment of 4414 nt of length for each one, all of them containing the 4160 nt of the complete s gene. the identity of the sequences obtained was 100% between them; therefore, to avoid redundant entries at genbank database, the sequences were submitted as a single entry under the accession number kt336490. the genetic identity of the sequence kt336490 (thereafter mentioned as pedv/cotopaxi/2014) was initially inferred from blastn analysis sharing 99% with more than 94 isolates or strains of pedv from us and korea. the sequence of the pedv usa/colorado/2013 isolate (accession number: kf272920) was selected to compare the sequence pedv/cotopaxi/2014 since it showed the highest score from the blastn analysis. thus, the sequence pedv/cotopaxi/2014 showed four nucleotide changes 1093axg, 2454cxt, 3051cxt, and 3607cxt when both sequences were compared. only two of these mutations led to amino acid changes when the deduced sequence was analyzed. the sequence pedv/cotopaxi/2014 showed the changes 360sxg and 1203lxf compared with the sequence of pedv isolate usa/colorado/2013. these mutations were not located at the neutralizing epitopes (ss2, ss6, or 2c10) [37] . therefore, associations with possible adaptive advantages caused by escaping to the immune response of the host cannot be suggested. the mutation 360sxg was located into the n-terminal domain (ntd) of s1 (figure 1 ). even though this domain has not been directly linked to pedv tropism and functionality as in transmissible gastroenteritis virus and murine hepatitis virus [38] , it is recognized that it can influence virus infectivity [30] . therefore, a mutation of serine in this domain could lead to an increase of the infectivity of the viral strains with this mutation, since serine is recognized as a catalytic residue of the trypsin (enzyme involved in the entry of the virus into the host cell in the digestive tract). nevertheless, further virulence studies will be required to verify this role. [30] ; chimera software v1.6.2 was used for visualization. domains s1 (orange) and s2 (blue) are denoted. the c-terminal rbd of the s1domain is represented in pink; n-terminal rbd of s1 domain is highlighted in yellow. the mutation 360sxg found in pedv/cotopaxi/2014 is denoted and represented in red. the best-fit model and the shape parameter of the gamma distribution (alpha) for the tree are indicated in the upper-left side. the numbers at a node are posterior probability values estimated. all different genogroups are denoted; the different clades ci and cii previously described by vlasova et al. [28] into the genogroup 2a are also denoted. the sequence of pedv/cotopaxi/2014 is highlighted in red. blue rectangles denote pedv strains previously classified as 2a in zhang et al. [31] (see figure 1b in zhang et al. [31] ; the strains were clearly grouped into genogroup 2b but were denoted as 2a). the us indel sequences were also denoted. the phylogenetic relationships among the pedv strains were reconstructed based on complete s gene by means of nj, ml, and bi analyses. all algorithms yielded congruent results showing the same topologies, which was supported by moderate to high confidence values given by the bootstrap percentage and the posterior probability (supplementary material figure s1 ). even though the tree yielded by bi was the best, the statistical support for this tree was not significantly different from the nj or ml trees (supplementary material table s2 ). thus, all topologies obtained showed two highly divergent genogroups (2a and 2b) (supplementary material figure s1 ). for a better visualization of the results, a short tree obtained from 60 pedv strains, representative of all genogroups or clades, was built by bi ( figure 2) . thus, the short tree showed the same distribution than the full tree (supplementary material figure s1 and figure 2 ). in figure 2 , two clades belonging to the pedv genogroups 2a and 2b can be clearly observed. in addition, from the genogroup 2a, two main clades (i and ii) previously described by vlasova et al. [28] were also obtained ( figure 2 ). the sequence from the ped-outbreak in ecuador pedv/cotopaxi/2014 was grouped into the clade ii of pedv genogroup 2a together with other sequences of isolates from mexico, canada, and united states ( figure 2 ). this group was highly supported by 0.93 posterior probability value ( figure 2 ). after the ped us outbreak, the virus rapidly spread to canada and mexico [4] . whilst contaminated food by spray-dried porcine plasma positive to pedv genome is pointed out as a possible way for introducing pedv from us to canada [18] , the legal movement of pigs is suggested as the source of introduction to mexico (http://www.thepigsite.com/swinenews/36693/mexico-reports-83-outbreaks-of-pedv-to-oie/). in the group of sequences in which pedv/cotopaxi/2014 was included, the nearest country to ecuador is mexico, indicating that the movement (legal or illegal) of animals between mexico and ecuador could be a possible source of introduction of the virus to ecuador. however, evidence about this possible link has not been found. phylogeographic reconstruction identified a specific location for the root of the tree with posterior probabilities for state sp = 0.81 for the locality of china (supplementary material figure s2 ). the phylogeographic study revealed the emergence of the chinese pedv strains followed by spreading to us in 2013 (figures 3(a) and 3(b), supplementary material video s1). huang et al. [17] previously observed this result describing the us ped-outbreak as an intercontinental transmission of the chinese strains. after introducing the chinese pedv strains to us the virus spread from us to korea (figure 3 (c), supplementary material video s1). s. lee and c. lee [39] reported the circulation of new pedv strains in south korea that were genetically like pedv strains that affected united states during 2013. this result suggested that the recent strains from south korea might have been originated in the united states, caused by the importation of pig breeding stock during or after the sudden emergence of pedv in the united states [39] . during 2010-2011, more than one-third of the total pig population in south korea was slaughtered as control measure to foot-and-mouth disease outbreaks; thus a huge importation of breeding pigs from us was carried out without a proper implementation of a vaccination policy [39] . the phylogeographic study also revealed the introduction of the virus in canada, mexico, and ecuador directly from the us (figure 3 (d) and supplementary material video s1). this result frames the most probable route of entry of pedv to ecuador from us. the sources of imports of live swine in ecuador in 2014 were mainly from chile and us (http://data.trendeconomy.com/trade/ecuador/import?com-modity=0103). thus, this movement of pigs is suggested as the principal way for introducing pedv to ecuador. because this is the first introduction of pedv in ecuador swine herds, a fast spread of the virus throughout the country is expected, especially because a vaccination policy against pedv has not been implemented yet. therefore, additional studies to decipher how the virus will be disseminated in ecuador and to other south american countries will be required. in this study, a rigorous measurement of the global phylogeographic approach for pedv strains was performed based on complete s gene sequences. the present work is the first study providing evidences that pedv strains are circulating into ecuador swine herds and revealing the molecular characteristic of the pedv isolate in the south american region. the spatial analyses suggested that these strains were introduced to ecuador by an importation from us. the authors declare that they have no conflicts of interest. table s1 . pedv sequences of the complete s gene used in the current study (xls). table s2 . comparison of topologies obtained for the complete s gene using ml, nj, and bi methods. figure s1 . phylogenetic tree obtained from the complete s gene and all sequences collected (table s1 ) (tiff). figure s2 . maximum credibility phylogeographic tree. the posterior probabilities for state were denoted (tiff). video s1. dynamics of spatial pedv diffusion. the most probable temporal distribution of pedv is provided; only rates supported by a bf of >5 were considered significant. 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zakhartchouk, alexander title: vaccines for porcine epidemic diarrhea virus and other swine coronaviruses date: 2016-12-02 journal: vet microbiol doi: 10.1016/j.vetmic.2016.11.029 sha: doc_id: 293458 cord_uid: jb7u9xn6 the recent introduction of the porcine epidemic diarrhea virus (pedv) into the north american swine herd has highlighted again the need for effective vaccines for swine coronaviruses. while vaccines for transmissible gastroenteritis virus (tgev) have been available to producers around the world for a long time, effective vaccines for pedv and deltacoronaviruses were only recently developed or are still in development. here, we review existing vaccine technologies for swine coronaviruses and highlight promising technologies which may help to control these important viruses in the future. coronaviruses were first described in the mid-1960s and subsequently isolated from a number of species including man, mice, swine and chicken. these viruses share a common morphological characteristic, a fringe of club-shaped projections, 12-24 nm long, around a pleomorphic 60-220 nm viral particle, having a resemblance to a solar corona (masters, 2013) . coronaviruses infect humans and various animal species, causing respiratory, gastrointestinal and neurological diseases as well as hepatitis. prominent examples include the severe acute respiratory syndrome virus (sars-cov), middle-eastern respiratory syndrome virus (mers-cov) and the feline infectious peritonitis virus (fipv), to name a few. swine coronaviruses can be divided into respiratory (prcov) and enteropathogenic coronaviruses such as transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv) and porcine deltacoronavirus (pdcov). the latter have similar epidemiological, clinical and pathological features. the family coronaviridae is currently divided into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. tgev and pedv belong to the alphacoronavirus genus, whereas pdcov belongs to genus of deltacoronaviruses. coronaviruses are enveloped, single-stranded, positive-sense rna viruses with the largest rna genome of approximately 30 kb reported to date. the genomic rna includes 5 0 and 3 0 untranslated regions (utr), and it is capped and polyadenylated. open reading frame (orf) 1a and orf1ab occupy the 5 0 two-thirds of the genome and encode two replicase polyproteins (pp1a and pp1ab). expression of pp1ab protein requires a ribosomal frameshift during translation of the genomic rna. produced polyproteins are proteolytically cleaved into 16 nonstructural proteins, nsp1 through nsp16 by the proteinase activity of nsp3 and nsp5. the 3 0 -proximal one-third of the genome encodes four structural proteins, including spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. some betacoronaviruses have an additional membrane protein, hemagglutinin esterase (he). interspersed between these genes are genes encoding accessory proteins. the number of these genes varies between different coronaviruses. for instance, tgev has 3 accessory genes, pdcov has 2, whereas pedv has only one (fig. 1) . the viral rna genome is packaged by the n protein into a helical nucleocapsid. in addition to the structural role, the n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, up-regulates interleukine-8 expression and antagonizes type i interferon production (ding et al., 2014; xu et al., 2013b) . the s protein, which forms peplomers on the virion surface, mediates binding to host receptors and membrane fusion. it can be divided into s1 and s2 domains. in some coronaviruses the s protein is processed into s1 and s2 fragments by cellular proteases or trypsin (belouzard et al., 2012; wicht et al., 2014) . the s protein is a major target for virus neutralizing antibodies (chang et al., 2002; reguera et al., 2012) . the m protein is the most abundant virion component and also contains conserved linear b-cell epitopes (zhang et al., 2012) . the e protein is responsible for the assembly of virion, and it causes endoplasmic reticulum stress and interleukin-8 expression up-regulation (xu et al., 2013a) . accessory genes are dispensable for virus growth in vitro, but they may play an important role in the virus survival in the infected host. indeed, the product of the tgev accessory gene orf7 reduces the expression of genes involved in the antiviral defense of the immune system, e.g. the interferon response, and inflammation (cruz et al., 2011) . the orf3 protein of pedv functions as an ion channel, and it is thought to be related with virulence of pedv (song et al., 2003; wang et al., 2012) . one of the pedv non-structural proteins, nsp1, was shown to be a type i interferon suppressor (zhang et al., 2016a) . interestingly, pdcov lacks the nsp1 gene. coronaviruses target predominantly type i and ii pneumocytes (prcov) or villous-and crypt enterocytes in the intestine (tgev, pedv and pdcov). pedv also infection goblet cells in the small intestine (jung and saif, 2015) . in addition, infection of alveolar macrophages and lamina propria macrophages has been shown for some but not all swine coronaviruses (laude et al., 1984; park and shin, 2014) . entrance of the virus into the target cells is mediated by a series of receptor ligand interactions including heparin sulfate (huan et al., 2015) and aminopeptidase n (apn) (chen et al., 1996; li et al., 2007) . importantly, the expression levels of aminopeptidase n appear to correlate with the level of infection, at least for pedv. the higher the expression levels the more severe is the infection (li et al., 2007; zhang and yoo, 2016) . as a result, it may be perceivable that piglets born with lower apn levels in the brush border may be more resistant to pedv than piglets with higher levels. enteric infections with tgev and pedv are characterized by severe diarrhea, vomiting and dehydration with high morbidity and mortality especially in piglets less than two weeks of age. in contrast, infections with respiratory coronaviruses cause very mild and transient disease in pigs of all ages, which often get unnoticed by the producer. unless complicated by concurrent infections, prcov infections are only short lasting with temporary phases of coughing and respiratory distress. however, prcov can become a more significant problem during co-infections with other pathogens such as the porcine reproductive and respiratory syndrome virus prrsv (jung et al., 2009) . infection of enterocytes with pedv results in villous atrophy which can lead to malabsorption, diarrhea and anorexia. within 24-48 h post infection, vomiting may occur, which typically does not last longer than 2-3 days post infection. diarrhea can be found within 24 to 36 h post infection depending on the dose of the virus and the age of the piglets. diarrhea typically lasts for about 5-8 days, but can last longer, and results in severe weight loss that often cannot be made up during the normal production cycle. viral shedding is highest between days 3-5, but can last for days to weeks post infection. surviving piglets start to recover around 6-8 days post infection, typically around the same time when proliferation of the crypt epithelium and regeneration of the villi occurs. similarly, tgev infects villous enterocytes and causes disease that clinically is indistinguishable from pedv. mortality rates are highest in young piglets, often reaching about 100%. in contrast, infection with pdcov causes milder infections in piglets between 3 and 5 weeks of age. diarrhea, vomiting and anorexia can be found in infected animals. in general, infected animals display much milder signs compared to infections with pedv and tgev. the innate immune response to enteric coronaviruses in pigs is characterized by a rapid antiviral response in the intestine, including the release of interferons, nuclear factor kb and other antiviral molecules (chattha et al., 2015; jung and saif, 2015; sang et al., 2010) . pigs can produce three types of interferons (sang et al., 2014) : type i interferons include well known interferons such as interferon a and b (ifn-a/b) and in pigs are encoded by as many as 17 different genes. the only type ii interferon in pigs is ifn-ɣ. type iii interferons include ifn-l1 (interleukin 29; il-29), ifn-l2 (il-28a), ifn-l3 (il-28b) and ifn-l4 (kotenko et al., 2003; park et al., 2012; prokunina-olsson et al., 2013; sheppard et al., 2003; zhang and yoo, 2016) . their functions are unknown in pigs. especially type i and iii interferons are used by the host to counteract viral infections. in response, most viruses including pedv and tgev have developed strategies to evade and interfere with the interferon response. several viral proteins, including structural and non-structural proteins have been identified for pedv and tgev that can suppress the interferon response. for an excellent review on the evasion of immunity by porcine coronaviruses please see (zhang and yoo, 2016) . the adaptive immune response to swine enteric coronaviruses is based on secretory antibodies and cytotoxic t cells. these include secretory iga antibodies (siga) that are produced by antibodysecreting cells in the lamina propria of the mucosal tissues and systemic antibodies such as igg and igm are found in serum and interstitial tissues and some isotypes can be transsudated across the mucosal epithelium into the lumen (chattha et al., 2015; horton and vidarsson, 2013) . the cellular response to swine coronaviruses is characterized by t helper cells that are supporting the production of antibodies and cytotoxic t cells that are targeting virus infected epithelial cells. in pigs, these are predominantly ɣɗcells, most of which can be found within the intraepithelial layer (bonneville et al., 2010) . the majority of t cell epitopes are located in the spike and nucleoprotein of coronaviruses (channappanavar et al., 2014; saif, 2004; sestak et al., 1999) . additionally, cd8 t cell epitopes have been found in the membrane protein of the human sars-cov (yang et al., 2006) in neonatal piglets, the main mechanism of protection is mediated by lactogenic immunity. during lactation, siga, igg and igm are passively transferred to the piglet via colostrum and milk (bohl and saif, 1975; saif and bohl, 1979, 1983; salmon et al., 2009) . colostrum contains predominantly igg, which is transudated from sow serum and absorbed by the piglet within the first 24-48 h of life. secretory iga is predominantly found in milk, after transitioning from colostrum to milk around 3-4 days of age (langel et al., 2016) . siga is produced by antibody secreting cells in the mammary gland, and it was shown many years ago by bohl and saif (bohl et al., 1972a ) that these cells migrate from the gut to the mammary gland at the end of pregnancy. this was confirmed by others in a variety of species and chemokines such as ccl28 and others have been found responsible for recruiting these antibody secreting cells to the mammary gland (bourges et al., 2008; lazarus et al., 2003; meurens et al., 2006 meurens et al., , 2007 wilson and butcher, 2004) . thus, in order to enhance the level of maternal immunity the oral route seems to be the most obvious choice for vaccinating the sow. indeed, most vaccines for enteric coronaviruses are designed to induce lactogenic immunity by vaccinating the sow, however, most of them are administered via systemic injection. in the absence of effective vaccines for pedv, many producers are currently using a lock-down of the barn combined with feeding back infectious live virus to pregnant sows. however, the duration of immunity often does not extent more than a few years (table 1) , depending on the type of vaccine being used with live vaccines typically providing longer lasting immunity. even after feed-back, immunity starts to wane after a relatively short period of time, often even less than a few months. for an excellent review of the role of lactogenic immunity for pedv see (langel et al., 2016) . in addition to antibodies, the colostrum also contains innate effector molecules such as defensins and antimicrobial peptides, interleukins and cytokines hlavova et al., 2014; mair et al., 2014; nechvatalova et al., 2011; salmon et al., 2009) . the level of cross-protection is somewhat unclear for coronaviruses. for pedv, goede et al. reported that 3-day old piglets born to sows that had been infected with a mild strain of pedv seven months previously, were protected against infection with a more virulent strain of pedv (goede et al., 2015) . in this experiment the sows were challenged with a more virulent pedv virus at day 109 of gestation, and orally re-challenged when the piglets were three days old. none of the sows displayed significant clinical symptoms. the piglets were orally challenged with 1 ml of mucus scrapings of the more virulent pedv. while mortality and morbidity rates varied significantly amongst the piglets in each group, the overall morbidity and mortality was reduced in piglets born to sows that had been pre-exposed to pedv. in the 90s, tgev was responsible for severe economic losses around the globe. several vaccine technologies were developed and commercialised. by administration to sows, the importance of lactogenic immunity was established (bohl et al., 1972b; saif and bohl, 1983) . however, with a disappearance of the disease in many parts of the world, fewer vaccines are now commercially available in north america and europe (table 2 ). most current commercial tgev vaccines are live attenuated vaccines that are given to the sow during gestation in order to provide lactogenic immunity to the newborn piglet. these vaccines are often available as bi-or trivalent vaccines combined with rotavirus, pedv and/or escherichia coli. experimental vaccines include novel dna vaccines, vectored vaccines and recombinant vaccines (table 1) . for example, the porcine adenovirus was used to deliver the tgev spike protein (tuboly and nagy, 2001) . yuan et al. used the swine pox virus to express the a epitope of the spike protein (yuan et al., 2015) . dna plasmids were generated for both pedv and tgev for the development of a dna vaccine (meng et al., 2013) . recombinant proteins (spike and nucleocapsid) have been extensively evaluated as recombinant vaccine following expression in bacteria, yeast and plants. many of these are being assessed for their potential to mucosal immunity after oral administration. oral delivery has not been demonstrated. the first vaccine for pedv in the us was developed by harrisvaccines tm in iowa, and conditionally licensed in 2013. the vaccine, initially called iped vaccine, was based on a truncated version of the pedv spike gene produced in the sirravax℠ rna particle technology platform , which is based on the a pvek replicon vector derived from the venezuelan equine encephalitis virus. the technology is a propagation defective, single cycle rna particle technology that is believed to target dendritic cells. a longer version of the spike genes was codon optimized and used in the second-generation vaccine called iped plus, which now is commercially available as porcine epidemic diarrhea vaccine, rna (ped rna). the vaccine induced immunity in young pigs after two doses given intramuscularly in a threeweek interval. vaccinated weaned pigs when challenged with homogenized gut tissue from a clinical isolate displayed significantly reduced severity of clinical signs (diarrhea) and reduced viral shedding for the first 72 h (mogler et al., 2014a) . vaccine efficacy was further tested in naïve sows. after three vaccinations at 8, 5, and 1 weeks pre-farrowing piglets from both groups were challenged between 2 and 6 days of age with 10 3 tcid 50 (pedv/co/ 2013). average litter mortality in the control group was 91%, while average mortality in the vaccinated groups was 69% (crawford et al., 2015) . similar results were found in sows previously exposed to pedv. after oral challenge of piglets within the first week average litter mortality was reduced from 59% in the control group to 45% in the vaccinated group. finally, greiner et al. (2015) evaluated the vaccine in 80 sows that had been previously exposed to pedv. vaccination induced higher titers against the s1 protein in the colostrum of vaccinated sows and reduced overall pig mortality by 3% (greiner et al., 2015) . a second vaccine for pedv in the us was developed by zoetis and made commercially available in 2014 under a conditional license. the vaccine consists of an inactivated whole virus formulated with an adjuvant. the vaccine was tested in pedv negative sows, which were vaccinated twice, 3 weeks apart, with the vaccine (n = 23) or an adjuvant placebo (n = 3). the vaccine was safe and immunogenic and induced neutralizing antibodies to both the whole virus and the spike protein . the vaccine was also tested under field conditions in a pedv positive commercial herd. sows (n = 120) were vaccinated at 5-and 2 weeks pre-farrowing, control sows (n = 120) received placebo. vaccination resulted in reduction of pre-weaning mortality due to pedv from 6.3% in piglets born to control sows versus 0.6% in piglets born to vaccinated sows. vaccination resulted in about 3 times higher neutralization antibody titers compared to the control group, and an additional 1.8 pigs per sows survived . a third vaccine was recently developed by the vaccine and infectious disease organization-intervac in canada. the vaccine is based on inactivated virus formulated with an adjuvant. when administered to seronegative sows 4 and 2 weeks prior to farrowing, high levels of neutralizing antibodies against pedv were found in colostrum and milk, as well as in the serum of piglets born to vaccinated sows. piglets were orally infected at 5 days of life with 300 pfu of pedv isolate co 025. it was found that 95% of all piglets (n = 83) born to vaccinated sows survived the infection and showed significantly reduced clinical symptoms, weight loss and viral shedding. in contrast, all piglets from unvaccinated sows displayed severe clinical symptoms including weight loss and dehydration, and 50% of these piglets died within 6 days post infection. these results were confirmed in two additional clinical trials. a large field trial involving > 600 sows was performed in three commercial units in saskatchewan, canada to assess the vaccine in different genetics, health statuses and management systems. the vaccine demonstrated to be completely safe to use; no adverse events including injection site reactions and reproductive complications were observed. vaccine efficacy was evaluated in 8% of these animals by transporting the pregnant sows a week before farrowing to the vido-intervac high containment facility. following oral challenge at 5 days of age, survival was significantly higher in piglets born to vaccinated sows than those from control sows (berube et al., 2015) . the assessment of duration of immunity is currently ongoing. in addition, an affinity tagged pedv s1 protein was expressed in the hek293 system to be used as subunit vaccine. when administered to pregnant sows the vaccine partially protected newborn piglets against infection with pedv (makadiya et al., 2016) . since early 1980s, pedv outbreaks have been reported in several asian countries, including china, japan, thailand, taiwan, the philippines, south korea and vietnam. in october 2010, a largescale outbreak of severe pedv was reported in china (wang et al., 2013) . in late 2013, pedv outbreaks occurred in japan, south korea and taiwan . phylogenetic analysis of pedv fulllength genomic sequences reveals that pedv can be genetically divided into 2 groups: g1 (classical) and g2 (field epidemic or pandemic). each group can be further divided into two subgroups: 1a and 1b, 2a and 2b, respectively. it is also possible that the low to moderate effectiveness of current pedv vaccines are due to genetic differences between vaccine and field epidemic strains (kim et al., 2015) . for disease control, an inactivated bivalent tgev and pedv vaccine was introduced in china in 1995. in march 2015, a trivalent attenuated vaccine (pedv, tgev and porcine rotavirus) was also approved. all these vaccines were based on the classical cv777 (g1-a) strain that can be grown to high titers in green monkey kidney vero cells. there are no data published on the efficacy of these vaccines. however, de arriba et al. (de arriba et al., 2002) orally inoculated 11-day-old conventionally reared piglets with two different doses of the attenuated cv-777 strain and challenged with the same virulent pedv strain three weeks later. the vaccinated pigs were partially protected against the challenge, and 25% of the low dose-and 50% of the high dose-exposed pigs did not shed virus after challenge. since winter of 2010, china experienced severe ped outbreaks with devastating damage to the swine industry. these outbreaks can be explained by re-emergence of new pedv strains. to answer industry demand, chinese researchers have developed a bivalent (pedv and tgev) attenuated vaccine that contained the pedv strain zj08 (g1-b) and a bivalent attenuated vaccine based on the aj1102 strain (g2-b). inactivated bivalent vaccine based on the g2b strain has also been developed. these vaccines are currently under clinical evaluation (wang et al., 2016) . in japan, pedv strain 83p-5 (g1-a) was attenuated after 100 passages in vero cells (sato et al., 2011) . subsequently, this strain has been employed as an intramuscular (im) live attenuated vaccine (p-5 v) in japan and south korea. in addition, two south korean virulent pedv strains (sm98-1 and dr-13) were attenuated by serial cell culture passages. the attenuated sm98-1 strain has been used as an im live or killed vaccine, whereas dr-13 was used as an oral live vaccine. this oral vaccine was registered and commercialised in the philippines in 2011. in south korea, the multiple dose vaccination program (3 or 4 im vaccinations in the following order: live-killed-killed or live-live-killed-killed, respectively) at 2-or 3-week intervals starting before farrowing is commonly recommended in pregnant sows (lee, 2015) . according to a south korean study, the administration of commercial vaccines increased the survival rate of piglets challenged with a virulent wild-type pedv from 18.2% to over 80%. however, all vaccines did not significantly reduce morbidity rate and virus shedding (lee, 2015) . also, song et al. (song et al., 2007) reported 60% reduction of the mortality rate of the suckling piglets born to the sows that were im vaccinated with dr-13 pedv vaccine. despite of nationwide use of commercially available vaccines, south korea experienced a devastated ped epidemic in 2013-2014. pedv g2-b strains were responsible for recent severe ped epidemics in asian countries and north america. considering this fact, south korean researchers (baek et al., 2016) tested an inactivated vaccine based on serially cultured g2-b strain kor/ knu-141112/2014. pregnant sows were immunized im with the inactivated adjuvanted vaccine at 6 and 3 weeks prior to farrowing. six-day old piglets were challenged with the homologous virulent virus. piglets born to vaccinated sows had reduced morbidity, mortality and quickly recovered daily weight gain. further studies are needed to evaluate efficacy of this vaccine in 1-or 2-day old piglets under field conditions. all commercially available vaccines that are in use in asia are traditional live attenuated or killed vaccines. however, researchers are working on the next-generation vaccines for pedv (table 1) . for instance, hou et al. (hou et al., 2007) expressed the pedv n protein on the surface of lactobacillus. oral and intranasal inoculations of recombinant l. casei into pregnant sow and mice resulted in high levels of n-specific serum igg and mucosal iga. similarly, liu et al. (liu et al., 2012) expressed s1 and n protein in recombinant l. casei and reported enhanced mucosal and systemic immune responses after oral immunization of mice. meng et al. (meng et al., 2013) evaluated the immunogenicity of recombinant dna plasmids expressing s genes from pedv and tgev in mice. the results showed that the recombinant dna plasmids increased the proliferation of t lymphocytes and the number of cd4+ and cd8+ t lymphocytes. in addition, the dna vaccines induced a high level of ifn-g in the immunized mice. at 35 days post-immunization, the recombinant dna plasmids bearing full-length s genes of tgev and pedv stimulated high levels of virus-neutralizing antibodies. zhang et al. (zhang et al., 2016b) have also constructed bivalent dna vaccine co-expressing s genes of tgev and pedv. attenuated salmonella typhimurium was used for oral delivery this vaccine into pigs. vaccinated pigs developed tgev and pedvspecific cellular and humoral immune responses; however, challenge experiment was not conducted in this study. there have also been reports on expressing recombinant pedv s protein fragments in plants (bae et al., 2003) and in a cell line (oh et al., 2014) . feeding mice with transgenic tobacco plants that express the s protein fragment containing the pedv neutralizing epitope induced pedv-specific antibody and cell-mediated immune responses. in another study, oh et al. (oh et al., 2014) established porcine cell line stably expressing s1 fragment of the pedv spike protein. pregnant sows were immunized im 3 times with the purified and adjuvanted protein 6, 4 and 2 weeks prior to farrowing. the sows developed pedv-specific neutralizing antibody response in serum and colostrum. one 4-to 5-day-old piglet was selected randomly from each farrowing sow for challenge with virulent pedv. the piglets born to vaccinated sows showed reduced morbidity, mortality and virus shedding. however, a low number of challenged piglets hamper the author's conclusion about efficient protection of neonatal piglets. with the disappearance of tgev around the world, the need for tgev vaccines has dropped over the last few years. for north america and europe, only two major international animal health companies continue to offer tgev vaccines. in contrast, many parts of asia including china and korea are still dealing with tgev outbreaks, and different types of vaccines are still available. the introduction of pedv into the north american herd in 2013-2014, however, has reversed this picture and has highlighted the global need for effective vaccines. coronaviruses represent an important group of animal pathogens that can have devastating impacts in a variety of species. for an industry to rely on biosecurity alone seems somewhat risky and, in case of an emerging disease, can lead to major economic losses, as witnessed in north america over the last two years. indeed, the phylogeny of coronaviruses demonstrates a great deal of diversity in antigenic variants, which may lead to limited cross-protection against infection with different strains. thus, it is important to continue to survey novel pedv variants that may emerge locally or globally through antigenic drift (point mutations) or antigenic shift (recombination events). effective prevention and control of pedv and other coronaviruses can only be achieved through the use of vaccines. an ideal vaccine prevents mortality and clinical disease in newborn piglets, the age group most seriously affected by the disease, as well as viral shedding. as lactogenic immunity is a key mechanism of protection, efforts to enhance the levels of antibodies in the milk through formulation (adjuvants) and delivery (mucosal) are critical. however, time is also of essence when dealing with a new strain, as traditional manufacturing vaccine methods like virus isolation, inactivation or attenuation can be time-consuming. therefore, research on next-generation vaccines such as rna particle, dna, sub-unit and viral-vectored approaches is critical for the prevention of future outbreaks of emerging coronavirus diseases. induction of antigen-specific systemic and mucosal immune responses by feeding animals transgenic plants expressing the antigen efficacy of an inactivated genotype 2b porcine epidemic diarrhea virus vaccine in neonatal piglets colostral antibodymediated and cell-mediated immunity contributes to innate and antigenspecific immunity in piglets mechanisms of coronavirus cell entry mediated by the viral spike protein development of a novel vaccine for porcine epidemic diarrhea virus passive immunity in transmissible gastroenteritis of swine: immunoglobulin characteristics of antibodies in milk after inoculating virus by different routes immunology of transmissible gastroenteritis antibody responses in serum, colostrum, and milk of swine after infection or vaccination with transmissible gastroenteritis virus gammadelta t cell effector functions: a blend of innate programming and acquired plasticity new insights into the dual recruitment of iga+ b cells in the developing mammary gland identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus t cell-mediated immune response to respiratory coronaviruses strategies for design and application of enteric viral vaccines p161, a murine membrane protein expressed on mast cells and some macrophages, is mouse cd13/aminopeptidase n protective efficacy of a replicon particle vaccine in both naïve and previously exposed gilts against porcine epidemic diarrhea virus coronavirus gene 7 counteracts host defenses and modulates virus virulence porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf3 and tbk1 safety and antibody response of pigs to an experimental porcine epidemic diarrhea virus (pedv) vaccine, killed virus previous infection of sows with a mild strain of porcine epidemic diarrhea virus confers protection against infection with a severe strain evaluation of a ped vaccine on piglet mortality and sow immunity the phenotype and activation status of t and nk cells in porcine colostrum suggest these are central/effector memory cells antibodies and their receptors: different potential roles in mucosal defense surface-displayed porcine epidemic diarrhea viral (pedv) antigens on lactic acid bacteria porcine epidemic diarrhea virus uses cell-surface heparan sulfate as an attachment factor porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections genetic characterization of porcine epidemic diarrhea virus in korea from ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex lactogenic immunity and vaccines for porcine epidemic diarrhea virus (pedv): historical and current concepts replication of transmissible gastroenteritis coronavirus (tgev) in swine alveolar macrophages a common mucosal chemokine (mucosae-associated epithelial chemokine/ccl28) selectively attracts iga plasmablasts outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus porcine aminopeptidase n is a functional receptor for the pedv coronavirus high-level mucosal and systemic immune responses induced by oral administration with lactobacillusexpressed porcine epidemic diarrhea virus (pedv) s1 region combined with lactobacillus-expressed n protein the porcine innate immune system: an update s1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen ) fields virology evaluation on the efficacy and immunogenicity of recombinant dna plasmids expressing spike genes from porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus expression of teck/ccl25 and mec/ccl28 chemokines and their respective receptors ccr9 and ccr10 in porcine mucosal tissues expression of mucosal chemokines teck/ccl25 and mec/ccl28 during fetal development of the ovine mucosal immune system vaccination of pedv-naïve dams with replicon rna particle vaccine protects suckling piglets from challenge development of an alphavirus rna particle vaccine against porcine epidemic diarrhea virus transfer of humoral and cell-mediated immunity via colostrum in pigs immunogenicity and protective efficacy of recombinant s1 domain of the porcine epidemic diarrhea virus spike protein porcine epidemic diarrhea virus infects and replicates in porcine alveolar macrophages il-29 is the dominant type iii interferon produced by hepatocytes during acute hepatitis c virus infection a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus field efficacy of an experimental porcine epidemic diarrhea (ped) vaccine administered to pregnant sows structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies passive immunity in transmissible gastroenteritis of swine: immunoglobulin classes of milk antibodies after oral-intranasal inoculation of sows with a live low cell culture-passaged virus passive immunity to transmissible gastroenteritis virus: intramammary viral inoculation of sows animal coronavirus vaccines: lessons for sars humoral and cellular factors of maternal immunity in swine differential expression and activity of the porcine type i interferon family molecular evolution of the porcine type i interferon family: subtype-specific expression and antiviral activity mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo active immunity and t-cell populations in pigs intraperitoneally inoculated with baculovirus-expressed transmissible gastroenteritis virus structural proteins il-28, il-29 and their class ii cytokine receptor il-28r differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf 3 oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr13 strain construction and characterization of recombinant porcine adenovirus serotype 5 expressing the transmissible gastroenteritis virus spike gene pedv orf3 encodes an ion channel protein and regulates virus production porcine epidemic diarrhea virus variants with high pathogenicity porcine epidemic diarrhea in china proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture ccl28 controls immunoglobulin (ig)a plasma cell accumulation in the lactating mammary gland and iga antibody transfer to the neonate porcine epidemic diarrhea virus e protein causes endoplasmic reticulum stress and up-regulates interleukin-8 expression porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin-8 expression long-lived effector/central memory t-cell responses to severe acute respiratory syndrome coronavirus (sars-cov) s antigen in recovered sars patients efficacy and immunogenicity of recombinant swinepox virus expressing the a epitope of the tgev s protein immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling identification of a conserved linear b-cell epitope in the m protein of porcine epidemic diarrhea virus suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp1 construction of a bivalent dna vaccine co-expressing s genes of transmissible gastroenteritis virus and porcine epidemic diarrhea virus delivered by attenuated salmonella typhimurium mucosal and systemic isotypespecific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus the authors declare no conflict of interest. key: cord-309359-85xiqz2w authors: song, daesub; moon, hyoungjoon; kang, bokyu title: porcine epidemic diarrhea: a review of current epidemiology and available vaccines date: 2015-07-29 journal: clin exp vaccine res doi: 10.7774/cevr.2015.4.2.166 sha: doc_id: 309359 cord_uid: 85xiqz2w porcine epidemic diarrhea virus (pedv), an alphacoronavirus in the family coronaviridae, causes acute diarrhea, vomiting, dehydration, and high mortality rates in neonatal piglets. pedv can also cause diarrhea, agalactia, and abnormal reproductive cycles in pregnant sows. although pedv was first identified in europe, it has resulted in significant economic losses in many asian swine-raising countries, including korea, china, japan, vietnam, and the philippines. however, from april 2013 to the present, major outbreaks of pedv have been reported in the united states, canada, and mexico. moreover, intercontinental transmission of pedv has increased mortality rates in seronegative neonatal piglets, resulting in 10% loss of the us pig population. the emergence and re-emergence of pedv indicates that the virus is able to evade current vaccine strategies. continuous emergence of multiple mutant strains from several regions has aggravated porcine epidemic diarrhea endemic conditions and highlighted the need for new vaccines based on the current circulating pedv. epidemic pedv strains tend to be more pathogenic and cause increased death in pigs, thereby causing substantial financial losses for swine producers. in this review, we described the epidemiology of pedv in several countries and present molecular characterization of current strains. we also discuss pedv vaccines and related issues. throughout europe. however, during the 1980s and 1990s, the number of ped outbreaks decreased markedly in the re gion. only a few severe outbreaks have been reported since the 1980s in europe. however, ped has become an endemic disease in asian pig farming countries, such as korea, china, vietnam, japan, the philippines, taiwan, and thailand [59] . until 2013, ped was thought to have been restricted to asian countries. however, an outbreak of pedv infection occurred in the united states in iowa in april 2013, and within 1 year, pedv had spread to canada and mexico [10] , which share borders with the united states. additionally, ped outbreaks occurred in korea and japan, across the pacific ocean, also within 1 year of the us outbreak [11, 12] . the pedv strain iso lated in the united states was genetically related to the chi nese pedv strain reported in 2012 [13] . interestingly, the ko rean and taiwanese pedv strains isolated after the us out break were genetically related to the us pedv strain [11] . after spring 2013, ped was no longer found only in asia. us scientists who had not researched pedv began to study this disease, which had previously not been a problem in ame ri can. from this, a ped vaccine reflecting the genetic charac teristics of the pedv strain isolated during the us outbreak was commercialized [14] . moreover, many veterinary scien tists have focused on the development of more effective ped vaccines because of the major economic losses associated with ped outbreaks. after the us outbreak, sporadic ped out south korean, piglets < 1 week of age died from severe watery diarrhoea after showing signs of dehydration. after the acute outbreak, piglets were anorectic, depressed, vomiting, and producing water faeces that did not contain any signs of blood. (c) necropsies of deceased piglets from the gimpo outbreak uncovered gross lesions in the small intestines, which were typically fluidic, distended, and yellow, containing a mass of curdled, undigested milk. atrophy of the villi caused the walls of the small intestines to become thin and almost transparent. (d) yellowish watery diarrhea in sucling piglets after acute infection of pedv. breaks occurred in germany. therefore, european countries were not isolated from the spread of the us pedv [15] . thus, the us outbreak was important turning point in ped research, and ped research can be said to be divided into two eras: be fore and after 2013. this review covers the vaccine, epidemiology, genetic struc ture, and characteristics of ped/pedv after 2013. this report may improve our understanding of this disease, which is cur rently the most fatal disease in pigs and one of the most costly health issues in animals. furthermore, this review may pro vide insight into important topics for investigation in ped re search. the genome of pedv is positivesense, singlestranded rna. the size of pedv genomic rna is about 28 kb. the organiza tion of the pedv genome is presented in table 1 . from the 5′ cap to the 3′ poly a tail, ped genomic rna contains seven open reading frames (orfs) encoding viral proteins. orf1a and orf1b encode the viral polymerase. orf3 encodes a non structural protein with an unknown function; this protein is thought to be related to viral pathogenicity [16] . additionally, the other orfs have specific names according to the proteins encoded in these regions, i.e., spike (s), envelope (e), matrix (m), and nucleocapsid (n) proteins [7, 17] . table 1 presents the characteristics of the pedv proteins as described in pre vious articles. of the pedv proteins, the s protein is considered the most antigenic. as summarized in table 1 , the s protein is respon sible for the interaction with host cellular receptor molecules [18, 19] . this interaction is crucial for the entry of the virus and is related to induction of neutralizing antibodies against the virus [18, 20, 21] . additionally, these critical characteristics of the s protein are used for analysis of the molecular epidemi ology of pedv. thus, pedv researchers have started to analyze the geno types of pedv using the s gene [22] . while other genes, such as the gene encoding the m protein and the gene encoded in orf3 [2325] have been used for phylogeny or molecular epi demiological studies, genetic diversity of the s gene is the fo cus of this review. one of the most interesting characteristics of the s gene is the diversity in this gene that has occurred from the end of 1970s to the present. genotypes in the s gene of pedv could be important because this gene may affect the pathogenicity of novel ped outbreaks based on variations in the s gene [3, 1012, 16, 2629] . moreover, the chinese ped out break in 2011 involved the presence of new variants based on the nucleotide similarities in the s gene. this variant pedv exhibited 93% similarity with the cv777 prototype pedv strain in the s gene [29] . moreover, the pedv isolated after the us outbreak also exhibited 89%92% similarity with the s protein of cv777 [26, 27] . phylogenetic analyses of pedv rna have been reported in many previous studies [7, 16, 26, 27, 30] . whether the 10% nucleotide difference often observed in the s gene can affect the pathogenesis of pedv has not been clarified. however, the similarities in the s gene, which is re sponsible for the host interaction and normalization, the s1 region in particular could be important in vaccine efficacy and development strategies [7, 17] . neutralizing epitopes of the pedv s protein have been investigated [20] . in particular, the co26k equivalent (coe) is a neutralizing epitope within the co26k collagenase fragment in transmissible gastroen teritis virus (tgev) [31, 32] . in the prototype pedv (cv777) and vaccine strains, certain coes are different from field iso lates that were isolated after 2011. the different coes are 549t and 594g in the prototype and vaccine strains, respectively (table 2) ; however, other coes that differ from that of cv777 have been shown to be identical to the vaccine strains 83p5 100th or attenuated dr13 [33] . in pedv isolates acquired af ter 2011 in china, these amino acids were changed to serine; similar observations were made for the pedv strain from the us outbreak [29] . the new variant pedv strains have similar mutational patterns for the neutralizing epitope. in contrast, pedv strains isolated from 2002 to 2009 exhibit different pat terns in 549t and 594g compared to strains isolated after 2011. interestingly, at amino acid position 549, some strains exhibit no changes; however, in most strains, amino acid 549 is chang ed to arginine (r). additionally, most strains exhibit a change from g to s at amino acid position 594, similar to current stra ins [22] . in order to elucidate the positive correlations between chan ges in the s protein and virulence or vaccine efficacy, further studies involving animal models are needed. in addition to the changes in the s protein described above, more variations have been discovered. for example, park et al. [34] reported large deletion in the s protein from amino acid 713 to amino acid 916, within the s1 and s2 domains. this virus was detected in 2008, and other regions of the s protein share 92.8%96.9% amino acid identity in the partial s1 domain with other strains isolated in korean in 2008 and 2009. in the united states, a deletion in the s gene was also reported after the united states outbreak. in december 2013, a strain with deletion of the s gene was detected and isolated in ohio [35] . this strain exhibited patterns that were different from those of the korean strain with the s gene deletion. the us deletion mutant contains a large deletion (197 amino ac ids in length, from amino acid 34 to amino acid 230) in the s1 domain [35] . the effects of deletion in the s protein should be further investigated and are reminiscent of porcine respirato ry coronavirus arising from s protein deletion in the genome of tgev [34, 35] . through pedv research, unique genetic characteristics other than those in the s gene have been reported. fulllength analysis of the orf3 gene of pedv revealed a 51bp deletion in the cellattenuated pedv strain dr13 [23] . this deletion in the orf3 gene has not been identified in wildtype pedv; however, in addition to the attenuated pedv dr13 strain, live pedv vaccine strains available in korea exhibit similar dele tion patterns by reverse transcription polymerase chain reac tion (rtpcr) comparisons [23] . this deletion pattern is clin ically useful for differentiation of live vaccine strains from wild type pedv casing diarrhea. the final genetic mutation we will discuss is deletion of the e gene in the cellattenuated pedv strain dr13. genetic anal ysis has focused on the s, n, and m genes. from analysis of the e gene, a unique deletion was found. moreover, compar ed to other pedv strains, including the wildtype and vaccine strains, only the attenuated pedv strain dr13 has been shown to have a 21bp deletion in this gene [30] . the e protein of the coronavirus is responsible for the assembly of the virus and cell stress responses [36] . the effects of the e gene deletion are under investigation. in the czech republic, rodak et al. [37] reported that 27 out of 219 fecal samples from diarrheic piglets (<21 days old) were positive for pedv. this outbreak occurred between may 2005 and june 2006 in an area densely populated with pigs in the po valley in northern italy [38] . some pedvpositive farms (35 out of 476) were detected between mid2006 and the end of 2007; however, the disease progressively disappeared [39] . during the period from 2007 to 2014, mild clinical signs were report in pigs of all ages, and mortality was observed in pig lets only in pedvpositive farms [40] . [46] , and the southern prov inces of vietnam [47] . in october 2010, a largescale outbreak of pedv was reported in several provinces in southern china. pedv also spread to other regions of the country, particularly in northwest [48] . pedv is now circulating in at least 29 chi nese provinces [49] . in october 2013, japan reported a pedv outbreak to the world organization for animal health (oie) [15] after a period of 7 years without an outbreak. according to the information provided by japan's national institute of animal health, pedv isolates from this outbreak are geneti cally related to the pedv isolates recovered from china and the united states in 2013. in addition, in late 2013, pedv out breaks were reported in south korea and taiwan [12, 50, 51] . the us pedv strains identified during the us outbreak were genetically related to the chinese strains (china/2012/ah2012) reported in 20112012 [13, 26] . pedv was first identified with in the united states in iowa in may 2013, although testing of historical samples showed that pedv occurred the month before in ohio. pedv rapidly spread throughout the country and was confirmed on farms from 32 states, including ohio, indiana, iowa, minnesota, oklahoma, illinois, and north car olina, by the end september 2014 [15] . pedv was detected in mexico for the first time in july 2013 [52] . in october 2013, pedv was identified for the first time in peru [53] . in novem ber 2013, pedv was also identified as the cause of outbreaks of diarrhea in farms in the espaillat province, dominican re public. by september 2014, ped outbreaks were reported in seven of the 31 provinces in the dominican republic [54] . in april 2014, canada reported outbreaks of pedv to the oie; these outbreaks started in january and affected 58 herds in four provinces [15] . an acute outbreak of diarrhea and death in lactating piglets was observed in columbia in march 2014. by september 2014, 54 samples from six departments were confirmed via laboratory testing [55] . several reports have described the development of rtpcr as a diagnostic technique for detection of both laboratory and field isolates [5659] . primers derived from the m gene can be used in an rtpcr system to obtain pedvspecific fragments [57] , and duplex rtpcr has been used to differ entiate between tgev and pedv [60] . within the past few years, several useful modifications of the basic rtpcr meth od have been reported. for example, it is possible to estimate the potential transmission of pedv by comparing viral shed ding load with a standard internal control dna curve [61] and by multiplex rtpcr to detect pedv in the presence of various viruses [62] -a technique that is particularly useful for rapid, sensitive, and costeffective diagnosis of acute viral gastroenteritis in swine. the commercial dual priming oligo nucleotide system (seegene, seoul, korea) (fig. 2) has been developed for the rapid differential detection of pedv. this system employs a single tube onestep multiplex rtpcr with two separate primer segments to block nonspecific priming [63] . recently, a proteinbased enzymelinked immunosor bent assay (elisa) system was developed to detect pedv. using this technique, a polyclonal antibody is produced by immunizing rabbits with purified pedv m gene after its ex pression in escherichia coli. immunofluorescence analysis can then be carried out with the antipedvm antibody in order to detect pedvinfected cells among other enteric vi ruses [64] . another useful reverse transcriptionbased diagnostic tool is rt loopmediated isothermal amplification. this assay, which uses 46 primers that recognize 68 regions of the tar get dna, is more sensitive than gelbased rtpcr and eli sa, largely because this method produces a greater quantity of dna [65] . immunochromatographic assay kits can be used at farms in order to detect the n (nucleocapsid) protein of pedv with 92% sensitivity and 98% specificity. moreover, a rapid technique for differential detection of pedv and por cine rotavirus (rv) has recently been commercialized and is now widely used in the field (fig. 3) . this technique is less sensitive than rtpcr, but allows for diagnosis within 10 min utes. thus, it is particularly effective for quickly determining quarantine or slaughter policies in the field. interestingly, some reports have commented on the detec tion of pedv genomic dna in sera. genomic detection in gno tobiotic piglets has been reported for serum viral rna con centrations ranging from 4.8 to 7.6 log10 genomic equivalents (ge)/ml after inoculation of the us pedv strain. similar de tection of the pedv genome has been observed in diarrheic pigs at age 1320 weeks (4.06.3 log10 ge/ml) [17] . however, no infectious pedv has been recovered from genomeposi tive sera samples. unfortunately, after intensive screening and trials to isolate the pedv genome from serum samples, we have not succeeded in this endeavor (data not shown). enteropathogenic viruses can be divided into two types (type i and ii) according to their infection site in the intestine [66] . viruses infecting villous enterocytes, including tgev, pedv, and rv, are type i viruses and can be suppressed by local gut associated immunity. diseases caused by type ii viruses, which infect crypt enterocytes basolaterally (e.g., canine parvovirus), can be prevented by inducing systemic or mucosal immuni ty. in this review, we discuss control strategies for reducing viral shedding, mortality, and the transmission of pedv in swine herds, such as transmission occurring from artificial oral exposure (i.e., the feedback method) and vaccines. in naïve swine herds, ped is characterized by vomiting and acute diarrhea and results in high mortality rates in piglets less than 2 weeks of age. neonatal pigs are born without ma ternal antibodies if they are not infected in utero, and they should receive passive lactogenic immunity (igg and iga) through intake of colostrum and milk to promote survival af ter birth. therefore, maternalderived immunity at an early age is critical for passive protection of neonatal pigs; for this purpose, immunization of the dam preparturition has been used successfully [66] . igg is the major immunoglobulin com ponent in pig colostrum, consisting of more than 60% of all immunoglobulins, but is not found in milk. iga accounts for a substantially reduced percentage of colostrum immunoglob ulin content; however, iga is more effective than igg or igm at protecting animals from orally infected agents because it is more resistant to the activity of proteolytic enzymes in the in testine and has a higher neutralizing ability than igg and igm [67] . bohl et al. [68] and saif et al. [69] demonstrated that oral inoculation of seronegative sows with live virulent tgev re sults in high rates of protection in suckling piglets. in these sows, passive protection is associated with high titers of se cretory iga (siga) in colostrum and milk. this investigation suggested the presence of a gutmammary glandsiga axis; that is, iga plasmablasts stimulated in the gut by virulent path ogens migrate to the gut lamina propria and mammary glands. several highly attenuated oral tgev vaccines, which repli cate lower in the gut, induce poor milk siga titers compared with virulent tgev in sows and result in lower protective effi cacy in piglets [68, 69] . this research could be employed to maternal immunization strategies for pedv. in areas where no effective ped vaccines are available, some veterinarians have recommended artificial infection of sows (i.e., the feedback method) during pregnancy to supply lacto genic immunity to their piglets [70] . the recommended feed back material is pooled feces collected from infected piglets during the first 18 hours of infection. every sow on the farm should be simultaneously administered feces containing high titers of pedv, allowing all sows and gilts to recover at approx imately the same time and stop shedding the virus. one of advantage of this type of feedback method is strong stimulation of mucosal immunity in the gut and a quick re sponse after immunization. after successful feedback, the piglets will be protected during the first few days after birth by passive antibodies through colostrum and milk. however, there is a potential risk of transmission of the contaminated viral or bacterial agents in the inoculum (e.g., porcine circovi rus type 2 [pcv2] infection, porcine reproductive and respi ratory syndrome virus [prrsv] , and salmonellosis) [71, 72] . additionally, it is possible that pedv may spread rapidly in pigs of all ages in the index farms. the severity of the disease may vary with unknown factors, such as stress, nutrition, or coinfection. in addition, there is a risk that the virulent virus used for feedback materials may spread to and produce dis ease in other herds. irregular immune responses in sows after feedback may also be a major concern for optimal induction of herd immunity for protection. all of these possibilities em phasize the need for a safe and effective pedv vaccine to pro tect both sows and piglets. for the prevention of pedv infection, several vaccines have been reported in asian countries; the predominance of vac cines in asian countries, but not in europe or america, is thou ght to be related to the occurrence of severe ped outbreaks and major economic losses in asia [7] . commercial pedv vaccines include live attenuated vaccines and binary ethyl enimine (bei) inactivated vaccines. some of these vaccines have been combined with vaccines for tgev (a bivalent vac cine) and porcine rv (a trivalent vaccine) and used in china and south korea [7, 25] . moreover, an attenuated virus vac cine using cell cultureadapted pedv has been administered to sows in japan since 1997. oral vaccination with a cellat tenuated vaccine has been used in south korea since 2004 and in the philippines since 2011 [7] . although these commer cial vaccines are considered effective and have been widely used, not all animals develop solid lactogenic immunity. sev eral factors are thought to be associated with the poor lacto genic immunogenicity of the commercial vaccine, including the immunizing route of the vaccine. song et al. [73] demon strated that oral inoculation of pedvseronegative pregnant sows with live attenuated pedv reduces the mortality of suck ling piglets more effectively rather than injection after chal lenge, and this protection is associated with elevated iga con centrations in colostrum and milk. despite the reduction in mortality rates in piglets delivered from orally vaccinated sows, there was no shortening of the duration of virus shedding and no reduced severity of diarrhea after challenge between vac cinated and control pigs. thus, some researchers may con clude that passive immunity by vaccination with the highly attenuated pedv strain dr13 does not prevent virus shedding after challenge. protection against virus challenge in conven tional pigs is related to the inoculation dose of the virus in the vaccine and the challenge dose of the virulent virus. at low doses of the attenuated pedv, 25% of pigs are protected against pedv challenge; however, this proportion increases to 50% when pigs are inoculated with a dose 20 times higher [74] . moreover, loss of body weight and the content of viral shed ding decrease in orally vaccinated pigs compared with those in intramuscularly vaccinated and unvaccinated pigs follow ing challenge with a low dose of virulent virus (1 ld50, 10 3.0 tcid50/dose). additionally, the lethal dose of pedv changes depending on the body weights of the infected piglets and in fection of sows with the challenge virus (data not shown). however, several publications have questioned the efficacy and/or safety of pedv vaccines used in asia [3, 28, 44, 75, 76] . in particular, after the us outbreak, the efficacy of commer cial ped vaccines in korea became controversial, and simul taneously, there was an urgent need for a new vaccine in or der to establish solid immunity in sow herds prior to farrow ing to protect piglets. furthermore, many groups have debat ed the appropriate standards for evaluation of the efficacy of vaccines after official challenge tests using currently available pedv vaccines in south korea in 2014. information on pedv mucosal immunity is limited. moreover, due to the complex characteristics of mucosal viral diseases, simple criteria, such as serum neutralizing antibodies, severity of diarrhea, and mortality after virulent challenge, are insufficient for the ac curate and optimal evaluation of pedv vaccine efficacy. for detailed identification of standards for the evaluation of pedv vaccines after virulent challenge, the following criteria, which may not be controllable, should be taken into consideration: characteristics of piglets used in the challenge test: con ventional or specific pathogenfree piglets, weight of pig characteristics of sows: parity number, existence of aga lactia or coinfection with other viral and bacterial patho gens, number of delivered piglets quantity of challenged virus: amount of viral load for the challenge virus may result in discrepancies in the observ ed mortality rates of piglets cohabitation with sows: the conditions of piglets challeng ed with virulent virus could be affected by the occurrence of viral shedding by sows duration of the challenge test the vaccine strains commonly used in korea display a max imum difference of 10% at the amino acid level compared with field viruses. additionally, using sn assays, the sm98 vaccine strain was shown to exhibit variable crossreactivity with several antisera against other vaccines and field viruses, implying that these vaccines would confer protection against the vaccine prepared using wildtype pedvs from the field [77] . however, the crossprotection between vaccine strains and field viruses should be elucidated through animal exper iments, which can show protection based on lactogenic im munity after vaccination. for the ideal development of pedv vaccines, new vaccine strains that are genetically related to field viruses are critical. furthermore, several criteria, includ ing factors related to the reduction in virus shedding and loss of body weight in piglets as well as the details of mucosal im munity and the relationships between protection and immu nity, should be considered and identified during the develop ment of nextgeneration ped vaccines. pedv research has become a hot topic in veterinary virology since the us outbreak in 2013. ped had been a regional dis ease primarily found in asian countries. however, ped was transmitted to the united states and subsequently to neigh boring countries, including mexico and canada. european countries have also encountered pedv. accordingly, with the spread of the ped outbreak area, research on pedv has in creased rapidly. veterinarians and veterinary scientists are striving to breakthroughs to combat current ped outbreaks worldwide, and remarkable advances have been made in the relatively short time after the 2013 outbreak. therefore, reasonable methods for ped vaccine evaluation, along with the development of new vaccines, are urgently need ed, and this complicated process could provide valuable in formation on pedv, immune responses to pedv infection, and pedv pathobiology. furthermore, rational evaluation procedures can help swine farmers understand and control ped more efficiently. a new coronaviruslike particle associated with diarrhea in swine isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of differ ent ages outbreak of porcine epidemic diarrhea in suckling pig lets letter to the editor cloning and sequence analysis of the m gene of porcine epidemic diarrhea virus ljb/03 isolation and se rial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate porcine epidemic diarrhoea virus: a com prehensive review of molecular epidemiology, diagnosis, and vaccines isolation of porcine epidemic diarrhea virus (pedv) in korea an outbreak of swine diarrhea of a newtype associated with coronaviruslike particles in japan distinct charac teristics and complex evolution of pedv strains complete genome sequence of k14jb01, a novel variant strain of por cine epidemic diarrhea virus in south korea uslike strain of por cine epidemic diarrhea virus outbreaks in taiwan origin, evolu tion, and genotyping of emergent porcine epidemic diar rhea virus strains in the united states ped vaccine gains conditional approval sci entific opinion on porcine epidemic diarrhoea and emerg ing porcine deltacoronavirus genetic variabil ity and phylogeny of current chinese porcine epidemic diarrhea virus strains based on spike, orf3, and mem brane genes porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunopro phylaxis the coro navirus spike protein is a class i virus fusion protein: struc tural and functional characterization of the fusion core complex the gprlqpy motif located at the carboxyterminal of the spike protein induces antibo dies that neutralize porcine epidemic diarrhea virus identification of the epit ope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus major recep torbinding and neutralization determinants are located within the same domain of the transmissible gastroenteri tis virus (coronavirus) spike protein sequence analysis of the partial spike glycoprotein gene of porcine epidemic diar rhea viruses isolated in korea cloning and further sequence analysis of the orf3 gene of wild and attenuatedtype porcine epidemic diarrhea viruses molecular characteriza tion and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china molecular epidemiology of porcine epidemic diarrhea virus in china isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states evaluation on the efficacy and immunogenicity of recombinant dna plasmids express ing spike genes from porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus complete ge nome sequence of a highly prevalent isolate of porcine epidemic diarrhea virus in south china new variants of porcine epidemic di arrhea virus, china molecular epidemiology and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea anti genic structure of the e2 glycoprotein from transmissible gastroenteritis coronavirus four major antigenic sites of the coronavirus transmissible gas troenteritis virus are located on the aminoterminal half of spike glycoprotein s bioinformatics insight into the spike glycoprotein gene of field porcine epidemic diarrhea strains during novel porcine epidemic di arrhea virus variant with large genomic deletion cell culture isolation and sequence analysis of genetically diverse us porcine epi demic diarrhea virus strains including a novel strain with a large deletion in the spike gene the coronavirus e protein: as sembly and beyond timized for porcine epidemic diarrhoea virus detection in faeces epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy comparison of enzymelink ed immunosorbent assay and rtpcr for the detection of porcine epidemic diarrhoea virus diagnosis and investiga tions on ped in northern italy genetic di versity of orf3 and spike genes of porcine epidemic diar rhea virus in thailand chineselike strain of porcine epidemic diarrhea virus impact of porcine epidemic diarrhea virus infection at different periods of pregnancy on subsequent reproductive perfor mance in gilts and sows one worldone health: the threat of emerging swine diseases. an asian perspective emerging and re emerging diseases in asia and the pacific with special em phasis on porcine epidemic diarrhoea heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolat ed in korea genetic characterization of porcine epidemic diarrhea vi rus (pedv) isolates from southern vietnam during 2009 2010 outbreaks outbreak of porcine epidemic diarrhea in piglets in gansu province the updated epidemic and controls of swine en teric coronavirus in china outbreakrelated porcine epidemic diarrhea virus strains similar to us strains, south korea comparative genome analy sis and molecular epidemiology of the reemerging por cine epidemic diarrhea virus strains isolated in korea two cases report of ped in different states in méxico porcine epidemic diarrhea outbreak in peru diarrea epidémica porcina (dep) en republica dominicana situacion de la diarrea epidemica porcina en colombia direct and rapid detection of porcine epidemic diarrhea virus by rt pcr rapid diagnosis of porcine epidemic diarrhea virus infection by polymerase chain reaction pedv leader sequence and junc tion sites identification and characteriza tion of new and unknown coronaviruses using rtpcr and degenerate primers differential detection of trans missible gastroenteritis virus and porcine epidemic diar rhea virus by duplex rtpcr fecal shedding of a highly cellcultureadapted porcine epidemic diarrhea virus af ter oral inoculation in pigs multiplex reverse transcrip tionpcr for rapid differential detection of porcine epide mic diarrhea virus, transmissible gastroenteritis virus, and porcine group a rotavirus onestep multiplex rt pcr for detection and subtyping of swine influenza h1, h3, n1, n2 viruses in clinical samples using a dual prim ing oligonucleotide (dpo) system development of a porcine epidemic diarrhea virus m proteinbased elisa for virus detection development of reverse transcription loopme diated isothermal amplification for rapid detection of por cine epidemic diarrhea virus enteric viral infections of pigs and strategies for in duction of mucosal immunity protection against rotavirusinduced gastroenteritis in a murine model by passively acquired gastrointestinal but not circulating antibodies antibody respons es in serum, colostrum, and milk of swine after infection or vaccination with transmissible gastroenteritis virus isolation of porcine immuno globulins and determination of the immunoglobulin class es of transmissible gastroenteritis viral antibodies exposing sows to pedv to build herd immunity national hog farmer detec tion of porcine circovirus 2 in mammary and other tissues from experimentally infected sows colostral transmission of porcine circovirus 2 (pcv2): reproduction of postweaning multi systemic wasting syndrome in pigs fed milk from pcv2in fected sows with postnatal porcine parvovirus infection or immunostimulation oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr13 strain mucosal and systemic isotypespecific antibody responses and protec tion in conventional pigs exposed to virulent or attenuat ed porcine epidemic diarrhoea virus isolation and characterization of a variant porcine epidemic diarrhea virus in china molecular characterization and phylogenetic analysis of new variants of the porcine epidemic diarrhea virus in gansu genetic characterization of porcine epidemic diarrhea virus in korea from key: cord-309693-f2htekhz authors: yu, meiling; wang, li; ma, sunting; wang, xiaona; wang, yusai; xiao, ya; jiang, yanping; qiao, xinyuan; tang, lijie; xu, yigang; li, yijing title: immunogenicity of egfp-marked recombinant lactobacillus casei against transmissible gastroenteritis virus and porcine epidemic diarrhea virus date: 2017-09-25 journal: viruses doi: 10.3390/v9100274 sha: doc_id: 309693 cord_uid: f2htekhz porcine transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) are the causative agents of highly fatal acute diarrhea in pigs, resulting in enormous losses in the pig industry worldwide. to develop an effective bivalent oral vaccine against tgev and pedv infection, the d antigenic site of the tgev spike (s) protein and the major antigen site (core neutralizing epitope—coe) of the pedv s protein were used as immunogens, and the enhanced green fluorescent protein (egfp) gene was used as a reporter to construct genetically engineered lactobacillus casei rlppg(f)-t7g10-egfp-6d-coe. the expression of proteins of interest by the recombinant l. casei was confirmed by confocal laser scanning microscopy and a western blot assay, and the immunogenicity of rlppg(f)-t7g10-egfp-6d-coe in orally immunized mice was evaluated. the results showed that levels of anti-pedv and anti-tgev serum immunoglobulin g (igg) and mucosal secreted immunoglobulin a (siga) antibodies obtained from the mice immunized with rlppg(f)-t7g10-egfp-6d-coe, as well as the proliferation levels of lymphocytes, were significantly higher than those in mice orally administered phosphate-buffered saline (pbs) or rlppg-t7g10. moreover, the serum igg antibodies showed neutralizing effects against pedv and tgev. our data suggest that the antibiotic resistance-free genetically engineered l. casei bivalent oral vaccine provides a safe and promising strategy for vaccine development against pedv and tgev. larger-scale outbreak in the united states in 2013. herds vaccinated with the cv777-inactivated vaccine were also infected, resulting in tremendous losses to the swine industry [5] [6] [7] . accumulating evidence indicates that this large-scale recurrence of ped was caused by highly virulent pedv variants [8] [9] [10] [11] . in addition, co-infection of tgev and pedv often causes higher morbidity and mortality in newborn piglets. therefore, the development of a safe and highly efficient vaccine against tge and ped would be of great importance. it is now clear that the gastrointestinal mucosas are the primary sites of pedv and tgev infection [12, 13] and that mucosal immunization is a promising way to prevent pedv and tgev infection. however, the existing live attenuated/inactivated vaccines administered parenterally cannot effectively induce mucosal immunity against pedv and tgev infection [14] [15] [16] [17] . although some vaccines for tge and ped developed in china can effectively stimulate intestinal mucosal immunity through houhai acupoint injection, this immunization procedure is more time-consuming and labor-intensive. therefore, vaccines that can induce a mucosal immune response against pedv and tgev would be of great significance, in particular those demonstrated to be safe, inexpensive, easy to use, and effective. the spike (s) glycoproteins of tgev and pedv possess antigen epitopes, and previous reports have demonstrated that the d antigen site (amino acids 378-392) in the s protein of tgev and the core neutralizing epitope (coe; amino acids 499-638) in the s protein of pedv can elicit neutralizing antibodies against tgev and pedv infection, respectively. this suggests that these are promising candidate antigens for the development of a genetically engineered vaccine [12, 18, 19] . lactobacillus casei (l. casei) is a potential delivery vehicle for oral vaccines, because it is a probiotic bacterium characterized by its safety and resistance to gastric acid and bile [20, 21] . previous reports have shown that recombinant l. casei live vaccine is able to colonize the murine intestines for five days [22] and the swine intestines for longer [23] . moreover, we have previously constructed a recombinant l. casei live vaccine expressing the d antigen site of the tgev s glycoprotein combined with muramyl dipeptide and tuftsin as adjuvants, suggesting the possibility of a promising oral vaccine against tgev challenge [12] . however, plasmid-mediated antibiotic resistance is commonly used as a selective marker for genetically engineered bacteria [24] [25] [26] . this could result in potential biosafety issues due to the transfer of antibiotic resistance from genetically engineered bacteria to environmental pathogens. enhanced green fluorescent protein (egfp) is a luminescent jellyfish protein with the amino acid substitutions necessary to generate a strong fluorescence signal when excited by ultraviolet or blue light. it is widely used in biological research, including in studies of cell differentiation, gene tracking, and protein localization and operation in vivo [27, 28] , providing a potential candidate for a screening marker to replace antibiotic resistance. in this study, a genetically engineered l. casei strain, rlppg f -t7g10-egfp-6d-coe, was constructed using the tgev s protein d antigen site and pedv s protein-neutralizing antigen epitope region coe as immunogens, l. casei 393 as an antigen delivery vehicle, and egfp as a selective marker, combined with a constitutive expression plasmid, ppg-t7g10. the immunogenicity of this strain when orally administered in mice was evaluated, suggesting a potential approach for the prevention of tgev and pedv infection. all applicable international and national guidelines for the care and use of animals were followed. approval (2016nefu-315, 13 april 2017) was obtained from the institutional committee of northeast agricultural university for the animal experiments. tgev strain th98 and pedv strain hlj-2012 were isolated by our laboratory from pedv/tgev-positive samples collected from pig farms in which a severe outbreak of acute diarrhea had been reported in piglets [29, 30] . l. casei atcc 393 was kindly provided by jos seegers (nizo institute, netherlands). african green monkey kidney cells (vero cells; atcc ccl-81) and swine testicle (st) cells were purchased from the china center for culture collection (wuhan, china) and were cultured in dulbecco's modified eagle medium (dmem; gibco, gaithersburg, md, usa) supplemented with 10% fetal bovine serum (fbs; gibco) at 37 • c with 5% co 2 . the details of all plasmids used in this study are listed in table 1 . the primers used for amplifying genes encoding 6d (a peptide of the the d antigenic site of the tgev spike (s) protein was repeated six times), coe, and egfp are listed in table 2 . the linker sequence (ggggs)3 was added in primers fs2 and re. a schematic diagram of the dna plasmid construction is shown in figure 1 . in brief, the gene encoding 6d was inserted into plasmid pmd18t-coe at saci and mlui sites, generating plasmid pmd18t-6d-coe; then, the fusion dna fragment 6d-coe, obtained from pmd18t-6d-coe by saci and apai digestion, was inserted into the corresponding sites of plasmid ppg-t7g10, generating recombinant plasmid ppg-t7g10-6d-coe. next, the gene encoding egfp was inserted into ppg-t7g10-6d-coe at saci and kpni sites, generating recombinant plasmid ppg-t7g10-egfp-6d-coe; finally, a chloramphenicol resistance (cm r ) gene selective marker in ppg-t7g10-egfp-6d-coe was deleted using restriction enzymes stui and ncoi, followed by blunt-end treatment and ligation, giving rise to recombinant plasmid ppg f -t7g10-egfp-6d-coe. all recombinant plasmids were identified by restriction enzyme digestion and sequencing. for construction of the recombinant lactobacillus strain, l. casei 393 competent cells were prepared according to a method previously described [34] , followed by electroporation. briefly, 50 ng of recombinant plasmid ppg f -t7g10-egfp-6d-coe was gently mixed with 200 µl of l. casei 393 competent cells at 4 • c for 1 min; then, the mixture was transferred into a pre-cooled gene pulser (bio-rad, hercules, ca, usa) disposable cuvette (inter-electrode distance of 0.2 cm) and subjected to a single electric pulse (1.5 v; 200 ω; 25 µf) with a gene pulser (bio-rad). after growth at 37 • c for 8 h, the recombinant lactobacillus strain with green fluorescence signal was collected through flow cytometry using a facscalibur (bd biosciences, san diego, ca, usa) at 488 nm and was grown on an de man-rogosa-sharpe (mrs) plate at 37 • c for 36 h. this was followed by pcr confirmation and a chloramphenicol sensitivity assay, giving rise to recombinant strain rlppg f -t7g10-egfp-6d-coe. the hereditary stability of recombinant l. casei strains was detected, and rlppg f -t7g10-egfp-6d-coe was analyzed for stability by serially transferring the cultures after 24 h of incubation into mrs medium at 37 • c (1% inoculum; 50 generations). plasmids were extracted from the cells, and pcr was used to confirm the presence of the gene egfp-6d-coe in the plasmid using the primers fe and rs2. plasmids ppg-t7g10-6d-coe and ppg f -t7g10-egfp-6d-coe were generated according to the steps indicated by the arrows. ① the gene encoding 6d was inserted into plasmid pmd18-t-coe at saci and mlui sites, generating plasmid pmd18-t-6d-coe; ② fusion dna fragment 6d-coe, obtained from pmd18-t-6d-coe by saci and apai digestion, was inserted into the corresponding sites of plasmid ppg-t7g10, generating recombinant plasmid ppg-t7g10-6d-coe; ③ the gene encoding enhanced green fluorescent protein (egfp) was inserted into ppg-t7g10-6d-coe at saci and kpni sites, generating recombinant plasmid ppg-t7g10-egfp-6d-coe; ④ the chloramphenicol resistance (cm r ) gene as a selective marker in ppg-t7g10-egfp-6d-coe was deleted using restriction enzymes stui and ncoi, followed by blunt-end treatment and ligation, giving rise to recombinant plasmid ppg f -t7g10-egfp-6d-coe. for analysis of the expression of proteins of interest by recombinant strains rlppg-t7g10-6d-coe and rlppg f -t7g10-egfp-6d-coe, the bacterial strains were grown in basal mrs broth at 37 °c for 16 h (optical density of sample measured at a wavelength of 600 nm (od600) ≈ 3.0) without shaking and were harvested by centrifugation at 10,000× g for 2 min. cells were washed twice with sterile phosphate-buffered saline (pbs; ph 7.4) and lysed using a mini-beadbeater (biospec, bartlesville, ok, usa). after centrifugation, the same quantity of total protein in the supernatants of each sample was isolated by sodium dodecyl sulfate 12% polyacrylamide gel figure 1 . schematic drawing of the construction of dna plasmids. plasmids ppg-t7g10-6d-coe and ppg f -t7g10-egfp-6d-coe were generated according to the steps indicated by the arrows. 1 the gene encoding 6d was inserted into plasmid pmd18-t-coe at saci and mlui sites, generating plasmid pmd18-t-6d-coe; 2 fusion dna fragment 6d-coe, obtained from pmd18-t-6d-coe by saci and apai digestion, was inserted into the corresponding sites of plasmid ppg-t7g10, generating recombinant plasmid ppg-t7g10-6d-coe; 3 the gene encoding enhanced green fluorescent protein (egfp) was inserted into ppg-t7g10-6d-coe at saci and kpni sites, generating recombinant plasmid ppg-t7g10-egfp-6d-coe; 4 the chloramphenicol resistance (cm r ) gene as a selective marker in ppg-t7g10-egfp-6d-coe was deleted using restriction enzymes stui and ncoi, followed by blunt-end treatment and ligation, giving rise to recombinant plasmid ppg f -t7g10-egfp-6d-coe. for analysis of the expression of proteins of interest by recombinant strains rlppg-t7g10-6d-coe and rlppg f -t7g10-egfp-6d-coe, the bacterial strains were grown in basal mrs broth at 37 • c for 16 h (optical density of sample measured at a wavelength of 600 nm (od 600 ) ≈ 3.0) without shaking and were harvested by centrifugation at 10,000× g for 2 min. cells were washed twice with sterile phosphate-buffered saline (pbs; ph 7.4) and lysed using a mini-beadbeater (biospec, bartlesville, ok, usa). after centrifugation, the same quantity of total protein in the supernatants of each sample was isolated by sodium dodecyl sulfate 12% polyacrylamide gel electrophoresis (sds-page) and was transferred onto polyvinylidene fluoride membranes, followed by development with mouse anti-6d monoclonal antibody/rabbit anti-coe polyclonal antibody (diluted at 1:500) prepared in our lab, or mouse anti-egfp monoclonal antibody (zsgb-bio, beijing, china; diluted at 1:4000). horseradish peroxidase (hrp)-conjugated goat anti-mouse/rabbit igg antibody (sigma, st. louis, mo, usa) was utilized as a secondary antibody, diluted at 1:5000. immunoblots were visualized with chemiluminescent substrate reagent (pierce, rockford, il, usa) according to the manufacturer's instructions. laser confocal microscopy was used to confirm the expression of fusion protein egfp-6d-coe on the surface of rlppg f -t7g10-egfp-6d-coe using rlppg-t7g10-6d-coe as a control. briefly, recombinant strains were cultured in mrs medium at 37 • c for 16 h; then 1 ml of culture was collected by centrifugation at 5000× g for 5 min. the pellets were washed three times with pbs, re-suspended in 1 ml of pbs, and smeared on a microscope slide. images were viewed by laser confocal microscopy (zeiss, oberkochen, germany). in order to evaluate the immunogenicity of recombinant strain rlppg f -t7g10-egfp-6d-coe used as an oral vaccine, 5-week-old female specific pathogen-free (spf) balb/c mice (derived from mus musculus) (n = 60) were obtained from liaoning changsheng biotechnology co., ltd. (liaoning, china) and kept under spf conditions for one week with free access to a standard chow diet and water, in accordance with institutional guidelines. prior to oral administration, the recombinant lactobacillus strains were cultured for 16 h in mrs medium without shaking, washed with sterile pbs, and re-suspended in pbs at a concentration of 10 10 cfu ml −1 . spf balb/c mice were randomly divided into four groups (15 mice per group): pbs, rlppg-t7g10, rlppg-t7g10-6d-coe, and rlppg f -t7g10-egfp-6d-coe. the immunization dosages are shown in table 3 . the mice were immunized once a day for 3 consecutive days and boosted twice at 2 week intervals. after immunization, serum samples were collected from the immunized mice on days 0, 7, 14, 21, 28 and 35, and were stored at −20 • c until they were required for use. mucosal lavage samples were obtained from the vaginas of the mice by washing with 200 µl of sterile pbs (ph 7.4) and were stored at −20 • c until analysis. in addition, fecal samples were collected and treated according to a method previously described [35] . briefly, a 0.1 g fecal pellet was suspended in 400 µl of pbs containing 1 mmol l −1 phenylmethylsulfonyl fluoride (sigma) and 1% bovine serum albumin (bsa) and was then incubated at 4 • c for 16 h. after centrifugation, the supernatants were stored at −20 • c until use. to detect tgev-and pedv-specific antibodies in the collected samples, polystyrene microtiter plates were coated with purified tgev/pedv for 12 h at 4 • c, using cultured st/vero cells as a negative antigen control. after blocking with 5% skim milk at 37 • c for 2 h and washing three times with pbs-0.1% tween 20 (pbst), serum and mucus samples serially diluted in pbs-1% bsa were added to wells in triplicate, and then plates were incubated for 1 h at 37 • c. after washing with pbst, hrp-conjugated goat anti-mouse igg or iga antibody (invitrogen, carlsbad, ca, usa) was added to each well (1:5000) and incubated for an additional 1 h at 37 • c. the substrate o-phenylenediamine dihydrochloride (sigma) was used for color development, and the absorbance was measured at 490 nm. the neutralizing capacities of serum antibodies obtained from the mice immunized with rlppg f -t7g10-egfp-6d-coe were determined. briefly, the 50% tissue culture infective dose (tcid 50 ) values of tgev and pedv were detected by the reed-muench method. serum antibodies collected from the vaccinated mice on day 35 post-immunization were diluted at 1:10-1:320 (in a total of 50 µl), mixed with an equal volume of pedv or tgev (100 tcid 50 per 100 µl) and incubated at 37 • c for 1 h. then, the treated viruses were added to a confluent monolayer of vero and st cells cultured in 24-well plates. the cells were overlaid with 1% methylcellulose, and the plates were incubated at 37 • c in a 5% co 2 atmosphere and examined daily for five days for tgev-and pedv-specific cytopathic effects (cpe). on day 35 post-immunization, splenocytes from three mice from each group were prepared for a lymphocyte proliferation assay as previously described [34] . briefly, 100 µl of splenocytes (5 × 10 7 cells ml −1 ) was suspended in roswell park memorial institute (rpmi) 1640 medium containing 10% fetal calf serum and then transferred to a 96-well flat-bottom plate; the cells were re-stimulated for 72 h with 1.0, 5.0 or 25 µg ml −1 tgev-6d protein and pedv-coe protein (produced by escherichia coli prepared in our lab), using 5.0 µg ml −1 concanavalin a (cona) and culture medium as positive and negative controls, respectively. the plates were supplemented with 10 µl of 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (mtt) per well and incubated for an additional 4 h, and then proliferation was measured using od 490 values. the experiment was carried out in triplicate, and the lymphocyte proliferation index in the spleen (si) was calculated as the mean reading of triplicate antigen stimulation wells divided by the mean reading of triplicate negative control wells. data are shown as the means ± standard errors of three replicates per test in a single experiment repeated three times. tukey's multiple comparison tests were used to analyze differences among the groups. a p-value of <0.05 was considered statistically significant, and p < 0.01 was considered highly significant. following flow cytometry screening and growth on antibiotic-free mrs plates, egfp-marked recombinant lactobacillus strain rlppg f -t7g10-egfp-6d-coe was obtained and identified with colony pcr (figure 2a) . as shown in figure 2b , the strain rlppg f -t7g10-egfp-6d-coe could not grow on mrs agar medium in the presence of chloramphenicol. the result of the hereditary stability of recombinant l. casei strains showed that rlppg f -t7g10-egfp-6d-coe is stable for more than 50 generations. for confirming the expression of the proteins of interest by the recombinant lactobacillus strain constructed in this study, the cell lysates of recombinant strains rlppg-t7g10-6d-coe and rlppg f -t7g10-egfp-6d-coe were analyzed by a western blot assay. as shown in figure 3 , the expected immunoblot bands of fusion protein pgsa-6d-coe of 80 kda expressed by rlppg-t7g10-6d-coe (figure 3a ,b) and the fusion protein pgsa-egfp-6d-coe of 110 kda expressed by rlppg f -t7g10-egfp-6d-coe were observed (figure 3a-c) , but these proteins were not expressed in rlppg-t7g10 or l. casei. moreover, green fluorescence could be observed by laser confocal microscopy on the surface of rlppg f -t7g10-egfp-6d-coe but not on rlppg-t7g10-6d-coe (figure 4) , indicating that egfp could successfully be used as a selective marker. for confirming the expression of the proteins of interest by the recombinant lactobacillus strain constructed in this study, the cell lysates of recombinant strains rlppg-t7g10-6d-coe and rlppg f -t7g10-egfp-6d-coe were analyzed by a western blot assay. as shown in figure 3 , the expected immunoblot bands of fusion protein pgsa-6d-coe of 80 kda expressed by rlppg-t7g10-6d-coe (figure 3a ,b) and the fusion protein pgsa-egfp-6d-coe of 110 kda expressed by rlppg f -t7g10-egfp-6d-coe were observed (figure 3a-c) , but these proteins were not expressed in rlppg-t7g10 or l. casei. moreover, green fluorescence could be observed by laser confocal microscopy on the surface of rlppg f -t7g10-egfp-6d-coe but not on rlppg-t7g10-6d-coe (figure 4) , indicating that egfp could successfully be used as a selective marker. for confirming the expression of the proteins of interest by the recombinant lactobacillus strain constructed in this study, the cell lysates of recombinant strains rlppg-t7g10-6d-coe and rlppg f -t7g10-egfp-6d-coe were analyzed by a western blot assay. as shown in figure 3 , the expected immunoblot bands of fusion protein pgsa-6d-coe of 80 kda expressed by rlppg-t7g10-6d-coe (figure 3a ,b) and the fusion protein pgsa-egfp-6d-coe of 110 kda expressed by rlppg f -t7g10-egfp-6d-coe were observed (figure 3a-c) , but these proteins were not expressed in rlppg-t7g10 or l. casei. moreover, green fluorescence could be observed by laser confocal microscopy on the surface of rlppg f -t7g10-egfp-6d-coe but not on rlppg-t7g10-6d-coe (figure 4) , indicating that egfp could successfully be used as a selective marker. anti-tgev/pedv-specific secreted iga (siga) and igg antibodies were assessed to evaluate the ability of rlppg-t7g10-6d-coe and rlppg f -t7g10-egfp-6d-coe to induce mucosal and systemic immune responses using balb/c mice as a model. as shown in figure 5 , anti-tgev/pedv-specific antibodies were detected at high levels 7 days post-immunization and were significantly increased after the booster immunization, peaking at 35 days post-immunization. the levels of anti-tgev/pedv-specific mucosal siga in mouse feces and vaginas, as well as the levels of anti-tgev/pedv-specific serum igg antibody in mice orally immunized with rlppg-t7g10-6d-coe/rlppg f -t7g10-egfp-6d-coe, were significantly higher (p < 0.01) than those in the pbs or rlppg-t7g10 groups. in addition, the levels of anti-tgev/pedv siga in the vaginas of mice orally immunized with rlppg f -t7g10-egfp-6d-coe 35 days post-immunization were significantly higher (p < 0.01) than those in the rlppg-t7g10-6d-coe group. there was no statistical difference (p > 0.05) observed in the pbs or rlppg-t7g10 groups before and after immunization. anti-tgev/pedv-specific secreted iga (siga) and igg antibodies were assessed to evaluate the ability of rlppg-t7g10-6d-coe and rlppg f -t7g10-egfp-6d-coe to induce mucosal and systemic immune responses using balb/c mice as a model. as shown in figure 5 , anti-tgev/pedv-specific antibodies were detected at high levels 7 days post-immunization and were significantly increased after the booster immunization, peaking at 35 days post-immunization. the levels of anti-tgev/pedv-specific mucosal siga in mouse feces and vaginas, as well as the levels of anti-tgev/pedv-specific serum igg antibody in mice orally immunized with rlppg-t7g10-6d-coe/rlppg f -t7g10-egfp-6d-coe, were significantly higher (p < 0.01) than those in the pbs or rlppg-t7g10 groups. in addition, the levels of anti-tgev/pedv siga in the vaginas of mice orally immunized with rlppg f -t7g10-egfp-6d-coe 35 days post-immunization were significantly higher (p < 0.01) than those in the rlppg-t7g10-6d-coe group. there was no statistical difference (p > 0.05) observed in the pbs or rlppg-t7g10 groups before and after immunization. the neutralizing capacities of the serum antibodies induced in mice orally immunized with recombinant strains against tgev (figure 6a ) and pedv (figure 6b ) showed inhibitory activity against viral infection. the anti-tgev neutralizing antibody titers were 10 −2 (1:100) and 10 −1.933 (1:85.7) and the anti-pedv neutralizing antibody titers were 10 −1.967 (1:92.7) and 10 −2.05 (1:112.2) in mice immunized with rlppg f -t7g10-egfp-6d-coe and rlppg-t7g10-6d-coe, respectively. these were significantly different from those obtained in the rlppgf-t7g10 and pbs groups (p < 0.05). bars represent the mean ± standard error of each group. * p < 0.05, ** p < 0.01 vs. phosphate-buffered saline (pbs) and vector control groups; # p < 0.05, ## p < 0.01 vs. rlppg f -t7g10-egfp-6d-coe group. the neutralizing capacities of the serum antibodies induced in mice orally immunized with recombinant strains against tgev (figure 6a ) and pedv (figure 6b ) showed inhibitory activity against viral infection. the anti-tgev neutralizing antibody titers were 10 −2 (1:100) and 10 −1.933 (1:85.7) and the anti-pedv neutralizing antibody titers were 10 −1.967 (1:92.7) and 10 −2.05 (1:112.2) in mice immunized with rlppg f -t7g10-egfp-6d-coe and rlppg-t7g10-6d-coe, respectively. these were significantly different from those obtained in the rlppgf-t7g10 and pbs groups (p < 0.05). the proliferation of spleen lymphocytes upon stimulation with purified coe or 6d proteins was analyzed by an mtt assay. the results showed that the proliferation levels of spleen lymphocytes from mice orally immunized with rlppg-t7g10-6d-coe and rlppg f -t7g10-egfp-6d-coe were significantly higher than those of spleen lymphocytes from mice in the rlppgf-t7g10 and pbs control groups (p < 0.01). there was no significant difference (p > 0.05) observed between the rlppg-t7g10-6d-coe and rlppg f -t7g10-egfp-6d-coe groups (figure 7 ). over the past decades, large-scale outbreaks of diarrhea in swine caused by pedv and tgev have occurred in america, europe, and asia, resulting in considerable economic losses to the pig industry; in particular, pedv infection has been responsible for many of these outbreaks [7, 8] . the pedv strain hlj-2012 used in this study was isolated from an outbreak of acute diarrhea in piglets in hegang, heilongjiang province, china [29] , and phylogenetic analysis based on the s gene revealed that hlj-2012 shares high homology with u.s. pedv strains. both belong to the giia subgroup, which represents epidemic and pandemic field strains [29, 36] . occasionally, changes in the antigenicity of pedv due to amino acid mutations have resulted in vaccination failure [5, 7] . the the proliferation of spleen lymphocytes upon stimulation with purified coe or 6d proteins was analyzed by an mtt assay. the results showed that the proliferation levels of spleen lymphocytes from mice orally immunized with rlppg-t7g10-6d-coe and rlppg f -t7g10-egfp-6d-coe were significantly higher than those of spleen lymphocytes from mice in the rlppgf-t7g10 and pbs control groups (p < 0.01). there was no significant difference (p > 0.05) observed between the rlppg-t7g10-6d-coe and rlppg f -t7g10-egfp-6d-coe groups (figure 7) . the proliferation of spleen lymphocytes upon stimulation with purified coe or 6d proteins was analyzed by an mtt assay. the results showed that the proliferation levels of spleen lymphocytes from mice orally immunized with rlppg-t7g10-6d-coe and rlppg f -t7g10-egfp-6d-coe were significantly higher than those of spleen lymphocytes from mice in the rlppgf-t7g10 and pbs control groups (p < 0.01). there was no significant difference (p > 0.05) observed between the rlppg-t7g10-6d-coe and rlppg f -t7g10-egfp-6d-coe groups (figure 7 ). over the past decades, large-scale outbreaks of diarrhea in swine caused by pedv and tgev have occurred in america, europe, and asia, resulting in considerable economic losses to the pig industry; in particular, pedv infection has been responsible for many of these outbreaks [7, 8] . the pedv strain hlj-2012 used in this study was isolated from an outbreak of acute diarrhea in piglets in hegang, heilongjiang province, china [29] , and phylogenetic analysis based on the s gene revealed that hlj-2012 shares high homology with u.s. pedv strains. both belong to the giia subgroup, which represents epidemic and pandemic field strains [29, 36] . occasionally, changes in the antigenicity of pedv due to amino acid mutations have resulted in vaccination failure [5, 7] . the over the past decades, large-scale outbreaks of diarrhea in swine caused by pedv and tgev have occurred in america, europe, and asia, resulting in considerable economic losses to the pig industry; in particular, pedv infection has been responsible for many of these outbreaks [7, 8] . the pedv strain hlj-2012 used in this study was isolated from an outbreak of acute diarrhea in piglets in hegang, heilongjiang province, china [29] , and phylogenetic analysis based on the s gene revealed that hlj-2012 shares high homology with u.s. pedv strains. both belong to the giia subgroup, which represents epidemic and pandemic field strains [29, 36] . occasionally, changes in the antigenicity of pedv due to amino acid mutations have resulted in vaccination failure [5, 7] . the s protein neutralizing antigenic epitopes of pedv have been reported to provide cross-protection against variant strains [9, 29] . therefore, we selected the coe of the pedv s protein as an immunogen for developing an effective vaccine against pedv infection. pedv and tgev infections initially occur on mucosal surfaces, especially the intestinal mucosal epithelial surface. therefore, mucosal vaccination is an effective strategy for preventing viral diarrheal diseases [12] . in this study, we used l. casei to deliver the tgev s protein protective d antigenic site (repeated six times) and the pedv s protein coe for developing an oral mucosal vaccine against pedv and tgev. our results suggested that the genetically engineered l. casei strain rlppg f -t7g10-egfp-6d-coe can be used as a bivalent oral vaccine for pedv and tgev, eliciting mucosal and humoral immune responses against both tgev and pedv via oral immunization. this was evidenced by significantly higher levels of virus-neutralizing antibodies, anti-pedv/tgev serum igg, and mucosal siga in mice orally immunized with rlppg f -t7g10-egfp-6d-coe, compared to the levels for the rlppg-t7g10 or pbs groups. moreover, the genetically engineered l. casei vaccine is safe and easy to administer, making it practical and convenient. mucosal immunity plays an important role in preventing viral diarrheal diseases, and siga antibodies from durable lactogenic immunity are a good way for piglets to obtain passive immunoprotection, indicating the importance of the siga antibody in the control of viral infection. at the same time, the level of siga can reflect the status of viral infection and the protective efficacy of vaccines [34] . in the present study, anti-tgev/pedv siga antibodies could be effectively induced at high levels in the feces and vaginas of mice orally treated with rlppg f -t7g10-egfp-6d-coe. iga is reported to peak at 6 weeks and decline at 8 weeks in piglets [26] ; thus, if durable immunity was present at 35 days post-immunization, newborn pigs could obtain immune protection. therefore, we assessed the levels of the siga antibody for 35 days post-immunization. our data showed that the levels of siga in the feces of mice orally immunized with rlppg f -t7g10-egfp-6d-coe gradually increased and peaked at 7, 21 and 35 days post-immunization, indicating that a mucosal immune response can be effectively elicited by rlppg f -t7g10-egfp-6d-coe after oral immunization. therefore, the developed vaccine provides a promising strategy for protecting piglets from pedv and tgev infection via oral immunization. moreover, the neutralizing activity of an antibody is an important index used for evaluating the immunoprotective efficacy of a vaccine. in this study, high levels of serum antibody were elicited following oral immunization with rlppg f -t7g10-egfp-6d-coe. this could effectively neutralize the pedv/tgev infection, and higher antibody titers reflected higher neutralizing activities. as a comparison, the neutralizing antibody titer induced by the recombinant l. casei oral vaccine developed in this study was higher than that induced by a previously developed dna vaccine [24] . therefore, oral immunization with genetically engineered l. casei strain rlppg f -t7g10-egfp-6d-coe may provide effective protection for piglets against pedv and tgev infection. in addition, our results showed that the egfp-marked recombinant lactobacillus oral vaccine rlppg f -t7g10-egfp-6d-coe (antibiotic-free selective marker vaccine) exhibited a similar immunogenicity to the antibiotic resistance marker vaccine rlppg-t7g10-6d-coe, as the levels of antibodies induced by the rlppg f -t7g10-egfp-6d-coe and rlppg-t7g10-6d-coe vaccines were not significantly different (p > 0.05). notably, the use of egfp as a selective marker in rlppg f -t7g10-egfp-6d-coe would avoid the main disadvantage of traditional plasmid expression systems by eliminating the use of antibiotic resistance genes as selective markers for genetically engineered bacteria [18, 24, 25] . moreover, rlppg f -t7g10-egfp-6d-coe was constructed with a constitutive expression plasmid developed by our lab, exhibiting a significant advantage to inducible gene expression systems that require the use of an inductive agent. additionally, a pgsa-derived anchoring matrix from bacillus subtilis [37, 38] was used to express the fusion proteins, which were displayed on the bacterial surface, eliciting good immunogenicity. the improved plasmid expression system used in this study therefore provides a powerful tool for the development of recombinant lactobacillus oral vaccines. in conclusion, an egfp-marked recombinant lactobacillus oral vaccine, rlppg f -t7g10-egfp-6d-coe, was constructed in this study and provides a promising strategy for the development of a bivalent oral vaccine against tgev and pedv infection. further investigations are underway to evaluate the immunogenicity of this vaccine following its oral administration in piglets and to optimize the immunization procedures for effective control of tge and ped. structure and genome expression a transmissible gastroenteritis in pigs isolation of a porcine 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gamma-glutamate synthetase complex of bacillus subtilis ter beek, a. rodz and pgsa play intertwined roles in membrane homeostasis of bacillus subtilis and resistance to weak organic acid stress the authors declare no conflict of interest. key: cord-302503-7s9f8wje authors: fu, yuguang; li, baoyu; liu, guangliang title: rapid and efficient detection methods of pathogenic swine enteric coronaviruses date: 2020-05-19 journal: appl microbiol biotechnol doi: 10.1007/s00253-020-10645-5 sha: doc_id: 302503 cord_uid: 7s9f8wje abstract: porcine enteric coronaviruses (covs) cause highly contagious enteric diarrhea in suckling piglets. these cov infections are characterized by clinical signs of vomiting, watery diarrhea, dehydration, and high morbidity and mortality, resulting in significant economic losses and tremendous threats to the pig farming industry worldwide. because the clinical manifestations of pigs infected by different covs are similar, it is difficult to differentiate between the specific pathogens. effective high-throughput detection methods are powerful tools used in the prevention and control of diseases. the immune system of piglets is not well developed, so serological methods to detect antibodies against these viruses are not suitable for rapid and early detection. this paper reviews various pcr-based methods used for the rapid and efficient detection of these pathogenic covs in swine intestines. key points: 1. swine enteric coronaviruses (covs) emerged and reemerged in past years. 2. enteric covs infect pigs at all ages with high mortality rate in suckling pigs. 3. rapid and efficient detection methods are needed and critical for diagnosis. the coronaviruses (covs) critically threaten human and animal health because infection with them results in respiratory or enteric tract diseases (woo et al. 2012) . for instance, pneumonia occurs in humans infected with the new cov that emerged in china in the middle of dec. 2019; the virus has subsequently spread to many countries worldwide where it threatens the health of humans, resulting in tremendous economic losses. covs are enveloped, positive-sense, singlestranded rna viruses that possess a genome ranging from 25.4 to 31.7 kb; they belong to the order nidovirales, family coronaviridae, and subfamily coronavirinae (woo et al. 2012) . based on antigenic relationships, the classification of coronaviruses was traditionally divided into 3 genera, but they were replaced by the following four genera: alphacoronavirus ( a l p h a -c o v ) , b e t a c o r o n a v i r u s ( b e t a -c o v ) , gammacoronavirus (gamma-cov), and deltacoronavirus (delta-cov), which are based on genetic and antigenic characteristics (woo et al. 2010 ). epidemiological surveys have indicated that bats and birds seem to be natural reservoirs for alpha-and beta-covs and gamma-and delta-covs, respectively (bolles et al. 2011; woo et al. 2012) . covs in four genera have been verified in a variety of species, e.g., canines, felines, and birds (chan et al. 2013) . six covs have been identified in swine (table 1) : porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), swine acute diarrhea syndrome coronavirus (sads-cov), and porcine respiratory cov (prcov) in the alpha-cov genus, porcine hemagglutinating encephalomyelitis virus (phev) in the beta-cov genus, and porcine deltacoronavirus (pdcov) in the delta-cov genus (jung et al. 2016; pan et al. 2017; pensaert and de bouck 1978; woo et al. 2012; zhang 2016; zhou et al. 2018 ). among the six swine covs, tgev, pedv, pdcov, and sads-cov are enteric viruses that cause diarrhea in the pig population, resulting in significant economic losses and tremendous threats to the pig industry worldwide. the four swine enteric covs causing highly contagious enteric diarrhea in neonatal and suckling piglets are clinically characterized by vomiting, watery diarrhea, dehydration, and high morbidity and mortality (gong et al. 2017; hsu et al. 2018) . because the clinical signs of pigs infected by these covs are very similar (table 1) , it is difficult to differentiate the specific pathogens based on clinical symptoms. effective high-throughput detection methods are needed for their differential determination and would represent powerful tools to prevent and control diseases. as far as we know, many standard detection methods can be used to distinguish between causative agents, including virus isolation, electron microscopy, virus neutralization, and indirect immunofluorescence assays. however, these methods are time-consuming, laborious, and not suitable for the early and rapid detection of the four swine enteric covs (carman et al. 2002; dulac et al. 1977; van nieuwstadt et al. 1988 ). the enzyme-linked immunosorbent assay is a powerful and highthroughput method for detecting specific antibodies, but the immune system of piglets is not well developed, so serological methods for detecting antibodies against these viruses are also not suitable for rapid and early detection. polymerase chain reaction (pcr) methods have been widely used to detect pathogens since the pcr was invented; pcr has proven to be powerful and convenient tools for precise detection of diarrheal pathogens in pig populations (ben salem et al. 2010; collins et al. 2008; kim et al. 2007 ). this paper reviews various pcr-based methods for the rapid and efficient detection of these pathogenic covs in swine intestines. pan-cov rt-pcr assay for the detection of covs figure 1 summarizes the basic workflow for the detection of the swine enteric coronaviruses from clinical samples. generally, porcine fecal or intestinal samples need to be suspended and homogenized in sterile pbs and centrifuged to remove debris. the supernatant should be collected and filtered through a 0.45-μm filter to remove the debris and some potential bacteria. the yield supernatant can be used to extract the total rnas by trizol reagent or rna extraction kit. the total rnas are used to reverse transcription by random primers to generate cdna. this cdna is employed as a template to do pcr amplification either by specific individual coronavirus or by pan-cov primers first then followed by specific primers targeting individual swine enteric coronavirus when necessary. the pcr products are eventually subjected to dna electrophoresis and analyzed under uv light to identify the desired bands. the pan-cov pcr method is a powerful tool for detecting all known and unknown covs; it is based on the conserved gene sequences among them (moes et al. 2005 ). this assay is widely used to detect covs. pan-cov rt-pcr was employed to detect all known covs in the human respiratory tract (vijgen et al. 2008) and to detect distinct alpha-covs in five different bat species (escutenaire et al. 2007; lazov et al. 2018; vijgen et al. 2008) . for swine enteric covs, pan-cov pcr also played an important role in identification. during the early stage of investigating cases of diarrhea in piglets in the usa caused by the pedv variant and pdcov, pan-cov rt-pcr was applied to identify the causative agent together with electron microscopy and sequencing stevenson et al. 2013 ). in addition, during the identification of piglet diarrhea disease caused by sads-cov in china, pan-cov rt-pcr was employed (pan et al. 2017) . in 2018, hu et al. reported an improved one-step pan-cov rt-pcr (hu et al. 2018) . though pan-cov pcr could detect all known covs in humans and animals, it could not make a differential detection, so it is not suitable for routine swine enteric cov detection. porcine epidemic diarrhea (ped) is an enteric disease caused by pedv that can infect pigs of all ages with different levels of clinical signs of vomiting, diarrhea, dehydration, and weight loss, but the disease is much more severe in suckling piglets (have et al. 1992; shibata et al. 2000; song and park 2012; sueyoshi et al. 1995) . ped was first reported in england in 1971, but the pedv was isolated for the first time in belgium in 1978 (pensaert and de bouck 1978; song and park 2012) ; however, the epidemic was not controlled in europe before 2000. in china, pedv was first identified in the 1980s, after which it was reported in some asian countries, e.g., japan and korea (kusanagi et al. 1992; song and park 2012; takahashi et al. 1983 ). in october 2010, a severe ped outbreak caused by a highly virulent pedv variant emerged in southern china with high mortality ranging from 70 to 100%; the result was devastating damage to the pig farm industry and tremendous economic losses, and later, the pedv variant spreads to other countries, e.g., usa, canada, and mexico ( for the early and rapid detection of pedv, different types of pcr methods have been developed. a real-time reverse transcription recombinase polymerase amplification assay (rt-rpa) based on the nucleocapsid gene of pedv was reported in 2018; the assay was able to detect 23 copies per reaction and was performed for 20 min at 40°c . based on the advantage of increased thermal conductivity of solid gold nanometal particles that could reduce nonspecific amplification and increase specific amplification, yuan et al. developed a nanoparticle-assisted pcr assay for the detection of pedv on the basis of the n gene in 2015, and the assay could detect 2.7 × 10 −6 ng/μl of pedv rna (yuan et al. 2015) . ren and li designed six primers according to the sequence of the n gene and established a reverse transcription loop-mediated isothermal amplification (rt-lamp) for the rapid detection of pedv (within 50 min) at 63°c. the detection limit of the method was 1 × 10 −4 μg pedv rna per reaction (ren and li 2011) . wang et al. designed five primers on the basis of the n gene sequence of pedv and established a reverse transcription cross-priming amplification-nucleic acid test strip (cpa-nast) for the detection of pedv. the method had high specificity for the detection of pedv and had the same sensitivity as traditional rt-pcr; the detection limit was a 10 −6 dilution of plasmid containing the target gene ). xing et al. developed an rna extraction and transcription-free, nanoparticle-based pcr (nbp-pcr) method to specifically detect pedv, and the sensitivity of the schematic diagram of the workflow for swine enteric coronavirus detection by rt-pcr. porcine fecal or tissue samples are homogenized in sterile pbs and centrifuged to remove debris. the supernatant is filtered through a 0.45-μm filter and used to total rna extraction. the total rnas are subjected to reverse transcription using random primer to generate cdna. with this cdna as template, the pcr amplification is carried out either by specific individual coronavirus or by pan-cov primers first then followed by specific primers targeting individual swine enteric coronavirus when necessary. the pcr products are subjected to dna electrophoresis and observed under uv light to identify the desired bands the method was 400-fold higher than that of conventional rt-pcr. in 153 fecal samples, the positive detection rate of nbp-pcr specific was much higher than that of conventional rt-pcr (5.88%) and sybr green real-time rt-pcr (xing et al. 2016 ). zhou et al. developed a conventional rt-pcr method, an sybr green i real-time rt-pcr, and taqman real-time rt-pcr reagents to detect the highly conserved m gene of pedv; the detection limit of the taqman real-time rt-pcr was 10 copies/μl of the target gene, and the sensitivity of the taqman real-time rt-pcr was 100-fold and 10,000-fold higher than that of sybr green i real-time rt-pcr and conventional rt-pcr, respectively ). in addition, there were variant pedv strains circulating in the field and the pcr methods for differentiating them had been established. song et al. analyzed pathogenicity and immunogenicity of pedv strain designated dr13 in piglets, which was a highly vero cell-adapted virus and could be employed as a vaccine candidate, and applied a restriction fragment length polymorphism (rflp) assay to differentiate dr13 from wild-type virus based on the difference of open reading frame (orf) 3 sequence (song et al. 2003) . in 2008, lee et al. reported an rt-pcr-based rflp assay targeting the n gene of pedv to distinguish field strains of pedv in korea from an attenuated-live vaccine j-vac, which was used in pig population to prevent pedv . for differentiation between attenuated-type pedvs including attenuated dr13, kped-9, and p-5v that were used as live virus vaccine and wild-type pedvs including cv777, brl/ 87, lzc, parent dr13, and field samples, park et al. established an rt-pcr assay based on the difference of orf 3 gene sequence of attenuated-and wild-type pedv, in which 51 nucleotide deletions were found in all live pedv vaccine . in may 2013, the virulent strain of pedv was verified in the usa resulting in significant economic losses in the swine industry. and a variant strain (oh851) of pedv emerged in the usa in december 2013, which differs from the virulent strains of pedv in the nucleotides of the 5′end of spike gene. to differentiate these two genotypes of pedv circulating in the usa, wang et al. reported a duplex probe-based real-time rt-pcr targeting the difference of spike gene among virulent and variant strains (wang et al. 2014b ). sequence analysis of pedv genome indicated that pedv attenuated vaccine strains (e.g., the cv777 and zj08 in china, the p-5v in japan, and kped-9 and dr13 in south korea in asia) have 49 base pair deletion in the open reading frame 3 (orf3); for differentiation of these cell-adapted vaccine strains from field strains, zhu et al. developed a nanoparticle-assisted rt-pcr assay targeting the orf3 (zhu et al. 2016b) . because three major pedv types have been identified in the usa after 2013 including the original us pedv strains, the spike gene insertion-deletion pedv strains, and the pc177 strain that possess a 197 amino acid-deletion in the s1region of spike protein, liu and wang developed a reverse transcription-pcr method to differentiate these variants on the basis of differences in the s1 gene (liu and wang 2016) . since 2010, pedv variants with base deletions and insertions in the s gene emerged in china and caused significant losses in piglets; zhao et al. developed a taqman probe-based real-time pcr method for the detection of different pedv variants and classical pedv strains based on the sequence difference of the pedv s gene, and the detection limit of the method was 5 × 10 2 target gene copies (zhao et al. 2014 ). su et al. established a duplex taqman probe real-time rt-qpcr method for detecting and differentiating classical and variant pedvs targeting the difference in the s gene; the detection limit of the method was 4.8 × 10 2 genome copies/reaction for both the classical and variant pedv. the results of clinical sample detection showed that the assay was more sensitive than conventional pcr, and variant pedv was prevalent in china (su et al. 2018) . due to the wide use of a live attenuated pedv vaccine, classical and wild variant strains circulating in pig farms are common; therefore, he et al. established a multiplex rt-pcr to differentiate these strains based on the difference in s gene sequences, and the detection limit of the method was 1 × 10 1.7 tcid 50 /100 μl for pedv (he et al. 2019 ). due to variant pedv strains that emerged in china since 2010 and attenuated pedv vaccines (e.g., cv777 strain) being widely used in china, liu et al. reported a taqman probe-based realtime pcr to differentiate these virulent strains and attenuated vaccine strains based on the orf3 deletion region to detect virulent pedv strains in vaccinated pig population (liu et al. 2019b ). transmissible gastroenteritis (tge) caused by tgev is an acute enteric diarrhea disease in pigs (garwes 1988) . tgev was isolated for the first time in 1946, and then outbreaks of the virus occurred in many countries in america, asia, and europe (doyle and hutchings 1946; kim et al. 2000; stevenson et al. 2013) . tgev causes severe enteritis in piglets before weaning, and the clinical signs include diarrhea, vomiting, dehydration, and high mortality, resulting in significant economic losses (ding et al. 2017; penzes et al. 2001; saif 1999) . for the early and rapid detection of tgev, different types of pcr-based methods have been established. chen et al. designed six specific primers targeting the nucleocapsid gene of tgev and developed an rt-lamp assay for the detection of tgev, which involved incubation at 60°c for 1 h. the sensitivity of rt-lamp was comparable to that of nested pcr described by rodríguez et al. in 2008 , and it was 10 times more sensitive than the pcr reported by paton et al. in 1997, which could detect 10 pg of rna per reaction paton et al. 1997; rodríguez et al. 2008) . rt-lamp for the detection of the tgev targeting the n gene sequence by incubation at 60°c for 30 min was reported by li et al., and the sensitivity of rt-lamp was much higher than that of a gel-based rt-pcr kit purchased from haigene company, china (li et al. 2012 ). vemulapalli et al. described a taqman probe-based real-time rt-pcr that specifically amplified conserved s gene sequences. the detection limit of the method was 1 tcid 50 /ml of tgev rna. the sensitivity of the assay was higher than that of the nested rt-pcr assay reported by kim et al. (kim et al. 2000; vemulapalli et al. 2009 ). wang et al. developed a rapid detection of tgev using real-time reverse transcription recombinase polymerase amplification (rt-rpa) based on the spike gene, and the method could detect 100 copies of tgev rna after 20 min at 40°c conditions . a real-time rt-pcr assay described by vemulapalli et al. was compared with the rt-rpa method in the analysis of 76 clinical samples, and the two methods generated the same results (vemulapalli et al. 2009; wang et al. 2018 ). in the mid-1980s, prcov, which has a deletion of nucleotides of the s gene that is present in tgev (laude et al. 1993) , was reported in europe, north america, and asia (pensaert et al. 1986; wesley et al. 1990 ). paton et al. developed an rt-pcr assay to differentiate tgev and prcv based on the difference in the s gene of the two viruses (paton et al. 1997) . pdcov is a novel swine enteric diarrhea virus that causes severe diarrhea, vomiting, and dehydration in piglets (janetanakit et al. 2016; song et al. 2015) . pdcov was first discovered in samples from the healthy pig that were collected in 2009 in hong kong when a molecular surveillance study was performed (woo et al. 2012) . in february 2014, the virus was detected in piglets with severe diarrhea in the usa (marthaler et al. 2014; wang et al. 2014a ). subsequently, pdcov was reported in south korea, mainland china, and thailand (chen et al. 2015a; janetanakit et al. 2016; lee and lee 2014; song et al. 2015) . to detect the virus with precision, pcr-based methods have been created. in 2014, pigs on 5 farms in ohio, usa, had clinical diarrheal disease. wang et al. developed a onestep rt-pcr targeting membrane and nucleocapsid gene of delta-cov and determined the causative agent as porcine delta-covs, but they did not evaluate the detection limit of the assay (wang et al. 2014a ). in 2015, song et al. established a nested rt-pcr method on the basis of the nucleocapsid gene sequence of the pdcov hku15 strain to identify the causative agent causing acute diarrhea in a pig farm in jiangxi, china, and the sensitivity of the assay was also not determined . to analyze the characteristics of porcine delta-covs in the usa, ma et al. established a real-time rt-pcr method for specific detection of the n gene, but the sensitivity of the assay was also not determined (ma et al. 2015) . for the analysis of pathogenicity and pathogenesis of a porcine delta-cov cell culture isolate, chen et al. developed an m gene-based real-time pcr method for detecting viral titers in different organs, but the sensitivity of the assay was not described in the publication (chen et al. 2015b ). marthaler et al. developed a one-step probe-based real-time rt-pcr method targeting the m gene sequence of pdcov, which was applied by the university of minnesota veterinary diagnostic laboratory. the detection limit of the assay was 2 viral rna copies per reaction, but the specificity of the assay was not evaluated (marthaler et al. 2014 ). the commercial reverse transcription-insulated isothermal pcr (rt-iipcr) pockit™ methods (pockit™ pedv reagent set and pockit™ pdcov reagent set, genereach usa, lexington, ma, usa) was used to analyze pedv and pdcov coinfection that occurred in diarrhea disease; zhang et al. described two singleplex rt-iipcr tests and a duplex real-time rt-pcr test for the detection of pedv and pdcov that were based on targeting the conserved m gene sequence. the detection limits of singleplex rt-iipcr were 21 rna copies per reaction for pedv and 9 rna copies per reaction for pdcov, and those of the duplex rt-iipcr were 7 rna copies and 14 rna copies per reaction for pedv and pdcov, respectively ). sads-cov, also designated seacov or peav, is a novel porcine enteric diarrhea virus that can cause severe and acute diarrhea and rapid weight loss in piglets (pan et al. 2017; zhou et al. 2018; zhou et al. 2017) . sads-cov was identified for the first time in southern china in late 2017, and it caused more than 24,000 piglet deaths, resulting in significant economic losses (gong et al. 2017) . the clinical signs of infection with sads-cov are similar to those of other known swine enteric covs: tgev, pedv, and pdcov (dong et al. 2015; sun et al. 2016) . for specific detection of sads-cov, zhou et al. established an sybr premix ex taqii-based real-time pcr on the basis of the rna-dependent rna polymerase (rdrp) gene for the detection of sads-cov; the sensitivity of which was not evaluated ). zhou et al. developed a taqman-based real-time pcr assay for sads-cov detection based on the conserved sequence within the n gene; the detection limit of the assay was 3.0 × 10 1 copies/μl, and the sensitivity of the method was 10-fold higher than that of conventional pcr, which also targeted the n gene (zhou et al. 2018 ). because the clinical signs caused by the four enteric covs in piglets are similar to each other, it is very difficult or timeconsuming to make a clear diagnosis of mixed infection in pigs using a single pcr method. therefore, it is critical to developing a multiplex polymerase chain reaction (mpcr) in which two or more loci can be simultaneously detected in the same reaction (chamberlain et al. 1988 ). tgev and pedv are traditional swine enteric diarrheal viruses, so several mpcr methods that can differentially detect the two viruses have been previously established. in 2001, kim et al. reported a duplex rt-pcr assay to detect tgev and pedv in one pcr tube targeting the s gene of the two viruses, and the detection limit of the assay was 2 tcid 50 /200 μl (kim et al. 2001) . in 2007, kim et al. established a multiplex realtime rt-pcr method based on the nucleocapsid (n) gene for the simultaneous detection and quantification of tgev and pedv, and the detection limits of this method were 90 copies and 70 copies for tegv and pedv, respectively (kim et al. 2007 ). zhu et al. designed two pairs of primers on the basis of the n gene sequences of tgev and pedv and established a nanoparticle-associated pcr assay; the sensitivity of the assay was 10-fold higher than that of conventional pcr (zhu et al. 2017) . apart from the four enteric covs, several enteric diarrheal viruses have been discovered, including porcine sapovirus (psav), porcine norovirus (pnov), porcine teschen virus (ptv), porcine kobuvirus (pkv), seneca valley virus (svv), porcine rotavirus (prv), porcine reovirus (reov), porcine bocavirus (pbov), and porcine astrovirus (pastv). therefore, some mpcr methods have been created to detect swine enteric diarrheal viruses, but they are not limited to enteric covs (ding et al. 2019) . ben salem et al. developed a nested rt-pcr method for the detection of pedv, tgev, and prv based on the sequence of the tgev purdue strain (accession no. nc_002306), the pedv strain cv777 (accession no. nc_003436), and the nsp5 gene of the prv-a osu strain (accession no. x15519), respectively. the detection limits of multiplex nested rt-pcr for tgev and pedv were 10 2 tcid 50 /ml and 27.2 μg/μl of rna, respectively (ben salem et al. 2010) . in 2013, zhao et al. developed a multiplex rt-pcr assay to identify pedv, tgev, prv-a, and porcine circovirus 2 (pcv2) in one reaction, and the sensitivity of the assay for the detection of tgev and pedv was 10-fold lower than that of a single rt-pcr method ). zhao et al. established a multiplex rt-pcr assay for rapid and differential diagnosis of pedv, tgev, prv-a, and pcv2 targeting the s gene, segment 6 region, and orf2 sequence, and the detection limits of the assay for tgev and pedv were 1.74 × 10 4 and 2.1 × 10 3 copies, respectively ). liu et al. developed a multiplex pcr assay to detect five diarrhea-related pig viruses: pedv (nucleoprotein), tgev (spike glycoprotein), prv-a, porcine group c rotaviruses (prv-c), and pcv2. the detection limits of the assay for pedv and tgev were 5 copies per reaction (liu et al. 2019a ). wen et al. developed a multiplex real-time pcr method based on evagreen fluorescent dye to simultaneously detect and distinguish pedv-nucleoprotein (n), tgev-spike glycoprotein (s), prv-a, prv-c, and pcv2, and the limits of detection ranged from 5 to 50 copies/μl (wen et al. 2019 ). there was no multiplex pcr method exclusively for differential detection of the four enteric covs until huang et al. developed a taqman-probe-based real-time rt-pcr method in 2019 targeting the m gene of pedv, the n gene of tgev, the m gene of pdcov, and the n gene of sads-cov. their multiplex real-time rt-qpcr assay could detect 10-100 copies of each target gene per pathogen (huang et al. 2019 ). as mentioned above, nested rt-pcr, rt-rpa, nanoparticleassisted pcr, rt-lamp, cpa-nast, sybr green-based real-time pcr, evagreen-based real-time pcr, and taqman probe-based real-time pcr represent single or multiplex pcr methods that have been developed to detect one, two, or four enteric pathogenic covs. in the field, the causative agents of swine enteric diarrhea are mixed; single rt-pcr methods are not suitable for rapid and efficient detection of covs even though they have higher sensitivity than multiplex rt-pcr. in addition, the pcr fragments of some single rt-pcr methods have to be subjected to agarose gel analysis to determine results, which is time-consuming. in particular, nested rt-pcr requires two pcr steps, resulting in an increased likelihood of contamination, so this method has not been widely used for pathogen detection. for rapid and efficient detection of pathogenic swine enteric covs, multiplex pcr methods, including conventional multiplex rt-pcr and multiplex real-time rt-pcr, are ideal options. although conventional multiplex rt-pcr can simultaneously differentially detect several different pathogens in one reaction, the method also possesses the disadvantages of single rt-pcr, e.g., risk of product contamination, and the inability to monitor developments in real time. the sensitivity of rt-pcr is 10-100 times lower than that of real-time rt-pcr, and the viral loads cannot be measured (keyaerts et al. 2006) . recently, real-time taqman probe-based rt-pcr methods have become increasingly used to detect targets because they own many advantageous characteristics: the ability to perform differential detection, high specificity, high sensitivity, high-throughput ability, high repeatability, quantification ability, and the ability to assess results in real time (slavov et al. 2016; teng et al. 2015; zhu et al. 2016a ). therefore, huang et al. in our lab developed a taqman-probe-based real-time rt-pcr for the differential detection of pedv, tgev, pdcov, and peav (huang et al. 2019) . although real-time taqman probe-based rt-pcr possesses many merits and can be used to rapidly and efficiently detect pathogenic swine enteric covs, the method requires a high-precision and sophisticated instrument, practical technicians, and a good laboratory; therefore, it cannot be used for detection in under-equipped laboratories or on-site. rapid, accurate, and more practical detection methods are of great significance for the surveillance, prevention, and control of enteric diseases in pigs, so novel assays are still deserved further development. for instance, test strip detection methods, which can be used by under-equipped laboratories or on-site and can be easily operated to quickly generate results, are urgently needed. as mentioned earlier, apart from the four enteric covs, many other pathogens causing diarrhea in pigs have been identified. and the causative agents of swine enteric diarrhea are mixed in the field. to rapidly determine whether the pathogens are enteric covs, pan-cov pcr is the best option to initially detect from clinical samples and followed by specific primers targeting individual swine enteric coronavirus for further identification when the result from pan-cov pcr is positive. however, when the pan-cov pcr results are negative, specific primers targeting other enteric diarrheal viruses are 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conventional rt-pcr, sybr green i and taqman real-time rt-pcr assays for the detection of porcine epidemic diarrhea virus development of taqman real-time reverse transcription-polymerase chain reaction for the detection and quantitation of porcine kobuvirus a sensitive duplex nanoparticle-assisted pcr assay for identifying porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus from clinical specimens establishment of a nanoparticle-assisted rt-pcr assay to distinguish field strains and attenuated strains of porcine epidemic diarrhea virus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-317496-6o2upns3 authors: pascual-iglesias, alejandro; sanchez, carlos m.; penzes, zoltan; sola, isabel; enjuanes, luis; zuñiga, sonia title: recombinant chimeric transmissible gastroenteritis virus (tgev)—porcine epidemic diarrhea virus (pedv) virus provides protection against virulent pedv date: 2019-07-25 journal: viruses doi: 10.3390/v11080682 sha: doc_id: 317496 cord_uid: 6o2upns3 porcine epidemic diarrhea virus (pedv) is an enteric coronavirus causing high morbidity and mortality in porcine herds worldwide. although both inactivated and live attenuated vaccines have been extensively used, the emergence of highly virulent strains and the recurrent outbreaks even in vaccinated farms highlight the need of effective vaccines. engineering of genetically defined live attenuated vaccines is a rational approach for novel vaccine development. in this line, we engineered an attenuated virus based on the transmissible gastroenteritis virus (tgev) genome, expressing a chimeric spike protein from a virulent united states (us) pedv strain. this virus (rtgev-rs-spedv) was attenuated in highly-sensitive five-day-old piglets, as infected animals did not lose weight and none of them died. in addition, the virus caused very minor tissue damage compared with a virulent virus. the rtgev-rs-spedv vaccine candidate was also attenuated in three-week-old animals that were used to evaluate the protection conferred by this virus, compared with the protection induced by infection with a virulent pedv us strain (pedv-nvsl). the rtgev-rs-spedv virus protected against challenge with a virulent pedv strain, reducing challenge virus titers in jejunum and leading to undetectable challenge virus rna levels in feces. the rtgev-rs-spedv virus induced a humoral immune response specific for pedv, including neutralizing antibodies. altogether, the data indicated that rtgev-rs-spedv is a promising vaccine candidate against virulent pedv infection. acute infectious diarrhea is a major cause of high morbidity and mortality in piglets worldwide. enteric infections in animals are frequently associated with viruses, including rotaviruses and coronaviruses (covs) [1] . a metagenomics analysis of diarrheic and healthy samples from china in 2012 found porcine covs in 77% of the diarrheic samples, and only in around 7% of the healthy samples, highlighting the potential relevance of covs as enteric porcine pathogens [2] . porcine epidemic diarrhea virus (pedv) was found in more than 50% of the diarrheic samples [2] , in agreement with the importance that this virus has for the porcine industry worldwide. pedv was first described in europe in the 1970s and the virus has, since then, remained endemic in the european porcine herds [3] . in asian countries, pedv was detected in the 1980s, with highly virulent outbreaks observed since viruses 2019, 11, 682 3 of 18 in piglets. subsequently, an attenuated chimeric vaccine candidate was developed by introducing duplication of transcription regulating sequences (trss), as previously described by our group [46] . this attenuated vaccine candidate conferred partial protection to pigs from a challenge with a virulent pedv. baby hamster kidney cells stably transformed with the gene coding for porcine aminopeptidase n (bhk-papn) [47] were grown in in dulbecco's modified eagle's medium (dmem) supplemented with 5% fetal calf serum (fcs) and g418 (1.5 mg/ml) as selection agent. vero cells were grown in dmem supplemented with heat-inactivated 10% fcs. a us virulent pedv strain (pedv-nvsl, genbank accession number kf267450) was kindly provided by ceva animal health. a recombinant virulent us pedv virus (rpedv), recovered from an infectious cdna engineered by our group (a. pascual-iglesias, l. enjuanes and s. zuñiga, unpublished data), was used as challenge virus in the animal experiments. pedv infectious cdna was maintained as a bac, including pedv usa/iowa/18984/2013 sequence (genbank accession number kf804028). pedv viruses and recombinant tgev viruses obtained in this work were grown in vero cells supplemented with infection medium (dmem supplemented with 0.3% tryptose phosphate broth, 0.02% yeast extract, and 10 µg/ml trypsin). the culture medium was supplied daily with 80% of the initial trypsin amount. a dna fragment containing the s gene sequence from virulent pedv usa/iowa/18984/2013 (genbank accession number kf804028) [48] was chemically synthesized and purchased from geneart (regensburg, germany). a pgem-t plasmid containing nucleotides 20,287 to 24,811 of tgev-sc11 genome (genbank accession aj271965) and engineered paci and mlui restriction sites [46] were used as an intermediate plasmid. overlapping polymerase chain reaction (pcr) was used to fuse pedv and tgev s gene sequences. the pedv s sequence encoding the ectodomain was amplified using purchased s sequence as template and oligonucleotides pac-s-vs (5 -ggattaattaagaagggtaagttgctcattagaaataatggtaagttactaaactttggtaa ccacttcgttaacacaccatgaagtctttaacctac-3 , paci restriction site in italics) and pedv-s-3952-rs (5 -ccacacataccaaggccacttgatgtatgtctc-3 ). tgev sequence encoding s protein transmembrane domain and endodomain was amplified using pgem-t-tgev-sc11 intermediate plasmid as a template and oligonucleotides tgev-s-4165-vs (5 -gagacatacatcaag tggccttggtatgtgtgg-3 ) and tgev-s-4439-rs (5 -tccaacgcgtaagtttag-3 , mlui restriction site in italics). in all cases, pedv sequences are indicated underlined. the obtained 4073 bp and 308 bp pcr products were used as templates and amplified with oligonucleotides pac-s-vs and tgev-s-4439-rs. the 4348 bp pcr product was digested with paci and mlui and cloned into the same sites of pgem-t-tgev-sc11 plasmid, generating pgem-tgev-spedv plasmid. this plasmid was digested with paci and mlui restriction enzymes and the fragment containing chimeric s gene was cloned into the same sites of pbac-tgev-sc11-p-m or pbac-tgev-sc11-rs [46] . all cloning steps were checked by sequencing of the pcr fragments and cloning junctions. for each mutant sequence, two independent cdnas were constructed. bhk-papn cells grown to 90% confluence in 35 mm plates were transfected using 4 µg of the corresponding pbac and 12 µl of lipofectamine 2000 (invitrogen, carlsbad, ca, usa), according to manufacturer's specifications. at 6 h post-transfection (hpt), bhk-papn transfected cells were trypsinized, washed twice with dmem, and plated over confluent vero monolayers grown in 35-mm-diameter plates. one hour later, trypsin was added to a final concentration of 10 µg/ml. after a 3-day incubation period, the cell supernatants were harvested (passage 0) [49] . the rtgevs were cloned by limiting dilution method, grown, and titrated as previously described [50] . twenty-one five-day-old suckling piglets, born from tgev and pedv seronegative sows, were randomly divided in two groups of nine piglets and one group of three animals. the nine-piglet groups were orally inoculated with 10 6 tcid 50 /animal of the different rtgevs in phosphate buffered saline (pbs). the three piglets in the other group were mock inoculated. infected animals were monitored daily to detect signs of enteric disease, and body weights were determined each day. three animals per group were euthanized and necropsied at days 2, 4, and 6 after inoculation. intestinal macroscopic lesions were evaluated. front, mid, and end sections of jejunum were collected in all cases. samples were kept frozen for subsequent virus titration, stabilized with rnalater stabilization reagent (life technologies, carlsbad, ca, usa) for rna extraction, or fixed in zinc formaline fixative (sigma-aldrich, st. louis, mo, usa) for histopathology. fixed jejunum sections were washed twice with pbs and stored in 70% ethanol at 4 • c. paraffin embedding, sectioning, and hematoxylin-eosin staining were performed by the histological laboratory (autopsy path kft., budapest, hungary). samples were examined with a zeiss axiophot fluorescence microscope. determination of the jejunum damage was obtained from unbiased measurement of three full-length perceived villi and crypt per location. at least two different locations per section per animal were measured. the villous height to crypt depth (vh/cd) ratio was calculated from the measurements. twenty-one 21-day-old piglets, born from tgev and pedv seronegative sows, were randomly divided in four groups ( table 1) . piglets of groups 1 and 2 were orally inoculated with 10 6 tcid 50 /animal of rtgev-rs-spedv or pedv-nvsl, respectively. piglets of groups 3 and 4 were mock inoculated. three weeks after the immunization, piglets of groups 1, 2, and 3 were challenged with 10 7 tcid 50 of rpedv per animal by oral route. infected animals were monitored daily to detect signs of enteric disease, and body weights were determined every 7 days. fecal swabs were collected at days 0, 7, 21, 25, 28, and 31 post-first inoculation. serum samples were taken at days 0, 21, 28, and 31 post-immunization. saliva samples were collected, using salivette tubes (sarstedt, nümbrecht, germany), at days 21, 28, and 31 post-first inoculation. three animals per group were euthanized and necropsied at days 4 and 10 post-challenge (25 and 31 post-immunization, respectively). intestinal macroscopic lesions were evaluated. front, mid, and end sections of jejunum were collected in all cases. samples were kept frozen for subsequent virus titration, stabilized with rnalater stabilization reagent (life technologies) for rna extraction, or fixed in zinc formaline fixative (sigma-aldrich) for histopathology. antibodies induced against tgev and pedv viruses were detected by elisa as described before [51] . briefly, elisas were performed using 0.75 µg per well of partially purified tgev, pedv-nvsl, or rpedv viruses. antigens were bound to 96-well microplates, saturated with 5% bovine serum albumin (bsa) in pbs for 2 h at 37 • c, and incubated with serial dilutions of the serum sample in wash buffer (0.1% bsa, 0.05% tween20 in pbs) for 90 min at 37 • c. microplates were washed three times with wash buffer. bound antibodies were detected by incubation with peroxidase-conjugated protein a, goat anti pig igg (fc domain), or goat anti pig iga (biorad, hercules, ca, usa), diluted 1:10,000 in pbs with 0.1% bsa. elisa was developed with k-blue tmb substrate (neogen, lexington, ky, usa) for 5 min at room temperature. reactions were stopped with 1.5 m h 2 so 4 , and the absorbance was read at 450 nm. the elisa values of the sera were expressed as sample to positive ratio [sp-ratio = (od of sample − od of negative control)/(od of positive control − od of negative control)]. heat-inactivated sera were incubated for 1 h at 37 • c in the presence of 100 pfus of pedv virus in dmem containing 5% fcs. serial dilutions of the mixtures were added to confluent vero cells in 96-well plates. after one hour of incubation at 37 • c, medium was removed and infection medium was added. after 72 h, cells were fixed with 10% formaldehyde in pbs, stained with a crystal violet solution, and virus titer was determined. the neutralization index was defined as the logarithm of the ratio of tcid 50 in the presence of medium or in the presence of serum sample. total intracellular rna was extracted from vero cells or tissue samples. total rna was purified with rneasy mini kit (qiagen, hilden, germany), according to the manufacturer's specifications. to isolate viral rna from fecal swabs, the swab was resuspended in 300 µl of pbs with antibiotics (40 u/ml penicillin-streptomycin, 200 µg/ml gentamicin, 1 µg/ml amphotericin b). rna was isolated from 140 µl of that solution using qiaamp viral mini kit (qiagen) following the manufacturers' instructions. in all cases, total cdna was synthesized using 100 ng of total rna as a template, random hexamers, and the high-capacity cdna transcription kit (life technologies), following the manufacturers' recommendations. the viral genome region from 3b gene to 3 utr (nt. 25856-28544) was sequenced in all cases. to that end, overlapping pcrs were performed using oligo pairs described in table 2 . the pcr fragments were then sequenced using specific oligonucleotides. pedv genomic rna (grna) levels were measured by rt-qpcr analysis using a custom taqman assay detecting a conserved orf1a region (taqman probe 6-fam-tgtact ggcttactggtgtt-mgb; forward primer 5 -tgttgctatgtttgtgcattgg-3 ; reverse primer 5 -tctgaatcactaggctgacctttg-3 ). tgev grna was evaluated using a custom taqman assay set up in our laboratory [52] . the porcine β-glucuronidase (gusb) gene (taqman code ss03387751_u1) was used as a reference housekeeping gene, since its expression remains constant in infected cells compared to that in non-infected cells [ [53] and data not shown]. data were acquired with a 7500 viruses 2019, 11, 682 6 of 18 real-time pcr system (applied biosystems, foster city, ca, usa) and analyzed with 7500 software v2.0.6. the relative quantifications were performed using the 2 −∆∆ct method [54] . all experiments and data analysis were miqe (minimum information for publication of quantitative real-time pcr experiments guidelines) compliant [55] . reverse size (bp) two-tailed, unpaired student t tests were used to analyze the difference in mean values between groups. all results were expressed as means ± the standard deviations of the means. p values <0.05 were considered significant. a chimeric s protein (spedv-tgev) was designed. the n terminus of the protein contained the exposed domain of pedv s protein from a virulent us strain ( figure 1a ), including the epitopes recognized by neutralizing antibodies, and the heptad-repeats domains (aa 1 to 1325). the c-terminus of the protein was that from the enteropathogenic tgev sc11 virus [36] , including the transmembrane domain and c-terminus (aa 1389 to 1448). the chimeric protein was cloned in the tgev infectious cdna [30] , replacing tgev s protein (rtgev-spedv, figure 1b ). in addition, chimeric spedv-tgev protein was cloned in an infectious cdna from an attenuated tgev, which contained duplications of transcription regulating sequences (trss) and engineered unique restrictions sites (rs) to avoid the overlapping of consecutive genes [46] (rtgev-rs-spedv, figure 1c ). recombinant viruses rtgev-spedv and rtgev-rs-spedv were recovered after transfection of vero cells with the wild-type and attenuated cdnas, respectively. these viruses required trypsin to efficiently infect vero cells, and form syncytia, similarly to pedv virus. growth kinetics in cell culture was analyzed by infecting vero cells at two multiplicities of infection (moi)moi of 0.1 and 0.001. both viruses reached peak titers at 28 or 48 hpi, depending on the moi ( figure 2a ). nevertheless, rtgev-rs-spedv virus peak titers were five-to 10-fold lower than those for parental rtgev-spedv virus ( figure 2a ). vero cells with the wild-type and attenuated cdnas, respectively. these viruses required trypsin to efficiently infect vero cells, and form syncytia, similarly to pedv virus. growth kinetics in cell culture was analyzed by infecting vero cells at two multiplicities of infection (moi)moi of 0.1 and 0.001. both viruses reached peak titers at 28 or 48 hpi, depending on the moi (figure 2a ). nevertheless, rtgev-rs-spedv virus peak titers were five-to 10-fold lower than those for parental rtgev-spedv virus ( figure 2a ). to analyze the virulence of control rtgev-spedv and attenuated rtgev-rs-spedv viruses, fiveday-old piglets were orally inoculated with 10 6 tcid50/animal. interestingly, despite the difference in viral titers observed in cell cultures, viral titers in the jejunum of infected piglets were similar for both viruses at 2 and 4 dpi ( figure 2b ). at 6 dpi, rtgev-spedv titers were slightly lower than those for rtgev-rs-spedv virus, suggesting a decreased number of gut epithelial cells available for reinfection. piglets inoculated with parental rtgev-spedv virus lost weight after infection (figure to analyze the virulence of control rtgev-spedv and attenuated rtgev-rs-spedv viruses, five-day-old piglets were orally inoculated with 10 6 tcid 50 /animal. interestingly, despite the difference in viral titers observed in cell cultures, viral titers in the jejunum of infected piglets were similar for both viruses at 2 and 4 dpi ( figure 2b ). at 6 dpi, rtgev-spedv titers were slightly lower than those for rtgev-rs-spedv virus, suggesting a decreased number of gut epithelial cells available for reinfection. piglets inoculated with parental rtgev-spedv virus lost weight after infection ( figure 3a ) and 70% died ( figure 3b ). this data indicates that the chimeric rtgev-spedv control virus was virulent. in contrast, animals infected with rtgev-rs-spedv virus maintained weight ( figure 3a ) and all of them survived ( figure 3b ), indicating that the virus was attenuated. interestingly, virus shedding in the feces of piglets infected with attenuated rtgev-rs-spedv virus was up to 50-fold lower than that observed in the animals infected with the virulent parental rtgev-spedv virus ( figure 3c ). 3a) and 70% died ( figure 3b ). this data indicates that the chimeric rtgev-spedv control virus was virulent. in contrast, animals infected with rtgev-rs-spedv virus maintained weight ( figure 3a ) and all of them survived ( figure 3b ), indicating that the virus was attenuated. interestingly, virus shedding in the feces of piglets infected with attenuated rtgev-rs-spedv virus was up to 50-fold lower than that observed in the animals infected with the virulent parental rtgev-spedv virus ( figure 3c ). in agreement with the observed clinical signs, epithelial degeneration and exfoliation in the different parts of the jejunum was observed in animals infected with the virulent rtgev-spedv virus, accompanied by inflammatory infiltration and shortening of the villi ( figure 4a ). interestingly, the jejunum damage was significantly lower in animals infected with the attenuated rtgev-rs-spedv virus ( figure 4b ). altogether, the data indicate that rtgev-rs-spedv virus was partially attenuated. in agreement with the observed clinical signs, epithelial degeneration and exfoliation in the different parts of the jejunum was observed in animals infected with the virulent rtgev-spedv virus, accompanied by inflammatory infiltration and shortening of the villi ( figure 4a ). interestingly, the jejunum damage was significantly lower in animals infected with the attenuated rtgev-rs-spedv virus ( figure 4b ). altogether, the data indicate that rtgev-rs-spedv virus was partially attenuated. the main concern for the use of modified live vaccines based on attenuated viruses is their biosafety. reversion to virulence or recombination with circulating strains is in the basis for the emergence of novel virulent pedv strains, especially in asia [29, 56] . the rtgev-rs-spedv virus genetic stability, which may influence its safety as vaccine candidate, was evaluated in cell culture. after 15 passages in cell culture, viral genome region comprising from 3b gene to 3 utr (nucleotides 25856 to 28544) was sequenced. no changes appeared in the passed virus compared with the parental one, strongly suggesting that the engineered virus was genetically stable in cell culture. to address virus stability in vivo, the 3 -ends (nt 25856 to 28544) of rtgev-spedv and rtgev-rs-spedv viruses were sequenced, using rna isolated from the jejunum of five-day-old infected piglets at six days post-infection. no modifications were found in the parental rtgev-spedv virus. in the rtgev-rs-spedv virus, modifications were only detected in the engineered trs duplicated sequences ( figure 5 ). small deletions and point mutations were found between e and m genes, eliminating the engineered fsei restriction site, but maintaining duplicated sequence ( figure 5 ). extensive deletion was observed between m and n genes, reverting the sequence to that of the wild-type virus ( figure 5 ). no changes were observed in the engineered mutations between n and 7 genes ( figure 5 ). interestingly, viruses 2019, 11, 682 9 of 18 when viral rna was isolated from feces of 21-day-old piglets at seven days post-vaccination (see below) and rtgev-rs-spedv virus was sequenced, the same modifications were observed. this data strongly suggests that the rtgev-rs-spedv genome recovered from piglets may represent the in vivo evolution of the engineered virus. it is worth noting that tgev attenuation was similar with the three trss duplications and with just the duplication between n and 7 genes [46] , suggesting that in vivo evolved rtgev-rs-spedv virus may still be attenuated. the main concern for the use of modified live vaccines based on attenuated viruses is their biosafety. reversion to virulence or recombination with circulating strains is in the basis for the emergence of novel virulent pedv strains, especially in asia [29, 56] . the rtgev-rs-spedv virus genetic stability, which may influence its safety as vaccine candidate, was evaluated in cell culture. after 15 passages in cell culture, viral genome region comprising from 3b gene to 3′utr (nucleotides 25856 to 28544) was sequenced. no changes appeared in the passed virus compared with the parental one, strongly suggesting that the engineered virus was genetically stable in cell culture. to address virus stability in vivo, the 3′-ends (nt 25856 to 28544) of rtgev-spedv and rtgev-rs-spedv viruses were sequenced, using rna isolated from the jejunum of five-day-old infected piglets at six days no changes were observed in the engineered mutations between n and 7 genes ( figure 5) . interestingly, when viral rna was isolated from feces of 21-day-old piglets at seven days postvaccination (see below) and rtgev-rs-spedv virus was sequenced, the same modifications were observed. this data strongly suggests that the rtgev-rs-spedv genome recovered from piglets may represent the in vivo evolution of the engineered virus. it is worth noting that tgev attenuation was similar with the three trss duplications and with just the duplication between n and 7 genes [46] , suggesting that in vivo evolved rtgev-rs-spedv virus may still be attenuated. pedv infects animals of all ages, although the severity of the clinical signs is age-dependent [57] . pregnant sows are the most suitable models to evaluate the effect of pedv vaccine candidates, as the target animals are neonatal piglets, and lactogenic immunity has an important role in protection [26] . nevertheless, availability, cost, and housing resources limited its use in preliminary vaccine candidate trials [16, 18] , and a young pig model has been proposed for preliminary vaccine efficacy trials [13, 16] . therefore, to evaluate the protection conferred by rtgev-rs-spedv virus, 21-day-old pigs were used. group 1 (g1) was vaccinated with rtgev-rs-spedv virus, group 2 (g2) was vaccinated with a virulent us pedv strain (pedv-nvsl), and two groups of animals (g3 and g4) were not vaccinated ( table 1) . twenty-one days after vaccination, animals from g1, g2, and g3 were challenged with a virulent recombinant pedv strain (rpedv) ( table 1) . as expected, due to the animal age, no clinical sings of enteric disease such as diarrhea, vomiting, or dehydration were observed after vaccination or challenge (data not shown). nevertheless, animals vaccinated with the virulent pedv-nvsl strain showed a delay in weight gain the first week after vaccination, compared with non-infected animals or those vaccinated with rtgev-rs-spedv virus ( figure 6 ). similar observations were reported for other virulent us isolates [13] . these data indicate that rtgev-rs-spedv virus was also attenuated in 21-day-old piglets. virus titers were evaluated in the jejunum of challenged animals. the challenge virus replicated efficiently in the gut of non-vaccinated animals ( figure 7a ). in contrast, the titers were reduced more than 25-fold or 50-fold in animals vaccinated with rtgev-rs-spedv and pedv-nvsl viruses, respectively ( figure 7a ). similar results were obtained when viral rna presence was evaluated, with up to 200-fold reduction in the accumulation of pedv rna ( figure 7b ). interestingly, after challenge, pedv virus was only shed by non-vaccinated animals ( figure 7c ). altogether, these data indicate that the challenge virus did not efficiently infect the vaccinated animals. furthermore, these results indicate that rtgev-rs-spedv protected the animals against virulent pedv challenge. pigs were used. group 1 (g1) was vaccinated with rtgev-rs-spedv virus, group 2 (g2) was vaccinated with a virulent us pedv strain (pedv-nvsl), and two groups of animals (g3 and g4) were not vaccinated (table 1) . twenty-one days after vaccination, animals from g1, g2, and g3 were challenged with a virulent recombinant pedv strain (rpedv) ( table 1) . as expected, due to the animal age, no clinical sings of enteric disease such as diarrhea, vomiting, or dehydration were observed after vaccination or challenge (data not shown). nevertheless, animals vaccinated with the virulent pedv-nvsl strain showed a delay in weight gain the first week after vaccination, compared with non-infected animals or those vaccinated with rtgev-rs-spedv virus ( figure 6 ). similar observations were reported for other virulent us isolates [13] . these data indicate that rtgev-rs-spedv virus was also attenuated in 21-day-old piglets. figure 6 . weight gain in vaccinated animals. three-week-old pigs were divided into four groups. animals from two groups were vaccinated with 10 6 tcid50/animal of rtgev-rs-spedv virus (red) or a virulent us pedv strain (pedv-nvsl, green). three weeks after vaccination (21 dpv indicated in red), non-vaccinated animals were either mock infected (mock, grey) or challenged with 10 7 tcid50/animal of a virulent pedv strain (non vaccinated, black). vaccinated animals were also challenged with the same dose as non-vaccinated animals. clinical signs and weight were observed daily after vaccination and challenge, and once per week during the course of the experiment. average daily gain represents the mean from six different animals. error bars represent the standard deviation for each value; p value, ** <0.01. virus titers were evaluated in the jejunum of challenged animals. the challenge virus replicated efficiently in the gut of non-vaccinated animals ( figure 7a ). in contrast, the titers were reduced more than 25-fold or 50-fold in animals vaccinated with rtgev-rs-spedv and pedv-nvsl viruses, respectively ( figure 7a ). similar results were obtained when viral rna presence was evaluated, with up to 200-fold reduction in the accumulation of pedv rna ( figure 7b ). interestingly, after challenge, pedv virus was only shed by non-vaccinated animals ( figure 7c ). altogether, these data indicate that the challenge virus did not efficiently infect the vaccinated animals. furthermore, these results indicate that rtgev-rs-spedv protected the animals against virulent pedv challenge. figure 6 . weight gain in vaccinated animals. three-week-old pigs were divided into four groups. animals from two groups were vaccinated with 10 6 tcid 50 /animal of rtgev-rs-spedv virus (red) or a virulent us pedv strain (pedv-nvsl, green). three weeks after vaccination (21 dpv indicated in red), non-vaccinated animals were either mock infected (mock, grey) or challenged with 10 7 tcid 50 /animal of a virulent pedv strain (non vaccinated, black). vaccinated animals were also challenged with the same dose as non-vaccinated animals. clinical signs and weight were observed daily after vaccination and challenge, and once per week during the course of the experiment. average daily gain represents the mean from six different animals. error bars represent the standard deviation for each value; p value, ** <0.01. the antibody response specific for pedv was evaluated in the sera from vaccinated and nonvaccinated animals. before challenge (21 dpv), vaccination elicited pedv-specific antibodies to a higher level in pedv-nvsl vaccinated animals than in rtgev-rs-spedv vaccinated ones ( figure 8a ). this result was expected, as the elisa test was developed against the whole pedv virus, and the chimeric rtgev-rs-spedv virus only expressed pedv s protein. after challenge at 28 and 31 dpv, pedv total antibodies increased in all challenged groups, as expected. interestingly, the increase was slower in the vaccinated pigs, in agreement with a decreased challenge virus replication in these animals. total anti-tgev antibodies were also evaluated by elisa. all animals were seronegative at the time of vaccination. when challenge was performed, the level of tgev antibodies was not significant in any group, and the all the animals remained seronegative after pedv challenge. this result was expected, as s protein is the main inducer of antibodies against tgev and it was not present in any of the viruses used for vaccination or challenge. in addition, it has been shown that the antibody response specific for pedv was evaluated in the sera from vaccinated and non-vaccinated animals. before challenge (21 dpv), vaccination elicited pedv-specific antibodies to a higher level in pedv-nvsl vaccinated animals than in rtgev-rs-spedv vaccinated ones ( figure 8a ). this result was expected, as the elisa test was developed against the whole pedv virus, and the chimeric rtgev-rs-spedv virus only expressed pedv s protein. after challenge at 28 and 31 dpv, pedv total antibodies increased in all challenged groups, as expected. interestingly, the increase was slower in the vaccinated pigs, in agreement with a decreased challenge virus replication in these animals. total anti-tgev antibodies were also evaluated by elisa. all animals were seronegative at the time of vaccination. when challenge was performed, the level of tgev antibodies was not significant in any group, and the all the animals remained seronegative after pedv challenge. this result was expected, as s protein is the main inducer of antibodies against tgev and it was not present in any of the viruses used for vaccination or challenge. in addition, it has been shown that there is no cross-reactivity between tgev and pedv [58] . to further analyze the humoral response specific for pedv, the levels of pedv-specific iggs ( figure 8b ) and igas ( figure 8c ) were evaluated. the data were similar to those obtained for total anti-pedv antibodies. it has been demonstrated that protection against pedv infection correlates with iga levels [21, 44, 59, 60] . it is worth noting that iga levels in the sera of vaccinated animals, after challenge, were significantly reduced compared with non-vaccinated animals ( figure 8c ), strongly suggesting that challenge virus did not elicit a potent iga response in the gut of vaccinated animals, most likely due to reduced replication in the enteric tract, correlating with protection. the neutralization capability of the antibodies elicited was evaluated. as expected, non-vaccinated animals only induced the production of neutralizing antibodies after challenge (figure 9 ). in contrast, pigs vaccinated with both pedv-nvsl and rtgev-rs-spedv virus induced a significant level of neutralizing antibodies before challenge, which were slightly increased after challenge (figure 9 ). the neutralization capability of the antibodies elicited was evaluated. as expected, nonvaccinated animals only induced the production of neutralizing antibodies after challenge (figure 9 ). in contrast, pigs vaccinated with both pedv-nvsl and rtgev-rs-spedv virus induced a significant level of neutralizing antibodies before challenge, which were slightly increased after challenge (figure 9 ). . neutralizing antibodies produced in vaccinated animals. virus neutralization assay was performed using serum samples, collected before (21 dpv) or after (31 dpv) challenge, from rtgev-rs-spedv virus (red) or pedv-nvsl (green) vaccinated animals, and from non-vaccinated and challenged animals (black). the values represent neutralization index from three different animals. error bars represent the standard deviation for each value. altogether, the data indicate that rtgev-rs-spedv virus conferred protection against pedv infection in the young pig model and could be the basis for an effective vaccine candidate. an attenuated chimeric rtgev virus expressing the ectodomain of a virulent us pedv s protein (rtgev-rs-spedv) was engineered as vaccine candidate for pedv and evaluated in a young piglet model system. the rtgev-rs-spedv virus elicited a neutralizing humoral response specific for pedv. in fact, vaccinated animals were protected against challenge with a virulent pedv virus. the attenuated live vaccine candidates are the best option for pedv vaccination because nonreplicative antigens, as in inactivated vaccines, do not induce mucosal immunity and protection [16, 26] . traditionally, attenuated live vaccines were developed for classical pedv strains, consisting of serial passages of the virulent virus in cell culture, as tissue culture adaptation led to attenuation in vivo. these vaccines were not effective to control the infection, as they were extensively used in countries were novel pedv strains emerged [14, 15] . as a consequence, many groups have developed traditionally attenuated live vaccines based on novel epidemic pedv strains. in general, it has been shown that more than 100 passages of the virulent virus in cell culture are required to attenuate pedv in vivo [61] [62] [63] [64] [65] [66] . it is worth noting that in our rtgev-rs-spedv vaccine candidate, the attenuating mutations were specifically designed and introduced in several locations of the viral genome. in contrast, traditionally attenuated vaccines contain non-controlled mutations throughout the genome figure 9 . neutralizing antibodies produced in vaccinated animals. virus neutralization assay was performed using serum samples, collected before (21 dpv) or after (31 dpv) challenge, from rtgev-rs-spedv virus (red) or pedv-nvsl (green) vaccinated animals, and from non-vaccinated and challenged animals (black). the values represent neutralization index from three different animals. error bars represent the standard deviation for each value. altogether, the data indicate that rtgev-rs-spedv virus conferred protection against pedv infection in the young pig model and could be the basis for an effective vaccine candidate. an attenuated chimeric rtgev virus expressing the ectodomain of a virulent us pedv s protein (rtgev-rs-spedv) was engineered as vaccine candidate for pedv and evaluated in a young piglet model system. the rtgev-rs-spedv virus elicited a neutralizing humoral response specific for pedv. in fact, vaccinated animals were protected against challenge with a virulent pedv virus. the attenuated live vaccine candidates are the best option for pedv vaccination because non-replicative antigens, as in inactivated vaccines, do not induce mucosal immunity and protection [16, 26] . traditionally, attenuated live vaccines were developed for classical pedv strains, consisting of serial passages of the virulent virus in cell culture, as tissue culture adaptation led to attenuation in vivo. these vaccines were not effective to control the infection, as they were extensively used in countries were novel pedv strains emerged [14, 15] . as a consequence, many groups have developed traditionally attenuated live vaccines based on novel epidemic pedv strains. in general, it has been shown that more than 100 passages of the virulent virus in cell culture are required to attenuate pedv in vivo [61] [62] [63] [64] [65] [66] . it is worth noting that in our rtgev-rs-spedv vaccine candidate, the attenuating mutations were specifically designed and introduced in several locations of the viral genome. in contrast, traditionally attenuated vaccines contain non-controlled mutations throughout the genome introduced during passages in cell cultures that, after passages in vivo, may lead to vaccine virus reversion to virulence. the engineered rtgev-rs-spedv vaccine candidate has the advantage of replicating in the jejunum of infected piglets as efficiently as a virulent control virus, without causing significant pathology, and most likely leading to a protective iga response. indeed, vaccinated animals were protected against virulent pedv challenge. in contrast, traditionally attenuated vaccines, as a consequence of adaptation to cell culture, have a dramatically reduced replication in the enteric tract [66] and, as a consequence, these vaccines do not actively replicate to induce good iga and lactogenic immune responses [1, 13, 26] . recently, a swine enteric coronavirus (secov) that is a recombinant between tgev and pedv has been described in europe [67] [68] [69] . the secov contains a genome background identical to tgev, but expresses the s protein from pedv. in addition, recombination led to extensive deletions in the coding sequence and in trs of tgev gene 3a, and, as a consequence, protein 3a may not be expressed by secov. it is worth noting that secov was isolated from diarrheic samples in all cases, suggesting that it is virulent. the engineered parental rtgev-spedv virus resembles the recombinant secov and, as shown by our data, is virulent in suckling piglets. in contrast, the engineered virus (rtgev-rs-spedv) used as a vaccine candidate was attenuated. there are some genetic differences that may eventually allow the discrimination between secov and the rtgev-rs-spedv vaccine candidate as (i) rtgev-rs-spedv virus did not contain the full pedv s gene sequence, (ii) it expressed tgev protein 3a, and (iii) because of the tgev strain used to engineer the infectious cdna, it did not express tgev protein 3b [70] . the rtgev-rs-spedv vaccine candidate was genetically stable in tissue culture; nevertheless, it evolved in vivo. therefore, its biosafety could be improved by adding an additional safety guard at the 5 -end of pedv virus genome (in the replicase gene), at a distal position from the attenuating mutations introduced by duplicating sequences closed to m, n and 7a genes ( figure 1 ) [71] [72] 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as vaccine candidate attenuation of an original us porcine epidemic diarrhea virus strain pc22a via serial cell culture passage porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus new chimeric porcine coronavirus in swine feces characterization of a novel chimeric swine enteric coronavirus from diseased pigs in central eastern europe in 2016 sequence motifs involved in the regulation of discontinuous coronavirus subgenomic rna synthesis identification of the mechanisms causing reversion to virulence in an attenuated sars-cov for the design of a genetically stable vaccine a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2'-o-methyltransferase activity mutagenesis of coronavirus nsp14 reveals its potential role in modulation of the innate immune response we thank ceva animal health for providing vero cells and pedv-nvsl virus. we are grateful to dora katona and máté halas (prophyl ltd., hungary) for in vivo experiments. we thank marga gonzalez (cnb-csic) for her technical assistance.conflicts of interest: z.p. is the global director of bio r&d at ceva animal health. the authors declare no other conflict of interest. key: cord-255773-b4re5bky authors: zhang, qingzhan; shi, kaichuang; yoo, dongwan title: suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp1 date: 2016-01-14 journal: virology doi: 10.1016/j.virol.2015.12.010 sha: doc_id: 255773 cord_uid: b4re5bky type i interferons (ifn-α/β) are the major components of the innate immune response of hosts, and in turn many viruses have evolved to modulate the host response during infection. we found that the ifn-β production was significantly suppressed during pedv infection in cells. to identify viral ifn antagonists and to study their suppressive function, viral coding sequences for the entire structural and nonstructural proteins were cloned and expressed. of 16 pedv nonstructural proteins (nsps), nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were found to inhibit the ifn-β and irf3 promoter activities. the sole accessory protein orf3, structure protein envelope (e), membrane (m), and nucleocapsid (n) protein were also shown to inhibit such activities. pedv nsp1 did not interfere the irf3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of irf3 and creb-binding protein (cbp) by degrading cbp. a further study showed that the cbp degradation by nsp1 was proteasome-dependent. our data demonstrate that pedv modulates the host innate immune responses by degrading cbp and suppressing isgs expression. porcine epidemic diarrhea (ped) is a highly contagious acute enteric disease characterized by vomiting, watery diarrhea, and severe dehydration of up to 80-100% mortality in suckling piglets (song and park, 2012; sun et al., 2012a; debouck and pensaert, 1980; junwei et al., 2006) . ped was first reported in england in feeder and fattening pigs during 1970s (wood, 1977) , and reemerged in asia since 2010 with greater virulence and economic losses li et al., 2012; puranaveja et al., 2009; yang et al., 2013) . in the us, pedv appeared for the first time in 2013 and severely affected most pig-producing states marthaler et al., 2013; mole, 2013; stevenson et al., 2013) . the causative agent is porcine epidemic diarrhea virus (pedv), which belongs to the alphacoronavirus genus in the family coronaviridae (http://ictvonline.org/virustaxonomy.asp). pedv is an enveloped virus with a single-stranded positive-sense rna genome of approximately 28 kb in length with the 5 0 -cap and the 3 0polyadenylated tail. the pedv genome is arranged with orf1a, orf1b, s, orf3, e, m, n, in order with both termini flanking with the 5 0 -and 3 0 -untranslated regions (utrs) (duarte et al., 1993) . orf1a codes for the large polyprotein pp1a, while orf1b is always expressed as a fusion protein pp1a/b with pp1a through a ribosomal frameshifting. pp1a and pp1a/b are further processed to 16 nonstructural proteins, nsp1 through nsp16. orf3 codes for an accessory protein which is likely an additional nonstructural protein, whereas s, e, m and n genes code for four structural proteins (song and park, 2012) . during viral infection, the sensing of foreign nucleic acids in the cytosol leads to the activation of an innate immune response to produce type i interferons (ifn-α/β) and establishes an antiviral state. the type i ifns and ifn-mediated response provide a first line of defense against viral infection. the host innate immune system deploys the pattern-recognition receptors (prrs) to sense and respond to the pathogen-associated molecular patterns (pamps) of virus (kawai and akira, 2011) . this recognition triggers the activation of retinoic acid-inducible gene i (rig-i) or melanoma differentiation gene 5 (mda5), which further binds to the mitochondrial adapter protein mavs/ips-1 and recruits tnf receptor-associated factor 3/6 (traf3 and traf6). traf3 activates iκb kinase (ikk)-related kinases such as tank-binding kinase 1 (tbk1) and ikkε for phosphorylation of interferon regulatory factors 3 and 7 (irf3/irf7) and type i ifn production (fitzgerald et al., 2003; sharma et al., 2003) . traf6 leads to tank1 activation, followed by nf-kb activation and cytokine production (rajsbaum and garcia-sastre, 2013) . upon tbk1 activation, phosphorylated irf3 undergoes homodimerization and unveils the nuclear localization signal leading to the nuclear translocation, where it forms a complex with the transcription co-activator creb (camp responsive element binding)-binding protein (cbp)/p300 (dragan et al., 2007; lin et al., 1998; panne et al., 2007) . the irf3-cbp/p300 complex further binds to the positive regulatory domain (prd) i-iv regions of the ifn-β promoter to assemble the enhanceosome together with nf-κb and other factors to turn on the transcription of type i ifn genes (honda and taniguchi, 2006) . the irf3-cbp/ p300 interaction is crucial for ifn transcription. following production and secretion, ifn molecules bind to the cell surface receptors and trigger the activation of janus kinase-signal transducers and activators of transcription (jak-stat) signaling cascade. phosphorylated stat1 and stat2 associate to form a heterodimer, which in turn recruits the ifn-regulatory factor 9 (irf9) to form the ifn-stimulated gene factor 3 (isgf3). isgf3 translocates to the nucleus and induces genes regulated by ifnstimulated response elements (isre), resulting in expression of hundreds of antiviral genes and establishment of an antiviral state (stark and darnell, 2012) . in turn, many viruses have evolved to counteract the host innate immune defense and such viral functions are often redundant. for nidoviruses, eleven and six viral proteins have been described as ifn antagonists for severe acute respiratory syndrome coronavirus (sars-cov) and porcine reproductive and respiratory syndrome virus (prrsv), respectively kindler and thiel, 2014; shi et al., 2014; sun et al., 2012b; totura and baric, 2012) . for betacoronaviruses, nsp1 has been reported as a multifunctional viral antagonist for innate immune response (huang et al., 2011b; narayanan et al., 2008; wang et al., 2010) . for pedv, the viral modulation of innate immune signaling is poorly understood. pedv infects vero cells, but these cells are type i ifndeficient due to a chromosomal deletion (desmyter et al., 1968 ). in the present study, we identified marc-145 cells as a suitable line of cells for pedv infection and for study of innate immune modulation. we showed that pedv suppressed the type i interferon production and isgs expression in these cells, and identified nsp1, nsp3, nsp7, nsp14, nsp15, nsp16, e, m, n and orf3 as the viral ifn antagonists. we showed that pedv nsp1 caused the cbp degradation by the proteasome-dependent pathway. the cbp degradation is a novel mechanism of coronavirus nsp1 for ifn suppression and our study provides a new insight into the immune modulation and evasion strategy of pedv. pedv replicates in the cytoplasm of villous epithelial cells of the small and large intestines (debouck and pensaert, 1980; sueyoshi et al., 1995) . the viral antigen is also detectable in the macrophages that infiltrated the lamina propria (lee et al., 2000) . histological studies showed that pedv replicates in the porcine respiratory tract in vivo and transformed alveolar macrophages (3d4) in vitro (park and shin, 2014) . vero cells are widely used for pedv for diagnosis, virus isolation, and research, but these cells are type i ifn-deficient due to the chromosomal deletion (desmyter et al., 1968) . to study a possible regulation of innate immune signaling by pedv, various cell lines were examined for susceptibility. cells were infected with pedv at an moi of 0.1 in various trypsin concentrations and cpe was examined daily for up to 5 days. in vero and marc-145 cells, apparent cpe of multinucleation was observed by 24 h post-infection ( fig. 1a and b, left panel). trypsin activates the cleavage of s protein and induces membrane fusion to trigger infection (park et al., 2011; wicht et al., 2014) . pedv infection was characterized by syncytia formation (hofmann and wyler, 1988) and infection foci were visualized by anti-pedv m and n antibodies, indicating the susceptibility of both cell types for pedv infection ( fig. 1a and b, middle and right panel). the viral proteins were detected using specific antibodies by western blot and the specific bands were corresponding to the m and n proteins, further confirming the productive infection of these cells by pedv (fig. 1c ). the optimal trypsin concentration for pedv propagation was 5 μg/ml and 2 μg/ml for vero and marc-145 cells, respectively. marc-145 cells have been used to study type i ifn signaling of porcine arterivirus (kim et al., 2010; overend et al., 2007; patel et al., 2010) , and thus infection of these cells one μg of each of the cloned genes was transfected to hela cells in 12-well plates, and protein expression was determined by immunofluorescence (b) and western blot (c) for each gene using anti-flag antibody. (d and e) regulation of poly(i:c)-induced ifn-β promoter activity by individual pedv proteins. hela cells were seeded in 12-well plates and co-transfected with pifn-β-luc along with individual pedv genes and prl-tk at a ratio of 1:1:0.1. since the expression levels of nsp3 and nsp16 were low, three-times more plasmids were transfected for these genes to ensure the comparable level of protein expression. prrsv nsp1α (p-nsp1α) is a known type i ifn suppressor, and the ifn-suppression of its mutant p-nsp1α(m) was lost. both constructs were included as controls. at 24 h post-transfection, cells were stimulated with poly (i:c) (0.5 μg/ml) for 12 h and the luciferase activities were measured. the reporter experiments were repeated three times, each time in triplicate. asterisks indicate the statistical significance. statistical analysis was performed by student's t test using gst as a control. n p o0.05, nn po 0.01 and nnn p o 0.001. (f) vsv-gfp bioassay. the cell culture supernatants for ifn-β promoter luciferase assays were collected and diluted serially by 2-folds up to 1:64. fresh marc-145 cells were grown in 96-well plates and incubated with each dilution of supernatants for 24 h, and then infected with vsv-gfp at an moi of 0.1 for 16 h. vsv replication was measured by monitoring the fluorescence by gfp expression using fluorescent microscopy. data were presented as log 2 sample dilution folds. (g) inhibition of irf3 promoter activation by pedv proteins. the ifn antagonists were further examined for irf3 activities by luciferase reporter assays. statistical analysis was performed by student's t test using gst as a control. by pedv allowed us to study ifn modulation and signaling cascade. to determine whether pedv infection antagonized the type i ifn response, ifn-β mrna was determined in virus-infected cells. marc-145 cells were infected with pedv and stimulated with poly (i:c) followed by qrt-pcr for ifn-β mrna using total rna. as shown in fig. 2a , pedv infection did not induce the level of ifn-β mrna expression whereas poly(i:c) alone induced the ifn-β gene expression effectively, indicating the suppression of ifn-β response by pedv. to further evaluate the ifn-β response in pedv-infected cells, a dual luciferase assay was performed. the results showed the suppression of ifn-β promoter activity in pedv-infected cells upon poly(i:c) stimulation (fig. 2b) , demonstrating the modulation of ifn production by pedv infection. irf3 was additionally examined for its role for pedv-mediated ifn-β suppression. the irf3 promoter activity was found to be inhibited (fig. 2c ). the suppression of ifn-β production was confirmed by bioassay using vsv-gfp. vsv is sensitive to type i ifn treatment and thus commonly used for ifn bioassays. culture supernatants were collected from pedv-infected cells and were uv-irradiated, followed by incubation with marc-145 cells and infection with vsv-gfp. vsv grew normally (fig. 2d) , whereas it did not grow with supernatants collected from poly(i:c)-treated cells for up to 1:8 dilution. vsv-gfp also grew normally with supernatants from both pedv-infected cells with or without poly(i:c) stimulation, confirming the suppression of type i ifn production by pedv. the viral ifn antagonism is often redundant, and at least 11 viral proteins have been identified as ifn antagonists for sars-cov (kindler and thiel, 2014; shi et al., 2014; totura and baric, 2012) . to identify such proteins for pedv, we cloned pedv genes representing nsps 1 through 16, and structural genes for s, e, m, and n including the orf3 accessory protein gene (fig. 3a ). among these, nsp11 is a small oligopeptide generated from pp1a when ribosomal frameshifting does not occur and so was not included in this study. each gene was inserted into the pxj41 expression vector with the flag tag at either n-or c-terminus, and examined for ifn suppression. the protein expression of cloned genes was examined by immunofluorescence (fig. 3b ) and western blot (fig. 3c ) using anti-flag antibody. all genes were expressed as anticipated. hela cells were then co-transfected with an individual gene along with pifn-β-luc and prl-tk, and reporter assays were conducted. prrsv nsp1α (p-nsp1α) is known as an ifn-β suppressor, and its cystine mutant p-nsp1α(m) (c28s) is an ifn suppression revertant (han et al., 2013; song et al., 2010) , and so they were included as positive and negative controls, respectively. poly(i:c) upregulated the ifn-β transcription in cells expressing pxj41, gst, and p-nsp1α (m), while p-nsp1α suppressed the ifn-β promoter activity as expected ( fig. 3d and e). of the nsps, nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were shown to down-regulate the ifn-β activity (fig. 3d ). for structural proteins, e, m, and n were found to suppress the ifn induction (fig. 3e) , and orf3 was additionally identified as an ifn suppressive protein (fig. 3e ). the findings from the reporter assays were validated by vsv-gfp bioassays. the dilution corresponding to 50% of cells exhibiting gfp expression was determined as the end-point inhibition. for pxj41, gst, and nsp9, gfp expression was evident and their end-point inhibitions were determined as 1:64 (fig. 3f ). in contrast, the viral proteins identified as the luciferase suppressors showed an apparent inhibition of vsv-gfp replication and their end-points were determined to be 1:4 to 1:16 (fig. 3f ). these titers represent 4-to 16fold lower than those of controls. taken together, these data demonstrate that pedv has the ability for ifn suppression, and nsp1, nsp3, nsp7, nsp14, nsp15, nsp16, orf3, e, m and n are the viral ifn antagonists. to determine the target for ifn inhibition, the irf3 pathway was examined for individual viral proteins using the irf3 luciferase reporter constructs. upon stimulation, the irf3-dependent luciferase expression was reduced by nsp1, nsp3, nsp7, nsp14, nsp15, nsp16, orf3, e, m, and n, comparing to those of pxj41 and gst (fig. 3g ). this suggests that the irf3 signaling pathway was interfered by these viral proteins for the suppression of the ifn-β production. pedv nsp1 antagonism is a nuclear event sars-cov is a betacoronavirus and its nsp1 triggers inhibition of type i ifn induction and downstream signaling, host mrna decay and cleavage, and inhibition of protein translation (huang et al., 2011b; lokugamage et al., 2012; narayanan et al., 2008; tanaka et al., 2012) . transmissible gastroenteritis virus (tgev) is an alphacoronavirus and its nsp1 inhibits host protein expression (huang et al., 2011a) . nsp1 of alphacoronavirus and betacoronavirus lacks the overall sequence similarity (narayanan et al., 2015) , and thus alphacoronavirus nsp1 may have a distinct basis for its biological function. since nsp1 appeared the most potent suppressor in our study on pedv, nsp1 was chosen to study the molecular basis for the ifn suppression. the subcellular localization was first examined by confocal microscopy in transiently expressing cells. the nsp1 distribution was evident in the both nucleus and cytoplasm ( fig. 4a) , which is consistent with tgev nsp1 (narayanan et al., 2015) . co-expression of nsp1 with either the endoplasmic reticulum or mitochondrial marker showed the site for cytoplasmic nsp1 in the endoplasmic reticulum (fig. 4b ). quantitative rt-pcr was conducted to evaluate ifn-β suppression in nsp1-gene transfected cells. the expression of nsp1 significantly suppressed the ifn-β mrna transcription (fig. 5a) , further validating the nsp1 antagonism against ifn-β production. subsequently, the ifnmediated antiviral gene expression was examined for isg15 and isg56 by qrt-pcr. pedv nsp1 reduced the poly(i:c)-stimulated mrna levels of both isg15 (fig. 5b ) and isg56 (fig. 5c ), indicating the suppression of ifn signaling by nsp1. the suppression of ifn-β, irf3, and nf-κb activations raises a possibility that nsp1 may target a component of the rig-i like receptor (rlr) signaling pathway. to examine this premise, nsp1 was co-expressed with one of the main components in the rlr signaling pathway, and ifn luciferase activities were determined at 24 h post-transfection. the over-expression of ips-1, or irf3 led to the robust activation of the ifn-β promoter as anticipated, whereas the activation was significantly inhibited by nsp1 ( fig. 6a fig. 4 . subcellular localization of pedv nsp1. hela cells were seeded on slides in 6-well plates and transfected with pedv nsp1 gene (a), or co-transfected with nsp1 and pdsred2-er or pdsred2-mito (b). at 24 h post-transfection, cells were fixed and permeabilized with triton x-100. cells were then incubated with rat anti-flag mab for 1 h, followed by alexa fluor 488-conjugated goat anti-mouse (green) secondary antibody to visualize nsp1. the er and mitochondrial proteins were fused with the er and mitochondria targeting sequence (clontech) and so directly visualized (red). nuclei (blue) were stained with dapi. images were collected using a zeiss lsm-510 meta confocal laser-scanning microscope and processed with the lsm image browser (zeiss). irf3 is a resident protein in the cytoplasm. when stimulated, it is phosphorylated and homodimerized, leading to the translocation to the nucleus (dragan et al., 2007) . to determine whether pedv nsp1 targeted the irf3-dependent pathway, the irf3 phosphorylation was first examined. nsp1-gene transfected cells were stimulated with poly(i:c), and the irf3 phosphorylation was examined by western blot. as anticipated, the poly(i:c) stimulation led to irf3 phosphorylation in pxj41-transfected cells, and similarly, in nsp1-expressing cells, the irf3 phosphorylation was evident and comparable to that of control (fig. 7a , top panel, lane 4), suggesting that pedv nsp1 exerts its suppression downstream of the irf3 phosphorylation. thus, the nuclear translocation of irf3 was next examined. prrsv nsp1α is known not to block the irf3 nuclear localization, and so was used as a control in this study. endogenous irf3 was normally diffused and distributed in the cytoplasm, but translocated to the nucleus when stimulated by poly(i:c) (fig. 7b, second panel) . similarly to prrsv nsp1αexpressing cells (fig. 7b , fourth panel), irf3 also localized normally in the nucleus after stimulation in pedv nsp1-expressing cells (fig. 7b, bottom panel) , suggesting that the ifn suppression by pedv nsp1 may be a nuclear event. the irf3 nuclear translocation in nsp1-expressing cells was further confirmed by cell fractionation and western blot analyses. while irf3 was phosphorylated and localized in the nucleus after stimulation, pedv nsp1 did not inhibit the irf3 phosphorylation and nuclear translocation (fig. 7c ), further indicating that the nsp1-mediated ifn suppression was a nuclear event. interruption of irf3 and cbp association by nsp1 after nuclear translocation, an irf3 dimer associates with the creb-binding protein (cbp). this complex then binds to the prd i-iii 6 . disruption of irf3-mediated ifn signaling by nsp1. hela cells were seeded in 12-well plates and co-transfected with pmavs/ips-1 (a) or pirf3 (b) along with the nsp1 gene, prl-tk, and ifn-β-luc reporter for 24 h. cells were harvested to measure the firefly and renilla luciferase activities. relative luciferase activity was defined as a ratio of the firefly luciferase to renilla luciferase activities. data are presented as mean value 7 standard deviation from three independent experiments. statistical analysis was performed by student's t test. n po 0.05, nn p o0.01, and nnn po 0.001. immunofluorescence staining for irf3 nuclear translocation by nsp1. hela cells were transfected with the nsp1-expressing plasmid (2 μg/well) in 6-well plates for 24 h and stimulated by poly(i:c) for 8 h. cells were fixed and incubated with rabbit anti-irf3 pab and rat anti-flag mab for 1 h. prrsv nsp1α does not inhibit the irf3 nuclear localization and was used as a control. alexa fluor 594-conjugated goat anti-rabbit and 488-conjugated goat anti-mouse secondary antibodies were used to visualize irf3 (red) and viral nsp1 (green), respectively. nuclei (blue) were stained with dapi. yellow arrows indicate irf3 localization in the nucleus in the absence of nsp1 expression. white arrows indicate irf3 localization in the nucleus in nsp1-expressing cells. (c) phosphorylation and nuclear localization of irf3 by nsp1. hela cells were transfected with the pedv nsp1 gene for 24 h, then stimulated with poly(i:c) for 8 h. cells were lysed for nuclear-cytoplasmic fractionations and subcellular distribution of irf3 and pirf3. hsp90 was used as a cytosolic protein marker and parp was used as a nuclear protein marker. regions of the ifn-β promoter to assemble the basal transcription machinery complex together with nf-κb and other transcription factors to turn on the transcription of type i ifn genes (honda and taniguchi, 2006) . thus, the irf3-cbp/p300 interaction for the assembly of enhanceosome is crucial for ifn expression. since pedv nsp1 did not block the irf3 phosphorylation and nuclear translocation in our study, it was hypothesized that nsp1 might disrupt the formation of enhanceosome in the nucleus. to address this, the irf3/ cbp association was first examined in nsp1-expressing cells. cells were transfected with the nsp1 gene and stimulated with poly(i:c) followed by co-immunoprecipitation using anti-irf3 antibody and immunoblot with anti-cbp antibody. in unstimulated cells, cbp was undetectable due to the absence of irf3/cbp association (fig. 8a , left lane), but irf3/cbp association became evident upon stimulation (fig. 8a, middle lane) . in nsp1-expressing cells however, the association of irf3 and cbp disappeared even upon stimulation (fig. 8a , right lane) and the detectable level of irf3 remained unchanged ( fig. 8a, second panel) . absence of the association of cbp/irf3 may occur when nsp1 binds to either irf3 or cbp, or when irf3 is unstable in the presence of nsp1. since pedv nsp1 was found to be a nuclear protein (figs. 4a, b and 7b, c), nsp1 in the nucleus might interact with either irf3 or cbp. however, neither the interaction between irf3 and nsp1, nor between cbp and nsp1 was observed by co-immunoprecipitation in our study. irf3 was also stable in the presence of nsp1 ( fig. 7a and c) , indicating that the absence of irf3/ cbp association was not due to the instability of irf3. interestingly, the level of cbp was found to decrease in nsp1-expressing cells (fig. 8a) , leading us to investigate the degradation of cbp by nsp1. some viruses including htlv, adenovirus, and an orthormyxovirus thogoto interact with cbp to modulate type i ifn induction, suppress protein expression, or promote virus infection (ferrari et al., 2014; jain et al., 2015; jennings et al., 2005; wurm et al., 2012; zhang et al., 2008) . degradation of cbp has been described for the porcine arterivirus prrsv as a strategy for ifn antagonism . since the level of cbp was found to decrease in pedv nsp1-expressing cells (fig. 8a) , cbp expression was validated in pedv-infected cells by co-staining using anti-cbp antibody and anti-pedv m pab. in uninfected cells, cbp was predominately localized in the nucleus in marc-145 and vero cells (fig. 8b, yellow arrows) . in contrast, cbp was depleted in virus-infected cells (fig. 8b , white arrows), demonstrating that the cbp was degraded by pedv. we further sought to study whether the cbp degradation by pedv was mediated by nsp1 protein. cbp was exclusively nuclear in control cells, whereas it was depleted in nsp1-expressing cells (fig. 9a) . prrsv nsp1α is known to degrade cbp in the nucleus , and in prrsv nsp1αexpressing cells, cbp was significantly depleted (fig. 9a ). the cbp degradation was quantified by examining the ratio of nsp1expressing cells showing cbp degradation out of the chosen number of nsp1-expressing cells (fig. 9b ). approximately 92% of prrsv nsp1α-expressing cells showed more than 80% reduction of cbp, which is in consistent with the previous report (han et al., 2013) . for pedv nsp1-expressing cells, approximately 90% cells showed more than 80% reduction of cbp, while no cbp reduction was observed in control cells. this finding was confirmed by western blot. in pedv nsp1-expressing cells, cbp degradation was evident compared to that of control cells (fig. 9c , top panel, lane 3). to eliminate a possibility that the reduction of cbp might be due to the short half-life of cbp, cyclohexamide (chx) treatment was conducted (fig. 9d) . at 24 h post-transfection, cells were treated with chx to shut down the new protein synthesis for indicated times followed by western blot. in nsp1-expressing cells, cbp reduction was evident at the beginning of chx treatment, and further decreased by 2 h post-treatment. the cbp degradation was complete by 4 h post-treatment, whereas nsp1 and β-actin remained stable (fig. 9d) . together, our data show that pedv nsp1 was the viral protein contributing to the cbp degradation. unlike prrsv nsp1α, pedv nsp1 does not contain a proteinase activity, and no direct interaction between cbp and nsp1 was identified in our study. it is thus unlikely that cbp would be a direct substrate of pedv nsp1. therefore, it was of interest to examine whether the cbp degradation was a proteasomedependent process. the treatment with mg132 blocked the cbp degradation by nsp1. as little as 5 μm of mg132 was sufficient to inhibit the cbp degradation, and 10 μm was able to restore the cbp level back to the control level (fig. 9e) . to eliminate the cbp degradation by nsp1 was cell-type specific, we further tested the cbp degradation by nsp1 in pig intestinal epithelial cell line (ipec-j2 cells), which reported to be susceptible to pedv (zhao et al., 2014) . cbp degradation in nsp1-expressing cells was evident comparing to control cells (fig. 9f, top panel) . additionally, the cbp degradation by nsp1 was also blocked by mg132 treatment in ipec-j2 cells (fig. 9f, bottom panel) . this study indicates that the cbp degradation by pedv nsp1 was proteasome-dependent in the nucleus. the innate immune system is the first line of host defense in response to viral infection. it initiates the production of type i ifns and proinflammatory cytokines through the recognition of pamps by prrs and establishes antiviral states which are highly effective on resisting and controlling infections. in turn, many viruses have developed strategies to counteract the host innate immune response to establish productive infection. previous studies have shown that pedv infection fail to induce the ifn-β promoter activation and that plp2 (papain-like proteinase 2) of pedv antagonizes the ifn response by deubiquitinating rig-i and sting (xing et al., 2013b) . the pedv n protein suppresses the irf3 and nf-κb activities and antagonizes the ifn-β production by disrupting the interaction between irf3 and tbk1 (ding et al., 2014) . on the contrary, a recent study shows that pedv infection induces nf-κb activation in intestinal epithelial cells with the n protein as the activator (cao et al., 2015b) . in the present study, we have identified marc-145 as pedv permissive cells, and used these cells as a model to study the innate immune modulation for pedv. we have shown the suppression of type i ifn production by pedv, which is consistent with the recent finding in iecs (cao et al., 2015a) . we also have identified multiple viral proteins responsible for this suppression. we have further determined pedv nsp1 as the viral component promoting cbp degradation in the nucleus via the proteasome-dependent pathway. many viruses in the order nidovirales are able to modulate the host innate response, which plays an important role for their pathogenesis. in the family arteriviridae, equine arteritis virus suppresses type i ifn production in equine endothelial cells (go et al., 2014) , and prrsv also suppresses ifn production (albina et al., 1998) . prrsv is susceptible to type i ifns in cells and the suppression of type i ifn varies for different isolates (albina et al., 1998; lee et al., 2004; overend et al., 2007) . mouse hepatitis virus (mhv), which is a betacoronavirus, induces a high level of ifn-α secretion by plasmacytoid dendritic cells (pdcs) during infection (cervantes-barragan et al., 2007) . however, other cell types infected by mhv such as macrophages, microglia, and oligodendrocytes produce extremely low-levels of type i ifns (li et al., 2010; roth-cross et al., 2008; zhou and perlman, 2007) . the mhv ns2 protein is dispensable for virus replication in cells but is required for induction of hepatitis in mouse (schwarz et al., 1990) . the 2 0 ,5 0 -phosphodiesterase (pde) activity of ns2 mediates the cleavage of 2 0 ,5 0 -oligoadenylate and prevents the activation of rnase l, while enhancing viral growth and pathogenesis, thus ns2 is a viral ifn antagonist (zhao et al., 2012) . sars-cov, which is another member virus in the genus betacoronavirus, impairs the ifn response in virus-infected cells, and an ifn therapy has been suggested to be efficacious for sars patients (cinatl et al., 2004; spiegel et al., 2005) . mers-cov is also a betacoronavirus, and both mers-cov and sars-cov do not induce a pronounced ifnresponse in polarized airway epithelial cells (calu-3), alveolar adenocarcinoma cells (a549) and human monocyte-derived macrophages (lau et al., 2013; zhou et al., 2014; zielecki et al., 2013) . even though the acute infection of tgev induces a highlevel of ifn-α in newborn pigs (la bonnardiere and laude, 1981) , protein 7 counteracts the host antiviral response and influences viral pathogenesis (cruz et al., , 2013 . the 7a protein of an alphacoronavirus feline infectious peritonitis virus is a type i ifn antagonist (dedeurwaerder et al., 2014) . type i ifns of chickens inhibits viral replication and respiratory illness of the gammacoronavirus infectious bronchitis coronavirus (ibv) (pei et al., 2001) . ibv delays the ifn response and the 3a and 3b accessory proteins have been identified as the ifn antagonists (kint et al., 2015) . thus, modulation of type i ifn response seems to be a common evasion strategy of viruses in the order nidovirales. we have shown in the present study the direct evidence that pedv indeed downregulates type i ifns production during infection. pedv suppresses the ifn-β and irf3 activities. since irf3 is a key element in the production of type i ifns, our finding leads to a hypothesis that pedv modulation of type i ifns production targets the irf3 signaling pathway. interestingly, pedv normally activates the nf-κb activity in vero e6 cells (xing et al., 2013b) . a recent study confirms that pedv infection in intestinal epithelial cells induces nf-κb activation (cao et al., 2015b) . in that study, nuclear localization of p65 increases by pedv after 12 h through 48 h. however, activation of nf-κb during viral infection is generally an early event. for prrsv, nf-κb is activated 30 min after infection (fu et al., 2012) . thus, how pedv modulates nf-κb activation during early time of infection needs to be further investigated. we have identified at least ten viral ifn antagonists and all ten proteins inhibit the irf3 activity. whether these ifn antagonists modulate the nf-κb activity needs to be further investigated. the pedv n protein suppresses sendai virus-induced nf-κb activity in a dose-dependent manner (ding et al., 2014) . in other study, n protein activates nf-κb in intestinal epithelial cells (cao et al., 2015b) . a possible explanation is that the nf-κb activation may be time-dependent and cell type-dependent. together, the irf3 signaling is likely the target by pedv for type i ifns modulation. at least eleven viral proteins have been identified as ifn antagonists for sars-cov (kindler and thiel, 2014; shi et al., 2014; totura and baric, 2012) , whereas ten proteins have been identified for pedv in our study. thus, coronaviruses seem to arm with multiple antagonists. a possible explanation for such a functional redundancy is that coronavirus genomes are the largest rna known to biology and undergo continuous genetic evolution. when a functional mutation occurs in a major antagonist, other antagonists may complement the function to ensure efficient replication and adaptation in hosts. for sars-cov, nsp1 is a multifunctional protein with the suppressive activity for ifn and blocks the phosphorylation of stat1 and degrades host cell mrna (totura and baric, 2012) . sars-cov nsp14 and nsp15 works as exoribonuclease and endoribonuclease, respectively, thus specific digestion of dsrnas and the consequent removal of rna-pamps may lead to an inadequate activation of ifn response (kindler and thiel, 2014) . sars-cov nsp16 contains 2 0 -omethlytransferase activity and modifies the cap of viral rnas in order to evade the detection by the host immune system (totura and baric, 2012) . the sars-cov m protein impedes the formation of traf3 á tank á tbk1/ikkϵ complex for suppression of type i ifn production (siu et al., 2009 (siu et al., , 2014 . the plp2 domain of sars-cov nsp3 negatively modulates type i ifn pathway and functions as a viral deubiquitinase. in our study, the full length pedv nsp3 indeed inhibit the ifn activity. all ten antagonists identified for pedv correspond to the respected antagonists of sars-cov. the corresponding proteins of pedv may share the similar motifs and functions with those of sars-cov. additionally, sars-cov encodes several accessory proteins. they are nonessential for viral replication but function as innate immune antagonists. for pedv, orf3 is the sole accessory protein, and a previous report shows that orf3 functions as an ion channel protein and is relevant to infectivity and pathogenicity (wang et al., 2012) . orf3 is nonessential for viral replication in vitro as shown by targeted rna recombination . in our study, orf3 is a potent ifn antagonist. the viral antagonists may target different pathways of the host innate immune signaling and their synergistic effects may shut down the host innate immune response more efficiently during the course of infection. cbp is a histone acetyltransferase and plays a key role in transcription regulation. the cbp/p300 coactivators interact with hundreds of transcription factors including stats, c-myc, pias1, p53, nf-κb, and irf family (bedford and brindle, 2012; goodman and smolik, 2000; long et al., 2004) . for ifn expression, the assembly of an enhanceosome consisting of nf-κb, irfs, atf2/c-jun, and the architectural protein hmg i(y) is required in response to virus infection. the ifn enhanceosome recruits cbp/p300 for synergistic activation of transcription (merika et al., 1998) . some viruses modulate the cbp activity for viral evasion. two distinct regions in the simian virus 40 t antigen can independently alter the levels and loading of cbp/p300 transcripts onto polysomes for cell immortalization and transformation (robles et al., 2013) . african swine fever virus nuclear protein a238l inhibits the expression of tnf-α by displacing the cbp/p300 coactivators (granja et al., 2006) , and herpes simplex virus 1 (hsv-1) icp0 protein recruits activated irf3 and cbp/p300 to the nuclear foci, which may result in reduced transcription of ifn-β and inhibition of the host response (melroe et al., 2007) . hsv-1 vp16 protein inhibits nf-κb activation and interferes the recruitment of irf3 to cbp to block the ifn-β production (xing et al., 2013a) . the ml protein of thogoto virus interferes with irf3 function without blocking its nuclear translocation but interrupts the association of irf3 with cbp (jennings et al., 2005) , which is similar to the function of pedv nsp1. the ml protein was later found to interact with the rna polymerase ii transcription factor iib (tfiib), however, this interaction hardly interferes the host general gene expression but strongly suppresses both the irf3-and nf-κbregulated promoter activities (vogt et al., 2008) . thus, it is hypothesized that the virus-mediated cbp degradation may play a specific and key role for ifn modulation with a little impact on general cellular gene transcriptions. the degradation of cbp is a novel strategy for ifn modulation and has been extensively studied in the family of arteriviridae, especially for prrsv . for prrsv nsp1α, cbp degradation is associated with the zinc-finger motif and is likely the key mechanism for ifn suppression (han et al., 2013) . for pedv, nsp1 is the most potent ifn suppressor among all viral antagonists without affecting the irf3 phosphorylation and nuclear localization. in line with this, pedv infection depletes the endogenous cbp. furthermore, pedv nsp1 disrupts the association of cbp-irf3 and degrades cbp in a proteasome-dependent manner. sars-cov nsp1 inhibits type i ifn production, induces host mrna degradation, and suppresses host protein translation (narayanan et al., 2008) . however, the domains of sars-cov nsp1 responsible for suppression of host gene expression and type i ifn production are absent in pedv nsp1 (huang et al., 2011b; narayanan et al., 2008) . even though nsp1s of alphacoronavirus and betacoronavirus share similar functions, they lack an overall sequence similarity and neither conserved motifs nor domains exist in viruses of alphacoronaviruses. thus, it is plausible that nsp1 of alphacoronaviruses may have a distinct function regulating host innate immune responses and gene expression. tgev nsp1 suppresses protein translation in cells and cell-free extracts. however, the suppression of protein translation by pedv nsp1 may not be a general event since the β-actin shows the similar level of expression after infection and transfection. the lack of association of cbp-nsp1 and irf3-nsp1 suggests that the cbp degradation by nsp1 is an indirect event that needs to be further determined. similar to tgev nsp1, the subcellular localization of pedv nsp1 is nuclear-cytoplasmic. the sequence of pedv nsp1 does not harbor any known nuclear localization signal, and thus nsp1 may piggy-bag a nuclear protein to enter the nucleus. the proteasome-dependent cbp degradation seems a unique viral tactic utilized to inhibit ifn-β production. it is of interest to study whether this is a common evasion strategy for coronaviruses. cbp localizes in the pml nuclear bodies, which are discrete nuclear foci that are disrupted in acute promyelocytic leukemia (boisvert et al., 2001; doucas et al., 1999; lamorte et al., 1998) . the pml nuclear bodies dynamically colocalize with numerous proteins including cbp, pml, p53, rb, sp100, daxx, eif4e, and sumo (jensen et al., 2001) . upon inhibition of proteasome activity, pml, sp100, ebna-5, sumo-1, and the 20s proteasome subunit move to the nucleolus, suggesting that proteasomal degradation occurs at the nuclear loci (boddy et al., 1996) . hausp, the ubiquitin-specific hydrolase in the pml nuclear bodies, removes ubiquitin moieties from proteins prior to proteasomal degradation (everett et al., 1998) . thus, pml nuclear bodies may represent the sites where ubiquitinated proteins are processed by enzymes such as hausp prior to degradation in the nucleolus (st-germain et al., 2008) . valproic acid, a histone deacetylase inhibitor, could induce cbp degradation through the ubiquitin-proteasome pathway, while increasing the colocalization of cbp with ubiquitin nuclear speckles and with pml nuclear bodies (st-germain et al., 2008) , suggesting that pml nuclear bodies may be the sites for the ubiquitin-dependent degradation of cbp. it is of interest to examine whether pedv nsp1 promotes ubiquitination of cbp for degradation in the nucleus and whether this degradation associates with pml nuclear bodies. pedv infects vero cells and marc-145 cells. porcine amino peptidase n (papn) has been identified as the major cell entry receptor for pedv (li et al., 2007; nam and lee, 2010) . transient expression of papn confers pedv non-permissive canine kidney cells (mdck) to be permissive for pedv infection. papn also increases the pedv infectivity in porcine small intestine epithelial cells (iecs) (cong et al., 2015) . the respiratory tract may support pedv infection in pigs and the virus infects and replicates in transformed alveolar macrophages (3d4) in vitro (park and shin, 2014) . primate apn or receptor-independent pathways in vero and marc-145 cells may complement the function of papn for pedv infection (taguchi and matsuyama, 2002) . in summary, we have shown the suppression of type i ifn production by pedv and have identified specific viral ifn antagonists. among these antagonists, nsp1 is the most potent protein and functions to degrade cbp in the nucleus. our data provides a novel insight into the understanding of the immune evasion strategy of pedv. hela cells (nih aids research and reference reagent program, germantown, md) and marc-145 cells (kim et al., 1993) reagent was purchased from invitrogen (carlsbad, ca). qiaamp viral rna mini kit and rneasy mini kit were purchased from qiagen (venlo, limburg). power sybr green pcr master mix was purchased from life technologies (carlsbad, ca). alexa fluor 594conjugated (goat anti-rabbit, red) and 488-conjugated (goat antimouse, green) secondary antibodies and pierce™ ecl western blotting substrate were purchased from thermo scientific (waltham, ma). the firefly luciferase genes were used as reporters with its expression under the control of various promoters as indicated below. the plasmid pifn-β-luc contains the entire ifn-β enhancer-promoter. the plasmid p4 â irf3-luc contains four copies of irf3 binding region prd i-iii of the ifn-β promoter. pifn-β-luc and p4 â irf3-luc were obtained from dr. stephan ludwig at heinrich-heine-universität, düsseldorf, germany (ehrhardt et al., 2004) . the renilla luciferase plasmid prl-tk (promega) contains the herpes simplex virus thymidine kinase (hsv-tk) promoter and was included in all experiments to serve as an internal control. active stimulator pmavs/ips-1 was obtained from dr. j. shisler (university of illinois, urbana, il). pirf3 was kindly provided by dr. b. gotoh (university of fukui, fukui, japan). pdsred2-er and pdsred2-mito were purchased from clontech. plasmids with the flag tag for expression of nsp1 through nsp16, and the s, s1, s2, orf3, e, m and n genes were cloned from the viral genomic rna by standard reverse transcription and pcr techniques using indicated primers (table 1) . twenty-three viral genes were amplified and cloned into the eukaryotic expression vector pxj41 using indicated restriction enzymes. the nsp1 to nsp16, orf3, and n genes were expressed as fusion proteins with the n-terminal flag tag, and the s, s1, s2, e, and m genes were expressed as fusion proteins with the c-terminal flag tag to avoid the functional disruption of the signal sequence. the constructs were confirmed by sequencing, immunofluorescence, and western blot. prrsv nsp1α and its cystine mutant p-nsp1α(m) (c28s) are described elsewhere (han et al., 2013; song et al., 2010) . hela cells were seeded in 12-well plates and grown to 80% confluency prior to transfection. individual viral protein genes, luciferase reporters, and prl-tk as an internal control were transfected at a ratio of 10:10:1 in a total of 1.05 μg/well using lipofectamine 2000 according to the manufacturer's instruction (invitrogen). at 24 h post-transfection, cells were stimulated by transfection with 0.5 μg/well of poly(i:c) for 12 h. cells were then lysed and luciferase assays were performed using the dual luciferease assay system according to the manufacturer's instructions (promega). values were normalized using the renilla luciferase activity as the internal control and presented in fold-changes. three independent assays were performed with each assay in triplicate. total rna was extracted from hela or marc-145 cells using rneasy mini kit according to the manufacturer's instructions (qiagen). the rna was treated with dnase i to remove contaminating genomic dna. reverse transcription (rt) reaction was performed with 1 mg of total rna using random primers and m-mlv reverse transcriptase (invitrogen). sybr green real-time pcr was conducted in the abi 7500 real-time pcr system according to the manufacturer's instructions (life technologies). the real-time pcr primers for ifn-β, isg15, isg56 and β-actin were listed in table 2 . for each sample, the β-actin gene was amplified and used as an internal control. specific amplification was confirmed by sequencing pcr products. the threshold cycle for target genes and the difference between their c t values (δc t ) were determined. the relative transcript levels of target gene are equal to 2 à δδct threshold method (livak and schmittgen, 2001) and are shown as fold changes relative to the respective untreated control samples. hela cells were seeded in 6-well plates and transfected with 2 μg of plasmid. at 24 h post-transfection, cells were stimulated by transfection with 1 μg of poly(i:c) for 12 h. supernatants were harvested for bioassay. for pedv, marc-145 cells were infected with pedv at an moi of 1 for 12 h prior to poly(i:c) stimulation. supernatants from virus-infected cells were uv-irradiated for 30 min to remove infectivity prior to bioassay. the supernatants were then serially diluted by 2-fold. marc-145 cells were freshly grown in 96-well plates and incubated with 100 μl of each dilution for 24 h. cells were then infected in 100 μl of vsv-gfp at 10 4 pfu/ ml for 16 h and gfp expression was examined by inverted fluorescence microscopy (nikon eclipse ts100, 10 â 10). each dilution was examined twice in triplicate each. indirect immunofluorescence assay (ifa) and confocal microscopy cells were seeded on coverslips and transfected with plasmids or infected with pedv. for transfection of hela cells, total 2 mg of individual plasmids were transfected for 24 h using lipofectamine table 1 primers used for the cloning of pedv nonstructural and structural genes (pedv strain usa/colorado/2013). restriction enzyme recognition sequences are underlined. the flag tag is italicized and underlined. primers used for relative quantitative real-time rt-pcr. ifn-β-f gatttatctagcactggctgg ifn-β-r cttcaggtaatgcagaatcc isg15-f caccgtgttcatgaatctgc isg15-r ctttatttccggcccttgat isg56-f cctccttgggttcgtctaca isg56-r ggctgatatctgggtgccta β-actin-f atcgtgcgtgacattaag β-actin-r attgccaatggtgatgac 2000 according to the manufacturer's instructions (invitrogen). cells were then either treated with poly(i:c) for 12 h or ifn-β for 40 min. cells were fixed with 4% paraformaldehyde in pbs overnight at 4°c and permeabilized using 0.1% triton x-100 for 15 min at room temperature (rt). after blocking with 1% bsa in pbs at rt for 30 min, cells were incubated with a primary antibody in pbs for 1-3 h. cells were then washed three times with pbs and incubated with alexa fluor 488-labeled anti-mouse secondary antibody, or alexa fluor 594-labeled anti-rabbit secondary antibody (thermo scientific) for 1 h at rt in the dark. cells were incubated with dapi for 5 min at rt for nuclear staining. after washing with pbs, cover slips were mounted on microscope slides using fluoromount-g mounting medium (southern biotech, birmingham, al), and visualized by fluorescence microscopy (nikon eclipse ts100). confocal microscopy was conducted as described elsewhere (kannan et al., 2009) . hela cells were seeded in 6-well plates to 80% confluency and transfected with 2 μg/well of nsp1 plasmid for 24 h. cells were stimulated with 1 μg of poly(i:c) for 12 h and fractionated using the nuclear/cytosol fractionation kit (biovision, milpitas, ca) with minor modifications. briefly, cells were washed with cold pbs and collected using cell scrapers in 1 ml of cold pbs. cell pellets were resuspended in 200 μl ceb-a buffer and incubated on ice for 10 min. after addition of ceb-b, tubes were vortexed and incubated on ice for 1 min. the cell lysates were then centrifuged at 4°c 5 min at 16,000g and supernatants were collected as the cytosolic fraction. the cell pellets were suspended in neb buffer and vortexed for 30 s and repeated 5 times every 10 min. the nuclear pellets were finally centrifuged for 10 min at 4°c 16,000g and kept the supernatants as the nuclear fraction. cells were harvested in ripa buffer [20 mm tris (ph 7.5), 150 mm nacl, 1 mm edta, 1 mm phenylmethanesulphonyl fluoride (pmsf), 0.1% sds, 0.5% sodium deoxycholate, 1% np-40] containing the proteinase inhibitors cocktail (promega). cells were frozen-thawed, collected in the pre-cold tubes, and centrifuged to remove insoluble components. total protein concentration was determined using pierce bca protein assay kit (thermo scientific). equal amounts of proteins were resolved by sds-page and blotted to pvdf membranes (millipore). after blocking with 5% nonfat dry milk in tbst (0.05% tween-20) for 1 h, membranes were incubated with a primary antibody in tbst containing 5% nonfat dry milk overnight at 4°c, followed by washing and incubation with horseradish peroxidase (hrp)-conjugated secondary antibody for 1 h at rt. the membrane was visualized using pierce ecl western blotting substrate (thermo scientific) and images were taken by fluorchem™ r system according to the manufacturer's instructions (proteinsimple). co-immunoprecipitation (co-ip) was performed as described previously with modifications (kim et al., 2010) . gene-transfected cells were lysed in lysis buffer [50 mm tris (ph 8.0), 150 mm nacl, 5 mm na 3 vo 4 , 1 mm pmsf, 100 mg/ml leupetin, 1% np-40, 10% glycerol] supplemented proteinase inhibitors cocktail (promega). cell lysates were clarified by centrifugation at 4°c for 10 min at 16,000g. supernatants were transferred to fresh tubes and incubated with either flag-or irf3-antibody at 4°c overnight, followed by incubation with protein g agarose beads (fast flow, millipore) at 4°c for 4 h. pellets were collected by centrifugation and washed for five times. the final pellets were eluted with laemmli sample buffer (bio-rad) and were subjected to western blot. student's t-test was used for all statistical analyses. asterisks indicate the statistical significance. *po0.05, **p o0.01 and ***p o0.001. interferon-alpha response 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immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus key: cord-321992-lk2ao6m8 authors: annamalai, thavamathi; saif, linda j.; lu, zhongyan; jung, kwonil title: age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs date: 2015-12-15 journal: vet immunol immunopathol doi: 10.1016/j.vetimm.2015.09.006 sha: doc_id: 321992 cord_uid: lk2ao6m8 porcine epidemic diarrhea (ped) is an enteric coronaviral infection that causes severe morbidity and mortality in suckling pigs, but less severe disease in older pigs. consequently, it causes significant economic losses to the pork industry. there are limited studies on the innate immune responses to ped virus (pedv) in pigs. the aims of our study were to investigate differences in innate immune responses to pedv infection in suckling and weaned pigs and to examine if disease severity coincides with reduced innate immune responses. weaned 26-day-old pigs (n = 20) and 9-day-old nursing pigs (n = 20) were assigned to pedv inoculated or uninoculated control groups. the pigs were observed daily for clinical signs, virus shedding and were euthanized at post-inoculation days (pids) 1 and 5 to assay immune responses. blood samples were collected at pids 1, 3 and 5. the natural killer (nk) cell frequencies, nk cell activities (lysis of target k562 tumor cells in vitro), cd3+cd4+ t cell and cd3+cd8+ t cell frequencies were measured in blood and ileum at pids 1 and 5. the pedv infected suckling pigs showed severe diarrhea and vomiting at pid 1, whereas the pedv infected weaned pigs showed milder clinical signs starting at pid 3. pedv infected suckling pigs had significantly higher diarrhea scores, earlier fecal pedv rna shedding and significantly higher viremia (viral rna in serum) compared to weaned pigs. there was no mortality in either infected suckling or infected weaned pigs. the control pigs not inoculated with pedv did not show any clinical signs and no detectable fecal or serum pedv rna. strikingly, pedv infected suckling pigs had significantly lower nk cell frequencies, undetectable nk cell activity and lower ifnγ producing nk cells in blood and ileum compared to pedv infected weaned pigs. pro-inflammatory cytokine profiles of pedv infected suckling pigs differed from those of pedv infected weaned pigs and coincided with onset of fecal pedv rna shedding and serum pedv rna titers. the infected suckling pigs have higher and earlier increases in serum ifnα, but lower serum il-8 and tnfα levels compared to infected weaned pigs. cd3+cd4+ t cell frequencies were significantly higher in ileum of suckling pigs than in weaned pigs, whereas there was no difference in cd3+cd8+ t cell frequencies. in conclusion, the observations of impaired lytic activity and ifn-γ production by nk cells in suckling pigs coincided with the increased severity of pedv infection in the suckling pigs compared with the weaned pigs. porcine epidemic diarrhea virus (pedv) is an enteric coronavirus (genus alphacoronavirus, family coronaviridae, order nidovirales) causing significant morbidity and mortality in suckling pigs. pedv was first diagnosed in the usa in may, 2013 (stevenson et al., 2013) and has spread throughout the usa and was also reported in mexico and canada (vlasova et al., 2014) . the estimated annual economic losses in the us from pedv is $900 million to $1.8 billion (paarlberg, 2014) . pedv causes severe enteric disease in suckling pigs (chen et al., 2014; stevenson et al., 2013) , but milder disease in older weaned pigs . this is similar to earlier observations for another enteric coronavirus infection of pigs, transmissible gastroenteritis (tge) (saif et al., 1994) . therefore, similar to tge virus (tgev) infection, biofeedback of intestinal contents of affected pigs to older pigs to build herd immunity is considered as an important method to reduce losses from pedv (jung and saif, 2015) . viral infections induce both innate and adaptive immunity. innate immunity involves production of cytokines and interferons as well as recruitment of innate immune cells such as http://dx.doi.org/10.1016/j.vetimm.2015.09.006 0165-2427/© 2015 elsevier b.v. all rights reserved. dendritic cells, macrophages and natural killer (nk) cells (rouse and sehrawat, 2010) . the innate immune response plays a significant role in controlling primary viral infections and in development of adaptive immune responses (aoshi et al., 2011; janeway and medzhitov, 2002) . nk cells are innate immune cells that display cytotoxic action against virus infected host cells and tumor cells (campbell and hasegawa, 2013; herberman et al., 1975; trinchieri, 1989) and thus play an important initial role in containing the viral infection. nk cells are also a major source of certain cytokines such as ifn␥ and tnf␣ (fauriat et al., 2010; vivier et al., 2008) . they play a key role in initial clearance of infection in viral diseases (brandstadter and yang, 2011) . cytokines are important in viral infections in that they are necessary for cell to cell communication for inflammation and immune responses (akira and kishimoto, 1992) . the early cytokines secreted during a viral infection help to modulate immune responses. the cytokines examined in this study are early cytokines that have mainly proinflammatory and antiviral action. interferons are a group of cytokines whose major function is antiviral activity (isaacs and lindenmann, 1957; wheelock, 1965) . ifn␣ is a type i interferon produced by most cells in response to viral infection, with the major source being innate immune cells such as monocytes and dendritic cells (trinchieri et al., 1978) . ifn␥ is a type ii interferon produced initially by innate immune cells such as macrophages, dendritic cells and nk cells, and later on by activated t cells (sen, 2001) . ifn␥ is important in enhancing the activities of phagocytic cells such as macrophages and nk cells (carnaud et al., 1999) . il-8 is produced by various cell types and is a proinflammatory cytokine due to its chemoattractive properties for inflammatory cells (arndt et al., 1996; huber et al., 1991) . il-17 is a proinflammatory cytokine secreted by th17 cells, as well as ␥␦t cells (innate immune cells in mucosa) (jin and dong, 2013) . it stimulates the inflammatory response to viral infections (ryzhakov et al., 2011) . il-12 is a proinflammatory cytokine produced mainly by phagocytic cells and is involved in activation of nk cell activity including ifn␥ production by nk cells (trinchieri, 1995) . tnf␣ is a proinflammatory cytokine secreted mainly by macrophages that regulates cell death, differentiation and inflammation (bradley, 2008) . studies of rotavirus infection of children showed that the cytokine responses varied depending on severity of clinical signs in individuals (jiang et al., 2003) . studies of human rotavirus infected gnotobiotic pigs showed similarly that the proinflammatory cytokine responses were more marked with virulent virus compared with attenuated virus infection (azevedo et al., 2006) . the above cytokines were examined in the present study to understand if differences in proinflammatory cytokine responses between suckling and weaned pigs may be involved in susceptibility of suckling pigs to severe disease by pedv infection. there is a lack of information on innate immune responses of young and older pigs to pedv infection that might explain some of the differences in disease severity between young and older pigs. in the present study, we investigated the innate immune responses such as cytokine and nk cell activity as well as changes in frequencies of t cells to examine if differences coincide with the higher disease severity of suckling versus weaned pigs. the virus inoculum used in this study was the wild-type virulent us pedv strain pc21a which was from the intestinal contents of a pedv positive field piglet, then serially passaged two times in gnotobiotic pigs (jung et al., 2014) . the original sample was negative by pcr for other enteric viruses such as tgev/porcine respiratory coronavirus (prcv), porcine deltacoronavirus, rotavirus groups a, b, and c, porcine enteric caliciviruses, st-valerien-like viruses, porcine astroviruses, enterovirus, kobuvirus, and bocavirus (amimo et al., 2013a,b; chung et al., 2005; jung et al., 2015b; kim et al., 2000; sisay et al., 2013; wang et al., 2011) . immune electron microscopy of the original sample using gnotobiotic pig hyperimmune serum to pedv showed only pedv particles. the gnotobiotic pig passaged pc21a intestinal contents were diluted in minimum essential medium (mem) and used as inoculum in this study as noted below. seronegative pregnant sows and 26-day-old, pedvseronegative weaned, large white × duroc crossbred pigs were obtained from a pedv-free specific pathogen free (spf) (confirmed by history, lack of qrt-pcr-pedv positive fecal samples and pedv antibodies) swine herd of the ohio state university. the spf osu herd was also seronegative for antibodies to porcine respiratory and reproductive syndrome virus, prcv, tgev and porcine circovirus type 2. the sows farrowed naturally and nursed their piglets until the end of the study. the four experimental groups in the study were as follows. group 1: pedv inoculated 9-day-old suckling pigs (n = 9); group 2: mem only inoculated 9-day-old suckling pigs (n = 11); group 3: pedv inoculated 26day-old weaned pigs (n = 11); group 4: mem only inoculated 26-day-old weaned pigs (n = 9). all experimental procedures on animals were approved by the institutional animal care and use committee of the ohio state university. pigs in pedv groups were inoculated orally with pedv inoculum [8.9 log 10 ge (genomic equivalents) (≈2.9 log 10 plaque forming unit)/pig] and pigs in mem only inoculated groups received mem. the inoculation dose was based on a previous pathogenicity study in our lab (jung et al., 2014) . following pedv inoculation, pigs were monitored for clinical signs daily until necropsy. diarrhea was assessed by scoring fecal consistency. fecal consistency was scored as, 0 = solid; 1 = pasty; 2 = semi-liquid; 3 = liquid, with scores of 2 or more considered diarrheic. inoculated and mock pigs (n = 3-4/group at each time-point) were euthanized for immunological studies at an acute stage on post inoculation day (pid) 1 and at a later stage (pid 5) of infection. blood samples were taken at pid 1 (n = 6-8 pigs per group), pid 3 (n = 4-8 pigs per group) and pid 5 (from euthanized pigs, n = 3-4 per group) and separate serum aliquots were prepared for cytokine analysis and viral rna quantification. rectal swabs were collected from all pigs on the designated pids to determine fecal ped viral shedding (pedv rna quantified by rt-qpcr). two rectal swabs were suspended in 4 ml mem (jung et al., 2014) . the rna was extracted from 50 l of serum or supernatants following centrifugation of the fecal suspensions (2000 × g for 30 min at 4 • c), using the mag-max viral rna isolation kit (applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. pedv rna titers in rectal swab supernatants and sera were determined by rt-qpcr as described previously (jung et al., 2014) . blood and ileum were collected on the day of euthanasia and processed for isolation of mnc as previously described (yuan et al., 1996) . the isolated cells were resuspended in rpmi medium (roswell park memorial institute medium) containing 8% fetal bovine serum, 2 mm l-glutamine, 1 mm sodium pyruvate, 0.1 mm suckling pigs had more severe clinical signs, earlier fecal pedv rna shedding and higher serum pedv rna compared to weaned piglets. following pedv inoculation, pigs were monitored every day and rectal swabs and blood for serum were collected at pid 1, 3 and 5. fecal consistency scores of 2 or more considered diarrheic. fecal shedding and serum pedv rna were determined by rtqpcr. the detection limit of rt-qpcr was 10 genomic equivalents (ge) per reaction, corresponding to 4.8 log10 ge/ml of rectal swab fluid or 3.8 ge/ml of serum. therefore, viral rna titers more than 4.8 log10 ge/ml in rectal swab fluid or 3.8 ge/ml in serum are considered positive. statistical analysis was done to compare suckling and weaned pigs (across rows) for different parameters measured. values with different alphabetical superscript within a parameter and time point are considered different at p < 0.05. the uninoculated pigs did not show any clinical signs. the rectal swab fluids and serum of uninoculated pigs were tested at similar time points and no pedv rna was detected. nonessential amino acids, 20 mm hepes and antibiotics (e-rpmi) and used for assays. k562 (human erythroleukemia cell line) tumor cells were used as target cells and the assay was done as described previously with a few modifications (cao et al., 2013; park et al., 2013) . the k562 cells were initially stained with carboxy fluorescein succinimidyl ester (cfse) (ebioscience, usa), washed and used for the assay. mncs from blood and ileum were used as effector cells. effector: target cell ratios of 25:1, 12.5:1 and 6.25:1 were used. the cells were mixed at the specified ratios and incubated overnight in e-rpmi at 37 • c. the cells were then incubated with 7-aminoactinomycin d (7-aad) (life technologies, usa) for 15 min at 4 • c to stain dead cells. the cells were examined by flow cytometry and the percentage of cfse positive cells that were also stained with 7-aad were assessed as dead k562 cells. cfse labeled k562 cells incubated without mncs and stained similarly with 7-aad were used as controls for spontaneous death of k562 cells. the procedure was followed as described previously (chattha et al., 2013; yuan et al., 2008 ) with a few modifications. mononuclear cells from blood and ileum were cultured for 18 h at 37 • c in e-rpmi. the protein transport inhibitor, brefeldin a (10 mg/ml; sigma-aldrich, usa), was added for the last 5 h to prevent secretion of ifn␥ produced by the cells. the cells were stained with cd3-fitc (fluorescein isothiocyanate) (clone ppt3; southern biotech, birmingham, al, usa), cd8-sprd (spectral red) (clone 76-2-11; bd biosciences, usa), and cd4-biotin followed by streptavidin apc (allophycocyanin) (bd biosciences, usa) as secondary antibody. samples were stained intracellularly with anti-porcine ifn-␥-pe (phycoerythrin) (clone p2g10; bd biosciences, usa). cd3-cd4-cd8+ ifn-␥+ cells were expressed as percentage of cd3-cd4-cd8+ nk cells. isotype antibody-labeled cells were used as controls. to determine the frequencies of t helper cells (cd3+cd4+), cytotoxic t cells (cd3+cd8+) and nk cells (cd3-cd4-cd8+), cell samples were stained with anti-porcine cd3-fitc, cd4-pe (clone 74-12-4; bd biosciences), and cd8-sprd for 15 min at 4 • c. the frequencies of t cells or nk cells were expressed as percentage of lymphocytes expressing the respective markers. cells stained with isotype antibodies were used as controls. serum was separated by centrifuging blood at 1850 × g for 15 min, and the collected serum was stored at −20 • c until tested. il-12, il-8, il-17 and ifn␣ were measured as previously described (azevedo et al., 2006; chattha et al., 2013) . for tnf␣, a porcine tnf␣ elisa kit was used per manufacturer's recommendations (kingfisher biotechnologies, st. paul, mn). all values are expressed as the means ± standard error of the means (sem). fecal consistency scores and viral rna titers in rectal fluids were analyzed and compared by a student's t-test using graphpad prism software (graphpad prism inc.). nk cell activity, nk cell numbers, t cell numbers and cytokine amounts were analyzed by one-way anova using graphpad prism software. a value of p < 0.05 was considered statistically significant. 3.1. suckling pigs had more severe clinical signs, earlier fecal pedv rna shedding and higher serum pedv rna titers compared to weaned pigs the suckling pigs showed severe diarrhea and vomiting at pid 1, whereas the weaned pigs showed milder clinical signs starting only at pid 3 ( table 1 ). the fecal consistency scores were significantly higher in suckling pigs compared to weaned pigs at all time points examined (p < 0.05) ( table 1 ). the fecal shedding pedv rna titer was high in suckling pigs starting from pid 1, whereas the weaned pigs started shedding pedv rna at pid 3 and shed at significantly higher titers at pid 5 compared to the suckling pigs (p < 0.05) ( table 1 ). the serum pedv rna titers were significantly higher in suckling pigs at all time-points sampled ( table 1 ). the higher severity of disease in suckling pigs coincided with the higher serum pedv rna titers in suckling pigs compared to weaned pigs. the mem only inoculated control pigs had no detectable pedv rna in either serum or feces. the suckling pigs had no detectable nk cell activity in blood and ileal mncs regardless of pedv infection, whereas the pedv infected and uninfected weaned pigs had low, but detectable nk cell activity (percentage of target lysis: 0.3-1.3) at pids 1 and 5 (fig. 1a-d) . suckling piglets had lower nk cell activity (% lysis of k562 cells) compared to weaned piglets in blood and ileal mononuclear cells at pid 1 (a and b) and pid 5 (c and d). blood and ileal mononuclear cells were co-cultured with cfse stained k562 cells at indicated ratios overnight. the dead cells were stained by incubating with 7-aad. the cells were observed by flow cytometry to obtain % of dead k562 cells. the groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. the bar graphs labeled with different alphabetical letters are significantly different (p < 0.01). the infected weaned pigs had significantly increased nk cell activity in ileum at pid 5 compared to uninfected weaned pigs (p = 0.01) (fig. 1d ). the nk cell activity was similar for infected versus uninfected weaned pigs at pid 1 ( fig. 1a and b ). this coincides with the delayed onset of virus shedding in the infected weaned pigs. the nk cell activity in ileum of uninfected weaned pigs was about 2-fold higher than in the blood of uninfected weaned pigs (p < 0.01). the nk cell activity in ileum of infected weaned pigs was about 5-fold higher than in the blood of infected weaned pigs at pid 1 (p < 0.05) ( fig. 1a and b) and about 7-fold higher at pid 5 (p < 0.0001) ( fig. 1c and d). there was no significant difference in the nk cell activity of blood mncs of infected and uninfected weaned pigs at pids 1 and 5 ( fig. 1a and c). the increase in nk cell activity was not correlated to the increase in nk cell frequencies in the ileal mncs. 3.3. weaned infected pigs had significantly higher nk cell frequencies in blood and ileum at pid 5 compared to suckling infected pigs the uninfected weaned pigs had significantly higher blood nk cell frequencies compared to uninfected suckling pigs (p < 0.05) ( fig. 2a) . the infected weaned pigs also had significantly higher nk cell frequencies in blood and ileum at pid 5 (p < 0.05) compared to infected suckling pigs ( fig. 2a and b) . at pid 1, the infected weaned pigs had similar nk cell frequencies in blood to uninfected weaned pigs. at pid 1, the infected suckling pigs showed a significant increase in nk cell frequencies in blood compared to uninfected suckling pigs (p < 0.05) (fig. 2a) . the ileal nk cell frequencies were similar across groups at pid 1 (fig. 2b) . however, at pid 5, the infected weaned pigs had significantly higher nk cell frequencies in ileum compared to infected suckling pigs, although there was an increase in nk cell frequencies in ileum of infected suckling and weaned pigs compared to uninfected suckling and weaned pigs, respectively (p < 0.05) (fig. 2b ). the nk cell frequencies of suckling and weaned pigs were higher in blood mncs than in ileal mncs at all time-points. 3.4. ifn producing cd3-cd4-cd8+ nk cell frequencies were significantly higher in blood of weaned pigs compared to suckling pigs at pid 1 and 5, regardless of pedv infection status and in ileum at pid 5 for pedv-infected pigs like for the nk cell activity ( fig. 1a and c) , the ifn␥ producing nk cells were undetectable in blood of suckling piglets (fig. 3a) . the infected weaned pigs had a significantly higher ifn␥ producing nk cell frequency in blood than uninfected weaned pigs at pid 1 (p < 0.03), although there was no difference between the two groups at pid 5. the ifn␥ producing nk cell frequency in ileum was similar in uninfected/infected suckling (pid 1) and weaned pigs (pid 1, 5) (fig. 3b) . at pid 1, the infected weaned pigs had similar ileal ifn␥ producing nk cell frequencies compared to infected suckling pigs. the infected suckling pigs had significantly lower ileal ifn␥ producing nk cell frequencies than the infected weaned pigs at pid 5 (p < 0.01). at pid 5, the ifn␥ producing nk cell frequency was significantly lower in the ileum of infected suckling pigs compared to uninfected suckling pigs (p < 0.05). however, in the weaned fig. 2 . weaned infected piglets had higher nk cell frequencies compared to suckling infected piglets in blood (a) and ileum (b) at pid 5. blood and ileal mncs were stained with anti-porcine cd3-fitc, cd4-pe and cd8-sprd. the nk cell (cd3-cd4-cd8+) frequency was expressed as percentage of lymphocytes. the groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. the different time points were also compared within an age group and tissue type. bar graphs labeled with no common alphabetical letter are significantly different (p < 0.05). infected pigs there was no significant reduction in the ileal ifn␥ producing nk cell frequency compared to uninfected weaned pigs. 3.5. pro-inflammatory cytokine profiles of pedv infected suckling pigs differed from pedv infected weaned pigs and coincided with fecal and serum pedv rna titers there was a marked induction of serum ifn␣ in infected suckling pigs at pid 1 which declined significantly thereafter. for infected weaned pigs, the response was highest at pid 3 (fig. 4a ). infected suckling pigs had significantly higher ifn␣ levels compared to infected weaned pigs at pid 1 (p < 0.001). at pids 3 and 5, there was no difference in the ifn␣ induction levels in infected weaned and suckling pigs. overall, the peak induction of serum ifn␣ in suckling pigs was at pid 1 and was much higher than the peak induction of ifn-␣ in weaned pigs which was at pid 3 (p < 0.01); both peaks coincided with the peaks of pedv rna shedding in feces and serum (suckling pigs) or serum (weaned pigs) and onset of diarrhea. the uninfected suckling and weaned pigs had similar low serum ifn␣ levels. serum il-12 levels followed a similar trend. il-12 levels of infected suckling pigs were highest at pid 1, whereas for infected weaned pigs, the induction was highest at pid 3 (fig. 4b) . there was no statistical difference in the peak il-12 levels between the infected suckling pigs at pid 1 and infected weaned pigs at pid 3. however, serum il-12 levels were significantly higher in infected suckling pigs compared to infected weaned pigs (p < 0.001) at pid 1. the infected weaned pigs had significantly higher serum il-12 fig. 3 . ifn␥ producing cd3-cd4-cd8+ nk cell frequencies were higher in weaned pigs compared to suckling pigs at pid 1 and 5 in blood (a) and at pid 5 in ileum (b). blood and ileal mncs were cultured in e-rpmi with protein transporter inhibitor, brefeldin a added. the cells were stained with cd3-fitc, cd8-sprd, and cd4-biotin followed by streptavidin apc as secondary antibody. samples were stained intracellularly with anti-porcine ifn␥-pe. flow cytometric analysis was done and the percentage of cd3-cd4-cd8+ nk cells that were also ifn␥ positive was calculated. the groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. the different time points were also compared within an age group and tissue type. bar graphs labeled with no common alphabetical letter are significantly different (p < 0.05). levels at pid 3 compared to infected suckling pigs (p < 0.01). the uninfected suckling and weaned pigs had similar serum il-12 levels at all days. overall, serum il-17 levels were much lower than for all other cytokines measured. serum il-17 levels were significantly higher only in infected weaned pigs compared to infected suckling pigs at pid 5 (p < 0.001) (data not shown). otherwise, there were no significant differences in the serum il-17 between weaned and suckling infected pigs at pids 1 and 3. the uninfected suckling and weaned pigs had similar serum il-17 levels. serum il-8 levels were significantly higher only in infected weaned pigs compared to infected suckling pigs at pid 5 (p < 0.01) (fig. 4c) . there was no difference in the serum il-8 between weaned and suckling infected pigs at pids 1 and 3. the uninfected suckling and weaned pigs had similar serum il-8 levels. serum tnf␣ levels also followed similar trends to ifn␣ including the pids of peak levels in infected weaned and suckling pigs. infected suckling pigs had highest tnf␣ levels at pid 1, whereas for infected weaned pigs, the response was highest at pids 3 and 5 (fig. 4d) . serum tnf␣ was significantly higher in weaned infected pigs than in infected suckling pigs at pids 3 and 5 (p < 0.001), whereas there was no significant difference in the serum tnf␣ between infected suckling and infected weaned pigs at pid 1. the peak tnf␣ level in weaned pigs at pid 5 was significantly higher than the peak tnf␣ level in suckling pigs at pid 1 (p < 0.01). in suckling and weaned pigs at pids 1, 3 and 5. blood samples were collected from the pigs at the specified time points and sera separated by centrifugation and frozen at −20 • c until use. the cytokine assays were done using standard assays. the groups that were compared were, infected suckling versus infected weaned at different time points, uninfected suckling or weaned versus infected suckling or weaned at different time points. the different time points within infected groups of suckling or weaned pigs were also compared. bar graphs labeled with no common alphabetical letter are significantly different (p < 0.01). the serum ifn␥, il-4 and tgf␤ levels were measured and there were no differences in their levels between the treatment groups (data not shown). 3.6. cd3+cd4+ t cell frequencies were higher in blood and ileum of suckling pigs than in weaned pigs, whereas there was no difference in cd3+cd8+ t cell frequencies in ileum of suckling and weaned pigs the cd3+cd4+ t cell frequencies in blood and ileum of uninfected suckling pigs were significantly higher than those in uninfected weaned pigs at pids 1 and 5 (p < 0.01) (fig. 5a and b) . however, the infected suckling pigs had statistically similar cd3+cd4+ t cell frequencies in blood at pid 1 compared to those of infected weaned pigs, whereas blood (fig. 5a) and ileum (fig. 5b ) of infected suckling pigs had higher cd3+cd4+ t cell frequencies compared to infected weaned pigs at pid 5 (p < 0.01). the cd3+cd4+ t cell frequency in blood did not differ between pids 1 and 5 within the infected or uninfected groups of suckling or weaned pigs. the cd3+cd4+ t cell frequencies in blood and ileal mncs were lower in infected suckling pigs compared to uninfected suckling pigs at pid 1 (p < 0.01) (fig. 5a and b) . the frequency fig. 5 . cd3+cd4+ cells were higher in ileum of suckling pigs than weaned pigs whereas there was no difference in cd3+cd8+ cells in ileum of suckling and weaned pigs. the graphs show the percentage of cd3+cd4+ cells in blood (a) and ileum (b) of suckling and infected pigs at pids 1 and 5, and the cd3+cd8+ cells in blood (c) and ileum (d) of suckling and infected pigs at pids 1 and 5. blood and ileal mononuclear cells were stained with cd3-fitc, cd4-pe and cd8-sprd for 15 min at 4 • c. the frequency of t cells or nk cells was expressed as percentage of lymphocytes. the groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. the different time points were also compared within an age group and tissue type. bar graphs labeled with no common alphabetical letter are significantly different (p < 0.05). of ileal cd3+cd4+ t cells was higher at pid 5 compared to pid 1 for suckling infected pigs (p < 0.05). the infected suckling pigs had transient relative leukopenia of cd3+cd4+ and cd3+cd8+ t cells at pid 1 compared to uninfected suckling pigs. there was no difference in the cd3+cd4+ t cell frequency in blood and ileal mncs in weaned infected and uninfected pigs at either time point. the cd3+cd8+ t cell frequency in blood mncs was significantly lower in infected suckling pigs compared to uninfected suckling pigs at pid 1 (p = 0.045). the infected weaned pigs had significantly higher frequency of cd3+cd8+ t cells in blood at pid 5 compared to pid 1. there was no difference in the cd3+cd8+ t cell frequency in blood mncs in infected and uninfected weaned pigs at either time point. there was no difference between the suckling and weaned uninfected pigs in cd3+cd8+ t cell frequencies in blood (fig. 5c ) and ileal (fig. 5d ) mncs. suckling infected pigs had significantly lower cd3+cd8+ t cell frequency in blood mncs compared to weaned infected pigs at pid 5. the cd3+cd8+ t cell frequency in ileal mncs was similar in uninfected suckling and weaned pigs. there was also no difference in the ileal cd3+cd8+ t cell frequency between the infected weaned and suckling pigs. the cd3+cd8+ t cell frequency in ileal mncs of infected weaned pigs was significantly higher compared to uninfected weaned pigs (p < 0.01) and the same was true for infected and uninfected suckling pigs (p < 0.01). the infected suckling pigs had significantly higher frequency of cd3+cd8+ t cells in ileum at pid 5 compared to pid 1. similarly, infected weaned pigs had significantly higher frequency of cd3+cd8+ t cells in ileal mncs at pid 5 compared to pid 1 (p < 0.05). in the present study, we investigated the differences between suckling and weaned pigs in innate immune responses to pedv infection and attempted to assess if these differences coincide with the greater severity of ped in suckling pigs. the major findings of this study were: (1) suckling pigs had earlier onset and more severe diarrhea and earlier fecal and higher serum pedv rna titers compared to weaned pigs; (2) pedv infected and uninfected suckling pigs had lower nk cell frequencies and no activity and lower ifn␥ producing nk cell frequencies compared to weaned pigs; (3) ifn␣ and pro-inflammatory cytokine profiles of pedv infected suckling pigs differed from pedv infected weaned pigs. in infected suckling and weaned groups, peak ifn␣, il-12 and tnf␣ levels coincided with onset of diarrhea and fecal pedv rna shedding and peak serum pedv rna titers (pids 1 and 3, respectively); and (4) frequencies of cd3+cd4+ t cells were significantly higher in ileum of suckling pigs than in weaned pigs, whereas there was no difference in frequency of cd3+cd8+ t cells. there was a transient relative leukopenia in cd3+cd4+ and cd3+cd8+ t cells in blood of suckling, but not weaned pigs at pid1. the nursing pigs showed severe clinical signs and fecal and serum pedv rna as early as pid 1 as reported previously (jung et al., 2014; stevenson et al., 2013) , but the weaned pigs showed mild and delayed clinical signs and delayed shedding of pedv rna in feces, in agreement with previous findings . in general, neonates of various species are more susceptible to severe disease than adults (camacho-gonzalez et al., 2013; rose, 1983; rosenberg et al., 1981; tregoning and schwarze, 2010; wohlfender et al., 2009 ). one of the reasons for the high susceptibility of neonates to severe disease from infections is deficient innate and adaptive immune responses/memory (levy, 2007; levy et al., 1999) . in the absence of previous exposure to pathogens, and therefore no adaptive immune responses, the newborn depends on innate immune responses to clear or reduce viral infection. in instances when the virus infection overwhelms the already weak innate immunity of the neonate, severe disease results (firth et al., 2005) . the virus levels in blood were directly related to severity of certain enteroviral diseases in infants (dagan et al., 1985; yen et al., 2007) as well as in other viral infections (maggi et al., 2003; vaughn et al., 2000) . interestingly in the present study, the moderate titer of pedv rna in serum of suckling pigs and 100-fold lower titers in weaned pigs coincided with the greater severity of disease in suckling pigs compared to weaned pigs. the severity of disease in suckling pigs also coincided with the early high titer pedv rna fecal shedding. porcine nk cells are identified as cd3-cd8+ cells (gerner et al., 2009) . suckling pigs had no detectable nk cell activity in blood or ileal mncs and the activity did not change with infection, although the suckling pigs had comparable nk cell frequencies in most instances (except pids of infected pigs) to weaned pigs. this is in confirmation of previous studies of nk cell frequency and cytotoxic activity of lymphocytes of healthy suckling pigs against k562 cells (onizuka et al., 1987; yang et al., 1987) . newborn pigs also did not have nk cell mediated cytotoxic activity against pk-15 cells persistently infected with tgev (cepica and derbyshire, 1984) . studies of tge showed that vaccination with a modified live tgev vaccine did not increase nk cell activity in newborn pigs (raymond and wilkie, 1998) , but in vitro stimulation of adult monocytes increased nk cell activity (charley et al., 1983) . in the present study, pedv infected weaned pigs showed increased frequencies as well as activity of nk cells in the ileum. although uninfected suckling pigs had comparable nk cell frequencies to weaned pigs, their frequencies were reduced in blood and slightly increased in ileum following infection, but nk cell activity remained undetectable. studies of human infants showed that neonates had significantly lower nk cell lytic activity and the activity was further reduced by infection or sepsis (georgeson et al., 2001; uksila et al., 1982) . nk cells of infants also had reduced antiviral activity such as defective killing of virus infected cells and reduced cytokine secretion (jacobson et al., 2013) . collectively, reduced nk cell activity and nk cell frequencies may have contributed to the early onset of virus shedding and the severe clinical signs in the suckling versus weaned pigs. coinciding with nk cell cytotoxic activity, the weaned pigs also had higher frequency of ifn␥ positive nk cells compared to suckling pigs. further, pedv infection reduced the frequencies of ifn␥ producing nk cells in ileum of suckling pigs. neonatal mncs are deficient in ifn␥ production in response to antigenic stimuli (wilson et al., 1986) . nk cells are the major source of ifn␥ and activation of nk cells by ifn␥ is essential for their lytic activity (wang et al., 2012) . studies have shown that the increase in frequency, activity of nk cells and ifn␥ production by nk cells is essential for reduction of viral load in mice infected with lymphocytic choriomeningitis virus or influenza virus (biron et al., 1983; mack et al., 2011; stein-streilein et al., 1988) . further, production of ifn␤ and ifn␥ in intestine of mice was essential and sufficient to induce innate immune cells to eliminate certain bacterial pathogens in mice (sotolongo et al., 2011) . since the suckling pigs showed reduced ifn␥ positive nk cell frequencies, this could result in reduced viral clearance contributing to the enhanced disease. similar to our observations, foot and mouth disease virus decreased the lytic activity of nk cells in swine (toka et al., 2009) . studies showed that il-18 production stimulates ifn␥ production and il-18 production is less in intestinal epithelium of neonatal pigs compared to older pigs (muneta et al., 2002) . thus, it appears that the failure to induce sufficient ifn␥ production by nk cells might be one reason for increased severity of disease and higher systemic load of pedv in suckling pigs. the weaned pigs had a delayed pro-inflammatory cytokine induction compared with suckling pigs which coincided with delayed infection, disease and shedding of pedv rna in feces of weaned pigs. ifn␣ is an antiviral cytokine produced in response to viral infection by monocytes and dendritic cells (hansmann et al., 2008; siegal et al., 1999) . consistent with fecal virus shedding and serum viral rna titers, the suckling pigs had the highest serum ifn␣ induction at pid 1. the higher systemic ifn␣ response in suckling pigs at the early stage of infection coincides with the serum pedv rna titer and very severe infection. collectively, lack of nk cell activity, reduced nk cell frequency and ifn␥ production might have contributed to the early onset of viral shedding, viremia (viral rna in serum) and severity of clinical signs. this is similar to the ifn␣ induction by tgev infection in suckling pigs (la bonnardiere and laude, 1981) . notably, treatment of newborn pigs with the interferon inducer, polyinosinic: polycytidylic acid complexed with poly-l-lysine, resulted in a delayed onset of clinical signs when pigs were infected with tgev (lesnick and derbyshire, 1988 ) and the ifn␣ treatment enhanced nk cell activity (charley et al., 1985) . also, treatment of tgev infected suckling pigs with oral human ifn␣ increased their survival rates (cummins et al., 1995) . in vitro studies of human dendritic cells stimulated with viruses show that neonatal cells are capable of producing ifn␣ responses similar to those of adults (renneson et al., 2009 ). the increased ifn␣ may be the result of infection in suckling pigs in the early stages and may have led to the reduced pedv rna shedding in feces at a later stage compared to the weaned pigs. the present studies showed that weaned infected pigs had higher il-8 at pid 5 and higher tnf␣ induction at pids 3 and 5 compared to suckling pigs. tnf␣ is a proinflammatory cytokine secreted by activated macrophages, nk cells, t cells and other cells (bradley, 2008) . serum levels of tnf␣ are often associated with severity of disease (jiang et al., 2003; waage et al., 1987) . studies of rotavirus infected children show that higher tnf␣ and lower ifn␥ are associated with more severe symptoms of infection (jiang et al., 2003) . similarly, studies of human rotavirus infection in a gnotobiotic pig model show that levels of cytokines like ifn␥ and tnf␣ are higher when pigs are infected with virulent virus compared to attenuated virus (azevedo et al., 2006) . human neonatal monocytes/macrophages stimulated in vitro with bacterial lipopolysaccharides or virus showed reduced tnf␣ responses when compared to monocytes/macrophages from adults (levy et al., 2004; valero et al., 2014) . in the present study, although the suckling pigs had more severe disease, their tnf␣ levels were similar to weaned pigs at pid1 and less than weaned pigs at pids 3 and 5. il-8 is a chemoattractant for neutrophils and is secreted by various cell types (bickel, 1993) . pigs infected with porcine respiratory and reproductive syndrome virus that subsequently cleared the infection had higher il-8 than pigs that did not clear the infection (kim and chae, 2003) . therefore, the higher il-8 in weaned pigs may have contributed to lower severity of disease. similar to the pattern of ifn␣, the serum levels of cytokines il-17 and il-12 were high in suckling pigs at pid 1 and in weaned pigs at pids 3 or 5 and coincided with the delay in onset of clinical signs and fecal pedv rna shedding in weaned pigs. studies of human neonates show that they are capable of producing certain pro-inflammatory cytokines in comparable levels to adults (schnurr et al., 2005) . therefore, suckling piglets could have a similar ability to produce these cytokines as the weaned pigs. thus, pro-inflammatory cytokine profiles of suckling and weaned pigs mirrored the severity of infection and viremia. the suckling pigs lacked innate immune activity to delay onset of viral shedding and viremia that resulted in more cells infected and higher ifn␣ (jung et al., 2015a) . in contrast, the robust innate immune activity in weaned pigs may have delayed the onset of viral shedding and viremia, resulting in a lower ifn␣ response. however, the innate immune response was not enough to prevent the higher fecal viral shedding at pid 5 in weaned pigs although there was reduced diarrhea compared to suckling pigs. the results also indicated the ability of suckling pigs to produce proinflammatory cytokines comparable to weaned pigs. cd4+ t cells represent t helper cells that aid in the humoral immune response and are responsible for th2 cytokine production. cd8+ t cells represent cytotoxic t cells that are important in killing virus infected cells (germain, 2002; gerner et al., 2009) . the cd3+cd4+ t cell frequency was higher in the uninfected suckling pigs compared to uninfected weaned pigs, whereas infected suckling pigs had transient relative leukopenia at pid 1 and then had increased cd3+cd4+ t cell frequencies at pid 5 compared to uninfected suckling pigs. neonates have a polarity toward th2 responses dominated by cd3+cd4+ t cells and this changes when they are given viral vaccines and it also changes with age (dowling and levy, 2014; kelly et al., 2007; kovarik and siegrist, 1998; siegrist et al., 1998) . the present study confirmed that the neonatal swine intestine has increased cd3+cd4+ t cells that can be altered by viral infection. the cd3+cd8+ t cell frequency was comparable in suckling and weaned uninfected pigs and infection increased the intestinal cd3+cd8+ t cell frequency in both groups. thus, the observed differences in pedv infectivity between suckling and weaned pigs might not be influenced by early cd3+cd8+ t cell frequency. since cd3+cd8+ t cells are involved in early antiviral adaptive immune responses, these cells are expected to play important roles in pedv infection of the intestine. longer observations are required to determine if there are differences in adaptive immune responses between the suckling and weaned pigs. in summary, 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neonatal cells. intrinsic and regulatory deficiencies diseases in neonatal foals. part 2: potential risk factors for a higher incidence of infectious diseases during the first 30 days post partum isolation and characterization of porcine natural killer (nk) cells viral load in blood is correlated with disease severity of neonatal coxsackievirus b3 infection: early diagnosis and predicting disease severity is possible in severe neonatal enterovirus infection systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease virus-specific intestinal ifn-gamma producing t cell responses induced by human rotavirus infection and vaccines are correlated with protection against rotavirus diarrhea in gnotobiotic pigs we thank dr. juliette hanson, andrew wright, megan strother, and ronna wood for assistance with care of experimental animals. we also thank xiaohong wang, kyle scheuer, dr sukumar kandasamy, bryan eyerly and john blakenship for technical assistance. salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development center, the ohio state university. this work was supported by a grant from the oardc seeds, grant # oaoh1536 (jung k, pi). key: cord-302819-oj33i2ma authors: pasick, j; berhane, y; ojkic, d; maxie, g; embury-hyatt, c; swekla, k; handel, k; fairles, j; alexandersen, s title: investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in canada date: 2014-08-07 journal: transbound emerg dis doi: 10.1111/tbed.12269 sha: doc_id: 302819 cord_uid: oj33i2ma summary: in january 2014, approximately 9 months following the initial detection of porcine epidemic diarrhea (ped) in the usa, the first case of ped was confirmed in a swine herd in south-western ontario. a follow-up epidemiological investigation carried out on the initial and 10 subsequent ontario ped cases pointed to feed as a common risk factor. as a result, several lots of feed and spray-dried porcine plasma (sdpp) used as a feed supplement were tested for the presence of pedv genome by real-time rt-pcr assay. several of these tested positive, supporting the notion that contaminated feed may have been responsible for the introduction of pedv into canada. these findings led us to conduct a bioassay experiment in which three pedv-positive sdpp samples (from a single lot) and two pedv-positive feed samples supplemented with this sdpp were used to orally inoculate 3-week-old piglets. although the feed-inoculated piglets did not show any significant excretion of pedv, the sdpp-inoculated piglets shed pedv at a relatively high level for ≥9 days. despite the fact that the tested pedv genome positive feed did not result in obvious piglet infection in our bioassay experiment, contaminated feed cannot be ruled out as a likely source of this introduction in the field where many other variables may play a contributing role. porcine epidemic diarrhea (ped) was first recognized in england in 1971 as an enteric disease affecting feeder and fattening pigs with less of an effect on suckling pigs (wood, 1977) . initially, it was this characteristic that distinguished it from transmissible gastroenteritis (tge). as the disease spread throughout europe, acute outbreaks of diarrhea began to be observed in pigs of all ages. in 1978, it was determined that this new type of epidemic viral diarrhea in pigs was caused by a coronavirus (debouck and pensaert, 1980) . this new coronavirus, porcine epidemic diarrhea virus (pedv), along with tge virus (tgev) have been classified into group 1 of the genus alphacoronavirus (song and park, 2012) . porcine epidemic diarrhea outbreaks were to 27 or more states (stevenson et al., 2013; chen et al., 2014; www.aasv.org/pedv/pedv_weekly_report_140319. pdf, accessed march 24, 2014). the initial usa pedv strain from april 2013 has been shown to phylogenetically cluster within subgroup iia along with a strain/lineage that was detected in china in 2012 (huang et al., 2013; stevenson et al., 2013; chen et al., 2014) . a second pedv strain, described by iowa state university (www.vetmed.iastate. edu/sites/default/files/vdpam/disease_ topics/swine-corona-viruses2-27-14_0.pdf, accessed march 7, 2014) and the ohio department of agriculture (wang et al., 2014) , was subsequently detected in late 2013/early 2014. based on the full-length spike (s) protein gene, this pedv strain clusters with another chinese lineage from 2010 to 2012 and is distantly related to the pedv strains that were initially detected in the usa, suggesting that multiple introductions, or a single introduction of multiple strains, of this virus have occurred. in late january 2014, the first case of ped was diagnosed in a swine herd in south-western ontario (manuscript accepted for publication). an epidemiological investigation of the ontario ped cases pointed to feed as a common risk factor. as a result, several lots of feed and spray-dried porcine plasma (sdpp) that was imported from the usa and used as a feed supplement were tested for the presence of pedv genetic material. several of these tested positive, supporting the possibility that contaminated feed may have been responsible for the introduction of pedv into canada (cfia news release, 2014; omaf, 2014; d. ojkic and g. maxie, personal communication) . this epidemiological link initiated bioassay trials to determine whether the sdpp supplement or the feed itself contained sufficient infectious pedv to infect weaned piglets under controlled laboratory conditions. nucleic acid extraction several nucleic acid extraction methods were employed depending on the sample matrix. all extractions included known positive and negative samples as well as a water negative control. for intestinal swab specimens taken at postmortem and placed in virus transport medium, 500-ll samples were extracted using the qiagen rneasy â mini kit (qiagen, mississauga, on, canada) following the manufacturer's instructions. this extraction method was used on the initial confirmatory submission received from the animal health laboratory, guelph. for rectal swab specimens taken from bioassay pigs and placed in phosphate-buffered saline (pbs), 50-ll samples were extracted using the mag-max tm -96 viral isolation kit (life technologies, burlington, on, canada) and a magmax tm express-96 instrument. this high-throughput extraction method was used to deal with the large number of samples that were generated during the bioassay experiment. for pelleted feed and sdpp samples, 10% (wt/vol) emulsions were first prepared in sterile pbs and then vortexed thoroughly until fully suspended/dissolved. three different methods were then evaluated for rna extraction: (i) magmax tm -96 viral isolation kit, (ii) qiagen rneasy â mini kit and (iii) tri-pure isolation reagent (roche diagnostic corporation, indianapolis, in, usa) . for the magmax tm -96 method, total rna was extracted from 50 ll of the unclarified 10% emulsion and eluted in 20 ll of nuclease-free elution buffer. for the qiagen rneasy â mini kit method, the 10% emulsion was clarified by brief centrifugation and total rna was extracted from 500 ll of the clarified supernatant and eluted in 50 ll of nuclease-free water. for the tripure method, 100 ll of unclarified emulsion was added to 900 ll of tripure isolation reagent and vortexed thoroughly. two-hundred microlitres of chloroform was then added, the mixture vortexed thoroughly for~30 s and incubated for 10 min. the aqueous and organic phases were separated by centrifugation and the aqueous phase added to an equal volume of isopropanol. the rna precipitate was then pelleted, and the pellet dried and dissolved in 20 ll of nuclease-free water. in an attempt to better determine the state of pedv in sdpp, a protocol using detergent treatment and differential centrifugation prior to magmax tm extraction was used. ten grams of sdpp was prepared as a 10% (wt/vol) suspension in pbs with or without 0.25% (vol/vol) nonidet p-40 (roche), a non-ionic, non-denaturing detergent. the suspension was first centrifuged at 10 000 g for 30 min at 4°c and the resulting supernatant was removed and centrifuged at 100 000 g for 1 h at 4°c. supernatants and pellets collected following both the 10 000 and 100 000 g centrifugation steps were then used for magmax extraction. total rna extracted by the various methods was screened using a real-time rt-pcr (rrt-pcr) assay that targets the nucleocapsid (n) gene of pedv (manuscript accepted for publication). the assay, consisting of forward primer pedn+2733-f: 5 0 -tatgctcagatcgccagt-3 0 , probe pedn+27354-pb: 5 0 -fam-gcaccaaatgttgcagcatt gct-bhq-1-3 0 , and reverse primer pedn-27395-r: 5 0 -cagccacattaccaccaaag-3 0 was run with two different chemistries -agpath-id tm one-step rt-pcr kit (life technologies) and the qiagen â one-step rt-pcr kit (qiagen) on two instrument platforms -smart cycler ii (cepheid, sunnyvale, ca, usa) and 7900ht sds (applied biosystems, foster city, ca, usa). each sample was amplified in a 25-ll rt-pcr reaction mix containing either 17 ll of agpath-id tm one-step rt-pcr mix or qiagen â one-step rt-pcr mix and 8 ll of rna. amplification was carried out for a total of 45 cycles. positive and negative controls as well as a notemplate control (water) were included in each run. conventional n and s gene rt-pcr followed by sequencing was used for confirmatory purposes on all diagnostic submissions and selected bioassay results. the primers used to amplify the partial n and s genes of pedv were based on those obtained from the national veterinary services laboratory (nvsl), ames, iowa with minor modifications. forward primer pedn253 -5 0 -ggcatttctactacc tcgga-3 0 and reverse primer pedn992 -5 0 -atagcct gacgcatcaacac-3 0 and forward primer peds218 -5 0 -gctagtggcgttcatggtat-3 0 and reverse primer peds442 -5 0 -taggcaattacgacctgttg were used to amplify 739 and 224 bp n and s1 gene products, respectively. these were then used directly for sequencing or gelpurified prior to sequencing or in some cases cloned prior to sequencing. for amplification of the full-length s gene of pedv, primer set s-f1 5 0 -tgctagtgcgtaataatgac-3 0 (pedv genome map position 20573-20592) and s-r1 5 0 -catctttgacaactgtgt-3 0 (pedv genome map position 24825-24842) described by huang et al. (2013) was used with a superscript ii rt/platinum taq one-step rt-pcr kit (invitrogen). the~4270-bp product was gelpurified and cloned using a clonejet tm pcr cloning kit (fermentas, burlington, on, canada). additional primer pairings that were also used to obtain s gene sequence information are as follows: s-f1 5 0 -tgctagtgcg taataatgac-3 0 (map position 20573-20592) and peds-2127-r 5 0 -acatatgcagcctgctctga-3 0 (map positions 22741-22760) that produced an~2187-bp amplicon and peds-mod-1581-f 5 0 -ccaaccttattgcatct-gac-3 0 (map positions 22214-22233) and s-r1 5 0 -cat ctt tgacaactgtgt-3 0 (map positions 24825-24842) that produced an~2628-bp amplicon. gel purification was carried out depending on the purity of the generated amplicons. sanger-sequencing reactions were performed using big dye terminator chemistry version 3.1 (foster city, ca, usa) and cycle-sequencing products were resolved on an applied biosystems 3130xl genetic analyser. virus isolation was carried out on vero cells as previously described (hofmann and wyler, 1988) with modifications. vero cells were cultured in dulbecco's minimal essential medium (dmem) high-glucose supplemented with 10% c-irradiated foetal bovine serum, 2 mm l-glutamine and 50 lg/ml gentamicin. confluent vero cell cultures in 25 cm 2 flasks were rinsed twice with pbs and then inoculated with 500 ll of the intestinal swab sample placed in virus transport medium or 10% (wt/vol) intestinal tissue emulsion. prior to inoculation, the intestinal swabs and intestinal tissue emulsions were treated with a 1009 antibiotic cocktail (109 final concentration) containing 10 000 international units/ml penicillin g, 10 mg/ml streptomycin, 10 mg/ml kanamycin, 5000 units/ml nystatin and 1500 units/ml polymyxin b sulphate at 1 : 10 for 30 min at room temperature. the 109 antibiotic cocktail treated swabs and emulsions were then clarified by centrifugation. after adsorbing the inoculum at 37°c with 5% co 2 for 1-2 h with continuous rocking, 4.5 ml of virus maintenance medium consisting of dmem supplemented with 2 mm l-glutamine, 0.33% (vol/vol) tryptose phosphate broth, 19 antibiotic cocktail and 1.25 lg/ml tpck-trypsin (sigma-aldrich, oakville, on, canada) was added to each flask. cultures were incubated at 37°c with 5% co 2 until 50-75% of the monolayer exhibited cytopathic effect (cpe) or for 4 days. isolation attempts involved a maximum of three passages. four percentage of paraformaldehyde was added to the tubes containing the plasma/feed, feces or the intestinal content samples taken from the bioassay pigs. the samples were then centrifuged for 20 min at 3800 g and negatively stained with 2% phosphotungstic acid. partial post-mortem examinations were performed and intestinal samples collected within 15 min (usually <5 min) following euthanasia. for each piglet, multiple sections of intestine (six sections of jejunum, six sections of ileum, one section of colon) were collected and fixed in 10% neutral phosphate-buffered formalin, routinely processed and stained with haematoxylin and eosin (he) for histopathological examination. for immunohistochemistry, 5-lm sections were cut, airdried overnight and placed into a 60°c oven for 1 h. the deparaffinized and rehydrated sections were quenched for 10 min in aqueous 3% hydrogen peroxide and rinsed in milliq water. epitopes were retrieved using dako target retrieval solution (dako, carpinteria, ca, usa) in a biocare medical decloaking chamber. once slides were cooled, they were placed into tris buffered saline plus tween (tbst; medimabs, montreal, qc, canada) for 5 min. the slides were incubated with mouse monoclonal antibody 66.31 (central veterinary institute wageningen ur, the netherlands) directed against the pedv s protein (van nieuwstadt and zetstra, 1991) at a dilution of 1 : 1500 for overnight incubation at 4°c. slides were rinsed with tbst and incubated for 30 min with an envision + anti-mouse kit (horseradish peroxidase labelled) (dako) and a tbst rinse. diaminobenzidine (dab; dako) was used as the substrate chromogen and the slides were counterstained with gill's haematoxylin. porcine epidemic diarrhea virus s protein antibodies were detected with a complex-trapping-blocking elisa that used monoclonal antibodies that were obtained from the central veterinary institute wageningen ur, the netherlands, and a protocol that was previously described by van nieuwstadt and zetstra (1991) with modifications. a british pedv isolate from 1987 (br1/87; have et al., 1992) , kindly provided by colleagues from the danish technical university-lindholm, denmark, was used to produce elisa antigen propagated in vero cells. samples with a blocking percentage >50% were considered positive, samples with <40% blocking were considered negative and samples between 40% and 50% blocking as dubious or suspicious. samples with initial results in the dubious/suspicious range were retested in the elisa and also tested by an immunofluorescence assay (ifa) using pedv br1/87 infected vero cells fixed with ice-cold methanol. forty 3-week old piglets were obtained from a single farm in manitoba (litters were weaned and mixed at the farm on the day of delivery but each animal was identified as to the sow of origin). piglets were randomly assigned (mixed on farm and selected at random at arrival) to four groups con-sisting of 12, 10, 10 and 8 piglets each. the groups were separately housed in containment level three animal cubicles at the national centre for foreign animal disease (ncfad) and cared for in accordance with canadian council on animal care guidelines and an animal use protocol approved by the institutional animal care committee. biosecurity features were used and standard operating procedures were followed to avoid cross-contamination among the four groups of pigs. each group of pigs was housed in physically separate animal cubicles with individually dedicated air systems. upon entry of each room, staff donned dedicated clothing and showered when exiting. additionally, staff worked in progressively 'infected' rooms, starting with the negative control room and finishing in the positive control room. following arrival but prior to inoculation, rectal swabs were taken from each piglet for pedv rrt-pcr testing and 5-ml blood taken to test for antibodies to pedv. a description of the different experimental groups is summarized in table 1 . feed and sdpp samples were processed as follows in preparation for inoculation. twenty-one grams of ground feed or sdpp was added to 210 ml pbs, mixed thoroughly and then divided into four aliquots of 50 ml for each piglet. the remaining 10 ml was stored at à70°c. animals received 25 ml of a feed or sdpp suspension via gastric tube while under light isoflurane anaesthesia and the remaining 25 ml orally after recovering from anaesthesia. three sdpp and two feed samples were tested with four piglets per sample as well as a positive and a negative control group. positive control animals received 50 ml (25 ml by gastric tube + 25 ml orally) of an intestinal tissue suspension positive for pedv. specifically, 2.0 ml of a 10% (wt/vol) tissue suspension of colonic tissue derived from the first-confirmed pedv case in ontario was diluted further in 500 ml pbs to give a final tissue dilution of 1 : 2500. negative control pigs received 50 ml pbs (25 ml by gastric tube + 25 ml orally). to prevent cross-contamination of the different inocula, the feed, sdpp and pedv positive intestinal tissue used as the positive control were all prepared in a biosafety cabinet that was thoroughly disinfected with a 1% virkon solution and allowed to clear for a minimum of 15 min between preparations. as an added precaution, the positive control inoculum from the pedv positive intestinal tissue was prepared last. at 7 days post-inoculation (dpi), the 10 negative control piglets were moved and allowed to co-mingle with the positive control (three contacts), sdpp (three contacts) and feed (four contacts) inoculated animals to assess transmission. piglets were observed daily for clinical signs, in particular vomiting or diarrhea, with the plan to euthanize any severely affected or moribund animals. rectal temperatures and swabs were taken daily from all animals. at 7 dpi, one piglet each from the positive control and the three sdpp inoculation groups were euthanized, postmortem examinations performed and various samples collected. at 12 dpi, one positive control piglet along with the three contacts (5 days post-contact) in that group were euthanized, post-mortem examinations performed and various samples collected. remaining animals were euthanized from 17 to 19 dpi and post-mortem examinations performed. a second bioassay experiment was run in parallel further evaluating the feed as a potential source of pedv (bioassay 2, table 2 ). these pigs were obtained from the same farm but housed separately from the first bioassay pigs and under similar conditions. forty-four 3-week-old piglets were randomly assigned to five groups, which included one group of 12 piglets that was mock inoculated and four groups of 8 piglets each that were inoculated with feed sample 1 as described above and with one of these four groups given the same feed (12.5 g per pig per day) as part of the normal daily feed for an additional 2 days. each of the four feed inoculated groups had an additional 4 piglets added in as contacts at day 2. in late january 2014, four suckling piglets that originated from a closed herd in south-western ontario were submitted to the animal health laboratory (ahl), university of guelph. this herd had a history of sudden onset of diarrhea in piglets less than a week of age and based on laboratory test results a diagnosis of ped was made (manuscript accepted for publication). briefly, intestinal swab specimens were tested for the presence of porcine respiratory coronavirus (prcv), transmissible gastroenteritis virus (tgev) and pedv by a real-time rt-pcr assay with all four swab samples producing strong positive results (c t s = 20.47, 21.85, 22.92 and 25.96) on the pedv n gene rrt-pcr assay. samples were forwarded to the national centre for foreign animal disease (ncfad) in winnipeg where the diagnosis of pedv was confirmed by pedv n and s gene conventional rt-pcr assays and sequencing. based on partial sequence information for the n and s genes, the ontario pedv isolate was found to be 99.8% identical to pedv isolated in the usa in 2013. an additional 10 cases that followed the ontario index case were submitted by ahl, guelph to ncfad, winnipeg for confirmation. all were confirmed by pedv n and s gene conventional rt-pcr assays with one of the submissions yielding a virus isolate. an epidemiologic investigation carried out on the index and 10 subsequent cases by the ontario ministry of agriculture and food (omaf) indicated feed as a common risk factor (cfia news release, 2014; omaf, 2014; d. ojkic and g. maxie, personal communication). this led ahl guelph to test lots of sdpp that were imported from the usa and the associated feed in which they were used as an additive for pedv nucleic acid by rrt-pcr assay. several of these gave positive results including the lot mentioned below (data not shown). five samples of sdpp from different pallets of a single lot and five feed samples that contained 6% (wt/wt) of the aforementioned plasma that were epidemiologically linked with the first cases of ped in south-western ontario, were tested by pedv n gene rrt-pcr and s gene conventional rt-pcr assays. at ncfad, all five sdpp samples gave weak positive reactions (c t s of 36. 35, 36.2, 36.97, 36.65 and 36.69) on the n gene rrt-pcr assay. these were also positive by s gene rt-pcr with three of the samples considered as moderately strong positives producing clearly visible bands of the correct size. 41.29, 40.55, 40 .21 on the second run. when the feed sample that was initially positive on the n gene assay was retested, only one of the five aliquots produced a c t value on first (39.85) and second (37.22) runs indicating that the levels of pedv nucleic acid in this sample are at the limit of detection. the~224 s1 gene amplicons from the five sdpp samples were sequenced and found to be 100% identical to one another. although the sequence obtained for the amplicon from the positive feed sample was a match to pedv (70-90% match to pedv sequences in genbank by using blast), the quality of the sequence was too poor (below 80% quality values) for accurate comparisons. attempts made to clone this amplicon to obtain better sequence data were not successful. in an attempt to better understand the state in which pedv exists in sdpp, a protocol involving treatment with the non-ionic, non-denaturing detergent nonidet p-40 followed by differential centrifugation was employed. ten grams of sdpp was used to produce a 10% (wt/vol) suspension in pbs containing or not containing 0.25% (vol/vol) nonidet p-40. this was initially centrifuged at 10 000 g and the resulting supernatant centrifuged at 100 000 g to sediment intact virus or viral nucleocapsids. on the sdpp sample that was tested, the following n gene rrt-pcr results were observed: c t of 35.84 for pbs supernatant after 10 000 g, c t of 36.74 for the pbs pellet after 10 000 g, c t of 38.83 for the pbs + nonidet p-40 comparison of s protein gene sequences obtained from bioassay piglets versus those of field cases. one-hundred and eighty-eight nucleotides of the~224-bp-small s gene conventional rt-pcr amplicon (primers excluded) obtained from rectal swab samples of bioassay piglets and canadian and usa field cases were aligned. ncfad 2014-022 is the identical sequence of the 5 sdpp samples sequenced directly. plasma #21-22, #25-26, #29-30 and #32 are from sdpp-inoculated bioassay piglets. ontindex is the ontario index case. ncfad2014-18 #1-10 are the 10 ontario field cases that followed the index case. pellet # 35 is from a rectal swab specimen taken at 3 dpi from piglet # 35 of the feed group. pei is from a field case from prince edward island. ncfad 2014-35 is from a field case from quebec. mafri is from a field case from manitoba and ncfad 2014-41 is from an environmental sample from manitoba. colorado (kf272920) and indiana (kf452323) are usa pedv isolates. supernatant after 10 000 g, c t of 36.74 for the pbs pellet after 100 000 g, and c t of 35.94 for the pbs + nonidet p-40 pellet after 100 000 g. c t values of 35.94 and 36.74 for both nonidet p-40 treated and untreated 100 000 g pellets are an indication that the plasma sample contained intact virions or at least viral nucleocapsids (risco et al., 1996; krempl and herrler, 2001) . finally, no coronavirus-like particles could be identified by direct electron microscopical examination of sdpp or feed samples. no clinical signs were observed in the negative control group. in contrast, animals in the positive control group were depressed, off feed and had diarrhea/soft feces beginning at 1 dpi and a small amount of vomiting noted at 11 dpi (4 days post-contact for contact animals). rectal temperatures, however, remained within the normal range (38.7-39.8°c). the piglet group inoculated with the three sdpp samples had a few piglets with diarrhea or soft feces beginning at 2 dpi onwards, however, rectal temperatures were also within the normal range. the piglet group inoculated with the two feed samples were mildly depressed with one or two animals with diarrhea or soft feces beginning at 1 dpi onwards and decreased feed intake from 3 to 7 dpi. rectal temperatures as with the other experimental groups were within the normal range. rectal swab samples collected from all of the piglets prior to inoculation were pedv n gene rrt-pcr negative. all of the negative control piglets remained n gene rrt-pcr negative up to and including 7 dpi at which time they were moved in with the other experimental groups as non-inoculated contacts. the eight piglets inoculated with the feed samples along with the four contact piglets introduced into this group beginning at 7 dpi also remained negative up to and including 17 dpi (day 10 post-contact). in contrast, piglets in the positive control and sdpp-inoculated groups were pedv n gene rrt-pcr positive beginning at 1-2 dpi and remained positive until 7 dpi for all piglets and beyond for many of them (fig. 1a,b) . some of the contact piglets introduced at 7 dpi became positive beginning at 3 days post-contact (fig. 1c,d) . no significant difference was observed in the kinetics of n gene rrt-pcr positivity in animals that were inoculated with the three sdpp samples that were tested, suggesting that each contained infectious virus. however, as these piglets were housed in the same animal cubicle, we cannot unequivocally state that all samples were infective but that at least one of the three samples contained infectious virus. negative contrast staining electron microscopy of fecal samples collected at 4 dpi from the sdpp-inoculated piglets and the positive control group piglets showed the presence of virus-like particles consistent with coronavirus virions. similar virus-like particles were also found in the content of the small intestine of a sdpp-inoculated piglet at 7 dpi and a positive control group contact piglet at 5 days post-contact. no virus-like particles were observed in fecal samples from the feed-inoculated group. the partial s1 gene rt-pcr was also carried out on rectal swabs collected from the sdpp and feed-inoculated piglets at 3 and 4 dpi to confirm the n gene rrt-pcr results described above. rectal swabs from all 12 sdppinoculated piglets were strongly positive while one of the feed-inoculated piglets (piglet 35) produced a very weakly visible amplicon of the correct size, and a few others produced weakly visible amplicons of a different size indicating non-specific amplification. seven amplicons from the sdpp-inoculated piglets (piglets 21, 22, 25, 26, 29, 30 and 32) at 3 dpi, the weakly visible amplicon from feed-inoculated piglet 35, and spurious bands from the remaining seven feed-inoculated piglets were gel-purified and sequenced. all 7 amplicons from the sdpp-inoculated piglets were pedv with sequences that were similar to the strain found in the usa since april/may 2013, detected in the initial ontario field cases and in the sdpp samples (fig. 2) . the sequence from feed-inoculated piglet 35 was also pedv but of poor quality and difficult to analyse. the seven spurious amplicons selected for sequencing were unrelated to pedv (e.g. thermoplasmatales archeon and candidatus methonomethylophilus using blast). the weak amplicon from piglet 35 was cloned and of the 8 clones sequenced, one had a typical pedv sequence (188/ 188 to pedv strain usa/indiana/17846/2013 genbank accession no. kf452323) similar to those from the sdppinoculated piglets, indicating that the 3 dpi rectal swab from piglet 35 contained pedv rna/virus, albeit at trace levels. sequence results are summarized in fig. 2 . the sequences are identical with the exception of a singlenucleotide polymorphism (snp) at nucleotide 20 961 (the s gene maps from nucleotide 20 634 to 24 794 of the pedv genome). individual sequences were found to have a c, a t or both at this site, consistent with a mixed population as indicated by some of the plasma-inoculated piglets. this snp results in either a histidine (c at 20 961) or a tyrosine (t at 20 961) at amino acid 100 of the s protein. the full s gene sequence was obtained from a rectal swab collected at 3 dpi from one of the sdpp-inoculated piglets (piglet 30; genbank accession no. km196110). comparison of this sequence to that obtained from the ontario index case (genbank accession no. km189366), the ontario pedv isolate (genbank accession no. km189367), a single case from prince edward island (genbank accession no. km189368) which was also epidemiologically linked to the same feed and sdpp as the ontario cases, and to those reported initially in the usa (e.g. usa/colorado/2013 and usa/indiana/17846/2013; genbank kf272920 and kf452323) showed them to be 99.8-99.9% identical at both the nucleotide and amino acid levels. comparison of the ontario index case and sdpp piglet 30 s gene sequences revealed 5 synonymous and 2 non-synonymous (h110y and s1099a) changes. comparison of the ontario pedv isolate and sdpp piglet 30 s gene sequences showed that they differed by 1 synonymous and 1 non-synonymous (h110y) change, while the prince edward island and sdpp piglet 30 s gene sequences were 100% identical at the nucleotide and amino acid levels. overall, this is consistent with a somewhat mixed population of genomes, but all with a very high degree of similarity to the sequences reported from the initial outbreaks in the usa. of the 3 piglets from the sdpp-inoculated group on which post-mortem examinations were performed at 7 dpi, piglet 26 had a significant enteropathy characterized by a small intestine that was flaccid, thin walled in some areas, and filled with fluid mixed with brown flecks. all three piglets had some degree of watery intestinal content, which primarily affected the lower jejunum and the ileum. microscopically, small intestinal villi were shortened and often had a 1 : 1 crypt to villus ratio (fig. 3a) . there was a mild increase in inflammatory cells in the lamina propria, dilation of lymphatics and submucosal edema. in some areas, enterocytes had cuboidal morphology and were vacuolated (fig. 3b) . pedv antigen was detected by ihc within enterocytes of all 3 animals with moderate numbers of positive enterocytes observed in piglet 32 and occasional positive enterocytes observed in piglets 22 and 26 (fig. 3c,d) . no pathological changes or pedv antigen staining were observed in the remainder of the piglets in the sdpp group that were examined at 18 dpi, which included contact animals at 11 days post-contact. of the animals in the feed-inoculated group that were examined at 7 dpi, the intestinal walls appeared normal although some of them had watery intestinal content. none of the contact animals in this group that were examined on post-contact day 12 had any significant lesions. microscopically, some sections of intestine showed normal length of the small intestinal villi (1 : 3 crypt to villus ratio), but in other areas, villi were shortened and there was a mild increase of inflammatory cells in the lamina propria. the positive control piglet examined at 7 dpi (piglet 12) had thin-walled small intestines that were flaccid and fluid filled. microscopically, the intestinal villi were blunted with a 1 : 1 crypt to villus ratio in some areas. the surface enterocytes were low columnar to columnar with an easily discernible brush border. a mild increase in inflammatory cells within the lamina propria was also observed. two of the positive control group contact animals examined on day 5 post-contact (piglets 8 and 9) had a significant enteropathy characterized by flaccid, distended fluid-filled intestines. microscopic lesions were similar to those described above for the sdpp-inoculated group except for the presence of significant necrotic debris in the lumen, which was only observed in the positive control day 5 post-contact animals (fig. 3e) . pedv antigen was detected extensively within enterocytes, primarily within the brush border but also within the cytoplasm of the cell body (fig. 3f) . although no significant gross pathological changes were observed in the intestinal wall of the positive control piglet examined at 12 dpi (piglet 17), the intestinal contents were still watery. no significant macroscopic lesions, microscopic changes, or pedv antigen staining were observed in the three positive control group pigs examined at 19 dpi. because the negative control group from this bioassay was co-mingled with the experimental groups, negative control piglets were not available for microscopic examination. thus, negative control piglets from the second bioassay experiment euthanized at 12 dpi were used for the histopathology and immunohistochemistry observations. microscopically, there were some areas where the small intestinal villi appeared shortened and there was some evidence of mild inflammation in the lamina propria (fig. 3g) . ihc was performed on tissues from 5 of the negative control animals, and pedv antigen was not detected (fig. 3h) . in summary, the only significant finding in relation to the bioassay was the presence of significantly shortened villi and the presence of pedv antigen in both the sdpp group of piglets at 7 dpi and the positive control contact animals at 5 days post-contact. overall, there was some indication of mild enteritis in some piglets from all groups. this latter finding is considered non-specific and could be due to a number of factors. however, it is important to note that in negative controls, this inflammation was not associated with pedv infection as no antigen was detected and all other test results showed these piglets to be negative for pedv. serum samples from all 40 piglets collected before inoculation were negative for antibodies to pedv as determined by complex-trapping-blocking elisa. the ten piglets in the negative control group were serologically negative at 7 dpi and remained so throughout the remainder of the experiment. all eight piglets in the feed-inoculated group were negative for pedv antibodies at 7, 14 and 17 dpi. in the sdpp group, 4 of 12 piglets (piglets 22, 24, 31 and 32) were dubious/suspicious reactors on day 7 and, of the two samples tested on day 14, one was positive by elisa and ifa (piglet 29) and the other a dubious/suspicious reactor in the elisa and positive in ifa (piglet 28). at 18 dpi, of the 9 piglets that were tested, 7 (piglets 21, 23, 24, 27, 28, 29 and 30) were positive while the remaining two (piglets 25 and 31) gave dubious/suspicious results on the initial test and, on retest, piglet 25 was a weak positive reactor in the elisa and positive by ifa. in the positive control group, one piglet (piglet 16) was positive and another (piglet 17) a dubious/suspicious reactor at 7 dpi. at 14 dpi, three of the four animals tested (piglets 13, 18 and 19) were positive for antibodies to pedv. by 19 dpi, all 8 animals tested (piglets 11, 13, 14, 15, 16, 18, 19 and 20) were seropositive although piglet 11 had a high variability and scored negative/dubious upon retest. of the 4 contact piglets introduced into the feed group at 7 dpi and tested at 10 days post-contact, one (piglet 2) scored as weak positive (50.75% blocking) by elisa. upon repeat testing, it was negative by elisa and ifa. the three contact piglets introduced into the sdpp-inoculated group and the three contact piglets introduced into the positive control group tested negative for pedv antibodies at 11 and 5 days postcontact, respectively. the practice of weaning piglets at 21 days of age or less has led to the use of complex diets containing a variety of supplemental ingredients that include sdpp. the procedure used to produce this feed supplement involves collecting blood from healthy pigs at the abattoir into tanks containing an anticoagulant. the pooled blood is then shipped to a processing facility where the cells and plasma are separated by centrifugation. following centrifugation, the liquid plasma, which is approximately 7% crude protein, is concentrated by evaporation, nano-filtration or ultra-filtration which results in crude protein levels that are in the 20-25% range. this material is then spray-dried to achieve a final crude protein concentration of at least 80%. studies have shown that the addition of sdpp to weaner pig diets can result in positive benefits including increased feed intake and weight gain and decreased requirement for antibiotics (reviewed by ferreira et al., 2009) . although blood from apparently healthy animals can be assumed to be sterile, the risk for virus contamination exists if blood from subclinically affected and viremic animals is collected or if the product becomes contaminated during any step of the process. viremia is not a known feature of pedv infection. blood collected at 7 dpi from three sdpp and one positive control piglet tested negative on the n gene rrt-pcr assay, as did 1 of the positive control piglets at 12 dpi, and three of the positive control group contact piglets at 5 days postcontact. although transport of pigs in inadequately cleaned trailers has been an implicated source of transmission of pedv (lowe et al., 2014) , other modes of transmission may also exist. our study indicates that pedv-contaminated sdpp may be one such mode for introducing the virus into a na€ ıve pig herd. while a typical pedv sequence could be obtained directly from the sdpp samples used for inoculation, only one of the two selected feed samples produced a pcr amplicon in the conventional s gene rt-pcr assay that was of the correct size. the sequence of this product, although consistent with pedv, was not of sufficient quality to make a definitive conclusion. using a pedv n gene rrt-pcr along with a number of ancillary tests, we demonstrated that piglets inoculated with contaminated sdpp could replicate and excrete the virus as well as transmit it to contact piglets. virus isolation was not used as a measure of virus excretion due to the inherent difficulties associated with cell culture isolation of this virus from clinical material. to illustrate this point, chen et al. (2014) have recently reported that of 33 fecal samples and 17 intestinal homogenates, only 2 pedv isolates were obtained giving a success rate of only 4%. moreover, none of the virus isolation attempts that they carried out on feces were successful. consequently, virus isolation for determination of pedv infectivity from clinical material is not viewed as a sensitive method. the duration of pedv shedding in experimentally infected 14-day-old piglets as determined by quantitative rt-pcr was previously reported to be 7 to 9 days (song et al., 2006) . the quantitative rt-pcr used in that study targeted the s gene of pedv generating a 651-bp amplicon. a two-step rt-pcr was employed in which the reverse transcription step was carried out separately to produce cdna. each cdna sample was then competed with 10 000 copies of an internal control dna during the pcr reaction as a means of quan-titating the amount of pedv rna present in the sample. in the study reported here, several piglets in the positive control and sdpp groups shed virus well beyond 9 days post-inoculation, with two animals in the positive control group and one animal in the sdpp group still producing positive fecal swab samples at 18 dpi and still capable of transmitting to some of the contact piglets that were introduced at 7 dpi. the longer duration of pedv shedding in our study could potentially be attributed to a greater sensitivity of the n gene rrt-pcr assay or to a more prolonged infection, perhaps caused by a lower initial infectious dose. regardless, more work needs to be performed in this area as an accurate assessment of the duration of shedding will have practical applications with respect to control of this disease. rectal temperatures of the inoculated piglets remained within normal range, and although some clinical signs in the form of depression, diarrhea and reduced feed intake were observed in the positive control group, similar signs were also observed, albeit less pronounced and in only a few piglets, in the other groups with the exception of the negative control group. however, such observations should not alone be interpreted as signs of pedv infection as piglets may have mild clinical illness due to many different causes including stress associated with moving and change in feed. the mild clinical signs observed in this study may be related to piglet age at the time of inoculation. using a field strain of pedv to inoculate specific pathogen-free pigs between the ages of 2 days and 12 weeks, shibata et al. (2000) reported that severe clinical disease and death only occurred in 2-7-day-old piglets. while rectal swabs from the negative controls and the feed-inoculated piglets remained negative throughout the study, the positive controls and the sdpp-inoculated piglets began shedding significant amounts of pedv beginning at 1 and 2 dpi. although negative by n gene rrt-pcr, a single piglet (piglet 35) from the feed-inoculated group had a weak band in the conventional s gene rt-pcr at day 3 and sequencing of this product was consistent with pedv although, similar to what was found for the feed sample, too weak for accurate determination. however, cloning of the pcr product and subsequent sequencing revealed that it was similar to the other pedv sequences obtained in this study as well as to those associated with canadian and initial usa field cases. electron microscopy of feces and intestinal content and immunohistochemistry on intestinal tissues of sdppinoculated and positive control piglets were consistent with coronavirus-like particles and the presence of pedv antigen in enterocytes, respectively. furthermore, at least one piglet from the sdpp-inoculated group examined at 7 dpi had an obvious enteropathy macroscopically and blunted small intestinal villi microscopically consistent with pedv infection. finally, seroconversion of animals in the positive control and sdpp-inoculated groups towards the end of the study provides further support of an active pedv infection. other studies have addressed the risk of transmitting viral contaminants, most notably porcine circovirus 2 (pcv-2), via spray-dried porcine plasma (patterson et al., 2010; pujols et al., 2008; shen et al., 2011) . one study (patterson et al., 2010) found that pcv-2 could be transmitted to na€ ıve pigs given pcv-2-contaminated sdpp by oral gavage. by contrast, the other two studies (pujols et al., 2008; shen et al., 2011) showed that weaned pigs that were fed pcv-2-contaminated sdpp neither developed clinical signs, became viremic or seroconverted. all three studies differed with respect to the health status of the animals used, the pooling of plasma from numerous animals, the pcv-2 dna load and the presence of anti-pcv-2 antibodies, all of which could contribute to the differences in the reported results. in conclusion, we have shown that the tested sdpp contains infectious pedv as demonstrated by a relatively high level of pedv excretion detected for ≥9 days by pedv n gene rrt-pcr. this was supported by s gene rt-pcr results and sequences and by seroconversion. moreover, the infection spread to 2 of 3 contact piglets introduced at day 7. the 12 piglets in the sdpp group were inoculated with three samples from different pallets of the same lot number in groups of 4 piglets. the kinetics of pcr positivity did not appear to differ among these piglets indicating that all three samples may likely have contained infectivity. however, as the piglets were kept together in the same room, we cannot unequivocally determine that all three samples were infective but can state that at least one of the three samples contained infectivity and that the results indicate that all 3 may likely have done so. inoculation with the tested feed samples did not produce any significant excretion of pedv although genetic material could be detected in the feed at trace levels and a single inoculated piglet at day 3 had traces of pedv genetic material in its rectal swab that was shown to be similar in sequence to the other samples tested. thus, as the tested feed did contain the sdpp shown to be infectious and did contain pedv genetic material and, moreover, could be detected in a single inoculated piglet at 3 dpi, we consider the tested feed as inconclusive or not possible to determine whether it is infectious or not by bioassay. the one feed that did produce a borderline result in a single piglet (feed sample 1) was subjected to a separate, second bioassay. although this bioassay included a larger number of piglets (n = 32), infectivity of the feed was not demonstrated. nevertheless, contaminated feed cannot and should not be ruled out as a potential source of infection as it is very possible that the limited bioassay studies described here are likely much less sensitive than what might occur under field conditions. many more piglets than used in this study coupled with larger amounts of feed, more stressful field conditions and ongoing infections may influence susceptibility of animals to pedv-contaminated feed. lastly, the feeds in question only contain 6% sdpp and, furthermore, had been in use for several weeks in the field prior to their evaluation in our bioassay experiments which, although stored under warehouse conditions, may have resulted in a further loss of any potential minimal infectivity present initially. cfia statement on porcine epidemic diarrhea virus in feed isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states experimental infection of pigs with a new porcine enteric coronavirus, cv 777 spray dried plasma for pigs weaned at different ages coronavirus infection in mink (mustela vison). serological evidence of infection with a coronavirus related to transmissible gastroenteritis virus and porcine epidemic diarrhea virus propagation of the virus of porcine epidemic diarrhea in cell culture 2013: origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states sialic acid binding activity of transmissible gastroenteritis coronavirus affects sedimentation behavior of virions and solubilized glycoproteins new variants of porcine epidemic virus use of two enzyme-linked immunosorbent assays to monitor antibody responses in swine with experimentally induced infection with porcine epidemic diarrhea virus porcine epidemic diarrhea investigation efficacy of experimentally produced spray-dried plasma on infectivity of porcine circovirus type 2 lack of transmission of porcine circovirus type 2 to weanling pigs by feeding them spray-dried porcine plasma the transmissible gastroenteritis coronavirus contains a spherical core shell consisting of m and n proteins commercially produced spraydried porcine plasma contains increased concentrations of porcine circovirus type 2 dna but does not transmit porcine circovirus type 2 to na€ ıve pigs isolation of procine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages porcine epidemic diarrhea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines use of an internal control in a quantitative rt-pcr assay for quantitation of porcine epidemic diarrhea virus shedding in pigs 2013: emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences usda technical note -porcine epidemic diarrhea (ped) new variant of porcine epidemic diarrhea virus an apparently new syndrome of porcine epidemic diarrhoea we gratefully acknowledge the hard and dedicated work of the animal care, laboratory, sample receiving and administrative staff involved in this project. we also acknowledge the contributions of dr. john copps and our collaborators at nvsl-ames, isu-ames, lindholm-denmark and lelystad-netherlands. key: cord-279813-mrei5kih authors: temeeyasen, g.; sinha, a.; gimenez-lirola, l.g.; zhang, j.q.; piñeyro, p.e. title: differential gene modulation of pattern-recognition receptor tlr and rig-i-like and downstream mediators on intestinal mucosa of pigs infected with pedv non s-indel and pedv s-indel strains date: 2017-12-14 journal: virology doi: 10.1016/j.virol.2017.11.024 sha: doc_id: 279813 cord_uid: mrei5kih porcine epidemic diarrhea virus (pedv) strains can be divided into non-s-indel and s-indel strains. pedv pathogenesis is strain-specific, and studies in neonatal pigs have demonstrated that the pedv non-s-indel strains are more pathogenic than the pedv s-indel strains. rna viruses, including pedv, can interact with a large number of pattern recognition receptors (prrs) in the intestinal mucosa, including toll-like receptors (tlrs) and rig-i-like receptors (rlrs). we investigated the differential gene modulation of tlrs, rig-i, and downstream mediators on the intestinal mucosa of neonatal pigs infected with pedv s-indel and non-s-indel strains. ten five-day-old piglets were inoculated orally with 10 ml of 10(4) tcdi(50)/ml of either pedv non-s-indel or s-indel strains. pedv s-indel infection induced pro-inflammatory cytokines through the non-canonical nf-κb signaling pathway by activating rig-i. in contrast, pedv non-s-indel infection suppressed the induction of pro-inflammatory cytokines and type 1 interferon production by down-regulation of tlrs and downstream signaling molecules. porcine epidemic diarrhea virus (pedv) belongs to the order nidovirales, family coronaviridae, and genus alphacoronavirus, and is an enveloped virus with a single-stranded positive rna genome. it contains four structural proteins-spike (s), membrane (m), nucleocapsid (n), and envelop (e)-responsible for viral infectivity and the induction of immune response kocherhans et al., 2001) . pedv causes enteric disease, resulting in significant morbidity and mortality in neonatal pigs, and has been reported as a major source of substantial economic losses in most swine producer countries (j. chen et al., 2010; q. chen et al., 2013; x. chen et al., 2012; cima, 2014; song and park, 2012; takahashi et al., 1983) . in 2014, a less pathogenic pedv strain was reported in the united states and several other swine producer countries vlasova et al., 2014; yamamoto et al., 2015) . this new strain presented insertions and deletions on the amino terminal region of the s protein. on the basis of differences in the s gene and virulence, emerging pedv strains can be divided into non-s-indel (s gene insertions and deletions) and s-indel strains (vlasova et al., 2014) . pedv pathogenesis is strain-specific, and pathogenesis studies in neonatal pigs have demonstrated that the pedv non-s-indel strain is more pathogenic than the pedv s-indel strain wang et al., 2016; yamamoto et al., 2015) . pedv pathogenesis is also inversely correlated with the age of the animals. in adult pigs, pedv infection is self-resolving regardless of previous pedv immune status. moreover, pedv s-indel was shown to be clinically relevant in neonates, but clinical disease could not be reproduced in pigs older than three weeks (annamalai et al., 2015; chen et al., 2016) . host pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the cytosolic retinoic acid-inducible gene-1 (rig-1)-like receptors, recognize pathogen-associated molecular patterns (pamps) during viral infection alexopoulou et al., 2001b; takeuchi and akira, 2010; uematsu and akira, 2008) . this interaction triggers the interferon regulatory transcription factor (irf3/7) and activates nf-κb, which modulates the expression of several pro-inflammatory cytokines and chemokines. type 1 interferon alpha/beta (ifnα/ifnβ) are the two essential cytokines that can control viral infections (kawai et al., 2005; seth, 2005; xu et al., 2005) . within nidoviruses, acute respiratory syndrome coronavirus (sars-cov) and middle-east respiratory syndrome coronavirus (mers-cov) can evade the host immune system by interfering with the nf-κb signaling pathway (dediego et al., 2014; matthews et al., 2014) , and the infectious bronchitis virus (ibv) inhibits the phosphorylation of kinases that are necessary to activate downstream signaling cascades (chen et al., 2013; devaraj et al., 2007; kint et al., 2015a; sun et al., 2012; zhou, 2007) . two signaling pathways, known as the classical (canonical) pathway and the alternative (non-canonical) pathway (kawai and akira, 2010; loo and gale, 2011) , lead to the activation of nf-κb. the canonical pathway includes the recruitment of the myeloid differentiation primary response gene 88 (myd88), containing the toll/interleukin-1 receptor (tir) domain for eventual activation of nf-κb and induction of type i interferons akira, 2007, 2010 ; thompson and locarnini, 2007) . in addition, endosomal receptor tlr3 contains exclusively tir-domain-containing adapter-inducing interferon-β (trif) adapter proteins that interact with traf6, which induces irf3/7 similar to the myd88 pathway (yamamoto et al., 2003; zhengfan, 2004; ) . finally, both the myd88 and trif pathways activate nf-κb and induce expression of the antiviral type i interferons (thompson and locarnini, 2007) . the non-canonical pathway is tlrindependent, and nf-κb can be modulated by rig-i-traf3 mediated through irf3 (devaraj et al., 2007) . several in vitro studies have tried to elucidate the role of pedv in innate immune response at the cellular level. it has been demonstrated that the nucleocapsid (n) protein of pedv, during infection of hek-293t cells, inhibited ifn-β production by annexing the vital interaction between irf3 and tbk1 (ding et al., 2014b) . other in vitro studies in porcine intestinal epithelial cells (iecs) determined that pedv infection impeded the production of ifn-β by inhibiting the rig-i pathway and hampering the activation of irf3 (cao et al., 2015a) . studies in vero cells showed that pedv infection degraded stat-1 and disrupted the ifn response . hence, it has been shown that pedv can regulate different immunological pathways in vitro; the main regulatory effect of pedv on mucosal innate immunity and its strain-dependence on viral pathogenicity is still unknown. the aim of this study was to investigate the differential gene modulation of pattern recognition tlr and rig-i-like receptors and downstream mediators on the intestinal mucosa of neonatal pigs infected with pedv non-s-indel and pedv s-indel strains. thirty 5-days-old conventional piglets were selected for the molecular evaluation of the gene modulation of pattern recognition tlr and rig-i-like receptors and downstream mediators on intestinal mucosa. the experimental design as well as information of the virus strains used in this study, kinetic of virus shedding, virus distribution in tissues, and pathogenicity was previously described in a pedv clinical pathogenesis study . in brief, pigs were injected intramuscularly with a dose of excede (zoetis, kalamazoo, mi) at time of delivery to iowa state university, laboratory animal resources facilities (ames, ia). all pigs were confirmed negative for pedv, pdcov, tgev, and porcine rotaviruses (groups a, b, and c) by virus-specific pcrs on rectal swabs, and seronegatives by pedv indirect immunofluorescent assay. after one day of acclimation, six-day-old pigs were inoculated orogastrically with 10 ml of 10 4 tcid 50 / ml of pedv non-s-indel (usa/ in19338/2013) or pedv s-indel (usa/il20697/2014), or 10 ml of virus-negative culture medium. five pigs from each group were euthanized at three and seven days post-inoculation (dpi), respectively. sections of distal small intestine of approximately 0.5 cm in length were snap frozen and saved at −80°c. frozen intestine tissue samples were aliquoted, placed in an rnalater™ stabilization solution (life technologies, carlsbad, ca), and kept at −80°c until further use. 2.2. rna extraction from porcine intestine tissue rna was extracted from 5 mg of porcine intestinal tissue using the ambion® magmax™ total rna isolation kit (life technologies) and a kingfisher® 96 magnetic particle processor (thermo fisher scientific, waltham, ma) following the manufacturer's instructions. rna was eluted into 70 µl of elution buffer and stored at −80°c. 2.3. expression of mrna for tlrs, inflammatory signaling pathways, and cytokines on porcine intestinal mucosa the relative quantification of gene expression of toll-like receptors tlr2, tlr3, tlr4, tlr7, tlr8, and tlr9 was evaluated on total rna extracted from porcine intestinal tissues. gene expression of inflammatory signaling pathway molecules, including rig-1, trif, myd88a, myd88b, irf7, traf6, nf-κb1 (p105) nf-κb1 (p50), and rela (p65), was also conducted by sybr-green rt-pcr on the rna extracted from intestinal pig mucosa. gene expression of cytokines ifnα, il-6, il-12, and tnf-α in intestinal mucosa was also investigated following the same protocol described herein for the rest of the genes. all reactions were performed in triplicate and the relative gene expression of each target gene was evaluated in reference to the expression of housekeeping genes gapdh and beta-actin. all primers were custom-synthesized (integrated dna technologies, inc., coralville, ia) to target amplicons, with sizes ranging between 95 and 120 nt base pairs according to the cdna sequence of each target gene, collected from the national center for biotechnology information (ncbi) database (table 1) . the mrna expression levels were quantified according to the δδct method (livak and schmittgen, 2001) . briefly, the difference in cycle times, δct, was determined as the difference between the tested gene and the reference housekeeping genes. the δδct was obtained by finding the difference between groups. the fold change was calculated as 2 -δδct . real-time rt-pcr was performed with eluted rna and primers, mixed with the commercial power sybr green rna-to-c t ™ 1-step kit (applied biosystems, foster city, ca), following the manufacturer's recommendations. the reverse transcription reaction was conducted at standard mode for 30 min at 48°c, enzyme activation was conducted at 95°c for 10 min using an applied biosystems™ 7500 realtime pcr instrument. the strands were denatured at 95°c for 15 s, then annealed and extended at 60°c for 1 min (40 cycles). a dissociation curve was obtained for each quantitative pcr run to assess its target specificity. the real-time rt-pcr was analyzed with a threshold fixed at a 0.1 setting. cycle threshold (ct) values ≤ 35 were considered positive for the housekeeping and tlr gene expressions. all samples were tested in triplicate and the results were expressed as fold changes relative to the control animals. data were analyzed for normality by kolmogorov-smirnov test. the statistical significance between the two treatment groups was determined by non-parametric statistical analysis using the mann-whitney test. significance was assessed at p < 0.05. data analysis was performed using graphpad prism ® (graphpad software inc., la jolla, ca). rna viruses, including pedv, can interact with a large number of pattern recognition receptors (prrs) in the intestinal mucosa, such us toll-like receptors (tlrs) and rig-i-like receptors (rlrs). this interaction plays a critical role in the activation of the innate immune response. tlrs are normally classified based on their anatomical location in membranes or cytoplasmic receptors. numerous tlrs are expressed in porcine enterocytes with different functions in molecule recognition. in addition, other intracytoplasmic prrs, such as retinoic acid-inducible gene (rig)-i-like receptors (rlrs) can sense and recognize double-stranded or single-stranded rna from a variety of pathogens. in this study, we evaluated gene modulatory effects on a group of prrs, including tlrs and rlrs, in the intestinal mucosa during early infection by pedv non-s-indel and s-indel strains. pigs infected with pedv s-indel showed a significant up-regulatory effect (p < 0.05) on tlr3, tlr4, and tlr7 gene expression by 3 dpi compared to non-infected control pigs and pigs infected with the pedv non-s-indel strain ( fig. 1b-d) . the regulatory effect observed in the pedv s-indel-infected group was transient, returning to basal control levels by 7 dpi. pedv s-indel infection had a significant downregulatory effect (p < 0.05) on tlr2 gene expression at 3 and 7 dpi (fig. 1a) . however, pedv s-indel infection did not affect gene modulation of tlr8 and tlr9 compared to the control group (fig. 1e , f). in contrast, infection with pedv non-s-indel was shown to have a downregulatory effect on the expression of tlr4, tlr7, tlr8, and tlr9 (p < 0.05) compared to the non-infected control and pedv s-indelinfected groups consistently at 3dpi (fig. 1c-f) . however, no statistically significant differences in tlr2 and tlr3 gene modulation levels (p > 0.05) were observed between the pedv non-s-indel and control groups at either 3 or 7 dpi (fig. 1a, b) . the modulation of rig-i was only affected in response to pedv s-indel infection at 3 dpi, with a significant increment in gene expression compared to the control and non-s-indel groups (p < 0.05). however, this modulatory effect was transient, with expression levels returning to the same levels observed for the control and pedv non-s-indel groups by 7 dpi (fig. 2) . fig. 7 summarize the differential modulatory effect on prrs genes induced by pedv s-indel vs non s-indel at dpi 3. prrs of the innate immune system initiate signal transduction cascades in response to ligation by microbial-associated molecular patterns (mamps), which leads to the transcriptional modulation of downstream signaling molecules. with the exception of tlr3, the myeloid differentiation primary response gene 88 (myd88) product is the most generally utilized tlr adapter, either by direct interaction (tlr5, 7-9) or via an intermediary interaction (tlr4) with the tir table 1 primers used for real-time pcr analysis of genes expression of pattern-recognition receptor tlr and rig-i-like and downstream mediators on pig intestinal mucosa. primer name primer sequence (5'−3") amplicon length accession number glyceraldehyde 3-phosphate dehydrogenase (gapdh); β-actin; toll-like receptor (tlr); retinoic acid-inducible gene-1 (rig-1)-like receptor; tir-domain-containing adapter-inducing interferon-β (trif); myeloid differentiation primary response gene 88 (myd88); nuclear factor (nf)-κb; nuclear factor nf-kappa-b p65 subunit (rela); interferon regulatory transcription factor (irf); tnf receptor associated factor (traf); interleukin (il); tumor necrosis factor (tnf); interferons (ifn). domain-containing adapter protein (tirap)/mal. engagement of myd88 leads to recruitment and assembly of the cytoplasmic il-1 receptor-associated kinases (irak) and the tnf receptor-associated factor (traf) 6 to form the irak-traf6 complex, which leads to the activation of the nuclear factor (nf)-κb pathway. the nf-κb transcriptional factor family is composed of nf-κb1 (p105), nf-κb1 (p50) and rela (p65), subunits that translocate into nuclei and regulate nf-κb. tram and trif mediate a signal transduction cascade downstream of both tlr3 and tlr4 (kawai and akira, 2010; thompson and locarnini, 2007) . the myd88-and trif-dependent pathways lead to the activation of interferon regulatory factors (irfs) and the secretion of type-i interferon (ifn) and pro-inflammatory cytokines by activated nf-κb signaling. despite the pedv s-indel strain inducing a positive modulatory effect on tlr3, tlr4, and tlr7 gene expression, no significant differences (p > 0.05) were observed in gene expression levels of trif, myd88 (subunits a and b) (fig. 3a, c, d) . moreover, the pedv s-indel strain appears to have a down-regulatory effect on traf6 and irf7 genes at 3 dpi (p < 0.05) (fig. 4a, b) . pedv s-indel-infected animals showed a differential gene modulation characterized by a significant up-regulatory effect in expression levels of nf-κb1 and (p50) genes (p < 0.05) at 7 dpi, and a down-modulatory effect on the expression of the rela/p65 gene compared to both the control and pedv non-s-indel-infected group at 3 and 7 dpi (fig. 5a, b) . contrary to the pedv s-indel strain, a down-regulatory effect of tlr4, tlr7, tlr8 and tlr9 genes was observed in response to infection with the pedv non-s-indel strain (fig. 1) , which also negatively affected the gene expression of downstream signaling molecules (p < 0.05), including trif, myd88 (subunits a and b), and traf6 (figs. 3a, c, d, 4a). although the expression levels of both p50 and p65 were down-regulated after infection with the pedv non-s-indel strain (fig. 5) , the down-regulatory effect on myd88 and trif gene pathways did not negatively affect the expression of nf-κb (p105) (fig. 3b) . fig. 7 summarize the differential modulatory effect of nf-κb through tlr downstream adapters induced by both pedv strains at dpi 3. pro-inflammatory cytokines and type i interferons (ifns) are produced at the local intestinal mucosal level as part of the innate immune response during the infection process. the modulatory effect of proinflammatory interleukins and ifn genes is the result of a long-elaborated pathway that includes the activation of prrs and downstream mediators, including myd88 and trif, via nf-κb activation. we evaluated whether the differential modulatory effect observed in prrs and downstream mediators in response to infection with the pedv s-indel versus the non-s-indel strain was also translated into a differential modulation in the expression of gene coding for pro-inflammatory cytokines and type i interferons. neither infection with pedv s-indel nor non-s-indel was demonstrated to have a modulatory effect on local production of il-6 (fig. 6a) . the local expression of il-12 was not affected at 3 dpi regardless of the pedv strain. however, a positive modulatory effect on il-12 gene expression was exerted by pedv s-indel at 7 dpi (fig. 6b) . the pedv non s-indel strain showed a transient negative modulatory effect of the tnf-α gene at 3 dpi, returning to basal control levels by 7 dpi. however, the pedv s-indel strain consistently up-regulated the expression of the tnf-α gene during the study (fig. 6c ). the expression of the ifn-α gene was negatively affected only in response to infection with the pedv non-s-indel strain at 3 dpi (p < 0.05) (fig. 6d ). fig. 7 summarize the effect of pro-inflammatory cytokine gene regulation by pedv s-indel and pedv non-s-indel strain at dpi 3. pathogen recognition by tlrs and rlrs activates the innate immune response through signaling pathways, resulting in the production of pro-inflammatory cytokines, type i interferons, and chemokines. intestinal mucosa are composed of a variety of specialized cells that play specific functions during the disease process (kawai and akira, 2006; kumar et al., 2009) . collectively, intestinal epithelium, dendritic cells, m cells, immune cells in the lamina propria, and peyer patches (e.g., lymphocytes and macrophages) play a role in the intestinal mucosa immune response against pathogens. prrs are constitutively expressed in all cell populations referred to above and may have differential roles in each cell type and component of the intestinal mucosa. initial prr-induced responses are critical in controlling infectious agents, but are also tightly regulated through time-, location-, and cell type-dependent mechanisms. positive and negative modulation interaction of signaling pathways is the main mechanism for maintaining inmate immune homeostasis. interferon production, especially type 1 interferon and anti-viral cytokines, is important for host protection against viral invasion. many viruses can evade the host immune system by regulating signaling pathways, resulting in the blockage of cytokine production (mccartney and colonna, 2009). porcine epidemic diarrhea virus (pedv) causes enteric diseases, resulting in significant economic losses. pedv pathogenesis is strainspecific, and pathogenesis studies in neonatal pigs have demonstrated that the pedv non-s-indel strain is more pathogenic than the pedv s-indel strain wang et al., 2016; yamamoto et al., 2015) . pedv s-indel was shown to be clinically relevant in neonates, but clinical disease could not be reproduced in pigs older than three weeks (annamalai et al., 2015; chen et al., 2016) . the molecular mechanism of innate immune modulation has only been evaluated in vitro, and thus available information is scarce (cao et al., 2015a (cao et al., , 2015b ding et al., 2014a; gao et al., 2015; xu et al., 2013) . however, it has been demonstrated that in vitro studies are highly dependent on virus strain and cell type, and are not valid for the evaluation of innate immune response against low-virulent strains (kint et al., 2015b) . therefore, in this study, we evaluated the differential modulation of intestinal mucosa prr signaling mounted against pedv non-s-idel and s-indel in there is limited information about the role of antiviral innate immunity in the pathogenesis of pedv infection. however, it is well known that viral infections are usually detected by cell membranes and endosomal-associated tlrs (e.g., tlr4 and tlr3, tlr7/8) and cytosolic rig-i-like receptors (rlrs), such as rig-i and mda5. activated tlr and rig-i/mda-5 signaling pathways initiate effective antiviral innate immune responses, in particular inducing type i infs (thompson and locarnini, 2007) . previous in vitro studies demonstrated that pedv strain cv777 infection of intestinal epithelial cells (iecs) modulates the nf-κb signaling pathway through up-regulation of tlr2, tlr3, and tlr9, but not rig-i (cao et al., 2015a) . however, our results showed that in vivo, pedv s-indel infection up-regulated the nf-κb signaling pathway through tlr3, tlr4, tlr7, and rig-i, resulting in increased expression levels of tnf-α. although no statistical significance was found, increased levels of ifn-α gene expression were also observed. interestingly, both pedv cv777 and pedv s-indel strains belong to genogroup 1, and both are associated with mild clinical disease. therefore, the differential modulation observed in the nf-κb signaling pathway could be more associated with in vivo-related conditions than genotypic characteristics of the virus strain used in this study. conversely, pedv non-s-indel down-regulated the nf-κb signaling pathway through a negative modulatory effect of tlr4, tlr7, tlr8, and tlr9, resulting in final attenuation of pro-inflammatory tnf-α and ifn-α gene expression. moreover, rig-i gene modulation after pedv non-s-indel remained unaffected. in a previous study, it was demonstrated that pedv non-s-indel had a significantly higher replication rate compared to the less virulent pedv s-indel strain . our study demonstrated that the ability of pedv to induce type i ifns is strain-specific. thus, type i ifns may play an important role in pedv replication and pathogenesis. the signaling pathway during ligand binding of single-stranded and double-stranded viral rna involves the cytoplasmic membrane tlr4 receptor and endosomal tlr7/8 and tlr-9, all of which are used as signaling pathway mediators, (myd88 and trif) for eventual activation of nf-kb akira, 2007, 2010 ; thompson and locarnini, fig. 3 . changes in toll/interleukin-1 receptor (tir), myeloid differentiation primary response gene 88 (myd88), and nuclear factor (nf)-κb genes mrna expression induced by porcine epidemic diarrhea virus (pedv) non s-indel and pedv s-indel strains in intestinal mucosa (a-d). ten pigs in each group were infected with pedv non s-indel, pedv s-indel or media (negative control) and five pigs from each group were necropsied at 3 and 7 days post-infection (dpi). the mrna levels of trif (a), nf-κb (b), myd88a (c), and myd88b (d) at the intestinal mucosa was determined individually in each animal by sybr-green qrt-pcr. all samples were tested in triplicate and the results are expressed as fold changes relative to the control animals data are presented as means ± standard errors. significant difference between pedv s-indel pedv non s-indel and control group are expressed with their p values. *p < 0.05; **p < 0.01, ***p < 0.001. 2007). however, endosomal receptor tlr3 (alexopoulou et al., 2001a) contains exclusively trif-adapter proteins that interact with traf6, which induces phosphorylation of irf3/7 similar to the myd88 pathway (yamamoto et al., 2003; zhengfan, 2004) . finally, both myd88 and trif pathways activate nf-kb and induce expression of the antiviral type i interferons (thompson and locarnini, 2007) . in this study, pedv non-s-indel infection had a negative gene modulation on membranes and endosomal tlrs. this negative modulatory effect was translated into a down-regulation of cytoplasmic mediator myd88 and trif genes. in vitro studies demonstrated that silencing trif and myd88 but not rig-i inhibited pedv-induced nf-kb activation, suggesting that the tlr signaling pathway is involved in pedv-induced nf-kb activation (cao et al., 2015b) . in addition, myd88 is a required component of the innate immune response to mouse-adapted sars-cov infection in vivo (totura et al., 2015) . although pedv s-indel induces nf-kb activation, tlr pathway mediators trif and myd88 were not significantly affected by the positive modulation of the cytoplasmic membrane and endosomal tlr genes in response to infection with pedv s-indel. these contradictory results suggest that trif-and myd88-independent pathways might be involved in nf-κb activation after pedv s-indel infection. traf6 is crucial for both rig-i-and tlr-mediated antiviral responses. the absence of traf6 resulted in enhanced viral replication and a significant reduction in the production of type i ifns after infection with the rna virus (konno et al., 2009 ). activation of nf-kb and irf7, but not irf3, is normally traf6-mediated. in this study, we fig. 4 . changes in tnf receptor associated factor (traf) 6 and interferon regulatory factor 7 (irf7) genes mrna expression induced by porcine epidemic diarrhea virus (pedv) non s-indel and pedv s-indel strains in intestinal mucosa (a-b). ten pigs in each group were infected with pedv non s-indel, pedv s-indel or media (negative control) and five pigs from each group were necropsied at 3 and 7 days post-infection (dpi). the mrna levels of traf 6 (a), and irf7 (b) at the intestinal mucosa was determined individually in each animal by sybr-green qrt-pcr. all samples were tested in triplicate and the results are expressed as fold changes relative to the control animals data are presented as means ± standard errors. significant difference between pedv s-indel pedv non s-indel and control group are expressed with their p values. *p < 0.05; **p < 0.01, ***p < 0.001. observed that both pedv s-indel and non-s-indel showed a negative modulatory effect on traf6, which was translated into a significant down-regulation of the irf7 gene only in pedv s-indel-infected animals. traf6 induced activation of irf7, while traf3 is thought to activate both irf3 and irf7 (konno et al., 2009 ). however, due to the differential modulation of irf7 observed in this study, the role of the traf6-dependent pathway cannot be fully elucidated, and perhaps irf3 plays a more important role in type i ifn production in pedv infection. the role of traf3 was not explored in this study but may likely play a role in irf7 and ifn-α gene modulation. in addition, the down-regulatory effect observed in traf6 gene expression for both pedv strains did not have the same modulatory effect on nf-kb gene expression. this study showed that nf-kb gene expression was pedv strain-dependent. although it may need further confirmation, the upregulation of nf-kb gene expression observed in response to pedv s-indel infection could be linked to the up-regulation of rig-i gene expression induced after infection with the pedv s-indel strain. signaling pathways (canonical and non-canonical) lead to the activation of nf-κb (kawai and akira, 2010; loo and gale, 2011) . modulation of nf-kb family members (hetero-dimer p50-p65) is affected through th tlrs, which unbound the inhibitory ikb proteins and allowed activation of the nf-kb canonical pathway (wietek and o'neill, 2007) . we observed that pedv non-s-indel exerted a down-regulatory effect on the p50-p65 hetero-dimer. the result observed during pedv non-s-indel infection is in agreement with a previous in vitro study that demonstrated that pedv-encoded nucleocapsid (n) protein can impede pro-inflammatory cytokine and type i interferon production by direct interaction with tank-binding kinase 1 (tbk1), resulting in inhibition of transcription factors, such as irf3 and irf7, causing final nf-κb interference (ding et al., 2014b) . moreover, it has been reported in vitro that pedv non s-indel nsp1 protein induced nf-κb suppression (zhang et al., 2017) . inhibition of the p50-p65 hetero-dimer blocks activation of the nf-κb canonical pathway and inhibits early tnf-α response (wietek and o'neill, 2007) . our results demonstrate that pedv non-s-indel could use this strategy to evade the host immune system in addition to exhibiting an ifn-α down regulatory effect. although in this study we observed that pedv s-indel down-regulated the p65 gene, the nf-κb pathway was up-regulated. it has been fig. 6 . changes in proinflammatory cytokines il-6, il-12, and tnf-α, and ifn-α genes mrna expression induced by porcine epidemic diarrhea virus (pedv) non s-indel and pedv s-indel strains in intestinal mucosa (a-d). ten pigs in each group were infected with pedv non s-indel, pedv s-indel or media (negative control) and five pigs from each group were necropsied at 3 and 7 days post-infection (dpi). the mrna levels of il-6 (a), il-12 (b), tnf-α (c), and ifn-α (d) at the intestinal mucosa was determined individually in each animal by sybr-green qrt-pcr. all samples were tested in triplicate and the results are expressed as fold changes relative to the control animals data are presented as means ± standard errors. significant difference between pedv s-indel pedv non s-indel and control group are expressed with their p values. *p < 0.05; **p < 0.01, ***p < 0.001. demonstrated that inhibition of the p50-p65 hetero-dimer or the p50 homo-dimer can be compensated for by the p52-relb hetero-dimer, resulting in the activation of the nf-κb non-canonical pathway (wietek and o'neill, 2007) . in vitro studies demonstrated that pedv protein e is capable of inducing p65 in intestinal epithelial cells and the activation of the nf-κb non-canonical pathway (xu et al., 2013) . moreover, further studies demonstrated that during pedv cv777 infection, nf-kb p65 was found to be translocated from the cytoplasm to the nucleus, and pedv-dependent nf-kb activity was associated with viral dose and active replication. in addition, the same study corroborated that upon pedv n protein overexpression in transfected iecs, p65 was detected in the nucleus (cao et al., 2015b) . the production of pro-inflammatory cytokines and type i interferon (ifn) at the local intestinal mucosal level is part of the innate immune response during viral infection. interleukin-6 (il-6) is associated with improving humoral and mucosal immune response (meng et al., 2013) . in this study, il-6 mucosal gene expression was not affected by pedv infection. il-6 expression levels are not necessarily correlated with the positive gene modulation of other pro-inflammatory cytokines. il-12 is the major type i cytokine produced by macrophages and dendritic cells. il-12 is believed to be responsible for enhancing th1 and s-iga response at the mucosal level (boyaka et al., 1999) . in this study, we observed that il-12 was up-regulated at 7 dpi by pedv s-indel. this modulatory effect is in agreement with a previous in vitro study, which also evaluated a low-virulent strain of cv777 (gao et al., 2015) . in this study, we observed a positive tnf-α gene regulatory effect likely associated with the up-regulation of nf-kb in response to pedv s-indel infection. we speculate that this might be the result of the rig-i noncanonical pathway of nf-κb, perhaps through the mitochondrial antiviral-signaling protein (mavs) and traf3 (not evaluated in this study) (bowie and unterholzner, 2008) . other coronaviruses, such as sars-cov, also showed a positive modulatory effect of tnf-α through the activation of nf-kb. conversely, pedv infection with the highvirulent non-s-indel strain induced a down-regulation effect on tnf-α gene expression. this negative modulatory effect on tnf-α could be associated with the severe pathological response characteristic of pedv non-s-indel strains. modulation of type i ifn response seems to be a common evasion strategy of viruses in the order nidovirales . sars-cov and mers-cov, both within the genus betacoronavirus, do not induce significant ifn response in respiratory cells in vitro. however, transmissible gastroenteritis epidemic virus (tgev) within the alphacoronavirus genus induces a high level of ifn-α in newborn pigs. pedv also exists within the alphacoronavirus genus; however, we found virus strain-related differences in ifn-α gene modulation. although no significant ifn-α gene modulation was observed after pedv s-indel infection, there was an increment consistent with previous reports on tgev. the down-regulation observed in pedv non-s-indel is more consistent with the effect observed with other members of the betacoronavirus genus. the differential modulatory effect in the tnf gene between pedv strains observed in this study is coincident with the severity in pathogenicity between pedv s-indel and non-s-indel. in summary, the aim of this study was to investigate the differential gene modulation of pattern recognition tlr and rig-i-like receptors and downstream mediators on the intestinal mucosa of neonatal pigs infected with pedv non-s-indel and pedv s-indel strains. our results suggest that pedv s-indel infection induces pro-inflammatory cytokines through the non-canonical nf-κb signaling pathway by activating fig. 7 . differential gene modulation of pattern-recognition receptor tlr and rig-i-like, and downstream mediators on intestinal mucosa of pigs infected with pedv non s-indel and pedv s-indel strains. this figure present differential gene modulation at day post-infection (dpi) 3. pedv non-s-indel infection suppressed the induction of the pro-inflammatory cytokine tumor necrosis factor alpha (tnf-α), and type 1 interferon production (ifn-α) through the down regulation of the cytoplasmic membrane and endosomal tlrs (tlr4, tlr7/8, tlr9), and tlr-downstream signaling molecules (myd88/trif and traf6). although the expression levels of both p50 and p65 were down-regulated after infection with the pedv non-s-indel strain, the down-regulatory effect on myd88 and trif gene pathways did not negatively affect the expression of nf-κb ( ). contrary, pedv s-indel infection induced a positive modulatory effect on tlr3, tlr4, and tlr7 gene expression. however, no significant modulatory effect was observed in the levels of trif, and myd88, genes. pedv s-indel infection induced the pro-inflammatory cytokines tnf-α, and interleukin (il)−12 through the non-canonical nf-κb signaling pathway by the activation of the intracytoplasmic rig-i receptor ( ). the rig-i receptor (fig. 7) . meanwhile, pedv non-s-indel infection suppresses the induction of pro-inflammatory cytokines and type 1 interferon production by down-regulation of the cytoplasmic membrane and endosomal tlrs, as well as tlr downstream-signaling molecules (canonical nf-κb pathway) (fig. 7) . these data present novel in vivo evidence supporting the notion that the evasion strategy of the pedv non-s-indel strain might provide important insights into the pathogenesis of this strain. based on our results, both low-virulent pedv s-indel and high-virulent pedv non-s-indel cause differential modulation on innate immune response pathways, which could be translated into differences in pathogenesis and clinical outcomes normally observed after pedv infection. pathogen recognition and innate immunity recognition of doublestranded rna and activation of nf-[kappa]b by toll-like receptor 3 recognition of doublestranded rna and activation of nf-kappab by toll-like receptor 3 age-dependent variation in innate immune responses to 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contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection toll-like receptors (tlrs) and their ligands distinct characteristics and complex evolution of pedv strains us variant porcine epidemic diarrhea virus: histological lesions and genetic characterization diversity and regulation in the nf-κb system visa is an adapter protein required for virus-triggered ifn-beta signaling porcine epidemic diarrhea virus e protein causes endoplasmic reticulum stress and up-regulates interleukin-8 expression role of adaptor trif in the myd88-independent toll-like receptor signaling pathway experimental inoculation of an s indel strain of porcine epidemic diarrhea virus in japan suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp1 inhibition of nf-κb activity by the porcine epidemic diarrhea virus nonstructural protein 1 for innate immune evasion toll-like receptor 3-mediated activation of nf-b and irf3 diverges at toll-il-1 receptor domain-containing adapter inducing ifn-beta mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded the authors would like to thank mary breuer for preparing fig. 7 , which was modified for use in this manuscript. we sincerely apologize to many scientists whose works were not cited in the reference list because of the space limitation. key: cord-321739-dnuu6jok authors: bowman, andrew s; krogwold, roger a; price, todd; davis, matt; moeller, steven j title: investigating the introduction of porcine epidemic diarrhea virus into an ohio swine operation date: 2015-02-15 journal: bmc vet res doi: 10.1186/s12917-015-0348-2 sha: doc_id: 321739 cord_uid: dnuu6jok background: porcine epidemic diarrhea virus (pedv) is a highly transmissible coronavirus that causes a severe enteric disease that is particularly deadly for neonatal piglets. since its introduction to the united states in 2013, pedv has spread quickly across the country and has caused significant financial losses to pork producers. with no fully licensed vaccines currently available in the united states, prevention and control of pedv disease is heavily reliant on biosecurity measures. despite proven, effective biosecurity practices, multiple sites and production stages, within and across designated production flows in an ohio swine operation broke with confirmed pedv in january 2014, leading the producer and attending veterinarian to investigate the route of introduction. case presentation: on january 12, 2014, several sows within a production flow were noted with signs of enteric illness. within a few days, illness had spread to most of the sows in the facility and was confirmed by rt-pcr to be pedv. within a short time period, confirmed disease was present on multiple sites within and across breeding and post weaning production flows of the operation and mortality approached 100% in neonatal piglets. after an epidemiologic investigation, an outsourced, pelleted piglet diet was identified for assessment, and a bioassay, where naïve piglets were fed the suspected feed pellets, was initiated to test the pellets for infectious pedv. conclusions: the epidemiological investigation provided strong evidence for contaminated feed as the source of the outbreak. in addition, feed pellets collected from unopened bags at the affected sites tested positive for pedv using rt-pcr. however, the bioassay study was not able to show infectivity when feeding the suspected feed pellets to a small number of naïve piglets. the results highlight the critical need for surveillance of feed and feed components to further define transmission avenues in an effort to limit the spread of pedv throughout the u.s. swine industry. porcine epidemic diarrhea virus (pedv) is a coronavirus of the genus alphacoronavirus. disease from pedv is characterized by vomiting, anorexia, and watery diarrhea in swine. the virus is particularly deadly for neonatal pigs for which malabsorption and dehydration [1] [2] [3] can result in mortality rates approaching 80%-100% [2, 4] . disease caused by pedv is clinically indistinguishable from transmissible gastroenteritis virus and cannot be diagnosed on presentation alone [4] . because attempts at virus isolation have only resulted in limited or temporary success, with virus isolation rates as low as 4% [5] , diagnosticians heavily rely upon rt-pcr tests to directly detect viral nucleic acid and diagnose pedv. pedv was first identified in belgium in 1978 and in the 1980s and 1990s, pedv was found throughout belgium, england, germany, france, the netherlands, and switzerland [6] . since the european emergence, pedv has affected the pork industries in philippines, south korea, and china [7] . in may 2013, the united states confirmed the first cases of pedv on farms in iowa and indiana [2] , after which the virus spread quickly throughout the country. while the mode of pedv introduction to the u.s. remains unknown, comparison of available sequence data indicates the pedv strains detected in the unites states have an ancestry linked to pedv strains detected in china. at the end of 2013, sequenced u.s. strains had greater than 99.0% sequence identity and several strains shared unique nucleotides with a chinese pedv strain isolated in the anhui province (ah2012) [2, 3] . unexpected genetic similarity of u.s. pedv strains to a bat coronavirus isolated in southeastern china may provide evidence for the role of cross-species transmission in the development of emergent strains that spread to the united states [3] . transmission of pedv occurs via the fecal-oral route [7] and fecal contamination of fomites may play a role in the introduction of the virus to swine. an investigation of 575 livestock trailers at 6 harvest facilities in the united states showed that all truck drivers stepped into the harvest facility at least once, and the proportion of pedv contaminated trailers increased from 6.6% before unloading to 9.2% after unloading [8] . these data indicate that contaminated transport vehicles and personnel could be associated with the rapid spread of the virus throughout the us. at present, pedv prevention and control in the u.s. are heavily dependent on biosecurity procedures. while transportation equipment might play a role in the spread of pedv, on-farm investigations into several pedv outbreaks in the united states have indicated that contaminated feed could be a pathway of viral introduction; however, scientific support of this route is regularly debated. dee et al. showed that material collected from the inside of feed bins during a pedv outbreak was infectious when concentrated and inoculated into pigs [9] . one canadian report showed spray-dried porcine plasma, a component used in some swine feed, was infectious to pigs, but the complete feed containing spray-dried porcine plasma was not infectious in an experimental setting [10] . on the other hand, a study team has provided contrary evidence with an unsuccessful attempt to infect pigs with spray-dried porcine plasma and data that indicates pedv is inactivated during spray-dried porcine plasma production process [11, 12] . in january 2014, an outbreak of pedv was confirmed in a multi-site, multiple flow swine operation in ohio. after a thorough epidemiologic investigation, contaminated feed was identified as the likely source of pathogen introduction, a finding supported by a positive rt-pcr result from testing the feed source. of note, rt-pcr detects viral rna, and thus can only confirm the presence of viral nucleic acid in a sample, not necessarily presence of viable and infectious virus. since pedv isolation is very difficult in numerous testing and research laboratories, virus isolation attempts from feed pellets could not be relied on to detect viable, infectious virus. consequently, a bioassay was initiated where samples of feed cryopreserved by the attending veterinarian during the outbreak were later fed to naïve piglets in an attempt to demonstrate feed infectivity. this report will discuss the aforementioned epidemiologic investigation and subsequent bioassay findings. the ohio swine operation (figure 1 ), consisting of 3 multi-site, farrow-to-finish production flows (referred to as flows a-c, each having two breed-wean sites) and a multiplier herd (referred to as d, with a single breedwean site) had no prior cases of pedv and was determined to have effective biosecurity measures in place evidenced by the absence of porcine reproductive and respiratory syndrome virus (prrsv) during more than the prior seven years. routine oral fluid testing of pigs in flow b on january 8, 2014 and surveillance testing in flow c in november and december 2013 were all negative for pedv. at time of weaning, pigs move from breed-wean premises to wean-to-finish barns for flow a and to nursery facilities and then finisher sites for flows b and c. weaned pigs from flow d, the multiplier herd, are raised in gilt developer units or wean-to-finish barns. on the morning of january 12, 2014, four lactating sows from the one of the breed-wean units in flow a (unit a1) were noted with vomiting and diarrhea. the illness spread rapidly and by 4:00 pm, 80 litters showed signs of diarrhea, vomiting, and dehydration. within a few days, 80% of sows on the site showed similar clinical signs. fecal samples taken january 15, 2014 were positive for pedv using rt-pcr. forty-two percent mortality was observed in piglets in the a1 farrowing unit. beyond the sow unit, a wean-to-finish barn in flow a that received pigs on january 10 th from both flow a breed-wean units (a1 and a2) reported loose stools on january 12 th and had confirmation of pedv with rt-pcr positive fecal samples collected the same day. also within flow a, one wean-to-finish barn that was filled with piglets from both flow a breed-wean units (a1 and a2) on january 10 th and 12 th had fecal samples test pedv rt-pcr positive on january 15, 2014. in addition, on january 15 th , a third wean to finish barn that received pigs from both flow a sow farms on january 9 th had fecal samples test pedv positive. a schematic of flow a is shown in figure 1a . while no pedv-like disease was observed in either breed-wean units in flow c, pigs in 3 nurseries within flow c did test pedv rt-pcr positive between january 14 th and january 20 th , 2014 ( figure 1c ). on january 22, 2014 one breed-wean unit within flow b (b1) began experiencing pedv-like disease. on that same day, an oral fluid sample from one of the nurseries in flow b that received pigs from both flow b breed-wean units (b1 and b2) on january 15 th , 17 th , and 20 th , 2014 tested pedv pcr positive ( figure 1b ). also on january 22 nd , two finishing barns in flow b had a pedv pcr positive oral fluid test. by january 25 th , the second breed-wean unit in flow b (b2) was also experiencing the disease. overall, mortality among neonatal piglets was close to 100% in flow b. five american association of swine veterinarians (aasv) pedv questionnaires were completed by a usda epidemiologist and an ohio department of agriculture veterinary medical officer in conjunction with swine operation representatives and the operation's local veterinarian. several potential pathways of pathogen introduction to the swine operation, including human introduction, delivery of contaminated supplies, aerosol spread, contaminated pig transport vehicles, and contaminated feed or feed ingredients were considered and evaluated. it is unlikely pedv was introduced to the operation by visitors or workers. there were no foreign visitors, and no employees had visited foreign countries within 10 days of the outbreak. nor did any employee have swine at their place of residence or associated farm enterprises. all swine operation employees and non-employee contractors follow meticulous biosecurity procedures to enter a facility, and movement of people from one facility to another within the same day is limited to production managers only, which typically occur only within the same flow. effectiveness of the biosecurity measures in place was evidenced by the absence of prrs cases for over seven years. veterinary, vaccine, and semen supplies delivered by supply vendors were also considered as a potential source of pedv introduction to the swine operation, but were subsequently ruled out as likely sources for several reasons. first, supplies are delivered to buildings separate from the swine housing areas and they are not shared among different flows. disease, however, broke out separately in geographically and personnel isolated units from 3 different flows. in addition, supplies were disinfected in a fume chamber within the enclosed room whereby the incoming materials were placed on an elevated metal grate and a mister system applied a quaternary ammonium/glutaraldehyde combination disinfectant (synergize, preserve international, reno, nv) and allowed to stand for 15 minutes before entry into site. this practice was considered to greatly reduce the likelihood of contaminated supplies as the potential route of pedv introduction. airborne spread could be considered with pedv [13] , as coronaviruses classified within the same group as pedv (group 1 coronaviruses) include those that cause enteric or respiratory infection. porcine respiratory coronavirus is a mutant of transmissible gastroenteritis virus and is an example of a group 1 coronavirus that is spread through droplets and aerosols [14] . aerosols or droplets are unlikely to be responsible for the spread of disease on this swine operation because most units are not located geographically close to each other, and disease broke on multiple separate units from 3 separate flows across a period of 13 days. additional factors that could be involved in virus transmission such as water supply and shavings used during transport of young pigs are not probable because pigs on all sites have equal exposure to these factors but not all sites were involved in the outbreak. lowe et al. have shown that contaminated transport vehicles are likely to be involved in rapid spread of pedv because it is common to transport pigs to harvest facilities on vehicles that have not been disinfected between loads [8] . in relation to the swine operation involved in this outbreak, it is improbable that contaminated vehicles were involved. first, the operation is closed, meaning no swine are brought on site unless they are owned by the entity and managed under the stringent biosecurity procedures displayed by the operation. second, the operation maintains 3 truck wash facilities where written protocols are followed to thoroughly clean, wash and disinfect all company trucks. the production company regularly audits the truck wash facilities and was actively testing trucks, trailers, drying equipment, and wash bays for pedv; all samples taken prior to the outbreak and during the first week of the outbreak were negative for pedv. cull animals are hauled on cull-only trailers controlled by the production system and are washed, disinfected, dried and inspected prior to use. cull animals are transferred to a neutral location where the animals are transferred onto a third party hauler's washed and disinfected trailer for market delivery. finisher trucks are not disinfected at company truck washes but rather at truck washes external to the production system. given lack of production system control over these external truck washes, finishing trucks are perceived as a higher biosecurity risk to the operation; however, trucks transporting finisher swine go only to harvest facilities and do not come in contact with pigs or sows from breedwean units within the operation. this operation primarily uses feed produced by the operation's on-site feed mill, with exception of an outsourced starter pellet fed to piglets at the time of weaning and a commercial meal mix used to start nursery pigs on pellets. it was determined that neither feed supplied by the operation's own mill, nor the commercial meal mix were likely to be involved in the transmission of pedv to pigs on the operation. prior to the outbreak, the same internal feed ingredients and commercial meal mix had been used with no ill effects. also, internal feed ingredients were used across all swine units within the operation, but not all swine units were involved in the outbreak. because the timing of the outbreak seemed to coincide with the switch to a new source of starter pellet feed, the attending veterinarian and farm officials suspected the new supplier's starter pelleted diet was the source of pathogen introduction. results of the epidemiologic investigation indicated pedv genetic material presence in the starter feed, validating this suspicion. starter feed pellets from the new supplier were offered to piglets in the a1 farrowing facility during the week of january 6, 2014. by january 12, 2014, clinical signs of pedv were present among sows and piglets in the a1 facility. starter feed pellets were subjected to standard biosecurity procedures to enter into the facility. in short, feed bags are placed into clean bins from the facility, and bins loaded with feed are disinfected in a fume chamber as they are transferred into the facility. following this protocol eliminates contamination from the outer surface of the bag as the source and indicates feed ingredients are likely the source of contamination. supporting the introduced pellets as a likely source, the a2 breed-wean site within the same flow, which never received the new supply of feed pellets, remained pedv negative. pigs in one nursery in flow b (bn1), 3 nurseries in flow c (cn5, cn6 and cn7 west barn), and one wean-to-finish unit in the multiplier herd (d) were also started on the new supplier's feed pellets. all of these sites were subsequently found to be pcr positive for pedv except the flow d wean-to-finish unit. it is thought that differences in pellet storage conditions may account for this inconsistency from the flow d unit. flows b and c store their feed pellets in a room separate from the barn. during winter, these rooms are estimated to be at 40°f (approximately 4°c). the units in flow d store their pellets within the barn where temperatures are around 80°f (approximately 27°c). storage in higher temperatures may have inactivated the virus. this hypothesis is supported in a study by jung and chae where storage of fecal samples at temperatures 21°c and greater resulted in a decline in pedv nucleic acid detection by rt-pcr when compared to those stored at 4°c [15] . another inconsistency was that two contract finisher facilities in flow b and both b flow breed-wean units also broke with disease or tested positive for pedv, even though these facilities did not receive the new supplier's pellets. because pedv is highly transmissible, spread of disease from the units where it broke to the breed-wean units by human error cannot be ruled out. of note, the same person does chores at the b1 farrowing unit and the bn1 nursery where pigs were fed the implicated pellets. also, the two flow b finisher facilities had just received pigs and do share a person who does chores between them, but that person did not have direct contact with any of the sow units. along with the timing of the outbreak that coincided with the switch to new supplier's feed pellets, strong evidence for these feed pellets as the source of the outbreak comes from pcr testing of the new supplier's pellets. pellets from the bn1 nursery tested pedv positive by rt-pcr on january 17, 2014 (c t value = 32.95). additionally, three lots of feed pellets from bn2 nursery, which had been cleaned and disinfected and was empty of pigs at the time, tested positive for pedv by rt-pcr (mean c t value = 32.58). pigs that had left this nursery and were now in a finisher facility tested negative for pedv, showing this nursery had been negative for pedv while it was housing pigs. similar findings result from testing of units within flow c. feeder pigs that left the facility at the end of december and beginning of january were tested and found to be pedv negative, confirming that no disease was present prior to january 12. one of the afflicted flow c nursery sites (cn7) consists of 2 barns labeled east and west. cn7 west barn received new pigs, pellets from the new supplier, and subsequently broke with pedv. at the same time, the cn7 east barn housed pedv negative pigs weighing approximately 50 lbs. from the previous placement. these pigs stayed pedv negative after moving offsite all the way through marketing. interestingly, cn7 west barn had pellets from both the old and new suppliers in the barn at the time of the outbreak. the test results showed swabs taken on the outside of both old and new suppliers' pellet feedbags were pedv rt-pcr positive (mean c t value = 31.93). therefore, pellet samples were collected with care to avoid contamination from the exterior of the bags. briefly, the top 25 cm of the feedbags were wiped with a 0.52% solution of sodium hypochlorite. bags were then opened by cutting the top of the bag off with a scalpel to ensure a minimum risk for potential dust contamination. feed samples were retrieved from the center of each bag by the attending veterinarian who was wearing a sterile obstetrical sleeve. the samples were placed into a sterile plastic bag, sealed, and submitted for testing. pedv rt-pcr was positive (mean c t value = 33.34) for the new supplier's pellets and pedv rt-pcr negative for the old supplier's pellets. these results were interpreted to mean that the exterior of the feedbags had become contaminated with pedv in the barn during the outbreak; however, since pedv was detected in the interior of the unopened bags of the new supplier's pellets, pedv contamination of this feed had to occur prior to delivery at the barn. building on the rt-pcr results from new supplier's feed pellets on the swine operation, back-up pellets of the same lots at the new supplier's manufacturing facility also tested rt-pcr positive (mean c t value = 32.97). testing of individual ingredients at the new supplier's facility yielded several positive results. strong evidence implicating pellets from the new supplier as the contamination source based on the pedv rt-pcr positive results is firmly supported by findings of the epidemiologic investigation. the epidemiologic investigation also concluded that virus isolation from pellets would be critical evidence that the pellets caused the outbreak. since pedv is very difficult to isolate, a bioassay was initiated to determine if the pellets in question could infect naïve piglets. during the outbreak at the swine operation, the attending herd veterinarian aseptically collected aliquots (as described above) of the rt-pcr positive pelleted feed from the farm and mixed them with sterile phosphate buffered saline to make a mash. these moistened, mash aliquots were stored at −20°c until the bioassay could be performed. ten, 10-day-old pigs, were obtained from a commercial sow herd. sows from the source herd, the facility where the bioassay was performed, and the piglets were all confirmed to be negative for pedv by rt-pcr at the start of the bioassay. serum, collected from the pigs prior to leaving the source farm, tested negative for pedv antibodies using an indirect immunofluorescence assay. during a 108 hour acclimation period, pigs were fed a commercial swine starter feed and rectal swabs from the pigs, feed samples, and environmental swabs were all collected on a daily basis. following the acclimation period, the pigs were provided ad libitum access to the rt-pcr positive mash along with dry pellets from the same lot for 7 days, and observed for clinical signs of pedv. feed samples, environmental swabs, and rectal swabs were collected each day of the study. after 7 days, the pigs were euthanized and intestinal tissues were submitted for diagnostic testing. the environment, starter feed, and pigs were pedv negative using rt-pcr prior to the study and during the 108 hour acclimation period. mash aliquots and pelleted feed obtained from the swine operation site tested weakly pedv positive with rt-pcr during the 7 day study (mean ct = 36.5). pigs were observed to be very healthy during the bioassay and no clinical signs of disease were observed in the pigs during the bioassay. environmental and rectal swabs collected daily during the study were negative for pedv using rt-pcr. microscopic examination of intestinal tissues collected from the piglets at the end of the study revealed no significant morphologic lesions. although the bioassay results did not confirm the feed pellets in question were infectious, feed cannot be ruled out as the cause of this outbreak. in the present study, the sensitivity of the bioassay was limited by the amount of feed the individual pigs and the small number of pigs collectively could consume during the trial period. even if infectious virus was present in the feed used for the bioassay, the mean c t value of 36.5 indicates it would be present at very low concentration. in addition, the pigs evaluated appeared healthy, with what was likely limited disease challenge resulting in little immune or digestive system compromise. in a field setting where there are thousands of pigs consuming tons of feed, and known, observable presence of unthrifty pigs with potentially compromised digestive or immune systems, it is conceivable that a very small amount of infectious pedv in a food source would be capable of initiating an outbreak that would rapidly spread through the population of susceptible animals. in addition, the present bioassay portion of the study may have been hindered by the 28 day lag from the time the feed was manufactured and the initiation of the bioassay. the time lag likely decreased the viability of any infectious pedv that was present in the feed at the time of delivery to the farm. because the timing of this outbreak coincided with a switch to new out-sourced feed pellets and due to the strong evidence provided by pedv positive rt-pcr results of these feed pellets at both the swine operation and the supplier, it is believed that contaminated feed pellets were the source of this outbreak. a study reported subsequent to completion of the present study proved that contaminated feed can serve as a vehicle to transmit pedv to naïve pigs [9] . the results of the epidemiologic investigation, proof of concept by other investigators and the presence of pedv rna from unopened bags of feed all support feed as the source of the outbreak. the inability of a bioassay to prove the feed pellets were infectious after the outbreak occurred must be considered, but the low sensitivity of this assay does not rule out feed as possible source. the results of the present and other studies demonstrate the need for strict biosecurity practices and thorough testing for feed and feed ingredients used in the pork industry for which, pedv outbreaks can cause devastating financial losses and pedv surveillance and prevention efforts are of the utmost importance. pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states fighting a deadly pig disease. industry, veterinarians trying to contain ped virus, new to the us isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines role of transportation in spread of porcine epidemic diarrhea virus infection, united states an evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of naive pigs following consumption via natural feeding behavior: proof of concept investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in canada the spray-drying process is sufficient to inactivate infectious porcine epidemic diarrhea virus in plasma porcine epidemic diarrhea virus rna present in commercial spray-dried porcine plasma is not infectious to naive pigs evidence of infectivity of airborne porcine epidemic diarrhea virus and detection of airborne viral rna at long distances from infected herds animal coronaviruses: what can they teach us about the severe acute respiratory syndrome? effect of temperature on the detection of porcine epidemic diarrhea virus and transmissible gastroenteritis virus in fecal samples by reverse transcription-polymerase chain reaction submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www we thank jody edwards for her expertise in scientific writing and editing and tim vojt for his medical illustration services. funding was provided by the national pork checkoff, pic north america and the u.s. department of agriculture. partial funding for open access was provided by the ohio state university open access fund. any mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the us department of agriculture. the contents herein are solely the responsibility of the authors and do not necessarily represent the official views of the national pork board, pic north america or the u.s. department of agriculture. the authors declare that they have no competing interests.authors' contributions asb performed the bioassay and drafted the manuscript. rak led the epidemiological investigation and assisted with preparation of the manuscript. tp and md performed field activities. sjm performed the bioassay and assisted with manuscript preparation. all authors read and approved the final manuscript. key: cord-336517-v7z62tld authors: chu, hsu-feng; chen, chiao-che; moses, david c.; chen, yau-hung; lin, chao-hsiung; tsai, ying-chieh; chou, chi-yuan title: porcine epidemic diarrhea virus papain-like protease 2 can be noncompetitively inhibited by 6-thioguanine date: 2018-08-20 journal: antiviral res doi: 10.1016/j.antiviral.2018.08.011 sha: doc_id: 336517 cord_uid: v7z62tld porcine epidemic diarrhea virus (pedv) is a coronavirus (cov) discovered in the 1970s that infects the intestinal tract of pigs, resulting in diarrhea and vomiting. it can cause extreme dehydration and death in neonatal piglets. in asia, modified live attenuated vaccines have been used to control pedv infection in recent years. however, a new strain of pedv that belongs to genogroup 2a appeared in the usa in 2013 and then quickly spread to canada and mexico as well as asian and european countries. due to the less effective protective immunity provided by the vaccines against this new strain, it has caused considerable agricultural and economic loss worldwide. the emergence of this new strain increases the importance of understanding pedv as well as strategies for inhibiting it. coronaviral proteases, including main proteases and papain-like proteases, are ideal antiviral targets because of their essential roles in viral maturation. here we provide a first description of the expression, purification and structural characteristics of recombinant pedv papain-like protease 2, moreover present our finding that 6-thioguanine, a chemotherapeutic drug, in contrast to its competitive inhibition on sarsand mers-cov papain-like proteases, is a noncompetitive inhibitor of pedv papain-like protease 2. discovered in the 1970s, porcine epidemic diarrhea virus (pedv) is a coronavirus that causes severe agricultural loss (chasey and cartwright, 1978; lee, 2015) . pedv infects the intestinal tract and causes diarrhea and vomiting in older pigs. the mortality of pedv-infected piglets can reach 100% due to extreme dehydration (stevenson et al., 2013) . recently, modified live attenuated vaccines for pedv genogroup 1 has been an important way to control the spreading of pedv in asia (song and park, 2012) . in 2013, a new vaccine-resistant pedv strain, appeared in the usa (stevenson et al., 2013) . since then, this new usa pedv strain, which was later assigned to genogroup 2a, has caused a continuous pandemic in north america, europe and asia (huang et al., 2013; song et al., 2015) . the resistance of the new pedv strain to the conventional vaccine highlights the issue that although vaccination is a powerful strategy, it can still fail due to the genetic diversity of epitopes between genogroups. as a result, it is necessary to develop another antiviral strategy for pedv. unlike two other highly pathogenic human covs, severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), which belong to the beta group, pedv belongs to the alpha group (chan et al., 2015) . like other covs, pedv depends on its own proteases, including main protease (m pro ) and papain-like protease (pl pro ) to cleave the polyprotein, and alpha covs have both papain-like protease 1 (pl1 pro ) and papain-like protease 2 (pl2 pro ) (lee, 2015; wojdyla et al., 2010) . polyprotein cleavage is required for viral maturation and thus these proteases are ideal antiviral targets (bacha et al., 2008; cheng et al., 2015; chou t et al., 2008; kumar et al., 2017; lin et al., 2018; park et al., 2012; ratia et al., 2008; wu et al., 2006) . furthermore, in contrast to highly variable spike proteins targeted by antibodies (li et al., 2005 (li et al., , 2017 wang et al., 2013; wu et al., 2009) , proteases with more conserved structure and catalytic function may serve as a general target across different covs bailey-elkin et al., 2014; chou et al., 2014; ho et al., 2015; ratia et al., 2006; yang et al., 2003) . like other coronaviral pl pro s, pedv pl2 pro is not only a deubiquitinating (dub) protease but also a multifunctional protein which plays a role in regulating host antiviral immune response (chaudhuri et al., 2011; clementz et al., 2010; mielech et al., 2014; xing et al., 2013; zheng et al., 2008) . dub activity of pl2 pro is required for the suppression of host immunity by blocking type 1 interferon signaling. however, up to now, the structural characteristics and the detailed catalytic mechanism of pedv pl2 pro are still unclear, as are strategies for its inhibition. according to sequence alignment (fig. s1 ) and homology modeling, pedv pl2 pro may be composed of four domains: ubiquitin-like (ubl), palm, thumb and fingers domains. the latter three domains form a catalytic core and may retain catalytic activity, like that of sars-and mers-cov pl pro s (chou et al., 2012; clasman et al., 2017; lei et al., 2014) . in the present study, we made a recombinant catalytic core pedv pl2 pro and demonstrated its secondary, tertiary and quaternary structural characteristics. further studies suggest that pedv pl2 pro exhibits much higher dub activity than that of sars-and mers-cov pl pro s and can be inhibited by the anti-leukemia drug 6-thioguanine (6tg). 6tg has been found to be able to inhibit multiple dub enzymes including human usp2 as well as sars-and mers-cov pl pro s (chen et al., 2009; cheng et al., 2015; chou et al., 2008; chuang et al., 2018; lin et al., 2018) . inhibition assays and docking simulations were used to clarify the detailed inhibition mechanism of 6tg against pedv pl2 pro . our findings increase understanding of the structure and catalytic activity of pedv pl2 pro and identify the first potent inhibitor against this enzyme. the coding sequence of the usa strain of pedv pl2 pro ubl and catalytic domain (genbank accession number ahc03501.1; polyprotein residues 1630-1922) was full-gene synthesized (genomics co., taiwan). cdna of pl2 pro digested by ndei and xhoi was then inserted into pet-28a and pet-29a expression vectors, respectively. for the catalytic core of pl2 pro (residues 1686-1922) , the primers 5′-ggctcc gcgaattgggattccc-3′ and 5′-aaaactcgagtcattcggacaccac cacattg-3′ were used for polymerase chain reaction. the product was digested with xhoi and then ligated into a phd vector with a 6× histag and a sumo (smt3) sequence at the n-terminus (lee et al., 2008) . the primer sequences for site-directed mutagenesis of the t39w mutant were 5′-gaatggccgtcgtgtgctgaaatggaccgataataattgc tgg-3′ and 5′-ccagcaattattatcggtccatttcagcacacgacggc cattc-3'. the reading frames of the above plasmids were verified by dna sequencing. the expression vector was transformed into e. coli bl21 (de3) cells (novagen). cultures were grown in lb medium at 37°c until the absorbance at 600 nm reached 0.6. the cells were then induced with 1 mm isopropyl-β-d -thiogalactopyranoside and incubated for 20 h at 20°c. after centrifugation, the cell pellet was suspended in lysis buffer (20 mm tris-cl, ph 8.5, 250 mm nacl, 5% glycerol, 0.2% triton x-100 and 2 mm β-mercaptoethanol) and lysed by sonication. next, the crude lysate was centrifuged at 12,000 rpm for 20 min. the soluble lysate was incubated with 2 ml of ni-nta agarose slurry (qiagen co., usa) at 4°c for 1 h. after the unbound lysate removed, the beads were washed with wash buffer (20 mm tris-cl, ph 8.5, 250 mm nacl, 8 mm imidazole and 2 mm β-mercaptoethanol). the beads with 6× his-tag protein bound were then transferred into 3.5 ml cutting buffer (20 mm tris-cl, ph 8.5, 250 mm nacl, and 2 mm β-mercaptoethanol), 0.1 mg ulp1 was added, and the mixture was incubated at 25°c for 4 h to remove the sumo fusion. finally, unbound pedv pl2 pro catalytic core was loaded onto an s-100 gel-filtration column (ge healthcare) equilibrated with running buffer (20 mm tris-cl, ph 8.5, 100 mm nacl and 2 mm dithiothreitol). each protein fraction was checked for purity using sds-page. fractions containing a protein band of the correct size were then pooled and concentrated using an amicon ultra-4 10-kda filter (millipore) to 20 mg/ml. the protein with 5% glycerol was flash-frozen with liquid nitrogen and stored at −80°c. the typical yield of protein was 3-5 mg per liter of cell culture. a jasco j-810 spectropolarimeter was used to analyze the secondary structure. recombinant pedv pl2 pro at a concentration of 1 mg/ml in 20 mm phosphate buffer (ph 6.5) was used for cd scanning from 250 nm to 190 nm at 20°c. the cuvette width was 0.1 mm. the far-uv cd spectrum data was analyzed using the cdsstr program at the dichroweb server (whitmore and wallace, 2008) . the normalized root mean square deviation was calculated to evaluate goodness of fit. the fluorescence spectrum of 0.5 μm protein dissolved in 20 mm phosphate buffer (ph 6.5) or 5.4 m guanidine hydrochloride was monitored at 25°c using a perkinelmer ls50b luminescence spectrometer. the excitation wavelength was 280 nm and the emission spectrum was scanned from 300 nm to 400 nm. measurements of maximal peak and intensity were used to identify conformational change. auc experiments were performed on an xl-a analytical ultracentrifuge (beckman coulter) using an an-60 ti rotor. sedimentation velocity (sv) experiments were performed using a double-sector epon charcoal-filled centerpiece at 20°c at a rotor speed of 42,000 rpm. a protein solution of 0.4 mg/ml pedv pl2 pro catalytic core and reference solutions (20 mm tris-cl, ph 8.5, 100 mm nacl) were loaded into the centerpiece. absorbance at 280 nm was monitored continuously with a time interval of 500 s and a step size of 0.003 cm. multiple scans at different time intervals were fitted to a continuous c(s) and c(m) distribution model using the sedfit program (schuck, 2000) . for dub assays, the reaction mixtures contained 0.25 μm of fluorogenic substrate ubiquitin-7-amino-4-trifluoro-methyl-coumarin (ub-afc) (boston biochem) and 0.17 μm of sars-or mers-cov pl pro or 0.004 μm of pedv pl2 pro in 20 mm phosphate buffer (ph 6.5) for a total volume of 0.5 ml. enzymatic activity at 30°c was determined by monitoring excitation and emission at 350 and 485 nm, respectively. to determine the inhibitory effect, velocity data at various concentrations of inhibitors was fitted to obtain ic 50 according to eq. (1): in which v is the initial velocity in the presence of inhibitor at concentration [i] and v 0 is the initial velocity in the absence of inhibitor, while n is the hill constant. the program sigmaplot 12.5 (systat software) was used for data analysis. the peptidyl substrates dabcyl-frlkggapikgv-edans and dabcyl-fkkkgggdvke-edans (synthesized by genscript) were used to measure the proteolytic activity of the pedv pl2 pro catalytic core and the t39w mutant. increases in fluorescence intensity were monitored at excitation and emission wavelengths of 329 and 520 nm, respectively, in a perkinelmer ls50b luminescence spectrometer. fluorescence intensity was converted to the concentration of hydrolyzed substrate using a standard curve determined by fluorescence measurements of defined concentrations of products. for kinetic analysis, the reaction mixtures contained 5-50 μm peptide substrate in 20 mm phosphate buffer (ph 6.5) for a total volume of 1 ml. after the addition of enzyme to the reaction mixture to a concentration of 3.85 μm, the fluorescence intensity was monitored at 30°c. the increase in fluorescence intensity was linear for 3 min and thus the slope of the line represented the initial velocity. steady-state kinetic parameters were determined by fitting the data to the michaelis-menten equation. enzyme kinetic assays were performed by a method similar to that described above at peptidyl substrate concentrations of 10-50 μm and inhibitor concentrations of 0-60 μm. the velocity data was found to best fit a noncompetitive inhibition model in accordance with eq. (2): in which k cat is the rate constant, [e], [s] and [i] denote the enzyme, substrate and inhibitor concentrations, respectively, and k m is the michaelis constant for the interaction between the peptide substrate and the enzyme. k is is the inhibition constant. the model structure of pedv pl2 pro was generated by swissmodeling (arnold et al., 2006) and molecular docking was performed using autodock vina (trott and olson, 2010) . several grid boxes of 27000 å 3 (30 å × 30 å x 30 å) with different centering coordinates were set to cover the entire putative structure. the docking parameters were set as default and the best 10 models in each coordinate set were listed for further inspection. the model that scored the best among these sets was selected as the final binding model. initially, attempts at expressing pedv pl2 pro including the ubl and catalytic domains (polyprotein residues 1630-1922) with either an nterminal or a c-terminal 6× his-tag were not successful. previous studies suggested that the ubl domain was not involved in the catalytic activity of sars-and mers-cov pl pro s (chou et al., 2012; clasman et al., 2017) . therefore we removed the ubl domain and applied a sumo fusion protein at the n-terminus of the catalytic core (residues 1686-1922) to enhance solubility. fortunately, it was possible to express the pedv pl2 pro catalytic core in e. coli and purify it following the removal of sumo. after further purification using size-exclusion chromatography, highly pure pl2 pro was obtained (fig. s2 , left panel, lane 6). mass spectrometry was performed to identify the sequence of the recombinant protein. in total, 19 peptides originating from pedv pl2 pro were identified (fig. s2, right panel) . alignment of these peptides with the pedv pl2 pro sequence shows 60% coverage, and both the n-terminus and c-terminus of the protein were confirmed (fig. s2, bottom panel) . as this is the first time that pure pedv pl2 pro has been obtained, its secondary, tertiary and quaternary structures were further analyzed. cd spectrometry and analysis of the spectrum by cdsstr (fig. s3a) showed that pedv pl2 pro consists of 27% α-helix, 29% β-sheet and 44% random coil. these proportions are similar to those of the sars-cov pl pro core (residues 1600-1858), which consists of 21% α-helix, 29% βsheet and 49% random coil (data not shown). for comparison, previous studies suggested that mers-cov pl pro consists of 23% α-helix, 36% βsheet and 38% random coil, where the higher content of β-sheet is because of the inclusion of the ubl domain (lin et al., 2014) . our results suggest that the three coronaviral pl pro s show similar secondary structural content. protein emission was used to reveal tertiary conformational change in the absence and presence of denaturant (fig. s3b) . the results demonstrated that the fluorescence emission of pedv pl2 pro in phosphate buffer (native form) shows a major broad peak at 330 nm which splits to two peaks at 310 nm and 360 nm in 5.4 m guanidine hydrochloride. the two peaks match the maximal emission wavelengths of tyrosine and tryptophan, respectively. this result suggests that the addition of denaturant induces exposure of the hydrophobic core of the protein. similar folding/unfolding change at maximal emission wavelength was also observed in the case of sars-cov pl pro , albeit the fluorescence intensity of denatured pedv pl2 pro is higher than that of the native enzyme (chou et al., 2012) . previous studies on the stability of the p53 core domain suggested that the higher fluorescence intensity of denatured p53 is because of aggregation (bullock et al., 1997) . sv experiments were carried out to determine the quaternary structure of pedv pl2 pro (fig. s4a ). using continuous c(s) and c(m) distribution analysis, we found one major peak with a sedimentation coefficient of 2.4 and molecular weight of 25 kda (figs. s4b and s4c ). this value is close to the predicted monomeric mass (25.8 kda). previous studies using the same sv experiment suggested that both the sars-and mers-cov pl pro s are also monomers lin et al., 2014) . overall, the secondary, tertiary and quaternary structures of the pedv pl2 pro catalytic core are similar to those of sars-and mers-cov pl pro s, even though their sequence identity is only 22-25% (fig. s1 ). next, in order to compare the dub activity of pedv pl2 pro with other coronaviral pl pro s, activity assays using ub-afc as the substrate were carried out. the activity was determined and then normalized to give the fold increase in dub activity of pedv pl2 pro (fig. 1) . the results showed that sars-and mers-cov pl pro s have similar dub activity. surprisingly, at a given substrate concentration, pedv pl2 pro shows activity comparable to that of the other two pl pro s at only onefortieth of the protein concentration. this indicates that pedv pl2 pro has considerably greater dub activity. furthermore, sequence alignment indicates that pedv pl2 pro may also have a zinc fingers motif like other coronaviral pl pro s (fig. s1 , green ovals) (bailey-elkin et al., 2014; lei et al., 2014; ratia et al., 2006; wojdyla et al., 2010) . previous studies have shown that the dub activity of coronaviral pl pro s can be inhibited by the addition of a chelator like edta, which removes intrinsic zinc ions (chou et al., 2008; lin et al., 2014) . in line with our expectations, inhibition of pedv pl2 pro by edta displays a similar dose-dependent pattern, suggesting that the removal of endogenous metal ions can inhibit pedv pl2 pro (fig. 1) . furthermore, like other coronaviral pl pro s, pedv pl2 pro can be inhibited by adding extra external zinc ions (chou et al., 2008; lin et al., 2014) . in addition, the proteolytic activity of pedv pl2 pro was investigated using fluorogenic peptidyl substrates (table 1) . for comparison, we used a peptidyl substrate with a sequence matching the p6 to p1 residues of the cleavage site of sars-cov pl pro , frlkgg, and a peptidyl substrate optimized for pedv pl2 pro whose cleavage site p6 to p1 residues are fkkkgg, based on the non-structural protein (nsp) 2 to 3 cleavage site (from genbank accession number ahc03501.1). interestingly, less saturation was observed while using the optimized peptidyl substrate (fig. s5a) . we failed to improve it due to the fact that the proteolytic activity at concentrations of substrate higher than 50 μm cannot be appropriately measured because of the inner-filter effect. after fitting the data to the michaelis-menten equation, k m of 18.6 μm and k cat of 0.065 min −1 for the sars-cov-derived substrate and the k m of 61.6 μm and k cat of 0.299 min −1 for the optimized substrate were determined (table 1 ). the optimized substrate shows a 1.4-fold higher k cat /k m , as a result of a 3.3-fold higher k m and 4.6-fold higher k cat , compared with the sars-cov-derived substrate. the dissimilar kinetic parameters for the two peptidyl substrates suggest that pedv pl2 pro may recognize various p4 residues between beta covs (l/f/v/g) and alpha covs (k/r/a). by contrast, sars-cov pl pro has a hydrophobic s4 subsite, with the result that it cannot cleave the pedv-optimized substrate and substrates of hcov-229e and ibv whose p4 residue is also a lysine han et al., 2005; lei et al., 2018; wojdyla et al., 2010) . furthermore, in contrast to its considerably greater dub activity, k cat of pedv pl2 pro for the optimized peptidyl substrate is 22fold lower than that of sars-cov pl pro (lin et al., 2014) . the inconsistent efficacy between dub and proteolytic activities indicates that pedv pl2 pro is more like a usp enzyme (avvakumov et al., 2006; renatus et al., 2006) . previous studies have suggested that pedv pl2 pro , but not pl1 pro , is an interferon antagonist via its dub activity (xing et al., 2013) . in addition, a recent review suggests that pl1 pro and pl2 pro of alpha covs may show different levels of efficiency for cleaving the nsp 1 to 2, 2 to 3 and 3 to 4 sites (lei et al., 2018) . previous studies of sars-cov pl pro demonstrated that the oxyanion is within hydrogen-bonding distance of the side chain of trp107 during catalysis (ratia et al., 2006) . although it is not conserved, mutation of the equivalent residue leu105 of mers-cov pl pro to tryptophan showed a 41-fold increase in k cat (lin et al., 2014) . again, according to sequence alignment, the equivalent residue of pedv pl2 pro is thr39 (fig. s1, purple oval) . to verify this, a t39w mutant was produced and its kinetic parameters were characterized by using the two peptidyl substrates (fig. s5b and table 1 ). interestingly, using the sars-covderived substrate produced a 9.3-fold increase in activity based on k cat / k m as a result of a 1.7-fold increase in k m and a 16-fold increase in k cat . this result demonstrates that the mutation enhances hydrolysis of the sars-cov-derived peptidyl substrate. in contrast, there is no significant difference in hydrolysis of the optimized substrate between wild-type pedv pl2 pro and the t39w mutant (table 1 ). this result indicates that the existence of residue thr39 may be quite enough to support proteolytic ability. further structural information on pedv pl2 pro in complex with ub or the optimized peptidyl substrate is required to demonstrate the detailed catalytic mechanism, especially for the formation of the oxyanion hole. several coronaviral pl pro inhibitors have been identified in previous studies (chen et al., 2009; cheng et al., 2015; chou et al., 2008; lin et al., 2018; ratia et al., 2008) . in the present study, these compounds were screened to determine whether they can inhibit pedv pl2 pro (table 2) . among the compounds, two thiopurine analogs, 6-mercaptopurine (6mp) and 6tg, were found to be able to inhibit the dub activity of pedv pl2 pro with ic 50 of 58.1 and 13.7 μm, respectively ( fig. 2 and table 2 ). ic 50 of 6tg is 4.2-fold lower than that of 6mp, suggesting that the amino group of 6tg may play the role of an active pharmacophore in the inhibition. in addition, two 6mp/6tg analogs, hypoxanthine, and 2-amino-6-methyl-mercaptopurine, were also used for structure-function relationship studies. we found that replacement of the thiocarbonyl group of 6mp/6tg with either a hydroxyl or a methylthio group resulted in compounds devoid of inhibitory activity, suggesting its importance in the inhibition (table 2) . due to its higher inhibition capability, 6tg was chosen for further investigation. kinetic assays at various concentrations of peptidyl substrates and 6tg were carried out to further investigate the inhibition mechanism (fig. s6) . interestingly, the observed kinetic parameters showed a decrease in the apparent k cat at increasing 6tg concentrations, whereas the apparent k m was not affected significantly. this is clearly indicative of a noncompetitive pattern of inhibition. indeed, the data was found to best fit to a noncompetitive inhibition model with the k is of 21.1 μm ( fig. 3 and table 2 ). this result suggests that 6tg and the peptidyl substrate may bind to different sites. for comparison, 6tg shows a competitive inhibitory effect against sars-and mers-cov pl pro s chou et al., 2008) but shows a noncompetitive inhibitory effect against human usp2 (chuang et al., 2018) , albeit their k is are close. these results indicate that 6tg can be a broad spectrum inhibitor against human and viral dub enzymes via different mechanisms. as 6tg is a noncompetitive inhibitor of pedv pl2 pro , recognition of the binding site of 6tg will allow us to understand its inhibitory mechanism more clearly. as no detailed structural information was . inhibition of pedv pl2 pro by 10-50 mm edta or 50 μm zn 2+ were also measured. activity data of each set was normalized to that of pedv pl2 pro . available, we generated a structural model of pedv pl2 pro and tried to discover a putative binding site of 6tg using in silico docking. interestingly but not surprisingly, we found that the putative binding site of 6tg with the highest affinity score is near the active site and on the left side of the blocking loop (residues 196 to 202), while the ubiquitin c-terminal tail is located on the right side (fig. 4a ). in our model, 6tg has polar interactions with residues asp195, gly197 (on the blocking loop), val230 and thr231 and shows hydrophobic contact with lys175 and pro229. binding of 6tg at this site may render the blocking loop less flexible and therefore disfavor catalysis. a series of noncovalent inhibitors such as compound grl0617 shows a similar blocking effect, although they are competitive inhibitors and bind to the s3-s4 subsite (ratia et al., 2008) . for comparison, besides residue val230, the alignment shows no sequence identity for the putative binding residues (fig. s1, red ovals) . furthermore, the same region is occupied by residue pro300 of sars-cov pl pro and residue lys306 of mers-cov pl pro (fig. 4b) , indicating that this binding site may only exist in pedv pl2 pro . in contrast, previous studies suggested that the binding site of 6tg for sars-and mers-cov pl pro s may be near the catalytic triad's cysteine residue due to its competitive pattern of inhibition chou et al., 2008) . in this study, we provide a first description of the expression, purification and structural properties of pedv pl2 pro as well as its potent inhibition by thiopurine analogs. the broad spectrum inhibitor 6tg was found able to inhibit not only the dub activity but also the proteolytic activity of pedv pl2 pro . these results shed light on the possibility of inhibition of pedv infection by small molecules instead of antibodies. furthermore, based on its noncompetitive inhibitory effect, we proposed an allosteric 6tg binding site which can stabilize the blocking loop near the active site, resulting in inhibition. the present study suggests that 6tg may be suitable as a lead compound for further (1)). b proteolytic activity at various concentrations of sars-cov-derived peptidyl substrate and 6tg was measured ( fig. s6 and fig. 3 ) and the apparent k is was determined from the best global fit of the data to a noncompetitive inhibition model (eq. (2)). r sqr was 0.964. c nd: ic 50 was not determined due to lack of inhibition at a compound concentration of 200 μm. (2)) and r sqr value is 0.964. the kinetic parameters from the best fit are shown in table 1 . the assay was repeated to ensure reproducibility. antiviral drug development. coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs the swiss-model workspace: a web-based environment for protein structure homology modelling amino-terminal dimerization, nrdp1-rhodanese interaction, and inhibited catalytic domain conformation of the ubiquitin-specific protease 8 (usp8) development of broad-spectrum halomethyl ketone inhibitors against coronavirus main protease 3cl(pro) crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papainlike protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression thermodynamic stability of wild-type and mutant p53 core domain middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sarslike disease 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inactivators of the severe acute respiratory syndrome 3cl protease crystal structure of nl63 respiratory coronavirus receptor-binding domain complexed with its human receptor the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production we would like to thank prof. ralph kirby for helpful suggestions. we also appreciate mrs. yen-su lin's help in the synthesis of compound grl0617. this research was supported by grants from the ministry of science and technology, taiwan, roc (104-2320-b-010-034, 105-2320-b-010-012 , 107-2320-b-010-045, and 106-2320-b-010-013) to cyc. hfc was supported by the ministry of education, taiwan, r.o.c. and bened biomedical co. supplementary data related to this article can be found at https:// doi.org/10.1016/j.antiviral.2018.08.011. key: cord-303186-2hxlx1j2 authors: won, hokeun; lee, dong-uk; jang, guehwan; noh, yun-hee; lee, seung-chul; choi, hwan-won; yoon, in-joong; yoo, han sang; lee, changhee title: generation and protective efficacy of a cold-adapted attenuated genotype 2b porcine epidemic diarrhea virus date: 2019-07-09 journal: j vet sci doi: 10.4142/jvs.2019.20.e32 sha: doc_id: 303186 cord_uid: 2hxlx1j2 the recent emergence and re-emergence of porcine epidemic diarrhea virus (pedv) underscore the urgent need for the development of novel, safe, and effective vaccines against the prevailing strain. in this study, we generated a cold-adapted live attenuated vaccine candidate (aram-p29-ca) by short-term passage of a virulent pedv isolate at successively lower temperatures in vero cells. whole genome sequencing identified 12 amino acid changes in the cold-adapted strain with no insertions and deletions throughout the genome. animal inoculation experiments confirmed the attenuated phenotype of aram-p29-ca virus in the natural host. pregnant sows were orally administered p29-ca live vaccines two doses at 2-week intervals prior to parturition, and the newborn piglets were challenged with the parental virus. the oral homologous prime-boost vaccination of p29-ca significantly improved the survival rate of the piglets and notably mitigated the severity of diarrhea and pedv fecal shedding after the challenge. furthermore, strong antibody responses to pedv were detected in the sera and colostrum of immunized sows and in the sera of their offspring. these results demonstrated that the cold-adapted attenuated virus can be used as a live vaccine in maternal vaccination strategies to provide durable lactogenic immunity and confer passive protection to litters against pedv. porcine epidemic diarrhea virus (pedv) is a member of the genus alphacoronavirus, belonging to in the family coronaviridae of the order nidovirales. pedv is a large, enveloped virus possessing a 5′ capped, single-stranded positive-sense rna genome of approximately 28 kb, with a 3′ polyadenylated tail [1, 2] . the pedv genome consists of seven canonical genes, including an open reading frame (orf) 3, flanked by 5′-and 3′-untranslated region (utrs). the first two large orfs comprise two-thirds of the genome and encode the replicase polyproteins, 1a and 1ab, which are then posttranslationally cleaved into 16 mature non-structural proteins (nsp1-16). the last one-third encodes four canonical coronavirus structural proteins in a fixed order, spike (s), membrane (m), envelope (e), and nucleocapsid (n), as well as a single accessory gene encoding orf3 between s and m [1] [2] [3] . the virus can be phylogenetically divided into two genotypes comprising two sub-genotypes: genogroup 1 (classical g1a and recombinant g1b) with low-pathogenicity, and genogroup 2 (local epidemic g2a and global epidemic or pandemic g2b) with high-pathogenicity [1, 2, [4] [5] [6] . pedv causes acute gastrointestinal symptoms characterized by a rapid onset of severe watery diarrhea, vomiting, fatal dehydration, and high mortality in newborn and suckling piglets [7] . in 1971, the virus was initially recognized in england and had since been geographically restricted and problematic in europe and asia during the past four decades [8] . however, in early 2013, pedv first emerged in the united states and rapidly spread to the adjacent north and south american countries, causing significant financial losses to their swine industries [9] [10] [11] . soon after, the us prototype-like highly virulent g2b pedv strains almost simultaneously invaded multiple asian nations, including south korea, taiwan, and japan, resulting in the recurrence of a massive nationwide ped epizootic [5, 12, 13] . pedv has now emerged or re-emerged as one of the most devastating porcine viruses, posing a tremendous threat to the global pork business. the challenges of substantial monetary damage caused by pedv highlight the exigent necessity for the establishment of optimal immunization strategies. in most cases of porcine enteric viruses that produce clinical disease in newborn piglets before the development of active immunity, the neonates are protected by passive lactogenic immunity derived from the dams [14] [15] [16] . prophylactic maternal immunization regimens with oral live viruses, including intentional infection and vaccination of sows during gestation, remain the most effective way to stimulate intestinal mucosal immunity that is subsequently transferred to piglets via mammary secretions [15, 17] . although intentional infection or feedback through controlled oral exposure is extensively used to induce herd immunity, there are several potential risks associated with this practices such as the wide dissemination of other microbial pathogens and the uncertainty of immune induction [1, 2, 15] . therefore, numerous research groups are attempting to develop safer and more effective live attenuated pedv vaccines to induce passive lactogenic immunity as an alternative to intentional infection. in south korea, a number of modified live virus (mlv) vaccines for classical g1a pedv came to be widely used throughout the country; however, they were incapable of controlling the impact of the recent massive g2b outbreaks in the domestic swine industry because of limited cross-protection between two genetic clusters [1, 2, 4, 18] . considering this efficacy issue, there is a high priority for the development of a next-generation mlv vaccine against g2b epizootic or related strains prevalent in the field, which can replicate to high titer in the gut and also boost lactogenic immune responses without giving rise to clinical illnesses. in general, the attenuated virus utilized to prepare the mlv vaccine can be achieved by traditional cell culture adaptation procedures of the virulent wild-type virus in non-host cell lines, but this process is impeded by difficulties in performing laboratory procedures, such as numerous time-consuming, and repetitive passages in non-host cell lines. in contrast, adaptation to growth at low (< 37°c) temperatures by short-term serial passages in vitro has been frequently used to generate several attenuated dna and rna viruses [19] [20] [21] [22] . in this study, we sought to create a cold-adapted attenuated g2b pedv low-passage strain by progressively decreasing growth temperatures to 32°c in vero cells and then attempted to evaluate its protective efficacy on neonatal piglets against virulent pedv challenge. vero cells (atcc ccl-81) were cultured in alpha minimum essential medium (α-mem; invitrogen, usa) with 5% fetal bovine serum (fbs; invitrogen) and antibiotic-antimycotic solutions (100×; invitrogen) and maintained at 37°c in a humidified 5% co 2 incubator. the isolation of a highly virulent korean pedv g2b strain, kor/aram/2014, was conducted from clinical fecal samples on vero cells in the presence of trypsin (usb, usa). virus isolation was confirmed by cytopathic effects (cpes) observation, immunofluorescence assay (ifa), and nucleotide sequencing as described previously [23] . a viral stock was prepared from the 5th passage in cell culture (aram-p5) and used as the parental and challenge virus in this study. pedv n protein-specific monoclonal antibody (mab) was obtained from choongang vaccine laboratories (cavac; korea). the pedv strain, aram-p5, was plaque-purified twice in vero cells, and the purified virus was serially passaged in vero cells by gradually reducing the incubation temperature from 37°c to 32°c as described previously with some modifications [24] . confluent vero cells grown in 100-mm diameter tissue culture dishes were washed with pbs and inoculated with pedv aram-p5 along with trypsin. after incubation at 37°c for 1 h, virus growth medium [bmpro-v medium (cell science & technology institute, inc., japan) supplemented with 5 μg/ml of trypsin] was added. the inoculated cells were maintained at 37°c under 5% co 2 and monitored daily for cpe. when > 70% of the cells showed cpe, the inoculated cells were subjected to three rounds of freezing and thawing. the culture supernatants were then centrifuged for 10 min at 400 × g (hanil centrifuge fleta5, korea) and filtered through a 0.45-μm pore-size filter (millipore, usa). the clarified supernatants were aliquoted and stored at −80°c as a viral stock (aram-p1-ca) for the next passage. in the same manner, aram-p1-ca was passaged 4 times with step-wise descending temperatures of 1°c per passage in vero cells up to 32°c. subsequently, aram-p5-ca was serially cultivated at 32°c for an additional 45 passages for cold adaptation, and virus stock were produced at 24, 28, 35, and 45 passages that were designated aram-p29-ca, p33-ca, p40-ca, and p50-ca. vero cells were infected with each aram virus stock in the presence of trypsin as described above. the culture supernatants were collected 48 h post-infection (hpi) when 70% cpe commonly developed. virus titers were measured by end-point titration in 96-well plates using 10-fold serial dilutions of the supernatant samples in triplicate for each dilution to determine the amount of virus required to produce cpe in 50% of the inoculated vero cells. the 50% tissue culture infectious dose (tcid 50 ) per ml of virus stock was calculated using the reed and muench method [25] . the full-length genomic sequences of the parental aram-p5 and cold-adapted aram-p29-ca strains were determined by traditional sanger methods. ten overlapping cdna fragments spanning the entire genome of each virus strain were amplified and sequenced as described elsewhere [5, 23, 24] . the 5′ and 3′ ends of the genome of the strain were determined by rapid amplification of cdna ends (race) as described previously [26] . the full-length genomic sequences were deposited in the genbank database under accession numbers mk559454 through mk559456. the sequences of 49 fully sequenced s genes and 39 complete genomes of global pedv isolates were independently used in sequence alignments and phylogenetic analyses. multiple sequence alignments were generated using the clustalx 2.0 program [27] and the percentages of nucleotide sequence divergences were assessed using the same software. phylogenetic trees were constructed from the aligned nucleotide or amino acid sequences using the neighbor-joining method and subsequently subjected to bootstrap analysis with 1000 replicates to determine the percentage reliability values of each internal node of the tree [28] . all phylogenetic trees were generated using mega 4.0 software [29] . the four in vivo swine studies described here were independently performed at the cavac animal facility under the guidelines established by its institutional animal care and use committee (iacuc, approval no. 160129-03). all animals were obtained from a conventional breeding farm with a good health record and either vaccination against pedv or no known prior ped outbreak and were tested to confirm that they were not infected with any porcine enteric viruses. piglet challenge trials was conducted to determine the infectious dose and virulence of a pedv aram strain using 3-day-old conventional suckling piglets delivered from commercial crossbred sows (great yorkshire × dutch landrace). thirty pigs were divided into six groups (group 1-group 6) of five animals and were fed commercial milk replacer frequently (4 times daily) with ad libitum access to water for the study (7 days). animals in groups 1-6 were allowed to acclimate for two days and then inoculated orally with 1 ml of 10 −2 , 10 −3 , 10 −4 , 10 −5 , and 10 −6 dilutions of the aram-p5 virus (10 5.0 tcid 50 /ml) and cell culture medium at 5 days of age, respectively. after inoculation, piglets were observed daily for clinical signs, including diarrhea and mortality. stool samples from pigs in all groups were collected prior to inoculation and daily with 16-inch cotton-tipped swabs and scored for fecal consistency for 5 days post-inoculation (dpi): 0 = normal; 1 = pasty; 2 = semi-liquid; 3 = liquid or watery. mean pig diarrhea dose (pdd 50 ) and lethal dose (ld 50 ) were determined as the reciprocal of the virus dilution at which 50% of the pigs developed watery diarrhea (score 3) or died at a given time point using the reed and muench method. to assess immunogenicity of cold-adapted derivatives, a total of 14 3-week-old conventional piglets delivered from commercial crossbred sows (great yorkshire × dutch landrace) were allocated into the cold adapted aram-p29-ca-inoculated (n = 3), p33-ca-inoculated (n = 3), p-45-ca-inoculated (n = 3), p50-ca-inoculated (n = 3), and sham-inoculated control (n = 2) groups. animals were immunized orally with a 1 ml dose of 10 4.5 tcid 50 /ml of each virus or sham inoculated with cell culture medium. pre-immune sera were collected at the immunization, and antisera were collected at 1 week intervals for 3 weeks. pathogenicity tests were performed using a total of 12 suckling piglets of 3 days of age obtained from commercial crossbred sows (great yorkshire × dutch landrace). pigs were randomly assigned to three experimental groups: the parental aram-p5-inoculated (n = 4), cold-adapted p29-ca-inoculated (n = 4), and sham-inoculated control (n = 4) groups. animals were fed commercial milk replacer frequently (4 times daily) and had ad libitum access to water for the duration of the study (9 days). after a 2-day acclimation period, piglets (5-day-old) in the virusinoculated groups received a 1 ml dose of 10 4.0 tcid 50 /ml (equivalent to 1000 ld 50 as determined in this study) of one of the viruses orally. the sham-inoculated pigs were administered cell culture medium as a placebo. animals were monitored daily for clinical signs of vomiting, diarrhea, and mortality throughout the experiment. rectal swabs were collected from all pigs and diluted with pbs to 10% (w/v) suspensions followed by centrifugation. the clarified supernatants were subjected to conventional reverse transcription polymerase chain reaction (rt-pcr) using an i-tge/ped detection kit (intron biotechnology, korea) and real-time rt-pcr to detect the presence of pedv shedding. a clinical significance score (css) was determined with the following scoring criteria based on visual examination for 7 dpi used to measure diarrheal severity: 0 = normal and no diarrhea (mean cycle threshold [ct] values of > 45); 1 = mild and fluidic feces; 2 = moderate watery diarrhea; 3 = severe watery and projectile diarrhea (mean ct values of < 20); 4 = death. piglets were necropsied upon death after challenge throughout the study, whereas all surviving pigs from the challenge and control groups were euthanized at 7 dpi for post-mortem examinations. swine vaccination and challenge experiments were conducted with a total of 6 commercial crossbred pregnant sows (great yorkshire × dutch landrace) with the same parity and expected farrowing date. animals were allocated randomly into three experimental groups: vaccinated group 1 (n = 3), challenge control group 2 (n = 2), and strict negative control group 3 (n = 1). a multiple-dose ped homologous vaccination schedule at 2-week intervals starting prior to farrowing was applied in the present protection study. three sows in group 1 were orally administered twice with a 1 ml dose of the aram-p29-ca with 10 4.5 tcid 50 /ml at 4 and 2 weeks pre-partum. the remaining sows in groups 2 and 3 were unvaccinated and served as controls. all sows were monitored daily for clinical alterations and adverse effects following vaccination. to mimic the field conditions of nursing piglets, all sows were allowed to farrow naturally and nurse their piglets freely for the duration of the study. ten 4-day-old suckling piglets per litter (a total of 50 newborn piglets) in groups 1 and 2 were challenged orally with a 1 ml dose of 10 4.0 tcid 50 /ml of virulent aram-p5 virus. the sham-inoculated piglets from group 3 were administered with cell culture medium as a placebo. animals were observed daily for clinical signs, including vomiting, diarrhea, and mortality. fecal specimens were taken from all groups and examined for pedv shedding as described above. a css was determined for 7 days postchallenge (dpc) as described above. blood samples were collected from sows before (at each vaccination), at, after farrowing (up to 2 weeks) and also from 5 representative piglets per litter from each dam at 0 and 7 dpc. colostrum was collected at the day of farrowing. viral rna was extracted from fecal suspensions prepared as described above using an i-tge/ ped detection kit according to the manufacturer's protocol. quantitative real-time rt-pcr was performed using a one step sybr primescript rt-pcr kit (takara, japan) as described elsewhere [24, 30, 31] . a pedv isolate with a known infectivity titer was 10-fold serially diluted to generate a standard curve in each pcr plate. the virus concentrations (tcid 50 /ml) in samples were calculated based on the standard curve. the mean ct values were calculated based on pcr positive samples, and the mean virus titers were calculated based on all pigs within the group. intestinal tissues and other major organs were grossly examined upon necropsy. small intestinal tissue specimens (< 3-mm thick) collected from each piglet were fixed with 10% formalin for 24 h at rt and embedded in paraffin according to standard laboratory procedures. the formalin-fixed paraffin-embedded tissues were cut in 5-8-μm thick sections on a microtome (leica, germany), floated in a 40°c water bath containing distilled water, and transferred to glass slides. the deparaffinized intestinal tissue sections were stained with hematoxylin and eosin (sigma, usa) to observe histopathological change or were subjected to immunohistochemistry (ihc) for the detection of pedv antigen using an mab specific for pedv n protein as described previously [23, 24] . pedv antigen detection was semiquantitatively measured throughout the ihc-stained jejunal sections based on the following scoring criteria as described previously [32] : 0 = no signal, 1 = 1%-10% of villous enterocytes within the section showing a positive signal, 2 = 11%-50% of villous enterocytes showing a positive signal, and 3 = greater than 50% of villous enterocytes showing a positive signal. the presence of pedv-specific neutralizing antibodies in serum and colostrum samples collected from pigs in all groups was determined using a serum neutralization (sn) test using pedv isolate aram-p5 as previously described [24] . the neutralization titer was calculated as the reciprocal of the highest dilution of serum that inhibited virus-specific cpe in duplicate wells. all values are expressed as the means ± standard deviation of the means (sdm). all statistical significances were evaluated by a student's t-test by using graphpad prism software version 5.0 (graphpad prism inc., usa). the p values of less than 0.05 were considered statistically significant. pedv isolation in cell culture is the first step toward the generation of a live attenuated vaccine. thus, we initially attempted to isolate pedv from clinical fecal suspensions and successfully propagated one korean pedv strain, designated as aram, in vero cells. the aram strain produced obvious cpe typical of pedv infection, such as cell fusion and multinucleated cell (syncytium) formation, in infected vero cells, which was verified by detecting pedv antigens by the ifa using a pedv n protein-specific mab (fig. 1a) . growth kinetics analysis indicated that aram replicated efficiently in vero cells, reaching a maximum viral titer of > 10 6 tcid 50 by 36 hpi (fig. 1b) virus titer (log tcid /ml) we subsequently determined the entire genomic sequences of the original fecal sample (aram-feces) and the cell culture-passaged aram-p5 virus using the sanger technology. both the identified genomes were identical in length [28,027-nucleotide (nt)], except for the 3′ poly(a) tails, and showed the typical genomic organization of alphacoronaviruses, consisting of the 292-nt 5′ utr, the 20345-nt orf1a/1b (nt 293 to 12601 for 1a and nt 12601 to 20637 for 1b), the 4152-nt s gene (nt 20634 to 24785), the 414-nt orf3 (nt 24785 to 25198), the 231-nt e gene (nt 25438 to 25668), the 681-nt membrane (m) gene (nt 25676 to 26356), the 1326-nt n gene (nt 26368 to 27693), and the 334-nt 3′ utr. compared to the complete genome sequence of the aram-feces, the aram-p5 virus had no amino acid (aa) substitution throughout the entire genome, suggesting genetic stability during cell passages. interestingly, the genome size of the aram strain was 11-nt shorter than that of most g2b field viruses; this was due to the presence of the unique 9-nt and 2-nt deletions (dels) at genomic positions 21,051-21,059 and 25,207-25,208, respectively. these dels are completely absent in the genome sequences of the other global g1 and g2 strains available in the genbank database. the former 9-nt del resulted in a 3-aa del at aa positions 140-142 in the n-terminal domain (ntd) of the s protein ( fig. 2a) , while the latter 2-nt (thymine residues) del at nt positions 414-415 ( 412 tattat 417 ) in orf3 caused premature termination 414-nt upstream from the authentic stop codon, thereby possibly producing a truncated orf3 without c-terminal 87-aa residues (fig. 2b) . (fig. 2b) . phylogenetic analysis based on the complete s protein clearly classified the pedv strains into two distinct genogroup clusters, g1 and g2, with two subgroups in each genogroup (fig. 3a) . the aram strains belonged to subgroup g2b, as it clustered closely with the contemporary domestic and global g2b isolates. subsequent whole-genome phylogeny showed the same grouping structure as that of an s gene-based phylogenetic tree (fig. 3b) . a pedv aram-p5 stock with a known cell culture infectious titer of 10 5.0 tcid 50 /ml was serially diluted 10-fold with cell culture medium, giving rise to theoretical infectious titers of 10 3 to 10 −1 tcid 50 /ml for the 10 −2 to 10 −6 dilutions, respectively, which were later used to determine the outcomes of infection in 5-day-old piglets ( table 1) . each group (groups 1-5) of five piglets was challenged orally with the 10-fold serially diluted (10 −2 -10 −6 ) aram-p5 virus, while control animals in group 6 were fed cell culture medium. all five piglets in the groups 1 to 3 (inoculated with virus dilutions 10 −2 to 10 −4 ) and three piglets in group 4 (inoculated with virus dilution 10 −5 ) developed moderate to watery diarrhea (scores 2-3) by 3 dpi and it lasted through the study period ( table 1) . by 5 dpi, 100% pigs of group 1, 60% (3/5) pigs of groups 2 and 3, and 20% (1/5) pigs of group 4 died from ped-related clinical signs. in contrast, all five pigs in groups 5 (inoculated with virus dilution of 10 −6 ) and the negative control group remained active and clinically unaffected throughout the 5-day experimental period. the pdd 50 and ld 50 of the aram virus were determined as 5.2 log 10 pdd 50 /ml and 4.0 log 10 ld 50 / ml, indirectly corresponding to theoretical cell culture infectious titers of 10 0.2 tcid 50 /ml and 10 1 tcid 50 /ml, respectively. the cold-adapted g2b pedv aram-ca strains were generated by a serial passage of aram-p5 at low temperatures commencing from 37°c to 32°c and additional adaptation for growth up 45 passages at 32°c in vero cells. we initially aimed to evaluate the immunogenicity of the cold-adapted aram derivatives in the natural host. antisera were collected from the pigs at 0-, 1-, 2-, and 3-week after oral administration of each virus and were tested for neutralizing activity against the aram isolate. the serum samples obtained from pigs immunized with the cold-adapted aram-p29-ca strain had higher neutralizing antibody titers, when compared with those from pigs in other ca virus-inoculated groups (fig. 4) . furthermore, the immune sera of pigs inoculated with aram-p40-ca or -p50-ca strain showed little or no neutralizing activity against pedv. since our data indicated that only aram-p29-ca can elicit robust antibody responses in immunized animals, this cold-adapted virus was selected for subsequent in vitro and in vivo studies. compared to 32°c, indicating impairment of growth at 37°c. this diminished growth at 37°c was confirmed by comparing growth kinetics in vero cells at both the temperatures. the aram-p29-ca strain exhibited efficient growth and had an infectious titer of > 10 6.5 tcid 50 / ml at 32°c by 2 dpi, but slower replication rates with a maximum titer of 10 2.5 tcid 50 /ml than that of the parental aram-p5 strain (a 10,000-fold reduction) at 37°c, indicating an adaptation to low temperatures. to identify the mutation that occurred during the cold adaptation of pedv, the genome of the cold-adapted virus was sequenced and compared to that of the wild-type virus. no additional indels were found in the genome after cold adaptation, and the genome size of the aram-p29-ca strain was determined to be 28,027-nt, which was identical to the parental genome size. two genetic del markers of the aram strain were also completely conserved in s and orf3 of the cold-adapted aram-p29-ca strain (fig. 2) . the 5′-and 3′-utrs of the cold-adapted virus remained unchanged, whereas the coding region contained a total of 16 nt substitutions (fig. 5) . among these mutations, 12 were non-synonymous, causing changes of 1-aa in nsp3, 1-aa in nsp5, 7-aa in s, 1-aa in e, and 2-aa in m. details in nt and aa differences between the parental aram-p5 and cold-adapted p29-ca strains are summarized in table 2 . further phylogenetic analysis based on the full-length s and the entire genome indicated that the cold-adapted aram-p29-ca strain was still clustered into subgroup g2b along with the parental and global epizootic strains (fig. 3) . next, animal inoculation experiments were performed to examine the in vivo phenotypic alterations associated with serial in vitro passage at low temperatures (a cold adaption tool for virus attenuation) of the virulent aram strain. the pathogenicity of the parental aram-p5 and its cold-adapted derivative, aram-p29-ca, was characterized in pigs. twelve piglets divided into three groups of four animals each were challenged orally with parental aram-p5 (group 1) or cold-adapted aram-p29-ca (group 2), and the remaining piglets in a control group received cell culture medium. clinical signs were recorded daily, and fecal swabs were collected pre-and post-challenge for the duration of the study. during the acclimation period, all piglets were active, showed no clinical symptoms, and had normal fecal consistency that did not contain any pedv genetic material. after the challenge, none of the negative-control piglets developed clinical presentations typical of pedv throughout the study. in contrast, aram-p5-challenged piglets (group 1) displayed clinical signs including loss of appetite and diarrheic feces by 1 dpi (mean css ± sdm of 0.67 ± 1.15) and underwent lethal watery diarrhea with vomiting thereafter (mean css of > 3.0) (fig. 6a) . all the inoculated animals in group 1 finally died by 5 dpi. remarkably, the cold-adapted aram-p29-ca virus-inoculated piglets in group 2 experienced neither pedv-related clinical symptoms nor mortality throughout the experiment. all animals in group 1 were positive for pedv, as determined by rt-pcr, by 1 dpi with a mean ct value of 28.55 (equivalent to 10 2.59 tcid 50 /ml) and shed significantly higher amounts of pedv in feces with ct ranging from 6.80-12.35 (10 4.97 -10 6.14 tcid 50 /ml) until death (fig. 6b) . likewise, fecal shedding of pedv was detected in all the piglets in group 2 by 1 dpi with the mean ct values of 24.59 (10 3.11 tcid 50 /ml), but thereafter decreased to the lowest levels thereafter followed by a slight increase at 5 dpi. overall, the quantities of viruses in the feces of animals of group 2 significantly declined compared to those in group 1, with wide ct ranges of 34.46-24.34 ( efficacy of cold-adapted attenuated pedv vaccine all animals in the parental aram-p5-infected group were necropsied upon death at 4 or 5 dpi, while piglets in the remaining groups were euthanized at the end of the study for postmortem examinations (fig. 7) . neither macroscopic nor microscopic intestinal lesions were evident in the negative control piglets (right panels c, f, and i). the virulent aram-p5-inoculated pigs macroscopically showed archetypal ped-like gross lesions. their small intestines were dilated with accumulated yellowish fecal fluid and had thin transparent walls as a result of villous atrophy (panel a), whereas the other internal organs appeared normal. in contrast, all animals infected with cold-adapted aram-p29-ca in group 2 displayed no remarkable visible pathological lesions in their gastrointestinal tracts with normal bowel wall thickness, comparable to those in the negative control group (panel b). microscopic assessment revealed that the small intestines from all dead piglets in group 1 were characterized by acute viral enteritis, with villous shortening and fusion, involving vacuolation of superficial epithelial cells, in the jejunum (panel d). furthermore, ihc staining showed intense antigen labeling in the cytoplasm of epithelial cells in atrophied intestinal villi with a immunosignal score of 2.3 ± 0.6 (mean ± sdm) in the jejunum (panel g). however, two out of four cold-adapted virus-inoculated pigs in group 2 exhibited mild villous atrophy in the small intestines, while the remaining pigs showed normal intestinal histopathologies (panel e), analogous to those of the negative control group (panel f). pedv antigen was rarely present in the small intestines in animals of group 2 with a significantly lower mean jejunal viral antigen score (0.3 ± 0.6) (panel h) than that in group 1 (panel g). altogether, our results indicate that cold-adapted aram-p29-ca showed noticeably weakened virulence with an attenuated phenotype in the highly susceptible piglets under experimental conditions. to evaluate the protective effectiveness of a cold-adapted pedv vaccine, our study employed an oral homologous prime-boost vaccination scheme at 2 weeks apart before farrowing. three sows in group 1 were orally primed and boosted with live vaccines at 4 and 2 weeks pre-partum, whereas 2 positive (group 2) and 1 negative (group 3) control animals were kept clinical significance score log tcid /ml unvaccinated. all sows in the vaccinated and unvaccinated groups experienced neither pedlike clinical symptoms, including pedv fecal shedding, nor adverse reactions to vaccines. at farrowing, there were no significant differences in reproductive performance between the vaccinated and unvaccinated sows. ten piglets per litter were allocated to each sow and were nursed by their dam. at day 4 post-farrowing, all neonates in groups 1 and 2 were orally exposed to the virulent aram-p5 virus. in contrast, nursing piglets in group 3 were mock-inoculated with mem. clinical signs were recorded daily, and fecal swabs were collected before and after the challenge for the duration of the study. during the pre-challenge period, all piglets were healthy and had no clinical symptoms showing normal fecal consistency without pedv shedding. following the challenge, pedv-exposed piglets from unvaccinated sows (group 2) developed clinical symptoms, including anorexia and diarrhea by 1 dpc (mean css of > 2.4) and suffered from severe watery diarrhea with vomiting thereafter (fig. 8a) challenge period. these results demonstrated that litters from vaccinated sows significantly mitigated diarrhea after the challenge compared to those from unvaccinated sows. pedv-associated mortality occurred in > 50% piglets (11 of 20) from unvaccinated sows by 4 dpi, and additional fatalities occurred until the end of the study, indicating a rate of > 90% mortality in the unvaccinated challenge control group 2 (fig. 8b) . in contrast, 10 of 30 piglets birthed by vaccinated sows (group 1) died from pedv exposure, showing an approximately 70% survival rate in the piglets. pedv fecal shedding was detected in all piglets from unvaccinated sows (group 2) by 1 or 2 dpc, and they persistently discharged high quantities (> 10 5.00 tcid 50 /ml) of pedv in feces until death (fig. 8c) . however, all animals from vaccinated sows (group 1) showed pedv shedding in feces by 3 dpi with a peak mean titer of 10 5.47 tcid 50 /ml but amounts of fecal shedding significantly declined thereafter. all piglets from the negative control sow (group 3) remained alive and had no viral shedding in rectal swabs for the duration of the study. considering that neutralizing antibodies are associated with protective immunity in neonates against pedv infection, we tested serum and colostrum samples collected from sows and their neonatal piglets for the presence of anti-pedv antibodies by an sn assay. as shown in fig. 9a , all sows developed anti-pedv neutralizing antibodies after the live prime vaccination. thereafter, the neutralizing antibody titers in sera of vaccinated sows gradually increased, likely resulting from a homologous boost. all the three sows maintained high neutralizing antibody titers for 2 weeks post-farrowing. consistent to the results from the serum samples, the neutralizing antibody level was significantly high in the colostrum of vaccinated sows (fig. 9b) . furthermore, the nursing piglets from each litter in group 1 had pre-challenge neutralizing antibody levels similar to those of their own dam and retained antibodies up to 7 dpi, indicating the protective effect of lactogenic immunity passively acquired from vaccinated sows (fig. 9c) . clinical significance score log tcid /ml (groups 2 and 3). taken together, these data demonstrate that oral vaccination with the coldadapted live attenuated virus efficiently elicits potent antibody responses in sows, which are subsequently transferred to the offspring via their milk to provide protection against pedv. a sow does not possess the means to transplacentally transmit maternal immunoglobulins to its fetus; hence, piglets are born without passive immune protection, being highly susceptible to a variety of infectious agents, particularly multiple enteric pathogens, such as pedv. as a substitute, the passive immunity is dependent on the supplement of maternal-derived immune components via mammary secretions (i.e., colostrum and milk) [15, 33] . therefore, maternal vaccination is the only crucial and effective tool to confer passive protection to newborn piglets through lactogenic immunity against pedv infection. this objective may be accomplished by the selection of an ideal vaccine candidate, the antigenicity of which is close to that of an epidemic strain, and virulence is minimal or absent in piglets. in south korea, several g1a pedv-based live vaccines have been extensively employed to prevent ped for decades. however, owing to partial protection of these historical vaccines against the contemporary g2b strains prevailing since the 2013-2014 pandemic, a demand for the development of new effective vaccines based on the dominant epidemic strain has increased. although efforts to pioneer a new g2b-based mlv vaccine have been made over the past several years, its success was delayed because of experimental hurdles such as a laborious long-term attenuation procedure in cell culture. it is acknowledged that cold adaptation of animal viruses at serially reduced temperatures for cultivation is the best method to derive a live virus vaccine line, as it is generally accompanied by loss of virulence [21] . in this study, we used a cold adaptation approach with a stepwise lowering of the cultivation temperature in vitro for pedv attenuation and aimed to determine the immunogenicity and pathogenicity of the cold-adapted strain and its efficacy as an attenuated live vaccine candidate under experimental conditions. as the pandemic of the g2b pedv strain ravages the global swine industry, the pedv field is moving towards the development of novel vaccines and vaccination strategies. to accomplish this task, we first isolated field g2b pedv that can be efficiently propagated in vitro. the g2b isolate, aram-p5, exhibited comparable growth characteristics with other g2b strains in regard to cytopathology, infectious titers, and replication kinetics in vero cells as reported previously [23] . genetic and phylogenetic analyses showed that the aram isolates are most closely related to the recent g2b strains prevalent worldwide. however, sequence comparisons with other pedv strains revealed outstanding genomic features in the aram strain, which naturally included two independent dels in s and orf3, respectively. more interestingly, due to the 2-nt del in orf3, aram is predicted to encode an alternative orf3 protein with a large 87-aa excised c-terminus. although the exact function of pedv orf3 remains unknown, a number of attenuated pedv strains contain their signature dels in several parts of orf3, suggesting its critical role in viral pathogenesis [24, 34, 35] . in particular, wang et al. [11] reported an attenuated pedv that encodes a c-terminal truncated orf3 protein of 91-aa residues, which is form the most analogous to the orf3 of the aram strain. however, experimental oral inoculation of newborn pigs with aram-p5 induced severe clinical presentations and macroscopic and microscopic intestinal lesions typical of acute pedv infection as reported previously [23] . in general, death rates average 50% in suckling piglets up to one week of age, often approaching 100% in neonates less than 3 days of age, and decrease to 10% thereafter [1, 2] . likewise, our study reproduced 100% mortality in 5-day-old conventional piglets inoculated orally with the 10 −2 diluted aram virus (calculated dose of 10 3 tcid 50 per pig). the data demonstrated that the aram isolate was highly enteropathogenic in neonatal piglets, implying that the orf3 product would be irrelevant to pedv virulence. since a growing body of evidence proposes that a combination of multiple genetic mutations in s, orf3, and functional nsps affects the virulence of pedv, we cannot exclude that the c-terminal defect in orf3 may be one of molecular and physiological factors that may simultaneously alter pathogenic mechanisms. however, consistent with previous works [24, 36] , growth kinetics of the aram strain in cell culture supports the notion that the accessory orf3 gene is dispensable for pedv replication in vitro. collectively, despite a redundant role in virus propagation in vitro, conservation of the complete orf3 in pedv field isolates still suggests its importance in causing natural infection in the animal host. to serve as a novel mlv vaccine strain, we generated an aram-derived attenuated pedv strain using a cold adaptation attenuation method. our approach for viral attenuation was to obtain a cold-adapted strain with impaired growth at physiological temperature (37°c), which can sufficiently induce mucosal immunity in the natural host. the cold-adapted aram-p29-ca strain was derived by 24 serial passages of the aram isolate at a low temperature (32°c). the aram-p29-ca strain developed unsuccessful in vitro infection at the physiological temperature, as a significant decrease in infectious units was observed. however, this virus showed significantly high immunogenicity in orally inoculated pigs. furthermore, when investigating the phenotype of aram-p29-ca in highly susceptible 5 day-old piglets, none of the inoculated animals became clinically sick and grossly abnormal. our experimental data indicate that the cold-adapted aram-p29-ca virus is attenuated virologically in vitro and clinically in vivo. subsequently, we sequenced the entire genome of the aram-p29-ca strain to decode the genetic mutations that emerged during sequential cell passages at 32°c. upon cold adaptation, a total of 12 non-silent mutations arose in orfs 1 through 5, except for orf3, without extra indels throughout the genome, whereas the two s and orf3 del signatures of the aram strain were entirely maintained. intriguingly, 7-aa substitutions were accumulated in the s protein, among which 4-and 3-aa substitutions were distributed in the n-terminus of s1 and s2 domains, respectively, suggesting that some of these changes are associated with viral attenuation. since the pedv s protein is heavily glycosylated, altered glycan motifs may be involved in viral pathogenesis by modifying the protein conformation and function [37] . however, no genetic changes were found in the putative n-glycosylation sites between the parental and cold-adapted strains. as the major antigenic determinant, the s glycoprotein possesses at least four neutralizing domains of pedv at aa positions 19-220 (ntd/s0), 499-600, 744-774, and 1371-1377 [38] . the aa residues that comprise these domains remained nearly unchanged throughout the cold adaptation, except for one n209y mutation in the ntd/s0 region. furthermore, no aa changes were found in additional discrete domains, including a c-terminal domain (residues 477-629) of s1 that can interact with cellular receptor(s) and a s2 fusion domain comprising of a hydrophobic fusion peptide (residues 891-908) and two heptad repeat regions (residues 978-1117 and 1274-1313). although the polygenic traits are likely associated with pedv virulence, it is of utmost importance that future work using reverse genetics technology should aim to address whether genetic drift, especially in the s protein, influences the pathogenicity of pedv. in addition, attenuated viruses often have the propensity to revert to virulent form under certain circumstances, which is a main drawback for their use as an mlv vaccine. indeed, the aram-p29-ca strain had an ability to replicate at 37°c since it denoted impaired, but not entirely halted, growth at 37°c, as the viral titer was determined to be 10 2.5 tcid 50 /ml. this incompletely abolished growth of aram-p29-ca at the physiological temperature implies a residual risk of reversion to virulence in vivo. thus, further safety studies will be necessary to evaluate the phenotypic stability of the attenuated aram-p29-ca virus in neonatal piglets. to assess the effectiveness of the cold-adapted live attenuated vaccine, a homologous primeboost sow immunization trial consisting of oral administration of a double-dose of the p29-ca virus (l/l vaccination) was implemented at intervals of 2 weeks before delivery. newborn piglets born to vaccinated and unvaccinated sow groups were inoculated orally with the parental virulent pedv strain. the difference in the burden of diarrheic disease as measured by mortality and morbidity was investigated between the challenged litters from vaccinated and unvaccinated sows. piglets born to unvaccinated sows presented a much higher mean css, as determined by the intensity of diarrhea, than those of vaccinated sows. after 2 dpc, the challenged animals born to vaccinated (group 1) sows had mild-to-moderate diarrhea, whereas the piglets born to unvaccinated (group 2) sows experienced fatal watery diarrhea. although nearly all the piglets in group 2 died by 7 dpc, group 1 also exhibited more than 30% neonatal mortalities during the experimental period. on the contrary, our previous trial has shown that a multiple-dose parenteral immunization with inactivated killed g2b vaccines (k/k vaccination) offered a more protective efficacy (93% survival rate) in piglets against g2b virus than that in the present study [39] . in contrast to piglets from unvaccinated sows that discharged high amounts of the virus in feces until death, pedv fecal shedding in animals from vaccinated sows was greatly reduced at 7 dpc and more than 50% of the piglets that survived shed no virus in stools at the end of the trial. collectively, the maternal l/l vaccination described in the present study is unlikely to be more efficacious than the k/k treatment in terms of the protective rate; however, it could help alleviate diarrheal severity, including the duration and quantity of fecal shedding of pedv. although our l/l vaccination of sows did not entirely avert morbidity in piglets upon a pedv challenge, its advantageous effects on relieving pedv shedding would lessen the possibility of the environmental contamination in the farrowing barns, thereby breaking the chain of secondary spread of the virus to other animals and herds during epidemics. furthermore, pedv transmission via the l/l vaccination is unlikely, as no pedv genetic material was detected in feces from any of the pregnant sows vaccinated with p29-ca. moreover, we were able to confirm the presence of satisfactory amounts of anti-pedv neutralizing antibodies in the sera and colostrum of l/l vaccinated sows at farrowing and post-farrowing stages, as well as in sera obtained from their offspring, indicating the piglet protection by transfer of maternal immunity via mammary secretions from the immunized dams. these data further strengthen the concept that an optimal vaccination regimen is associated with retaining high levels of neutralizing antibodies in the lactogenic secretions of vaccinated sows, which in turn are correlated with colostrum and milk iga antibody titers to offer protective immunity against pedv [1, 2, 15, 40] . in summary, this is the first report describing the development of a cold-adapted mlv vaccine based on a virulent g2b pedv strain. the present study demonstrated the loss of virulence of a highly pathogenic pedv strain, with 12 genetic mutations throughout the entire genome, after cold adaptation. vaccination-challenge studies revealed that sows immunized with a homologous oral l/l maternal vaccination regimen provides protective lactogenic immunity to piglets, thereby reducing mortality, morbidity, and fecal shedding of the virus. in south korea, multiple-dose vaccination programs including l/k/k at 2-or 3-week intervals before delivery have been recommended nationwide in pregnant sows for decades [1, 2] . due to the absence of a new safe and effective g2b live vaccine, controlled oral exposure (feedback) for prime-boost vaccination strategies is mostly implemented as a countermeasure in the field since the 2013-2014 ped epidemics. despite certain positive contributions of feedback to naïve herds, intentional infection using an undefined inoculum may cause undesirable negative consequences, which include insufficient immunity induction, unexpected virus transmission, amplified viral recombination and diversity; and more importantly, dissemination of other microbial pathogens conceivably evolving to develop into a new disease agent. once the cold-adapted aram-p29-ca-based mlv vaccine is supplied in the market, gradual withdrawal of the feedback practice will be imminent. since oral administration does not have the capacity to ensure that all animals are immunized with a proper dose, this demerit should be ameliorated to easily use the live vaccine under field conditions. with the availability of inactivated g2b pedv vaccines in the domestic market, customized vaccination programs, such as the application of heterologous oral prime-parenteral boost l/k/k or l/l/k schemes, could be established based on various herd circumstances, including naïve, epidemic, and endemic statuses. it is noteworthy that maternal vaccination has to be implemented in conjunction with additional intervention strategies involving intensive biosecurity, husbandry, and hygiene practices for ped prevention and eradication. porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus porcine viruses: from pathogenesis to strategies for control fields virology heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea immunogenicity and protective efficacy of recombinant s1 domain of the porcine epidemic diarrhea virus spike protein porcine epidemic diarrhoea: new insights into an old disease a new coronavirus-like particle associated with diarrhea in swine deadly pig virus slips through us borders emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences distinct characteristics and complex evolution of pedv strains us-like strain of porcine epidemic diarrhea virus outbreaks in taiwan molecular characterization of pig epidemic diarrhoea viruses isolated in japan from antibody responses in serum, colostrum, and milk of swine after infection or vaccination with transmissible gastroenteritis virus lactogenic immunity and vaccines for porcine epidemic diarrhea virus (pedv): historical and current concepts does circulating antibody play a role in the protection of piglets against porcine epidemic diarrhea virus? previous infection of sows with a "mild" strain of porcine epidemic diarrhea virus confers protection against infection with a "severe" strain genetic characterization of porcine epidemic diarrhea virus in korea 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epidemic diarrhea virus porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-β production by inhibiting irf3 activation in immortalized porcine alveolar macrophages pathogenesis of porcine epidemic diarrhea virus isolate (us/iowa/18984/2013) in 3-week-old weaned pigs viral diarrhea of man and animals cloning and further sequence analysis of the orf3 gene of wild-and attenuated-type porcine epidemic diarrhea viruses pedv orf3 encodes an ion channel protein and regulates virus production manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo the s2 glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes efficacy of an inactivated genotype 2b porcine epidemic diarrhea virus vaccine in neonatal piglets characterization of anti-porcine epidemic diarrhea virus neutralizing activity in mammary secretions key: cord-321814-vt6yio6x authors: pan, yongfei; tian, xiaoyan; li, wei; zhou, qingfeng; wang, dongdong; bi, yingzuo; chen, feng; song, yanhua title: isolation and characterization of a variant porcine epidemic diarrhea virus in china date: 2012-09-12 journal: virol j doi: 10.1186/1743-422x-9-195 sha: doc_id: 321814 cord_uid: vt6yio6x an outbreak of diarrhea in pigs started in guangdong, south china in january 2011. cases were characterized by watery diarrhea, dehydration and vomiting, with 80–100% morbidity and 50–90% mortality in suckling piglets. the causative agent of the diarrhea was ultimately identified as porcine epidemic diarrhea virus (pedv). in this study, we isolated a pedv strain designated chgd-01 from piglet intestines using vero cell cultures, and its specific cytopathic effects were confirmed in susceptible cells by direct immunofluorescence testing and electron microscopy. the complete genome of chgd-01 was shown to be 28,035 nucleotides in length, with a similar structure to that of pedv reference strains. phylogenetic analyses based on the whole genome revealed that chgd-01 shared nucleotide sequence identities of 98.2–98.4% with two other chinese isolates reported in the same year, thus constituting a new cluster. amino acid sequence analysis based on individual virus genes indicated a close relationship between the spike protein gene of chgd-01 and the field strain knu0802 in korea. its orf3 and nucleoprotein genes, however, were divergent from all other sequenced pedv isolate clusters and therefore formed a new group, suggesting a new variant pedv isolate in china. further studies will be required to determine the immunogenicity and pathogenicity of this new variant. porcine epidemic diarrhea virus (pedv) is the causative agent of porcine epidemic diarrhea (ped), an enteric disease characterized by vomiting, watery diarrhea, and dehydration in swine. this disease was first reported in feeder and grower pigs in the uk in 1971 [1] , after which the virus was identified [2, 3] . the disease has subsequently been reported in a number of european countries [4, 5] , and more recently in china, korea, japan, thailand and vietnam [6] [7] [8] [9] [10] [11] [12] . pedv is an enveloped rna virus belonging to group 1a, genus coronavirus, family coronaviridae, within the order nidovirales. the viral genome is a single-stranded positive-sense rna of approximately 28 kb in size, containing six genes: the replicase (rep), spike (s), orf3, envelope (e), membrane (m), and nucleoprotein (n) genes, arranged in the order 5'-rep-s-orf3-e-m-n-3' [13] [14] [15] . as a coronavirus, pedv comprises three corresponding major viral structural proteins: the s (180-220 kda), m (27) (28) (29) (30) (31) (32) , and n (55-58 kda) proteins [16, 17] . the s protein plays a pivotal role in determining viral-cellular fusion activity and in inducing an immune response in the natural host [18] [19] [20] . the m protein plays an important role in the virus-assembly process, and induces antibodies that neutralize virus in the presence of complement [21] [22] [23] . the n protein of coronaviruses forms a helical ribonucleoprotein with the virus genomic rna and is the predominant antigen produced in coronavirus-infected cells, thus making it a major viral target [24, 25] . unlike the structural proteins, little is known about the functions of the accessory proteins. the recently-identified orf3 gene has been demonstrated to be a potentially important determinant of virulence in this virus [26, 27] . pedv can generally be controlled using the vaccine strategy, and vaccination with killed or attenuated pedv vaccine has been widely carried out in china, where ped usually manifests a mild and enzootic pattern (lower mortality in suckling piglets). however, a severe acute diarrhea outbreak associated with high morbidity (80-100%) and mortality (50-90%) was observed in suckling piglets at approximately 10 premises in guangdong, china, in early 2011. although most sow herds had previously been vaccinated with both killed and attenuated pedv vaccines based on cv777, some of these were still infected, showing transient diarrhea and anorexia, but not death. pedv field isolates thus need to be isolated and their molecular epidemiology investigated in order to better control and prevent future pedv outbreaks. in this study, a pedv strain was isolated from sick piglets during this outbreak and grown in vero cells. molecular characterization of the virus identified it as a variant pedv emerging in china. for pcr/rt-pcr detection of viruses, intestinal and fecal samples from 33 sick piglets were first examined for the presence of tgev, porcine rotavirus, porcine reproductive and respiratory syndrome virus (prrsv), pcv2, porcine kobuvirus and pbov. all assays were negative except for three pcv2-positive samples. all specimens were subsequently examined for pedv, and all samples were positive (data not shown). all samples were tested for common pathogenic intestinal bacteria, such as salmonella and pathogenic escherichia coli, but no significant numbers of salmonella or e. coli were isolated from sick piglets from different premises. a distinct cpe was noted after seven passages in vero cells. the cpe was characterized by cell fusion, syncytia formation and eventual detachment from the plastic surface ( figure 1b) . the virus isolate designated chgd-01 was biologically cloned by three rounds of plaque purification in vero cells prior to further virus characterization. in addition, cells tested positive by ifa ( figure 1d ). electron microscopy of a negatively-stained sample revealed the presence of medium-sized viral particles of approximately 80-120 nm in diameter. in some of the virions, surface projections characteristic of coronaviruses were evident ( figure 1e ). phylogenetic analyses of the s protein amino acid sequences revealed that all pedv strains in this study could be separated into two groups: chgd-01 belonged to group 2, which also contained the two japanese isolates kawahira and nk, eight korean field strains (chinju99, knu-0801, knu-0802, and knu-0901-knu-0905) and two chinese strains (bj-2011-1 and ch/fjnd-3/2011), which were deposited in genbank in 2011 ( figure 3b ). the orf3 gene of chgd-01 was determined to be 675 nucleotides in length, coding for a polypeptide of 225 amino acids. the orf3 gene of chgd-01 shared 93.3-97.3% amino acid identity with other pedv strains, and the highest identity with the korean virulent dr13. based on phylogenetic analyses of the orf3 amino acid sequences, all the pedv strains could be divided into four groups (figure 3c ). group 1 comprised cv777, br1/87, sm98 and lzc strains; group 2 consisted of vaccine strains (attenuated dr13); group 3 was made up of eight korean field strains and 11 chinese strains. these results showed some similarities with the previous report by park et al. [28] . interestingly, phylogenetic analysis showed that chgd-01 formed a fourth group, clearly different from other pedv isolates. each group had unique differences in its sequences. group 2 had a 17-amino acid deletion (at positions 82-98), which is a marker of attenuated vaccine. the m protein of chgd-01 was found to be 226 amino acids long and highly conserved. it shared 96.9-99.5% amino acid identity with the other pedv strains. the highest amino acid identity (99.5%) was with the vietnamese field isolates (vn116m5 and vn116m6), which were isolated from southern vietnam during the 2009-2010 ped outbreaks. phylogenetic analyses demonstrated that all pedv strains were similarly divided into two distinct genetic groups ( figure 3d the n gene of chgd-01 was 1326 nucleotides in length, coding for a polypeptide of 441 amino acids. the amino acid sequence had 95.9-97.7% identity with other pedv strains, and the highest identity (97.7%) with the ch/s and vaccine strains (attenuated dr13). alignment analysis indicated that the entire nucleocapsid protein of chgd-01 was generally highly conserved, but had 16 amino acid substitutions compared to cv777. it was predicted to contain eight potential serine (s)-linked phosphorylation sites and six potential threonine (t)-linked phosphorylation sites, including two protein kinase c phosphorylation sites, one casein kinase ii phosphorylation site, and one camp-and three cgmp-dependent protein kinase phosphorylation sites. based on analyses of the amino acid sequences of the pedv n proteins, all pedv strains could be divided into four groups (figure 3e ). chgd-01 differed from all other pedv isolates, and formed a new separate group. there were unique changes in the deduced amino acid sequences among the groups. two amino acid changes ( a 145 t , v 216 m ) were found in group 2; k 252 r , n 255 s and q 397 l occurred only in group 3; groups 3, 4 shared two specific amino acid changes ( s 142 t , h 242 l ); and g 84 a was found in groups 2, 3 and 4. noticeably, six amino acid mutations ( a 145 s , n 157 s , k 248 l , a 293 t , i 322 v , q 397 p ) were observed only in chgd-01 (figure 2c ). the clinical signs from the outbreak in guangdong, along with both macroscopic and microscopic examinations of the lesions, were strongly suggestive of pedv or tgev infection. tgev and other possible pathogens were excluded and pedv was therefore focused on as the only significant causative agent, followed by attempts to isolate it from piglets with the disease. pedv was isolated successfully and propagated in vero cell cultures in the presence of trypsin, according to the method described by hofmann and wyler, and by kusanagi et al. [29, 30] . however, the cpe caused by the pedv isolate was not detected in vero cells until after seven passages, despite being detectable by pcr. this was in contrast to the results of hofmann and wyler, who observed a cpe even at passage one in the same cells, and a prominent effect from virus passage five [29] . kusanagi et al. reported that a characteristic cpe was detected at passage two, and became more evident with subsequent passages of vero cells [30] . this discrepancy may be attributable to the relative susceptibilities of vero cells to different isolates of pedv. to characterize the virus isolate, we determined the complete genomic sequence of chgd-01 and analyzed the phylogenetic relationships among pedv strains at the genomic and individual gene levels. the most variable regions were located in the s and orf3 genes. the s protein is involved in receptor binding and virus entry, the induction of neutralizing antibodies, and host-cell fusion [18] [19] [20] . it is also an important target for monitoring the genetic diversity of coronavirus isolates [31, 32] . most pedv reference isolates prior to 2007, including all chinese strains, were partitioned into the first cluster (group1; g1), whereas the three chinese strains (chgd-01, bj-2011-1 and ch/fjnd-3/2011) belong to the second cluster (group 2; g2). all these three strains were isolated in 2011 when severe ped outbreaks were rampant in some areas of china. all korean field strains belong to this cluster (except the japanese isolates kawahira and nk), most of which were isolated in 2008 and 2009. interestingly, these three chinese strains formed a unique cluster with the highest amino acid identities to knu-0802 (96.5%, 96.9% and 96.2%, respectively). in this study, the same amino acids insertions and deletion of the s gene were observed among three chinese (chgd-01, bj-2011-1 and ch/fjnd-3/2011) and two korean (knu-0802 and knu-0902) strains. both knu-0802 and knu-0902 were isolated in south korea during 2008-2009. these results suggest that the recently isolated chinese strains may have originated from korean ones. the orf3 gene is situated between the s and e genes in the pedv genome, but its function is unknown. previous studies found that a continuous 49 or 51 nucleotide region was deleted within the orf3 gene when pedv was repeatedly passaged in cell culture, indicating a possible involvement in viral pathogenicity [33, 34] . recent investigations demonstrated that all the reported pedv isolates could be classified into three groups, based on phylogenetic analyses of the orf3 genes [28, 34] . our study notably revealed that the amino acid sequence of orf3 in the chgd-01 strain was clearly different from those of all other pedv isolates. this difference could not be attributed to the effects of limited cellular passaging, because the orf3 gene sequence of chgd-01 passage 0 cells (i.e. intestinal fecal sample) was almost identical to that of passage 10 cells (data not shown). chgd-01 fell out with the three previously identified groups, and thus constitutes a new group. the chgd-01 isolate showed 93.3-95.1%, 97.6%, and 96.0-97.3% amino acid sequence identities with members of groups 1, 2 and 3, respectively. genomic recombination is known to occur at high frequency between heterogenous genomes of coronaviruses. however, recombination analysis provided no evidence to suggest that chgd-01 was derived directly from the recombination of known pedv strains. further molecular epidemiological evidence is needed to determine the origin and evolution of the chgd-01 genome. the variations within the chgd-01 genome, especially in the orf3 and s genes, place it in a new cluster with bj-2011-1 and ch/fjnd-3/2011, based on phylogenetic analysis of the pedv genomes. ped was generally considered to be under control or had only mild effects in swine herds in china before the beginning of 2011. since then, however, ped has unexpectedly devastated many swine farms, including those were killed and attenuated vaccines based on cv777 were being used, suggesting that these vaccines were no longer able to confer protection. this raised the question of whether any obvious changes in antigenicity and/or virulence are associated with the genetic variations demonstrated in chgd-01. the results of this study suggest that there is an urgent need for more fundamental research aimed at understanding the basic biology of this virus strain, as well as the mechanisms of immunogenicity and pathogenesis of pedv. the present study isolated and identified a pedv chgd-01 strain from infected piglets in guangdong in 2011. analysis of the genetics and evolution of chgd-01 demonstrated significant genetic diversity compared to other pedv reference strains, suggesting the presence of a new variant pedv in china. outbreaks of diarrhea were observed at a number of pig farms at different locations between january and march 2011. the cases were characterized by watery diarrhea, dehydration and vomiting, with 80-100% morbidity and 50-90% mortality in suckling piglets, whereas affected sows were characterized by diarrhea, anorexia, and depression, but recovered within 1 week. breeding herds had been immunized with attenuated or killed ped-tge combined vaccine produced in china in the fall and winter of the previous year. the pedv component of this vaccine was based on the cv777 strain. sick or dead piglets aged 1-21 days were submitted for laboratory investigation. necropsy examination of all piglets revealed that the small intestines were congested and filled with fluid, and were thin-walled as a result of severe mucosal atrophy. microscopically, marked cytoplasmic vacuolation and exfoliation of enterocytes with subsequent shortening of villi were noted. tissues and feces and/or blood were collected from live or dead animals submitted for polymerase chain reaction (pcr) detection to perform a surveillance. samples from feces and intestine tissues were subjected to virological investigations for common viral swine pathogens such as pedv, transmissible gastroenteritis virus (tgev), porcine rotavirus (prv), porcine circovirus 2 (pcv2) [35] , porcine kobuvirus [36] , and porcine bocavirus (pbov) [37] , using previously described methods. growth medium (gm) was dulbecco's modified eagle's medium (dmem, gibco, usa) supplemented with 10% heat-inactivated fetal calf serum, 0.3% tryptose phosphate broth (tpb), and antibiotics. maintenance medium (mm) consisted of dmem supplemented with 0.3% tpb and 10 μg/ml trypsin (gibco). virus isolation was performed using vero cells (ccl-81™, atcc), as described previously [29, 30] with minor modifications. briefly, intestinal samples positive for pedv by reverse transcription (rt)-pcr were further filtered through a 0.22-μm syringe filter (millipore, usa) and used as inoculum. gm was removed from confluent monolayer cell cultures, which were then washed twice with dmem and inoculated with the filtered intestinal content suspensions. after adsorption for 60 min at 37°c, the cells were washed with dmem and mm was added. the vero cell cultures were observed for 5 days for cytopathic effects (cpe). immunofluorescence assay (ifa) and electron microscopy were used to detect pedv in the infected cells. the ifa utilized a 1:1000 dilution of mouse anti-s monoclonal antibody (cat no: 9191, jbt, korea) specific for pedv and a 1:100 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse igg (cat no: 02-18-06, kpl, usa). for electron microscopy, infected cell culture supernatants were partially purified by ultracentrifugation through a 20% (wt/wt) sucrose cushion, negatively stained with 2% ammonium molybdate, and examined with an electron microscope (jem-1400, jeol ltd., japan). viral rnas were extracted from chgd-01-infected vero culture supernatant using trizol ls reagent (invitrogen, usa), according to the manufacturer's instructions. twelve pairs of oligonucleotide primers were used to amplify the different regions of the chgd-01 genomes, and were designed based on the sequences of pedv strain cv777. the pcr products were purified and cloned into pmd18-t vector (takara, japan) and sequenced using an automated genome sequence (genetic analyzer 3730xl; applied biosystems, usa). the terminal sequences were acquired using a kit for rapid amplification of cdna ends (race) (clontech, japan). all primers are listed in table 1 . sequence data were assembled and analyzed using lasergene software (dnastar inc., usa). multiple sequence alignments were performed using clustal x 2.1 [38] . phylogenetic analyses were carried out using the mega 4 program [39] . phylogenetic trees based on the amino acid sequences of the s, orf3, m and n proteins were elaborated using the neighbor-joining method, with bootstrapping over 1,000 replicates. the pedv strains utilized in the present study including complete genome, s, orf3, m and n genes are listed in table 2 , along with their genbank accession numbers. protein kinase-specific phosphorylation sites were identified using the prediction tool kinasephos program through the web server (http://kinasephos.mbc. nctu.edu.tw/predict.php) [40] . the gene sequence was scanned for possible recombination events using the software package simplot (v 3.5.1), according to the methods described previously [41] . the genome sequence of chgd-01 was registered in genbank under the accession number jx261936. the authors declare that they have no competing interest. authors' contributions yfp, xyt, wl carried out most of the experiments and drafted the manuscript. fc, yhs critically revised the manuscript and the experimental design. qfz, ddw and yzb contributed to the interpretation of the findings and revised the manuscript. all of the authors read and approved the final manuscript. letter to the editor. pig farming a new coronavirus-like particle associated with diarrhea in swine virus-like particles associated with porcine 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kinase-specific phosphorylation sites full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination isolation and characterization of a variant porcine epidemic diarrhea virus in china this work was supported by grants from the special project from guangdong science and technology department (no. 2010b090301020). submit your next manuscript to biomed central and take full advantage of: key: cord-330035-0d6w8xyd authors: jeon, ji hyun; lee, changhee title: cellular cholesterol is required for porcine nidovirus infection date: 2017-09-07 journal: arch virol doi: 10.1007/s00705-017-3545-4 sha: doc_id: 330035 cord_uid: 0d6w8xyd porcine reproductive and respiratory syndrome virus (prrsv) and porcine epidemic diarrhea virus (pedv) are porcine nidoviruses that are considered emerging and re-emerging viral pathogens of pigs that pose a significant economic threat to the global pork industry. although cholesterol is known to affect the replication of a broad range of viruses in vitro, its significance and role in porcine nidovirus infection remains to be elucidated. therefore, the present study was conducted to determine whether cellular or/and viral cholesterol levels play a role in porcine nidovirus infection. our results showed that depletion of cellular cholesterol by treating cells with methyl-β-cyclodextrin (mβcd) dose-dependently suppressed the replication of both nidoviruses. conversely, cholesterol depletion from the viral envelope had no inhibitory effect on porcine nidovirus production. the addition of exogenous cholesterol to mβcd-treated cells moderately restored the infectivity of porcine nidoviruses, indicating that the presence of cholesterol in the target cell membrane is critical for viral replication. the antiviral activity of mβcd on porcine nidovirus infection was found to be predominantly exerted when used as a treatment pre-infection or prior to the viral entry process. furthermore, pharmacological sequestration of cellular cholesterol efficiently blocked both virus attachment and internalization and, accordingly, markedly affected subsequent post-entry steps of the replication cycle, including viral rna and protein biosynthesis and progeny virus production. taken together, our data indicate that cell membrane cholesterol is required for porcine nidovirus entry into cells, and pharmacological drugs that hamper cholesterol-dependent virus entry may have antiviral potential against porcine nidoviruses. nidovirales is a large order of enveloped positive-sense, single-stranded rna viruses that consists of the families arteriviridae, coronaviridae, roniviridae, and mesoniviridae, whose members infect a broad range of hosts including humans and other mammals, birds, fish, insects, and crustaceans [10, 38, 47, 51] . although the genome sizes and virion morphologies of nidoviruses are strikingly different, the genome organization and replication strategy are comparable across the order. the nidovirus genome is composed of two large open reading frames (orfs), 1a and 1b, encompassing the 5′-proximal two-thirds of the viral genome that encode non-structural proteins (nsps) and the remaining orfs located in the 3′-proximal genome part that code for structural proteins [25, 50] . the initial translation from replicase orf1a and orf1b yields large polyprotein (pp) precursors, pp1a and pp1ab, via a -1 ribosomal frameshift (rfs), which then undergo autoproteolysis by viral proteases to eventually produce functional nsps, including the viral rna-dependent rna polymerase (rdrp) [3, 47, 59] . the membrane-bound rdrp-containing replication complex engages in viral genomic rna replication and subgenomic (sg) mrna transcription. the latter finally generates a 3′ co-terminal nested set of sg mrnas that are used to express nidoviral structural proteins [25, 49, 50] . porcine reproductive and respiratory syndrome virus (prrsv) is a pathogenic macrophage-tropic arterivirus of swine that results in reproductive failure in pregnant sows abstract porcine reproductive and respiratory syndrome virus (prrsv) and porcine epidemic diarrhea virus (pedv) are porcine nidoviruses that are considered emerging and re-emerging viral pathogens of pigs that pose a significant economic threat to the global pork industry. although cholesterol is known to affect the replication of a broad range of viruses in vitro, its significance and role in porcine nidovirus infection remains to be elucidated. therefore, the present study was conducted to determine whether cellular or/and viral cholesterol levels play a role in porcine nidovirus infection. our results showed that depletion of cellular cholesterol by treating cells with methyl-β-cyclodextrin (mβcd) dosedependently suppressed the replication of both nidoviruses. conversely, cholesterol depletion from the viral envelope had no inhibitory effect on porcine nidovirus production. the addition of exogenous cholesterol to mβcd-treated cells moderately restored the infectivity of porcine nidoviruses, indicating that the presence of cholesterol in the target cell membrane is critical for viral replication. the antiviral activity of mβcd on porcine nidovirus infection was found to be predominantly exerted when used as a treatment pre-infection or prior to the viral entry process. furthermore, pharmacological sequestration of cellular cholesterol efficiently blocked both virus attachment and internalization and, accordingly, markedly affected subsequent post-entry steps of the replication cycle, including viral rna and protein biosynthesis and progeny virus production. taken together, and acute or chronic respiratory illnesses in pigs of all ages. prrsv primarily replicates in porcine alveolar macrophages (pams) and can establish persistent infection in lymphoid tissues of infected pigs that lasts for several months. as a result, prrsv infection suppresses normal macrophage function and immune responses and is often associated with severe disease outcomes, including increased pre-weaning mortality in growing pigs resulting from secondary bacterial or viral infections, thereby affecting the swine production system [17, 37, 49] . porcine epidemic diarrhea virus (pedv) is a pathogenic enterocyte-tropic swine coronavirus that causes acute enteritis with high mortality rates in neonatal piglets. pedv infection is characterized by severe villous atrophy in the small intestine that results in watery diarrhea followed by fatal dehydration and death in newborn piglets [27, 45] . although pedv outbreaks have been reported in europe and asia, the most serious epizootics for nearly the past three decades have occurred in asia. however, since the virus first emerged in the united states in 2013 [52] , pedv has become recognized globally as a highly contagious and deadly virus. these two viruses, prrsv and pedv, represent emerging and re-emerging porcine nidoviruses that continue to threaten pork-producing countries around world, leading to huge financial losses to the global swine industry [20, 27] . lipid rafts, which are enriched in cholesterol, sphingolipids, and associated proteins, are unique liquidordered microenvironments in the plasma membrane and are involved in a variety of cellular processes as well as in multiple stages of the virus life cycle. cholesterol, a major constituent of lipid rafts, maintains the tight packaging of sphingolipids, and several proteins are partitioned into these microdomains. cholesterol depletion destroys this structural order, leading to disorganization of lipid raft microdomains and dissociation of bound proteins [2, 22] . therefore, plasma membrane cholesterol plays important roles in the infection processes of various non-enveloped and enveloped viruses [5, 34, 36, 56] . in particular, enveloped virus entry requires cholesterol in either the viral or cellular membrane or both [1, 4, 13, 15, 16, 35, 43, 57, 62] . however, there are few reports on the potential relationship between cholesterol and the replication of porcine nidoviruses, although cellular membrane cholesterol has been shown to be a determinant of prrsv entry in african monkey kidney marc-145 cells [21, 55] . in the present study, therefore, we investigated the requirement for cholesterol and its mechanism of action in porcine nidovirus infection. independent depletion of cholesterol from the plasma membrane of target cells by treatment with methylβ-cyclodextrin (mβcd) significantly impaired prrsv and pedv infection. these inhibitory effects on viral replication were partially reversible by replenishment with exogenous cholesterol. in contrast, porcine nidoviruses were shown to be resistant to pharmacological reduction of the cholesterol content of the viral envelope. our data indicate that cholesterol-enriched microdomains are essential for prrsv and pedv in the cellular membrane, but not in the viral membrane. further experiments revealed that pharmacological depletion of cellular cholesterol primarily interferes with virus binding and penetration and subsequently influences post-entry stages of the prrsv and pedv replication cycle, including viral genomic and sg rna synthesis, viral protein expression, and virus production. altogether, our results suggest that cholesterol in the cellular membrane is critical for porcine nidovirus entry and that disruption of the cholesterol-dependent entry process may be an excellent therapeutic option for nidovirus infection in human or veterinary subjects. pam-pcd163 cells [31] were cultured in rpmi 1640 medium (invitrogen, carlsbad, ca) supplemented with 10% fetal bovine serum (fbs, invitrogen), antibioticantimycotic solution (100×, invitrogen), 10 mm hepes (invitrogen), 1 mm sodium pyruvate (invitrogen), and nonessential amino acids (100×, invitrogen) in the presence of 50 μg of zeocin (invitrogen) per ml. vero cells were cultured in alpha minimum essential medium (α-mem, invitrogen) with 10% fbs and antibiotic-antimycotic solution. st-papn cells [39] were cultured in α-mem with 10% fbs and antibiotic-antimycotic solution in the presence of 200 μg of g418 (invitrogen) per ml. the cells were maintained at 37 °c in a humidified 5% co 2 incubator. prrsv strain vr-2332 was propagated in pam-pcd163 cells as described previously [29] . pedv strain sm98-1 was kindly provided by the korean animal and plant quarantine agency and propagated in vero cells as described previously [19, 39] . mβcd and water-soluble cholesterol were purchased from sigma (st. louis, mo) and dissolved in ethanol and phosphate-buffered saline (pbs), respectively. these compounds were diluted to the desired concentrations in maintenance medium. prrsv n and pedv n protein-specific monoclonal antibodies (mab) were obtained from choogang vaccine laboratory (cavac; daejeon, south korea). antibodies to porcine cd163 (pcd163) and β-actin were purchased from abd serotech (raleigh, na) and santa cruz biotechnology (santa cruz, ca), respectively. the polyclonal antibody recognizing porcine aminopeptidase n (papn) obtained from balb/c mice immunized with purified papn (sigma) was a gift from bang-hun hyun (animal and plant quarantine agency, gimcheon, south korea). the cytotoxic effects of reagents on pam-pcd163 and vero cells were analyzed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay (sigma) that allows detection of cell viability. briefly, pam-pcd163 and vero cells were grown at 1 × 10 4 cells/well in 96-well tissue culture plates with mβcd or water-soluble cholesterol treatment for 48 h. after 2 days of incubation, 50 μl of mtt solution (1.1 mg/ml) was added to each well and the samples were incubated for an additional 4 h. the supernatant was then removed from each well, and 150 μl of dmso was added to dissolve the formazan crystals produced by mtt. the absorbance of the solution was measured at 540 nm using an enzyme-linked immunosorbent assay plate reader. all mtt assays were performed in triplicate. pam-pcd163 and vero cells grown on microscope coverslips placed in 6-well tissue culture plates were pretreated with mβcd or ethanol for 1 h and mock infected or infected with prrsv and pedv, respectively, at a multiplicity of infection (moi) of 1. virus-infected cells were then grown in the presence of mβcd or vehicle for 48 h, fixed with 4% paraformaldehyde for 10 min at room temperature (rt) and permeabilized with 0.2% triton x-100 in pbs at rt for 10 min. the cells were blocked with 1% bovine serum albumin (bsa) in pbs for 30 min at rt and then incubated with n-specific mab 7 for 2 h. after washing five times in pbs, the cells were incubated for 1 h at rt with a goat anti-mouse secondary antibody conjugated with alexa fluor 488 (molecular probes, carlsbad, ca), followed by counterstaining with 4′,6-diamidino-2-phenylindole (dapi; sigma). the coverslips were mounted on glass microscope slides in mounting buffer (60% glycerol and 0.1% sodium azide in pbs), and cell staining was visualized using a leica dm il led fluorescence microscope (leica, wetzlar, germany). in addition, pam-pcd163 and st-papn cells stably expressing pcd163 and papn, respectively, were grown in the presence of mβcd or vehicle for 24 h, fixed, and subsequently subjected to ifa with anti-pcd163 or anti-papn antibody as described above. cell staining was analyzed using a confocal laser scanning microscope (carl zeiss, gattingen, germany). quantification of virus-infected cells after treatment with mβcd was analyzed by flow cytometry. pam-pcd163 and vero cells were pretreated with mβcd, infected with virus, and maintained as described above. virus-infected cells were trypsinized at 48 h postinfection (hpi) and centrifuged at 250 × g (hanil centrifuge fleta 5) for 5 min. cell pellets were washed with cold washing buffer (1% bsa and 0.1% sodium azide in pbs), and 10 6 cells were resuspended in 1% formaldehyde solution in cold wash buffer for fixation at 4 °c in the dark for 30 min, followed by centrifugation and incubation of the pellets in 0.2% triton x-100 in pbs at 37 °c for 15 min for permeabilization. after centrifugation, the cell pellets were resuspended in a solution of primary anti-n mab, and the mixture was incubated at 4 °c for 30 min. the cells were washed and allowed to react with an alexa fluor 488-conjugated anti-mouse igg secondary antibody at 4 °c for 30 min in the dark. the stained cells were washed again and analyzed on a facsaria iii flow cytometer (bd biosciences). expression of the viral receptor on the cell surface upon cholesterol depletion was also evaluated by facs analysis as described previously with some modifications [16] . briefly, pam-pcd163 and st-papn cells were trypsinized at 48 h post-seeding. the detached cells were fixed and then reacted with the primary antibody or normal mouse igg1 (santa cruz biotechnology), followed by incubation with secondary antibody as described above. the stained cells were either left untreated or were treated with mβcd at 37 °c for 1 h and analyzed using a flow cytometer. pam-pcd163 and vero cells were infected with prrsv or pedv and treated with mβcd or vehicle. the culture supernatants were collected at different time points (6, 12, 24, 36 , and 48 hpi) and stored at -80 °c. the prrsv titer was measured by limiting dilution on pam-pcd163 cells in duplicate by ifa as described above, and the 50% tissue culture infectious dose (tcid 50 ) per ml was calculated using the spearman-kärber method [14] . the pedv titer was determined by plaque assay using vero cells as described previously [39] and was expressed as plaque-forming units (pfu) per ml. viral stocks were treated with mβcd at various concentrations at 37 °c for 1 h followed by ultracentrifugation to remove the mβcd. the mβcd-treated virus supernatants were purified through a 20% sucrose cushion (wt/vol) prepared in te buffer [10 mm tris-hcl (ph 8.0), 1 mm edta] by centrifugation at 36,900 rpm for 1 h at 4 °c in a p70at rotor (model cp100wx; hitachi, hitachinaka, japan). the virion cholesterol content was determined using fluorescence intensity analysis. briefly, 96-well plate wells were coated with 50 ng of the purified viruses in 50 mm sodium bicarbonate buffer (ph 9.6) and incubated at 4 °c overnight. plates were washed three times with washing buffer (0.05% tween-20 in pbs) and blocked with 5% powdered skim milk (bd biosciences, belford, ma) in pbs at 37 °c for 2 h. after washing, filipin iii (cayman chemical, ann arbor, mi) was added in triplicate for 1 h in the dark. the plates were washed, and fluorescence intensity was measured with a spark 10m multimode microplate reader (tecan, männedorf, switzerland). in parallel, the purified samples were used to infect pam-pcd163 or vero cells for 48 h, and the virus-infected cells were independently subjected to facs analysis and virus titration to determine prrsv or pedv infection as described above. pam-pcd163 and vero cells were first preincubated with vehicle or mβcd at various final concentrations for 1 h and then supplemented with or without 100 μg/ml or 40 μg/ml exogenous cholesterol, respectively, and incubated for 1 h. the cells were then inoculated with prrsv or pedv as described above. the virus inoculum was removed, and the infected cells were maintained in fresh medium containing mβcd and exogenous cholesterol. at 48 h dpi, the virusinfected cells were harvested and subjected to facs analysis to assess the infectivity of prrsv and pedv as described above. in parallel, the cellular cholesterol content was determined using a cholesterol cell-based detection assay kit (cayman chemical) according to the manufacturer's instructions. briefly, virus-infected cells were cultivated in the presence of mβcd and exogenous cholesterol for 48 h, fixed with cell-based assay fixative solution (cayman chemical) for 10 min at rt, and then washed three times for 5 min each with cell-based assay wash buffer (cayman chemical). the cells were incubated with filipin iii for 1 h in the dark. after washing three times in wash buffer, the cells were counterstained with dapi, and filipin staining was visualized using a fluorescent leica dm il led microscope. pam-pcd163 and vero cells were infected with prrsv and pedv, respectively, at an moi of 1 as described above. at -1, 0, 1, 2, 4, 6, 8, 10, 12, or 24 hpi, mβcd was added to maintain the indicated final concentration over the remainder of the time course experiment. the virus-infected and inhibitor-treated cells were further maintained and trypsinized at 48 hpi, followed by centrifugation. the harvested cells were subjected to facs analysis to assess the presence of prrsv or pedv infection as described above. binding and internalization assays were performed as described previously with some modifications [8] . pam-pcd163 and vero cells grown in 6-well culture plates were pretreated and infected with prrsv and pedv, respectively, at an moi of 1 at 4 °c for 1 h in the presence of mβcd. unbound viruses were then removed by washing with pbs, and the cells were either incubated at 4 °c (allowing virus binding only) or 37 °c (permitting virus binding and internalization) in the presence of mβcd for 1 h. in the latter case, the cells were further treated with proteinase k (0.5 mg/ml) at 37 °c for 45 min to remove bound but uninternalized virus particles. the prrsv-infected cells were then serially diluted in rpmi medium and inoculated onto fresh pam-pcd163 cell monolayers in 96-well tissue culture plates. at 48 h post-incubation, bound or internalized viruses were titrated by ifa as described above, and the tcid 50 was determined. for pedv, the serially diluted infected cells were inoculated onto uninfected vero cells, and, after 48 h, viruses were titrated using plaque assay and quantified as pfu per ml. pam-pcd163 and vero cells were incubated with mβcd for 1 h prior to infection and then inoculated with prrsv or pedv at an moi of 1 for 1 h at 37 °c. the virus inoculum was subsequently removed, and the infected cells were maintained in fresh medium containing mβcd for 48 h. total rna was extracted from lysates of the infected cells at 48 hpi using trizol reagent (invitrogen) and then treated with dnase i (takara, otsu, japan) according to the manufacturer's protocols. the concentrations of extracted rna were measured using a nanovue spectrophotometer (ge healthcare, piscataway, nj). quantitative real-time rt-pcr was conducted using a thermal cycler dice real time system (takara) with gene-specific primer sets as described previously [23] . the rna levels of viral genes were normalized to that of mrna for the β-actin or glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene, and relative quantities (rq) of mrna accumulation were determined using the 2 -δδct method. to detect alterations in genomic rna and sg mrna levels in the presence of mβcd during porcine nidovirus infection, the results obtained from drug-treated cells were compared with those from vehicle-treated cells. pam-pcd163 and vero cells were grown in 6-well tissue culture plates for 1 day and were mock infected or infected with prrsv and pedv, respectively, at an moi of 1 in the presence of mβcd. at the indicated times, cells were harvested in 80 μl of lysis buffer (0.5% triton x-100, 60 mm β-glycerophosphate, 15 mm ρ-nitrophenyl phosphate, 25 mm mops, 15 mm mgcl 2 , 80 mm nacl, 15 mm egta [ph 7.4], 1 mm sodium orthovanadate, 1 μg of e64 per ml, 2 μg of aprotinin per ml, 1 μg of leupeptin per ml, and 1 mm pmsf) and sonicated on ice five times for 1 s each. homogenates were lysed for 30 min on ice and clarified by centrifugation at 15,800 × g (eppendorf centrifuge 5415r, hamburg, germany) for 30 min at 4 °c. the protein concentrations of the cell lysates were determined by bca protein assay (pierce, rockford, il). the cell lysates were mixed with 4× nupage sample buffer (invitrogen) and boiled at 70 °c for 10 min. the proteins were then separated on a nupage 4-12% gradient bis-tris gel (invitrogen) under reducing conditions and electrotransferred onto immobilon-p (millipore, billerica, ma). the membranes were subsequently blocked with 3% powdered skim milk in tbs (10 mm tris-hcl [ph 8.0], 150 mm nacl) with 0.05% tween-20 (tbst) at 4 °c for 2 h and reacted at 4 °c overnight with primary antibodies against prrsv n, pedv n, or β-actin. the blots were then incubated with secondary horseradish peroxidase (hrp)-labeled antibody (santa cruz biotechnology) at a dilution of 1:5,000 for 2 h at 4 °c. proteins were visualized using enhanced chemiluminescence (ecl) reagents (amersham biosciences, piscataway, nj) according to the manufacturer's instructions. to quantify the viral proteins produced, band densities of prrsv n and pedv n proteins were quantitatively analyzed using a computer densitometer with the wright cell imaging facility (wcif) version of the imagej software package (http://www.uhnresearch.ca/facilities/wcif/imagej/), based on the density value relative to that of the β-actin gene. all statistical analyses were performed using student's t-test, and p-values less than 0.05 were considered statistically significant. to investigate whether cholesterol plays a role in viral infection, we used mβcd, which is the most common cholesterol-sequestering agent used for plasma membranes. to examine the effect of mβcd on porcine nidovirus replication, prrsv and pedv were selected because they are economically important viral pathogens in the pork industry. based on mtt assay, none of the doses of mβcd tested in the current study caused detectable levels of pam-pcd163 or vero cell death (fig. 1a) . pam-pcd163 and vero cells were pretreated with mβcd at concentrations of 0.5 to 2 mm or with ethanol as a vehicle control for 1 h prior to infection. mβcd or vehicle was present throughout infection. virus production was initially measured by monitoring cytopathic effect (cpe) after infection and then confirmed by immunofluorescence using the respective anti-n protein mab at 48 hpi (fig. 1b) . in vehicle-treated control cells, visible cpe appeared at 24 hpi (data not shown) and became predominant by 48 hpi, and virus-specific staining was pronounced in many cell clusters, indicating infection and spread of the viruses to neighboring cells. in contrast, mβcd had an obvious inhibitory effect on porcine nidovirus propagation. as shown in fig. 1b , the cholesterol-sequestering compound dramatically diminished virus-induced cpe (first and fourth panels) and expression of prrsv and pedv genes in a dose-dependent manner. based on the quantification of n protein by flow cytometry, the proportion (%) of virus-infected cells was noticeably reduced after mβcd treatment. a maximum of ~80% inhibition of both viruses was observed in response to 1.5 mm mβcd ( fig. 1c and d) . in addition, the effective doses for inhibiting 50% (ed 50 ) of the replication of prrsv and pedv were determined to be about 770 μm and 820 μm, respectively. taken together, these data show that cholesterol depletion of target cells efficiently suppresses the replication of porcine nidoviruses. we then investigated the effects of cholesterol depletion on the envelopes of porcine nidoviruses. each viral stock was treated with mβcd up to 2 mm prior to inoculation of the respective target cells. the virion cholesterol content after mβcd treatment was measured using filipin iii as a fluorescent polyene antibiotic that binds to cholesterol. as shown in fig. 2a , viral cholesterol levels were significantly reduced in mβcd-treated viruses compared to those in vehicle-treated viruses. however, in contrast to depletion of cellular cholesterol, the removal of cholesterol from virions resulted in no significant reduction in the replication of prrsv and pedv, even at the highest concentration used (fig. 2b) . furthermore, the titers of both prrsv and pedv remained unchanged upon treatment of each virus with mβcd (fig. 2c) . our results indicate that the viral cholesterol content is irrelevant to prrsv and pedv infection in vitro. to verify the importance of cellular cholesterol in porcine nidovirus infection, we first examined whether replenishment of exogenous cholesterol restored mβcd-induced inhibition of porcine nidovirus infectivity. to accomplish this, cholesterol-depleted cells were treated with 100 μg or 40 μg of exogenous cholesterol per ml, which is the highest noncytotoxic concentration for pam-pcd163 or vero cells, respectively, before virus inoculation. both mβcd and exogenous cholesterol were supplied throughout the course of infection. the addition of exogenous cholesterol to mβcd-treated and virus-infected cells was found to significantly reverse the antiviral activity of mβcd through depletion of cellular cholesterol. incubation with mβcd alone greatly reduced prrsv production to 12% and 5% at 1.5 mm and 2 mm, respectively, whereas supplementation with exogenous cholesterol enhanced virus production to 32% and 23% at the same concentrations of mβcd (fig. 3a) . likewise, although pedv infection declined to 48%, 19%, and 2% in the presence of mβcd alone at 1 mm, 1.5 mm, and 2 mm, respectively, virus production increased to 59%, 26%, and 10% at the same concentrations of mβcd when exogenous cholesterol was added (fig. 3b ). to verify these results, we also investigated alterations in cellular cholesterol content in cells treated with mβcd and exogenous cholesterol using a fluorescent filipin iii. cellular cholesterol levels specifically decreased in virus-infected and mβcd-treated cells compared to those in virus-infected and untreated cells. supplementing exogenous cholesterol distinctly elevated the cholesterol level in virus-infected and mβcd-treated cells (fig. 4) . altogether, the data reveal that cellular cholesterol content plays a pivotal role in porcine nidovirus infection. to determine the point at which mβcd acts during porcine nidovirus infection, pam-pcd163 and vero cells were treated with mβcd at various time points postinfection. at 48 hpi, the levels of prrsv or pedv replication were measured indirectly by quantifying the cells that expressed viral n protein using flow cytometry (fig. 5 ). treating cells with 2 mm mβcd at -1 and 0 hpi resulted in an approximately 94% and 79% decrease in prrsv production, respectively, in comparison with control levels (vehicletreated cells). strikingly, the addition of mβcd at 1 hpi and thereafter (post-entry periods) had no significant inhibitory effect on prrsv infectivity compared to the control levels. similarly, treatment of pedv-infected cells with 2 mm mβcd up to 0 hpi suppressed viral production by 99-95%, whereas exposure to the compound at 1-24 hpi resulted in no reduction in pedv infectivity. these data showed that mβcd had to be present pre-infection or at an early stage of viral infection to exert its antiviral effect as a cellular cholesterol depletion reagent. therefore, there is an important effect of cholesterol in the pre-entry period during porcine nidovirus infection. next, we sought to pinpoint the step(s) in the replication cycle of porcine nidoviruses that were precisely targeted by pharmacological depletion of cellular cholesterol. to address this, the earliest steps, the two stages of virus entry (virus attachment and penetration), were examined using an internalization assay after treatment with mβcd. pam-pcd163 and vero cells were inoculated with prrsv and pedv, respectively, at 4 °c for 1 h to allow only virus attachment and were further maintained either at 4 °c or 37 °c to restrict or permit virus internalization, respectively, in the presence of mβcd. the samples incubated at 37 °c were subsequently treated with proteinase k to remove remaining viral particles from the cell surface. serially diluted infected cells were then subjected to an infectious center assay on uninfected pam-pcd163 and vero cell monolayers, and virus titers were measured 2 days later by ifa or plaque assay. as shown in fig. 6 , the titers of prrsv and pedv were reduced in a dose-dependent manner in cells treated with mβcd maintained at 4 °c to permit virus binding but prevent internalization, indicating that cholesterol depletion has an inhibitory effect on virus attachment to these cells. moreover, production of both viruses was diminished in mβcdtreated cells incubated at 37 °c to allow virus entry to proceed, which suggested that cholesterol sequestration disturbs internalization of prrsv and pedv. in addition, we analyzed the amount of pcd163 or papn expressed on the cell surface after mβcd treatment and found that the surface expression level of the viral receptor in cells treated with 2 mm mβcd was similar to that of on vehicle-treated cells (fig. 7) . taken together, these results indicate that pharmacological depletion of cholesterol hinders virus attachment and its subsequent penetration event without altering viral receptor expression and that cellular membrane cholesterol is indispensable for the porcine nidoviral entry process. like other positive-sense rna viruses, following virus entry, the nidovirus genome is released into the cytoplasm and promptly serves as a template for translation of viral proteins by hijacking the host translational machinery. early nidoviral translation produces the replicase polyproteins that are proteolytically processed into nsps, which subsequently drive de novo synthesis of nidoviral rna. therefore, we focused on the post-entry phases of the viral life cycle to investigate the functional mechanisms of sequestration of cellular cholesterol in pedv infection. because nidoviral infection generates genomic and sg rna species, we first tested whether the removal of cellular cholesterol specifically affected genome replication and sg mrna transcription. for this purpose, relative levels of both genomic rna and sg mrna were assessed by quantitative real-time strand-specific rt-pcr in the presence or absence of mβcd following porcine nidovirus infection. as shown in fig. 8a , mβcd almost completely inhibited the synthesis of prrsv genomic rna and sg mrna at a concentration of 2 mm when compared to untreated infected cells. furthermore, an analogous effect of mβcd on genome replication and sg mrna transcription of pedv was observed. little pedv genomic rna and sg mrna was detected in cells treated with 1.5 mm mβcd (fig. 8b) . the decreases in viral rna levels caused by mβcd did not reflect nonspecific inhibition of transcription because the internal control (β-actin or gapdh) mrna level remained unchanged in all samples (data not shown). taken together, these results indicated that treatment with mβcd subsequently suppressed synthesis of nidoviral genomic rna and sg mrna. since nidoviral structural proteins are translated from their respective sg mrna transcripts late in the infectious cycle, it is conceivable that suppression of viral protein expression is a consequence of cascade-like inhibition of viral rna synthesis. thus, we examined whether viral protein translation was affected by depleting cholesterol from cell plasma membranes. to accomplish this, pam-pcd163 and vero cells were exposed to mβcd for 1 h prior to infection, and the compound was allowed to remain in the culture medium during infection and subsequent incubation. the expression levels of prrsv and pedv n proteins in the presence or absence of mβcd were evaluated at 48 hpi by (fig. 9 ). densitometric analysis of the western blots revealed that the intracellular expression of both n proteins was dramatically reduced by mβcd, with a maximum of more than 95% inhibition at the highest concentration (fig. 9) . these data suggested that the sequestration effect of cellular cholesterol on viral protein expression is attributable to its specific preceding actions on viral rna biosynthesis during nidoviral replication. at 48 hpi, cell lysates were prepared, resolved by sds-page, transferred to a nitrocellulose membrane, and immunoblotted using antibodies that recognize the prrsv n protein or pedv n protein. the blot was also reacted with mouse mab against β-actin to verify equal protein loading. viral protein expression was quantitatively estimated by densitometry and expressed as the density value relative to that of the β-actin gene, and mβcd-treated sample results were compared to those of the vehicle control. the values shown are the means from three independent experiments, and the error bars denote standard deviations. **, p < 0.001 in addition, virus yields were determined during pharmacological depletion of cellular cholesterol to investigate whether endogenous cholesterol is necessary for production of infectious viral progeny. after infection, viral supernatants were collected at 48 hpi, and viral titers were measured. as illustrated in fig. 10a , the presence of mβcd suppressed the growth of viral progeny in a dose-dependent manner. the peak viral titer was determined to be 10 6.05 tcid 50 /ml and 10 7.10 pfu/ml in the vehicle-treated control for prrsv and pedv, respectively. however, the addition of 2 mm mβcd reduced titers of prrsv and pedv to 10 3.82 tcid 50 /ml and 10 5.08 pfu/ml, respectively (representing a more than 2-log reduction compared to control levels). examination of the growth kinetics also indicated that porcine nidovirus replication was markedly delayed when the cells were treated with mβcd at its optimal concentrations for each virus (fig. 10b) . these findings confirmed that cellular cholesterol content is integral in optimal progeny virus production from host cells. the order nidovirales is a monophyletic group of enveloped, positive-strand rna viruses with human and various animal hosts that produce a 3′ co-terminal nested set of sg mrnas during infection. this order unites the four distantly related families arteriviridae, coronaviridae, roniviridae, and mesoniviridae, based on a number of common properties such as genome organization, predicted proteomes, and synthesis of genomic and sg viral rnas, and it also separates them into large-(coronaviruses, toroviruses, and roniviruses), intermediate-(mesoniviruses), and small-genome (arteriviruses) nidoviruses to stress the clear genome size differences [18, 25, 26, 50] . research on porcine nidoviruses is necessary not only for developing strategies to control these viruses in pig populations but also for understanding the molecular biology of human or veterinary-important nidoviruses. despite extensive attention and research investment, two porcine nidoviruses, prrsv and pedv, continue to plague pig-producing countries, causing a significant economic impact on the swine industry worldwide. this is partially attributable to the lack of efficient vaccines that can confer full protection against nidoviral infections and the lack of antiviral agents to treat these infections. although cholesterol is required for optimal infectivity of diverse non-enveloped and enveloped viruses [48, 56] , its contribution to and specific function in porcine nidovirus replication are currently unknown. the present study showed that pharmacological sequestration of cholesterol in the cellular membrane but not the viral envelope exerts an efficient antiviral effect against prrsv and pedv in vitro. this effect could be counteracted by the addition of exogenous cholesterol, indicating the importance of membrane cholesterol for porcine nidovirus infection. depletion of cellular cholesterol using the drug mβcd primarily affected the virus attachment and internalization stages, significantly affecting post-entry steps in the replication of porcine nidovirus. altogether, our data indicate that cellular membrane cholesterol plays a critical role in entry of prrsv and pedv into target cells. because viruses are obligate intracellular parasites, they have evolved elaborate relationships with their host cells and developed the ability to modulate lipid composition, lipid synthesis, and host cell signaling pathways [6] . in particular, cholesterol-rich microdomains appear to be important in the entry of various viruses. indeed, many viruses have been shown to exploit cholesterol, which is present in either the viral envelope [22, 54] , cellular membrane [4, 36] , or both [40, 48] , for maximal virus entry. furthermore, accumulating evidence indicates that cholesterol is an essential component in the life cycle of several nidoviruses. the depletion of cellular cholesterol inhibits the entry of coronaviruses, including mouse hepatitis virus [12] , severe acute respiratory syndrome coronavirus (sars-cov) [32] , human coronavirus 229e [42] , and avian infectious bronchitis virus [22] as well as arteriviruses, including equine arteritis virus [41] and prrsv [21, 55] . on the other hand, transmissible gastroenteritis virus (tgev) and canine coronavirus require cholesterol both in the target cell membrane and in the viral envelope [43, 44] . the current study revealed that prrsv and pedv are sensitive to the depletion of cholesterol only in the cell membrane. although cholesterol depletion has been shown to inhibit prrsv entry into african green monkey kidney-derived marc-145 cells, whether plasma membrane cholesterol is involved in either virus attachment or penetration or both is unknown [21, 55] . in the present study, we used a continuous prrsv-permissive pam cell line that is considered the primary cell target for prrsv in the natural host, making it a good system for studying virushost interactions [31] . in a previous study, yin et al. [61] suggested that cholesterol is critical for a post-adsorption step in the entry of tgev, another porcine alphacoronavirus. porcine cd163 and apn have been shown to confer permissiveness of non-susceptible cell lines to prrsv and pedv, respectively, and have been identified as key molecules in the entry of porcine nidoviruses [9, 31, 39] . a previous report showed that cholesterol depletion did not alter cd163 expression in marc-145 cells [21] . likewise, the present study indicated that pharmacological depletion of cellular cholesterol had no effect on the levels of pcd163 and papn expression in porcine cells. recent studies have indicated that apn is not a functional cellular receptor for pedv, suggesting that the presence of the authentic virus receptor is essential for viral entry [33, 46] . therefore, it is still possible that cellular cholesterol is quantitatively related to a hitherto unidentified receptor for pedv. based on our results, nevertheless, we propose that cellular cholesterol is important in both the binding and internalization stages of prrsv and pedv entry into susceptible cell lines. these findings are striking in that these two viruses are known to use different cell entry mechanisms for the initiation of virus infection: prrsv enters pam cells via receptor-mediated endocytosis followed by ph-dependent fusion between viral and endosomal membranes [60] , whereas pedv enters target cells via virus-receptor interactions, followed by direct ph-independent fusion of the viral and plasma membranes [27] . because cholesterol is an essential lipid component of cell membranes, its depletion has the potential to inhibit virus entry via several mechanisms. cholesterol is a critical structural component of lipid rafts, together with sphingolipids. these lipids influence viral infection by regulating viral and/ or cellular membranes and thus can function by preferentially partitioning into specific membrane microdomains [6] . cholesterol may affect virus entry by modifying interactions between virus particles and host cell membranes, and lipid recognition by certain viral constituents may be essential for virus entry [58] . in the case of sars-cov, cholesterol in the plasma membrane plays an important role in interactions of the viral spike protein and cellular receptor angiotensinconverting enzyme 2 for optimal infection [16] . by analogy, it is feasible that cellular cholesterol depletion might disturb binding of prrsv and pedv to specific cellular receptors. secondly, the level of cholesterol is important for maintaining biological membrane fluidity, and its removal could reduce the potential for lateral diffusion in the membrane [7] . this reduction in membrane fluidity could have an influence on the entry of prrsv and pedv. thirdly, the lipid environment, including the cholesterol level, is known to contribute to the charge of ion channels formed in cellular membranes [11] . considering this issue, ion channel alterations in response to the lack of cellular cholesterol may affect the entry process of porcine nidoviruses. lastly, cholesterol removal has been shown to result in the inhibition of cellular signaling pathways [53] . we previously found that prrsv and pedv activate specific intracellular signaling networks such as mitogen-activated protein kinase (mapk) cascade pathways to favor replication of these viruses, but these signaling pathways are irrelevant to virus internalization [24, [28] [29] [30] . based on these previous data, cellular cholesterol does not appear to act through mapk signaling pathways. although our analysis did not provide clear evidence of the mechanism by which cholesterol promotes porcine nidovirus entry, we assume that the mechanism may differ among viruses and that cholesterol-dependent virus entry might be dependent on more than one of the aforementioned pathways simultaneously. in conclusion, our findings indicate that optimal infectivity of porcine nidoviruses requires cholesterol in the plasma membrane and that this is critical for the entry of prrsv and pedv. however, cholesterol depletion resulted in a reduction, but not abolishment, of virus infectivity, indicating that virus entry may still occur with lower levels of cholesterol but that increased cholesterol content makes this process more efficient. future work should address the question of whether cholesterol facilitates prrsv and pedv entry through interactions between viral attachment proteins and cellular receptors and/or by affecting membrane fluidity. the current study indicates that cellular cholesterol is a key player in the early stages of porcine nidovirus infection, including attachment and penetration. impeding porcine nidovirus entry is a viable antiviral strategy because it likely acts on extracellular targets, thereby limiting cell damage, and these viruses might be used as surrogate models for testing antiviral agents against human nidoviruses. although further studies based 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any studies with animals performed by any of the authors. key: cord-322683-wkrj6n1d authors: zhang, pengfei; yu, linyang; dong, jianguo; liu, yanling; zhang, leyi; liang, pengshuai; wang, lei; chen, bin; huang, li; song, changxu title: cellular poly(c) binding protein 2 interacts with porcine epidemic diarrhea virus papain-like protease 1 and supports viral replication date: 2020-07-13 journal: vet microbiol doi: 10.1016/j.vetmic.2020.108793 sha: doc_id: 322683 cord_uid: wkrj6n1d porcine epidemic diarrhea virus (pedv) belongs to the alphacoronavirus genus in the coronaviridae family. similar to other coronaviruses, pedv encodes two papain-like proteases. papain-like protease (plp)2 has been proposed to play a key role in antagonizing host innate immunity. however, the function of plp1 remains unclear. in this study, we found that overexpression of plp1 significantly promoted pedv replication and inhibited production of interferon-β. immunoprecipitation and mass spectrometry were used to identify cellular interaction partners of plp1. host cell poly(c) binding protein 2 (pcbp2) was determined to bind and interact with plp1. both endogenous and overexpressed pcbp2 co-localized with plp1 in the cytoplasm. overexpression of plp1 upregulated expression of pcbp2. furthermore, overexpression of pcbp2 promoted pedv replication. silencing of endogenous pcbp2 using small interfering rnas attenuated pedv replication. taken together, these data demonstrated that plp1 negatively regulated the production of type 1 interferon by interacting with pcbp2 and promoted pedv replication. porcine epidemic diarrhea virus (pedv) was identified as the causative agent of porcine epidemic diarrhea (ped) in 1978 (pensaert and de bouck, 1978) and has had catastrophic impacts on the global pig industry. the clinical signs and symptoms of ped include severe enteritis, vomiting, watery diarrhea, and high mortality. in 2010 china experienced and outbreak of a mutant pedv, leading to huge economic losses (sun et al., 2012a) . pedv has a positive-sense single stranded rna genome that is approximately 28 kb in size and contains six open reading frames (orfs). orf1ab encodes polyprotein (pp)1a and pp1ab, which are further cleaved into non-structural protein (nsp)1-16. the structural spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins are encoded by orf2, orf4, orf5 and orf6, respectively. orf3 encodes the accessory protein orf3 (kocherhans et al., 2001) . nsp3 is the largest nsp, and comprises papain-like protease (plp)1 and plp2 domains (lei et al., 2018) . the innate immune response is the first line of host defense against viral infection (o'neill and bowie, 2010) . type 1 interferons (ifns) play a key role in host resistance to viral infections. rna viruses induce the production of ifns through toll-like receptor 3 (tlr3) and retinoic acid-inducible gene (rig)-i-like receptor-dependent pathways (kawai and akira, 2007) . the virus can evade host innate immune response in two major ways: by modifying or hiding pathogen-associated molecular patterns (pamps), and by encoding specific proteins to block immune responses. coronaviruses have evolved specific mechanisms to evade or inhibit host antiviral innate immune j o u r n a l p r e -p r o o f responses (g devaraj et al., 2007; zhou and perlman, 2007) . many pedv proteins are involved in escaping the innate immune response. nsp1, nsp3, nsp7, nsp14, nsp15, and nsp16, as well as the structural e, m and n proteins, demonstrated ifn antagonism . the mechanisms underlying the ifn antagonism of nsp1, plp2, nsp5, and n protein have been elucidated (wang et al., 2016; xing et al., 2013; zhang et al., 2016) . pedv nsp1 inhibits production of type i ifns by degrading cyclic adenosine monophosphate responsive element-binding protein-binding protein and inhibiting immune stress granule expression (dragan et al., 2007; zhang et al., 2016) . pedv nsp5 is a 3c-like protease that proteolytically cleaves the nuclear transcription factor kappa b (nf-κb) essential modulator (nemo) at glutamine 231, impairing the ability of nemo to activate ifn production (wang et al., 2016) . the pedv n protein interacts with the tank-binding kinase, blocking its association with interferon regulatory factor 3 (irf3) and thus inhibiting irf3 activation and type i ifn production (ding et al., 2014; hu et al., 2018) . pedv can also resist immune responses by co-opting host cell proteins. poly(c) binding protein 2 (pcbp2) belongs to a class of proteins that bind poly(c) sequences in both rna and dna and are involved in maintaining mrna stability, regulating translation and cellular antiviral responses (makeyev and liebhaber, 2002) . pcbp2 expression is induced following viral infection and acts as a negative regulator of mitochondrial antiviral signaling protein (mavs), triggering its degradation (you et al., 2009) . it was previously reported that pcbp2 interacted with porcine reproductive and respiratory syndrome virus (prrsv) nsp1β and supported viral replication (beura et j o u r n a l p r e -p r o o f wang et al., 2012) . pcbp2 also antagonized vesicular stomatitis virus growth by affecting viral gene expression . it is unclear whether pcbp2 is also involved in pedv replication. although significant progress has been made in understanding pedv evasion of innate immune responses, the mechanism of interaction between viral and host cell proteins remains unclear. in the present study, we demonstrated that plp1 interacted with pcbp2 to inhibit ifn-β production and promote pedv replication. human embryonic kidney (hek) 293t cells, porcine intestinal epithelial ipec-j2 cells and african green monkey vero e6 kidney cells were maintained in dulbecco's modified eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (fbs), 100 units/ml penicillin and 100 μg/ml streptomycin. pedv strain gdgh (genbank accession number: mg983755) was isolated and stored in our laboratory. a dna sequence encoding pedv plp1 protein was cloned into pcmv-ha as previously described to yield the pcmv-plp1-ha expression vector (yu et al., 2019) . pcbp2 and pcbp1 were amplified by pcr and cloned into the pecmv-3×flag-n vector using the primers listed in table 1 . all plasmids were verified by dna sequencing. hek293t cells were seeded on coverslips in 24-well plates and cotransfected with j o u r n a l p r e -p r o o f 100 ng of ifn-luc, 5 ng of tk-luc and 500 ng of pcmv-plp1-ha or empty pcmv-ha vector. twenty-four hours post-transfection (hpt), cells were stimulated with 10 ng/ml tumor necrosis factor (tnf)-α for 6 h. cell lysates were prepared for analysis of luciferase activity using a luciferase enzyme assay system in accordance with the manufacturer's instructions (promega, beijing, china). total cellular rna was extracted using the minibest universal rna extraction kit (takara, japan) in accordance with the manufacturer's protocol. total rna was reverse transcribed to cdna using prime script™ rt master mix (takara, japan) and relative gene expression levels were quantified by quantitative rt-pcr using sybr® green real-time pcr master mix (toyobo, japan) using the cycle threshold method. βactin was used as the internal control. all primers were designed using primer premier 6.0 software and are listed in table 1 . hek293t cells were grown to 80% confluence in a 100 mm dish and transfected with empty pcmv-ha vector or pcmv-plp1-ha using lipofectamine ltx reagent (thermo scientific, china) and following the manufacturer's instructions. at 24 hpt, cells were washed with phosphate-buffered saline (pbs) and lysed with lysis buffer (20 mm tris, ph 7.5, containing 150 mm nacl, 1% triton x-100, sodium pyrophosphate, β-glycerophosphate, ethylenediaminetetraacetic acid, na3vo4, and leupeptin) supplemented with 1 mm phenylmethylsulfonyl fluoride. lysates were centrifuged at j o u r n a l p r e -p r o o f 12,000 g for 10 min at 4°c. the supernatant was collected and incubated with 20 µl of anti-ha agarose (pierce® ha tag ip/co-ip kit, thermo scientific) at 4°c with shaking overnight. immunoprecipitates were washed with tbst buffer (25 mm tris-hcl, ph 7.2, containing 0.15 m nacl and 0.05% tween 20) and eluted in non-reducing sample buffer. the samples were separated by sds-page and either transferred to a nitrocellulose membrane for western blotting or silver stained for mass spectrometry (pierce tm, thermo scientific). the silver stained gel was analyzed, and one differentially expressed protein spot was selected for liquid chromatography-mass spectrometry (lc-ms/ms) analysis. briefly, the excised band was cut into small cubes approximately 0.5-1 mm 3 in size, decolorized in a 37°c water bath, further decolorized in decolorizing solution (30 mm potassium ferricyanide and 100 mm sodium thiosulfate) in a 37°c water bath, reduced with 50 mm dithiothreitol at 56°c for 1 h, then alkylated with 100 mm iodoacetamide for 40 min at room temperature in the dark. the samples were digested with trypsin overnight. tryptic peptides were analyzed using easy-nl c1200 and q-exactive (thermo scientific, waltham, ma). trapped peptides were separated on an analytical c18 column (75 μm × 25 cm, thermo scientific). the mobile phases consisted of 2% acetonitrile (acn) a and 80% acn b, both containing 0.1% formic acid. a gradient of 5%-100% solvent b was used to elute the peptides at a constant flow rate of 300 nl/min for 90 min. data were acquired using a ms scan range (m/z) of 350-1300, j o u r n a l p r e -p r o o f acquisition mode dda and a resolution of 70,000. thermo xcalibur 4.0 software (thermo) was used for data acquisition. protein identifications were assigned using the ncbi nr databank and swissprot/uniprot databank. cells were harvested and lysed with lysis buffer. protein samples were mixed with sds sample loading buffer, electrophoresed on 10% or 12% polyacrylamide gels and transferred to a nitrocellulose membrane. the membrane was incubated with primary antibodies for 2 h at 37°c. plp1 was detected using a primary anti-ha-tag rabbit monoclonal antibody (cell signaling technology, danvers, ma). an anti-flag mouse antibody (sigma-aldrich, st. louis, mo) was used to detect flag-tagged proteins and an anti-tubulin antibody (cell signaling technology) was used to detect tubulin. rabbit polyclonal pcbp2 antibody (proteintech, chicago, il) was used to detect endogenous pcbp2 protein. horseradish peroxidase-conjugated anti-mouse igg and goat anti-rabbit igg antibodies were used as secondary antibodies. chemiluminescence was detected using the gel imaging system tanon-5200multi (tanon, shanghai, china). vero e6 cells were grown in glass slides and cotransfected with pcmv-plp1-ha, pecmv-3×flag-pcbp2 or empty vector (pecmv-3×flag). at 36 hpt, the cells were washed three times with pbs, fixed with 0.4% paraformaldehyde for 1 h at room temperature, and blocked with 1% bovine serum albumin for 1 h. subsequently, the cells were permeabilized with 0.5% triton x-100 for 5 min, and then incubated at 37°c primers for pcbp2 quantitative rt-pcr are shown in table 1 . anti-tubulin antibody and rabbit polyclonal pcbp2 antibody (proteintech) were used to assess expression levels of tubulin and pcbp2 proteins. all results were presented as the means ± standard errors of the means of three independent experiments. data were analyzed using graphpad prism 6.0 (graphpad software, inc., la jolla, ca). differences between and among groups were assessed j o u r n a l p r e -p r o o f using the student's t-test and two-way analysis of variance, respectively. values of p < 0.05 were considered statistically significant and were indicated as follows: *p< 0.05, **p< 0.01, and ***p< 0.005. ifn-α/β is a key component of the host innate immune response to viral infection. pedv plp2 has been proposed to play a key role in antagonizing innate immunity (xing et al., 2013) . to investigate the effect of plp1 on pedv replication, vero e6 to investigate the effect of plp1 on type i ifn promoter activation, hek293t cells were cotransfected with ifn-luc and pcmv-plp1-ha or empty pcmv-ha vector. tk-luc was cotransfected as an internal control. at 24 hpt, the cells were stimulated with 10 ng/ml tnf-α for 6 h and prepared for luciferase analysis. as shown in fig. 1b , tnf-α significantly activated the ifn promoter and plp1 overexpression significantly inhibited tnf-α induced ifn-luc activity. to investigate the effects of plp1 on type i ifn production, hek293t cells were stimulated with tnf-α (10 ng/ml), then cells were cotransfected with pcmv-plp1-ha or empty pcmv-ha vector. at 24, 36 and 48 hpt, cells were harvested for total j o u r n a l p r e -p r o o f rna extraction and ifn-β mrna abundance was assessed by quantitative rt-pcr. as shown in fig. 1c , tnf-α stimulated ifn-β mrna transcription, whereas overexpression of plp1 significantly inhibited tnf-α-induced transcription of ifn-β mrna. these results indicated that plp1 acted as a negative regulator of ifn-β, thus augmenting pedv infection. to further investigate the mechanism through which pedv plp1 inhibited expression of ifn-β, we identified cellular interaction partners of plp1 using immunoprecipitation and mass spectrometry. hek293t cells were transfected with pcmv-plp1-ha or pcmv-ha empty vector. at 24 hpt, the cells were lysed and proteins were immunoprecipitated using the pierce® ha tag ip/co-ip kit (thermo scientific, china). the eluent was collected and analyzed by sds-page. silver staining showed multiple and distinct protein bands immunoprecipitated in plp1 overexpressing cells but not in cells transfected with empty vector. we chose the band with the largest expression difference for mass spectrometry analysis (fig. 2b ). gene ontology (go) and kyoto encyclopedia of genes and genomes (kegg) analysis was performed to analyze the functions of target proteins (fig. 3) . the identified host proteins were classified based on molecular function (mf), cellular component (cc) and biological process (bc) (fig. 3a) . the proteins were involved in cellular processes, metabolic processes and biological regulation. the majority of proteins pulled down with plp1 are listed in table 2 . analysis of the top 20 pathways showed that the majority of proteins played a role in organismal systems, human diseases and viral j o u r n a l p r e -p r o o f infection (fig. 3b) . identification of cellular interaction partners showed that pcbps were involved in the immune response against pedv infection. on the basis of the mass spectrometry results, we initially identified two proteins (pcbp1 and pcbp2) related to immune responses for further study. the genes encoding these two proteins were amplified and cloned into pecmv-3×flag, and hek293t cells were cotransfected with pcmv-plp1-ha and pecmv-3×flag-pcbp1 or pecmv-3×flag-pcbp2. at 24 hpt, cells were lysed and immunoprecipitated with mouse anti-flag antibody. the results showed that pcbp1 and pcbp2 were both pulled down, and that pcbp2 interacted most strongly with plp1 ( fig. 4) . therefore, pcbp2 was selected for further studies. to examine the co-localization of pcbp2 with plp1, vero e6 cells were cotransfected with pcmv-plp1-ha and pecmv-3×flag-pcbp2. at 24 hpt, the cells were stained with anti-ha and anti-flag antibodies and analyzed by confocal microscopy. overexpressed plp1 and pcbp2 were mainly co-located in the cytoplasm (fig. 5a) . we further analyzed the co-localization of plp1 and endogenous pcbp2 in vero e6 cells. the cells were transfected with pcmv-plp1-ha or pcmv-ha, then analyzed by confocal microscopy following staining with anti-ha and anti-pcbp2 antibodies. as shown in fig. 5b , plp1 was mainly co-localized with endogenous pcbp2 in the cytoplasm. to study whether plp1 promoted pcbp2 upregulation, vero e6 cells were transfected with different concentrations of pcmv-plp1-ha or empty pcmv-ha vector and western blotting was performed using anti-ha, anti-pcbp2, and anti-tubulin antibodies. plp1 overexpression enhanced endogenous accumulation of pcbp2 protein in a dose-dependent manner (fig. 6) . to investigate the effect of endogenous pcbp2 on viral replication, expression of pcbp2 was silenced using sirnas. three sirnas (1 to 3) were designed. following transfection of ipec-j2 cells, the effect of knockdown was assessed by western blotting. levels of pcbp2 protein were significantly decreased in all sirna-treated groups compared with the control group, and sirna1 showed the most efficient pcbp2 silencing (fig. 7a) . next, ipec-j2 cells were treated with sirna1 or a negative control (nc) sirna for 24 h and then infected with pedv at a moi of 0.1. levels of pcbp2 mrna and pedv rna were determined at 24 and 48 hpi by quantitative rt-pcr. as shown in fig. 7b and 7c, pcbp2 mrna levels in sirna-treated ipec-j2 cells were significantly reduced compared with nc-sirna-treated cells, and pedv replication was significantly suppressed (fig. 7b and 7c ). to further investigate whether overexpression of pcbp2 would affect pedv replication, ipec-j2 cells were transfected with pecmv-3×flag-pcbp2 or empty vector. at 24 hpt, the cells were infected with pedv at a moi of 0.1 and pedv viral loads were determined at 24, 48 and 60 hpi by quantitative rt-pcr. we found that pcbp2 overexpression enhanced pedv replication (fig. 7d) . these results indicated j o u r n a l p r e -p r o o f that plp1 acted as a negative regulator of ifn-β, and plp1 functions to augment pedv infection by interacting with pcbp2. this work was supported by the national key technologies r&d program nsp3 is the largest protein encoded in the coronavirus genome and comprises two subdomains (plp1 and plp2), which are mainly responsible for cleavage of nsp1/nsp2 and nsp2/nsp3, respectively (barretto et al., 2005; harcourt et al., 2004) . not all coronaviruses have a plp1 domain, while the plp2 domain is conserved in all coronaviruses. plp proteins encoded by various viruses have been shown to inhibit innate immunity (zheng et al., 2008) . severe acute respiratory syndrome coronavirus plp inhibits irf3 activation by blocking ubiquitination of rig-i, tnf-receptor associated factor 3, and stimulator of interferon genes (a lindner et al., 2007) . transmissible gastroenteritis virus plp1 and prrsv plp2 rely on deubiquitination activity to antagonize ifn production hu et al., 2017; j o u r n a l p r e -p r o o f 2012b). similar to other coronaviruses, pedv can also inhibit the production of type 1 ifns (zheng et al., 2008) . pedv plp2 has been shown to have deubiquitination activity and is an ifn antagonist, but plp1 has no deubiquitination activity (xing et al., 2013) . our results showed that plp1 is also an ifn antagonist. overexpression of plp1 enhanced pedv infection (fig. 1a) . using a dual fluorescent reporter, we found that plp1 could inhibit tnf-α-induced production of ifn-β. in hek293t cells transfected with plp1 expression vectors, induction of ifn-β by tnf-α was inhibited (fig. 1c) . these results suggested that plp1 could enhance pedv infection by inhibiting ifn-β production. studies have shown that the plp1 of pedv cv777 strain has no ifn antagonism (zheng et al., 2008) . there are 10 amino acid substitutions distinguishing the plp1s of pedv gdgh and cv777 strains (data not shown). we suspect that these substitutions may lead to differences in plp1 function. future studies should investigate the effects of amino acid mutations at these sites on plp1 function. the innate immune response is an important line of defense, and type 1 ifns are essential for host defense against viral infection. the double-stranded rna (dsrna) molecules produced by viral rna replication viral are pamps and can be recognized by pattern recognition receptors (prrs) (matzinger, 2002) . tlrs and rig-i-like receptors are two crucial prrs that recognize pathogens and stimulate downstream signaling to activate immune responses (huang et al., 2009; kawai and akira, 2007) . mavs is a downstream adaptor protein of rig-1. rig-1 mediates the activation of nf-κb and irf3 by interacting with the caspase-recruiting-like domain of mavs to inhibit the production of type i ifns (biacchesi et al., 2009; kawai et al., 2005; meylan et al., j o u r n a l p r e -p r o o f seth et al., 2005) . pcbp2 is a negative regulator of mavs whose expression is induced following viral infection and results in mavs degradation (you et al., 2009) . we found that overexpression of pcbp2 promoted pedv replication (fig. 7d) . pedv replication could be inhibited by silencing pcbp2 expression (fig. 7c ). pcbp2 has been reported to play a role in a variety of viral infections luo et al., 2014; wang et al., 2012) . we found that plp1 interacted with pcbp2 ( fig. 4 and fig. 5 ), and that overexpression of plp1 induces the expression of pcbp2 (fig. 6) . these results indicated that plp plays an important role in the antagonism of ifn responses. therefore, the study of plp proteins is important for understanding viral escape from host immune responses. compared with plp2, the function of plp1 has received less attention. the plp2s of many coronaviruses have deubiquitination activity, and can interfere with host antiviral responses (barretto et al., 2005) . we found that pedv plp1 also has ifn antagonism and contributes to viral replication. our results indicate that plp1 interacted with pcbp2, thereby helping pedv escape from host immune responses. our findings reveal a new mechanism evolved by pedv to circumvent the host antiviral response, and contribute to our understanding of the role of coronavirus selectivity in isg15 and ubiquitin recognition by the sars coronavirus papain-like protease the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity cellular poly(c) binding proteins 1 and 2 interact with porcine reproductive and respiratory syndrome virus nonstructural 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targeted by hepatitis c virus sensing and signaling in antiviral innate immunity a new coronavirus-like particle associated with diarrhea in swine identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 outbreak of porcine epidemic diarrhea in suckling piglets nonstructural protein 2 of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene 15 porcine epidemic diarrhea virus 3c-like protease regulates its interferon antagonism by cleaving nemo interaction of cellular poly(c)-binding protein 2 with nonstructural protein 1beta is j o u r n a l p r e -p r o o f beneficial to chinese highly pathogenic porcine reproductive and respiratory syndrome virus replication the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase pcbp2 mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip4 porcine epidemic diarrhea virus nsp4 induces pro-inflammatory cytokine and chemokine expression inhibiting viral replication in vitro suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp1 plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by key: cord-340438-9q3ic0ye authors: zhang, jianqiang; yim-im, wannarat; chen, qi; zheng, ying; schumacher, loni; huang, haiyan; gauger, phillip; harmon, karen; li, ganwu title: identification of porcine epidemic diarrhea virus variant with a large spike gene deletion from a clinical swine sample in the united states date: 2018-02-21 journal: virus genes doi: 10.1007/s11262-018-1542-7 sha: doc_id: 340438 cord_uid: 9q3ic0ye two genetically different porcine epidemic diarrhea virus (pedv) strains have been identified in the usa: us prototype (also called non-s indel) and s indel pedvs. in february 2017, a pedv variant (usa/ok10240-8/2017) was identified in a rectal swab from a sow farm in oklahoma, usa. complete genome sequence analyses indicated this pedv variant was genetically similar to us non-s indel strain but had a continuous 600-nt (200-aa) deletion in the n-terminal domain of the spike gene compared to non-s indel pedvs. this is the first report of detecting pedv bearing large spike gene deletion in clinical swine samples in the usa. porcine epidemic diarrhea virus (pedv) is the causative agent of porcine epidemic diarrhea (ped) that was first recorded in europe in the 1970s [1, 2] . pedv spread to asia during the 1980s and 1990s and became endemic in pigs in asian countries [3] . in 2010, a severe ped outbreak occurred in china characterized by high morbidity in pigs of all ages and high mortality in neonatal piglets [4, 5] . in 2013, ped outbreaks were reported for the first time in the usa [6] and caused substantial economic losses [7] . subsequently, us-like pedvs were identified in other american countries and also emerged or re-emerged in some asian and european countries [8] . global pedvs exhibit significant genetic diversities. recently, lin et al. [8] proposed to categorize global pedv strains into classical, s indel, emerging north american non-s indel, and emerging asian non-s indel strains. in the usa, at least two genetically different pedv strains have been identified: the highly virulent pedv first identified in april 2013 associated with severe ped outbreaks was referred to as 'us prototype' or 'us original' or 'non-s indel' strain [9, 10] ; a clinically milder pedv variant identified in the usa in january 2014 which was different from the original highly virulent pedv strains, as reflected by insertions and deletions in the spike (s) gene, was designated as 's indel' pedv [10, 11] . in this case report, we describe, for the first time, identification of a pedv variant with a large spike gene n-terminal domain deletion from a clinical swine sample in the usa. at the iowa state university veterinary diagnostic laboratory (isu vdl), a nucleocapsid (n) gene-based real-time rt-pcr (rrt-pcr) is routinely used for the screening detection of pedv from clinical specimens [12] [13] [14] . if positive, a spike gene-based multiplex rrt-pcr can be further used to differentiate non-s indel from s indel pedv strains. in february 2017, rectal swabs collected from a edited by juergen a richt. genbank accession numbers: the complete genome sequences of two porcine epidemic diarrhea viruses described in this study have been deposited in genbank under accessions mg334554 and mg334555. sow farm in oklahoma, usa, were submitted to the isu vdl for pedv pcr testing. the samples were positive for pedv by the n gene-based rrt-pcr. subsequent pedv s gene-based differential rrt-pcr revealed that these samples were negative for s indel pedv (c t > 40) but positive for non-s indel pedv. generally, the pedv s gene-based differential rrt-pcr gave 2-3 c t higher than the n genebased rrt-pcr on the same sample. however, the sample #8 gave unexpected results: strong positive by n gene-based rrt-pcr (c t 15.5) but weak positive for non-s indel pedv by the differential rrt-pcr (c t 36.8). to determine the possible reasons for this observation, the sample #8 and another control sample #6 (c t 18.2 by n gene-based rrt-pcr and c t 20.8 for non-s indel by the s gene-based differential rrt-pcr) were sequenced using next-generation sequencing technology following previously described procedures [15, 16] . the pedv in the sample #6 (usa/ ok10240-6/2017) and the sample #8 (usa/ok10240-8/2017) had whole genome sequences of 28,038 and 27,438 nucleotides in length, respectively. the sequences of these two pedvs have been deposited into genbank (mg334554 and mg334555). phylogenetic analyses based on the whole genome sequences and the spike gene indicated that both ok10240-6 and ok10240-8 belong to the us non-s indel cluster (fig. 1) . however, compared to the ok10240-6 and other non-s indel pedv strains, the ok10240-8 pedv had a large continuous deletion of 600-nt (200-aa) in the spike gene/protein (nt ∆91-690; aa ∆31-230; fig. 2 ). the remaining genome of the ok10240-8 pedv, other than the s deletion region, had approximately 99.7% nt identity to other non-s indel pedv strains. a gel-based rt-pcr [17] was used to differentiate the ok10240-8 pedv from non-s indel pedv. twenty more samples were collected from the same farm; all of them contained non-s indel pedv but none of them contained ok10240-8-like pedv, indicating the prevalence of ok10240-8-like pedv in swine populations may be very low. virus isolation attempts on the sample #8 in vero cells (atcc ccl-81) were unsuccessful. the remaining sample #8 (250 µl diluted in 2250 µl culture medium) was orally inoculated into two 10-day-old pedv-negative piglets (10 ml/pig) but did not result in active infection. pedv spike (s) protein is a type i membrane glycoprotein with a signal peptide (amino acid residues 1-18), a large [18] . the s protein assembles into homotrimers that form the clubshaped projections (spikes) on the virion surface. pedv s protein has multiple functions including (1) mediating receptor binding through its s1 subunit (aa 1-729) and fusion of the viral and cellular membranes during cell entry through its s2 subunit (aa 730-1386); (2) harboring neutralization epitopes. specifically, the n-terminal domain (aa 19-233) exhibits sialic acid binding activity; the receptor-binding domain (aa 501-629) is believed to interact with a protein receptor; and a fusion peptide domain (aa 891-908) mediates virus-cell membrane fusion during cell entry [18] . neutralization epitopes have been reported within the amino acid residues 1-219, 499-638, 636-789, and 1371-1377 [19] [20] [21] [22] . the aminopeptidase n protein (apn) serves as a receptor for several alphacoronaviruses such as canine coronavirus type ii, feline coronavirus type ii, transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), and human coronavirus 229e [18] . porcine apn was considered to be the putative receptor of pedv with some supporting evidence [23] [24] [25] [26] ; however, some recent studies indicate that porcine apn may not be a functional receptor for pedv [27, 28] . the n-terminal domain of pedv s protein is one of the most variable regions in the pedv genome. the insertions and deletions of s indel pedv strains and the large deletion (aa ∆31-230) of the pedv variant (ok10240-8) identified in this study are all located in the n-terminal domain region. it is predicted that deletion of 200-aa at this region would not interfere with either the protein receptor binding or the neutralization epitopes 499-638, 636-789, and 1371-1377. 3-d structural analyses of the s protein also suggest that this 200-aa deletion may not interfere with trimer formation. however, the neutralization epitope within residues 1-129 and the sialic acid binding activity of the virus may be affected by this 200aa deletion. in fact, some studies have shown that pedv strains having variations in the n-terminal domain of s protein exhibited different sialic acid binding activities [18, 29] . it remains to be determined whether the activity of sialic acid binding by the s protein affects virus entry into cells, replication in cells, and pathogenicity in pigs. in addition, 200-aa deletion in the ok10240-8 pedv variant may affect virus virulence and pathogenicity. construction of a recombinant pedv carrying 200-aa deletion in this it was previously reported [30] that a cell cultureadapted us pedv isolate tc-pc177-p2 contained 591nt (197-aa) deletion in the s protein (aa ∆34-230) but such deletions were not present in the original clinical sample oh/pc177/2013 (fig. 2) . a japanese pedv strain tottori2/2014, identified in a clinical sample, contained 582-nt (194-aa) deletion in the s protein (aa ∆23-216) [31] . a korean pedv strain mf3809/2008, identified in a clinical sample, contained a 612-nt (204-aa) deletion in the s protein but in a different location (aa ∆713-916) [32] . a recent study reported the coexistence of pedv with a large s gene deletion and pedv with intact s gene in domestic pigs in japan [33] . it has been demonstrated that the usa/ tc-pc177-p2 and jpn/tottori2/2014 isolates harboring a large s gene deletion are less virulent than non-s indel pedvs in experimentally inoculated pigs [8, 29, 34] . in terms of tgev, a large (224-aa) deletion in the spike gene changed the viral tropism from intestinal to respiratory and this tgev mutant was later renamed as prcv [35] . in contrast, the pedv variant tc-pc177-p2 with large s gene deletion did not change intestinal tropism [8, 29] . in summary, a new pedv variant strain (usa/ ok10240-8/2017) belonging to the non-s indel cluster but with a 600-nt deletion (200-aa deletion) in the n-terminal domain of the s gene was identified in this study. this is the first report of a pedv strain with a large deletion in the s gene identified in clinical swine samples in the usa. this pedv with large s gene deletion was present on the same farm where non-s indel pedv with intact s gene was detected but it appeared that the prevalence of ok10240-8-like pedv in swine populations may be low. additional molecular epidemiological studies are needed to monitor the emergence of novel pedv variants and determine their prevalence levels in us swine. pig farm acknowledgement this study was supported by the iowa state unikey: cord-307110-eiobmxp2 authors: zhao, shan; li, wentao; schuurman, nancy; van kuppeveld, frank; bosch, berend-jan; egberink, herman title: serological screening for coronavirus infections in cats date: 2019-08-13 journal: viruses doi: 10.3390/v11080743 sha: doc_id: 307110 cord_uid: eiobmxp2 coronaviruses (covs) are widespread among mammals and birds and known for their potential for cross-species transmission. in cats, infections with feline coronaviruses (fcovs) are common. several non-feline coronaviruses have been reported to infect feline cells as well as cats after experimental infection, supported by their ability to engage the feline receptor ortholog for cell entry. however, whether cats might become naturally infected with covs of other species is unknown. we analyzed coronavirus infections in cats by serological monitoring. in total 137 cat serum samples and 25 fcov type 1 or type 2-specific antisera were screened for the presence of antibodies against the s1 receptor binding subunit of the cov spike protein, which is immunogenic and possesses low amino acid sequence identity among coronavirus species. seventy-eight sera were positive for antibodies that recognized one or more coronavirus s1s whereas 1 serum exclusively reacted with human coronavirus 229e (hcov-229e) and two sera exclusively reacted with porcine delta coronavirus (pdcov). we observed antigenic cross-reactivity between s1s of type 1 and type 2 fcovs, and between fcov type 1 and porcine epidemic diarrhea virus (pedv). domain mapping of antibody epitopes indicated the presence of conserved epitope(s) particularly in the cd domains of s1. the cross-reactivity of fcov type 1 and pedv was also observed at the level of virus neutralization. to conclude, we provide the first evidence of antigenic cross-reactivity among s1 proteins of coronaviruses, which should be considered in the development of serological diagnoses. in addition, the potential role of cats in cross-species transmission of coronaviruses cannot be excluded. coronaviruses (covs) are enveloped viruses with a positive-stranded rna genome and classified into four genera (alpha-, beta-, gamma-and deltacoronavirus) within the subfamily orthocoronavirinae in the family coronaviridae of the order nidovirales. covs are found in a variety of mammals and birds, in which they can cause respiratory, enteric and systemic infections [1] [2] [3] . additionally, covs have proven ability for cross-species transmission, exemplified by the emergence of severe acute respiratory syndrome (sars) coronavirus in 2002/2003, and of the middle-east respiratory syndrome (mers) coronavirus in 2012 [4] . both viruses belong to the betacoronavirus genus and have an animal origin. sars coronavirus crossed over from bats via intermediate hosts to humans, became human-adapted and quickly spread worldwide before its containment. mers coronavirus recurrently enters the human population via its dromedary camel reservoir host, with limited, non-sustained human-to-human transmission particularly in healthcare settings [5] [6] [7] . apart from sars-and mers-cov, all four globally endemic human covs (hcov-oc43, hcov-nl63, hcov-229e and hcov-hku1) originate viruses 2019, 11, 743 2 of 16 from animals [8] [9] [10] [11] . in addition, cross-species transmission potential of covs is also illustrated by the occurrence of chimeric coronaviruses that resulted from recombination events between feline covs (fcov) and canine covs (ccov) [12, 13] . in order to get insight into the frequency of interspecies transmission of coronaviruses within and between animal and human populations and the risk of subsequent development of a pandemic, it is useful to screen for coronavirus infections in animal species; especially those that are in close contact with humans. serological assays that can detect virus-specific antibody responses against infection play an important role in these epidemiological studies [14] . cats live in close contact with humans and often roam around freely in the environment. hence cats are an interesting species to study for infections with coronaviruses. infections with feline coronaviruses (fcovs) are recognized and widespread [15, 16] . fcovs are classified into two types, type 1 and type 2, based on the genetic and antigenic difference of their spike (s) protein [17] . in the field, the majority of fcov infections are caused by fcov type 1, while fcov type 2, derived from recombination events of type 1 fcovs and ccovs obtaining the s gene and some flanking regions of ccovs, is less prevalent [18, 19] . depending on the virulence of the fcov strain and the immune response of the cat, the clinical presentation can range from apparently asymptomatic, through diarrhea, to full-blown feline infectious peritonitis [20] . fcovs are members of the genus alphacoronavirus, to which also hcov-229e, porcine transmissible gastroenteritis virus (tgev), and ccov belong. the latter three viruses and fcov type 2 have been proven to use feline aminopeptidase n (fapn) as a functional receptor in vitro [21] . the receptor for type 1 fcov has still not been identified [22] . notably, previous studies have shown that hcov-229e and ccov could infect cats after experimental inoculation, causing an asymptomatic infection [23, 24] . thus, cats might potentially become naturally infected with covs of other species which may lead to virus-host adaptation e.g., mutation or recombination, resulting in emergence of novel coronaviruses and potentially new diseases [19, 25] . the extent to which infections with covs of other species occur in the field, has not been explored in previous epidemiological studies of cov infections in cats [15, [26] [27] [28] . being the main envelope protein of coronaviruses, the spike (s) protein mediates cell attachment and membrane fusion to allow viral entry. s functions as the main determinant of cell-, organ-and host-tropism. additionally, it is also the major target of neutralizing antibodies. spike comprises two functionally interdependent subunits, s1 and s2, with s1 responsible for receptor binding and s2 for membrane fusion [3, 29] . the s1 subunit is the least conserved and the most variable immunogenic antigen between coronavirus species [30] . therefore, the s1 subunit is well suited as an antigen to screen for coronavirus type specific antibodies [31] . in this study, covs infection in cats were detected through profiling antibody presence in serum samples from cats. recombinant cov spike s1 subunits of different animal and human covs were expressed in a mammalian expression system and used for screening of cat sera for the presence of antibodies against the respective proteins. positive samples were also tested by virus neutralization assays to support the specificity of the reaction [32] [33] [34] . this investigation intends to extend our knowledge of cov epidemiology, potential reservoirs, and cross-species transmission. specific fcov type 1 and fcov type 2 sera were obtained from specific pathogen free (spf) cats previously infected with strain uu2 or rm and fipv-1146 respectively [35, 36] . in addition, for the serological survey, 137 feline sera were retrieved from the serum bank in our lab. these had all been collected from cats in the netherlands. most of the samples (>80%) were from a study on antibody titer testing for feline panleukopenia virus. the other samples were send to our lab for fip or felv-fiv diagnostics. sera of uninfected spf cats were included as negative controls. all samples were stored at −20 • c until analysis. african green monkey kidney cells (vero-ccl81), human hepatoma cells (huh7), pig kidney epithelial cells (llc-pk1), human embryonic kidney 293 cells stably expressing the sv40 large t antigen (hek-293t) were maintained in dulbecco modified eagle medium (dmem, lonza, basel, switzerland) supplemented with 10% fetal bovine serum (fbs, bodinco, alkmaar, the netherlands). virus strains used in this study have been described previously [37] [38] [39] . briefly, recombinant porcine epidemic diarrhea virus (pedv) (rpedv-s dr13 -gfp) was propagated and titrated in vero cells, and hcov-229e in huh7 cells. pdcov was propagated and titrated in llc-pk1 cells, but supplemented with 1 µg/ml tpck-treated trypsin (sigma-aldrich, inc., st louis, mo, usa) in dmem. synthetic sequences of 12 coronavirus spike s1 subunits (hcov-hku1 (gb: yp_173238.1), mers-cov (gb:yp_009047204.1), sars-cov (gb: aax16192.1), hcov-oc43 (gb: aar01015.1), hcov-229e (gb: np_073551.1), hcov-nl63 (gb: yp_003767.1), tgev (gb: abg89325.1), pedv (gb: aog30832.1), bcov (gb: p15777.1), pdcov (gb: aml40825.1), fcov type 1 (gb: fj938060.1), fcov type 2 (gb: ay994055.1)) and different domains of pedv s1 subunit (s1 0 and s1 a-d , as identified and described in [40] ) were cloned into pcaggs expression plasmids as described previously [41] . similarly, the expression constructs encoding chimeric proteins in which s1s were fused to the fc domain of mouse igg2a. for protein production, hek-293t cells were transfected with plasmid dna conjugated to polyethyleneimine (polysciences, inc., warrington, pa, usa). at 8-16 h post transfection, inoculum was removed and the transfection mixture was replaced by 293 sfm ii expression medium (gibco®, life technologies inc., grand island, ny, usa). at 6-7 days post transfection, cell supernatants were harvested and proteins were collected by protein a sepharose beads (ge healthcare bio-sciences ab, uppsala, sweden). proteins were then eluted with 0.1 m citric acid, ph 3.0 and neutralized with 1 m tris-hcl, ph 8.8. concentrations of proteins were assessed by nanodrop spectrophotometry (thermofisher scientific inc., waltham, ma, usa) and confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page) with bovine serum albumin (bsa, bioivt, west sussex, uk) as standard. typical yields for proteins were 0.2-0.5 mg/ml. for long term storage, proteins were stored at −80 • c upon usage. to study the potential cross-reaction between fcov type 1 and 2 in more detail, models of fcov type 1 (strain: uu2; genbank accession no.: fj938060.1) and fcov type 2 (strain: 79-1146; genbank accession no.: ay994055.1) s proteins were generated via the automated protein structure swiss-model homology modelling server (https://swissmodel.expasy.org/) [42] using the elucidated hcov-nl63 cryo-em structure (pdb code: 5szs) as the input model. figures were made with pymol (the pymol molecular graphics system, version 1.0 schrödinger, llc.). fcov s1 domains of both type 1 and 2, namely s1 0-cd , were expressed as murine fc fusion proteins in hek-293t cells as described above. high binding microtiter plates (greiner bio-one bv, alphen aan den rijn, the netherlands) were coated overnight at 4 • c with equal molar amount of protein (0.25 pmol per well, diluted in phosphate buffered saline (pbs, ph 7.4)). after three washes with washing buffer (pbs containing 0.05% tween-20), the plates were blocked for 2 h at 37 • c with blocking buffer (pbs containing 5% milk powder (protifar, nutricia, zoetermeer, the netherlands), 0.05% tween-20). protein coating efficiency was assessed by binding of anti-mouse igg antibodies in a direct elisa, and confirmed the equimolar coatings of all proteins. to detect antigenic reaction with serum samples, sera were tested in duplicate at a 1:200 dilution in blocking buffer, and then incubated in the plates at 37 • c for 1 h. after washing, plates were incubated with a 1:4000 diluted horseradish peroxidase (hrp)-conjugated goat anti cat igg (rockland immunochemicals, inc., pottstown, pa, usa) at 37 • c for 1 h. the peroxidase reaction was then visualized via adding tmb super slow one component hrp microwell substrate (biofx®, surmodics ivd, inc., eden prairie, mn, usa) for 10 min. reaction was stopped with 12.5% sulfuric acid and optical densities (od) were measured at 450 nm. negative sera (from uninfected spf cats) were included to determine the elisa cut-off values; sera with od values higher than 5-fold the od of negative sera were considered positive. all 12 s1 proteins were coated on the same elisa plates making it easy to screen and compare the od values of individual sera in one assay. hereby we excluded the sera that give high background od values against all proteins being considered false positive. neutralization assays were performed with some of the covs to support the specificity of elisa results. cat sera were serially diluted 2-fold in dmem and mixed 1:1 with rpedv-s dr13 -gfp, hcov-229e or pdcov (2000 50% tissue culture infective doses [tcid 50 ]/ml). these mixtures were then incubated at 37 • c for 1h, and 100 µl of each mixture was used for inoculation with vero, huh7 and llc-pk1 cell monolayers in 96-well plates, respectively. for pdcov infection, tpck-treated trypsin (sigma-aldrich, inc., st louis, mo, usa) was supplied to llc-pk1 tissue culture medium at a final concentration of 1 µg/ml. at 2-5 days post infection, cytopathic effect (cpe) could be observed via microscopy. virus neutralization titers (vnt) were expressed as the highest serum dilution resulting in 90% reduction of cytopathic effect (hcov-229e and pdcov) or virus-induced fluorescent cells (pedv). before virus neutralization, sera were inactivated through incubation at 56 • c for 30 min. experiments were performed in triplicate. feline sera (n = 137) were screened by indirect elisa for antibody reactivity against 12 cov s1 antigens. the od values against these 12 antigens are shown in figure 1 . in total, 78 of the 137 sera (56.9%) contained anti-cov antibodies, while 43 sera showed reactivity against more than one cov s1 antigen. none of the samples had to be discarded because of reactivity against all of the proteins indicating a potential false positive result. the frequency of different combinations of cov-s1 reactive samples is summarized in table 1 . reactivity against eight out of 12 cov s1 antigens could be observed, whereas none of the sera recognized the s1 protein of hcov-hku1, mers-cov, sars-cov and hcov-oc43. different s1 antigens were grouped by amino acid sequence phylogeny (left panel) using mega7. each column of the heat map represents an individual sample, and columns were arranged in a descending order based on elisa-reactivity against feline coronavirus (fcov) type 1. table 1 . numbers of positive cat samples and different combinations of reactivity found. the number of positive sera against each individual s1 is shown in the bottom row. positive elisa reactions are colored in orange. cut-off value was determined as the 5-fold over the od450 of negative sera. fcov type1-s1 fcov type2-s1 pdcov-s1 tgev-s1 229e-s1 bcov-s1 different s1 antigens were grouped by amino acid sequence phylogeny (left panel) using mega7. each column of the heat map represents an individual sample, and columns were arranged in a descending order based on elisa-reactivity against feline coronavirus (fcov) type 1. table 1 . numbers of positive cat samples and different combinations of reactivity found. the number of positive sera against each individual s1 is shown in the bottom row. positive elisa reactions are colored in orange. cut-off value was determined as the 5-fold over the od450 of negative sera. number of cats (total = 137) fcov type1-s1 fcov type2-s1 pedv-s1 pdcov-s1 tgev-s1 229e-s1 nl63-s1 bcov-s1 as expected, many sera were positive for fcov s1, with 75 sera (54.7%) positive for fcov type 1 and 26 sera (19.0%) for fcov type 2 s1 ( figure 1 ). all of the fcov type 2 s1 positive sera also tested positive for fcov type 1 s1, while 15 of 26 fcov type 2 s1 positive sera also reacted with tgev s1. the fcov type 2 s1 and tgev s1 elisa reactivities showed a strong nonparametric spearman correlation (spearman r = 0.84, p < 0.0001). with respect to this, we suggest that the tgev s1 positivity was due to cross-reactivity of fcov type 2 s1, as fcov type 2 shows close antigenic and genetic relationship with tgev (s1 shares 70.4% amino acid sequence identity). the remaining 11 fcov type 2 s1 positive but tgev s1 negative sera do react with fcov type 1 s1. an explanation might be the cross-reactivity between fcov type 1 and type 2. remarkably, 40 feline sera were reactive with s1 proteins from human, porcine and bovine covs (table 1) , including hcov-229e (16/137), hcov-nl63 (2/137), pedv (27/137), pdcov (8/137) and bcov (1/137). od values of feline sera positive for hcov-nl63 s1 and bcov s1 were relatively low ( figure 1a ). elisa reactivity towards non-feline cov s1 proteins might be explained by infection with the respective or related covs or by the presence of cross-reacting antibodies, although there was low sequence identity (< 32.8%) between s1 proteins of fcov type 1 and related non-feline coronaviruses (for the complete comparison of s1 sequence identities, see table 2 ). yet, all of pedv-s1 positive sera were also positive for fcov type 1 s1 ( figure 1b , table 1 ). the elisa results of fcov type 1 s1 and pedv s1 showed a strong nonparametric spearman correlation (spearman r = 0.83, p < 0.0001). thus, this might indicate the occurrence of antibody cross-reactivity against fcov type 1 and pedv s1 antigens. many of the hcov-229e and pdcov s1 positive sera also reacted with fcov type 1 s1, but no strong nonparametric spearman correlation was observed (hcov-229e, r = 0.216; pdcov, r = 0.307). one feline serum only reacted with hcov-229e viruses 2019, 11, 743 7 of 16 s1, and two feline sera only recognized pdcov s1. ( figure 1b , table 1 ). this observation led us to hypothesize that cross-reactivity may not play a role in elisa reactivity of these three sera, but that the three cats had been infected with these viruses or related viruses. 1 fcov type1-s1 (fj938060.1) 2 fcov type2-s1 (ay994055.1) 28.5 3 pedv-s1(aog30832.1) 32.8 30. 2 4 pdcov-s1 (aml40825. in our screening, 43 samples were shown to be positive for two or more s1 proteins including fcov type 1. the data prompted us to test different hypotheses which may explain this phenomenon: specific reaction through natural virus infection or reaction due to cross-reactivity with fcov-s1 antigens. to explore this further, we employed 25 fcov type 1 specific sera derived from specific pathogen free (spf) cats that had been experimentally infected with fcov type i strain rm (n = 9) or strain uu2 (n = 16). these sera were tested for their elisa reactivity against seven cov s1 proteins (excluding tgev s1) that showed positive reactivity in the previous serological screening. as expected, all 25 sera were positive for fcov type 1 s1 in our elisa; interestingly, four samples also reacted with fcov type 2 s1, and five samples with pedv s1. no positive elisa-reactivity was detected with s1 of hcov-229e, pdcov, hcov-nl63 or bcov (table s1 ). thus, fcov type 1 infection could lead to the generation of antibodies that cross-react in the s1-elisa with fcov type 2 and pedv s1 proteins. the elisa cross-reactivity of fcov type 1 specific sera with fcov type 2 s1 antigens prompted us to map the domains responsible for cross-reaction within the s1 subunit. hence, to identify domain borders within s1, we built homology-based models of both fcov type 1 and type 2 spike using the related elucidated hcov-nl63 cryo-em structure as the template model. as shown in figure 2a , continuous structural domains can be identified for the s1 subunit of both spikes, namely s1 0 , and s1 a through s1 d . amino acid sequence identities of these domains between fcov type 1 and type 2 differ, ranging from 22.4% to 57.4% ( figure 2b ). several s1 proteins for both type 1 and type 2 fcov-s1 comprising one or two domains were expressed and purified ( figure 2c ). fcov type 1 specific sera (n = 4 for strain rm and n = 3 for strain uu2) and type 2 (n = 6, strain 79-1146) specific sera were then tested against these proteins in elisa format. the four fcov type 1 specific sera that cross-reacted with s1 of fcov type 2 again showed binding to fcov type 2 s1. but the fcov type 2 specific sera showed little to no reactivity against fcov type 1 s1. as shown in figure 3 , the type specific antisera reacted with all of the homologous s1 domains, with s1 and s1 b of both type 1 and type 2 displaying the strongest reaction. interestingly, the cd domain showed the highest level of cross-reactivity between fcov type 1 and 2, in agreement with its highest sequence identity among s1 domains (figure 3) . the other three domains showed little to no cross-reactivity. antisera reacted with all of the homologous s1 domains, with s1 and s1 b of both type 1 and type 2 displaying the strongest reaction. interestingly, the cd domain showed the highest level of crossreactivity between fcov type 1 and 2, in agreement with its highest sequence identity among s1 domains ( figure 3 ). the other three domains showed little to no cross-reactivity. the s1 subunit of one protomer are colored, with s1 0 shown in cyan, s1 a in blue, s1 b in green, and the domains s1 cd in red. the s2 part of the protomer is marked in light gray. (b) schematic presentation of the fcov type 1 (strain uu2) and type 2 (stain 79-1146) s protein with the signal peptide (sp), the s1 subunit (the domains are colored as described in the legend of figure 2a ) and the s2 subunit (the c-terminal transmembrane domain is indicated by a black box). amino acid sequence identities between fcov type 1 and type 2 s1 domains are indicated. (c) diagram of the different s1 subdomains sequence. all s1 subdomains were c-terminally tagged with the fc part of mouse igg2a (not shown in the figure) and expressed as fc fusion proteins. the s1 subunit of one protomer are colored, with s1 0 shown in cyan, s1 a in blue, s1 b in green, and the domains s1 cd in red. the s2 part of the protomer is marked in light gray. (b) schematic presentation of the fcov type 1 (strain uu2) and type 2 (stain 79-1146) s protein with the signal peptide (sp), the s1 subunit (the domains are colored as described in the legend of figure 2a ) and the s2 subunit (the c-terminal transmembrane domain is indicated by a black box). amino acid sequence identities between fcov type 1 and type 2 s1 domains are indicated. (c) diagram of the different s1 subdomains sequence. all s1 subdomains were c-terminally tagged with the fc part of mouse igg2a (not shown in the figure) and expressed as fc fusion proteins. viruses 2019, 11, x for peer review 9 of 16 figure 3 . elisa-reactivity of fcov specific antisera against different s1 subdomains of fcov type 1 and 2. equimolar amount of purified s1 proteins and the four s1 subdomains were coated onto 96well plates and antibody binding was determined by elisa. the fcov type 1 and 2 specific antisera used in the screening were derived from experimentally infected specific pathogen free (spf) cats and are indicated at the right side of each panel, absorbance values and antigens in use are shown on the y-and x-axis, respectively. graphs represent the mean values from three independently performed experiments. standard deviations are indicated as error bars. because the fcov type 1 specific cat sera also showed elisa reactivity with pedv-s1 (table s1 ), we analyzed the reaction of the five pedv-s1 positive cats in more detail. samples were analyzed via elisa using antigens comprising different pedv-s1 domains, as described in our previous study [40] . cat sera taken pre-and post-fcov infection were collected and tested. as indicated in figure 4 , all five cats had developed pedv-s1 reactivity to different extent after fcov type 1 inoculation. noticeably, all sera showed the highest od values with the cd domain, while the other domains, including the s1 b containing the presumed receptor binding domain (rbd) [40] , were non-reactive ( figure 4 ). on the other hand, the swine pedv positive control serum exhibits strong reactivity against all pedv-s1 domains. the next question we asked was whether fcov type 1 specific sera could neutralize pedv infection in tissue culture, as they showed no reactivity with the s1 b of pedv spike. as shown in figure 5 , pedv neutralizing antibodies were detected in three out of five fcov type i specific cat sera. figure 3 . elisa-reactivity of fcov specific antisera against different s1 subdomains of fcov type 1 and 2. equimolar amount of purified s1 proteins and the four s1 subdomains were coated onto 96-well plates and antibody binding was determined by elisa. the fcov type 1 and 2 specific antisera used in the screening were derived from experimentally infected specific pathogen free (spf) cats and are indicated at the right side of each panel, absorbance values and antigens in use are shown on the y-and x-axis, respectively. graphs represent the mean values from three independently performed experiments. standard deviations are indicated as error bars. because the fcov type 1 specific cat sera also showed elisa reactivity with pedv-s1 (table s1) , we analyzed the reaction of the five pedv-s1 positive cats in more detail. samples were analyzed via elisa using antigens comprising different pedv-s1 domains, as described in our previous study [40] . cat sera taken pre-and post-fcov infection were collected and tested. as indicated in figure 4 , all five cats had developed pedv-s1 reactivity to different extent after fcov type 1 inoculation. noticeably, all sera showed the highest od values with the cd domain, while the other domains, including the s1 b containing the presumed receptor binding domain (rbd) [40] , were non-reactive (figure 4 ). on the other hand, the swine pedv positive control serum exhibits strong reactivity against all pedv-s1 domains. the next question we asked was whether fcov type 1 specific sera could neutralize pedv infection in tissue culture, as they showed no reactivity with the s1 b of pedv spike. as shown in figure 5 , pedv neutralizing antibodies were detected in three out of five fcov type i specific cat sera. the experiment was carried out in duplicate and repeated three times. error bars indicate standard deviations. sera were collected from spf cats prior (cat 91-131p) and after (cat 91-131) experimentally inoculated with fcov type 1. positive serum: pedv positive swine serum collected from the field; negative serum: serum from fcov negative spf cat. several serum samples from field cats, but not virus-specific serum samples from fcov inoculated spf cats, were found to be elisa positive for hcov-229e (n = 16) and pdcov s1 (n = 8) ( figure 1a) . also, a few feline sera displayed unique elisa positivity for s1 of hcov-229e (n = 1) or pdcov (n = 2) ( figure 1b, table 1 ). this could indicate that these antibodies were induced upon infection with these specific viruses. to corroborate the possibility of a natural infection in these cats with hcov-229e or hcov-229e-like viruses, we tested sera neutralization antibody titers. the results showed that one of the hcov-229e s1 reactive feline sera was able to neutralize hcov-229e infection (vnt = 32); no neutralization of pdcov was detected for the all pdcov-s1 positive sera. coronavirus infections are endemic and ubiquitous in feline populations. two viral types, type 1 and 2, are distinguished and both of them could well sustain themselves in the cat reservoir [15, 43] . both have been shown to have worldwide distribution, with the seropositivity rate up to 90% among animal shelter populations and in multi-cat households [20, 44] . the majority of natural infections are caused by type 1 fcovs, while in the field type 2 fcovs are less common and mainly occur in asia [15, 28, [45] [46] [47] . covs are generally considered to be host-specific; however, cross-species transmission does occur which may lead to incidental infections like the spillover of mers-cov from dromedary camel to humans, where humans function as an incidental and ultimately dead-end host [4] . but covs might also adapt to the new host exemplified by the animal origin of all four endemic human covs (hcov-oc43, hcov-nl63, hcov-229e and hcov-hku1) [8] [9] [10] [11] . whereas in cats infections with fcov are well recognized, studies regarding possible natural infections with other animal and human coronaviruses are lacking to the best of our knowledge. knowing the genetic variability of coronaviruses and the use of orthologous receptors by non-feline covs, studies on cross-species transmission are desirable. this may provide insight regarding whether cross-species transmission does occur. in the present study we used the highly immunogenic s1 antigens to screen cat sera for the presence of antibodies against feline and non-feline coronaviruses, as a first indication of possible infections with these viruses. in our study, 78 of the 137 cat sera were shown to be seropositive for coronaviruses. the seropositive rate (54.7%) against s1 of fcov type 1 is consistent with previous studies [15, 47] . all of the fcov type 2 s1 positive sera of naturally infected cats were also positive for fcov type 1 s1, which might be the result of cross reaction between the two proteins, despite their low amino acid identity. elisa with specific antisera from experimentally fcov type 1 and type 2 infected cats showed that sera of several fcov type 1 infected cats could cross-react with fcov type 2 s1. domain mapping elisa results showed that fcov type 1 specific sera react to different levels with the s1 domains of fcov type 1 s1 protein, and also reacts with fcov type 2 s1 cd . vice versa, fcov type 2 specific sera also reacted with s1 cd of fcov type 1. these observations pose a potential two-way cross-reactivity between s1 cd domains. interestingly, in parallel with our findings on feline coronaviruses, we identified a number of samples that were seropositive against the s1 of pedv, a viral pathogen that mainly replicates in the porcine intestinal epithelium. to study the possibility of cross-reaction, samples derived from preand post-fcov infected cats were screened against pedv s1 in elisa. the reactivity found against pedv s1 with fcov specific sera shows that cross-reaction can occur at the level of domain s1 cd ; the other pedv s1 domains showed no reaction with the fcov positive sera. judging from these observations, it seems that s1 cd plays an important role in cross-reaction between fcov type 1 and 2, and also fcov and pedv. as s1 cd is the most conserved domain among fcov and also between fcov and other alphacoronaviruses (for a systematic assessment of sequence identities, see table 3 ), it is reasonable to hypothesize that antibodies can develop against conserved epitopes within this region and subsequently cause cross-reaction. this should be taken into account when developing and interpreting serological assays. table 3 . identities of amino acid sequences of fcov type 1 (strain: uu2) s1 and s1 domains compared with the amino acid sequences of other alphacoronaviruses. (identities are shown in %; na: not available) the genbank accession numbers of these viruses are as follows: fcov type 1 (uu2), fj938060.1; fcov type 1 (rm), fj938051.1; fcov type 2, ay994055.1; tgev, abg89325.1; pedv, aog30832.1; hcov-229e, np_073551.1; hcov-nl63, yp_003767.1. amino acid % identity to fcov type 1(uu2) s1 s1 0 s1 a s1 b s1 cd noticeably, elisa reactivity among cat sera towards the n-terminal fcov s1 domains 0 and a was less consistent and generally lower compared to whole s1, which seems to correlate with the higher antigenic variation in those domains found among fcov type 1 strains [48] (figure 3 ). especially the sera from fcov-rm infected cats (cat 91, 93, 95 and 115) showed lower od values against s1 a . this phenomenon could be explained as the samples displaying higher reactivity were from cats inoculated with fcov-uu2 (cat 089, 131 and 129), the particular strain from which the s1 region was used as an antigen in the elisa studies. in the meantime, the possibility of the variable elisa reactivity might be due to the difference in individual antibody levels. in principle, the distinct antigenic reactivity of s1 0 and s1 a between the two fcov types might facilitate the development of a specific elisa method which allows the serological discrimination of fcov type 1 and type 2 infections in cats. in order to provide further insight regarding cross-reactivity between fcov type 1 and pedv, we performed virus neutralization assays. cross-neutralization of pedv infection could be observed for some of the feline fcov type 1 post-infection sera, in contrast to the pre-infection serum counterparts. since fcov specific pedv neutralizing sera did not react with pedv s1 0 , s1 a or s1 b , it is likely that the cross-neutralizing antibodies are targeting conserved epitopes in the s1 cd domain or the s2 subunit of the pedv spike protein [49] . given the unknown tgev infection background of the pedv positive pigs, the cross-reaction of pedv specific sera against fcov type 1 could not be explored in our study, as tgev positive pig samples would certainly influence the outcome [12, 18, 50] . of note, our findings cannot exclude the possibility that field cats might incidentally get naturally infected with pedv or pedv-like viruses, as there had been one report showing the detection of pedv in one stray cat via pcr assay [51] . it would be interesting to include more sera of cats from pig farms in future studies. considering the fact that cats play an important role in human society and have constant interaction with humans, it is of interest to conduct serological surveys for possible reverse zoonosis of human pathogens. in our study s1 antigens of several human coronaviruses were included and this led us to identify hcov-229e seropositive feline samples in our elisa survey (table 1) ; one serum in particular reacted solely with hcov-229e s1 but not with any other coronavirus. of the hcov-229e s1 reactive feline sera one showed low neutralizing activity against hcov-229e infection. this might suggest that positive cats were indeed exposed to hcov-229e or related viruses. mers seropositivity is also seen in other species besides the dromedary host [52] . rare cases of seropositivity might be considered as spill-over infections from the dromedary camel reservoir. similar (perhaps dead-end) spill-over infections of 229e from the human reservoir to cats might also occur. a similar principle could also apply for pdcov, a porcine pathogen that emerged rather recently. both hcov-229e and pdcov use apn as their receptor and have been reported to also be able to use feline apn for cellular entry [21, 37] . although reports are lacking regarding the natural infection of these two viruses in cats, hcov-229e was shown to cause a priming effect of fcov antibody in experimentally fcov infected cats suggesting that infection occurred [24] . therefore, the detection of antibodies against s1 of hcov-229e in a portion of the cats might be specific and due to the exposure to hcov-229e through daily interaction with humans. eight cats were seropositive for pdcov of which two cats were seropositive only for pdcov and not for any other covs. this could be caused by infection with pdcov or pdcov-related viruses through avian sources, considering the fact that cats are natural avian predators and the presumed avian origin of pdcov [2, 37] . our findings emphasize the potential role of cats as incidental hosts for non-feline coronaviruses and the need of in-depth study of naturally infected pathogens in cats. besides serological studies, efforts should also focus on isolation and identification of these viruses in cats. in conclusion, we presented a thorough serological survey in cats using s1 proteins of different animal and human coronaviruses. we demonstrated, despite the low amino acid identity, cross-reactivity between s1 proteins of fcov type 1 and 2, and between that of fcov type 1 and pedv. this should be considered when developing fcov serological assays as well as interpreting the results. our observation that some feline sera displayed antibody reactivity exclusively against non-feline cov s1 proteins warrant further research into the epidemiology and cross-species transmission of coronaviruses in cats and other animals that are in close contact with humans. further large scale serological studies regarding coronaviruses infection across animal species using arrays of cov s1 antigens can shed light into the hitherto unresolved host promiscuity of coronaviruses and the risk of cross-species transmission. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/11/8/743/ s1, table s1 : elisa reactivity (od450 values) of 25 fcov type 1 specific antisera against s1 antigens of different coronaviruses. the authors declare no conflict of interest. pre-fusion structure of a human coronavirus spike protein discovery of seven novel mammalian and 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infection in non-camelid domestic mammals this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-342923-prgorr3d authors: li, zhonghua; zeng, wei; ye, shiyi; lv, jian; nie, axiu; zhang, bingzhou; sun, yumei; han, heyou; he, qigai title: cellular hnrnp a1 interacts with nucleocapsid protein of porcine epidemic diarrhea virus and impairs viral replication date: 2018-03-13 journal: viruses doi: 10.3390/v10030127 sha: doc_id: 342923 cord_uid: prgorr3d the nucleocapsid (n) protein is a major structural component of porcine epidemic diarrhea virus (pedv), which is predicted to be a multifunctional protein in viral replication. heterogeneous nuclear ribonucleoprotein a1 (hnrnp a1) is a cellular protein participating in the splicing of pre-mrna in the nucleus and translation regulation in the cytoplasm. according to our previous proteomic study about pedv infection in vivo, hnrnp a1 was thought to be a cellular factor influencing pedv replication. in this report, pedv n protein was discovered to colocalize with cellular hnrnp a1 in perinuclear region of pedv infected cells. co-immunoprecipitation (co-ip) results clearly demonstrated that pedv n protein could bind to human hnrnp a1. replication of pedv was inhibited by silencing the expression of hnrnp a1 in ccl-81 cells, suggesting the positive effect of hnrnp a1 on pedv infection. porcine epidemic diarrhea virus is the most important viral agent which can cause diarrhea in pigs. serious threat has been posed on the world pig industry as a result of the high morbidity and mortality caused by pedv in piglets [1] [2] [3] [4] [5] [6] [7] . pedv, a single-stranded positive sense rna virus, is a member of coronavirus. four structural proteins, including spike protein (s), membrane protein (m), envelope protein (e) and nucleocapsid protein (n), and the genome constitute the virion. pedv n protein has multiple functions. firstly, as a structural protein, n protein along with the genomic rna forms the nucleocapsid of pedv. furthermore, n protein plays an important role in pedv rna synthesis and enhancing the pedv transcription and virion assembly [8] [9] [10] . however, the effects of n protein on pedv infection usually depend on its interaction with some host factors. for example, pedv n protein binds with phosphoprotein nucleophosmin (npm1) and then protects it from proteolytic degradation by caspase-3, enhancing cell survival and pedv growth [11] . in addition, it has been proved that interaction between pedv n protein and tbk1 could inhibit irf3 activation and type i ifn production, further to causing the circumvention of the host's antiviral immunity [12] . more than 20 heterogeneous nuclear ribonucleoproteins (hnrnps) have been discovered and among them hnrnp a1 is the best-characterized one [13, 14] . hnrnp a1 is a rna-binding protein which functions as binding to pre-mrna to form hnrnp particles in eukaryotic cells [15] . this protein contains two rna-binding domains (rbds) and a glycine-rich domain responsible for protein-protein interaction [16, 17] . it has been reported that hnrnp a1 selectively interacts with different rna-binding proteins through its glycine-rich domain [18] . previous studies have demonstrated hnrnp a1 could interact with n proteins of sars coronavirus and mouse hepatitis virus (mhv) [14, 19] . since pedv is also a member of coronavirus and pedv n protein is a rna binding protein, it is hypothesized that pedv n protein might also be able to interact with hnrnp a1 during pedv infection. our previous work has proved that hnrnp a1 underwent different regulations in jejunum tissues of piglets infected with pedv virulent strain and its attenuated strain [20] . therefore, we assume that hnrnp a1 may play a role in the life cycle of pedv. in this study, it is found that hnrnp a1 could interact with pedv n protein and pedv replication could be inhibited by silencing the hnrnp a1. the pedv yn144 (genbank accession no. kt021232), yn13 (genbank accession no. kt021228), and cv777 (genbank accession no. af353511.1) strain were used throughout this study. yn13 and yn144 strains were obtained by passaging the yn strain, a variant strain isolated from the intestine of a piglet with diarrhea, for 13 and 144 generations, respectively. our previous study has demonstrated that yn13 was a virulent strain and yn144 was an attenuation strain [20] . cv777 strain, a classical pedv strain, was provided by chengdu tecbond biological product co., ltd. (chengdu, china). the ccl-81 cell line and hek293t cell line were purchased from american type culture collection (atcc) and cultured in dulbecco's modified eagle's medium (dmem), supplemented with 10% fetal bovine serum (invitrogen, carlsbad, ca, usa) at 37 • c with 5% co 2 . the ccl-81 cell line was used for virus growth, infection, and cell lysate preparation. hek293t cell line was used for co-ip analysis. the rabbit anti-hnrnp a1, anti-flag polyclonal antibody (pab) and mouse anti-β-actin, anti-flag mono-antibody (mab) were purchased from abclonal (wuhan, china). mouse mab against pedv n protein was purchased from youlong biotech (shanghai, china). mouse mab against pedv spike protein was established by our laboratory. application of this mab has been described in some previous studies [21, 22] . alexa fluor 488-conjugated anti-rabbit, anti-mouse and alexa fluor 594-conjugated anti-mouse antibodies were purchased from antgene biological (wuhan, china). the cdna expression construct encoding yn144 n protein was pcr amplified and cloned into pcaggs-flagc, which encode a c-terminal flag. details of this part have been described in our previous study [20] . briefly, twelve piglets were randomly divided into three groups of four. the piglets in different groups were orally administrated with 4.22 ml yn13 and 4.22 ml yn144 with the same titer of 10 5.375 median tissue culture infective dose (tcid 50 ) ml −1 and 4.22 ml dmem, respectively. all piglets were euthanized and necropsied when diarrhea was observed in the piglets in yn13-infected group. jejunum tissues were separated rapidly, washed with ice-cold pbs buffer, snap-frozen in liquid nitrogen, and kept at −80 • c for subsequent proteome study. for the proteome study, itraq labeling coupled with lc-ms/ms was chose to analyze whole cell changes of jejunum of piglets, infected with pedv yn13 strain and yn144 strain. were precleared with protein a/g agarose (beyotime, shanghai, china) for 2 h then centrifuged at 3000 rpm for 5 min at 4 • c. supernatants were incubated with mono-antibody against flag for 12 h, then protein a/g beads were added and incubated at 4 • c for 6 h. the beads were then washed with ip lysis buffer five times and boiled in sample buffer, and the proteins were subjected to sds-page, followed by immunoblotting analysis with anti-flag pab or anti-hnrnp a1 pab. ccl-81 cells grown on coverslips were infected with pedv yn144 strain, yn13 strain and cv777 strain, respectively, at a multiplicity of infection (moi) 0.001. at 12 h post infection (hpi), the cells were fixed with 4% paraformaldehyde for 10 min followed by being treated with methanol. fixed cells were blocked with 5% bovine serum albumin and incubated with anti-hnrnp a1 pab and mab against pedv n protein. alexa fluor 488-conjugated anti-rabbit and alexa fluor 594-conjugated anti-mouse antibodies were served as the secondary antibody. cell nucleus were stained with 4 ,6-diamidino-2-phenylindole (dapi). the localization of pedv n protein, hnrnp a1 and cell nucleus were then observed on a zeiss confocal microscope (zeiss, oberkochen, germany). ccl-81 cells were transfected with sirnas targeting to hnrnp a1 with lipofectamine 2000 reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. the sirnas targeting hnrnp a1 were synthesized by gene pharma (shanghai, china). effect of rna interference was tested by western blot at 60 hpt. ccl-81 cells seeded in a 24-well plate were transfected without sirna or with 20 nm nonspecial control sirna or sirna1 targeting to hnrnp a1. at 60 hpt, cells were infected with or without pedv. at 12 hpi (yn144) or 24 hpi (yn13 and cv777), the cells were fixed with 4% paraformaldehyde followed by treating with methanol. then the fixed cell was blocked with 5% bovine serum albumin and then incubated with mab against pedv s protein. alexa fluor 488-conjugated anti-mouse antibody was applied to detect the primary antibodies. dapi was selected to stain the cell nucleus. rna was extracted by using tripure isolation reagent (roche, in, usa) following the manufacturer's instructions. the cdna was obtained by rt-pcr using the primescript™ rt master mix (takara, tokyo, japan). the real-time rt-pcr assay for quantifying pedv genome used the following primer and probe sequences: pedv forward primer: 5 -cgtacaggtaagtcaattac-3 , pedv reverse primer: 5 -gatgaagcattgactgaa-3 , pedv taq-man ® probe: fam-ttcgtcaca gtcgccaagg-tamra. cells were lysed with lysis buffer (beyotime, shanghai, china) containing 1 mm pmsf. these proteins were subjected to sds-page and then the separated protein bands were transferred onto pvdf membrane using a trans-blot (bio-rad, berkeley, ca, usa). the membrane was incubated in blocking buffer (tris-buffered saline (tbs), containing 0.05% tween-20 (tbst) and 5% skim milk) for 2 h at room temperature followed by washing three times by pbst. then the membrane was incubated with the corresponding primary antibodies for 2 h at room temperature. after being washed three times by pbst, the membrane was incubated with (hrp)-conjugated goat-anti mouse/rabbit igg at room temperature for 1.5 h. finally, the protein bands were visualized using the clarity™ western ecl blotting substrate (bio-rad, hercules, ca, usa). according to the result of lc-ms/ms, hnrnp a1 was down-regulated in an attenuated pedv strain (yn144) infected jejunum tissues, while showed no apparent change in a virulent pedv strain (yn13) infected jejunum tissues [20] . western blot assay was applied to confirm the changes of hnrnp a1. as shown in figure 1 , regulations of hnrnp a1 were consistent with the result of lc-ms/ms. hnrnp a1 has been proved to be involved in the replication process of other coronaviruses [14, 23] . therefore, we assumed that hnrnp a1 may play a role in pedv replication and influence the pathogenicity of pedv in vivo. according to the result of lc-ms/ms, hnrnp a1 was down-regulated in an attenuated ped rain (yn144) infected jejunum tissues, while showed no apparent change in a virulent pedv stra n13) infected jejunum tissues [20] . western blot assay was applied to confirm the changes rnp a1. as shown in figure 1 , regulations of hnrnp a1 were consistent with the result of l s/ms. hnrnp a1 has been proved to be involved in the replication process of other coronavirus 4, 23] . therefore, we assumed that hnrnp a1 may play a role in pedv replication and influen e pathogenicity of pedv in vivo. previous studies have demonstrated that n protein of some coronaviruses, such as mhv an rs-cov, could interact with hnrnp a1. in this study, we determined to investigate whether otein can be colocalized with hnrnp a1 during pedv infection. ccl-81 cells were infected wi e yn144, yn13, and cv777 strain of pedv, respectively, and the localization of np and hnrnp a as observed by confocal microscopy at 12 hpi. as shown in figure 2 , hnrnp a1 was main calized in the nucleus without pedv infection. however, some hnrnp a1 was transported fro e nucleus to the cytoplasm and colocalized with pedv n protein during pedv infectio rthermore, pedv n protein was localized in cytoplasm and no differences were found in t calization of n protein of these pedv strains. the immunofluorescence assay demonstrated th dv n protein and hnrnp a1 colocalized in cytoplasm. previous studies have demonstrated that n protein of some coronaviruses, such as mhv and sars-cov, could interact with hnrnp a1. in this study, we determined to investigate whether n protein can be colocalized with hnrnp a1 during pedv infection. ccl-81 cells were infected with the yn144, yn13, and cv777 strain of pedv, respectively, and the localization of np and hnrnp a1 was observed by confocal microscopy at 12 hpi. as shown in figure 2 , hnrnp a1 was mainly localized in the nucleus without pedv infection. however, some hnrnp a1 was transported from the nucleus to the cytoplasm and colocalized with pedv n protein during pedv infection. furthermore, pedv n protein was localized in cytoplasm and no differences were found in the localization of n protein of these pedv strains. the immunofluorescence assay demonstrated that pedv n protein and hnrnp a1 colocalized in cytoplasm. the colocalization of pedv n protein and hnrnp a1 demonstrated that a certain interaction may exist between these two proteins. thus, co-immunoprecipitation (co-ip) was performed to testify this phenomenon. 293t cells transfected with plasmids expressing the flag-tagged pedv n protein were subjected to immunoprecipitation using anti-flag antibody. interaction of pedv n protein with host protein hnrnp a1 was analyzed by immunoblotting with anti-flag antibody and anti-hnrnp a1 antibody. as shown in figure 3 , cellular hnrnp a1 protein was only detected in the presence of flag tagged pedv n by co-ip. these results indicate that n protein interacted with hnrnp a1. the colocalization of pedv n protein and hnrnp a1 demonstrated that a certain interaction may exist between these two proteins. thus, co-immunoprecipitation (co-ip) was performed to testify this phenomenon. 293t cells transfected with plasmids expressing the flag-tagged pedv n protein were subjected to immunoprecipitation using anti-flag antibody. interaction of pedv n protein with host protein hnrnp a1 was analyzed by immunoblotting with anti-flag antibody and anti-hnrnp a1 antibody. as shown in figure 3 , cellular hnrnp a1 protein was only detected in the presence of flag tagged pedv n by co-ip. these results indicate that n protein interacted with hnrnp a1. cell lysates were also applied to confirm the expression of proteins (input). in order to investigate whether hnrnp a1 participates in the replication of pedv, three sirnas targeting to hnrnp a1 were synthesized to silence the expression of this protein. sequences of the sirnas were shown in table 1 . ccl-81 cells were transfected with sirnas against hnrnp a1 expression, cells were collected at 60 hpt to analyze the silencing efficiency of hnrnp a1 at the protein level. as shown in figure 4 , sirna-1 showed the best performance for silencing hnrnp a1 and was chosen for the following research. in order to investigate whether hnrnp a1 participates in the replication of pedv, three sirnas targeting to hnrnp a1 were synthesized to silence the expression of this protein. sequences of the sirnas were shown in table 1 . ccl-81 cells were transfected with sirnas against hnrnp a1 expression, cells were collected at 60 hpt to analyze the silencing efficiency of hnrnp a1 at the protein level. as shown in figure 4 , sirna-1 showed the best performance for silencing hnrnp a1 and was chosen for the following research. ccl-81 cells were transfected with sirna-1 for 60 h followed by infection with pedv yn144 strain. these cells were subjected to western blot, indirect immunofluorescence assay (ifa) or real-time pcr at 12 hpi figure 5 . according to the results, a conclusion was reached that knockdown of hnrnp a1 reduced the replication of yn144. hek293t cells were transfected with a vector expressing pedv n protein with a flag tag (flag-pedv-n) or empty vectors (flag-pcaggs) and the whole-cell lysates obtained at 24 hpt were immunoprecipitated with anti-flag mab. after separation by sds-page, proteins were detected by immunoblotting with the indicated antibodies (ip: flag). cell lysates were also applied to confirm the expression of proteins (input). in order to investigate whether hnrnp a1 participates in the replication of pedv, three sirnas targeting to hnrnp a1 were synthesized to silence the expression of this protein. sequences of the sirnas were shown in table 1 . ccl-81 cells were transfected with sirnas against hnrnp a1 expression, cells were collected at 60 hpt to analyze the silencing efficiency of hnrnp a1 at the protein level. as shown in figure 4 , sirna-1 showed the best performance for silencing hnrnp a1 and was chosen for the following research. ccl-81 cells were transfected with sirna-1 for 60 h followed by infection with pedv yn144 strain. these cells were subjected to western blot, indirect immunofluorescence assay (ifa) or realtime pcr at 12 hpi figure 5 . according to the results, a conclusion was reached that knockdown of hnrnp a1 reduced the replication of yn144. sequences (5′-3′) sirna-1 ggaagaguuguggaaccaatt sirna-2 ggauuugguaaugauggaatt sirna-3 gcgguggaggucaauacuutt negative control sirna (nc) uucuccgaacgugucacgutt the pedv virulent strain yn13 and pedv classical strain cv777 were further applied to test the positive effects of hnrnp a1 on pedv replication. as shown in figures 6 and 7 , similar results were obtained. these results demonstrated that hnrnp a1 is a positive regulator of pedv replication. sirna-1 ggaagaguuguggaaccaatt sirna-2 ggauuugguaaugauggaatt sirna-3 gcgguggaggucaauacuutt negative control sirna (nc) uucuccgaacgugucacgutt the pedv virulent strain yn13 and pedv classical strain cv777 were further applied to test the positive effects of hnrnp a1 on pedv replication. as shown in figures 6 and 7 , similar results were obtained. these results demonstrated that hnrnp a1 is a positive regulator of pedv replication. figure 5 . knockdown of hnrnp a1 expression inhibits yn144 replication. ccl-81 cells were transfected without sirna (a) or with nc (b) or sirna-1 (c). at 60 hpt, cells were infected with yn144 strain at a moi of 0.001 or mock infected (d). virus suspensions were harvested at 12 hpi to calculate the virus titer by real-time rt-pcr. the virus copies per ml suspension was calculated (a). cells were harvested at 12 hpi to analyze the expression of pedv n protein by western blot (b) and the numbers of cells infected with pedv by ifa (original magnification 100×) (c). pedv n protein levels were quantified by measuring band intensities and normalized with respect to the amount of β-actin. data are shown as means ± the standard errors of the mean (sem) of at least three independent experiments, with the error bars representing the standard deviations. the pedv virulent strain yn13 and pedv classical strain cv777 were further applied to test the positive effects of hnrnp a1 on pedv replication. as shown in figures 6 and 7 , similar results were obtained. these results demonstrated that hnrnp a1 is a positive regulator of pedv replication. the virus titer by real-time rt-pcr. the virus copies per ml suspension was calculated (a). cells were harvested at 24 hpi to analyze the expression of pedv n protein by western blot (b) and the numbers of cells infected with pedv by ifa (original magnification 100×) (c). pedv n protein levels were quantified by measuring band intensities and normalized with respect to the amount of β-actin. data are shown as means ± sem of at least three independent experiments, with the error bars representing the standard deviations. in this study, interaction between hnrnp a1 and pedv n protein was verified by co-ip and immunofluorescence assay. our results established that n protein interacts with hnrnp a1 in pedv infected cells and 293t cells expressing pedv n protein. hnrnp a1 not only mainly localizes in the cell nucleus, but also shuttles between the nucleus and the cytoplasm [24, 25] . however, this protein underwent a relocalization to cytoplasm during pedv infection, suggesting a possible functional link between hnrnp a1 and pedv infection. this phenomenon is very similar to a previous study about mhv [23] . furthermore, we found that the pedv n protein and hnrnp a1 co-localized predominantly in the perinuclear region of pedv infected cells, in which active coronavirus replication/transcription complexes reside. this indicates both pedv n protein and hnrnpa1 might participate in constituting the pedv replication/transcription complex and their interaction may be involved in regulation of pedv replication. it is well known that hnrnp a1 is one part of replication/transcription complex of both sars-cov and mhv [8, 14, 23, 26, 27] . overexpression of hnrnpa1 facilitates mhv replication while inhibition of hnrnp a1 expression results in the reduction of mhv replication [26] . since pedv is in this study, interaction between hnrnp a1 and pedv n protein was verified by co-ip and immunofluorescence assay. our results established that n protein interacts with hnrnp a1 in pedv infected cells and 293t cells expressing pedv n protein. hnrnp a1 not only mainly localizes in the cell nucleus, but also shuttles between the nucleus and the cytoplasm [24, 25] . however, this protein underwent a relocalization to cytoplasm during pedv infection, suggesting a possible functional link between hnrnp a1 and pedv infection. this phenomenon is very similar to a previous study about mhv [23] . furthermore, we found that the pedv n protein and hnrnp a1 co-localized predominantly in the perinuclear region of pedv infected cells, in which active coronavirus replication/transcription complexes reside. this indicates both pedv n protein and hnrnpa1 might participate in constituting the pedv replication/transcription complex and their interaction may be involved in regulation of pedv replication. it is well known that hnrnp a1 is one part of replication/transcription complex of both sars-cov and mhv [8, 14, 23, 26, 27] . overexpression of hnrnpa1 facilitates mhv replication while inhibition of hnrnp a1 expression results in the reduction of mhv replication [26] . since pedv is also a member of coronavirus, hnrnp a1 may be involved in the process of pedv infection. in order to test this hypothesis, we silenced the hnrnp a1 and then studied its effect on pedv infection. similar to the result of mhv following silencing hnrnp a1, replication of three pedv strains were all inhibited, suggesting a positive effect of hnrnp a1 on pedv infection. replication of coronavirus depend on the generation of nested subgenomic mrnas (sgmrnas) with a common capped 5 leader sequence [28] . optical transcription of sgmrnas requires the interaction between its 5 leader sequence and the intergenic (ig) sequences of each orf. it has been reported that hnrnp a1 can bind to both the 5 terminal leader sequences and ig sequences [29] . so, inhibition of pedv by silencing hnrnp a1 is likely due to the break of the interaction between 5 terminal leader sequences and ig sequences. however, till now we have not got evidence to support it. in addition, silencing of hnrnp a1 might also be able to reduce its interaction with pedv n protein. the function of their interaction on pedv infection is under the investigation of our laboratory. our previous study has demonstrated that hnrnp a1 was downregulated in the jejunum of pedv strain yn144 infected group, but no apparent change in group infected with yn13 [20] . since yn13 caused diarrhea in piglets while yn144 could not, therefore, downregulation of hnrnp a1 was one of the reasons responsible for the lower pathogenicity of yn144 than yn13 in vivo. from the field to the lab-an european view on the global spread of pedv porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis new variants of porcine epidemic diarrhea virus, china porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines outbreak-related porcine epidemic diarrhea virus strains similar to us strains distinct characteristics and complex evolution of pedv strains first detection, clinical presentation and phylogenetic characterization of porcine epidemic diarrhea virusin austria the coronavirus nucleocapsid is a multifunctional protein molecular characterizations of subcellular localization signals in the nucleocapsid protein of porcine epidemic diarrhea virus the identification and characterization of two novel epitopes on the nucleocapsid protein of the porcine epidemic diarrhea virus nucleocapsid interacts with npm1 and protects it from proteolytic cleavage porcine epidemic diarrhea virus 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strain-like strains of porcine epidemic diarrhea virus comparative genomic analysis of classical and variant virulent parental/attenuated strains of porcine epidemic diarrhea virus the nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein a1 in vitro and in vivo a1-mediated translational regulation of the g quadruplexcontaining ron receptor tyrosine kinase mrna linked to tumor progression cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein a1 controls translation initiation of specific mrnas heterogeneous nuclear ribonucleoprotein a1 regulates rna synthesis of a cytoplasmic virus multiple type a/b heterogeneous nuclear ribonucleoproteins (hnrnps) can replace hnrnp a1 in mouse hepatitis virus rna synthesis blocking eif4e-eif4g interaction as a strategy to impair coronavirus replication nuclear proteins hijacked by mammalian cytoplasmic plus strand rna viruses the authors have declared no conflict of interest. key: cord-330475-mameyzih authors: shi, da; lv, maojie; chen, jianfei; shi, hongyan; zhang, sha; zhang, xin; feng, li title: molecular characterizations of subcellular localization signals in the nucleocapsid protein of porcine epidemic diarrhea virus date: 2014-03-13 journal: viruses doi: 10.3390/v6031253 sha: doc_id: 330475 cord_uid: mameyzih the nucleolus is a dynamic subnuclear structure, which is crucial to the normal operation of the eukaryotic cell. the porcine epidemic diarrhea virus (pedv), coronavirus nucleocapsid (n) protein, plays important roles in the process of virus replication and cellular infection. virus infection and transfection showed that n protein was predominately localized in the cytoplasm, but also found in the nucleolus in vero e6 cells. furthermore, by utilizing fusion proteins with green fluorescent protein (gfp), deletion mutations or site-directed mutagenesis of pedv n protein, coupled with live cell imaging and confocal microscopy, it was revealed that, a region spanning amino acids (aa), 71–90 in region 1 of the n protein was sufficient for nucleolar localization and r87 and r89 were critical for its function. we also identified two nuclear export signals (nes, aa221–236, and 325–364), however, only the nuclear export signal (aa325–364) was found to be functional in the context of the full-length n protein. finally, the activity of this nuclear export signal (nes) was inhibited by the antibiotic lepomycin b, suggesting that n is exported by a chromosome region maintenance 1-related export pathway. porcine epidemic diarrhea (ped) was first recognized as a devastating enteric disease in feeder and fattening pig, resembling transmissible gastroenteritis (tge) in pigs in the united kingdom, in 1971. it is a member of coronavirinae, which are single-stranded, positive-sense rna viruses with the largest genome that are known to infect humans, other mammals, and birds, usually causing subclinical or respiratory and gastrointestinal diseases. the porcine epidemic diarrhea virus (pedv) subgenomic mrnas, which are transcribed from the genome, produce viral proteins, such as the spike (s, 180-220 kda), envelope (e, ~8.8 kda), membrane (m, 27-32 kda), nucleoprotein (n, [55] [56] [57] [58] , and several other proteins of unknown function [1] [2] [3] . among the proteins, n, as the rna-binding protein, play an important role in both virus rna synthesis and modulating host cell processes, and phosphorylation may regulate these processes by exposing various functional motifs [4, 5] . several other functions have been postulated for the coronavirus n protein throughout the virus life cycle, including encapsidation, packaging, correct folding of the rna molecule, the deregulation of the host cell cycle [6] [7] [8] , inhibition of interferon production [9, 10] , up-regulation of cox2 production [11, 12] , up-regulation of ap1 activity [13] , induction of apoptosis [14] [15] [16] , association with host cell proteins [17] , and rna chaperone activity [18] . therefore, it is clear that n is a multifunctional protein involved in biological processes related to the survival of pedv. the nucleolus was one of the first subcellular structures to be identified by early users of the light microscope, appearing as a highly refractive black dot(s) in the nucleus of the cell [19] . the nucleolus is a highly specialized structure that participates in regulation of several host cell processes, including ribosome subunit biogenesis, rna processing, control of cell growth and response [20] . interestingly, a cytoplasmic-nucleolar distribution pattern has been reported for the n proteins of several coronaviruses, including representative members of alphacoronavirus (transmissible gastroenteritis virus, tgev), betacoronavirus (mouse hepatitis virus, mhv; and severe acute respiratory syndrome coronavirus, sars-cov), and gammacoronavirus (infectious bronchitis virus, ibv) [2, 21, 22] . further study indicated that n protein co-localize with major nucleolar proteins, including nucleolin, fibrillarin, and nucleophosmin [23] [24] [25] . how viral and cellular proteins traffic to the nucleolus and what determines their sub-nucleolar localization is not clearly understood, but proteins that localize to the cytoplasm and nucleus or nucleolus contain multiple signals that determine their subcellular localization [26] , such as nucleolar localization signal (nols). active nuclear import of proteins is mediated by nuclear localization signals (nlss), which are then recognized by proteins of the importin super-family (importin α and β) that mediate the transport across the nuclear envelope using ran-gtp [27] . similar to nuclear import, export of a protein from nucleus depends on the presence of a specific nuclear export signal (nes) [28] . the chromosome region maintenance 1 (crm1; also known as exportin 1 or xpo1) has been identified as an export receptor that interacts with the predominant nes, the so-called leucine-rich nes, which is found in a large variety of nucleocytoplasmic shuttling proteins [29, 30] . in fact, some of these ness are not necessarily leucine rich but rather characterized by several hydrophobic residues. the pharmacological compound leptomycin b (lmb) directly interacts with crm1 and blocks nes-mediated protein export [31] . therefore, the proteins can shuttle between the nucleus and the cytoplasm with their subcellular localization signals. it was reported previously that n protein nucleolar localization is a common feature in coronaviruses, however, there are different results regarding n subcellular localization in a strain of sars-cov [4] . within the alphacoronavirus coronaviruses, the precise nols and nes of pedv n and its traffic mechanism are still elusive. therefore, we have attempted to characterize these signals, and the molecular mechanism responsible for its subcellular localization. in this study, we examined the intracellular localization of the pedv n protein in pedv-infected and transfected cell lines using mouse polyclonal antisera and confocal microscopy. by generating a series of deletion and mutagenesis constructions, we found that amino acids 71-90 in region 1 were sufficient for nucleolar retention and we also identified two ness (aa221-236 and 325-364), but only the nes (aa325-364) was found to be functional in the context of the full-length n protein. the nucleocytoplasmic shuttling of n and the nuclear export of gfp-nes could be blocked by lmb, an inhibitor of the crm1, which is the receptor for exportin-1-dependent nuclear export. a polyclonal antibody specifically against the n protein was produced to determine its intracellular localization. we generated anti-n mouse antisera using an e coli-produced fusion protein as the antigen. the antigenicity of the recombinant n protein was confirmed by immunoreactivity with pedv pigs sera using elisa assay, which showed high sensitivity and specificity (data not show). to examine the reactivity and specificity of the mouse antiserum, blot results demonstrated that mouse antisera notably reacted with n protein from cv777 strain pedv, and the cell lysate from vero e6 cells transfected with pcdna3.1-n showed a band with the same molecular mass to n protein, whereas no band was detected from samples of cells uninfected pedv and transfected with an empty vector alone ( figure 1a ,b). our results showed that n protein was localized predominantly in the cytoplasm in pedv cv777-infected cells, while in a few cells fluorescence was also observed in the nucleus (or nucleolus) ( figure 2a ). no significant fluorescence was observed in uninfected cells (data not shown). a similar observation was also found in vero e6 cells transfected with plasmid expressing full length n protein ( figure 2b ). the n protein was observed to localize mainly in the cytoplasm with some protein in the nucleus (or nucleolus). the results suggested that the n protein localized to a subnuclear structure and may contain functional signals. to identify predicted nuclear (or nucleolar) localization signals, and whether they participate in this process or not, we conducted further experiments. after 24 h, infected or transfected, cells were fixed and analyzed by indirect immunofluorescence using mouse anti-n polyclonal antibody (green) and stained with pi (red) to visualize the nuclear dna. differentially fluorescing images were gathered separately using confocal microscopy. images were obtained with a 63× oil objective. to identify whether there were subcellular localization signals in pedv n protein, we first conducted a bioinformatics analysis on the protein using existing motif prediction algorithms. predict nls [32] and psort ii [33] were used to identify potential nlss, and the nes predictor (net nes) [29] was used to identify potential ness. predict nls found no nlss, whereas psort ii indicated that pedv n protein contained a pat7 motif (261pkknksr267). net nes found no nes. in other studies, coronaviruses, such as tgev, mhv, ibv, and sars-cov showed a common characteristic, that of nls-rich in c-terminal. so through amino acid sequence comparison among groups we also found a basic amino acid-rich short peptide (383rkkekknkre393) in c-terminal. we presumed it might play a role in n protein localization as a new localization signal, and named it patx. although n protein contains putative nuclear (or nucleolar) localization signals (n (or no) ls), it is not known whether they are functional or not. to investigate whether these and other unknown signals operated to determine the subcellular trafficking of n protein, we utilized acgfp as a fusion marker to observe the localization characteristics of n and truncated n protein in live cells. the b23.1-dsred fusion protein was used to tag the nucleolus, so we could analyze nucleolar localization properties and colocalization in cotransfected cells by live cell imaging (direct fluorescence) or confocal microscopy. at 24 h post-cotransfection, live cell imaging indicated that as previously shown, acgfp evenly distributed throughout the cytoplasm and the nucleoplasm but not the nucleolar, on the contrary acgfp-n protein localized to both the cytoplasm and nucleolus but not the nucleus in vero e6 cells ( figure 3 ). from the above results we hypothesize that there is a nols in n, which can guide exogenous protein to the nucleolus. to determine the exact nols, the acgfp-tagged truncated different regions of the n protein were transfected into vero e6 cells, which did not destroy the integrity of the signals. in our observation, acgfp-nr1 mainly localized to nucleolar structures, acgfp-nr2 localized predominantly to the cytoplasm and appeared also to accumulate in the nucleolus to the low level as the cytoplasm, whereas acgfp-nr3 predominantly localized to the cytoplasm; acgfp-nr1+2 protein same to acgfp-n and localized to the cytoplasm and nucleolus, acgfp-nr2+3 protein localized predominantly in cytoplasmic ( figure 3 ). this evidence demonstrated that region 2 targeted to the nucleolus with weaker enrichment, while region 1 localized to the nucleolus with stronger enrichment. thus, we speculated that region 1 have an effect on the nucleolar localization of n. this data also suggested that region 2 and 3 may contain ness because region 2 and 3 was mainly directed to the cytoplasm. interestingly, although region 2 and 3 contained predicted pat7 and patx motifs, respectively, they could either have been submissive to the nes or not functional. none of these fusion proteins had a distribution similar to acgfp only. to further identify the sequence for nucleolar localization in detail, a series of expression constructs containing fragments of region 1 were constructed. vero e6 cells were cotransfected with, either pacgfp-nr1 1-50 , pacgfp-nr1 51-100 , or pacgfp-nr1 101-147 , and pdsred-b23.1, analyzed using live cell imaging and confocal microscopy at 24 h post-transfection. the data indicated that acgfp-nr1 51-100 colocalized with b23.1, whereas the other two fusion proteins did not ( figure 4 ). to further refine the amino acids involved in nucleolar localization, 20 amino acid overlapping motifs encompassing amino acids 51-100 were cloned into downstream of acgfp, creating plasmids pacgfp-nr1 51-70 , pacgfp-nr1 61-80 , pacgfp-nr1 71-90 , and pacgfp-nr1 81-100 for the expression of recombinant fusion proteins. vero e6 cells were cotransfected with these constructs and pdsred-b23.1, at 24 h post-cotransfection analyzed using live cell imaging and confocal microscopy ( figure 5 ). the data indicated that acgfp-nr1 71-90 localized to the nucleolus and colocalized with b23.1. therefore, the amino acids at positions 71-90 in pedv n protein were capable to localize in the nucleolus. comparison of the pedv n protein nols with known cellular and viral nolss showed that the r87 and r89 of the pedv n protein nols might be conserved, although some cellular and viral nolss in this site did not contain basic amino acids ( figure 6 ). current work is directed at further resolving pedv nols sequence, including the contribution of individual amino acid residues. red squares indicate the amino acids of conservation. the cellular and viral nolss are described in nols ibv n protein [34] , nols prrsv n [20] , nols htlv-1 rex [35] , nols hsv gamma1 34.5 [36] , nols mdm2 [37] , nols mdv meq [38] , nols nf-kappa [39] , nols nuclear vcp-like protein (nvl2) [40] , nols p 120 [41] , nols surviving-deltaex3 [42] , nols (ggnnv) protein alpha [43] , bhv-1 bicp27 [44] , earning-associated protein 1-19 [45] , hsv-1 icp27 [46] , nols angiogen [47] , nols fibroblast growth factor-2 [48] , nols herpes-mareks meq [38] , nols hic p40 [49] , nols hiv-1 rev [50] , nols hiv-1 tat [51] . to map the amino acid sequence of region 2 responsible for its nuclear export, similar to the approach used to identify the nols in region 1, region 2 was subdivided into two distinct components. amino acids 148-220 and 221-294 were placed downstream of acgfp, creating expression vectors pacgfp-nr2 148-220 and pacgfp-nr2 221-294 . these expression plasmids were transfected into vero e6 cells, the nuclear was stained with dapi at 24 h post-transfection indicated that amino acids 148-220 directed acgfp to the cytoplasm and nucleus and had a subcellular localization similar to acgfp. in contrast, amino acids 221-294 directed acgfp to the cytoplasm; further investigation revealed that amino acids 241-260 and 261-294, when fused to acgfp, directed this protein to the cytoplasm and nucleus, whereas amino acids 221-240 directed acgfp to the cytoplasm (figure 7) . to further define the amino acids involved in cytoplasm trafficking, we conducted a tetra-alanine substitution mutagenesis of amino acids 221-240. these were placed down stream of acgfp, creating expression plasmids, pacgfp-nr2 221-224dlva-aaaa , pacgfp-nr2 225-228avkd-aaaa , pacgfp-nr2 229-232alks-aaaa , pacgfp-nr2 233-236lgig-aaaa and pacgfp-nr2 237-240enpd-aaaa . therefore, in some cases, the wild-type alanine was not substituted. the data indicated that substituting 221-224dlva-aaaa, 229-232alks-aaaa, and 233-236lgig-aaaa abolished cytoplasm trafficking. the remaining tetra-alanine substitutions had no effect on cytoplasm trafficking (figure 8 ), indicating that amino acids 221 dlvaavkdalkslgig 236 were involved in cytoplasm trafficking. to test whether this amino acid sequence was involved in directing the cytoplasm trafficking of n protein, this motif was deleted in the context of full-length n protein tagged to acgfp (plasmid pacgfp-n ∆221-236 ). this plasmid was transfected into vero e6 cells and the subcellular localization of the resulting fusion protein acgfp-n ∆221-236 investigated using confocal microscopy. there was no difference at 24 h post-transfection ( figure 9 ) compared with cells expressing acgfp-n protein. this data also indicated that the nes identified in region 2 was not necessary for cytoplasm trafficking in pedv n protein. as region 3 of pedv n protein localized to the cytoplasm, thus, the similar approach was used to identify the nes in region 3. the data indicated that amino acids 295-394 directed acgfp to the cytoplasm; further investigation revealed that amino acids 325-364 directed acgfp to the cytoplasm ( figure 10 ). taken together, we proposed that amino acids 325-364 are necessary and sufficient to direct n protein to the cytoplasm, and no other signals are involved. to determine if the pedv n protein was exported via the crm1-mediated pathway, the subcellular localization of the pacgfp-n, pacgfp-nr3 or pacgfp-nr3 325-364 , was compared between vero e6 cells left untreated or treated with 2.5 ng/ml lmb. as shown in figure 11 , the nucleocytoplasmic shuttling of pedv n, nr3 and nr3 325-364 was completely inhibited by lmb. the finding indicates that pedv n shuttling activity was affected by lmb and, thus, suggests that pedv n protein was transported via the classical crm1-dependent pathway. pedv is an important pathogen causing viral diarrhea in the swine industry. although much research has been carried out on the general characteristics of pedv, few reports have been reported on the functions of the pedv structural proteins, especially n protein. n protein plays an important role in virus replication and modulation of host cellular machinery, which should be a result of its self-interaction and interaction with other viral and cellular proteins and with virus and host cell nucleic acids. therefore, it is important to understand the subcellular localization properties of the pedv n protein. figure 11 . the nuclear export mechanism of pedv n. vero e6 cells were transiently transfected with plasmids encoding pacgfp-n, pacgfp-nr3, pacgfpnr3 365-394 , with or without treatment with lmb, and examined live 24 h after transfection by confocal microscopy. each image is representative of the majority of the cells observed in the same cells. the nucleolus (no) is arrowed where appropriate. to enter and export from the nucleus, all molecules or cargoes must traverse a large macromolecular structure called the nuclear pore complex (npc), which is located in the nuclear envelope. small molecules up to 40-60 kda or less than 10 nm in diameter can passively diffuse through the npc, but if proteins are larger than this size-exclusion limit and/or are required to move against a concentration gradient, then transport requires energy-driven mechanisms [52] . in this case, most proteins should contain the appropriate trafficking motifs, such as nls. the rules and signals that govern the nuclear localization of proteins are well defined. nlss can be classified into several categories, including the pat4 and pat7 motifs and bipartite nlss, are composed of basic amino acids of a given sequence length. the pat4 motif consists of a continuous stretch of (usually) four basic amino acids, and the pat7 motif starts with a proline residue and is followed by six amino acids [32, 53] . by contrast, the signals that govern nucleolar localization and retention are not well defined [26] . the motifs involved are usually rich in arginine and lysine residues; however there is no immediately obvious consensus sequence or structure. proteins that localize to the nucleolus can also have nuclear-import motifs. the nolss that have been identified so far can be grouped into those that contain single motif and those that contain multiple motifs [54] . in this study, the living cells fluorescence microscopy and confocal microscopy were employed for investigating the subcellular localization and nuclear import and export mechanisms of pedv n protein. it is well known that living cells fluorescence microscopy and confocal microscopy has an advantage over conventional in vitro nuclear transport assays in that cells are not physically damaged by microinjection, detergent, or mechanical perforation, this means that cellular components important for trafficking, such as nuclear import and export receptors, the mt network, intact [55, 56] . our results indicated that pedv n protein mainly distributes throughout the cytoplasm with localization to a substructure within the nucleolus in infected cells. similar results were also obtained in transfected cells. to eliminate the influence of charged protein migration to the nucleolus post-fixation, and to investigate the nuclear (or nucleolar) localization of pedv n protein and the functions of these motifs in more detail, we generated constructs that express the protein (or parts of the protein) as a fusion with enhanced green fluorescent protein. the protein could then be detected by direct fluorescence using both live-cell and confocal microscopy. no difference in the localization of either protein was observed between different cell lines, and the presence of a fluorescent tag at either the n terminus or c terminus of n protein did not affect the localization of the fusion protein compared with native protein [2, 4] . furthermore, the b23.1 gene was amplified from the vero e6 cells and then fused with dsred in order to visualize the nucleolus. transfection assay results indicated that acgfp localized predominately to the cytoplasm and the nucleus, but not the nucleolus. the characteristics of acgfp-n protein localized to either the cytoplasm alone or the cytoplasm and nucleolus, with a maximum of 60% of transfected cells exhibiting this phenotype at 24 h. our studies indicated that fusing acgfp with pedv n protein increased the molecular weight of this protein (82 kda) above the size exclusion limit of the nuclear pore complex, and it could not diffuse passively through the npc. this result suggested that there must be some signals in n protein that determined the nucleolar localization. a number of viruses and viral proteins can disrupt nucleolar architecture [57] , and the n protein of coronavirus can also localize to the nucleolus in a cell cycle dependent manner and this may be related to dynamic trafficking [58] . a previous study of sars-cov indicated the n protein inhibited b23 phosphorylation and might influence ribosome biogenesis to suppress host gene expression and create a more favorable milieu for virus survival [25] , so the n protein of pedv might take part in some cellular process. to investigate whether pedv n protein contained a nols, the protein was expressed as a series of single and overlapping regions. this preliminary analysis indicated that pedv n protein contained a nols in region 1. deletion mutagenesis delineated amino acid pedv n 71-90, a 20 aa motif that modulated nucleolar localization. furthermore, comparison with cellular and viral nolss, the data indicated that r87 and r89 may be critical for the nucleolar localization. for the site of replication and virus assembly is the cytoplasm, if the subcellular localization of the nucleocapsid protein is in the nucleolar, then the protein is not available for rna synthesis, encapsidation and assembly, and therefore progeny virus production might be less efficient. however, if such proteins do target the nucleolar as part of a virus replication strategy then they will contain appropriate targeting signals. thus, these will include not only nlss and nolss, but perhaps more importantly nes. by using a series of n deletion mutants fused to the acgfp and tetra-alanine substitution mutagenesis, we identified two nes site in region 2 (aa221-236) and region 3 (aa325-364), but nes in region 2 was deleted in the context of full length n protein did not affect cytoplasm trafficking, suggesting that the nes presented in region 2 submissive to the nes in region 3 or not function. furthermore, pedv n protein nucleocytoplasmic shuttling was specifically blocked by lmb treatment suggested that pedv n protein is transported via the classical crm1-dependent pathway. vero e6 was grown and maintained in dulbecco's modified eagle's medium supplemented with 10% heat-inactivated fetal calf serum and penicillin-streptomycin, and incubated at 37 °c in 5% co 2 . vero e6 cells were infected with pedv strain cv777 kindly provided by pensaert m b at moi = 1, and were seeded and after 24 h for both indirect immunofluorescence assay and western blot analysis. all enzymes used for cloning procedures were purchased from takara (dalian, china) except t4 dna ligase from new england biolabs (ipswich, ma, usa). pdsred-b23.1 encodes a fusion of dsred protein to the amino terminus of b23.1 and pacgfp-n encodes a c-terminally gfp-epitope-tagged version of pedv n and has been previously described [59] . the regions and subregions were amplified by pcr using pacgfp-n as a template. primers incorporated 5' bamh i site and 3' xho1 used for cloning into pet30a; primers incorporated 5' kpn i site and 3' xho i site used for cloning into pcdna3.1 and the other primers incorporated 5' xhol i site and 3' kpn i site used for cloning into pacgfp-c1. the cloning procedures for constructing most of recombinant plasmids were similar and all primers are listed in table 1 . at all times, numbers used in primer or construct names denoting amino acid numbers refer to their position on the full length n protein. for the tetra-alanine substitution, each pair of oligonucleotide strands was annealed, and the resultant double-stranded dna fragments were cloned into the vector pacgfp-c1. additionally, the nes of nr2 was deleted in the context of full-length n protein by overlapping pcr using forward primer 1-u and reverse primer 1-l (table 1) to generate one pcr product and forward primer 2-u and reverse primer 2-l (table 1) to generate the second pcr product. a second round of pcr was performed using both pcr products as templates and 1-u and 2-l as the forward and reverse primer, respectively. the resulting product was subcloned into pacgfp-c1. all plasmid were confirmed by sequencing analysis. the recombinant 6× his-tagged n protein was expressed in e. coli bl21 (de3) cells after induction with 1 mm iptg for 5 h in lb-medium at 37 °c . the recombinant protein was purified by a gravitraptm affinity column (ge healthcare, bio-sciences, piscataway, nj, usa) according to the manufacturer's instructions. balb/c mice were immunized with 0.1~0.2 mg of purified recombinant n protein injected subcutaneously at multiple sites on the back. booster injections were given two and four weeks later. blood was drown from the mouse at the fifth week following the immunization, and the blood was allowed to clot at 4 °c and the antiserum was recovered by centrifugation at 5,000× g for 10 min at 4 °c . a control serum was made by injecting normal saline under the same conditions. the animal experiment was approved by harbin veterinary research institute and performed in accordance with animal ethics guidelines and approved protocols. the animal ethics committee approval number is heilongjiang-syxk-2006-032. on the day before transfection, 0.5~2 × 10 5 cells grown on 35 mm culture dishes so that 60%-80% confluent cell monolayers were transfected with plasmid dna with lipofectamine™ 2000 (invitrogen, groningen, netherlands) according to the instructions of the manufacturer. protein samples were collected 48 h after transfection by direct lysis of the cells in 5× sds protein sample buffer with 5% 2-mercaptoethanol. the samples were boiled for 5 min, resolved with 12% sds-page, and blotted with the following antibodies. for the infection and transfection protein samples: the n protein polyclonal antiserum was the first antibody and irdyetm700dx conjugated affinity purified anti-mouse igg (h&l)(mouse) (li-cor biosciences, lincoln, ne, usa) was the second antibody; the images were acquired by the odysseytm infrared imaging system (li-cor). vero e6 cells were grown on coverslips and fixed 24 h post-infection (or post-transfection) with 50% methanol-50% acetone for analysis by indirect immunofluorescence using mouse anti-pedv n polyclonal sera (1:50 dilution) followed by fluorescein isothiocyanate (fitc)-labeled goat anti-mouse antibody (sigma, st louis, mo, usa). the cells were washed twice with phosphate-buffered saline (pbs) and subjected to perforation using 0.2% triton x-100. then the cells were stained with propidium iodide (pi) (50 μg/ml) (sigma) to visualize nuclear dna. samples were analyzed using fluorescence microscopy. for acgfp and dsred fusion expression constructs, co-transfection analyses were carried out. at 24 h post-transfection, subcellular localization properties in living cells were analyzed by laser confocal scanning microscope (leica laser technik, heidelberg, germany) with appropriate filters. in order to better display the results at the time of identify the nes in the regions of nr2 and nr3, at 24 h after transfection, cells on glass cover slips were rinsed with pbs and subjected to fixation using 50% methanol-50% acetone for 30 min and permeabilized with 0.2% triton x-100. then the nuclear was stained with 4',6-diamidino-2-phenylindole (dapi) (0.05μg /ml) (sigma) and analyzed by confocal microscope. cells were transfected with pacgfp-n, pacgfp-nr3, or pacgfp-nr3 325-364 . at 18 h post-transfection, cells were washed with pbs followed by either treated with 2.5 ng/ml lmb diluted with medium or left in unsupplemented cell culture medium for a further 6 h, then analyzed by used confocal microscope. a nols (aa71-90) and a functional nes (aa325-364) of pedv n protein were identified for the first time. additionally, the n protein was demonstrated to transport between the nucleolus and the cytoplasm through the nes by crm1-dependent pathway. characterization of the structural proteins of porcine epizootic diarrhea virus, strain cv777 the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus coronavirus genome structure and replication subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein trafficking motifs in the sars coronavirus nucleocapsid protein sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus detection of the nucleocapsid protein of severe acute respiratory syndrome coronavirus in serum: comparison with results of other viral markers the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclincyclin-dependent kinase complex and blocks s phase progression in mammalian cells inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor-3 potent inhibition of sars associated coronavirus (scov) infection and replication by type i interferons (ifn-alpha/beta) but not by type ii interferon (ifn-gamma) nucleocapsid protein of sars-cov activates the expression of cyclooxygenase-2 by binding directly to regulatory elements for nuclear factor-kappa b and ccaat/enhancer binding protein severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists activation of ap-1 signal transduction pathway by sars coronavirus nucleocapsid protein the nucleocapsid protein of the sars coronavirus is capable of self-association through a c-terminal 209 amino acid interaction domain the sars coronavirus nucleocapsid protein induces actin reorganization and apoptosis in cos-1 cells in the absence of growth factors sars-cov nucleocapsid protein induced apoptosis of cos-1 mediated by the mitochondrial pathway nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a coronavirus nucleocapsid protein is an rna chaperone the nucleolus 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 localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division nuclear/nucleolar localization properties of c-terminal nucleocapsid protein of sars coronavirus interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rna-associated protein fibrillarin the nucleocapsid protein of sars-associated coronavirus inhibits b23 phosphorylation to be or not to be in the nucleolus nucleocytoplasmic transport: navigating the channel nucleocytoplasmic transport nesbase version 1.0: a database of nuclear export signals orfeome cloning and global analysis of protein localization in the fission yeast schizosaccharomyces pombe crm1 is an export receptor for leucine-rich nuclear export signals finding nuclear localization signals psort: a programme for detecting sorting signals in proteins and predicting their subcellular localization delineation and modelling of a nucleolar retention signal in the coronavirus nucleocapsid protein sequence requirements for nucleolar localization of human t cell leukemia virus type i px protein, which regulates viral rna processing signals that dictate nuclear, nucleolar, and cytoplasmic shuttling of the gamma (1) 34.5 protein of herpes simplex virus type 1 identification of a cryptic nucleolar-localization signal in mdm2 nucleolar and nuclear localization properties of a herpesvirus bzip oncoprotein cytosolic, nuclear and nucleolar localization signals determine subcellular distribution and activity of the nf-kappa b inducing kinase nik nvl2 is a nucleolar aaa-atpase that interacts with ribosomal protein l5 through its nucleolar localization sequence characterization of the nuclear and nucleolar localization signals of bovine herpesvirus-1 infected cell protein 27 identification of the nuclear and nucleolar localization signals of the protein p120 identification of a novel nucleolar localization signal and a degradation signal in survivin-deltaex3: a potential link between nucleolus and protein degradation a viral protein inhibits the long range signaling activity of the gene silencing signal identification of nuclear/nucleolar localization signal in aplysia learning associated protein of slug with a molecular mass of 18 kda homologous protein identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein icp27 novel properties of the nucleolar targeting signal of human angiogenin nuclear and nucleolar localization of 18-kda fibroblast growth factor-2 is controlled by c-terminal signals sequence requirement for the nucleolar localization of human i-mfa domain-containing protein (hic p40) identification of sequences important in the nucleolar localization of human immunodeficiency virus rev: relevance of nucleolar localization to function effects of a highly basic region of human immunodeficiency virus tat protein on nucleolar localization classical nuclear localization signals: definition, function, and interaction with importin alpha transport into and out of the nucleus. microbiol ppar γ mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance function of dynein and dynactin in herpessimplex virus capsid transport p53 is associated with cellular microtubules and is transported to the nucleus by dynein rna viruses: hijacking the dynamic nucleolus cell cycle dependent localization of the coronavirus nucleocapsid protein co-localization analysis between porcine epidemic diarrhea virus nucleocapsid protein and nucleolar phosphoprotein b23.1 this work was supported by grants from the national natural science foundation of china (31172350), the natural science foundation for distinguished young scholars of heilongjiang province (jc201118), the agricultural scientific and technological transformative project (2011gb23260003), and higher school science and technology innovation team project of heilongjiang province (2011td001). the sponsors had no role in study design, collection, analysis, and interpretation of the data, writing the report, and in the decision to publish the results of the study. the authors declare no conflicts of interest. key: cord-339924-tsmnkuhw authors: jung, kwonil; wang, qiuhong; scheuer, kelly a.; lu, zhongyan; zhang, yan; saif, linda j. title: pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs date: 2014-04-17 journal: emerg infect dis doi: 10.3201/eid2004.131685 sha: doc_id: 339924 cord_uid: tsmnkuhw to understand the progression of porcine epidemic diarrhea virus infection, we inoculated gnotobiotic pigs with a newly emerged us strain, pc21a, of the virus. at 24–48 hours postinoculation, the pigs exhibited severe diarrhea and vomiting, fecal shedding, viremia, and severe atrophic enteritis. these findings confirm that strain pc21a is highly enteropathogenic. in june 2013, intestinal contents were obtained from a 1-day-old pig with diarrhea on a farm in ohio, usa. pedv strain pc21a was detected in the sample by reverse transcription pcr (rt-pcr) selective for the nucleocapsid gene (229-557 nt). the partial nucleocapsid gene sequence of pc21a was identical to that of 2 us pedv outbreak strains from colorado, usa: usa/colorado/2013 (genbank accession no. kf272920) and 13-019349 (genbank accession no. kf267450). only coronavirus-like particles were observed in the fecal sample by electron microscopy (figure 1 ). the sample was negative for rotavirus groups a and c and for transmissible gastroenteritis virus/porcine respiratory coronavirus by rt-pcr (7, 8) . the sample was bacteriologically sterilized by using 0.22-µm syringe filters and then prepared as inoculum. nearterm gnotobiotic pigs were delivered aseptically by hysterectomy from a specific pathogen-free sow (9) . six 10-to 35-day-old pigs were randomly assigned to a pedv-infected group (pigs 1-5) or a negative control group (pig 6). information about inoculation and inocula pig-passage number is described in table 1 . pigs 1-3 and 5 were inoculated orally and/or intranasally with 6.3-9.0 log 10 genomic equivalents (ge) of pedv strain pc21a; pig 4 was exposed to the virus by indirect contact with inoculated pig 3. for each sample, the quantity of pedv rna ge was ≈10 6 times higher than plaque assay results for a cell-adapted pedv strain, pc22a. clinical signs were monitored hourly. pig 4 was monitored for longer-term clinical signs and virus shedding. pigs were euthanized for pathologic examination at 3 stages of infection: acute, mid, and later stages (<24 h, 24-48 h, and >48 h, respectively, after onset of clinical signs). the ohio state university institutional animal care and use committee approved all animal-related experimental protocols. fecal or rectal swab samples were prepared as described (9) . virus rna was extracted by using the mag-max viral rna isolation kit (applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. titers of virus shed in feces were determined by taqman real-time rt-pcr using the onestep rt-pcr kit (qiagen, valencia, ca, usa) as reported (10) , with modifications in the forward primer and probe to provide a 100% match to the us strains: forward 5′-cg-caaagactgaacccactaac-3′ and probe fam-tgyyaccayyaccacgactcctgc-bhq. a standard curve was generated by using the pcr amplicon (pedn 229/557) of strain pc21a. the detection limit was 10 ge per reaction, corresponding to 4.8 log 10 and 3.8 log 10 ge/ml of fecal and serum samples, respectively. small and large intestine tissues, lung, liver, heart, kidney, spleen, and mesenteric lymph node were examined grossly and histologically. mean jejunal vh:cd was measured by using pax-it software (paxcam, villa park, il, usa) as described (11) . the frozen tissues were prepared and tested by immunofluorescence staining, as described (12) , for the detection of pedv antigen, using monoclonal antibody 6c8-1 against the spike protein of pedv strain dr13 (provided by daesub song, korea research institute of bioscience and biotechnology, daejeon, korea). acute, severe watery diarrhea and vomiting developed in all inoculated pigs. clinical signs developed 24-48 h after inoculation, regardless of the inoculum dose or number of inoculum pig passages (table 1 ). pig 4, which was followed longer, also exhibited dehydration, loss of bodyweight, and lethargy, but it consumed most of the milk that was offered. however, ≈120 h after onset of clinical signs, pig 4 collapsed after showing signs of disorientation and emaciation. immune electron microscopy, using a gnotobiotic pig hyperimmune serum to pedv, showed only pedv particles in the intestinal contents. for the pig-passaged pc21a strain, rt-pcr/pcr results were negative for transmissible gastroenteritis virus/porcine respiratory coronavirus (7), rotavirus groups a-c (8), caliciviruses (13, 14) , astroviruses (15) , circoviruses, enterovirus, kobuvirus, and bocavirus. for pigs 1 and 2, the detection of fecal virus shedding 24-48 h after inoculation coincided with the onset of clinical signs; for pigs 3 and 4, fecal shedding occurred before the onset of clinical signs (table 1) . by macroscopic examination, all infected pigs exhibited typical pedv-like lesions, characterized by thin and transparent intestinal walls (duodenum to colon) and accumulation of large amounts of yellowish fluid in the intestinal lumen (figure 2, panel a) . the stomach was filled with curdled milk, possibly due to reduced intestinal peristalsis. the other internal organs appeared normal. histologic lesions included acute diffuse, severe atrophic jejunitis (figure 2 , panel b) and mild vacuolation of superficial epithelial cells and subepithelial edema in cecum and colon ( figure 2 , panel c). these findings were similar to those in conventional pigs naturally infected with asian or us strains of pedv and in caesarean-derived, colostrum-deprived pigs experimentally infected with cv777 (2,3,5,6). the mean jejunal vh:cd of the 5 infected pigs ranged from 1.2 to 3.4, probably depending on the stage of infection (table 1) , and that of the negative control pig was 6.3 (±0.2). vh:cd for pig 4, which was euthanized at a later stage of infection, was 1.5 (±0.2), a ratio indicative of continued cellular necrosis. neither clinical signs nor lesions developed in the negative control pig during the experiment. immunofluorescence-stained cells were observed mainly in the epithelium of atrophied villi of small (duodenum to ileum) and large intestines (table 2; figure 2 , panels d-f), as reported in other studies (2, 3, 5) . the immunofluorescence was confined to the villous epithelial cells (figure 2 , panels d-f). a few immunofluorescence-stained cells were detected infrequently in the peyer patches of pig 4. lung tissues of the infected pigs did not show immunofluorescence staining, indicating that pedv does not infect figure 1 . electron micrograph of a us porcine epidemic diarrhea virus (pedv) particle detected in a field fecal sample collected during a 2013 outbreak of ped on a farm in ohio, usa; the fecal sample from which pedv strain pc21a in this study was obtained was from a pig on the same farm during the same outbreak. the sample was negatively stained with 3% phosphotungstic acid. scale bar = 50 nm. lung tissues under the conditions tested. although pc21a strain replicated in cecum and colon epithelial cells, cellular necrosis and villous atrophy were not evident. whether pedv infection of the large intestine contributes to the severity of ped is unclear. all infected pigs tested at acute or later stages of infection had viral rna titers of 4.8-7.6 log 10 ge/ml in serum samples (table 1) . these titers were similar to those for field samples tested by real-time rt-pcr; 11 (55%) of 20 acutephase serum samples collected from 13-to 20-week-old pigs with diarrhea from ohio had viral rna titers of 4.0-6.3 ge/ml. the early, severe diarrhea and vomiting and the pedv fecal shedding at high titers may be accompanied by viremia. no infected pigs had detectable viral rna in serum samples obtained before inoculation, and no negative control pig had detectable viral rna during the experiment. (4); -, no cells showed staining. ‡at 24 days of age, noninoculated pig 4 was exposed by indirect contact to pig 3 (at pih 0) through small holes drilled into the stainless steel divider panel located between the 2 pigs in the shared pig tub isolator unit. clinical signs and virus shedding were monitored after indirect contact. diarrhea and vomiting developed in pig 4 approximately 2-4 h after clinical signs developed in pig 3 (i.e., in pig 4, signs developed 46-50 h after indirect contact with inoculated pig 3); pig 4 was euthanized 120 h after the onset of clinical signs. in 2013, the first us outbreaks of the rapidly spreading porcine virus, pedv, caused a high number of pig deaths and substantial economic losses (1,2); however, little was known about progression of the disease. our data confirm that us pedv pc21a is highly enteropathogenic and acutely infects the entire intestine, but the jejunum and ileum are the primary sites of infection. pc21a infection causes severe atrophic enteritis accompanied by viremia that leads to severe diarrhea and vomiting. fighting a deadly pig disease: industry, veterinarians trying to contain ped virus, new to the us emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences the pathogenesis of an enteric infection in pigs, experimentally induced by the coronaviruslike agent, cv-777 pathology of experimental cv777 coronavirus enteritis in piglets. i. histological and histochemical study an immunohistochemical investigation of porcine epidemic diarrhoea in situ hybridization for the detection and localization of porcine epidemic diarrhea virus in the intestinal tissues from naturally infected piglets development of a reverse transcription-nested polymerase chain reaction assay for differential diagnosis of transmissible gastroenteritis virus and porcine respiratory coronavirus from feces and nasal swabs of infected pigs detection and genetic diversity of porcine group a rotaviruses in historic (2004) and recent (2011 and 2012) swine fecal samples in ohio: predominance of the g9p[13] genotype in nursing piglets the effects of simvastatin or interferon-alpha on infectivity of human norovirus using a gnotobiotic pig model for the study of antivirals multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus the effects of transplacental porcine circovirus type 2 infection on porcine epidemic diarrhoea virus-induced enteritis in preweaning piglets porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections prevalence and molecular characterization of porcine enteric caliciviruses and first detection of porcine kobuviruses in us swine characterization and prevalence of a new porcine calicivirus in swine, united states novel astroviruses in insectivorous bats we thank james e. collins and doug marthaler for kindly providing us pedv sequences for primer design and testing for enterovirus, kobuvirus, and bocavirus; j. hanson, g. meyers, and r. mccomick for assistance with animal care; x. wang, m. lee, s.s. wagner, a. veeramani, and chun-ming lin for technical assistance; andrea kaszas for assistance with electron microscopy; and v. anastasia for helpful discussion.salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development center, the ohio state university. this work was supported in part by a grant from the national pork board (no. 13-222 to q.w. and l.j.s.).dr jung is a veterinary pathologist at the ohio state university. his major research interests include diagnostic molecular pathology, pathogenesis, and immune responses to enteric viral infections, using germ-free animal models. key: cord-347475-ttmactz0 authors: mesquita, j. r.; hakze‐van der honing, r.; almeida, a.; lourenço, m.; van der poel, w. h. m.; nascimento, m. s. j. title: outbreak of porcine epidemic diarrhea virus in portugal, 2015 date: 2015-09-07 journal: transbound emerg dis doi: 10.1111/tbed.12409 sha: doc_id: 347475 cord_uid: ttmactz0 an outbreak of porcine epidemic diarrhea virus (pedv) in the south of portugal in january 2015 and the spread of pedv northwards in the territory are described. comparative analysis of the amplified sequences showed a very high (99.0%) identity with the pedv variant most recently reported in the united states and also show complete (100%) identity to the strains recently reported in germany, supporting the hypothesis that a unique strain is currently circulating in europe. the origin of this pedv variant still needs to be elucidated and further studies in the remaining european countries may contribute to the knowledge. porcine epidemic diarrhea (ped) is an acute and highly con-tagious enteric disease of pigs. typical clinical symptoms of ped include watery diarrhea, vomiting, dehydration, and porcine epidemic diarrhea (ped) is an acute and highly con-tagious enteric disease of pigs. typical clinical symptoms of ped include watery diarrhea, vomiting, dehydration, and porcine epidemic diarrhea (ped) is an acute and highly con-tagious enteric disease of pigs. typical clinical symptoms of ped include watery diarrhea, vomiting, dehydration, and porcine epidemic diarrhea virus (pedv; family coronaviridae, subfamily coronavirinae, genus alphacoronavirus) is a highly contagious virus responsible for enteric disease in swine characterized by an acute onset of symptoms including severe watery diarrhea, vomiting, dehydration, and high mortality in suckling piglets (ictv, 2012; song and park, 2012) . upon its first description in 1971 in the united kingdom, this disease was initially termed "epidemic viral diarrhea" due to the quick spread across europe (wood, 1977; song and park, 2012) . since then pedv has caused substantial economic losses, predominantly in asia, but in may 2013 a new pedv variant has emerged and rapidly spread in the us (stevenson et al., 2013) . from this time to early june 2014, pedv outbreaks had been reported in 33 states of the united states causing approximately 7 million piglet deaths, and resulting in severe economic losses to the swine industry (us department of agriculture, 2015). in early 2014, a novel variant of pedv (oh 851) was identified in ohio, containing insertions and deletions in the s gene (s indel), causing mild clinical signs and lower mortality rates in suckling piglets (wang et al., 2014) . more recently, an epidemic of severe watery diarrhea in southern germany with typical clinical signs of pedv was reported and found to be caused by a novel pedv, closely related to the us strain oh851, causing concern regarding on the possible circulation of a novel more virulent strain (hanke et al., 2015) . in the present work we describe an outbreak of severe watery diarrhea in swine caused by pedv in portugal, early 2015, and the spread northwards in the country. in january 2015 a pig farm in the south region of portugal reported diarrhea in all animals 2 days after introducing new animals from a different producer. diarrhea lasted for 1 week and a high mortality in piglets was observed. from january to april 2015 another 43 pig farms (4 farms from the south and 39 farms from the center of portugal) have reported similar epidemic diarrheas. a total of 84 fecal samples were collected from all 44 farms and submitted to analysis. stools were diluted (10% phosphate buffered saline), and viral nucleic acid was extracted from centrifuged stool suspensions using high pure rna isolation kit (roche applied sciences, mannheim, germany) and nucleospin rna virus (machery nagel, haerdt, france). nucleic acids were tested for the presence of pedv using a commercial real-time rt-pcr kit according to the manufacturer's instructions that targets the nucleocapsid protein gene (n gene) (viroreal â kit pedv; ingenetix, vienna, austria). a total of four samples positive for pedv rna by realtime rt-pcr (two from a farm in the south and two from a farm in the center of portugal) were further tested by conventional pcr using primers s1-univ-f (5 0 -tac tta caa ctc cac tg ttt -3 0 ) and s1-univ-r (5 0 -cca ttg ata gta gtg tca ga -3 0 ) that amplify a 440 bp region of the s protein. for the cdna synthesis, 0.2 ll of the s1univ-r primer (10 lm), 1 ll of dntp (10 mm), 6.8 ll of h 2 o and 5 ll of rna were incubated for 5 min at 65°c and placed on ice before being added 1 ll of superscript â ii reverse transcriptase (200 u/ll) (invitrogen), 1 ll of rnasin (40 u/ll) (promega), 1 ll 0.1 m dithiothreitol and 4 ll first strand buffer (59) (invitrogen). the rt mix was incubated for 60 min at 50°c. for the pcr, 2 ll of cdna were added to 2 ll of clontech buffer (109), 0.4 ll 509 dntp, 0.4 ll 509 polymerase mix, 1 ll of both s1-univ-f (10 lm) and s1-univ-r(10 lm) primers and 13.2 ll of h20. the pcr reactions were carried out under the following program: 5 min at 94°c followed by 40 cycles of 30 s at 94°c, 30 s at 54°c, 30 s at 68°c and a final extension of 10 min at 68°c. after electrophoresis appropriately sized bands (440 bp) were excised and purified using gel dna recovery kit (zymo reserch, ca, usa), and sequenced in both directions. sequence editing and multiple alignments were performed using bionumerics version 6.6 (applied maths, kortrijk, belgium). the pedv positive samples selected for genetic characterization were also screened for rotavirus group a (viroreal â rotavirus (a); ingenetix, vienna, austria) and transmissible gastroenteritis virus (tgev) (viroreal â tgev; ingenetix) accoding to the manufacturers instructions. in this study we describe an outbreak of severe watery diarrhea in swine in portugal, early 2015, with a rapid spread northwards in the territory. (pedv portugal 2015) . phylogenetic analysis was performed using mega version 6.0 software (tamura et al., 2013) . farms from the center. from the total 84 studied diarrheic stools, pedv was found in 55. four of these samples (two were from a farm in the south and two were from a farm in the center of portugal) were tested by conventional rt-pcr and amplified products (440 bp) were subjected to sequencing in order to obtain information about their genetic relatedness with pedv reference strains (fig. 1) . comparative analyses of these amplicons showed that all amplified sequences were identical (100%), showing that a single strain was responsible for this outbreak. analysis also showed that the amplified sequences share a very high (99.0%) identity with the new pedv variant oh851 (genbank accession no. kj399978) of the united states that affects sows (wang et al., 2014) . interestingly, the amplified sequences showed to be identical (100%) to the strains recently reported in germany pedv/ger/l00719/ 2014 (genbank accession no. lm645058) and pedv/ ger/l00721/2014 (genbank accession no. lm645057). the pedv positive samples selected for genetic characterization showed to be negative for rotavirus group a and tgev. in conclusion, pedv infection was confirmed in a pig herd in the south of portugal in january 2015 and found to be spreading northwards affecting a total of 32 farms. comparative analyses of a 440 bp region of the spike protein gene showed that the isolates were identical to the ones reported in 2015 in germany. the findings of an identical pedv strain in the south of europe, substantially distant from germany where the novel strains have been reported seem to indicate that a single strain (different from the american) is circulating in europe. as with germany, in portugal there is no active surveillance scheme for pedv, hence we cannot state with confidence that this strain has not been circulating in portugal for a longer time. also, the origin of this pedv variant still needs to be elucidated and further studies in the remaining european countries may contribute to the knowledge. the re-emergence of pedv in europe with altered virulence seems to be a relevant issue in swine health and may justify active surveillance by the official entities. comparison of porcine epidemic diarrhea viruses from germany and the united states virus taxonomy: classification and nomenclature of viruses; ninth report of the international committee on taxonomy of viruses porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines 2013: emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences mega6: molecular evolutionary genetics analysis version 6.0 federal order: reporting, herd monitoring and management of novel swine enteric coronavirus diseases new variant of porcine epidemic diarrhea virus an apparently new syndrome of porcine epidemic diarrhea the authors would like to acknowledge all pig farmers that participated in this study. this work was supported by european union funds (qren/feder) under the project ovislab ict-2013-05-004-5314 id-64757. key: cord-354729-dpaz01np authors: huan, changchao; pan, haochun; fu, siyao; xu, weiyin; gao, qingqing; wang, xiaobo; gao, song; chen, changhai; liu, xiufan title: characterization and evolution of the coronavirus porcine epidemic diarrhoea virus hljby isolated in china date: 2019-08-22 journal: transbound emerg dis doi: 10.1111/tbed.13321 sha: doc_id: 354729 cord_uid: dpaz01np a strain of porcine epidemic diarrhoea virus (pedv), namely hljby, was isolated in heilongjiang province, china. to provide insight into the understanding of the phylogenetic and the current epidemiological status of pedv, pedv hljby was compared with cv777 and other pedv strains deposited in the genbank. the homology between the entire genomic nucleotide sequences of pedv hljby and cv777 was 97.7%. the homology of m gene was the highest (99.0%). however, the homology of orf3 gene was 97.7%, and protein of orf3 was 90.1%. in addition, hljby showed the highest nucleotide identity (99.9%) with pedv‐sx/china/2017 strain and lowest similarity (91.2%) to pedv/belgorod/dom/2008 strain. we analysed the changes in s gene and its protein of pedv hljby with 65 historic pedv strains. the highest nucleotide identity was 99.9% compared with pedv‐sx/china/2017 strain, and the lowest nucleotide identity was 60.0% compared with pedv/belgorod/dom/2008 strain. the length of deduced amino acid sequences of s proteins varied from 1,372 to 1,390 amino acids (aa). compared with most aa sequences of s proteins, hljby exhibited 5 aa deletions (position 55, 59–61, 144). analysis and comparison of open reading frame 3 (orf3) proteins between hljby strain and other pedv strains were also focused in this study. we revealed that the length of deduced amino acid sequences of orf3 proteins was 80–224 aa among tested strains and the identity of hljby orf3 amino acids with other pedv strains was 71.4%–98.9%. orf3 protein of both hljby strain and pedv‐sx/china/2017 strain consists of 91 aa, with 133 aa deletions at their c' end in relation to the other tested pedv strains. the phylogenetic tree based on different proteins or genes resulted in different phylogenetic groups. for pathogenicity evaluation of pedv hljby strain, colostrum deprivation piglets were challenged with pedv hljby, and pedv reference strain cv777 as a control, the results showed that animals challenged with either of these pedv strains developed diarrhoea, and histopathological examination of small intestines of challenged animals showed acute viral enteritis with villous atrophy in either pedv hljby‐p10 or pedv cv777‐p8 inoculated piglets. cluding a positive-sense single-strand rna genome, which can cause a devastating enteric disease characterized with dehydration and watery diarrhoea (pensaert & de bouck, 1978) . pedv is the causative agent of porcine epidemic diarrhoea (ped), which has high mortality in suckling piglets (debouck & pensaert, 1980; pijpers, nieuwstadt, terpstra, & verheijden, 1993) . this disease was initially documented in the united kingdom in 1971. since 1990s, ped was not serious. however, outbreak of ped suddenly occurred in the united states, canada and mexico causing huge economic losses (mole, 2013; stevenson et al., 2013; vlasova et al., 2014) . in addition, ped caused tremendous economic losses to the swine industry in europe and asia, including china, korea and japan (kocherhans, bridgen, ackermann, & tobler, 2001; sun et al., 2012) . pedv is a nonsegmented and infectious rna virus. the genome of pedv is 27-33 kb in length containing a 5'cap and a 3'polyadenylated tail (pensaert & de bouck, 1978) . in addition, the genome includes seven open reading frames (orfs) encoding three nonstructural proteins (replicase 1a,1b and orf3) and four structural proteins (the spike (s), envelope (e), membrane (m) and nucleoprotein (n)). these proteins arrange in the order of 5'-replicase(1a/1b)-s-orf3-e-m-n-3' (kocherhans et al., 2001) . to reveal the characteristic and the diversity between pedv strains currently circulating in china and other pedv strains outside, the complete genomic sequence of pedv hljby strain was determined and analysed, and the pathogenicity of pedv hljby strain in newborn piglets was also evaluated. pedv hljby strain was isolated from the intestinal contents of a piglet with diarrhoea from heilongjiang province, china at 2011. vero cells were grown in dulbecco's modified eagle's medium (dmem, hyclone) supplemented with 8% foetal bovine serum (fbs, gibco) and were maintained in maintenance medium (dmem supplemented with 2% fbs) at 37 °c in a 5% co 2 incubator. strain hljby was passaged ten times in vero cells. pedv n-specific antibody used in immunofluorescence assay (ifa) was gifted by professor xiang mao. pedv reference strain cv777 was purchased from china institute of veterinary drug control. when pedv-infected vero cells showed 70%-80% cytopathic effect (cpe), cell culture flasks were frozen and thawed three times, and cell debris was pelleted by centrifugation for 30 min at 12,000 rpm. culture supernatants were collected and used for preparation of viral rna. total rna was extracted using trizol reagent (vazyme biotech) according to the manufacturer's instructions. total rna was used for synthesis of cdna with hiscript reverse transcriptase (hiscript ii 1st strand cdna synthesis kit; vazyme biotech) according to the manufacturer's instructions. specific primers for pedv were designed based on pedv-cv777 genome (table 1) . pcr was performed to amplify for the six overlapping dna fragments using lamp dna polymerase (vazyme biotech). the expected bands in agarose electrophoresis of pcr products were excised, and a genclean column gel extraction kit (generay biotech) was used to purify the synthesized dna, and the dna products were cloned into pjet1.2 vector (thermo). the positive clone was sequenced by biotechnology co, ltd. the validated genome sequence of pedv hljby strain was submitted to genbank and acquired accession number: kp403802.1. sequence data was employed to assemble and analyse by dnastar software package (dnastar inc. genes of pedv strains were used for sequence alignments and phylogenetic analyses. nucleotide sequences of full-genomes, orf3 genes and s genes of pedv strains were aligned using the clustalx 2.0 program (thompson, gibson, plewniak, jeanmougin, & higgins, 1997 ). vero cells grown on coverslips were infected with 0. fifteen newborn piglets without colostrum were purchased from one pig farm and were free of pedv, transmissible gastroenteritis virus, porcine deltacoronavirus, and porcine rotavirus. the piglets were divided randomly into three groups: the sham-inoculated control group (n = 5), cv777-p8-inoculated group (n = 5), and hljby-p10-inoculated group (n = 5). the piglets were fed commercial milk replacer (8 times daily). the piglets in the challenge groups received an oral 1 ml dose of 10 7.0 tcid 50 /ml of pedv cv777 or hljby. the sham-inoculated pigs were given dmem medium (1 ml) orally. all animals were monitored for mortality and signs of vomiting and/or diarrhoea (observed and recorded for every 3 hr during whole experiment). all piglets were euthanized at 7 d post-challenge and checked for macroscopic and microscopic lesions. to reveal the characteristics of pedv hljby strain and determine more precisely the relationship among the pedv strains currently circulating in china and those from other nations, the full-length genome sequence of strain hljby was deduced by combining the sequences of several overlapping cdna fragments. the genome sequence of strain hljby was 27,953 nucleotides (nt) in length, excluding the 3' poly(a) tail. the genomic organization was typical of previously sequenced pedv strains and was summarized as 5'utr-orf1a/1b-s-orf3-e-m-n-3'utr ( figure 1 ). of note, compared with classical pedv cv777, the genome of hljby contains four deleted nucleotides or regions including 72 nt, 89-93 nt, 3403-3426 nt, 21092-21094 nt, respectively. the four deleted nucleotides or regions were located at 5'utr, orf1a/1b, s, orf3, respectively. to investigate the molecular characteristics of pedv hljby strain, utr (5' and 3') and the nucleotide and predicted amino acid sequences of the nonstructural and structural proteins (replicase orf1a/1b, s, orf3, e, m, n) of pedv hljby strain were compared with cv777. as shown in figure 2a and table 3 , the nucleotide of 5'utr of pedv hljby had 5 nt deletions and 1 nt insertion and the identity is 96.0% compared with cv777. 24 nt deletions were found in orf1a/1b of pedv hljby (figure 2b ), and the nucleotide sequence identity was 98.0%, and the amino acid sequence identity was 98.0% (table 3) . 3 nt deletions showed in s of pedv hljby ( figure 2c ), and the nucleotide sequence identity was 97.0%, and the amino acid sequence identity was 96.0% (table 3) . 399 nt deletions exhibited in orf3 of pedv hljby (figure 2d ), and nucleotide sequence identity was 91.7%, and the amino acid sequence identity was 90.1% (table 3 ). in protein e, the nucleotide sequence identity was 97.0%, and the amino acid sequence identity was 97.0% (table 3) , resulting in amino acid changes in e (11val to ala and 76 val to ile) ( figure 2e ). in protein m, the nucleotide sequence identity was 99.0% and the amino acid sequence identity was 98.0% (table 3) , resulting in 4 amino acid changes in m ( figure 2f ). in protein n, the nucleotide sequence identity was 98.0% and the amino acid sequence identity was 98.0% (table 3) , resulting in 9 amino acid changes in n ( figure 2g ). the nucleotide sequence identity of 3'utr was 97.0% (table 3) . the complete genome sequences of pedv strains from different locations and years were compared, and the results revealed that hljby had a nucleotide identity of 99.9%-91.2% with other entire pedv genomes available in genbank ( the orf3 protein (an accessory protein) was located between s and to investigate the evolution of pedv, phylogenetic analysis based on the entire genomic nucleotide sequences of pedv hljby strain the group ⅲ was further divided into subgroup ⅲa and ⅲb. the the group ⅱ included the other pedv strains (figure 7) . to in recent years, pedv has re-emerged as one of the deadliest and most contagious pathogens in swine, causing large economic (li et al., 2012) . therefore, the relationship of (wang et al., 2012) . orf3 may contribute to the virulence of pedv. the orf3 was a key gene for pedv culture in vitro. orf3 gene was usually used to differentiate between field and vaccine-derived isolates and altered the virulence of pedv (park et al., 2008) . in addition, the pedv s protein is an important viral gene for studying genetic relatedness among isolates and epidemiology of pedv (chen et al., 2014; gerber et al., 2014; lee, 2015; lee et al., 2010; oh et al., 2014) . nucleotide sequencing of s gene analysis revealed that 3 nt deletions were found in s gene of pedv hljby compared with cv777 ( figure 2c ) and the nucleotide sequence identity is 97.0%. the amino acid sequence identity of s protein was 96.0% between pedv hljby and cv777. 3 nt deletions of s gene were located in the amino terminus of s gene, which was suitable to analyse the significant differences in epidemiology of pedv (li et al., 2012; temeeyasen et al., 2014; vlasova et al., 2014) . s gene functions as a receptor-binding domain (belouzard, millet, licitra, & whittaker, 2012) . and epidemiology of pedv. our study will provide more information about diversity, evolution, and in particular, the epidemic characin the phylogenetic tree of the whole genome, s aa and orf3 aa, the phylogenetic tree based on both the whole genome and s aa was similar, but the phylogenetic tree of orf3 aa was different from the phylogenetic trees for the s protein and genomic nucleotide sequences. all pedv strains were divided into three groups based on the phylogenetic tree of the whole genome and s protein. in the phylogenetic tree of the whole genome and s protein, pedv/belgorod/ dom/2008 strain located at group ⅰ, and pedv hljby belonged to the group ⅱ. in the phylogenetic tree of orf3 protein, the group ⅰ only harboured pedv/belgorod/dom/2008 and hljby. the phylogenetic tree based on different proteins implied the different evolution events. this will provide further information for virus evolution. the phylogenetic trees suggested that pedv hljby might originate from a genetic recombination event between pandemic strains and classical strains. we need to explore the mechanism and/or cause of the different phylogenetic trees based on different proteins or genes. this will lay a foundation for the recombination and evolution of pedv. biological characteristics of pedv hljby including the cytopathic effect, and pathogenicity, indicated that pedv hljby induced an apparent and classical cytopathic effect (cpe) in vero cells, and caused acute viral enteritis with villous atrophy in small intestine of challenged piglets. but all hljby-p10 or cv777-p8 inoculated piglets had no deaths during the challenge experiment. these results suggested that pedv hljby might have similar pathogenicity in experimentally infected piglets compared with that of pedv reference strain cv777. in summary, we found that the evolutionary trees are different based on different proteins or genes of pedv strains. this phenomenon needs further study. pathogenicity evaluation of pedv hljby indicated that pedv hljby potentially possessed equal pathogenicity compared with that of pedv reference strain cv777. this study might provide a reference for the evolutionary and variation of pedv. we thank james allen, phd, for editing the english text of a draft of this manuscript. this work was supported by the natural the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. all experimental protocols were approved by the animal care and mechanisms of coronavirus cell entry mediated by the viral spike protein prevalence of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus infection in korean pigs. the veterinary record isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states experimental infection of pigs with a new porcine enteric coronavirus, cv 777 detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence porcine epidemic diarrhea virus: an emerging and reemerging epizootic swine virus heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea new variants of porcine epidemic diarrhea virus role of transportation in spread of porcine epidemic diarrhea virus infection, united states deadly pig virus slips through us borders immunogenicity and protective efficacy of recombinant s1 domain of the porcine epidemic diarrhea virus spike protein impact of porcine epidemic diarrhea virus infection at different periods of pregnancy on subsequent reproductive performance in gilts and sows cloning and further sequence analysis of the orf3 gene of wild-and attenuated-type porcine epidemic diarrhea viruses a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs. the veterinary record emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences outbreak of porcine epidemic diarrhea in suckling piglets genetic diversity of orf3 and spike genes of porcine epidemic diarrhea virus in thailand the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools distinct characteristics and complex evolution of pedv strains pedv orf3 encodes an ion channel protein and regulates virus production characterization and evolution of the coronavirus porcine epidemic diarrhoea virus hljby isolated in china key: cord-344309-6c2wttxg authors: lin, huixing; zhou, hong; gao, lu; li, bin; he, kongwang; fan, hongjie title: development and application of an indirect elisa for the detection of antibodies to porcine epidemic diarrhea virus based on a recombinant spike protein date: 2018-08-20 journal: bmc vet res doi: 10.1186/s12917-018-1570-5 sha: doc_id: 344309 cord_uid: 6c2wttxg background: as the major causative agent of swine viral diarrhea, porcine epidemic diarrhea virus (pedv) has caused massive losses to the economies of swine raising countries. accordingly, the serological detection of corresponding antibodies would be beneficial to diagnose pedv indirectly to control the disease. in this study, an indirect enzyme-linked immunosorbent assay (elisa) based on the recombinant truncated spike (s) protein of pedv was developed and validated. results: the reaction conditions of the developed indirect elisa were optimized. this indirect elisa was compared to indirect immunoinfluscent assay (ifa), and the overall coincidence rate was 96.74% based on testing 368 clinical serum samples with different pedv antibody levels. no cross-reactivity with other common swine pathogens was detected for the developed s1 indirect elisa. finally, the s1 indirect elisa was applied to detect serum antibodies of 3304 field samples collected from different pig farms in eastern china, and it presented an overall substantial agreement on the pedv infection status. conclusions: this established s1 indirect elisa is capable of detecting serum antibodies against pedv, and due to its high sensitivity and specificity, it could be applied for serological evaluation and indirect diagnosis of pedv infection. porcine epidemic diarrhea (ped) is a highly contagious swine enteritis caused by porcine epidemic diarrhea virus (pedv), which belongs to the order nidovirales and family coronaviridae. the typical symptoms of ped are diarrhea, vomiting, and dehydration, which can be especially dangerous to suckling piglets [1, 2] . the mortality of neonatal piglets younger than 5 days old can approach 100% [3] [4] [5] . ped first appeared in britain in 1971, followed by an outbreak of diarrhea in several pig farms in belgium in 1977 [6] . these outbreaks led to identification of a coronavirus-like particle named cv777, which is now recognized as the classic pedv strain. in recent years, ped epidemics have become prevalent in swine-raising countries in asia, including south korea, china, japan, and vietnam, and can cause enormous economic loss [7, 8] . pedv is a single-stranded rna virus composed primarily of four structural proteins: the spike protein (s, 180-220 kda), membrane protein (m, 27-32 kda), envelope protein (e, 7 kda) and nucleocapsid protein (n, 55-58 kda). s protein is located on the surface of the virus particle. it is categorized as a type i membrane fusion protein and has the significant biological effect of binding to target cell receptors and entering the cell through plasma membrane fusion [9, 10] . the s protein has higher antigenicity than any of the other pedv proteins, and anti-s antibodies detected in pedv-infected pigs persist longer than anti-n antibodies [11] . the s protein can be separated into the s1 (1-789 aa) subunit and the s2 (790-1383 aa) subunit [12] . the s1 subunit is the extracellular domain and can recognize and bind to target cell receptors [13] , and it is closely linked to the formation of neutralizing antibodies. therefore, this study selected a gene fragment within the s1 subunit as a coating antigen to develop an indirect enzyme-linked immunosorbent assay (elisa) method for the detection of pedv antibodies. the pedv yc2014 strain was isolated on a breeding farm in yancheng city in 2014 (genbank: ku252649.1). the prokaryotic expression vector pet-28a(+) was purchased from biovector ntcc inc. (beijing, china). the hrp-goat anti-pig iga, hrp-goat anti-pig igg, and fitc-goat anti-pig iga was purchased from abcam plc. (shanghai, china). the standard pedv negative serum were collected from specific pathogen free (spf) pigs. the standard pedv positive serum were collected from experimentally pedv immunized spf pigs, at 7, 14, 21, 28, 35, 42 and 49 day post-inoculation (dpi). these standard serum were identified of pedv-specific antibodies positive by both indirect immunoinfluscent assay (ifa) and seroneutralization assay (sn) as previously described [14, 15] . the swine porv antibody elisa kit and the swine tgev elisa kit were obtained from ingenasa (madrid, spain). the gene sequence of truncated spike protein (named s1) was amplified from the genomic rna of pedv yc2014 strain (genbank: ku252649.1) by reverse-transcriptase (rt)-pcr. the forward primer was 5' cgcggat ccgtcactaggtgccagtccactattaa-3'and the reverse primer was 5'-cccaagctttcaattgtaaa tatccactttaagaaaacaataa-3′. underlined portions represent bamh i and hind iii restriction sites, respectively. the target gene was 1068 bp in length and subcloned into the prokaryotic expression vector pet-28a(+), then transformed into a strain of competent e. coli cells, dh5α. transformed colonies were selected from luria-bertani (lb) agar plates containing kanamycin (50 μg/ml) and were identified by pcr. the resulting recombinant expression plasmid was named 28a-s1 and was identified by double enzyme digestion and dna sequence analysis. subsequently, the recombinant expression plasmid 28a-s1 was transformed into e. coli bl21 (de3). the positive transformants were cultured in lb medium containing 50 μg/ml kanamycin with vigorous shaking at 37°c until the 600 nm optical density (od 600 ) of bacteria cultures reached approximately 0.5. then, by adding isopropyl-β-d-thiogalactopyranoside (iptg), the recombinant protein s1 was induced for 6-8 h at 37°c. the expression of recombinant proteins was analyzed by 12% (v/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and the gels were stained with coomassie brilliant blue. induced cells were pelleted and washed in phosphate-buffered saline (pbs) three times, then lysed by sonication in an ice-water bath. the lysed cells were centrifuged at 12,000 g for 10 min, then the precipitate (inclusion body) was dissolved with binding buffer containing 8 m urea. the supernatant and the precipitate were then subjected to sds-page analysis. the recombinant protein was purified through affinity chromatography using a ni-nta spin column following the manufacturer's recommendations. purified recombinant protein s1 was subjected to 12% (v/v) sds-page, and the gel was prepared for western blotting as follows. recombinant proteins separated in the gel were electrically transferred to a nitrocellulose membrane and the membrane was blocked overnight at 4°c with tris-buffered saline containing tween-20 (tbst) which contained 5% (w/v) skimmed milk powder. the composition of tbst is as follows: 20 mm tris-hcl, 150 mm nacl, and 0.05% tween-20. the membrane was then incubated with pig anti-pedv polyclonal antibody (1:300 dilution in blocking buffer) for 1 h at 37°c on a plate shaker. following this incubation, the membrane was washed three times with tbst buffer and reacted with hrp-goat anti-pig igg (1:2000 dilution in tbst) at 37°c for 45 min. after three washes, the final color reaction was developed with a solution of 3,3′-diaminobenzidine (dab). conventional indirect elisa was performed with the following steps. elisa plates with 96 wells (costar, usa) were coated with 100 μl purified recombinant s1 protein in bicarbonate buffer (ph = 9.6) for 2 h at 37°c. then, plates were washed three times with pbst (pbs containing 0.05% tween-20) and blocked with 5% skimmed milk in pbs for 2 h at 37°c. after plates were washed, 100 μl porcine serum samples diluted in pbs containing 5% (w/v) skimmed milk was added and incubated for 45 min at 37°c. plates were washed four times and reacted with 100 μl diluted secondary antibody (hrp-goat anti-pig iga or hrp-goat anti-pig igg) for 30 min at 37°c for the purpose of detecting iga or igg against pedv in serum samples. plates were then washed four times and 100 μl tetramethylbenzidine (tmb) substrate solution was added to each well for a chromogenic reaction at room temperature for 15 min in complete darkness. the color reaction was stopped by the addition 50 μl of 2 m h 2 so 4 to each well. finally, the od 450 was measured and recorded immediately using an infinite 200 pro microplatereader (tecan, männedorf, switzerland). the optimal dilution of recombinant protein s1 and standard serum was determined by a checkerboard titration based on the method mentioned above. briefly, the concentration of s1 protein was gradually reduced in the following series: 10, 7.5, 5, 2.5, 1.0, and 0.5 μg/ml. the standard pedv positive and negative serum were serially diluted in a 2-fold series from 1:20 to 1:320. when the od 450 ratio of positive serum to negative serum was highest, and the od 450 of positive serum was closest to 1.0, the corresponding dilutions of coated antigen and serum sample were considered optimal. in addition to optimal protein dilution, the coating conditions, blocking solution, and reaction time of various materials was explored. furthermore, the optimal concentration of hrp-goat anti-pig iga was tested using the following dilutions: 1:2000, 1:5000, 1:10000, 1:15000, and 1:20000. two hundred and seventy serum samples were collected for the purpose of determining the positive-negative cut-off value, of which 90 serum samples were collected from 90 spf pigs, 180 serum samples were collected from grow-finish pigs from five farms between 2009 and 2012. these five farms were located in areas with no previous history of enteric signs compatible with viral diarrhea, and were pedv rna negative by real-time rt-pcr based on a single collection. these serum samples were confirmed pedv-negative by both ifa and sn assays as previously described [14, 15] , and then were used to define the cut-off value in the s1 indirect elisa. the od 450 value of these pedv-negative serum samples obtained in this s1 indirect elisa were recorded to calculate the cut-off value. the mean od 450 value of these negative samples (n) + 3 × standard deviations (sd) was defined as the cut-off value. serum samples showing od 450 value greater than or equal to this cut-off were considered pedv-seropositive. to assess the accuracy of this developed s1 indirect elisa, 368 serum samples from different pig farms were tested using this elisa method. as a comparison, ifa was applied to test these samples and act as a reference method to distinguish positive or negative samples. briefly, vero cells grown on 96-well plates were infected with the pedv yc2014 strain at multiplicity of infection (m. o. i) of 5. at 48 h post-infection, cells were washed three times with pbst and fixed with cold methanol for 10 min at − 20°c. cells were then washed three times with pbst and blocked with 10% bovine serum albumin (bsa) at 37°c for 1 h. after been double diluted for six consecutive dilutions in dilution buffer (1% bsa in pbst), the 368 serum samples with varied pedv antibody status were added in the wells of 96-well plate, and were incubated for 1 h at 37°c. after three washes with pbst, cells were treated with a fitc-conjugated goat anti-pig iga (thermo scientific) at a 1:500 dilution with pbs for 30 min at 37°c. after a final four washes with pbst, all wells were examined using fluorescence microscopy (axio observer z1, zeiss, germany). the pedv antibody titers of the serum samples were expressed as the highest dilution of serum samples producing green fluorescent in the wells of 96-well plates. the results of this two methods were compared, and the sensitivity and specificity of detection were calculated to evaluate the accuracy of the s1 indirect elisa. sensitivity was defined as the ratio of positive tests from the developed s1 indirect elisa to the positive tests from the ifa. specificity was defined as the ratio of negative tests from the developed elisa to the negative tests from the reference ifa. serum cross-reactivity of s1 indirect elisa to other pathogens to validate the cross-reactivity, this s1 indirect elisa was utilized to test porcine serum positive for other swine pathogens, namely, porcine transmissible gastroenteritis virus (tgev), swine rotavirus (porv), porcine kobuvirus (pkv), porcine bocavirus (pbov), porcine norovirus (pnov), porcine circovirus type 2 (pcv2), porcine reproductive and respiratory syndrome virus (prrsv), and enterotoxigenic e. coli (etec), jerson prand of the small intestine, clostridium welchii type c. the positive sera were prepared by our lab, by immunizing the specific pathogen free (spf) piglets with purified virus or bacteria. thirty positive serum samples for each virus were tested, and each sample was repeated in triplicate. to test the repeatability of this elisa, 255 serum samples with different pedv antibody levels were chosen. for inter-assay variability, each sample was tested in 5 replicates on plates of different occasions. for intra-assay variability, each sample was tested in 5 replicates on plates within the same occasion. the results were presented as the coefficient of variation (cv), which is the ratio of the standard deviation (sd) to the mean od 450 value of each group of samples (s). a cv value criterion of 10% was used to meet the repeatability requirement of the test. this s1 indirect elisa was applied to seroepidemiological analysis of a total of 3304 clinical swine serum samples collected from thirty seven farms in eastern china. the pedv infection status of a given farm was determined based on demonstration of pedv rna in fecal samples by real-time rt-pcr and presence of enteric signs [16] . one thousand one hundred and twenty five serum samples were collected from nursery and grow-finish pigs from ten farms between 2011 and 2015 at 3-6 weeks after the start of pedv outbreaks in these farms. four hundred and eighty two serum samples were collected from nursery and grow-finish pigs from five farms between 2012 and 2015. these five farms were located in areas with no previous history of enteric signs compatible with viral diarrhea, were pedv rna negative by real-time rt-pcr based on a single collection, and were considered non-exposed to pedv. three hundred and forty four serum samples from nursery pigs without pedv exposure were collected from six farms between 2011 and 2015. these samples were confirmed to be positive for anti-porv antibodies (3 farms, n = 149) or anti-tgev antibodies (3 farms, n = 195) by both ifa and commercial elisa kits (obtained from ingenasa). one thousand three hundred and fifty three porcine serum samples with unknown pedv exposure status were randomly selected from sixteen farms from nursery and grow-finish pigs between 2011 and 2014. the gene of pedv truncated s1 fragment (67-1134 nt) was amplified by rt-pcr (fig. 1a) . a 1068 bp pcr product was obtained and subcloned to prokaryotic fig. 1 amplification, sds-page and western blotting analysis of the recombinant protein s1. a rt-pcr amplification of the truncated s1 gene fragment. lane m, dl2000 dna marker. lane 1 and 2, the truncated s1 gene fragment. b identifiction of the recombinant expression plasmid 28a-s1 by double enzyme digestion. lane m, dl5000 dna marker. lane 1, the recombinant expression plasmid 28a-s1 digested by bamh i/sal i. c sds-page analysis of s1 protein. lane m, prestained protein molecular weight standard. lane 1, transformed cells of bl21/pet-28a(+) after iptg induction for 6 h. lane 2, transformed cells of bl21/28a-s1 after iptg induction for 6 h. lane 3, purified recombinant protein s1 by affinity chromatography of ni-nta spin column. d western blotting analysis of s1 protein. lane m, prestained protein molecular weight standard. lane 1, e. coli bl21 with empty vector pet-28a(+) reacted with polyclonal mouse anti-pedv antibody. lane 2, purified s1 protein reacted with polyclonal mouse anti-pedv antibody. a prominent band with the expected size 42 kda appeared after incubation expression vector pet-28a(+), and the inserted gene was sequenced to ensure the correctness of the reading frame. as shown by sds-page (fig. 1c) , the recombinant protein s1 was expressed in the form of inclusion body, resulting in a 6 × his-tag fusion protein whose molecular mass was approximately 42 kda. sonicated lysates from recombinant e. coli were harvested, and the precipitate was dissolved in 8 m urea and purified by affinity chromatography of ni 2+ -nta agarose. the immunoreactivity of s1 protein was examined by western blotting. an obvious band revealed that s1 protein was specifically bound by pig anti-pedv polyclonal antibody (fig. 1d) . as expected with checkerboard titration, with concentrations of antigen and serum regularly decreasing, absorbance values of corresponding samples declined. the optimal dilution of coated antigen s1 protein was measured at 0.25 μg/well (2.5 μg/ml), and optimal serum sample dilution was 1:40 (table 1) . furthermore, other reaction conditions of the developed elisa were optimized. in brief, the optimum coating condition was 2 h at 37°c. the best blocking solution was selected as 5% skimmed milk in pbs. the optimal reaction times for serum, secondary antibodies, and tmb solution were 45 min, 30 min and 15 min, respectively. finally, the best working dilution of the hrp-goat anti-pig iga was 1:10,000. to determine the cut-off value of the s1 indirect elisa, 270 pedv-seronegative samples, verified by both ifa and sn assays, were tested by this elisa method. the average optical density of these negative serum samples (n) was calculated as 0.185, and the standard deviation (sd) of these samples was 0.0337. consequently, the cut-off threshold value of s1 indirect elisa was calculated to be 0.286, indicating that the sample od 450 ≥ 0.286 was identified as pedv-seropositive and vice versa. this developed s1 indirect elisa was applied to 368 serum samples with varied pedv antibody status ( table 2 ). in these samples, the s1 indirect elisa detected 213 pedv-positive samples, of which 206 tested pedv-positive by ifa. on the other hand, of the remaining 155 samples that tested pedv-seronegative by this s1 indirect elisa, 150 of them were tested pedv-negative by ifa. hence, the sensitivity of s1 indirect elisa was 96.71% among pedv-seropositive individuals, and the specificity was 96.77% among pedv-seronegative individuals using ifa as standard evaluation method. in summary, the overall coincidence rate of the s1 indirect elisa to ifa was 96.74%. to test the cross-reactivity of this s1 indirect elisa, other viruses known to cause swine diarrhea were examined. the average od 450 of positive serum samples for tgev, porv, pkv, pbov, pnov, pcv2, prrsv, etec, jerson prand of the small intestine, and clostridium welchii type c were 0.193, 0.121, 0.098, 0.147, 0.150, 0.182, 0.178, 0.188, 0.145 and 0.124, respectively. the results showed that these serum samples were pedv-seronegative and non-cross-reactive with this s1 indirect elisa, indicating that the established elisa was an effective method for detecting pedv antibodies. the repeatability of s1 indirect elisa intra-assay variability of the s1 indirect elisa was assessed by testing 255 swine serum samples, each with 5 replicates. this analysis produced cvs ranging from 2.2-3.7%, with an average value of 2.8%. inter-assay variability of four batches using identical samples produced cvs ranging from 2.6-4.5%, with an average value of 3.2%. the results demonstrated that this elisa method yielded low levels of variation, and its repeatability was in the credible range. this s1 indirect elisa method was used on 3304 swine serum samples of different pedv exposure status collected from 37 farms (table 3 this s1 indirect elisa was applied to test the sera of pedv immunized pigs at 7, 14, 21, 28, 35, 42 and 49 day post-inoculation (dpi). the results (fig. 2) showed that, both the iga and the igg were positive at 7 dpi. at early infection stage (7 dpi), the iga titer was significantly higher than the igg titer; at 14 dpi the iga titer was equivalent to the igg titer; and after 21 dpi, the iga titer was significantly lower than the igg titer. the igg could exist in the sera of infection recovered stage for more time than iga. since december 2010, a large-scale outbreak of diarrhea has been observed in swine farms in china. accumulated evidence indicates that this large-scale outbreak of diarrhea were caused by highly virulent pedv variants [17, 18] . serological assays can quickly detect large numbers of samples with both high sensitivity and specificity. several indirect elisa have been developed based on either whole pedv preparations or recombinant viral proteins [19] [20] [21] . the s protein of pedv has numerous epitopes and highly antigenic index regions that induce the production of neutralizing antibodies [10, 22, 23] , and anti-s antibodies detected in pedv-infected pigs persist longer than anti-n antibodies [11] , thus, we choose s protein as the diagnostic antigen. the recombinant s1 protein was applied to establish an indirect elisa, and its reaction conditions were optimized. as pedv is an enteric virus, it directly infects and damages enterocytes. mucosal iga, but not systemic igg, plays a crucial role in protection [24, 25] . in this research, the titers of iga in the serum were tested for serological evaluation and indirect diagnosis of pedv infection. of the 1125 serum samples which were pedv exposed, the overall positive rate of the antibody is 91.29%, which were varied from 78 to 100% between 10 farms. of the 482 serum samples which were pedv non-exposed, the overall positive rate of the antibody is 6.43%, which were varied from 5.1 to 7.8% between 5 farms. the evaluated elisa presented an overall substantial agreement on the pedv infection status of the field swine serum samples. the intra-and inter-assay variability tests proved that this elisa method had good repeatability. when testing other positive serum related to swine viral pathogens, this established elisa demonstrated no cross-reactivity to them. further, the overall rate of coincidence of this elisa was calculated at 96.74% compared with ifa, proving that the diagnostic sensitivity and specificity of this elisa method were favorable. of the numerous pathogens which can cause swine viral diarrhea, pedv, tgev, and porv account for the largest proportion [26] . in the present study, 16/149 anti-porv antibody positive sample and 27/195 anti-tgev antibody positive samples were tested pedv antibody positive, which indicated there may be co-infection of pedv and other virus. in conclusion, this established s1 indirect elisa is capable of detecting serum antibodies against pedv, and due to its high sensitivity and specificity, it could be applied for serological evaluation and indirect diagnosis of pedv infection. fig. 2 determination of iga and igg in the sera of pedv immunized pigs. the sera of pedv immunized pigs were tested by this s1 indirect elisa at 7, 14, 21, 28, 35, 42 and 49 day post-inoculation (dpi). both the iga and the igg were positive at 7 dpi. the igg could exist in the sera for longer time than iga. different letters (a, and b) indicate significant difference between the groups porcine epidemic diarrhea in europe: in-detail analyses of disease dynamics and molecular epidemiology nursery pig growth performance and tissue accretion modulation due to porcine epidemic diarrhea virus or porcine deltacoronavirus challenge in situ hybridization for the detection and localization of porcine epidemic diarrhea virus in the intestinal tissues from naturally infected piglets poly (d,l-lactide-coglycolide) nanoparticle-entrapped vaccine induces a protective immune response against porcine epidemic diarrhea virus infection in piglets cross protective immune responses in nursing piglets infected with a us spike-insertion deletion porcine epidemic diarrhea virus strain and challenged with an original us pedv strain an apparently new syndrome of porcine epidemic diarrhoea isolation of porcine epidemic diarrhea virus during outbreaks in south korea heterogeneity in membrane protein genes of porcine epidemic diarrhea viruses isolated in china structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus s2 fusion protein identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus an elisa for detection of antibodies against porcine epidemic diarrhoea virus (pedv) based on the specific solubility of the viral surface glycoprotein spike protein region (aa 636789) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus strains comparison of serum neutralization and enzyme-linked immunosorbent assay on sera from porcine epidemic diarrhea virus vaccinated pigs. the veterinary quarterly epidemic strain yc2014 of porcine epidemic diarrhea virus could provide piglets against homologous challenge multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus new variants of porcine epidemic diarrhea virus, china sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection development and application of an elisa for the detection of porcine deltacoronavirus igg antibodies detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa phage-displayed peptides having antigenic similarities with porcine epidemic diarrhea virus (pedv) neutralizing epitopes identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis does circulating antibody play a role in the protection of piglets against porcine epidemic diarrhea virus? antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains the data analyzed during the current study are available from the corresponding author on reasonable request. authors' contributions hl, kh and hf designed the study. hl, hz, lg and bl performed and collected data from experiment and analyzed data. hl, hz wrote the manuscript. all authors read and approved the final 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. all experimental protocols were approved by the institutional animal care and use committee of nanjing agricultural university (no. syxk2015-0057) and performed accordingly. samples were collected only from animals for laboratory analyses, avoiding unnecessary pain and suffering of the animals. the owners gave their written consent for sample collection, and the locations where we sampled are not privately owned or protected in any way. the studies did not involve endangered or protected species. not applicable. the authors declare that they have no competing interests. key: cord-346457-2mq2aije authors: wang, zhilin; li, xuerui; shang, youjun; wu, jinyan; dong, zhen; cao, xiaoan; liu, yongsheng; lan, xi title: rapid differentiation of pedv wild-type strains and classical attenuated vaccine strains by fluorescent probe-based reverse transcription recombinase polymerase amplification assay date: 2020-06-22 journal: bmc vet res doi: 10.1186/s12917-020-02424-1 sha: doc_id: 346457 cord_uid: 2mq2aije background: porcine epidemic diarrhea virus (pedv), an intestinal coronavirus that causes acute diarrhea and high mortality in suckling piglets, can result in high economic losses in the swine industry. in recent years, despite the use of china’s current vaccine immunization strategy, multiple types of pedv strains were still found in immunized swine herds. our research aims to explore a new rapid differentiation method to distinguish the different types of pedv strains and assess the safety evaluation of classical attenuated vaccine strains in swine herds. results: in the study, a differential one-step quantitative real-time fluorescent reverse transcription recombinase polymerase amplification (real-time rt-rpa) method based on the pedv universal real-time rt-rpa assay was established according to the orf1 deletion sequences of three classical attenuated vaccine strains (pedv attenuated vaccine kc189944, attenuated cv777 and dr13) and five vero cell-adapted isolates (js2008, sdm, sq2014, sc1402, hljby), which could effectively differentiate pedv classical attenuated vaccine strains from wild-type strains (pedv classical wild strains and variant strains). the detection limits of pedv rna in the both pedv real-time rt-rpa assays were 300 copies within 20 min at 39 °c, and the detection limits of classical attenuated vaccine strain cv777, vero-cell-adapted isolate js2008, and pedv wild-type strain dx were 10(0.5) tcid(50)/100 μl, 10(1.1) tcid(50)/100 μl, and 10(1.2) tcid(50)/100 μl, respectively. both assays were highly specific for pedv, showing no cross-reactivity with other enteral viruses. conclusion: this rpa method we developed is simple, time-effective, and safe and provides a reliable technical tool for the differential diagnosis and clinical epidemic surveillance of pedv classical attenuated vaccine strains and wild-type strains. porcine epidemic diarrhea virus (pedv) is a serious pathogen which is characterized by severe diarrhea, vomiting, and dehydration in pigs [1, 2] . pedv is an enveloped, single-stranded, positive-sense rna virus belonging to the alpha coronavirus genus of the coronaviridae family [1] . the entire genome sequence of pedv is approximately 28 kb, which consists of seven open reading frames, encoding four structural proteins [spike protein (s, 150-220 kda), membrane protein (m, 20-30 kda), envelope protein (e, 7 kda), and nucleocapsid protein (n, 58 kda)], and three non-structural proteins (replicases 1a and 1b and orf3) [3] [4] [5] . ped was first reported in the united kingdom in 1971 [6] . in 1978, pedv was isolated in belgium and named cv777 [1] . in china, a diarrheal disease caused by pedv was first observed in 1973 [7] . since the winter of 2010, large-scale outbreaks of ped caused by highly pathogenic pedv strains have resulted in the death of a large number of pigs in south china [8, 9] . subsequently, this highly pathogenic pedv strains were discovered in other countries including the united states and south korea, etc. [10] [11] [12] [13] . since the 1990s, both inactivated and live attenuated pedv vaccines have been widely used to prevent dissemination of the virus in asia, including cv777 strain-based inactivated or attenuated live vaccines and bivalent inactivated vaccine using attenuated pedv and tgev in china [14, 15] , the kped-9 and dr13 strainbased attenuated live vaccines in south korea [11, 16] , and the p-5 v strain-based live attenuated vaccine in japan [17] . however, despite the emergence of china's current vaccine immunization strategy, multiple types of pedv strains were still found in immunized swine herds [15, [18] [19] [20] [21] . an analysis of pedv whole-genome differences whose sequences were available in genbank indicated that four hypervariable regions were present in pedv, comprising the c terminus of the nsp2 gene and the n terminus of the nsp3 gene, the spike gene, the open reading frame 3 (orf3), and the n gene region [22] . these gene mutations might alter the antigenicity of vaccines derived from classical attenuated vaccine strains and consequently resulted in inefficient vaccination in many pig farms [23] . at present, a variety of strains already exists in china, including classical strains (classical wild strains and classical attenuated vaccine strains, genotype 1) [23, 24] , highly virulent strains (genotype 2) [24] , the s-indel-pedv strains (genotype 1b) [7, 25, 26] , recombinant variants of attenuated vaccine strains and wild-type strains [27, 28] , and the variant with a large deletion in the s1 n-terminal domain, etc. [29] . moreover, multiple types of pedv strains coexisted in the same environment and even co-infected the same pig [23] , with a potential risk of recombination between wild-type strain and the classical attenuated vaccine strain [27, 28] . this could potentially result in the enhancement of the recombinant attenuated vaccine strains in virulence and increase the difficulty of identifying different types of strains. therefore, it was particularly important to establish a new detection method to distinguish pedv classical attenuated vaccine strains and wild-type strains and to monitor the prevalence of classical attenuated vaccine strains in pigs and evaluate attenuated vaccine safety. in recent years, the sequence characteristics of the spike (s) gene in pedv strains, including insertion and deletion in the s gene (s-indel) and only the s1 deletion gene, have been used as genetic markers to distinguish to distinguish different types of pedv strains [25, 29] . the research showed that nucleotide deletions of the orf3 gene in the vero cell-adapted attenuated vaccine strain were used to distinguish pedv attenuated vaccine strains and wild-type strains [16, 30] . based on the variation characteristics of the s gene and orf3 gene, many methods including traditional pcr [23] , real time rt-pcr [24, 31, 32] and nanoparticleassisted rt-pcr [21] have been established. however, there are difficulties in the monitoring, diagnosis and prevention of different types of pedv strains. in the present study, compared with the genome sequence of 38 pedv wild-type strains and 8 vero cell-adapted strains whose sequences were available in genbank ( fig. 1 and table s1), we found that three vero cell-adapted classical attenuated vaccine strains (pedv attenuated vaccine kc189944, attenuated cv777 and dr13) derived from classical strains and five vero cell-adapted isolates (js2008, sdm, sq2014, sc1402, hljby) have a 24-nucleotide deletion sequence of nonstructural protein 3(nsp3) gene in the orf1. these eight vero cell-adapted strains were artificially cell-passaged and did not naturally exist in the field unless they were used as attenuated live vaccines to be inoculated into pigs. based on these discoveries, a real time rt-rpa method was developed to effectively differentiate pedv classical attenuated vaccine strains from wild-type strains. this method contained not only a pedv universal real-time rt-rpa assay targeting the nucleocapsid gene that can identify all types of pedv strains but also included another pedv vaccine real-time rt-rpa assay targeting the 24nucleotides deletion sequence in the orf1 of three classical attenuated vaccine strains that specifically identified pedv classical attenuated vaccine strains. this method was shown to be an excellent alternative tools for the preliminary differentiation of pedv classical attenuated vaccine strains and wild-type strains, with potential use as a diagnostic method in clinical samples. the specificity, sensitivity, and repeatability analysis of the pedv real-time rt-rpa method as shown in fig. 2 , pedv wild-type strain dx, classical attenuated vaccine strain cv777, and vero-cell-adapted isolate js2008 showed fluorescent signals in the pedv universal real-time rt-rpa assay, and only the pedv classical attenuated vaccine strain cv777 and vero-celladapted isolate js2008 showed fluorescent signals in the pedv vaccine real-time rt-rpa assay. no fluorescent signals were obtained for tgev, pcv-2, pdcov, ppv or pkv, indicating the high specificity of the two assays. pedv universal real-time rt-rpa and pedv vaccine real-time rt-rpa standard curves were established using different copy numbers of standard rna as templates for sensitivity analysis. the detection limits of both the pedv universal real-time rt-rpa assay (fig. 3 ) and the pedv vaccine real-time rt-rpa assay (fig. 4) were 3.0 × 10 2 copies/reaction. as shown in table 1 , the detection limits of both pedv standard rnas were 3.0 × 10 2 rna copies/reaction. repeatability was evaluated using two standard rnas of the pedv attenuated vaccine strain cv777 n gene and the orf1 region, respectively, with coefficients of variation 0.68-1.47 (table 1 ). for the viruses that infect in vero-e6, the detection limits of pedv wild-type strain dx, classical attenuated vaccine strain cv777, and vero-celladapted isolate js2008 were 10 1.2 tcid 50 /100 μl, 10 0.5 tcid 50 /100 μl, and 10 1.1 tcid 50 /100 μl, respectively. the viral load originally contained in the sample was calculated as y u = − 1.27x + 14.20 (r 2 = 0.993) (fig. 3b) for the pedv universal real-time rt-rpa assay and y v = − 1.31x + 15.04 (r 2 = 0.996) (fig. 4b) for the pedv vaccine real-time rt-rpa assay. evaluation of pedv real-time rt-rpa method, real-time rt-pcr, and one-step rt-pcr assays with clinical samples to verify the reliability of the established real time rt-rpa method, a total of 80 suspected pedv samples were assessed by pedv real-time rt-rpa method, real-time rt-pcr assay, and rt-pcr assay, giving positive rates of 80.00, 81.25, and 77.50% for pedv wild-type strains, and 8.75, 8.75, and 7.50% for pedv classical attenuated vaccine strains, respectively (table 2) . of the 12 samples that tested negative in the rt-pcr assay, 9 samples were negative and the other 3 were positive (one classical attenuated vaccine strain and two wild-type strains) as tested by the real time rt-rpa method, and 8 samples were negative and the other 4 were positive (one classical attenuated vaccine strain and three wild-type strains) as tested by the real time rt-pcr assay. all positive amplified products were sequenced, which confirmed the presence of pedv in the samples and the viral load of all pedv positive samples was measured by real-time rt-rpa method in this study (fig. 5 ). the pedv real-time rt-rpa method has highly positive diagnosis agreement with real-time rt-pcr (98.6%) and rt-pcr assays (95.8%). these indicated the high specificity and sensitivity of these assays. in addition, transmissible gastroenteritis virus (tgev) and porcine kobuvirus (pkv) were detected in the 8 pedvnegative samples (data not shown). the growth curve for the pedv classical attenuated vaccine strain cv777 in vero-e6 cells determined by pedv real-time rt-pcr, pedv universal real-time rt-rpa, and pedv vaccine real-time rt-rpa indicated that viruses replicated rapidly during the first 24 h post-infection (hpi), achieving the highest titer at approximately 36 hpi (fig. 6) . the virus titer gradually decreased because vero-e6 cells breakdown after 36 h. these results showed that real-time rt-pcr and real-time rt-rpa are two alternative assays for the differentiation and characterization of pedv properties in vero-e6 cells. phylogenetic tree analysis of pedv strains in china demonstrated that the entire pedv genomes evolved into two separate genogroups, gi (classical strains, gi-a and gi-b) and gii (variant strains) [25, 33] . classical cv777 (accession number: af353511) and dr13 (accession number: jq023161) belonged to the gi-a subgroup. the gi-b subgroup included vero cell-adapted vaccine strains (pedv attenuated vaccine kc189944, attenuated cv777 and dr13) derived from classical strains, a recombinant vero cell-adapted isolate (js2008) of pedv attenuated vaccine and mutants [27] , and four other vero cell-adapted isolates (sdm, sq2014, sc1402, hljby) [20, 25, [33] [34] [35] [36] . pedv strains in the gi-b subgroup not only have nucleotide variations in the orf3 or spike gene [25, 29, 33] but also have 24-nt deletions of the nsp3 gene in the orf1 region in our study (fig. 1) . these nucleotides deletion in the orf1 may have occurred in cell-adapted viruses during adaptation and attenuation through serial passage in vero cells, which is identical to nucleotide deletion of spike or orf3 genes in the cell-adapted isolates and attenuated live vaccines [13, 16] , and thus would be unlikely to be detected in wild-type strains. based on the above findings, a differential pedv real-time rt-rpa method was established to distinguish pedv classical attenuated vaccine strains from the wild-type strains. this method contained two real-time rt-rpas, a universal real time rt-rpa assay targeting nucleocapsid gene was used to detect all types of pedv strains and another pedv vaccine real time rt-rpa could identity pedv classical attenuated vaccine strains according to orf1 24-nucleotides deletion region of three classical attenuated vaccine strains (pedv attenuated vaccine kc189944, attenuated cv777 and dr13) and five vero cell-adapted isolates (js2008, sdm, sq2014, sc1402, hljby) genome sequences, which were compared to 38 pedv wild-type strains published by genbank (fig. 1 ). the two real-time rt-rpa assays have good specificity, with fluorescent signals only visible for pedv positive rna amplicons. the pedv detection limits of both real-time rt-rpa assays were 10 2 copies, reflecting good sensitivity. clinical samples tests indicated that the pedv real-time rt-rpa method has a highly overall agreement with real-time rt-pcr (98.6%) and rt-pcr assays (95.8%) ( table 2) , respectively, but require less than half the time of them, suggesting that the real-time rt-rpa method could be used as an alternative detection method. sequencing results of 7 samples that were positive in the pedv vaccine real time rt-rpa assay were the same as the nucleotide deletion positions of the orf1 and orf3 fragments in classical attenuated vaccine cv777. it is worth noting that among the seven classical attenuated vaccine strains in all samples, six were from the piglets that were orally inoculated with the classical attenuated vaccine cv777, and none of the live pedv strain was successfully isolated in vero-e6 cells. this may be because the stool samples contained less viral load or only contains nucleic acids. due to orf1 of some vaccine candidates have not been reported, especially the vaccine candidates from highly virulent strains (genotype 2a) emerged after 2010, we are not sure whether these vaccine candidates derived from non-classical attenuated vaccine strains have the same 24 nucleotides deletion of nsp3 gene in the orf1. if these vaccine candidates do not have the 24 nt deletions pattern, they can not be detected by our method. similarly, if the wild-type virus may repair the nsp3 gene 24-nt-deletion region of classical attenuated vaccine strain in a co-infection event, the 24 nucleotides-repaired strain not be detected by our method. nevertheless, commercial vaccines widely used in pig farms of china are developed based on classical attenuated strains, while vaccines derived from non-classical attenuated vaccine candidates are being developed but not yet commercialized, our method is safe, accurate and reliable for the detection and identification of classical attenuated vaccine strains in pig farms. therefore, our method can be used to distinguish classical attenuated vaccine strains and wild-type strains, while vaccine candidates derived from other nonclassical attenuated vaccine strains may not be detected. this real-time rt-rpa method was established to distinguish between classical attenuated vaccine strains that were artificially vaccinated and wild-type strain during epidemiological surveillance, thereby obtaining more accurate epidemiological data. additionally, because the real-time rt-rpa is more rapid than real-time rt-pcr and isothermal loop-mediated isothermal amplification (lamp) technology. it is also more efficient, requiring only a pair of primers and a low running temperature (30-45°c) for a short period (20-40 min) [37] [38] [39] . this compares with lamp requirements of 4-6 primers and a high running temperature (60°c) for 1 h [40] [41] [42] , and real-time rt-pcr of a pair of primers and a high running temperature (95°c) for over 1 h [43] . additionally, there are no melting temperature requirements for rpa primers and probes because their annealing and elongation are enzyme-mediated rather than temperature-driven [39, 44] . moreover, the combination of rpa and real-time fluorescence quantification with gel electrophoresis results in high simplicity, specificity, and accuracy [38, 39, 45] . finally, the portability of the assays means that they can be used in the field and in areas where resources are limited [46] . in conclusion, we developed a simple, rapid, and reliable real time rt-rpa method for the differentiation of pedv classical attenuated vaccine strains and wild-type strains. these assays were analyzed using fluorescent dyes and shown to be highly specific and sensitive. they provide a reliable technical tool for the differentiation of the pedv classical attenuated vaccine strains and wildtype strains, as well as the surveillance of the clinical epidemic status of the disease. pedv attenuated vaccine strain cv777 and vero-cell-adapted isolates js2008 were passaged in vero e6 cells. pedv wild strain dx, transmissible gastroenteritis virus (tgev), porcine circovirus type 2 (pcv-2), porcine deltacoronavirus (pdcov), porcine kobuvirus (pkv), and porcine parvovirus (ppv) were maintained in our laboratory. fecal samples were collected from 80 piglets suspected of being infected with pedv from five pig farms in dingxi, jiayuguan, linxia, and tianshui, gansu province, china. clinical samples were centrifuged at 4000 g for 15 min, and the supernatant was stored at − 80°c. viral rna and dna were extracted using the takara minibest viral rna/dna extraction kit ver. 5.0 (takara co., ltd., dalian, china) according to the manufacturer's instructions, and then quantified using an nd-2000c spectrophotometer (thermo scientific, wilmington, de, usa). for clinical samples, extracted viral rna was eluted in 30 μl of rnase-free water. all rna and dna templates were stored at − 80°c until required. real-time rpa primers and probes (synthesized by sangon biotech, shanghai, china) were designed and verified by blast analysis (https://blast.ncbi.nlm.nih.gov/ blast.cgi) according to the nucleocapsid gene conserved sequence and replicase gene orf1 region (containing a 24-nucleotide deletion) of the pedv classical attenuated vaccine cv777 strain (table 3) (table 3 ) from pedv classical attenuated vaccine cv777 cdna and named pedv-v/qrt-rpa. synthesis of the first-strand pedv classical attenuated vaccine strain cv777 cdna was performed by reverse transcription using the primescript™ 1st strand cdna synthesis kit (takara, dalian, china), and the pcr conditions were as follows:95°c for 5 s, followed by 35 cycles of 95°c for 5 s, 55°c for 30 s, and 72°c for 30 s, with a final extension at 72°c for 10 min. the amplified pcr product was then linked to the pgem-t easy vector to construct the standard plasmid for use in the pedv vaccine real-time rt-rpa assay. both plasmids were sequenced by tsingke biological technology, linearized by digestion with nde i (takara co., ltd.), purified using the takara minibest dna fragment purification kit ver. 4.0 (takara co., ltd.), and transcribed in vitro with the ribomax large scale rna production system-t7 (promega, madison, wi, usa). agarose gel electrophoresis was used to verify the length and integrity of transcribed standard pedv rna in vitro. the number of standard rna copies was calculated using an nd-2000c spectrophotometer and the rna copy number was calculated as follows: (6.02 € y 10 23 copy number/mole number) × ( rna concentration)/(340 × base number). the real-time rt-rpa assay was performed using twis-tamp® exo rt (twistdx) in the following assay reaction system: 420 nm (2.1 μl) of each rpa primer (10 μm), 120 nm (0.6 μl) exo probe (10 μm), 14 mm (29.5 μl) rehydration buffer, 1 μl of viral rna or 4 μl sample rna, 1 μl of rnase inhibitor, and ddh 2 o to a total volume of 47.5 μl. after mixing, this was added to the recombinase reaction pellet with 2.5 μl 280 mm magnesium acetate and mixed well. the sample was vortexed, briefly centrifuged, and the tubes were immediately placed in the agilent technologies mx3000p thermocycler device (life technologies, carlsbad, ca, usa) to start the reaction at 39°c for 20 min (20 s per cycle, a total of 60 cycles). real-time rt-pcr was performed to identify pedv wildtype strains and classical attenuated vaccine strains in an agilent mx3000p thermocycler machine (life technologies) using the primers and probe based on the pedv spike gene [31] with the one step primescript® rt-pcr kit (perfect real time; takara co., ltd). the assay was performed as follows: 42°c for 5 min, then 95°c for 10 s, followed by 40 cycles of 95°c for 5 s and 60°c for 31 s. the specificity and sensitivity analysis of pedv real-time rt-rpa method a total of 10 ng of rna or dna extracted from pedv dx, cv777, and js2008, and tgev, pkv, pdcov, ppv, and pcv-2 was used as a template to analyze the specificity of pedv real-time rt-rpa method on an agilent mx3000p thermocycler machine (life technologies). this was repeated three times. sensitivity analysis of two pedv real-time rt-rpa assays was conducted using 10-serial dilutions of standard rna as the original template ranging from 3 × 10 8 to 3 × 10 1 copies. a total of 1 μl of each serial dilution was used to evaluate the dynamic detection range of the two pedv real-time rt-rpa assays. meanwhile, 10 3.5 tcid 50 /100 μl of classical attenuated vaccine strain cv777, 10 4.1 tcid 50 /100 μl of vero-cell-adapted isolate js2008, and 10 5.2 tcid 50 /100 μl wild dx strain were 10-fold serially diluted with modified eagle medium (mem) and used as original templates in the rt-rpa method to determine the detection limitation. each run was repeated four times, and a probabilistic regression analysis was performed with an agilent mx3000p thermocycler machine (life technologies) to determine the limits of the assay. the standard curve was calculated using graphpad prism 5.0 software (graphpad software inc., san diego, ca, usa). two pairs of primers, f1-v/r1 and f1-c/r1 [32] , were used for rt-pcr with the primescript™ one step rt-pcr kit ver. 2.0 (takara co., ltd., dalian, china) to detect 80 samples with suspected pedv infection from four locations in gansu province, china. the 80 fecal samples were obtained from five pig farms that have a history of inoculating the attenuated vaccine cv777, six of which from the piglets orally inoculated with the attenuated vaccine cv777 and show no clinical symptoms of diarrhea, and other 74 fecal samples were collected from piglets with symptoms of diarrhea. all fecal samples underwent pedv real-time rt-rpa, pedv real-time rt-pcr, and rt-pcr assays. finally, all pedv-positive products were sequenced by tsingke biological technology. supplementary information accompanies this paper at https://doi.org/10. 1186/s12917-020-02424-1. additional file 1 : table s1 . the name, accession number, and search website of the pedv strain in the study. a new coronavirus-like particle associated with diarrhea in swine outbreak of porcine epidemic diarrhea in suckling piglets further analysis of the genome of porcine epidemic diarrhoea virus sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf completion of the porcine epidemic diarrhoea coronavirus 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diagnostic applications loop-mediated isothermal amplification (lamp): principle, features, and future prospects lava: an open-source approach to designing lamp (loop-mediated isothermal amplification) dna signatures development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus evaluation of two real-time polymerase chain reaction assays for porcine epidemic diarrhea virus (pedv) to assess pedv transmission in growing pigs dna detection using recombination proteins real-time reverse transcription recombinase polymerase amplification assay for rapid detection of porcine epidemic diarrhea virus factors influencing recombinase polymerase amplification (rpa) assay outcomes at point of care publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank aje english editing experts for revising the grammar and language. authors' contributions x l collected the samples in gansu province and designed the study. zlw, z d, and jy w performed the experiments. zl w, xr l, yj s, xa c and ys l analyzed the data. zl w drafted the manuscript. all the authors read and approved the final paper. this study was funded by national key research and development program (2016yfd0500703). the design of this study, sample collections, data analysis, and the writing of this manuscript were supported by this funding (2016yfd0500703). all data generated or analysed during this study are included in this published article (and its additional file table s1). not applicable. the authors declare that they have no competing interests. the work is an original paper and is not under consideration in other journals.author details key: cord-332811-kjgah8ts authors: lee, do hyun; jeon, young-soo; park, choi-kyu; kim, seungjoon; lee, du sik; lee, changhee title: immunoprophylactic effect of chicken egg yolk antibody (igy) against a recombinant s1 domain of the porcine epidemic diarrhea virus spike protein in piglets date: 2015-06-23 journal: arch virol doi: 10.1007/s00705-015-2494-z sha: doc_id: 332811 cord_uid: kjgah8ts porcine epidemic diarrhea virus (pedv) is a highly contagious enteric pathogen of swine causing high mortality rates in piglets. pedv outbreaks have occurred continuously in most swine-producing asian countries and have recently emerged in the united states, leading to large economic losses for both the asian and us pig industries. the spike (s) protein of pedv consists of the s1 and s2 domains, responsible for virus binding and fusion, respectively. the involvement of the s1 domain in specific high-affinity interactions with the cellular receptor and induction of neutralizing antibodies in the natural host makes it a logical target for the development of effective vaccines and therapeutics against pedv. passive immunization by oral administration of egg yolk antibodies (igy) obtained from immunized chickens provides an alternative source of specific antibodies for the prevention and treatment of pedv in newborn piglets. in this study, we produced an igy against the pedv s1 protein and investigated its immunoprophylactic effect in neonatal piglets. a codon-optimized pedv s1 gene consisting of amino acid residues 25–749 was synthesized and used to establish a stable porcine cell line constitutively expressing a recombinant pedv s1 protein containing the chicken immunoglobulin fc fragment at its c-terminus. the purified recombinant s1 protein was found to mediate potent immune responses in immunized hens. we next tested the ability of oral passive immunization with anti-pedv s1 igy to protect piglets against pedv. specific chicken igy against the s1 protein was orally administered to neonatal piglets, and their responses subsequent to a virulent pedv challenge were monitored. the results showed that oral administration of anti-pedv s1 igy efficiently protects neonatal piglets against pedv, suggesting its potential as a prophylactic or therapeutic agent against acute pedv infection. porcine epidemic diarrhea (ped) is characterized by acute enteritis and watery diarrhea followed by severe dehydration, leading to high mortality rates in neonatal piglets [5, 29, 33] . the disease was initially recognized in england in 1971 [27] , and the causative agent, ped virus (pedv), was not identified until 1978 [28] . ped epidemics were first reported in asia in 1982, and ped has since continued to threaten swine health, causing substantial economic losses for the asian swine industry [3, 11, 21, 30, 38] . in early 2013, sudden ped outbreaks occurred in the united states sweeping through the pork industry across the country [24, 35] . subsequently, starting in late 2013, large-scale outbreaks of ped rapidly recurred in korea, taiwan, and japan; a us-strain-like pedv was found to be responsible for the recent outbreaks in those countries, raising global concerns regarding control measures for ped prevention [17] [18] [19] 22] . pedv is a large enveloped virus possessing a singlestranded, positive-sense rna genome of approximately 28 kb with a 5 0 cap and a 3 0 polyadenylated tail, belonging to the genus alphacoronavirus within the family coronaviridae of the order nidovirales [28, 33] . the spike (s) protein of pedv is a type i membrane glycoprotein consisting of 1,383 to 1,386 amino acids (aa), depending on the strain, that can be divided into the s1 (aa 1-735) and s2 (736-end) domains based on homology to the s proteins of other coronaviruses [6, 9, 15, 36] . similar to other coronavirus s proteins, the pedv s protein plays a critical role by interacting with the cellular receptor to mediate viral entry and inducing neutralizing antibodies in the natural host [1, 2] . more precisely, the s1 domain has been reported to contain the receptor-binding region and the main neutralizing epitopes [16, 37] . furthermore, the s1 region has been established as a suitable target for determining genetic relatedness among pedv isolates and for developing differential diagnostic assays and effective vaccines [4, 7, 15, 26] . in korea, both modified live and killed vaccines against pedv have been developed for disease control and use in the domestic market. however, the continued occurrence of ped nationwide has caused enormous financial losses for the korean pork industry, raising questions regarding the efficacy of these commercial vaccines. this phenomenon appears to be due to genetic and antigenic differences between the vaccine strain and the strains prevalent in the field [15, 26] . thus, the lack of effective vaccines increases the need for next-generation field-virus-based vaccines or control measures against pedv infection. passive immunization by oral administration of specific antibodies represents an attractive approach against gastrointestinal pathogens in both humans and animals [23] . egg yolk immunoglobulin (igy) from immunized chickens has been demonstrated to be a convenient large-scale source for specific antibodies; it has also been shown to be safe and effective against pedv in newborn piglets [12] . the aim of the present study was to produce an igy against the pedv s1 protein and assess its immunoprophylactic properties in neonatal piglets. a codon-optimized pedv s1 gene was used to establish a stable porcine cell line constitutively expressing a recombinant s1 protein. the recombinant s1 protein was capable of inducing efficient cytokine and antibody responses in immunized hens. moreover, oral passive immunization using chicken igy raised against the pedv s1 protein was found to control and prevent ped post-challenge in suckling piglets. cells, virus, and antibodies hek-293t cells (crl-1573) were purchased from the american type culture collection (atcc; manassas, va, usa) and cultured in dulbecco's modified eagle medium (dmem) with high glucose (invitrogen, carlsbad, ca, usa) supplemented with 10 % fetal bovine serum (fbs; invitrogen) and antibiotic-antimycotic solution (1009; invitrogen). pk-15 cells were grown in rpmi 1640 medium (invitrogen) containing 10 % fbs and antibiotic-antimycotic solution. vero cells were cultured in alpha minimum essential medium (a-mem; invitrogen) with 10 % fbs and antibiotic-antimycotic solution. the cells were maintained at 37°c in a humidified 5 % co 2 atmosphere. the sm98-1 pedv vaccine strain was obtained from the korean animal and plant quarantine agency and propagated in vero cells as described previously [8] . challenge pedv was prepared from intestinal contents obtained from a field case that was found to be free of other common etiologic agents of neonatal porcine diarrheal diseases, as described previously [26] . briefly, a 4-day-old suckling piglet was inoculated orally with a small-intestine homogenate containing the field virus. small-intestine tissues were collected and homogenized in a 10 % suspension with a-mem using a magna lyser (roche diagnostics, mannheim, germany) by three repetitions of 15 s at 7,000 rpm, and tissue suspensions were clarified by centrifugation for 10 min at 4,5009g (hanil centrifuge fleta5, incheon, south korea). the clarified supernatant was filtered through a 0.22-lm-pore-size syringe filter (millipore, billerica, ma, usa), aliquoted, and stored at -80°c until use as the crude challenge virus. horseradish peroxidase (hrp) or fluorescein isothiocyanate (fitc)-conjugated secondary antibodies were purchased from jackson immunoresearch laboratories (west grove, pa, usa), and alexa fluor 488-conjugated secondary antibody was obtained from molecular probes (carlsbad, ca, usa). the pedv s-protein-specific monoclonal antibody (mab) was a kind gift from sang-geon yeo (kyungpook national university, daegu, south korea), and the nucleocapsid (n)-protein-specific mab was obtained from choongang vaccine laboratory (cavac; daejeon, south korea). dna manipulation and cloning were performed according to standard procedures [34] . escherichia coli strain dh5a (rbc bioscience, taiwan) was used as the host for general cloning. the pfb-neo-pedv-rs1-ig plasmid encoding a full-length, codon-optimized pedv s1 gene (aa 24-735) was described previously [26] . the pcdna3.1/chicken fc plasmid encoding the fc domain of chicken igg was a kind gift from hyung-kwan jang (chonbuk national university, jeonju, south korea). the chicken fc (cfc) fragment from pcdna3.1/chicken fc was subcloned into pfb-neo-pedv-rs1-ig by replacing the fc domain of human igg1 to construct the gene expression plasmid, pfb-neo-pedv-rs1-cfc, which produces a chicken fc-tagged fusion protein, rs1-cfc. the constructed plasmid was verified by nucleotide sequencing. generation of a stable pk-15 cell line expressing pedv rs1-cfc the retrovirus gene transfer system (agilent technologies, santa clara, ca, usa) was used to generate a cell line constitutively expressing the recombinant pedv rs1-cfc gene or an empty retroviral vector as described elsewhere [15, 25] . antibiotic-resistant continuous cell clones were examined by rt-pcr to verify the presence of the fulllength rs1-cfc gene, and the positive clones (pk-rs1-cfc) were then amplified for subsequent analysis. pk-rs1-cfc cells were grown on microscope coverslips placed in 6-well tissue culture plates. at 48 h post-seeding, the cells were fixed with 4 % paraformaldehyde for 10 min at room temperature (rt) and permeabilized with 0.2 % triton x-100 in phosphate-buffered saline (pbs) at rt for 10 min. the cells were subsequently blocked with 1 % bovine serum albumin (amresco, solon, oh, usa) in pbs for 30 min at rt and then incubated with a goat antichicken igg antibody conjugated to fitc. finally, the cells were counterstained with 4 0 ,6-diamidino-2-phenylindole (dapi; sigma, st. louis, mo, usa), and cell staining was visualized using a leica dm il led fluorescence microscope (leica, wetzlar, germany). pk-rs1-cfc cells were grown in 100-mm-diameter tissue culture dishes to 90 % confluency in serum-free medium (optipro sfm; invitrogen). at 72 h post-seeding, the protein-containing cell culture supernatants were harvested, and soluble proteins were immunoprecipitated with chicken igy precipitating resin (genscript, piscataway, nj, usa) according to the manufacturer's protocol in the presence of protease inhibitors at 4°c for 16 h. the beads were collected by centrifugation at 5,0009g (eppendorf centrifuge 5415r, hamburg, germany) for 5 min at 4°c and washed three times with 0.5 m nacl in pbs. the samples were subsequently eluted with 50 mm sodium citrate/50 mm glycine (ph 2.0) and neutralized with 1 m tris-hcl (ph 8.0). the purified proteins were concentrated with amicon ultracentrifugal filters 100k (millipore). protein concentration was measured using a pierce bca protein assay (thermo scientific, rockford, il, usa), and the final products were analyzed by western blotting to confirm target protein purification. the protein-containing cell culture supernatants or purified proteins as described above were mixed with nupage 49 lds sample buffer (invitrogen) and boiled at 70°c for 10 min. the proteins were separated on a nupage 4-12 % gradient bis-tris gel (invitrogen) under reducing conditions and stained using simplyblue safestain (invitrogen) according to the manufacturer's instructions or electrotransferred onto immobilon-p polyvinylidene fluoride (pvdf) membranes (millipore). the membranes were then blocked with 3% powdered skim milk (bd biosciences, belford, ma, usa) in tbs (10 mm tris-hcl [ph 8.0], 150 mm nacl) with 0.05 % tween-20 (tbst) at 4°c for 2 h and then reacted directly with the goat anti-chicken igg hrp-conjugated secondary antibody or with anti-pedv s mab followed by the corresponding hrp-labeled secondary antibody at a 1:2,000 dilution for 2 h at 4°c. finally, the proteins were visualized using enhanced chemiluminescence reagents (ge healthcare, piscataway, nj, usa) according to the manufacturer's protocol. twenty 10-week-old white leghorn hens were randomly allocated into three groups and immunized by intramuscular injection into the breast muscle with either the binary ethylenimine (bei)-inactivated sm98-1 pedv vaccine strain, 200 lg of the purified pedv rs1-cfc protein resuspended in pbs, or pbs with a mineral oil-based adjuvant (montanide isa 70 vg; seppic, puteaux, france) ( table 1 ). the hens were then boosted three times with the same immunogens emulsified with adjuvant at 3-week intervals. blood samples were collected prior to immunization, at each boost, and three weeks subsequent to the eggs collected from immunized hens were washed with diluted sodium hypochlorite solution (yuhanclorox, seoul, south korea), disinfected with 70 % ethanol, and used for egg yolk fractionation. the separation of the yolk from the albumen and chalaza was performed as described previously [13] . the egg yolk material was mixed with distilled water (dw) at a 1:7 (v/v) ratio immediately followed by the addition of strong acid electrolytic water (ph \ 2.0) at 0.02 % of the total volume. samples were thoroughly whisked using a hand blender for approximately 10 min. the egg yolk mixture was kept at 4°c for 24 h, and supernatants were then separated and clarified by centrifugation for 20 min at 3,0009g (hanil centrifuge fleta5) to thoroughly remove lipids. the supernatants containing igy was collected, lyophilized using a spray dryer and stored at 4°c for further experiments. peripheral blood mononuclear cells (pbmcs) were isolated from whole blood using a standard gradient centrifugation purification protocol with histopaque 1077 (sigma) according to the manufacturer's instructions. total rna was extracted from the pbmcs of hens using trizol reagent (invitrogen) and treated with recombinant dnase i (takara, otsu, japan) according to the manufacturer's protocols. the concentrations of the extracted rna were measured with a nanovue spectrophotometer (ge healthcare). quantitative real-time rt-pcr was done using a thermal cycler dice real time system (takara) with a one step sybr primescript rt-pcr kit (takara) and the following gene-specific primer sets as described previously [20, 31, 32] : chicken ifn-a forward, 5 0 -atcctgct gctcacgctccttct-3 0 ; chicken ifn-a reverse, 5 0 -gg tgttgctggtgtccaggatg-3 0 ; chicken ifn-b forward, 5 0 -gcctccagctccttcagaatacg-3 0 ; chicken ifn-b reverse, 5 0 -ctggatctggttgaggaggctgt-3 0 ; chicken ifn-c forward, 5 0 -agctgacggtggacc-tattatt-3 0 ; chicken ifn-c reverse, 5 0 -ggctttgcgc tggattc-3 0 ; chicken il-6 forward, 5 0 -caaggtgac ggaggaggac-3 0 ; chicken il-6 reverse, 5 0 -tggcgag gagggatttct-3 0 ; chicken il-8 forward, 5 0 -ccaag-cacacctctcttcca-3 0 ; chicken il-8 reverse, 5 0 -gcaaggtaggacgctggtaa-3 0 ; chicken tnf-a forward, 5 0 -gaagcagcgtttgggagt-3 0 ; chicken tnf-a reverse, 5 0 -gttgtgggacagggtagg-3 0 ; chicken glyceraldehyde-3-phosphate dehydrogenase (gapdh) forward, 5 0 -ggtggtgctaagcgtgttat-3 0 ; chicken gapdh reverse, 5 0 -acctctgtcatctctccaca-3 0 . the steady-state mrna levels of each cytokine gene were normalized against the level of chicken gapdh mrna, and the relative quantity (rq) of mrna accumulation was evaluated using the 2 -ddct method. the relative fold change in the expression of each gene was then calculated by comparing pre-immune and immunized sera. pedv-specific neutralizing antibodies in the serum and igy samples collected from hens in all groups were determined using a serum neutralization test in 96-well microtiter plates with the sm98-1 pedv vaccine strain as previously described [14] . briefly, individual virus stocks were diluted in serum-free a-mem to a concentration of 200 plaque-forming units in a volume of 50 ll. the diluted virus was then mixed with 50 ll of a twofold serial dilution of individual inactivated sera or igy solution dissolved in dw (100 mg/ml) in 96-well plates, and the mixture was incubated at 37°c for 1 h. next, approximately 1 9 10 4 vero cells in 100 ll of a-mem with 10 % fbs were added to each well, and the mixture was further maintained at 37°c in a 5 % co 2 incubator for 3 to 4 days. the neutralization titer was calculated as the reciprocal of the highest serum dilution that inhibited virus-specific cytopathic effects in all duplicate wells. the in vivo swine experiments described here were performed at the improah animal facility under the guidelines established by the institutional animal care and use committee. a total of 18 newborn piglets were obtained from seronegative pregnant sows at a commercial pig farm with no known prior ped outbreak or vaccination with pedv. all animals were determined to be free of antibodies to pedv, as well as to transmissible gastroenteritis virus and porcine reproductive and respiratory syndrome virus. no other animals aside from those included in the study were housed at the facility for the duration of the experiment. the design for the present pig experiment is outlined in table 2 . piglets were randomly divided into four groups: group 1, administration of igy against pedv; group 2, administration of igy against pedv rs1-cfc; group 3, administration of igy against placebo; group 4, control. the groups were housed in separate rooms, and no physical contact was allowed between the groups. all neonatal piglets except for animals in the control group were inoculated orally with 1 ml of the small-intestine homogenate containing 10 4 tcid 50 /ml pedv field virus determined using real-time rt-pcr as described previously [10] . following challenge exposure, piglets were administered orally with 2 ml of the corresponding igy solution dissolved in dw (250 mg/ml) at 1 and 2 days post-inoculation (dpi). clinical signs of diarrhea and the mortality in challenged piglets were monitored daily throughout the study. stool samples from all groups were collected daily with 16-inch, cotton-tipped swabs and subjected to rt-pcr using an i-tgev/pedv detection kit (intron biotechnology, seongnam, south korea) to detect the presence of pedv. fecal shedding of pedv was measured by quantitative real-time rt-pcr as described above, and the results were analyzed using the system software as described previously [32] . all piglets from the challenged and control groups were euthanized at 5 dpi for post-mortem examination. smallintestinal tissue specimens collected from each piglet (\3 mm thick) were fixed with 10 % formalin for 24 h at rt and then embedded in paraffin according to standard laboratory procedures. the formalin-fixed paraffin-embedded tissues were cut into 5-to 8-lm-thick sections on a microtome, floated in a 40°c water bath containing dw, and transferred onto glass slides. the tissues were then deparaffinized in xylene for 20 min and washed in decreasing concentrations of ethanol (100 %, 95 %, 85 %, 70 %, and 50 %) for 3 min each. the deparaffinized intestinal tissue sections were stained with hematoxylin and eosin (h&e; sigma) to observe histopathological lesions of pedv infection and subjected to ifa using the pedv n-specific mab and goat anti-mouse igg secondary antibody conjugated to alexa fluor 488 as described above. the student's t-test was used for all statistical analyses and p-values of less than 0.05 were considered statistically significant. generation of stable porcine-origin cell lines expressing the full-length, codon-optimized s1 protein previously, we synthesized a full-length, codon-optimized s1 gene and confirmed that codon optimization greatly enhanced the expression level of s1 upon transient transfection [26] . in this study, the codon-optimized s1 gene was used for stable transfection to produce preparative amounts of the s1 protein. to accomplish this, sublines of pk-15 cells were established that stably expressed the recombinant codon-optimized s1 under the control of a retroviral ltr promoter. ten generated cell clones were initially collected and subjected to rt-pcr and western blot analysis to examine s1 expression at the mrna and protein level, respectively (data not shown). based on the results of the western blot analysis, one pk-rs1-cfc cell clone that consistently expressed the highest level of s1 was chosen for subsequent studies. to characterize the pk-rs1-cfc cells, intracellular and extracellular expression levels of s1 were examined by immunofluorescence and western blotting. as shown in fig. 1a , specific cell staining was clearly evident when pk-rs1-cfc cells were reacted with the anti-chicken igg antibody, confirming a consistent high expression level of the s1 protein. western blot analysis of cell culture supernatants revealed that the pk-rs1-cfc cells stably expressed and cumulatively secreted high levels of the approximately 180-kda s1. the recombinant s1 protein expressed in the supernatants of stable pk-rs1-cfc cells was purified using chicken igy precipitation resin beads. the purified s1 protein was detectable at a high level by simplyblue staining and was confirmed by immunoblotting with antichicken igg antibody (fig. 1b) . using our purification and concentration procedures, we were able to purify an average yield of 30 lg of s1 protein per 6 ml of pk-rsi-cfc cell culture supernatant cultivated in a 100-mm tissue culture dish for 72 h. in addition, the overall growth kinetics of s1 gene-expressing pk-15 cells were found to be similar to those of the parental pk-15 cells, indicating that s1 expression has no effect on cell growth (data not shown). to produce igy, hens assigned to the three groups were immunized intramuscularly, as outlined in table 1 . blood samples were collected before immunization (pre-immune), at each boost, and 3 weeks after the final boost. pbmcs were prepared from blood samples obtained during the first ifn-a, ifn-b , ifn-c, il-6, and il-8, was significantly altered by immu-nization of inactivated pedv or s1-cfc compared to levels in the placebo-inoculated group (fig. 2) . these data indicate that the immunogens used in this study efficiently stimulated immune responses in chickens. serum samples collected at each collection time were subjected to a serum neutralization test against the pedv vaccine strain. non-immunized hens showed only minimal neutralizing antibody titers, whereas immunized hens exhibited gradually increasing neutralizing antibody titers (fig. 3a) . furthermore, hens immunized with inactivated pedv (group 1) and s1-cfc protein antigen (group 2) at 3-week intervals produced similar neutralizing antibody titers ranging from 1:32 to 1:64 subsequent to the final immunization. in addition, egg yolk samples from each group were found to contain neutralizing antibody titers comparable to those in the corresponding serum samples, indicating the presence of neutralizing igy antibodies (fig. 3b) . to evaluate the immunoprophylactic efficacy of anti-pedv s1 igy, neonatal 4-to 5-day-old pedv-seronegative piglets were divided into four groups and challenged orally with wild-type pedv followed by the respective antibody treatment at days 1 and 2 post-challenge. animals in group 4 were not challenged with pedv and given igy orally from non-immunized chickens at the same time points as the other groups. clinical observations of mortality and diarrhea in challenged piglets are summarized in table 3 . none of the piglets from group 4 died or developed any clinical signs of diarrhea. additionally, fecal shedding of pedv was also detected in the rectal swabs of these animals for the duration of the study (fig. 4) . in contrast, all piglets from the challenged groups (1-3), regardless of igy administration, exhibited diarrhea that began at 1 or 2 days post-challenge and lasted throughout the challenge experiment. in group 3, one piglet died; the remaining piglets experienced severe watery diarrhea and shed pedv in their feces, with a mean cycle threshold (ct) value of 16.9 (range 14.8-18.6) during the study (fig. 4) . although none of the piglets from groups 1 and 2 treated with igy died during the challenge experiment, the number of piglets exhibiting diarrhea post-challenge varied depending on the group. all piglets from these groups showed mild-to-severe diarrhea lasting for the entire experiment but recovered from the diarrhea by the end of the study. furthermore, fecal shedding of pedv from these piglets was significantly lower than in group 3 piglets; pedv rna was detected in the fecal swab samples of all pigs in groups 1 and 2, with mean ct values of 24.1 (range fig. 1 constitutive expression of the recombinant s1 protein in pk-rs1-cfc cells. (a) immunofluorescence assay for the rs1 protein. pk-15 or pk-rs1-cfc cells grown in a 6-well tissue culture plate were fixed with 4 % formaldehyde at 48 h post-seeding and incubated with anti-chicken igg antibody (top panels). the cells were then counterstained with dapi (bottom panels) and examined using a fluorescent microscope at 4009 magnification. (b) purification of the rs1 protein. the recombinant s1 protein was purified from serum-free medium of pk-rs1-cfc cells grown in a 100-mm tissue culture dish. the cell culture supernatant and the purified rs1 protein were resolved on a 4-12 % gradient bis-tris gel and stained with simplyblue solution (left panel) or electrotransferred onto a pvdf membrane (right panel). the membrane was blotted with a chicken igg-specific antibody 23.1-24.8) and 21.9 (range 20.0-26.5), respectively (fig. 4) . however, despite the similar levels of diarrhea, the group 2 piglets shed slightly higher amounts of pedv in their feces compared to those in group 1. gross intestinal lesions consistent with viral enteritis, including thin-walled and fluid-content-dilated small intestines, were typically observed in all group 3 piglets; less-severe lesions were observed in piglets given either igy (data not shown). likewise, the majority of enterocytes over the entire villi in the control piglets with normal igy treatment (group 3) were affected by pedv, showing moderate-to-severe villous atrophy and destruction, while piglets from groups 1 and 2 exhibited mild intestinal lesions comparable to those in the non-challenged group, and viral antigens were only detected in their small intestines (figs. 5 and 6 ). however, anti-pedv s1 igy treatment (group 2) was more efficacious than treatment with igy raised against the whole virus (group 1) in reducing the overall severity of macroscopic and microscopic intestinal lesions in the piglets. collectively, all immunoprophylactic methods used in this study were capable of protecting neonatal piglets against mortality and disease severity after challenge with a virulent pedv. vaccination against pedv is one of the most important and effective prevention measures by passively transferring specific neutralizing antibodies present in vaccinated sows to their litters through colostrum and milk. despite the availability of commercial attenuated and inactivated vaccines in korea, pedv continues to plague the domestic pork industry, raising issues regarding their protective efficacy. recently, severe outbreaks of pedv have reemerged in korea and have been estimated to affect over 40 % of pig farms, causing serious economic impact on producers and customers [17, 18] . our previous studies suggested that antigenic and genetic variations between the vaccine virus and field pedvs may be the cause of the incomplete efficacy or failure of vaccination, which appears to be responsible for the periodic outbreaks in domestic herds [15, 26] . furthermore, a comparison of the amino acid sequence of the s1 domain of the pedv s protein, which is associated with viral binding to host cell receptors and contains neutralizing epitopes [16, 37] , has shown a difference of over 10 % between the vaccine strain and field isolates, suggesting the need for nexteffect of igy against the pedv s1 protein 2203 generation vaccine development [15, 17] . in addition to vaccination, artificial passive immunization using igy has been used commercially as an alternative method for controlling ped by providing supportive immunity to neonatal piglets exposed to acute infection in korea. however, opinions differ among swine practitioners and producers regarding the efficacy of this immunoprophylactic strategy. a similar debate regarding the commercial use of igy may arise since the vaccine strain is also used to immunize chickens for the production of anti-pedv igy in korea. we have previously demonstrated that the s1 protein of pedv could be considered a potential candidate antigen for vaccination [26] . in the present study, the s1 protein of the field pedv was used as an immunogen to inoculate hens, thereby producing anti-pedv s1 igy. we first aimed to stably express the full-length, codon-optimized s1 gene of pedv in porcine-origin cells and to evaluate the immunogenicity and efficacy of igy against the recombinant s1 protein. subsequently, we were able to successfully generate a stable pk cell line continuously producing large amounts of the codon-optimized s1 protein tagged with cfc. following the purification and concentration processes, approximately 30-40 lg of the recombinant s1-cfc protein could be consistently harvested from the culture supernatants of pk-rs1-cfc cells grown in a 100-mm culture dish. since humoral immunity is an important indicator for evaluating the effect of the s1-based immunogen used in this study, we immunized chickens with the s1-cfc antigen prepared from the cell culture supernatants of pk-rs1-cfc cells and investigated whether they developed cytokine and antibody responses. all of the proinflammatory cytokines tested, except for tnf-a, were stimulated in chickens immunized with either the whole virus or the s1-cfc antigen. of these, the expression levels of il-6 and il-8 genes were distinctly enhanced in the s1-cfc-inoculated group compared with the inactivated pedv-inoculated group. furthermore, the chicken sera raised against s1-cfc contained higher levels of neutralizing antibody than those raised against the sm98-1 vaccine virus. however, the final levels of anti-pedv igy were lower in the eggs of the s1-cfc-immunized chickens compared to those of the sm98-1-inoculated group. these inconsistent results may be attributed to the use of the heterogeneous sm98-1 virus in the serum neutralization assay, which possesses a high degree of genetic variation in field isolates. nevertheless, our data indicated that the recombinant s1 protein efficiently elicits immune responses in chickens. subsequently, we tested the prophylactic efficacy of each igy in suckling piglets post-challenge-exposure. our data showed that, regardless of the igy administered, all neonatal piglets treated with igy survived after challenge with virulent pedv, suggesting that the anti-pedv s1 igy provides effective passive immunity to prevent mortality comparable to whole-virus-based igy. despite the lower levels of virus shedding in the feces of animals treated with anti-pedv igy, the anti-s1 igy-based strategy resulted in more-efficient protection than the anti-pedv igy procedure, as determined by the duration and severity of diarrhea and histopathological lesions. these results indicated that the administration of anti-s1 igy could significantly reduce the mortality and clinical signs of piglets, suggesting that tissue specimens were collected from the small intestines of piglets from each group at the time of necropsy. the formalin-fixed and paraffin-embedded tissue sections were deparaffinized and stained with hematoxylin and eosin. the sections were examined using a light microscope at 409 magnification. the inset images are enlarged versions of parts of the picture effect of igy against the pedv s1 protein 2205 application of anti-s1 igy is capable of partially blocking the virus from invading the small intestine. a more promising approach would be to use the entire field virus or its full-length s protein to produce igy instead of using only the s1 domain, since the s protein contains multiple functional domains and neutralizing epitopes to efficiently stimulate non-susceptible hosts. for this purpose, the production and application of igy against a korean field isolate are currently under investigation. in conclusion, to the best of our knowledge, this is the first study to evaluate the immunoprophylactic effect of igy against the recombinant s1 protein of the field virus in a pig model. the results presented here indicate that the recombinant s1 protein can elicit cytokine and antibody responses and induce neutralizing antibodies in chickens. furthermore, challenge experiments revealed that administration of anti-s1 igy efficiently protected suckling piglets against field pedv by providing passive immunity. pedv infection causes high mortality rates (90-100 % in neonatal piglets under 7 days of age), and epidemiological observations indicate the rapid spread of the disease. in this circumstance, the preliminary results of the present study showed the potential of anti-pedv s1 igy application as a considerable measure against pedv. further experiments to optimize production procedures will be required to achieve higher titers of igy; field studies on farms will be needed to better evaluate the efficacy of anti-s1 igy. these studies will provide additional practical information for the future use of this alternative igy method as a supplement to passive immunity against ped and other economically important viral diseases. fig. 6 detection of pedv in small intestine tissues of piglets. tissue specimens were prepared from small intestine of piglets from each group at the time of necropsy. the formalin-fixed and paraffinembedded tissue sections were deparaffinized and subjected to immunofluorescence staining with an anti-pedv n antibody. the sections were then counterstained with dapi and examined using a fluorescence 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in immortalized porcine alveolar macrophages human telomerase reverse transcriptase-immortalized porcine monomyeloid cell lines for the production of porcine reproductive and respiratory syndrome virus diseases of swine molecular cloning: a laboratory manual emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences proteolytic cleavage of peplomeric glycoprotein e2 of mhv yields two 90k subunits and activates cell fusion spike protein region (aa 636789) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan acknowledgments this research was supported by technology development program for bio-industry, ministry for agriculture, food and rural affairs, republic of korea (311007-05-1-hd120). conflict of interest the authors declare that they have no conflict of interest.ethical approval all procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted. key: cord-331542-wy068c6o authors: kong, ning; meng, qiong; jiao, yajuan; wu, yongguang; zuo, yewen; wang, hua; sun, dage; dong, sujie; zhai, huanjie; tong, wu; zheng, hao; yu, hai; tong, guangzhi; xu, yongjie; shan, tongling title: identification of a novel b-cell epitope in the spike protein of porcine epidemic diarrhea virus date: 2020-04-03 journal: virol j doi: 10.1186/s12985-020-01305-1 sha: doc_id: 331542 cord_uid: wy068c6o background: porcine epidemic diarrhea virus (pedv) infection causes an acute enteric tract infectious disease characterized by vomiting, anorexia, dehydration, weight loss and high mortality in neonatal piglets. during pedv infection, the spike protein (s) is a major virion structural protein interacting with receptors and inducing neutralizing antibodies. however, the neutralizing b-cell epitopes within pedv s protein have not been well studied. methods: to accurately identify the important immunodominant region of s1, the purified truncated s1 proteins (sa, sb, sc, sd and se) were used to immunize balb/c mice to prepare polyclonal antibodies. the antisera titers were determined by indirect elisa, western blot and ifa after four immunizations to find the important immunodominant region of s1, and then purified the immunodominant region of s1 protein and immunized mice to generate the special antibodies, and then used recombinant peptides to determine the b-cell epitopes of monoclonal antibodies. results: five antisera of recombinant proteins of the spike protein region of pedv were generated and we found that only the polyclonal antibody against part of the s1 region (signed as se protein, residues 666–789) could recognize the native pedv. purified se protein was used to immunize balb/c mice and generate mab 2e10. pepscan of the se protein demonstrated that se16 ((722)sstfnstrel(731)) is the minimal linear epitope required for reactivity with the mab 2e10. further investigation indicated that the epitope se16 was localized on the surface of pedv s protein in the 3d structure. conclusions: a mab 2e10 that is specifically bound to pedv was generated and identified a specific linear b-cell epitope (se16, (722)sstfnstrel(731)) of the mab. the epitope region of pedv s1 localized in the different regions in comparison with the earlier identified epitopes. these findings enhance the understanding of the pedv spike protein structure for vaccine design and provide a potential use for developing diagnostic methods to detect pedv. porcine epidemic diarrhea (ped) is an acute enteric tract infectious disease characterized by vomiting, anorexia, dehydration, weight loss and high mortality in neonatal piglets [1, 2] . the disease was reported in european and asian pig industries over the last 30 years, with the virus firstly appearing in england and belgium in the early 1970s [3, 4] . porcine epidemic diarrhea virus (pedv), although the etiologic agent of ped has become a severe problem in many asian countries, including china, korea, japan and thailand [5] [6] [7] [8] . since 2010, the virulent pedv has become prevalent in swine herds and incurred huge economic losses to the swine industry [9] [10] [11] . due to the lack of effective vaccines, ped is still circulating in the worldwide. pedv belonging to the genus alphacoronavirus, family coronaviridae, has an approximately 28 kb genome of single-stranded, positive-sense rna [1] . the pedv genome encodes two large polyproteins, an accessory protein and four structural proteins. the structural proteins contain glycosylated spike (s), envelope (e), glycosylated membrane (m) and rna-binding nucleocapsid (n) proteins [12] . the spike gene can be divided into s1 and s2 domains, as in other coronaviruses and it has multiple functions that can interact with cellular receptors and regulating viral entry and containing neutralizing epitopes to induce neutralizing antibodies [13, 14] . in the present study, we expressed and purified the recombinant truncated pedv s1 constructs (sa-se) to immunize balb/c mice and found that se, one of the s1 construct (residues 666-789), was the immunodominant region of s1 protein. furthermore, we utilized the se protein to immunize balb/c mice and obtained one se specific mab, 2e10. a novel linear b-cell epitope, ( 722 sstfnstrel 731 ), was subsequently identified using the se specific mab 2e10. these results provide valuable information for virus diagnosis and vaccine design. african green monkey kidney cells (vero e6) and sp2/0 myeloma cells were cultured in a humidified 5% co 2 atmosphere at 37°c. all the culture media were dulbecco's modified eagle's medium (dmem, hyclone) supplemented with 10% fetal bovine serum (fbs) and antibiotics (0.1 mg/ ml of streptomycin and 100 iu/ml of penicillin). pedv strain js-2013 was obtained from the shanghai veterinary research institute (caas, china). plasmid dna (pcold-tf) containing the s1 gene (1-2367 bp) of pedv strain js-2013 was constructed by our own laboratory. the five overlapping fragments, comprising partial length of pedv s1 gene, were constructed and designated as sa, sb, sc, sd and se. a bamh i site and sixteen extra bases that were homologous to the terminal sequence of the vector were added to the 5′ end. the sequences of the primers used for amplification of the gene in this study are shown in table 1 . all the recombinant plasmids were constructed by clonexpress ii one step cloning kit (vazyme biotech, c112-02), according to the manufacturer's instructions. the truncated segments sa-se were cloned into pcold-tf vector and the confirmed recombinant plasmids were transformed into e. coli bl21 and induced by isopropyl-β-d-thiogalactoside (iptg) at 16°c for 24 h. the truncated pedv s1 proteins were analyzed by sodium dodecylsulfatepolyacrylamide gel electrophoresis (sds-page) and western blot. all the recombinant proteins were purified by using nickel magnetic beads (biotool, shanghai, china) to prepare polyclonal antibodies. groups of five 8-week-old female balb/c mice were intraperitoneally immunized with 50 μg purified truncated pedv s1 proteins. antigens were emulsified in the same volume of complete freund's adjuvant (sigma, usa) for the initial immunization, then emulsified in incomplete freund's adjuvant on subsequent immunizations at 2week intervals for 6 weeks. phosphate-buffered saline (pbs) was used for the controlled trial with the same procedures. three days after the final boosting, the mice were narcotized and their blood samples were collected from the caudal vein. the collected antisera were diluted 1000-fold and used for indirect elisa, western blot and ifa to detect the immunoactivity of truncated proteins. female 8-week-old balb/c mice were immunized with 50 μg purified protein emulsified in the same volume of complete freund's adjuvant via intraperitoneal injection. this procedure was followed by three additional injections at 2-week intervals with the same dose of antigen emulsified in incomplete freund's adjuvant. three days after the final injection, spleen cells from immunized mice were fused with sp2/0 myeloma cells using polyethylene glycol 1450 (peg1450, sigma, usa), as previously described [15] . then the hybridoma cells were seeded into 96-well plates and selected in hypoxanthine-aminopterin-thymidine (hat) medium and hypoxanthine-thymidine (ht) medium. the cell culture supernatants of surviving clones were determined by indirect elisa for antibody reactivity and specificity. positive hybridomas were cloned four times by limiting dilution. ascites fluids were produced in pristane induced balb/c mice. indirect elisa was used to identify the immune reactivity of the truncated proteins and the screen of positive hybridoma cells. the elisa plates were plated with purified pedv s1 protein or synthesized peptides (400 ng/well) in carbonate bicarbonate buffer (15 mm na 2 co 3 , 35 mm nahco 3 [ph 9.6]) and coated at 4°c overnight. the plates were blocked for 1 h at 37°c using 5% non-fat dry milk in phosphate buffer with 0.05% tween-20 (pbst). after being washed thrice, the plates were incubated with 100 μl diluted anti-sera or antibodies at 37°c for 1 h. the plates were incubated with horseradish peroxidase (hrp) -conjugated goat anti-mouse igg (proteintech group, china) with 1:20,000 dilution in pbst at 37°c for 1 h after being washed thrice in pbst. then, plates were washed with pbst and incubated with 50 μl/well of tmb liquid (amresco, solon, ohio, usa) for 15 min at room temperature with protection from light. the results were read with od450 values after being stopped by 2 m h 2 so 4 (50 μl/well). to analyze the anti-sera or antibodies specificity interacted with pedv, vero cells were infected with pedv (multiplicity of infection, moi = 1), or mock infected with the medium, and then incubated for indicated times as previously described [16] . the cells were harvested and lysed using ripa lysis buffer (thermo, usa) containing protease inhibitor cocktail (bimake, usa) and phosphatase inhibitor cocktail (bimake, usa) on ice for 5 min. the cell lysates were then separated by 10% sds-page and transferred to the nitrocellulose (nc) membrane (ge healthcare, usa). the membranes were blocked with 5% non-fat dry milk in tbst (tbs with 0.1% polysorbate-20) for 1 h at room temperature (rt). the membranes were subsequently incubated with hrp-conjugated goat anti-mouse igg (1:6000 dilution in tbst) for 1 h at rt. proteins were visualized by using supersignal west pico chemiluminescent substrate (thermo fisher scientific, usa) according to the manufacturer's instructions. vero cells were plated in a six-well plate and infected with pedv when the cells reached approximately 90% confluence. at 24 h postinfection, the cells were fixed with 4% paraformaldehyde (sigma-aldrich) for 15 min and permeabilized with 0.1% triton x-100 (sigma-aldrich) for 10 min at room temperature. after being washed three times in phosphate-buffered saline (pbs), the cells were blocked with 10% bovine serum albumin (bsa) in pbs for 1 h at 37°c and then incubated with the primary antibody for 1 h. after three washes with pbs, cells were incubated with alexa fluor 488 donkey anti-mouse igg (h + l) antibody in the dark for 1 h at 37°c. following several washes, the fluorescence was visualized by using an olympus® ix73 inverted microscope. the spatial position of the identified epitope was analyzed by mapping the location on the 3d structure model of pedv s [17] by using pymol software [18, 19] , and the secondary structure of amino acid sequences of the identified epitope was also analyzed by protean software (dnastar's lasergene, inc., madison, wi, usa) [20] . all results are representative of three independent experiments. statistical analysis was performed using prism 5.0 software (graphpad). significance was determined by two-tailed student's t test. statistical significance: *p < 0.05, **p < 0.001. immunodominant region of s1 protein to accurately identify the immunodominant region of s1, the purified truncated s1 proteins (sa, sb, sc, sd and se) were used to immunize balb/c mice to prepare fig. 1 detection of recombinant proteins' antigenicity for polyclonal antisera (pcabs) by indirect elisa, western blot and ifa. a reactivity of antisera against the recombinant s1 protein by indirect elisa. b western blot analysis of the recombinant s1 protein with pcabs. the recombinant s1 protein (approximately 140 kda) was transferred to the nitrocellulose (nc) membrane, followed by the different pcabs (negative control, sa polyclonal antisera, sb polyclonal antisera, sc polyclonal antisera, sd polyclonal antisera and se polyclonal antisera) as primary antibody. c immunofluorescence analysis of se polyclonal antisera against pedv. vero cells were plated in six-well plates and inoculated with pedv (0.01 moi). twenty-four hours later, cells were fixed and incubated with se polyclonal antisera or normal mouse serum (negative control), and then incubated with alexa fluor 488 donkey anti-mouse igg (h + l) antibody. scale bars: 200 μm polyclonal antibodies. the antisera titers were determined by indirect elisa, western blot and ifa after four immunizations. the indirect elisa and western blot analysis showed that the se polyclonal antisera had the highest antibody titers against pedv s1 protein ( fig. 1a and b) , suggesting that se protein was the important immunodominant region of s1. the immunofluorescence signal for the pedv s1 protein was also detected by the se polyclonal antisera (fig. 1c) , further confirming that se polyclonal antisera could recognize the native pedv. collectively, these results indicated that the region of se (666-789 aa) was the immunodominant region of s1 protein. purified se protein was used to immunize balb/c mice to prepare mabs, and then determined the antisera titers using indirect elisa after four immunizations. the mouse with the highest antibody titers against se protein was used for cell fusion. after being subcloned by limiting dilution and screening for four times, one positive mab against se protein was identified and named 2e10. the mab 2e10 cell clone was used to prepare ascites containing mabs. the ascites was collected and purified using nab protein g spin columns (thermo fisher scientific, rockford, il). western blot analysis (fig. 2a) and immunofluorescence assay (ifa) (fig. 2b) were used to identify the specificity of the mabs against pedv. the results suggested that the mab 2e10 could be specifically reacted with native pedv protein. to identify the antigenic epitope recognized by mab 2e10, four truncated and overlapping 6 × his-tagged peptides (se1-se4) spanning the se fragments were designed (fig. 3a) and expressed using the bacterial system. the sequences of the primers were also shown in table 1 . all fusion proteins were predominantly expressed in soluble form in bacterial cells. subsequently, western blot was used to determine immune reactivity between the mab 2e10 and these se fragments. the results showed that mab 2e10 reacted with fragments spanning aa 690 to 736 (se2) and 707 to 761 (se3) but not aa 667 to 711 (se1) or 742 to 798 (se4), suggesting that the epitope recognized by 2e10 was located in aa 707 to 736 of pedv s1 protein (fig. 3b) . furthermore, we generated two deletion constructs of se (se5, se6) that span the overlaps of se2 and se3 (fig. 3a) to identify the antigenic epitope. as demonstrated by western blot, se6 was recognized by mab 2e10 (fig. 3c) , which suggested that se6 ( 717 isslssstfnstrelpgffy 736 ) epitope may be a harbored antigenic epitope. in order to further minimize the epitope of se6, two shortened peptides (se7-se8) were synthesized via solid-phase peptide synthesis (table 2) . using indirect elisa, we found that se8 showed a strong reaction with mab 2e10 as se6 did (fig. 4a) . the result suggested that se8 was the essential region for recognition by mab 2e10. according to the results, four shortened peptides (se9-se12) by deleting three amino acids at either the amino or the carboxy terminus in sequence from the se8 were synthesized ( table 2 ). in elisa, only se11 could be recognized by mab 2e10, which indicates that the 2e10-specific epitope was 722 sstfnstrelpg 733 (fig. 4a) . with the same method, the other four shortened peptides (se13-se16) were synthesized according to the peptide sequence of se8. the results showed that the peptide se16 ( 722 sstfnstrel 731 ) was strongly recognized by the mab 2e10 (fig. 4a) . taken together, these results demonstrate that se16 ( 722 sstfnstrel 731 ) is the minimal linear epitope required for reaction with the mab 2e10. to localize the identified epitope se16, a 3d structural model of pedv s was obtained from the protein data bank (pdb, id: 6u7k), and the spatial distribution was analyzed by pymol software. the structural visualization revealed that the identified epitope recognized by 2e10 was exposed on the surface of pedv s structure (fig. 4b) and partial peptide formed a beta-sheet structure (fig. 4c) . moreover, the identified epitope se16 had high antigenic index and hydrophilicity (fig. 4d) . it was suggested that the epitope can easily explore and induce host immune response in those infected animals. pedv s protein, one of the most important glycoproteins, contains multiple neutralizing epitopes to induce neutralizing antibodies. the s protein is divided into s1 (residues 1-789) and s2 (residues 790-1383) domains which are defined by the conserved nonamer and the gxcx motifs in coronavirus group ii members [21] . based on the information of other coronaviruses, we found that the s1 domain is thought to contain multiple virus neutralization epitopes and receptor bindingdomains [22] . the s2 domain forms the trans-membrane structure of the s protein, but can not induce neutralizing antibodies [23] . these properties make it possible that the s1 could be a suitable candidate for screening and identifying antigenic epitopes. epitopes are important antigenic elements of virus structural proteins, which could induce antibody production and cell-mediated immunity against viruses. therefore, epitopes are essential to develop epitope-based vaccines and diagnostics. preparation of mab is required to identify the epitopes of the s1 protein. we had tried to use intact s1 protein to immunize mice, but it was unsuccessful to get the mice serum with immune activity. because of unidentified but complex factors, we chose to divide the s1 protein into five fragments (sa, sb, sc, sd, se). the five truncated proteins were expressed and the immunogenicity of the proteins was identified. these results indicated that the se protein (666-789 aa) had good reactivity as the immunodominant region of s1 protein. then the se protein was selected as an immunogen to elicit the formation of monoclonal antibody. after cell fusion and four times of selection, mab 2e10 was chosen because of its specific reactivity with the se protein as well as the native s protein pedv. several domains containing neutralizing epitopes within the s protein were identified, such as residues 499-638 [24] , residues 636-798 [25] , residues 592-607 [26] and residues 575-639 [27] . these mocking epitopes had antigenic similarities with the pedv neutralizing epitopes. in this study, we expressed a series of truncated proteins (se1-se6) to map the epitopes of se protein. the se6 protein could be recognized by mab 2e10. generally, linear epitopes consist of six to nine or more continuous amino acid residues. so, the pepscan method was used to fig. 4 pepscan of the epitopes se6 and localization of the epitopes recognized by the mab 2e10. a elisa analysis of the truncated peptides se6-se16 with mab 2e10 and pbs. the surface b and cartoon c from se16 was labeled in the sequence chain view picture of pedv s, obtained from the protein data bank (pdb, id: 6u7k). d the structural features of se16 were predicted by protean software. the epitope se16 was shown in the boxes truncate the epitope se6 by deleting three amino acids at either the amino or the carboxy terminus in sequence respectively. a total of ten sub-segments (se7-se16) was synthesized and the results of elisa suggested that se16 could react with mab 2e10. above all these results, the epitope se16 is the immunodominant region of pedv s protein. the epitope se16 ( 722 sstfnstrel 731 ) was highly conserved among different strains of virulent pedv. further, the 3d structural visualization showed that the epitope se16 presented on the surface of pedv s and had a feature of high antigenic index and hydrophilicity. the character of this location makes this epitope easily explore and induce host immune response in the infected animals. we prepared a mab 2e10 that is specifically bound to pedv and identified a specific linear b-cell epitope (se16, sstfnstrel 731 ) of the mab. the epitope region of pedv s1 localized in the different regions in comparison with the earlier identified epitopes. therefore, the identified region is a novel b-cell antigenic epitope region of pedv s protein and the identified epitope has potential use for developing diagnostic reagent and effective vaccines for pedv. a new coronavirus-like particle associated with diarrhea in swine threedimensional sequential study of the intestinal surface in experimental porcine cv 777 coronavirus enteritis virus-like particles associated with porcine epidemic diarrhoea an apparently new syndrome of porcine epidemic diarrhoea chinese-like strain of porcine epidemic diarrhea virus molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china an outbreak of swine diarrhea of a newtype associated with coronavirus-like particles in japan prevalence of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus infection in korean pigs new variants of porcine epidemic diarrhea virus, china emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences phylogenetic and antigenic characterization of newly isolated porcine epidemic diarrhea viruses in japan porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines expression of synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic b subunit of escherichia coli heat-labile enterotoxin in tobacco plants coronavirus spike proteins in viral entry and pathogenesis monoclonal antibody to bone marrow stromal cell antigen 2 protein of swine suppression of virulent porcine epidemic diarrhea virus proliferation by the pi3k/akt/gsk-3alpha/beta pathway the 3.1 a cryo-em structure of the porcine epidemic diarrhea virus spike protein in the prefusion conformation identification of a novel linear b-cell epitope in nonstructural protein 11 of porcine reproductive and respiratory syndrome virus that are conserved in both genotypes identification of novel epitopes targeting nonstructural protein 2 of prrsv using monoclonal antibodies identification of two novel epitopes targeting glycoprotein e of pseudorabies virus using monoclonal antibodies furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry assembly of coronavirus spike protein into trimers and its role in epitope expression cooperative involvement of the s1 and s2 subunits of the murine coronavirus spike protein in receptor binding and extended host range identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus spike protein region (aa 636789) of porcine epidemic diarrhea virus is essential for induction of neutralizing antibodies identification of a novel linear b-cell epitope within the collagenase equivalent domain of porcine epidemic diarrhea virus spike glycoprotein identification of neutralizing monoclonal antibodies targeting novel conformational epitopes of the porcine epidemic diarrhoea virus spike protein publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank mr. yongjun chen for his assistance in animal feeding. availability of data and materials data generated and analyzed in this study are presented in this manuscript. data can be obtained by contacting the corresponding author. the authors declare that they have no competing interests.author details key: cord-334218-bkjfy66e authors: lin, jung-da; lin, chuen-fu; chung, wen-bin; chiou, ming-tang; lin, chao-nan title: impact of mated female nonproductive days in breeding herd after porcine epidemic diarrhea virus outbreak date: 2016-01-15 journal: plos one doi: 10.1371/journal.pone.0147316 sha: doc_id: 334218 cord_uid: bkjfy66e porcine epidemic diarrhea virus (pedv) is an important pathogen that has a significant economic impact on the swine industry by imposing a high rate of mortality in suckling piglets. however, limited information on the productivity values of gilts and sows infected with pedv is available. here, we evaluate the productivity index in gilts and sows during the 1-year period before (19 january 2013 to 18 january 2014) and after (19 january 2014 to 18 january 2015) a pedv outbreak from a 2000-sow breeding herd in taiwan. the farrowing rate (fr), return rate (rr), total pigs born per litter (tb), pigs born alive per litter (ba), weaning pigs per litter (wpl), pre-weaning mortality, percentage of sows mated by 7 days after weaning, weaning to first service interval (wfsi), mated female nonproductive days (npds), replacement rate of sows and sow culling rate were compared using productive records. the fr (-9.6%), rr (+9.8%), tb (-1.6), ba (-1.1), wpl (-1.1), sows mated by 7 days after weaning (-6.9%), wfsi (+0.8 days), npds (+6.9 days) and sow culling rate (+7.2%) were significantly different between the 1-year pre-pedv outbreak period and the post-pedv outbreak period. impacts of the pedv infection on the reproductive performance were more severe in pregnant gilts than in sows. in conclusion, these findings indicate that the outbreak of pedv caused an increase in the rate of npds in breeding herds. porcine epidemic diarrhea (ped) is an important swine disease that causes a significant impact in most pig-producing countries [1] . the causative agent, the ped virus (pedv), belongs to the genus alphacoronavirus, family coronaviridae, and order nidovirales [1] . although pedv was first observed in europe in the early 1970s [2] , it has become an increasing problem worldwide, including in the americas [3] [4] [5] , asia [6] [7] [8] [9] [10] [11] and europe [12, 13] . the devastating effect of pedv infection is mainly due to the acute watery diarrhea and dehydration induced in infected pigs that not only leads to high (80-100%) mortality in neonatal piglets [3, 9] but also impairs the health and performance of the surviving pigs [14] . the impact of pedv infection on the reproductive performance of gilts and sows depends on the period of pregnancy, during which females are exposed to the pathogen and the parity number [15] . the farrow rate (fr) (-3.8%), percentage of stillborn piglets per litter (+1.8%), and percentage of mummified fetuses per litter (+1.1%) were significantly different during the 4-month period of the pedv outbreak compared with the same period in the year before the outbreak [15] . however, limited information on the productivity index of the gilts and sows that were exposed to the pedv during the 1-year period of the pedv outbreak is available. mated female nonproductive days (npds) in the herd with the pedv outbreaks have not been reported. npds are the days that a mated sow or gilt is present in the herd and is neither gestating or lactating [16] . the formula for calculating npd is npd = 365−[(litter/female/year) × (gestation days + lactation days)]. several factors affect the npds [16] : i) replacement gilt days, entry to first service, entry to culling and entry to death; ii) weaning-to-first service days (the number of days from weaning until a female is mated again); iii) first to repeat service interval (days to find re-cycling females after breeding); iv) weaning to removal period; and v) death loss and gestation days that do not result in farrowing. therefore, npds represent key performance indicators of breeding herd performance. the objectives of the present study were to investigate the effects between a 1-year period before and after pedv outbreak on a sow's reproductive traits on a commercial pig farm in taiwan. this was a retrospective study that did not require direct intervention, retrieval of clinical specimens or animal experiments. the pig owners provided written consent for data collection and publication. no specific permissions were required for the location of the data because the data were collected with swine management software. the present study was conducted on a 2000-sow commercial pig farm in the central region of taiwan. the name of the pig farm is mai-chung pig farm with the following geographical coordinates (latitude/ longitude): 23°46 0 14.2@n and 120°14 0 56.0@e. on average, the productivity index of the study herd before the outbreak of pedv was superior to that recorded in other breeding herds in taiwan. the health of the herds was monitored by the herd veterinarian and the animal disease diagnostic center (addc), national pingtung university of science and technology (npust). the majority of the females were crossbreed landrace × yorkshires that were produced from their own grandparent stock. the veterinarian recommended vaccinating gilts against foot-and-mouth disease virus (fmdv), classical swine fever virus (csfv), aujeszky's disease (adv), porcine parvovirus (ppv), porcine circovirus type 2 (pcv2) and atrophic rhinitis between 24 and 30 weeks of age in replacement gilts. mass vaccinations for adv, pcv2 and fmdv were conducted in the sows every 4 months, 6 months and year, respectively. vaccinations of sows against csfv and porcine reproductive and respiratory syndrome virus (prrsv) were conducted on weaning day. no sows exhibited adv, pcv2, ppv, prrsv or bacterial abortion (streptococcus suis, erysipelothrix spp, leptospira interrogans) in this breeding herd during the study period as monitored by the herd veterinarian and the addc, npust through molecular diagnosis and serological surveillance. the target replacement rate of the sows by gilts was approximately 48% annually. sow culling due to age was planned to occur after the sixth parity, after the second return or vulva discharges at 14-21 days postservice. on 18 january 2014, pedv infection was confirmed from this pig farm by addc, npust [9] . farm immunization was performed using twice feedback with a 2-week interval for gilts and sows with approximately 10 ml of the homogenized intestines collected from pedv-infected suckling piglets (1 piglet for 20 sows on average). after the first feedback, more than 90% of the gilts and sows showed clinical signs of anorexia, diarrhea and vomiting. stool specimens were positive for pedv using real-time pcr assays conducted by addc, npust. less than 5% of the gilts and sows showed clinical signs of mild diarrhea and vomiting in the second feedback. suckling piglet mortality reduced and pedv-signs of gilts and sows stopped within 4 weeks after the first feedback immunization. until early august 2014, pig owners worried about the reemergence of pedv. therefore, feedback of gilts and sows was conducted with approximately 10 ml of the homogenized frozen intestines from pedv-infected suckling piglets (1 piglet for 100 sows on average) for three consecutive days. after this feedback event, more than 80% of the gilts and rarely sows showed clinical signs of mild anorexia, diarrhea and vomiting. no feedback immunization has been performed in this pig herd since august 2014. there were 20 and 8 sows (approximately 60% of them are primiparous sows) that were observed to have endemic pedv infections in early december 2014 and september 2015, respectively. these pedv infections were confirmed by addc, npust. during the post-pedv period, all of the gilts and sows were exposed to pedv by natural or/and feedback routes. data on gilt and sow reproductive traits were obtained from the swine management software of the herd from 19 january 2013 to 18 january 2015 (porcitec 2009 version, agritec). the collected data included sow identities, mating dates, mating results, number of days until the sows returned to estrus after mating, fr, return rates (rr), litters/mated female/year (lmfy), percentage of sows mated by 7 days after weaning, weaning to first service intervals (wfsi), farrowing intervals (fi), npds, replacement rate of sows, sow culling rate, total pigs born per litter (tb), pigs born alive per litter (ba), weaning pigs per litter (wpl) and pre-weaning mortality. the reproductive data from before and after the pedv outbreak were collected during periods from 19 january 2013 to 18 january 2014 and from 19 january 2014 to 18 january 2015, respectively. npds of the different parities (1st, 2nd and >2nd) during the 1 year pre-(2013) and post-(2014) pedv outbreak were compared using a two-way anova for multiple comparisons. the average number of mated females, average parity of farrowed sows, number of matings, number of farrowings, fr, rr, number of abortions, lmfy, percentage of sows mated by 7 days after weaning, wfsi, fi, npds, replacement rates of sows and sow culling rates of preand post-pedv outbreak periods were compared using a mann-whitney test. p values < 0.05 and <0.01 were considered to be statistically significant and highly significant, respectively. the productivity index of the sows in the herd during the 1-year period pre-and post-pedv outbreak is presented in table 1 . the number of matings showed a 58-point increase during the post-pedv outbreak period; however, the number of farrowings showed a 214-point decrease during this period. the average parity of the farrowed sows was significantly higher post-pedv outbreak compared with the period before the pedv outbreak (3.8 vs. 3.5). one year before the pedv outbreak, the fr, rr and number of abortions in the herd were 90.5%, 8.1% and 20, respectively (table 1) . however, a 9.6 percentage point decrease in fr (p<0.001) ( table 1 and fig 1a) , 9.8 percentage point increase of rr (p<0.001) ( table 1 and fig 1a) and a 4.8% increase of abortion rates (p = 0.0288) ( table 1) were observed after the pedv outbreak (table 1) . additionally, accounting for parity, the influence of the pedv outbreak on the fr and rr was more pronounced in pigs having their initial pregnancies (fig 1b and 1c ). interestingly, the number of abortions rapidly increased during the pedv outbreak period (19 january to 18 february 2014) (fig 2) . the abortion rate (ar) after the pedv outbreak was significantly higher than the ar before the pedv infection (+4.8%, p = 0.028) ( table 1) . together, these results indicated that the reduction in reproductive performance was more severe in pregnant gilts than in pregnant sows during the post-pedv outbreak period. litter size at birth and weaning tb, ba, total weaning pigs, wpl and pre-weaning mortality during the 1-year period pre-and post-pedv outbreak are shown in table 2 . tb (p<0.001), ba (p<0.001) and wpl (p<0.001) decreased significantly after the pedv outbreak compared with the 1-year period before pedv infection. the suckling piglets infected with pedv during the disease outbreak period (19 january to 18 february 2014) had a 41.7 percentage point increase in pre-weaning mortality (17.3% vs. 59%) compared with the same period in the year before the outbreak (19 january to 18 february 2013) (data not shown). * and ** were considered statistically significant and very highly significant, respectively. npds, replacement rate of sows and culling rate the percentage of sows mated within 7 days post-weaning, wfsi, fi, npds, lmfy, replacement rates of sows and sow culling rates are listed in table 1 . one year after the pedv outbreak, we recorded a 6.9 percentage point decrease in the sows mated within 7 days after weaning (p = 0.0121) ( table 1) , 0.8 percentage point increase in wfsi (p = 0.0131) ( table 1) , 3-day increase in fi (p = 0.0035) and 6.9-day increase in npds (p = 0.0126) ( table 1) , whereas the influence of the pedv outbreak on the lmfy (p = 0.0681), replacement rate of sows (p = 0.9206) and sow culling rate (p = 0.0575) was not significantly different between the preand post-outbreak periods. in addition, when parity was taken into account, the influence of the pedv outbreak on the npds was more pronounced in pigs with initial pregnancies (fig 3) . the percentage of sows mated within 7 days post-weaning was related to the wfsi, which is one of the factors that affected the npds. interestingly, the percentage of sows mated within 7 days post-weaning declined (from 86.7% to 45.6%) during the pedv outbreak period (19 january to 18 february 2014) compared with the same period in the year before the pedv outbreak (19 january to 18 february 2013) (fig 4) . the wfsi was highly variable during the post-pedv outbreak period compared with the period 1 year before the outbreak (fig 5) . in addition to this pig farm, we also analyzed the productivity index during the 1-year period before and after pedv outbreaks from one taiwanese farrow-to-finish herd (500 sows). the results showed that there were impacts on reproductive performance after pedv infection. we recorded a 6.2-day increase in npds during the post-outbreak periods (data not shown). taken together, these results indicate that there was a significant increase of the npds after the pedv outbreaks occurred, especially in pregnant gilts. the devastating effect of pedv infection is primarily due to the acute watery yellowish diarrhea and dehydration, with mortality rates ranging from 80 to 100% in suckling piglets under 2 weeks of age [9] . however, only a few studies have attempted to assess the impact of pedv infection on the reproductive and growth performance of sows [15] and surviving pigs [14] , respectively. in the present study, we compared the productivity index of gilts and sows between 1 year pre-and post-pedv outbreak in a taiwanese breeding herd. comparison of the fr (80.9% vs. 87.5%), rr (17.9% vs. 5.0%), ar (6.5% vs. 2.7%), tb (-1.6 vs. +0.3), ba (-1.1 vs. -0.1) and pre-weaning mortality (59% vs. 49.2%) revealed that post-pedv period effects were more severe than those observed in a study conducted in thailand [15] . this finding may be due to the following: i) the different observation periods (a 1-year period in the present study vs. a 4-month period in the thailand study); ii) the endemic pedv outbreak was present in this herd after the pandemic outbreak of pedv and iii) the different strains of pedv (us-like strain in taiwan vs. chinese-like strain in thailand) [9, 15, 17] . overall, these two studies consistently observed that the influence of the pedv outbreak on fr and rr was more pronounced in pigs that are early in their pregnancy. npds are key performance indicators of breeding herd performance. some factors that may affect npds include the following: i) replacement gilt timing, ii) weaning-to-first service days, iii) first service to repeat service intervals, iv) weaning to removal intervals, and v) death losses. to our knowledge, this is the first report showing the influence of pedv on npds in gilts and sows. factors that contributed to prolonging npds include increases in rr, number of abortions, percentage of sows mated within 7 days after weaning, wfsi and fi. in general, lactation levels declined during the pedv outbreak, especially in infected herds with high suckling mortality. incomplete uterine involution and tissue repair in early weaned sows contributed to the increased embryo loss in infected herds. the percentage of sows mated within 7 days after weaning rapidly declined (86.7% from 19 january to 18 february 2013 vs. 45.6% from19 january to 18 february 2014) during the pedv outbreak period, resulting in an increase in wfsi. a significant increase in ar was also observed by pijpers et al. [18] and olanratmanee et al [15] , although the mechanism underlying rr and fr is not known. previously, studies have shown that lactation intervals not only affect the average number of days from weaning to estrus but also the pregnancy rates and number of live embryos per female [19] . this fact explains why the fr and tb significantly decreased after the pedv outbreak in the present study. our results revealed that the influence of the pedv outbreak on npds was more pronounced in primiparous sows. the primiparous sows exhibited severe clinical signs of anorexia, diarrhea and vomiting when infected with pedv, while young sows were still utilizing nutrients for both growth and maintenance of the reproductive function. previous studies have reported that i) increasing feed intake during lactation can increase luteinizing hormone secretion and reduce the weaning-to-estrous and farrowing-to-estrous intervals in primiparous sows [20] , ii) protein (lysine) restriction throughout lactation alters circulating concentrations of somatotropic hormones and insulin at the end of lactation and has a negative impact on the post-weaning ovulation rate in primiparous sows [21] , iii) low lysine levels in primiparous lactating sows impaired follicular development and reduced the ability of follicles to support oocyte maturation [22] and iv) low-parity sows were more sensitive to lactational feed intake than high-parity sows in terms of wfsi [23] . we recorded a 0.8-day increase in wfsi post pedv infection (table 1) , occurring prominently in primiparous sows (fig 5) . these facts may explain why the influence of the pedv outbreak on npds was more pronounced in primiparous sows. additionally, maximizing feed intake during lactation is critical to improve the overall sow reproductive performance including productivity and longevity [24] . until now, pedv has not been considered as direct cause of reproductive problems. therefore, inadequate feed and nutrient intake when pedv infection may be cause the excessive body weight loss that can lead to short-term reproductive problems such as extended wfsi and smaller subsequent litter size. in the long run, problems such as higher culling rate of the breeding herd will result in low average parity; reduced pigs weaned per reproductive lifetime and increased production cost. in general, all phases of the reproductive cycle are related [24] . overall, these facts may provide an explanation to why pedv infection has such long-term impact on sow reproductive performance. the impact of pedv on npds in gilts and sows may be controlled or improved by the following methods: i) reducing the abortion rate (performance of the feedback immunization must be avoided when the gilts and sows are in their first month of pregnancy [15] ), ii) decreasing lactation length [25] , iii) decreasing the percentage of gilts in the breeding herd inventory [25] , iv) decreasing the female culling rate [25] [26] [27] and v) increasing the percentage of multiple matings [25] [26] [27] . the pig owners did not alter the gilt and sow management, including lactation length (25.6 days vs. 25.7 days)( table 1) , percentage of multiple matings (99.7% vs. 98.7%)( table 1) , parity of culled sows (sow culling due to age was planned to occur after the sixth parity), and percentage gilts in the breeding-female inventory (gilt pool: 1019 vs. 971), etc. (table 1) . therefore, the reproduction indices were normal until the second year after the first pedv outbreak. we recorded a 91.7 percentage of sows mated within 7 days post-weaning, 12.9 percentage of rr, 5.8-day of wfsi and 40.5-day of npds during the second year of the pedv outbreak (19 january 2015 to 18 november 2015) (s1 table) . overall, the control and improvement of impact of pedv on npds in gilts and sows may alter the management of females. outbreaks of pedv not only lead to high mortality in neonatal piglets and a poorer performance of surviving pigs but also impair the productivity index in gilts and sows. this is the first report to show the influence of pedv on npds in gilts and sows. these findings should contribute to an understanding of the effects of pedv outbreak post-infection on sow herds and to an identification of ways to curtail losses as a result of this disease. porcine epidemic diarrhea: a review of current epidemiology and available vaccines letter to the editor emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in canada distinct characteristics and complex evolution of pedv strains molecular characterization of the porcine epidemic diarrhea virus tw4/2014 in taiwan comparative genome analysis and molecular epidemiology of the reemerging porcine epidemic diarrhea virus strains isolated in korea outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea us-like strain of porcine epidemic diarrhea virus outbreaks in taiwan molecular characterization of pig epidemic diarrhoea viruses isolated in japan from epidemiology and vaccine of porcine epidemic diarrhea virus in china: a mini-review comparison of porcine epidemic diarrhea viruses from germany and the united states emergence of porcine epidemic diarrhea virus in southern germany impact of porcine epidemic diarrhea on performance of growing pigs impact of porcine epidemic diarrhea virus infection at different periods of pregnancy on subsequent reproductive performance in gilts and sows diseases of the reproductive system chinese-like strain of porcine epidemic diarrhea virus porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs effect of lactation duration on reproductive performance of sows influence of lactation length and feed intake on reproductive performance and blood concentrations of glucose, insulin and luteinizing hormone in primiparous sows protein (lysine) restriction in primiparous lactating sows: effects on metabolic state, somatotropic axis, and reproductive performance after weaning impact of dietary lysine intake during lactation on follicular development and oocyte maturation after weaning in primiparous sows feed intake pattern during lactation and subsequent reproductive performance of sows nutrition of the sow management factors associated with swine breedingherd productivity in the united states six component intervals of nonproductive days by breeding-female pigs on commercial farms by-parity nonproductive days and mating and culling measurements of female pigs in commercial breeding herds key: cord-346872-k5d5793a authors: yuan, peng; yang, zhou; song, han; wang, kai; yang, yang; xie, luyi; huang, shilei; liu, jia; ran, lin; song, zhenhui title: three main inducers of alphacoronavirus infection of enterocytes: sialic acid, proteases, and low ph date: 2018-09-03 journal: intervirology doi: 10.1159/000492424 sha: doc_id: 346872 cord_uid: k5d5793a transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) are similar coronaviruses, causing diseases characterized by vomiting, diarrhea, and death from severe dehydration in piglets. thus, they have caused huge losses to the swine-breeding industry worldwide. nowadays, they are easily transmitted among the continents via vehicles, equipment, and cargo. both viruses establish an infection in porcine enterocytes in the small intestine, and their spike (s) proteins play a key role in the virus-cell binding process under unfavorable conditions when the intestine with a low ph is filled with a thick layer of mucus and proteases. sialic acid, proteases, and low ph are three main inducers of coronavirus infection. however, the details of how sialic acid and low ph affect virus binding to the host cell are not determined, and the functions of the proteases are unknown. this review emphasizes the role of three factors in the invasion of tgev and pedv into porcine enterocytes and offers more insights into alphacoronavirus infection in the intestinal environment. two porcine coronaviruses, transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv), are clustered as different species into the alphacoronavirus genus. they both are important viral pathogens in piglets, causing similar pathological characteristics with acute diarrhea and dehydration [1, 2] , leading to massive losses in the modern swine-breeding industry worldwide. although their transmission route is limited to the fecal-oral route, as economic globalization increases rapidly and transportation develops remarkably, vehicles, equipment, and cargo have been convenient for these viruses to spread to all continents. tgev and pedv replicate in enterocytes of the small intestine and are the causative agent of a fatal diarrhea in newborn piglets. doyle and hutchings [3] described the hku12, and munia coronavirus hku13. both γ-and δ-covs have also been found in mammals [10] . α-, β-, γ-, and δ-covs are 4 genera of coronaviridae that are clustered together based on numerous studies and serological and genotypic criteria (table 1) . each genus has its own representative cov species. tgev, pedv, and human coronaviruses (hcov-229e and hcov-nl63) are typical viruses of the genus α-cov. the representative species of β-cov are mouse hepatitis virus and severe acute respiratory syndrome coronavirus (sars-cov). infectious bronchitis virus is currently the most studied virus in the γ-cov genus [11] . little is known about δ-cov. this review emphasizes the role of three factors (sialic acid, proteases, and low ph) in the invasion of tgev and pedv into porcine small intestine epithelial cells and provides information with respect to α-cov infection that brings new insights into virus research. the spike (s) protein of covs is essential for the interaction with receptors and the fusion of the viral particles and for cellular membranes. it also plays a crucial role in the interspecies transmission of covs. the interactions of cov s glycoproteins with receptors on the cell surface determine the host range and tissue tropism of covs [12, 13] . virus infection begins with the interplay between the fig. 1 . phylogenetic tree of the coronavirinae subfamily. the phylogenetic tree was built on the basis of the nucleotide sequences of complete spike genes from 213 coronaviruses. the nucleotide sequence alignment and the construction of the phylogenetic tree were completed using the mega5.0 program with a proper substitution model: d = transitions + transversions, and all other settings were maintained as default. the final depiction of the phylo-genetic tree was completed using itol on the internet. as the map shows, the coronavirus family is divided into 4 main groups, and 11 coronaviruses are regarded as representatives, which are shown in various colors. interestingly, the spike gene of pedv isolate strain zj14hz030301 is separated into a single group in the phylogenetic tree. s protein and its specific receptors, followed by penetration into the cells by a fusion event [14, 15] . the s protein, a class i fusion protein, is a membrane protein. it is the largest glycoprotein of covs [16] , projecting out from the surface of cov particles and forming a homotrimer structure called a peplomer. the s protein is responsible for the corona-like appearance of the surface projections in the electron microscope. the peplomer includes a globular portion and a protein stalk. by adopting the helical structure that is characteristic of class i virus fusion proteins, the protein stalk connects the globular portion to the transmembrane domain [17] . the nterminal s1 domain constitutes the globular region, and the stalk is made up of the membrane-proximal s2 domain. the n-terminal s1 domain and c-terminal s2 domain of the s protein play a similar role in all covs, the s1 region is related to receptor binding, and the s2 domain plays a role in the membrane fusion process. in addition, the s1 domain contains two subdomains, an nterminal domain (ntd) and a c-terminal domain (ctd) (fig. 2) . the two subdomains, called rbds (receptor binding domains), bind with specific cell receptors, including a series of proteins and sugars. thus, determinants in the s1 domain are not only crucial for initiating virus entry into cells, they also determine the cell and host tropism of covs [12] . tgev and pedv s proteins have many similarities in their secondary structure. the tgev s protein is produced as a 1,447-amino acid precursor polypeptide with a 16-residue signal peptide. according to analysis of the s protein sequences of covs with different biological phenotypes, there are 4 antigenic sites (c, b, d, and a in that order) in the n-terminal half of the s protein, among which the a site can induce neutralizing antibodies [4] and is highly conserved in tgev and porcine respiratory coronavirus (prcov), a respiratory variant of tgev. the s protein encoded by prcov lacks about 200 amino acids in the n-terminal region that contain determinants related to the enteropathogenicity of tgev [18] . pedv has a 150-to 220-kda spike glycoprotein with a homotrimeric structure. the pedv s protein is also a type i transmembrane glycoprotein that contains 5 regions, a signal peptide (residues 1-20), an s1 region (residues 21-793), an s2 region (residues 794-1385), a transmembrane domain (residues 1335-1358), and a cytoplasmic tail (residues 1359-1385) [19] . the s1 region is responsible for virus particle binding to the cellular receptors, whereas the s2 region participates in membrane fusion of the virus and host cells. like other covs, the s1 region possesses two subdomains comprising an ntd (residues 21-324) and a ctd (residues 253-638). the ctd of the s1 domain binds to a functional cellular receptor for pedv infection. sequence analyses of pedv prototype and variant strains reveal that the n terminus of the s protein changes more easily than the c terminus. in addition, a previous report suggested an interaction of ntd of the s1 domain with a coreceptor [20, 21] . simplified graphic of the structural domains of the main coronaviruses' spike proteins. the spike protein structure can be divided into the s1 and s2 domains, and the structural domains in the spike protein are located in the order (from c to the n terminus) as: transmembrane (tm), heptad repeats (hrs) in the s2 domain, c-terminal domain (ctd), and n-terminal domain (ntd) in the s1 domain as well as the signal peptide (sp). in the graphic, compared with tgev, prcov lacks an ntd, which is associated with enteric tropism. the rbd in the sars spike protein is shown as discontinuous in the graphic. the α-cov rbd comprises about 150 residues that adjoin the ctd in the s1 region ( fig. 2) . previous studies on the ctd concluded that the structure had the characteristic of independent expression of the s protein and preservation of its native structure maintained the binding specificity [4] . several structural studies indicate that the α-cov rbd adopts a β-barrel fold with 2 highly twisted β-sheets, in which 3 β-strands (β1, β3, and β7) run parallel and 3 disulfide bonds exist in the 3 β-structures [22] . the rbd of tgev is located within the ctd of the s1 domain [23] . in the tgev rbd crystal structure, the bent-strand β5 crosses both β-sheets. n-linked glycans are concentrated at one side of the β-barrel; the opposite side is not glycosylated and might be closer to other s protein domains. the n-and c-terminal ends of the rbd are located on the same side of the domain (terminal side); at the opposite side, 2 β-turns form the tip of the barrel in the tgev rbd [12] . like tgev, the crystal structure of a single domain unit in the prcov rbd adopts a β-barrel fold with 2 highly twisted β-sheets located in the ctd of the s1 domain and engages in binding to the host cell surface receptor. compared with the tgev s domain, the related prcov lacks the ntd, which is related to enteric tropism. the tgev or prcov rbd tips consist of 2 protruding β-turns (β1-β2 and β3-β4), each having a solvent-exposed aromatic residue (tyrosine or tryptophan) [20] . in the tertiary structure of the prcov rbd, 3 loops (β1-β2, β3-β4, and β5-β6) at the tips of the β-barrel domains are responsible for receptor binding. some researchers found that single amino acid mutations in the 3 loops completely or significantly reduced the ability of prcov rbd to bind to host receptors, and mutations outside the rbd had no effect on receptor recognition [24] . to date, there have been few reports on the rbd structure of pedv. it is interesting that 3 receptor binding mutant proteins, rbm1-1, rbm2-1, and rbm3-2 proteins, did not significantly reduce pedv papn-binding activities in virus infection [4] , suggesting the pedv s protein uses a receptor-binding mechanism different from tgev and prcov. pedv is further confirmed to have a broader receptor range than other α-covs [21] . during the progress of evolution and adaptation to diverse hosts, covs have evolved to use various receptors to enter host cells. different hosts or virus strains produce evolutionary diversity in the same virus family, and the binding of covs to susceptible cells seems to show variation in the receptors used that correspond to viral groups and species. these covs recognize distinct cellular receptors and coreceptors, such as proteins and sugars, to facilitate their penetration into cells [8] . currently, there are 4 main protein receptors: apn, angiotensin-converting enzyme 2 (ace2), carcinoembryonic antigen-related cell adhesion molecule 1 (ceacam1), and dipeptidyl peptidase 4 (dpp4). most members of α-cov use apn as the receptor for infecting host cells, such as tgev, pedv, and hcov-229e. apn, also known as cd13, is a 150-kda type ii transmembrane protein that belongs to a membrane-bound metalloprotease family [24] . interestingly, hcov-nl63, as an α-cov, shares the same cell entry receptor, identified as ace2, with sars-cov, which is a β-cov. ace2 is a type i integral membrane glycoprotein with an n-terminal extracellular domain comprising 2 α-helical lobes, between both of which there is a catalytic site with a coordinated zinc ion [25] . by contrast, the β-cov mouse hepatitis virus utilizes ceacam1 as a cell surface receptor for the s protein. ceacam1, the first identified cov receptor [26] , is a type i transmembrane multifunctional protein and a member of the immunoglobulin superfamily termed igsf. middle east respiratory syndrome-related cov, belonging to β-cov, has been shown to use dpp4 as a cell entry receptor. dpp4 (also known as cd26), a type ii membrane protein, is a multifunctional membrane-bound serine protease that forms homodimers on the surface of host cells. the dpp4 ectodomain comprises about 730 amino acids and has 2 domains, an α/β-hydrolase domain and an 8-bladed propeller [27] . among these cellular receptors and coreceptors, apn is a major cell entry receptor for covs. apn exists on the epithelial cell surface of different tissues. in particular, it is expressed abundantly in the brush border membrane of the small intestine, the kidney, and the respiratory tract [28] . most α-covs use apn as cell entry receptor. for example, previous studies showed that tgev uses porcine (p) apn as the receptor in the entry process [24] , whereas human (h) apn is a receptor for the infection by hcov-229e [29] . the reason why tgev uses apn as an entry receptor has not been clarified. it might be linked to its abundance on the surface of epithelial cells rather than its biological function, which seems to be dispensable for cov binding capacity [30, 31] . in the small intestine mucosa, apn occupies about 8% of the total protein content of the differentiated enterocytes [32] . for pedv, although there are many articles in which papn was proposed as the recep-doi: 10.1159/000492424 tor in pedv infection, this view has been questioned due to the lack of robust direct evidence. the characteristic structure of apn is a large glycosylated ectodomain with a zinc metal ion at the active site, which functions as a zinc-dependent protease responsible for cleavage of the n-terminal amino acids, mediated by the helah motif [33] . the enzymatic function of the papn catalyzes the removal of amino acid residues from the n termini of oligopeptides, and apn has been termed the "moonlighting enzyme" because of its many cell functions. apn can be cleaved into n-terminal (95 kda) and c-terminal (50 kda) subunits by trypsin digestion and comprises 4 domains (di-div) [29, 32] . it is heavily glycosylated and forms dimers through extensive div-div interactions. sequence conservation in the rbd tip of α-covs exerts a crucial function in which the apn recognition mode is highly conserved [29] . moreover, the specificity of apn with the recognition structure in the s protein is linked to the structure of the apn n-linked glycan and fusion with the rbd β1-β2 turn. in addition, the covs tyrosine and tryptophan residues are critical in forming the tgev rbd-apn structure. hcov-229e does not have a tyrosine in its rbd β1-β2 turn, hence it recognizes the human apn that lacks this form of glycosylation [20, 34] , meaning that hcov-229e recognition of apn must be unique. there have been some studies that determined the structure of a protruding tip for binding to small apn cavities in this human α-cov. the s proteins of pedv and tgev share high homology, but they have different host preferences. in addition, pedv has been verified to use a different receptor recognition model compared with tgev, prcov, and hcov-nl63. the n-terminal region in the pedv s1 domain binds to sugars, which are regarded as its coreceptor [20] . in addition to binding to defined protein receptors, some covs show a sialic acid-binding activity. at present covs in α-, β-, and γ-cov have developed variant sialic acid binding activities [35] . according to current research, 3 types of sugars have been characterized as receptors or coreceptors for cov entry into host cells: 5-nacetylneuraminic acid (neu5ac), 5-n-glycolylneuraminic acid (neu5gc), and 5-n-acetyl-9-o-acetyl neuraminic acid (neu5, 9ac2) [36] . recognition of sugars as co-receptors of tgev and pedv seems to be a strategy by which these viruses adapt to the living organism, meaning that tgev and pedv bind to sialic acid to survive under unfavorable intestinal tract conditions. tgev was first described to have a sialic acid binding activity in 1996 [37] . the sialic acid binding activity resides in the n-terminal portion of the s1 subunit that has been linked to the enteropathogenicity of tgev and that is absent from the s protein of prcov. the sialic acid preferentially recognized by tgev is n-glycolylneuraminic acid (neu5ac) [37] . the spike protein has a trimeric structure and retains its sialic acid binding activity in soluble forms of the protein [38, 39] . tgev recognizes and binds the sugar moieties of glycoconjugates that are highly o-glycosylated which promotes binding but is not sufficient for initiation of infection. it is believed that abundant sialic acid in mucins aids tgev to penetrate the mucus layer and then to get access to papn on the surface of the intestinal epithelial cells. thus, the efficiency of infection can be enhanced under unfavorable conditions. binding to papn and sialic acid are two independent processes. interestingly, binding to papn is more efficient in the absence of sialic acid [40] . there are few studies on the binding of pedv to sialic acids. pedv has been proven to have the ability to bind sialic acids. neu5ac presented the highest binding affinity with pedv s1 in experiments using a glycan array screening. moreover, the sialic acid binding region of pedv was confirmed as being in the ntd of the s protein (residues 1-320), similar to other covs [21] . however, it is unknown how the binding of sialic acid to the s protein can affect pedv entry into cells. increasing numbers of proteases have been demonstrated to participate in pedv and tgev infection of host cells in mechanisms where they do not act as receptors. these proteases are reportedly involved not only in adaptation of virus to innate immune response, but also in proteolytic processing of the s protein. covs always produce two types of cysteine proteases, a chymotrypsin-like main protease and papain-like proteases (pl1pro and pl2pro). in general, they are essential for replicase polyprotein processing and viral replicationtranscription. pl1pro, a nonstructural protein of tgev, resides in the nonstructural protein nsp3 subunits of tgev [41] . the structure of tgev pl1pro resembles a right hand with 3 distinct regions, the palm, thumb, and fingers (fig. 3) cysteine residues binding the zinc ion, and a catalytic triad formed by residues cys32, his183, and asp196 [42] [43] [44] . the tgev pl1pro protein, which contains a socalled usp-like binding site, exhibits deubiquitinating enzyme activity in vitro [45] . deubiquitinating enzymes play essential roles in the innate immune response with interferon (ifn) secretion into the intestine mucosa. in the intestine, the tgev entry is restricted by ifns in epithelial mucosa. the pathogen-associated molecular patterns of tgev, virus rnas, are sensed by pattern recognition receptors in host cells [46] , triggering the ifn expression. tgev arouses a early ifn-α production in intestinal secretions [47] , ifn-α can improve b-cell response to provide protection against reinfection [48] . ifn-λ is the main mucosal antiviral cytokine responsible for resisting virus invasion in the gut, such as tgev and pedv [49] , together with interleukin-22, which forms the tissue barrier in intestinal epithelial cells to reduce viral infection [50] . tgev pl1 protease possesses deubiquitinating activity and hydrolyzes the peptide that binds both lys48-and lys63-linked polyubiquitin chains [41] . regulation of signaling molecules by ubiquitin has a significant function in the activation of the ifn response. tgev pl1 binds and deubiquitinates retinoic acid-induced gene rig1 and stimulator of interferon gene sting, which are regulators in the ifn signaling pathway, and then the levels of phosphorylated ifn regulatory factor 3 exhibit reduced activity [51] , which counteracts ifn regulatory factor 3 translocating into the nucleus to activate the transcript of ifns, such as ifn-α/β and ifn-λ. tgev adopts the strategy to interfere and inhibit ifn secretion into the intestine gut to improve the efficiency of virus invasion. previous reports confirm that proteolytic processing of the s protein contributes to the fusion of the viral membrane with cellular membranes and is necessary for virus entry. proteases in virus entry are capable of cleaving the s protein. these proteases in the pig small intestine potentially facilitate pedv infection of host cells [52] . pedv successfully infects african green monkey kidney (vero) cells in vitro with extracellular trypsin [53] . the use of trypsin improves the possibility of pedv entry, because trypsin facilitates s protein-mediated fusion with the plasma membrane to deliver viral genomes into host cells. it has been shown that trypsin is helpful for syncytium formation in pedv infection of mdck cells [54] interestingly, pedv can also propagate without trypsin, suggesting that trypsin might be relevant for cellcell fusion rather than viral envelope-cell membrane fusion [55] . for pedv entry into vero cells under the trypsin-free conditions, endogenous proteases in endosome may adopt trypsin-like function, prompting pedv s-mediated fusion. ttsp is a type of trypsin-like serine protease termed type ii transmembrane serine protease. ttsp is confirmed to cleave and activate proteins on the surface of influenza viruses and covs, allowing multicycle replication in the absence of trypsin. ttsps are reportedly involved in the release of pedv virions [56] . tmprss2 and mspl are members of ttsps. tmprss2 and mspl exhibit trypsin-like features in the amplification of pedv in vitro in the absence of trypsin and play a vital role in cellcell fusion and virus-cell fusion. it is found that pedv s protein is colocalized with tmprss2 and mspl extensively and cleaved by coexpression with tmprss2 or mspl. tmprss2 and mspl could cleave pedv s protein into two fragments of the same size. the two ttsps interact with the pedv s protein to promote viral entry into cells by promoting cell-cell fusion and virus-cell fusion. moreover, mspl exhibited the strongest effect in the replication of pedv compared to tmprss2. interestingly, the adaptive capacity and the growth of pedv in vero cells expressing tmprss2 and mspl are higher than those in cells treated with trypsin [57] . it is comfirmed that pedv requires serine and serinelike proteases (fig. 4) for its entry through endocytosis in the early stages of infection. for cell-cell fusion, serine proteases are involved in pedv entry in an acidic phindependent manner [58] . cellular serine proteases posfig. 3 . ribbon drawing of the tgev pl1pro [40] . the entire structure resembles a hand, with the palm, thumb, and fingers represented in yellow, green, and blue, respectively. the catalytic triad is presented as magenta spheres, and the zinc ion is a gray sphere. color version available online doi: 10.1159/000492424 sess a catalytic triad of amino acids comprising his, asp, and ser, which are located in similar positions at the 3-dimensional structure. the nucleophilic ser is responsible for cleavage and is often replaced by a functionally and spatially equivalent cys in viral trypsin-like proteases [42, 44] . the asp of the active-site residues can be substituted by an equivalent glu. serine or serine-like proteases in the cytoplasm are required for the fusion between the pedv envelope and the host endosomal membrane. serine proteases activate pedv entry in a low ph-independent manner. new evidence has emerged that papn in the small intestine acts as a protease for pedv, which contrasts with the idea that papn plays role as a cell surface receptor. the structure of apn is shown in figure 5 . with regard to the similarities between pedv and tgev and the accordance of the pathology of pedv infection with the tissue distribution of papn, papn used to be regarded as a receptor for pedv, which was supported by some indirect evidence [24, 59] . however, pedv can also infect and propagate in papn-negative vero cells [53, 58] , indicating that there is no direct evidence to support the view that papn is a receptor for pedv. in current research, by employing cpk cells to express porcine homologs of papn, ace2, ceacam1, and dpp4, results were obtained showing that cpk cells were susceptible to tgev, but not to pedv. in addition, pedv infection was not affected by soluble papns, suggesting that pedv utilizes different receptors compared with other covs. by contrast, another study showed that when papn was overexpressed in porcine cpk cells, papn enabled pedv multiplication [59, 60] . another study showed that nonpermissive st cells expressing a papn gene supported productive infection of pedv of the host cells, showing that constitutive overexpression of papn directly promotes pedv multiplication [29] . in summary, papn more likely functions as a protease in pedv infection rather than as a receptor, which is supported by research evidence; however, the mechanism by which papn promotes pedv infection remains unknown. it is unquestionable that ph is important for the process of cov entry into cells. low ph is necessary for conformational changes of viral glycoproteins and proteolytic activation of viral glycoproteins by endosomal proteases [61] . it is reported that ph-dependent conformational changes occur in the cov peplomer [62] , and the s1 domain is released from peplomers on the surface of the virus. inside the endosome, endogenous proteases participating in membrane fusion are active in a low-ph environment. tgev and pedv tend to multiply at a low ph. the amount of tgev in the medium at ph 6.5 was 10-fold greater than that at ph 7.2, and the yield was almost 100-fold higher than that at ph 8.0 [63] . tgev binds to apn and enters the host cell via endocytosis. upon attaching to the porcine apn receptor, tgev is incorporated into the cell membrane and enters by way of caveola-dependent endocytosis [64] . subsequent membrane fusion is promoted by active cellular proteases in the endosome in a low-ph environment [65, 66] . using mdck cells overexpressing porcine apn, it has been demonstrated that acidification of the intracellular compartment promotes membrane fusion inside the endosome and cellular proteases are activated in a phdependent manner to facilitate membrane fusion [59, 65] . 1hxe) . the x-ray diffraction method was used to test the structure. the structure comprises 4 α-helixes (α1-α4) and 8 β-sheets (β1-β8) presented in diverse colors in the light of different domains. there is an na + site close to β2 (yellow), shown in stick form. different from the caveola-mediated uptake of tgev, pedv enters cells using endocytosis via clathrin-coated pits. after the first step of interacting with receptors on the cell surface, pedv penetration is facilitated by viral envelope fusion with the host cell plasma membrane, which happens inside a ph-dependent endosomal compartment. the endosomal cellular elements are very important for the activation of low phdependent proteases, rather than the virus needing an acidic environment to trigger its entry [41, 67] . the acidic conditions do not affect pedv itself, suggesting that acidic ph is not the only factor that affects viral infection of host cells. interaction between the s protein and the host cells is an indispensable step for viral entry into host cells. in the process of tgev and pedv entry into cells, the environmental conditions in the intestinal gut are complex and unfavorable. a thicker layer of mucus containing sialic acid covers the intestinal epithelium, and the small intestine generally has a low ph. in addition, the intestine is filled with proteases from the stomach and intestinal wall. these conditions make it hard for viruses to infect intestinal cells compared with infection of cultured cells. the selection of tgev and pedv rbds interacting with cell surface receptors is the result of pressure from immune surveillance. sialic acid usage by tgev and pedv promotes the efficient invasion into host cells. pedv and tgev invade enterocytes in the intestinal epithelium by exploiting proteases in the small intestine. tgev induces pl1pro to hijack cellular canonical pathways to prevent viral protein degradation, facilitating later virus entry into targeted cells. tmprss2 and mspl interact with the pedv s protein to promote viral entry into cells by promoting cell-cell fusion and virus-cell fusion. recently, porcine apn has been observed to function as a protease for pedv, not as a receptor, to promote viral multiplication during pedv entry into the cell, the mechanism of which requires further study. low ph in the initial stage of pedv entry into intestinal epithelial cells allows structural changes and protease activation during binding to surface receptors. molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus (tgev) and porcine respiratory coronavirus (prcv) field isolates co-circulating in a swine herd porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines a transmissible gastroenteritis in pigs antigenic modules in the n-terminal s1 region of the transmissible gastroenteritis virus 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spike fusion protein by trypsin in cell culture role of proteases in the release of porcine epidemic diarrhea virus from infected cells tmprss2 and mspl facilitate trypsin-independent porcine epidemic diarrhea virus replication in vero cells isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate conformational change of the coronavirus peplomer glycoprotein at ph 8.0 and 37 degrees c correlates with virus aggregation and virus-induced cell fusion porcine aminopeptidase n mediated polarized infection by porcine epidemic diarrhea virus in target cells clathrin-and serine proteases-dependent uptake of porcine epidemic diarrhea virus into vero cells monoclonal antibodies to the peplomer glycoprotein of coronavirus mouse hepatitis virus identify two subunits and detect a conformational change in the subunit released under mild alkaline conditions the influence of ph on the growth and stability of transmissible gastroenteritis virus in vitro evidence for a putative second receptor for porcine transmissible gastroenteritis virus on the villous enterocytes of newborn pigs the coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment a lysinemethionine exchange in a coronavirus surface protein transforms a retention motif into an endocytosis signal inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry we thank zhangcheng li (college of animal science, southwest university) for his guidance in constructing the evolutionary tree. this project has been supported by the fundamental research funds for the central universities (xdjk2017d082). no potential conflict of interest is reported by the authors. key: cord-316908-8ti75mru authors: wei, xiaona; she, gaoli; wu, tingting; xue, chunyi; cao, yongchang title: pedv enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway date: 2020-02-10 journal: vet res doi: 10.1186/s13567-020-0739-7 sha: doc_id: 316908 cord_uid: 8ti75mru with the emergence of highly pathogenic variant strains, porcine epidemic diarrhea virus (pedv) has led to significant economic loss in the global swine industry. many studies have described how coronaviruses enter cells, but information on pedv invasion strategies remains insufficient. given that the differences in gene sequences and pathogenicity between classical and mutant strains of pedv may lead to diverse invasion mechanisms, this study focused on the cellular entry pathways and cellular transport of the pedv gi and gii subtype strains in vero cells and ipec-j2 cells. we first characterized the kinetics of pedv entry into cells and found that the highest invasion rate of pedv was approximately 33% in the ipec-j2 cells and approximately 100% in the vero cells. to clarify the specific endocytic pathways, systematic research methods were used and showed that pedv enters cells via the clathrinand caveolae-mediated endocytosis pathways, in which dynamin ii, clathrin heavy chain, eps15, cholesterol, and caveolin-1 were indispensably involved. in addition, lipid raft extraction assay showed that pedv can also enter cells through lipid raft-mediated endocytosis. to investigate the trafficking of internalized pedv, we found that pedv entry into cells relied on low ph and internalized virions reached lysosomes through the early endosome–late endosome–lysosome pathway. the results concretely revealed the entry mechanisms of pedv and provided an insightful theoretical basis for the further understanding of pedv pathogenesis and guidance for new targets of antiviral drugs. as a type of alphacoronavirus, porcine epidemic diarrhea virus (pedv) has caused enormous economic loss to the global pork industry, especially after the emergence of highly pathogenic pedv variant strains in 2010. pedv was first reported in 1971 in the uk [1] , and afterward was also discovered in europe and asia [2] [3] [4] [5] . although pedv has persisted in asian swine-producing countries, it does not attract enough global attention. after october 2010, severe ped outbreaks occurred even in chinese pig farms that were already vaccinated with cv777inactivated or live-attenuated vaccines [2, [6] [7] [8] . in 2013, the first pedv outbreak occurred in the us and rapidly spread across the entire country [9] . molecular epidemiological results show the genetic differences between classical (gi subtype) and new pedv variant strains (gii subtype) [2, 3, 5, 6, 8, 9] . pedv is presently recognized worldwide due to dramatic changes observed in its epidemic character, pathogenic properties, and gene drift [10] . studies focused on its pathogenesis [11, 12] , immune evasion [13] and developing effective vaccines [14, 15] are progressing. a virus is a non-cellular life form that must rely on cells to complete its life cycle. the first step in virus infection is successful entry into cells. most enveloped viruses enter cells through cellular endocytosis [16, 17] . the endocytic pathways utilized by viruses vary, including clathrin-mediated endocytosis (cme), caveolaemediated endocytosis, lipid raft-mediated endocytosis, and macropinocytosis, among others. cme is the most classical and well-known endocytic pathway utilized by viruses. after binding to cell surface receptors, the virus is packaged by clathrin-coated pits (ccps) and transported to clathrin-coated vesicles (ccvs), in which virus particles as the "cargo" will be transported to the early endosomes [18, 19] . caveolae is a plasma-specific invagination structure with a diameter of 50-100 nm. when viral particles interact with receptors, caveolae coated with caveolin-1 invaginates and pinches off plasma membrane, then the caveolae vesicles mature into caveosomes and deliver "cargoes" to early endosomes [20, 21] . lipid raft are plasma membrane microdomains enriched in sphingolipids and cholesterol that participate in the lateral organization of the cell surface. raft-mediated endocytosis is the process of internalization of ligands and receptors by these domains [22] . the mechanisms of some coronaviruses entry into cells have already been studied, such as severe acute respiratory syndrome coronavirus (sars-cov), murine hepatitis virus (mhv), and human coronavirus (hcovs). entry of sars-cov into hepg2 and cos7 cells is clathrindependent while entry into vero e6 cells is clathrin-and caveolae-independent [23, 24] , but the lipid raft plays an important role in the process [25] . mhv entry into cells needs clathrin [26] [27] [28] , the same as hcov-nl63 [29] . for hcov-229e, caveolae-mediated endocytosis is utilized to enter human fibroblast cells [30] . sars-cov and mhv-cov can induce continuous micropinocytosis, but this occurs in the later phase during infection and is not associated with virus entry [31] . coronaviruses enter host cells via various endocytic pathways after viral spike glycoprotein (s) interacts with receptors and then initiates the endocytic process. internalized viruses are trafficked like cargoes to membrane fusion sites through specific transport routes. different covs have varying fusion sites [32] . the fusion site of the middle east respiratory syndrome coronavirus (mers-cov) takes place in the early endosome, while mhv and the feline infectious peritonitis virus (fipv) are transported to the lysosome to fuse. although there have been many studies on the invasion mechanism of cov, the invasion strategy of pedv has not yet been fully elucidated. in 2014, park et al. [33] revealed that pedv entry followed clathrin-mediated endocytosis and was dependent on a low ph for successful entry into cells. in their research, one pedv strain was studied in vero cells in the presence of trypsin and only the chemical inhibitors and confocal method were used to reveal the pedv entry. however, there are still important questions to address. considering that covs take advantage of different pathways to enter cells, whether different subtypes of pedv invade cells by different ways and whether pedv enter different types of cells through different ways remains to be determined. to concretely clarify the entry and transportation routes of pedv, we used vero and ipec-j2 cells as models for pedv entry and chose cv777-like strain gds09 and highly pathogenic variant strain gds01 to compare the invasion strategies of different pedv subtypes. our results will advance the understanding of the pathogenesis and immune evasion of pedv. vero cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (fbs, gibco) and antibiotics (100 u/ml penicillin and 100 μg/ml streptomycin). ipec-j2 cells were grown in dmem/nutrient mixture f-12 (dmem/f12) supplemented with 5% fbs and antibiotics. the pedv strains used in this study were cv777-like strain gds09 (gi subtype, genbank id: mh726408.1) and highly pathogenic variant strain gds01 (gii subtype, genbank id: km089829.1). exogenous trypsin (10 μg/ml) was added to proliferate pedv strains in vero cells and 5 μg/ ml trypsin was added in ipec-j2 cells. sbti (soy bean trypsin inhibitor type i; sigma no. t6522). the endocytic inhibitors used included dynasore (sigma-aldrich, no. 324410), chlorpromazine (cpz, sigma-aldrich, no. c0982), methyl-β-cyclodextrin (mβcd, sigma-aldrich, no. c4555), nystatin (sigma-aldrich, no. 475914), ammonium chloride (nh4cl, sigma-aldrich, no. a9434), and bafilomycin a1 (baf a1, sigma-aldrich, no. 196000). antibodies against clathrin heavy chain, caveolin 1, eea1, rab7, and lamp1 coupled with secondary goat anti-rabbit alexa fluor 488 and goat anti-mouse alexa fluor 594 were purchased from abcam. the mouse anti-pedv-s monoclonal antibody [34] and anti-pedv-n polyclonal antibody (prepared in our laboratory) were used in the immunofluorescence analysis and western blotting analysis, respectively. the overexpression plasmids of wild-type and mutant dynamin ii (gfp-dyn-wt and gfp-dyn-m), eps 15 (gfp-eps15-wt and gfp-eps15-m), and caveolin-1 (gfp-cav-wt and gfp-cav-m) were provided by prof. mark mcniven, mayo center for biomedical discovery (rochester, mn, usa). to investigate the trypsin dependency of pedv strains, vero cells were seeded in 6-well plates until confluence. after washed with pbs, cells were infected with pedv strains at a multiplicity of infection (moi) of 0.5 with or without trypsin (10 μg/ml) or with trypsin and 25 μg/ml sbti for 12 h before the quantification of the viruses by qrt-pcr. to test the dynamics of pedv internalization, vero or ipec-j2 cells were seeded in 12-well plates until confluence. the cells were pre-chilled for 10 min and inoculated with pedv at a moi of 0.5 at 4 °c for 1 h for virus binding. the cells were washed three times with ice-cold pbs to remove unbounded viruses and immediately warmed to 37 °c to initiate internalization. after incubation for the indicated time intervals, the cells were treated with proteinase k (1 mg/ml) at 4 °c for 30 min and then washed with pbs to inactivate and remove the non-internalized pedv particles. the control cells were then washed with pbs. the cells were collected and subjected to qrt-pcr analysis [34] . to test the effect of inhibitors on pedv internalization, it was necessary to evaluate the cytotoxicity of cell inhibitors. the cells were seeded in 96-well plates at a density of 2 × 10 5 cell/well, grown for 24 h, and treated with endocytic inhibitors at the indicated concentration for 4 h. then 10 μl of cck-8 solution was added to each well and incubated at 37 °c for 1 h. an absorbance of 450 nm was measured. the experiments were repeated three times independently. the concentration of each used inhibitor did not cause significant cytotoxicity to the cell viability. to test the effect of inhibitors on pedv internalization, the cells were pre-treated with different concentrations of drugs for 1 h and then infected with gds01 or gds09 strains at moi = 1 in the presence of drugs for 1 h. after washing with citrate buffer (ph 3.0) [35] and pbs, the cells were incubated with medium containing trypsin for 6 h or 9 h at 37 °c and collected for qrt-pcr and western blotting analysis, respectively. the expression of pedv n protein was detected by qrt-pcr and western blotting with gapdh as the reference. total rna was extracted using trizol (invitrogen) according to the manufacturer's instruction and cdna was synthesized with a revertra ace qpcr rt master mix with gdna remover kit (toyobo, osaka, japan). qpcr reaction was performed using a sybr premix ex taq ii kit (takara, tokyo, japan) using a light cycler 480 real-time pcr system (roche diagnostics, indianapolis, in, usa). for the western blotting analysis [36] , the cells were washed with pbs and lysed in ripa lysis buffer on ice for 30 min. after sds-page electrophoresis, proteins were transferred onto polyvinylidene fluoride (pvdf) membrane via the semidry method and immunoblotted with the corresponding antibodies. transfection of vero cells and ipec-j2 cells with the overexpression plasmids of wild-type and mutant dynamin ii (gfp-dyn-wt and gfp-dyn-m), eps 15 (gfp-eps15-wt and gfp-eps15-m), and caveolin-1 (gfp-cav-wt and gfp-cav-m) were performed using lipofectamine 2000 (invitrogen) transfection reagents according to the manufacturer's protocol. the cells were seeded in 12-well plates until 80% confluence. 24 h after transfection, the cells were infected with pedv at moi = 1 for 1 h. virus was moved with citrate buffer and pbs and replaced with fresh medium containing trypsin, and virus internalization was evaluated by confocal fluorescence microscope. for the rna interference assay, sirnas against dynamin ii (sidyn, sus scrofa: 5′-cac ctc atg atc aat aac a-3′, chlorocebus sabaeus 5′-cct aca tca aca cga acc a-3′), clathrin heavy chain (sichc, sus scrofa 5′-ccc ata cca tga ctg atg a-3′, chlorocebus sabaeus 5′-gat gaa cct tat gca tgc a-3′), eps 15 (sieps15, sus scrofa 5′-cct gtg gat att ctt gga a-3′, chlorocebus sabaeus 5′-ccc aga aac agc aag tac a-3′), and caveolin-1 (sicav, sus scrofa 5′-caa cat gca gaa aga aat a-3′, chlorocebus sabaeus 5′-cct tca ctg tga cga agt a-3′) were designed and synthesized based on the corresponding full-length mrna sequences of sus scrofa and chlorocebus sabaeus, sir-nas against rab7a (sirab7, 5′-gat ggt gga tga cag act a-3′), and vps39 (sivps39, 5′-gct tca aga gag act act a-3′) were designed and synthesized based on the corresponding mrna homologous sequences of sus scrofa and chlorocebus sabaeus. the control sirna (sicontrol) was designed and synthesized irrelevantly to all the known genes of sus scrofa and chlorocebus sabaeus genome, respectively, by ribobio (guangzhou, china). the cells were seeded in 12-well plates until 80% confluence. to ensure transfection efficiency, a second transfection was carried out at 24 h after the first transfection. at 48 h post-first transfection, the cells were infected with pedv at moi = 1 for 1 h. virus was moved with citrate buffer and pbs and replaced with fresh medium containing trypsin, and virus internalization was evaluated by qrt-pcr and western blotting at 6 hpi and 9 hpi, respectively. alexa-594 labeled transferrin (trf ) or alexa-555 labeled cholera toxin b subunit (ctb) were diluted at 1:500 and mixed with pedv at moi = 10. the cells were washed three times with pbs and added to the mixture of pedv and trf or ctb at 4 °c for 1 h and then incubated at 37 °c for 30 min for internalization. after washing with pbs, the cells were fixed, permeabilized, blocked, incubated with mouse anti-pedv-s monoclonal antibody, incubated with alexa 488-conjugated goat anti-mouse igg (h + l), stained with dapi, and analyzed using a confocal fluorescence microscope. light exposure was avoided throughout this experiment. cells cultured in glass-bottom dishes for 12 h were washed with ice-cold pbs and incubated with pedv at 4 °c for 1 h. cold viruses were replaced with pre-warmed medium, and the cells were immediately shifted to 37 °c. at specific time points, the cells were fixed in 4% paraformaldehyde at rt for 15 min after washing three times with pbs. permeabilization was carried with 0.5% triton x-100 at rt for 15 min. after washing with pbs, the cells were blocked with 5% bsa in pbst at rt for 60 min to block unspecific binding sites. the specific primary antibodies against chc, eea1, caveolin-1, rab7, lamp1, and anti-pedv-s antibody were used to probe the cells at 4 °c overnight. the cells were incubated with secondary antibodies (goat anti-rabbit igg antibody conjugated to alexa fluor 488 and goat anti-mouse igg antibody conjugated to alexa fluor 594) at 37 °c for 1 h. fluorescent images were acquired using the light-scanning module of a leica tcs sp8 sted 3× confocal microscope. the cells (5 × 10 7 ) were incubated or not incubated with pedv at 37 °c for 1 h, washed three times with ice-cold pbs, and lysed in 1 ml tne buffer (25 mm tris, 150 mm nacl, 5 mm edta, and ph 7.5) containing 1% triton x-100 and 1% phenylmethanesulfonyl fluoride (pmsf) on ice for 30 min. the homogenized cell lysates were centrifuged at 4 °c for 5 min at 1000 g and the supernatant was mixed with isometric 1 ml containing 80% sucrose in tne buffer. the lysates-sucrose mixture was placed at the bottom of ultracentrifugal tubes and overlaid with 7 ml 30% and 3 ml 5% sucrose in tne buffer. the cell lysates were ultracentrifuged at 4 °c for 16 h at 20 000 g in a sw41 rotor (beckman). after centrifugation, twelve 1 ml fractions were collected from the top to the bottom of the tubes. the fractions were concentrated with 6% peg at 4 °c overnight, and the pellets were resuspended in 100 μl of tne buffer after centrifuging at 4 °c for 30 min at 10 000 g. the localizations of lipid raft-associated protein caveolin-1 and pedv n protein were analyzed by western blotting. all the graphs were created with graphpad prism 6 software. all the data are presented as the means ± standard deviations (sds) from at least three independent experiments. significance was estimated using one-way anova with multiple comparisons to control. p values less than 0.05 were defined as the threshold for statistical significance. p values between 0.05 and 0.01 were marked with one asterisk, p values between 0.01 and 0.001 were marked with two asterisks, p values between 0.001 and 0.0001 were marked with three asterisks, and p values less than 0.0001 were marked with four asterisks. coronavirus entry is inextricably linked with proteolytic processing of the s protein. in most cases, pedv is trypsin dependent. thus, we investigated the trypsin dependency of both strains used in our research. as shown in figure 1a , gds01 strain needed trypsin while gds09 strain is trypsin independent. so, we added trypsin in the following assays to explore the invasion mechanism of pedv. the dynamics of viruses invading different kinds of cells vary, and there may be differences among various subtypes of the same virus. thus, it is necessary to know the entry dynamics of pedv before studying the endocytic pathways. the cells were incubated with pedv at moi = 0.5 at 4 °c for adsorption and shifted to 37 °c to initiate internalization. the adsorbed but not internalized virions were removed with proteinase k, and the pedv invasion rates at different time points were detected using qrt-pcr. the invasion kinetics ( figure 1 ) showed that most of the pedv particles were detached from the cells by proteinase k at the beginning of invasion. after 60 min in the vero cells ( figure 1b ) and 45 min in the ipec-j2 cells ( figure 1c ), nearly maximum proportions of viral particles completed the internalization. approximately 95% of the pedv particles entered the vero cells, while only 30% entered the ipec-j2 cells. notably, the gds01 strain demonstrated less efficient invasion than the gds09 strain, but there was no significant difference. dynamin ii plays an essential role in cellular membrane fusion during vesicle formation due to its gtpase activity, and it is necessary for clathrin-and caveolae-mediated endocytosis [35, 37] . thus, we explored the essentiality of dynamin ii in pedv entry using specific chemical inhibitors, overexpression of domain-negative mutants of dynamin ii, and sirna interference. dynasore [38] , a cell-permeable non-competitive inhibitor of dynamin ii, was used to pre-treat cells at different concentrations to analyze the effect on pedv entry. the cytotoxicity test showed that 50 μm of dynasore had no effect on the viability of the vero and ipec-j2 cells (additional file 1). cells were pre-treated with 30 μm and 50 μm dynasore for 1 h before pedv infection. dmso was used as a negative control. the effect of dynasore on pedv entry was quantified by qrt-pcr at 6 hpi. pedv invasion was significantly inhibited by dynasore. at a concentration of 50 μm, the invasion rates of the gds01 and gds09 strains into the vero cells were approximately 17% and 12%, and the invasion rates into the ipec-j2 cells were 8% and 63%, respectively ( figure 2a) . a comparison of the invasion rates of the gds01 and gds09 strains indicated that the gds01 strain was more sensitive to dynasore than the gds09 strain when invading the ipec-j2 cells but there was no significant difference between them when invading the vero cells. many studies used the overexpression of dominant negative mutants to explore the role of dynamin ii in virus entry [39, 40] . mutation of dynamin ii from 44k to 44a can inhibit gtpase activity and reduce endocytosis [41] . cells were transfected with wild-type and mutant types of dynamin ii respectively and infected with gds01 and gds09 strains at 24 h after transfection. the confocal results showed that the vero and ipec-j2 cells overexpressing wild-type dynamin ii (gfp-dyn-wt) were infected with pedv while the cells overexpressing mutant dynamin ii (gfp-dyn-m) were barely infected ( figure 2b ). sirna interference was also used to identify the importance of dynamin ii on virus entry [23, 35, 42] . sus scrofa and chlorocebus sabaeus sirnas of dynamin ii (sidyn) were designed and synthesized. the interference efficiency of sirna on the dynamin ii expression in the vero and ipec-j2 cells was obvious at both the mrna and protein levels (additional file 1). cells were infected with pedv after transfection twice and the internalized virions were quantified at 6 hpi and 9 hpi by qrt-pcr and western blotting assay, respectively. the qrt-pcr results ( figure 2c ) showed that the knockdown of dynamin ii expression reduced the pedv internalization. the internalization rates of the gds01 and gds09 strains into vero cells were approximately 50% and 57%, the internalization rates into the ipec-j2 cells were approximately 48% and 60%, respectively, but there was no significant difference between the gds01 and gds09 strains in the two cells ( figure 2c ). the same results were confirmed by western blotting ( figure 2d ). taken together, the results suggested that pedv entry relies on dynamin ii. clathrin-mediated endocytosis is the most commonly used and classical endocytic pathway for virus entry. to identify whether pedv utilized cme to enter cells, we co-inoculated the vero and ipec-j2 cells with pedv and trf, which is the most typical biomolecule that uses cme to enter cells [43] . the co-inoculation results figure 1 trypsin-dependency and kinetics of pedv entry into cells. a vero cells were seeded in 6-well plates until confluence. cells were washed with pbs and infected with pedv strains (moi = 0.5) without trypsin or in the presence of trypsin (10 μg/ml) or trypsin and 25 μg/ml sbti. cells were collected for qrt-pcr at 12 hpi. b, c vero cells (b) and ipec-j2 cells (c) were incubated with pedv gds01 and gds09 strains, respectively, at 4 °c for 1 h and shifted to 37 °c immediately to initiate internalization. at 0, 15, 30, 45, 60, 75, 90, 105, and 120 min after incubation, the cells were treated with proteinase k (1 mg/ml) at 4 °c for 30 min to inactivate the non-internalized virions. the control cells were washed with pbs. the invasion rates were calculated by qrt-pcr analysis. ****p < 0.001. dynamin ii involved in pedv entry. a cells were pre-treated with 30 μm and 50 μm dynasore at 37 °c for 1 h, respectively, and incubated with gds01 or gds09 strain for 1 h. dmso was used as a negative control. the cells were collected at 6 hpi for qrt-pcr assay to test the invasion efficiency of pedv. b vero and ipec-j2 cells were transfected with gfp-dyn-wt and gfp-dyn-m, respectively, and infected with pedv strains at 24 h after transfection. the cells were fixed at 12 hpi and stained for confocal analysis. c, d vero and ipec-j2 cells were transfected with sidyn twice and infected with pedv strains at 24 h after the second transfection. the invasion rates of pedv into the cells were detected at 6 hpi and 9 hpi for qrt-pcr and western blotting analysis, respectively. ctrl means control. scale bars indicate 25 μm. **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001. showed that pedv co-located with trf in the two types of cells ( figure 3a ), which means that pedv might utilize clathrin-mediated endocytosis to enter cells. to prove this conjecture, we used specific chemical inhibition, the overexpression of domain negative mutants of eps 15, the knockdown expression of chc and eps 15 by sirna, and the location of pedv in the cells to estimate the role of clathrin-mediated endocytosis in pedv entry. cpz is a specific chemical inhibitor used to block the cme pathway by preventing the assembly of ccps at the plasma membrane [44] . the cytotoxicity test showed that 30 μm cpz have no effect on the viability of the vero cells and 50 μm cpz have no effect on the viability of the ipec-j2 cells (additional file 2). vero and ipec-j2 cells were treated with cpz at different concentrations for 1 h and then infected with pedv. the internalization rates of pedv after cpz treatment were quantified by qrt-pcr and western blotting at 6 hpi and 9 hpi, respectively. the qrt-pcr results (figure 3b ) showed that pedv invasion was significantly inhibited by cpz. the invasion rates of the gds01 and gds09 strains at the highest drug concentrations were nearly 48% and 23% in the vero cells and 24% and 50% in the ipec-j2 cells, respectively. notably, there were no significant differences between the gds01 and gds09 strains in the vero cells but the gds01 strain was more sensitive than the gds09 strain in the ipec-j2 cells, reflecting the gds01 strain's significantly decreased invasion rates ( figure 3b ). the same results were also observed by western blotting ( figure 3c ) and ifa assay (additional files 3, 4) . the role of cme in endocytosis was also identified by the overexpression of gfp-tagged dominant negative mutants of eps 15 [45] . eps 15 is a critical component of ccps by interacting with adaptor protein 2 (ap-2), a major clathrin adaptor complex [46] . cells transfected with wild-type (gfp-eps15-wt) and mutant eps 15 (gfp-eps15-m) were infected with pedv strains at 24 h after transfection. the confocal results of the pedv invasion showed that the cells overexpressing wild-type eps 15 were infected with pedv while few infections were observed in the overexpressed gfp-eps15-m cells ( figure 3d ). sirna was also used to explore the role of cme in pedv entry by interfering with the expression of clathrin heavy chain (chc) and eps 15. chc and clathrin light chain form a triskelion shape, which is a key component for regulating the formation and disassembly of the clathrin lattice [47] . cells were infected with pedv strains after transfection twice, and the invasion rates of the viruses were assessed using qrt-pcr and western blotting assay at 6 hpi and 9 hpi, respectively. the quantitative experiments showed that knockdown of the expression of chc significantly reduced the invasion rates of pedv. the invasion rates of the gds01 and gds09 strains were 50% and 62% in the vero cells and 61% and 65% in the ipec-j2 cells, respectively, and there was no significant difference between the gds01 and gds09 strains ( figure 3e ). knockdown of the expression of eps 15 also significantly reduced the invasion rates of pedv. the invasion rates of the gds01 and gds09 strains were 61% and 65% in the vero cells and 51% and 66% in the ipec-j2 cells, respectively, and there was no significant difference between the gds01 and gds09 strains ( figure 3g ). the significant inhibition of sichc and sieps15 on pedv entry was also observed by western blotting assay (figures 3f, h) . to estimate whether pedv directly entered the cells through cme, we analyzed the localization of pedv and chc in the vero and ipec-j2 cells, respectively. pre-cooled cells were incubated with pedv at 4 °c for 1 h for adsorption and shifted to 37 °c for internalization. five min later, the cells were washed and fixed for observation using an ultrahigh-resolution laser confocal microscope. the confocal results showed that pedv particles co-located with chc protein in the vero and ipec-j2 cells ( figure 3i ), but some virions were not co-localized with chc. the results indicated that pedv can enter cells through the cme pathway, but cme may not be the only pathway utilized by pedv. cholesterol, an important component of cell membranes, embeds phospholipid bilayers and plays a crucial role in the fluidity of cell membranes [48] . many studies showed that most enveloped virus relied on cholesterol to invade cells [49, 50] . if a virus invades cells, depending on the presence of cholesterol, it will be sensitive to cholesterol extractants. mβcd can eliminate cholesterol on the plasma membrane of cells [51] . nystatin can bind to the cholesterol-enriched regions of cell membrane and then decompose cholesterol and impair cholesterol synthesis [52] . the cytotoxicity test showed that the maximum tolerance concentrations of vero and ipec-j2 cells to mβcd were 3 mm and 1.5 mm, respectively, and the maximum tolerance concentrations to nystatin were 30 μm and 50 μm, respectively (additional file 5). cells were pre-treated with different concentrations of mβcd and nystatin for 1 h, then infected with pedv. the effects of drugs on pedv entry were estimated by qrt-pcr and western blotting at 6 hpi and 9 hpi, respectively. mβcd showed a significant inhibition of pedv entry. the internalization rates of the gds01 and gds09 strains after mβcd treatment were approximately 4% and 6% in the vero cells and approximately 5% and 35% in the ipec-j2 cells. there were no significant differences between the gds01 and gds09 strains in the mβcd-treated vero figure 3 pedv entry relies on the cme pathway. a vero cells and ipec-j2 cells were incubated with mixture of alexa-594 labeled trf (red) and pedv (green) at 4 °c for 1 h, and then shifted to 37 °c for 30 min. the cells were fixed and stained for pedv using monoclonal antibody against pedv s protein. the cellular localizations of trf and pedv were observed with a confocal fluorescence microscope. light exposure was avoided throughout this process. b, c. the vero cells were pre-treated with 10 μm and 30 μm of cpz, and the ipec-j2 cells were pre-treated with 30 μm and 50 μm of cpz, respectively, at 37 °c for 1 h and incubated with gds01 or gds09 strains for 1 h. double-distilled water was used as a negative control. the cells were collected at 6 hpi and 9 hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. d the vero cells (left) and ipec-j2 cells (right) were transfected with gfp-eps15-wt and gfp-eps15-m, respectively, and infected with pedv strains at 24 h after transfection. the cells were fixed at 12 hpi and stained for confocal analysis. e-h the vero cells and ipec-j2 cells were transfected with sichc and sieps 15 and infected with pedv strains at 24 h after the second transfection. the cells were collected at 6 hpi and 9 hpi for qrt-pcr and western blotting analysis, respectively. i the cells were pre-cooled at 4 °c for 15 min, incubated with pedv strains at 4 °c for 1 h, shifted to 37 °c for 5 min to initiate internalization, and washed for three times to remove un-internalized viral particles. the cells were fixed and stained with anti-pedv-s (red) and anti-chc (green) primary antibodies. ctrl means control. scale bars indicate 50 μm in a, 25 μm in d, and 5 μm in i. **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001. cells but in the ipec-j2 cells, the gds01 strain was more sensitive to mβcd ( figure 4a ). the results were confirmed by western blotting ( figure 4b ) and ifa assay (additional files 3 and 4). pedv entry was significantly inhibited by nystatin treatment. the internalization rates of the gds01 and gds09 strains after nystatin treatment were approximately 75% and 25% in the vero cells and approximately 65% and 45% in the ipec-j2 cells. the gds09 strain was more sensitive to nystatin than the gds01 strain in both the vero and ipec-j2 cells ( figure 4c ). the same results were confirmed by western blotting ( figure 4d ) and ifa assay (additional files 3 and 4). to further evaluate the importance of cholesterol, cells pre-treated with mβcd were supplemented with exogenous cholesterol and then infected with pedv, and the changes in the viral invasion rates were quantified by qrt-pcr at 6 hpi. the results showed that adding exogenous cholesterol could significantly increase the invasion rate of pedv. the average invasion rate of the gds01 and gds09 strains increased from 10% to over 90% in the vero cells and from 10% and 50% to approximately 95% in the ipec-j2 cells ( figure 4e ). the results indicated that pedv invading and entering cells depended on cholesterol but the two pedv subtypes showed different degrees of dependence on cholesterol when entering the vero and ipec-j2 cells. as both caveolae and lipid raft are rich in cholesterol, they are sensitive to cholesterol inhibitors [53, 54] . to identify whether caveolae-mediated endocytosis was involved in pedv entry, cells were co-inoculated with pedv and ctb, which entered the cells after interactions with specific receptors [55] . the localization results showed that pedv co-localized with ctb in vero and ipec-j2 cells ( figure 5a ), which means pedv may utilize caveolae-mediated endocytosis to enter cells. as the caveolae is mainly coated with caveolin-1 [56] , knocking down the expression and separating the interaction factors with caveolin-1 blocked the caveolae-mediated endocytic pathway. overexpression of the domain-negative mutant of caveolin-1 [57] blocked its interaction with the interaction factors. cells were transfected with wild-type caveolin-1 (gfp-cav-wt) and mutant caveolin-1 (gfp-cav-m), then infected with pedv at 24 h after transfection, and fixed for confocal observation at 12 hpi. the results showed that pedv infected gfp-cav-wt-overexpressing cells but barely infected gfp-cav-m-overexpressing vero or ipec-j2 cells ( figure 5b ). sirnas (sicav) were designed and synthetized to knockdown caveolin-1 expression. cells were transfected with sicav twice and then infected with pedv. after the second transfection, the invasion rates of pedv were measured by qrt-pcr and western blotting at 6 hpi and 9 hpi, respectively. the qrt-pcr results showed that the knockdown of caveolin-1 expression reduced the internalization of pedv. the inhibition rates in the gds01 and gds09 strains were 53% and 32% in the vero cells and 33% and 40% in the ipec-j2 cells, respectively (figure 5c) . compared with the gds09 strain, the gds01 strain showed a higher degree of reduction in the invasion rate in the vero cells but there was no significant difference in the ipec-j2 cells ( figure 5c ). the same results were confirmed by western blotting assay ( figure 5d ). to identify the role of caveolae in pedv entry, we investigated the cellular localization of pedv with caveolin-1. pre-cooled cells were incubated with pedv at 4 °c and then shifted to 37 °c for internalization. the cells were then washed and fixed for 10 min for observation with an ultrahigh-resolution laser confocal microscope. the cellular localization results showed that pedv was colocated with caveolin-1 in the vero and ipec-j2 cells ( figure 5e ). pedv can enter cells through the caveolaemediated pathway. if pedv can enter cells through the lipid raft pathway, the viral components should be contained in lipid raft enrichment layer after isolated by sucrose density gradient centrifugation [58] . after incubation with pedv, the cells were lysed and subjected to sucrose gradient centrifugation. the products were extracted from the top down and a total of 12 fractions were obtained for western blotting analysis. caveolin-1 was used as the protein marker representing the lipid raft layer [58] . the results showed that pedv could be detected in the upper lipid raft enrichment layer. almost all the virions were concentrated in the lipid raft enrichment layer in the vero cells ( figure 6a ) and virions were detected in both the upper and lower components in the ipec-j2 cells ( figure 6b ). the differences between the gds01 and gds09 strains were the proportion of virions in the upper and lower components in the ipec-j2 cells. the gds01 particles were mainly present in the upper layer while amounts of gds09 particles were present in the lower layer (figure 6b) . the results indicated that pedv utilized lipid rafts to enter cells. viruses that enter cells via endocytosis are usually trafficked by endocytic vesicles to early endosomes for sorting and are transported to late endosomes or fused with early endosomes [59] . if viruses do not fuse in the early endosomes and release genomes into the cytoplasm, they will enter the late endosomes with the further acidification and maturation of the early endosomes. similarly, figure 4 pedv entry relies on cholesterol. a, b vero cells and ipec-j2 cells were pre-treated with 1.5 mm and 3 mm and 1 mm and 1.5 mm mβcd, respectively, at 37 °c for 1 h and incubated with gds01 or gds09 strains for 1 h. double-distilled water was used as a negative control. the cells were collected at 6 hpi and 9 hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. c, d the vero cells and ipec-j2 cells were pre-treated with 10 μm and 30 μm and 30 μm and 50 μm of nystatin, respectively, at 37 °c for 1 h and incubated with gds01 or gds09 strains for 1 h. dmso was used as a negative control. the cells were collected at 6 hpi and 9 hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. e the vero cells and ipec-j2 cells were pre-treated with different concentrations of mβcd at 37 °c for 1 h, supplemented with 400 μg/ml of soluble cholesterol at 37 °c for 1 h, and infected with pedv strains for 1 h. the cells were collected at 6 hpi for qrt-pcr assay to test the invasion efficiency of pedv. *p < 0.05; **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001. a vero cells and ipec-j2 cells were incubated with a mixture of alexa-555 labeled ctb (red) and pedv (green) at 4 °c for 1 h, and then shifted to 37 °c for 30 min. the cells were fixed and stained for pedv using monoclonal antibody against s protein. the cellular localizations of ctb and pedv were observed with a confocal fluorescence microscope. light exposure was avoided throughout this process. b vero cells (up) and ipec-j2 cells (down) were transfected with wild-type caveolin-1 (gfp-cav-wt) and domain negative mutant of caveolin-1 (gfp-cav-m), respectively, and infected them with pedv strains at 24 h after transfection. the cells were fixed at 12 hpi and stained for confocal analysis. c, d the vero cells and ipec-j2 cells were transfected with sicav twice and infected with pedv strains at 24 h after the second transfection. the cells were collected at 6 hpi and 9 hpi for qrt-pcr and western blotting analysis, respectively. ctrl means control. e cells were pre-cooled at 4 °c for 15 min, incubated with pedv strains at 4 °c for 1 h, shifted to 37 °c to initiate internalization for 10 min, and washed for three times to remove viral particles that were not internalized. the cells were fixed and stained with anti-pedv-s (red) and anti-caveolin-1 (green) primary antibodies. scale bars indicate 50 μm in a, 25 μm in b, and 5 μm in e. *p < 0.05; **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001. if the ph environment in the late endosomes does not meet the requirements for conformational changes of viral glycoproteins to cause membrane fusion, viruses will enter the lysosomes with the transportation of the late endosomes and finally achieve membrane fusion. viruses require acidic ph to traffic between endosomes. therefore, it is necessary to clarify whether pedv relies on a low ph environment. nh4cl is an inhibitor of endosome acidification [60] . baf a1 is a v-atpase inhibitor that can block the traffic of endocytic cargoes from early endosomes to late endosomes and inhibit the stability of low ph environments in the lysosome lumen [61] . cells were pre-treated with different concentrations of nh4cl and baf a1 (additional file 6) and infected with pedv (additional file 4). the invasion rates of pedv were measured by qrt-pcr and western blotting at 6 hpi and 9 hpi, respectively. the results showed that cells pretreated with nh4cl significantly inhibited pedv entry. the invasion rates in the gds01 and gds09 strains were approximately 30% and 15% in the vero cells and 50% and 73% in the ipec-j2 cells ( figure 7a ). this significant inhibition was also confirmed by western blotting (figure 7b ) and ifa assay (additional files 3 and 4). baf a1 can significantly inhibit pedv entry. the invasion rates in the gds01 and gds09 strains were approximately 75% and 5% in the vero cells and 41% and 30% in the ipec-j2 cells ( figure 7c ). western blotting ( figure 7d ) and ifa assay (additional files 3, 4) also confirmed the inhibition of baf a1. the results proved that pedv entry requires low ph. early endosomes mature into late endosomes by increasing intraluminal acidity through proton pump activity. late endosomes can become larger vesicles by fusing with the same type of endosomes, so they mostly exist in the form of a multivesicular body (mvb). late endosomes release rab5, incorporate rab7, and prepare to fuse with lysosomes [62] . to explore whether pedv particles are trafficked after internalization, we interfered with the expression of rab7 [63] involved in late endosomes and vps39 [32] involved in late endosometo-lysosome maturation and identified whether viral particles co-located with early endosomes, late endosomes and lysosomes. knockdown of the expression of rab7 (sirab7) can significantly inhibit the invasion efficiency of pedv. the invasion rates of the gds01 and gds09 strains were 49% and 51% in the vero cells and 82% and 38% in the ipec-j2 cells ( figure 8a) . similarly, the knockdown of the expression of vps39 (sivps39) also significantly inhibited the invasion efficiency of pedv. the invasion rates of the gds01 and gds09 strains were 68% and 56% in the vero cells and 45% and 50% in the ipec-j2 cells ( figure 8b ). for cellular location by pedv observation, pre-cooled cells were incubated with pedv at 4 °c and then shifted to 37 °c for internalization. the cells were washed and fixed at different time points after shift for observation using an ultrahigh-resolution laser confocal microscope. cellular localization assays showed that internalized pedv could co-locate with eea1, the early endosome protein marker, in the vero were incubated or not with pedv at 37 °c for 1 h and then lysed in tne buffer containing 1% triton x-100 and 1% pmsf on ice for 30 min. after mixing with isometric 80% sucrose, the homogenized cell lysates were subjected to ultracentrifugation after being overlaid with 30% and 5% sucrose. after centrifugation, a total of 12 fractions were collected from the top to the bottom of the tubes. the localizations of the lipid raft-associated protein caveolin-1 and pedv n protein were analyzed by western blotting after being concentrated with 6% peg6000. and ipec-j2 cells 30 min after endocytosis ( figure 8c ) and could co-locate with rab7, the late endosome protein marker, 40 min after endocytosis ( figure 8d ), while the late endosomes were mostly in the form of mvb. colocalization of pedv with lamp1 (lysosomal associated membrane protein 1), an important lysosome membrane component, was also observed 50 min after endocytosis in the two types of cells ( figure 8e ). the results demonstrated that pedv was trafficked to the lysosomes after entering the cells through endocytosis, and there were no differences between the two pedv genotypes and cells. since highly pathogenic variant strains emerged in 2010, pedv has attracted global attention. many studies have reported that vaccines based on cv777 or cv777-like strains have low protection efficiency against re-emerging variant strains [3, 4, [6] [7] [8] . genotyping showed that pedv strains can be sorted into two genotypes, gi subtypes (classical) and gii subtypes (variant). the nucleotide sequence of s1 subunit of s protein is 75-90% similar between gi and gii, which shows high variability [10, 64] . as the main antigen of pedv, the s protein incubated with gds01 or gds09 strains for 1 h. double-distilled water was used as a negative control. the cells were collected 6 hpi and 9 hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. c, d the vero cells and ipec-j2 cells were pre-treated with 200 nm and 400 nm baf a1 at 37 °c for 1 h and then incubated with gds01 or gds09 strains for 1 h. dmso was used as a negative control. the cells were collected 6 hpi and 9 hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001. vero cells and ipec-j2 cells were transfected with sirab7 and sivps39 twice, respectively, and then infected with pedv strains at 24 h after the second transfection. the cells were collected at 6 hpi for qrt-pcr analysis. ctrl means control. c-e the vero cells and ipec-j2 cells were pre-cooled at 4 °c for 15 min, incubated with pedv strains at 4 °c for 1 h, shifted to 37 °c to initiate internalization. the non-internalized viral particles were removed by washing. 30 min after shifting, the cells were fixed and stained with anti-pedv-s (red) and anti-eea1 (green) primary antibodies (c). 40 min after shifting, the cells were fixed and stained with anti-pedv-s (red) and anti-rab7 (green) primary antibodies (d). 50 min after shifting, the cells were fixed and stained with anti-pedv-s (red) and anti-lamp1 (green) primary antibodies (e). scale bars indicate 5 μm in c-e. *p < 0.05; **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001. plays an important role in inducing immune responses and viral entry. mutation of the s gene may lead to different mechanisms of virus invasion, which may help elucidate the pathogenesis and immune evasion of pedv. as sars-cov utilizes varying endocytic routes to invade different cells [23, 24] , we wondered whether pedvs can enter cells through different pathways. burkard et al. clarified that coronavirus entered cells through the endosome/lysosome pathway and was proteolytic dependent. the furin cleavage site just upstream of the fusion peptide (fp) of the s protein was the key to determining the fusion site of the viral membrane [32] . while the s protein of pedv does not have the furin cleavage site, a conserved arginine just upstream of the putative fp as the potential cleavage site can be cleaved by trypsin [65] . whether the theory mentioned above is applicable to pedv thus needs further study. here, we explored the gi and gii subtype pathways of pedv entry into vero and ipec-j2 cells, respectively, and the transportation route after internalization. our results showed that two the subtypes of pedv utilized clathrin-, caveolae-, and lipid raft-mediated endocytosis to enter the vero and ipec-j2 cells, but the utilization efficiency of each endocytic pathway varied depending on the different genotypes and types of cells. to describe the dynamic curve of pedv entry, the appropriate viral scavenger is extremely important to remove virus particles effectively adsorbed on the cell surface. we compared the effects of citrate buffer (ph 3.0) with proteinase k (1 mg/ml), and the latter exhibited a stronger capacity to remove viruses. the results of dynamic invasion showed that the virus invaded vero cells 100% within 60 min, while the invasion efficiency of the ipec-j2 cells was only approximately 30%. although ipec-j2 cells are considered the host cells of pedv, the cell lines cultured in vitro lost their polar growth state in vivo [66] , which possibly affects the viral recognition and reduces the infection efficiency of the virus. gds01 showed a lower invasion rate than gds09, but there was no significant difference. dynamin ii, a gtpase, plays an important role in endocytosis by pinching the endocytic vesicles off the plasma and is necessary in clathrin-and caveolae-mediated endocytosis. we found that pedv entry is dynamin iidependent, although the sensitivity of the gds01 and gds09 strains to dynasore varied. considering the low specificity of chemistry inhibitor, dominant negative mutant and sirna-mediated knockdown of dynamin ii were carried out to evaluate dynamin ii for pedv infection. both the gds01 and gds09 strains were inhibited by sidynamin ii with no significant difference. clathrin-mediated endocytosis is a classical and commonly used pathway for most enveloped viruses. many coronaviruses use cme to enter cells, such as sars-cov entry into hepg2 cells and cos7 cells and phev entry into neuro-2a cells [23, 35] . to ascertain whether pedv utilized cme to enter cells, the chemistry inhibitor cpz was used to prevent clathrin assembly and further block cme. clathrin is composed of light chain (clc) and heavy chain (chc) which form clathrin lattices under the interaction of ap-2 and eps 15. both cpz pre-treatment and sirna-mediated knockdown of chc and eps 15 can significantly reduce the invasion rate of pedv into cells, with no significant difference between the gds01 and gds09 strains. dominant negative mutants can provide a more specific method to study endocytic pathways by separating the prototype protein from their interaction regulatory factors. in this study, we also showed that cells overexpressing dominant negative mutant of eps 15 were barely infected with pedv but cells overexpressing wild-type eps 15 were infected as normal. however, the inhibition of endocytosis by overexpressing dominant negative mutants may be compensated through other clathrin-independent endocytic pathways. when viruses enter cells through cme, they are carried by clathrincoated vesicles. co-localization of viral particles and chc indicated that pedv entry relies on cme. although caveolae and lipid rafts have the same components, such as caveolin-1, gm1, and cholesterol, they are two completely different endocytic pathways. before investigating whether pedv can use these two pathways to enter cells, we first examined whether pedv invasion depends on cholesterol. the cholesterol inhibitors nystatin and mβcd had significant inhibitory effects on pedv entry. nystatin had higher inhibitory effects on gds09 entry than gds01. the inhibitory effects of mβcd on vero cells were similar for gds09 and gds01 strains. mβcd effects were lower on gds09 than gds01 in ipec-j2 cells. we hypothesized that gds01 cell invasion mainly depended on cholesterol on the cell surface, while gds09 depended on the presence of cholesterol on the cell surface and cholesterol synthesis. exogenous cholesterol supplementation also confirmed the importance of cholesterol in pedv entry. endocytic vesicles formed in the caveolae-mediated pathway were coated with caveolin-1, which plays a critical role in the process. dominant negative mutant, rna interference, and the cellular co-localization of caveolin-1 with viral particles provided further evidence that pedv entry needed caveolin-1. collectively, both subtypes of pedv entered the vero and ipec-j2 cells through caveolae-mediated endocytosis. however, park et al. [33] showed that pedv entry was independent of caveolae-coated pit assembly by treating vero cells with nystatin. the different results may be explained by different operational details. firstly, nystatin was used before and during the incubation of pedv in this study, while only before incubation in the research of park et al. the concentrations of nystatin used in the two studies were different. the highest concentration of nystatin used in park's research was 20 μm [33] , while the highest concentration we used was 30 μm in vero cells. when the concentration is 10 μm, nystatin does not inhibit the entry of pedv, which is consistent with park's results. secondly, park et al. added methyl cellulose to block second-cycle infection [33] , while we added nothing except trypsin in medium. whether these reasons cause two different results needs further study. lipid raft acted as a platform for cell signal transduction and viral invasion, distributed in an island form on the plasma membrane of cells, and was isolated by sucrose density gradient centrifugation. western blotting analysis showed that pedv n protein located in the lipid raft (upper layer) with caveolin-1 in the cells. as shown in figure 6b , pedv n protein is also present in the bottom layer when infected with ipec-j2 cells, which may be due to the different composition of the plasma membranes of the two kinds of cells. we demonstrated that pedv gi subtype gds09 and gii subtype gds01 strains could enter vero and ipec-j2 cells via the clathrin-, caveolae-, and lipid raft-mediated endocytosis pathways. furthermore, we also found that the invasion efficiency of the two strains was different with different endocytosis pathway. these differences between gds01 and gds09 strains may be due to the difference of s gene especially the s1 region of s gene (homology was about 92%), which is responsible for cell entry and membrane fusion by binding with receptor. the difference of gene may lead to the difference of binding ability or affinity between s protein and receptor, thus leading to the different utilization or initiation efficiency of different endocytosis pathways. however, whether the different gene sequence causes different invasion efficiency between gds01 and gds09 strains needs further study. after internalization, viral particles are transported by specific endosomes for membrane fusion. the classical transit route is the endo-/lysosomal pathway, in which endocytic cargoes are transported along endocytic vesicles, early endosomes, and late endosomes-lysosomes. park et al. have confirmed that nh 4 cl and baf-a1 could inhibit pedv entry [33] , which is consistent with our results, but needs to be confirmed by different methods. in this study, in addition to chemical inhibitors, we also used sirna interference and cellular localization of virus particles to identify the role of ph and endosomes. the results of this study revealed that pedv entry relied on low ph, which means that internalized pedv particles are transported to endosomes and lysosomes, as demonstrated by the co-localization of viral particles with eea1, rab7, and lamp1. liu et al. [67] reported that pedv s protein was activated by lysosomal cysteine proteases to activate pedv entry. however, based on the data, we could not conclude that membrane fusion occurred at the lysosomes; more technical methods are necessary to demonstrate the mechanism. in conclusion, studying the internalization and intracellular trafficking mechanism of pedv are important to understand viral pathogenesis and benefit to the development of future therapies strategies. this study demonstrated that both the gi and gii subtypes of pedv enter vero and ipec-j2 cells via the clathrin-, caveolae-, and lipid raft-mediated endocytosis pathways, but the efficiency of each endocytosis pathway varies depending on the different genotypes and types of cells. the internalized pedv entered the lysosomes through the early and late endosomes. the results of this study provide a theoretical basis for the further understanding of pedv pathogenesis to find new targets of antiviral drugs. virus-like particles 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isolation and recombinant analysis of variants of porcine epidemic diarrhea virus strains from beijing oral administration of coated pedv-loaded microspheres elicited pedv-specific immunity in weaned piglets a flagellin-adjuvanted ped subunit vaccine improved protective efficiency against pedv variant challenge in pigs virus entry at a glance virus entry paradigms mechanisms of clathrin-mediated endocytosis mechanisms of endocytosis virus entry by endocytosis endocytosis via caveolae regulation of raft-dependent endocytosis clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ace2 with the cytoplasmic tail deleted sars coronavirus entry into host cells through a novel clathrin-and caveolaeindependent endocytic pathway lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle role of endocytosis and low ph in murine hepatitis virus strain a59 cell entry endosomal proteolysis by 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springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank prof. qiang yang, college of veterinary medicine, nanjing agricultural university, china, for providing us with ipec-j2 cells. we would also like to thank prof. mark mcniven, mayo center for biomedical discovery, usa, for providing us with overexpression vector of wild-type and domain negative mutant of dynamin ii and caveolin-1. supplementary information accompanies this paper at https ://doi. org/10.1186/s1356 7-020-0739-7. and ipec-j2 cells were treated with different concentrations of dynasore at 37 °c for 4 h. cck-8 solution was added to each well at 37 °c for 1 h, and absorptions of 450 nm were detected. dmso was used as a negative control. (b) the vero cells and ipec-j2 cells were transfected with sidyn, and the second transfection was carried out at 24 h after the first transfection. the inference efficiency was detected by qrt-pcr and western blotting at 48 h after the first transfection. ctrl means control. **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001.additional file 2. clathrin-mediated endocytosis is involved in pedv entry. (a) vero cells and ipec-j2 cells were treated with different concentrations of cpz at 37 °c for 4 h. cck-8 solution was added to each well at 37 °c for 1 h, and absorptions of 450 nm were detected. double-distilled water was used as a negative control. (b, c) the vero cells and ipec-j2 cells were transfected with sichc and sieps15, and the second transfection was carried out at 24 h after the first transfection. the inference efficiency was detected by qrt-pcr and western blotting at 48 h after the first transfection. ctrl means control. *p < 0.05; **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001. cells were seeded in 12-well plates until confluence. cells were pre-treated with 30 μm cpz, 3 mm mβcd, 30 μm nystatin, 50 mm nh4cl and 200 nm baf a1 respectively, at 37 °c for 1 h and incubated with gds01 (a) and gds09 (b) strains for 1 h. double-distilled water was used as a negative control. the cells were collected at 9 hpi and detected by immunofluorescence staining against the pedv s protein (green). nuclei were stained with dapi (blue). scale bars indicate 100 μm. cells were seeded in 12-well plates until confluence. cells were pre-treated with 50 μm cpz, 1.5 mm mβcd, 50 μm nystatin, 50 mm nh4cl and 400 nm baf a1 respectively, at 37 °c for 1 h and incubated with gds01 (a) and gds09 (b) strains for 1 h. double-distilled water was used as a negative control. the cells were collected at 15 hpi and detected by immunofluorescence staining against the pedv s protein (green). nuclei were stained with dapi (blue). scale bars indicate 100 μm. the authors declare that they have no competing interests.received: 5 october 2019 accepted: 7 january 2020 key: cord-331919-6kistim2 authors: song, daesub; park, bongkyun title: porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines date: 2012-01-22 journal: virus genes doi: 10.1007/s11262-012-0713-1 sha: doc_id: 331919 cord_uid: 6kistim2 the porcine epidemic diarrhoea virus (pedv), a member of the coronaviridae family, causes acute diarrhoea and dehydration in pigs. although it was first identified in europe, it has become increasingly problematic in many asian countries, including korea, china, japan, the philippines, and thailand. the economic impacts of the pedv are substantial, given that it results in significant morbidity and mortality in neonatal piglets and is associated with increased costs related to vaccination and disinfection. recently, progress has been made in understanding the molecular epidemiology of pedv, thereby leading to the development of new vaccines. in the current review, we first describe the molecular and genetic characteristics of the pedv. then we discuss its molecular epidemiology and diagnosis, what vaccines are available, and how pedv can be treated. porcine epidemic diarrhoea (ped), which was first observed among english feeder and fattening pigs in 1971 [1] , is a devastating enteric disease that manifests as sporadic outbreaks during the winter, leading to damage on breeding farms. characterised by watery diarrhoea, ped resembles transmissible gastroenteritis (tge), but has less of an effect on suckling pigs (\4-to 5-week old); this is what allowed ped to first be distinguished from the tge virus and other recognized enteropathogenic agents. as it spread through europe, the disease was named 'epidemic viral diarrhoea (evd) .' unlikely what the disease used to outbreak in fattening pigs, different types of evd caused acute diarrhoea in pigs of all ages in 1976. this type of evd was classified as evd type 2 [1] , different from the previously recognized type 1 [2] . evd type2 was turned out to be caused by a coronavirus-like agent in 1978 [3, 4] using experimentally designed cv777 which caused enteropathogenic infection in both piglets [3] and fattening swine. this was when the disease started to be called as 'porcine epidemic diarrhoea (ped)' [4] . both transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhoea virus (pedv) are classified into group 1 of the genus coronavirus. pedv ranges in diameter from 95 to 1990 nm (mean diameter: 130 nm), including its projection. as in many particles with a tendency to a round shape, the pedv contains a centrally located electronopaque body; it also possesses widely spaced club-shaped projections measuring 18-23 nm in length. the internal structure of the virus remains unknown. the pedv is sensitive to ether and chloroform and has a density in sucrose of 1.18 g/ml. the virus possesses a glycosylated peplomer (spike, s) protein, poll (p1), envelope (e), glycosylated membrane (m) protein, and an unglycosylated rna-binding nucleocapsid (n) protein [5] . cell cultureadapted pedv loses its infectivity when heated to c60°c for 30 min, but is moderately stable at 50°c; further, the virus is stable between ph 5.0 and 9.0 at 4°c and between ph 6.5 and 7.5 at 37°c [6] . pedv shows no haemagglutinating activity [6] . the pedv propagates by orally inoculating piglets, after which, during the early stages of diarrhoea, it collects in the tissues and contents of the small intestine [3] . vero (african green monkey kidney) cells support the serial propagation of pedv and grow successfully in laboratory conditions; however, growth of the virus depends on the presence of trypsin in the cell culture medium. cytopathic effects consist of vacuolation and formation of syncytia. during the 1980s and 1990s, ped was prevalent throughout europe, in countries such as belgium, england, germany, france, the netherlands, and switzerland (table 1) . ped is currently a source of concern in asia, where outbreaks are often more acute and severe than those observed in europe. in this respect, and in their high mortality rates, these resemble tgev outbreaks. for example, japanese outbreaks between september 1993 and june 1994 resulted in 14,000 deaths, with mortality ranging from 30 to 100% in suckling pigs. during these epidemics, adult pigs showed only temporary decreases in appetite and milk production [7] . another ped epidemic occurred in the winter of 1996, during which 39,509 of 56,256 infant farrow-to-finish piglets died after experiencing diarrhoea. between january 1992 and december 1993, 56.3% of viral enteric cases in infant pigs surveyed in korea were attributable to pedv, rather than tgev. the vast majority of outbreaks (90%) involved piglets \10-day-old [8] . the clinical lesions of pedv in the small intestine of piglets were similar to those of tgev. lesions are confined to the small intestine, which is distended with yellow fluid (fig. 1 ). ped outbreaks also occurred in thailand from 2007 to 2008. most of the affected farms reported that the disease first occurred in farrowing barns; 100% of newborn piglets were subsequently lost. between august 1997 and july 1999, 50.4% of 1,258 enteric cases across 5 korean provinces were diagnosed as ped [9] ; further, a korean abattoir serosurvey found pedv seroprevalences of 17.6-79% (mean of 45%) in samples from 469 pigs from seven provinces. cumulatively, these results suggest that the virus had become endemic in some areas [10] ( table 1) . however, recent outbreaks seemed to be concentrated in certain countries where pork industry is prevalent, such as philippines, south korea and china. pedv is an enveloped virus possessing an approximately 28 kb, positive-sense, single-stranded rna genome with a 5 0 cap and a 3 0 polyadenylated tail [11, 12] . the genome comprises a 5 0 untranslated region (utr), a 3 0 utr, and at least seven open reading frames (orfs) that encode 4 structural proteins [spike (s), envelope (e), membrane (m), and nucleocapsid (n)] and three non-structural proteins (replicases 1a and 1b, and orf3); these are arranged on the genome in the order 5 the polymerase gene consists of 2 large orfs, 1a and 1b, that cover the 5 0 two-third of the genome and encode the non-structural replicase polyproteins (replicases 1a and 1b). genes for the major structural proteins s (150-220 kda), week of age died from severe watery diarrhoea after showing signs of dehydration. after the acute outbreak, piglets were anorectic, depressed, vomiting, and producing water faeces that did not contain any signs of blood. necropsies of deceased piglets from the kimpo outbreak uncovered gross lesions in the small intestines, which were typically fluidic, distended, and yellow, containing a mass of curdled, undigested milk. atrophy of the villi caused the walls of the small intestines to become thin and almost transparent virus genes (2012) 44:167-175 169 e (7 kda), m (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) , and n (58 kda) are located downstream of the polymerase gene [15, 18, 20] . the orf3 gene, which is an accessory gene, is located between the structural genes. it encodes an accessory protein, the number and sequence of which varies among different coronaviruses [20] . the pedv s protein is a type i glycoprotein composed of 1,383 amino acids (aa). it contains a signal peptide (1-18 aa), neutralising epitopes (499-638, 748-755, 764-771, and 1,368-1,374 aa), a transmembrane domain (1,334-1,356 aa), and a short cytoplasmic domain. the s protein can also be divided into s1 (1-789 aa) and s2 (790-1,383 aa) domains based on its homology with s proteins of other coronaviruses [21] [22] [23] [24] [25] [26] . like other coronavirus s proteins, the pedv s protein is a glycoprotein peplomer (surface antigen) on the viral surface, where it plays a pivotal role in regulating interactions with specific host cell receptor glycoproteins to mediate viral entry, and stimulating induction of neutralising antibodies in the natural host [15, 21-23, 26, 27] . moreover, it is associated with growth adaptation in vitro, and attenuation of virulence in vivo [28, 29] . thus, the s glycoprotein would be a primary target for the development of effective vaccines against pedv. additional studies of this structure are essential for understanding the genetic relationships between, and diversity of, pedv isolates, the epidemiological status of pedv in the field, and the association between genetic mutations and viral function [29] [30] [31] [32] [33] . it was reported that aminopeptidase n is the receptor of tgev, human coronavirus 229e (hcov-229e) and feline coronavirus (fecov) which all belong to group i coronavirus including pedv [34] . the pedv m protein, the most abundant envelope component, is a triple-spanning structural membrane glycoprotein with a short amino-terminal domain on the outside of the virus and a long carboxy-terminal domain on the inside [35] . the m protein not only plays an important role in the viral assembly process [36, 37] but also induces antibodies that neutralise the virus in the presence of its complement [37, 38] . the m protein may play a role in a-interferon (a-ifn) induction [39] . coexpression of m and e proteins allowed the formation of pseudoparticles, which exhibited interferogenic activity similar to that of complete virions [40] . additional work on the m glycoprotein should increase our understanding of the genetic relationships between, and the diversity of pedv isolates and the epidemic situation of pedv in the field [30, [41] [42] [43] [44] [45] . the n protein, which binds to virion rna and provides a structural basis for the helical nucleocapsid, is a basic phosphoprotein associated with the genome [5, 16, 18, 46] . as such, it can be used as the target for the accurate and early diagnosis of pedv infection. it has been suggested that n protein epitopes may be important for induction of cell-mediated immunity (cmi) [38] . whereas the genes encoding the structural proteins have been thoroughly investigated for most coronaviruses, little is known about the functions of the accessory proteins, which are not generally required for virus replication in cultured cells [46] [47] [48] [49] . on the contrary, their expression might lead to decreases of viral fitness in vitro, and mutants with inactivated accessory genes are easily selected during serial passage through cell cultures [50] [51] [52] [53] . in general, accessory genes are maintained in field strains [50, 54] , and their loss mainly results in attenuation in the natural host [55] [56] [57] . in the case of pedv, the only accessory gene is orf3, which is thought to influence virulence; cell culture adaptation has been used to alter the orf3 gene in order to reduce virulence [52] , as has been done for tgev [53] . differentiation of orf3 genes between the highly cell-adapted viruses and field viruses could be a marker of adaptation to cell culture and attenuation of the virus [52, 58, 59] . thus, measures of variation in orf3 gene differentiation could be a valuable tool in molecular epidemiology studies of the pedv [42, 45, 52, 59] . genetic and phylogenetic analyses based on the s, m, and orf3 genes have been used to determine the relatedness of pedv isolates, both within korea and among various countries in which pedv has surfaced. research on part of the s gene, and on the entire m gene, have suggested that pedvs can be separated into three groups (g1, g2, g3), which have three subgroups (g1-1, g1-2, g1-3) [32] . according to analysis of the partial s genes, the g1 pedvs had 95.1-100% nucleotide sequence similarities with each other, and they had 93.5-96.7 and 88.7-91.5% sequence identities with the g2 and g3 pedvs, respectively. the g2 pedvs had 96.7-99.8% similarities with each other, and they had 91.8-93.0% similarities with the g3 pedvs [32] . these results reflect the existence of genetic diversity among the korean pedv isolates (fig. 3) . the majority of the korean pedv isolates are closely related to chinese strains [32] . the chinese pedv clade also contains all strains isolated from several outbreaks of pedv that have occurred in thailand since late 2007. these classifications have been based on the phylogenetic relationship of the s genes, and support the results of park et al. [32] . recently, after analyzing the full s gene-based phylogenetic tree [31] reported that all pedvs can be separated into 2 clusters, and that korean field isolates are more closely related to each other. in 2006, an analysis of the m gene of 6 pedvs isolated from the faeces of chinese piglets indicated that the isolates compose a separate cluster with chinese strain js-2004-02 [60] . these results demonstrated that there may be a new prevailing pedv genotype in china [60] . phylogenetic relationships of complete m gene nucleotide sequences indicate that recent thai pedv isolates are closely related to isolates from china [30] . likewise, most korean pedv isolates have been found to be closely related to chinese strains [45] , and belong to the third of 3 pedv groups containing all pedv isolates [45] . 3 relationships among pedvs isolated from various countries based on the partial s gene including epitope region. the phylogenetic tree was constructed using the neighborjoining method in mega version 5.05 with pairwise distances [99] . bootstrap values (based on 1,000 replicates) for each node are given if [60%. the scale bar indicates nucleotide substitutions per site. asterisk represents pedv isolate whose sequence available in genbank database was shorter as compared to that of other reference strains. pedvs isolated from various countries were marked with various colors: europe (black), korea (blue), china (red), japan (olive green), thailand (green) and viet nam (purple) (color investigations of the orf3 gene have revealed the reemergence of pedv in immunised swine herds since early 2006 [42] . orf3 genes have been used to divide chinese field strains and pedv reference strains into 3 groups; further, chinese field strains appear to be closely related to korean strains, but genetically different from pedv vaccine strains. another report revealed that pedv has caused enteric disease with devastating impact since the first identification of pedv in 1992 in korea, and recent, prevalent korean pedv field isolates are closely related to chinese field strains but differ genetically from european strains and vaccine strains [45] . a diagnosis of ped cannot be made on the basis of clinical signs and histopathological lesions [61] [62] [63] [64] . due to the similarities in causative agents of diarrhoea, differential diagnosis is necessary to identify the pedv in the laboratory [64, 65] . many techniques have been used for the detection of pedv, including immunofluorescence (if) tests, immunohistochemical techniques, direct electron microscopy, and enzyme-linked immunosorbent assays (elisa). however, these techniques are time-consuming and are low in sensitivity and specificity [66] . kim et al. [67] compared three techniques (rt-pcr, immunohistochemistry and in situ hybridization) for the detection of pedv. they concluded that although rt-pcr identified the presence of pedv more frequently than the other methods, when only formalin-fixed tissues are submitted, immunohistochemistry and in situ hybridization would be useful methods for the detection of pedv ag and nucleic acid. the pedv leader sequence was used to develop a reverse transcriptase polymerase chain reaction (rt-pcr) diagnostic technique [68, 69] that has successfully been used to detect both laboratory and field isolates [70, 71] . m gene-derived primers can be used in an rt-pcr system to obtain pedv-specific fragments [69] , and duplex rt-pcr has been used to differentiate between tgev and pedv [66] . the past few years have seen several useful modifications of the basic rt-pcr method. for instance, it is possible to estimate the potential transmission of pedv by comparing viral shedding load with a standard internal control dna curve [72] , as well as to perform multiplex rt-pcr to detect pedv in the presence of various viruses [73] -a technique that is particularly useful for rapid, sensitive, and cost-effective diagnosis of acute swine viral gastroenteritis). the commercial dual priming oligonucleotide (dpo) system (seegene, seoul, korea) was also developed for the rapid differential detection of pedv. it employs a single tube 1-step multiplex rt-pcr with two separate primer segments to block a non-specific priming [74] . another useful reverse transcription-based diagnostic tool is rt loop-mediated isothermal amplification (rt-lamp). this assay, which uses 4-6 primers that recognize 6-8 regions of target dna, is more sensitive than gel-based rt-pcr and elisa, in large part because it produces a greater quantity of dna [75] . immunochromatographic assay kits can be used at farms in order to detect pedv s proteins with 92% sensitivity and 98% specificity. this technique is less accurate than rt-pcr, but allows diagnosis within 10 min. thus, it is particularly effective for quickly determining quarantine or slaughter policies in the field. especially, endemic situation of ped infection brought the several commercialised ped virus detection systems using diagnosis techniques including conventional duplex rt-pcr (intron biotechnology, inc, korea), real time rt-pcr (kogenebiotech, kore), dpo based multiplex rt-pcr (seegene, seoul, korea), and immunochromatography (bionote, korea) in korea. recently, a protein-based elisa was developed to detect pedv. in this technique, a polyclonal antibody is produced by immunising rabbits with purified pedv m gene after its expression in escherichia coli. if analysis with anti-pedv-m antibody is then able to detect pedvinfected cells among other enteric viruses [76] . elisa blocking and indirect if have been used to detect pedv antibodies at 7 and 10-13 days postinoculation, respectively [77] . for all tests, the second (convalescent) serum sample should be collected and examined no sooner than 2-3 weeks after the onset of diarrhoea. pedv antibodies, detected by the elisa-blocking and if-blocking tests, have been found to persist for at least 1 year. due to the special features of the porcine mucosal immune system, the presence of serum antibodies against gastroenteric pathogens is not always correlated with protection; rather, detection of these antibodies only proves that individuals had contact with infectious microorganisms [78] [79] [80] . additionally, ha et al. [81] recently reported that colostrum iga concentration is a better marker of protection from pedv infection than serum neutralising (sn) titre from serum samples; however, sn titres may still be useful in determining herd infection status [81] . until they are 4-to 13-day old, piglets are protected against pedv by specific igg antibodies from the colostrum and milk of immune sows [82] ; the length of immunity depends on the titre of the mother. after antigenic sensitisation in the gut, iga immunocytes migrate to the mammary gland, where they localise and secrete iga antibodies into colostrum and milk. this 'gut-mammary' immunologic axis is an important concept in designing optimal vaccines to provide effective lactogenic immunity [83] . pigs that regularly suckle the immune mother are constantly inoculating their lumens with milk-bound iga antibodies, a process that confers passive immunity. igg accounts for more than 60% of colostrum immunoglobulin content. however, iga is more effective at neutralising orally infectious pathogens than either igg or igm because it is more resistant to proteolytic degradation in the intestinal tract and has a higher virus neutralising ability than igg and igm [84] . therefore, only passive transfer of iga from an immunised mother effectively induces immune responses in suckling piglets [85] . however, these antibodies do not protect against intestinal infection with pedv. several pedv vaccines, which differ in their genomic sequence, mode of delivery, and efficacy, have been developed. a cell culture adaptation of the cv777 strain had a strikingly different genomic sequence [18] , was associated with much lower virulence in new born caesarean-derived piglets, and caused much less severe histopathological changes. however, in europe, the disease caused by pedv was not of sufficient economic importance to start the vaccine development. therefore, the trial of vaccine development was mainly accomplished in asian countries where the pedv outbreaks have been so severe that the mortality of the new born piglets was increased. an alternative vaccine for suckling piglets may be an attenuated form of the virus derived from serial passage (passage level: 93) of the pedv [86] . in japan, a commercial attenuated virus vaccine of cell cultureadapted pedv (p-5v) has been administered to sows since 1997. although these vaccines were considered efficacious, not all sows developed solid lactogenic immunity [87] . oral vaccination with attenuated pedv dr13 (passage level: 100) has recently been proven to be more efficacious than injectable vaccine. further, this vaccine candidate remained safe even after three back passages in piglets [88] . piglet mortality can be reduced by orally inoculating pregnant sows with the dr13 strain. the viral strain was licensed, and used as an oral vaccine in south korea from 2004 (patent no. 0502008). and the oral vaccine was registered and commercialised in philippine at 2011. despite the documented benefits of the dr13 vaccine, it does not significantly alter the duration of virus shedding-an indication of immune protection [79, 89] in challenged piglets. shorter periods of virus shedding, as well as reduced severity and duration of diarrhoea in piglets, result from higher titres of serum antibodies; complete protection from pedv infection prevents shedding after exposure to viral challenge [90] . oral immunisation with highly attenuated pedv confers partial protection against virulent challenge in conventional pigs, a result that is related to inoculation dose. at low doses of the attenuated pedv, 25% of pigs are protected against pedv challenge, but this proportion increased to 50% when pigs were inoculated with a dose 20 times stronger [91] . however, viral shedding may be difficult to measure accurately, as it is varies depending on viral strain and sensitivity of the detection tool [72] . therefore, for the ideal and perfect development of vaccines, several criteria including the factors related the reduction of virus shedding in piglets, and the details of the mucosal immunity of pedv should be focused in the course of development of next generation vaccines. information on pedv mucosal immunity has typically been limited. de arriba et al. used the enzyme linked immunospot (eli-spot) technique to characterise the isotype-specific antibody secreting cells in mucosal and systemic-associated lymphoid tissues in pigs inoculated with pedv. after infection with pedv, levels of antibody secreting cell (asc) in the gut were similar to those observed in response to tgev and rotavirus infection; igg ascs were more prevalent than iga ascs. in pedv-infected pigs, a limited number of igm ascs were detected at post infection day (pid) 4, and memory b cells appeared at pid 21 in mesenteric lymph nodes, spleen, and blood. finally, the authors noted correlations between protection and both serum isotype-specific antibody and asc response in gut-associated lymph tissues and blood on the challenge day [90] [91] [92] . there have also been reports of immune responses by transgenic plants and lactic acid bacteria that express the pedv antigen [85, 93, 94] . the transgenic tobacco plants that express the s protein corresponding to the neutralising epitope of pedv was tested whether feeding the plants induced the immune response in murine model. and the efficacy of orally administered antigen gene transgenic carrot and lettuce were tested after codon optimization and application of viral expression systems [85] . in mice, induced antibodies have neutralising activity against pedv. no neutralising antibodies were detected in either mice or pigs given mucosal immunizations with recombinant lactobacillus casei expressing pedv n (nucleoprotein) on its surface. however, this treatment elicited high levels of mucosal iga and circulation igg immune responses against the pedv n protein. before this vaccine can be commercialised, further studies are needed; for instance, it will be necessary to understand discrepancies between test results of the first lab scale vaccine and large-scale pilot vaccines. research into this and other potential vaccines should be made a priority, as pedv-mediated diarrhoea causes significant economic losses in the swine industry. however, there is also a potential drawback to the use of live-attenuated vaccines. recently, a survey conducted in china indicated close phylogenetic relationships between a chinese pedv field strain (ch/gsjiii/07) and two vaccine strains, suggesting that live vaccines can evolve into more infectious forms in the field [42] . during the european outbreak of pedv, pregnant sows were deliberately exposed to the intestinal contents of dead infected pigs, thus artificially stimulating lactogenic immunity and, hopefully, shortening the duration of outbreaks at farms [12] . however, several complications arose from this treatment. because the intestinal contents did not have homogenous titres of pedv, the induction of immunity-including solid lactogenic immunity-might not be expected. diseases may be spread via contamination with viral agents, such as prrsv and pcv2. immunoprophylactic agents may also be used to treat pedv. for instance, anti-pedv chicken egg yolk immunoglobulin (igy) and colostrums from immunized cows have been found to increase survival rates of virally challenged piglets [95, 96] . mouse monoclonal single chain variable fragment (scfv) antibodies to neutralised pedv, which can be expressed in e. coli, are as potent as parental antibodies and block pedv infection into target cells in vitro [97] . thus, it is possible that recombinant e. coli cells expressing scfv can be used as prophylactic agents against pedv infection. epidermal growth factor (egf), which stimulates the proliferation of intestinal crypt epithelial cells and promotes recovery from atrophic enteritis in pedvinfected piglets [98] , has also been proposed as a potential novel therapy to promote intestinal villous recovery in piglets with pedv infections; it may also be useful in other species with viral atrophic enteritis. drawbacks of this treatment include its high price and questionable safety. pig farming veterinary virology disease of swine clinical histopathological and immunohistochemical findings nidoviruses the coronaviridae coronavirus immunogens revue canadienne de recherche veterinaire revue canadienne de recherche veterinaire disease of swine development of an elisa for the detection fo antibody isotypes against porcine epidemic diarrhoea virus (pedv) in sow's milk key: cord-311255-zaa8i9vh authors: kim, youngnam; lee, changhee title: porcine epidemic diarrhea virus induces caspase-independent apoptosis through activation of mitochondrial apoptosis-inducing factor date: 2014-07-31 journal: virology doi: 10.1016/j.virol.2014.04.040 sha: doc_id: 311255 cord_uid: zaa8i9vh abstract the present study sought to investigate whether porcine epidemic diarrhea virus (pedv) induces apoptosis and to elucidate the mechanisms associated with apoptotic cell death after pedv infection. pedv-infected cells showed evidence of apoptosis in vitro and in vivo. however, experimental data indicated that the caspase cascade is not involved in pedv-induced apoptotic cell death. interestingly, mitochondrial apoptosis-inducing factor (aif) was found to translocate to the nucleus during pedv infection, and aif relocalization was completely abrogated by the presence of cyclosporin a (csa), an inhibitor of cyclophilin d (cypd) that is an essential component of the mitochondrial permeabilization transition pore (mptp) complex. csa treatment resulted in significant inhibition of pedv-triggered apoptosis and suppressed pedv replication. furthermore, direct inhibition of aif strongly impaired pedv infection and virus-induced apoptosis. altogether, our results indicate that a caspase-independent mitochondrial aif-mediated pathway plays a central role in pedv-induced apoptosis to facilitate viral replication and pathogenesis. porcine epidemic diarrhea (ped) is a devastating disease of swine that was initially recognized in the united kingdom in 1971 and that has then spread to swine-producing european countries (oldham, 1972; pensaert et al., 1981) . since the 1990s, the disease has become rare in europe and is more often associated with postweaning diarrhea in adult pigs (saif et al., 2012) . ped epidemics were first reported in asia in 1982 and since then, ped has continued to threaten swine health causing substantial financial losses in the asian swine industry (chen et al., 2008; kweon et al., 1993; li et al., 2012; puranaveja et al., 2009; takahashi et al., 1983) . in 2013, ped outbreaks suddenly appeared in the united states and have swept the national pork industry with high mortality in newborn piglets, posing significant economic and public health concerns (mole, 2013; stevenson et al., 2013) . in 1978, the etiological agent of this disease was identified as a coronavirus, ped virus (pedv), which belongs to the genus alphacoronavirus within the family coronaviridae of the order nidovirales (pensaert and de bouck, 1978; saif et al., 2012) . pedv is a large, enveloped virus possessing a single-stranded positivesense rna genome of approximately 28 kb with a 5 0 cap and a 3 0 polyadenylated tail (pensaert and de bouck, 1978; saif et al., 2012) . the pedv genome is composed of a 5 0 untranslated region (utr), at least 7 open reading frames (orf1a, orf1b, and orf2 through 6), and a 3 0 utr (kocherhans et al., 2001) . the two large orf1a and 1b cover the 5 0 two-thirds of the genome and encode the nonstructural replicase genes. the remaining orfs in the 3 0 terminal region code for four major structural proteins, the 150-220 kda glycosylated spike (s) protein, 20-30 kda membrane (m) protein, 7 kda envelop (e) protein, and 58 kda nucleocapsid (n) protein (duarte et al., 1994; saif et al., 2012) . pedv is a pathogenic enterocyte-tropic coronavirus of swine, and its infection is marked by acute enteritis with mortality approaching 100% in suckling piglets up to one week of age (debouck and pensaert, 1980; saif et al., 2012) . the virus is transmitted via the fecal-oral route and the virus replicates in the cytoplasm of villous epithelial cells throughout the small intestine. pedv infection results in severe villous atrophy, leading to a reduction in the villous height:crypt depth ratio from the normal 7:1 to 3:1. this interrupts digestion and absorption of nutrients and electrolytes, thereby causing malabsorptive watery diarrhea followed by serious and fatal dehydration in piglets (saif et al., 2012) . in addition, pedv can be propagated in vero (african green monkey kidney) cells in vitro, where the cytopathic effects contents lists available at sciencedirect journal homepage: www.elsevier.com/locate/yviro (cpes) consist of vacuolation and formation of syncytia with up to 100 nuclei (hofmann and wyler, 1988) . however, the mechanisms inducing cell death in pedv-infected target cells, both in vitro and in vivo, remain poorly understood. apoptosis is a tightly regulated mechanism of cell death that is triggered by various extracellular (extrinsic) or intracellular (intrinsic) stimuli and modulated by anti-and pro-apoptotic cellular factors (galluzzi et al., 2012) . cells undergoing apoptosis are accompanied by characteristic morphological changes, including rounding-up of the cell, chromatin condensation, nuclear fragmentation, and plasma membrane blebbing (kroemer et al., 2009) . apoptosis is considered to be a host innate defense mechanism that disrupts viral replication by eliminating virus-infected cells. therefore, viruses have developed strategies to avoid apoptosis, which, in turn, prevent premature cell death and thus maximize progeny viral production (thomson, 2001) . however, many viruses have the ability to actively induce apoptosis as a response to viral replication, thereby facilitating the release and dissemination of viral progeny to neighboring cells. this pro-apoptotic event is one of the cytolytic properties of viral infections causing cpe in vitro or/and plays a pathogenic role contributing to cell damage, tissue injury, and disease severity in vivo dediego et al., 2011; eleouet et al., 1998; favreau et al., 2012; lan et al., 2013; lee and kleiboeker, 2007; st-louis and archambault, 2007; sarmento et al., 2006; suzuki et al., 2008) . at present, it is unknown whether pedv triggers apoptotic cell death, and if so, whether virusinduced apoptosis aids or worsens viral replication and pathogenicity. therefore, in this study, we aimed to determine if pedv induces apoptosis following infection in vitro and in vivo and to define the specific pathways involved in apoptotic death of virus-infected cells. apoptotic cell death occurred after in vitro and in vivo infections of pedv, suggesting that pedv-induced apoptosis may play an important role in cytotoxicity and pathogenesis. treatment with a pancaspase inhibitor failed to suppress pedv-induced apoptosis and pedv infection. however, pedv replication and virus-triggered apoptosis were significantly impaired by inhibitors of cyclophilin d (cypd) and apoptosis-inducing factor (aif). these data indicate that pedv promotes aif-mediated and caspase-independent apoptosis, which is essential for the replication of pedv. overall, our study will improve the understanding of the correlation between pedvinduced apoptosis and its pathogenic mechanisms and show the potential of anti-apoptotic therapeutic strategies for combating pedv infection. to investigate whether apoptotic cell death was triggered by pedv, virus-infected cells were first examined for the presence of dna fragmentation, which is a hallmark of cells undergoing apoptosis. vero cells were infected with pedv, and cpe was monitored after infection. light microscopy of the pedv-infected vero cells indicated that typical cpes characterized by vacuolation and syncytia formation were visible starting at 24 hpi and became prominent by 48 hpi (fig. 1a , upper panels). to detect intracellular fragmented dna, cellular dna from mock-or virus-infected cells was harvested and subjected to a dna laddering assay. consistent with the cpe observation results, intracellular dna fragmentation appeared at 24 hpi, and the dna ladder pattern was apparent at 48 hpi (fig. 1a, lower panel) . this characteristic of apoptosis was further confirmed using a tunel assay. as shown in fig. 1b , compared to mock-infected cells, tunel-labeled cells were only observed within virus-infected cells. the process of pedv-induced apoptosis was then assessed using annexin v/pi flow cytometry. virus-or mock-infected cells were stained with annexin v and pi and then examined using facs flow cytometry to quantitatively determine the percentage of viable, apoptotic, and dead cells. pedv infection produced a high level of apoptosis (annexin v positive/pi negative) appearing at 6 hpi, and the percentage of early apoptotic cells increased with infection time, reaching a maximum of 87.4% at 48 hpi (fig. 1c ). in addition, we sought to determine whether pedv induces apoptosis in the natural host. to accomplish this, a tunel assay was performed to detect and quantify apoptosis in small intestine sections prepared from piglets experimentally inoculated with pedv. immunofluorescence using n protein-specific mab revealed that abundant viral antigens were primarily present in enterocytes over the entire villi in all areas of the small intestine at 3 dpi and increasingly dominant in the interior of the villi at 5 dpi; the majority of virus-infected cells were tunel-positive (fig. 2) . in contrast, no tunel-positive enterocytes were observed in the tissue specimens prepared from a non-infected animal (fig. s1 ). taken together, these data demonstrate that pedv infection induces apoptosis in vitro and in vivo. caspases, a family of aspartate-specific cysteine proteases, play critical roles in the execution phase of apoptosis of the cell leading to morphological and nuclear changes . in order to elucidate the mechanism and the type of apoptotic cell death, we initially assessed whether the broad-spectrum pancaspase inhibitor, z-vad-fmk, blocks pedv-induced apoptosis. based on the results of the mtt assay, none of the doses of z-vad-fmk tested in the present study resulted in any change in cell viability (data not shown). vero cells were treated with z-vad-fmk at concentrations of 100 μm followed by viral infection. at the indicated time points post-infection, virus-infected and z-vad-fmk-treated cells were analyzed based on facs quantification of annexin v binding. notably, treatment with z-vad-fmk was incapable of protecting cells from pedv-induced apoptosis (fig. 3a) . the percentage of early apoptotic cells in the presence of z-vad-fmk was almost similar to that in vehicle (dmso)-treated cells (second panel) and pedv-infected cells during the course of infection (compared to fig. 1c ). these results indicated that caspases are not associated with pedv-triggered apoptosis. for further confirmation, we tried to determine the activity of caspase-3 upon pedv infection by western blot analysis. caspase-3 is the main executioner caspase activated by the both extrinsic and intrinsic apoptotic pathways causing the morphological features of apoptosis. as shown in fig. 3b , no detectable level of cleaved caspase-3 was observed in pedv-infected cells up to 48 hpi (first panel), whereas the n protein was first detected at 6 hpi, and its production greatly increased thereafter, indicating pedv replication (third panel). on prolonged exposure of the western blot, however, it was apparent that a small amount of activated caspase-3 appears only at 48 hpi (second panel), which seems to be a basal level normally present at late time of infection. we next investigated if the replication of pedv is also affected by the same caspase inhibitor. vero cells were pretreated with z-vad-fmk at concentrations of 10-100 μm or with dmso as a vehicle control for 1 h prior to infection. z-vad-fmk was present during the entire period of infection. viral production was measured by monitoring cpe and confirmed by immunofluorescence at 48 hpi (fig. 3c ). apparent cpe and n-specific staining were equally evident in cells treated with z-vad-fmk when compared to vehicle-treated control cells. furthermore, pedv replication, as quantified by the growth kinetics results, was not reduced by z-vad-fmk treatment, indicating that activation of caspases is irrelevant to pedv replication (fig. 3d ). altogether, our data reveal that caspase cascades are not activated by pedv and their chemical inhibition diminished neither pedv-induced apoptotic cell death nor viral replication, suggesting that caspase activation is dispensable for the process. since caspases appear to be non-essential factors in pedvtriggered apoptosis, we investigated an alternative caspaseindependent apoptotic pathway mediated mainly by aif in relation to apoptotic cell death induced by pedv. aif is a flavoprotein embedded in the outer surface of the mitochondrial inner membrane. following apoptotic stimuli, aif is proteolytically cleaved and truncated aif is released from the mitochondria and translocates to the nucleus, where it causes high-molecular weight dna fragmentation and chromatin condensation independently of caspases (cregan et al., 2002; daugas et al., 2000; galluzzi et al., 2012; joza et al., 2001; lu et al., 2013; yuste et al., 2005) . mitochondria plays a central role in the intrinsic apoptotic pathway by releasing proapoptotic factors, such as aif and cytochrome c (cytc), through the mitochondrial permeability transition pore (mptp), which is permeabilized in response to intracellular stress (galluzzi et al., 2012) . in order to study the potential role of mitochondrial aif in the caspase-independent pathway of pedvinduced apoptosis, thus, we first aimed to determine the subcellular localization of aif in pedv-infected cells using immunofluorescence confocal microscopy. although aif continued to localize broadly in the mitochondria after pedv infection shown by its colocalization with a mitochondrial-specific marker, the majority of cells infected with pedv also displayed a condensed aif staining pattern within the nucleus, and its intensity was for the dna fragmentation assay, dna was extracted from mock-or pedv-infected cells and nucleosomal dna fragmentation of the cells was then analyzed by agarose gel electrophoresis (bottom panel). as a positive control, cells were treated with staurosporine for 24 h to induce apoptosis. lane m represents a 1-kb ladder as a dna molecular-weight size marker. (b) tunel labeling of pedv-infected cells. mock-infected control and pedv-infected cells fixed at 48 hpi were labeled with tunel (green) and sequentially stained with an anti-pedv-n antibody (red). the cells were then counterstained with dapi and photomicrographs of tunel labeling and n protein staining in virus-infected cells was observed using a confocal microscope at 400 â magnification. in a merged image, all tunel-positive cells were localized within the nuclei of corresponding pedv-infected cells. (c) cell death analysis by flow cytometry with dual annexin v-pi cell labeling. pedv-infected cells collected from different time periods were dually labeled with annexin v and pi and analyzed by facs. the lower left quadrants represent intact cells (annexin v negative/pi negative); the lower right quadrants represent early apoptotic cells (annexin v positive/pi negative); the upper right quadrants indicate late apoptotic and/or necrotic cells (annexin v positive/pi negative); the upper left quadrants indicate necrotic cells (annexin v negative/pi positive). the figure is representative of three independent experiments. the graph on the right represents the percentage of each quadrant and the non-significant percentages of annexin v-positive and pi-negative cells were excluded. tissue specimens were collected from the duodenum, jejunum, and ileum of infected animals at the indicated days postinoculation. the formalin-fixed and paraffin-embedded tissue sections were deparaffinized and sequentially labeled with tunel (green) followed by an anti-pedv-n antibody (red). the sections were then counterstained with dapi and examined using a confocal microscope at 200 â magnification. yellow regions indicate merged images, where cells are positive for both tunel and pedv infection. significantly elevated at 48 hpi ( fig. 4a , arrows). this observation was confirmed by western blot analysis of mitochondrial and nuclear extracts. as shown in fig. 4b , an uncleaved 62 kda aif was detected in both extracts during the course of infection, whereas a cleaved form of aif was observed only in nuclear extracts of pedvinfected cells. we then analyzed the translocation of another proapoptotic protein, cytc, which is released to the cytoplasm by mitochondrial outer membrane permeabilization to execute caspase proteolytic cascade-dependent intrinsic apoptosis. in contrast to aif, however, cytc was sustainedly located in the mitochondria of pedv-infected cells, as demonstrated by the colocalization of cytc with mitotracker and the detection of cytc in mitochondrial fractions (fig. 4c and d) . this cytc retention in the mitochondria during pedv infection was concurrent with the caspase-independent apoptotic cell death induced by pedv as described above. consequently, our findings demonstrate that pedv infection specifically triggers mitochondrial-nuclear translocation of aif but not the release of mitochondrial cytc. cyclophilin d (cypd) is localized to the mitochondrial membrane matrix and is a main component in the process of mptp opening (baines et al., 2005; schinzel et al., 2005) . therefore, we aimed to determine whether inhibition of cypd could prevent apoptotic cell death induced by pedv. cells were treated with csa, a chemical inhibitor of cypd, to inhibit mptp formation upon pedv infection. the dna laddering assay indicated that the presence of csa in cells infected with pedv completely abolished intracellular dna fragmentation (fig. 5a, lane 4) , while cells infected with pedv alone or combined with z-vad-fmk clearly displayed a dna laddering pattern (fig. 5a, lanes 2 and 3) . upon csa treatment, pedv-induced apoptosis was then quantitatively evaluated by annexin v/pi flow cytometry. as shown in fig. 5b , treatment with csa notably decreased the percentage of early apoptotic cells induced by pedv (second panel), ranging from 19.1% to 29.9% during the course of pedv infection (compared to fig. 3a ). these results demonstrate that inhibition of cypd efficiently suppresses apoptotic cell death induced by pedv, indicating that pedv infection triggers the mitochondrial apoptotic pathway. to examine the effect of csa on pedv replication, cells were pretreated with csa at concentrations of 1-10 μm, and csa was present during the entire period of infection. pedv replication was determined by monitoring virus-specific cpe and verified by immunofluorescence at 48 hpi (fig. 6a) . in contrast to vehicletreated control cells, csa had a strong inhibitory effect on pedv propagation by dramatically decreasing virus-induced cpe and viral gene expression even at the lowest concentration used. the number of cells expressing viral antigen, as quantified by n protein staining results, was also reduced during csa treatment, resulting in almost complete inhibition in response to 10 μm (fig. 6b ). to further assess the antiviral activity of csa against pedv replication, viral yield was determined during treatment with csa. upon infection, viral supernatants were collected at 48 hpi, and viral titers were measured. as fig. 6c shows, the presence of csa significantly reduced the release of viral progeny in a dosedependent manner. the peak viral titer was determined to be 10 6.68 pfu/ml in the vehicle-treated control, whereas the addition of 10 μm csa declined the titer of pedv to 10 1.69 pfu/ml (an almost 5-log reduction compared to the control). the growth kinetics study further demonstrated that the overall process of pedv replication was significantly delayed when cells were treated with csa (fig. 6d ). in addition, we investigated whether viral protein translation and viral rna synthesis were affected by csa. the expression level of the pedv n protein in the presence or absence of csa was evaluated at 48 hpi by western blot analysis. densitometric analysis of the western blot revealed that intracellular expression of the viral protein was entirely prevented by csa at a concentration of 10 μm (fig. 7a) . similarly, levels of both genomic rna and sg mrna were greatly diminished in a dosedependent manner and their synthesis was fully abolished at the highest csa concentration (fig. 7b ). taken together, our data strongly indicate that csa proficiently suppresses the replication of pedv, which is correlated with inhibition of pedv-induced apoptosis by csa. the present study shows that mitochondrial aif is translocated to the nucleus following pedv infection. because cypd is involved in alteration of mitochondrial membrane permeability, followed by release of proapoptotic factors, it is conceivable that inhibition of cypd would block aif nuclear translocation induced by pedv, leading to impaired pedv-induced apoptosis and viral replication. we therefore determined whether csa specifically affects the nuclear translocation of aif mediated by pedv infection. immunofluorescence confocal microscopy results demonstrated that aif nuclear translocation in pedv-infected cells was distinctly hampered following cypd inhibition by csa, exhibiting a cytoplasmic distribution that colocalized with the mitochondrial marker during the entire course of pedv infection (fig. 8a) . mitochondrial retention of aif in the presence of csa was confirmed by a cell fractionation assay. as expected, the level of truncated aif in the nuclear fractions was barely detectable in the presence of csa, demonstrating that inhibition of cypd failed to cause the relocalization of aif from the mitochondria to the nucleus (fig. 8b) . altogether, these results suggest that upon pedv infection, cypddependent mptp directly modulates apoptotic cell death by relocating aif to the nucleus, resulting in successful viral replication. considering that aif is a key proapoptotic protein released from the mitochondria in response to pedv infection to trigger apoptosis, we sought to determine whether chemical inhibition of aif could control pedv-induced apoptosis and viral replication. to accomplish this, we first used an aif inhibitor, n-phmi, that prevents aif-induced dna fragmentation (susin et al., 1996; wang et al., 2007) . treatment with n-phmi resulted in substantial reduction of the percentage of apoptotic cells during pedv infection (fig. 9) . addition of n-phmi retained an average cell viability of over 80% during the entire time of pedv infection, demonstrating that inhibition of aif robustly represses apoptosis induced by pedv. to further confirm the importance of aif in apoptosis triggered by pedv infection, we examined whether this drug alters the nuclear relocalization of aif following pedv infection by confocal microscopy analysis. as depicted in fig. 10a , n-phmi treatment completely abrogated aif nuclear translocation induced by pedv, leading to mitochondrial accumulation of aif. western blot analysis provided further support by showing the absence of truncated aif in the nuclear fractions of pedv-infected cells in the presence of n-phmi (fig. 10b) . these results strongly indicated that nuclear targeting of aif is required for pedv-induced apoptotic cell death. finally, to determine whether aif function is necessary for pedv replication, vero cells were treated with n-phmi for 1 h prior to infection, and the drug was allowed to remain during infection and subsequent incubation. at 48 hpi, the level of pedv replication was measured indirectly, as viral antigen production, by quantifying cells expressing the n protein through ifa (fig. 11a) . treatment of cells with 5 μm n-phmi resulted in a 490% decrease in pedv production compared to the untreated vehicle control. to further examine the inhibitory effect of n-phmi on pedv infection, virus production was measured using viral supernatants propagated for 48 h under each condition. as shown in fig. 11b , inhibition of aif by n-phmi markedly decreased the titer of pedv in a dosedependent manner, and the lowest viral titer was determined to be 10 2.61 pfu/ml at the concentration of 5 μm (an almost 4-log reduction compared to the control). furthermore, the replication process of pedv severely interfered with the presence of n-phmi (fig. 11c) . taken together, our data show that aif is an essential host determinant in a caspase-independent apoptosis process activated by pedv, which is indispensable for pedv replication. apoptosis is a tightly controlled multistep process of cell death that occurs in response to a wide range of stimuli, including viral infections. viruses possess various mechanisms to inhibit apoptosis that allow them to evade the innate immune defenses, which restrict viral infection by eliminating infected cells through the interactions at different stages of the apoptotic pathway. however, some viruses induce apoptosis to facilitate the release and dissemination of viral progeny for further invasion, which are important biological parts in viral pathogenesis and disease processes that promote cell death and tissue injury. although various viruses are known to modulate apoptosis as a critical armament to complete their replication cycle, it remains so far even undetermined whether pedv induces apoptosis. in the present study, we characterized pedv-induced cytotoxicity and demonstrated that apoptotic cell death is triggered in pedv-infected cells in vitro, displaying biochemical features of apoptosis including oligonucleosomal dna fragmentation and ps exposure. this finding strongly suggests that the cytopathology of pedv infection, represented by vacuolation and syncytia formation in vitro, is associated with the apoptotic process. the capability of pedv to induce apoptosis was also verified by an in vivo study using the small intestinal tissues of piglets experimentally inoculated with virulent pedv. the results showed that the great part of the villus enterocytes in all segments of the small intestine infected with pedv experienced apoptotic cell death at 3 dpi, and thereafter, cells undergoing apoptosis were more evident inside the small intestine (fig. 2) . since a specific hallmark of pedv infection in neonatal piglets is the severe destruction of the villi in the small intestine causing lethal diarrhea and dehydration, pedv-induced massive apoptosis in vivo appears to be one of the viral pathogenic mechanisms to destroy the target enterocytes leading to villus atrophy or vacuolation. the morphological characteristics of apoptosis are typically caused by the sequential activation of caspases, which are normally present in mammalian cells as inactive precursors . upstream initiator caspases are first activated by both extrinsic and intrinsic apoptotic pathways and subsequently, are responsible for processing and activation of downstream corresponding effector caspases to mediate apoptosis galluzzi et al., 2012) . to understand the molecular mechanisms underlying apoptosis caused by pedv, we focused on the potential involvement of caspases in apoptotic cell death upon pedv infection. in the present study, caspase-3, the primary effector caspase, was not proteolytically cleaved to trigger the execution phase of apoptosis during the course of pedv infection, although its inactive precursor was continuously present in cells infected with pedv. however, our western blot data, after long exposure, showed only slight levels of caspase-3 activation at 48 hpi (fig. 3b, second panel) . this observation ruled out the possibility that the absence of active caspase-3 was caused by problems with the antibody used or by technical issues. likewise, chemical suppression of caspase activation was found to be absolutely incapable of abrogating pedv-induced apoptosis and viral replication. these results strongly indicate that the failure of the pan-caspase inhibitor to protect cell survival is likely a consequence of caspase-independent cell death, which is indispensable for the replication of pedv. our data here reveal that caspases are irrespective of both pedv-induced apoptosis and viral replication. thus, we sought to investigate other pro-apoptotic factors that could be involved in pedv-induced apoptosis, independent of caspases. in response to various intracellular stress conditions, both pro-and antiapoptotic signals converge toward a mitochondrion-centered control mechanism to trigger intrinsic apoptosis. under this circumstance, mitochondrial outer membrane permeabilization (momp) can begin directly at the outer mitochondrial membrane due to the pore-forming activity of the pro-apoptotic bax subfamily that translocate from the cytoplasm to the mitochondria or can result from the mitochondrial permeability transition at the inner mitochondria membrane caused by the opening of the mptp (brenner and grimm, 2006; galluzzi et al., 2012; tait and green, 2013) . this phenomenon permits the release of several proteins normally retained within the mitochondrial intermembrane space to the cytosol (galluzzi et al., 2012) . among these, cytosolic cytc participates with other pro-apoptotic factors in the formation of the apoptosome, which triggers the caspase-dependent proteolytic cascade (galluzzi et al., 2012; li et al., 1997) , whereas aif, a ubiquitously expressed flavoprotein, critically functions in caspase-independent apoptosis by relocating to the nucleus and promoting large-scale dna fragmentation and cell death (galluzzi et al., 2012; joza et al., 2001; susin et al., 1999) . we demonstrate here that the translocation of mitochondrial aif to the nucleus was actively manifested during pedv infection. conversely, displacement of cytc from the mitochondria to the cytoplasm was completely absent in pedv-infected cells. this result further supports the hypothesis that pedv triggers apoptosis in a caspase-independent manner. the association of pedv-induced apoptosis with aif translocation to the cytosol, without the release of cytc followed by caspase activation, indicates that aif is the sole pro-apoptotic factor released via altered mitochondrial membrane permeability and primarily acts directly to execute apoptotic cell death in pedv infection. the unique relocation of aif, but not cytc, leads us to speculate that momp may be regulated by the opening process following the blot was also reacted with anti-β-actin antibody to verify equal protein loading. pedv n protein expression was quantitatively analyzed by densitometry in terms of the relative density value to the β-actin gene, and csa-treated sample results were compared to dmso-control results. (b) total cellular rna was extracted at 48 hpi, and strand-specific viral genomic rnas (black bars) and sg mrnas (white bars) of pedv were amplified by quantitative real-time rt-pcr. viral positive-sense genomic rna and sg mrna levels were normalized to monkey gapdh mrna, and relative quantities (rq) of mrna accumulation were evaluated. csa-treated sample results were compared with dmso-treated results. values are representative of the mean from three independent experiments and error bars denote standard deviations. *, p ¼0.001 to 0.05; †, po 0.001. mptp formation during pedv infection, in which cypd functions as a major inducing factor to constitute the mptp complex in the mitochondrial membrane. the data clearly show that inhibition of cypd by csa blocked apoptosis induced by pedv and was accompanied by 70-80% cell survival after pedv infection, comparable to mock-infected cells. furthermore, csa could exert the antiviral activity via potent inhibition of multiple steps of the pedv life cycle, including viral translation, rna synthesis, and spread of viral progeny. notably, chemical suppression of cypd impaired aif nuclear translocation, leading to absolute mitochondrial retention of aif. thus, cypd-mediated mptp modulation plays a pivotal role in caspase-independent apoptotic cell death by controlling aif translocation upon pedv infection. numerous putative cellular proteins are known to bind with cypd to promote mptp formation. bax is one such factor, which has been shown to be displaced toward the mitochondria and to interact subsequently with cypd to facilitate the formation of pores in the mitochondrial membrane in response to apoptotic stimuli, including viral infection (favreau et al., 2012; kumarswamy and chandna, 2009 ). although it is unclear whether bax is directly involved in pedv-induced apoptosis, this mechanism is less plausible because pedv infection was irrelevant to mitochondrial cytc release, which is mainly mediated by mitochondrial translocation of bax. similarly, direct inhibition of aif with a chemical inhibitor completely confined aif protein in the mitochondria without nuclear localization, and robustly diminished pedvinduced apoptotic cell death and pedv replication. the inhibitory activity of n-phmi on pedv infection was less efficient than that of csa, although treatment with n-phmi also exhibited significant suppression of pedv replication in its own way. this difference can be attributed to the wide-spectrum antiviral effect of csa via multiple simultaneous mechanisms; csa is known to inhibit the replication of several rna viruses, including nidoviruses, by affecting the function of various members of the cellular cyclophilin protein family (de wilde et al., 2013; kambara et al., 2011; nakagawa et al., 2004; pfefferle et al., 2011; qing et al., 2009; ) . moreover, it was recently demonstrated that csa fully blocks arterivirus replication by suppressing viral rna synthesis (wilde et al., 2013) . we also showed here that treatment of cells with csa dramatically reduces synthesis of both genomic rna and sg mrna pedv-specific cpes were observed daily and were photographed at 48 hpi using an inverted microscope at a magnification of 200 â (first panels). at 48 hpi, virus-infected cells were subjected to ifa with anti-pedv n antibody (second panels) followed by dapi counterstaining (third panels) and examined using a fluorescent microscope at 200 â magnification. viral production in the presence of n-phmi was assessed exactly as described in the legend to fig. 6b. (b) progeny virus release in the presence of n-phmi. vero cells were pretreated with dmso or n-phmi for 1 h and were mock-or pedv-infected (moi of 0.1). at 48 hpi, the virus supernatants were collected and viral titers were measured. (c) growth kinetics of pedv upon n-phmi treatment. at the indicated time points post-infection, culture supernatants were harvested and viral titers were determined. results are expressed as the mean values from three independent experiments and error bars represent standard deviations. †, p o 0.001. (fig. 7b) . therefore, csa appears to synergistically elicit its antiviral activity on the replication of pedv through inhibition of apoptotic cell death and interference with viral rna synthesis. in summary, our findings described here reveal that pedv induces apoptotic cell death, which represents a cytolytic mechanism causing cpes in vitro and contributes to pedv pathogenicity by damaging the villous enterocytes of the small intestine, leading to malabsorptive diarrhea. however, pedv-induced apoptosis was neither inhibited by z-vad-fmk nor accompanied by the activation of caspase cascades and the concomitant release of cytc from the mitochondria, strongly indicating that this cell death event by pedv is caspase-dispensable. on the other hand, we demonstrated the essential roles of cypd and aif in pedv-induced apoptosis; in response to pedv infection, momp occurs through opening of the mptp dependent on cypd, which collapses the mitochondrial potential, resulting in the characteristic release of aif to the nucleus and thereby mediating large-scale dna fragmentation. furthermore, inhibition of either cypd or aif robustly abrogated the life cycle of pedv at subcytotoxic doses. to our knowledge, this is the first report of cypd-involved, aif-indispensable apoptosis, independent of both caspase activation and cytc release following viral infection. although mitochondrial cypd and aif necessarily contribute to pedv-induced apoptosis, the upstream intracellular regulatory signal(s) and the viral protein(s) that are associated with this mitochondrion-centered apoptotic pathway mediated by pedv infection are still unclear and accordingly, identification of such cellular and viral factors would be the next issue to be addressed in future studies. in conclusion, the results presented here indicate that pedv induces apoptotic cell death via a caspaseindependent, aif-mediated pathway that plays a critical role in pedv replication and pathogenesis and suggest that the antiapoptotic approach may be one of appropriate strategies for the development of an anti-pedv compound to combat ped. vero cells were cultured in alpha minimum essential medium (α-mem; invitrogen) with 10% fetal bovine serum (fbs; invitrogen) and antibiotic-antimycotic solutions (100 â ; invitrogen). the cells were maintained at 37 1c in a humidified 5% co 2 incubator. pedv strain sm98-1 was kindly provided by the korean animal and plant quarantine agency and propagated in vero cells as described previously (hofmann and wyler, 1988) . staurosporine was purchased from cayman chemical company and used at a concentration of 1 μg/ml to induce apoptosis. z-vad-fmk (r&d systems), cyclosporin a (csa; cell signaling technologies), and nphenylmaleimide (n-phmi; sigma-aldrich) were dissolved in dimethyl sulfoxide (dmso) to yield a 20 mm, 10 mm, or 1 m stock, respectively. the pedv n protein-specific monoclonal antibody (mab) was a kind gift from sang-geon yeo (kyungpook national university, daegu, korea). antibodies specific for aif, cytc, sp1, and β-actin and horseradish peroxidase (hrp)-conjugated secondary antibodies were obtained from santa cruz biotechnology. the caspase-3, voltage-dependent anion channel (vdac), and αtubulin antibodies were purchased from sigma-aldrich. the cytotoxic effects of reagents on vero cells were analyzed using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay (sigma-aldrich) to detect cell viability. briefly, vero cells were grown at 1 â 10 4 cells/well in a 96-well tissue culture plate with z-vad-fmk, csa or n-phmi treatment for 24 h. after 1 day of incubation, 50 μl of mtt solution (1.1 mg/ml) was added to each well, and the samples were incubated for an additional 4 h. the supernatant was then removed from each well, after which 150 μl of dmso was added to dissolve the colored formazan crystals produced by the mtt. the absorbance of the solution was measured at 540 nm using an enzyme-linked immunosorbent assay plate reader. all mtt assays were performed in triplicate. vero cells were grown at 3.5 â 10 5 cells/well in 6-well tissue culture plates for 1 day and then mock infected or infected with pedv at a multiplicity of infection (moi) of 0.1. in addition, cells were pretreated with z-vad-fmk or csa for 1 h followed by pedv infection. at the indicated times, cells were harvested, washed with pbs, and then incubated in a cell lysis buffer (10 mm tris, ph 7.5, 1 mm edta, and 0.2% triton x-100) containing 500 μg/ml protease k for 24 h at 55 1c. the dna was then extracted twice with phenol/chloroform, precipitated with isopropanol, and resuspended in distilled water. next, the purified dna was incubated with 20 μg/ml ribonuclease a for 1 h at 37 1c, electrophoresed on a 1.2% agarose gel containing midori green advanced dna stain (nippon genetics), and photographed. vero cells were grown in 6-well tissue culture plates for 1 day and mock infected or infected with pedv at an moi of 0.1. to examine the effect of each inhibitor on pedv-induced apoptosis, cells were treated with z-vad-fmk, csa, or n-phmi and then infected with pedv. the virus-inoculated cells were further propagated in the presence of z-vad-fmk (100 μm), csa (5 μm), n-phmi (5 μm) or dmso (vehicle control). phosphatidylserine exposure was determined by measuring annexin v binding at the indicated times using an alexa fluor 488 annexin v/dead cell apoptosis kit (invitrogen), according to the manufacturer's protocol. in brief, cells were harvested, washed with cold pbs, and suspended in 100 μl 1 â annexin-binding buffer. the cells were then incubated with alexa fluor 488-conjugated annexin v and propidium iodide (pi) at room temperature (rt) for 15 min in the dark. following the incubation period, 400 μl of annexin-binding buffer was added to each sample, and the samples were mixed gently and kept on ice. the fluorescent signals of annexin v and pi were detected at channels fl-1 and fl-2, respectively, and analyzed using a fluorescence-activated cell sorter (facs) aria iii flow cytometer (bd biosciences). cells negative for pi uptake and positive for annexin v were considered apoptotic. vero cells were grown on microscope coverslips placed in 6well tissue culture plates and mock infected or infected with pedv at a moi of 0.1. the virus-infected cells were fixed at 48 h postinfection (hpi) with 4% paraformaldehyde for 25 min at 4 1c and permeabilized with 0.2% triton x-100 in pbs at rt for 5 min. for tissue labeling, two 4 day-old piglets were orally inoculated with 1 ml of small intestine homogenate containing 10 5 tcid 50 of a pedv field isolate as described previously (oh et al., in press) , and tissue specimens were collected from the duodenum, jejunum, and ileum of piglets at 3 and 5 days post-inoculation (dpi) at necropsy. each small intestinal tissue (o3 mm thick) was fixed with 10% formalin for 24 h at rt and embedded in paraffin according to standard laboratory procedures. the formalin-fixed, paraffin-embedded tissues were cut to slices 5-8 μm thick on a microtome, floated on a 40 1c water bath containing distilled water, and transferred onto glass slides. the tissues were then deparaffinized in xylene for 5 min and washed in decreasing concentrations of ethanol (100%, 95%, 85%, 70%, and 50%), for 3 min each. the sections were fixed with 4% paraformaldehyde for 15 min, permeabilized with 20 μg/ml proteinase k solution at rt for 10 min, and fixed again with 4% paraformaldehyde for 5 min. a terminal deoxynucleotidyl transferase-catalyzed deoxyuridine phosphate-nick end labeling (tunel) assay was performed using a deadend fluorometric tunel system kit (promega) according to the manufacturer's instructions with some modifications. briefly, pedv-infected vero cells and the deparaffinized intestinal tissue sections were rinsed twice with pbs, and the tunel reaction mixture was added, incubated for 60 min at 37 1c, immersed in 2 â ssc buffer for 15 min, and washed 3 times in pbs. tunellabeled cells were subjected to an immunofluorescence assay using n-specific mab and alexa fluor 594-conjugated goat antimouse antibody as described below. the samples were mounted on microscope glass slides in mounting buffer (60% glycerol and 0.1% sodium azide in pbs) and analyzed under a confocal laser scanning microscope (carl zeiss) using an excitation wavelength in the range of 450-500 nm and an emission wavelength in the range of 515-565 nm. vero cells grown on microscope coverslips placed in 6-well tissue culture plates were pretreated with z-vad-fmk, csa or n-phmi for 1 h and mock infected or infected with pedv at a moi of 0.1. the virus-infected cells were subsequently grown in the presence of inhibitors until 48 hpi, fixed with 4% paraformaldehyde for 10 min at rt and permeabilized with 0.2% triton x-100 in pbs at rt for 10 min. the cells were blocked with 1% bovine serum albumin (bsa) in pbs for 30 min at rt and then incubated with nspecific mab for 2 h. after being washed five times in pbs, the cells were incubated for 1 h at rt with a goat anti-mouse secondary antibody conjugated to alexa fluor 488 (invitrogen), followed by counterstaining with 4 0 ,6-diamidino-2-phenylindole (dapi; sigma-aldrich). the coverslips were mounted on microscope glass slides in mounting buffer and cell staining was visualized using a fluorescent leica dm il led microscope (leica). for study of colocalization, mitotracker red cmxros (200 nm; invitrogen) was added to viable vero cells and left for 45 min at 37 1c prior to fixation. the cells were then stained with aif-or cytc-specific antibody as described above, and cell staining was analyzed using a confocal laser scanning microscope (carl zeiss). vero cells were grown in 6-well tissue culture plates for 1 day and were mock infected or infected with pedv at an moi of 0.1. at the indicated times, cells were harvested in 50 μl of lysis buffer (0.5% tritonx-100, 60 mm β-glycerophosphate, 15 mm ρ-nitro phenyl phosphate, 25 mm mops, 15 mm, mgcl 2 , 80 mm nacl, 15 mm egta [ph 7.4], 1 mm sodium orthovanadate, 1 μg/ml e64, 2 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 mm pmsf) and sonicated on ice 5 times for 1 s each. homogenates were lysed for 30 min on ice, and clarified by centrifugation at 15,800 â â â g (eppendorf centrifuge 5415r) for 30 min at 4 1c. for cell fractionation, inhibitor-treated, pedv-infected vero cells were fractionated using a nuclear/cytosol or mitochondria/cytosol fractionation kit (biovision) according to the manufacturer's manuals. the total protein concentrations in the supernatants were determined using a bca protein assay (pierce). equal amounts of total protein were separated on a nupage 4-12% gradient bis-tris gel (invitrogen) under reducing conditions and electrotransferred onto immunobilon-p (millipore). the membranes were subsequently blocked with 3% powdered skim milk (bd biosciences) in tbs (10 mm tris-hcl [ph 8.0], 150 mm nacl) with 0.05% tween-20 (tbst) at 4 1c for 2 h and incubated at 4 1c overnight with the primary antibodies. the blots were then incubated with corresponding secondary hrp-labeled antibodies at a dilution of 1:5000 for 2 h at 4 1c. proteins were visualized using enhanced chemiluminescence (ecl) reagents (ge healthcare) according to the manufacturer 0 s instructions. to quantify viral protein production, band densities of pedv n proteins were quantitatively analyzed using a computer densitometer with the wright cell imaging facility (wcif) version of the imagej software package (http://www.uhnresearch.ca/facilities/wcif/imagej/) based on the density value relative to the β-actin protein. vero cells were pedv infected and treated with z-vad-fmk, csa, n-phmi or dmso as described above. the culture supernatant was collected at different time points (6, 12, 24, 36, and 48 hpi) and stored at -80 1c. the pedv titer was determined by a plaque assay using vero cells and quantified as plaque-forming units (pfu) per ml. vero cells were pedv inoculated with treatment of csa for 1 h at 37 1c. the virus inoculum was subsequently removed and the infected cells were maintained in fresh medium containing csa for 48 h. total rna was extracted from lysates of the infected cells at 48 hpi using trizol reagent (invitrogen) and treated with dnase i (takara) according to the manufacturer 0 s protocols. the concentrations of the extracted rna were measured using a nanovue spectrophotometer (ge healthcare). quantitative real-time rt-pcr was conducted using a thermal cycler dice real time system (takara) with gene-specific primer sets described previously (kim and lee, 2013) . the rna levels of viral genes were normalized to that of mrna for the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene, and relative quantities (rq) of mrna accumulation were evaluated using the 2 à δδct method. to detect alteration of genomic rna and subgenomic (sg) mrna levels in the presence of csa during pedv infection, the results obtained using csa-treated samples were compared with vehicle-treated results. all statistical analyses were performed using student 0 s t-test, and p-values of less than 0.05 were considered statistically significant. loss of cyclophilin d reveals a critical role for mitochondrial permeability transition in cell death the permeability transition pore complex in cancer cell death molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china fas-mediated apoptotic signaling in the mouse brain following reovirus infection apoptosis in animal models of virus-induced disease apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death apoptosis-inducing factor (aif): a ubiquitous mitochondrial oxidoreductase involved in apoptosis experimental infection of pigs with a new porcine enteric coronavirus, cv 777 severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis cyclophilin inhibitors block arterivirus replication by interfering with viral rna synthesis sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway human coronavirus-induced neuronal programmed cell death is cyclophilin d dependent and potentially caspase dispensable mitochondrial control of cellular life, stress, and death propagation of the virus of porcine epidemic diarrhea in cell culture essential role of the mitochondrial apoptosis-inducing factor in programmed cell death involvement of cyclophilin b in the replication of japanese encephalitis virus completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence classification of cell death: recommendations of the nomenclature committee on cell death putative partners in bax mediated cytochrome-c release: ant, cypd, vdac or none of them? mitochondrion isolation of porcine epidemic diarrhea virus (pedv) in korea ribavirin efficiently suppresses porcine nidovirus replication porcine hemagglutinating encephalomyelitis virus induces apoptosis in a porcine kidney cell line via caspase-dependent pathways porcine reproductive and respiratory syndrome virus induces apoptosis through a mitochondria-mediated pathway new variants of porcine epidemic diarrhea virus cytochrome c and datp-dependent formation of apaf-1/caspase-9 complex initiates an apoptotic protease cascade calcium flux and calpain-mediated activation of the apoptosis-inducing factor contribute to enterovirus 71-induced apoptosis specific inhibition of hepatitis c virus replication by cyclosporin a immunogenicity and efficacy of recombinant s1 domain of the porcine epidemic diarrhea virus spike protein letter to the editor a new coronavirus-like particle associated with diarrhea in swine an immunoelectron microscopic and immunofluorescent study on the antigenic relationship between the coronavirus-like agent, cv 777, and several coronaviruses the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors. plos pathog chinese-like strain of porcine epidemic diarrhea virus cyclosporine inhibits flavivirus replication through blocking the interaction between host cyclophilins and viral ns5 protein coronaviruses rabies virus-induced apoptosis involves caspase-dependent and caspase-independent pathways cyclophilin d is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences the equine arteritis virus induces apoptosis via caspase-8 and mitochondria-dependent caspase-9 activation molecular characterization of mitochondrial apoptosis-inducing factor bcl-2 inhibits the mitochondrial release of an apoptogenic protease equine coronavirus induces apoptosis in cultured cells mitochondrial regulation of cell death. cold spring harbor perspect an outbreak of swine diarrhea of a newtype associated with coronavirus-like particles in japan viruses and apoptosis actinomycin d enhances trail-induced caspase-dependent and -independent apoptosis in sh-sy5y neuroblastoma cells cysteine protease inhibition prevents mitochondrial apoptosis-inducing factor (aif) release supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2014.04.040. key: cord-345940-adg264vb authors: wanitchang, asawin; saenboonrueng, janya; srisutthisamphan, kanjana; jongkaewwattana, anan title: characterization of influenza a virus pseudotyped with the spike protein of porcine epidemic diarrhea virus date: 2018-08-22 journal: arch virol doi: 10.1007/s00705-018-4001-9 sha: doc_id: 345940 cord_uid: adg264vb the coronavirus spike protein and the influenza virus hemagglutinin are class i viral membrane fusion proteins. while the two proteins display strong structural conservation and the mechanisms underlying membrane fusion are similar, they share no sequence similarity. whether they are functionally interchangeable is currently unknown. in this study, we constructed sciav-s, a single-cycle influenza a virus pseudotyped with the spike protein of porcine epidemic diarrhea virus (pedv), and demonstrated that this virus could infect cultured cells and trigger massive syncytium formation. treatment with endocytosis inhibitors did not affect syncytium formation by infected cells. moreover, the infectivity of sciav-s was associated with the degree of cell adaptation of pedv-s. intriguingly, sciav-s lacking functional neuraminidase (na) exhibited substantially higher infectivity, suggesting a pivotal role of the sialic acid in the binding/entry of pedv. together, sciav-s offers a robust platform for the investigation of the entry mechanism of pedv or, possibly, of other coronaviruses. porcine epidemic diarrhea virus (pedv), a member of the genus alphacoronavirus, is a pleomorphic enveloped virus that contains a large (approximately 28 kb), positive-sense, single-stranded rna genome. it is a causative agent of ped, a highly contagious gastrointestinal disease in pigs with mortality reaching 100% in suckling piglets. while pedv replicates efficiently in porcine enterocytes in vivo, it grows poorly in cultured cells unless adapted through multiple passages [6] . like those of other coronaviruses (covs), the pedv spike protein (pedv-s) is responsible for both receptor recognition and fusion between the virus and host cells [10, 15] . notably, the identity of the receptor used by pedv is still a subject of controversy. while earlier studies have suggested that pedv uses porcine aminopeptidase n (papn) as a cellular receptor [9, 12, 22, 24] , more-recent studies have supported a contrary view based on several lines of evidence [17, 29] . cov-s proteins, including pedv-s, are members of the class i viral membrane fusion proteins, which also include hemagglutinin (ha) from influenza a virus (iav) [3, 14] . both cov-s and ha fold into a metastable prefusion conformation and require proteolytic cleavage (priming) by host proteases to make them fusogenic. this cleavage produces a receptor-binding subunit (ha1 of influenza virus or s1 of cov-s) and a membrane-fusion subunit (ha2 or s2), which remain associated through non-covalent interactions. while details of the prefusion structure of pedv-s are lacking, recent studies of the spike proteins of the betacoronaviruses mhv and hku1 have suggested that the overall prefusion structures of cov-s and ha are similar [14] and that they might share a common mechanism of membrane fusion. however, a few fundamental differences between the two proteins should also be considered. while the fusion peptide of ha is located at the n-terminus of ha2, cov-s has an internal fusion peptide located downstream of the n-terminus of s2. consequently, two proteolysis sites are usually required for the conformational change of s2: one at the s1/s2 junction and the other at the so-called s2' site located at the n-terminus of the fusion peptide [1, 2] . in addition, unlike ha, many cov-s, proteins probably including that of pedv, are not sufficiently primed by proteolysis during viral packaging release and thus require further proteolysis by extracellular proteases, cell-surface proteases, or lysosomal proteases [14] . considering these common and unique properties of the two proteins, it would be interesting to investigate whether they could functionally replace each other in a virus infection context. recombinant retrovirus pseudotyped with pedv-s has been generated and employed to investigate the pedv entry mechanism [20, 33] . however, cells transduced by pseudotyped retrovirus usually display characteristics that are distinct from those of cells that are infected with pedv. for example, cell-cell fusion is rarely observed in transduced cells. notably, with the successful development of a system to construct single-cycle influenza a virus (sciav) [23, 36] , it is now possible to assess whether sciav can be generated when pedv-s is provided in trans instead of ha. the resulting pseudotyped virus (sciav-s) will be beneficial especially for studying entry mechanism mediated by pedv-s. the present study was undertaken to test the hypothesis that pedv-s can functionally replace ha to drive replication of iav by constructing sciav-s and examining its ability to infect pedv-permissive cells. we also showed, using our pseudotyped virus system, that sialic acid is critical for mediating pedv entry. in addition, we demonstrated that iav carrying pedv-s derived from cell-adapted and field-isolated strains exhibited distinct characteristics in infected cells, suggesting different modes of entry among pedv strains. human embryonic kidney (hek) 293t, veroe6-apn, and madin-darby canine kidney (mdck) cells were maintained at 37 °c in opti-mem (thermo scientific) supplemented with 10% heat-inactivated fetal bovine serum (fbs) and 100 iu of penicillin and 100 mg of streptomycin per ml in humidified 5% co 2 incubators. all sciav-pedv-s used in this study were generated in the hek293t cells and stored at −80 °c until use. recombinant pedv avct12 was generated by reverse genetics and propagated in veroe6-apn cells as described previously [8, 37] . influenza a virus (a/ pr/8/34) was generated and titrated as described previously [34] . the full-length pedv s derived from pedv avct12 (gen-bank lc053455.1), pedv yn144 (genbank kt021232.1) and pedv g2 (field isolate) were codon-optimized for expression in mammalian cells and synthesized (genscript and synbio tech). subsequently, each construct was subcloned into the pcaggs expression plasmid. to ensure optimal surface expression, er retention signals at the c-terminal end were removed from all constructs. the phw-ha-mcherry and phw-δm2 plasmids were constructed as described previously [36] . to construct phw-δna, phw2000 encoding the na gene of a/pr/8/34 was subjected to site-directed mutagenesis to introduce two consecutive stop codons at amino acids 164 and 165. all plasmids were subjected to nucleotide sequencing to ensure that no unwanted mutations were inadvertently introduced. sciav-s expressing the mcherry protein were rescued using a strategy similar to one described in a previous study [36] . briefly, hek293t cells in a six-well plate were transfected with 0.5 μg each of phw2000 plasmids encoding the seven segments (pb2, pb1, pa, np, na, m, and ns) from a/ pr/8/34 and phw-ha-mcherry together with 2 μg of the pcaggs plasmid encoding each construct of pedv-s, using fugene hd (promega). to construct sciav-s lacking na, phw-δna was used instead of phw2000-na. likewise, sciav-s lacking m2 was constructed by using phw-δm2 instead of phw2000-m. at 72 h after transfection, cell supernatants were harvested and adsorbed directly onto veroe6-apn cells for further analysis. western blot assays were carried out according to the published procedure with some modifications [35] . transfected cells were collected and lysed in 200 μl of mammalian cell lysis buffer (50 mm tris [ph 8.0], 5 mm edta, 100 mm nacl, 1% np-40 and protease inhibitor mixture) for 30 min on ice. after centrifugation at 10,000 × g for 5 min, lysates were separated on 10% sds-page gels and subsequently transferred to nitrocellulose membranes (bio-rad), followed by blocking with 5% non-fat milk in tbs-t for 1 h. membranes were probed with one of the primary antibodies, including anti-pedv-s mouse polyclonal antibody (a kind gift from dr. qigai he), and anti-β-actin mouse monoclonal antibody clone c-4 (santa cruz biotechnology) followed by goat anti-mouse antibodies conjugated to hrp (biolegend). the signals were visualized with western blotting detection reagent (bio-rad). veroe6-apn cells were grown on a lab-tek ii chamber slide (thermo scientific) in opti-mem supplemented with 10% fbs for 12 h at 37 °c. the adherent cells were inoculated with sciav-s for 1 h at 37 °c. after three washes with pbs, cells were cultured in serum-free opti-mem in the presence of trypsin (2 μg/ml) for 24 h. cells were fixed in 80% chilled acetone for 10 min and blocked in 10% fbs/1%bsa/pbs for 30 min. the slides were subsequently incubated with mouse anti-influenza-a-virus np antibodies (clone 2c9; southern biotech) for 1 h, washed three times with pbs, and incubated with fitc-conjugated anti-mouse igg antibodies. after additional washes, the slides were mounted with antifade mounting medium with dapi (vector laboratories). cells were examined using an olympus ix51 fluorescence microscope. data were expressed as mean ± sd. statistical tests were performed using the student t-test in graphpad prism 5.0 (graphpad software). recently, we established a reverse genetics system to construct sciav in which the ha gene was replaced by the mcherry gene. the virus was recovered in hek293t cells and subsequently propagated in mdck cells stably expressing the ha protein [36] . using a similar strategy, we attempted to rescue sciav-s avct12 by transfecting hek293t cells with plasmids necessary for iav reverse genetics together with codon-optimized s avct12 as depicted in fig. 1a . notably, we did not add trypsin to transfected cells during virus rescue to avoid the detachment of hek293t cells. moreover, since cells expressing s avct12 underwent rapid cell-cell fusion, cells stably expressing s avct12 could not be obtained for the propagation of sciav-s avct12 . alternatively, the trypsin-free viruses released from hek293t cells were directly inoculated onto veroe6-apn cells treated with trypsin 12 h after infection. at 24 after infection, mcherry expression was assessed by fluorescence microscopy. unexpectedly, not only did we observe a strong mcherry signal, but we also detected extensive syncytia in sciav-s avct12 -infected cells (fig. 1b) . in the absence of trypsin treatment, we could still detect a few cells with an mcherry signal but no syncytium formation (fig. 1b) , suggesting that sciav-s avct12 might be able to enter cells but trypsin is essential for induction of cell-cell fusion. it should also be noted that control supernatants from hek293t cells transfected with pcaggs expressing s avct12 alone did not yield detectable syncytium formation in veroe6-apn cells, suggesting that the detected syncytia were not due to pedv-s expressed from residual plasmids (data not shown). to characterize sciav-s avct12 , we initially tried to purify rescued viruses from the supernatants and perform western blot analysis to detect co-expression of pedv-s and iav nucleoprotein (anp). unfortunately, despite several attempts, we could not obtain sufficient purified virus for the assay (data not shown). alternatively, we performed immunofluorescence staining to examine the expression of anp in the syncytium of sciav-s avct12 -infected veroe6-apn cells and detected strong expression of anp (fig. 1c) . to further test whether the mcherry expression in infected cells required active influenza a virus polymerase activity, we attempted to construct sciav-s avct12 by omitting the plasmid encoding the pb2 polymerase and examined its infectivity in veroe6-apn cells. as expected, in the absence of pb2, neither an mcherry signal nor syncytium formation could be detected in infected cells (fig. 1d) . these results suggest that sciav can be pseudotyped with pedv-s and that the pseudovirus can efficiently infect pedv-permissive cells. it is also notable that the massive syncytium formation in sciav-infected cells was unexpected, and this observation has never been reported elsewhere. this points to the possibility that sciav-s might enter cells by a mechanism similar to that of pedv. given that sciav-s avct12 -infected cells formed large syncytia in the presence of trypsin (fig. 1b) and that s avct12 was not synthesized de novo in sciav-s avct12 -infected cells, we speculated that the observed syncytium formation might have been mediated by s avct12 remaining at the cell surface following internalization of sciav-s avct12 . in other words, replacing ha with s avct12 might have switched the mode of cell entry of sciav-s avct12 from endocytosis to direct fusion at the plasma membrane. moreover, since the m2 ion channel is essential for the uncoating of iav in host cell endosomes, entry of sciav-s avct12 by direct fusion with the plasma membrane would bypass viral-endosomal fusion, rendering the proton channel function of m2 unnecessary. to test this hypothesis, we constructed sciav-s avct12 lacking a functional m2 ion channel (sciavδm2-s avct12 ) and assessed its infectivity in veroe6-apn cells. as shown in fig. 2 , we detected undisturbed mcherry expression with large syncytium formation in sciavδm2-s avct12 -infected cells when infected cells were treated with trypsin. notably, we have shown previously that sciavδm2 carrying iav ha showed no mcherry signal in mdck cells [36] . previously, it was reported that pedv strain kpedv-9 is likely to enter host cells via endocytosis, as infectivity was remarkably impaired when cells were treated with endocytosis inhibitors [26] . it is still not known however, whether pedv can also fuse directly with the plasma membrane without being internalized by endocytosis, as reported previously for other covs. given that sciav-s avct12 lacking the functional m2 ion channel could infect veroe6-apn cells and form syncytia without de novo synthesis of s avct12 , we thus hypothesized that sciav-s avct12 and pedv avct12 could enter host cells by direct fusion at the plasma membrane. to this end, veroe6-apn cells were pretreated with hypertonic sucrose (0.45 m) to impair the formation of both coated and uncoated pits at the plasma membrane [4] before being infected with sciav-s avct12 and recombinant pedv avct12 . in contrast to what has been reported previously [26] , we detected no changes in either syncytium formation or mcherry expression in infected cells regardless of hypertonic sucrose treatment (fig. 3a) . likewise, veroe6-apn cells treated with nh 4 cl (50 mm for 2 h) to neutralize the intracellular ph also displayed strong mcherry expression and extensive syncytium formation when infected with sciav-s avct12 (fig. 3a) . in line with the observation in sciav-s avct12 -infected cells, infection of veroe6-apn cells with pedv avct12 was also hardly affected by treatment with hypertonic sucrose or nh 4 cl solution (fig. 3b) . it is also notable that pretreatment of veroe6-apn or mdck cells with hypertonic sucrose or nh 4 cl could substantially inhibit replication of influenza a virus (fig. 3c) . these results collectively suggest that at least pedv avct12 can enter host cells via a mechanism that is independent of the clathrin-mediated endocytosis pathway. several studies have shown that naturally isolated pedv strains, mostly in the g2 genogroup, replicate poorly in vero cells [25] . accumulated mutations in the s gene are strongly associated with cell adaptation [11, 18, 31] . it was therefore of interest to assess the infectivity of sciav pseudotyped with s derived from pedv with various degrees of cell adaptation. s avct12 is closely related to the classical pedv strain sm98-1, which is considered a highly cell-adapted strain. in addition, a variant (yn144) derived from a non-adapted strain after the 144 th passage in vero cells [5] is deemed to be an intermediately adapted strain. we therefore synthesized a codonoptimized s gene (s g2 ) based on a pedv isolated from intestinal tissues of a piglet experiencing severe watery diarrhea during the outbreak in thailand in 2014. blast analysis of the s g2 protein revealed that the amino acid sequence of our protein was 99.12% identical to that of ch/gxnn/2012 (genbank afo42860.1). moreover, a codon-optimized s gene of strain yn144 was synthesized. as mentioned previously, both synthetic constructs were designed to exclude c-terminal er retention motifs (yxxφ and kxhxx). when plasmids expressing each pedv-s were used to transfect veroe6-apn cells in the presence of trypsin, only those expressing s avct12 and s yn144 displayed massive syncytium formation (fig. 4a) . the failure to form syncytia in pedv-s g2 -transfected cells was not due to defects in protein expression in transfected cells (fig. 4a) . we next attempted to construct sciav pseudotyped with s g2 (sciav-s g2 ) and with s yn144 (sciav-s yn144 ) and evaluated their infectivity in veroe6-apn cells. as a control, sciav-s avct12 was also generated and examined. while abundant mcherry-expressing cells and large syncytia were detected in the cell cultures infected with sciav-s avct12 and sciav-s yn144 , those infected with sciav-s g2 displayed considerably fewer positive cells with punctate expression of mcherry and no apparent syncytium formation (fig. 4b) . the absence of syncytia in sciav-s g2 -infected cells may be due to an intrinsic property of pedv-s g2 , as cells transfected with pcaggs-s g2 also displayed less syncytium formation than those transfected with pcaggs-s avct12 and -s yn144 (fig. 4a) . taken together, these results suggest that pedv strains with different degrees of adaptation might utilize distinct pathways for internalization into host cells. several lines of evidence have indicated that pedv utilizes sialic acid, possibly neu5ac, as a co-receptor for attachment and entry [13, 19] . moreover, pretreatment of host cells with recombinant na has been shown to substantially veroe6-apn cells. the mean ± sd from three independent experiments is presented. n.s., not statistically significant. c. veroe6-apn or mdck cells were mock-treated or treated with 0.5 m sucrose or 50 mm nh 4 cl prior to infection with influenza a virus (a/pr/8/34; moi = 0.01). infected cells were maintained in opti-mem supplemented with tpck-trypsin (1 μg/ml). at the indicated time points after infection, supernatants were collected and the amount of infectious virus (tcid 50 ) was determined in mdck cells. the mean ± sd from three independent experiments is presented. n.s., not statistically significant; **, p < 0.01 reduce pedv infectivity, and the n-terminal domain (ntd) of pedv-s (residues 1-249) has been proposed to be critical for sialic acid binding [9] . unlike other previously described pseudotyped viruses, sciav-s, produced in this study, contains active na on its particles. it is therefore ideal for investigating the role of na in infection by sciav-s. since sciav-s does not require na for release, we were able to construct an sciav-s variant bearing a defective na by introducing two stop codons in the na gene (δna). of note, the na deletion did not compromise the packaging signal of the na gene, and therefore, packaging of the na segment should be undisturbed. as expected, in the absence of na, both sciav-s avct12 and -yn144 could infect veroe6-apn cells with substantially higher infectivity (fig. 5a) . while sciav-s g2 with δna did not trigger syncytium formation in infected cells, we observed significantly more cells displaying an mcherry signal when compared to cells infected with the virus with intact na (fig. 5b) . taken together, these results indicate that the na activity of sciav-s might hinder the attachment of the pedv-s on the sciav-s particles with sialic acid on the cell surface. constraints associated with in vitro culture of natural isolates of pedv have considerably hindered the study of their replication, and, consequently, they have not yet been well characterized. in particular, the mechanism underlying how pedv enters a host cell is not well understood. we demonstrate in this study that iav particles could be efficiently pseudotyped with pedv-s, and such pseudoviruses could fig. 4 infectivity of sciav bearing pedv-s derived from viruses with different levels of cell adaptation. a. veroe6-apn cells were transfected with pcaggs encoding s avct12 , s yn144 or s g2 (2 μg each). transfected cells were maintained in opti-mem supplemented with trypsin (2 μg/ml). transfected cells were examined 24 h later for syncytium formation. arrows denote syncytia. likewise, hek293t cells were transfected with pcaggs encoding pedv-s using the same conditions. at 48 h after transfection, cells were harvested, lysed and subjected to western blot analysis using anti-pedv-s1 antibodies. beta actin was used as a loading control. b. sciav psedotyped with s avct12 , s yn144 or s g2 was constructed, and its infectivity in veroe6-apn cells was evaluated. at 24 hpi, infected cells were examined for syncytium formation and mcherry expression under a fluorescent microscope. scale bars, 50 μm (20 μm for sciav-s g2 -infected cells) be employed to investigate the receptor binding and cellular entry of pedv. using a strategy similar to the one used with our previously described sciav system [36] , we successfully rescued sciav carrying the mcherry gene in hek293t cells co-expressing pedv-s derived from various strains and demonstrated that sciav-s avct12 could infect veroe6-apn cells and produce a strong mcherry signal. unfortunately, due to the high fusogenicity of s avct12 , we could not generate cells stably expressing the protein as a platform to propagate sciav-s avct12 in the current study. however, s avct12 is now being modified to be less fusogenic without compromising its receptor-binding function. one of the most intriguing findings is our observation that sciav-s avct12 -infected cells could form massive syncytia similar to those produced by pedv-infected cells. since s avct12 is not synthesized de novo in sciav-s avct12infected cells, the syncytium formation observed at neutral ph is likely to be due to interaction between the receptor and s avct12 remaining on the cell surface after internalization of the virus. our data thus underscore the likelihood that sciav-s avct12 can enter cells by direct fusion at the plasma membrane. it is worth noting that some studies have shown that treatment with trypsin allows some covs to bypass the endosome-dependent pathway for entry [1, 21, 30] . however, sciav-s avct12 was recovered in the absence of trypsin, and virus inoculation was carried out using trypsin-free viruses. notably, unless trypsin was added at 12 hpi, we could not detect any syncytium formation. indeed, syncytium formation was entirely unexpected, as pedv has been reported to enter cells by clathrin-mediated endocytosis even in the presence of large amounts of trypsin during infection [26] . we also showed that sciav-s avct12 lacking the m2 proton channel could infect veroe6-apn cells and trigger formation of large syncytia similar to those observed in sciav-s avct12 -infected cells. in contrast to our previous report that sciav-ha requires functional m2 for infectivity [36] , our findings here suggest that sciav-s avct12 can undergo replication without the need for m2-mediated internal virion acidification in the endosome. that pedv can enter permissive cells by both direct fusion with the plasma membrane and endocytosis is not surprising as similar phenomena have also been observed with other covs [7, 30, 32] . in this study, we also speculated that differences in the s proteins of different pedv strains might play a role in determining the mode of entry. in line with our hypothesis, park and colleagues have recently demonstrated that the cleavage of the s protein during viral assembly appears to affect the mode of mers-cov entry. if s is cleaved at the s1/s2 junction during biogenesis by host proteases such as furin or proprotein convertases in producer cells, the released virions are primed and able to enter target cells by fusing at the plasma membrane. alternatively, virions released with uncleaved s need to be processed further by endocytic proteases in the target cell and therefore require endocytosis for entry [27] . although pedv-s, like other alphacoronaviruses, is a. sciav pseudotyped with s avct12 or s yn144 in the presence or absence of a functional na gene in segment 6 was constructed. syncytium formation was assessed in veroe6-apn cells (in a six-well plate) infected with each sciav-s construct. the mean + sd from three independent experiments is presented. **, p < 0.01. b. sciav pseudotyped with s g2 in the presence or absence of a functional na gene in segment 6 was constructed. the expression of mcherry was assessed in infected veroe6-apn cells at 24 hpi. scale bars, 20 μm believed to be present in an uncleaved form [15] , data showing the cleavage of s in different pedv strains are lacking. s proteins of cell-adapted pedv such as pedv avct12 or pedv yn144 may be cleaved during morphogenesis of the virion, whereas those of pedv g2 may require further processing in the target cells. further analysis of cleavage of the s protein in cell-adapted and field-isolated strains will be critical for better understanding of the mechanisms underlying pedv entry. another important point worth mentioning is that we showed, using sciav-s, that sialic acid moieties located on the cell surface plays a role in pedv attachment. although it has been demonstrated that the ntd of pedv-s could bind to sialic acid, most data of the available have been based on assays using recombinant s proteins [13] . because the construction of recombinant pedv bearing specific mutations in the s gene can be technically challenging, the available data concerning the binding of pedv s to sialic-acid-containing glycoproteins in the context of infection have so far been limited to the use of natural isolates such as the pedv-uu strain [15] . with a well-established iav reverse genetics system, sciav-s serves as a robust platform to assess binding of the s protein to sialic acid in the context of infection. furthermore, since many covs, such as mers-cov [16] , and transmissible gastroenteritis coronavirus (tgev) [28] , have been reported to bind to sialic acids, it is also possible to construct sciav pseudotyped with cov-s of interest to investigate this interaction in more detail. it is noteworthy that cells infected with sciav-s display massive cell-cell fusion that is very similar to what is observed in cells infected with pedv but does not occur in cells transduced with other pseudoviruses. moreover, we showed here that pedv-s derived from a field isolate, despite its poor growth in vitro, can be cloned into the expression vector and subsequently used to construct sciav-s for further investigation. sciav thus serves as a new platform not only for investigating the mechanism by which pedv-s mediates cell entry but also to gain insights into the entry mechanisms utilized by cell-adapted and naturally isolated pedv. activation of the sars 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virus counterpart in promoting virus replication the role of orf3 accessory protein in replication of cell-adapted porcine epidemic diarrhea virus (pedv) acknowledgements we thank dr. qigai he for the kind gift of the anti-pedv-s antibodies.funding this study was funded by national center for genetic engineering and biotechnology (biotec)'s fellow grant, national science and technology development agency (p-15-51261) and giga impact initiative grant (p16-51674). human and animal rights statement neither animal nor human testing was involved in this study. key: cord-353245-es7b1rs0 authors: song, deping; huang, dongyan; peng, qi; huang, tao; chen, yanjun; zhang, tiansheng; nie, xiaowei; he, houjun; wang, ping; liu, qinglan; tang, yuxin title: molecular characterization and phylogenetic analysis of porcine epidemic diarrhea viruses associated with outbreaks of severe diarrhea in piglets in jiangxi, china 2013 date: 2015-03-19 journal: plos one doi: 10.1371/journal.pone.0120310 sha: doc_id: 353245 cord_uid: es7b1rs0 porcine epidemic diarrhea (ped), caused by porcine epidemic diarrhea virus (pedv), is a highly contagious, acute enteric viral disease of swine characterized by vomiting, watery diarrhea, dehydration and death. to identify and characterize the field pedvs associated with the outbreaks of severe diarrhea in piglets in jiangxi, 2013, the complete genome sequences of two representative strains of pedv, designated ch/jx-1/2013 and ch/jx-2/2013, were determined and analyzed. the genome sequences of both emergent jiangxi pedv strains, ch/jx-1/2013 and ch/jx-2/2013, were 28,038 nucleotides in length excluding 3’ poly (a) tail. compared to the pedv cv777 strain, ch/jx-1/2013 and ch/jx-2/2013 had some unique genetic characteristics in the proximal region of the 5´-utrs. phylogenetic analysis of the complete genomes and the structural proteins revealed that ch/jx-1/2013 and ch/jx-2/2013 had a close relationship with post-2010 chinese pedv strains and us strains identified in 2013. the nucleotide identity between the two jiangxi strains (ch/jx-1/2013 and ch/jx-2/2013) and 30 strains of pedv identified ante-2010 and post-2010 ranged from 96.3–97.0% and 97.3–99.7%, respectively. multiple nucleotide and deduced amino acid mutations were observed in the orf1a/b, s, orf3, e, m and n genes among the current field pedv strains when compared to the cv777 strain. some of the mutations altered the amino acid charge and hydrophilicity, and notably, there was an amino acid substitution in the middle of one neutralizing epitope (l1371i) of the s gene of both ch/jx-1/2013 and ch/jx-2/2013. taken together, the accumulated genetic variations of the current field pedv strains might have led to antigenic changes of the viruses, which might confer the less effectiveness or failure of the cv777-based vaccines currently being widely used in jiangxi, china. the ethics committee of jiangxi agricultural university approved the animal protocol for this study (protocol number p-2013-03). all the procedures involving animals in this study were carried out in accordance with the care and use guidelines of experimental animals established by the ministry of agriculture of china. intestinal and fecal samples were collected according to the approved procedures. the intestinal samples used in this study were obtained from the dead piglets and the fecal samples were non-invasively collected immediately after excretion from healthy and diarrheal pigs from premises with ped outbreaks in jiangxi, china (s1 table) . fecal and intestinal samples (n = 125) were collected from < 10-day-old suckling piglets with severe watery diarrhea, vomiting and dehydration from different premises in jiangxi province, china (25-29°n and 114-118°e) in 2013. all the samples were examined by a rt-pcr established in our laboratory. briefly, the total rna was extracted from the samples using rnaplus reagent (takara, japan) following the manufacturer's instructions. the one-step rt-pcr for determination of pedv-positive samples was carried out using 50-200 ng of extracted rna, forward primer (5´-gtattggtggtgagcggaat-3´), reverse primer (5´-cctgttcc gccattctatca-3´) and onestep rt-pcr kit (qiagen, valencia, ca, usa) according to the manufacturer's protocol. to eliminate possible co-infections caused by porcine transmissible gastroenteritis virus (tgev), porcine rotavirus (porv), and other common pathogenic intestinal pathogens, differentiation assays were performed using standard protocols. ninety-six out of 125 samples were tested positive for pedv, and all samples had been confirmed to be free of tgev, porv and other common enteric pathogens (data not shown). to address the genetic/antigenic variations, and phylogenetic characteristics of pedv strains associated with 2013 jiangxi ped outbreaks, two representative pedv strains, designated ch/jx-1/2013 and ch/jx-2/2013 (the genbank accession numbers are kf760557 and kj526096, respectively), were used for sequencing the full-length genome. total rnas were extracted from the feces and small intestinal homogenates by rnaplus reagent (takara, japan) according to the manufacturer's instructions. the concentrations of the extracted rnas were measured by nanodrop 2000 spectrophotometer (thermo scientific, usa) and then stored at −80°c until use. the first-strand cdna synthesis was performed at 42°c for 50 min and then 95°c for 5 min to inactivate the m-mlv reverse transcriptase (takara, japan) and followed by 4°c for 5 min. the entire pedv genome was amplified by 33 pairs of primer designed with primer 3 software (http://primer3.ut.ee/) based on the conserved regions determined by a multiple alignment analysis of the reference strains and cv777 (s2 table) . fragments were amplified on the conditions of a denaturation at 94°c for 4 min, 35 cycles (94°c x 45 sec, 53°c x 45 sec, 72°c x 1.5 min), and then with a final extension at 72°c for 10 min. pcr products obtained were subjected to gel purification using a gel extraction kit (takara, japan), and afterwards cloned into pmd 18-t vectors (takara, japan) following the manufacturer's protocols. five positive clones of each amplicon were submitted to a commercial sequencing company (sangon biotech, shanghai, china) for sequencing at both directions by sanger sequencing methodology. the 5'-and 3'-race for the determination of the terminal sequences of both ch/jx-1/2013 and ch/jx-2/2013 were performed by using 5'/3' smarter race kit (clontech, beijing, china) following the manufacturer's instructions. the raw sequence fragments were imported to seqman in dnastar lasergene v 7.10 (dnastar, inc., madison, wi) for assembly and annotation. nucleotide and deduced amino acid (aa) sequences of both ch/jx-1/2013 and ch/jx-2/2013 and 30 reference pedv sequences retrieved from genbank were comparatively analyzed. a summary of the background information of pedvs used in this study is shown in table 1 . the complete genome sequences of ch/jx-1/2013 and ch/jx-2/2013 were deposited into genbank. phylogenetic trees based on the entire genomes, and deduced aa sequences of s, orf3, e, m, and n genes were constructed using the neighbor-joining method of mega 5.2.2 (http://www.megasoftware.net/) with a bootstrap of 1,000 replicate datasets. primary sequences of 5´-proximal region of 5´-utrs (nt 42 to 133) were pairwise compared between the two jiangxi strains (ch/jx-1/2013 and ch/jx-2/2013) and the reference strains. antigenicity and hydrophilicity analyses based on the aa sequence from 1 to 350 at n-terminal of the s proteins were carried out by protean software of dnastar lasergene v7.10 (dnastar, inc., madison, wi). table) . a total of 26 unique nucleotide substitutions were identified between two jiangxi pedv strains (ch/jx-1/2013 and ch/jx-2/2013) and 30 reference pedv strains, which resulted in 14-aa changes, and most of them were located in orf1a, orf1b and s gene. two of the nucleotide substitutions led to aa changes along with the charge variations ( table 2 ). the phylogenetic tree based upon the full-length genome sequence of ch/jx-1/2013, ch/jx-2/2013 and 30 reference pedvs indicated that the pedv strains could be divided into two groups, designated for group1 (g1) and group 2 (g2): g1 could be further divided into three subgroups, i.e., 1a, 1b and r, a tentative cluster consisting of virulent dr13 isolated in south korea in 1999 and ch/s isolated in china in 1986; and g2 was split into two subgroups, 2a and 2b (fig. 1a) . notably, all the strains identified from 2011 to 2013 were fallen into g2; while the prototype of cv777, together with three korean strains (sm98, virulent dr13 and attenuated dr13), and four earlier chinese strains (js2008, js2008new, lzc and ch/s) were fallen into another group, i.e., group 1. genetic characteristics were observed between the two groups: 1) compared to genome sequences of the members in g1, four insertions, 20803g, 20810cagggtgtcaa20820, 20830g, 21042aat21044 and two deletions, 20842a, 21097cgtgat21102, existed in the n-terminal domain (ntd) of the s protein in g 2 members; 2) the three field pedv strains of js2008, js2008new and sd-m together with two attenuated pedv strains, dr13 and vaccine_kc189944, were clustered into subgroup 1b. all of the five pedv strains aforementioned had 24-nt deletions in nsp3 of orf1a and 49-nt deletions in the c terminus of orf3; 3) ch/jx-1/2013 and ch/jx-2/2013 along with three chinese strains (gd-b, js-hz2012 and bj-2011-1) were clustered into an independent clade, and ch/jx-1/2013 and ch/jx-2/2013 had 99.7%, 99.7% and 99.6% nucleotide identity with the three chinese strains, respectively. these strains shared six additional unique nucleotide substitutions (t227a, c2342t, g2346t, t2724c, t6331c, c7233t) with the rest of reference pedv strains. of which, one led to an aa change (t682m in orf1a, from hydrophilic polarity t to hydrophobic polarity m). the full-length of s gene of ch/jx-1/2013 and ch/jx-2/2013 was 4,161 nt in size, which was 9nt longer than that of the prototype of pedv cv777 strain. the results from nucleotide sequence comparisons of the s gene of 34 strains of pedv, including ch/jx-1/2013, ch/jx-2/2013 and other 32 reference pedv strains from china, united states, uk, south korea, belgium, france and japan showed that the two jiangxi strains had a 99.8% nucleotide identity with each other, and 96.7% nucleotide identity with cv777. ch/jx-1/2013 and ch/jx-2/2013 shared 97.3-99.6% nt identity and 93.1-99.1% aa identity with the post-2010 pedv strains, respectively. by contrast, the two jiangxi strains showed only 93.5-95.0% nt identity, and 92.5-94.8% aa identity with the ante-2010 pedv strains and attenuated vaccine strains, respectively (s4 table) . the phylogenetic tree based on the aa sequences of the s protein of 34 strains of pedv showed that they could be classified into two major groups, and each group contained two subgroups (fig. 1b) . both ch/jx-1/2013 and ch/jx-2/2013 belonged to subgroup 2b, which also included five newly identified us strains (13-019349, co/13, ia1, isu13-22038-ia-homogenate and isu13-22038-ia-p9) and 13 chinese strains identified at the same period or later than 2010. compared to g1, there were two insertions (61vnst64 and 136n) and one deletion (155dg156) in the g2 members. amino acid differences were also present between g1 and g2, and most of them were located in the area of aa 1 to 350 at n-terminal of the s protein ( table 3 ). the analysis based on aa from positioned 1 to 350 of the s protein suggested that the antigenicity and hydrophilicity of s protein of ch/jx-1/2013, ch/jx-2/2013 might have already changed due to the mutated amino acids with changes of charges and polarities (fig. 2) . notably, an amino acid substitution was found in the middle of one neutralizing epitope phylogenetic trees based on the complete genome, aa sequences of structural proteins and orf3 of pedv strains. the trees were constructed by the distance-based neighbor-joining algorithm using mega 5.2.2 software. bootstrap was set in 1,000 replicates with a value >70% to assess the significance of the tree topology. a bar of 0.002/0.005 indicates nucleotide or amino acid substitutions per site. "•" indicates the strains identified in this study, "" indicates the strains from china, "◆" indicates the strains from belgium, "■"indicates the strains from usa, "▲"indicates the strains from south korea, "▼"indicates the strains from france, "◇"indicates the strains from japan. 1a: phylogenetic tree generated on the basis of nucleotide sequences of the complete genome of 33 pedvs. 1b to 1f: phylogenetic trees based on deduced amino acid sequences of s glycoprotein genes, orf3, envelope, membrane, and nucleocapsid genes, respectively. table 4 ). the phylogenetic tree based on the deduced aa sequences of the orf3 of the earlier european strains, cv777, korean strains, us strains and chinese strains identified ante or post-2010 revealed that those strains were grouped into two different groups (fig. 1c) . cv777 and two early strains sm98 and lzc, and two cell-adapted strains sd-m and attenuated dr13 were classified into group 1; while the 24 strains, including ch/jx-1/2013, ch/jx-2/2013, 15 chinese pedv strains, a korean strain (virulent dr13), five newly determined us strains, and (from negative charge/hydrophilic polarity e to neutral charge/ hydrophobic polarity q) of the n terminus of the m protein, which might have an impact on its antigenicity/immunogenicity. the entire n gene of ch/jx-1/2013 and ch/jx-2/2013 was 1,326 nt long, encoding a protein of 441-aa. sequence analyses revealed that the aa homology between ch/jx-1/2013 and ch/jx-2/2013 and other pedv strains used in this study varied from 95-98.9%. the nucleotide and amino acid sequences of n protein were highly conserved and neither insertion nor deletion was found in all these pedv strains analyzed in the study except a few sporadic mutations. multi-alignment results of the aa sequences of the pedv n proteins indicated that all the strains could be divided into two groups, i.e., group 1 and group 2. each group contained two subgroups (fig. 1f) . the phylogenetic topology of n protein was similar to that of orf3. three specific aa changes were present between group 1 and group 2, at the residue 142 (a142t), 242 (h242l, from hydrophilic polarity i to hydrophobic polarity t) and 397 (q397l). five aa substitutions at the residue 84 (g84a), 205 (n205k, from neutral charge n to positive k), 381(l381p), 395 (l395q, from hydrophobic l to hydrophilic q), and 398 (h398n, from hydrophilic h to hydrophobic n) existed in subgroup 2a when compared with other three subgroups, i.e., subgroup 2b, subgroup1a and subgroup 1b. the 5´utrs of ch/jx-1/2013 and ch/jx-2/2013 were 292 nt in length, which was 4-nt shorter than that of cv777, resulting from 5-nt deletion and one nt insertion in the proximal region of 5´-utrs of the two jiangxi strains (fig. 3) . the core sequence (cuaaac) of the pedv leader transcription-regulating sequence (trs) was extremely conserved with no nucleotide substitutions in all the pedv strains. an 'a' deletion was observed immediately following the core sequence of these pedv strains except the strains of cv777 and lzc. in comparison with cv777 and early isolated strains of sm98 and lzc, a 'u' insertion in loop 2 was found in both ch in other four pedv strains isolated earlier (cv777, attenuated vaccine, lzc, and sm98). compared to cv777, a 4-nt deletion (uucc) existed in the stem region of sl4 of ch/jx-1-/2013, ch/jx-2/2013 and other six pedv strains. however this deletion would not impact the secondary structure of the sl4 as demonstrated by an analysis (data not shown). the 3'utrs of both ch/jx-1/2013 and ch/jx-2/2013 were 334 nt in size, and no insertion and deletion was observed (table 2 ). ped affected massive pig farms in jiangxi, china in 2013, causing substantial economic losses in the pig industry of jiangxi. to elucidate the molecular characterization and phylogeny of the pedv field strains associated with 2013 jiangxi ped outbreaks, the entire genome sequences of two representative pedv strains confirmed in jiangxi were determined and analyzed. phylogenetic analyses demonstrated that ch/jx-1/2013 and ch/jx-1/2013 defined in this study along with the strains determined post-2010 were clustered into group 2, whereas the strains detected ante-2010 were clustered into group 1. the results of phylogenetic analyses of those pedv strains examined in this study were similar to that of described elsewhere [22] . ch/jx-1/2013 and ch/jx-1/2013 showed the highest nucleotide identity (>99%) with six newly confirmed us strains (ia1, co/13, usa/indiana, isu13-22038-ia-homogenate, isu13-22038-ia-p9, and 13-019349) and five chinese strains (gd-b, ah2012, bj-2011-1, ch/zmd/zy, and ch-hz/2012) identified post-2010, suggesting these pedv strains might have evolved from the same origin although the mechanisms of the evolution of the viruses are roughly unknown yet. three field pedv strains, js2008, js2008new and sd-m together with two attenuated pedv strains, dr13 and vaccine_kc189944, were clustered into an independent cluster and showed 24-nt deletions in nsp 3 of orf1a and 49-nt deletions in the c terminus of orf3. these unique characteristics suggested that the three field pedv strains might derive from the same source, and a recombination event might have occurred between these three viruses and the two vaccine strains in the same subgroup. interestingly, a comparison analysis revealed that ch/jx-1/2013 and ch/jx-1/2013 had 99.7% nucleotide identity to the strain of gd-b (genbank accession no.jx088695) isolated from guangdong, an adjacent province of jiangxi, in which the severe ped outbreaks emerged in october 2010 [11, 23] . pigs are frequently traded between jiangxi and guangdong, which might be one of important factors causing the cross dissemination of pedvs in this region. however, further study needs to be performed. the findings from this study demonstrated that the 5´-utr and orf3 protein of ch/jx-1/2013 and ch/jx-2/2013 and all other pedv strains analyzed were highly conserved, which were consistent with the studies reported previously [5, 9] . in general, strict conservation of these regions is essential for the pedv life cycle. it is known that the 5´-utrs of coronaviruses form conserved rna structural elements which are critical for viral replication, sgmrna transcription, and translation [24] . studies on the betacoronavirus have indicated that the stemloop 2 (sl2) in 5´utr is extremely conserved in all coronaviruses, and plays a cis-acting reaction on transcription and replication, and more importantly the replication of the virus requires the conservative sequence and a firm number of nucleotides with specific properties in sl2 in 5´utr [25] . although the 'u' insertion in sl2 does not alter the rna secondary structure, it might slightly affect the efficiency of virus replication, which needs to be addressed in the future studies. the core sequence (5´-cuaaac-3´) in trs of pedv, which was also present in ch/jx-1/2013 and ch/jx-2/2013, has reported to be a determinant factor in transcriptional regulation in coronavirus because the synthesis of sgmrna requires the appropriate tertiary structure of core sequence [26] . both pedv and tgev belong to the alphacoronavirus genus within the coronaviridae family and possess the conserved core sequence structure. studies based on the infectious genomic tgev cdnas have proved that the nucleotides immediately flanking the trs sequence could apparently affect the expression of the sgmrna. and the nucleotides adjacent to core sequences of the leader trs (cs-l) by the 3´region are more decisive for mrna synthesis than nucleotides in the 5´region. the motif of 5´-gaaa-3´within 3´cs-l (5´-cuaaacgaaa-3´) shows a higher expression than that of other motifs [27] . in respect of the pedv, an 'a' deletion immediately followed the cs-l sequence in the 5´utr of ch/jx-1/2013 and ch/jx-2/2013 as well as other post-2010 pedv strains analyzed in the study and thus, this deletion formed a four base oligonucleotide (5´-gaaa-3) adjacent to the cs-l by the 3´region, which might up-regulate the sgmrna expression in pedv [24] . the s protein of pedv is known to play pivotal roles in viral entry and inducing the neutralizing antibodies in natural hosts, and thus makes it become a primary target for the development of effective vaccines against pedv [28] [29] [30] [31] . it was demonstrated that there were significant genetic variations in the s gene between the newly determined pedv field strains and early isolates [20, 32] . the results in this study agreed with previous documentations. additionally, ch/jx-1/2013 and ch/jx-2/2013 showed extremely high nucleotide and aa identity with each other but less to cv777 and attenuated strains (attenuated vaccine_kc18944 and attenuated dr13). there were significant aa differences between the g1 and g2 members, and most of them were located in ntd of the s protein of pedvs. when compared to the group 1 strains of pedv, ch/jx-1/2013 and ch/jx-2/2013, members of group 2, showed significant antigenic and hydrophobic differences, and some of which were located in the neutralizing epitope regions. those genetic variations made the ch/jx-1/2013 and ch/jx-2/2013 and post-2010 field pedv strains different from the ante-2010 strains, especially the cv777. it might explain why the recent ped outbreaks were significantly different from the previous sporadic outbreaks. present ped became a devastating enteric viral disease causing substantial economic losses in the pig industry in major pig-raising countries in the world, and recently made it become a reportable disease by usda [33] . moreover, the present pedv field strains displayed considerable genetic variations from cv777-based vaccine strain, a strain being widely used for production of pedv vaccines in china for many years. notably, leucine, a highly hydrophobic aa residue in the middle of one neutralizing epitope (l1371i) of the s gene of cv777 was substituted by an isoleucine, a higher hydrophobic index aa residue in both ch/jx-1/2013 and ch/jx-2/2013 strains. this variation might provide a possible mechanism for a poor protection on swine vaccinated with the cv777-based vaccines. park et al. [29] have reported that a reduced cell-culture-adapted pedv strain (attenuated dr13, passage 100) has a truncated orf3 of a 51-nt deletion, suggesting that this gene may be involved in cell tropism and is essential for the virulence of pedvs. by contrast, ch/jx-1/2013 and ch/jx-2/2013, together with the post-2010 variant pedv strains had an intact orf3 of 675 nucleotides encoding a protein of 224 aa. the phylogenic analysis demonstrated that the orf3-based tree had a similar topology with the one generated from the entire genome sequences. and thus the orf3 of pedv may serve as a useful target gene for phylogenetic analysis of newly emerging field pedv strains since its small size. as recognized previously [34, 35] , there is a large deletion region existing in pedv isolates attenuated dr13 (51-nt at the position of 245-295), sm98 (210 nt at the position of 1-210) and sd-m (51-nt at the position of 245-295), a chinese strain propagated only four passages on cell lines. in summary, the 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chinese porcine epidemic diarrhea virus strains based on spike, orf3, and membrane genes cloning and further sequence analysis of the orf3 gene of wild-and attenuated-type porcine epidemic diarrhea viruses xiangbai hua and jiaxiang li are greatly appreciated for their help in sampling campaign. key: cord-339871-jso21mbx authors: lee, sunhee; lee, changhee title: genomic and antigenic characterization of porcine epidemic diarrhoea virus strains isolated from south korea, 2017 date: 2018-05-16 journal: transbound emerg dis doi: 10.1111/tbed.12904 sha: doc_id: 339871 cord_uid: jso21mbx porcine epidemic diarrhoea virus (pedv) is a globally emerging and re‐emerging enteric coronavirus in pigs causing serious economic threats to the world swine industry. since the re‐emergence of massive pedv outbreaks in south korea in 2013−2014, domestic pig farms have continued to experience ped epidemics or endemics. this study represents the molecular characterization of pedv isolates identified in diarrhoeic animals collected across the country in 2017. initial sequencing analysis of the full‐length s genes revealed that 70% of the 2017 isolates (7/10) belong to the g2b subgroup, while the remaining isolates were classified as g1b. the data indicated that both variant g1b and global epidemic g2b strains were responsible for current ped outbreaks in south korea. the 2017 g1b and g2b isolates shared 98.7%–99.4% and 98.1%–99.2% amino acid sequence identity at the s gene level and 99.3% and 99.0%–99.6% nucleotide sequence homology at the genome level compared to the corresponding korean prototype g1b and g2b strains, respectively. in an interesting manner, one g2b‐like knu‐1705 strain was found to possess a large 39‐nucleotide deletion in the orf1a region theoretically encoding nonstructural protein 3. phylogenetic analysis based on the entire genome and spike protein sequences indicated that the 2017 isolates were most closely related to other global g1b or g2b strains but formed different branches within the same genogroup. these results indicate that pedvs undergo continuous evolution in the field. in addition, one 2017 pedv strain, kor/knu‐1705/2017, was successfully isolated and propagated in vero cells. the antisera raised against the korean prototype 2014 g2b strain efficiently neutralized knu‐1705 virus infection, suggesting antigenic homology between the 2014 and 2017 pedv strains. our data advance the understanding of the molecular epidemiology and antigenicity of pedv circulating in south korea. polyadenylated tail and consists of seven canonical coronaviral genes, including open reading frame (orf) 3, in the following conserved order: 5 0 untranslated region (utr)-orf1a-orf1b-s-orf3-e-m-n-3 0 utr (kocherhans, bridgen, ackermann, & tobler, 2001) . the first two large orfs, orf1a and 1b, encompass the 5 0 -proximal two-thirds of the genome and code for nonstructural proteins (nsps). orf1a translation yields a replicase polyprotein (pp) 1a, whereas orf1b is expressed by a à1 ribosomal frame shift that c-terminally extends pp1a into pp1ab. these pp1a and pp1ab are proteolytically matured by internal viral proteases to generate 16 processing end-products, named as nsp1-16. the remaining orfs in the 3 0 -proximal region of the genome encode four canonical structural proteins, spike (s), envelope (e), membrane (m) and nucleocapsid (n), as well as one accessory gene, orf3 (duarte et al., 1994; kocherhans et al., 2001; lai, perlman, & anderson, 2007; lee, 2015; saif et al., 2012) . porcine epidemic diarrhoea was first observed in feeder and fattening pigs in england in 1971 (oldham, 1972) and caused widespread epidemics in multiple swine-producing countries in europe during the 1970s (opriessnig, 2016) . a marked decrease in acute ped epizootics occurred in europe in the 1980s and 1990s, and only sporadic outbreaks have occurred in recent years (opriessnig, 2016) . in asia, ped was first reported in 1982, and unlike in europe, it has since posed a huge economic threat to the asian pork industry (chen et al., 2008; kweon et al., 1993; li et al., 2012; puranaveja et al., 2009; takahashi, okada, & ohshima, 1983) . in may 2013, ped outbreaks suddenly appeared in the united states and swiftly spread across the nation, as well as to adjacent countries. this outbreak caused the death of more than 8 million newborn piglets in the united states alone during a 1-year-epidemic period, leading to annual losses in the range of $900 million to $1.8 billion (langel, paim, lager, vlasova, & saif, 2016; mole, 2013; ojkic et al., 2015; stevenson et al., 2013; vlasova et al., 2014) . the us emergent strain-like viruses further reached east asian countries, resulting in nationwide ped disasters (lee, 2015; lin et al., 2014; maff, 2018) . during the 2013-2014 pandemics, ped rapidly swept across mainland south korea and jeju island, killing hundreds of thousands of piglets in domestic herds , 2017 . since then, ped epizootics or enzootics have regionally occurred through provinces in south korea with intensive swine industries. to investigate the diversity of pedvs responsible for the ongoing outbreaks in south korea, in this study, we determined the full-length sequences of the s proteins of field isolates and complete genome sequences of representative strains identified throughout 2017. in addition, we isolated and serially propagated a kor/knu-1705/2017 strain and assessed the antigenic cross-reactivity between 2014 and 2017 pedv field isolates. the small intestine or stool specimens were collected from piglets showing acute watery diarrhoea at various swine farms located in eight different provinces from march through december 2017. intestinal homogenates were prepared as 10% (wt/vol) suspensions in phosphate-buffered saline (pbs) using a magna lyser (roche diagnostics, mannheim, germany) by three repetitions of 15 s at a speed of 8,000 g. faecal samples were also diluted with pbs to 10% (wt/vol) suspensions. the suspensions were then vortexed and centrifuged for 10 min at 4,5009 g (hanil centrifuge fleta5, incheon, south korea). the clarified supernatants were initially subjected to rt-pcr using a tge/ped detection kit (intron biotechnology, seongnam, south korea) according to the manufacturer's instructions. pedv-positive samples were filtered through a 0.22-lm-poresize syringe filter (millipore, billerica, ma) and stored at à80°c until subsequent sequencing analysis and virus isolation. the s glycoprotein gene sequences of the virus isolates were determined by the traditional sanger method. two overlapping cdna fragments spanning the entire s gene of each isolate were amplified by rt-pcr as described previously (lee, park, kim, & lee, 2010) . the individual cdna amplicons were gel-purified, cloned into a pgem-t easy vector system (promega, madison, wi) and sequenced in both directions using two commercial vector-specific t7 and sp6 primers and gene-specific primers. the full-length s sequences of 10 pedv, designated knu-1701 to -1710, were deposited in the gen-bank database under the accession numbers shown in figure 1a . in addition, the complete genomes of representative pedv field strains were sequenced by the traditional sanger method. ten overlapping cdna fragments spanning the entire genome of each virus strain were rt-pcr-amplified as described previously lee et al., 2015 , and each pcr product was sequenced as described above. the 5 0 and 3 0 ends of the genomes of individual isolates were determined by rapid amplification of cdna ends (race) as described previously . general procedures for dna manipulation and cloning were performed according to standard procedures (sambrook & russell, 2001) . the complete genomic sequences of the 2017 viruses were deposited in the gen-bank database under the accession numbers shown in figure 1b. the sequences of the 48 fully sequenced s genes and 31 complete genomes of global pedv isolates were independently used in sequence alignments and phylogenetic analyses. multiple sequence alignments were generated with the clustalx 2.0 program (thompson, gibson, plewniak, jeanmougin, & higgins, 1997) , and the percentages of nucleotide sequence divergences were further assessed using the same software program. phylogenetic trees were constructed from the aligned nucleotide or amino acid sequences using the neighbour-joining method and subsequently subjected to bootstrap analysis with 1,000 replicates to determine the percentage reliability values of each internal node of the tree (saitou & nei, 1987 pedv isolation was conducted from faecal suspensions on vero cells in the presence of trypsin (usb, cleveland, oh) as described previously . virus isolation was confirmed by cytopathic effect (cpe) observation, immunofluorescence assay (ifa) and nucleotide sequencing as described previously . the isolated pedv strain was propagated for serial passages in vero cells, and virus titres were determined as described previously . the cross-reactivity of antisera collected from sows inoculated with a korean pandemic g2b strain knu-141112 isolated in 2014 (baek et al., 2016) was evaluated by a serum neutralization (sn) test in 96-well microtiter plates against the past 2014 and present 2017 isolates as previously described oh, lee, choi, & lee, 2014) . the neutralization titre was calculated as the reciprocal of the highest dilution of serum that inhibited virus-specific cpe in all duplicate wells. the pedv s glycoprotein is a suitable viral gene for investigating genetic relatedness among isolates and the molecular epidemiology of pedv (chen et al., 2014; gerber et al., 2014; lee, 2015; lee et al., 2010; oh et al., 2014) . based on the s gene sequences, therefore, pedv can be genetically separated into two genogroup clusters, genogroup 1 (g1, classical and recombinant: low-pathogenic) and genogroup 2 (g2, field epizootic or panzootic: high-pathogenic), which are further divided into subgroups 1a and f i g u r e 1 phylogenetic analysis based on nucleotide sequences of the spike genes (a) and full-length genomes (b) of porcine epidemic diarrhoea virus strains. a region of the spike protein and complete sequence of tgev were included as an outgroup in each tree. multiple sequence alignments were performed using the clustalx program, and the phylogenetic tree was constructed from the aligned nucleotide sequences using the neighbour-joining method. numbers at each branch represent bootstrap values greater than 50% of 1,000 replicates. names of the strains, countries, years of isolation, genbank accession numbers, and genogroups and subgroups proposed in this study are shown. the pedv isolates identified in this study are indicated by solid circles. scale bars indicate nucleotide substitutions per site lee and lee | 951 1b as well as 2a and 2b (lee, 2015; figure s1 ). in an interesting manner, the g1b virus s genes were well-conserved, sharing 99.0%-99.7% aa identity with each other, whereas the 2017 g2b s genes were relatively variable, exhibiting 97.1%-99.8% aa homology with each other (table 1) (table s1 ). the number of nt/aa differences and percent identity shared between the 2017 isolates and genogroup representative strains is summarized in table s2 . to establish the genetic relationships involved, phylogenetic analyses were carried out using the nucleotide sequences of the s gene and full-length genome of the 2017 isolates, which were determined in this study and are available from genbank (figure 1) . consistent with previous studies (lee, 2015; lee et al., 2015) , phylogenetic analysis based on the pedv s genes revealed clear separation among the g1a, g1b, g2a and g2b subgroups. all g2b strains identified in 2017 were grouped within the g2b clade; however, they were in different branches from the emergent us strains and past re-emergent korean field isolates (figure 1a) . the 2017 g1b isolates were most closely clustered together, forming an independent branch within the g1b subgroup. furthermore, a phylogenetic tree subsequently reconstructed from the complete genome showed the same grouping structure as the s gene-based tree (figure 1b) . as shown previously (lee, 2015; lee et al., 2015) , the entire genome-based phylogenetic tree revealed that the g1b strains including knu-1702 were grouped within the g2 clade because of the similarity between the g1b and g2b genomes, except for the nthe percent nucleotide identity was shown in the upper right and the percent amino acid identity was presented in the lower left. lee and lee | 953 which were less than 1-log 2 lower but not significantly different compared to those against knu-141112. taken together, our data indicate that the antisera cross-reacted well between the homologous g2b field isolates, suggesting antigenic similarity between the 2014 and 2017 pedv strains. pedv has emerged or re-emerged as one of the deadliest and most contagious viral pathogens in swine, leading to large financial losses in the global swine industry. along with strict biosecurity, vaccination is a fundamental tool for managing and eradicating pedv during epidemic or endemic outbreaks. although g1a-based vaccines against pedv were developed and used to combat this disease in south korea over the past decade, their efficacy in the field, as well as the advantages and disadvantages of their use, is continuously debated. furthermore, a growing body of evidence suggests that their incomplete effectiveness may result from antigenic, genetic (>10% aa variation between respective s proteins) and phylogenetic (g1 versus g2) differences between vaccine and field epidemic strains (lee et al., 2010; oh et al., 2014; kim et al., 2015; lee et al., 2015; lee, 2015) . the advent of the 2013-2014 pedv pandemic led to a breakthrough in the development of g2bbased vaccines phenotypically and genotypically homologous to field strains responsible for global ped epidemics, and these g2b vaccines are currently applied to prevent pedv in south korea. another important policy for controlling pedv is to operate a monitoring and surveillance system (moss) to monitor genetic diversity among field isolates and surveil the emergence of novel variants in the field, which will contribute to preventing future outbreaks. to provide insight into the understanding of the current epidemiological status of pedv in south korea, the present study aimed to investigate the genetic, phylogenetic and antigenic characteristics of pedvs responsible for regional outbreaks in south korea in 2017. nucleotide sequencing analysis revealed that two different pedv genotypes, low-pathogenic g1b and high-pathogenic g2b, caused regional outbreaks in south korea, with the latter genotype more 1% nucleotide sequence variations at the genome level with the 2013-2014 pandemic strains. however, field g2b isolates with nearly 2% amino acid sequence divergence compared to previous g2b strains at the s gene level were identified in the present study. furthermore, mutations within the s protein were randomly and extensively distributed in the s1 and s2 regions among the 2017 isolates ( figure s1 ). replication. however, this unique del is in the glu-rich acidic region, which does not affect the authentic roles of nsp3 and thus is nonessential for coronavirus replication (lei, kusov, & hilgenfeld, 2018) . although the virus can tolerate the large nsp3-del which is dispensable for pedv replication as shown in figure conditions. therefore, the timeline of this situation is unclear and it is unknown whether antigenic differences among pedv epidemic strains will contribute to the failure of current g2b vaccines. to counteract the prospective scenario, further studies are critical for securing culturable pedv epidemic strains that are genetically, phenotypically and antigenically characterized in the laboratory. in summary, genetic and phylogenetic analyses indicated that the 2017 epidemic-related isolates are closely related with corresponding global g1b or g2b strains identified in previous outbreaks and that the virus continues to evolve in its host environment. despite their genetic diversity, antigenicity currently seems to remain unchanged among g2b strains, indirectly confirming the efficacy of g2b-based vaccines against homologous g2b pedvs responsible for current epidemics. because the virus is assumed to undergo an evolutionary process to accumulate mutations to ensure viral fitness in the field, new genotypes or variants of pedv, against which the current g2b vaccine may provide partial protection, will eventually emerge. furthermore, this circumstance may advent earlier than expected if pedv outbreaks fade from our attention following sporadic or endemic outbreaks without serious economic problems. therefore, it is important to execute mandatory notification of ped-like outbreaks essentially followed by activating an moss, including early diagnosis, to survey forthcoming pedv strains that may emerge locally or globally through genetic drift (e.g., nonsilent point mutations) or genetic shift (e.g., recombination events) and obtain and characterize epidemic field isolates to predict and prepare for future epizootics or panzootics. we would like to acknowledge the swine veterinarians and choon the authors declare that they have no conflict of interest. lee http://orcid.org/0000-0002-5930-5461 efficacy of an inactivated genotype 2b porcine epidemic diarrhea virus vaccine in neonatal piglets nidovirales: a new order comprising coronaviridae and arteriviridae isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein 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swine chinese-like strain of porcine epidemic diarrhea virus diseases of swine the neighbor-joining method: a new method for reconstructing phylogenetic trees molecular cloning: a laboratory manual emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan mega4: molecular evolutionary genetics analysis (mega) software version 4.0 the clustalx windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools distinct characteristics and complex evolution of pedv strains additional supporting information may be found online in the supporting information section at the end of the article. how to cite this article: lee s, lee c. genomic and antigenic characterization of porcine epidemic diarrhoea virus strains isolated from south korea key: cord-339012-4juhmjaj authors: hou, wei; liu, fei; van der poel, wim h.m.; hulst, marcel m. title: rapid host response to an infection with coronavirus. study of transcriptional responses with porcine epidemic diarrhea virus date: 2020-07-28 journal: biorxiv doi: 10.1101/2020.07.28.224576 sha: doc_id: 339012 cord_uid: 4juhmjaj the transcriptional response in vero cells (atcc® ccl-81) infected with the coronavirus porcine epidemic diarrhea virus (pedv) was measured by rnaseq analysis 4 and 6 hours after infection. differential expressed genes (degs) in pedv infected cells were compared to degs responding in vero cells infected with mammalian orthoreovirus (mrv). functional analysis of mrv and pedv degs showed that mrv increased the expression level of several cytokines and chemokines (e.g. il6, cxcl10, il1a, cxcl8 [alias il8]) and antiviral genes (e.g. ifi44, ifit1, mx1, oasl), whereas for pedv no enhanced expression was observed for these “hallmark” antiviral and immune effector genes. pathway and gene ontology “enrichment analysis” revealed that pedv infection did not stimulate expression of genes able to activate an acquired immune response, whereas mrv did so within 6h. instead, pedv down-regulated the expression of a set of zinc finger proteins with putative antiviral activity and enhanced the expression of the transmembrane serine protease gene tmprss13 (alias mspl) to support its own infection by virus-cell membrane fusion (shi et al, 2017, viruses, 9(5):114). pedv also down-regulated expression of ectodysplasin a, a cytokine of the tnf-family able to activate the canonical nfkb-pathway responsible for transcription of inflammatory genes like il1b, tnf, cxcl8 and ptgs2. the only 2 cytokine genes found up-regulated by pedv were cardiotrophin-1, an il6-type cytokine with pleiotropic functions on different tissues and types of cells, and endothelin 2, a neuroactive peptide with vasoconstrictive properties. furthermore, by comprehensive datamining in biological and chemical databases and consulting related literature we identified sets of pedv-response genes with potential to influence i) the metabolism of biogenic amines (e.g. histamine), ii) the formation of cilia and “synaptic clefts” between cells, iii) epithelial mucus production, iv) platelets activation, and v) physiological processes in the body regulated by androgenic hormones (like blood pressure, salt/water balance and energy homeostasis). the information in this study describing a “very early” response of epithelial cells to an infection with a coronavirus may provide pharmacologists, immunological and medical specialists additional insights in the underlying mechanisms of coronavirus associated severe clinical symptoms including those induced by sars-cov-2. this may help them to fine-tune therapeutic treatments and apply specific approved drugs to treat covid-19 patients. the lack of knowledge for treating hospitalized sars-cov-2 infected patients is one of the pressing problems of the current covid-19 pandemic. the sars-cov-2 virus shows a close genetic similarity to the in april 2003 identified sars virus (sars-cov-1) and to other sars-related coronaviruses isolated from humans and bats. sars-cov-2 induces clinical respiratory symptoms familiar to the 2003 virus, mostly in persons with underlying diseases like copd, heart failure, diabetes and obesity (1: wu et al. 2020) . despite the 2003 sars-cov-1 virus has been extensively studied in the last two decades, there are no vaccines available yet, neither there are effective prophylactic and therapeutic treatment regimens with drugs that work equally well for each individual patient with sars-induced respiratory problems. such treatments might prevent development of severe disease patterns like "acute respiratory distress syndrome" (ards) and other, often fatal complications, and may decrease the case-fatality rate of sars-cov-2 infections. in our lab we study the alpha-coronavirus pedv. pedv was first detected in pig herds in 1977 in europe (2: pensaert and de bouck 1978) . however, this virus reemerged in the spring of 2013 in north america causing a massive outbreak among pig herds, resulting in the death of about 30% of the suckling piglets due to severe diarrhea and dehydration ( although several studies concluded that these clinical symptoms were caused by mrv itself, in concordance with the co-existence of mrv3 in pedv infected piglets, also other mrv serotypes were isolated from hospitalized patients with airway problems diagnosed positive for sars-cov-1 (11: cheng et al. 2009 , 12: duan et al. 2003 , 13: zuo et al. 2003 . recently, a cross-family recombinant coronavirus was isolated in china from bat faeces in which an rna sequence originating from the s1 segment of mrv was inserted in the coronavirus genome between the n and ns7a genes, indicating that both viruses were replicating simultaneously in a single cell in bats (14: huang et al. 2016) . a prevalence study showed that this cross-family recombinant coronavirus circulated in an isolated bat colony in a cave in china (15: obameso et al. 2017 ). this cooccurrence of mrv with coronaviruses raised the questions whether a synergistic effect between both viruses exists and if such coexistence plays a role in viral pathogenesis. therefore we studied the host response in cultured cells early (4 and 6 hours) after pedv and mrv infection using rnaseq. our original goal was to identify early factors and processes induced by pedv or mrv that could stimulate or influence the replication and pathogenesis of the other virus. the host, tissue and cell tropism of pedv differs from sars-cov-1 and -2. however, the genomic organization, replication strategy and function of a part of the viral nonstructural proteins share common features among all coronaviruses (16: brian and baric 2005) . this applies particularly for interactions in infected cells of nonstructural coronavirus proteins with specific host proteins. host proteins that are recruited or silenced to support virus replication, assembly and release. in our experiment we used vero cells (cercopithecus aethiops epithelial kidney cell line; atcc® ccl-81) because these cells support efficient infection and replication of both mrv and pedv. vero cells are susceptible for many coronaviruses, including sars-cov-1 and -2 (17: chu et al. 2020). they originate from epithelial tissue, in part resembling nasal and bronchial epithelium cells, the prime target cells infected by sars-cov-2 in the airways of humans. recent research showed that sars-cov-2 is also able to replicate in epithelial cells of human small intestinal organoids (18: lamers et al. 2020) . a disadvantage of vero cells is a deletion in the type i interferon (ifn) gene cluster on chromosome 12 (19: osada et al. 2014 ). therefore, these cells lack expression of type i ifns important for activation of antiviral defense mechanisms. however, research has shown that vero cells by-pass this ifnactivation route and could mount an antiviral response mediated by interferon regulatory factor 3 (20: chew et al. 2009 ). single infections with pedv or mrv3 alone and simultaneous (double) infections of vero cells with both viruses were performed using a maximum multiplicity of infection (moi) to achieve a synchronized infection of all cells. by rnaseq measured expression levels of mrna transcripts/genes in infected cells were compared to rnaseq profiles measured from similar treated mock-infected cells harvested at the same time point after infection. the detected sets of differential expressed genes (degs) for pedv and mrv were analyzed by gene set enrichment analysis (gsea) using functional bioinformatic programs to retrieve biological processes (pathways and gene ontology terms [go-term]) and associations with chemical compounds, including drugs. in addition, we searched the literature for functional information of the pedv-degs to find possible associations with sars-cov-2 pathogenesis. because of the covid-2 pandemic we gave priority to publish the results of this functional bioinformatical analysis and datamining for the single infected vero cells with pedv separate from the results of the double infections with mrv3. in this report we focused on the "very early" host response of epithelial cells to an infection with the coronavirus pedv and pay less attention to the role of specific viral proteins in this host response to pedv. in part our results were in agreement with results of a previous rnaseq study comparing sars-cov-2 and influenza host responses by rnaseq (21: blanco-melo et al. 2020). but we also found associations with biological processes, and pivotal genes/proteins acting in these processes, that had not been recognized before. this information may contribute to the search for novel or alternative preventive or therapeutic drugs and treatment protocols for this devastating covid-19 disease. a time-dependent infection experiment was performed with cultured vero cells. details are described in supplementary file 1 (material and methods) and visually displayed in this file. briefly, overnight cultured vero cells grown in 2 cm 2 wells were mock-infected, infected with mrv3 strain wbvr (7: hulst et al. 2017) or pedv strain cv777 (2: pensaert and de bouck 1978, 22: rasmussen et al. 2018] ) with a multiplicity of infection of ≥1 for 30 min at 4°c. for pedv and corresponding mockinfected cells, 10 µg/ml of trypsin in serum-free medium was used to facilitate infection of vero cells during the whole experiment. all virus and mock-infected timepoints were performed in quadruplicate. after incubation for 30 min at 4°c, virus was discarded and cells were washed twice and supplied with fresh culture medium. cells were incubated for 0, 2, 4, 6, and 16h at 37°c and 5% co2. after incubation for the indicated times, cells were placed on ice before total rna was isolated from three of the quadruplicate wells. the replication of both viruses in vero cells was monitored using virus-specific rt-qpcr tests ( fig.1 : methods and primers used for pcr are provided in supplementary file 1). in addition, cells in one of the quadruplicate wells incubated for 16h were fixated and stained with antibodies directed against the s2 spike protein of pedv and the s1 attachment protein (α1) of mrv3. a decrease in ct-values for pedv was not observed before 6 h post inoculation (6 h.p.i), indicating that replication in pedv infected cells started later than was observed for mrv (at 4 h.p.i). staining of the cells after 16h indicated that nearly all vero cells were infected with mrv3 and more than 50% with pedv. also more than 50% of the cells in 16hwells appeared as fused cells (syncytia), confirming that more than 50% of the cells were infected with pedv. quality control of the total rna isolated from infected cells using an agilent bioanalyzer showed that rnas isolated from pedv infected wells at 16 h.p.i. were partially degraded (rin values below 9), making them unsuitable for rnaseq analysis. therefore, only 0, 4 and 6h timepoints were analyzed using rnaseq. stained with a monoclonal antibody directed against the s2 spike protein. mrv and mock infected cells were stained with a polyclonal rabbit serum raised against a peptide sequence of the s1-attachment protein of mrv serotype 3. nuclei were stained blue with the hoechst, 4',6-diamidino-2-phenylindole dye. equal amounts of total rna isolated from triplicate wells were pooled and subjected to rnaseq analysis by genomescan b.v.(leiden, the netherlands) using next generation sequencing (ngs) (see supplementary file 2a for details). mapping of ngs reads to the cercopithecus aethiops reference genome and preparation of datafiles with calculated fold change (fc) of expression levels of mapped mrnas, were performed for each comparison at 0, 4, and 6h by genomescan (see supplementary file 2b). from these datafiles we extracted lists of degs with a fc>2 and p-value of <0.05. after accessing the ncbi, panther or kegg databases for human orthologs, not annotated cercopithecus aethiops degs were annotated with an hugo official gene symbols (http://www.genenames.org). in supplementary file 3 sheet "pedv-mrv degs fc>2", lists of all annotated pedv and mrv degs are presented with their fc. in a separate sheet "pedv-degs functional info" all 266 individual degs regulated by pedv at 4 and 6 h.p.i. are presented with their fc, information about their function and the types of human cells in which expression of the gene is relatively high compared to other human cells (retrieved from the "primary cell atlas" dataset of biogps: http://biogps.org/). note that all tables in these excel sheets of supplementary file 3 are sortable using the headers. in all results paragraphs beneath information about the biological function of degs was retrieved by consulting the "genecards" (weizmann institute of science: https://www.genecards.org/) and ncbi gene reports (entrez gene: https://www.ncbi.nlm.nih.gov/gene/), and literature linked to these reports (for references about these biological functions of genes/proteins we refer to publications cited in these reports: "genecards" weblinks are provided in supplementary file 3). sets of pedv and mrv degs were analyzed using the gsea program geneanalytics (lifemap sciences, inc.) and pathways (for mrv and pedv), go-terms (not for mrv), and associations with compounds/drugs (not for mrv) with a high or medium score (p-value <0.05) were retrieved and listed in 3 separate sheets in supplementary file 3 (sheets "mrv-pedv pathways", "pedv g0-terms" and "pedv compounds"). similar and related pathways retrieved for both pedv and mrv, and remarkable pedv pathways, go-terms and compound associations are summarized in table 1 . for pedv all degs within these pathways are provided with their regulation, up (green) or down (red). for mrv only degs in common with pedv-degs were listed in table 1 (see sheet "mrv-pedv pathways" in supplementary file 3 for all mrv-degs acting in these pathways). subsets of pedv-degs were selected matching the terms "chemokines-cytokines", "antiviral" , and terms related to the pathogenesis of covid-2 (explained below) using the genotyping program varelect (lifemap sciences, inc.) and displayed in supplementary file 3 in separate sheets: "chemokines-cytokines", "(anti)-viral", etc. based on these selections we prepared a set of pedv key-degs consisting of genes regulated with a fc of >10 (up) or <-10 (down) or playing an important role in biological processes induced by pedv and related to covid-19 pathology. in beneath results sections we tried to give as much as possible meaningful information about the function of key-degs for which we found an association with sars-cov-2 infections. we emphasize that further dedicated experimental and in-silico research is necessary to confirm the involvement of the proteins encoded by these genes for pathogenesis of this viral disease. table 1 . enriched pathways, go-terms and compound associations of pedv-degs. *pedv enriched pathways (a), go-terms (b), and associations with compounds and drugs (c) with a high and medium score and with at least 2 matching genes were retrieved from geneanalytics. common pathways for mrv were included in table 1a . a full list of pathways with degs, retrieved for mrv at 4 and 6h, is provided in supplementary file 3 (sheet mrv-pedv pathways). a possible function or process related to specific degs, pathways, go-term, or compounds/drugs is provided in blue text between brackets. $ official gene-symbols (hugo abbreviations) are listed for degs. down-regulated degs were colored red and up-regulated degs were colored green. in section a the number of degs regulated by mrv in a pathway and the common degs are provided between brackets. degs regulated by both pedv and mrv are underlined. compared to mrv, only a few genes involved in "cytokine/chemokine signaling" were regulated at 4 and 6h by pedv. in fig. 2a the regulation of cytokines/chemokines in pedv and mrv infected vero cells are displayed. this indicated that mrv increased the transcription of a broad set of cytokines/chemokines, including interferonmediated cytokines like cxcl10 and cxcl8 (alias il8), already at 4 h.p.i., whereas pedv did not, even not when replication of pedv rna was detected by rt-qpcr at 6 h.p.i.. for mrv, this cytokine/chemokine response at 4 h.p.i. was followed by high up-regulation of "hallmark" antiviral genes at 6 h.p.i. (see fig. 2b : e.g. interferoninduced genes [ifi] and oasl) and chemokines that attract t cells, monocytes, granulocytes, including basophils (e.g. cxcl8, cxcl11 and ccl2). pedv infection up-regulated only a few genes coding for proteins with cytokine activity (ctf1 and edn2), and also did not elevated gene expression of these "hallmark" antiviral genes. in contrast, pedv down-regulated expression of 6 genes (out of 9 in total) coding for zinc finger proteins (out of 9 in total), all 6 with an antiviral activity towards herpes simplex virus 1 ( fig 2c) binding of thrombin to f2rl2 reduces inflammation, activates platelets and increases vasodilation and permeability of the vascular wall (see also below in the section "platelets activation"). csf1r is a receptor for the cytokine colony stimulating factor 1, a cytokine that regulates differentiation and function of macrophages, and in the cns, the density and distribution of microglia cells. the blnk gene codes for a cytosolic protein that passes on b-cell receptor signals in the signaling cascade that activates b-cell development and function. gene expression of genes coding for essential components of this b-cell signaling, like "spleen associated tyrosine kinase" (syk) and "lyn proto-oncogene"(lyn) were not regulated by pedv, nor by mrv. genes involved in amino acid, protein translation and metabolism of immuno-active compounds. pedv degs coding for enzymes involved in the metabolism of the non-essential amino acids histidine, phenylalanine, tryptophan and proline were found enriched in the pedv dataset (see supplementary file 3, sheet pedv-compounds). remarkable were the degs coding for amine oxidases involved in the catabolism of the biogenic amines histamine, tryptamine and phenylethylamine, their derivates and related substrates/products of these enzymes (fig. 3, aoc1 , maoa, il4i1). none of these amino oxidase genes were regulated by mrv. using the genotyping program varelect, pedv-degs with an association with these biogenic amines were retrieved (supplementary file 3, sheet "biogenic amines"). three enzymes clustered in the "histidine metabolism" pathway (https://www.kegg.jp/keggbin/show_pathway?hsa00340+4128) with histamine and reaction products generated from this biogenic amine (fig. 3) . also most association of pedv-degs were found by varelect for histamine. the gene coding for the amine oxidase "interleukin 4 induced 1" (il4i1) was strongly down-regulated (30-fold) 4h after infection with pedv. besides catalysis of l-phenylaniline into phenylpyruvate (fig.3) , il4i1 also fulfills an important role in signaling in "synaptic clefts" formed between antigen presenting cells (apc) and t cells (so-called "immune cleft": see also below) ( expression of the prostaglandin-endoperoxide synthase 2 (ptgs2) gene was upregulated by mrv at 6h p.i. ptgs2 synthesizes prostaglandin endoperoxide h2 (pgh2), an compound with a short half-life and the precursor of many biological active prostaglandins: e.g. thromboxane-a2 (mediates activation of platelets), pgi2 and pge2. in contrast, pedv increased the expression of the gene coding for prostaglandin e synthase (ptges) which converts pgh2 into pge2. pge2 is a direct vasodilator, but does not inhibit platelet aggregation. pge2 also suppresses t cell receptor signaling. pedv decreased expression of the gamma-glutamyltransferase 1 gene (ggt1) after 4h (2-fold), but increased expression of this gene 2 hours later to a 4-fold level compared to mock infected cells. ggt synthesizes leukotriene d4 (ltd4) from ltc4. ige-activated mast cells may secrete ltd4 and ltc4, together with histamine and platelets activating factor (paf). this vesicle mediated secretion by mast cells (degranulation) results in stimulation of mucus production, and similar to histamine, increases the permeability and smooth muscle contraction of the vascular wall. in persons suffering from asthma this degranulation leads to an immediate allergic response (bronchospasm, airflow obstruction and forming of edema). genes involved in "cilia and synaptic cleft" formation. gsea detected "axon guidance" as the go-term with the highest score for pedv (see table 1 ). in addition, pedv-degs were enriched coding for proteins involved in calcium ion-dependent exocytosis from vesicles into the "synaptic clefts" between two cells (e.g. between axons and dendrites), and degs coding for proteins involved in formation of cilia. cilia protruding from cells are found in many forms. they can have a static (structural) function, e.g. in forming of clefts between two cells (see fig. 4 ), or a motile function. motile cilia on the surface of ciliated cells lining up the epithelial layers in the nose, trachea and bronchia sweep out superfluous mucus containing dirt from the airways. pedv degs matching the terms "cilia" and "synaptic cleft" retrieved form the genotyping program varelect were further examined by consulting functional information in the ncbi gene and genecards reports in order to evaluate their association with these processes (see supplementary file 3, sheet "cilia and synaptic cleft"). based on this analysis we identified genes in the set of pedv degs which can i) negatively regulate cell adhesion (rnd1 and sema5a), ii) inhibit formation of cilia (kinases mak1 and cdk20, highly up-regulated at 6h), and iii) regulate cytoskeleton rearrangements that facilitate axon growth and growth and stabilization of dendritic spines (f2rl2, regulation of genes involved in histamine/biogenic amines (see above) and formation of cilia/clefts suggested that gene expression related to this intersynaptic signaling between immune cells could be affected in response to infection with pedv (fig 4) . in particular, the highly down-regulated gene il4i1 (30-fold at 4h p.i.) is of interest (see also above). il4i1 is believed to be secreted from apc's (e.g. dc's) in the immune cleft formed with t cells (31: molinier-frenkel et al. 2019). the mechanism how il4i1 transmits its signal to t cells is not completely understood. it could bind to a receptor that concentrates this amino oxidase in the cleft, resulting in elevated h2o2 and ammonia production, phenylalanine depletion and phenylpyruvate production in the cleft space. these alteration in the concentration of these chemicals in the cleft are sensed by the t cell. the paralog of il4i1, amino oxidase maoa (down-regulated 11-fold by pedv) could also play a similar role in this signaling process. remarkable was also the strong down-regulation of genes coding for the olfactory receptor family 2 subfamily a member 14 (or2a14; 17-fold at 4h) and anoctamin 2 (ano2, alias cacc;14-fold at 4h). ano2 is a calcium-activated chloride channel imbedded in the basal membranes of neurons that harbor apical membrane receptors like or2a14 that sense odorants. by importing chloride ions into the cytosol ano2 contributes to the depolarization of these neurons (https://www.kegg.jp/kegg-bin/show_pathway?hsa04740+57101). loss of smell and taste is one of the first noticeable symptoms of covid-19. genetic defects in the ano2 gene are associated with von willebrand disease, a bleeding disorder due to defective platelet aggregation ( . also disease incidence in adult males is significantly higher than in females of the same age. to assess whether pedv-degs relate to these pathological symptoms, the genotyping program varelect was used to identify genes matching the terms "ards", "cardiomyopathy", "obesity (diabetic)", and "platelets activation". detailed information about all matching degs is provided in supplementary file 3 in separate sheets for all 4 queried terms. degs matching to more than one query term are displayed in fig. 5 . remarkable associations of degs with these terms are mentioned in sections beneath. . edn2 and agt2 are vasoactive peptides and binding of edn2 and agt2 to their receptors on granular cells of the juxtaglomerular apparatus in the kidney raises free calcium levels in the cytosol, leading to inhibition of camp-mediated secretion of the aspartylprotease renin (ren), the key regulator of renin-angiotensin-aldosterone system (raas) (https://www.kegg.jp/kegg-bin/show_pathway?hsa04924+1907). ren converts pre-angiotensinogen (agt) to the endocrine peptide-hormone agt1 (https://www.kegg.jp/kegg-bin/show_pathway?hsa04614+5972). agt1 is further cleaved to variants with specific endocrine activity by angiotensin i converting enzymes (e.g. ace and ace2: ). on the surface of bronchial epithelial cells ace2 was identified as entry receptor for sars-cov-1 and -2 (43: hoffmann et al. 2020). the octamer peptide agt2 stimulates secretion of the mineralocorticoid hormone aldosterone by the adrenal glands. aldosterone, and the agt and edn peptide hormones regulate an array of physiological processes in the body, e.g. vascular smooth muscle contraction, blood pressure, fluid and electrolyte homeostasis (44: agapitov et al. 2002) . all processes that are important for proper functioning of the vascular system, heart muscles and kidneys. ctf1 is directly involved in the pathology of numerous cardiovascular diseases by promoting cardiac myocyte hypertrophy (41: wollert et al. 1996) , which may lead to the onset of heart-diseases like "hypertrophic cardiomyopathy" or "dilated cardiomyopathy", and eventually, to (lethal) heart failure. pedv induced a strong down-or up-regulation of several other genes directly involved in the function of cardiomyocytes. sodium voltage-gated channel subunit 4 (scn4b) was strongly upregulated (15-fold) and mylk2 (see above), citrate synthase (cs; down-regulation 24-fold) and ankyrin repeat domain 1 (ankrd1) were strongly down-regulated. down-regulation of cs may reduce oxidative capacity in cardiomyocytes. gene expression of ankrd1 was down-regulated 12-fold in response to mrv infection at 4 h.p.i, but reverted to a 24-fold up-regulation 2h later. ankrd1 is a putative transcription factor involved in regulation of gene expression in hypertrophic myocytes (https://www.wikipathways.org/index.php/pathway:wp516) regulation of cytosolic calcium levels in the cytosol of cardiomyocytes, e.g. by binding of col1a1 and col2a (up-regulated by pedv, see above) to integrin subunit alpha on the surface of cardiomyocytes or after import of calcium ions mediated by calcium voltage-gated channels (e.g. by cacna1h; down-regulated 5-fold at 4h by pedv) may also trigger myocyte hypertrophy. pedv strongly down-regulated gene expression of a potassium voltage-gated channel (kcnq4;16-fold). this in contrast to a strong up-regulation of the sodium symporter scn4b. for the potassium channel cacna1h (alias kv7.4) it was reported that this channel regulates the membrane potential and ca 2+ permeability of mitochondria located in the vicinity the sarcoplasmic reticulum in rat cardiomyocytes (45: testai et al. 2016). all three above mentioned ion channels are also involved in the process of excitation, contraction/relaxation and repolarization of cardiac myocytes. mrv also downregulated gene expression 3 to 4-fold for the potassium (kcnq4) and calcium channel cacna1h, but did not increased expression of the sodium symporter gene scn4b or orthologs of this gene. chronic hypertension and heart disease/failure is a complication frequently observed in obese/diabetic patients. in accordance with this, 16 out of the 65 pedv degs matching the term "obesity" also matched with the term "cardiomyopathy" (see additional remarkable pedv-degs. highly up-or down-regulated pedv-degs not mentioned in the text, and to our opinion interesting with regard to coronavirus infection, are briefly described in table 2 . among these degs several genes coding for transcription factors and genes transcribed in antisense rna's that inhibit translation of their coding counterparts. for more functional information about these degs we refer to the weblinks provided in supplementary file 3, sheet "pedv key degs". table 2 . remarkable pedv-degs not mentioned in the text. in this report we measured the transcriptional response of vero cells shortly after infection with the coronavirus pedv. the function of the responding host genes and the biological processes in which they act were studied in detail by us to find plausible relations to covid-19 pathology. because of the differences in genomic organization and expression of viral proteins between sars-cov-2 and pedv, we paid less attention to couple the response of specific host genes to the function of specific coronavirus proteins. we were able to infect the majority of vero cells (>50%) with pedv and mrv synchronically. this resulted in a unique set of highly up-and down-regulated degs for pedv. not more than 14% of the 266 pedv-degs (n=37) were similar to mrv-degs (total of 727 mrv-degs). in contrast to mrv, we observed no typical response of antiviral genes and related cytokine/chemokine genes in vero cells within 6 h.p.i. for pedv. for mrv these processes started already before 4 h.p.i.. we have to notice that pedv replication started 2h later than mrv3 replication, which could in part be the reason for not detecting transcriptional regulation of specific cytokine, chemokine and antiviral genes for pedv. longer incubation times than 6h were not planned in the original design of our experiments and would have resulted in a set of pedv-degs dominated by genes involved in syncytia forming and apoptotic/necrotic cell death. nevertheless, at 6h replication of pedv rna was detected by rt-pcr, indicating that dsrna was present in the cells and could have be sensed by cytosolic pattern recognition receptors of the rig-i-like family to initiate an antiviral and related cytokine/chemokine response. similar as observed in another study with pedv and vero cells, and in analogy with sars-cov-1 and -2, we observed a high up-regulation of the transmembrane serine protease gene (tmprss13) that acts as a co-factor in the infection process of cells ( we observed a reduced expression of eef1a1, as part of a transcription factor-complex that binds and activates the promotor of ifnγ, and of the cytokine eda and its receptor (edaradd) involved in activation of canonical-nfkb transcription of antiviral cytokine-chemokine genes like cxcl8 (alias il8) an cxcl10. this reduced expression of eef1a1 and eda and its receptor may play a crucial role in delaying or downgrading an ifn-mediated antiviral and cytokine/chemokine response in our vero cell system. the elevated transcription of many cytokine and chemokine genes in vero cells by mrv suggests that replication of this rna virus in epithelial cells induces secretion of these immune effectors (more information about the genes/processes that responded to mrv infection will be published elsewhere). pedv, and also the mrv3 strain we used both replicate in enterocytes lined up in the intestinal mucosal layer. in the intestinal and bronchial epithelial layer, microfold (m) cells are imbedded between these lined up epithelial cells. m-cells sense and engulf foreign pathogens/antigens from the lumen to present them to residing immune cells. according to pathway analysis, most t cell related immune genes regulated in response to mrv infection were part of the pathways "t-helper 17 (th17) differentiation/activation" and "il17 signaling". antigen presentation by m cells to th17 cells in the submucosal layers stimulate secretion of different types of il17 cytokines (il17a-d, il25 and il17f) resulting in activation of different types of innate immune cells and t cells, including th1/th2 cells. dysregulation of this pathogen-induced il17 response may disturb the balance between th1/th2-cell mediated immune responses, resulting in excessive inflammation, damage to the epithelial layer and on the long term, to autoimmune reactions. tf rorc (or specific isoforms of this tf, see above) plays a pivotal in controlling il17 expression and secretion by th17 cells. pedv strongly up-regulated gene expression of rorc in vero cells whereas mrv down-regulated expression of this tf. this difference in regulation of tf rorc suggests that virus-induced activation or suppression of il17 secretion by th17 cells in submucosal layers of airway epithelium can be an important mechanism to dysregulate the activation of t cell responses. therefore, tf rorc might be a potential target for drug treatment/development. drugs affecting expression of rorc, like the fluorinated steroid "dexamethasone" and the synthetic tetracycline derivative "doxycycline" (http://ctdbase.org/basicquery.go?bqcat=gene&bq=rar+related+orphan+receptor +c) are already under investigation in relation to sars-cov-2 pathogenesis. transcriptional regulation of a set of genes coding for enzymes involved in biogenic amine metabolism was unique for pedv, and not observed for mrv. most associations of these pedv-degs were found with histamine, a compound produced by mast cells and basophils, and released by these cells in response to allergens and pathogen-induced inflammation. the 10-fold up-regulation of the enzyme aoc1 suggests that histamine is converted to imidazole-acetaldehyde (see fig. 3 ). however, without data of intra-and extracellular concentrations of the chemicals this remains speculative. recent reports indicated that submucosal mast cells in the lungs were triggered by sars-cov-2 infection to release pro-inflammatory cytokines (il1, il6 and tnf-alpha) . mdscs infiltrate these tumors and inflamed tissues to suppress the local activity of specific immune cells. therefore, the role of infiltrating mdscs at inflammatory sites in the lungs of covid-19 patients, as part of an sars-cov-2 immune-evading strategy, and the role of il4i1 in this process, is worthwhile to investigate in more detail. expression of genes that promote or inhibit the formation and motility function of cilia were time-dependently regulated by pedv. the 20-fold up-regulation of fez1 gene expression at 4h (20-fold) descended within 2h to a moderate 6-fold upregulation. this descend occurred simultaneously with elevation of gene expression of the kinases mak and cdk20, both involved in inhibition of cilia formation. because pedv efficiently replicates in enterocytes that carry ciliated membrane protrusions (microvilli) on their luminal surface, regulation of these genes could be related to structural changes in the cytoskeleton of cells imposed by virus replication (e.g. syncytia forming in pedv infected vero cultures). likewise, sars-cov-2 replication could also impose structural changes in cilia protruding from the surface of upperairway epithelial cells (nose, trachea) and bronchi. based on our results we cannot pinpoint a specific process in which these cilia-regulating genes act. possible processes can either be formation of an immune cleft, a virological cleft to promote more effective infection of neighboring cells, or cytoskeleton rearrangements to support virus replication, morphogenesis and budding from cells. interestingly, two recent studies revealed a high level of expression of the sars-cov-2 entry receptor ace2 and its co-receptor tmprss2 in ciliated airway epithelial cells ( . these processes deserve more attention, and may also be considered as possible target-processes for interference with drugs. within our set of pedv-degs, and the biological processes deduced from this set, we found associations with diverse aspects of covid-19 pathogenesis, i.e. "ards", "cardiomyopathy", "obesity (diabetic)", and "platelets activation". however, it is unknown whether the proteins encoded by these pedv-degs indeed play a role in the biological processes underlying the symptoms and complications observed in hospitalized persons infected with sars-cov-2. nevertheless, a part of these genes/processes may be starting points for further dedicated research. research to fine tune drug treatment protocols that are already applied for covid-19 patients, or research that provides new insights for treatments with alternative prophylactic and therapeutic approved drugs. in table 3 . we summarized the pedv-degs that are, to our opinion, of interest for modulation of the biological processes underlying the pathogenesis of covid-19. table 3 . possible target genes for covid-19 therapy. colophon. the overwhelming amount of data published recently made it impossible for us to oversee all (novel) facts about the sars-cov-2 virus and pathology of the covid-19 disease. some important genes and related processes imbedded in our set of pedv-degs may have been overlooked by us. therefore, we encourage researchers, especially medical, immunological and pharmaceutical specialists, to study this set of degs in detail. the users-friendly supplementary file 3 with functional information about degs and related biological processes can be down-loaded from the web. by publishing these pedv data ahead of our complete study, we hope that some of the gene targets and cognate processes we have identified for the coronavirus pedv will contribute to a better understanding how hospitalized covid-19 patients can be treated and cured. a more condensed version of this manuscript, focusing on the original goal of our study, will be submitted to a peer-reviewed virological journal soon. risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease a new coronavirus-like particle associated with diarrhea in swine origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis a novel pathogenic mammalian orthoreovirus from diarrheic pigs and swine blood meal in the united states identification of a novel reassortant of a mammalian orthoreovirus in faeces of diarrheic pigs in the netherlands: presentation at the 11th annual 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subpopulations with distinct t cellsuppressive activity sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes sars-cov-2 receptor ace2 and tmprss2 are primarily expressed in bronchial transient secretory cells mechanisms of innate immune evasion in re-emerging rna viruses supplementary file 3: interactive excel file with sortable tables in separate sheets. please, first read the sheet "read me" for an explanation and instructions for the use of the tables. excel sheets contain tables with i) pedv and mrv degs extracted from rnaseq data files, ii) functional information about the pedv-degs, iii) gsea extracted pathways (for mrv and pedv), go-terms (only for pedv) and compound associations (only for pedv), and iv) associations of pedv-degs with the terms "cytokines-chemokines", "(anti)-viral", "biogenic amines", "cilia and synaptic clefts", and the disorders "ards, "cardiomyopathy", "obesity", and "platelets activation" (a-c-o-p). the authors declare that they have no competing interests.authors' contributions. key: cord-343780-084lq92r authors: hsu, tien-huan; liu, hao-ping; chin, chieh-yu; wang, chinling; zhu, wan-zhen; wu, bing-lin; chang, yu-chung title: detection, sequence analysis, and antibody prevalence of porcine deltacoronavirus in taiwan date: 2018-07-26 journal: arch virol doi: 10.1007/s00705-018-3964-x sha: doc_id: 343780 cord_uid: 084lq92r porcine deltacoronavirus (pdcov) was initially documented in hong kong and later in the united states, south korea, and thailand. to investigate if pdcov is also present in taiwan, three swine coronaviruses—pdcov, porcine epidemic diarrhea virus (pedv), and transmissible gastroenteritis coronavirus (tgev)—were tested using real-time reverse transcription polymerase chain reaction (rrt-pcr) in 172 rectal swab samples from piglets exhibiting diarrhea between january 2016 and may 2017 on 68 pig farms in taiwan. the rrt-pcr results were positive for pdcov (29/172, 16.9%), pedv (36/172, 20.9%), tgev (2/172, 1.2%), and coinfections (16/172, 9.3%). after cloning and sequencing, pdcov nucleocapsid genes were analyzed. phylogeny results indicated that the nucleotide sequences of all isolates were like those reported in other countries. to further trace pdcov in the period of 2011 to 2015, an enzyme-linked immunosorbent assay (elisa) was used to detect antibodies against pdcov. the results showed that 279 of 1,039 (26.9%) sera were positive for the pdcov nucleocapsid protein, implying that pdcov might have existed in taiwan before 2011. porcine deltacoronavirus (pdcov) is an enveloped, positive-sense single-stranded rna virus that belongs to the family coronaviridae. pdcov was first discovered in hong kong, china in 2012 [1] . it was subsequently reported in the united states [2] [3] [4] and south korea [5] in 2014, followed by thailand and mainland china in 2015 [6, 7] . currently, there are at least three members of the family coronaviridae that can cause diarrhea in pigs: transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), and porcine deltacoronavirus (pdcov) [8] . both tgev and pedv belong to the genus alphacoronavirus, whereas pdcov belongs to the new genus deltacoronavirus. pedv is an important enteric pathogen that causes piglet diarrhea worldwide, and it has caused significant economic losses in the swine industry in taiwan from 2013 to 2014 [9] . however, pdcov and pedv share a similar clinical manifestation, and many studies have shown that coinfection with pedv and pdcov is common in piglets [2] [3] [4] . thus, the purpose of this study was to identify the viruses responsible for causing diarrhea in piglets and to specifically investigate the prevalence of pdcov infection in taiwan. in this study, 172 rectal swabs of piglets that suffered from diarrhea from 68 pig farms located in central and southern taiwan were collected between january 2016 and may 2017. all rectal swabs were transported in phosphate-buffered saline with 5% glycerol. the total nucleic acid content was then extracted from the rectal swabs using a labprep™ dna/rna mini kit (taigen biotechnology, taiwan). the isolated nucleic acid samples were tested for the presence of swine enteric coronaviruses-tgev, pedv, and pdcovusing the idexx™ realpcr ® test kits. pdcov-positive samples were further examined by traditional reverse transcription polymerase chain reaction (rt-pcr) with primers pdcov-n1 (5'-acc atc gct cca agt cat tcttg-3') and pdcov-n2 (5'-gag tgg agt tgg gtg ggt tta reverse-transcription reaction was performed at 50 °c for 30 minutes, and then a standard polymerase chain reaction was performed: 30 cycles of 94 °c (30 seconds), 55 °c (30 seconds), and 72 °c (1 minute). after electrophoresis and gel elution, amplified products were cloned and sequenced. the nucleotide sequence data were analyzed using chromas lite, and the deduced amino acid sequences of the open reading frames were compared to other pdcov sequences using blast. the sequences of three complete pdcov nucleocapsid (pdcov-n) genes, taiwan 22, taiwan 33, and taiwan 36, were obtained and deposited in genbank (accession numbers ky586147 to ky586149). multiple sequence alignment and phylogenetic tree construction were then performed using the mega7 program [10] . a pdcov-n clone (taiwan 22) was further subcloned into the protein expression vector pet-32a(+) using primers pdcov-n-f (5'-acc gga tcc atg gct gca cca gta gtccc-3') and pdcov-n-r (5'-cac aag ctt cta cgc tgc tga ttc ctgct-3'). the pdcov-n protein was then expressed by adding isopropyl-β-d-thiogalactoside (iptg) (final concentration 0.3 mm) to a bacterial culture and was purified using immobilized metal affinity chromatography for a subsequent enzyme-linked immunosorbent assay (elisa). a total of 1,039 serum samples collected from 34 pig farms in taiwan during 2011 to 2015 were tested, and 27 specific-pathogen-free (spf) pig sera were obtained from the animal technology institute taiwan to be used negative controls. the elisa was conducted following the modified procedures described in a previous study [11] . in brief, 96-well microtiter plates were coated with purified his-tagged pdcov-n (100 ng/well) in 50 mm carbonate buffer (ph 9.6) at 4 °c for 12-14 hours and blocked with blocking buffer (3% bovine serum albumin in tris-buffered saline (tbs): 50 mm tris-cl, ph 7.5, 150 mm nacl) at room temperature for 1 hour. after washing three times with tbst (tbs containing 0.05% tween-20), 100 μl of the tested sera (1:100 dilution in blocking buffer) were added to the wells and incubated at room temperature for 1 hour. after incubation, the plate was washed three times with 200 μl of tbst and incubated at room temperature for 1 hour with 100 μl of goat α-porcine ig antibody conjugated with horseradish peroxidase at a concentration of 1:4000 dilution in blocking buffer. after washing, 100 μl of 3,3',5,5'-tetramethylbenzidine (tmb) substrate solution was added, and the plate was incubated at room temperature for 10 minutes. the development reaction was stopped by adding 50 μl of 2 m h 2 so 4 , and the absorbance at 450 nm wavelength was measured. all tests included a blank coated with antigen only, a second antibody control, and six spf sera as negative controls. if the detection value was lower than (mean neg + 2× sd), the serum was considered negative. on the other hand, a serum detection value larger than [(mean neg + sd) × 2] was considered positive. of these tested samples, 29 were positive for pdcov (16.9%), 36 were positive for pedv (20.9%), and only two were positive for tgev (1.2%). regarding coinfection rates, only one of the 172 specimens (0.6%) was positive for all three coronaviruses, one was positive for pedv and tgev (0.6%), and 14 of them (8.1%) were positive for pdcov and pedv. based on the real-time rt-pcr (rrt-pcr) detection results, the percentage of pig farms that were positive for at least of one of the coronaviruses was 25% for pdcov (17/68), 22.1% for pedv (15/68), and 2.9% for tgev (2/68). we compared the sensitivity of the idexx rrt-pcr kit and the traditional rt-pcr used for cloning the n protein of pdcov in this study. all 29 pdcov-positive samples were re-examined by conventional rt-pcr, which showed that only eight (8/29, 27.6%) were positive, accounting for 4.7% (8/172) of total tested samples. the relatively low positive rate determined by conventional rt-pcr was comparable to that determined by a similar method in a recent study, in which pdcov was detected exclusively in nursing piglets, with an overall prevalence of approximate 1.28% (5/390) in southern china [12] . it is plausible that for the specimens containing pdcov genomic rna of low quantity or quality, most of the commercial rrt-pcr kits, which are designed to amplify short target sequences, can achieve higher sensitivity in detection compared with conventional rt-pcr. in addition, four of the eight positive samples were cloned and sequenced, and the sequence data matched the pdcov-n reference sequences. all of these four sequences covered the complete coding sequences of pdcov-n, but one of them, taiwan 34, had a nonsense mutation within the pdcov-n gene (data not shown). such a mutation might arise from an occasional change in rna sequence occurring during serial propagation of pdcov [13] . despite the mutation, the result revealed the presence of the pdcov genome in the specimen in which taiwan 34 was cloned, while it remained to be clarified if mutations occurred in other pdcov-encoded genes as well. phylogeny analysis of pdcov-n genes showed that pdcovs found in taiwan were highly similar in their nucleotide sequences to isolates from the united states, mainland china, and other countries (fig. 1) . however, the aminoacid-based phylogeny results of the pdcov-n proteins revealed that taiwan isolates can be clustered into different groups (fig. 2) . this might be due to missense mutations in the 66 th (f → y), 198 th (e → k), 234 th (g → r/t), 271 th (f → s), and 275 th (g → e/d) amino acid residues of the pdcov-n protein (fig. 3) . the charges of amino acids were changed in three residues (198 th , 234 th , and 275 th ), and the remaining residue changes had altered polarity. the phylogeny results imply that pdcovs isolated in taiwan might have existed for a long time. to determine if pdcov had already existed in taiwan before 2016, the pdcov-n protein was cloned, expressed, purified, and coated onto a 96-well microtiter plates for retrospective testing. swine sera collected between 2011 and 2015 were tested for their reactivity to a pdcov-n protein. the elisa results showed that 279 of 1,039 (26.9%) sera were able to react with the pdcov-n protein when [detected value > (mean neg +sd) × 2] was used as positive threshold, but only 48 of 1,039 (4.6%) sera were positive when [detected value > (mean neg +sd) × 3] was set as the threshold. the data indicated that pdcov has existed in taiwan since 2011. similar elisa tests were also used to investigate the presence of pdcov in other studies [7, 11, 14, 15] . based on our findings, we confirm that infection of pdcov and its coinfection with pedv in pigs have existed in taiwan between 2011 and 2017. it is known that antibodies against pedv, tgev, or pdcov provide no crossprotection against either of the other two coronaviruses [14, [16] [17] [18] [19] . coinfection with pedv and pdcov might explain why some efforts have been ineffective in the pedv vaccination program. therefore, a divalent vaccine to control pdcov and pedv is desperately needed. 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 rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus detection and genetic characterization of deltacoronavirus in pigs porcine coronavirus hku15 detected in 9 us states complete genome characterization of korean porcine deltacoronavirus strain kor/knu14-04 porcine deltacoronavirus in mainland china pathogenecity of 2 porcine deltacoronavirus strains in gnotobiotic pigs us-like strain of porcine epidemic diarrhea virus outbreaks in taiwan mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets a recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus occurrence and sequence analysis of porcine deltacoronaviruses in southern china isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the united states development and application of an elisa for the detection of porcine deltacoronavirus igg antibodies retrospective testing and case series study of porcine delta coronavirus in us swine herds antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains two-way antigenic crossreactivity between porcine epidemic diarrhea virus and porcine deltacoronavirus evaluation of serological cross-reactivity and cross-neutralization between the united states porcine epidemic diarrhea virus prototype and s-indel-variant strains reactivity of porcine epidemic diarrhea virus structural proteins to antibodies against porcine enteric coronaviruses: diagnostic implications funding this study was supported by grants 105as-10.1.2-bq-b1 and 106as-9.1.2-bq-b1 from the bureau of animal and plant health inspection and quarantine (baphiq), council of agriculture in taiwan. tien-huan hsu has received grants from the bureau of animal and plant health inspection and quarantine. all authors declare no conflict of interest.ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. rectal swabs were collected from clinically diarrheic piglets in pig farms in taiwan, while swine sera were obtained from the animal disease control centers of various counties and the animal technology institute taiwan. key: cord-348669-mizygp4j authors: beall, anne; yount, boyd; lin, chun-ming; hou, yixuan; wang, qiuhong; saif, linda; baric, ralph title: characterization of a pathogenic full-length cdna clone and transmission model for porcine epidemic diarrhea virus strain pc22a date: 2016-01-05 journal: mbio doi: 10.1128/mbio.01451-15 sha: doc_id: 348669 cord_uid: mizygp4j porcine epidemic diarrhea virus (pedv) is a highly pathogenic alphacoronavirus. in the united states, highly virulent pedv strains cause between 80 and 100% mortality in suckling piglets and are rapidly transmitted between animals and farms. to study the genetic factors that regulate pathogenesis and transmission, we developed a molecular clone of pedv strain pc22a. the infectious-clone-derived pedv (icpedv) replicated as efficiently as the parental virus in cell culture and in pigs, resulting in lethal disease in vivo. importantly, recombinant pedv was rapidly transmitted to uninoculated pigs via indirect contact, demonstrating virulence and efficient transmission while replicating phenotypes seen in the wild-type virus. using reverse genetics, we removed open reading frame 3 (orf3) and replaced this region with a red fluorescent protein (rfp) gene to generate icpedv-δorf3-rfp. icpedv-δorf3-rfp replicated efficiently in vitro and in vivo, was efficiently transmitted among pigs, and produced lethal disease outcomes. however, the diarrheic scores in icpedv-δorf3-rfp-infected pigs were lower than those in wild-type-virusor icpedv-infected pigs, and the virus formed smaller plaques than those of pc22a. together, these data describe the development of a robust reverse-genetics platform for identifying genetic factors that regulate pathogenic outcomes and transmission efficiency in vivo, providing key infrastructural developments for developing and evaluating the efficacy of live attenuated vaccines and therapeutics in a clinical setting. dustry in asia (13, 14) . during this ongoing outbreak, new strategies are desperately needed to understand pathogenic mechanisms and the functions of viral genes and to provide new technologies to combat this disease. pedv appears to recognize cd13, an aminopeptidase n protein, as a receptor for entry into pig cells, as well as the sugar coreceptors heparan sulfate and n-acetylneuraminic acid (15, 16) . pedv can infect multiple cell types in vitro, including swine, human, primate, and bat cells, suggesting the possibility of adaptation and spread to other species (15) . the pedv genome is composed of 28,000 nucleotides (nt) encoding seven known open reading frames (orfs) expressed from both genomic and subgenomic mrnas (17) . subgenomic mrnas are arranged in a nested fashion from the 3= end of the genome. pedv encodes the traditional coronavirus structural proteins: a receptor-binding spike glycoprotein (s), the envelope protein (e), the membrane glycoprotein (m), and the nucleocapsid protein (n). the spike glycoprotein is a type i membrane glycoprotein composed of s1 and s2 external domains, a transmembrane domain, a c-terminal cytoplasmic domain, and a signal peptide. the s protein plays a role in virulence, growth adaptation, receptor binding, and viruscell membrane fusion (18, 19) . e of pedv (pedv-e) upregulates stress pathways in the host cell, induces antiapoptotic factors, and is important for viral budding (20) . pedv-n induces cell stress and prevents apoptosis through similar pathways and prolongs the host cell's s phase (21) . additionally, pedv contains at least three additional orfs: orf1a, orf1b, and orf3. orf1a and -b encode two viral proteases that process these large precursor polyproteins into 16 nonstructural proteins, including the viral replicase and associated rna-modifying enzymes that are critical for full-length and subgenomic positive-and negative-strand rna synthesis (22) . orf3 regulates virus production and encodes an ion channel important for viral fitness but is not required for viral replication in vitro (23, 24) . in this study, we generated the first infectious cdna clone of a virulent north american pedv strain, pc22a (25) . parental genomic and orf3 deletion recombinant viruses were generated using the infectious cdna clone system; the latter was also engineered to express red fluorescent protein (rfp). both recombinant viruses are replication competent in vitro and pathogenic in neonatal gnotobiotic (gn) piglets. parental and recombinant viruses were efficiently transmitted to uninoculated pigs via indirect contact, allowing for genetic studies into the molecular mechanisms regulating virus transmission and pathogenesis. the availability of an infectious clone for pedv will allow us further opportunities to understand gene function and genetic variants in pedv pathogenesis and transmission, leading to better-informed design of vaccines and therapeutics. we have developed molecular clones for several highly pathogenic swine and human coronaviruses, using class ii restriction endonucleases, to directionally assemble a full-length cdna viral genome from a set of sequentially designed smaller cdnas (26) (27) (28) (29) (30) (31) . to develop a molecular clone for pedv, the highly virulent pc22a strain (fig. 1a ) was sequenced and synthesized as six contiguous pedv subclones designated a to f (fig. 1b) . subclones a/b, b/c, c/d, and d/e are joined by unique sapi restriction endonuclease cleavage sites (at nucleotide positions 4071, 8287, 12016, and 16941, respectively) that allow for directional assembly into a full-length cdna without alteration of the viral amino acid sequence. subclones e and f are joined at a unique bsai site at nucleotide position 22504. in subclone f, a single bsai restriction site in pedv-pc22a was removed by introducing a silent mutation at position 24337, effectively marking the recombinant genome (fig. 1c) . thus, each fragment contains a unique set of class ii restriction enzyme sites flanking the genomic sequence that allow for unique 3-nt overhangs between each fragment. this specificity allows for systematic, efficient, and directional assembly of the complete pedv genome by in vitro ligation. the pedv a fragment contains a t7 start site, whereas the f fragment terminates in 22 a residues, allowing for in vitro transcription and capping of a polyadenylated full-length transcript. pedv-orf3 is an accessory orf encoding a putative ion channel protein that is oftentimes deleted from some natural isolates or following in vitro passage, suggesting that it encodes nonessential functions in vitro and/or in vivo (24) . to generate a fluorescently marked pedv genome mutant, orf3 in the pedv f fragment was replaced with red fluorescent protein (rfp), named tomato-red (fig. 1d) . the mutant was created using native restriction enzyme recognition sequences that allowed for the preservation of the orf3 transcription regulatory sequence (trs), which regulates subgenomic rna expression (fig. 1d) . recovery of recombinant viruses. to isolate recombinant wild-type and rfp-expressing recombinant pedvs, each plasmid fragment was digested with noted restriction enzymes, purified, and ligated to create a full-length pedv cdna genome. using the t7 rna polymerase, full-length transcripts were synthesized in vitro as previously described by our group (26, 30) . as previous swine and human coronavirus infectious clones displayed improved recovery rates and replication efficiency in the presence of the supplemented n gene transcript (26, 30) , capped pedv-n gene transcripts were mixed with the full-length genomic transcripts prior to their electroporation into vero cells. within 24 to 48 h postelectroporation, subgenomic recombinant-virus mrna could be detected via reverse transcription-pcr (rt-pcr). after we isolated recombinant virus from pig intestinal contents after inoculation, wild-type and recombinant viruses replicated to titers that approached or exceeded 1 ϫ 10 4 pfu/ml in vero cells, equivalent to titers commonly reported in the literature ( fig. 2a) . a recombinant infectious-clone-derived pedv (icpedv) produced a plaque morphology (fig. 2b ) similar to that of the parental strain and formed syncytia characteristic of pedv in culture (fig. 2c) . notably, icpedv-⌬orf3-rfp displayed a reduced plaque size compared to that of either pc22a or icpedv (fig. 2b) , indicative that orf3 may be important for in vitro growth of the virus and suggestive of possible attenuation of the orf3 deletion mutant. at 48 and 72 h postelectroporation with icpedv-⌬orf3-rfp, fluorescent red cells were seen in cell culture, both within individual cells and within larger syncytia (fig. 2d) . characterization of recombinant viruses. to evaluate protein expression in our recombinant pedv, we cloned and expressed pedv-s and pedv-n in venezuelan equine encephalitis virus strain 3526 virus replicon particles (vrp) and isolated vrp-pedv-s and vrp-pedv-n. the vrp were inoculated into the footpads of mice, and polyclonal pedv-n and pedv-s antisera were collected after a day 21 boost. using western blot techniques, we confirmed the presence of the 180-/90-and~50-kda pedv-s and pedv-n proteins, respectively, in cells infected with icpedv, icpedv-⌬orf3-rfp, and parental pedv in vitro (fig. 3a) . thus, molecularly derived viruses have protein expression phenotypes similar to those of the parental virus. to further confirm the presence of the recombinant virus postelectroporation, we reverse transcribed genomic rna from virions in the culture media and then sequenced them to demonstrate the presence of the distinguishing bsai cloning site in infectiousclone viruses (fig. 3b) . additionally, we sequenced the leadercontaining subgenomic mrna transcripts to ensure that both our recombinant viruses and their parental strain shared the wild-type transcription regulatory sequence (trs) required for normal coronavirus replication and growth kinetics (fig. 3c ). together, these data definitively demonstrate that both recombinant clones generated replicating recombinant virus in vitro. icpedv replication and pathogenesis in gn piglets. pedv pc22a is highly pathogenic in newborn piglets and is rapidly transmitted to littermates. to determine if icpedv replicated parental pedv pc22a in vivo pathology and transmission phenotypes, gnotobiotic (gn) pigs were orally inoculated with icpedv (passage 0 [p0]), icpedv-⌬orf3-rfp (p0), or pc22a (p3) and housed with uninfected indirect-contact pigs ( table 1) . challenged animals demonstrated fecal viral rna shedding, and diarrhea started 1 to 3 days postinfection (dpi) in all three virus groups ( fig. 4a ; table 1 ). importantly, uninoculated indirect-contact pigs within each group demonstrated both robust virus shedding and diarrhea, confirming the transmissibility of both pc22a and the recombinant virus (fig. 4b ). all three viruses replicated to similar peak titers (11 to 13 log 10 genomic equivalents [ge]/ml). however, pc22a and icpedv group pigs had more-severe diarrhea (highest fecal score of 3) than icpedv-⌬orf3-rfp group pigs (highest score of 2). the day of harvest for each piglet was dependent on clinical fecal scores or occurred upon the death of the piglet. notably, days of harvest varied both between virus types and within virus groups. because of the animal numbers in each group and because of the variation within each pedv group, the day of harvest cannot be used as a reliable indicator of the relative degree of viral virulence or attenuation. the pathogenesis of icpedv in gn pigs also replicated the pathogenic phenotype of pedv strain pc21a, which had been collected from the same swine farm on the same day as pc22a (4) . histopathological examination showed severe villous atrophy in pedv pc22a-and icpedv-inoculated pigs and moderate-tosevere villous atrophy in icpedv-⌬orf3-rfp-infected pigs (fig. 5a ). the villous height/crypt depth (vh/cd) ratio of the jejunum of a mock-inoculated pig was significantly higher than those of pc22a-, icpedv-, and icpedv-⌬orf3-rfp-infected pigs (p ͻ 0.05) ( table 1) . immunohistochemistry (ihc) for pedv-n confirmed the presence of recombinant virus throughout the small intestine (duodenum, jejunum, and ilium) in both icpedv-and icpedv-⌬orf3-rfp-infected pigs ( fig. 5b ; table 2 ). in addition, icpedv antigens were detected in the large intestine (colon). these results indicate that the cell culture supernatants of icpedv and icpedv-⌬orf3-rfp contained infectious recombinant virus particles that replicated well in gnotobiotic pigs. while the recombinant icpedv replicated the clinical phenotypes of parental pc22a in vivo, icpedv-⌬orf3-rfp infection resulted in a partial attenuation in pigs based on lower diarrhea scores. the rapid infection of contact pigs suggests efficient transmission of icpedv and icpedv-⌬orf3-rfp, replicating both parental pc22a and circulating u.s. strain transmission phenotypes. emerging viruses pose a considerable threat to humans and society by causing morbidity and mortality in human populations or causing significant losses in important food sources and trade, leading to economic instability and loss of critical protein sources, especially in poor rural populations. porcine epidemic diarrhea virus is a serious livestock pathogen that is causing significant economic losses in the swine industry internationally. to date, over a billion piglets have died globally. live vaccine has been used historically to combat pedv outbreaks in asia; however, live vaccines available today are ineffective in preventing outbreaks of circulating pandemic strains, including u.s. outbreak strains, and have not significantly reduced the global disease burden (14) . other important nidovirus infections of swine include transmissible gastroenteritis virus (tgev), its related respiratory variant designated porcine respiratory coronavirus (prcv), and porcine reproductive and respiratory disease virus (prrsv), which have caused major economic losses to the swine industry since the late 1980s (32) (33) (34) . in addition to pedv, an emerging coronavirus, porcine deltacoronavirus, has recently been reported in swine, demonstrating the possibility of continued emergent threats to this important food industry (6, 35) . given the apparent increase in the number of new swine viruses identified over the past 30 years, it seems clear that management practices and/or other changes in the ecosystem are providing an environmental setting that promotes the emergence of new viral pathogens for the swine industry. if so, these data document the need for the development of new, rapid-response intervention platforms for disease control in critical livestock populations that are centrally linked to human health. in this article, we describe the first molecular clone for a highly pathogenic u.s. strain of pedv, pc22a, isolated from an outbreak in ohio in june 2013 (25) . both the parental pedv pc22a strain and its derivative recombinant cloned virus were genetically stable and fully pathogenic in neonatal gnotobiotic pigs, demonstrating that icpedv provides not only a strategy that allows for the systematic evaluation of the role of viral genes in pathogenesis, tropism, and virulence but also a translational platform for the development of rationally attenuated live virus vaccines. in addition, we have constructed a recombinant pedv that bears an indicator gene, the rfp gene, which allows for rapid evaluation of antiviral efficacy and neutralizing antibody levels by means of high-throughput cell culture systems. recently, a molecular clone for a high-growth tissue culture variant of a 2010 thai isolate, designated pedv avct12 , was reported in the literature (36) . in contrast to our findings, fulllength recombinant pedv avct12 could not be isolated unless orf3 expression had been ablated, either by naturally occurring deletions or by insertion of an indicator gene in this location. interestingly, naturally occurring deletions also removed 7 amino acids from the c terminus of the s protein, similar to deletions described with other tissue culture strains, like pedv strain chm2013 (36) . at this time, the discrepancy between the two laboratory results is intriguing and most likely is directly related to the backbone sequence of the two isolates and/or the difficulties associated with culturing clinical isolates of pedv in vitro. tissue culture pedv avct12 replicates 2 to 3 logs more efficiently than wild-type pedv pc22a and icpedv in culture, and trypsin is also required to culture the latter isolates in vitro. future studies may well reveal the emergence of similar tissue culture adaptations during serial passage of our highly virulent pedv pc22a and icpedv in culture. importantly, pathogenic outcomes in vivo were not evaluated using the heavily tissue culture-adapted pedv avct12 strain, so the utility of this recombinant virus in evaluating pathogenic outcomes and/or the role of tissue cultureadaptive mutations in virulence are uncertain at best. although an exact infectious dose of our recombinant viruses was not determined from these studies, ͻ1 pfu of pedv pc22a is sufficient to cause disease in piglets (37) . little information is available regarding the molecular mechaa pigs 2, 5, 7, and 10 were exposed by indirect contact to the pigs which were housed in the same isolator through small holes drilled into the stainless steel divider. the panel was located between the 2 pigs in the shared pig tub isolator unit. b nd, not done because pig 1 was used for hyperimmune serum production; na, not available; vh/cd, villous height/crypt depth. c fecal scores were as follows: 0, normal; 1, pasty stool; 2, semiliquid diarrhea; and 3, liquid diarrhea. nisms governing efficient coronavirus pathogenesis and transmission between hosts. importantly, icpedv and icpedv-⌬orf3-rfp are efficiently transmitted to cohoused littermates, providing a potential platform for investigating the genetic mechanisms regulating efficient transmission between hosts. while similar studies using highly pathogenic influenza viruses in ferrets are highly controversial because of potential human-pandemic concerns (38) , identifying genetic factors that attenuate transmission frequency offer a powerful tool to improve the safety and efficacy of live attenuated coronavirus vaccines, especially given the highanimal-density manufacturing approaches used in the swine industry. such studies may also provide significant insights into the fundamental principles and genetic functions that influence the transmission efficiency of other highly pathogenic human coronaviruses, like severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). pedv infection is most devastating in neonatal and suckling piglets, necessitating vaccines that target lactogenic immunity through the vaccination of pregnant sows and gilts. piglets do not attain passive immunity preparturition but instead receive iggand iga-based lactogenic immunity from colostrum and milk, respectively. with both tgev and pedv, sows infected with live virulent virus transferred more protective immunity against viral challenge in their nursing piglets than sows infected with attenuated or inactivated virus (39) . the usda has granted conditional licenses to two pedv manufacturers to date. the harris vaccine uses an attenuated venezuelan equine encephalitis virus (veev) vaccine strain replicon particle expressing the pedv spike protein (40) . the second is a parenterally killed virus vaccine made by zoetis (41) . both are used to immunize pregnant sows and gilts. their efficacy and ability to protect against various circulating u.s. strains are still under evaluation. we note that the vrp platform described in our paper was based on a biosafety level 2 (bsl2) nonselect agent, a veev strain designated 3526, which has been used in animal and human trials (42) (43) (44) . in contrast to other veev replicon platforms, veev 3526 retains wild-type e1 and e2 glycoproteins, which efficiently target dendritic cells (45, 46) , but lacks e3 sequences. this deletion of e3 confers an attenuated phenotype in vivo. veev 3526 expressing appropriate s glycoprotein genes provides robust protection against other coronaviruses, like sars-cov, mers-cov, and hku5-s (47-49). using veev 3526 structural genes allows for recovery of high-titer vrp encoding pedv-s and/or -n under bsl2 conditions. it remains unclear whether the vrp 3526 platform will prove sufficiently robust to induce in sows lactogenic immunity capable of protecting suckling piglets (49) . future experiments will have to be designed and implemented to test the relative efficacies of vrp vaccines, killedwhole-virus vaccines, or future live attenuated-virus vaccines. importantly, no live-virus vaccine is currently available in the united states, and historical live vaccines have not been effective in combating current u.s. or asian strains (14) . robust studies using sars-cov have identified several viral genes, including the e protein, the exon nsp14 rna proofreading machinery, and the 2-o-methyltransferase nsp16 replicase, as high-priority targets for rational attenuation of coronaviruses (50) (51) (52) . because coronaviruses undergo rna recombination at high frequency and encode an exonuclease function (53, 54) , recombination repair and reversion to wild-type virus is a pressing concern when designing live attenuated coronavirus vaccines. however, our laboratory has developed strategies to prevent recombination repair that limit the capacity of rationally designed live attenuated virus to revert to the wild-type virus sequence (55) . effective vaccines are increasingly important, as new strains are identified in the united states and circulating strains continue to devastate herds. the infectious clone platform allows for rapid construction of genetically modified pedv variants to evaluate the function of antigenic variation on neutralization phenotypes and can be used for the rational design of a live virus vaccine. this platform also allows for incorporation of genetic changes to enhance the replication of the virus in vitro for more efficient production of attenuated vaccines. globally, humans have experienced coronavirus outbreaks with increasing frequency, including outbreaks of two new human coronaviruses in the last 15 years, notably, sars-cov and mers-cov (56) . human and animal coronaviruses share similar structural proteins and replication dynamics. currently, no transmission model for these important human pathogens is available. the neonatal pig model described in this study can provide a surrogate transmission model for human coronaviruses. separately, the study of coronavirus transmission in its original host in a bsl2 climate affords the opportunity to safely and accurately research a cohoused with an inoculated piglet(s) to determine transmission. all animals succumbed to illness or were euthanized due to illness at their final time points (pedv, 1 dpi; icpedv, 4 dpi; icpedv-⌬orf3-rfp, 7 dpi; contact control, 4 dpi). all images show representative histological slides of jejunum specimens from mock-infected or infected animals. histology of mock-, icpedv-, or icpedv-orf3-rfp-infected animals, showing h&e staining and immunohistochemistry (ihc) staining (b) for pedv nucleocapsid (n) protein using mouse anti-pedv-n. a specimen from a contact piglet infected by icpedv is also shown. cell fusion and vacuolation were noted at the villus tips (arrows). ihc staining of icpedv-or icpedv-⌬orf3-rfp-infected animals whose results are represented in panel a is shown. icpedv 2 5 0 3 3 1 3 4 2 3 3 1 4 5 1 3 2 2 5 5 1 3 2 1 icpedv-⌬orf3-rfp 6 2 0 1 1 0 7 7 1 2 3 0 8 3 0 2 2 0 a the ihc signal of pedv antigen was scored as 0 to 3 according to the percentage of villous enterocytes within the section showing a positive signal. a score of 0 means that there were no positive cells, and scores of 1, 2, and 3 mean that ͻ30%, 30 to 60%, and ͼ60% of villous enterocytes showed a positive signal, respectively. family of viruses that is devastating animal and human populations. it is possible that genetic manipulation of recombinant pedvs will enable studies that can significantly enhance our understanding of the role of coronavirus genetics in the transmission, virulence, and pathologies that are central to both animal and human health and disease prevention. in this article, we describe a reverse-genetics platform for a highly virulent u.s. pedv strain that causes lethal disease in newborn piglets, allowing for the future identification of attenuating mutations and virulence alleles. in parallel, we have developed indicator viruses that can be used for high-throughput neutralization assays or to evaluate the impact of antivirals. this reversegenetics system will allow for quick and robust pedv genetic manipulation in a clinical north american isolate, allowing for in-depth study of viral replication and pathogenesis, which are essential for the development of safe and robust live attenuatedvirus vaccines. the wild-type pc22a strain of pedv (passage 4) was cultured on vero cells, as described previously (25) . cells were grown in growth medium containing dulbecco's modified eagle medium (dmem) (life technologies) supplemented with 5% fetal bovine serum (life technologies) and 1% antibiotic-antimycotic (gibco). virus was grown in vero cells in maintenance medium, which was dmem supplemented with 10 g/ml trypsin (life technologies), 0.3% tryptose phosphate broth (sigma), and 1% antibiotic-antimycotic (life technologies). cells were kept in a humidified incubator at 37°c and 5% co 2 . assembly of full-length recombinant pedv. the icpedv clone was designed using six separate fragments (ordered from bio basic) flanked with unique flanking class ii restriction sites that leave nonpalindromic overhangs. sequences were ordered based on pc22a passage 4 sequence (genbank accession number km392224.1). all cdna subclones were grown in the pcxl-topo vector. in fragment e, a naturally occurring bsai site was removed by introducing a silent mutation in order to prevent interference with assembly of the full-length infectious clone. all pedv fragments were sequenced after transfection into bacterial culture to ensure sequence fidelity. the pedv fragments were digested using restriction sites designated in fig. 1 , run on a 1% agarose gel, excised, and purified using a qiaquick gel extraction kit (qiagen). the pedv fragments were mixed and ligated overnight at 4°c using t4 dna ligase (roche). ligated fragments were phenol-chloroform extracted, and fulllength t7 rna transcripts were generated as described in the mmessage mmachine manufacturer protocol (ambion), but we allowed the reaction to run at 30°c for 3 h and then at room temperature for 2 h. in addition, sp6 pedv-n gene transcripts were generated from the pcr-purified pedv-n gene sample using a 4:1 ratio of cap to gtp (ambion). to generate the orf3 deletion rfp construct, the tdtomato gene was amplified by pcr with flanking pedv sequence and then inserted using native restriction sites into pedv-f. pedv-f-⌬orf3-rfp was cultured and sequenced to ensure seamless replacement of orf3 with rfp containing the orf3 trs. in vitro transfection. genome-length and n rna transcripts were mixed with 800 l of vero-bi cells (1 ϫ 10 7 cells/ml) in phosphatebuffered saline (pbs) and then added to an electroporation cuvette. three pulses of 450 v at 50 f were used to transfect the cells with a gene pulser ii electroporator (bio-rad). the cells were allowed to recover for 10 min at room temperature and then were transferred to a 75-cm 2 flask in growth medium at 37°c for 2 h, after which time the cells were washed and incubated in cell culture medium. trypsin was added to the culture at 5 g/ml 12 h postelectroporation to assist in virus recovery and spread. sequence analysis identification of marker mutations. virus harvested from small intestinal contents was grown in vero-bi cell culture for 48 h. virion rna was harvested from the supernatant using the qiaamp viral rna minikit (qiagen). after purification, viral cdna was generated with superscript ii reverse transcriptase (life technologies) as previously described by our group (26) . to demonstrate the presence of the marker mutation, the icpedv bsai mutation site was amplified by pcr using primers 5= tccaagccatttctagttctatt 3= and 5= tgacacaac aaagatgagaaca 3=. pcr amplicons were gel purified and then sequenced using primers 5= tcaggctagcaggaagttag 3= and 5= ag gtcaactagtgtgttgttgatat 3=. western blot and transcript analysis. virus from infected animals was cultured in vero-bi cell culture for 48 h and washed with pbs, and intracellular rna was harvested from cells using np-40 buffer (150 mm nacl, 1% triton x-100, 50 mm tris, ph 8.0) for western blots or trizol (life technologies) for rna analysis. cdna from viral rna transcripts was generated using superscript ii reverse transcriptase (life technologies) and pcr amplified using primer pairs from the pedv leader sequence and nucleocapsid gene. pcr products were separated on a 1% agarose gel and visualized on a dark reader transilluminator (clare chemical research). for western blot analysis, protein from infected cells was denatured in 4ϫ laemmli buffer (bio-rad) at 95°c for 6 min and then separated on gradient 4 to 15% mini-protean precast gels (bio-rad) prior to electrophoretic transfer of the proteins to polyvinylidene difluoride (pvdf) membranes (bio-rad). to detect pedv antigens, blots were first blocked with 5% milk in tris-buffered saline with tween 20 (tbst) and then probed with a polyclonal mouse serum (diluted 1:200) from mice which had been immunized with venezuelan equine encephalitis virus replicon particles (vrp) expressing pedv nucleocapsid (n) or spike (s) glycoprotein. blots were developed using ge healthcare amersham ecl western blotting detection reagents and exposed to film for imaging. animal studies. four groups of 2-to 3-week-old gnotobiotic (gn) pigs were used to examine the replication and pathogenesis of icpedvand icpedv-⌬orf3-rfp-derived viruses in vivo and compared with pc22a-and mock-infected positive and negative controls, respectively (table 1) . piglets were orally inoculated with 2 ml of icpedv or icpedv-⌬orf3-rfp culture supernatants after transfection (p0) (ͻ1.0 ϫ 10 2 pfu/ml) or with the tissue culture-adapted pc22a strain at passage level 3 (p3) at a dose of 5.8 log 10 pfu/pig. to investigate transmission, pigs 2, 5, 7, and 10 were cohoused in the same isolator as infected pigs but were separated by a stainless steel divider that contained small holes which allowed only indirect contact between the groups. animals were monitored daily for clinical signs of disease, including diarrhea and vomiting. rectal swabs were collected for scoring fecal denseness (scores: 0 ϭ normal; 1 ϭ pasty stool; 2 ϭ semiliquid diarrhea; and 3 ϭ liquid diarrhea) and for enumerating fecal viral rna shedding by rt-quantitative pcr (rt-qpcr). except for one pig in the icpedv group, which was kept long term for the production of hyperimmune serum, the gn pigs were euthanized at acute infection phase (within 5 days postinoculation [dpi] or 7 days postcontact with the inoculated pigs [dpc]) for histopathological examinations. at necropsy, small and large intestinal contents were collected and tested by rt-qpcr for viral rna levels and for infectious virus by plaque assay. the different sections of the small intestine (duodenum, jejunum, ilium) and large intestine (cecum and colon) were collected for histopathological examination and stained with hematoxylin and eosin (h&e) stain. the derivation and maintenance of gn pigs, sample collection and testing, and histopathology were performed as previously described (25, 57) . all the animal use protocols employed in this study were reviewed and approved by the agricultural animal care and use committee of the ohio state university. ihc staining. the immunohistochemistry (ihc) staining procedure was optimized as described previously (37) using the non-biotinpolymerized horseradish peroxidase (hrp) system (biogenex laboratories, san ramon, ca). briefly, intestinal tissue sections from each pig were deparaffinized and rehydrated in graded ethanol to pbs (ph 7.4). antigen retrieval and unmasking were performed by treatment with 0.05% pronase e (sigma-aldrich, st. louis, mo) for 20 min. the endogenous per-oxidase activity was quenched with 3% hydrogen peroxide (sigma) for 20 min. then, the sections were incubated in power block solution (bio-genex) for 30 min at room temperature. mouse monoclonal antibody anti-pedv nucleocapsid protein (n) (sd6-29; a gift from e. nelson and s. lawson, south dakota state university) was applied to each section at 4°c overnight. after two washes in pbs, a commercial super sensitive ihc detection system (biogenex) was used. finally, these sections were counterstained with mayer's hematoxylin (biogenex) and dehydrated, and coverslips were added. the ihc signal of pedv antigen was scored as 0 to 3 according to the percentage of villous enterocytes within the section showing a positive signal. scores were as follows: 0 indicated that there were no positive cells and 1, 2, and 3 indicated that ͻ30%, 30 to 60%, and ͼ60% of villous enterocytes showed a positive signal, respectively. identification of new respiratory viruses in the new millennium. viruses emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences pathogenesis of porcine epidemic diarrhea 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epidemiology and available vaccines receptor usage and cell entry of porcine epidemic diarrhea coronavirus porcine epidemic diarrhea virus uses cellsurface heparan sulfate as an attachment factor genome organization of porcine epidemic diarrhoea virus mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo receptor-bound porcine epidemic diarrhea virus spike protein cleaved by trypsin induces membrane fusion porcine epidemic diarrhea virus e protein causes endoplasmic reticulum stress and up-regulates interleukin-8 expression porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin-8 expression completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence pedv orf3 encodes an ion channel protein and regulates virus production manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus systematic assembly of a full-length infectious clone of human coronavirus nl63 development of mouse hepatitis virus and sars-cov infectious cdna constructs reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus 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 live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction transmissible gastroenteritis virus infection: a vanishing specter background paper. the appearance of the porcine respiratory coronavirus has created new problems and perspectives detection and genetic characterization of deltacoronavirus in pigs genetic manipulation of porcine epidemic diarrhea virus (pedv) recovered from a full-length infectious cdna clone determination of the infectious titer and virulence of an original us porcine epidemic diarrhea virus pc22a strain avian influenza virus transmission to mammals resolution of the cellular proteome of the nucleocapsid protein from a highly pathogenic isolate of porcine reproductive and respiratory syndrome virus identifies parp-1 as a cellular target whose interaction is critical for virus biology usda licenses first vaccine for porcine epidemic diarrhea. usda animal and plant health inspection service usda grants zoetis a conditional license for porcine epidemic diarrhea vaccine improved mucosal protection against venezuelan equine encephalitis virus is induced by the molecularly defined, live-attenuated v3526 vaccine candidate venezuelan equine encephalitis virus vaccine candidate (v3526) safety, immunogenicity and efficacy in horses environmental hazard assessment of venezuelan equine encephalitis virus vaccine candidate strain v3526 infected dendritic cells are sufficient to mediate the adjuvant activity generated by venezuelan equine encephalitis virus replicon particles role of dendritic cell targeting in venezuelan equine encephalitis virus pathogenesis rapid generation of a mouse model for middle east respiratory syndrome a mouse model for betacoronavirus subgroup 2c using a bat coronavirus strain hku5 variant successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2=-omethyltransferase activity severe acute respiratory syndrome coronaviruses with mutations in the e protein are attenuated and promising vaccine candidates recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission coronaviruses: an rna proofreading machine regulates replication fidelity and diversity rewiring the severe acute respiratory syndrome coronavirus (sars-cov) transcription circuit: engineering a recombination-resistant genome molecular pathology of emerging coronavirus infections pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs we thank j. hanson, r. wood, and j. ogg for animal care assistance, and t.oka and x. wang for technical assistance. special thanks to steven lawson and eric nelson (department of veterinary and biomedical sciences, south dakota state university) for providing mouse anti-pedv nucleocapsid protein monoclonal antibody sd6-29.salaries and research support were provided by state and federal funds appropriated to ohio agricultural research and development center (oardc), the ohio state university.the authors acknowledge the key financial support from the national institutes of health, center for diagnostics and discovery (u19 ai109761) to r.s.b. and grant 2015-67015-23067 from the usda to q.w., l.s., and r.s.b. key: cord-344558-1jgqofbr authors: kocherhans, rolf; bridgen, anne; ackermann, mathias; tobler, kurt title: completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence date: 2001 journal: virus genes doi: 10.1023/a:1011831902219 sha: doc_id: 344558 cord_uid: 1jgqofbr the sequence of the replicase gene of porcine epidemic diarrhoea virus (pedv) has been determined. this completes the sequence of the entire genome of strain cv777, which was found to be 28,033 nucleotides (nt) in length (excluding the poly a-tail). a cloning strategy, which involves primers based on conserved regions in the predicted orf1 products from other coronaviruses whose genome sequence has been determined, was used to amplify the equivalent, but as yet unknown, sequence of pedv. primary sequences derived from these products were used to design additional primers resulting in the amplification and sequencing of the entire orf1 of pedv. analysis of the nucleotide sequences revealed a small open reading frame (orf) located near the 5′ end (no 99–137), and two large, slightly overlapping orfs, orf1a (nt 297–12650) and orf1b (nt 12605–20641). the orf1a and orf1b sequences overlapped at a potential ribosomal frame shift site. the amino acid sequence analysis suggested the presence of several functional motifs within the putative orf1 protein. by analogy to other coronavirus replicase gene products, three protease and one growth factor-like motif were seen in orf1a, and one polymerase domain, one metal ion-binding domain, and one helicase motif could be assigned within orf1b. comparative amino acid sequence alignments revealed that pedv is most closely related to human coronavirus (hcov)-229e and transmissible gastroenteritis virus (tgev) and less related to murine hepatitis virus (mhv) and infectious bronchitis virus (ibv). these results thus confirm and extend the findings from sequence analysis of the structural genes of pedv. porcine epidemic diarrhoea virus (pedv) is a causative agent for diarrhoea in pigs, particularly in neonates. the disease has been recognised for approximately thirty years, but the causative virus was only first described in 1978 [1] , while another ten years elapsed before a method was developed for propagation of the virus in cell culture [2] . during this time, outbreaks of the disease were reported from numerous european countries as well as korea, china and japan. the epidemiology and pathogenesis of the disease have been well described by pensaert [3] . the biological behaviour, electron microscopic appearance and polypeptide structure of pedv resulted in its provisional classification as a coronavirus [2, 4, 5] . coronaviruses belong to the taxonomic order of nidovirales and contain a single stranded rna genome of positive polarity, which is approximately thirty kilobases in length. the genes encoding the structural proteins are located at the 3 0 end of the genome. an astonishing two-thirds of the genome consist of the replicase gene, which is located at the 5 0 end of the genome. the replicase proteins are encoded by orf1a and orf1b. these two long, slightly overlapping orfs are connected by a ribosomal frame shift site in all coronaviruses sequenced to date. this regulates the ratio of the two polypeptides encoded by orf1a and the readthrough product orf1ab. about 70±80% of the translation products are terminated at the end of orf1a, and 20±30% continue to the end of orf1b. the polypeptides are post-translationally processed by viral encoded proteases [reviewed by 6]. these proteases are encoded within orf1a; the polymerase-and the helicase-function are encoded by orf1b. we have previously completed the sequencing of the nucleocapsid-(n), membrane-(m), small membrane-(e), orf3 and spike-(s) genes of the pedv strain cv777 [7±9]. the alignment of the deduced amino acid sequences indicated that pedv occupies an interesting intermediate position between the two well-characterized members of the group i coronaviruses, transmissible gastroenteritis virus (tgev) and human coronavirus (hcov)-229e. in this study, we have continued to determine and analyse nucleotide sequences of pedv. to our knowledge, only two group i coronaviruses have been sequenced completely, hcov-229e and tgev [10, 11] . in addition, two strains of mouse hepatitis virus (mhv), jhm and a59 belonging to the group ii coronaviruses, and infectious bronchitis virus (ibv) have been completely sequenced [12±15] . therefore, the sequence presented in this paper is the sixth sequence of a coronavirus covering the entire genome. growth of cell adapted pedv strain cv777 was performed essentially as has been described elsewhere [2, 8] , except that virus-infected cells were harvested at approximately 18 h post infection. cells were freeze-thawed three times and cell debris removed by low speed centrifugation. virus was pelleted by centrifugation for 2 h at 22,000 rpm and 4 c in a sw28 rotor of a beckman centrifuge. virus pellets prepared from two 175 cm 2 flasks were pooled and resuspended in 1 ml trizol tm (gibco-brl), and rna was prepared as recommended by the manufacturer. in order to obtain the first partial pedv specific sequences, the predicted amino acid sequences of the hcov-229e and tgev polymerase orfs were aligned and homologous regions identified. the homologous regions were used to design degenerate primers [9] that were used for rt-pcr amplifications. these initial amplicons were cloned and sequenced [9] . later, a mixture of up to six antigenome sense primers based on pedv specific sequences or the degenerate primers and random hexamer primer (purchased from schmidheini ag; balgach, switzerland) was used for first strand cdna synthesis. rna prepared from two 175 cm 2 flasks of virus-infected cells was denatured for 10 min at 65 c and first strand cdna was performed in a 20 ml total reaction volume using superscriptii tm (gibcobrl; basel, switzerland) according to the manufacture's protocol. this was modified to create the longer reverse transcription products by including a denaturation step at 95 c for 5 min following the first 1 h incubation at 42 c, followed by the addition of 1 ml superscriptii tm and a second prolongation step of 1 h at 42 c. template rna was digested by adding 1 ml rnaseh (gibcobrl; basel, switzerland) to the reaction mix and incubating at 37 c for 20 min. pcr amplification was performed as described elsewhere. in brief, pfu dna polymerase (stratagene; basel, switzerland) was used for the amplifications, which were performed on a dna engine (mj research) machine. pcr fragments were subsequently cloned into pbluescript 1 ii ks or puc19 vectors using standard procedures. the nucleotide sequence was determined on these cdna clones. direct sequencing was performed on a rt-pcr product (see fig. 1b ), which was cleaned through an agarose gel. the contigs of the sequence determinations were constructed using seqman (dna*, lasergene, madison wi, usa). we previously reported the determination of the pedv leader sequence on the mrna encoding the n-gene [16] . this sequence was used for the primer design in order to amplify the 5 0 end of the genome. the leader sequence was used for the in silico construction of the genomic rna sequence, which is available on genbank database (accession number af353511). virus sequences covering replicase genes were obtained from the genembl sequence database. the files with the accession numbers x69721, z34093, af029248, and m95169 for hcov-229e, tgev (purdue 115), mhv-a59, and ibv (beaudette) respectively were used. the deduced amino acid sequences were compared as indicated in the text using pileup and gap (gcg package version 10.0; madison, wi, usa). the files generated by pileup were used in distances (gcg package version 10.0; madison, wi, usa) to determine the kimura protein sequence distances, which were subsequently used for the construction of unrooted dendrogram using treegen on the cbrg server (http://cbrg.inf.ethz.ch/) the cloning approach we used previously to clone the pedv m and n genes involved designing primers based on conserved regions of the coronavirus m and n genes to amplify the equivalent to the unknown pedv sequence. in this study, we employed this technique to clone parts of the orf1 of pedv. such a method is useful for viruses which do not grow to high titre, avoids lengthy screening of clones and could potentially be applied to the cloning of any group i coronavirus. however, the large size of orf1 and the paucity of sequence data from other coronaviruses made this an ambitious objective. a number of conserved functional domains were identified in the predicted orf1 products, but these domains are mainly located in the orf1b region and leave large regions of the orf1a product with no known function and only a low level of sequence conservation between different coronavirus genomes. in order to clone and determine the sequences for the pedv orf1, the predicted amino acid sequences of the hcov-229e and tgev orf1 were aligned and homologous regions identified. the hcov-229e and tgev orfs were sufficiently closely related to allow complete alignment of the predicted expression products. in contrast, the mhv and ibv sequences were much more divergent, and could only be aligned with the group i sequences in some of the conserved regions. degenerate primers were designed from regions conserved between the hcov-229e and tgev and, where possible, mhv and ibv orf1. these primers were used both to prime reverse transcription and for the pcr amplifications. sequence data derived from these pcr products allowed us to design sequence-specific primers which were then used to amplify the entire orf1 (see fig. 1b ). numerous small cdna clones, five large cdna clones and one rt-pcr product covering the 5 0 twothirds of the pedv genome were used to determine the nucleotide sequence of the pedv orf1 (fig. 1 ). this analysis completes the nucleotide sequence of pedv, and thereby the sixth entire sequence determined from a coronavirus genome [10±13,15] . the genome of pedv (cv777) excluding the poly a-tail is 28033 nt in length. analysis of the newly determined nucleotide sequence revealed a pattern of orfs typical of coronaviruses. a small orf with the potential to code for a 12-amino acid peptide was found at the 5 0 end of the genome from nucleotide position 99±137. such small orfs (uorfs) are present in all coronaviruses sequenced so far. the uorfs of hcov-229e [17] and ibv [15] are found to be eleven codons in length, while that of mhv is eight codons long [18, 19] . that of tgev can only encode a three-amino acid peptide [20] . two long orfs of 12354 and 8037 nt, which overlap by 46 nt, covered most of the newly determined sequence. by analogy to published coronavirus sequences [15, 17, 20] , the orfs were designated orf1a and orf1b. the predicted orf1a of fedv extended from nucleotide 297 to 12650. this resulted in a 4117-codon orf. the overlapping orf1b starting at nucleotide 12605 and ending at nucleotide 20641 had the capacity to code for 2678 amino acids. it has been proposed for coronaviruses and other members of the order nidovirales [21] that the nucleotide sequences in the overlapping regions of orf1a and orf1b are able to fold into a pseudoknot tertiary structure [22, 23] . this region allows the ribosome shifting of the reading frame during translation of the orf1a and subsequently continues the translation in orf1b. the function of these rna structures as ribosomal frame shift sites was demonstrated for the analogous sequences of ibv [24] and hcov-229e [25] . it seems likely that the translation of the pedv orf1b is mediated by such a ribosomal frame shifting. the nucleotide sequences of pedv, hcov-229e, and tgev covering the ribosomal frame shift site are more conserved to each other than to mhv-a59 or ibv. in order to identify the sequence which could be involved in the formation of the tertiary structure, the nucleotide sequences covering the end of orf1a and the beginning of orf1b from hcov-229e [25] and tgev [20] were aligned with the corresponding sequence of pedv. fig. 2a shows the predicted frame shift region of pedv based on this comparison. the so-called slippery site (uuuaaac) at which frame shifting occurs is identical in all coronaviruses sequenced so far. the stems and loops required to provide the tertiary structure of the frame shift regions of tgev and hcov-229e were compared and fig. 2b shows the predicted tertiary structure required for the frame shift of pedv based on this comparison. pairwise comparison of the deduced amino acid sequences (using gap) revealed that orf1b of pedv is more conserved than orf1a to corresponding sequences of other coronaviruses. the percentage of similarities and identities is shown in table 1 . the putative protein sequence of orf1a was most similar to the sequence of orf1a of hcov-229e (59.4%) and less similar to the corresponding orf1a of tgev (52.1%), mhv-a59 (39.5%) and ibv (38.7%). the same relationship, but at a higher level of similarity, was true for the deduced amino acid sequence of the predicted pedv orf1b. it was most similar to the amino acid sequence of hcov-229e orf1b and tgev orf1b (83.2% and 80.3%, respectively). the similarity to the orf1b from mhv-a59 and ibv was around 64%. the deduced amino acid sequences of orf1a and orf1b from pedv were aligned with the corresponding sequences of hcov-229e, tgev, mhv-a59, and ibv using pileup. the degrees of amino acid homologies are graphically presented as dendrograms (fig. 3a,b) . the multiple sequence alignments revealed several putative functional domains common to coronavirus sequences [23, 26] located on the deduced amino acid sequence of orf1ab of pedv. some of these had been used to design the primers for the rt-pcr amplification. in the orf1a region the following motifs were observed. two motifs indicative of papain-like proteases (plp) were present at amino acid positions 1077±1266 and 1716±1917. the plp motif is found twice in the replicase genes of hcov-229e, tgev and mhv, but only once in that of ibv. in this respect, pedv resembles hcov-229e, tgev and mhv rather than ibv. a highly conserved region (x-domain) was found between the two plp motifs. despite this motif being present in all coronavirus sequences, its function is not yet known. a picornavirus 3c-like (3c1) protease domain is located between amino acids 2998 and 3299 of the pedv orf1a. all corona-and arteriviruses encode this motif, which is the main protease for the coronavirus mediated processing of the polyproteins. three markedly hydrophobic domains conserved among coronaviruses are found in orf1a. the first is located after the second plp motif and the others flank the 3cl motif. finally, a growth factorlike (gfl) domain was located close to the end of orf1a (amino acid position 3965±4000). in the orf1b region, three structural protein motifs could be recognized, which all play a role in viral replication. a sub-sequence at amino acid position 4636±4939 containing the characteristic tripeptide orf1 of pedv sdd (or gdd in most rna viruses) [26] is probably the active site for the rna dependent rna polymerase. a metal ion-binding domain covering amino acids 5027±5103 and a helicase motif at amino acid positions 5309±5624 were also observed in the pedv orf1b product. alignments of the deduced amino acid sequences of the 3cl protease and the polymerase motif from five different coronaviruses are shown in fig. 4a and 4b, respectively. the findings concerning conserved domains are summarised in fig. 1a . a deletion of about 180 amino acids located between the x-domain and the second plp motif in the putative orf1a sequence of tgev compared to that of hcov-229e was reported by eleouet et al. [20] . this additional sequence was present in the pedv orf1a product. the alignment (using gap) of the hcov-229e and pedv amino acid sequences revealed 42.5% similarity and 31.5% identity in this region. earlier sequence analysis of pedv based on the structural protein sequences has shown that pedv is most closely related to hcov-229e and tgev [ 7± 9,27] , less related to mhv-a59, and least related to ibv. however, it was not possible to determine the relative similarities of hcov-229e, tgev and pedv. in this study, the similarities and identities of the amino acid sequence alignments based on orf1a and orf1b show clearly that pedv is most closely related to hcov-229e and, moreover, that hcov-229e is more similar in sequence to pedv than it is to tgev. in addition to the sequence analysis, the presented work offers various possibilities for future research on coronaviruses. functional analysis and processing of the as yet uncharacterised pedv orf1 is now possible. recently, almazan et al. and yount et al. achieved the generation of infectious tgev from cdna [28, 29] and thiel et al. suceeded in generating full length cdna clones of hcov-229e and ibv in a recombinant vaccinia virus system [30] . the sequence and the cdna clones covering the entire genome of pedv would allow the development of a mini-genome system to study viral replication or the generation of an assembled, infectious cdna clone. bearing in mind the close relationship of pedv and hcov-229e, the latter approach could be used to exchange functional parts of these viruses to gain new insights into the biology of these viruses. furthermore, the porcine epidemic diarrhea virus virus infections of porcines a reverse genetic system for coronaviruses the authors thank christa meyer for excellent technical assistance. these studies were supported by the swiss national science foundation, grant #31-43503.95. key: cord-327000-oyg3oyx1 authors: li, shasha; yang, jinping; zhu, zixiang; zheng, haixue title: porcine epidemic diarrhea virus and the host innate immune response date: 2020-05-11 journal: pathogens doi: 10.3390/pathogens9050367 sha: doc_id: 327000 cord_uid: oyg3oyx1 porcine epidemic diarrhea virus (pedv), a swine enteropathogenic coronavirus (cov), is the causative agent of porcine epidemic diarrhea (ped). ped causes lethal watery diarrhea in piglets, which has led to substantial economic losses in many countries and is a great threat to the global swine industry. interferons (ifns) are major cytokines involved in host innate immune defense, which induce the expression of a broad range of antiviral effectors that help host to control and antagonize viral infections. pedv infection does not elicit a robust ifn response, and some of the mechanisms used by the virus to counteract the host innate immune response have been unraveled. pedv evades the host innate immune response by two main strategies including: 1) encoding ifn antagonists to disrupt innate immune pathway, and 2) hiding its viral rna to avoid the exposure of viral rna to immune sensors. this review highlights the immune evasion mechanisms employed by pedv, which provides insights for the better understanding of pedv-host interactions and developing effective vaccines and antivirals against covs. porcine epidemic diarrhea virus (pedv) is the etiological agent of porcine epidemic diarrhea (ped) that causes an acute and highly contagious enteric disease of swine characterized by vomiting, diarrhea, dehydration, and anorexia in pigs of all ages, especially resulting in severe diarrhea and high mortality rate in piglets. in serious cases, outbreak of ped even leads to a mortality rate of 100% in pigs [1] [2] [3] . the causative agent of ped was first described in the 1970s in england [4] . in 1976, an unrecognized enteric disease was reported in several european countries [5] . the viral diarrhea was collectively designated "ped" in 1982 [6] . endemic ped had been described in both developed and developing countries, but with a low impact on the swine industry until 2010. in 2010, outbreaks of ped caused by highly pathogenic variant pedv strains occurred in china, and this was immediately reported in other asian counties, causing up to 100% mortality in suckling piglets [1, [7] [8] [9] . in april 2013, pedv entered the united states (us) for the first time and the virulent strains spread rapidly across the us [10]. apart from almost 100% mortality rate in suckling piglets, pedv infection also damaged the growth performance of finishing pigs [11] . the highly pathogenic strains of pedv spread worldwide, resulting in serious problems to the swine industry and substantial economic losses [7, 10, 12, 13] . vaccination used to be the main strategy to prevent and control the rate of pedv infection [14] , however, the current available pedv vaccines cannot provide complete protection for the pigs affected by the highly pathogenic strains. the optimal vaccines should induce efficient maternal antibodies in sows that could be transmitted to the offspring and protect neonatal suckling piglets from pedv. the vaccination 1 (s1) protein [34] . apart from apn, pedv is able to bind to sialic acids [36] . it remains unknown whether pedv uses sugar coreceptors during viral infection [34, 36] . pedv infects multiple cell lines from different species including bat and primate (human and non-human) in vitro. the ability of pedv to infect the cells of different species indicates that the virus utilizes the evolutionarily conserved cell components as receptors, thereby enhancing the potential for cross-species and potentially, zoonotic transmission [37, 38] . the highly pathogenic variant strains of pedv were identified in 2010 and caused a high morbidity of up to 100% in piglets, and since then, these strains become dominant, leading to most of the acute outbreaks of ped worldwide [1, 7, 8] . the high virulence of these strains is critically associated with the immune evasion mechanisms employed by the virus. pedv has evolved different strategies to delicately manipulate and damage the host innate immune system for their multiplication. clarification of these mechanisms is critical for understanding the host range, tropisms, pathogenesis, and for developing effective vaccines and antiviral drugs to curb the spread of pedv in pigs. in this review, we provide an overview of different mechanisms used by pedv to evade host innate immune responses. pedv is an enveloped, positive-sense, single-stranded rna virus. the pedv genome constitution represents a standard cov arrangement. the viral genome is approximately 28 kb in length, containing a 5 terminal cap, a 3 poly (a) tail, as well as seven open reading frames (orfs), including the orf1a, orf1b, s, orf3, e, m, and n genes ( figure 1 ) [39] [40] [41] . the n terminal orf1a and orf1b encode two large replicase polyprotein precursors (pp1a and pp1ab), which are subsequently processed into 16 nonstructural proteins (nsp1 to 16). orf1a encodes pp1a which is cleaved by viral proteases into 11 nonstructural proteins (nsp1-nsp11). the orf1b generates five additional nonstructural proteins (nsp12-16) that are proteolytically cleaved by the viral proteases from pp1ab [42] . nsp3 contains two papain-like protease domains (plp1 and plp2 or pl pro ) that cleave the nsp1-4 region of the replicase polyprotein at three sites. nsp5, a chymotrypsin-like enzyme also known as 3c-like protease, cleaves the polyprotein at remaining cleavage sites [43] . the c terminus of viral genome contains five orfs, encoding four structure proteins (spike protein (s), small envelope glycoprotein (e), membrane glycoprotein (m), and nucleocapsid protein (n)), as well as a hypothetical accessory protein orf3 [44] [45] [46] . the 16 nsps, together with the n protein, and several host proteins, form a large replication and transcription complex (rtc) that engages in the minus-strand rna synthesis, using viral genomic rna. these nsps play important roles in virion structure modification and the replication and transcription of pedv [47] . the c terminus of viral genome contains five orfs, encoding four structure proteins (spike protein (s), small envelope glycoprotein (e), membrane glycoprotein (m), and nucleocapsid protein (n)), as well as a hypothetical accessory protein orf3 [44] [45] [46] . the 16 nsps, together with the n protein, and several host proteins, form a large replication and transcription complex (rtc) that engages in the minus-strand rna synthesis, using viral genomic rna. these nsps play important roles in virion structure modification and the replication and transcription of pedv [47] . the viral proteins of pedv perform different biological functions during viral entry, replication cycle and propagation (table 1) . pedv s protein, a type i membrane glycoprotein protein located on the envelope of the virus, consists of an n-terminal signal peptide, a large extracellular region, a single transmembrane domain, as well as a short cytoplasmic tail [48, 49] . the ectodomain of s protein comprises s1 and s2 subunits. the n-terminal s1 region, containing n-and c-terminal domains (s1-ntd and s1-ctd), is mainly responsible for receptor binding [50] . the c-terminal membrane-anchored s2 region is mainly involved in triggering the fusion of the viral envelope with host cell membranes [48, 49] . the interaction of the cov s protein with its host cell surface receptor is a key determinant for host tropism. the s1-ctd of most known members of α-cov genus, including pedv, interacts with aminopeptidase n (apn) to entry into the target cell [32, 36, [51] [52] [53] [54] . in addition, s protein contains the epitopes that are the major targets of the neutralizing antibody. n protein is the most abundant viral protein during the early phase of infection in cov-infected cells [55] . similar to the n proteins of other covs, the pedv n protein has multiple functions, such as acting as a structural protein that forms nucleocapsid with viral genomic rna, playing important roles in viral replication, transcription, and assembly [56, 57] . the expression of the n protein in intestinal epithelial cells extends the s-phase of cell cycle, causes endoplasmic reticulum (er) stress, and upregulates interleukin-8 expression [44] . moreover, the n proteins of several α-covs and β-covs, including pedv, pdcov, sars-cov, and mouse hepatitis virus (mhv), have been identified as innate immunity antagonists [58] [59] [60] [61] . however, the involved antagonistic mechanisms are particularly different. pedv m protein participates in virion assembly and virus budding through collaboration with other viral proteins, and engages in the induction of neutralizing antibodies against pedv [62, 63] . pedv m protein is distributed throughout the cytoplasm. it induces the cell growth retardation in intestinal epithelial cells (iec) and arrests the cells in s-phase [64] . in addition, pedv m protein is identified as an interferon (ifn) antagonist with an unrecognized mechanism [65] . pedv e protein is important for the virus packaging and budding [66] . it is predominantly localized in the er, having no effect on cell growth, cell cycle and cyclin a expression in iec. however, it causes er stress and activates the nuclear factor-κb pathogens 2020, 9, 367 5 of 27 (nf-κb) pathway which is responsible for the up-regulation of il-8 and the anti-apoptotic protein bcl-2 expression [67] . orf3 has been predicted to possess multiple transmembrane domains [68] , while it is predominantly distributed in the cytoplasm [69] . orf3 also detains cells at s-phase, facilitating vesicle formation, and thus promoting pedv multiplication [69] . a recent study suggests that orf3 interacts with s protein during pedv assembly and consequently benefits viral replication [70] . cov nsps play multiple roles in the synthesis or processing of viral rna, or in virus-host interactions aiming to create an optimal environment for virus replication, such as facilitating viral entry, viral gene expression, rna synthesis, and virion release. nsp1 is a n-terminal cleavage product of orf1a polyprotein [71] , a 9-kda protein, that exists only in α-covs and β-covs [72] . the nsp1 of α-covs is not very similar to β-covs nsp1 with regard to sequence homology and size [73, 74] . based on the sequence alignment analysis of the genomes of different covs, the viral nsp1 can be regarded as a genus-specific marker [75] . moreover, β-covs nsp1 has been widely reported to inhibit host protein expression. however, the biological functions of α-covs nsp1 remain largely unknown. despite the lack of overall sequence similarity, the nsp1 of different covs shares a similar function to interfere with host protein expression [76] . these studies suggest the importance of nsp1 in the life cycle of different lineages of covs. it is shown that tgev nsp1 inhibits host gene expression and is critical for viral virulence [77] . pedv nsp1 induces the degradation of cbp and nf-κb to abate ifn response [78] , but the detailed mechanisms remain unclear. the sizes and amino acid sequence identity of nsp 2 are variable among different covs [76] . nsp2 of mhv and sars-cov are involved in viral rna synthesis [79] . pedv nsp2 has unknown functions in replication, and may implicate the virus-host interactions and virulence. nsp3 is the largest nsp protein, containing two papain-like protease (plp1 and plp2) domains, of which pedv plp2 acts as a viral deubiquitinase (dub), to negatively regulate type i ifn signaling [80] . covs plps domains exhibit multiple functions, serving as a viral protease, dub, as well as an ifn antagonist [81] . covs, like other positive-stranded rna viruses, induce membranous rearrangements of varying morphologies that are essential for rtcs anchoring [82, 83] . the cov-induced replicative structures consist of double-membrane vesicles (dmvs) and convoluted membranes (cms), which form a large reticulovesicular network that are critical for viral replication and transcription [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] . among the cov nsps, nsp3, nsp4, and nsp6 include the hydrophobic transmembrane domains engaging in anchoring the viral rna synthesis components to the membranes [94] . for mers-cov and sars-cov, co-expression of nsp3 and nsp4 is required to induce dmvs [95] . sars-cov nsp6 has membrane proliferation ability as well, which also contributes to dmvs formation [96] . the structure and functions of α-cov nsp3 are largely unknown [97] . nsp4 is also a marker for cov-induced membrane structures; some results indicate that the nsp4-10 of pp1a act as a large complex through multidomain structure or scaffold during viral rna replication progress, before its cleavage into individual products [98] . covs nsp5 encodes a 3c-like proteinase (3cl pro ). the polyproteins pp1a and pp1ab are processed into individual elements of replicase by 3c-like protease and plps [99] . moreover, pedv nsp5 plays a crucial role in virus replication and also blocks host innate immune responses [100] . crystallographic or nuclear magnetic resonance structures have shown that nsp3, nsp5, nsp7, nsp8, nsp9, and nsp10 have the plprob and the adp-ribose 1 -phosphatase (adrp) activity [101] [102] [103] [104] [105] [106] [107] . the crystal structure of sars-cov nsp9 suggests that nsp9 is dimeric and it is able to bind to single-stranded rna [108] . the crystal structure of sars cov nsp10 protein suggests that nsp10 is a zinc-finger protein, which is existent exclusively in covs so far [107] . moreover, nsp7-10 have rna binding activity and nsp12 encodes a single rna-dependent rna polymerase (rdrp). the biochemical characterization and crystallization of sars cov nsp7 and nsp8 manifests that eight copies of nsp8 and eight copies of nsp7 form a supercomplex [106] . the complex is supportive for nucleic acid binding and may be associated with the processivity of viral rdrp [102, 106] . recently, structural studies have described that the sars-cov nsp12 polymerase binds to the nsp7 and nsp8 complex [109] that may increase the polymerase activity of nsp12 rdrp [110] . cov nsp13, a ntpase/helicase, is also determined to play essential roles in viral replication [111] . cov nsp14 is a multifunctional protein with 3 -5 pathogens 2020, 9, 367 6 of 27 exoribonuclease activity and n-7-methyltransferase [mtase] activity [112, 113] . nsp14 catalyzes the n7-methylation of gppp-rna to form a cap-0 structure. cov nsp15 encodes an endoribonuclease (endou), performing functions through a hexamer in many covs [114] [115] [116] . the endoribonuclease activity of nsp15 is not essential for cov replication [117, 118] . for covs, the 5 end of the viral genomic rna and subgenomic mrna (sgmrna) is supposed to have cap structures: an n-7 methylated guanosine nucleoside (m7gpppn) (cap 0) and a methyl group at the 2 -o-ribose position (cap 1) of the first nucleotide [119] . these cap structures enhance the initiation of translation of viral proteins, protect viral mrnas against cellular 5 -3 -exoribonuclease and limit the recognition of viral rna by host innate system [120, 121] . nsp13 is proposed to catalyze the first step of the 5 -capping reaction of viral rnas [122] . the methylation of the two sites in the 5 cap are catalyzed by three nsps; nsp14 (the n-7-mtase), nsp16 (the 2 -o-methyltransferase), and nsp10 [112, [123] [124] [125] [126] . in addition, the 3 -5 exoribonuclease activity of nsp14 is involved in a replicative mismatch repair system during rna synthesis, which improves the replication fidelity of cov [42] . although these nsps have been demonstrated to play essential roles in viral replication, transcription and/or post-translational polyprotein processing [127] , the nsp12-16 of pedv and other covs are poorly characterized to date, except for sars-cov. nsp7 is an ifn antagonist; nsp8 inhibits type iii ifn response; nsp9 is involved in nucleic acid binding; nsp10 enhances the inhibitory effect of nsp16 on ifn-β production. [65, [131] [132] [133] nsp12 encoding a rdrp; viral replication [134] nsp13 a ntpase/helicase that is essential for viral replication [111] nsp14 n-7-mtase; catalyzing n7-methylation of gppp-rna to form a cap-0 structure; 3 -5 exoribonuclease activity involves in a replicative mismatch repair system during rna synthesis; ifn antagonist [42, 65, 112, 113 host cells generally defend against virus infection by mounting an innate antiviral immune response to prevent the spread of the infection and aid in initiating an adaptive immune response which eventually removes the viruses from host. therefore, the first barrier to restrain viral infections is the host innate immune system, which is related to multiple proteins and mechanisms, including ifns, inflammatory cytokine, apoptosis, autophagy, and so on. the activation of type i ifn responses is composed of three stages: (1) recognition of pathogen-associated molecular pattern (pamp) by prrs; (2) secretion of type i ifns through paracrine or autocrine pathways; and (3) expression of numerous antiviral ifn-stimulated genes (isgs) which bring the host into the antiviral state [136] . at least three important prrs have been identified in recognition of viral nucleic acids, including retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) (detection of viral rna in the cytoplasm) [137] , the membrane-bound toll-like receptors (tlrs) (recognition of viral rna or dna in the endosome) [138] , as well as a structurally unrelated group of viral dna sensors (e.g., cgas (cyclic gmp-amp synthase) and ifi16) localized in the host cytoplasm and/or nucleus [139] . in the cytosol, the formation of specific secondary structure of viral rna is closely related to viral rna delivery and replication. these molecular signatures are detected by rlrs including rig-i (also known as ddx58), melanoma differentiation-associated gene 5 (mda5), and laboratory of genetics and physiology-2 (lgp2) [140, 141] . prrs recognize pamps, inducing an intracellular signaling cascade, thus leading to the activation of transcription factors such as irfs and nf-κb, which in turn induce the production of ifns [142] . both rig-i and mda5 have two n-terminal card domains to interact with the card domains of the downstream adaptor proteins, and a dead/h-box rna helicase domain for rna binding. however, lgp2 does not have the n-terminalcard domains, and the involved functions remain unclear [143] [144] [145] . dsrna, as a specific secondary structure of viral rna, can be sensed by rig-i/mda5 to induce ifn-α/β production through the cascade activation of the rlr pathway [146] [147] [148] . activated rig-i/mda5 forms homo-oligomers and recruits the adaptor mitochondrial antiviral signaling (mavs) to induce the formation of signaling complex of mavs with other proteins on mitochondria. tnf receptor-associated factor 6 (traf6) associates with tnfr1-associated death domain protein (tradd), tripartite motif 14 (trim14), and pyruvate carboxylase (pc), resulting in the activation of iκb kinases (ikks). ikks (ikkα, ikkβ and ikkγ) phosphorylate nf-κb inhibitor (iκb), leading to the ubiquitination of iκb and its subsequent degradation. nf-κb is then activated and translocated into the nucleus to turn on the expression of proinflammatory and type i ifn. mavs also recruits and activates tbk1/ikkε by trafs that are pre-associated with tbk1/ikkε, via direct interaction between the sdd domain of tbk1/ikkε and the coiled-coil domain of trafs [149] . activated tbk1/ikkε phosphorylates ifn regulatory factors (irf3/irf7), that further dimerize and import into the nucleus to promote type i ifn production. on the other hand, mavs interacts with mita (also known as sting), that is located in mitochondria and the endoplasmic reticulum (er) membrane. mita interacts with the trap complex, which may be involved in recruiting tbk1 and ikkε to phosphorylate irf3. ubiquitination and deubiquitination are decisive in the regulation of rlr pathways activation [150] . upon binding to viral dsrna, rig-i and mda5 undergo conformational changes and release the n-terminal tandem card domains [151] [152] [153] . the card domains of rig-i are modified by lysine 63 (k63) polyubiquitin chains through the ubiquitin ligases trim25, rnf135, and riplet. this modification is crucial for rig-i to recruit mavs [154, 155] . in addition to the ubiquitination of rlrs, the polyubiquitylation of traf3 and traf6 also play an important role in the regulation of innate immune signaling by activation of tbk1 and ikks, respectively. the k63 polyubiquitin chains can be removed by dubs such as the tumor suppressor protein cyld, duba and a20, providing a mechanism to downregulate immune responses [156] . ubiquitination and deubiquitination are in a dynamic equilibrium to maintain immune homeostasis. type i ifns are secreted by secretory cells and peripheral cells through self-secretion or paracrine secretion manners. extracellular type i ifns bind to a heterodimeric complex composed of subunits of ifn-α receptors 1 (ifnar1) and 2 (ifnar2) located on the cell surface, which activates the tyrosine kinase 2 (tyk2)/janus kinase 1 (jak1) signal transducer. tyk2/jak1 subsequently induces the phosphorylation of transcription factors stat1 and stat2, which dimerize and in turn recruit ifn-regulatory factor 9 (irf9), to form stat1-stat2-irf9 trimerized complex (isgf3). this complex then translocates to the nucleus, where it binds to the ifn-stimulated response elements (isre motif; conserved sequence is tttcnntttc) [157] . the binding of isgf3 to isre finally triggers expression of ifn-stimulated genes (isgs) that directly or indirectly exert antiviral effects in host cells [157] . three types of ifns (types i, ii and iii) have been identified. type i and type ii ifns have been widely reported. type ii ifns only contain ifn-γ. ifn-γ is produced by natural killer (nk) cells and activated cd4 + and cd8 + t cells in response to the cytokines such as interleukin-12 (il-12) and il-18 [158] . ifn-γ binds to the type ii ifn receptor composed of two subunits, ifngr1 and ifngr2. ifngr1 and ifngr2 induce the formation of stat1-stat1 homodimers. stat1-stat1 homodimers translocate to the nucleus and bind to the promoter of the ifn-γ-activation site (gas) elements, to initiate the transcription of ifn-γ-regulated genes [159] . type iii ifns have been explored in recent years, to unravel the underlying mechanisms that manipulate host innate immune responses. type iii ifns in humans contains ifn-λ1 (interleukin 29 ), ifn-λ2 (il-28a), ifn-λ3 (il-28b), and ifn-λ4 [160] [161] [162] . their expression profiles, signaling pathways, and gene expression programs resemble those of type i ifns. the production of type i and iii ifns are both initiated through the recognition of pamps or damage associated molecular patterns (damps) by prrs [163] . despite the fact that the same transcriptional factors are required for the activation of promoters of type i and iii ifns, the nf-κb pathway is a pivotal regulator in ifn-λ production, whereas the irfs pathway dominates type i ifns expression. the promoter of ifn-λ1 includes more nf-κb binding sites compared with in the ifn-β promoter [164] . the signal transduction of type iii ifns depends on the ifn-λ-specific receptor, il-28ra chain and il-10r2 chain [161, 165] . synthetic ifn-λ binds to il-28rα and induces a conformational change within the receptor subunits, that triggers the activation of the receptor-associated tyrosine kinases (tyk2 and jak1), which then phosphorylate stat1 and stat2. stat1 and stat2 are heterodimerized and interact with irf9 to form the isgf3 transcription complex that binds to isre in the promoters of isgs, to induce the expression of hundreds of proteins with antiviral functions [166] . induction of ifn-α/β is the most rapid and effective mechanism for hosts to initiate antiviral innate immune responses. sars-cov, mhv and many other covs are sensitive to ifns. a great number of viral dsrnas intermediates are generated during covs infection that contribute to ifn production, but these covs remain highly pathogenic. as a matter of fact, covs have developed a set of elaborate mechanisms to evade or inhibit the host antiviral innate immune response during virus evolution [134, 167] . the evasive strategies utilized by pedv are classified into four major types: (1) inhibition of rlrs-mediated ifn production pathways, (2) inhibition of the activation of transcription factors responsible for ifn induction, (3) disruption of the signal cascades induced by ifn, and (4) hiding its viral rna to avoid the exposure of viral rna to immune sensors. in the past decade, accumulating evidence demonstrates that pedv n protein, nsp1, plp2, nsp5, nsp15, and nsp16 antagonize type i ifn or type iii ifns production [58, 65, 78, 80, 100, 132, 135, 168] . this explains why only weak ifns' and cytokines' expression is detected in pedv-infected cells [169, 170] . n protein, as an abundantly produced structural protein within cov-infected cells, has multiple functions, including virus replication, transcription, and assembly [56, 134] . pedv n protein has been identified as an ifn antagonist that blocks the expression of ifn-β and isgs by suppression of the pathogens 2020, 9, 367 9 of 27 irf3 and nf-κb activities [58] . pedv n protein inhibits the activation of the ifn-β promoter induced by tbk1 and its upstream rig-i, mda-5, visa, and traf3, while not affecting the activation of the ifn-β promoter driven by irf3. further experiments confirm that n directly interacts with tbk1 to obstruct the association between tbk1 and irf3, which inhibits tbk1-induced irf3 phosphorylation and ifn-β production [58] . moreover, the effect of pedv n protein on type iii ifn production has also been evaluated [168] . n protein inhibits polyinosinic-polycytidylic acid (poly(i:c))-induced ifn-λ3 production by blocking the nuclear translocation of nf-κb, but does not antagonize the type i or type ii ifn expression induced by poly(i:c) in ipec-j2 cells [168] . recent studies show that sars-cov n protein inhibits type i ifn production through suppressing trim25-mediated rig-i ubiquitination [171] . the mers-cov n protein also blocks ifn production by interacting with trim25 [171] . in addition, both mhv and sars-cov n proteins perturb the function of cellular protein activator of protein kinase r (pact), which can bind to rig-i and mda5 to activate ifn production, and thus antagonize type-i ifn signaling [61] . these results indicate the important function of the covs n protein in modulating host innate immune response. whether pedv n protein targets trim25 or pact should be investigated. although several studies have been performed to understand the pathogenicity of pedv, there remains limited information about the interaction between viral proteins and host cell factors during viral infection. cov n protein is a vital viral protein involved in virus replication. current researches have indicated that n protein interacts with many host proteins, such as hcypa [172] , proteasome subunit p42 [173] , smad3 [174] , hnrnp-a1 [175] , and the chemokine cxcl16 [176] . in the host cells, a large number of host proteins reveal various functions. however, for the virus, the genome only encodes several limited viral proteins. therefore, these viral proteins have to be multifunctional, which is pivotal to virus replication and existence. pedv nsp1 is the n-terminal cleavage product from polyproteins pp1a and pp1a/b processed by nsp3 and nsp5 [177] and is about 110 amino acids in length [74, 178] . nsp1 of many α-cov and β-cov exhibits both functional conservation and mechanistic diversity in suppressing host gene expression and ifn signaling. for sars-cov, nsp1 triggers the decay and cleavage of host mrna and inhibits host protein translation, subsequently inhibiting type i ifn production [179, 180] . sars-cov nsp1 also blocks the expression of ifn-inducible genes, by restraining the signal transduction during virus infection [181, 182] . the tgev nsp1 considerably suppresses host protein expression during viral infection [77] . structural studies show that the core structure of pedv nsp1 is highly similar to those of sars-cov nsp1 and tgev nsp1 [183] . pedv nsp1 inhibits host gene expression and three motifs (amino acids 67 to 71, 78 to 85, and 103 to 110) form a stable functional region for inhibition of host protein synthesis, differing considerably from sars-cov nsp1 [183] . pedv nsp1 has been identified as an ifn antagonist, which constrains poly (i:c)-induced ifn-β promoter activity [65] . nsp1 significantly inhibits the activation of ifn-β promoter triggered by irf3, whereas it does not inhibit irf3 phosphorylation and its nuclear translocation. nsp1 interrupts the association of irf3 with creb-binding protein (cbp), by promoting cbp degradation in the nucleus via the proteasome-dependent pathway. cbp/p300, the transcription co-activator camp responsive element binding protein (creb), forms a complex with the activated irf3 in nucleus. the irf3-cbp/p300 complex binds to the positive regulatory domain (prd) regions of the ifn-β promoter, to assemble the enhanceosome with nf-κb and other factors, which ultimately turn on the transcription of type i ifn genes [184] [185] [186] . therefore, pedv nsp1 blocks type i ifn production in the nucleus. activated nf-κb induces the production of type i ifns and proinflammatory cytokines and is important for inhibiting viral infection. pedv nsp1 has been shown to interfere with the nf-κb activity [78] and is the most potent suppressor of proinflammatory cytokines at early infection. it inhibits the phosphorylation and degradation of iκbα, and blocks p65 nuclear translocation, leading to the suppression of both ifn and the early production of pro-inflammatory cytokines [78] . moreover, pedv inhibits type iii ifn production and nsp1, nsp3, nsp5, nsp8, nsp14, nsp15, nsp16, orf3, e, m, and n are identified as type iii ifn antagonists. among these antagonists, nsp1 is the most potent suppressor [130] . pedv nsp1 blocks the nuclear translocation of irf1 and decreases the amounts of peroxisomes and then suppresses irf1-mediated type iii ifns. the conserved residues of pedv nsp1 protein are crucial for ifn suppression [130] . multiple effects of nsp1 on modulating innate immune response during pedv infection suggest the vital role of nsp1 in the pedv replication cycle. the antiviral innate immune signaling pathways are regulated by several posttranslational modifications (ptms), such as phosphorylation, ubiquitination, glycosylation, neddylation and sumoylation [187] , of which ubiquitination is a critical modification to modulate the stability and activity of prrs and other components of innate immune signaling pathways. during viral infection, a reciprocatory action (occurrence of ubiquitination and deubiquitination) helps maintain the homeostasis of host immune responses. hence, deubiquitinases (dubs) are indispensable in the regulation of virus-induced type i ifn signaling [188] . many host dubs have been reported engaging in the regulation of innate immune signaling pathways [189] [190] [191] . in recent years, a variety of viral dubs have been discovered to target key components of type i ifn pathway during various rna virus infections. for example, foot-and-mouth disease virus leader proteinase (fmdv lb pro ) [192] , and porcine reproductive and respiratory syndrome virus nsp2 (prrsv nsp2) possess ubiquitin-deconjugating activity to deubiquitinate key host components [193, 194] . to counteract host antiviral response, covs likely take advantage of dub activity to break host innate immunity. indeed, the plps of mouse hepatitis virus a59 (mhv-a59) [195] , sars [196] , and human cov nl63 have dub activity and antagonize ifn induction [197] . pedv plp2 has been reported as having a deubiquitinase activity as well, and it can be co-immunoprecipitated by rig-i and sting. as mentioned above, fmdv lb pro , mhv plp2 and sars plps all counteract host innate immune response through blocking the ubiquitination of the components of rlrs pathways. similarly, pedv plp2 removes the ubiquitinated conjugates from rig-i and sting by its dub activity, to negatively regulate type i ifn production. pedv plp2 probably interacts with rig-i and sting, which prevents the activation of rig-i and sting by hindering the recruitment of downstream signaling molecules. as expected, the interference with the ubiquitination of rig-i and sting by plp2 clearly benefits pedv replication [80] . pedv nsp3 contains two core domains of plps (plpl and plp2). it is determined that pedv plp2, but not plp1, inhibits the ifn-β promoter activation in hek293t cells. the dub activity of plp2 is highly dependent on its catalytic activity. three catalytically inactive mutants of pedv plp2 (c1729a, h1888a and d1901a) are defective in the deubiquitination of its targets and fail to impair virus-induced ifn-β production. sars-cov plp2 interacts with mdm2 (mouse double minute 2 homolog) to deubiquitinate and stabilize mdm2, approving the degradation of p53 and the suppression of ifn signaling [198] . pedv infection degrades p53 by upregulation of mdm2 expression [198] . pedv plp2 may be responsible for targeting the p53 pathway and inhibiting p53-dependent apoptosis, leading to immune evasion. a recent study determined that tgev pl1 inhibits the ifn-β expression and interferes with the rig-1and sting-mediated signaling pathway through a viral dub activity [195] . it suggests that different viral proteins are involved in the deubiquitination of host proteins for different covs. however, these studies offer a probability to design a common therapeutic against different viral dubs to reduce the replication and pathogenesis of covs. therefore, further studies are required to understand more about the substrate specificity of these viral dubs and clarify the precise functions of cov protease/dub activity. notably, 3c pro is a critical ifn antagonistic protein identified in multiple different families of viruses. the 3c pro of picornaviruses, including fmdv [199] , hepatitis a virus (hav) [200] , enteroviruses (ev71, ev-d68) and coxsackieviruses (cvb3, cv-a16, cv-a6) [201] [202] [203] [204] , antagonize innate immune signaling by targeting the critical components of the ifn pathways for proteolysis. a newly emerged picornavirus, seneca valley virus (svv), has also evolved an effective mechanism to escape host antiviral innate immune using its 3c pro . moreover, 3c pro cleaves the signaling components (mavs, trif, and tank) of type i ifn pathway and induces the degradation of the transcription factors irf3 and irf7 to constrain host antiviral response [205, 206] . cov nsp5 is called 3c-like protease (3cl pro ), that resembles the 3c pro of other rna viruses. for cov, the polyprotein precursors (pp1a and pp1b) are mainly processed to generate mature nonstructural proteins by 3cl pro . to date, the 3cl pro of covs, including pedv and pdcov, have been confirmed to antagonize type i ifn production by the cleavage of nf-κb essential modulator (nemo) and stat2 [100, 207, 208] . nemo is essential for rna virus-induced activation of nf-κb, irf3, and irf7 [209] . nemo is required for mavs-induced ikkα/β activation and is also crucial for the activation of tbk1/ikkε [149] . to establish successful infections, pedv targets nemo to subvert host innate immune responses. pedv nsp5 significantly inhibits sendai virus (sev)-induced ifn-β synthesis and the process depends on its protease activity [100] . further experiments show that pedv nsp5 inhibits rig-i/mda5 signaling and targets the upstream of tbk1. the cleavage of nemo by nsp5 is identified as responsible for this inhibitive effect. the pedv nsp5-mediated cleavage of nemo efficiently blocks nemo-mediated downstream signaling. the cleavage site within nemo that is grasped by nsp5 has been determined. of these reported immune evasion strategies employed by covs, the cleavage of innate immune adaptors is a particularly effective manner to disrupt antiviral responses. nsp5 is essential for the life cycle of pedv and other covs [210, 211] . it is a potential target for the development of anti-coronaviral therapeutics. although pedv nsp5 does not target stat2 mediated type i ifn signaling pathway, pedv nsp7 has been reported to inhibit the stat1 and stat2 induced activation of isre [212] . nsp7 competes with karyopherin α (kpna1), which is an adaptor mediating nuclear translocation of isgf3, in combination with stat1, to block isgf3 nuclear transport. however, the expression and phosphorylation of stat1 and stat2 are not affected by pedv nsp7. in fact, pedv infection degrades stat1, leading to the inhibition of ifn signaling [170] . therefore, other pedv encoded proteins likely target ifns mediated signaling. covs belong to rna viruses, which produce several rna species, such as dsrna intermediates and rna with a 5 -triphosphate during replication. these rna intermediates are potent stimulators of prrs and are associated with the organelles of viral rna replication, dmvs [213, 214] . dmvs formed from membranous rearrangements seem to sequester the replication intermediates using membrane-bound vesicles or invaginations to keep away from prrs. therefore, the form of dmvs may be a strategy for pedv to escape innate immune recognition in the cytosol (figure 2 ). however, whether dmvs alone are sufficient to shield rna from prrs remains unknown. besides, the replication organelles, the endoribonuclease activity and viral 5 end rna capping/protection mechanisms are also the critical ways of avoiding rna recognition or protecting it from degradation [132, 135] . (1) pedv nsp15 cov nsp15 has endou catalytic activity that was initially thought to play a vital role in virus replication. however, the catalytic-defective endou of mhv shows only a subtle defect in viral replication compared to wt virus in fibroblasts [215] . similar results are found for the nsp15 mutants of sars-cov and hcov-229e [216] . these findings suggest that the endou activity of nsp15 is not required for rna synthesis. recently, nsp15 has been demonstrated to act as a new ifn antagonist of covs [117, 118, 217] . recent reports indicate that covs' endou activity is essential for prevention of rna recognition by mda5, protein kinase r (pkr), and oas/rnase l system [118] . pkr and oas/rnase l recognize and destroy foreign rna in the cytosol to defend viral infections. to counteract the function of pkr and oas/rnase l, the virus hides or modifies its viral rna, to avoid the exposure of viral rna to these molecules. in all covs, the endou catalytic domain in nsp15 is highly conserved. pedv endou activity has been indicated as having an antagonistic effect on ifn signaling [135] . the endou activity of pedv nsp15 not only inhibits the type i ifn response in porcine macrophages, but also antagonizes the type iii ifn response in porcine epithelial cells. the replication of endou-mutant pedv (icpedv-enumt) is considerably impaired in porcine epithelial cells compared to the wild type pedv (icpedv-wt). the icpedv-enumt clearly induces early and robust type i and type iii ifns production, as well as isgs' expression compared with that induced by icpedv-wt. the endou-deficient pedv infected animals also show reduced viral shedding and mortality. these results indicate that the endou activity of pedv nsp15 plays a vital role in evading host antiviral innate immunity (figure 2 ) [135] . (2) pedv nsp16 cov nsp16 is a 2 -o-methyltransferase (2 -o-mtase). to evade recognition by the host immune sensors, many covs encode methyltransferases involved in the capping of viral rna. this modification makes the viral rna indistinguishable with host cell mrna, which is important to avoid the recognition of viral rna by mda5 ( figure 2 ) [121, 218] . methylation of the two sites in the 5 cap is catalyzed by three nsps, including nsp14 (the n-7-mtase), nsp16 (the 2 -o-mtase), and nsp10 [112, [123] [124] [125] [126] . for example, sars-cov nsp16 acts as a 2 -o-mtase to prevent innate immune recognition and promote viral proliferation [113, 121, 123] . pedv nsp14 and nsp16 have been identified as the viral ifn antagonists [65, 132] . the overexpression of nsp14 or nsp16 remarkably inhibits ifn-β production, but nsp16 appears to play a more important role in innate immunity regulation than nsp14 [132] . nsp16 is a highly conserved methyltransferase which contains an invariant kdke motif within the methyltransferase core [219] . this kdke motif is required to mediate its activity. notably, the mutation of any of the kdke active sit has been shown to abolish the 2 -o-mtase activity [123] . pedv nsp16 kdke motif plays a critical role in the inhibition of type i ifn production, suggesting the important role of the 2 -o-mtase in pedv-mediated immune evasion. pedv nsp16 negatively regulates rlr-mediated signal pathway activation, and inhibits the expression of the ifn-stimulated ifit family members (ifit1, ifit2, ifit3), which in turn promotes pedv replication. taken together, these results demonstrate that pedv nsp16 negatively regulates cellular antiviral response to promote viral replication [132] . screening inhibitors targeting the 2 -o-mtase of nsp16 might be a prominent strategy to inhibit cov infections and develop antivirals for treatment of the diseases caused by covs. additionally, covs nsp14 also includes the 3 -to-5 exoribonuclease (exon) activity [113] . a mutation of tgev nsp14 exon generates lower levels of dsrna than wildtype tgev and thus triggers a reduced antiviral response [220] . nsp14 exon activity is also critical for the resistance of host innate immune response in mhv-infected cells [221] . the role of nsp exon activity of pedv in counteracting host antiviral response should be investigated to uncover more functions of pedv nsps. these data suggest that pedv has evolved multiple evasive mechanisms to circumvent viral rna recognition or prevent rna degradation to establish a successful infection in the host. activity is also critical for the resistance of host innate immune response in mhv-infected cells [221] . the role of nsp exon activity of pedv in counteracting host antiviral response should be investigated to uncover more functions of pedv nsps. these data suggest that pedv has evolved multiple evasive mechanisms to circumvent viral rna recognition or prevent rna degradation to establish a successful infection in the host. heat shock protein 27 (hsp27) belongs to the small heat shock proteins family, which has been identified as a multifunctional protein involved in cytoskeletal stability, proinflammatory processes, and the inhibition of apoptosis [222, 223] . several hsps have been reported to be implicated in pedv infection in vitro and in vivo [224, 225] . indeed, the infection of many viruses up-regulates hsp27 expression by different mechanisms to delay cellular apoptosis, then supplies sufficient time for viral replication [226, 227] . however, pedv infection significantly induces the decreased expression of hsp27 in vero cells [225] and marc-145 cells [228] . hsp27 activates the phosphorylation of nf-κb, and thus promotes the mrna expression of ifn-β in marc-145 cells. as hsp27 is an upstream regulator of antiviral immune signaling, overexpression of hsp27 significantly inhibits the pedv replication. pedv has developed a strategy via mediating the suppression of hsp27 production to escape from host innate immune response [228] . hsp70, the most conserved hsp, is also important for the multiplication of several covs. the recruitment of hsp70 is thought to be a viral survival strategy for several viruses in their hosts [229] . the relationship between hsp70 and pedv should be exploited further in future. viral infection triggers host immune response to induce ifns' and inflammatory cytokines' production. released ifns elicit the expression of numerous isgs which limit viral replication in the infected cells. however, the release of excessive amounts of ifns and inflammatory cytokines will lead to autoimmune and auto-inflammatory diseases. the concomitant uncontrolled apoptosis is also one outcome that is harmful to the host. to maintain the reaction in a proper balance, hosts have evolved a series of effective mechanisms to control the antiviral innate immune response [230] . in contrast, viruses often break this balance, causing improper apoptosis reaction, which benefits viral replication. pedv infects various host cells including vero, pk-15 and marc-145 and cause obvious cytopathic effects. pedv-induced apoptosis of the infected cell has been demonstrated both in vitro and in vivo [231] . apoptosis is induced through the activation of apoptotic caspases, including caspase-2, -3, -6, -7, -8, -9, and -10 [232] . pedv infection results in obvious caspase-3 and caspase-8 activation, as well as the cleavage of apoptosis-inducing factor mitochondria-associated 1 (aifm1) and poly adp-ribose polymerase (parp), which leads to apoptotic nuclear fragmentation. pedv spike protein s1 significantly elicits host cell apoptosis, while the nsp1-16 and other structural proteins (m, n, e, s2, and orf3) have none or few effects on cell apoptosis. therefore, s1 protein is probably the critical protein mediating the apoptosis induced by pedv [128] . the multiple stages of cov replication cycle are closely associated with cellular membrane compartments, especially the endoplasmic reticulum (er). the shape and functions of er can be influenced by different physiological states and environmental conditions. when protein synthesis amounts surpass the folding capacity, the accumulation of a large amount of unfolded proteins in the er leads to er stress. consequently, cells manifest a corresponding biological reaction that is widely known as the unfolded protein response (upr) [233] . once the upr is induced, it alleviates the problems by host protein translation inhibition (by the transducer pkr-like er protein kinase (perk)-induced phosphorylation of eif2a), stimulating protein folding. if homeostasis cannot be re-established, apoptosis eventually is triggered. indeed, the activation of upr regulates a wide variety of signaling pathways, such as apoptosis, autophagy, mitogen-activated protein (map) kinase activation, and innate immune response [234] . furthermore, α-cov and β-cov may induce er stress in the infected cells [235] . pedv orf3, as the only accessory protein encoded by pedv, is thought to be related to virus production and virulence of pedv [68] . a series of studies suggest that orf3 plays multiple roles, in addition to acting as an ion channel during pedv replication. recent studies show that pedv orf3 consists of four transmembrane domains (tmds) and localizes in the cytoplasm in the aggregation manner [236] . orf3 is a transmembrane protein, and the confocal microscopy analysis indicates that the aggregated orf3 localizes in the er to induce the er stress associated with either apoptosis or autophagy. however, pedv orf3 induces the autophagy via driving conversion of lc3-i to lc3-ii, but not influencing the apoptosis. orf3-induced autophagy is dependent on er stress response. pedv orf3 triggers er stress response via the up-regulation of grp78 protein expression and the activation of the perk-eif2α signaling pathway. moreover, orf3 protein is identified as an ifn antagonist to block ifn response by an unknown mechanism in pedv-infected cells [65] . the functions of pedv orf3 should be further exploited. pedv has caused epidemic and endemic infections in pig populations in many countries and has become a major economic threat to the swine industry. previous studies have identified viral factors that target key signaling molecules in the rlrs' pathways, as well as viral factors that target the downstream signaling pathways responsible for isgs induction. of the 23 pedv-encoded proteins, at least 10 viral proteins have been identified as type i ifn antagonists [65, 78] . the mechanisms utilized by pedv nsp1, plp2, nsp5, and n protein to antagonize type i ifn production have been clarified (figure 3 , [58, 65, 78, 80, 129] ). however, the specific mechanisms of other viral proteins to inhibit type i ifn production remain largely unknown. at present, 11 pedv proteins have been identified as type iii ifns' antagonists. the suppression of type iii ifn signaling by n protein, nsp1, as well as nsp15, has been reported, while the mechanisms utilized by these viral proteins need to be further investigated. in addition, the covs replication cycle may induce the changes of er stress, cell apoptosis, autophagy pathways, which contain intricate virus-host interactions and cross-talk relationships. thus, more researches for pedv are needed to truly reflect viral evasions from innate immune defenses. the findings of pedv-host interactions will help prevent and control pedv spreading. have been clarified (figure 3 , [58, 65, 78, 80, 129] ). however, the specific mechanisms of other viral proteins to inhibit type i ifn production remain largely unknown. at present, 11 pedv proteins have been identified as type iii ifns' antagonists. the suppression of type iii ifn signaling by n protein, nsp1, as well as nsp15, has been reported, while the mechanisms utilized by these viral proteins need to be further investigated. in addition, the covs replication cycle may induce the changes of er stress, cell apoptosis, autophagy pathways, which contain intricate virus-host interactions and cross-talk relationships. thus, more researches for pedv are needed to truly reflect viral evasions from innate immune defenses. the findings of pedv-host interactions will help prevent and control pedv spreading. removes ubiquitinated conjugates from rig-i; pedv nsp5 induces cleavage of nemo; pedv n protein directly interacts with tbk1 to obstruct the association between tbk1 and irf3; pedv nsp1 causes degradation of cbp and iκbα, as well as inhibition of iκbα phosphorylation and p65 activation. pedv nsp16 inhibits type i ifn production and nsp10 enhances the inhibitory effect of nsp16 on type i ifn production. for type iii ifn, pedv n protein blocks the nuclear translocation of nf-κb; pedv nsp1 blocks the nuclear translocation of irf1 and reduces the amounts of peroxisomes. pedv nsp15 inhibits the type i ifn and type iii ifn responses by unknown mechanisms. pedv nsp7 interacts with stat1 and stat2 to block nuclear translocation of isgf3. removes ubiquitinated conjugates from rig-i; pedv nsp5 induces cleavage of nemo; pedv n protein directly interacts with tbk1 to obstruct the association between tbk1 and irf3; pedv nsp1 causes degradation of cbp and iκbα, as well as inhibition of iκbα phosphorylation and p65 activation. pedv nsp16 inhibits type i ifn production and nsp10 enhances the inhibitory effect of nsp16 on type i ifn production. for type iii ifn, pedv n protein blocks the nuclear translocation of nf-κb; pedv nsp1 blocks the nuclear translocation of irf1 and reduces the amounts of peroxisomes. pedv nsp15 inhibits the type i ifn and type iii ifn responses by unknown mechanisms. pedv nsp7 interacts with stat1 and stat2 to block nuclear translocation of isgf3. author contributions: the manuscript was prepared by s.l., j.y., z.z., and h.z., and reviewed by the co-authors. all authors have read and agreed to the published version of the manuscript. outbreak of porcine 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caspases control antiviral innate immunity porcine epidemic diarrhea virus induces caspase-independent apoptosis through activation of mitochondrial apoptosis-inducing factor caspase function in programmed cell death signal integration in the endoplasmic reticulum unfolded protein response coronavirus infection, er stress, apoptosis and innate immunity regulation of stress responses and translational control by coronavirus porcine epidemic diarrhea virus orf3 protein causes endoplasmic reticulum stress to facilitate autophagy this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we are thankful to everybody who participated in the studies. the authors declare no conflict of interest. key: cord-355465-qjtifwhd authors: van diep, nguyen; sueyoshi, masuo; norimine, junzo; hirai, takuya; myint, ohnmar; teh, angeline ping ping; izzati, uda zahli; fuke, naoyuki; yamaguchi, ryoji title: molecular characterization of us-like and asian non-s indel strains of porcine epidemic diarrhea virus (pedv) that circulated in japan during 2013–2016 and pedvs collected from recurrent outbreaks date: 2018-03-14 journal: bmc vet res doi: 10.1186/s12917-018-1409-0 sha: doc_id: 355465 cord_uid: qjtifwhd background: since late 2013, porcine epidemic diarrhea virus (pedv) has reemerged in japan and caused severe economic losses to the swine industry. although pedv vaccines have been used widely, the disease has swept rapidly across the county, and is commonly observed in ped-vaccinated farms, and has recurred in domestic herds. to better understand pedvs responsible for the reemerging outbreaks in japan, full-length spike (s), membrane (m), and nucleocapsid (n) genes of 45 pedvs collected in japan during 2013–2016, were sequenced and analyzed. results: phylogenetic analysis based on s gene sequences revealed that all the recent field pedvs were genetically distinct from the classical japanese strains, and were classified into three genotypes: north american (na), s indel, and asian non-s indel. our data suggested a possibility that multiple parental pedv strains were introduced into japan from abroad at the same time or similar times. the newly identified japanese strains showed the closest relationship to the us strains. two sublineages of japanese strains circulating in japan were similar to two sublineages identified in the us, suggesting common ancestors for these strains. in comparison with two vaccine strains used in japan, the field strains had various changes in epitope regions, glycosylation sites, and phosphorylation sites. these substitutions, particularly observed in epitope regions of the s (521, 553, 568, and 570), m (5), and n (123, 252, and 255) proteins, may have affected antigenicity and vaccine efficacy, resulting in an unsuccessful pedv control. sequence comparisons between pedvs collected from primary and secondary outbreaks in three herds revealed that the disease has developed to an endemic stage in which pedv could persist for nearly two years in the herds or local regions, causing subsequent epidemics. conclusions: these results elucidate the genetic characteristics, origin, and molecular epidemiology of pedvs circulating in japan, as well as the pedv strains causing recurrent outbreaks. this study provides a better insight into the pedvs responsible for recent outbreaks in japan, and could potentially help to develop measures for controlling and preventing the disease. electronic supplementary material: the online version of this article (10.1186/s12917-018-1409-0) contains supplementary material, which is available to authorized users. porcine epidemic diarrhea virus (pedv) is the etiologic agent of porcine epidemic diarrhea (ped), an acute enteritis disease that is characterized by vomiting and watery diarrhea and usually leads to high morbidity and mortality, especially in piglets [1] . the disease was first recognized in england in 1971 [2] , and the prototype virus, which was designated as pedv cv777, was identified in belgium [3] . subsequently, pedv has been reported in several other european countries, causing only sporadic outbreaks. since the 1980s, it has been widespread in asia and has become an economic concern for the swine industry in many asian countries such as japan, china, south korea, thailand, and taiwan [1, 4] . in late 2010, new chinese strains of pedv were detected. these new strains were clinically more severe than the classical strains, resulting in 80%-100% illness among infected swine herds and a 50%-100% mortality rate among infected suckling piglets [5] . in april 2013, a pedv outbreak was confirmed in the us for the first time. it swept across more than 30 states in the country, causing deaths of more than 8 million newborn piglets during the one-year-epidemic period, and subsequently spread throughout north america, including canada and mexico [6] . afterward, us-like ped epidemics have been reported in south korea, taiwan, japan, germany, france, and belgium [4, 7] . belonging to the coronaviridae family in the nidovirale order, pedv has a single-stranded positive-sense rna genome of approximately 28 kb in size that encodes four structural proteins (s, e, m, and n) and nonstructural proteins (1a, 1ab, and orf3) [1] . among the viral proteins, the s glycoprotein is the most diverse [8] , and plays a critical role in the process of inducing neutralizing antibodies and binding specific receptors [9] . at least two b cell epitopes (ss2 and ss6) and two regions containing neutralizing epitopes (coe and 2c10) have been identified on this protein [10] [11] [12] . the s gene is also associated with growth adaptation in vitro and attenuation of virulence in vivo [13] , and was used as an important component in studies for understanding genetic relatedness among pedv isolates, the epidemiological status, and vaccine development [14, 15] . the m protein is the most abundant component of viral protein in the envelope, and is responsible for morphogenesis, assembly, and budding [16] . the n protein of coronavirus interacts with viral genomic rna, serving as the critical basis for the helical nucleocapsid during the viral assembly [17] . two antigenic epitopes (nep-d4 and nep-d6) have been identified in the n protein of pedv [18] . in japan, the ped-like disease was observed from late 1982 to early 1983, but was not reported again until the outbreaks that occurred between september 1993 and june 1994 [19] . in an epidemic of diarrhea caused by a pedv infection in 1996, more than 39,000 suckling pigs died [20] . afterward, there were no ped cases reported from 2006 until the first half of 2013 [21, 22] . ped outbreaks first reemerged in october 2013 from a southern location (okinawa island) and spread rapidly throughout the country. until may 2016, pedv infections were reported from more than 1000 farms in 39 of 47 prefectures; the infections affected over 1.5 million pigs and led to the death of half a million pigs, according to the ministry of agriculture, forestry, and fisheries of japan (http://www.maff.go.jp). currently, pedv infections and piglet mortality has decreased significantly. however, ped still occurred and frequently recurred in pig farms in japan. therefore, better insight into the causes for the reemergence of ped outbreaks is required for more efficient control and prevention of the disease. toward this goal, we sequenced and analyzed the full-length s, m, and n genes of the japanese field pedvs in comparison with those of vaccine strains used in japan, classical japanese pedv strains, and isolates from other countries. sample collection, rna extraction, and pedv detection small intestine or stool specimens were obtained from suckling piglets, post-weaning piglets, and sows presenting acute watery diarrhea at pig farms in japan, between december 2013 and february 2016. intestinal samples were collected from dead piglets, and fecal samples were non-invasively collected immediately after excretion. thus, no aggressive actions toward the pigs were carried out for sampling purpose. samples of two major vaccine strains that have been employed in japan, p5-v (nisseiken co., ltd) and 96-p4c6 (kaketsuken co., ltd., japan), were collected from commercial vaccine bottles used in the swine farms. sample preparation, rna extraction, and pedv detection were performed as previously described [23] . japanese field samples may contain both pedv variants with large deletions in the 5′terminus of the s gene and pedv strains with an intact s gene [24] . due to the complexity in discriminating, isolating, and individually sequencing pedv strains, only specimens containing pedvs with an intact s gene were used in our study. finally, 45 pedv-positive field samples were collected from 37 farms, located in five different prefectures in japan. these samples, along with the two vaccine samples described above, were sequenced to determine the full-length sequence of the s, m, and n genes ( table 1) . all these field pedvs were confirmed to possess an intact 5′-terminus of the s gene, showing only the expected prominent single dna band from pcr products on a 1.2% agarose gel [24] . after producing cdna, as previously described [23] , the full-length s gene of pedv was amplified using the primer pair fs-f/fs-r ( table 2 ) and kod fx kit (toyobo co., japan). the pcr products were used as templates for nested pcr reactions that amplified five dna fragments spanning the entire s gene using 5 primer sets (cs1-cs5), as previously reported [24] . the m and n genes of the pedv were also amplified from the cdna using two newly designed primer sets, fmf/fmr and fnf/fnr ( table 2 ). emeraldamp max pcr master mix kit (takara bio, japan) was used for pcr amplifications of the cs1-cs5 fragments, as well as the m and n genes. the amplified pcr products were purified using a fastgene gel/pcr extraction kit (nippon genetics co., ltd., japan) according to the manufacturer's protocol. all sequencing reactions were carried out in duplicate, and sequences were determined in both directions using bigdye® terminator v3.1 cycle sequencing kits (applied biosystems, ca, usa). products were analyzed using abi prism 3130xl genetic analyzers (applied biosystems). nucleotide (nt) and deduced amino acid (aa) sequences were edited, assembled, and aligned using geneious v9.1.6 software (http://www.geneious.com), and percentage sequence divergences at the nt and aa levels were further calculated by using the same software. the obtained nt sequences were deposited in genbank under the following accession numbers: ky619734-ky619839 as shown in table 1 . unrooted phylogenetic trees were constructed using molecular evolutionary genetics analysis (mega) software, version 6.06 [25] , with the maximum likelihood method and bootstrap tests of 1000 replicates. the best-fit nt substitution models for analysis were assessed. phylogenetic trees based on the nt sequences of the full-length s and n genes were generated using the tamura-nei substitution model with a discrete gamma distribution (tn93 + g). a phylogenetic tree based on the nt sequences of the m gene was created using the kimura 2-parameter substitution method with a discrete gamma distribution (k2 + g). n-glycosylation sites were predicted using a service available on http://www.cbs.dtu.dk/services/ netnglyc. phosphorylation sites were predicted using the netphos 3.1 server (http://www.cbs.dtu.dk/services/netphos) and netphosbac 1.0 server (http:// www.cbs.dtu.dk/services/netphosbac-1.0). prediction of antigenic regions was performed using the emboss protein analysis tool [26] integrated into the geneious software. identical nt sequences of the s gene were distinguished and excluded, resulting in the identification of 34 individual sequences from the 45 total collected field strains. phylogenetic analysis based on the s gene sequences from the japanese strains and reference strains identified from various countries revealed two major clusters: genogroup g1 divided into subgroups g1a and g1b, and genogroup g2 divided into subgroups g2a and g2b (fig. 1 ). all 45 japanese field pedvs in the present study fell into 3 subgroups g2a, g2b, and g1b. the most dominant strains belonged to g2b, which comprised 42 of 45 current japanese strains, along with the highly virulent (north american type) pedvs isolated in the us, canada, mexico, south korea, and taiwan. two pedv of the 45 field pedvs, 14jm-01 from miyazaki and 16 other strains from aomori, aichi, kagoshima, and miyazaki were grouped into a monophyletic branch designated as ped-j1, and they shared high nt and aa sequence identities (99.69%-100% and 99.35%-100%, respectively) with each other. another subclade, designated as ped-j2, including 14 japanese strains collected in miyazaki were also clustered into a segregated branch as shown in fig. 1 the sequence data revealed that s genes from the japanese field pedvs are of 4152-4161 nt long, and encode proteins with 1381-1386 aa residues. this consequence was due to the presence of several deletions or insertions that accumulated primarily in the n-terminus of the s protein. multiple alignments of the s gene demonstrated that 14jm-140 and 14jm-144 exhibited insertions and deletions (1-nt, 11-nt, and 3-nt deletions at positions 167, 176, and 416, respectively, and a 6-nt insertion between positions 474 and 475), which are typical for s indel strains [28, 29] . compared to the two vaccine strains, all japanese field pedvs were highly conserved in epitopes 2c10 and ss2, except for strain 13jm-293 that had an aa substitution (l1375i) within 2c10 (fig. 2) . additionally, compared to the vaccine strains, all the japanese field pedvs had two aa changes (l768s and d770s) in the epitope ss2, and three serine substitutions (a521s, t553s, and g598s) in the neutralizing domain coe (excluding 13jm291 and 14jm-268). notably, the aa substitutions at positions 521 and 553 resulted in predictable phosphorylation sites in the s protein of the japanese field strains. furthermore, different amino acids were found at the 12 sites within the coe domain (fig. 2) . regarding the prediction of high-specificity n-glycosylation sites in the s protein, most japanese strains belonging to g2b exhibited eight n-glycosylation sites over the entire s protein (additional file 1: table s1 ). 14jm-268 had one additional n-glycosylation site at residue 557, and 14jm-242 possessed an additionally unique n-glycosylation site at position 382 of the aa chain. two japanese s indel strains (14jm-140 and 14jm-144) had seven n-glycosylation sites, while 13jm-291 had eight n-glycosylation sites. compared with the vaccine strain, 96-p4c6, most of the japanese non-s indel strains in g2b lost three n-glycosylation sites (at 132, 557, and 1233) and gained another nglycosylation site (at 60). compared with p-5 v, these g2b japanese strains lost three n-glycosylation sites (at 117, 132, and 515) and gained four other nglycosylation sites (at 60, 119, 325, and 1262). the m gene sequences from all the japanese field pedvs and the two vaccine strains consisted of 681 nucleotides, encoding a protein of 226 aa. identical nt sequences were distinguished and excluded, resulting in the identification of seven unique sequences from the 45 total collected pedvs (table 2) . a phylogenetic tree based on the m gene of the japanese strains and reference strains revealed that all the sequences were divided into 2 groups (g1 and g2); g2 was further divided into two subgroups g2a and g2b (fig. 3a) . all the current japanese pedvs fell into g2, and showed the closest relatedness to the na strains and emerging strains identified in europe, south korea, and china. these field pedvs were phylogenetically distant from the vaccine strains and a classical japanese strain (jme2) of g1. the m genes of field pedvs had 99.56%-100% nt identity to each other and 97.5%-98.24% nt identity with the vaccine strains. the b cell epitope, wafyvr [30] , located at position 195-200 of the m protein, was conserved in all field and vaccine strains. compared with the vaccine strains, all japanese field strains had an aa substitution (a214s) belonging to an antigenic region (aa 205-223), as predicted by the emboss protein analysis tool (additional file 2: figure s1a ). strain 14jm-236 had a different aa (s213 n) leading to the loss of a predicted serine phosphorylation site. in another predicted epitope region (aa 5-15), the field pedvs had one different aa (v12i) compared to p-5 v, and two aa substitutions (f5s and q13e) compared to 96-p4c6. these substitutions (f5s) resulted in gaining a predicted high-specificity n-glycosylation site. the n gene of all the field pedvs and vaccine strains was 1326 nt in length encoding a protein of 441 aa. identical nt sequences of the n gene were distinguished and excluded, resulting in the identification of 19 unique sequences. phylogenetic analysis based on the n genes from the japanese strains and reference strains revealed the classification of pedv strains into three groups: g1, g2, and g3 (fig. 3b) . all japanese field strains were closely clustered together into subgroup g3 with other us and us-like strains. the japanese field pedvs shared 98.72%-100% n gene nt sequence identity to each other and 95.70%-96.76% to the vaccine strains. strain 13-jm127 from the first outbreak in miyazaki had 100% nt identity to eight other field strains collected in miyazaki, aichi, and kagoshima, and this is likely to be the most dominant sequence of n gene among the field strains. compared to the vaccine strains, all the field strains in the present study had 9 aa substitutions (additional file 2: figure s1b ); the substitution k123 n resulted in forming a predicted n-glycosylation site on the n protein from the field strains, while the aa substitutions a142t and n255s observed in field strains formed two predicted phosphorylation sites. of note, the substitutions k123 n and n255s belong to the reported antigenic epitopes nep-d4 (aa 18-133) and nep-d6 (aa 252-262), respectively [18] . there were three swine farms where ped outbreaks first occurred in 2014, and then recurred in 2016, as shown in table 3 . farm 1 is located in aomori prefecture, while farms 2 and 3 are located in the same town in miyazaki prefecture. phylogenetic analysis based on the s gene sequences revealed that three pedv strains (14jm-168, 16jm-334, and 16jm-339) collected from two different outbreaks on farm 1 were closest to each other and segregated into a distinct minor branch. pedvs from the primary ped outbreaks on farm 2 (14jm-179, 14jmon farm 1, the s, m, and n genes of pedvs identified from the recurrent outbreak (16jm-334 and 16jm-339) shared the highest (99.66%, 100%, and 99.55% respectively) nt sequence identities to a strain (14jm-168) collected table 3 information of primary and recurrent ped outbreaks occurred in the three pig farms in this study farm mortality rate of piglets less than two weeks-old; b mortality rate of the piglets less than one-week-old all the information for mortality and morbidity was kindly supplied by the veterinarians responsible for these farms from the primary outbreak. the s genes of pedvs from the primary outbreak on farms 2 and 3 had high (99.92%-99.98%) nt identity to each other. these strains also had identical sequences for the m and n genes. in the secondary outbreaks, pedvs from farms 2 and 3 also shared highly homologous genes. the s and m genes of two strains (16jm-319 and 16jm-323) from farm 3 shared 100% nt sequence identities with those of a strain (16jm-325) from farm 2. compared with the strain collected from the primary outbreak on farm 1, two strains from the recurrent outbreak had 11 aa substitutions in their s proteins (additional file 3: figure s2) . notably, the substitution s722 n lead to the loss of a predicted serine phosphorylation site. these recurrent strains also had two aa substitutions (r166s and t413 n) in the n protein; the r166s substitution formed a predicted serine phosphorylation site. comparison to pedvs from the primary outbreak on farms 2 and 3, pedvs from the recurrent outbreaks had 13 aa substitutions in the s proteins, in addition to a single aa change (a42v) in the m protein and two aa changes (l18i and s31f) in the n protein. notably, the substitutions l18i and s31f occurred in the reported epitope nep-d4 of the n protein. since late 2013, massive ped outbreaks have recurred in japan causing tremendous financial losses in the swine industry. despite the nationwide use of available attenuated vaccines developed decades ago [21] , pedv has rapidly swept across the county. our sequencing data revealed that the prevailing japanese pedvs are genetically heterogeneous and can be classified into 3 genotypes: na, s indel, and asian non-s indel. both the na and s indel types, which have been identified in recent studies in japan [21] [22] [23] 31] were also responsible for recent outbreaks in the us and south korea. in our study, besides the two previously reported pedv types, we identified another type of japanese pedv (designated as asian non-s indel) that is closely related to the vietnamese, thai, and chinese strains. the asian non-s indel type was collected from an early ped epidemic that occurred in southern japan where the recent ped pandemic began spreading. moreover, the asian non-s indel type appeared at a similar time to the appearance of the na and s indel types in japan [21] . together with the absence of ped cases from 2007 to 2012 in japan [21, 22] , we speculated a possibility that the three types of japanese pedv originated from one source and emerged at the same or similar time in the southern region of japan before spreading across the country. the result of this study revealed that all the field pedv strains (except 13jm-291) were closely related to emerging strains identified in the us, south korea, taiwan, and in european countries. of note, the japanese pedvs were genetically most closely related to the us strains; some even had identical s gene. since the recent ped pandemics in japan started to reemerge six months after those in the us, the close genetic relationship indicated that the current japanese pedv might have been introduced directly from the us, or both the us and japanese pedvs were derived from one source of origin. since april 2013, the na type of pedv has emerged in the us. the na strains were identified subsequently in south korea, taiwan, and japan from late 2013. another pedv type possessing typical insertions and deletions in the n-terminus of the s gene when compared to the prototype na type was first discovered in ohio in the us [29] . this pedv type, therefore, was designated as s indel. the s indel pedvs were also identified later in recent outbreaks in japan, south korea, canada, belgium, france, germany, portugal, slovenia, and the netherlands [4] . both highly virulent and s indel pedvs prevailing in the us are supposed to be derived from chinese pedvs as a result of recombinant events [4, 28, 32] . in accordance with these findings, our sequence analysis showed that the s indel strains, such as ch/hbqx/10 and js120103, had already appeared in chinese ped outbreaks during 2010-2012 [33, 34] . with the prevalence of both na and s indel strains in the us, south korea, and japan since 2013, we suggested that there are two major possibilities of origin for the reemerging japanese pedv strains. first, us-like strains have been directly transmitted to japan from the us or south korea. second, the same source of the us strains could have introduced the pedv into japan, from china, shortly after the us outbreaks. however, our study revealed the prevalence of the asian none-s idel pedv strain in japan, which has not been reported in the us or south korea. thus, we incline toward the possibility that multiple parent pedv strains have been introduced into japan, from china or china's neighboring countries (such as the southeast asian region), causing the recent ped pandemic. further investigation and surveillance are required to specifically identify the source of origin for the reemerging japanese pedvs. sixteen pedv strains from four different prefectures including miyazaki, kagoshima, aomori, and aichi formed a well-supported subclade (ped-j1). fourteen pedv strains from various farms in miyazaki prefecture were also grouped into a distinct subclade (ped-j2). these results suggested that strains of those japanese sublineages (ped-j1 and ped-j2) may derive from two common ancestral pedvs whose progeny have spread in the regions. two genetic sublineages of us strains, namely ia1-co/13 and mn-ia2 [32] , were identified from the us outbreaks in 2013. notably, the japanese pedv of the japanese sublineage ped-j1 in this study showed a close relationship with the us sublineage ia1-co/13, and pedvs of the japanese sublineage ped-j2 showed close relatedness with the us sublineage mn-ia2. this data revealed the common pattern of genetic diversity as well as the close relationship between the japanese strains and the us strains, suggesting a common ancestor for these strains. in our previous study [23] , by sequencing a partial s gene containing a neutralizing epitope region (coe domain), we suggested that 14jm-140 may belong to the s indel type that has been circulating in japan together with the highly virulent us-like strains. in this study, by sequencing the full-length s gene, we confirmed 14jm-140 to be an s indel strain. we also identified another strain that belonged to the s indel type, 14jm-144, although it was not predicted to be an s indel pedv in the previous study. this discrepancy arose due to the region of the partial s gene utilized in the previous report that lack the typical insertions and deletions of the s indel type. our data revealed the circulation of s indel strains in ped outbreaks occurring in japan, but these strains compose only a minor population of the reemerging japanese pedvs. these results are consistent with two recent reports of japanese pedv [21, 22] . phosphorylation of viral proteins is a reversible posttranslational modification that can have major impacts on viral infection, replication, and cytotoxicity in a host cell [35] . for infectious bronchitis virus, belonging to the coronaviruses, the phosphorylation of the nucleocapsid protein has a dramatic influence on rna binding capability and the recognition of viral rna from nonviral rna in the process of viral replication [36] . along with phosphorylation, glycosylation is another posttranslational modification that often strongly affects the protein functions of viruses. n-glycosylation sites in the spike gene of the severe acute respiratory syndrome coronavirus were demonstrated to be crucial for viral infection [37] . on the other hand, mutations observed in the neutralizing regions have been proposed as presenting a viral evolution, allowing escape from antibodies developed against vaccine or classical pedv strains [38] . in this study, we characterized differences in epitope regions, high-specificity n-glycosylation and phosphorylation sites, between the vaccine and recent field strains. various substitutions observed in the s (521, 553, 568, and 570), m (5) , and n (123, 252, and 255) proteins may have affected the antigenicity, conferring the capacity for immune evasion in the pedv field strains, and consequently, influencing the efficacy of the vaccines. in fact, despite vaccination, pedv infections still spread rapidly and are commonly observed on ped-vaccinated farms in japan [23] . vaccination showed very low efficacy and could not mitigate the severe losses caused by ped [39] . based on the antigenicity analysis of the vaccine and field strains, as well as the recent performance of the ped vaccines on the japanese pig farms, we suggest that the development of novel vaccines based on reemerging pedvs is necessary to control current ped outbreaks. the first ped epidemic on farm 1 was a severe occurrence in may 2014 (table 3) . afterward, obvious clinical symptoms and death caused by ped were not observed in pigs of the herd until february 2016 when the subsequent outbreak recurred with less severity than the first. although the pigs did not show any obvious symptoms of ped, the presence of pedv on this farm was detected in piglets several times by rt-pcr during the period from june 2014 to january 2016, according to the information supplied by veterinarians responsible for this farm. phylogenetic analysis based on the sequences of the s and n genes showed that pedv strains from the primary and secondary outbreaks formed monophyletic branches. they also shared with each other the highest sequence identity of the s, m, and n genes compared to other strains used in this study. additionally, ped outbreaks were not observed on other farms within a 5 km radius of farm 1, and many strict measures for establishing a high level of security were implemented on this farm. taken together, we speculated that pedv strains causing the primary outbreak induced partial protective immunity but persisted in the herd for nearly two years. subsequently, the persisted pedvs caused the secondary epidemic. this is the first report of a two-year-long persistence of pedv in a pig herd. it has been proposed that the popular circulation of pedv variants with large deletions in the s gene is associated with the persistence of the pedv on the infected farms in japan [24] . however, we did not detect the presence of the large s deletion variants in the fecal and intestinal samples collected from this farm. other factors, including the genetic variation of causative pedv strains, may play a more important role in the disease recurrence. on farms 2 and 3, ped first occurred in may 2014, despite all the sows receiving antepartum pedv vaccinations in 2013. afterward, the disease disappeared until early 2016 when the subsequent outbreaks recurred. there was no detection of the pedv during the interval between these two outbreaks. phylogenetic analysis based on the s and n genes revealed that the strains collected from the first outbreaks on farms 2 and 3 formed a monophyletic branch. moreover, these two farms were located close to each other with a distance of 0.5 km (separated by a small mountain), and the outbreaks occurred at the same time. this suggests that the pedv strains introduced onto these farms that induced the first outbreaks were derived from a common source of pedv. likewise, in the secondary outbreaks, the strains collected from these two farms also have the highest genetic identity to each other and formed minor monophyletic branches, as shown in the phylogenetic trees for the s and n genes. all the strains collected from the primary and recurrent outbreaks on these farms were closely clustered into the subclade ped-j2, together with other strains collected from other farms located in miyazaki prefecture (fig. 1) . this data suggested that the pedv strains causing the subsequent outbreaks on farms 2 and 3 evolved from pedvs that had been circulating in the miyazaki region since 2014. there are two possibilities for the origin of the strains that caused the subsequent outbreaks on farms 2 and 3. first, pedvs may have persisted silently on these farms since 2014, evolving, and then causing the subsequent outbreaks when suitable conditions arrived in early 2016. another possibility is that pedvs may have persisted on other pig farms, circulating in the miyazaki region since the 2014 ped occurrence, after which the pedvs were introduced again onto farms 2 and 3, causing the secondary epidemics. more data is needed to elucidate exactly the origin of these pedvs strains. the occurrence of ped in these herds has partially indicated the failure of using the current pedv vaccines, which are based on old seed stock of classical japanese strains. besides, the repeated outbreaks in these herds indicate that the disease has developed to an endemic stage. these results could be used to elucidate the prevalence of pedvs and to contribute to the prevention of ped in the region. in conclusion, our study revealed that japanese field pedvs causing ped outbreaks during 2013-2016 could be classified into three types, and these pedvs might have been introduced from overseas at the same time or during similar periods. the disease has developed to an endemic stage in which the pedv can persist for a long time in these herds or these local regions causing subsequent epidemics. the distant genetic relationships and various aa substitutions between the japanese field pedvs and vaccine strains may be responsible for the unsuccessful ped control in japan. our findings will be useful in understanding the origin and molecular epidemiology of the pedv, and helping to develop measures for the control of the disease. additional file 1: table s1 . highly-specific n-glycosylation sites in the spike protein of the vaccine and field strains. (docx 13 kb) additional file 2: figure s1 . amino acid substitutions in the m and n proteins between the field and vaccine strains. porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines letter to the editor a new coronavirus-like particle associated with diarrhea in swine evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains new variants of porcine epidemic diarrhea virus, china cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus detection and molecular diversity of spike gene of porcine epidemic diarrhea virus in china the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus the gprlqpy motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea genetic diversity of orf3 and spike genes of porcine epidemic diarrhea virus in thailand incorporation of spike and membrane glycoproteins into coronavirus virions the coronavirus nucleocapsid is a multifunctional protein the identification and characterization of two novel epitopes on the nucleocapsid protein of the porcine epidemic diarrhea virus an immunohistochemical investigation of porcine epidemic diarrhoea porcine epidemic diarrhea: its diagnosis and control molecular characterization of pig epidemic diarrhoea viruses isolated in japan from isolation and molecular characterization of porcine epidemic diarrhea viruses collected in japan in 2014 us-like isolates of porcine epidemic diarrhea virus from japanese outbreaks between novel porcine epidemic diarrhea virus (pedv) variants with large deletions in the spike (s) gene coexist with pedv strains possessing an intact s gene in domestic pigs in japan: a new disease situation mega6: molecular evolutionary genetics analysis version 6.0 emboss: the european molecular biology open software suite emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences distinct characteristics and complex evolution of pedv strains new variant of porcine epidemic diarrhea virus identification of a conserved linear b-cell epitope in the m protein of porcine epidemic diarrhea virus isolation and experimental inoculation of an s indel strain of porcine epidemic diarrhea virus in japan origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states genetic variation analyses of porcine epidemic diarrhea virus isolated in mid-eastern china from 2011 to 2013 molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus field strains in central china during 2010-2012 outbreaks phosphorylation events during viral infections provide potential therapeutic targets role of phosphorylation clusters in the biology of the coronavirus infectious bronchitis virus nucleocapsid protein specific asparagine-linked glycosylation sites are critical for dc-sign-and l-sign-mediated severe acute respiratory syndrome coronavirus entry bioinformatics insight into the spike glycoprotein gene of field porcine epidemic diarrhea strains during 2011-2013 in guangdong, china measure to mitigate loss caused by ped (porcine epidemic diarrhea) using maternal immunity the work was conducted with funding from the university of miyazaki (grant number 2014-70) and japan society for the promotion of science (kakenhi program, grant number 17h04639). the funders had no role in the design of the study and collection, analysis, and interpretation of data and in preparation of the manuscript. data supporting the conclusions of this article are included within the article and its additional files table s1, figure s1 , and figure s2 . sequence data supporting the conclusions of this article are available in the genbank repository under the accession numbers ky619734-ky619839.authors' contributions ry and nd designed the study. ry, ms, zn, and th provided the viral samples and other materials. nd, nf, at, ui, and om conducted experiments. nd designed the primers, interpreted the sequencing data and wrote the manuscript draft. ry, ms, zn, th, at, ui, nf, and om analysis the data and revise the manuscript. all authors prepared and approved the final manuscript. in this study, the intestinal samples were collected after the death of pigs due to the virus infection in the pig farms. no pig was culled for sampling purpose in this study the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-355991-4zu69e0y authors: piñeyro, pablo enrique; lozada, maria inez; alarcón, laura valeria; sanguinetti, ramon; cappuccio, javier alejandro; pérez, estefanía marisol; vannucci, fabio; armocida, alberto; madson, darin michael; perfumo, carlos juan; quiroga, maria alejandra title: first retrospective studies with etiological confirmation of porcine transmissible gastroenteritis virus infection in argentina date: 2018-09-24 journal: bmc vet res doi: 10.1186/s12917-018-1615-9 sha: doc_id: 355991 cord_uid: 4zu69e0y background: in 2014, a notification of porcine transmissible gastroenteritis virus (tgev) was made by the national services of animal health of argentina (senasa) to the world organization of animal health (oie). the notification was based on a serological diagnosis in a small farm with a morbidity rate of 2.3% without enteric clinical signs. in order to determine if tgev was circulating before the official report, a retrospective study on cases of neonatal diarrhea was performed. the selection criteria was a sudden increase in mortality in 1to 21-day-old piglets with watery diarrhea that did not respond to antibiotics. based on these criteria, three clinical cases were identified during 2010–2015. results: all animals that were evaluated presented histological lesions consistent with enteric viral infection. the feces and ultrathin sections of intestine that were evaluated by electron microscopy confirmed the presence of round particles of approximately 80 nm in size and characterized by finely granular electrodense nucleoids consistent with complete particles of coronavirus. the presence of the tgev antigen was confirmed by monoclonal specific immunohistochemistry, and final confirmation of a metabolically-active virus was performed by in situ hybridization to detect a tge mrna encoding spike protein. all sections evaluated in this case were negative for pedv and rotavirus a. conclusions: this is the first case series describing neonatal mortality with etiological confirmation of tgev in argentina. the clinical diagnosis of tgev infections in endemic regions is challenging due to the epidemiological distribution and coinfection with other enteric pathogens that mask the clinical presentation. electronic supplementary material: the online version of this article (10.1186/s12917-018-1615-9) contains supplementary material, which is available to authorized users. there are five coronaviruses (covs) known to infect swine, and the clinical disease is mainly associated with neonatal diarrhea, but respiratory and neurological signs have also been reported [1] [2] [3] [4] . porcine transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) belong to the coronaviridae family, coronavirinae subfamily, and genus alphacoronavirus [5] . a new coronavirus genetically distinct from tgev and pedv, porcine deltacoronavirus (pdcov) (genus deltacoronavirus), has recently been associated with enteric disease in pigs [6] . enteric porcine coronaviruses including tgev, pedv and pdcov are characterized by acute diarrhea and anorexia with rapid dissemination in naïve populations. the severities of clinical diarrhea, vomiting, and anorexia can vary based on the age of the affected pigs [7, 8] . without adequate passive lactogenic immunity, the mortality rate in neonatal piglets can reach up to 100% [1, [9] [10] [11] . etiological diagnosis relies mainly on molecular tools like pcr and serology, as the clinical signs and enteric lesions associated with tgev and pedv are indistinguishable [8, 12, 13] . the epidemiology of tgev is rather complex, and infection in neonates can arise from multiple sources. in addition to swine, it has be documented that cats, dogs, and foxes can host tgev [14] . the virus can be shed in feces for approximately 18 months, and milk shedding from infected sows can result in vertical transmission. historically, tgev infection has followed a seasonal pattern, becoming more prevalent during winter months perhaps due to increased viral survival in colder temperatures and with less exposure to sunlight. tgev is susceptible to most commercial disinfectants, but resistant to digestive bile and stable at ph 3 [1] . only one tgev genotype has been described, however differences in pathogenicity among strains has been reported in field outbreaks, although not confirmed by an experimental study [12] . infection with tgev has two different clinical presentations: epidemic and endemic. in epidemics, tgev enters a naïve herd and all pig categories are affected, particularly piglets that are 1-2 weeks old. the duration of the clinical presentation is short, approximately 3 weeks, and in small, farrow-to-finish herd, the infection can be self-limiting [1, 14] . endemic disease scenarios, those occurring after the epidemic phase, are observed in farms with incomplete aiao management or in breeding farms that have a continuous flow of naïve gilts. in breeding herds, the varying levels of humoral and lactogenic immunity lowers piglet mortality, but may lengthen the course of the disease [1] . since the first description provided in the united states [15] , tgev infections have been reported all over the world. in south america, it has been reported in colombia [16] , venezuela [17] , bolivia, and is currently seen in brazil [18] . in argentina, an episode of high pre-weaning mortality related to isospora suis infection alone or in association with an unknown enteric virus was reported in 1998 [19] . further studies using negative stain electron microscopy demonstrate the presence of viral particles consistent with coronavirus in feces of pre-weaning diarrheic (34.4%) and post-weaning (10%) piglets [20] . a retrospective histopathological study performed on cases of neonatal diarrhea at our laboratory during 2013 showed that 29% of neonatal diarrhea cases had lesions consistent with viral enteritis [21] . in 2014, a notification of tgev infection was reported by the national services of animal health of argentina (senasa) to the world organization of animal health (oie). it was detected by serology in a small farm with an apparent morbidity rate of 2.3% without clinical signs [22] . this is an unusual presentation of tgev infection and might be related to passed infection or interspecies transmission [1] . in order to clarify the situation prior to the first official report of tgev infection in argentina, a retrospective study was performed on cases suspected of tgev-like disease recorded at the laboratory of special veterinary pathology at the college of veterinary sciences, la plata university. benchmarking analyses of epidemiological behavior and clinical histories were the criteria for herd selection. etiological diagnosis was confirmed by electron microscopy (em), immunohistochemistry (ihc), and in situ hybridization (ish-rna) of archived paraffin blocks. clinical, pathological and etiological findings case 1 thirteen 1-to 7-day-old piglets with body weights ranging from 1 to 1.5 kg were submitted for pathological investigation. pigs with clinical diarrhea were dirty, wet, had stained perinea, and showed moderate dehydration characterized by sunken eyes and diffusely pale mucosa. their small intestinal walls were thin with unremarkable mesenteric lymphatic vessels, and were distended by gas or occasionally contained yellow watery digesta with a ph of 5-6 ( fig. 1a) . their stomachs were empty or contained floccules of undigested milk. no other macroscopic findings were observed. the histopathological evaluation showed a shortening of intestinal villi with a crypt-villous ratio of 1:2 ( fig. 1b) , that villi were fused and lined by vacuolated cuboidal or attenuated epithelium, and that the lamina propria was expanded by moderate edema. microscopic lesions were limited to the jejunum and ileum. immunohistochemistry and ish-rna against tgev showed strong staining in the epithelium of sections that presented minimal epithelial villous changes (fig. 1c, d) . conversely, sections with the most severe epithelial damages were ihc negative. all sections evaluated in this case were negative for pedv and rotavirus. a gross evaluation of five piglets between 2 and 3 days old showed marked dehydration, wet and stool-stained perinea, and poor body conditions (1.15 kg average body weight) (fig. 2a) . the small intestine contained abundant yellow-watery diarrhea with a ph of 5-6 in two pigs and alkaline ph in three pigs (fig. 2b) . a histopathology examination of multiple sections of jejunum and ileum showed mild to moderate villous shortening and fusion (fig. 2c) . the villous enterocytes showed a marked cytoplasmic vacuolization (fig. 2d) . the lamina propria was minimally infiltrated by lymphocytes and plasma cells, and was expanded by edema. the superficial villous enterocytes in the ileum showed strong hybridization signals characterized by active-replicating tgev (fig. 2e) . no microscopic lesions were seen in the sections of colon. all sections evaluated in this case were negative for pedv and rotavirus a. in the ultrathin sections, rounded particles measuring approximately 80 nm in diameter were located in the cytoplasm of the intestinal epithelial cells. the particles were characterized by finely granular electrodense nucleoids with electron lucent centers compatible with complete particles of coronavirus (fig. 2f ) . three neonatal piglets that died naturally presented with fecal stained perinea and were markedly dehydrated. the stomachs were empty, the small intestinal wall was thin/translucent, and scant yellow watery contents were sometimes apparent. the histopathology examination of the jejunum and ileum showed moderate villous fusion and shortening, and the villi were lined by low-cuboidal to flattened/attenuated epithelium (fig. 3a) . the lamina propria was infiltrated by numerous lymphocytes and plasma cells, was expanded by edema, and exhibited lymphangiectasia (fig. 3b) . the tgev antigen was detected by ihc in multiple sections of the jejunum and ileum (fig. 3c) . no significant lesions were observed in the section of colon. all sections evaluated in this case were negative for pedv and rotavirus a. the epidemiological and clinical presentations of outbreaks of neonatal mortality associated with enteritis and the detection of tgev started in the gestation units. both gilts and sows showed anorexia, diarrhea, and vomiting before enteric signs were observed in neonatal piglets. however, the prevalence of clinical signs in the breeding stock was low and no mortality was reported. when tgev enters in a naïve herds, an epizootic form characterized by a 100% mortality of pre-weaning piglets due to diarrhea and dehydration is normally observed [1, 14] . although in the present study, the farms had a high prevalence of diarrhea in suckling pigs, only farms a and c showed almost 100% neonatal mortality, while in farm b had approximately 20% neonatal mortality. the clinical presentation and epidemiological pattern observed in farm b resembled the tge endemic form. although no other etiological diagnosis was confirmed, the low mortality associated with tgev that was observed in farm b might be the result of previous exposure to prcv. prcv infection can confer cross-protection against tgev, reducing the enteric clinical signs and pre-weaning mortality [12] . therefore, herds concomitantly infected with prcv and tgev develop less severe clinical signs, making the clinical differentiation from other enteric infections such as rotavirus or e. coli infections more challenging [1, 14] . another potential reason for the low mortality rate due to tge infection that was observed in farm b could be the intermittent viral exposure of the breeding stock that provided partial immunity to the neonatal piglets [14] . in all herds in this study, it was suspected that the virus entered the farms through the subclinically infected replacement animals although the pre-weaning mortality rate in farm b was lower than that in farm a and c, the pre-weaning mortality was higher in this farm than the mortality rate seen in similar production systems due to other enteric causes such us i suis [19, 23] , c. perfringes type a [24] , or c. difficile [25] . according to a previous study, the pre-weaning mortality due to diarrhea should not exceed 20% [26] . usually, in porcine covs infections, the course of the clinical disease is short and normally does not exceed 3-4 weeks [14] due to the establishment of a rapid herd immunity, as early as one week post-infection. however, in farm b the clinical presentation persisted for approximately 2 months. potential reinfection due to poor husbandry and incomplete aiao management are just few potential causes of viral persistence in the environment that can predispose reinfection of the farrowing units. in clinically affected litters, most of the pigs are dirty, wet, and dehydrated, with diminished body weights. diarrhea is watery, yellowish, and with an acidic smell from the presence of undigested milk [23] . the mortality rate is inversely correlated with the age of the piglets, reaching 100% in 2-7 day-old piglets. this predisposition is due to the slow replacement rate of villus epithelial cells (around 10 days) in neonatal piglets compared with the replacement rate of 3-week-old piglets (around 2-4 days) [14] . in this study, mortality varied from 20 to 100%. the jejunum and ileum are the target segments of the small intestine for virus multiplication. however, tgev infection is segmental, so multiple segments should be included for histopathology or etiological diagnoses in situ such as ihc or ish-rna. due to the retrospective nature of this study, fixed tissue was used to confirm the presence of tgev and the morphological changes consistent with viral infection in the intestinal mucosa. it is important to highlight that although tgev was detected by different means in each farm, due to the segmental nature of the lesions and viral distribution, viral arn or viral antigen was not detected in all of the intestinal segments that were evaluated. in piglets that are less than 2 weeks old, the reduction of villus length and the fusion of jejunum and proximal ileum are the main histological changes [27] . the normal villous/crypt length ratio is 7:1, however, after 24-48 h post-infection, the villous/ crypt ratio can be reduced to 2:1 or 1:1 [27] . since tgev replicates in mature absorptive epithelial cells [28] , a false negative diagnosis can be observed by ihc or ish in specimens that display villous shortening. few absorptive vacuolated cells are seen normally in the intestine, however, during tgev infection, they are found in great numbers with a cuboidal shape [27] . in endemic tgev infection, histopathological diagnosis is more difficult because only 25% of pigs display typical tge lesions. in addition, immunofluorescence tests and ihc often fail in endemic farms due to a low number of enterocytes in the tgev-infected because of partial protection conferred by colostral antibodies [23, 29] . different diagnostic techniques have been used to detect tgev infection such as ihc, ish, electron microscopy, and immunoelectron microscopy and pcr [13] ; however, histopathology remains the most useful tool for screening diagnosis [18] . in this study, although all cases were selected using clinical features and epidemiological information, the histological evaluation consistently showed lesions compatible with viral infection. the application of ihc and ish-rna on archived paraffin blocks from cases of neonatal diarrhea with high morbidity and mortality allowed retrospective identification of tgev infection. diagnosis of tgev infection in endemic regions, such as in argentina, is complicated due to the epidemiological distribution and clinical signs that might be masked with other enteric infections. further studies are necessary to determine the true prevalence of this pathogen and the correlation with neonatal enteric cases observed in confined production systems. case selection was based on the clinical history reported by the farm managers and referring veterinarians. the selection criteria was a sudden increase in mortality that included, significant increment of more than 2 sd from average pre-weaning mortality and last for a period of a week. in addition reported mortality should be associated with the presence of watery diarrhea in 1-to 21-day-old piglets that did not respond to antibiotics. first screening of cases was done by histopathological evaluation, and only cases presenting features of viral enteritis with no other detected pathogen were included. based on these criteria, three clinical cases were identified from 2010 to 2015. a 170-sow farrow-to-finishing herd located in buenos aires served as the first case. the farm produced its own replacement breeding stock, however, two months before the outbreak, gilts were introduced from a breeding company. the parity of the breeding stock was distributed as 40% gilts while the rest of the reproductive stock parity varied from 2 to 6. on september 2011, approximately 10% of the pregnant sows presented acute vomiting while 30% of the pregnant sows presented acute diarrhea. during the period when the sows showed gastro-enteric clinical signs, 2-to 4-day-old piglets presented vomiting (75-80%) and diarrhea (90%), and the mortality rate of suckling pigs reached 90%. the course of the disease in both breeder stock and piglets lasted for approximately three weeks. case 2 involved a one-site herd of 350 sows with its own replacement gilts and the following parity distribution: 20% gilts, 40% parity 1-2, and 27.3% distributed amongst parity 3-5. boars were purchased from a breeding company and were incorporated into the reproductive herd without quarantine. the farm was located within a few miles of a swine slaughterhouse in buenos aires. in february 2012, the pregnant sows showed anorexia (14-30%) and diarrhea (1%) associated with heat returns and abortions (3.3%). in the farrowing houses, approximately 100% of the lactating sows presented with anorexia. pre-weaning mortality associated with the presence of diarrhea varied from 16.5% at the beginning of the outbreak to 27.9% 3 to 4 weeks after the initial clinical signs. an anatomopathological evaluation showed that 93.6% of the total pre-weaning mortality was due to diarrhea. a one-site herd of 400 sows was the subject of case 3. in july 2013, two boars were located close to the gestation unit. a week later, gestation sows showed anorexia (16.8%) and diarrhea (5.3%). thereafter, in the gilts, diarrhea was evident in the nursery (3-7%) and fattener (5-23%). two-day-old piglets showed watery diarrhea (100%) with a mortality rate of 95%. affected piglets died from severe dehydration within two days of the onset of clinical signs. the course of the disease lasted approximately two months with an overall pre-weaning mortality of 50% during that period. at the onset of the outbreak, clinically affected suckling pigs were submitted for postmortem examination. tissue samples from different organs, including multiple segments of small intestine (duodenum, jejunum, and ileum) and large intestine, were fixed in 10% buffered formalin, processed for routine histopathologic examination, and stained with hematoxylin and eosin. etiological diagnosis in tissues and feces by immunohistochemistry, in situ hybridization, and electron microscopy immunohistochemestry ihc was carried-out briefly to differentiate tgev [30] from pedv [31] . monoclonal antibodies against tgev (osu: #.14e3-3c) and pedv (osu 6c8) at dilutions of 1:8000 were used. antigen retrieval was performed with humid heat and revealed with peroxidase (novocastra a , leica biosystems, il, usa). to rule out other potential viral enteritis, rotavirus a was evaluated in all sections by ihc [32] . rotavirus ihc was performed using a monoclonal antibody against rotavirus a (santa cruz: sc-101363) at a 1:2000 dilution. antigen retrieval was performed with epitope retrieval solution 2 for 20 min, as programmed on leica bond iii, and revealed with powervision poly-hrp anti-mouse (leica pv 6113). all samples were tested in duplicate and sections were controlled appropriately for tgev, pedv and rotavirus a with positive and negative controls (additional file 1: figure s1 ). in situ hybridization ish-rna was developed through the rnascope platform (advanced cell diagnostics, inc., ca), targeting the specific reverse complementary nucleotide sequence of the tge viral mrna (716-1859 region of spike gene, genbank: kc609371.1). therefore, positive hybridization signals represent a metabolically-active virus characterized by the tge mrna encoding spike protein. unstained paraffin tissue sections were processed as previously described [33] . briefly, tissues were deparaffinized and treated with hydrogen peroxide at room temperature for 10 min. the slides were hybridized using a hybridization buffer, and sequence amplifiers were added. the red colorimetric staining detected the tge hybridization signal, and counterstaining occurred with hematoxylin. representative sections of small intestine were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 m, ph 7.4 phosphate buffer (pbs) and post-fixed in 1% osmium tetroxide in pbs. after dehydration in an alcohol series, the fragments were embedded in epoxy resin, quetol 812 (nisshin em co., ltd., tokyo). ultrathin sections were cut, double-stained with uranyl acetate-lead citrate, and observed under a jem-1200ex (jeol co. ltd., tokyo). diseases of swine new variant of porcine epidemic diarrhea virus hemagglutinating encephalomyelitis coronavirus infection in pigs porcine respiratory coronavirus: molecular features and virus-host interactions a comparative sequence analysis to revise the current taxonomy of the family coronaviridae pathogenicity and pathogenesis of a united states porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets retrospective testing and case series study of porcine delta coronavirus in u.s. swine herds porcine epidemic diarrhea: a review of current epidemiology and available vaccines emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences porcine epidemic diarrhea virus 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gastrointestinal tract of pigs infected with transmissible gastroenteritis mechanisms of porcine diarrheal disease transmissible gastroenteritis in endemically infected breeding herds of pigs in east anglia immunohistochemistry of transmissible gastroenteritis virus antigens in fixed paraffin-embedded tissues monoclonal antibody-based immunohistochemical detection of porcine epidemic diarrhea virus antigen in formalin-fixed, paraffin-embedded intestinal tissues immunohistochemical detection of porcine rotavirus using immunogold silver staining (igss) rnascope: a novel in situ rna analysis platform for formalin-fixed, paraffin-embedded tissues additional file 1: figure s1 . panel of pathogens used as control for detection of tgev by immunohistochemistry. row one include immunostaining of tgev clinical cases against tgev, pedv, and rotavirus specific antibodies. a moderate to severe immunostaining is observe only reacting against tgev. in row two include positive controls for each pathogen detected by immunohistochemistry. row three present section tested negative by pcr for tgev, pedv, and rotavirus that were used as negative control of the immunohistochemistry techniques. ( the authors declare that all the data supporting the findings of this report is available in the article.authors' contributions pep, cjp, maq performed histological examination, data analysis and conclusion and were the major contributors in writing the manuscript; dmm, performed ihc; fv, performed ish; mil, rs, jac, emp, aa, lva field data collection and case identification. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. 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-340202-ikptxviu authors: van diep, nguyen; norimine, junzo; sueyoshi, masuo; lan, nguyen thi; hirai, takuya; yamaguchi, ryoji title: us-like isolates of porcine epidemic diarrhea virus from japanese outbreaks between 2013 and 2014 date: 2015-12-02 journal: springerplus doi: 10.1186/s40064-015-1552-z sha: doc_id: 340202 cord_uid: ikptxviu since late 2013, outbreaks of porcine epidemic diarrhea virus (pedv) have reemerged in japan. in the present study, we observed a high detection rate of pedv, with 72.5 % (148/204) of diarrhea samples (suckling, weaned, and sows) and 88.5 % (77/87) of farms experiencing acute diarrhea found to be positive for pedv by reverse transcription pcr. sequencing and phylogenic analyses of the partial spike gene and orf3 of pedv demonstrated that all prevailing japanese pedv isolates belonged to novel genotypes that differed from previously reported strains and the two pedv vaccine strains currently being used in japan. sequence and phylogenetic analysis revealed prevailing pedv isolates in japan had the greatest genetic similarity to us isolates and were not vaccine-related. unlike vaccine strains, all prevailing field pedv isolates in japan were found to have a number of amino acid differences in the neutralizing epitope domain, coe, which may affect antigenicity and vaccine efficacy. the present study indicates recent pedv isolates may have been introduced into japan from overseas and highlights the urgent requirement of novel vaccines for controlling pedv outbreaks in japan. porcine epidemic diarrhea (ped) is a highly contagious and devastating viral enteric disease characterized by vomiting, acute onset of severe watery diarrhea, and dehydration. ped has a high infectivity and a particularly significant mortality in piglets (pensaert and de bouck 1978) . the porcine epidemic diarrhea virus (pedv), an enveloped, single-stranded rna virus belonging to the alphacoronavirus genus of the coronaviridae family, is responsible for ped. the pedv genome is approximately 28 kb in length and is composed of seven open reading frames (orf) that encode four structural proteins, namely, spike (s), envelope (e), membrane (m), and nucleocapsid (n), and three major non-structural proteins, including replicases 1a and 1b, and orf3 (song and park 2012) . of the structural protein, the pedv s protein plays a pivotal role in regulating interactions with specific host cell receptors to mediate viral attachment and entry. moreover, the s protein is associated with the induction of host neutralizing antibodies, growth adaptation in vitro, and attenuation of virulence in vivo (song and park 2012) . thus, study of the s glycoprotein has been essential in understanding the genetic relationships between pedv strains, the epidemiological status of pedv in the field, and the development of vaccines (song and park 2012; chen et al. 2012) ; (temeeyasen et al. 2014) . in addition to the s glycoprotein gene, the orf3 gene has received a large amount of attention in the aspect of pedv virulence. orf3 gene plays a role in encoding an ion channel protein (wang et al. 2012 ) and it has been suggested to be an important determinant for virulence of this virus (song and park 2012) . the virulence of ped can be reduced by altering the orf3 gene through cell culture adaptation (park et al. 2008) , and variation in orf3 was reported to be associated with viral attenuation in the natural host (song et al. 2003) . also, vaccine-derived isolates with unique continuous deletions of 49 and 51 orf3 nucleotides have been confirmed (chen et al. 2010; park et al. 2008) . therefore, these unique deletions in the orf3 gene can be used to differentiate between field and attenuated vaccine strains. moreover, orf3 gene variation may represent a useful tool in molecular epidemiological studies of pedv (park et al. 2008 (park et al. , 2011 song et al. 2003; chen et al. 2010) . in japan, the first outbreak of ped-like disease was reported in late 1982 and early 1983 (kusanagi et al. 1992; takahashi et al. 1983) , and was followed by pandemics between late 1993 and 1996 (sueyoshi et al. 1995; tsuda 1997) . afterwards, there have been sporadic ped outbreaks in intervals of several years. since late 2013, numerous diarrhea epidemics, suspected to be caused by ped, have occurred in pigs throughout japan. these epidemics were characterized by severe diarrhea, dehydration, and vomiting in pigs of all ages. mortality rates were particularly high among suckling pigs. up to the end of august 2014, more than 410,000 of 1,286,000 pigs from 817 infected farms have died of ped in japan based from the report of ministry of agriculture, forestry and fisheries (maff) (http://www.maff.go.jp). however, there have been few studies investigating the re-emergence of pedv in japan. this study aimed to evaluate the genetic characteristics and molecular epidemiology of the emergent japanese pedv isolates using genome analysis and phylogenetic analysis of the partial s gene and orf3. a total of 72.5 % (148 of 204) of samples (suckling, weaned, and sows) from 77 pig farms (88.5 %) experiencing acute diarrhea in six prefectures were found to be positive for pedv by rt-pcr. pedv-positive samples were identified from the following prefectures: miyazaki (n = 107), kagoshima (n = 9), aichi (n = 15), akita (n = 1), hokkaido = 7), and aomori (n = 9). to investigate the heterogeneity of the recent japanese isolates and their genetic relationship with modified live vaccines, in addition to 2 pedv vaccine strains (p5-v and 96-p4c6) used in japan, representative isolates were selected for sequencing of the partial s gene and full orf3 gene. the partial s gene, including the co-26k equivalent (coe) domain, of 80 pedv samples from 69 pedvinfected farms were amplified, purified, and sequenced. the partial s sequences were aligned at nucleotides 1477-2116 (amino acids 493-705) of the full s gene. identical nucleotide sequences were distinguished and excluded, resulting in the identification of 23 individual sequences from the total of 80 field pedv isolates (table 1) . however, sequencing revealed high genetic variation between nucleotides 1815 and 1944 (amino acid residues 605-648). a total of 20 nucleotide substitutions were detected, leading to 13 amino acid changes, within the partial s gene (fig. 1) . the coe domain (amino acids 499-638) of the s protein consists of 140 aa and contains epitopes that are capable of inducing pedv-neutralizing antibodies (chang et al. 2002) . compared to the two vaccine strains (p-5v and 96-p4c6), all japanese field strains had 3 different amino acids at positions 517 (a → s), 549 (t → s), and 594 (g → s) within the coe domain. furthermore, differences in amino acids were found at the following 10 sites within the coe domain of the s protein: 500 , and 632 (l → f) as shown in fig. 1 . to investigate the heterogeneity of prevailing pedv strains in japan, phylogenetic tree of 23 partial s genes of pedv field strains and two vaccine strains were constructed together with 4 previously reported japanese pedv strains (nk, kh, mk, 83p-5) and reference strains from other countries available in genbank. consistent with previous reports (park et al. 2007; puranaveja et al. 2009; temeeyasen et al. 2014 ), phylogenetic tree based on partial s gene sequences in this study demonstrated that pedv strains can be divided into three groups: g1, g2, and g3. group g1 can be further divided into 3 subgroups: g1-1, g1-2 and g1-3 ( fig. 1) . notably, all field pedv isolates circulating in japan were found to cluster closest to isolates from the usa (usa/ iowa/16465/2013, oh851, iowa106, mn, and ia2, usa/colorado/2013, isu13-22038-ia-homogenate), and us strain-like pedv from south korean (knu-1401, knu-1406-1, knu-1310, knu-1311) collected from 2013 to 2014 ); they were found cluster within the same subgroup g1-1 (fig. 2) . further, isolate 14jm-140 clustered with s indel strains (oh851, iow106, knu-1406), forming a distinct minor branch within the cluster. on the other hand, vaccine strains p5-v and strain nk clustered within group g2, while the vaccine strain 96-p4c6 and old strains mk, kh, and 83p-5 belonged to subgroup g1-2. these results demonstrated distantly genetic relationships between field isolates and vaccine strains, in addition to previously reported pedv strains in japan. pairwise alignment of field japanese pedv isolates demonstrated high nucleotide and amino acid sequence identity between strains (99.1-100.0 and 97.2-100 %, respectively). notably, 42 field pedv isolates collected from 3 prefectures in this study had identical partial s gene sequence (100 % nucleotide homology). japanese to investigate the genetic relationship between recent japanese field isolates, and modified live vaccine strains and reference strains, the nucleotide sequences of the orf3 genes of 28 recent pedv isolates and 2 vaccine samples (p5-v and 96-p4c6) were sequenced and analyzed (table 1) . sequencing data revealed the orf3 genes of all 28 pedv samples were 675 bp in length and (table 1 ). sequence analysis revealed that the orf3 genes of the japanese field isolates were relatively well-conserved. only 5 point substitutions were observed at nt 24, 51, 189, 302, and 501, with only the substitution at nt 302 resulting in a non-synonymous substitution of t to i at residue 100. phylogenetic analyses revealed that, based on the orf3 gene, all pedv isolates could be divided into three groups namely: g1, g2, and g3 (fig. 3) . notably, all japanese field isolates clustered closely with us isolates and recent south korea isolates, forming a separate subcluster within g1. on the other hand, the vaccine strains, p5-v and 96-p4c6, which have been used to prevent pedv infection in japan, were found within g3, which are therefore genetically distant from prevailing field isolates. dna sequence homology of 99.6-100 % was observed between orf3 genes of recent japanese isolates that had the highest dna identity (99.4-100 %) with the commonly observed on ped-vaccinated farms, leading to the loss of high numbers of pigs. as a result, the genetic characteristics and origins of prevailing pedvs in japan, the efficacy of pedv vaccines being used in japan in protecting previously well pigs from prevailing pedvs, and the genetic differences between vaccine strains and field pedv isolates remain critically important questions that have yet to be fully elucidated. we therefore performed this study to address these important issues regarding ped. in this study, 88.5 % (77 of 87) of pig farms in six prefectures (miyazaki, kagoshima, aichi, hokkaido, aomori, and akita) were confirmed as infected with pedv, and 72.5 % (148 of 204) of samples were found to be positive for pedv. this result demonstrates a high prevalence of pedv infection in japanese pig herds. the pedv spike glycoprotein has a high degree of variability and contains several epitopes (chang et al. 2002; sun et al. 2008 ). among these epitope sites, the coe domain (aa 499-638) is an important region capable of inducing pedv-neutralizing antibodies (chang et al. 2002) . in comparison with vaccine strains (p-5v and 96p4c6) commonly used in japan, all the field isolates were found to have 3-7 different residues in the coe domain, particularly at these 3 positions (517, 549, and 594). notably, the amino acid at these three sites were all serine, one of few major amino acids that are capable of generating new o-linked glycosylation or phosphorylation. netphos 2.0 server (http://www.cbs.dtu.dk/services/ netphos) and netphosbac 1.0 server (http://www.cbs. dtu.dk/services/netphosbac-1.0) were used for prediction of phosphorylation site. the result showed that phosphorylation was generated from serine residues at the position 517 and 549 of the field japanese isolates. on the other hand, using bepipred 1.0 server (http://www. cbs.dtu.dk/services/bepipred) to predict the location of linear b cell epitopes, no remarkable difference between the field pedv isolates and the vaccine strains were found. therefore, further research is needed to determine whether these amino acid differences may affect the antigenicity of prevailing pedv isolates and consequently influence the efficacy of the vaccines currently used on japanese pig farms. in may 2013, pedv was detected for the first time in the united states. subsequently, us strain-like pedvs were reported in south korea in late 2013 , and germany in may 2014 (hanke et al. 2015) . the result of phylogenetic and genetic analysis of the partial s gene demonstrated that prevailing japanese pedv isolates had been previously unreported in japan, shared the greatest similarity with us strainlike strains, and may have been introduced into japan via unknown routes. to date, two distinct pedv strain types have been identified in us: the highly virulent us pedv (us prototypes) (stevenson et al. 2013 ) and the s indel pedv variant, which contains insertions and deletions in the n-terminal region of the s protein, reported to cause milder disease in the field (vlasova et al. 2014; . interestingly, isolate 14jm-140 was grouped in the same cluster as the s indel variant (oh851, iow106, knu-1406) with 100 % nucleotide identity observed between these strains. clinical signs recorded on pedv-infected farms demonstrated that only 24 piglets (out of a total of 400 pigs on the farm) had disease manifesting as diarrhea, with 4 piglets dying at the time of ped onset. this finding suggests the japanese field isolate 14jm-140 may, in fact, be the s indel pedv variant. this pedv variant is prevalent and associated with low morbidity and mortality in ped outbreaks in japan, although more extensive genome sequencing is required to clarify this finding. orf3 is an accessory gene thought to influence virulence and cell culture adaptation and has been used as a viral target in attempts to reduce pedv virulence. generally, orf3 has utility as a valuable tool in the study of pedv molecular epidemiology and for differentiating between field and vaccine-derived isolates. our study revealed that the orf3 gene of the japanese vaccine strains, p5-v and 96p4c6, have unique deletions (49 and 4nt, respectively) that can lead to reading frame-shift and coding of truncated polypeptides. this genetic characteristic can be used to differentiate between field and attenuated-derived vaccine pedv. moreover, the finding of 49nt deletion in orf3 gene of p5-v in this study is different from the result of a previous study (park et al. 2008) in which authors reported that p5-v have 51nt deletion in orf3 gene. this difference may be due to the genetic variation of p5-v during the time of the study. the results of the present study demonstrated that no deletion were observed in the japanese field isolates, suggesting that they are not vaccine-related. phylogenetic analysis further demonstrated that japanese field isolates had greatest genetic similarity with us strains. this study demonstrated a high detection rate of pedv on pig farms in japan. all recent japanese pedv isolates were found to have previously unreported genotypes that differed from japanese strains prior to 2013 as well as vaccine strains currently being used in japan and had the greatest genetic similarity with us isolates, as compared with other countries. these findings suggest that prevailing pedv isolates may have been introduced into japan from overseas. the distant genetic relationship and amino acid differences in the neutralizing epitope coe domain between recent pedv isolates and the vaccine strains may be responsible for unsuccessful ped control in japan. therefore, the development of new vaccines with greater protection against pedv outbreaks in japan is required. a total of 204 samples were collected from suckling pigs, weaned pigs, and sows at 87 pig farms (farrow-to-finish and farrow-to-wean) experiencing acute diarrhea from six prefectures from north to south of japan between december 2013 and october 2014. the number of samples from each prefecture was as follows: miyazaki (n = 134), kagoshima (n = 14), aichi (n = 28), akita (n = 3), hokkaido (n = 10), aomori (n = 15). one to 10 fecal samples, intestinal samples, or intestinal contents, were obtained from each outbreak of diarrhea. fecal samples were taken from animals showing signs of diarrhea at the time of collection. samples of intestine and intestinal content were collected from animals that have died due to severe diarrhea within 3 h. all the samples were temporarily preserved in ice boxes at time of collection, kept in icebox containing dry ice during transportation, and stored at freezer (−70 °c) when it arrived at the laboratory of veterinary pathology, univeristy of miyazaki, using cryogenic freezing systems. samples of two vaccine strains, p-5v (produced by nisseiken co., ltd) and 96-p4c6 (produced by kaketsuken co., ltd), were collected from commercial vaccine bottles used in pig farms in japan. specimens from sick pigs were homogenized and diluted five times in dulbecco's modified eagle's medium with a low concentration of glucose. samples were then centrifuged at 5000 rpm for 10 min at 4 °c. supernatants were stored and subsequently used for rna extraction. for each pedv sample, total rna was extracted from 100 to 300 µl aliquots of supernatant using reliaprep ™ rna cell miniprep kits (promega corpoation, wi, usa) in accordance with the manufacturer's instructions. the presence of pedv in samples was detected by reverse transcription polymerase chain reaction (rt-pcr) using a previously published primer pair (kim et al. 2001) . briefly, nucleotide strands of these primers are 5′-ttctgagtcacgaacagcca-3′ (p1, forward), 5′-catatgcagcctgctctgaa-3′ (p2, reverse). the size of amplified product was 651 bp. the amplified genomic region of the s gene contains the neutralizing epitope region, co-26k equivalent (coe) (chang et al. 2002) . one tube rt-pcr reaction was performed using accessquick ™ rt-pcr system kits (promega corpoation, wi, usa). exactly, 4 µl of rna template was mixed with a reaction mixture, which contained 12.5 µl of accessquicktm master mix (2×), 0.5 µl of each specific primer (10 µm), and 0.5 µl of amv reverse transcriptase (5 u/µl). then, 7 µl of nuclease-free water was added to reach the total volume reaction of 25 µl. the rt-pcr reaction were done using takara pcr thermal cycler (japan). following a reverse transcription step of 45 °c for 45 min and an incubation step of 94 °c for 2 min, 35 cycles were performed as follows: 94 °c for 30 s, 53 °c for 30 s, and 72 °c for 1 min. cycles were followed by a terminal 10 min extension step at 72 °c. the last stage was preserving the pcr products at 4 °c. the rt-pcr products were visualized by electrophoresis in a 1.5 % agarose gel containing ethidium bromide. a primer pair was designed for amplifying the full orf3 gene of pedv with the following sequences: forward primer (orf3-f), 5′-gtcctagacttcaaccttac-gaag-3′; and reverse primer (orf3-r), 5′-aactac-tagaccattatcattcac-3′. the predicted size of the orf3 pcr product was 740 bp. for the application of the partial s gene and the orf3 gene of pedv, rt was first performed using random primers and oligodt primers from reverse transcription system kits (promega, madison, wi, usa). complimentary dna was immediately used to amplify the partial s gene (primer pair p1/ p2) and orf3 gene (primer pair orf3-f/orf3/r) using gotaq ® green master mix kits (promega, madison, wi, usa) under the following condition: denaturation at 94 °c for 2 min; 35 cycles of denaturation at 94 °c for 30 s, annealing at 53 °c for 30 s, and extension at 72 °c for 1 min. pcr products were purified using fastgene gel/pcr extraction kits (nippon genetics co., ltd, japan), according to the protocol of the commercial kit's instruction. all sequencing reactions were carried out in duplicate and sequences were determined in both direction with bigdye ® terminator v3.1 cycle sequencing kits and an abi prism 3130xl genetic analyzers (applied biosystems, ca, usa). the resultant nucleotide sequences were deposited in genbank under the following accession numbers: kt968486-kt968518. nucleotide and deduced amino acid sequences were edited, aligned (muscle algorithm), and analyzed using bioedit version 7.2.5 and molecular evolutionary genetics analysis (mega) software version 6.0 (tamura et al. 2013) . phylogenetic trees based on the nucleotide sequences of the partial s gene and orf3 gene were constructed with maximum likelihood method using hasegawa-kishino-yano substitution model with discrete gamma distribution, and bootstrap tests of 1000 replicates in the mega v.6 program. identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus molecular epidemiology of porcine epidemic diarrhea virus in china molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) samples from field cases in fujian, china comparison of porcine epidemic diarrhea viruses from germany and the united states differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex rt-pcr isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate outbreak-related porcine epidemic diarrhea virus strains similar to us strains full-genome sequence analysis of a variant strain of porcine epidemic diarrhea virus in south korea sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea cloning and further sequence analysis of the orf3 gene of wild-and attenuated-type porcine epidemic diarrhea viruses molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea a new coronavirus-like particle associated with diarrhea in swine chinese-like strain of porcine epidemic diarrhea virus porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf 3 emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences an immunohistochemical investigation of porcine epidemic diarrhoea identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan mega6: molecular evolutionary genetics analysis version 6.0 genetic diversity of orf3 and spike genes of porcine epidemic diarrhea virus in thailand porcine epidemic diarrhea: its diagnosis and control distinct characteristics and complex evolution of pedv strains pedv orf3 encodes an ion channel protein and regulates virus production the work was conducted with funding from the university of miyazaki. the authors declare that they have no competing interests. there is no human or animal subject involved in the experiment.received: 29 june 2015 accepted: 24 november 2015 authors' contributions ry, ms, and th collected the samples and epidemiological information as well as pathology findings. nvd and ry designed, conducted molecular tests, and did sequencings. nvd, ry, jn and ntl designed, interpreted the sequencing findings and wrote the paper. all authors prepared the manuscript. all authors read and approved the final manuscript.author details 1 laboratory of veterinary pathology, department of veterinary, faculty of agriculture, university of miyazaki, 1-1 gakuenkibanadai-nishi, miyazaki 889-2192, japan. 2 faculty of veterinary medicine, vietnam national university of agriculture, hanoi, vietnam. key: cord-319460-n4ezxnjc authors: bertasio, cristina; giacomini, enrico; lazzaro, massimiliano; perulli, simona; papetti, alice; lavazza, antonio; lelli, davide; alborali, giovanni; boniotti, maria b. title: porcine epidemic diarrhea virus shedding and antibody response in swine farms: a longitudinal study date: 2016-12-15 journal: front microbiol doi: 10.3389/fmicb.2016.02009 sha: doc_id: 319460 cord_uid: n4ezxnjc the porcine epidemic diarrhea virus (pedv) causes an acute and highly contagious enteric disease characterized by severe enteritis, vomiting, watery diarrhea, and a high mortality rate in seronegative neonatal piglets. in the last few years, ped had a large economic impact on the swine industries in asia and the us, and in 2014, the pedv also re-emerged in europe. two main pedv variants circulate worldwide but only the s indel variant, considered a mild strain, is spreading in europe. to gain insights into the pathogenicity of this variant, its viral load and temporal shedding pattern were evaluated in piglets from infected farms. quantitative real-time pcr (qpcr) targeting the spike gene, was validated according to the minimum information for quantitative real-time pcr experiments guidelines. the qpcr was applied to longitudinal studies conducted in four swine farms naturally infected with the pedv s indel variant. clinical data, fecal swabs, and blood samples were collected from 103 piglets at 15–30-day intervals for 2–5 months. on all four farms, diarrhea was observed in sows during gestation and in farrowing units, and the mortality rates of piglets were 18, 25, 30, and 35%. different clinical pictures (0-50% of diarrhea positivity), viral titer levels (mean 5.3-7.2 log(10) genome copies/ml), and antibody conditions (30-80% of positivity) were registered among sows on the four farms. the percentage of qpcr positive piglets varied greatly from the beginning (63–100%) to the end (0%) of the infection course. clinical signs were present in 96% of the qpcr positive animals. viral loads ranged from 8.5 log(10) to 4 log(10) genome copies/ml in suckling pigs at 3–6 days of age and were not statistically different among farms, despite the different patterns observed in sows. after 2–3 weeks, only a few piglets still showed detectable viral levels and clinical signs, and they developed antibody responses. moreover, co-infections with other pathogens and biosecurity procedures limiting the circulation of the virus could have influenced the severity of ped infection. qpcr and clinical data were useful in understanding the dynamics of pedv infections and, therefore, in implementing appropriate control measures. porcine epidemic diarrhea virus (pedv) causes an acute and highly contagious enteric disease, which is characterized by severe enteritis, vomiting, watery diarrhea and a high mortality rate in neonatal piglets. belonging to the family coronaviridae, genus alphacoronavirus, pedv has a single-stranded, positive-sense rna genome of ∼28 kb that encodes four structural proteins, spike (s), envelope, membrane, and nucleocapsid, and three nonstructural proteins (kocherhans et al., 2001) . in particular, the s protein is important in regulating interactions with specific cell receptor glycoproteins to mediate viral entry, inducing neutralizing antibodies (bosch et al., 2003) , growth adaptation in vitro and in virulence attenuation in vivo (sato et al., 2011) . since 2010, ped has caused large economic losses in the swine industries of asia and north america (usa and canada) (stevenson et al., 2013; kochhar, 2014) . based on the nucleotide sequence of the s1 spike gene (lee, 2015) , two main genetic variants have been detected: a "highly virulent" strain, called "non-s indel (insertions and deletions)" (vlasova et al., 2014; sun et al., 2015) , and a "mild" strain, called "s indel", identified in pigs with mild clinical signs and no mortality (vlasova et al., 2014; wang et al., 2014) . since 2014, in europe, the non-s indel pedv strain has been detected only in the ukraine (dastjerdi et al., 2015) , while the s indel strain has spread throughout many countries including germany, france, belgium, portugal and italy (grasland et al., 2015; hanke et al., 2015; mesquita et al., 2015; theuns et al., 2015; boniotti et al., 2016) . all of the s indel pedv european strains share 99% nucleotide identity with s indel pedv oh851, but in contrast with field observation in the usa , outbreaks with high mortality rates in suckling piglets have been reported in germany and portugal (mesquita et al., 2015) . in italy, ped has been documented since 1997, with a few cases appearing per year. however, between 2005 and 2006, a severe pedv epidemic occurred in italy (martelli et al., 2008) , which was characterized by mortality rates in neonatal piglets of up to 34%, whereas it was very low in adults. during 2007-2012, only sporadic confirmed clinical cases of ped were reported (boniotti et al., 2016) . during this period, two swine coronavirus clades were identified. the first resembled the oldest global pedv strain cv777 (pedv/italy/7239/2009) and the second resembled a new transmissible gastroenteritis coronavirus/pedv recombinant variant (secov/italy/213306/2009). during summer 2014, animals on two farms displaying mild clinical signs were detected as positive for pedv by pcr (boniotti et al., 2016) , and at the beginning of 2015 a new severe epidemic wave occurred (efsa ahaw panel, 2014) . recently, the virulence of the s-indel strain has been evaluated and compared to the non-s indel strain through experimental infections (lin et al., 2015; yamamoto et al., 2015; chen et al., 2016) . in one study, 5-day-old piglets inoculated with an s indel pedv strain (usa/il/2014/20697) did not develop clinical signs and had only mild histopathological lesions (chen et al., 2016) . lin et al. (2015) showed that the virulence of the s indel pedv strain was lower than the non-s indel us pedv based on a longer incubation time, a shorter duration of diarrhea, a lower percentage of infected enterocytes and a lower piglet mortality rate. however, the severity of clinical signs and mortality rates (0-75%) varied greatly among litters inoculated with the s indel strain. these variations were associated with piglet birth weights and the sow's health and lactation status (lin et al., 2015) . to evaluate differences in morbidity and the virulence of the s indel strains, field data are extremely important, as many concurrent factors, not reproducible in an experimental infection, can determine the infection severity. presently, the limited available field observations mesquita et al., 2015; stadler et al., 2015) indicated that the virulence level of s indel pedv varied greatly. the different immunological states of infected piglets, due to lactogenic immunity, is a likely explanation of such variations. viral load is an important indicator in evaluating the virulence of a strain and the susceptibility of infected animals, and in understanding the mechanisms of viral transmission and circulation within the different farm units. at the moment, very limited data have been published on the pedv viral load during an outbreak under field conditions (bjustrom-kraft et al., 2016) . in this study, we describe acute outbreaks of ped in three farrow-to-finish and one farrow-to-wean farms in northern italy. we conducted a longitudinal study by sampling the feces and blood of piglet groups from each farm at fixed intervals during a 2-5 months period, and then we determined pedv shedding and the antibody presence. in particular, the quantification of pedv in fecal samples was used to better describe the infection dynamics under field conditions. three farrow-to-finish and one farrow-to-wean farms located in the north of italy were chosen for this study, which occurred from january to may 2015. farms were selected based on the following criteria: location in the high density swine production area of the po valley, a short distance from the diagnostic laboratory to facilitate the conservation of samples and quick delivery, sudden onset of enteric clinical signs, mortality in newborn piglets clearly referable to ped and an absence of an anamnesis of ped on the farm. in table 1 , data on the four farms are summarized. the ped clinical and epidemiological characteristics, as well as the disease course, for the four farms are recorded in supplementary table 1 . at each sampling time, clinical evaluations of the farms were determined by vets from our institute, recording the percentages, in four ranges, low (0-5%), medium (6-20%), high (21-50%), and very high (>50%), of diarrheic pigs among the different farm units (supplementary table 2 ). fecal consistency was visually evaluated and scored using the following criteria: 0 = normal, 1 = formed and soft, 2 = semi-solid, 3 = watery, and 4 = presence of mucus and blood. the maximum observed fecal score attributed to each farm unit was recorded at each sampling time and reported in supplementary table 2. at 3-28 days from the first appearance of clinical signs on the four farms (f1−f4), 10 sows per farm, and 19, 24, 30, and 30 newborn piglets from f1, f2, f3, and f4, respectively, were selected from symptomatic litters, identified by ear tag and sampled every 2 weeks (samplings 1-3), 4−5 weeks (samplings 4−5) and after 7 weeks (sampling 6). the number of sampled animals decreased as the study continued due to mortality and animal sales (supplementary table 3 ). the sows were sampled only once at the beginning of the study. the presence of diarrheic piglets was recorded at each sampling time (supplementary table 3 ). fecal and blood samples were collected from sows, whereas blood and rectal swabs were collected from piglets. feces were diluted 1:10 (w/v) in minimum essential media (mem). rectal swabs were suspended in 1 ml of mem, vortexed and incubated at 4 • c for 30 min to allow the release of the feces from the cotton. fecal suspensions were clarified by centrifugation for 10 min at 4,000 × g to eliminate fecal debris. viral rna was extracted from 200 µl of sample using a commercial kit (nucleomag r vet kit, macherey-nagel, düren, germany), according to the manufacturer's instructions. an exogenous internal control rna (ic) (qiagen, hilden, germany), was added to specimens prior to rna extraction to verify the success of the procedure and the absence of inhibitors. the extraction was carried out on the biosprint 96 instrument (qiagen) using the nucleomag vet 200 protocol. nucleic acids were eluted into 100 µl of elution buffer and immediately subjected to rt-pcr or stored at −80 • c until used. extracted viral rna was subjected to a "one-step" rt-pcr assay using the commercial quantifast pathogen rt-pcr kit (qiagen) with primers and probe targeting the s1 gene of pedv that were previously developed at the university of minnesota veterinary diagnostic laboratory (2014), and are reported in table 2 . the optimum concentrations of primers and probe were deduced by titration experiments and are reported in table 2 . pcr reactions were performed on 5 µl of extracted viral rna in a final volume of 25 µl, which also contained 5 µl of 5× pcr-master mix, 2.5 µl of a 10× internal control assay, 2.5 µl of each forward and reverse primer (final concentration 500 nm), 0.5 µl of ped_s probe (final concentration 200 nm), and 0.25 µl of enzyme mix. the pedv rna was reverse transcribed at 50 • c for 20 min, followed by 1 cycle of taq polymerase activation at 95 • c for 5 min. amplification consisted of 45 cycles at 95 • c for 15 s and 60 • c for 30 s. amplification were performed on a cfx96 touch real-time pcr detection system (bio-rad laboratories) and data were analyzed with the software bio-rad cfx manager 3.1, using the single threshold method for the cq determination. an experiment was accepted when the cq of the "no template control" (ntc) was >40 and the ic of a negative control was <35. we cloned a fragment of the s1 gene (nucleotide positions 1503-2153; genbank dq985739.1) into a pcr r 2.1-topo r vector (topo ta cloning r kit, invitrogen) according to the manufacturer's instructions. plasmid dna was linearized by restriction enzyme digestion and then subjected to transcription using the ribomax large scale rna production system (promega) to produce an rna transcript. the rna concentration was determined using an infinite r 200 nanoquant spectrophotometer (tecan). a 10-fold serial dilution in nuclease-free water of ssrna transcripts (2 × 10 6 -2 × 10 2 copies/µl) was used to generate a standard curve and to quantify pedv rna in the samples. triplicates of each dilution were run in each assay. the following equation: where x represents the genome copies/µl, was used to transform the samples' cq values into estimates of genome copies of pedv rna per ml of fecal homogenate. the qpcr assay used to quantify pedv rna was validated according to the minimum information for quantitative realtime pcr experiments guidelines (bustin et al., 2009) . validation results are summarized in table 3 . to ascertain the specificity of the qpcr used in this study, we tested eight samples that were negative for pedv and positive for different viral agents, including the limit of detection (lod) and limit of quantification (loq) were determined by testing 10 replicates of a 10-fold serial dilution of ssrna transcript, from an initial concentration of inhibition assay 3.15 cycles r 2 , linear correlation index; lod, limit of detection; loq, limit of quantification. 2 × 10 5 to a final concentration of 2 × 10 −2 genome copies/µl (supplementary table 4 ). the lod was calculated as the lowest concentration at which 100% of the positive samples are detected. the loq was defined as the lowest concentration of viral rna that can be determined with acceptable precision (relative standard deviation ≤25%) under the stated conditions of the test. the linear correlation index (r 2 ) and the slope of the calibration curve were calculated using mean values from three replicates of four different runs. the reaction efficiency (e) under our experimental conditions was determined by the following formula: the dynamic range was determined by testing three replicates of a 10-fold serial dilution of ssrna transcript, from an initial concentration of 2 × 10 8 to a final concentration of 2 × 10 0 genome copies/µl (supplementary figure 1) . repeatability was evaluated by analyzing three pedv positive samples in triplicate in the same extraction and qpcr run (supplementary table 5 ). intra-assay variance was expressed as the range of the relative standard deviation (rsd%) associated with copy number/µl. reproducibility was determined by testing three pedv-positive samples having different concentration levels. samples were extracted and quantified on four different days (supplementary table 6 ). inter-assay variance was expressed as mean rsd% with minimum and maximum values of copy numbers at each concentration level, calculated for the different concentration levels. to verify the absence of contaminants that can cause the inhibition of qpcr assays after the rna extraction procedure, the extracted rnas of 10 specimens were diluted 1:10 (v/v) in water and subjected to qrt-pcr. inhibition was evaluated by calculating the differences in mean cq values between diluted and undiluted samples. the s1 gene sequences of pedv positive samples on farm 1, were obtained from one sow and one piglet at the first sampling and from three piglets at 61 days of age, as described by boniotti et al. (2016) (accession number: ky009940-ky009941). an in-house pedv whole virus elisa was used. in brief, a pedv strain cv777-based elisa was developed and validated at izsler based on the previously described double-antibody sandwich elisa protocol (sozzi et al., 2010) . the elisa microplates were coated with the 1f12 capture monoclonal antibody (mab). serum samples diluted 1:2 or 1:4 were mixed with equal volumes of whole pedv inactivated with ß-propiolactone and pre-incubated in an auxiliary microplate for 1 h at 37 • c. then, 50 µl of the pre-incubated mixtures were transferred into the 1f12 mab-coated plate and the conjugated horseradish peroxidase mab 4c3 was added. following a further 1h incubation at 37 • c, the plate was washed. the colorimetric reaction was performed, and optical densities (od) were measured at 492 nm using an elisa plate reader. results were calculated by determining the absorbance value reduction, expressed as percentage of inhibition (pi) using the control wells as the reference. the antibody-blocking reaction was considered positive if the pi was ≥60%. for differential diagnoses and to investigate concomitant infections, fecal and serum samples were further investigated to detect transmissible gastroenteritis coronavirus, porcine deltacoronavirus, swine rotavirus a, b, c, and h, porcine reproductive and respiratory syndrome virus; influenza a virus, escherichia coli, clostridium perfringens, and salmonella typhimurium, as previously described (kim et al., 2001; marthaler et al., 2014a,b) . statistical analyses were performed on quantitative data from fecal specimens of the first sampling both in sows and piglets, using the kruskal−wallis test and dunn's multiple comparisons test (graphpad instat prism 6.0 software). at the beginning of the s indel pedv epidemic wave in northern italy (january 2015), four farms with symptoms referable to ped were selected for a longitudinal study. farm production type, onset of outbreak, mortality rate in suckling pigs and sampled animals for each farm, were described in table 1 table 2 ). watery diarrhea with presence of mucus and blood was observed in piglets of the four farms, but it was also present in gestation and farrowing sows. growing and fattening animals from f1, f2, and f3 also showed severe clinical signs including watery diarrhea and anorexia. vomiting was observed in fattening animals on f1. dehydration was observed in litters on f1 and f3 and cachexia in litters on f1 and f4. agalactia was present in the sows from f1, f2, and f3. for differential diagnoses and to investigate concomitant infections, which potentially could impact the evolution of clinical signs and the course of the disease, fecal and serum samples were further investigated to detect other viral and bacterial pathogens. rotavirus a was present in both the sows and piglets of f3, e. coli, expressing the virulence factor f4, in f2 and f3, c. perfringens in f1 and f3, and s. typhimurium in f3. however, no clinical signs, referable to a specific enteric disease, were present before the beginning of the longitudinal study. symptoms at the first sampling, watery diarrhea or soft diarrhea was observed in 30% (3/10), 0% (0/10), 50% (5/10), and 40% (4/10) of the sows from f1, f2, f3, and f4, respectively (table 4) . at the same time high percentages (68-100%) of 3-6-day-old piglets were observed to have diarrhea on all of the farms ( table 5) . clinical symptoms in piglets progressively disappeared at the following sampling times. however, a second outbreak of diarrhea was observed in 3 out of 16 animals (19%) at two months of age on f1. samples were only taken from sows at the initial sampling time, which occurred at 7, 28, 3, and 19 days after the onset of symptoms on f1, f2, f3, and f4, respectively. pedv rna was detected in 8/10 sows on f1, 8/8 sows on f2, 10/10 on f3 and 7/10 on f4 ( table 4 ). the highest fecal pedv rna shedding titers were detected on f2 and f3 (8.3 and 7.6 log 10 genome copies per ml of fecal homogenate), with a mean titer among shedding animals of 6.5 and 7.2 log 10 copies/ml, respectively ( figure 1a) . mean lower titers were observed in f1 and f4 animals (5.3 and 5.4 log 10 copies/ml, respectively) where the highest titers were 6.4 and 6.5 log 10 copies/ml, respectively. the fecal viral shedding in piglets from the four farms is summarized in table 5 and figure 1b . all of the farms had a similar virus-shedding pattern, with high percentages of pedv pcr positive 3-6-day-old animals (sampling 1) that decreased in 14-18-day-old animals (sampling 2) and was no longer present in 30-36-day-old animals (sampling 3) to the end of the study period (table 5) . furthermore, on f1, a second peak of viral shedding was detected in weaning piglets at 61 days of age (sampling 4). two weaning piglets did not show detectable viral shedding in the previous samplings. interestingly, the presence of different strains was assessed by sequencing the s1 gene of positive samples collected at the first and forth samplings. two genetic variants with a single amino acid substitution (156e > g) were detected at samplings 1 and 4. on f2, 87.5% of 3-day-old piglets were pcr positive, four of them (16.7%) were positive at 18 days of age (sampling 2) and only one animal was still positive at 32-days old (sampling 3). moreover, one pig showed intermittent pedv shedding, being pcr positive at the first sampling (3 days after birth), negative at 18-and 32-days old (samplings 2 and 3, respectively) and again positive at 67-days old (sampling 4) (supplementary table 3 ). on f3, all of the 4day-old piglets were pcr positive using rectal swab samples but only three of them remained positive at 17-days old (sampling 2). at the following sampling times, when they were 36-and 72days old, they were all pcr negative. on f4, 86.7% of 3-day-old piglets were pcr positive but viral shedding was not detected in 30-day-old piglets through the end of the study. the highest fecal pedv rna shedding titer was observed in 3-6 day-old piglets with mean values (among shedding animals) of 5.9, 5.6, 5.6, and 6.2 log 10 copies/ml on f1, f2, f3, and f4, respectively ( figure 1b; supplementary table 3 ). no significant differences were statistically evident among the farms. the titer values observed in 14-18-day-old piglets were 5.9 (one animal), 4.8 (mean value of two animals), 4.9 (mean value of three animals) log 10 copies/ml on f2, f3, and f4, respectively (supplementary table 3 ). pedv-antibodies were detected in 8/10, 7/10, 3/10, and 3/9 sows on f1, f2, f3, and f4, respectively ( table 4 ). in newborn piglets, pedv-antibodies were detected in 26, 4, 0, and 20% of animals from f1, f2, f3, and f4, respectively ( table 5 ). in total, 54% of the sows had anti-pedv antibodies at delivery but only a few piglets (3%) showed detectable antibodies and a lack of clinical signs at 3-6 days of age. most of the piglets on the four farms developed antibody responses within 3 weeks of age, and they remained stable until the end of the study (60-100 days of age). in the last few years, ped had a large economic impact on the swine industries in asia and the us, and in 2014, the pedv +, presence/positive; −, absence/negative; na, not available (missing sample). *animals died or were not available (na). the number of sampled animals decreased as the study continued due to mortality, earmarks lost (na a ), and animal sales (na b ). also re-emerged in europe. two main pedv variants circulate worldwide but only the s indel variant, considered a mild strain, is spreading in europe. during summer 2014, animals with mild clinical signs on two farms were detected as pcr positive for pedv in northern italy (boniotti et al., 2016) . thereafter, a new severe ped epidemic wave occurred. to gain insights into the pathogenicity of this variant, we described the results of a longitudinal study conducted during acute outbreaks of ped in three farrow-to-finish (f1−f3) and one farrow-to-wean farm (f4), occurred in the beginning of 2015. on the four farms, the mortality rates of suckling piglets were high (25, 30, 35 and 18% on f1, f2, f3 and f4, respectively) ( table 1) . however, they did not reach the percentages observed in the us, caused by the original pedv strain (>50%) (alvarez et al., 2015) . moreover, the four farms showed high percentages (21−50, >50%) of diarrheic animals, in particular, in all the units of the f1, in piglets of f2 and in sows and piglets of f3 and f4, but differences among farms were also observed. the course of ped was particularly severe on f1, where we observed a high percentage of animals with diarrhea in suckling, weaning and fattening animals (supplementary table 2 ). moreover, the virus appeared to circulate longer on this farm since positive animals were detected even 60 and 100 days after the first pedv detection within the farm. in particular, at 60 days of age a second peak of viral shedding was observed in three animals. the s1 gene sequences of the strain identified in these animals showed a the different patterns and severity of clinical signs observed on the four farms may have resulted from the concurrent effects of co-infections with other pathogens. in particular, rotavirus, with a well-known pathogenic aptitude, which is mainly dosedependent, could have had a synergistic effect with pedv on f3, causing a slight change in the course of the disease in sows and litters after the first week, and predisposing animals to secondary infections, such as those caused by e. coli f4, c. perfrigens, and s. typhimurium. in this study, we determined the fecal pedv shedding in sows and in their piglets from 3 to 6 days to 60−100 days of age. in particular, we selected groups of 10 sows and 19-30 piglets on each farm. none of the farms had reported pedv infections prior to this study, and thus, we can surmise that the sows had been recently infected at the time of their enrollment in the study, while piglets were infected after birth, through contact with infected sows. more than 80% of the sows showed detectable levels of pedv at the first sampling but only the 30% showed diarrhea. the severity of clinical signs caused by pedv is agedependent (jung et al., 2016) . in adult animals, clinical signs are usually milder or completely absent. moreover, in our study, pedv exposure dose and the gestational stage of the sows could have influenced their health status at the moment of delivery in terms of clinical signs, viral loads and pedv antibody level. on f1 and f2, where the onset of pedv symptoms occurred in the fattening unit, 8 and 7 out of 10 sows, respectively, had pedv antibodies. on f3, where the onset of pedv symptoms occurred during the gestation period, 5 out of 10 sows had diarrhea but only 3 had already developed pedv antibodies. on this farm, all of the sows showed high mean viral loads (7.2 log 10 copies/ml) as determined by pedv rna. sows on f3 probably had less time to develop antibodies and transmit them to their litters through the colostrum. in fact, all of the piglets were infected and showed high titers of pedv rna in fecal materials. similarly, on f4, where the onset of the outbreak took place in the delivery room, 3 out of 9 sows showed detectable levels of pedv antibodies. at the first sampling, a high percentage (63-100%) of the 3-6-day-old piglets from all of the farms were positive by qpcr. despite the different immunity levels among the sows on the four farms, and the different proportions of infected piglets, the quantitative pedv rna results in piglets were not statistically different among farms. as proposed by other authors, we assumed that once the pigs were infected and viral replication began, the initial viral dose appeared to have little impact, at the group level, on the average amount of fecal shedding (thomas et al., 2015) . at 2 weeks of age, the proportion of pcr positive piglets decreased to 10-22.7%, and at 1 month of age only one animal was positive. the pedv rna titer also decreased with time, confirming that ped is characterized by high pedv fecal shedding titers a few days post infection and the titers tend to decrease after 1 week. intermittent shedding can be observed until 60 days post infection, as shown in f1 and f2, and such variations in excretion levels and viral loads could be determined by poor management and a lack of biosecurity measures, but it could also be due to a new introduction of the virus, as likely occurred on f1. for immunity responses, 100% of the animals on f1 showed detectable levels of pedv antibodies up to the end of the study (145 days of age). on f2, f3, and f4, the percentage of seropositive animals decreased slightly starting from 80 days of age. the longer and biphasic circulation of the virus on f1 might have favored subsequent exposures of the animals to the virus, and thus, a more durable immunity. the likely outcome of a massive outbreak of pedv within one herd is a diffuse and long-lasting immunity. indeed, especially in farrow-to-finish farms, such herd immunity and, particularly, the maintenance of seropositive sows could be an important tool to prevent severe cases of ped in newborn piglets by transferring passive maternal immunity with colostrum. goede et al. (2015) reported that durable lactogenic immunity was present in sows previously exposed (7 months) to a s indel strain of pedv and that this immunity induced cross-protection to an original virulent pedv. cumbersomely, in this study we did not plan to investigate the presence of maternal antibodies (igg and iga) in the colostrum or to register the piglets' birth weight, two important factors influencing the protective maternal immunity. however, considering the high percentage of diarrheic and pedv positive piglets, and the presence of clinical signs in animals of different ages, including sows, we can suppose that these farms were originally naïve with regard to pedv infection, and thus the lactogenic antibodies were not present at all, or anyway sufficient to effectively protect piglets from infection. thus, future management of pedv infection in farrow-to-finish or farrow-to-wean farms cannot disregard to periodically check and evaluate the iga and igg titers in sow sera and in the colostrum. determining the viral loads and shedding rates of pedv in real field situations during outbreaks is important in evaluating the virulence of a strain and in predicting the susceptibility of infected animals, at different ages and in the various farm units, within a herd. at the moment, very limited data have been published on the pedv viral load during an outbreak under field conditions (bjustrom-kraft et al., 2016) . the qpcr assay used in this study was validated according to the minimum information for quantitative realtime pcr experiments guidelines (bustin et al., 2009; table 3 ; supplementary table 7 ). the method validation is an important requirement to allow the comparison of quantitative results from different studies, which would be otherwise difficult to achieve. moreover, understanding the mechanisms of viral transmission and circulation within the farm can be useful in implementing appropriate control measures on the farm to limit the infections spread. considering the ability of pedv to be dispersed rapidly through fecal contamination, particular attention should be paid to biosecurity and hygiene measures, such as the proper disinfection of equipment and sites, manure disposal, suitable procedures for the movements of animals within the farm, and the use of disposable clothes and shoes for personnel, staff and visitors. longitudinal field studies examining natural infections are comparatively uncommon amongst reports of pedv in comparison to the several experimental studies already performed. in fact, many unforeseen events can adversely affect the success of this kind of study: dead of animals, lost of the earmark, selling or moving of the animals due to unexpected need of the farmer. these practical hitches could negatively influence the sampling procedures (e.g., respect of time points) and data registration. therefore, by planning these "observational" studies, so tight inclusion criteria cannot be established and limited field data could be successfully registered. on the other hand, the longitudinal studies, directly conducted in natural outbreaks, are inclusive of the several "farm factors and field effects" which are very difficult to reproduce in experimental trials, and thus, the obtained results, being more reliable and adherent to real conditions could be effectively used in risk analysis and for defining control strategies. in conclusion, longitudinal studies conducted under field conditions during and after a ped outbreak could be useful in determining the level of immunity acquired at the herd level and may be integrated with the data acquired from experimental infections. they provide an added value, in the possibility to study and evaluate the effects of cofactors, such as other infectious agents, and management and environmental conditions, on the evolution and epidemiology of the disease. the study was exempt of ethical approval procedures because animal samplings were performed during the 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evaluation of a duplex real-time rt-pcr for detection and differentiation of virulent and variant strains of porcine epidemic diarrhea viruses from the united states isolation and experimental inoculation of an s indel strain of porcine epidemic diarrhea virus in japan we thank anna mangeli for the skilled technical assistance and federico scali for the statistical analysis. thanks also to science docs for the english language editing of this manuscript. the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.02009/full#supplementary-material key: cord-354052-x4ckzw64 authors: li, chunhua; li, zhen; zou, yong; wicht, oliver; van kuppeveld, frank j. m.; rottier, peter j. m.; bosch, berend jan title: manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination date: 2013-08-02 journal: plos one doi: 10.1371/journal.pone.0069997 sha: doc_id: 354052 cord_uid: x4ckzw64 porcine epidemic diarrhea virus (pedv) causes severe economic losses in the swine industry in china and other asian countries. infection usually leads to an acute, often lethal diarrhea in piglets. despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. we have established a reverse genetics system for pedv based on targeted rna recombination that allows the modification of the 3′-end of the viral genome, which encodes the structural proteins and the orf3 protein. using this system, we deleted the orf3 gene entirely from the viral genome and showed that the orf3 protein is not essential for replication of the virus in vitro. in addition, we inserted heterologous genes (i.e. the gfp and renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the orf3 gene. we demonstrated the expression of both gfp and renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. the new pedv reverse genetics system will enable functional studies of the structural proteins and the accessory orf3 protein and will allow the rational design and development of next generation pedv vaccines. porcine epidemic diarrhea virus (pedv) causes diarrhea and dehydration in newborn piglets. the virus infects the epithelial cells of the small intestine resulting in severe mucosal atrophy and consequent malabsorption. pedv is common and the cause of serious problems, particularly in pigs in asia. the disease usually appears in winter during which it can cause high fatalities in suckling piglets (see for a recent review [1] ). from 2010, an outbreak of pedv has swept china with over 1 million fatalities among newborn piglets causing substantial economic losses in the swine industry [2] . the characteristics of the infection and its epidemiology were quite dramatic with morbidity and fatality approaching 100% in one-week old piglets, despite the use of commercial, inactivated vaccines. virus transmission occurs via the fecal-oral route and possibly also by vertical transmission through lactation [2] . currently there is no efficient way of treatment of the disease. prevention of the infection usually relies on vaccination with cell culture adapted live-attenuated or inactivated viruses although the efficacy of current vaccines has been questioned [2, 3] . pedv belongs to the alphacoronavirus genus within the coronavirinae subfamily of the coronaviridae family. coronaviruses are important pathogens of concern for human and animal health. they occur in almost any species, usually causing respiratory or intestinal infections. interest in these viruses has increased significantly as a result of the sars epidemic in 2002 and 2003. coronaviruses are enveloped viruses and possess a positive-sense rna genome ranging from 26 to 32 kilobases, which is the largest viral rna genome known (fig. 1a) . the 59 two-third of the viral genome contains two large open reading frames (orfs), 1a and 1b, which encode two non-structural polyproteins, pp1a and pp1ab, that direct genome replication and transcription. the remaining part of the genome contains orfs specifying structural and non-structural proteins. they are expressed via a 39-terminal nested set of subgenomic messenger rnas, the transcription of which is regulated by conserved six-nucleotides transcriptionregulating sequences (trss; in pedv xua(a/g)ac [4] ). these subgenomic mrnas encode at least four structural proteins, three membrane anchored proteins called the spike (s), membrane (m) and envelope (e) protein, and the nucleocapsid (n) protein that encapsidates the genomic rna. the non-structural proteins expressed from the subgenomic mrnas encode one or more accessory proteins, which are specific for each coronavirus genus. the genome structures of alphacoronaviruses including pedv and related members such as the human coronavirus (hcov) strains 229e and nl63 show the typical set of essential core genes but they share only one accessory gene, orf3, located between the s and the e gene (fig. 1a) . the pedv orf3 gene encodes a 224 amino acids (aa) long protein with three to four predicted transmembrane domains [5] . entry of coronaviruses into their host cells is mediated by the approximately 200 kda large s glycoprotein. trimers of s form the characteristic spikes on the viral surface which interact with the host receptor and mediate membrane fusion. pedv was reported to utilize the porcine aminopeptidase n as a receptor [6] . yet, pedv is usually propagated in vero cells, which are derived from the african green monkey kidney, indicating that pedv can utilize non-porcine receptors for cell entry. propagation of pedv in cell culture requires addition of trypsin which is believed to prime or activate the s protein for membrane fusion during virus cell entry and syncytia formation [7] recently it was demonstrated that trypsin cleavage may also play a role in detachment of the virus from infected cells [8] . interestingly, a cell culture adapted strain was reported to replicate in the absence of trypsin [9] , which suggests that the virus acquired mutations in the s protein conferring its trypsin-independence. the s protein also stimulates the induction of neutralizing antibodies and hence is an important target in developing effective vaccines. research on the molecular biology and pathogenicity of pedv has been severely hampered by the lack of a reverse-genetic system. here we report the first reverse genetic system for pedv based on targeted rna recombination. establishment of the reverse genetic system included two stages (fig. 1b) . one was the generation of the chimeric virus mpedv, a pedv derivative carrying spikes derived from the murine coronavirus mouse hepatitis virus (mhv), hence growing only in murine cells. in the second stage the mpedv virus was used as a recipient virus to reintroduce the pedv spike along with other genome alterations, in casu the deletion of the orf3 gene or the insertion of foreign, reporter genes. the generated pedv derivatives now carrying again pedv spikes could be easily selected by their regained tropism for non-murine cells. to set up a targeted rna recombination system for pedv we first created a recombinant pedv virus carrying mhv spikes (mpedv). to this end a transfer vector p-mpedv was construced ( fig. 2a) that was composed of a 59-terminal genomic cdna fragment ligated to a cdna representing the entire 39-terminal part of the genome starting within orf1b, except for the s gene. this gene was replaced by a hybrid gene encoding a chimeric s protein composed of the 1,263 aa long ectodomain from mhv s and the transmembrane domain plus cytoplasmic tail (61 aa) from pedv s. rna was transcribed from the t7 promotor of this vector and electroporated into pedv-infected vero cells after which the cells were overlaid onto a murine cell (l cells) monolayer. the recombinant mpedv virus generated during subsequent incubation was cloned by two rounds of plaque selection on l cells. the identity of purified mpedv viruses was checked at a genetic level by rt-pcr sequencing of the orf1b-s gene junction (data not shown) and at the protein level by an immunofluoresence assay (fig. 3a ). all mpedv infected cells stained positive both with the polyclonal mhv serum and with the monoclonal antibody directed against the pedv nucleocapsid protein confirming the purity and the identity of the chimeric virus. in contrast to the parental virus, mpedv displayed the ability to induce syncytia in the absence of trypsin (fig. 3a) . as predicted, cell-cell fusion mediated by mpedv could be inhibited by a mhv s specific, peptidic fusion inhibitor (fig. 3b) . the generated mpedv virus was used as a recipient virus to reintroduce by similar procedures the pedv spike along with other genome modifications by targeted rna recombination. candidate recombinant viruses carrying the pedv spikes can be selected by their regained ability to replicate in vero cells. apart from the wild-type recombinant virus (r-wtpedv) we aimed at constructing a virus lacking the orf3 gene (pedv-dorf3). a number of cell culture adapted viruses including the strain used in this study have each acquired during passaging an identical 51 nucleotide in-frame deletion in the orf3 gene, giving rise to a 17 amino acid deletion (aa 82-98) in their orf3 protein [10] . we constructed a transfer vector (ppedv-dorf3, fig. 2a ) from which the entire orf3 gene was deleted. donor rnas transcribed from the ppedv and ppedv-dorf3 transfer vectors were electroporated into mpedv-infected l cells after which we were able to recover and purify the r-wtpedv and pedv-dorf3 viruses in vero cells. rt-pcr analysis confirmed the intended loss of the orf3 gene from the viral genome ( fig. 4a ) and the genetic identity of the orf3 lacking virus was further verified by sequencing of the rt-pcr product (data not shown). the pedv-dorf3 grew unimpaired in cell culture (fig. 4b) , demonstrating that the orf3 gene product is not required for virus propagation in vitro. in addition, the successful deletion of the orf3 gene from the viral genome demonstrated the feasibility of the mpedv-based targeted rna recombination system to manipulate the 39 end of the viral genome. we next explored the possibilities of expressing heterologous proteins from the pedv genome by inserting reporter genes at different genomic positions. transfer vectors were made with the renilla luciferase gene (936 nt) and the gfp gene (720 nt) at the position of orf3, creating the ppedv-dorf3/rluc and ppedv-dorf3/gfp vectors ( fig. 2a) . these marker genes are under the transcriptional control of the trs of orf3 (ctagac) which is located in the 39end of the s gene, 46 nucleotides upstream of the orf3 gene. the renilla luciferase gene was also inserted as an extra expression cassette between the orf1b and s gene, creating the ppedv-rluc vector. to this end the otherwise overlapping orfs 1b and s were first separated and a unique bamhi restriction site was introduced (p-rpedv vector, fig. 2a and b), which did not hamper the generation of a viable virus (data not shown). the renilla luciferase gene was subsequently cloned into the bamhi site of the p-rpedv vector under control of the trs in orf1b (gtaaac) originally driving s gene expression, whereas the s gene was provided with a new trs (gtaaac; fig. 2b ). the pedv-dorf3/gfp, pedv-dorf3/rluc and pedv-rluc recombinant viruses were successfully recovered by the targeted rna recombination procedure. rt-pcr analyses confirmed the insertion of both reporter genes at the intended positions ( fig. 5a) , which was further confirmed by sequencing. we studied the luciferase expression by the 2 recombinant viruses carrying a rluc gene as well as the expression kinetics of one of these viruses, pedv-rluc, upon infection of vero cells at three different moi's. the result shows (fig. 5b ) that luciferase expression levels were linearly related to the moi during the early phase of infection until 12 hours p.i. whereas at 24 hours p.i. luciferase values converged due to reinfections. similar kinetics of luciferase expression, but to higher levels, was observed for the pedv-dorf3/rluc recombinant virus (fig. 5b ). next we studied the gfp expression of the pedv-dorf3/gfp virus upon infection of vero cells at two moi's. gfp expression in pedv-dorf3/gfp virus infected cells could be seen starting from 9 hours p.i. and became clearly evident at 12 hours p.i. (fig. 5c ). the cell adapted pedv dr13 p100 strain can propagate in the absence of trypsin in the growth medium but does not form syncytia when trypsin is absent. yet the clustered appearance of gfp-positive cells suggests that the virus predominantly spreads locally from cell to cell which may correlate with the reported cell surface attachment of progeny viruses released from infected cells in the absence of trypsin [11] . the early detection of the luciferase and gfp reporter proteins during infection can be applied to develop a more rapid pedv neutralization diagnostic test. the readout of the classical virus neutralization assay with wild-type pedv is based on the visual inspection of cytopathic effect and can only be done after a multicycle infection which takes at least 2-3 days. thus, the pedv-dorf3/gfp and pedv-dorf3/rluc virus were preincubated with dilutions of serum obtained from an experimentally pedv-infected pig and control serum, and the mixtures were subsequently added to vero cells and incubated after which the gfp and renilla luciferase expression was recorded at 9 and 6 hours p.i., respectively (fig. 5d) . in contrast to the control serum, the pedv antibody-positive serum was able to neutralize pedv infection as reflected by the reduction of gfp positive cells and luciferase activity. the results demonstrate that neutralization of the pedv-dorf3/gfp and pedv-dorf3/rluc virus can already be scored within a single replication cycle, thereby significantly speeding up the assay time. this type of assay is additionally preferred as it avoids the subjectivity that is associated with scoring of cytopathic effects. here we describe the first reverse genetics system for pedv. as we illustrate, this system now enables the manipulation of the 39proximal ,8 kilobases of the pedv genome including the structural protein genes. generation of pedv recombinants was based on the well-known high efficiency of rna recombination of coronaviruses in combination with host cell tropism switching for selection of the recombinant viruses. similar recombination systems have been successfully developed for mhv and fipv coronaviruses by the masters and rottier laboratories [12, 13] . for a number of coronaviruses genetic engineering of the full length genome has also become accomplished by the development of infectious cdna clones [14] [15] [16] [17] [18] [19] [20] . the ability to manipulate the pedv genome will be extremely valuable to study the molecular and biological features of pedv infections as well as to develop new tools and strategies for prevention and therapy of this important veterinary pathogen. unlike most other coronaviruses, the pedv genome contains only a single accessory gene, the orf3 gene, which encodes a multispanning 224-aa long membrane protein. intriguingly, propagation of pedv isolates in tissue culture cells readily leads to deletions within orf3 suggesting a dispensable role, at least for the parts deleted from the orf3 protein, for viral replication in vitro. in all these adapted viruses a shorter orf3 gene product is still translated with a minimal size of 91 amino acids [10] . the orf3 gene of the cell-adapted dr13 vaccine strain (genbank accession no.: jq023162.1) employed in our study has a 49 nucleotide deletion compared to that of the parental dr13 virus (genbank accession no.: jq023161.1), but still encodes the nterminal 81 residues long orf3 protein part including the first transmembrane domain, after which it gets out of frame due to the deletion. the deletion of the entire orf3 gene from the genome the function of the pedv orf3 product remains enigmatic. recently it was shown that the protein exhibits ion channel activity and modulates virus production [5] . sirna knockdown of orf3 gene in pedv infected cells reduced the number of particles released from the cells [5] . the question remains here why passaging of pedv in cell culture would lead to the functional loss of a gene beneficial for virus propagation in vitro, unless the 91residue truncated protein still provides that function. homologues of the orf3 protein are found in all other alphacoronaviruses. the orf3 protein of hcov-nl63 was shown to be nglycosylated at the amino terminus and incorporated into virions [21] . yet, deletion of the orf3 gene from the viral genome had little effect on virus replication in cell culture [22] . like for pedv, loss of orf3 genes of the alphacoronaviruses tgev and hcov-229e (here named orf4) is associated with unimpaired virus passaging in cell culture [23, 24] . despite a non-essential role in cell culture, the maintenance of the orf3 gene in alphacoronavirus field isolates strongly points to an important role of the orf3 protein in natural infection in the animal host. consistently, the loss of virulence of life-attenuated pedv vaccine strains has been associated with mutations in the orf3 gene resulting from cell culture adaptation [10, 25] although a contribution of the numerous additionally acquired mutations in other genes such as the spike gene can obviously not be excluded [26, 27] . the specific function of the orf3 protein (and other viral proteins in the 39 genome region) in pedv replication and pathogenesis can now be further investigated using the reverse genetics system. the introduction of foreign genes at different genomic positions without apparent great fitness loss of the virus in vitro (data not shown) once more illustrates the remarkable genome plasticity of the coronavirus genome [28, 29] . the insertion of reporter genes like for gfp and luciferase will be very useful for the study of various molecular and virological aspects of pedv infection. in addition, as we demonstrate here, these reporter properties may also be exploited for applications such as the establishment of convenient virus neutralization assays that provide answers within hours rather than days. furthermore, genomic insertion of genes encoding foreign antigens using the reverse genetics system opens avenues to the development of pedv as a vaccine vector for protection against other relevant porcine pathogens in addition to pedv. l [12] and vero ccl81 cells (purchased from atcc) were maintained as monolayer cultures in dulbecco's modified eagle medium containing 10% fetal calf serum, 100 iu of penicillin/ml, and 100 mg of streptomycin/ml (all from life technologies, ltd., paisley, united kingdom). pedv (isolated from a commercial vaccine of greencross, south korea) was propagated in vero cells in the absence of trypsin. virus was harvested by three cycles of freeze-thawing the infected cells and supernatant followed by removal of cell debris by centrifugation at 3,0006g for 20 minutes. virus infectivity in the supernatant was measured by an end-point dilution assay on vero cells and 50% tissue culture infectious doses (tcid 50 ) were calculated. mhv (strain a59) was propagated in mouse l cells as described previously [12] . the rabbit anti-mhv serum k135 raised against purified mhv has been described elsewhere [30] . the monoclonal antibody (mab) 3f12 recognizing the pedv nucleocapsid protein was obtained from bionote, korea. polyclonal pedv serum from a pig experimentally infected with pedv (strain cv777) was kindly provided by dr. kristin van reeth (gent university). pedv antibody-negative control serum was obtained from a newborn piglet deprived of colostrum. ppedv vector. a cdna clone encompassing the 39-terminal 7,832 nt part of the pedv genome starting within orf1b was obtained by reverse transcription-pcr (rt-pcr) with viral genomic rna isolated from virions as a template and primers 4922 and 4815 as plus-and minus-strand primers (for primer sequences see table 1 ), respectively. the overhang of primer 4922 and primer 4815 contained a bglii and a paci restriction site, respectively. the bglii-paci digested fragment was cloned into the bamhi-paci digested pmh54 vector [12] , creating the plasmid ppedv-1b-3t. the 59-terminal 605 nt of orf1a was amplified using primers 4884 and 4885. primer 4884 contains a t7 polymerase recognition site, as well as a bglii restriction site and primer 4885 contained a bamhi restriction site. the bglii-bamhi digested fragment was ligated into the bamhi site of the ppedv-1b-3t plasmid, resulting in the ppedv vector. p-rpedv vector. a transfer vector was constructed in which the partly overlapping orf1b and s gene were separated by introduction of a unique bamhi site to facilitate further cloning. the stop codon of orf1b was mutated to taa to knock out the overlapping atg start codon of the spike gene. first, the forward primer 5127 containing the bamhi site and a trs (taaac), and the reverse primer 4815 containing a unique paci site were used to amplify the 39 proximal 7,332 nt of the pedv genome starting with the spike gene. this fragment was cloned into the bamhi-paci site of pmh54 vector, creating the ppedv-s-3t vector. second, primers 4884 and 4885 containing a bglii and bamhi site, respectively, were used to rt-pcr amplify the orf1a fragment which was introduced into the bamhi digested ppedv-s-3t vector creating the ppedv-1a-s-3t plasmid. third, primers 4922 and 4923 that contain a bglii and bamhi in the overhang, respectively, were used to amplify the orf1b fragment by rt-pcr. this fragment was cloned into the bamhi site of the ppedv-1a-s-3t vector, creating the p-rpedv vector. p-mpedv vector. first, the plasmid ptums [31] encoding the mhv spike was used as an intermediate vector to construct a chimeric spike composed of the ectodomain of mhv and the transmembrane and cytoplasmic domain of pedv. for the construction of the hybrid gene, a styi restriction site was used that is located in both s genes at the transition between the protein's ectodomain and transmembrane domain. the forward primer 4814 (styi site in overhang) and reverse primer 4924 (eagi site in overhang) were used to amplify the 39 end of the pedv s gene and downstream sequences and cloned into the styi-eagi digested ptums plasmid, creating the ptums(mp) vector. second, to create the p-mpedv vector, the pedv s gene in the p-rpedv 0.1 and fluorescence images were taken at different times p.i. nuclei of cells were stained with dapi (blue). (d) a rapid virus neutralization assay based on recombinant pedvs expressing reporter proteins. pedv-dorf3/rluc and pedv-dorf3/gfp (8,000 tcid50) were mixed with subsequent dilutions of serum positive for pedv antibodies and a negative control serum (n.c.) for 30 minutes at room temperature. mixtures were incubated with vero cells and renilla luciferase (left panel) or gfp (right panel) expression was measured at 8 and 9 hours p.i., respectively. doi:10.1371/journal.pone.0069997.g005 vector was replaced by the chimeric mhv-pedv spike gene by cloning the bamhi-pmli digested fragment of ptums(mp) into the bamhi-pmli digested p-rpedv vector. ppedv-dorf3 vector. primers 5300 and 5301 were used to amplify the e gene and downstream sequences using the ppedv vector as a template. the forward primer 5300 contained a pmli and an ecorv restriction site and the reverse primer 5301 contained an restriction econi site to facilitate further cloning. the pmli-econi digested pcr fragment was cloned into the pmli-econi digested ppedv vector to create the ppedv-dorf3 vector. ppedv-rluc and ppedv-dorf3/rluc vector. the renilla luciferase gene was excised from the prlnull vector (promega) using enzymes nhei and xbai, blunted with dnapolymerase i large (klenow) fragment and ligated into the bamhi digested and blunted p-rpedv vector or the ecorv digested ppedv-dorf3 vector, resulting in the ppedv-rluc and ppedv-dorf3/rluc transfer vector, respectively. ppedv-dorf3/gfp vector. the gfp gene was excised from the pegfp-n1 plasmid (clontech) with enzymes ncoi and noti, blunted with dna-polymerase i large (klenow) fragment and ligated into the ecorv digested ppedv-dorf3 vector yielding the ppedv-dorf3/gfp transfer vector. the identity of all generated transfer vectors was verified by sequencing. a targeted recombination system was established for pedv in a two-stage process as outlined in fig. 1b . stage 1 generation of mpedv. introduction of the hybrid mhv-pedv s gene into the pedv genome by targeted rna recombination was carried out essentially as described previously for mhv and fipv [12, 13] . briefly, capped runoff donor rna transcripts were synthesized from the paci-linearized p-mpedv vector using a t7 rna polymerase kit (ambion) as specified by the manufacturer. donor rna was electroporated (gene pulser electroporation apparatus [bio-rad]; two consecutive pulses of 0.3 kv/975 mf) into pedv-infected (multiplicity of infection [moi] of 0.4) vero cells (2610 7 cells) at 8 hours post infection (p.i.). the electroporated cells were co-cultured in a 25-cm 2 flask with 5610 6 murine l cells. after 48-60 h of incubation at 37uc, when syncytia could be detected in the murine l cells, progeny virus in the culture supernatant was harvested and mpedv recombinant virus was purified by two consecutive cycles of plaque purification on l cells at 37uc. stage 2 generation of recombinant pedvs. the construction of pedv recombinant viruses that had regained the pedv s gene was carried out in a reverse process by using ppedv-derived donor rnas and mpedv as the recipient virus. capped runoff transcripts were synthesized from paci-linearized ppedv, ppedv-rluc, ppedv-dorf3, ppedv-dorf3/rluc, or ppedv-dorf3/gfp, respectively, with a t7 rna polymerase kit (ambion) as specified by the manufacturer. the donor transcripts were electroporated (as specified above) into murine l cells (2610 7 cells) that had been infected 4 h earlier with mpedv (moi = 1). these cells were then plated onto a monolayer of vero cells. after 4-5 days of incubation at 37uc progeny virus in the culture supernatant was harvested by freeze-thawing and candidate recombinant viruses were purified by two rounds of end-point dilutions on vero cells. recombinant genotypes were confirmed by rt-pcr on purified genomic rna and subsequent sequencing. (immuno)fluorescence microscopy l cells and vero cells were inoculated with mhv, mpedv or pedv (moi = 0.05). after 2 hours of incubation the cells were washed with pbs and incubated in culture medium. at 6.5 hours p.i., the cells were rinsed with pbs and fixed with 3.7% formaldehyde for 20 min at room temperature. the cells were washed three times with pbs and incubated with the k135 rabbita-mhv serum and the 3f12 mouse mab a-pedv-n. after 30 min at room temperature, the cells were rinsed three times with pbs and stained with goat a-rabbit fitc-conjugated and donkeya-mouse cy3 conjugated secondary antibodies (cappel). nuclei were stained with dapi (molecular probes) for 10 min at room temperature. finally, the cells were washed three times with pbs and fluorescence was viewed with an evos-fl fluorescence microscope (advanced microscopy group) at 106 magnification. the evos-fl was also used to view gfp fluorescence from pedv-dorf3/gfp infected cells after paraformaldehyde fixation. vero cell monolayers were infected as described above with the pedv-rluc and pedv-dorf3/rluc viruses at indicated moi's. at indicated times post infection, cell lysate samples were assayed for luciferase activity using the renilla luciferase assay system (promega) according to the manufacturer's instructions, and the relative light units (rlu) were determined with a berthold centro lb 960 plate luminometer. pedv-dorf3/rluc or pedv-dorf3/gfp were mixed with serial dilutions of positive or negative piglet serum or with cell culture medium. the inoculum was incubated for 30 minutes at room temperature to allow virus neutralization before inoculating vero cell monolayers as described above. cells were either lysed at 8 hours post infection and assayed for renilla luciferase activity as described above or subjected to fluorescence microscopy as described above at 9 hours post infection. porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. virus genes outbreak of porcine epidemic diarrhea in suckling piglets molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea pedv leader sequence and junction sites pedv orf3 encodes an ion channel protein and regulates virus production porcine aminopeptidase n is a functional receptor for the pedv coronavirus propagation of the virus of porcine epidemic diarrhea in cell culture role of proteases in the release of porcine epidemic diarrhea virus from infected cells derivation of attenuated porcine epidemic diarrhea virus (pedv) as vaccine candidate cloning and further sequence analysis of the orf3 gene of wild-and attenuated-type porcine epidemic diarrhea viruses enhanced cell fusion activity in porcine epidemic diarrhea virus adapted to suckling mice retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier switching species tropism: an effective way to manipulate the feline coronavirus genome engineering the largest rna virus genome as an infectious bacterial artificial chromosome reverse genetics system for the avian coronavirus infectious bronchitis virus infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus a reverse genetics approach to study feline infectious peritonitis reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus systematic assembly of a fulllength infectious cdna of mouse hepatitis virus strain a59 strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model human coronavirus nl63 open reading frame 3 encodes a virion-incorporated n-glycosylated membrane protein systematic assembly of a full-length infectious clone of human coronavirus nl63 human coronavirus 229e encodes a single orf4 protein between the spike and the envelope genes efficacy of a transmissible gastroenteritis coronavirus with an altered orf-3 gene oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr13 strain cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus dr13 mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo coronaviruses as vectors: position dependence of foreign gene expression coronaviruses maintain viability despite dramatic rearrangements of the strictly conserved genome organization translation of three mouse hepatitis virus strain a59 subgenomic rnas in xenopus laevis oocytes nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex key: cord-341469-7guojyay authors: park, seong-jun; kim, hye-kwon; song, dae-sub; moon, hyoung-joon; park, bong-kyun title: molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea date: 2011-01-06 journal: arch virol doi: 10.1007/s00705-010-0892-9 sha: doc_id: 341469 cord_uid: 7guojyay porcine epidemic diarrhea virus (pedv) has caused enteric disease with devastating impact since the first identification of pedv in 1992 in korea. in this study, we investigated molecular epidemiology, showed genetic diversity, and analyzed phylogenetic relationships of korean pedv field isolates with other pedv reference strains. genetic analysis of the complete m and orf3 genes showed that each pedv group had several unique characteristics, and this indicated that specific groups of pedvs may be differentiated from the other pedvs by specific nucleotide differences. especially, orf3 gene analysis can be used for discrimination between vaccine and wild-type pedvs. sequence and phylogenetic analysis showed that recent, prevalent korean pedv field isolates have close relationships to chinese field strains and differ genetically from european strains and vaccine strains used in korea. these results raise questions as to whether a new type of pedv vaccine may be necessary for preventing pedv infection more effectively in korea. porcine epidemic diarrhea virus (pedv), a member of the family coronaviridae, subfamily coronavirinae, genus alphacoronavirus, is an enveloped, single-stranded rna virus. pedv was first reported in belgium and the united kingdom in 1978 [21] . since the first identification of pedv, outbreaks of pedv infections have been reported in many swine-producing countries, notably in europe and asia [22] . porcine epidemic diarrhea (ped), caused by pedv, is an acute, highly contagious, and devastating enteric disease that is characterized by severe diarrhea, dehydration and significant mortality in swine, resulting in severe economic losses in the european and asian swine industry [22] . coronaviruses have a genome organization with a common set of five genes arranged in a conserved order [7] . the polymerase gene, occupying 70% of the genome, encodes the replicase polyproteins. the genes for structural proteins s, e, m, and n are located downstream of the polymerase gene [7] . in addition, a variety of genes encoding accessory proteins whose number and sequence vary among different coronaviruses are studded between the structural genes, and are called accessory genes [16] . one of the four structural proteins, the m protein, a structural membrane glycoprotein and the most abundant envelope component, is a triple-spanning membrane protein with a short amino-terminal domain on the exterior of the virion and a long carboxy-terminal domain on the inside [29] . in addition to playing an important role in the viral assembly process [5, 17] , the m protein induces antibodies that neutralize virus in the presence of complement [23, 24] . the m protein has also been proposed to play a role in a-interferon (a-ifn) induction [14] . it has been demonstrated that coexpression of m and e proteins allows the formation of pseudoparticles, which exhibit an interferogenic activity similar to that of complete virions [1] . unlike the structural proteins, in most cases, little is known about the functions of the accessory proteins, and in general they are not required for virus replication in cultured cells [4, 25, 31, 32] . quite the opposite, their expression might lead to a decrease in viral fitness in vitro, and mutants with inactivated accessory genes are easily selected during serial passage in cell cultures [11, 15, 26, 30] . in field strains, however, accessory genes are generally maintained [8, 11] , and their loss mainly results in attenuation in the natural host [6, 10, 18] . especially, in the case of pedv, the orf3 gene is the only accessory gene, and it has been suggested to be an important determinant for virulence of this virus. virulence of pedv can be reduced by altering the orf3 gene through cell culture adaptation [26] in a manner similar to tgev [30] , and differentiation of orf3 genes between the highly cell-adapted viruses and field viruses could be a marker of adaptation to cell culture and attenuation of virus [19, 26] . in addition, differentiation of the orf3 gene could be a valuable tool for molecular epidemiology studies of pedv [2, 19, 26] . in korea, pedv was first isolated in 1992 [12] . it has been detected frequently in many provinces and has become one of the most important viral enteric diseases. in spite of using the vaccine strategy at present, damage caused by pedv infection is continuous and serious in korea. to better control and prevent pedv infection, it is necessary for us to investigate the molecular epidemiology of pedv field isolates in korea. in this study, therefore, we investigate the molecular epidemiology and genetic diversity of korean pedv field isolates and analyze phylogenetic relationships of the korean pedv field isolates to other pedv reference strains reported previously. the present study focused on the m and orf3 genes because of their characteristics described above. porcine intestinal and fecal samples were collected between october 2002 and june 2007 from 26 swine farms in six provinces of korea. all pigs from the 26 farms showed signs of watery diarrhea and dehydration at the time of sample collection. fresh samples were collected from individual pigs, placed into a sterile specimen container, and submitted to the department of veterinary medicine virology laboratory, college of veterinary medicine, seoul national university. these intestinal and fecal samples were confirmed to be positive for pedv by reverse transcription-polymerase chain reaction (rt-pcr) [20] . the attenuated dr13 strain and its parent strain were obtained from our laboratory [26] . the attenuated dr13 strain was derived from the parent strain by 100 serial passages in vero cells and was used for manufacture of the korean ped oral vaccine by the green cross veterinary product co., ltd. (yongin, korea). the kped-9 strain, used for manufacture of the korean live pedv vaccine, was kindly provided by the green cross veterinary product co., ltd. (yongin, korea), and the p-5 v strain for manufacture of the japanese live pedv vaccine was provided by the nisseiken regional distributor in korea. pedv-positive fecal samples were prepared as 10% (v/v) fecal suspensions in phosphate-buffered saline (pbs; 0.1 m, ph 7.2), and positive intestinal contents were also prepared as 10% (v/v) intestinal suspensions in pbs by homogenization. the sample suspensions were vortexed and centrifuged for 10 min at 4800 x g. rna was extracted from a 250-ll starting volume of the centrifuged 10% sample suspensions using trizol ls (invitrogen corp., carlsbad, ca, usa) according to the manufacturer's instructions. viral rnas were extracted from attenuated dr13, kped-9 and p-5v strains as described above. reverse transcription (rt) was carried out using random hexamer primers (takara bio inc., otsu, japan), and the cdna was immediately used for amplification or stored at -20°c. primers were designed based on the published sequences of the e and n genes to cover the complete m gene of pedv. the primers were pedm1 (forward), 5'-gtcttacatg cgaattgacc-3', and pedm2 (reverse), 5'-ggcata gagagataatggca-3'. the size of the amplified product was predicted to be 808 bp. pcr was carried out using a commercial amplification system (perkin-elmer, applied biosystems, foster city, ca) as described previously [20] with simple modifications. the pcr was performed at 94°c for 5 min, followed by 35 cycles of 94°c 30 s, 50°c 30 s, 72°c 30 s, and a final extension at 72°c for 7 min, and the sample was then held at 4°c. the primer pair orf3-1/orf3-2 [26] , targeting the s/e regions (corresponding to nt 24,741-25,570 of cv777; 830 bp) of pedv, was used for generating the complete orf3 gene of pedv, and pcr was performed using the protocol described previously [19] . the rt-pcr products for each m and orf3 gene were analyzed by 1.5% agarose gel electrophoresis and visualized by ultraviolet illumination after ethidium bromide staining. bands of the correct size were excised and purified using a qiaquick gel extraction kit (qiagen gmbh, hiden, germany) according to the manufacturer's instructions. the purified rt-pcr products corresponding to the full-length m and orf3 genes of pedv were cloned into pdrive cloning vector (qiagen gmbh, hiden, germany) as described previously [20] , and the cloned plasmids were purified using a qiaprep ò spin miniprep kit (qiagen gmbh, hiden, germany) before sequencing. sequencing of plasmid dna was carried out at least twice in both directions at the genotech institute (genotech co., ltd., korea) using t7 and sp6 primers and an automated dna sequencer (abi system 3700, applied biosystems inc., foster city, usa). the nucleotide sequences of the full-length m and orf3 genes of korean pedv field isolates were aligned using clustalx version 1.83 [28] , edited using bioedit version 7.0.5 (http://www.mbio.ncsu.edu/bioedit/bioedit.html), and compared with those of pedv reference strains in the genbank database as well as in previous papers. sequence similarity analysis was performed for the aligned nucleotide and amino acid sequences using megalign software (dnastar inc., madison, wi, usa). phylogenetic analysis of the korean pedv field isolates with other pedv reference strains based on the nucleotide alignments was done by the neighbor-joining method and the minimum-evolution method of molecular evolutionary genetics analysis (mega version 4.1) using pairwise distances [27] . the korean pedv field isolates and the other pedv reference strains used for sequence alignment, sequence analysis and phylogenetic analysis are indicated in the figure legends. phylogenetic analysis based on a complete m gene fragment of korean pedv field isolates, together with other pedv reference strains, confirmed that all pedvs, including korean field isolates, fell into three groups, and all pedvs isolated from korea belonged to group 3 ( fig. 1 ). more precisely, as shown in fig they did not have nucleotide deletions or insertions but did have point mutations. these isolates had a conserved ataaac sequence at 11 nucleotides upstream of the initiator atg, as previously recognized in br1/87 [9] . sequence analysis of the complete m genes showed that all pedvs, including the korean field isolates, fell into three groups, and group 3 had four subgroups (3-1, 3-2, 3-3, and 3-4), as can be seen in the phylogenetic tree ( fig. 1) . each group had unique differences in its sequences. group 1 had seven specific nucleotide changes (g?c at 139, t?c at 255, c?t at 387, a?g at 444, a?c at 570, g?a at 574, and a?c at 621) that were not found in the other groups, and two of the seven changes lead to amino acid changes (v?l at 47 and g?s at 192). group 2 had one specific nucleotide change (c?t at 618). the specific differences between group 3 and other groups are as follows: subgroup 3-1 exhibited one specific nucleotide change (t?a at 588). in particular, in the case of subgroups 3-2, 3-3 and 3-4, which include all of the korean field isolates, subgroups 3-2 and 3-3 shared one specific nucleotide change (c?t at 198), and subgroups 3-3 and 3-4 shared two specific nucleotide changes (t?c at 285, and t?a at 348). subgroup 3-4 had one specific nucleotide change (c?a at 597). these results indicated that specific groups of pedvs may be differentiated from all other pedvs, including korean field isolates, by specific nucleotide differences, although more pedvs need to be analyzed for more accurate analysis. sequence homology analysis of the m gene sequence homology results based on the complete m gene of all pedvs, including the korean pedv field isolates, phylogenetic analysis of the orf3 gene phylogenetic analysis based on a complete orf3 gene fragment of korean pedv field isolates, together with other pedv reference strains, confirmed that all pedvs, including korean field isolates, fell into three groups (fig. 2) . one group comprised the cv777, br1/87, and lzc strains. the second group consisted of vaccine strains (the attenuated strains dr13, kped-9, p-5v and cv777 vs), the ch/gsjiii/07 strain, and the dbi865 korean field isolate. the third group was made up of 25 korean field isolates, chinju99, the parent strain dr13, and 11 chinese strains. the third group had two subgroups (3-1 and 3-2). nine korean field isolates from 2007 and ten chinese strains formed one subgroup (3-1), and 15 korean field isolates from 2003-2007 formed a second subgroup together with chinese strain ch/s, korean strain chinju99, and the parent strain dr13. sequences of complete orf3 genes of korean pedv field isolates were determined and compared to those of other pedv reference strains. all korean pedv field isolates except dbi865 had a single orf of 675 nucleotides encoding a protein of 224 amino acids, with a predicted mr of 25.1-25.3 kda. on the other hand, dbi865 had a single orf of 276 nucleotides encoding a protein of 91 amino acids, with predicted mr of 10.2 kda because of a 49-nucleotide deletion at position 245-293. all korean pedv field isolates, including dbi865, had a conserved sequence (ctagac) at 46 nucleotides upstream of the initiator atg, similar to that described above for the m gene. sequence analysis of the complete orf3 genes showed that all pedvs, including the korean field isolates, fell into three groups, and group 3 had two subgroups (3-1 and 3-2), as can be seen in the phylogenetic tree (fig. 2) . each group had differences in its sequences. group 1 had eight specific nucleotide changes (c?t at 62, a?g at 160, a?g at 235, t?c at 243, a?g at 301, c?t at 360, t?c at 450 and g?a at 497) that were not found in the other groups, and four of the eight changes led to amino acid changes (a?v at 21, i?v at 54, t?a at 101 and s?n at 166). group 2 showed one specific nucleotide change (c?a at 339) and had two classes of large nucleotide deletions at position 245-295, which are predicted to produce truncated proteins. the specific differences between group 3 (containing all korean field isolates except dbi865) and the other groups are as follows: group 3 had three specific nucleotide changes (t?g at 238, c?t at 264 and c?t at 274), and two of three changes led to amino acid changes (f?v at 80 and l?f at 92). in particular, subgroup 3-1, which includes nine korean field isolates from 2007 and ten chinese strains, was differentiated from subgroup 3-2 by one specific nucleotide change (c?t at 237). these results showed that these specific nucleotide differences may be used to differentiated specific groups of pedvs from other pedvs, including korean field isolates. however, more pedvs need to be analyzed. in addition, complete orf3 gene analysis can be used for differentiation between vaccine-and wild-type pedvs because all vaccine-type pedvs had a large nucleotide deletion in the orf3 gene. sequence homology analysis of the orf3 gene sequence homology results based on the complete orf3 gene of all pedvs, including korean pedv field isolates, in this study, the complete m and orf3 genes of korean pedv field isolates were amplified by rt-pcr, cloned and sequenced to investigate their molecular and epidemiological characteristics and their genetic diversity. in addition, phylogenetic relationships between korean pedv field isolates and other pedv previously reported reference strains were also analyzed. all korean pedv field isolates (excluding dbi865) have only point mutations in the m and orf3 genes, and all of them, including dbi865, have ataaac and ctagac sequences at 11 and 46 nucleotides upstream of the initiator atg of the m and orf3 genes, as recognized previously [9] . these sequences are hexameric motifs that are common to coronaviruses and are similar to the hexameric motifs xua(a/g)ac found adjacent to other pedv orfs. these hexameric motifs have been proposed to be a start site for the transcription of subgenomic mrnas [13] . the dbi865 isolate has a 49-nucleotide deletion at position 245-293 of the orf3 gene, resulting in a truncated protein of 91 amino acids in size. this size of this deletion is exactly the same as the one present in the vaccine strain cv777 and the chinese field strain ch/gsjiii/07 [2] . genetic analysis based on complete m and orf3 genes showed that each pedv group had several unique characteristics, and these results indicated that specific groups of pedvs may be differentiated from other pedvs, including korean field isolates, by specific nucleotide differences, but more pedvs need to be analyzed for more accurate analysis. especially, complete orf3 gene analysis can be used for discrimination between vaccine and wild-type pedvs because only vaccine-type pedvs had large deletions in the orf3 gene. according to sequence and phylogenetic analysis using the complete m gene, all pedvs isolated in korea are ped vaccines are commonly used in korea as losses caused by pedv infection increase. however, most of the korean pedv field isolates analyzed in this study differ from members of the group that includes the vaccine strains, and only a few isolates belonged to that group. these results may be attributable to immune pressure due to widespread vaccine use in korea. in addition, this reflects the existence of genetic diversity among the korean pedv field isolates and raises questions as to whether a new type of pedv vaccine may be necessary for more effective prevention of pedv infection. the dbi865 isolate has high sequence identity and a close phylogenetic relationship to the vaccine strains according to genetic and phylogenetic analysis of the complete orf3 gene, and this result implies that it might be derived from a vaccine strain. although the mechanism by which the dbi865 isolate emerged is unclear, two possibilities are suggested: (1) the dbi865 isolate might have evolved from a vaccine strain; (2) dbi865 isolate might have been produced in nature through recombination between a pedv field isolate and a vaccine strain. of these two possibilities, the latter is more likely because the partial s and orf3 genes of the dbi865 isolate are closely related to those of g1-1 (which is genetically different from the vaccine strains) [20] and group 2 (which are genetically similar to the vaccine strains). this is similar to what was shown for other korean field isolates used in this study. in the case of the e1642 and m2366 isolates, partial s [20] and orf3 genes were different from those of the vaccine strains, and the m genes were similar to those of the vaccine strains. the partial s gene of the e1697 isolate differs from that of the vaccine strains [20] , and the m gene is closely related to those of the vaccine strains. however, despite trying several times to amplify the complete m gene of the dbi865 isolate, we did not obtain any amplicons from this isolate, and for that reason, further studies, such as full-length sequencing as well as complete m gene sequencing of the dbi865 isolate, are needed to determine how the dbi865 isolate originated. the present study allows a better understanding of the molecular epidemiology, genetic diversity, and phylogenetic relationships of korean pedv field isolates with other pedv reference strains. we expect that the results of this study will help to prevent and control pedv infection more effectively. coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes molecular epidemiology of porcine epidemic diarrhea virus in china table 2 nucleotide and deduced amino acid sequence similarity based on the full-length orf3 genes of the korean pedv field isolates and those upper and lower triangles indicate percent nucleotide (nt) and amino acid (aa) sequence similarity, respectively a european strains (cv777 and br1/87) and chinese lzc strain b vaccine strains (attenuated strains dr13, kped-9 d chinese strains ch/s, korean strains (chinju99 and parent dr13) and 15 korean field isolates (bi976, bi981, bi1108, m1595, e1642, m1763, m2227, m2366, v2501, mf78, bif118, vf131, cpf259, cpf531 and pff1051 genetic characteristics of porcine epidemic diarrhea virus isolated in korea heterologous gene expression from transmissible gastroenteritis virus replicon particles 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 the genome organization of the nidovirales: similarities and differences between arteri-, toro-, and coronaviruses human coronavirus 229e encodes a single orf4 protein between the spike and the envelope genes sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf live, attenuated coronavirus vaccines through the directed deletion of groupspecific genes provide protection against feline infectious peritonitis the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf7a/7b transcription unit of different biotypes isolation of porcine epidemic diarrhea virus (pedv) in korea coronavirus: organization, replication and expression of gene single amino acid changes in the viral glycoprotein m affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus luxury at a cost? recombinant mouse hepatitis viruses expressing the accessory hemagglutinin esterase protein display reduced fitness in vitro coronavirus accessory proteins protein interactions during coronavirus assembly transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence cloning and further sequence analysis of the orf3 gene of wild-and attenuated-type porcine epidemic diarrhea viruses sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhea the coronavirus membrane protein coronavirus immunogens murine coronavirus nonstructural protein ns2 is not essential for virus replication in transformed cells differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf 3 mega4: molecular evolutionary genetics analysis (mega) software version 4.0 the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools identification of proteins specified by porcine epidemic diarrhoea virus efficacy of a transmissible gastroenteritis coronavirus with an altered orf-3 gene in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice acknowledgments this work was supported by a grant (code #20070401034009) from biogreen 21 program, rural development administration, republic of korea. key: cord-344845-52rehsd5 authors: opriessnig, tanja; gerber, priscilla f.; shen, huigang; de castro, alessandra marnie m. g.; zhang, jianqiang; chen, qi; halbur, patrick title: evaluation of the efficacy of a commercial inactivated genogroup 2b-based porcine epidemic diarrhea virus (pedv) vaccine and experimental live genogroup 1b exposure against 2b challenge date: 2017-10-26 journal: vet res doi: 10.1186/s13567-017-0472-z sha: doc_id: 344845 cord_uid: 52rehsd5 porcine epidemic diarrhea virus strains from the g1b cluster are considered less pathogenic compared to the g2b cluster. the aim of this study was to compare the ability of g1b-based live virus exposure against use of a commercial g2b–based inactivated vaccine to protect growing pigs against g2b challenge. thirty-nine pedv naïve pigs were randomly divided into five groups: exp-im-1b (intramuscular g1b exposure; g2b challenge), exp-oral-1b (oral g1b exposure; g2b challenge), vac-im-2b (intramuscular commercial inactivated g2b vaccination; g2b challenge), pos-control (sham-vaccination; g2b challenge) and neg-control (sham-vaccination; sham-challenge). pigs were vaccinated/exposed at 3 weeks of age (day post-vaccination 0, dpv 0), vac-im-2b pigs were revaccinated at dpv 14, and the pigs were challenged at dpv 28. among all groups, vac-im-2b pigs had significantly higher anti-pedv igg levels on dpv 21 and 28 while exp-oral-1b pigs had significantly higher anti-pedv iga levels on dpv 14, 21, 28 and 35. exp-oral-1b also had detectable iga in feces. intramuscular pedv exposure did not result in a detectable antibody response in exp-im-1b pigs. the fecal pedv rna levels in vac-im-2b pigs were significantly lower 5–7 days after challenge compared to the pos-control group. under the study conditions a commercial inactivated g2b-based vaccine protected pigs against g2b challenge, as evidenced by reduction of pedv rna in feces for 3–4 logs during peak shedding and a shorter viral shedding duration. the oral, but not the intramuscular, experimental g1b-based live virus exposure induced a high anti-pedv iga response prior to challenge, which apparently did not impact pedv shedding compared to pos-control pigs. clinical porcine epidemic diarrhea and its causative virus pedv were discovered in european pigs in the 1970s [1, 2] , spread to asia during the 1980s and 1990s [3] , and became endemic in pigs on both continents [2, 3] . approximately 10 years ago pedv re-emerged as an important enteric disease of suckling and growing pigs [4] . in 2013, pedv was introduced for the first time to north america [5] causing major disease and mortality [6] . pedv can be differentiated into genogroups [7] . on the basis of spike (s) gene sequences, pedv isolates can be divided into g1a, g1b, g2a and g2b [7, 8] . g1a includes historic pedv isolates such as cv777 and attenuated variants distributed in europe and asia, whilst g1b includes the so called s-indel strains which can be found in europe, asia and north america. g2a isolates are restricted to asia whereas g2b isolates are present in asia and the ukraine [9] , and since us introduction in 2013 are widespread in the us and considered the us prototype [8, 10] . differences in pathogenicity between representative isolates of different genogroups have been demonstrated [10] [11] [12] , with g2b isolates usually being more pathogenic compared to g1b isolates. partial crossprotection between pedv g1b and g2b isolates has been demonstrated experimentally [12] . in january 2014 the first conditional licensed pedv vaccine was introduced to the north american pig market [13] , and today an rna particle-based vaccine and an inactivated pedv vaccine are available in the us to immunize sows against pedv [13] . while the use of these vaccines is often beneficial in previously exposed herds, they often fail in naïve herds [14] . one reason for the variable vaccine efficacy observed under field conditions may be the usage of inactivated vaccines given intramuscularly rather than live virus vaccines given orally to induce a strong local enteric immunity. it would be risky to use a known pathogenic g2b live vaccine virus in a pig population; however, using a less virulent variant such as g1b instead may be safe and efficacious. the objectives of this study were to compare the efficacy of heterologous g1b and homologous g2b based vaccines in protecting growing pigs against g2b challenge. specifically, an experimental g1b-based live vaccine, administered orally or intramuscularly and a commercial g2b-based inactivated vaccine administered intramuscularly were compared side by side. the experimental protocol was approved by the iowa state university institutional animal care and use committee (approval number: 5-14-7804-s). thirty-nine, 2-week-old, colostrum-fed, arbitrarilyselected, crossbred, pedv naïve weaned pigs were randomly assigned to one of five rooms and groups, with 7-8 pigs in each group (table 1) . all groups were fed ad libitum with a balanced, age-appropriate, pelleted feed ration. at 3 weeks of age or dpv 0, exp-im-1b, exp-oral-1b and vac-im-2b groups were vaccinated with different vaccines and routes as outlined in table 1 , whereas pos-control and neg-control pigs were sham-vaccinated with saline. vac-im-2b pigs were revaccinated at 5 weeks of age (dpv 14). at day postchallenge (dpc) 0 or dpv 28, when the pigs were 7 weeks old, they were challenged as shown in table 1 . the pos-control group served as a challenge control group while the neg-control was sham-challenged and served as unvaccinated, unchallenged group. half of the pigs in each group were necropsied at dpv 31/dpc 3 and the remainder at dpv 42/dpc 14. the experimental design and sample collection details are summarized in figure 1 . blood was collected in serum separator tubes on a weekly basis (fisher scientific, pittsburgh, pa, usa), centrifuged at 3000 × g for 10 min at 4 °c, and the serum was stored at −80 °c until testing. rectal swabs were collected at dpv 0, 7, 14, 21 and 28 followed by daily collection until dpv 41/dpc 13 using polyester swabs and stored in 5 ml plastic tubes containing 1 ml of sterile saline solution at −80 °c until testing. individual fecal samples were collected in 50 ml plastic tubes and frozen immediately at −80 °c until testing. at 3 weeks of age (dpv 0), the exp-im-1b and the exp-oral-1b pigs were vaccinated with a g1b (us s-indel-variant) live pedv isolate 14-20697 at the 7 th cell culture passage [10, 15] as indicated in table 1 . after cell culture adaption this virus was used to infect 5-day old pigs previously and had reduced pathogenicity compared to g2b isolates [10] . for the intramuscular vaccination, 2 ml of the g1b virus stock with a titer of 5 × 10 4 50% tissue culture infectious dose (tcid 50 ) per ml was mixed with 0.4 ml adjuplex ™ vaccine adjuvant (lot number slbp5255 v; sigma-aldrich, st louis, mo, usa) prior to injection. the same g1b cell culture adapted virus stock used in this study has been shown to have moderate to severe enteric pathogenicity in 5-day old pigs [10] . each pig in the exp-im-1b group received 2.4 ml intramuscularly into the neck, with a total pedv dose of 1 × 10 5 tcid 50 . for the oral vaccination route, each exp-oral-1b pig was administered 10 ml of the g1b virus stock with a titer of 6.8 × 10 3 tcid 50 per ml by slowly dripping the vaccine into the mouth of each pig with a total dose of 6.8 × 10 4 tcid 50 . adjuvant was not used for the oral vaccination route. pigs in the vac-im-2b group were vaccinated intramuscularly with 2 ml of a commercial conditionally-licensed inactivated pedv vaccine based on a g2b strain (zoetis; serial number 117962) into the right neck. the vac-im-2b group was revaccinated 2 weeks later (dpv 14) with another 2 ml of the vaccine as recommended by the manufacturer. the pos-control group was sham-vaccinated intramuscularly in the neck with 2.4 ml saline and the neg-control group was sham-vaccinated orally with 10 ml saline (table 1) . the 8 th passage of virulent pedv g2b strain 13-19338e [10, 16] was grown to a final titer of 6.8 × 10 4 tcid 50 per ml. at 7 weeks of age, exp-im-1b, exp-oral-1b, vac-im-2b and pos-control pigs (table 1) the neg-control group was sham-vaccinated orally with saline and sham-inoculated with saline and served as unvaccinated and unchallenged control group. slowly dripping the inoculum into the mouth with a total dose of 6.8 × 10 5 tcid 50 . pigs in the neg-control group were sham-inoculated with 10 ml saline orally. all pigs were weighed at dpv 0, at dpv 28/dpc 0 and at dpv 42/dpc 14 ( figure 1 ). the average daily gain (adg) from dpv 0 (vaccination 1) to dpv 42/dpc 14 (necropsy 2) was calculated. after pedv challenge the fecal consistency was scored for each pig daily, ranging from 0 to 3 with 0 = solid, 1 = semisolid, 2 = pasty, and 3 = liquid. all pigs were examined daily for other signs of illness including lethargy, respiratory disease, inappetence and lameness. all serum samples were tested for the presence of pedv igg and iga antibodies by an in-house pedv g2b s1 protein based indirect elisa [17, 18] . for igg detection, a sample-to-positive (s/p) ratio of > 0.2 was considered positive, between 0.14 and 0.2 as suspect, and < 0.14 as negative. for the iga elisa an s/p ratio above or equal to 0.14 was considered positive. in addition, fecal samples collected at dpv 0, dpv 28, and at necropsy at dpv 31/dpc 3 or dpv 42/dpc 14 were also tested for presence of pedv iga antibodies [18] . modifications for this assay included that samples were diluted 1:2 and the secondary antibody was diluted 1:2000. the positive cutoff for this assay was s/p ratio equal or greater than 0.14. serum samples at dpv 28 were titrated for anti-pedv virus neutralizing antibodies by an immunofluorescence assay as previously described [15] . serum was diluted two-fold starting from 1:20 to 1:1280. titers were given as the reciprocal of the last dilution giving a positive result. total nucleic acids were extracted from all rectal swabs using the magmax ™ pathogen rna kit (applied biosystems, life technologies, carlsbad, ca, usa) on an automated nucleic acid extraction system (thermo scientific kingfisher ® flex, thermo fisher scientific, pittsburgh, pa, usa) according to the instructions of the manufacturer. all rna extracts were tested for the presence of pedv rna by a quantitative real-time pcr [19] . samples were considered negative when no signal was observed within 40 amplification cycles. half of the pigs in each group were necropsied at dpv 31/dpc 3 and the remaining pigs were necropsied at dpv 42/dpc 14. the pigs were humanely euthanized by intravenous pentobarbital sodium overdose (fatal plus ® , vortech pharmaceuticals, ltd, dearborn, mi, usa). gross lesions were assessed by a veterinary pathologist and eight sections of small intestines, three sections of large intestines and one section of mesenteric lymph node were collected, fixed in 10% neutral-buffered formalin, and routinely processed for histological examination. microscopic lesions were evaluated by a veterinary pathologist blinded to the treatment groups. sections of small intestines were evaluated for the presence of villus atrophy and scored from 0 (none) to 3 (severe). pedvspecific antigen was detected by immunohistochemistry (ihc) using a monoclonal antibody specific for pedv (bionote, hwaseong-si, gyeonggi-do, korea) [5, 20] . the amount of pedv antigen was scored by a pathologist blinded to treatment status. scores ranged from 0 to 3 with 0 = no signal, 1 = 1-10% of villous enterocytes within the section showing a positive signal, 2 = 11-50% of villous enterocytes showing a positive signal, and 3 = more than 50% of villous enterocytes showing a positive signal. for data analysis, jmp ® software version 11.0.0 (sas institute, cary, nc, usa) was used. summary statistics were calculated for all the groups to assess the overall quality of the data set including normality. statistical analysis of the data was performed by one-way analysis of variance (anova) for continuous data. a p value of less than 0.05 was set as the statistically significant level. pairwise test using tukey's adjustment was subsequently performed to determine significant group differences. real-time pcr results (copies per ml of fecal swab suspension) were log 10 transformed prior to statistical analysis. the area under the curve (auc) of viral shedding of each animal and the total auc for each group was calculated using the log transformed values of the viral loads from dpv 29 to 41/dpc 1 to 13. one-way anova and a bonferroni post hoc test were used to compare groups. non-repeated nominal data were assessed using a nonparametric kruskal-wallis one-way anova, and if significant, pairwise wilcoxon tests were used to evaluate differences among groups. clinical signs in the pedv-infected pigs were limited to diarrhea. three days after vaccination, 4/8 exp-oral-1b pigs had semisolid feces and 7 days later all pigs in this group had pasty feces. none of the pigs in the other groups had any fecal consistency changes and all pigs remained normal until pedv challenge. liquid fecal consistency was observed in 4/7 exp-im-1b pigs by dpv 31/dpc 3 and feces remained fluid in the majority of the pigs until dpv 35/dpc 7 before becoming pasty-to-solid. in the remaining groups individual pigs had liquid feces for 1-2 days of duration (data now shown) with no differences among groups. the overall adg is summarized in table 1 . there were no significant differences among groups. all pigs were negative for anti-pedv igg antibodies in serum samples by elisa at dpv 0 and neg-control pigs remained seronegative for the duration of the study. anti-pedv igg antibodies were first detected in 2/8 vac-im-pedv pigs at dpv 7 ( figure 2 ). by dpv 14, 3/8 pigs in this group were seropositive and 3/8 were suspect. all vac-im-2b pigs were anti-pedv igg positive by dpv 21. in the exp-oral-1b group, 6/8 pigs were positive for anti-pedv igg antibodies by dpv 14, 7/8 were positive at dpv 21 and at dpv 28, 3/4 pigs were positive by dpv 35/dpc 7, and at termination of the study 3/4 were positive and 1/4 was suspect ( figure 2 ). the exp-im-1b group remained anti-pedv igg negative until dpv 35/ dpc 7 at which time all three remaining pigs in this group were positive (figure 2 ). one of four pos-control pigs seroconverted to pedv by dpc 7 and all 4 pigs in this group were seropositive by dpc 14. anti-pedv neutralizing antibodies were not detected in the exp-im-1b, pos-control and neg-control groups at dpv 28. neutralizing antibody titers ranged from 40 to 320 with a geometric mean of 89.7 in the exp-oral-1b group, and ranged 40-1280 with a geometric mean of 320 in the vac-im-2b group. all pigs were negative for anti-pedv iga antibodies in serum samples by elisa at dpv 0 and 7 and neg-con-trol pigs remained seronegative for the duration of the study. anti-pedv iga antibodies in serum samples were first detected in 8/8 exp-oral-1b pigs at dpv 14 ( figure 3 ). one week later at dpv 21, anti-pedv iga in sera were also detected in 5/8 vac-im-2b pigs; however, in this group antibody levels decreased, and by dpv 28/ dpc 0 only 1/8 pigs were anti-pedv iga positive. iga antibodies against pedv in the exp-im-1b group were detected by dpv 35/dpc 7 in 2/3 pigs and by dpv 42/dpc 14 in 3/3 pigs (figure 3 ). one of four pos-control pigs had detectable anti-pedv iga antibodies by dpc 7 and 4/4 pigs were seropositive by dpc 14 (figure 3 ). by dpv 28/dpc 0, one exp-oral-1b pig had detectable iga levels in feces and by dpv 31/dpc 3 one additional pig in this group was positive for pedv iga in feces (data not shown). by 14 dpc, pedv iga antibodies in feces were present in 2/3 exp-im-1b pigs, 3/4 exp-oral-1b pigs, 2/4 pos-control pigs and 1/4 vac-im-2b pigs (data not shown). all pigs were negative for pedv rna in fecal swabs on dpv 0 and neg-control pigs remained negative for the duration of the study. after vaccination with a live g1b strain, fecal shedding was detected in 8/8 exp-oral-1b pigs by dpv 7 and in 7/8 pigs by dpv 14 (figure 4 ). in addition, 5/8 exp-oral-1b pigs had detectable amounts of pedv rna in serum by dpv 7 (data not shown). on the day of challenge, 1/8 exp-oral-1b pigs shed low amounts of pedv in feces. pedv rna was detected in rectal swabs of 2/8 exp-im-1b pigs by dpv 7; however, pedv rna was never detected in serum (data not shown). after challenge, 3/7 exp-im-1b pigs shed virus by dpv 29/dpc 1. viral shedding in rectal swabs was first detected by dpv 30/dpc 2 in 5/8 exp-oral-1b pigs, in 1/8 vac-im-2b pigs and in 2/8 pos-control pigs. group mean genomic copies of pedv rna in rectal swabs are summarized in figure 5 . the average duration of pedv shedding was calculated by adding the number of consecutive pedv pcr positive days of each pig that remained in the study until dpv 42/dpc 14 divided by all pigs in a group. the average duration of pedv shedding and the auc are summarized in table 1 . at dpv 31/dpc 3, pedv-infected pigs regardless of vaccination status had hyperemic intestines that were fluidfilled. specifically, liquid intestinal content was noted in 3/4 exp-im-1b pigs, in 2/4 exp-oral-1b pigs, in 2/4 vac-im-2b pigs and in 1/4 pos-control pigs. at dpv 42/dpc 14, 1/4 pos-control pigs had fluid filled intestines and a dilated colon without remarkable lesions in any of the other pigs. microscopic lesions were seen in 2/4 exp-im-1b pigs, 1/4 exp-oral-1b pigs, 1/4 vac-im-2b pigs and 2/4 pos-control pigs which had mild to severe atrophic enteritis by dpv 31/dpc 3. there were no lesions in any of the other pigs. five of the six pigs with microscopic lesions also had moderate-to-high amounts of pedv figure 2 group mean anti-pedv igg response in serum samples. the samples were collected at the day of initial vaccination (dpv 0), and dpv 7, 14, 21, 28, 35 and 42 and tested by an in house igg pedv elisa. pigs were exposed to live g1b pedv at 3 weeks of age (dpv 0) or were vaccinated at 3 (dpv 0) and 5 (dpv 14) weeks of age with a commercial inactivated pedv g2b vaccine. pigs were challenged with pedv g2b at 7 weeks of age (dpv 28/day post challenge or dpc 0). data presented as mean group elisa sample-to-positive (s/p) ratios ± sem. significantly different values for a dpc are indicated by different superscripts ( a,b,c ). the significance level was set to p < 0.05. seropositive pigs/total number of pigs per group for groups that contained at least one seropositive pigs can be seen next to the group mean. antigen associated with the lesions (two exp-im-g1b pigs, scores 3 and 3; a exp-oral-g1b pig, score 2; a vac-im-g2b pig score 3; and a pos-control pig, score 3). there were no significant differences in antigen levels or severity of microscopic lesions among groups. no microscopic lesions nor pedv antigen were observed at dpv 42/dpc 14. vaccination strategies to protect against pedv are challenging, as the most vulnerable population is suckling pigs. vaccine efficacy studies using pregnant sows are difficult and costly. to select novel pedv vaccine candidates and to generate preliminary data, the growing pig model has been used [21] . in this study growing pigs were used to test and compare the efficacy of live or inactivated vaccines to protect pigs against challenge with a highly virulent g2b pedv isolate. pig veterinarians and producers often prefer intramuscular administration to assure each pig gets vaccinated with the appropriate dose. intramuscular administration is known to induce a systemic immune response [22] . in this study, vac-im-2b pigs had a strong anti-pedv igg response in serum which was significantly higher compared to exp-oral-1b pigs. this could be due to the adjuvant amphigen ® used in the commercial product or due to the booster dose that the vac-im-2b pigs received. in contrast to live virus exposure, inactivated vaccines are almost always given in 2 dose regimens; hence in this study the vac-im-g2b group received a booster dose whereas the exp-im-g1b and exp-oral-g1b pigs did not. in contrast to oral exposure to a live virus, pigs vaccinated with the commercial inactivated virus had a weak anti-iga response in serum and no anti-pedv iga response in feces. this is not surprising as inactivated vaccines often do not induce effective mucosal immunity in naïve pigs whereas oral exposure elicits better gut immunity [22] . it has been shown that iga levels in serum correlates with iga measured in feces from experimentally infected piglets [18] and in serum and colostrum and milk samples of sows orally immunized [13] . these studies indicate that measuring iga levels in serum samples may be a marker of protection. for safety reasons, veterinarians and producers often prefer inactivated vaccines. however, for some viruses such as porcine reproductive and respiratory syndrome figure 3 group mean anti-pedv iga response in serum samples. the samples were collected at the day of initial vaccination (dpv 0), and dpv 7, 14, 21, 28, 35 and 42 and tested by an in house iga pedv elisa. pigs were exposed to live g1b pedv at 3 weeks of age (dpv 0) or were vaccinated at 3 (dpv 0) and 5 (dpv 14) weeks of age with a commercial inactivated pedv g2b vaccine. pigs were challenged with pedv g2b at 7 weeks of age (dpv 28/day post challenge or dpc 0). data presented as mean group elisa sample-to-positive (s/p) ratios ± sem. significantly different values for a dpc are indicated by different superscripts ( a,b,c ). the significance level was set to p < 0.05. seropositive pigs/total number of pigs per group for groups that contained at least one seropositive pigs can be seen next to the group mean. virus (prrsv), it has been shown that inactivated vaccines are largely ineffective [23] . prrsv requires live virus to migrate to the lung and replicate at low levels to induce protection. similarly, pedv may also require local activation of the gut-associated mucosal system. in asia, where pedv vaccines have been available for decades, attenuated g1a-based intramuscular vaccines are commonly used [4, 24] . we attempted to inject a g1b isolate intramuscularly with an adjuvant. under the study conditions, except for 2/7 exp-im-1b pigs with low levels of pedv rna in rectal swabs at 7 dpv, there was no sign of infection in this group based on lack of seroconversion and lack of detectable pedv rna in serum or feces. the two pedv rna positive exp-im-1b samples were retested and results confirmed (data not shown). the pigs that were vaccinated intramuscularly with a commercial g2b vaccine were protected against homologous g2b challenge as evidenced by reduction of the amount of pedv rna in feces by 3-4 logs during peak shedding between dpc 5-7 (dpv 33-35) and shortening of the duration of viral shedding. viral titers to determine infectivity were not determined, but it has been shown previously that contact pigs can be infected for up to 14 days after initial infection of a seeder pig group [25] . in this study a homologous g1b challenge for pigs vaccinated with the experimental g1b live vaccine was not included due to space and cost reasons. in addition, g2b isolates, considered to be more pathogenic compared to g1b isolates [10, 12] , appear to be the primary cause of clinical disease associated with pedv under field conditions and are more widely distributed compared to g1b isolates. pigs orally vaccinated with an experimental heterologous g1b live vaccine had a tendency for a shortened viral shedding duration; whereas pigs vaccinated intramuscularly with an experimental heterologous g1b live vaccine were not protected. it has been shown that piglets orally inoculated with a virulent cv777 strain were fully protected after challenge, while protection was not complete in pigs orally inoculated with an attenuated cv777 strain [26] . prior to usage the g1b stock was passaged seven times which could have resulted in a low degree of attenuation. it is worth noting that pigs orally immunized with the g1b live vaccine presented mild diarrhea and shed high levels of virus for at least 2 weeks after immunization. this could pose risks of infection and potentially more serious clinical signs in younger piglets. results from studies on cross-protection between genogroups have been contradictory. a previous study showed that although g1a-based vaccines (cv777 and dr13 strains) could provide protection against homologous challenge, they were not protective against contemporary chinese g2b strain yc2014 [27] . it has been suggested that sows naturally-infected with a g1b strain produce heterologous lactogenic protective immunity against g2b strains 7 months after initial infection [28] . however, infection of 3-4 day old piglets with g1b strain provided variable protection against a g2b challenge 21-29 days later and the extent of protection was shown to be litter-dependent (mortality 0 to 75%) [12] . additionally, the antigen concentration in the commercial (10 6 -10 8 tcid 50 /dose) and experimental (10 4 -10 5 tcid 50 /dose) intramuscular vaccines may have contributed to differences in the protection observed in the current study. the dose of experimental vaccine was limited by the g1b virus titer achieved after propagation. under the conditions of this study, a commercial inactivated g2b-based pedv vaccine administered intramuscularly protected pigs against homologous challenge. in contrast, an experimental g1b-based live virus vaccine given intramuscularly was not protective. the same virus given orally induced a high iga response but the virus shedding pattern after challenge mimicked that of the pos-control group suggesting limited protection. this could perhaps indicate that induction of a genotype specific humoral and/or cellular immune response may be important for pedv protection. . pigs were exposed to pedv at 3 weeks of age (dpv 0) with live pedv g1b or vaccinated at 3 (dpv 0) and 5 (dpv 14) weeks of age with a commercial inactivated pedv g2b vaccine. pigs were challenged with pedv g2b at 7 weeks of age (dpv 28/dpc 0). a. mean group log 10 pedv genomic copies per ml of fecal swab suspension ± sem. significant different values for a sample type and dpc are indicated by different superscripts ( a,b,c ). the significance level was set to p < 0.05. there were no significant differences among groups on dpv 0 and 21 and on dpc 0, 8, 11 and 13 a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhea: a retrospect from europe and matters of debate porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines porcine epidemic diarrhea: a review of current epidemiology and available vaccines emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences ped virus reinfecting us herds. virus estimated to have killed 7 million-plus pigs porcine epidemic diarrhea virus: an emerging and reemerging epizootic swine virus origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states porcine epidemic diarrhea virus among farmed pigs pathogenesis comparison between the united states porcine epidemic diarrhoea virus prototype and s-indel-variant strains in conventional neonatal piglets isolation and experimental inoculation of an s indel strain of porcine epidemic diarrhea virus in japan experimental infection of a us spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original us pedv infection lactogenic immunity and vaccines for porcine epidemic diarrhea virus (pedv): historical and current concepts status of vaccines for porcine epidemic diarrhea virus in the united states and canada evaluation of serological cross-reactivity and cross-neutralization between the united states porcine epidemic diarrhea virus prototype and s-indel-variant strains isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa detection of immunoglobulin (ig) a antibodies against porcine epidemic diarrhea virus (pedv) in fecal and serum samples porcine epidemic diarrhea virus rna present in commercial spray-dried porcine plasma is not infectious to naive pigs monoclonal antibody-based immunohistochemical detection of porcine epidemic diarrhea virus antigen in formalin-fixed, paraffin-embedded intestinal tissues an inactivated vaccine made from a us field isolate of porcine epidemic disease virus is immunogenic in pigs as demonstrated by a dose-titration strategies for design and application of enteric viral vaccines inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction comparison of serum neutralization and enzyme-linked immunosorbent assay on sera from porcine epidemic diarrhea virus vaccinated pigs evaluation of porcine epidemic diarrhea virus transmission and the immune response in growing pigs mucosal and systemic isotype-specific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus epidemic strain yc2014 of porcine epidemic diarrhea virus could provide piglets against homologous challenge previous infection of sows with a "mild" strain of porcine epidemic diarrhea virus confers protection against infection with a "severe" strain the authors thank kelsey oakly and eve fontanella for assistance with the animal work and gustavo de-sousa-e-silva, marcelo nunes de almeida and will alberto lopez for assistance with the necropsies. the study was funded by the iowa livestock health advisory council (ilhac). the authors declare they have no competing interests. to performed the experiments, analysis of the data (including statistical analysis) and drafting of the manuscript. pfg performed analysis of samples and data. hs, ammgc, jz and qc performed the experiment. jz and qc provided the inoculum stocks. pgh performed the animal studies and necropsies, edited and finalized the manuscript. all authors read and approved the final manuscript.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-331652-oc5s1if2 authors: trudeau, michaela p.; verma, harsha; sampedro, fernando; urriola, pedro e.; shurson, gerald c.; mckelvey, jessica; pillai, suresh d.; goyal, sagar m. title: comparison of thermal and non-thermal processing of swine feed and the use of selected feed additives on inactivation of porcine epidemic diarrhea virus (pedv) date: 2016-06-24 journal: plos one doi: 10.1371/journal.pone.0158128 sha: doc_id: 331652 cord_uid: oc5s1if2 infection with porcine epidemic diarrhea virus (pedv) causes diarrhea, vomiting, and high mortality in suckling pigs. contaminated feed has been suggested as a vehicle of transmission for pedv. the objective of this study was to compare thermal and electron beam processing, and the inclusion of feed additives on the inactivation of pedv in feed. feed samples were spiked with pedv and then heated to 120–145°c for up to 30 min or irradiated at 0–50 kgy. another set of feed samples spiked with pedv and mixed with ultracid p (nutriad), activate da (novus international), kem-gest (kemin agrifood), acid booster (agri-nutrition), sugar or salt was incubated at room temperature (~25°c) for up to 21 days. at the end of incubation, the virus titers were determined by inoculation of vero-81 cells and the virus inactivation kinetics were modeled using the weibull distribution model. the weibull kinetic parameter delta represented the time or ebeam dose required to reduce virus concentration by 1 log. for thermal processing, delta values ranged from 16.52 min at 120°c to 1.30 min at 145°c. for ebeam processing, a target dose of 50 kgy reduced pedv concentration by 3 log. all additives tested were effective in reducing the survival of pedv when compared with the control sample (delta = 17.23 days). activate da (0.81) and kem-gest (3.28) produced the fastest inactivation. in conclusion, heating swine feed at temperatures over 130°c or ebeam processing of feed with a dose over 50 kgy are effective processing steps to reduce pedv survival. additionally, the inclusion of selected additives can decrease pedv survivability. porcine epidemic diarrhea virus (pedv) is a pleomorphic, enveloped rna virus, classified as a coronavirus under the family coronaviridae [1] . the virus was first identified during an outbreak of diarrhea on a belgian swine breeding farm in 1978 [2] . upon infection with pedv, suckling pigs experience diarrhea, vomiting, and high mortality [3] . since the initial identification of pedv, it has been reported in canada, korea, china, thailand, italy, hungary, and vietnam [4] . it is important to note that china and vietnam are two of the top swine producing countries in asia and that they were both impacted by pedv [5] . in the united states, the virus was first detected in april 2013 and has since caused high piglet mortality in over 17 states [6] . some have suggested that pedv is transmitted via contaminated feed [7] . although research has been conducted to show the survival of pedv in feed and feed ingredients [8] [9] [10] there is limited data comparing feed processing treatments and their efficacy in inactivating the virus in complete swine feed. in the feed and ingredient industry, temperature and time conditions vary with the processing method used e.g., pelleting, rendering, pasteurization, or spray drying. moisture content of feed and ingredients is also impacted by the type of processing method used. the interactions between temperature, time, and moisture play a role in virus inactivation in feed. ingredients of porcine origin have been thought to have the greatest risk for disease transmission [9] . when processing rendered ingredients for animal feed, the processing conditions vary based on the composition of the raw material. in general, the national renderers association suggests temperatures between 115°c and 145°c for 40 to 90 min for most rendering systems. such temperatures are deemed effective in inactivating pathogens such as salmonella spp. [11] . similarly, the enveloped classical swine fever rna virus is readily inactivated in pasteurization processes after only 1 min at 71°c [12] . high temperatures (around 100°c) are also used in other processes including micronization to prepare cereal grains [13, 14] . the high temperatures used in processing rendered products and cereal grains may also be effective in inactivating pedv in contaminated feed, but no research has been conducted to compare pedv survival at temperatures greater than 80°c. other processing procedures such as ionizing irradiation have been shown to reduce the survivability of pathogens in feed, specifically listeria monocytogenes in poultry feed [15] . more recent applications of ionizing radiation technology such as electron beam (ebeam) are now used to routinely pasteurize foods and decontaminate animal feeds from pathogens [16, 17] . the main characteristics of ebeam technology include its non-thermal nature (thereby reducing potential nutrient losses), utilization of commercial electricity (does not rely on radioactive isotopes), high speed processing, and the ability to precisely control the dose delivered [17, 18] . the us food and drug administration (fda) has recognized this process as a treatment method for food and animal feed for doses up to 50 kgy [19] . all of these attributes make the ebeam technology attractive for a wide variety of applications, including pasteurization of animal feed. in addition to thermal and ebeam treatments, certain organic acid feed additives including propionic, formic, and butyric acid have been used in the past for their antimicrobial properties. these additives have previously been used as a method of controlling pathogens such as salmonella spp. and escherichia coli in poultry feed and other matrices [20, 21] . however, the effects of organic acids and other additives including sugar and salt in feed have not been investigated. therefore, the objective of this study was to determine if thermal and non-thermal methods of microbial inactivation, as well as the use of selected feed additives, are effective in reducing the survival of pedv in experimentally contaminated swine feed. the nvsl (national veterinary service laboratory, ames, ia) strain of pedv was propagated and titrated in vero-81 (african green monkey kidney, atcc 1 ccl-81™) cells. we have found that both vero-76 and vero-81 cells are susceptible to pedv. we chose to use vero-81 in this study. the cells were grown in dulbecco's modified eagle medium (dmem; mediatech, herndon, va, usa) containing 8% fetal bovine serum (fbs; gibco, life technologies, grand island, ny, usa), 50 μg/ml gentamicin (mediatech), 150 μg/ml neomycin sulfate (sigma, st. louis, mo, usa), 1.5 μg/ml fungizone (sigma), and 455 μg/ml streptomycin (sigma). the cells were washed three times with phosphate buffered saline (pbs; ph 7.2). after virus inoculation, the cells were incubated at 37°c for 1 h, allowing virus adsorption using maintenance medium (dmem with gentamicin, neomycin sulfate, fungizone, streptomycin and 10 μg/ml trypsin). the cells were washed again 60 minutes after inoculation. the washing medium was hanks' balanced salt solution containing no trypsin. however, the maintenance medium did contain trypsin. inoculated cells were incubated at 37°c under 5% co 2 . cells were examined daily under an inverted microscope for the appearance of virus-induced cytopathic effects (cpe) for up to five days post-infection. the virus was harvested by subjecting the infected cells to three freeze-thaw cycles (-80°c/25°c) followed by centrifugation at 2500 × g for 15 min at 4°c. the supernatant was collected and aliquoted into 50 ml tubes followed by storage at -80°c until used. in all experiments, the surviving virus after a certain treatment was recovered in an eluent consisting of a 3% solution of beef extract solution (lab scientific, highlands, nj) in 0.05m glycine (sigma, st. louis, mo), ph 7.5. following elution, the eluate was centrifuged for 10 min at 2500 x g to remove organic matter/debris. the supernatants were used to determine the amount of surviving virus, if any. for titration of pedv in virus stock and other samples, serial ten-fold dilutions of the eluates prepared in dmem (maintenance medium) were inoculated into vero-81 monolayers contained in 96-well microtiter plates (nunc, ny, usa) using 100 μl/well and three wells per dilution. inoculated cells were incubated at 37°c under 5% co 2 for up to seven days until the cpe appeared. the highest dilution showing cpe was considered the end point. virus titers were calculated as a median tissue culture infectious dose (tcid 50 /ml) by the karber method [22] . the amount of surviving virus was compared with the starting virus titer to calculate the amount of inactivated virus and was expressed in log scale (log 10 tcid 50 /ml). a complete phase ii pig starter feed (cgi, enhanced np-nt, batch no. 831458) was obtained and confirmed to be pedv negative by rt-pcr. the proximate analysis of this feed sample is displayed in table 1 . aliquots of feed (5 g amounts) were prepared in glass beakers, which were the survival of porcine epidemic diarrhea virus (pedv) in complete feed then placed into drying ovens set at 120, 130, 140, and 145°c. the feed aliquots remained in the ovens for 30 min to reach oven temperature. once the feed reached the indicated temperature, the beakers were immediately removed and spiked with 1 ml of pedv (6.8x10 3 tcid 50 / ml) that was previously tempered at room temperature (~25°c). after mixing, beakers were placed back in the ovens at the appropriate temperatures and incubated for 0, 5, 10, 15, 20, 25, and 30 min. after the incubation period, the samples were removed from the oven, actively cooled with a fan for 15 min, the virus eluted from the feed using the 3% beef extract eluent solution, and the number of surviving viral particles titrated. triplicate samples of each temperature were combined for a single titration and inoculation into cells. the experiment was performed in duplicate. preliminary dose mapping experiments were performed to calculate the dose uniformity within the samples. a dose-uniformity ratio was calculated by maximum dose/minimum dose for each experiment. under ideal circumstances, the dose-uniformity ratio (dur) should be about 1.0. during commercial processing of animal feed using ebeam technology, the size of the final package needs to be optimized to assure dose uniformity. the optimization involves adjusting the dimensions and weight to ensure adequate penetration of the ebeam electrons as well as dose uniformity within the package. under commercial processing conditions, the goal is to try to attain as uniform a dose as possible. at the ebeam facility on the tamu campus, the dur of a commercial pet food product is 1.78. aliquots of complete phase ii pig starter feed (5 g amounts) were prepared in several 50 ml plastic centrifuge tubes followed by the addition of 1 ml of pedv to each tube. after mixing, the virus-spiked feed was placed into individual plastic bags. the bags were flattened to remove all of the air and create a thin, even layer of the feed sample. the bags were triple-sealed to meet the biosafety procedures at the ebeam irradiation facility. the sealed bags were shipped on ice via overnight shipping to the national center for electron beam research at texas a&m university, college station, tx. preliminary dose delivery trials were performed to determine the appropriate conveyor speed and other specifications to achieve the target doses. on the day of arrival, the samples were exposed to target ebeam doses of 10, 20, 30, and 50 kgy. a control sample was also shipped with the irradiated samples, but it was not exposed to ebeam irradiation at the facility. after treatment, the samples (including the controls) were shipped back to the university of minnesota (st. paul) on ice via overnight shipping. upon arrival, the samples were immediately eluted using the aforesaid eluent solution. this experiment was performed only once. aliquots of feed in 5 g amounts were placed in plastic scintillation vials. the amount of feed additive used in the 5 g sample was determined based on the recommended dosage from the manufacturers. the following additives were added to separate aliquots of feed: 0.015 g ultracid p (nutriad, dendermonde, belgium), 0.02 g activate da (novus international, st. charles, mo), 0.01 g of acid booster (agri-nutrition, deforest, wi), 0.01 g kem-gest (kemin agrifoods, des moines, ia), 0.02 g of granulated sugar (shoppers value, eden prairie, mn) and 0.02 g of commercial salt (essential everyday, eden prairie, mn). another set of vials was used as paired controls without the addition of any feed additive. after the additives were added to the appropriate vials, 1 ml of pedv was added to each vial followed by vortexing. all vials were incubated at room temperature (around 25°c during the season which this experiment was performed). after incubation for 0, 1, 3, 5, 7, 14, and 21 days, the surviving virus was eluted from the samples using the 3% beef extract eluent solution. the experiment was performed in duplicate. each experiment used a set of triplicate samples that were combined and used for a single titration and inoculation into cells. after completion of the trial, the ph of each feed sample solution with and without the additive was measured. the ph was measured by adding 50 ml of deionized water to 5 g of feed followed by mixing continuously on a magnetic stirrer for 15 min at room temperature. after mixing, a ph probe was used to measure the ph of the liquid. the ph measurement experiment was performed in triplicate. in addition to measuring ph, the active ingredients for activate da (2-hydroxy-4-methylthiobutanoic acid), kem-gest (phosphoric, fumaric, lactic, and citric acids), sugar (sucrose), acid booster (phosphoric, citric, and lactic acids), salt (sodium chloride), and ultracid p (orthophosphoric, citric, fumaric, and malic acids) were compared. inactivation kinetic data (log tcid 50 /ml) were analyzed by ginafit, a freeware add-in for microsoft excel developed by geeraerd et al. [23] . the traditional log-linear model that assumes a linear relationship between the virus concentration and processing time was developed by bigelow and esty [24] and used to characterize the survival curves of pedv by using the following equation: where n is the surviving virus titer after the treatment (expressed as tcid 50 /ml), n 0 is the initial virus titer (tcid 50 /ml), k is the kinetic parameter (min -1 or day -1 ), and t is the treatment time (min or days). the kinetic parameter k is usually expressed as d, also known as the decimal reduction time (time required at a certain temperature to reduce 90% or 1 log of the initial virus titer) and was calculated as follows: the weibull distribution function has been used to describe non-linear inactivation patterns of various microorganisms after different thermal and non-thermal processing [25] . assuming that the temperature resistance of the virus is governed by a weibull distribution, mafart et al. developed the following weibullian equation: where n is the surviving virus after the treatment expressed as (tcid 50 /ml), n 0 is the initial virus titer (tcid 50 /ml), δ is the time of the first logarithm decline for the virus titer population (min or days), and n is the shape parameter. the n value provided a general description of the form of the curve; if n > 1, the curve is convex (it forms shoulders), if n < 1, the curve is concave (it forms tails), and if n = 1, the curve is a straight line and can be described by a linear model. two valid replicates were used to evaluate how well the model fit with the experimental data by calculating the adjusted r 2 (adj. r 2 ) as follows: where m is the number of observations, j is the number of model parameters, and ssq is the sum of squares. an anova test using the mixed procedure of sas (sas inst., cary, nc) was used to determine statistical differences across treatments. least squared means with tukey adjustment was used to determine differences between treatment means, with p < 0.05 considered to be significantly different. the experimental unit was the value obtained from the combined triplicate vials. the fixed effects were temperature or feed additive used. table 2 . the pedv showed a high thermal resistance in the dry feed samples and it was completely inactivated (3.0 log reduction) at each of the tested temperatures within 30 min. the original moisture content of the feed sample was 8.57% (w/v). after adding 1 ml of virus media the moisture increased to 23.8% (w/v). this change in moisture content potentially increased the ability of heat to reduce virus concentration, as heat inactivation is more efficient in higher moisture content as previously demonstrated by the higher sensitivity of bovine parvovirus to moist heat vs. dry heat [26] . the shape parameter (n) indicates the shape of the curve with a value n > 1 forming shoulders and being convex, n < 1 forming tails and being concave, and n = 1 being linear. the virus concentration in feed (control sample) and feed with additives is shown in table 3 . the addition of the additives to the feed sample decreased the initial virus titer. the d and delta values of the log-linear and weibull models respectively, are shown in table 4 . the use of the weibull model resulted in greater adj. r 2 values than the log-linear model, and therefore, was used to characterize the survival of pedv with the addition of additives during storage at room temperature. delta values for all the additive-containing samples, except ultracid p and salt, were significantly lower than the control sample (p < 0.05) indicating faster inactivation kinetics (table 4 ). after 21 days of incubation at room temperature (25°c), a 3 log reduction was observed in samples containing kem-gest, sugar, and salt. in the same 21-day incubation period, a 2 log reduction was observed in activate da, and <2 log reduction was observed in the control, ultracid p, and acid booster. the comparison of all feed additives in terms of active ingredient and ph value is shown in table 5 . in general, the additives containing phosphoric acid were more effective at reducing virus concentration. the only additive containing 2-hydroxy-4-methylthiobutanoic acid (activate da) was the most effective in reducing virus concentration. there were differences in the ph values of the feed samples after the addition of additives. feed was suggested as a vehicle of transmission of pedv when the first canadian swine farm tested positive for the virus [9] . a follow-up epidemiological investigation suggested that a the shape parameter (n) indicates the shape of the curve with a value n > 1 forming shoulders and being convex, n < 1 forming tails and being concave, and n = 1 being linear. doi:10.1371/journal.pone.0158128.t004 batch of spray-dried porcine plasma (sdpp) used in swine nursery feed was the potential source of pedv infection. in a bioassay experiment, a contaminated sdpp sample was able to infect pigs, however, when added to the complete feed no additional pigs were infected. this was potentially caused by a dilution effect or an extended time period between virus contamination and bioassay [9] . a similar conclusion that feed or feed ingredients could be spreading pedv between farms was made during an investigation of a pedv outbreak in ohio. in this situation, virus was detected in the feed through rt-pcr on a farm with infected sows. in a follow up bioassay, none of the pigs developed an infection after consuming the contaminated feed [7] . in this study, the source of feed contamination was not found, although the rt-pcr test was positive for pedv. dee et al. [10] showed that feed experimentally contaminated with pedv rna could cause an active infection in pigs when feed was consumed via natural feeding behavior. the collection of references indicates that if complete feed happens to be contaminated with pedv, it is possible for the contaminated feed to cause an active infection on a farm. in summary, these results suggested that additional research is needed to evaluate potential mitigation strategies to control transmission of pedv through feed processing methods. the survival of pedv in feed that was thermally treated at 120 to 145°c for 0 to 30 minutes was first evaluated. previous research has focused on the survival of pedv at temperatures below 80°c. one study found that a cell culture-adapted strain of pedv was moderately stable at 50°c for 30 min, with only a reduction of 0.4 log 10 pfu/ml at 50°c when compared to the control [1] . a further investigation indicated that treating pedv at temperatures between 60 to 80°c for 30 min caused a complete loss of infectivity, but the log reduction in virus concentration was not reported [1] . in another study, the survival of a wild type strain of pedv in fecal samples required only 10 min of exposure at 71°c to be inactivated to the extent that it was not able to cause infection in live pigs [27] . this variability in virus inactivation could be possibly due to differences in moisture content between the different samples tested. the moisture content will ultimately have an impact on virus survivability during heat treatment. although the exposure times and temperatures varied in these studies, their results correspond with previous data indicating that pedv is a low thermally stable virus [4] . our hypothesis was that the thermal treatment of feed would reduce the survival of pedv in swine feed. this hypothesis was confirmed by our experimental results which demonstrated that a 3 log (99.9%) reduction of the pedv concentration is achieved within 25 min of exposure at 120°c. these results also highlight the finding that temperatures over 130°c don't increase the inactivation of pedv. this is an important observation because excessive heating of high protein feed ingredients results in reductions of amino acid digestibility through maillard reactions [28] . in addition to the log reduction of pedv in feed at high temperatures, the production of a shoulder in the inactivation curve produced by the weibull model also help determine pedv survival at high temperatures. the observed shouldering phenomenon observed in the inactivation curves may indicate that virus is resistant to a certain temperature-time level combination and a threshold needs to be surpassed to see a significant level of inactivation. another possible reason for this shoulder in the curve could be the possibility that the feed was cooled when the room temperature inoculum was added. this would mean the first few min the feed was placed back in the oven was spent heating back up to oven temperature, which could explain the plateau in virus concentration. overall, the results from our experiment confirm that the concentration of pedv in complete feed can be reduced through the application of heat. when exploring non-thermal treatment processes to reduce pedv in feed, an ebeam irradiation dose of 50 kgy was found to be effective in reducing virus concentration by 3 log (99.9%). currently, the food and drug administration has stipulated an upper dose limit of 50 kgy of ionizing irradiation doses in animal feeds [19] . the use of ebeam processing of animal feeds and diets has been shown to be an effective process for inactivating pathogens in the us [16, 17] . animal feed and bags (weighing about 20 kg) are routinely treated using ebeam processing. process controls are in place to measure the maximum and minimum doses in these sample bags to meet customer needs and adhere to regulatory limits. though we have shown that ebeam treatment is effective in reducing the concentration of pedv in swine feed, followup studies are needed to verify the results seen in this study with different types of feed. our experimental data, suggesting that a target dose of 50 kgy will achieve approximately 3 log 10 reduction in pedv, are important in that it highlights the criticality of ensuring low viral bioburden during feed formulation by adhering to good manufacturing processes. these results suggest that if the initial pedv bioburden is greater than 4 log 10 tcid 50 , the fda mandated 50 kgy dose upper limit may not be effective in complete inactivation of virus in these feeds. furthermore, if the maximum titers of pedv in commercial swine feed are known, then the dose can be calibrated to theoretically achieve the total elimination of pedv in commercial swine feed. in addition to more research investigating maximum pedv titers, it may also be necessary to investigate the use of irradiation on individual feed ingredients. our experiment only measured the impact of irradiation on complete swine feed, and so it cannot be confirmed if the same results would be seen in individual feed ingredients. finally, we evaluated the survival of pedv in feed when various feed additives were included at manufacturers' recommended doses. the additives experiment was only performed at room temperature. on a swine farm, feed can be stored at a variety of temperatures based on season, barn layout, bagged feed vs. bulk feed, barn temperature, and other variables. room temperature was used in this experiment as a feasible average temperature, though it may not accurately apply to every situation. feed is suggested to be stored in a cool, dry place and so it is likely some swine feed will be stored at temperatures below the approximately 25°c tested in this experiment. the addition of organic acid feed additives in the diet has been shown to increase nutrient digestibility by lowering ph and modifying gut microflora, which ultimately improve growth performance and provide an alternative to antibiotic use in weaned pig diets [29] . organic acids have been used to reduce the prevalence of bacterial pathogens such as salmonella in poultry feed [21] . in an additional experiment, the antimicrobial product salcurb, which is used to control salmonella in feed, was tested for its ability to decrease the presence of viable pedv in feed. in this experiment, a bioassay showed that the feed treated with the salcurb product was not able to cause an infection in naïve pigs [30] . a proposed method for how the salcurb inactivated the pedv was not reported. differences in pedv survival in feed has not only been observed in the presence of additives, but also in the presence of different feed ingredients. in an experiment testing 18 different feed ingredients, it was determined that the survival of pedv in these ingredients was ingredient specific with an extended survival being observed in soybean meal [31] . this indicates that variation as small as changes in ingredients could change virus survival in feed. it has been reported that pedv is stable at a ph of 6.5 to 7.5 at a temperature of 37°c [32] . because intestinal viruses must survive the acidic conditions of the stomach, they are generally more resistant to acidic ph. with this being said a change in ph outside of ideal conditions for an extended period could cause an increase in virus inactivation. a previous review reported that the ph of the diet was decreased from an average ph of 5.96 to 4.71 when organic acids (fumaric, propionic, formic, citric, sodium citrate, hydrochloric, sodium fumaric, or malic acid) were added to feed [33] . this ph decrease of the diet was hypothesized to decrease the survival of pedv in the presence of additives. the results from our study did not necessarily support this hypothesis. activate da and kem-gest were the most effective in reducing virus survivability and also had the most acidic ph values when added to feed, which confirm the original hypothesis. however, ultracid p did not result in a different diet ph compared with kem-gest, yet it had the highest delta value of all additives evaluated. in addition to this, the feed sample would require a type of liquid to be added to the feed for ph to even play a role, as a dry substrate will not have a ph. in the experiment, 1ml of virus media was added to the sample and mixed. this means it could be possible for the ph of the media to change because of the additive when the virus was initially added. if this single change were to explain the differences in survival for our experiment, there would be a more prominent pattern between the ph of the feed solution and the delta value observed. with a lack of a clear pattern between ph and virus survival, and in some cases no difference between the ph of the solution with the control and with some of the additives tested, there is another aspect causing the changes in virus survival between the feed additives used. there are other components of feed additives that have the capability to reduce pedv survivability, as suggested by the inability to explain the decrease in pedv survivability due to change in ph alone. we hypothesized that specific active ingredients play a role in differences in virus survivability among additives. many of the less effective additives contained citric acid and lactic acid as active ingredients. it has been previously shown that citric and lactic acids only have moderate antimicrobial activity because they are solely effective at a low ph in the environment [34] . because most swine feed samples have a neutral ph, it is not surprising that additives containing citric acid as an active ingredient may not have been as effective in inactivating pedv. in contrast, fumaric acid has been shown to be a very effective antimicrobial, and is extremely effective in reducing the survivability of e. coli [35] . no studies have been published to show that fumaric acid is an effective anti-viral compound, but its presence in kem-gest, which resulted in the second lowest pedv survivability in our study, implies it may also be an effective ingredient in reducing survivability of pedv. the active ingredient of activate da, the most effective additive in our study, is 2-hydroxy-4-methylthiobutanoic acid. this ingredient has been researched exclusively as a cost effective supplement in low methionine diets for swine [36] . although it has not been approved for use as an antimicrobial or antiviral agent, the results from our study suggest it could have potential in reducing virus survivability. overall, careful selection of additives containing the most effective active ingredients is necessary for achieving reduced pedv survivability in complete swine feeds. further research is needed to confirm the relative effectiveness of each active ingredient and how they may interact with each other. one limitation in this experiment is the unknown variable of water activity. by adding liquid media to the samples the moisture content was already dramatically increased. this increase in moisture potentially caused changes in the inactivation of the virus when exposed to heat that may not be observed in the low moisture conditions of animal feed. it has been demonstrated that pedv inactivation using heat treatments and changes in ph differs in liquid media compared with a dried plasma product [37] . because our experiments investigated both change in ph through an additive and change in heat treatment, our increased moisture from inoculating the sample could cause different inactivation kinetics than what would be seen in dry feed without media. in addition to this, the humidity in the environment was not controlled and the water activity of the samples were not measured. without measuring these variable there is no way to tell the role they played in the inactivation of pedv in the feed samples. in addition to unknown water activity, this experiment also had the limitation of low starting virus titers. this made it impossible to reach the standard of complete inactivation (typically 7 log reduction) because the starting virus titers were only 4 logs. our results show that pedv survivability can be reduced using ebeam processing or thermal processing at temperatures greater than 130°c. the use of some feed additives was also effective in reducing virus levels, but future investigations are needed to determine the prevalence and maximum titers of pedv in commercial swine feeds to determine the most appropriate treatment parameters (time and temperature of thermal processing, dose of ebeam processing, and feed additive selected) to be most effective in inactivating pedv. feed has been suggested as a possible vehicle of pedv transmission. this has created a need to evaluate new and existing feed processing methods to reduce pedv survivability in feed. results from this study have shown that both thermal (130°c for at least 15 min) and non-thermal (50 kgy ebeam dose) feed processing technologies can eliminate 99.9% of pedv infectivity when working with low virus titers (6.8x10 3 tcid 50 /ml). additionally, the survivability of pedv can be reduced by the use of selected acidifiers and organic acids in swine feeds. quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (pedv) a new coronavirus-like particle associated with diarrhea in swine emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences porcine epidemic diarrhea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines pig production in subtropical agriculture origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states investigating the introduction of porcine epidemic diarrhea virus into an ohio swine operation risk assessment of feed ingredients of 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produce: a handbook of best-practice examples, innovative commercial solutions and case studies electron beam pasteurization and complimentary food processing methods sterilization methods and the comparison of e-beam sterilization with gamma radiation sterilization food and drug administration. 21 c.f.r. 579 feed additives to control salmonella in poultry the use of organic acids to combat salmonella in poultry: a mechanistic explanation of the efficacy fifty percent endpoint calculation ginafit, a freeware tool to assess non-log-linear microbial survivor curves the thermal death point in relation to typical thermophylic organisms on calculating sterility in thermal preservation methods : application of the weibull frequency distribution model further studies on thermal resistance of bovine parvovirus against moist and dry heat methods for inactivating pedv in hog trailers. in: twenty-second annual swine disease conference for swine practitioners amino acid digestibility of heat damaged distillers dried grains with solubles fed to pigs acidifier as an alternative material to antibiotics in animal feed an evaluation of a liquid antimicrobial (sal curb 1 ) for reducing the risk of porcine epidemic diarrhea virus infection of naïve pigs during consumption of contaminated feed an evaluation of porcine epidemic diarrhea virus survival in individual feed ingredients in the presence or absence of a liquid antimicrobial diseases of swine critical review of acidifiers microbial ecology of foods addition of fumaric acid and sodium benzoate as an alternative method to achieve a 5-log reduction of escherichia coli o157:h7 populations in apple cider the relative effectiveness of 2-hydroxy-4-(methylthio) butanoic acid and dl-methionine in young swine 1 sensitivity of porcine epidemic diarrhea virus (pedv) to ph and heat treatment in the presence or absence of porcine plasma we thank mickey speakmon for his assistance with the ebeam irradiation experiment. key: cord-316134-lkd2mj27 authors: sungsuwan, suttipun; jongkaewwattana, anan; jaru-ampornpan, peera title: nucleocapsid proteins from other swine enteric coronaviruses differentially modulate pedv replication date: 2020-01-15 journal: virology doi: 10.1016/j.virol.2019.11.007 sha: doc_id: 316134 cord_uid: lkd2mj27 porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev) and porcine deltacoronavirus (pdcov) share tropism for swine intestinal epithelial cells. whether mixing of viral components during co-infection alters pathogenic outcomes or viral replication is not known. in this study, we investigated how different coronavirus nucleocapsid (cov n) proteins interact and affect pedv replication. we found that pdcov n and tgev n can competitively interact with pedv n. however, the presence of pdcov or tgev n led to very different outcomes on pedv replication. while pdcov n significantly suppresses pedv replication, overexpression of tgev n, like that of pedv n, increases production of pedv rna and virions. despite partial interchangeability in nucleocapsid oligomerization and viral rna synthesis, endogenous pedv n cannot be replaced in the production of infectious pedv particles. results from this study give insights into functional compatibilities and evolutionary relationship between cov viral proteins during viral co-infection and co-evolution. porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev) and porcine deltacoronavirus (pdcov) belong to the family coronaviridae (enjuanes, 2000) . pedv and tgev have been classified into the alphacoronavirus genus, whereas pdcov belongs to the deltacoronavirus genus (jung et al., 2016a; jung and saif, 2015a) . they share similar genome architectures, with a 25-30 kb positivesense, single-stranded rna genome. the 5′ two-thirds of the viral genome encodes non-structural proteins from open reading frames (orf) 1a and 1b necessary for viral genome replication. the rest of the genome encodes a number of unique accessory proteins such as pedv orf3, tgev 3a/3b/7, pdcov ns6/ns7, and four common structural proteins, namely the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins (kocherhans et al., 2001; lee and lee, 2014; penzes et al., 2001) . these enteric swine coronaviruses (covs) infect epithelial cells lining the small intestine and cause villous atrophy, resulting in malabsorption and severe diarrhea (jung et al., 2016a ). an outbreak of these viruses, especially pedv, can lead to up-to-100% mortality in neonatal piglets, prompting huge economic losses in the swine production industry worldwide. unless they are examined by laboratory-level diagnosis, these covs produce almost indistinguishable pathogenesis. co-infection of enteric pathogens are common. tgev and pdcov have been found to co-circulate with pedv in the field (song et al., 2015; wang et al., 2016) . in pdcov-positive samples, the rate of pedv co-infection as detected by rt-pcr varies from 33% to 50% (jung et al., 2016a; jung and saif, 2015a) . although tgev infection has become rarer nowadays, it has still been detectable in samples in china, and often together with pedv and/or pdcov (dong et al., 2015; wang et al., 2013) . despite substantial epidemiological evidence of co-infection, the effects of these events on disease outcomes have not yet been formally described. since these enteric swine covs share cell tropism, co-infection of these viruses can theoretically cause mixing of viral components in the same cellular compartments, possibly leading to direct or indirect effects on viral replication kinetics or pathogenic outcomes. to the best of our knowledge, there are currently no reports on studies at molecular or cellular levels on how viral components from different cov species interact with or affect other viruses. investigation of possible molecular interactions between components of pedv, pdcov and tgev and their influence on replication of each virus would provide a crucial insight into comprehensive understanding of these covs. of all viral proteins, we have chosen to start with the n protein, as it is among the most abundant and ubiquitous structural proteins in infected cells. the cov n protein is functionally conserved across the family coronaviridae (chang et al., 2009; cong et al., 2017) , with its primary function being to form a scaffold for packaging viral genomic https://doi.org/10. 1016/j.virol.2019.11.007 received 20 september 2019; received in revised form 4 november 2019; accepted 5 november 2019 rna (grna) into the internal core of virions (de haan and rottier, 2005) . besides scaffolding, other functions of the cov n protein (primarily based on studies of common representatives of the family like severe acute respiratory syndrome-cov (sars-cov) or mouse hepatitis virus (mhv)) include acting as rna chaperones (zuniga et al., 2007 (zuniga et al., , 2010 , promoting viral genome transcription or replication (hurst et al., 2010 (hurst et al., , 2013 masters et al., 1994; zuniga et al., 2007 zuniga et al., , 2010 , facilitating viral assembly (de haan and rottier, 2005; kuo et al., 2016) , suppressing antiviral rna-interference activity from their hosts (cui et al., 2015) , and suppressing host immunity (ding et al., 2014 (ding et al., , 2017 xu et al., 2013; zhang et al., 2018) . based on sequence alignment and limited structural data from some representative covs, all cov n proteins are predicted to consist of three structural domains: the n-terminal domain (ntd), linker region (lkr) and c-terminal domain (ctd) (chang et al., 2014; mcbride et al., 2014) . ntd binds rna through electrostatic interaction with its charged amino acids as well as interaction between conserved aromatic residues in the proteins and nucleotide bases in the rna (chang et al., 2014; huang et al., 2004; tan et al., 2006) . lkr is a disordered domain between ntd and ctd. studies of sars-cov n indicate roles for lkr in rna binding, virion assembly and self-association (chang et al., 2009 (chang et al., , 2013 he et al., 2004a) . although it is also reported to have rna binding capacity, ctd is a more hydrophobic domain mainly responsible for self-association to form stable dimers and subsequent oligomers of cov n (chen et al., 2007; yu et al., 2006) . studies of n proteins from sars-cov and mhv revealed that n dimerization followed by multimerization is a common process in the formation of viral ribonucleoprotein that initiates viral genome packaging in the virion assembly process among covs (cong et al., 2017; fan et al., 2005; ma et al., 2010; yu et al., 2006) . examples from other viruses suggest that cross-association between viral nucleocapsid proteins could determine different outcomes during co-infection. for instance, mixed infection between two types of plant tospoviruses results in interspecies interaction between their n proteins and more severe symptoms compared to single infections, suggesting synergy between these viruses (bag et al., 2012; tripathi et al., 2015) . on the other hand, intertypic interference between types a and b influenza viruses could be partially explained by the inhibitory effect of type b influenza virus nucleoprotein exerted on its type a counterpart (aoki et al., 1984; jaru-ampornpan et al., 2014) . given structural similarities, we expect that cov n proteins would likely cross-interact with each other during a co-infection event. whether this cross-interaction has beneficial or detrimental effects on viral replication has never been explored. investigation of the interaction between cov n proteins could lead to insights into virus evolution, anti-cov drug development and vaccine design (chang et al., 2016; lo et al., 2013) . human embryonic kidney (hek) 293t cells, wild-type veroe6 cells and veroe6-based cell lines stably expressing cov n were maintained in optimem supplemented with 10% fetal bovine serum (fbs) and antibiotics at 37°c with 5% co 2 . the virus pedv-avct12-mcherry (pedv-mcherry), its infectious clone (psmart-bac-mcherry-pedv avct12 [ppedv-mcherry]) and pcaggs-pedv n-myc, a plasmid for c-terminally myc-tagged pedv n expression, have been described previously (jaru-ampornpan et al., 2017; jengarn et al., 2015) . tgev and pdcov n encoding genes were codon-optimized for high expression in mammalian cells and to avoid expression of pdcov ns7 embedded in the pdcov n gene ) (genbank: aag30228.1, afd29191.1, respectively). pcr products of the corresponding n genes with indicated tags at the c-termini were ligated into pcaggs vectors to give plasmids for expression of tagged cov n (pcaggs-tgev n-flag and pcaggs-pdcov n-ha). pcaggs-ha-pedv n for n-terminally ha-tagged pedv n expression was similarly constructed based on the same pedv n dna sequence in pcaggs-pedv n-myc. pgex-4t3-pedv n was constructed by in-frame insertion of the pedv n gene sequence, codon-optimized for bacterial expression based on the sequence from avct12 strain (accession number lc053455), into the pgex-4t3 vector (amersham) following the coding sequence of n-terminal gst. all plasmids were verified by dna sequencing. cells were lysed with lysis buffer (25 mm tris-hcl ph 7.4, 150 mm nacl, 1 mm edta, 1% np-40 and 5% glycerol, supplemented with a protease inhibitor cocktail (halt™ protease inhibitor cocktail, thermo scientific)). proteins in the cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), and then transferred onto nitrocellulose membranes (bio-rad laboratories). the membrane was blocked in 5% skim milk prior to incubation with indicated primary antibodies. horseradish peroxidase (hrp)-conjugated goat anti-mouse igg (biolegend) or (hrp)-conjugated donkey antirabbit igg (biolegend) was used as secondary antibodies. primary antibodies used in this study include mouse-anti-myc (thermo scientific), rabbit-anti-flag (cell science technology), rabbit-anti-ha (cell science technology), mouse-anti-s1 (a kind gift from dr. qigai he, huazhong agricultural university), and mouse-anti-pedv n (sd 6-29, medgene labs). hek293t cells were co-transfected with pcaggs-pedv n-myc (or the empty pcaggs vector for negative controls) and either pcaggs-ha-pedv n, pcaggs-tgev n-flag or pcaggs-pdcov n-ha using fugene hd (promega) according to the manufacturer's instructions. at 48 h post-transfection (hpt), cell lysates were prepared as described previously and pre-adsorbed to control agarose beads (thermo scientific) for 1 h at 4°c to reduce non-specific binding. then, the treated lysates were incubated with anti-myc-conjugated agarose beads (thermo scientific) at 4°c overnight. the mixtures were washed three times with lysis buffer supplemented with 250 mm nacl. ip complexes were eluted by boiling the bead mixtures in 2× non-reducing sample buffer (thermo scientific) for 5 min and supplemented with 2 mm dtt before sds-page and western blot analysis. gst and gst-pedv n were expressed in bl21 (de3*) cells. after 3h protein induction by 0.5 mm isopropyl ß-d-1-thiogalactopyranoside, bacteria were lysed by sonication in 1× phosphate-buffered saline (pbs). the lysates were clarified and stored in 100-μl aliquots at -80°c until use. hek293t cells were transfected with plasmids as indicated and were lysed at 48 hpt with lysis buffer. the bacterial lysate containing the bait protein (gst/gst-pedv n) and the hek293t cell lysate containing the prey protein (ha-pedv n, tgev n-flag, pdcov n-ha, or combinations thereof) were incubated with pre-equilibrated gsh ff resin (amersham) overnight and washed twice in high-salt gst buffer and four times in low-salt gst buffer (20 mm tris-hcl ph 8.0, 20% glycerol, 1 mm edta, 0.1% np-40 and 1 m/100 mm nacl) (nguyen and goodrich, 2006) . the bound proteins were released by boiling the resin in 2× sds-page loading buffer and analyzed by coomassie staining (for bait) and western blotting with indicated antibodies (for prey). figures were representatives of three independent experiments. for fig. 2 , quantification of eluted protein bands in each gst pulldown reaction was performed using the biorad image lab software 6.0.1 and expressed as the ratios of pulled-down pedv n (western blot with anti-ha) to eluted gst-pedv n (coomassie stain). because of variability between independent experiments, these ratios were normalized to the conditions without extra cov n protein performed side-by-side in each experiment before statistical comparison. for the rnasea experiment, the combined lysates were treated with 10 μg of rnasea (thermo scientific) or pbs buffer for 30 min at 4°c to avoid protein precipitation at high temperature. to verify the absence of rna after the treatment, total rnas were purified from parts of treated lysates by rna purification kit (thermo scientific) and were used at equal volumes as templates for rt-pcr. the rt-pcr products were visualized by ethidium bromide staining. veroe6 cell lines expressing pedv n-myc, tgev n-flag and pdcov n-ha were constructed using the same protocol as veroe6-pedv n cells as described previously (liwnaree et al., 2019) . briefly, lentiviruses carrying each cov n gene were generated by co-transfection of psin-csgw-ubem carrying the inserted cov n gene with a packaging plasmid encoding gag, pol, rev and tat (pcmv-δr8.91) and a plasmid expressing lentiviral vsv envelope glycoprotein (pmd2.g) into hek293t cells. at 48 hpt, supernatants containing the lentiviruses were harvested and filtered through a 0.45-μm filter. the filtered supernatants were then used to transduce veroe6 cells. a single clone of the transduced cells expressing the corresponding n protein was selected based on similar levels of the transgene protein expression. uniform expression of exogenous n proteins in the engineered veroe6 cell lines was verified by immunofluorescence analysis. briefly, cells were grown in an eight-well slide. at 24 h, cells were fixed with 80% cold acetone for 10 min. after washing twice with pbs, cells were blocked with 10% fbs, 1% bovine serum albumin (bsa) in pbs for 1 h at room temperature. cells were then incubated with mouse anti-pedv n antibodies (sd 6-29) or anti-pdcov n (sd55-197, medgene labs) or anti-tgev n (211.56, median diagnostics, republic of korea). after washing twice with pbs, cells were incubated with goat antimouse igg alkaline phosphatase antibodies (abcam). fluorescence was developed by adding alkaline phosphatase substrate (immpact™ vector® red). fluorescence images were taken under a fluorescence microscope (olympus). the n-deficient pedv infectious clone (ppedv-mcherry-δn) was constructed using a strategy described previously (jengarn et al., 2015; wanitchang et al., 2019) . a frameshift mutation was used to silence n expression by mutating the original start codon in the n orf, atggct, to atatgt in an intermediate plasmid, ptz-gh, containing a pedv genome fragment (designated 'gh'; fig. 6a ) with the 5′ end of the n gene as described previously (jengarn et al., 2015) . the site-directed mutagenesis was performed in the ptz-gh cloning vector. the mutated fragment was amplified by primers that facilitated subsequent in-fusion ligation (in-fusion hd, clontech) into a pre-digested psmart-bac plasmid containing the rest of the pedv genome (jengarn et al., 2015) to yield psmart-bac-pedv-mcherry-δn (designated ppedv-mcherry-δn). hek293t cells were transfected with 2 μg of the indicated infectious clone (or co-transfected with the indicated amount of a cov n expressing plasmid) using fugene hd (promega) according to the manufacturer's instructions. at 72 hpt, cell lysates were prepared for western blot analysis as described previously, and supernatants were transferred for adsorption on veroe6 cells for 1 h at 37°c. for viral rescue of pedv-mcherry-δn, veroe6 cells stably expressing pedv n was further transfected with 2 μg of pcaggs-pedv n for 24 h before virus adsorption to ensure sufficient expression of pedv n in the infected cells. the inocula were then removed, and the cells were washed once with pbs and supplied with 2 ml fresh optimem with 0.1% tryple (thermo scientific). at indicated time points, infected cells were imaged under a fluorescence microscope (olympus), and the supernatants were collected for subsequent experiments or viral titer analysis by tcid 50 assay. veroe6-based cell lines (5×10 5 cells/ml) were plated in a six-well plate. at 24 h, the cells were washed once with pbs and treated with 1 ml of virus at the indicated multiplicity of infection (moi). at 1 h post-adsorption, the inoculum was removed, and the cells were washed once with pbs and supplied with 2 ml of fresh optimem containing 0.1% tryple. extent of pedv infection was monitored by mcherry fluorescence under a fluorescence microscope. cell lysates or supernatants were harvested at the indicated time points for further analysis. for viral infection in the transient cov n expression experiment, veroe6 cells were transfected with 1 or 2 μg of pcaggs-pedv n-myc, pcaggs-pdcov n-ha, pcaggs-tgev n-flag, or the empty pcaggs vector and were incubated for 24 h to allow for protein expression. cells were then infected with pedv-mcherry (moi = 0.0001) as described above. monolayers of veroe6 cells in 96-well plates were washed once with pbs. one hundred microliters of 10-fold serially diluted virus in optimem with 0.1% tryple was added to the cells (8 wells per each dilution). at 72 h post-infection (hpi), the infected cells were scored by mcherry expression under a fluorescence microscope. tcid 50 titers were calculated using the reed-muench method (reed and muench, 1938) . for analysis of viral rna synthesis, total rna was extracted from veroe6 cell lines expressing various n infected with pedv-mcherry (moi = 0.0001) at indicated time points using the rna extraction kit (thermo scientific). dnasei (fermentas) was used to treat the rna (15 min at 37°c) before inactivation with edta (10 min at 65°c). onestep rt-qpcr was performed with the luna universal one-step rt-qpcr mix (new england biolabs) as described previously (liwnaree et al., 2019) . relative quantities of rna accumulation were evaluated using the 2 -δδct method normalized against viral rna levels from infected veroe6 cells at 24 hpi. values are reported as averages ± sem from three independent experiments. during co-infection, viruses can influence the course of infection of other viruses via direct physical interactions between viral components. to explore the possibility that swine cov n proteins might affect replication of other co-infecting covs, we first investigated cross-species interaction between these n proteins. specifically, we asked if n proteins from tgev or pdcov can interact with pedv n and possibly affect pedv replication. we first assessed interspecies protein-protein interaction by co-immunoprecipitation. hek293t cells were co-transfected with pcaggs-pedv n-myc and pcaggs expressing ha-pedv n, other cov n proteins bind competitively to pedv n. gst pulldown by gst-pedv n was performed with hek293t cell lysates containing ha-pedv n and varying amounts (0-2 μg transfected plasmids) of pdcov n-ha or tgev n-flag. the input and eluates were separated by sds-page. coomassie staining was used to detect gst-pedv n, and western blotting was used to detect haand flag-tagged proteins. the figure is representative of three independent experiments. quantification was performed using the biorad image lab software 6.0.1 as described in materials and methods. the pulldown ratios from the conditions without pdcov or tgev n proteins in each independent experiment were set to one. *p < 0.05, **p < 0.01 compared to the conditions without pdcov or tgev n proteins. fig. 3 . effect of rna on homo-and hetero-oligomerization of pedv n. gst pulldown by gst-pedv n was performed with hek293t cell lysates containing ha-pedv n, pdcov n-ha or tgev n-flag. the lysate mixtures were treated with 10 μg rnasea (+) or buffer (-) for 30 min at 4°c prior to incubation with gsh ff resin. the input and eluates were analyzed with sds-page (coomassie staining for gst-pedv n) and western blotting for the other n proteins. rna was extracted from a portion of the lysate mixtures, treated with dnasei, and subjected to rt-pcr with primers specific to the nucleocapsid genes studied in each experiment. expected rt-pcr products were about 1200 bpand were analyzed by agarose gel electrophoresis. pdcov n-ha or tgev n-flag. at 48 hpt, lysates were prepared and incubated with agarose beads coupled to anti-myc antibodies prior to elution and analysis by western blotting. as expected, ha-tagged pedv n could be precipitated with myc-tagged pedv n but not with empty beads, indicating specificity of the homo-oligomeric interaction (fig. 1a) . interestingly, both n proteins from related covs also displayed specific interaction with pedv n, both co-eluting with myctagged pedv n (fig. 1a) . nevertheless, we observed slight non-specific binding in the case of pdcov n-ha despite stringent washing conditions. therefore, we utilized a gst pulldown assay as an alternative and independent verification of these results. for gst pulldown, the bait proteins, gst and gst-pedv n, were expressed in bacterial bl21 (de3*) cells, and the prey proteins, ha-pedv n, pdcov n-ha and tgev n-flag, were expressed in hek293t cells. bacterial and hek293t cell lysates were incubated with gsh ff resin overnight to allow for bait-prey interaction. after removing nonspecific binding by multiple rounds of washing, the bound proteins were released and analyzed by sds-page. bait and prey proteins were detected by coomassie staining and western blotting with indicated antibodies, respectively. ha-pedv n could only be observed when pulled down with gst-pedv n, while no ha-pedv n was observed when pulled down with gst, demonstrating the specificity of the assay (fig. 1b) . next, we performed the gst pulldown experiment with pdcov n and tgev n as prey proteins. similarly, we found that pdcov n-ha and tgev n-flag only co-eluted with gst-pedv n, indicating that n proteins from both tgev and pdcov bound to pedv n specifically (fig. 1b) . both co-immunoprecipitation and gst pulldown results indicated that interspecies hetero-oligomers of cov n proteins are possible, suggesting conservation of the oligomerization motifs and function of nucleocapsid proteins among swine covs. these results encouraged us to further probe the factors affecting the nature of these interactions. the cross-association capability of pdcov n or tgev n could possibly interrupt homo-oligomerization between pedv n and lead to competitive binding between pedv n and other cov n proteins. using the gst pulldown assay, we further probed the interplay between these cov n proteins to see how pedv n homo-oligomers would be affected in the presence of other cov n proteins. hek293t cells were cotransfected with pcaggs-ha-pedv n and varying amounts of pcaggs-pdcov n-ha or pcaggs-tgev n-flag. at 48 hpt, lysates were prepared and incubated with gst-pedv n-containing bacterial lysate and gsh ff resin overnight. gst pulldown revealed that, upon increasing amounts of co-eluting pdcov or tgev n, co-eluting hatagged pedv n decreased reciprocally (fig. 2) . this indicates, to a certain level, a competition between homo-oligomerization of pedv n and hetero-oligomerization with nucleocapsid proteins from other covs, and suggests that these cov n proteins utilize the same motifs or binding interfaces in oligomeric complex formation or harness common factors such as rna that can mediate oligomeric interactions. one of the primary functions of cov n proteins is to bind and organize viral rna genomes for viral assembly (mcbride et al., 2014) . however, there have been conflicting reports about the necessity of viral rna in promoting or assisting cov n self-oligomerization. some reports observed rnasea susceptibility of the oligomeric complex (narayanan et al., 2003; verheije et al., 2010) , while others detected intact higher cov n oligomers despite the absence of rna (cong et al., 2017; jayaram et al., 2006; ma et al., 2010) . to investigate whether the presence of rna influences interspecies association between these swine enteric cov n proteins, gst pulldown experiments were performed with or without rnasea treatment. first, however, we tested whether or not rna affected pedv n homo-oligomerization in this assay setup. hek293t cell lysate containing ha-tagged pedv n was mixed with gst-pedv n bacterial lysate and split into two equal parts. the lysate mixtures were either treated with rnasea or pbs buffer for 30 min at 4°c. the absence of rna after the treatment was assured by performing rt-pcr reactions with primers specific to the pedv n gene (fig. 3) . the lysate mixtures were then incubated with gsh ff resin overnight to compare the amount of ha-pedv n pulled down by gst-pedv n in the presence or absence of rna. our results across three independent experiments consistently showed a slight but noticeable increase in gst-pedv n in the elution fraction after rnasea treatment (fig. 3) . moreover, the amount of ha-pedv n pulled down in the absence of rna seemed to be greater than that observed in the presence of rna, suggesting that rna interferes with the ability of pedv n to bind to each other. we also consistently observed a slight decrease in the level of tagged pedv n in the input fraction after the lysates were treated with rnasea, implying decreased overall stability of pedv n in the absence of rna. similar decreases in protein levels were not observed with tgev or pdcov n proteins (see below), suggesting specificity of the phenomenon. interestingly, when the experiments were performed on lysates containing pdcov n-ha or tgev n-flag, a completely opposite trend was observed for hetero-oligomerization. while increased gst-pedv n was still similarly observed in the eluted fraction in the absence of rna, both pdcov n and tgev n were pulled down by gst-pedv n much less efficiently in the absence of rna (fig. 3) . it should be noted that the total amount of the tagged cov n proteins did not decrease, arguing against the possibility that tgev or pdcov n proteins were more prone to degradation without the bound rna (fig. 3, 'input' lanes) . these results suggest that the presence of rna immensely helps strengthen hetero-oligomerization between pedv n and other cov n proteins but renders homo-oligomerization between pedv n weakened. although not an absolute requirement for interspecies complex formation, rna could act as a bridge for n proteins from different species to form the complex, implying the heterologous protein-protein interactions are significantly weaker or less specific than the homologous interaction. even though the levels of rna purified from rnasea-treated lysates were below the detection limit of spectrophotometer (data not shown) and could not produce visible rt-pcr products, we could not rule out the possibility of leftover small rna fragments mediating heterologous cov n protein binding, giving rise to much fainter tgev n or pdcov n bands that co-eluted with gst-pedv n. in summary, the results in this section demonstrate conserved association capability among different cov n proteins but suggest some differentiating features, especially the influence of rna on each complex, between homo-and hetero-oligomers involving pedv n. hetero-oligomer formation as demonstrated in the previous section formed a basis for our investigation into n-mediated virus-virus interaction. as it has been shown for other viruses, it is not unreasonable to assume that the specific and competitive binding between n proteins of pedv and other covs could interrupt regular functions of pedv n during viral growth. to investigate how this protein-protein interaction might affect pedv replication, we transiently transfected veroe6 cells with varying amounts of the pcaggs plasmid expressing n proteins from either pdcov or tgev for 24 h before infection with pedv-mcherry (moi = 0.0001) and followed the course of viral replication for each condition. the levels of cov n expression were verified by western blotting against tags attached to each n protein (fig. 4a) . cell culture supernatants were collected at 24, 48 and 72 hpi to determine virus titers by tcid 50 assay (fig. 4b-d) . the results showed that veroe6 cells transiently transfected with either pedv n or tgev n showed a dose-dependent increase, up to an order of magnitude, in pedv-mcherry titers compared to veroe6 cells transfected with a blank vector ( fig. 4b and d) . interestingly, veroe6 cells transiently transfected with pdcov n showed suppression on pedv-mcherry growth, especially at high levels of pdcov n expression (fig. 4c ). due to relatively low transfection efficiencies in veroe6 cells and the uncontrollable proportion of exogenous n expression and pedv infection occurring in the same cells, the actual effects of these cov n proteins on pedv growth might be even more pronounced than observed from the transfection experiments. to test this, we constructed veroe6-based cell lines stably expressing n from pedv, pdcov and tgev by lentivirus transduction. veroe6 clones expressing relatively equal amounts of n proteins from either pedv, pdcov and tgev were selected based on western blot analysis (fig. 5a) . uniform expression of exogenous n proteins in these engineered veroe6 cells was also assessed by immunofluorescence to eliminate the concern from the transient transfection experiment (fig. 5a) . equal numbers of cells were plated to confluence in 24-well plates and infected with pedv-mcherry (moi = 0.0001). spread of pedv infection and syncytium formation was monitored daily by fluorescence microscopy. supernatants were collected at 24, 36 and 48 hpi to monitor pedv growth by tcid 50 assay. veroe6 cells expressing n from pedv and tgev clearly accelerated pedv syncytia formation and spread. by 36 hpi, most cells displayed signs of profuse infection; by 48 hpi, infected cells were dead and detached compared to infection in wild-type veroe6 cells (fig. 5b) . interestingly, pdcov n stably expressed in veroe6 cells significantly suppressed pedv spread; even at 48 hpi, no extensive syncytia had formed (fig. 5b) . quantification of supernatant pedv-mcherry titers by tcid 50 assay also reflected the extent of syncytium formation. at 36 hpi, veroe6 cells expressing tgev n or pedv n yielded about a hundred times more infectious pedv virus particles than veroe6 cells, while those expressing pdcov n resulted in an infectious titer about ten times lower. by 48 hpi, pedv replication in wild-type veroe6 cells had started to catch up and closed the gap to about one order of magnitude. however, veroe6-pdcov n cells still showed significantly slower pedv replication kinetics (fig. 5c ). as expected, we noted that the suppressive effect from veroe6-pdcov n cells was stronger than that observed in transiently transfected veroe6 cells. this could be because the stable cell line homogeneously expresses pdcov n which can directly exert an effect on the infecting virus, while only a fraction of infected cells was transfected (resulting in the effect being diluted out by cells that were infected but not transfected). previously, we observed a significant enhancement of pedv rna synthesis and increased viral titers in veroe6-pedv n cells (liwnaree et al., 2019) . to explore if similar mechanisms were employed by tgev n and pdcov n on pedv replication, we then probed viral rna production inside these veroe6-based cell lines using rt-qpcr. total rna was extracted from cells infected with pedv-mcherry at 24, 36 and 48 hpi. specific primers were used to quantify viral genomic rna (orf1a gene; grna) and subgenomic rna (5′utr-s gene; sgrna) production. the levels of each rna species were normalized to the levels of gapdh mrna from the same conditions and were expressed as relative to rna levels at 24 hpi from infected veroe6 cells. as previously observed, veroe6-pedv n cells drastically increased sgrna and grna production at 36 and 48 hpi (fig. 5d ). on the other hand, veroe6-pdcov n cells displayed stunted pedv rna transcription and replication; even at 48 hpi, the levels of sgrna and grna were still lower than those observed in veroe6 cells at 24 hpi (fig. 5d ). veroe6-tgev n cells moderately enhanced pedv rna synthesis compared to wild-type veroe6 cells but did not reach the same level as veroe6-pedv n cells (fig. 5b ). according to the results, the presence of extraneous n proteins posed greater effects on grna replication than sgrna transcription. these data corroborated with visual observation and quantitative measurement of infectious viral particle production. together, they strengthened the findings in the transient expression experiments and suggest opposing directions of influence on pedv replication mediated by n proteins derived from related covs. based on their ability to cross-associate and affect pedv rna synthesis and replication, we speculated that n proteins from other covs, especially tgev, may have interchangeable functions with pedv n in pedv replication. in an attempt to answer which roles and functions of pedv n could be replaced by other cov n proteins, we constructed an n-deficient pedv infectious clone, ppedv-mcherry-δn, and determined whether supplying cov n proteins in trans could replace the missing pedv n protein from the pedv's viral genome. since the nucleotide fragment of the n gene in the viral genome may play other important roles in viral genome packaging or transcription, we employed a frameshift mutation strategy to silence n expression from the infectious clone while minimally disturbing the nucleotide sequence of the n gene. the original start codon in the n orf, atggct, was mutated to atatgt, thereby shifting the start codon by two nucleotides, resulting in a +2 frameshift during translation (fig. 6a) . the mutated sequence theoretically produced a short unrelated peptide of 21 amino acids with early termination. we intentionally kept the mcherry gene in the construct as a visual indicator for sub-genomic mrna transcription and protein expression in the virus rescue process. to verify the lack of n expression during pedv reverse genetics rescue, we transfected the n-deficient infectious clone into hek293t cells. western blot analysis confirmed that n expression was completely diminished in cells transfected with the n-deficient infectious clone compared to the wild-type ppedv-mcherry (fig. 6b) . with the n-deficient infectious clone in hand, we next investigated pedv reverse genetics rescue in the presence of other cov n proteins. s. sungsuwan, et al. virology 540 (2020) 45-56 hek293t cells were co-transfected with ppedv-mcherry-δn and a pcaggs plasmid expressing cov n protein. expression of mcherry from the infectious clone was used as a visual proxy for production of viral rna and proteins in hek293t cells during the reverse genetics step. no detectable fluorescence was observed from co-transfection with the empty pcaggs vector, confirming the role of pedv n protein during at least the first round of viral rna transcription and viral protein translation (fig. 7a ). co-transfection with pcaggs-pedv n-myc yielded a substantial fraction of red cells, while slightly lower amount of mcherry protein expression was observed from cells cotransfected with pcaggs-tgev n-flag. co-transfection with pcaggs-pdcov n-ha resulted in very low, yet detectable, numbers of mcherrypositive cells (fig. 7a ). cell lysates were prepared at 72 hpt and probed for s expression using anti-s antibody to corroborate visual inspection. as a negative control, cells transfected with ppedv-mcherry-δn and pcaggs vector displayed no detectable trace of s (fig. 7b) . cells co-transfected with pcaggs expressing cov n proteins showed varying levels of s expression, with pedv n-myc giving the highest s expression and pdcov n-ha giving the lowest (fig. 7b) . tgev n-flag expression resulted in noticeably lower s expression compared to pedv n-myc but still higher than that of pdcov n-ha (fig. 7b) . the levels of spike protein expression from the n-deficient clone in each condition correlated with the level of mcherry expression as observed by fluorescence microscopy (fig. 7a and b) . these results suggested that cov n proteins from alphacoronaviruses can aid viral rna and protein production from infectious pedv clones more efficiently than that from deltacoronavirus. to determine if pedv rescue successfully produced viable infectious particles with help from various cov n proteins in trans, hek293t cell supernatants were collected at 72 hpt to inoculate veroe6-pedv n cells pre-transfected with pcaggs-pedv n. we found that additional transfection could ensure sufficient pedv n expression in infected cells and facilitated pedv replication better than untreated veroe6-pedv n cells (data not shown). limited syncytium formation was observed only in veroe6-pedv n cells inoculated with the supernatant derived from hek293t cells co-transfected with pcaggs-pedv n-myc (fig. 7c) . however, the infecting viruses could not continuously propagate to produce extensive syncytia as routinely observed with the rescue of ppedv-mcherry. this is probably due to insufficient pedv n protein levels as supplied by the stable cell line compared to virally encoded pedv n. remarkably, supernatants from hek293t cells co-transfected with a plasmid encoding tgev n or pdcov n did not contain infectious progeny from virus rescue, displaying no second-round pedv-mcherry replication in veroe6-pedv n cells as determined by mcherry expression or syncytia formation (fig. 7c) . these results suggest that, despite some conserved function of n proteins among porcine enteric covs in self-oligomerization and assistance during viral rna synthesis or protein expression, n is not fully interchangeable among covs for generating pedv infectious virus progeny. these results imply that the suppressive or enhancing effects on pedv replication in cells expressing pdcov n or tgev n may reflect their effects on viral rna replication and/or viral protein expression, rather than their abilities to supply complementary n proteins during rnp core formation or virion assembly. although the swine industry has focused on pedv alone as one of its largest threats, few have attempted to understand the implications of swine enteric cov co-infection despite multiple epidemiological studies. as the true impact of co-infection on disease outcomes still awaits further investigation, these viruses could predictably exert influence on each other, considerably shaping the course of their infection or evolution. molecular interactions underlying these possible viral interferences also remain to be identified and characterized. in this work, we tried to understand how different enteric swine cov n proteins, one of the most abundant structural proteins, interact and affect functions of pedv n and replication kinetics of pedv. first, we provided evidence of cross-interaction between cov n proteins despite low sequence similarity among them. amino acid sequence analyses on pedv, tgev and pdcov n proteins revealed that the proteins can be divided roughly into two structural domains (ntd fig. 6 . construction of the n-deficient pedv infectious clone, ppedv-mcherry-δn. (a) schematic representation of a frameshift mutation strategy to silence n expression from the ppedv-mcherry-derived infectious clone to yield ppedv-mcherry-δn. cmv, cytomegalovirus immediateearly promoter; hdv, hepatitis delta virus ribozyme self-cleavage site; bgh, bovine growth hormone termination; t, transcription terminator. (b) western blot analysis of lysates prepared from hek293t cells transfected with ppedv-mcherry or ppedv-mcherry-δn (2 μg each) at 72 hpt. and ctd) with relatively high conservation and three intrinsically disordered regions (idrs) with relatively low conservation located in the middle and at the two termini (fig. 8) . this domain analysis is consistent with previous crystallographic studies on sars-cov and mhv n, which also suggested conserved general structural organization of cov n (chang et al., 2009; cong et al., 2017; ma et al., 2010; yu et al., 2006) . moreover, immunological data documenting cross-reactivity of antibodies against n proteins from tgev or pdcov with pedv n further implied shared topological similarity between these cov n proteins (gimenez-lirola et al., 2017; lin et al., 2015; ma et al., 2016) . nevertheless, precise molecular mechanisms mediating interspecies oligomerization of cov n proteins remain to be determined. structural and biochemical studies of sars-cov n strongly suggest a role of ctd as a major mediator for cov n dimerization. moreover, ctds of n proteins from sars-cov and porcine reproductive and respiratory syndrome virus (prrsv), despite diverse sequences, could adopt well-conserved structures, implying similar approaches to oligomerization, at least at a gross structure level (yu et al., 2006) . however, since cov n ctds formed intricate molecular interaction networks during dimerization (chang et al., 2014) , it is unlikely that cov n proteins from different species could replace all these specific molecular interactions. consistent with our results that hetero-oligomer formation is highly sensitive to the presence of rna, a more likely possibility is that rna-protein interactions play a more dominant role in interspecies cov n interactions than protein-protein interactions. next, we asked if n proteins from different covs can replace pedv n function during viral rna and protein synthesis. during the rescue of the n-deficient pedv infectious clone, we showed that supplying n protein from any cov in trans can initiate first-round sgrna transcription and protein expression. most likely, this function stems from the shared intrinsic ability of cov n to bind viral rna and act as an rna chaperone that initiates rna replication and/or transcription processes (zuniga et al., 2007 (zuniga et al., , 2010 . these results support previous notions that cov n proteins play important roles during cov rescue. for instance, rescue of tgev infectious rna required at least co-transfection of mrna encoding the n protein (yount et al., 2000) . schelle et al. demonstrated that an n-deficient hcov229e rna vector replicon carrying the gfp gene can produce sgrna and express gfp from the vector when co-transfected with n from pedv, a closely related virus, but not from mhv, a more distantly-related one (schelle et al., 2005) . we noted fig. 7 . other cov n proteins can partially function to replace pedv n. for pedv rescue experiments, hek293t cells were co-transfected with ppedv-mcherry-δn and 1 μg of pcaggs plasmid expressing n from pedv, pdcov or tgev. at 72 hpt, (a) cells were imaged under fluorescence microscopy to monitor mcherry expression from ppedv-mcherry-δn, and (b) cell lysates were prepared for western blot analysis with an anti-s antibody. supernatants from (a) were adsorbed onto veroe6-pedv n cells, which were additionally transfected with 2 μg pcaggs-pedv n to ensure sufficient n expression. at 72 hpi, (c) cells were imaged under fluorescence microscopy to monitor mcherry expression and syncytium formation as a sign for successful pedv infection into veroe6-pedv n cells (scale bar, 100 μm). a similar preference in our experiments. between tgev n and pdcov n, the former is more effective in replacing the indigenous pedv n than the latter. since both pedv and tgev belong to the alphacoronavirus genus, while pdcov is classified as a deltacoronavirus, tgev n would be expected to be functionally and structurally closer to pedv n, and hence able to form replication/transcription complexes and participate in pedv replication with higher compatibility than pdcov n. although both tgev n and pdcov n can oligomerize and perform some functions during g/sgrna transcription, they alone cannot totally replace all pedv n's functions. without native pedv n, infectious pedv virions could not be detected in the presence of tgev or pdcov n, implying that other functions are not interchangeable. these critical functions might involve viral core assembly as cov n participates in many steps during viral core or virion assembly (mcbride et al., 2014) . for instance, several reports showed that cov n is involved, directly or indirectly, with recognition of specific packaging signals for genome packaging (hsieh et al., 2005; hsin et al., 2018; kuo et al., 2014) . since rna packaging signals among covs are distinct in terms of length and sequence, even closely related cov n might not be able to recognize pedv packaging signals. similar compatibility issues have been raised in the case of viral interference between different types of influenza viruses (baker et al., 2014) . another critical step that could be disrupted by heterologous cov n is viral assembly through interactions between n and m proteins. it is widely accepted that cov n and m proteins interact to form the viral core, but different covs utilize different regions of n in binding to their own m proteins. for instance, while mhv utilizes the very c-terminal end of n to bind m, sars-cov n uses the middle disordered linker for the interaction (he et al., 2004b; narayanan et al., 2000) . in addition, although both tgev and mhv n proteins used their c-termini to bind m, the n binding sites on tgev m and mhv m are very distinct (hurst et al., 2005; kuo and masters, 2002) , pointing to high specificity for each virus. it could be argued that our use of c-terminally tagged cov n proteins might have contributed to failure in rescuing infectious pedv from ppedv-mcherry-δn, possibly due to interference with n-m protein interaction. nevertheless, our results showed that addition of a myc tag at the c-terminus of pedv n did not completely ruin protein-protein interaction required for viral assembly as pedv n-myc remained capable of producing infectious virions. moreover, using untagged pedv n and tgev n during reverse genetics rescue of ppedv-mcherry-δn gave similar results to those presented in fig. 7 (data not shown) , suggesting that the inability of tgev n or pdcov n to rescue infectious pedv lacking endogenous nucleocapsid protein stemmed largely from incompatibility of interspecies protein interaction rather than the interference from epitope tags. in all, it is conceivable that not all of n functions could be replaced by the protein originating from other viral species, even though n proteins from pedv, tgev and pdcov can cross-assemble, form highorder oligomers and aid in rna synthesis processes. notably, we observed completely opposite effects of tgev and pdcov n proteins on pedv replication. we noticed that, in the presence of rna, homologous binding between pedv n itself is weakened and cross binding between pedv n and the other n proteins is strengthened. during co-infection, where viral grna is ubiquitous, the rna could act as a tethering bridge to stabilize the formation of heterologous oligomers, which may not be a productive core structure for pedv virion assembly and may interrupt the optimal processes in viral fig. 8 . analysis of cov n sequences reveals low sequence similarity but conserved structural organization. multiple amino acid sequence alignment was performed with t-coffee (notredame et al., 2000) . the color scheme denotes consistency between different alignment methods ranging from bad (low agreement; blue and green) to good (high agreement; pink). bold and normal lettering denote ordered and disordered regions as predicted by the prdos program (ishida and kinoshita, 2007) . (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) replication that prefer the homo-oligomer of n. such interruption could explain the suppressive effect of pdcov n. nevertheless, the existence of interspecies cov n oligomers might not be the only factor dictating the effect of extraneous cov n proteins on pedv replication. tgev n also forms rna-dependent interspecies oligomers with pedv n and could have resulted in similar suppression. instead, we observed a small enhancement effect on pedv replication in the presence of tgev n. this could be the net outcome of other oligomerization-independent roles of tgev n that indirectly enhance pedv replication. a study by zhao et al. showed that, when compared to single-virus infection, coinfection of tgev and pedv could synergistically enhance rupture to tight and adherens junctions of the intestinal epithelial cells ipec-j2 by suppressing expression of proteins that help form the junction (zhao et al., 2014) . such altered barrier integrity is possibly caused by cov n and facilitate viral replication. another possible role of pedv or tgev n that could lead to enhancement of pedv replication is to act as a viral suppressor of rna silencing (cui et al., 2015) . using mhv n as a model, they found that cov n can bind and suppress host antiviral rnai, leading to increased viral protein expression and subsequent viral replication. more remarkably, they found that n proteins of alphacoronaviruses, including tgev and pedv, also have this ability. indeed, among different cov n proteins, they found that tgev n was among the most active and mers-cov n was the least active (cui et al., 2015) . therefore, n from tgev may also employ this mechanism in promoting pedv replication, while some other cov n proteins might not be capable of this function. these are interesting hypotheses currently under investigation, which could reveal more complexity of co-infection between these viruses. in summary, this work shows how other swine enteric cov n proteins can substitute for endogenous pedv n in some functions but not in others, demonstrating both conservation and specificity among related viruses in the coronaviridae family. furthermore, co-expression experiments revealed interesting phenomena in which different cov n proteins exert vastly different effects on pedv replication. these results raise even more questions. for example, in the context of co-infection, in which all other viral components are present, what would be the net outcomes on replication kinetics of each virus? are there effects from other viral components? considering the opposing effects on pedv replication of n proteins from tgev, an older virus emerging prior to pedv, and pdcov, a newly emerged one, is it possible that nucleocapsid proteins could act as one of the factors in permitting or limiting emergence of enteric swine covs? if so, what is the effect of pedv n on newer covs such as swine acute diarrhea syndrome coronavirus (sads-cov), which has been detected predominantly in farms encountering pedv epidemics (zhou et al., 2018) , and vice versa? further investigation into these questions would give valuable insights for us to better understand co-infection events and their implications for the emergence of novel enteric swine viruses. all authors of this manuscript have no conflict of interest to declare. mechanism of interference between influenza a/wsn and b/kanagawa viruses complementation between two tospoviruses facilitates the systemic movement of a plant virus silencing suppressor in an otherwise restrictive host influenza a and b virus intertypic reassortment through compatible viral packaging signals transient oligomerization of the sars-cov n protein-implication for virus ribonucleoprotein packaging the sars coronavirus nucleocapsid protein-forms and functions multiple nucleic acid binding sites and intrinsic disorder of severe 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and up-regulates interleukin-8 expression strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model crystal structure of the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona-and arteriviridae type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp 1 in irf1 signaling transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j2 cells coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription coronavirus nucleocapsid protein is an rna chaperone the authors would like to thank dr. qigai he for anti-pedv s antibody, and benjamas liwnaree for technical assistance. we thank the members of virology and cell technology research team, especially dr. samaporn teeravechyan, for critical comments and language help on the manuscript. this work was supported by biotec's fellow grant [grant number p-15-51261]. key: cord-355119-sdg9zdc1 authors: lin, huixing; chen, lei; gao, lu; yuan, xiaomin; ma, zhe; fan, hongjie title: epidemic strain yc2014 of porcine epidemic diarrhea virus could provide piglets against homologous challenge date: 2016-04-22 journal: virol j doi: 10.1186/s12985-016-0529-z sha: doc_id: 355119 cord_uid: sdg9zdc1 background: porcine epidemic diarrhea virus (pedv) is the main causative agent of porcine epidemic diarrhea (ped). since december 2010, a large-scale outbreak of diarrhea has been observed in swine farms in china. accumulated evidence indicates that this large-scale outbreak of diarrhea were caused by highly virulent pedv variants. methods: a pedv strain, yc2014, was isolated from intestinal samples of suckling piglets with acute diarrhea in 2014. the complete genomic sequence of yc2014 and the nucleotide sequence of s gene were aligned with sequences of published isolates using mega 5.1 software. the immune protective efficiency of yc2014 were determined by testing pedv neutralizing antibodies in sera, the colostrum and the milk on 7th day after farrowing of the immunized sows. the diarrhea symptoms of piglets after challenge were also observed. results: phylogenetic analysis of the complete genomic sequence of yc2014 and the nucleotide sequence of s gene demonstrated that the yc2014 pedv strain was clustered with the pedv epidemic strains, with >99 % nucleotide identity to these pedv strains. the s gene sequence of yc2014 shared only 93.9 % ~ 94.4 % identities with classical cv777, dr13 and js2008 strains, with 15 nucleotide insertion in three sites and three nucleotide deletion in one site. the amino acid (aa) sequence of s gene of yc2014 shared only 92.8 % ~ 93.4 % identities with classical cv777, dr13 and js2008 strains, with 5 aa insertion in two sites and 1 aa deletion in one site. in the immune protective efficiency tests, the neutralizing antibody titers in sera, the colostrum and the milk on 7th day after farrowing of the inactivated yc2014 pedv strain immunized group were significantly higher than the inactivated cv777 immunized group and the inactivated dr13 immunized group (p < 0.05). the traditional inactivated pedv vaccines made from cv777 or dr13 could not protect piglets from yc2014 challenge, while inactivated yc2014 could provide piglets with 100 % protection against yc2014 challenge. conclusions: the results showed that, great antigenicity variation had occurred to this yc2014 pedv strain. the yc2014 pedv strain could provide piglets against homologous challenge. it is critical for future pathogenic and antigenic studies, as well as for the development of effective preventive and control vaccines against pedv. porcine epidemic diarrhea virus (pedv) is an enveloped, single-stranded, positive-sense rna virus that is taxonomically classified within the family coronaviridae, genus alphacoronavirus. pedv is the main causative agent of porcine epidemic diarrhea (ped), a devastating enteric disease that is characterized by watery diarrhea, vomiting, dehydration and significant mortality in piglets. approximately 80 to 100% of pedv-infected piglets die within 24 h of being infected with virulent pedv strains, resulting in tremendous economic losses to the swine industry [1, 2] . since december 2010, a large-scale outbreak of diarrhea, characterized by watery stool, dehydration, and vomiting, with 80 to 100 % morbidity and 50 to 90 % mortality in suckling piglets, has been observed in swine farms in china [3, 4] . accumulated evidence indicates that this large-scale outbreak of diarrhea may be caused by highly virulent pedv variants [5, 6] . in the present study, a pedv strain, yc2014, was isolated from intestinal samples of suckling piglets with acute diarrhea in 2014, the evolutionary characteristics and the immune protective efficiency of yc2014 were also determined. in the pedv isolation and propagation experiment, the partial gene of nucleocapsid protein was analyzed by rt-pcr using primers n1/n2, which amplified an approximately 1 kb nucleocapsid gene fragment present in the isolated yc2014 infected vero cells, but not in the blank control vero cells (fig. 1a) . the isolated yc2014 pedv strain was detected in the cytoplasm of infected vero cells by an anti-pedv n protein polyclonal antibody. red fluorescence could be observed in the yc2014 strain-infected vero cells (fig. 1b) . the isolated yc2014 pedv strain was confirmed to be negative for other porcine enteric viruses, such as rotavirus groups a, b and c, tgev, prcv, calicivirus and porcine deltacoronavirus by rt-pcr. the growth kinetics study showed that yc2014 replicated rapidly and efficiently in vero cells, reaching a maximum titer >10 7 tcid 50 /ml by 48 hpi (fig. 2) . a total of 28,077 nucleotides were sequenced in the isolated yc2014 pedv strain, including the polyprotein, s, orf3, e, m, and n protein-encoding genes. the sequence of yc2014 was submitted to genbank (accession no. ku252649). the complete genomic sequence of yc2014 and the nucleotide sequence of s gene were aligned with sequences of published isolates using mega 5.1. phylogenetic analysis demonstrated that the yc2014 pedv strain was clustered with the pedv epidemic strains (g2 cluster, fig. 3a and b), with >99 % nucleotide identity to these strains. the complete genome nucleotide sequence shared 96.8 %~97.8 % identities with classical cv777, dr13, js2008, ah-m and sd-m strains (g1 cluster). the s gene sequence of yc2014 shared only 93.9 %~94.4 % identities with the pedv g1 cluster, such as cv777, dr13 and js2008 strains, with 15 nucleotide insertion in three sites (167 bp, 175~185 bp, 418~420 bp) and three nucleotide deletion in one sites (470~472 bp). the amino acid (aa) sequence of s gene of yc2014 shared only 92.8 %~93.4 % identities with classical cv777, dr13 and js2008 strains, with 5 aa insertion in two sites (aa59~62, aa140) and 1 aa deletion in one site (aa160). nucleocapsid protein specific antibodies tests showed that a antibody response was detected in all the inactivated pedv strains immunized groups 7 days after the first immunization (fig. 4a) . the antibody levels of all the inactivated pedv strains immunized groups maintain high levels at all the testing time points, but the antibody titers of these three groups were not significantly different (p > 0.05). the results of pedv neutralizing antibodies tests showed that the neutralizing antibody levels of the inactivated pedv strains immunized groups gradually increased until 28 days after the first immunization, and still maintain high level at 7 days after farrowing. the neutralizing antibody titer in sera samples of the yc2014 pedv strain immunized group was significantly higher than the other three groups (p < 0.05, fig. 4b ). the neutralizing antibody titer in the colostrum and the milk on 7th day after farrowing of the yc2014 pedv strain immunized group was also significantly higher than the other three groups (p < 0.05, fig. 4c ). after yc2014 challenge, piglets in group one, group two and group four showed significant acute diarrhea. weight gain was reduced, loss of appetite and mental uneasiness persisted, while piglets in group three, group five and group six showed no obvious diarrhea symptoms. two days after challenge, mortality of group one, group two and group four were 100 %. no mortality or obvious clinical symptoms were observed in group three, group five and group six ( table 1 ). ped can generally be controlled using a vaccine strategy. vaccination with killed or attenuated pedv vaccine has been widely carried out in china and other swine raising countries, where ped usually manifests as a mild and enzootic pattern (lower mortality) some years ago. however, severe acute diarrhea outbreaks associated with high morbidity (80-100 %) and mortality (50-90 %) were observed in suckling piglets in most areas of china since december 2010, although most sow herds had previously been vaccinated with traditional inactivated pedv vaccines based on cv777 or dr13. in april 2013, ped was diagnosed in the eastern midwest region of the united states, subsequently, it spread rapidly to 30 neighboring states by june 2014. accumulative evidence indicates that this large-scale outbreak of diarrhea may be caused by highly virulent pedv variants [7] [8] [9] . the s protein makes up the large surface projections of the virion and plays a pivotal role in determining viral-cellular fusion activity and activating the immune system [10] [11] [12] . the variations in amino acid sequence likely changed the immunogenicity of the s protein and led to immunization failure of current commercial vaccines. in this study, we successfully isolated the yc2014 pedv strain from porcine intestinal samples in dead piglets during outbreaks of acute diarrhea. the s gene nucleotides analysis of yc2014 indicated that it was clustered with the pedv epidemic strains, with 15 nucleotide insertion in three sites and three nucleotide deletion in one site compared to classical pedv vaccine strains cv777 and dr13. the variations of the s gene sequence and deduced amino acid sequence of the yc2014 strain compared to traditional pedv vaccine strains may be the reason why some swine farms had well pedv vaccine immunizations but still had sustained epidemic diarrhea which caused huge economic losses. vaccination is one of the most effective ways in preventing ped infection. immunization of sows with pedv vaccines at 20-30 days before production will provide substantial passive immunity to the newborn piglets [13] . in this study, the nucleocapsid protein specific antibody levels of three different inactivated pedv strains immunized groups gradually increased at all the testing time points. however, the antibody titers of these three groups are not significantly different. immunization with inactivated yc2014 could protect piglets from acute diarrhea against homologous strain challenge. since yc2014 was clustered with the pedv epidemic strains, with >99 % nucleotide identity to most of these epidemic strains, the inactivated yc2014 may protect other variant strains challenge in some extent. however, it needs further refine animal studies to assess this suppose. the results showed that, great antigenicity variation had occurred to this yc2014 pedv strain under the selective pressure of vaccines. therefore, it is important to investigate pedv variants currently circulating in sow herds to assess their ability to allow for cross-protection against highly virulent pedv strains and prevent pedv epidemics. the results showed that, great antigenicity variation had occurred to this yc2014 pedv strain. the yc2014 pedv strain could provide piglets against homologous challenge. it is critical for future pathogenic and c neutralizing antibodies detection in the colostrum and the milk on 7th day after farrowing. * indicates the neutralizing antibody titer of the yc2014 pedv strain immunized group was significantly higher than the other three groups (p < 0.05) antigenic studies, as well as for the development of effective preventive and control vaccines against pedv. in may 2014, porcine intestinal tracts, intestinal contents and fecal samples were collected from dead piglets during outbreaks of diarrhea on a breeding farm. this farm keeps more than 1800 sows and located in yancheng city within the jiangsu province. clinical signs were characterized by acute vomiting, anorexia, and watery diarrhea, with high mortality in piglets less than 10 days old. the diarrhea outbreaks occurred throughout the year and re-occurred at 2-3 weeks interval. virus isolation was performed as described previously [14] . the rna of the isolated yc2014 pedv strain cultures were extracted using the viral rna mini kit (geneaid biotech, taiwan) according to the manufacturer's instructions. the presence of pedv in the vero cell culture was confirmed by reverse transcription pcr (rt-pcr) with one pairs of primers to amplify approximately 1 kb partial sequence of nucleocapsid protein, n1: gcaaacggg tgccattatctc, n2: ctagctcacgaacagccac attac. the samples of pedv in the vero cell culture were confirmed to be negative for rotavirus a, b and c, transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), caliciviruses and porcine deltacoronavirus via rt-pcr as previously described [15] [16] [17] [18] [19] . the pedv yc2014 strain was then identified with immunofluorescence assay (ifa). briefly, vero cells grown on a 6-well plate were infected with the pedv yc2014 strain. at 48 h post-infection, cells were washed twice with pbs and fixed with cold methanol for 10 min at −20°c. cells were then washed three times with pbst and blocked with 10 % bovine serum albumin (bsa) at 37°c for 1 h. preparations were incubated for 1 h at 37°c with mouse anti-pedv nucleocapsid protein polyclonal antibody in dilution buffer (1 % bsa in pbst), this mouse anti-pedv nucleocapsid protein polyclonal antibody, prepared by our laboratory, was collected from serum of icr mice immunized with purified prokaryotic expressed n protein. after three washes with pbst, cells were treated with a rhodamineconjugated goat anti-mouse igg (cwbio, china) at a 1:5000 dilution with pbs for 30 min at 37°c. after a final four washes with pbst, all wells were examined using fluorescence microscopy (axio observer z1, zeiss, germany). after ten passages on vero cells, the one-step growth curve of yc2014 strain in vero cells was monitored at 8 h interval after infection. based on the sequence of the cv777 pedv strain (genbank: af353511.1), seven pairs of oligonucleotide primers ( table 2 ) were designed to amplify the different regions of the yc2014 genome. the pcr products were cloned into the puc19 vector using clonexpress entry one step cloning kit (vazyme) and sequenced by invitrogen biotechnology (shanghai, china). the 5′ and 3′ ends of the genome of yc2014 were validated using the rapid amplification of cdna ends (race) cdna amplification kit (clontech, japan). all fragments were sequenced in both directions in triplicate. the complete genomic sequence of yc2014 and the nucleotide sequence of s gene were aligned with sequences of published isolates using mega 5.1 software. phylogenetic trees were constructed using the maximum likelihood method and supported with a bootstrap test of 1000 replicates. genomic sequences of the isolated yc2014 pedv strain were submitted to genbank under accession no. ku252649. all experimental protocols were approved by the laboratory animal monitoring committee of jiangsu province and performed accordingly. twelve commercial high-health sows (large white) were randomly divided into four groups (table 3) after immunization, sera samples were collected from sows at 7-day intervals until 35 days after immunization (7 days after farrowing) for detection of nucleocapsid protein specific antibodies with commercial indirect elisa kits (biovet, canada) and pedv neutralizing antibodies using yc2014 as indicator virus as described previously [20] . briefly, 50 μl of pedv strain yc2014 (2.0 × 10 3 tcid 50 /ml) was added to an equal volume of the sera samples and incubated for 1 h at 37°c. the mixture was then inoculated to a 96-well plate containing confluent vero cells. 2 h later, the culture plate was washed with d-hank's three times, and then added 100 μl of dmem containing 2 % fetal bovine serum (fbs). 48 h later, the culture plate was fixed with cold methanol for 10 min at −20°c, and incubated with mouse anti-pedv nucleocapsid protein polyclonal antibody for 1 h at 37°c, and then stained with fitc-labeled rabbit antimouse igg (santa cruz biotechnology). the serum titers were determined as the reciprocal of the last serum dilution at 70 % or greater fluorescent focus reduction in the infected cell cultures under a fluorescent microscope. the colostrum and the milk on 7th day after farrowing of each sow were also collected and used for the detection of pedv neutralizing antibodies. the average number of sows farrowing was 13, piglets in each group was coded and chosen randomly, respectively. on 7th day after farrowing, thirty piglets were chosen (table 4 ), ten from group one (inactivated cv777 immunized group), ten from group two (inactivated dr13 immunized group), five from group three (inactivated yc2014 immunized group), and five from group four (saline treated group). these piglets were divided into six groups (table 4) , each group were housed in a separate room and were artificial feeding with milk. piglets in group one to group four were challenged orally with 1.0 × 10 4 tcid 50 of yc2014. group five were challenged orally with 1.0 × 10 4 tcid 50 of cv777. group six were challenged orally with 1.0 × 10 4 tcid 50 of dr13. piglets were observed daily after challenge about the diarrhea symptoms. all data were analyzed using one-way anova and values of p < 0.05 were considered statistically significant. submit your next manuscript to biomed central and we will help you at every step: pathogenesis of porcine epidemic diarrhea virus isolate (us/iowa/18984/2013) in 3-week-old weaned pigs epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy new variants of porcine epidemic diarrhea virus, china molecular characterization and phylogenetic analysis of new variants of the porcine epidemic diarrhea virus in gansu complete genome sequence of a chinese virulent porcine epidemic diarrhea virus strain sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states complete genome sequence of porcine epidemic diarrhea virus strain usa/colorado/2013 from the united states emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus the gprlqpy motif located at the carboxyterminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr13 strain propagation of the virus of porcine epidemic diarrhea in cell culture detection and genetic diversity of porcine group a rotaviruses in historic (2004) and recent (2011 and 2012) swine fecal samples in ohio: predominance of the g9p[13] genotype in nursing piglets detection of group b and c rotaviruses by polymerase chain reaction detection and differentiation of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus in clinical samples by multiplex rt-pcr detection of transmissible gastroenteritis virus by rt-pcr and differentiation from porcine respiratory coronavirus pcr-based retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in us swine construction and immunogenicity of recombinant adenovirus expressing the capsid protein of porcine circovirus 2 (pcv2) in mice this study was supported by program from the jiangsu province science and technology support program (be2013433), the jiangsu agriculture science and technology innovation fund (cx (15)1056 the authors declare that they have no competing interests.authors' contributions hxl and hjf designed the study. lc and lg performed the virus isolation. hxl and zm performed the animal test. all authors read and approved the final manuscript. key: cord-341521-dntkdwkj authors: luo, yi-ran; zhou, shu-ting; yang, liang; liu, yuan-ping; jiang, sheng-yao; dawuli, yeliboli; hou, yi-xuan; zhou, tian-xing; yang, zhi-biao title: porcine epidemic diarrhoea virus induces cell-cycle arrest through the dna damage-signalling pathway date: 2020-03-24 journal: j vet res doi: 10.2478/jvetres-2020-0024 sha: doc_id: 341521 cord_uid: dntkdwkj introduction: porcine epidemic diarrhoea virus (pedv) infection causes watery diarrhoea, vomiting, anorexia, and weight loss, especially among neonatal piglets, inflicting on them morbidity and mortality potentially reaching 90%–100%. despite it being known that certain mammalian cell phases are arrested by pedv, the mechanisms have not been elucidated, and pedv pathogenesis is poorly understood. this study determined the effect of an epidemic pedv strain on cell cycle progression. material and methods: we observed the effect of the pedv shpd/2012 strain on an infected vero cell cycle through flow cytometry and western blot, investigating the interrelationships of cell-cycle arrest, the dna damage–signalling pathway caused by pedv and the phosphorylation levels of the key molecules chk.2 and h2a.x involved upstream and downstream in this pathway. results: pedv induced vero cell-cycle arrest at the g1/g0 phase. the phosphorylation levels of chk.2 and h2a.x increased with the prolongation of pedv infection, and no significant cell-cycle arrest was observed after treatment with atm or chk.2 inhibitors. the proliferation of pedv was also inhibited by treatment with atm or chk.2 inhibitors. conclusion: pedv-induced cell-cycle arrest is associated with activation of dna damage–signalling pathways. our findings elucidate the molecular basis of pedv replication and provide evidence to support further evaluation of pedv pathogenesis. porcine epidemic diarrhoea virus (pedv) is the pathogen responsible for porcine epidemic diarrhoea (ped). ped causes watery diarrhoea, vomiting, anorexia, and weight loss, especially among neonatal piglets, and the morbidity and mortality of ped in neonatal piglets can reach 90%-100%. pedv first appeared in england (21) and belgium (15) in the early 1970s, and outbreaks later occurred in the czech republic, china, korea, vietnam, and the united states. since 2010, new mutant strains of pedv have been discovered in china (4, 8, 12-17, 29, 30) . these strains spread to the united states, south korea, and southeast asia and caused significant economic losses to the pig industry. the pathogenesis of pedv is not clear, and the mechanism for infection of cells by pedv remains largely unknown. recently, pedv infection has been shown to cause s-phase arrest of porcine small intestinal epithelial cells (iec) (24, 25) . in a study of porcine transmissible gastroenteritis virus (tgev), it was also found that pedv can cause s-phase arrest of st cells (2) . furthermore, mouse hepatitis virus (mhv), infectious bronchitis virus (ibv), severe acute respiratory syndrome-coronavirus (sars-cov), and other coronaviruses can cause g0/g1-or g2/m-phase arrest (3, 7, 19, (26) (27) (28) . nevertheless, the mechanisms by which epidemic strains of pedv affect the mammalian cell cycle have not been elucidated. in this study, vero cells were infected with the epidemic shpd/2012 pedv strain to determine the effect on cell cycle progression, to explore the molecular mechanisms producing the effects of pedv on the cell cycle, and to provide theoretical support for a better definition of the pathogenesis of pedv. cell and virus cultures. the epidemic pedv strain shpd/2012 and vero cell line were isolated and preserved by the zoonotic disease and comparative medicine laboratory of shanghai jiao tong university (22) . vero cells were cultured in dulbecco's modified eagle's medium (dmem, gibco, grand island, ny, usa). the cells were infected with the epidemic shpd/2012 pedv strain at a multiplicity of infection (moi) of 0.5 in t25 culture flasks. afterwards, 4 ml of maintenance cell culture solution (containing 10 μg/ml of trypsin and 2% dmem) was added, and cells were incubated for 4-5 days, during which period the cytopathic effect (cpe) was observed daily. when cpe reached 80%, the cells were frozen at −80°c in a forma 900 series freezer (thermo fisher scientific, waltham, ma, usa). after freezing and thawing, culture samples were centrifuged (5810r, eppendorf, hamburg, germany) at 9,000 g for 5 min and the supernatants were collected. vero cell cycle synchronisation and preparation for flow cytometry. initially, after pedv infection, the cell cycles of the vero cells were synchronised by serum starvation in serum-free medium, locking most cells in the g1/g0 phase (9) . then, the cells were seeded in 6-well culture plates at a concentration of 3 × 10 5 cells per well and were incubated at 37°c with 5% co2 for 24 h. after 24 h, the culture medium was carefully replaced with serum-free dmem, and the cells were incubated at 37°c with 5% co2 for a further 36 h. dry bromodeoxyuridine (brdu) powder (becton, dickinson and company, franklin lakes, nj, usa) was dissolved in dimethylsulphoxide (dmso, sigma-aldrich, st. louis, mo, usa) to make a 10 mm stock solution (1,000 ×). almost 4 h before staining for flow analysis, brdu solution was added to the infected and control cells at a final concentration of 10 μm. cells were incubated with brdu at 37°c for 4 h, after which the medium was replaced with dmem. cells were digested with trypsin (sigma-aldrich), collected in centrifuge tubes, and centrifuged at 1,000 g for 5 min to pellet cells. after centrifugation, the supernatant was discarded, and the cells were resuspended in chilled pbs solution with ph 7.2. cells were counted with a cell counter (countess, invitrogen, waltham, ma, usa). for each sample, 1 × 10 6 cells were added to the centrifuge tube. cells were then resuspended in 70% ethanol and fixed at 4°c for 12 h. they were then washed and resuspended in 1 ml chilled pbs and next centrifuged at 1,500 g for 10 min, and the supernatant was discarded. anti-brdu monoclonal antibody was diluted 1:5 in pbs solution containing 1% foetal bovine serum and 0.5% triton x100 (sigma-aldrich), 100 μl of antibody dilution was added to each cell sample, and the samples were incubated at room temperature for 20 min. after incubation, each sample was washed and resuspended in 1 ml of chilled pbs, then centrifuged at 1,500 g for 10 min, and the supernatant was discarded. fitc-conjugated fluorescent secondary antibody (jackson immunoresearch laboratories, west grove, pa, usa) was diluted 1:1,500 in pbs, 100 μl of diluted secondary antibody was added to each cell sample, and these were then incubated at room temperature for 30 min in the dark. after incubation, each sample was again washed and resuspended in 1 ml chilled pbs, then centrifuged again at 1,500 g for 5 min, and the supernatant was discarded. a total of 100 μl of propidium iodide (pi -sigma-aldrich) stain was added to each sample, and samples were incubated at room temperature for 30 min in the dark. cells were washed and resuspended in 1 ml chilled pbs, then analysed by flow cytometry (beckman fc 500 mpl, beckman coulter, pasadena, ca, usa). flow cytometric cell cycle analysis. flowjo 9.0 software (flowjo, ashland, or, usa) was used to analyse the flow cytometric data to detect differences in cell cycle distribution of vero cells. the intact cell population was identified based on the forward scattersideways scatter diagram of each sample, and the middle of the cell grouping with a relatively concentrated central region was identified as r1. from the r1 population, pi intensity was plotted on the x axis (red channel), and brdu (fitc, green channel) was plotted on the y axis to present the periodic distribution scatter of double staining for cell cycle analysis. the s (r3), g1/g0 (r2), and g2/m (r4) phase cells were distinguished based on the intensity of the fitc fluorescence signal, and cells in the g1/g0 or g2/m phases were discriminated according to the intensity of the pi fluorescence signal; the proportion of cells in each phase was determined for the r1 population. we analysed the cell cycle flow cytometry data of the pedv-infected samples and mock control samples at 0 h, 12 h, 24 h, 28 h, and 32 h post infection, according to the methods above, and determined the proportion of cells in each phase of the cell cycle. analysis of changes in cyclin expression by western blot. flow cytometry analysis of the cell cycle can determine the cell cycle changes, but for revealing the molecular mechanism of the cell-cycle arrest induced by pedv infection in more detail, we evaluated the time-dependent changes in expression of cyclin proteins in mock-infected and infected cells. antibodies against pedv n protein (6), cyclin d1 (abcam, cambridge, uk), anti-cyclin e1 (cell signaling technologies, danvers, ma, usa), and cyclin b1 (abcam) were used as the primary antibodies. horseradish peroxidase (hrp)-conjugated secondary antibodies (sigma-aldrich), and other targets were used to evaluate protein expression of samples at 6 h, 12 h, 24 h, and 48 h after infection, to determine the changes in expression of relevant signalling molecules. protein expression was visualised by enhanced chemiluminescence reagents (thermo fisher scientific). relative quantification of cyclin expression was determined by the width of the slip in western blot. evaluation of dna damage pathway as related to pedv infection. phospho-specific antibodies for each molecule of interest in the pathway were used in this experiment, and the relative phosphorylation content of the major signalling molecules was detected by western blot. additionally, inhibition of specific signalling molecules in the dna damage pathway by chemical agents enabled us to confirm whether cellcycle arrest caused by pedv infection involved activation of the dna damage-signalling pathway. since the activation of the dna damage pathway would be ascertained by evaluating phosphorylated proteins, phosphatase inhibitors were added to prevent removal of phosphate moieties during the preparation of the protein sample. treatment of cells with dna damage pathway inhibitors. the following chemical inhibitors of the dna damage-signalling pathway were used: the atm inhibitor, which was 2-morpholin-4-yl-6thianthren-1-yl-pyran-4-one; and the chk.2 inhibitor which was 2-(4-(4-chlorophenoxy)phenyl)-1h-benzimidazole -5-carboxamide (cell signaling technologies). both drugs were dissolved in dmso to a working concentration of 10 μm. an equivalent volume of dmso was added to the blank control cells. we used inhibitors of atm and chk.2 to block the dna damage-signalling pathway and to confirm whether pedv infection can lead to cell-cycle arrest. cells were seeded in 6-well culture plates at a concentration of 5 × 10 5 cells/well, and incubated with dmso, atm inhibitors, or chk.2 inhibitors for 2 h. after seeding, cells were infected with pedv at an moi of 0.5, received the addition of complete medium, and were incubated for 48 h. a portion of the cells was digested according to the procedure described in the preparation for flow cytometry, then fixed and stained for flow detection; the other portion was prepared for quantitative fluorescence pcr to detect the number of virus copies (10) and to evaluate the effect of dna damage pathway inhibitors on viral replication, according to these methods. the overall picture of the detected differences in cell cycle distribution is shown in figs. 1a and b . the proportion of s-phase cells in the mock-infected group gradually increased, and in the infected group it significantly decreased over time. g2 cells represented a smaller proportion in the infected group than in the mock group at all times, and in the latter group, cells in the g1/g0 stage became gradually fewer than those in the infection group over time. in mock cells, due to the addition of complete medium, cells were stimulated by growth factors in fbs, and as vero cells recovered and continued to divide, the proportion of cells in the g1/g0 phase gradually decreased; the rate of reduction was highest between 12 and 24 h (fig. 2) . likewise, cells in the s phase increased over time, and the rate of addition was highest between 12 and 24 h (fig. 3) . although the proportion of cells in the g2/m phase was lower in the infected and control groups, the proportion and incremental increase of g2/m-phase cells in the control group was higher than that in the infected group (fig 4) . in co-treated infected cells, the addition of fbs resulted in a slight decrease in cells in the g1/g0 phase. however, 24 h later, infected cells in this phase were more numerous than those of the control group, according to the proliferation of the virus (fig. 2) . the proportion of cells in the s phase in the infected group increased up to 24 h but began to decrease thereafter (fig. 3) . cyclin protein expression changes. at 24 h and 48 h of virus proliferation, expression of cyclin e1 was significantly lower in the infected group than in the mock group. cyclin d1 had decreased by 48 h in the infected group. in contrast, the expression of cyclin b1 in the infected group was higher than in the mock group at 12 h and 24 h, showing an increasing trend, although it also began to gradually decrease at 48 h (fig. 5) . cell-cycle arrest and pedv proliferation dependency on activation of the dna damage pathway. after 12 h and 18 h the phosphorylation levels of chk.2 were higher in the pedv infection group than in the control group, and followed a gradually increasing and then decreasing trend (fig. 6) . the phosphorylation of h2a.x increased at 12 h, 18 h, and 24 h in the pedv infection group samples, and correlated with pedv proliferation in vero cells, as indicated by pedv n expression (fig. 6) . these data indicate that infection with pedv activates the dna damage-signalling pathway in vero cells, and suggest that since h2a.x is the downstream mediator of chk.2, the phosphorylation of chk.2 occurs earlier than phosphorylation of h2a.x. there were no significant differences in cell cycling or cell-cycle arrest between the mock-infected cells treated with dmso and those treated with the atm and chk.2 inhibitors (fig. 7) . however, for cells infected with pedv and treated with dmso, the proportion of cells in the g1 and g0 phases was significantly increased, and the proportion of cells in the s phase and the g2/m phase was decreased; for cells treated with the atm and chk.2 inhibitors, the proportion in the g1/g0 phase was slightly increased (fig. 7) . at 48 h after infection, cells treated with dmso had the highest virus proliferation of nearly 500,000 copies/ml; at 48 h after infection, cells treated with the atm inhibitor and chk.2 inhibitor exhibited 50,000 virus copies/ml, which is dramatically lower than that of cells treated with dmso (fig. 8) . we therefore conclude that pedv proliferation in vero cells could be inhibited by chemical blockade of the dna damagesignalling pathway. several viruses cause cell-cycle arrest in host cells, and different viruses have distinct ways and mechanisms of inducing it (4, 7, 19, (26) (27) (28) . in this study, we used flow cytometry and western blot to investigate the mechanism of cell-cycle arrest in vero cells after infection with pedv. we evaluated cell-cycle proportions using brdu-pi bicolour-labelled flow cytometry, which determines the proportion of s-phase cells by inserting brdu into newly replicated dna molecules, and indicates cells in the g1/g0 and g2/m phases by pi bound to chromosomes in all stages of the cell cycle. this method is more accurate and better reflects the proportions of a cell population in the different phases of the cell cycle than traditional pi single-staining methods. compared with the control group, infection of vero cells with the pedv shpd/2012 strain induced a significant increase in the proportion of cells in the g1/g0 phase after 24 h. in order to investigate the effect of pedv infection on cell-cycle arrest further, we evaluated changes in the expression of several cyclins at different time periods in the infected and control groups. the results of flow cytometric cell-cycle analysis and western blot indicated that pedv infection caused g1/g0 cell-cycle arrest in vero cells. since dna damage signalling is a major pathway for regulating cell-cycle arrest, we predicted that cycle arrest caused by infection of vero cells with pedv is due to the activation of the dna damagesignalling pathway. we evaluated expression of the phosphorylated forms of atm and chk.2 to detect activation of the dna damage-signalling pathway in the infected and control groups. we also treated cells with chemical inhibitors of atm and chk.2 to determine if cell-cycle arrest after pedv infection was linked with activation of the dna damage-signalling pathway. control vero cells treated with the atm or chk.2 inhibitors did not undergo cell-cycle arrest because the dna damage-signalling pathway is not normally activated in these cells. in the infected group, the cells treated with dmso showed increased g1/g0 cell-cycle arrest, while the cells treated with atm and chk.2 inhibitors did not, indicating that these two inhibitors block the dna damage-signalling pathway, and do not arrest the g1/g0 cell cycle. these results revealed that cell-cycle arrest after infection of vero cells with pedv was caused by activation of the dna damage-signalling pathway. in this study, we found that the phosphorylation of chk.2 and h2a.x in the dna damage-signalling pathway is significantly enhanced along with the proliferation of pedv. we demonstrate that the inhibitors of chk.2 and h2a.x can reduce the g1/g0phase arrest of vero cells infected by pedv, indicating that the effect of pedv infection on vero cell cycles is diminished with a blockade of the dna damagesignalling pathway. additionally, proliferation of pedv was reduced after the dna damage-signalling pathway was blocked. these data indicate that g1/g0 arrest caused by pedv infection in vero cells is dependent on the activation of the dna damage-signalling pathway, and the activation of this pathway also affects the proliferation of pedv in vero cells. previously, some studies reported that cov infection or viral proteins could cause g1/g0-phase arrest, but the mechanisms varied from species to species. for example, researchers found that the expression of cyclin d3 protein and the phosphorylation of rb in cells were reduced by eukaryotic expression of sars-cov 3a protein, resulting in the arrest of both cells in the g1 phase (28) . similarly, sars-cov protein 7a had the same impact on cyclin d3 and rb, and the 44-82 amino acid region of the orf 7a gene may be involved in this function (27) . eukaryotic expression of egfp-tagged 3b protein in vero, hek-293 and cos-7 cell lines has been found to block the g1/g0 phase of host cells. in addition, the n protein of sars-cov also plays a role in arresting the host cell cycle (18) , by binding to the cyclin-dependent kinase (cdk) complex to inhibit the synthesis of genomic dna (blocking the s phase). further studies confirmed that the n protein could also bind to cyclin d directly, inhibit the activity of the cdk4-cyclin d complex, and downregulate the expression of cdk2 and the activity of the cdk2 complex. additionally, the downregulation of e2f1 was also observed in vero e6 cells transfected with the n protein (18) . some studies have shown that the non-structural p28 protein encoded by the ero1 gene of mhv could increase the activity of the rb protein, p53, and p21 cip1 molecules in cells, thereby blocking the cell at the g1/g0 phase (11). by upregulating the phosphorylation level of p53, mhv p28 protein can increase the transcriptional activity of p21 cip1 so as to inhibit the activity of the cdk2-cyclin e complex. ultimately, mhv p28 protein can inhibit the phosphorylation of the rb protein, making a large number of cells at the g1/g0 phase. researchers found that in virus-infected cells, levels of g1-phase regulators such as cyclin d1 and d2 decrease with ibv replication, eventually leading to g2/m-phase arrest (7) . some other researchers further showed that the blockade induced by ibv could be achieved in the absence of p53 in host cells, while their studies also showed that the nsp13 protein of ibv could activate the dna damage-signalling pathway, which also leads to downstream cell-cycle arrest (23). ding et al. (5) showed that st and pk-15 cell lines infected by tgev could induce s-phase and g2/mphase arrest. western blot results showed that tgev infection promoted the expression of p21 and downregulated cyclin b, cdc2, cdk2, and proliferating cell nuclear antigen molecule. furthermore, by adding the inhibitor of the p53 molecule, it was found that such inhibitory effect would weaken the cycle-arrest effect of tgev infection on st and pk-15 cells, thus leading to speculation that tgev infection activates the signal pathway of the p53 molecule, on the activation of which depends the cycle-arrest phenomenon (1) . the n protein of pedv can cause s-phase prolongation of the infected cells (24) . the researchers who made that finding generated a porcine small intestinal epithelial cell line stably expressing n protein. flow cytometry showed that n protein expression could block the cells in the s phase, but the mechanism was not fully elucidated. in this study, we demonstrated that pedv arrests the cell cycle by activating the dna damage-signalling pathway, which is beneficial to the proliferation of the virus. the authors declare that there is no conflict of interests regarding the publication of this article. respiratory syncytial virus matrix protein induces lung epithelial cell cycle arrest through a p53 dependent pathway expression of protein n of porcine transmissible gastroenteritis in swine intestinal epithelial cells and effect and influence on cell cycle murine coronavirus nonstructural protein p28 arrests cell cycle in g0/g1 phase comparative genomic analysis of classical and variant virulent parental/attenuated strains of porcine epidemic diarrhea virus transmissible gastroenteritis virus infection induces cell cycle arrest at s and g2/m phases via p53-dependent pathway preparation and characterization of monoclonal antibodies against the n protein of porcine epidemic diarrhoea virus shpd/2012 strain cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china synchronization of cell populations in g1/s and g2/m phases of the cell cycle development and application of a taqman real-time rt-pcr assay for the detection of a porcine epidemic diarrhea virus strain in china hepatitis c virus ns3/4a protein interacts with atm, impairs dna repair and enhances sensitivity to ionizing radiation genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from henan, china phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field strains in central china based on the orf3 gene and the main neutralization epitopes molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field strains in south china a new coronavirus-like particle associated with diarrhea in swine epidemiology and vaccine of porcine epidemic diarrhea virus in china: mini-review outbreak of porcine epidemic diarrhea in suckling piglets the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks s phase progression in mammalian cells the dynamics of cell cycle regulation porcine epidemic diarrhea virus variants with high pathogenicity an apparently new syndrome of porcine epidemic diarrhea characteristics of vero cells infected by shpd/2012 strain of porcine epidemic diarrhea virus coronavirus infection induces dna replication stress partly through interaction of its nonstructural protein 13 with the p125 subunit of dna polymerase delta porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin-8 expression porcine epidemic diarrhea virus m protein blocks cell cycle progression at s-phase and its subcellular localization in the porcine intestinal epithelial cells g0/g1 arrest and apoptosis induced by sars-co v 3b protein in transfected cells sars coronavirus 7a protein blocks cell cycle progression at g0/g1 phase via the cyclin d3/p rb pathway g1 phase cell cycle arrest induced by sars-cov 3a protein via the cyclin d3/prb pathway occurrence and investigation of enteric viral infections in pigs with diarrhea in china isolation and identification of a variant porcine epidemic diarrhea virus yi-ran luo, and shu-ting zhou contributed equally to this study. key: cord-344421-rmnck42f authors: theuns, sebastiaan; vanmechelen, bert; bernaert, quinten; deboutte, ward; vandenhole, marilou; beller, leen; matthijnssens, jelle; maes, piet; nauwynck, hans j. title: nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric virus date: 2018-06-29 journal: sci rep doi: 10.1038/s41598-018-28180-9 sha: doc_id: 344421 cord_uid: rmnck42f enteric diseases in swine are often caused by different pathogens and thus metagenomics are a useful tool for diagnostics. the capacities of nanopore sequencing for viral diagnostics were investigated here. first, cell culture-grown porcine epidemic diarrhea virus and rotavirus a were pooled and sequenced on a minion. reads were already detected at 7 seconds after start of sequencing, resulting in high sequencing depths (19.2 to 103.5x) after 3 h. next, diarrheic feces of a one-week-old piglet was analyzed. almost all reads (99%) belonged to bacteriophages, which may have reshaped the piglet’s microbiome. contigs matched bacteroides, escherichia and enterococcus phages. moreover, porcine kobuvirus was discovered in the feces for the first time in belgium. suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding (10(6.42)–10(7.01) copies/swab) and diarrheic signs was not observed during a follow-up study. retrospective analysis showed the widespread (n = 25, 56.8% positive) of genetically moderately related kobuviruses among belgian diarrheic piglets. minion enables rapid detection of enteric viruses. such new methodologies will change diagnostics, but more extensive validations should be conducted. the true enteric pathogenicity of porcine kobuvirus should be questioned, while its subclinical importance cannot be excluded. metagenomics is a valuable asset for diagnostics in pigs, leading to discovery of novel viruses and identification of porcine viral enteric disease complexes. although standardized procedures have been developed to study viral metagenomes in fecal samples, they still require an extensive sample preparation, including random or targeted pre-amplification of viral genomes present in the sample 13 . most sequencing platforms still require capital investments and high sample turnover rates to be cost-effective. performing the necessary analyses often results in long time periods between sample arrival and diagnostic reporting, since results can only be processed after finishing the sequencing run. third-generation sequencing using minion (oxford nanopore technologies, ont) might be a useful and affordable diagnostic tool for swine veterinary medicine as it allows rapid sample preparation and real-time sequence analysis. the flowcells used for sequencing consist of a membrane containing multiple csgg nanopore proteins from escherichia coli 14 . an ion current is established through this pore resulting in typical current changes upon passage of specific nucleotides. this signal is converted into a nucleotide sequence by computational algorithms (basecalling). since the release of minion technology, major advances have been made in terms of the number and the quality of reads generated 15 . in the field of virology, the technology has mainly been applied in human medicine. using nanopore sequencing, it was possible to distinguish three poxviruses with 98% nucleotide similarity at strain level 16 . minion has also been used as a diagnostic tool during recent ebolavirus outbreaks in west africa, allowing fast on-site characterization of circulating strains 17, 18 . coupled to a laptop-based bioinformatics workflow, minion was able to detect chikungunya virus, ebola virus and hepatitis c virus in less than 6 hours using earlier versions of the technology 19 . a multiplex pcr method for complete on-site zikavirus genome sequencing in samples with low viral loads has recently been developed by quick and coworkers 20 . partial dengue virus genomes were isothermally amplified followed by sequencing, allowing classification of strains in serotypes 21 . in veterinary virology, the use of nanopore sequencing is growing. a novel species of papillomavirus was identified in warts from giraffes, using rolling-circle amplification and nanopore sequencing 22 . the entire genome of a parapoxvirus isolated from a seal was obtained by combining data from illumina next-generation sequencing with nanopore sequencing data 23 . one study has reported the detection of venezuelan equine encephalitis virus from unamplified cdna created from poly-a tailed rna using cell culture grown viruses 24 . to the author's knowledge, the present study is the first using minion as an aid in porcine health management. this study was aimed to explore the possibilities of minion as a rapid and easy-to-use diagnostic tool in pig health management for diagnosis of viral enteric disease complexes. the ability to detect high loads of cell culture-grown rotavirus and coronavirus, mimicking shedding quantities observed in diarrheic piglets, was evaluated. in a second case, the ability to detect (novel) viruses in diarrheic feces of a one-week-old piglet with diarrhea was investigated. no gene-specific or random pre-amplification of viral nucleic acids was conducted to challenge the minion's sensitivity. a porcine kobuvirus was discovered in the latter case and a longitudinal field study was conducted hereafter to elucidate the shedding patterns of this virus. moreover, archival (2014) fecal samples from diarrheic suckling piglets less than two weeks old were investigated for the presence of kobuviruses, to study their epidemiology in belgium. be performed for 243,313 reads with a mean length of 740 nucleotides. reads with a q-score lower than 7 were filtered out, resulting in 179,015 remaining sequences (mean length 816 nt) for use in downstream analyses. results of the sequencing run, including taxonomical classification and mapping of reads against pedv and rotavirus a (rva) reference genomes are shown in fig. 1a . after 24 hours of sequencing, a total of 15,232 reads were classified as viral by sensitive tblastx comparison against a complete viral database. of these, 39.3% (n = 5,985) and 10.3% (n = 1,564) were assigned to viral families comprising porcine epidemic diarrhea virus (family coronaviridae) and rotavirus (family reoviridae, subfamily sedoreovirinae), respectively. a fraction of the reads (29.3%, n = 4,468) were assigned to order caudovirales. these reads originated from the lambda phage dna used in a previous control run on the same flowcell. at 7.5 and 24.2 seconds after the start of sequencing, respectively, the first reads matching pedv and rva were translocated through a nanopore. most reads were generated in the first twelve hours of sequencing and read accumulation was most exponential in the first three hours of sequencing (fig. 1b) . pedv and rva sequences were extracted from the dataset and mapped against viral reference genes to calculate sequencing depths over time (fig. 1c) . after one hour, sequencing depths were higher for pedv (43.0x) than for rva (4.9 to 22.1x). high sequencing depths were acquired after three hours of sequencing for pedv (103.5x) and for most rva gene segments (19.2 to 48.2x). de novo assembly was executed on the quality-filtered reads prior to identification (tblastx) to recover viral genomes. this resulted in the recovery of the almost complete pedv genome and rva gene segments with identities varying between 95 and 99% compared to the reference genes (table 1) . higher assembly accuracies (97 to 99%) were obtained when only the reads matching against rotavirus and pedv were included for de novo assembly (table 1) . however, execution of de novo assembly prior to taxonomical classification (tblastx) reduced the time to identify entire viral genomes in the dataset. virome composition of a young diarrheic piglet using nanopore sequencing. a total of 30,088 reads were generated by sequencing the diarrheic fecal sample for three hours. of these, 25,466 reads (q-score >7, mean read length 653 nt) were used for further analyses. different methods were used to compare the reads against a viral database using the hpc cluster of ghent university and results are shown in fig. 2 . comparison against a complete viral database resulted in the detection of 6,781 to 8,677 potential viral reads, depending on the blast settings. blastn resulted in rapid taxonomical identification of reads at almost similar sensitivity compared to tblastx. however, there was a very high difference between wall times on the hpc cluster, with only 26 seconds of analysis time for blastn, versus almost 24 hours for tblastx. the majority of sequences were assigned to bacteriophages within the order caudovirales and families siphoviridae (n = 3,213 to 4,163 reads), podoviridae (n = 2,506 to 3,002 reads) and myoviridae (n = 912 to 1,202 reads). a de novo assembly was executed on the basecalled, quality filtered reads and the resulting contigs were used as input material for virsorter analysis. nineteen contigs were classified as sure (n = 4; category 1), somewhat sure (=14; category 2) and not so sure (=1; category 3) to be phage-like contigs (fig. 2b) . comparison of these contigs against the genbank database using blast allowed classification into four different groups. ten contigs showed moderate to high nucleotide similarities to the bacteroides phage b124-14, suggesting that they all belonged to one phage genome. this was also supported by the fact that all these contigs mapped nicely distributed across the reference genome of bacteroides phage b124-14 (data not shown). the longest contig with a size of 39,069 nucleotides, together with four other contigs showed similarities (95% nt identity) to different escherichia phages. as they also mapped nicely distributed across the reference genome of escherichia phage vb_ecop_phapec7, it seems that they must also belong to one phage genome (data not shown). two contigs showed poor similarity to both the enterococcus phage vb_efas_ime_196, isolated from hospital sewage in china from an enterococcus faecalis strain, and the enterococcus hirae bacterial genome. the latter might be a prophage inserted in the bacterial genome. interestingly, three contigs were identified for which no similarities were found with existing viruses in genbank, but contig 0105 mapped to the reference genome of the enterococcus phage vb_efas_ime196 (data not shown). these might be novel phages or divergent variants from existing phages present in genbank. three eukaryotic porcine viruses, porcine kobuvirus (n = 18 to 22 reads), enterovirus g (n = 5 to 9 reads) and astrovirus (n = 4 reads) were found at much lower abundancies. the genera kobuvirus and enterovirus belong to the family picornaviridae, whereas the genus mamastrovirus belongs to the family astroviridae. kobuvirus reads were mapped against a european reference strain s-1/hun/2007/hungary, as shown in fig. 2c . however, full-genome coverage at high sequencing depth was not obtained. shedding of porcine kobuvirus and rotaviruses in suckling piglets. the shedding of porcine kobuvirus, rva and rotavirus c (rvc) was quantitatively investigated in 5 suckling pigs of the same farm from which the diarrheic feces originated. the fecal shedding patterns of the different viruses and presence of diarrheic signs are shown in fig. 3a . all piglets started shedding porcine kobuvirus at the end of the first week after parturition. in two piglets (a and d) the shedding was sustained and lasted for at least 2 weeks (above the limit of quantification). peak shedding titers of the porcine kobuvirus varied between 6.42 and 7.01 log 10 copies/swab, which is generally lower than peak shedding observed for typical enteric viruses such as rotavirus and pedv. moreover, the peak of shedding was not related to diarrheic episodes, questioning the role of this virus in the pathogenesis of diarrhea on the farm. diarrheic signs were only noticed in two piglets (a and b). in piglet b, an association between high rvc shedding and diarrheic episodes was observed. in contrast, there was no direct association between peak shedding of kobuvirus and diarrheic episodes. interestingly, a peak in kobuvirus shedding was observed in piglet c at day 11 post-farrowing. this animal died shortly hereafter, but it was unclear if this can be attributable to the kobuvirus infection. acute rva shedding was observed at the end of the suckling period in three of five piglets, even though all sows were vaccinated before farrowing using a bovine inactivated rotavirus vaccine. retrospective analysis of porcine kobuviruses shedding in belgian diarrheic suckling pigs and phylogenetic analysis. a total of 44 diarrheic fecal samples collected in 2014 were screened for the presence of kobuvirus using the new rt-qpcr. of these, 25 samples (56.8%) tested positive and 18 samples showed quantifiable viral loads (4.31 to 6.83 log 10 copies/swab). seven samples were positive, but viral loads were too low to allow accurate quantification. the presence of rva and rvc had been quantitatively assessed in these samples in a previous study and the occurrence of co-infections between rotaviruses and kobuvirus is shown in fig. 3b 25 . kobuvirus was found in equal ratios in rotavirus-negative and -positive samples. twelve samples contained a single rotavirus infection with a high rva load and in four of these, a high kobuvirus load (5.16 to 5.42 log 10 copies/g) was observed. a single rvc infection was found in seven samples and in four of these tested positive for kobuvirus at high loads (4.31 to 5.59 log 10 copies/g). a dual rva/rvc infection was seen in two samples, but neither contained quantifiable kobuvirus loads. many (n = 10) of the rotavirus-negative samples contained high kobuvirus loads. strain 17v079 showed high similarity to other belgian porcine kobuvirus isolates from 2014 (92.1 to 94.0% nucleotide sequence identity) and the hungarian reference strain s-1/hun/2017 (93.4%). furthermore, there was a high level of genetic variability between the 2014 belgian porcine kobuvirus isolates, with nucleotide sequence identities ranging between 90.1 and 97.2%. a phylogenetic analysis, using the 3d gene of 17v079 and twelve belgian isolates from 2014 ( fig. 3c) , shows the belgian strains clustering between strains from different geographical locations. prevention and treatment of enteric disease problems in young piglets is frequently hampered by a lack of diagnostic tools. veterinarians are restricted to a short list of known viruses, bacteria, parasites and management factors to define a differential diagnosis. only the most likely cause(s) of the disease will be diagnostically investigated, often leading to negative, inconclusive or incomplete results. however, metagenomics studies have indicated the existence of viral enteric disease complexes, potentially involving multiple known and novel viruses 3, 4, 6, 7, 9, 10 . detection of nucleic acids from pathogens using ngs-based metagenomics approaches is a partial solution to diagnostic testing problems and can provide a complete readout of viruses and other pathogens present in a sample. however, most ngs platforms require large investments and processing of the reads can only start at the end of the sequencing run. viral metagenomics also requires extensive laboratory preparations, including centrifugation, filtration and nuclease treatment to discard bacterial and host nucleic acids that make up to the bulk of all nucleic acids present 13 . furthermore, the amount of viral nucleic acids in a sample is very low, requiring targeted or random amplification of these genomes before ngs analysis. amplification may induce bias and hampers the development of a fast diagnostic pipelines due to considerable time loss. all these factors lead to a long turnover time between sample collection and diagnostic reporting. the third-generation sequencing device minion (ont), holds promise as a diagnostic platform, as it allows real-time sequencing and analyses of all dna/rna in a sample, theoretically without needing pre-amplification of viral nucleic acids. it was the aim of the present study to evaluate this technology for use as a rapid tool for porcine viral enteric disease complex identification, without the conduction of viral nucleic acid amplification. in a first experiment, cell culture-grown pedv and rva, known to induce diarrhea in young pigs, were pooled at high loads mimicking shedding quantities in diarrheic piglets. sequencing of this pooled sample with the minion resulted in rapid identification of both viruses. real-time analysis of the sequencing reads was not conducted, but is achievable as previously demonstrated by greninger and colleagues using the surpi analysis pipeline for rapid identification of human viruses from different clinical matrices 19 . interestingly, the first reads matching pedv and rva were generated respectively after 7 and 24 seconds of sequencing. high sequencing depths (43.0x) were acquired within one hour of sequencing for pedv and within three hours for most of the eleven rva gene segments (19.2-48.2x). overall, higher sequencing depths were generated for pedv that could indicate that sequencing of longer viral genomes is favored over smaller gene segments, as pedv has a genome size of approximately 28 kb, and rva gene segments are shorter (0.6 to 3.3 kb). this bias might have been introduced during the ligation of the sequencing adapters to the viral nucleic acids. it can be hypothesized that adapters are more easily attached to longer dna fragments, and bias should be avoided by standardization of viral nucleic acid input length. rapid read generation allows flexible use of the sequencing platform and sequences can be read until enough genome information of the viruses of interest is available. while the technology can be useful for giving fast readouts of viruses (<3 hours) present in a sample, thorough validation, using well-defined virus stocks, spiking experiments in matrices (e.g. feces) and real clinical samples is necessary to make sure that all members of the porcine viral enteric disease complex are accurately being diagnosed. furthermore, the accuracy of the technology needs further improvement, as error rates of contigs from de novo assemblies still ranged between 1 and 5%, hindering the precise analysis of subtle but important mutations in the viral genome. after the successful identification of the cell culture-grown viruses, the performance of the minion was further explored by analyzing a diarrheic fecal sample of a one-week-old suckling piglet. real-time pcr analyses were conducted for rva, rvc, pedv and tgev. enterococcus hirae was isolated at a private diagnostic laboratory, but this bacterial species is not considered a typical cause of diarrheic disease in pigs 26 . viral metagenomics was conducted on this sample using the minion and two different blast search algorithms were used to taxonomically identify the reads by comparing them against a complete viral database. overall, tblastx with an e-value of 10 −3 was able to identify the most viral reads compared to other search options conducted. however, blastn search options also reached high sensitivity, but at much lower time cost: 26 seconds instead of almost 24 hours. for rapid read analysis and searching for closely related non-divergent viral sequences, blastn or another fast methodology should thus be preferentially used. however, tblastx might pick up more divergent or novel viruses, improving overall sensitivity. three porcine viruses, including porcine kobuvirus, porcine mamastrovirus and enterovirus g, were identified in sample 17v079. astro-and enteroviruses have been detected earlier in both diarrheic and non-diarrheic feces of belgian pigs and in feces from pigs around the globe 9,10,27 . in a recent study from thailand, the difference in prevalence of astrovirus in diarrheic (8.4%) versus non-diarrheic (4.6%) piglets less than 4-weeks-old was not statistically significant. also other studies have shown that the role of porcine astrovirus in the pathogenesis of pig diarrhea is not completely clear 28 . in contrast, associations between diarrhea and human astrovirus infections have been made 29 . a recent study in 5 european countries (hungary, spain, germany, austria and sweden) have indicated the widespread of porcine astroviruses in the swine population. a one hundred procent prevalence of astrovirus was found in diarrheic and non-diarrheic pigs from austria and spain. porcine astroviruses have recently also been linked to outbreaks of neurological disorders in weaned piglets from hungary, and in 5-week-old pigs and sows in the united states 30, 31 . the gut might be a hypothetical entry port for such neurological astrovirus infections. enteroviruses have been more generally linked to neurological disorders in pigs, although they are commonly found in feces as well 11, 12, [32] [33] [34] . in a study from vietnam, no significant correlation was found between diarrhea status and presence of enterovirus g in feces 35 . the involvement of both astro-and enteroviruses in the pathogenesis of enteric disorders might be questioned here, but cannot be completely ruled out. furthermore, while sensitive tblastx searches were used here, there is still a possibility that a completely novel virus might be present in the dark matter of the sequencing reads. however, reporting of a porcine kobuvirus in belgian piglets with minion is unique. in belgium, kobuviruses had previously only been found in diarrheic samples of calves and young cattle in belgium 36 . in the present study, a novel rt-qpcr assay, targeting the conserved 3d gene encoding the rna-dependent-rna-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions. similar kinetics were also analyzed for porcine rotavirus a and c. while suckling piglets started shedding porcine kobuvirus from one week of age, an association between peak viral shedding (6.42 to 7.01 log 10 copies/swab) and diarrheic signs was not observed. in one pig, an association was made between diarrheic episodes and the peak of rotavirus c shedding, a well-known enteric pathogen 37, 38 . very interestingly, kobuvirus fecal loads were typically lower than those reported of well-described enteric viruses of which the pathogenicity has been proven using piglet infection models, such as pedv and rotavirus [39] [40] [41] . similar viral loads for porcine kobuvirus were also found in case (4.60 ± 1.76 copies/qpcr reaction) and control pigs (4.79 ± 1.72 copies/qpcr reaction) during a recent danish study to evaluate the role of viruses in the pathogenesis of the new neonatal porcine diarrhea syndrome. the study demonstrated that kobuvirus, astrovirus, rotavirus a, porcine teschovirus, porcine norovirus and porcine coronaviruses were not involved in the pathogenesis of the syndrome 42 . the finding of low kobuvirus loads in feces casts doubt over the true enteric pathogenic tropism of the virus. hypothetically, its replication is likely not distributed across the whole villus but limited to either enterocytes at the villus' tips or to immune cells present in the gut. the presence of kobuvirus rna in serum has also been demonstrated in hungarian pigs, but it was not known if the virus is also replicating in other organs 43 . both the oro-fecal route and the feeding of milk to sucklings pigs could be involved in virus transmission. highest rates of infection were observed in suckling piglets, compared to older pigs, in other countries [44] [45] [46] . in our study, relatively long shedding of porcine kobuvirus was observed in three out of five animals, which may indicate that this virus may induce persistent infections. a 2011 brazilian study demonstrated the presence of kobuvirus rna in serum from 3-day-old piglets, which had disappeared by day 21, indicating viral clearance from the blood and excluding systemic persistence 45 . a complete lack of pathogenicity cannot be excluded, as porcine kobuviruses might play a role as a subclinically important virus. such subclinical, yet immunosuppressive, properties have been attributed to the economically important swine pathogen porcine circovirus 47 . of interest, one of the piglets died at the peak of kobuvirus shedding, although it was not clear if there was any causality between virus replication and the piglet's death. in vivo animal experiments in a model of neonatal, conventional kobuvirus-negative piglets should be conducted to elucidate the pathogenesis of porcine kobuviruses. attempts were made to isolate the virus in different cell lines (ma104, st and sk), and peripheral blood mononuclear cells. there was no evidence of cytopathogenic effect after several days of incubation. antibodies to visualize antigen expression were not available and therefore the possibility of replication without scientific reports | (2018) 8:9830 | doi:10.1038/s41598-018-28180-9 evident cytopathogenic effect cannot be ruled out. efforts will be made to isolate the virus in porcine primary enterocyte cultures, once available. to assess more broadly the prevalence of kobuvirus in the belgian swine industry, a retrospective analysis of diarrheic samples from suckling piglets less than two weeks old was conducted. a high proportion (40.9%) of the samples (n = 44) contained quantifiable viral loads ranging between 4.31 to 6.83 log 10 copies/g feces. viral loads found were thus comparable to the loads excreted by piglets in the longitudinal analysis and the above-mentioned study from denmark, demonstrating the endemic presence of the virus in the belgian swine population 42 . in the present study, non-diarrheic piglets were not included and therefore no association between kobuvirus prevalence and disease can be made. however, the prevalence of kobuvirus has been widely described in pigs from several european countries (the netherlands (16.7%), slovakia (63.4%), hungary (81.0%), czech republic (87.3%), austria (46.2%), italy (52.4%), germany (54.5%) and sweden (45.0%)), american countries (the united states (21.9%) and brazil (53.0%)), african countries (kenya (14.9%) and uganda (15.5%)) and asian countries (thailand (99%), south korea (52.1%) and vietnam (29.3%)) [44] [45] [46] [48] [49] [50] [51] [52] [53] . in a small proportion of these studies, statistically significant associations between prevalence of kobuvirus and diarrhea in pigs were demonstrated, such as in hungary (54.5% prevalence in healthy pigs vs 92.3% prevalence in diarrheic pigs), spain (47.5% healthy vs 74.4% diarrheic), brazil (41% vs 78.4%), thailand (19.3% vs 84.5%) and vietnam (27.6% to 40.9%) 35, 45, 46, 52 . indeed, it is difficult to make correlations between prevalence of the virus and diarrhea, as the pathogenicity of the virus could be largely influenced by other factors such as co-infections with other enteric viruses, microbiota and management factors. belgian isolates showed genetic moderate to high genetic variability, with nucleotide identities between 90.1 and 97.2%. furthermore, they clustered diffusely between strains from different countries around the world, indicating that strains are not distinguishable based on their geographical origin. because most (99%) of the reads generated during sequencing of the fecal sample 17v079 matched bacteriophages upon analysis with blast, bacteriophages may have played an important role in the pathogenesis of the diarrheic disease. de novo assembled contigs were analyzed using virsorter, a software package for mining viral signals from microbial genomic data. such tools allow maximizing the possibility of detecting dsdna phages 54 . several contigs showed high similarities to the bacteroides phage b124-14, found in municipal wastewater and human fecal samples. it was shown to be absent in 30 samples collected from different animal species, including pigs, and is therefore considered a human-specific phage 55, 56 . the finding of several contigs, genetically similar to phage b124 and likely belonging to one phage genome, indicates that this phage found in the pig fecal sample may also replicate in the microbiome of the young pig gut and not solely in humans. however, it is possible that the phage's replication ability in the pig's gut is age-dependent and that very young age groups were not sampled in previous studies. interestingly, several of the contigs found also showed similarities to escherichia phages. two of the contigs were similar to escherichia phages phapec5 and phapec7, isolated from belgian rivers in the neighborhood of poultry houses and known to cause lytic infections in avian pathogenic escherichia coli. electron microscopic images of the phages phapec5 and phapec7 indicated that they belonged to the family podoviridae 57 . two other contigs were similar to two closely related escherichia phages, st11ph5 and g7c, found in sewage and horse feces, respectively 58 . finally, one contig showed limited similarity to an enterococcus phage, isolated from hospital sewage in china, while a last contig showed moderate similaraties to the bacterial enterococcus hirae genome. this region may be a prophage, inserted in the bacterial genome. the phages found in this piglet may have reshaped the gut microbiota, allowing opportunistic bacteria such as enterococcus hirae to proliferate and to start secreting toxins. it is also possible that a phage infection of bacteria in the pig's gut led to a stress status for these bacteria, prompting the secretion of toxins. the new neonatal diarrhea syndrome described above shows high similarities to the disease described in the case 17v079 and it may be that bacteriophages are involved in the pathogenesis of this syndrome. so far, the role of phages has not been considered in the pathogenesis of several enteric disorders, but given the high abundance here, it should be in future studies. it is clear that new technologies will change the way diagnostics are be performed in the near future. pricing might currently be an aspect hampering high-troughput analysis of samples in swine veterinary medicine, but as the technology evolves fast, this might become very soon less relevant. complete overviews of all viruses and other pathogens in a sample will be given in a single readout instead of requiring different diagnostic assays. however, care should be given to the interpretation of such results, as they should only be analyzed by trained veterinarians. viruses. porcine rotavirus a (rva) strain rva/pig-tc/bel/12r046/2012/g9p [23] was isolated from a diarrheic piglet and grown for three successive passages in ma104 cells to an infectious virus titer of 10 7.8 ccid 50 / ml. the nucleotide sequences of the 11 gene segments of this strain were resolved earlier using sanger sequencing (genbank accession numbers: km82070 (vp1), km820707 (vp2), km827014 (vp3), km820720 (vp4), km820728 (vp6), km820735 (vp7), km820742 (nsp1), km820672 (nsp2), km820679 (nsp3), km820686 (nsp4) and km820693 (nsp5)) 59 . a porcine epidemic diarrhea virus strain (pedv, cv777) isolated in belgium in the 1970s was adapted for growth in vero cells in the 1980s 60 . in our laboratory, the virus was grown to an infectious virus titer of 10 6.0 ccid 50 /ml (genbank accession number: af353511). origin of a fecal sample from diarrheic suckling piglets. a diarrheic fecal sample was collected from a belgian pig on a farm housing a total of 620 sows and using a 2-week batch-production system, with a weaning age of 23 days. topigs norsvin sows were crossed with piétrain boars, producing 32. toxins (suiseng, hipra). rotavirus a vaccination was done off-label with an inactivated bovine rotavirus a vaccine (lactovac, zoetis). until recently, diarrheic problems were rarely present in suckling piglets and also very low mortality percentages (6.2-7.1%) were observed. since the spring of 2017, enteric disease started causing more severe problems accompanied with mortality on this farm, mainly in 7-days-old suckling piglets. a diarrheic fecal sample of such a piglet was investigated at a private diagnostic laboratory (dialab, belsele, belgium) and labeled 17v079. no virological cause was found to explain the diarrheic problems on the farm. the only isolated bacterium was enterococcus hirae. this bacterium was thereon added to the sow vaccination schedule (inactivated autovaccine). no other pathogens were found in this sample. as the clinical picture hinted at a viral cause for the disease, the sample was sent to the laboratory of virology at the faculty of veterinary medicine (ghent university) for further analysis. the sample tested negative for rva, rvc, pedv and tgev using in-house rt-qpcr assays 25, 61, 62 . therefore, it was decided to perform a metagenomics analysis with minion described in this study. purification of viral nucleic acids. first, viral enrichment was done based on the netovir protocol to obtain pure viral nucleic acids for sequencing library preparation 13 . minion analyses of cell culture grown viruses rva and pedv were conducted at the laboratory of clinical virology (rega institute, ku leuven), whereas the diarrheic fecal sample was analyzed at the laboratory of virology (faculty of veterinary medicine, ghent university). rva and pedv stocks were centrifuged at 17,000 × g for 3 min. the supernatant of both suspensions was diluted to 6 log 10 ccid 50 /ml and 500 µl of each suspension was mixed to reach an equal concentration of both viruses. this mixture was filtered using a 0.8 µm polyethersulphone filter for 1 min at 17,000 × g, followed by a nuclease treatment for 2 hours at 37 °c to digest free nucleic acids in the suspension: 250 µl of the sample was added to 14 µl of home-made buffer (1 m tris, 100 mm cacl 2 and 30 mm mgcl 2 , ph 8), 4 µl of benzonase nuclease (millipore) and 2 µl micrococcal nuclease (neb) as described earlier 13 . fourteen microliters of edta were added to stop the reaction, followed by extraction of nucleic acids from the viral particles using the qiaamp viral rna mini kit (qiagen). the manufacturer's instructions were followed but no carrier rna was added and elution was done in 30 µl of ave to concentrate the viral nucleic acid extract. the diarrheic fecal sample 17v079 was processed similarly as the cell culture grown viruses, with some minor modifications. a 10% w/v suspension of the diarrhea was made in minimum essential medium and centrifuged. the supernatant was filtered through a 0.45 µm syringe filter (sarstedt) and treated with benzonase nuclease for 1 hour to speed up the diagnostic pipeline. viral nucleic acids were extracted using the qiaamp cador pathogen mini kit according to the manufacturer's instructions without addition of carrier rna. elution was done in a volume of 50 µl. cdna and second strand synthesis for nanopore sequencing. nucleic acids were heated at 95 °c for 2 min and chilled on ice to resolve secondary rna structures and to denature double-stranded rna. superscript iv reverse transcriptase (thermoscientific) was used to generate cdna. ten microliters of template nucleic acids were mixed with 0.5 µl random hexamer primers (random primer 6, new england biolabs), 1 µl dntp mix (neb) and 2.5 µl nuclease-free water. primer annealing was conducted at 65 °c for 5 min, after which 4 µl superscript iv reaction buffer (thermoscientific), 1 µl dithiothreitol (thermoscientific) and 1 µl superscript iv reverse transcriptase (thermoscientific) were added in a total reaction volume of 20 µl. the reaction conditions were as follows: 23 °c for 10 min, 50 °c for 10 min, 80 °c for 10 min and an infinite hold step at 10 °c. a second strand of dna was generated from single stranded (c)dna molecules using the nebnext second strand synthesis kit (neb). twenty microliters cdna reaction mixture were added to 10 µl nebnext second strand synthesis reaction buffer, 5 µl nebnext second strand synthesis enzyme mix and 45 µl nuclease-free water (80 µl total reaction volume). isothermal amplification was done at 16 °c for 1 h and double-stranded nucleic acids were purified using 144 µl of magnetic ampure xp beads (beckman coulter). two washing steps with freshly prepared 70% ethanol were conducted before eluting in 52 µl nuclease-free water. nanopore sequencing library preparation. a deoxyadenosine was ligated to the 3′-end of double-stranded nucleic acids to allow binding of complimentary sequencing adapters. fifty microliters of (un) amplified dna were mixed with 7 µl ultra ii end-prep reaction buffer (new england biolabs) and 3 µl ultra ii end-prep enzyme mix (new england biolabs), and incubated at 20 °c for 5 min and 65 °c for 5 min. next, nucleic acids were purified using 60 µl ampure xp beads and eluted in 31 µl nuclease-free water. sequencing adapters, provided with the ligation sequencing kit 1d (r9.4) (sqk-lsk108, ont), were ligated to the da-tailed nucleic acids. end-prepped dna (30 µl) was mixed with 20 µl adapter mix (amx, ont) and 50 µl blunt/ta ligation master mix (new england biolabs) in a total reaction volume of 100 µl and incubated at room temperature for 10 min. the sequencing library, containing double-stranded dna with adapters ligated to the 3′ ends, was then purified using 40 µl ampure xp beads. two washing steps were conducted using 140 µl adapter bead binding buffer (abb, ont) before eluting in 15 µl of elution buffer (elb, ont). (exp-llb001, ont), 12 µl adapted and tethered library and 12.5 µl nuclease-free water. sequencing was done using the software programme minknow software (ont). bio-informatics analyses. raw reads were produced by minknow. live basecalling was enabled for the first experiment using minknow version 1.5.5. in the second experiment, basecalling was done after the sequencing run using albacore (version 1.2.5., ont). quality scores and read lengths were visualized using nanoplot, followed by quality filtering with nanofilt 63 . reads with a q-score lower than 7 were omitted. sequences were then analyzed using different blast methods including blastn and tblastx (blast version 2.6.0; e-value cut-off 1e −3 -1e −10 ) to compare sensitivity and run-times to detect viral sequences among the reads. a complete viral database was composed of all virus sequences in genbank (taxonomy id 10239, containing sequences up to 17th of september 2017). the best hit (lowest e-value) was visualized using kronatools 64 . reads matching viruses were extracted using seqtk (https://github.com/lh3/seqtk) and used in downstream analyses. graphmap (version 0.5.2) and samtools (version 1.6) were used for mapping of reads against reference sequences, while canu 1.6 was used for de novo assembly of viral genomes [65] [66] [67] [68] . virsorter was run using the 'viromes' database to look for phages, with the virome decontamination mode on to identify phage contigs 54 . bio-informatics analyses were executed on a local computer cluster and the high-performance computing facilities of ghent university. the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. sanger sequencing of porcine kobuvirus polymerase gene. a porcine kobuvirus was discovered in the sample 17v079 using the minion. the sequence of the 3d gene of porcine kobuvirus encodes the polymerase and is considered to be most conserved among different strains. the exact nucleotide sequence of this virus was verified using reverse transcripion polymerase chain reaction followed by sanger sequencing, as low coverage was obtained with minion. rt-pcr was executed using the onestep rt-pcr kit (qiagen) with the newly designed primers kobu_6049fw and kobu_7524rv (idt dna technologies) ( table 2 ). the rt-pcr reaction contained 5 µl 5 × qiagen onestep rt-pcr buffer, 1 µl dntps, 3 µl of each primer (10 µm), 7 µl nuclease-free water, 1 µl onestep rt-pcr enzyme mix and 5 µl template rna or water (total reaction volume of 25 µl). rt-pcr conditions were as follows: 50 °c for 30 min, 95 °c for 15 min, followed by 30 cycles of amplification (94 °c for 30 s, 50 °c for 30 s and 72° for 90 s) and a final extension step at 72 °c for 1 min. reactions were held at 10 °c prior to loading 5 µl pcr product with 1 µl of loading dye in a 1.5% agarose gel. electrophoresis was conducted for 30 min at 100 v and pcr product was visualized by ethidium bromide staining and uv light. the amplicon was sent to gatc (constance, germany) for sanger sequencing using an abi 3730xl dna analyzer system. quality control of the raw chromatograms was done using 4peaks (nucleobytes bv, the netherlands) and blastn (ncbi, united states). specific rt-qpcr primers (table 2) for the porcine kobuvirus polymerase-encoding gene were designed using primerquest and oligoanalyzer (idt dna technologies) to allow exact quantification in feces of piglets. each rt-qpcr reaction consisted of 10 µl precisionplus onestep qrt-pcr mastermix containing sybr green, rox and an inert blue pipetting dye (primerdesign, southampton, united kingdom), 0.4 µl of each primer (200 nm) and 6.2 µl nuclease-free water. three microliters of template rna or water were added to each tube containing 17 µl mastermix. a synthetic rna positive control (175nt) was generated by rt-pcr using the primers kobu3d_qpcr +t7_fw and kobu3d_qpcr_rv, followed by in vitro transcription of this pcr product using a t7 rna polymerase. the positive control was measured using nanodrop and used to setup a standard curve over a linear dynamic range (ldr) from six to one log 10 copies/reaction. reaction conditions were as follows: 55 °c for 10 min and 95 °c for 2 min, followed by 40 cycles of denaturation (95 °c for 10 s) and annealing (58 °c for 60 s). detection of sybr green fluorescence was done at the end of each annealing phase. a melt curve analysis was executed to assess specificity of the amplicons generated. each dilution point in the standard curve and each sample was tested in duplicates. amplicons were analyzed once on an agarose gel to assess the correct length of the amplicon and sanger sequencing was conducted to confirm the amplification of the partial porcine kobuvirus polymerase gene. assays were valid if the efficiency over the ldr was between 90 and 110%, and r 2 of the standard curve replicates was >0.99. quantification of the viral loads was possible if the cq-values of two qpcr replicates fell within the ldr of the assay. both replicates had to be positive for a sample to be considered as positive. if the cq-values of specific amplicons have fallen behind the lowest point of the standard curve, the sample was considered positive but not quantifiable. longitudinal investigation of kobuvirus and rotavirus shedding in suckling piglets. upon characterization of the virome with the minion, a longitudinal follow-up study was setup between august and september 2017. to warrant the health status of the pig stock, entrance to the farm was strictly regulated. sampling was performed by the farmer. detailed instructions and sampling materials were provided to the farmer. sample collection in the longitudinal field study was done in agreement with the european legislation on animal experiments. sample collection was approved by and done in accordance to the requirements of the local ethical committee of the faculty of veterinary medicine and bioscience engineering of ghent university. one day after parturition of the sows, five litters were selected at random. within each litter, one piglet was identified for longitudinal follow-up during the entire suckling period. a dry cotton rectal swab (copan) was collected from each individual piglet at days 1, 5, 8, 11, 14, 17, 20 and 22 after birth. the swab was placed immediately in 2 ml of viral transport medium (phosphate buffered saline containing 1000 u/ml penicillin (continental pharma, puurs, belgium), 1 mg/ml streptomycin (certa, braine l′alleud, belgium), 1 mg/ml gentamicin (life technologies) and 0.01% v/v fungizone (bristol-myers squibb, braine l′alleud, belgium)) in a sterile 15 ml falcon tube (sarstedt) and stored at −20 °c. every week, samples were collected from the farm and transported to the laboratory of virology. the farmer was asked to mark the tube of each sample for presence or absence of diarrheic signs. upon arrival in the laboratory of virology, the samples were thawed and placed on a shaker for 30 min at 4 °c to release viral particles in the transport medium. samples were extracted using the qiaamp cador pathogen mini kit according to the manufacturer's instructions and purified nucleic acids were eluted in 100 µl of ave and stored at −70 °c until rt-qpcr analysis. rt-qpcr analysis was conducted, as described above, to quantify porcine kobuvirus genome copies per swab. furthermore, rva and rvc shedding was assessed using previously described in-house rt-qpcr assays 25, 61 . belgian suckling pigs. fecal samples (n = 44) of diarrheic suckling piglets less than 2 weeks old were sent to a private laboratory by veterinarians (dialab, belsele, belgium) for etiological diagnosis, as described earlier. these samples were collected in 2014 and stored at −70 °c in the laboratory. they had previously been evaluated for the presence of rotaviruses using rt-qpcr 25 . rna extraction was conducted using the qiaamp cador pathogen mini kit (qiagen) as described above and rt-qpcr was done to quantify the load of kobuvirus rna copies. samples with a quantifiable viral load were subjected to rt-pcr to amplify the 3d polymerase gene, after which sanger sequencing was performed. the sequences encoding the polymerase of 11 belgian porcine kobuvirus isolates were deposited into genbank with accession numbers mh184664-mh184674. the sequences were used to conduct a multiple sequence alignment together with other porcine kobuvirus strains in mega 7 using the clustalw plug-in 69 . a maximum-likelihood phylogenetic tree was constructed with raxml using a general time reversible model with gamma distribution (20 cats, alpha: 0.121, loglk = 14938.461) and heuristic branch swapping 70 . tree editing was done using affinity designer (serif). pairwise distances were calculated using the p-distance model in mega with bootstrap values set at 500 replicates. porcine epidemic diarrhea: a retrospect from europe and matters of debate porcine rotaviruses: 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snp calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data the sequence alignment/map format and samtools mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets raxml version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies this study was funded by the belgian federal public service health, food chain safety and environment (rf17/6312) and an agricultural trajectory of the flemish agency innovation and entrepreneurship (vlaio, la140955). bv (1s28617n) and lb (1s61618n) were supported by a phd scholarship of the research foundation flanders (fwo vlaanderen). pieter ganseman is acknowledged for his excellent technical assistance. philip vyt (dialab, belsele) is acknowledged for providing the diarrheic fecal sample of the suckling piglet. we appreciate the collaboration with the farmer from the case farm and are grateful for providing us fecal samples from his pigs. 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-339546-m7rqr886 authors: de arriba, m.l; carvajal, a; pozo, j; rubio, p title: isotype-specific antibody-secreting cells in systemic and mucosal associated lymphoid tissues and antibody responses in serum of conventional pigs inoculated with pedv date: 2002-01-01 journal: vet immunol immunopathol doi: 10.1016/s0165-2427(01)00386-5 sha: doc_id: 339546 cord_uid: m7rqr886 an enzyme-linked immunospot (elispot) has been developed to detect porcine epidemic diarrhea virus (pedv)-specific antibody secreting cells (asc) in gut associated lymphoid tissues (duodenum and ileum lamina propria and mesenteric lymph nodes) and systemic locations (spleen and blood) of conventional pigs so as to characterise the mucosal and systemic antibody response generated by the infection with pedv. a total number of 28 eleven-day-old conventional pigs were orally inoculated with the field isolate of the pedv strain cv-777. diarrhea was observed in 32% of the pigs and virus shedding was demonstrated in 100% between postinoculation day (pid) 1 and 8. serum igg and iga antibodies to pedv were detected by isotype elisa from pid 12 and 15, respectively, reaching maximum values at pid 32 (igg) and 21 (iga). pedv specific igm asc occurred in all the tissues between pid 4 and 7, with the strongest response in the intestinal lamina propria. iga and igg asc responses were evident in the intestinal lymphoid tissues from pid 21, the highest number of specific asc corresponded to the duodenum lamina propria. in the systemic lymphoid tissues the number of igg and iga asc detected were lower than in the mucosal tissues, however, in the blood, presence of iga asc was constantly detected from pid 14 until the end of the experiment. memory antibody response to the pedv was also studied by secondary in vitro stimulation of the mononuclear cells (mnc) isolated from mesenteric lymph nodes, spleen and blood. the memory b cell response was prominent at pid 21 and 25 and consisted in igg and iga asc. to our knowledge, this is the first report to research into the presence and distribution of specific asc in different locations of the systemic and the gut associated lymphoid tissues after a pedv infection as well as the presence of memory b cells. porcine epidemic diarrhoea virus (pedv) is an important pathogen causing severe gastroenteritis with a clinical picture similar to that of transmissible gastroenteritis (tge). pedv has been classi®ed in group i of the coronaviridae family, which also includes transmissible gastroenteritis virus (tgev), feline infectious peritonitis virus (fipv) and human respiratory coronavirus 229e (hcv229e) (cavanagh et al., 1994; murphy et al., 1999) . porcine epidemic diarrhoea (ped) was ®rst described in great britain in 1971 (oldham, 1972) , and nowadays the virus is widely distributed throughout europe and asia, although has not been detected in america. in spain, a serological survey carried out in 1993±1994 detected antibodies against the virus in 54% of the breeding herds (carvajal et al., 1995b) . moreover, over the last 4 years the virus has been found in a high percentage of the clinical cases of diarrhoea analysed at our laboratory. these data indicate that pedv is currently one of the most important causes of gastroenterical disorders in pigs in spain, which seems to be the same case in other countries (van reeth and pensaert, 1994) . clinically, the disease can appear in two forms, ped type i, which affects only pigs older than 4±5 weeks, and ped type ii, which affects pigs of all ages. both are characterised by profuse, watery diarrhoea, depression and anorexia. morbidity is high, close to 100%, but mortality rate is relatively low in adult pigs (3%), whereas not in suckling piglets among which the severity of the disease increases and mortality can reach 90% (pensaert, 1999) . the epithelium of the small intestine and the colon is the site for virus replication and although the presence of viral antigen in the mesenteric lymph nodes has been described, there is no evidence of virus replication in tissues other than in those in the gastroenteric tract. infection of the enterocytes causes vacuolisation and ®nally destruction, which leads to villous atrophy and watery diarrhoea due to malabsorcion. so far, there is no effective vaccine or speci®c treatment available, and the only measures to control the disease are those directed to preventing the entrance of the virus on the farm (pensaert, 1999) . the development of immunological strategies in order to induce protection would be desirable, mainly those involving the protection of suckling piglets less than 2±3 weeks old. little has been reported relating to the immunological aspects of the disease other than detection of serum antibodies against the virus in convalescent animals (carvajal et al., 1995a; van niewstadt and zetstra, 1991; debouck and pensaert, 1984) . however, due to the special features of the mucosal immune system of the pigs, the presence of serum antibodies against gastroenteric pathogens is not always correlated with protection (saif and wesley, 1999; saif, 1996; saif et al., 1994 , to ã et al., 1998 ward et al., 1996) and only proves the contact with the microorganism. in the present work, we have tried to contribute to the information on the immune mechanisms occurring after pedv infection by making a ®rst approach to the characterisation of the antibody response generated by the virus. considering the fact that in the enteric infections in porcine is the local immunity which plays the main role in protection instead of the systemic immunity, as previously mentioned, the ®rst step was to develop techniques to investigate the immune response to pedv in different tissues. finally, we tried to emulate a natural infection by inoculating conventional pigs with a virulent strain of pedv and monitorised the humoral response in different locations of the lymphoid system, involving gut associated lymphoid tissues as well as systemic tissues. the wild type isolated of the cv-777 strain of pedv, kindly provided by dr. peansert (gent, belgium) was ampli®ed by passages in conventional 1-week-old piglets without antibodies against pedv. animals were orally inoculated and sacri®ced in the acute phase of diarrhoea, collecting the intestinal contains and the small intestine. the small intestine from each animal was macerated in pbs (1:2 (w/v)) and, like the intestinal contains, clari®ed by centrifugation at 5000 â g for 20 min at 4 8c. finally, all the fractions were pooled and stored at à70 8c. the cell-culture adapted pedv, strain cv-777, was propagated in vero cells as previously described (hofmann and wyler, 1988) . brie¯y, vero cells were grown with eagle's minimum essential medium (gibco, life technologies) buffered with bicarbonate and supplemented with 5% (v/v) foetal calf serum (gibco), 0.04% (w/v) yeast extract (difco, mi, usa), streptomycin (10 mg/l) and penicillin (10,000 ui/l) (penicillin-streptomycin, gibco). con¯uent monolayers were infected by removing the growth medium and adding the viral inoculum diluted in medium without foetal calf serum but containing 10 ml/ml trypsin (difco). in order to standardise the elispot, three conventional 4-week-old pigs were hyperimmunisated and used as a source of lymphocytes primed against pedv. pigs were orally inoculated with a suspension of the virulent, wild type, pedv and boostered 4 and 12 weeks later by peritoneal injection of the same inoculum concentrated by ultracentrifugation at 10 000 â g and diluted (1:1) in freund's adjuvant (complete in the ®rst injection and incomplete in the second) with antibiotics (gentamycin: 500 mg/ml (gibco), streptomycin: 20 mg/ml, penicillin: 20,000 ui/ml (penicillin-streptomycin, gibco)). serum samples were taken weekly after the ®rst inoculation to monitorise the antibody production. once a high serum antibody titter was reached, blood samples were collected periodically in 25% (v/v) acid citrate glucose to obtain the mononuclear cells (mnc). finally, the pigs were sacri®ced and the spleen and mesenteric lymph nodes were aseptically collected. as a negative control, blood samples from pedv seronegative conventional pigs were collected in the same manner. a total number of 28 conventional pigs, seronegative to pedv and from a herd with no previous history of the disease, were weaned at 11±12 days of age and maintained in isolation facilities. pigs were inoculated orally with 3 ml of the virulent cv-777, a dose previously established which assesses a high rate of infection and the effective development of the immune response but without causing mortality among the pigs. the animals were observed daily for clinical signs and rectal swabs from all of them were taken for 11 days after the inoculation. blood samples were also collected twice a week until the end of the experiment. at postinoculation days (pid) 4, 7, 14, 21, 25 and 31, 4±5 selected pigs were euthanised by a sodic penthotal injection (eutalender, normon, madrid, spain). the small intestine (duodenum and ileum), spleen, mesenteric lymph nodes and blood were aseptically collected for isolation of mnc. three conventional, seronegative, pigs served as negative controls and were sacri®ced without previous inoculation. an elisa that combines the use of two monoclonal antibodies (mab) against the s protein of pedv (cvi-pedv 66.31 and 66.49) and a blocking step with rabbit-anti pedv hyperimmune serum or gut-origin pedv, was carried out as previously described (carvajal et al., 1995a) to detect speci®c antibodies in serum or viral antigen in rectal swab samples, respectively. antibody isotypes igg and iga to pedv in serum samples were detected and titrated using an indirect elisa as previously described (de arriba et al., 1994) . antigen was obtained from pedv infected cell culture supernatants that were lysated, concentrated 50 times by ultracentrifugation (100 000 â g, 4 8c, 2 h) and semi-puri®ed by ultracentrifugation under the same conditions through 20% sucrose. mock-infected cultures were given the same treatment in order to obtain a control antigen. viral or control antigens were immunocapturated in polystyrene microtiter plates (costar, ma, usa) previously coated with the mab cvi-pedv-66.31. in the next step, serial 2-fold dilutions, starting at 1:20, of each serum sample were incubated in paired wells, containing viral or control antigen. two biotinylated mabs 3h7 (80 ng/ml) and 6d11 (65 ng/ml) against porcine igg and iga, respectively (paul et al., 1989) , followed by horseradish peroxidase-conjugated streptavidin (kpl, md, usa) and abts substrate were used for the detection of both isotypes of antibodies. titres were expressed as the reciprocal of the lowest positive sample dilution and titres <20 were assigned a value of 10 for calculation of geometric mean titre (gmt). mnc were isolated from mln, blood, spleen and the lamina propria of the small intestine (duodenum and ileum) by using modi®cations of previously described methods (chen et al., 1995; van cott et al., 1993; yuan et al., 1996) . blood was collected aseptically in 25% (v/v) acid citrate glucose and the peripheral blood lymphocytes were isolated by ficoll-paque (ficoll-paque research grade, pharmacia biotech., upsala, sweden) density gradient centrifugation. lymphocytes collected from the interface were washed twice in hanks' balanced salt solution and resuspended in enriched medium (rpmi 1640 containing 8% fetal calf serum, 2 mm l-glutamine, 1 mm sodium pyruvate, 0.1 mm nonessential aminoacids, 20 mm hepes, and 20 mg of ampicillin and 100 mg of gentamicin per millilitre). spleen, mesenteric lymph nodes and two fragments of the small intestine (one from duodenum and the other from ileum) were collected aseptically and placed in ice-cold wash medium (rpmi 1640 containing 10 mm hepes and 200 mg of gentamicin and 20 mg ampicillin per ml). spleen and mesenteric lymph nodes were homogenised pressing through stainless steel screens (80 mesh) of a cell collector (cell-selector, e-c apparatus, fla, usa). cell suspensions were placed in a 30% percoll (pharmacia biotech.) solution and centrifugated at 1200 â g for 30 min at 4 8c. the resulting pellets were subjected to a discontinuous gradient centrifugation in percoll: the cells were resuspended in 43% percoll, underlined with 70% percoll and centrifuged at 1800 â g for 20 min at 4 8c. finally, the mnc were aspirated from the percoll interface, washed twice with wash medium and suspended in enriched medium. fragments of approximately 20 g of duodenum and ileum were cut in small pieces, washed twice with wash solution and twice with hanks' balanced salt solution. in order to remove epithelial cells, the tissues were placed in hank's balanced salt solution containing 1 mm dithiothreitol and 5 mm edta and vigorously shaken for 30 min. tissues were then digested for two 31-min periods at 37 8c in gentle shaking with enriched medium containing 400 ui of type ii collagenase (sigma, mo, usa) per millilitre and 5 mm edta. digested supernatants were collected and the remaining tissues were pressed through the stainless steel 80-mesh screens. the single cellular suspensions obtained were pooled with the digested supernatants and subjected to the gradient centrifugation in percoll as described for mesenteric lymph nodes and splenic mnc. viability of all mnc preparations was proved by the trypan blue exclusion test, being in any case higher than 95%. two different kind of plates were assayed to set up the elispot, on one hand pedv infected and ®xed cell monolayers and on the other, semi-puri®ed antigen that was immunocaptured in plates previously coated with a mab against the s protein of the virus. vero cells, grown in 96-well tissue culture plates, were inoculated with the cell culture adapted pedv as described (hofmann and wyler, 1988) . after incubation at 37 8c in a humid incubator with 5% co 2 , plates were ®xed in 80% acetone in pbs for 20 min at room temperature and stored at à20 8c. different dilutions were tested in order to ®nd the optimal multiplicity of infection to yield over 90% of infected cells. control plates were mock-inoculated and treated in the same way. optimal antigenic expression was veri®ed in every batch of plates by indirect immunouorescence using the mab 113b, directed to the s protein of the pedv and kindly provided by dr. m. ackermann (zurich, switzerland). only the plates showing more than 80% of¯uorescent surface were used to perform the elispot. ninety-six-well microtiter plates were coated and dilutions of viral or control antigen were immunocaptured as described for the isotype elisa. the elispot technique was based on previously published methods (chen et al., 1995; czerkinsky et al., 1983, sedgwick and holt, 1983; van cott et al., 1993; yuan et al., 1996) modi®ed and adapted by us for the detection of pedv-speci®c antibody secreting cells (asc). fixed cell plates were thawed and rehydrated by incubation with pbs for 5 min at room temperature while antigen captured plates were washed three times before use in the elispot. different amounts of mnc (5 â 10 3 , 5 â 10 4 and 5 â 10 5 ) from each tissue were added to duplicate wells of the ®xed-pedv infected or mock-infected cell plates or to the plates with the immunocaptured antigen (viral and control antigen). plates were centrifuged at low speed 50 â g for 5 min and incubated at 37 8c with 5% co 2 for different periods. in order to remove the cells and between steps, the plates were washed 5 times with 0.05% tween 20-pbs (pbst). the antibody production was detected by using biotinylated mouse mabs 3h7 (80 ng/ml) and 6d11 (65 ng/ml) against porcine igg and iga, respectively (paul et al., 1989) and goat anti-porcine igm serum (1:20,000, kpl) diluted in pbst. after 2 h incubation at room temperature, horseradish peroxidase-conjugated streptavidin (kpl) was added (1:20,000) and incubated for 1 h at room temperature. finally, the spots were developed by tetramethylbencidine (tmb) with h 2 o 2 membrane peroxidase substrate system (kpl) and counted under an optic microscope. counts were averaged from the duplicated wells at the dilutions showing less than 40 spots per well and were expressed relative to 5 â 10 5 mnc. working conditions were optimised to detect the highest number of speci®c spots against pedv. the in vitro viral stimulation technique was modi®ed from published methods (van cott et al., 1993 . mnc puri®ed from spleen, blood and mesenteric lymph nodes were diluted in enriched medium containing 50 mm 2-mercaptoethanol (2me-enriched medium) (sigma) to 5 â 10 6 mnc per millilitre. 750 ml of each cell preparation were added to two consecutive wells of a 12-well tissue culture plate and stimulated with semi-puri®ed pedv viral antigen, prepared as described for the isotype elisa and diluted in 750 ml of the 2me-enriched medium. plates were maintained in a humid incubator with 5% co 2 for 5 days and, from the second day on, 500 ml per well of fresh 2me-enriched medium were added. on the ®fth day, mnc were harvested, rinsed twice with wash medium, suspended in 2me-enriched medium and tested by elispot (testing 5 â 10 2 , 5 â 10 3 and 5 â 10 4 mnc per well). kruskal-wallis non-parametric analysis of variance was used to prove statistically signi®cant differences in the number of asc between different days. antibody gmt were compared by means of student t-test at each point in time. signi®cance was assessed at p < 0:05. for the analysis the systat for windows v.5.03 (systat) and the spreadsheet microsoft excel v.7.0 (microsoft) were used. pigs inoculated with the virulent isolate of the pedv strain cv-777 exhibited moderate signs of the disease, mainly semi-liquid diarrhoea, shown in 32% of the pigs. the onset of the diarrhoea was between pid 2 and 4 and its average duration was 1.7 days. rectal virus shedding was detected by blocking-elisa in 100% of the pedv exposed animals. viral antigen was present in faecal samples from pid 1 to 8, but most of the pigs shed the virus in faeces between pid 3 and 6. the average duration of the shedding period was 5.4 days and the peak of the gmt of viral antigen in rectal swabs was at pid 5 ( fig. 1) . after inoculation with virulent pedv, seroconversion was demonstrated in 100% of the pigs by blocking elisa. speci®c antibodies were detected in 52.6% of the pigs at pid 4, in 96% at pid 7 and in 100% at pid 12. the isotype-speci®c elisa antibody titters in the serum of inoculated piglets are shown in fig. 2 . titters of igg increased signi®cantly from pid 12 over the duration of the experiment, reaching the maximum at that moment (pid 32, gmt 2650). iga serum optimal antigenic expression with the infected and ®xed cell monolayers was obtained by inoculating 2:8 â 10 3¯u orescent focus-forming units of viral inoculum per well and ®xing the plates after 12±15 h of incubation. these conditions guaranteed a citopatic effect between 80 and 90%, which gave a very high rate of¯uorescence, but keeping the integrity of the monolayer. plates with the immunocaptured antigen yielded the best results when the semi-puri®ed viral antigen diluted 1:25 was used (2.05 mg of protein per well) and the plates were incubated for 4 h at 37 8c or overnight at 4 8c. using infected and ®xed cell monolayers unspeci®c spots were non-detected either in the plates with mock-infected cell monolayers or with mnc from naõ ève piglets and, therefore, every spot detected was considered a positive result. on the other hand, plates with the immunocaptured antigen showed some unspeci®c spots in the control wells and also when mnc from negative pigs were tested. moreover, a higher number of spots were usually detected when the assay was performed over infected and ®xed cell monolayers. consequently, due to the high speci®city shown for the ®xed pedv infected cell plates and its higher sensitivity, we decided to use these plates for the rest of the experiments. in every assay mock-infected plates were included as negative control to assess the speci®city. in order to evaluate the distribution of pedv-speci®c asc, mnc from mesenteric lymph nodes, lamina propria of duodenum and ileum, spleen and blood were recovered at various pid from pedv exposed pigs and tested for antibody production by elispot. kinetics of those responses are summarised in figs. 3±5 and table 1 . no virus-speci®c asc were detected by elispot either with the control pigs or when the assay was performed with mock-infected cell plates. pedv-speci®c igm asc were the predominant response between pid 4 and pid 7. at pid 4 speci®c igm asc were found in all the tissues with the only exception in the blood, the strongest response corresponded to the intestinal lamina propria, where the number of igm asc was 6±7-fold greater than in mesenteric lymph nodes and spleen. at pid 7, the number of igm asc declined in the duodenum and the ileum, but not in mesenteric lymph nodes and systemic tissues (spleen and blood) and at pid 14 blood was the only tissue in which these cells were detected. although some iga asc were detected as early as pid 7 in duodenum lamina propria, the igg and iga asc responses in the intestinal lymphoid tissues were evident from pid 21 on. igg asc were demonstrated in the three tissues at pid 21, being maximum in the duodenum (fig. 4, 23 .5 igg asc per 5 â 10 5 mnc). from this day and until the end of the experiment, the number of igg asc declined in the duodenum, but not in the mesenteric lymph nodes, where maximum was reached on pid 32. although no signi®cant statistical difference was demonstrated, the number of iga asc detected in the intestinal lymphoid tissues by elispot was, in general, lower than the number of igg asc. the highest iga asc response was also observed in the duodenum, where these cells were shown on pid 7, 21 and 25. the lowest response was obtained in ileum, in which iga asc were demonstrated only in a limited number at pid 25 (<1 iga asc per 5 â 10 5 mnc) (fig. 5) . the number of igg asc in spleen and blood was lower than in the intestinal lymphoid tissues and these cells were evidenced later, between pid 25 and 32. a slow number of iga asc were detected in the spleen, but only at pid 14. however, in the blood, the presence of iga asc was proved from pid 14 until the end of the experiment on pid 32. due to the individual variability in the responses observed and mainly to the limited number of pigs used at each point in time, statistical comparison among tissues or days showed no signi®cant differences. mnc were stimulated in vitro with different amounts of virus and over different periods. the best results were obtained when culturing the cells for 5 days with 4.6 ml (238.6 ng of protein) of the semi-puri®ed viral antigen per 5 â 10 5 mnc. in vitro stimulation of the cells was only possible with the mnc from mesenteric lymph nodes, spleen and blood, but not with those from the intestinal lamina propria since they had an irregular survival due to their frequent contamination with the enteric microbial ora. results from the secondary immune response obtained after the in vitro stimulation are summarised in table 2 . even though low numbers of igm asc were detected on pid 4, secondary antibody response was more prominent on pid 21 and 25, consisting of igg and iga asc. the number of speci®c asc increased from 38 to 1137 times after the secondary stimulation and in a similar way to the primary responses, igg asc were predominant in the systemic tissues (spleen and blood), whereas not in the mln, in which the secondary iga asc response was higher at pid 21. in vitro stimulation of naõ ève mnc from the control group did not yield virus-speci®c asc after 5 days of culture. table 1 numbers of isotype-specific asc to pedv (per 5 â 10 5 mnc) in duodenum and ileum lamina propria, mesenteric lymph nodes (mln), spleen and blood from pigs experimentally exposed to the virulent isolate of the pedv strain cv-777 and sacrificed on pid 4, 7, 14, 21, 25 in the present work, we have made a characterisation of the humoral immune response occurring after the infection with pedv. the study was carried out with conventional pigs, infected with a virulent, wild type isolate of the virus, with the aim of emulating the natural conditions in which the infection took place. experimental models were 12-day-old, pedv seronegative piglets. no older animals were used due to the high growth rhythm of porcines, which makes it very dif®cult to handle a large number of pigs for an extended period. however, these young animals, in spite of having a non-totally-mature-immune system, are immunocompetent from the birth and are capable of developing a complete immune response (roth, 1999) . virulent pedv caused moderate to severe diarrhea in 30% of the inoculated piglets with a average duration of 1.7 days. the incidence and severity of the disease in our experiment was lower than previously described by other authors (carvajal et al., 1995a; debouck and pensaert, 1984; debouck et al., 1981) who found that almost 100% of the inoculated piglets, gnotobiotic and conventional, developed diarrhea over an average period of 7±10 days. this difference may be explained by the low dose of virus used in our experiment. this dose was experimentally determined in previous assays in order to guarantee infection but without causing severe disease, in order that immune response could be developed and detected in the piglets. however, viral antigen was detected in faeces of all the pigs for the average period of 5.4 days, starting between pid 1 and 3, as it has been described in similar experiments (carvajal et al., 1995a) . speci®c antibodies against pedv were demonstrated by blocking elisa in 100% of the inoculated pigs between pid 4 and 12. this result is closely related with previous studies which report seroconversion 1 week after pedv infection (carvajal et al., 1995a; van niewstadt and zetstra, 1991) . although pedv-igg antibodies were always the predominant response in blood, in consistence with the patron for distribution of the different immunoglobulin isotypes, an important iga response was detected in blood from pid 15±25, simultaneous to the detection of iga asc in blood. these results indicate that even blood is not considered an important source of the typically mucosal-associated table 2 numbers of isotype-specific asc to pedv (per 5 â 10 5 mnc) after in vitro secondary stimulation of the mnc isolated from mesenteric lymph nodes (mln), spleen and blood of conventional pigs experimentally exposed to the virulent isolate of the pedv strain 7, 14, 21, 25 igm iga igg igm iga igg igm iga igg 4 11 (44) 0.7 (±) 0 (±) 22 (36) 0 (±) 0 (±) 8 (±) 0 (±) 0 (±) 7 0 (±) 0 (±) 0 (±) 0 (±) 0 (±) 0 (±) 0 (±) 0 (±) 0 (±) 14 0 (±) 0 (±) 0 (±) 0 (±) 0 (±) 0.8 (±) 0 (±) 0 (±) 0 (±) 21 0 (±) 44 (35) 38 (14) 0 (±) 220 (±) 586 (±) 0 (±) 29 (46) 267 (2136) 25 0 (±) 156 (±) 1137 (247) 0 (±) 7 (±) 97 (34) 0 (±) 49 (163) 1038 (478) a asc after secondary in vitro stimulation/asc after primary in vivo stimulation is given within the parenthesis. immunoglobulin. its detection is possible in the early stages after infection, probably in relation to the lymphocyte homing process (corthesy and kraehenbuhl, 1999; kagnoff, 1996; kantele et al., 1997; salmi and jalkanen, 1997) . in order to standardise elispot, two different kinds of antigenated plates were compared, infected and ®xed cell culture plates and semi-puri®ed antigen immunocaptured plates. in spite of the fact that no statistical studies were carried out, elispot performed with infected cell culture plates gave better results in terms of sensitivity and speci®city. in the immunocaptured plates we used a mab directed against s-protein of pedv and, therefore, most of the viral antigen captured in the plate could be fragments of the spike more than the complete viral particle. on the other hand, viral inoculum for peritoneal immunisation of the animals was concentrated using ultracentrifugation and may then induce a high immune response against n and m protein which could be better detected with the infected and ®xed cell plates. using the elispot, we demonstrated the early development of a primary speci®c immune response against pedv, formerly igm asc followed by igg and iga asc responses. pedv-speci®c igm asc were detected in all tissues between pid 4 and 7, with this response being higher and faster in duodenum and ileum lamina propria than in the other tissues, including mesenteric lymph nodes. the earlier presence of speci®c asc in these tissues could be related to its anatomic proximity to the virus replication site in the enterocytes of the small intestine. the antigen could be delivered immediately to the lamina propria where it would stimulate the local immune response whereas its diffusion to the mesenteric lymph nodes would be more delayed (corthesy and kraehenbuhl, 1999; kagnoff, 1996; kraehenbuhl and neutra, 1992; . after this initial response, there was no important detection of pedv-asc in the intestine or mesenteric lymph nodes until pid 21. maximum values of asc were reached between pid 21 and 32, at the end of the experiment. similar experiences with other porcine enteric viruses such as tgev or rotavirus (chen et al., 1995; van cott et al., 1993; yuan et al., 1996) showed consistent iga and igg asc responses as early as pid 12, with the maximum amounts of cells detected also being higher than those described in the report. this difference could be due to a lower sensitivity of the elispot performed for pedv which would not detect low numbers of speci®c-iga and igg asc present in the ®rst weeks after the infection. we also have to consider that experiences with rotavirus were carried out with gnotobiotic pigs in which the antigen-speci®c asc/total asc ratio is higher than in conventional animals. however, at least for tgev, we cannot rule out the possibility of a higher antigenic stimulation as compared with pedv. although duodenum registered the highest number of speci®c-igg and iga asc of the three mucosal associated lymphoid tissues, we consider that this result is more related to the distribution and organisation of the intestinal lymphoid tissue than to real differences in the immune response between tissues. mnc puri®ed from duodenal lamina propria belong to the diffuse lymphoid tissue, in which most of the cells, mainly the lymphocytes, are mature and active (kraehenbuhl and neutra, 1992; pescovitz, 1999) . on the other hand, mnc from ileum and mesenteric lymph nodes also include large numbers of naive cells from the germinal centres of the lymph nodes or the peyer patches that were not removed from the ileum (heel et al., 1997; pescovitz, 1999) . as a constant in our results, the number of igg asc in gut associated tissues was higher than the number of iga asc. this fact has also been described in previous reports (van cott et al., 1993; yuan et al., 1996 yuan et al., , 1998 studying immunity by elispot in tgev and rotavirus infections. even though there is no clear explanation for this fact, the possibility of extraintestinal stimulation of the immune system has been proposed. however, pedv has only been located in mesenteric lymph nodes apart from the intestinal mucosa (debouck et al., 1981; pensaert, 1999) and the possibility of systemic stimulation is very low. further investigation is necessary to elucidate this question. with regard to the response detected in the systemic lymph tissues and mainly in the blood, our results showed the presence of speci®c iga asc at pid 14, earlier than in mucosal associate lymphoid tissues. as mentioned previously, we explain this ®nding as a consequence of the lymphocyte homing, a process in which the lymphocytes migrate through the blood to the efector sites, such as the intestinal lamina propria, after completing its maturating in distal sites of the mucosal immune system (corthesy and kraehenbuhl, 1999; kagnoff, 1996; kantele et al., 1997; salmi and jalkanen, 1997) . thus, at least shortly after the infection, the pedv speci®c asc present in the blood could be considered more related to the response in mucosal associated lymph tissues than to the systemic response. secondary in vitro stimulation of the mnc from inoculated piglets was performed in order to determine the potential b-cell memory response after pedv infection. no speci®c-asc was evidenced after the secondary in vitro stimulation of mnc obtained from control animals with no previous contact with pedv. this result con®rms, as it has been described for other virus (berthon et al., 1990; van cott et al., 1993; yuan et al., 1996) , that only previously in vivo stimulated cells are capable of developing a speci®c response when stimulated in vitro with pedv antigen. moreover, a limited number of igm asc were detected at pid 4, memory b cells appeared consistently at pid 21 in mesenteric lymph nodes, spleen and blood from pedv infected pigs. similar results have been found after oral inoculation of pigs with porcine rotavirus while in tgev infected animals speci®c b memory cells were detected from pid 12 (van cott et al., 1993) . again, the number of pedv±asc per 5 â 10 5 mnc after secondary in vitro stimulation were lower than reported by van cott et al. (1993) in similar studies for tgev. as we have suggested for the in vivo response, this difference could be attributed either to a lower sensitivity of the elispot performed with pedv or to differences in the immune response against each virus. in agreement with other authors (berthon et al., 1990; van cott et al., 1993 and as described for the in vivo stimulated asc, clear predominance of pedv-speci®c igg asc was observed in systemic lymphoid tissues, blood and spleen, but not in the mesenteric lymph nodes where, at pid 21, iga asc outnumbered igg asc although reverse circumstances occurred at pid 25. to sum up, a ®rst attempt has been made to study the immune response developed in conventional piglets after infection with pedv. this report shows the presence of speci®c-asc from the different isotypes and in several locations of gut associated and systemic lymphoid tissues and also describes the iga and igg kinetics in serum. in addition, our study shows the presence of speci®c memory b cells from the third week after infection. present results have important implications for future studies of immunity and protection against pedv infection and consequently in the development and evaluation of future immunopro®lactic strategies to prevent and control the disease. kinetics of the in vitro antibody response to transmissible gastroenteritis (tge) virus from pig mesenteric lymph node cells, using the elisaspot and elisa tests evaluation of a bloking elisa using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies seroprevalence of porcine epidemic diarrhea virus infection among different types of breeding swine farms in spain revision of the taxonomy of the coronavirus, torovirus and arterivirus genera enumeration of isotype-specific antibody-secreting cells derived from gnotobiotic piglets inoculated with porcine rotaviruses antibody-mediated protection of mucosal surfaces a solid-phase enzyme-linked immunospot (elispot) assay 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porcine epidemic diarrhea immunology of the pig the immune system mucosal immunity: an overview and studies of enteric and respiratory coronavirus infections in a swine model of enteric disease transmissible gastroenteritis and porcine respiratory coronavirus immunity to transmissible gastroenteritis virus and porcine respiratory coronavirus infections in swine how do lymphocytes know where to go: current concepts and enigmas of lymphocyte homing a solid phase immunoenzymatic technique for enumeration of specific antibody-secreting cells serum and intestinal isotype antibody responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease isotype-specific antibody-secreting cells to transmissible gastroenteritis virus and porcine respiratory coronavirus in gut-and bronchus-associated lymphoid tissues of suckling pigs contribution of antibody-secreting cells induced in mucosal lymphoid tissues of pigs inoculated with respiratory or enteric strains of coronavirus to immunity against enteric coronavirus challenge use of two enzime-linked inmunosorbent assays to monitor antibody responses in swine with experimentally induced infection with porcine epidemic diarrhea virus prevalence of infections with enzootic respiratory and enteric viruses in feeder pigs entering fattening herds development of mucosal and systemic lymphoproliferative responses and protective immunity to human group a rotavirus in a gnotobiotic pig model systemic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease antibody-secreting cell responses and protective immunity assessed in gnotobiotic pigs inoculated orally or intramuscularly with inactivated human rotavirus we wish to thank dr. l.j. saif and dr. m. ackermann for providing mabs and dr. m.b. pensaert for providing the wild type isolated of the pedv strain cv-777. we also wish to thank g.f. bayo ân for her excellent technical assistance. this work was funded by the comisio ân interministerial de ciencia y tecnologõ âa (cicyt) project no. agf-960486. salaries were provided by the excelentisima diputacio ân provincial de leo ân. key: cord-343132-qqhivgkq authors: chang-liao, wan-ping; lee, an; chiu, yu-han; chang, hui-wen; liu, je-ruei title: isolation of a leuconostoc mesenteroides strain with anti-porcine epidemic diarrhea virus activities from kefir grains date: 2020-07-15 journal: front microbiol doi: 10.3389/fmicb.2020.01578 sha: doc_id: 343132 cord_uid: qqhivgkq swine grown under commercial conditions are vulnerable to environmental exposure to several viruses, which may cause infectious diseases and spread easily and rapidly, resulting in significant economic losses in animal husbandry. previous studies have suggested that probiotics seem to be a new and promising alternative to vaccinations to protect animals against potential viral infections. in this study, we used the vero cell culture model of infection to study porcine epidemic diarrhea virus (pedv). we screened lactic acid bacteria (lab) with anti-pedv potential from kefir grains, which are starter cultures used to ferment milk into kefir. twenty-nine lab strains were isolated and identified as enterococcus durans, lactobacillus kefiri, lactococcus lactis, and leuconostoc mesenteroides, according to 16s ribosomal rna (rrna) and rpoa gene sequence analyses. the anti-pedv activities of the lab intracellular extracts were compared, and the intracellular extracts of ln. mesenteroides showed higher anti-pedv activities than that of the other species. among the ln. mesenteroides strains, a strain designated ypk30 showed a higher growth rate than that of the other strains and was further evaluated for its anti-pedv activity. the results showed that the intracellular extracts of ln. mesenteroides ypk30 possessed in vitro prophylactic, therapeutic, and direct-inhibitory effects against pedv in the vero cell model. the expression levels of type 1 interferon (ifn)-dependent genes, including myxovirus resistance 1 (mx1) and interferon-stimulated gene 15 (isg15), were significantly increased after treatment with intracellular extracts of ln. mesenteroides ypk30 for 24 h. such expression suggests that the anti-pedv activity of ln. mesenteroides ypk30 could be attributed to its up-regulatory effect on the expression of mx1 and isg15 genes. these results suggested that ln. mesenteroides ypk30 has the potential to provide some levels of host protection against pedv infections. in order to increase swine and poultry production, it is common to raise animals in high-density populations. swine grown under commercial conditions are vulnerable to environmental exposure to several viruses. some viruses may cause infectious diseases, which are spread easily and rapidly cause significant economic losses in animal husbandry. vaccination is one of the most efficient strategies to prevent viral diseases and control infections and is the current industry standard. efficacious vaccines have been developed and applied successfully for the prevention of several infectious viral diseases in swine, such as porcine parvovirus (ppv), foot-and-mouth disease virus (fmdv), porcine circovirus type 2 (pcv2), and transmissible gastroenteritis virus (tgev; meng, 2012; gerdts and zakhartchouk, 2017) . however, some of the vaccination methods require the operator to handle each animal, induce the stress on the animal, and are time-consuming and costly (marangon and busani, 2007) . although efficacious vaccines are available to reduce the impact of the infectious viruses mentioned above, unfortunately, substantial challenges remain in obtaining safe and efficacious vaccines for a variety of newly emerging and re-emerging viruses, such as african swine fever virus and porcine epidemic diarrhea virus (pedv; lee, 2015) . pedv is a member of the genus alphacoronavirus in the family coronaviridae of the order nidovirales and has emerged as a significant pathogen causing lethal watery diarrhea, vomiting, and dehydration in nursing piglets. highly pathogenic strains of pedv have mortality rates of 50-90% in neonatal piglets, which has resulted in huge economic losses to the swine industry worldwide (koonpaew et al., 2019; wang et al., 2019) . accumulated evidence indicates that pedv encodes defensive mechanisms to evade virus recognition by host pattern recognition receptors (prrs) present on antigen-presenting cells, inhibit interferon (ifn) induction, and antagonize ifn signaling and antiviral effector machinery (hao et al., 2019; koonpaew et al., 2019) . pedv has been studied extensively and some vaccines have been developed against pedv, but the efficacy of these vaccines in the field remains questionable (wang et al., 2019) . probiotics, which are live microorganisms that when administered in adequate amounts confer a health benefit on the host, have long been used as feed additives because of their abilities to normalize gut microbiota, boost the immune system, prevent diarrhea, and improve feed conversion efficiency (fontana et al., 2012; alonso and guarner, 2013) . the effect of probiotics is achieved mainly through the intervention on gut microbiota, which increases the levels of beneficial bacteria and decreases the pathogenic populations in the gastrointestinal tract (liu et al., 2015; yadav and shukla, 2019) . in addition to the microbiota-modulatory properties, recent studies showed that the immunomodulatory activity of probiotics is another important mechanism of action of probiotics for the inhibition of pathogens (llewellyn and foey, 2017; azad et al., 2018) . those immunoregulatory probiotics provide host protection against pathogenic infections by modulating innate and adaptive antiviral immune responses (villena et al., 2016) . several probiotic strains, most of them belonging to lactobacillus and bifidobacterium genera, have been shown to perform antiviral activities (villena et al., 2016; arena et al., 2018) . these antiviral activities could be mediated by the immunomodulatory properties of probiotics because it was observed that administration of probiotics induced the expression of ifn and interferonstimulated genes (isgs), which are crucial components of the ifn responses and play a key role in establishing an antiviral state for virus clearance and restriction of spread (zelaya et al., 2015; villena et al., 2016; arena et al., 2018; eguchi et al., 2019) . if probiotics have antiviral activity, it seems to be a new and promising alternative to vaccinations to protect animals against potential viral infections (al kassaa et al., 2014) . kefir is an acidic and mildly alcoholic fermented milk product that is believed to have many beneficial activities, such as hypocholesterolemic activity, antibacterial and antifungal activities, antitumor activity, immunomodulatory activity, and quickening of wound healing (bourrie et al., 2016) . traditionally, kefir is produced from milk fermented with a mixed microflora confined to a matrix of discrete kefir grains, which are a combination of bacteria and yeasts in a symbiotic matrix (marshall and cole, 1985) . various bacteria and yeasts have been identified in kefir grains. the bacteria present in kefir grains may include acetobacter, bifidobacterium, lactobacillus, lactococcus, leuconostoc, oenococcus, and streptococcus, while the yeasts present in kefir grains may include candida, kluyveromyces, and pichia (lin et al., 1999; wang et al., 2012; bourrie et al., 2016) . numerous bacterial strains with specific properties, such as hypocholesterolemic effect, antiallergenic effect, immunoregulatory effects, and antipathogenic activities, have been isolated from kefir grains (prado et al., 2015) . however, to the best of our knowledge, following a thorough review of the relevant literature, there has been no study to isolate bacterial strains with antiviral activities from kefir grains. in the present study, we used the vero cell culture model of infection by pedv to screen lactic acid bacteria (lab) with anti-pedv activities from kefir grains. the anti-pedv activities of the lab strains were evaluated in prophylactic, therapeutic, and direct-inhibitory models. the strains with anti-pedv activities were further studied to define their effects on the expression of type 1 ifn-dependent genes, including 2'-5'-oligoadenylate synthetase 1 (oas1), myxovirus resistance 1 (mx1), and interferon-stimulated gene 15 (isg15) in vero cells. a total of 29 lab strains were isolated from kefir grains according to the method described by lin et al. (1999) . the lab isolates were cultured in de mann, rogosa, sharpe (mrs) broth (oxoid, basingstoke, uk) at 37°c for 16 h without shaking. the bacterial concentrations were measured by optical density readings at 600 nm or by traditional colony counting methods (vinderola et al., 2000) . for colony morphological observation, the lab isolates were cultivated on mrs agar plates (oxoid) or blood agar plates frontiers in microbiology | www.frontiersin.org (merck, darmstadt, germany) at 37°c for 24 h and then observed. for cell morphological observation, the lab isolates were cultured in mrs broth (oxoid) at 37°c for 16 h without shaking. the bacterial cells were harvested by centrifugation at 5,000g for 20 min at 4°c, stained with gram stain (sigma-aldrich, st. louis, mo, usa) according to the manufacturer's instructions, and observed under a microscope. unstained bacteria were observed under a phase-contrast microscope. molecular identification of the bacterial isolates was performed by the methods described by weisburg et al. (1990) and naser et al. (2005) . genomic dna of the bacterial isolates was isolated using the dneasy blood & tissue kit (qiagen inc., valencia, ca, usa). the standard 16s ribosomal rna (rrna) gene primers and rna polymerase α subunit gene rpoa primers were used for pcr to amplify the 16s rrna gene and rpoa gene sequences of the bacterial isolates, respectively (weisburg et al., 1990; naser et al., 2005) . the resultant pcr products were sequenced by an automatic sequencing service provided by genomics biotech inc., (new taipei city, taiwan). the nucleotide sequences of the 16s rrna and rpoa genes were aligned using the national center for biotechnology information's basic local alignment search tool (blast). for the determination of biochemical characteristics, the carbon source use profiles of the lab isolates were determined using an api 50 ch system (biomerieux, inc., marcy l'etoile, france) according to the manufacturer's instructions. for the determination of physiological characteristics, the lab isolates were cultured at 10°c, 37°c, ph 4.8, or in the presence of 10% ethanol according to the methods described by lin et al. (1999) . before the anti-pedv activity assay, the cytotoxicity of the lab isolates on the african green monkey kidney cell line vero was evaluated according to the method described by mosmann (1983) . the vero c1008 cells were purchased from the bioresource collection and research center of food industry research and development institute (bcrc, hsinchu, taiwan) and were routinely grown at 37°c in a humidified atmosphere of 95% air and 5% co 2 in dulbecco's modified eagle's medium (dmem, gibco, grand island, ny, usa) supplemented with 10% fetal bovine serum (fbs; moregate biotech., queensland, australia). for evaluation of cytotoxicity, a 1.0-ml aliquot of the 16-h culture of each bacterial strain was centrifuged at 9,000g for 10 min at 4°c. the bacteria were collected, washed twice with sterile phosphate-buffered saline (pbs; 0.1 m, ph 7.0), resuspended in 1.0 ml of sterile pbs, and sonicated for 10 min with an ultrasonicator (model xl, misonix, farmingdale, ny). the sonicated bacteria were fractioned into intracellular extracts and cell-wall pellet fractions by subsequent centrifugation at 13,000g for 20 min at 4°c. the intracellular extracts were harvested, and the cytotoxicity on vero cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (mtt) assay according to the method described by mosmann (1983) . briefly, vero cells were seeded at a density of 1.5 × 10 5 cells/well on a 24-well plate in 500 μl of dmem. after incubating at 37°c for 24 h, 100 μl of the bacterial intracellular extracts were added into each well and incubated at 37°c for another 24 h. after washing twice with sterile pbs, the cells were incubated with 500 μl of mtt (5 mg/ml in pbs) at 37°c for 2 h. after the incubation, the medium was removed, and 200 μl of dimethyl sulfoxide (dmso) were added into each well to dissolve the formazan crystals. the absorbance was measured at 570 nm using a microplate reader (victor 3 , perkinelmer inc., waltham, ma, usa), and percentages of cell metabolic activity were calculated as follows: absorbance of sample % / where the absorbance of sample is the absorbance of cells treated with test sample and the absorbance of control is the absorbance of cells treated with dmso. the pedv taiwan pintung 52 strain was isolated in early 2014 from the intestinal homogenate of a 7-day-old suckling pig in taiwan and adapted to vero cells as previously described by chang et al. (2017) . viral infection and propagation were performed in vero cells according to the method described by chang et al. (2017) . before the anti-pedv activity screening experiments were conducted, the viral titers of pedv were adjusted to 200 fifty-percent tissue culture infective dose (tcid 50 )/ml, and the intracellular extracts of bacterial isolates were prepared as described above. vero cells were seeded at a density of 3 × 10 4 cells/well on a 96-well plate in 100 μl of modified postinoculation (pi) medium containing dmem (gibco, grand island, ny, usa) supplemented with tryptose phosphate broth (0.3%), yeast extract (0.02%), and 10 μg/ml of trypsin. after incubating at 37°c for 24 h, 100 μl of the bacterial intracellular extracts were added into each well and incubated at 37°c for another 24 h. after washing the cells twice with pi medium, 200 μl of pi medium containing 200 tcid 50 /ml of the pedv was added into each well and incubated at 37°c for 1 h. after 1 h of incubation, the supernatants were replaced by fresh pi medium and the cells were maintained at 37°c for 48 h. after washing twice with sterile pbs (0.1 m, ph 7.0), the cell metabolic activity was determined using the mtt assay as described previously. effects of lab intracellular extract, cell-wall fraction, and extracellular supernatant against pedv a 1.0-ml aliquot of the 16-h culture of each lab strain was centrifuged at 9,000g for 10 min at 4°c. the resultant extracellular supernatants and bacterial cells were harvested separately. the bacterial cells were washed twice with sterile pbs, resuspended in 1.0 ml of sterile pbs, and sonicated for 10 min with an ultrasonicator (model xl, misonix, farmingdale, ny, usa). the sonicated bacterial cells were fractioned into intracellular extracts and cell-wall pellet fractions by subsequent centrifugation at 13,000g for 20 min at 4°c. the extracellular supernatants, intracellular extracts, and cell-wall fractions of each bacterial strain were harvested, and the anti-pedv activities were evaluated as described above. vero cells were seeded on a 96-well plate and incubated at 37°c for 24 h. afterward, the cells were washed with pi medium, and 200 μl of pi medium containing 200 tcid 50 /ml of pedv were added into each well and incubated at 37°c for 1 h. after 1 h of incubation, the supernatants were replaced by 100 μl of fresh pi medium and 100 μl of the bacterial intracellular extracts. the cells were maintained at 37°c for 48 h. after washing twice with sterile pbs, the cell metabolic activity was determined by mtt assay as described previously. . glyceraldehyde 3-phosphate dehydrogenase (gapdh) was chosen as an internal control, and all relative gene expression levels were normalized to gapdh by the comparative c t method. the primers used for the relative quantification are provided in table 1 . the data were analyzed using spss version 25 software (ibm spss, new york, ny, usa). one-way analysis of variance (anova) followed by duncan's multiple range test was used to detect the differences among the means of the different treatment groups, and a value of p less than 0.05 was considered significant. student's t-test was used to detect the differences between the treatment and control groups, and a value of p less than 0.05 was considered significant. each experiment was conducted in triplicate, and all results were expressed as means ± standard deviations. twenty-nine lab strains were isolated from kefir grains. according to the 16s rrna and rpoa gene sequence analysis, three isolated strains belong to the species enterococcus durans, 16 isolated strains might belong to the species lactobacillus kefiri, five isolated strains might belong to the species lactococcus lactis, and five isolated strains might belong to the species leuconostoc mesenteroides ( table 2 ). the in vitro prophylactic effects of the lab strains on pedv were evaluated in the vero cell model. vero cells were pretreated with the intracellular extracts of lab for 24 h. after the removal of the bacterial intracellular extracts, the vero cells were infected with pedv. if the intracellular extracts of lab possessed an in vitro prophylactic effect against pedv, the bacterial pretreated vero cells would be infected with less pedv and thus would show higher cell metabolic activity than the un-pretreated cells. the in vitro prophylactic effects of the bacterial intracellular extracts of different lab species against pedv were compared, and human ifn-α2b was used as a positive control. vero cells pretreated with ifn-α2b prior to pedv infection showed a significantly higher cell metabolic activity and less cytopathic effects than the un-pretreated cells (figures 1, 2) , indicating that ifn-α2b possessed an in vitro prophylactic effect against pedv. among the cells pretreated with the intracellular extracts of different lab species, the cells pretreated with the intracellular extracts of ln. mesenteroides showed significantly higher cell metabolic activity than those pretreated with the intracellular extracts of the other lab species (figure 1 ). in addition, vero cells pretreated with the intracellular extracts of ln. mesenteroides also showed less cytopathic effects than the un-pretreated cells (figure 2) , indicating that ln. mesenteroides possessed an in vitro prophylactic effect against pedv. the in vitro prophylactic effects of the five strains of ln. mesenteroides isolated from kefir grains on pedv were further compared with each other. as shown in figure 3 , the metabolic activity of vero cells pretreated with the intracellular extracts of ln. mesenteroides, regardless of which strain, were similar to those pretreated with ifn-α2b (p > 0.05) but were significantly higher than the un-pretreated cells (p < 0.05), indicating that all the ln. mesenteroides strains isolated from kefir grains possessed in vitro prophylactic effects against pedv. in vitro prophylactic effects of ln. mesenteroides ypk30 intracellular extract, cell-wall fraction, and extracellular supernatant against pedv among the ln. mesenteroides strains, a strain designated ypk30 showed a higher growth rate than the other strains (data not shown) and was further evaluated for its basis of anti-pedv activity. we compared the in vitro prophylactic effects of intracellular extracts, cell-wall fractions, and extracellular supernatants of ypk30 against pedv. as shown in figure 4 , the metabolic activity of vero cells pretreated with the intracellular extracts and extracellular supernatants of ypk30 were similar to each other and were significantly higher than those of un-pretreated cells or those pretreated with the cell-wall fractions of ypk30 (p < 0.05), indicating that both of the intracellular extracts and extracellular supernatants of ypk30 possessed in vitro prophylactic effects against pedv. vero cells were infected with pedv for 1 h, and then the remaining pedv was removed. the pedv-infected cells were treated with the intracellular extracts of ypk30 or ifn-α2b for 48 h, and then the metabolic activities of vero cells were determined. as shown in figure 5 , pedv-infected vero cells treated with the intracellular extracts of ypk30 for 48 h showed higher cell metabolic activity than the untreated cells or those treated with ifn-α2b (p < 0.05), indicating that the intracellular extracts of the ypk30 strain possessed an in vitro therapeutic effect against pedv. vero cells were co-incubated with pedv and the intracellular extracts of ypk30 or ifn-α2b for 1 h. after removal of the pedv and intracellular extracts of ypk30, the vero cells were incubated for 48 h, and then the metabolic activities of the vero cells were determined. the metabolic activities of the vero cells treated with the intracellular extracts of ypk30 and pedv simultaneously were significantly higher than those treated with pedv alone or those treated with pedv and ifn-α2b (figure 6 ; p < 0.05). these results suggested that the intracellular extracts of ypk30 possessed an in vitro direct-inhibitory effect against pedv in vero cells. in order to elucidate the antiviral mechanisms of the ypk30 strain, the expression levels of type 1 ifn-dependent genes, including oas1, mx1, and isg15, were quantified in vero cells after 0, 24, or 48 h of treatment with the intracellular extracts of ypk30. as shown in figure 7 , ifn-α2b, which served as the positive control, significantly increased the expression levels of oas1, mx1, and isg15 genes in vero cells at 24 and 48 h. the intracellular extracts of ypk30 also significantly increased the expression levels of mx1 and isg15 genes but did not affect the expression levels of the oas1 gene in vero cells at 24 h. however, the expression levels of oas1, mx1, and isg15 genes in vero cells did not differ between the untreated and ypk30-treated groups at 48 h. according to the 16s rrna and rpoa gene sequence analysis, ypk30 exhibited 99.93 and 99.57%, respectively, identity with ln. mesenteroides (table 2) , and its 16s rrna and rpoa gene sequences were deposited in the ncbi genbank database under accession number mt293805 and mt333858, respectively. macroscopic observation showed that ypk30 exhibited a smooth and grayish white colony morphology and did not possess hemolytic capacity on blood agar plate. the cells of ypk30 appeared purple after gram staining, indicating the strain ypk30 was gram positive. microscopic observation showed the cells of ypk30 were observed as spherical or lenticular forms. analysis on the basis of phenotypic (including gramstain-positive, catalase-negative, nonmotile, and asporogenous) and physiological characteristics (including growth at 10 or 37°c but not growth at ph 4.8 or in 10% ethanol) indicated that ypk30 was closely related to species ln. mesenteroides. to further confirm the identification of ypk30 with the species ln. mesenteroides, the biochemical characteristics of ypk30 were compared with those of ln. mesenteroides subsp. cremoris atcc 19254, ln. mesenteroides subsp. dextranicum atcc 19255, and ln. mesenteroides subsp. mesenteroides atcc 8293 by using the api 50 ch system. analysis of carbon all data are expressed as mean ± sd (n = 3). bars marked with a star or double stars mean that they are significantly different from the control (cells treated with pbs) at the 5 or 1% confidence level, respectively. source utilization profiles indicated that both ypk30 and ln. mesenteroides subsp. dextranicum atcc 19255 grew on six out of 49 carbohydrates, including n-acetyl glucosamine, d-fructose, d-glucose, d-mannose, saccharose, and d-trehalose. distinct variation was observed between ypk30 and ln. mesenteroides subsp. cremoris atcc 19254 in the metabolism of the sugars d-fructose, d-mannose, and d-trehalose. additionally, distinct variation was observed between ypk30 and ln. mesenteroides subsp. cremoris atcc 19254 in the metabolism of the carbohydrates amygdaline, l-arabinose, cellobiose, esculine, d-galactose, β-gentiobiose, d-lactose, maltose, α-methyl-d-glucoside, d-raffinose, ribose, d-turanose, and d-xylose. therefore, the carbon source use characteristics of ypk30 were similar to those of ln. mesenteroides subsp. dextranicum. according to the results of microscopic observations, biochemical characteristics, and the 16s rrna and rpoa gene sequence analysis, the features of ypk30 were consistent with those of ln. mesenteroides subsp. dextranicum, as described in bergey's manual of systematic bacteriology (vos et al., 2009) . the vero cell line is one of the most commonly used cell lines for pedv isolation and propagation (koonpaew et al., 2019) . in this study, we used a vero cell culture model to evaluate the in vitro prophylactic effects of lab against pedv. four lab species, including e. durans, lb. kefiri, lc. lactis, and ln. mesenteroides , were isolated from kefir grains, and the in vitro prophylactic effects of the intracellular extracts of these four species against pedv infection in vero cells were compared. among these four lab species, the intracellular extracts of ln. mesenteroides showed a higher in vitro prophylactic effect against pedv than the other species did (figure 1) . in addition to the in vitro prophylactic effect, the intracellular extracts of ln. mesenteroides ypk30 also possessed in vitro therapeutic effect and in vitro direct-inhibitory effects against pedv in vero cells (figures 4-6) . vero cells have a major deletion in the type 1 ifn gene cluster, which results in ifn deficiency (desmyter et al., 1968; koonpaew et al., 2019) . although vero cells do not secrete type 1 ifns when infected by viruses, they still have the type 1 ifn receptors and respond normally to type 1 ifns. therefore, vero cells were widely used to compare virus-mediated ifn antagonism specific to the ifn signaling pathway (simmons et al., 2010) . in the in vitro prophylactic and therapeutic models, the intracellular extracts of ln. mesenteroides ypk30 did not directly interact with pedv by physical contact. therefore, the in vitro prophylactic and therapeutic effects of the intracellular extracts of ln. mesenteroides ypk30 against pedv in vero cells seem not be attributed to the direct interaction of bacterial components or metabolites with virus. since the ifn pathway is crucial in initiating viral resistance, we suggest that the in vitro prophylactic and therapeutic effects of the intracellular extracts of ln. mesenteroides ypk30 against pedv in vero cells could be attributed to its effect on the ifn signaling pathway in vero cells. stimulation of innate immune responses by probiotics could be one of the mechanisms responsible for the protection provided by probiotics against viral infection. other proposed mechanisms include inhibition of virus adsorption and penetration into cells as a result of direct interaction of probiotics and virus, competition between probiotics and virus for epithelial cell receptors, and secretion of metabolites with antiviral activity (mousavi et al., 2018; biliavska et al., 2019) . previous studies have shown that specific probiotic bacteria bind to and inactivate rotaviruses and vesicular stomatitis viruses, which lead to blocking of the virus adsorption on the cell (salminen et al., 2010; kanauchi et al., 2018) . besides the direct interaction between probiotics and viruses, specific probiotic bacteria could interact with epithelial and mucosal cells and compete with pathogens for attachment to cell receptors, thereby preventing invasion into the cells by a virus (fernandez-duarte et al., 2018; mousavi et al., 2018) . in addition, specific probiotic bacteria could synthesize some antiviral metabolites, such as lactic acid, hydrogen peroxide, or bacteriocins (al kassaa et al., 2014) . in the present study, the intracellular extracts of ln. mesenteroides ypk30 possessed an in vitro direct-inhibitory effect against pedv in vero cells (figure 6) . future studies will be aimed at identifying the mechanisms of the in vitro direct-inhibitory effect of the intracellular extracts of ln. mesenteroides ypk30 against pedv in vero cells. numerous mechanisms for the immunomodulatory properties of probiotics have been proposed. some extracellular polysaccharides produced by specific probiotic bacteria possess immunomodulatory activities, which induce an increase in the expression of ifn-α, ifn-β, and the antiviral factors mx1 and rnase l in porcine intestinal epithelial cells (kanmani et al., 2018) . beside extracellular polysaccharides, some cellular components, such as dna and bacterial cell-wall components, including peptidoglycan, s-layer proteins, teichoic acids, capsule, and pellicle, as well as other released peptides could modulate the innate antiviral immune response (quinteiro-filho et al., 2015) . in the present study, pretreatment of vero cells with the extracellular supernatants or intracellular extracts of ln. mesenteroides ypk30 for 24 h before infection with pedv showed higher cell metabolic activities than those of un-pretreated cells, indicating that both of the intracellular extracts and extracellular supernatants of ln. mesenteroides ypk30 possessed in vitro prophylactic effects against pedv in vero cells. however, pretreatment of pedv cells with the cell-wall fractions of ln. mesenteroides ypk30 for 24 h before infection with pedv did not impede pedv replication. according to these observations, we suggested that the immunomodulatory activity of ln. mesenteroides ypk30 seems not to rely on the structural cell components. future studies should be aimed at assessing the molecular mechanism(s) responsible for the observed effects. type 1 ifns exert their antiviral activities though the induction of hundreds of isgs (lenschow et al., 2005) . classical isgs responsible for inhibition of viral infection include oas1, mx1, and isg15 (schoggins, 2014) . oas1, which belongs to the oas enzyme family, is activated by double-stranded rna binding, catalyzes the formation of 2′-5′ oligoadenylates to activate cellular rnase l, which in turn, degrade cellular and viral rna (choi et al., 2015) . mx1 is a dynamin-like gtpase that appears to target viral nucleocapsids, resulting in the inhibition of viral rna polymerase activity, effectively blocking both transcription and replication of the virus (schoggins, 2014) . isg15 is a small, ubiquitin-like molecule that has numerous antiviral functions, including inhibition of virus release, isgylation of both viral and host proteins, and immunomodulatory cytokinelike properties in its unconjugated form (schoggins, 2014) . in the present study, we determined the effects of intracellular extracts of ln. mesenteroides ypk30 on the expression levels of oas1, mx1, and isg15 genes in vero cells and found that treatment of vero cells with the intracellular extracts of ln. mesenteroides ypk30 did not affect the expression levels of the oas1 gene but significantly increased the expression levels of mx1 and isg15 genes 24 h after treatment (figure 7) , indicating that the anti-pedv activity of the intracellular extracts of ln. mesenteroides ypk30 could be attributed to its up-regulatory effect on the expression of mx1 and isg15 genes in vero cells. according to the results of microscopic observations, biochemical characteristics, and the 16s rrna and rpoa gene sequence analysis, ypk30 was identified as ln. mesenteroides subsp. dextranicum. ln. mesenteroides are commonly associated with foods, such as fermented dairy products (e.g., cheese, yogurt, and kefir), fermented vegetables (e.g., sauerkraut and kimchi), and fermented meats (holland and liu, 2011) . the long history of safe consumption of ln. mesenteroides in traditional fermented foods has led to the conclusion that it is generally regarded as safe (gras; flórez et al., 2016) . several ln. mesenteroides strains are reported to have anti-listerial, antiviral, or immunomodulatory activities (seo et al., 2012; shao et al., 2020) . since the production of exopolysaccharides and bacteriocins are important properties of ln. mesenteroides, the probiotic characteristics of ln. mesenteroides could be attributed to their production of exopolysaccharides and bacteriocins. several studies demonstrated that some bacteriocins produced by ln. mesenteroides have anti-pathogenic activities (stiles, 1994; holland and liu, 2011; arakawa et al., 2016) , and some exopolysaccharides produced by ln. mesenteroides showed antiviral and immunomodulatory activities (nácher-vázquez et al., 2015; mahdi et al., 2019) . in our study, we found that the intracellular extracts of ln. mesenteroides ypk30 possessed in vitro prophylactic, therapeutic, and direct-inhibitory effects against pedv in vero cells, which occur in part through the up-regulation of mx1 and isg15 expression in vero cells. to the best of our knowledge, based on a thorough review of the relevant literature, this scientific report is the first of anti-pedv potential for ln. mesenteroides. future research will be conducted to evaluate the protection efficiency of ln. mesenteroides ypk30 against pedv infections in piglet infectious challenge models. the lab strain ypk30 isolated from kefir grains was genotypically and phenotypically characterized as belonging to ln. mesenteroides subsp. dextranicum. ln. mesenteroides subsp. dextranicum ypk30 displayed in vitro prophylactic, therapeutic, and direct-inhibitory effects against pedv in vero cells via up-regulation of mx1 and isg15 expression in vero cells. these findings suggest that ln. mesenteroides subsp. dextranicum ypk30 has a potential to acts as an antiviral agent for protection against pedv infections. the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found in the article/supplementary material. j-rl, w-pc-l, and h-wc contributed to conception and design of the study. w-pc-l, al, and y-hc carried out the experiments and did the data analysis. w-pc-l and j-rl wrote the first draft of the 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interferon-stimulated genes: roles in viral pathogenesis evaluation of leuconostoc mesenteroides yml003 as a probiotic against low-pathogenic avian influenza (h9n2) virus in chickens the probiotic, leuconostoc mesenteroides, inhibits listeria monocytogenes biofilm formation short communication: antiviral activity of porcine ifn-λ3 against porcine epidemic diarrhea virus in vitro a determinant of sindbis virus neurovirulence enables efficient disruption of jak/stat signaling bacteriocins produced by leuconostoc species intestinal innate antiviral immunity and immunobiotics: beneficial effects against rotavirus infection viability of probiotic (bifidobacterium, lactobacillus acidophilus and lactobacillus casei) and nonprobiotic microflora in argentinian fresco cheese bergey's manual of systematic bacteriology investigation of microorganisms involved in biosynthesis of the kefir grain emerging and re-emerging coronaviruses in pigs 16s ribosomal dna amplification for phylogenetic study recent systems biology approaches for probiotics use in health aspects: a review nasal priming with immunobiotic lactobacillus rhamnosus modulates inflammation-coagulation interactions and reduces influenza virus-associated pulmonary damage this research was conducted using funds partially provided by grants from the ministry of science and technology (grant nos. most 108-2313-b-002-012 and most 108-2321-b-002-064) and academia sinica (grant nos. as-kpq-108-itar-td03 and as-kpq-109-itar-td03). key: cord-330772-i7cfmw9x authors: peng, ju-yi; horng, yi-bing; wu, ching-ho; chang, chia-yu; chang, yen-chen; tsai, pei-shiue; jeng, chian-ren; cheng, yeong-hsiang; chang, hui-wen title: evaluation of antiviral activity of bacillus licheniformis-fermented products against porcine epidemic diarrhea virus date: 2019-12-03 journal: amb express doi: 10.1186/s13568-019-0916-0 sha: doc_id: 330772 cord_uid: i7cfmw9x bacillus licheniformis (b. licheniformis) is commonly used as probiotic and its secondary metabolites are attractive anti-microbial candidate. in the present study, we aimed to evaluate the antiviral activity of crude extracts from b. licheniformis against porcine epidemic diarrhea virus (pedv), a highly contagious enveloped porcine virus that has caused great economic loss in pigs. in vivo, pedv-infected piglets supplemented with air-dried solid state fermentative cultivate containing b. licheniformis-fermented products (blfp) showed milder clinical symptoms and decreased viral shedding. importantly, no significant systemic pathological lesions and no reduction in average daily gain were noted in pigs supplemented with the blfp, which suggests that it is safe for use in pigs. in vitro experiments revealed that while b. licheniformis crude extracts exhibited no toxicity in vero cells, co-cultivation of b. licheniformis crude extracts with pedv significantly reduced viral infection and replication. summarized current results suggest that the b. licheniformis-fermented products could be a novel candidate food additive for reducing the impact of ped on the swine industry. beginning in 2013, outbreaks of new variants of porcine epidemic diarrhea virus (pedv) have caused high mortality and morbidity in piglets, leading to great economic losses (lee 2015) . the virus causes porcine epidemic diarrhea (ped), which is characterized by watery diarrhea, vomiting, and severe dehydration in pigs of all ages, especially suckling piglets (lee 2015; song and park 2012) . this highly contagious disease has spread quickly in porcine industries in several countries (jung and saif 2015; lee 2015) . furthermore, pedv infection causes severe perturbations of gut microbiota, reducing probiotic bacterial abundance, enriching pathogenic bacteria, and even impairing the growth performance of pedv-surviving pigs (alvarez et al. 2015; deping song et al. 2017) . the development of effective protective agents against pedv infection is urgently needed. probiotics is one of the choices to be an alternative to antibiotic growth promoter, agp (abudabos et al. 2017) . our recent studies reported that lactobacillus species and clostridium butyricum-fermented probiotics product alleviate diarrhea incidence and reduce the gut pathogens in weaning piglets ). on the other hand, bacillus subtilis (b. subtilis) and b. licheniformisfermented products could increase growth performance and mitigate clostridium perfringens-induced necrotic enteritis in broilers (cheng et al. 2018; lin et al. 2019) . enhancement of nutrient digestibility and lactobacillus counts of feces by feeding b. subtilis and b. licheniformis has also been reported in pigs (lan and kim 2019) . it has been demonstrated that b. licheniformis exhibiting antimicrobial activity against pathogens might be due to the production of antibacterial biosurfactants (lin et al. 2019) . furthermore, our experiment results also demonstrated that b. licheniformis-fermented products exhibit antibacterial activities against clostridium perfringens and staphylococcus aureus in vitro (lin et al. 2019 ). in the present study, the antiviral effect of the blfp crude extract against pedv were evaluated in pigs. the surfactin-like peptide in the blfp crude extract was identified from the secondary metabolite of b. licheniformis fermentative cultivates. the in vitro toxicity and antiviral ability of the surfactin-like peptide in the blfp crude extract against pedv were evaluated using the vero cells. vero c1008 cells (american type culture collection (atcc) no. crl-1586) were maintained in dmem (gibco, ny, usa) supplemented with 10% fetal bovine serum (hyclone, utah, usa) and 1% penicillin-streptomycin-amphotericin b (gibco, ny, usa). the passage 6 pedv-pintung 52 strain (pedvpt-p6) viral stock was used at a titer of 1 × 10 5 tcid 50 /ml. air-dried solid-state fermentative cultivates of b. licheniformis (weigmann) chester (atcc ® 27811 ™ ) were suspended in distilled water and heated at 30 °c for 30 min. supernatants were harvested after centrifugation at 13,000 rpm for 30 min. after adjusting the ph value to 2.0 with 6 n hcl for protein precipitation, the precipitates were dried in a freeze dryer after washing twice with distilled water. this b. licheniformis-fermented products (blfp) was used in animal study. to characterized the blfp, the surfactin derived from bacillus subtilis (sigma-aldrich, st louis, usa) was used as standard substance. the content of blfp in the fermentative product was determined by high-performance liquid chromatography (hplc) as previously described (schneider and marahiel 1998) . briefly, after filtration, samples were subjected to analysis via spd-10a hplc (shimadzu, japan) with a pre-packed lichrospher 100 rp-18 column (merck, darmstadt, germany). the mobile phase was a mixture of 3.8 mm trifluoroacetic acid and 200 ml ddw with 800 ml 100% methanol. elution was performed at a flow rate of 1 ml/min and determined with a uv detector (10a vp, shimadzu, tokyo, japan) at 210 nm. the gradient strategy was as follows: 0-3.5 min, 60% a to 93% a; 3.5-20 min, keeping 93% a and 7% b (a, acetonitrile; b, ultrapure water); min pressure 0 bar, max pressure 400 bar, pressure stability 10 bar; injection volume 10 μl; syringe speed 8 μl/s; flush volume 800 μl. liquid chromatography-mass spectrometry (lc-ms) full scan positive mode was performed with m/z ranging from 200 to 2000. fifteen 4-week-old, pedv-fecal rna and pedv seronegative, large white × duroc crossbred pigs were acquired from a conventional pig farm with no known history of ped. treatments were: (1) control (n = 5); (2) pedv (n = 5); and (3) pedv + blfp (n = 5). these pigs were fed a commercial diet mixed with or without 5 kg/l blfp as feed additives for 7 days prior to the viral challenge, and were challenged with or without 5 × 10 5 tcid 50 of the virulent pedvpt-p6 (p6) at 5 weeks of age (table 1) . each group was housed in a separate fenced area. clinical symptoms, fecal consistency scoring, and fecal viral shedding were recorded and tested daily. the clinical scores were recorded using a 4-scale: 0, normal feces; 1, pasty; 2, semi-fluid; 3, fluid. all piglets were weighed weekly and sacrificed 19 days post-infection (dpi) for safety assessment by pathological examination. the animal experiment was approved by the institutional animal care and use committee of national taiwan university (taipei, taiwan, ntu105-el-00087). real-time quantitative rt-pcr (qpcr) was performed based on a previously described method with modification (chang et al. 2017) . the viral rna was extracted from 200 μl of culture supernatants using cador ® pathogen 96 qiacube ® (qiagen, chatsworth, ca, usa) according to the manufacturer's instructions. reverse transcription was performed using the quantinova reverse transcription kit (qiagen) according to the manufacturer's protocol. for qpcr assay, the quantinova probe master mix of fecal swabs were used to determine the genomic equivalents (ge). the detection limit of the real-time rt-pcr was 60 ge of dna using the plasmid encoding the pedv n gene as a standard (data not shown). all pigs were euthanized and necropsy was performed at 19 dpi to evaluate the safety of blfp in pigs. representative tissue samples were collected, fixed in 10% neutral-buffered formalin, processed routinely, sliced into 5-μm-thick sections, and stained with hematoxylin and eosin. the histopathological observations were recorded and assessed blindly by one veterinary pathologist. to evaluate the cytotoxicity of blfp crude extract in vitro, vero cells were first grown in a 96-well microplate (corning life sciences, corning, ny, usa) at a density of 20,000 cells per well 1 day prior to the experiment. after removing the culture supernatant, ten-fold serially diluted blfp crude extract in pi medium (dmem supplemented with 0.3% tryptose phosphate broth (sigma-aldrich, mo, usa), 0.02% yeast extract (acumedia, michigan, usa), and 10 μg/ml of trypsin (sigma-aldrich, mo, usa) was added to the cell monolayer. after 48 h, the culture supernatant was removed and 100 μl alamarblue ™ was added (g-biosciences, st. louis, usa; 10% in pbs). after 2 h of incubation at 37 °c, the plate was read with the excitation wavelength at 575 nm and with the emission wavelength at 590 nm. the od value of each well was calculated to obtain the percentage reduction of alamarblue according to the manufacturer's instructions. additionally, an untreated group g1, which consisted of vero cells without any treatment, was included to represent 100% reduction of alamarblue. all data were further calculated as a percentage of the untreated group g1. normalized data were plotted against concentrations of blfp crude extract and fitted to a non-linear regression curve using graph-pad prism (graphpad software, san diego, ca). the 50% cytotoxicity concentration (cc 50 , the concentration of blfpat which cellular viability was reduced to 50%) was calculated accordingly. to study the antiviral activity of blfp crude extract against pedv, the biosurfactants were added at different time points during the viral infection. vero cells were seeded in a 96-well microplate (20,000 cells/well) 1 day before the experiment. blfp crude extract at 150 ppmin pi medium were added to each well in different orders to treat 200 tcid 50 /ml pedv infections in cells. the experiment included five groups (as illustrated in fig. 4a to further investigate the antiviral mechanism in the post-drug group (g6), the replication kinetics of pedv in vero cells with or without crude extract of blfp were compared. vero cells were seeded in a 96-well microplate (20,000 cells/well) 1 day before the experiment. the crude extract of blfp at 150 ppm in pi medium was added to 200 tcid 50 /ml pedv-infected cells. culture supernatants and cells were collected separately at the 2, 8, 12, 24, and 48 h post-infection. viral titers in culture supernatants were determined using the reed-müench method (reed 1938) and are expressed as the 50% tcid 50 /ml. virus-specific rna in culture supernatants and in cells was quantified by real-time reverse transcription-pcr. virus titration was performed in the culture supernatants. briefly, the first 10-fold dilution and the subsequent 10-fold serial dilution of the supernatants were added to 20,000 cells/well vero cells on 96-well microplates. after 1 h of incubation, the inoculants were aspirated, and the cells were washed with pi medium two times and then cultured in pi medium. after 72 h, the plate was subjected to cytopathic effect (cpe) observation and the titer of pedv was determined using the reed-müench method (reed 1938) and is expressed as the 50% tcid 50 /ml. to determine the ic 50 , vero cells were seeded in 96-well microplates (20,000 cells/well) one day before the experiment. ten-fold serially diluted crude extract of blfp in pi medium was added to vero cells 1 h after 200 50% tissue culture infectious dose (tcid 50 ) pedv infection. after 48 h, the percent reduction of alamarbluestaining was examined by the alamarblue ™ assay as described above. all normalized data were plotted and fitted to a nonlinear regression curve in graphpad prism (graphpad software) to generate the ic 50 . to elucidate the direct virucidal activity of crude extract of blfp against pedv, the pedvpt-p6 viral stock at a titer of 1 × 10 5 tcid 50 /ml was mixed with or without 150 ppm blfp for 1 h at 4 °c. the mixtures were serially diluted 10-fold in pi medium and added to vero cells. after 72 h, the viral titers were determined as described above. all data were analyzed and plotted using graphpad prism (graphpad software). all error bars represent standard deviation (sd). the significance of the differences among groups in the cell-based study was determined using student's t test or one-way anova with tukey's multiple comparison. a p-value of < 0.05 was interpreted as statistically significant. the results of the average daily gain (adg) and fecal rna shedding in the animal experiments were analyzed and the variables among groups were compared using a non-parametrical kruskal-wallis test, with p < 0.05 considered significant. all data were analyzed using graphpad prism software (graphpad prism inc.) as shown in fig. 1 , hplc analysis of the b. licheniformis extract showed a single peak (fig. 1b) at the retention time of 16-17 min, which is identical to the standard substance surfactin derived from bacillus subtilis (fig. 1a) . (fig. 1) . to assess the efficacy of the antiviral ability of blfp against pedv, clinical symptoms after viral challenge were recorded daily (fig. 2) . the fecal conditions of pigs in all groups were scored as normal (score = 0) before pedv inoculation. in general, pigs supplemented with blfp exhibited milder symptom compared to piglets supplemented with controls. in the control group, all five pigs (5/5) exhibited normal clinical signs (score = 0) during the study. in the pedv group in which pigs were supplemented with control food, different severities of pedv-associated clinical signs were first detected 2 days post infection (dpi). two of these five pigs (2/5) exhibited semi-fluid feces (score = 2) at 2 dpi, while 3/5 pigs showed typical semi-fluid (score = 2) to watery diarrhea (score = 3) at 2 to 8 dpi, and eventually recovered after 8 dpi. in the pedv + blfp group, most of the pigs exhibited pasty feces (score = 1) at 2-5 dpi. only one pig exhibited transient typical watery diarrhea (score = 3) at 6 dpi. compared to the pedv-infected group with control food, the severity and duration of typical diarrhea (scores 2-3) were reduced in the pedv + blfp group. to quantify pedv-associated fecal viral shedding to evaluate the antiviral efficacy of blfp against pedvpt-p6, rt-qpcr was used to detect fecal viral shedding. in the pedv group (the grey line in fig. 3) , fecal viral shedding was first detected (3.92 ± 3.88 log 10 ge) at 2 dpi, found to gradually increase to peak viral load (5.71 ± 3.38 log 10 ge) at 5 dpi, and was continuously detected until 12 dpi. in the pedv + blfp group (the black line in fig. 3) , the pattern of viral shedding was similar to but lower than that inthe pedv group during the study, although the difference was not statistically significant. all pigs were weighed weekly in order to evaluate their growth performance with and without biosurfactants as a feed additive. no significant difference in the average daily gain (adg) of pigs was noted among all groups in each week (fig. 4) . a b fig. 2 clinical scoring of fecal consistency. compared to pedv-infected groups, the appearance and duration of typical diarrhea (score 2-3) were reduced in the pedv + blfp group to evaluate the safety of blfp as a feed additive for pigs, necropsy was performed in all piglets 3 weeks postinfection for gross and histopathological examination. for the gross and histopathological examinations, no macroscopic or microscopic lesions were noted in any of the groups. these findings suggest that blfp as a feed additives at 5 kg/ton are safe in conventional pigs up to 26 days of feeding. to determine the median cytotoxic concentration (cc 50 ) of blfp crude extract in vero cells, the blfp crude extract was serially diluted 10-fold from 150 to 0.015 ppm and added to cells. 48 h post-incubation (hpi), no cytotoxicity due to blfp crude extract was observed in vero cells (fig. 5) . to determine the maximal inhibitory concentration, the post-drug group g6 was used to evaluate the maximal inhibitory effect of blfp crude extract by cocultivating fig. 3 fecal shedding of pedvpt-p6 detected in piglets fed < 5 kg/ton blfp in each group (n = 5). the pattern of viral shedding in the pedv + blfp group was similar to but lower than that of the pedv group during the study, with no significant difference detected. changes in the mean values of genomic equivalents (ge)/ml are presented as log10 values ± sd. the viral rna loads of inoculated groups and the control group were compared using a non-parametrical kruskal-wallis test fig. 4 average daily gain of pigs in each group. all pigs were weighed weekly in order to evaluate their growth performance with or without blfp as feed additives. no statically significant difference in the average daily gain was noted among all groups each week blfp crude extract with pedv-infected cells during the whole study. the results indicated that the antiviral activity of blfp crude extract is dose-dependent, and the inhibition of pedv-induced cpe was calculated with an ic 50 value of 0.07 ± 0.45 ppm (fig. 6) . to elucidate the antiviral activity of blfp crude extract against pedv, 150 ppm of blfp crude extract was added to the culture at different time points during the viral infection. as shown in fig. 6a , typical pedv-induced cpe was characterized by formation of syncytial cells, cell death, and detachment from the culture plate in pedv-infected cells. the percent reductions of alamar-blue staining in g3-g5-cells treated with blfp crude extract 1 h before infection, at the same time as infection, and 1 h after viral infection, then replaced with fresh pi medium-were calculated as 50.7 ± 16.2%, 26.0 ± 20.1%, and 44.3 ± 5.8%, respectively, and showed no significant dose-dependent effect of blfp crude extract against pedv. the half-maximal inhibitory concentration (ic 50 ) of blfp crude extract against pedv in vero cells is 0.07 ± 0.45 ppm. data are presented as the mean ± sd out of three test replicates differences from those in the pedv-infected group g2 (fig. 7b) . for the post-drug group g6, significant inhibition of pedv-induced cpe, with an 81.5 ± 7.61% reduction of alamarblue staining, was observed compared to that in g2 and other groups (g3-g5). similarly, the amount of viral rna in supernatants from g6, ranging from 6.26 to 7.39 log 10 ge, was significantly lower than that of g2-g5, ranging from 8.41 to 9.26 log 10 ge (fig. 7c) . additionally, the viral titer of supernatants in the g6 group, which was less than 10 tcid 50 /ml, was lower than that of g2-g5, ranging from 1 × 10 4 to 1 × 10 5 tcid 50 /ml. moreover, when evaluating the direct virucidal activity of blfp crude extract against pedv, a reduction of the viral titer of pedv from 10 5 to 10 4 tcid 50 /ml was observed. to further investigate the antiviral mechanism in the post-drug group (g6), the replication kinetics of pedv in vero cells in the presence or absence of blfp crude extract were determined. compare to pedv-infected vero cells cultured without blfp crude extract, which showed viral titers from 1.78 × 10 5 at 24 hpi to 3.16 × 10 6 tcid 50 /ml at 48 hpi (fig. 8a) , cells treated with blfp crude extract had an undetectable extracellular viral titer, which was significantly lower than that of cells without blfp crude extract. similarly, extracellular viral rna levels in pedv-infected cells cultured with biosurfactants were significantly lower than those without blfp crude extract 24 and 48 hpi (fig. 8b) . however, no significant differences in intracellular viral titers (fig. 8c ) or intracellular viral rna (fig. 5d) were noted in pedv-infected cells treated with or without blfp crude extract. since late 2013, new variants of pedv have been identified in several counties and have caused a devastating disease and economic loss. this viral threat has prompted a global effort to develop antivirals against new variant pedvs in vitro (song and choi 2011; kwon et al. 2013) and in vivo (cho et al. 2012; kim et al. 2015) . herein, we demonstrated that: (1) blfp crude extract presents no cell toxicity to vero cells, and exhibits significant antiviral ability against pedvpt-p6 in vero cells when biosurfactants are co-cultivated with pedv; (2) piglets supplemented with blfp at concentrations of 5 kg/ton or less exhibited milder clinical symptoms and lower viral shedding compared to piglets supplemented with control food, and blfp caused no significant toxicity. therefore, blfp is suggested to be a promising novel feed additive that can act as an additive against pedv in the field. the main antiviral mechanism of blfp crude extract is thought to be related to its unique biochemical structure that increases membrane permeability to disrupt and lyse microbial membranes (huang et al. 2006 performing a direct-virucidal study of blfp crude extract against pedv, a ten-fold reduction of the viral titer was able to confirm the directed-antiviral ability of blfp crude extract. according to our hplc analysis, a peak of surfactin-like peptide was identified in the blfp crude extract. several different potential antiviral mechanisms of biosurfactants have been previously reported (vollenbroich et al. 1997; wang et al. 2017) . lipopeptide biosurfactants have been previously suggested to possess probiotic attributes for animal and human use (schneider 1998; sen 2010) . a previous study demonstrated that the antiviral ability of surfactin from b. subtilis was achieved through competition with the tgev entry receptor . the inhibition of viral enzymes such as proton-atpase that are required for the entry of some viruses into cells by surfactin from b. subtilis (vollenbroich et al. 1997 ) has also been reported. it is worth noting that not only did co-cultivation of the surfactin-like peptide in the blfp crude extract with pedv-infected vero cells significantly reduce viral infection and replication in the study, but blfp crude extract-treated pedvinfected cells also exhibited an undetectable extracellular viral titer, suggesting that blfp crude extract may play an important role in reducing the release of virions. these results suggest additional mechanisms of blfp crude extract against pedv in vero cells. although further study of the antiviral mechanisms of blfp crude extract is needed in the future, observation of the diverse antiviral activities of blfp crude extract will lead to further development of this new therapeutic candidate. previous studies demonstrated that lipopeptides produced by various bacillus spp. were safe in mice (youn-hwan hwang et al. 2008 ) and weaned piglets (torres et al. 2017) when administered under a certain concentration. in order to use blfp as a feed additive for its antiviral properties, assessment of its antiviral and safety profile in pigs is essential. our results demonstrated that piglets supplemented with blfp show milder symptoms fig. 8 replication kinetics of pedv in vero cells treated with or without blfp crude extract. a extracellular viral titers in the supernatants of pedv-infected vero cells treated with and without blfp crude extract were determined by viral titration in vero cells using the reed-müench method and expressed as the 50% tcid 50 /ml. b extracellular viral rna levels in the supernatants of pedv-infected vero cells treated with or without blfp crude extract were determined by real-time reverse transcription (rt)-pcr. c intracellular viral titers in pedv-infected vero cells treated with or without blfp crude extract were determined by viral titration in vero cells. d intracellular viral rna levels in the supernatants of pedv-infected vero cells treated with or without blfp crude extract were determined by real-time rt-pcr. statistical analysis was performed using student's t-test, and statistically significant differences were labeled with *(p < 0.05) and have reduced viral shedding. importantly, no significant systemic pathological effects and no changes in average daily gain were noted. these results suggest that the biosurfactants at concentrations of 5 kg/ton or less will be very safe in pigs in future applications. in a previous study, it was found that administration of surfactin from b. subtilis at over 500 kg/ton leads to necrosis of hepatocytes in rats (hwang et al. 2009 ). in the future, animal experiments using higher dosages of biosurfactants would be necessary to validate its maximal safety dosage. in the present study, blfp was investigated as an antiviral candidate against pedv. in vitro, blfp crude extract exhibited antiviral activities against pedv when incubated long-term with pedv-infected cells. in vivo, we first validated the safety and antiviral ability against of blfp as a feed additive less than 5 kg/ton against pedv after a long period of pedv inoculation. our results suggest that blfp could be a novel candidate feed additive for reducing the titer of pedv in the swine industry. effect of organic 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epithelial cells evaluation of genetic and developmental toxicity of surfactin c from bacillus subtilis bc1212 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions j-yp, y-bh, and c-yc have drafted, revised the work, and conducted the experiments; y-bh and y-hc provide specimens and substantial contributions to the conception and analysis; h-wc, y-cc, p-st, and c-rj help to interpret data; y-hc and h-wc. design the work and approve the submitted version. all authors read and approved the final manuscript. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. the animal experiment was approved by the institutional animal care and use committee (iacuc) of national taiwan university (taipei, taiwan, ntu105-el-00087). not applicable. the authors declare that they have no competing interests. key: cord-329227-sqetz7h6 authors: hou, yixuan; meulia, tea; gao, xiang; saif, linda j.; wang, qiuhong title: deletion of both the tyrosine-based endocytosis signal and the endoplasmic reticulum retrieval signal in the cytoplasmic tail of spike protein attenuates porcine epidemic diarrhea virus in pigs date: 2018-11-07 journal: journal of virology doi: 10.1128/jvi.01758-18 sha: doc_id: 329227 cord_uid: sqetz7h6 porcine epidemic diarrhea virus (pedv) causes high mortality in neonatal piglets. the pedv spike (s) protein contains two intracellular sorting motifs, yxxφ (tyrosine-based motif yevf or yeaf) and kvhvq at the cytoplasmic tail, yet their functions have not been fully elucidated. some vero cell-adapted and/or attenuated pedv variants contain ablations in these two motifs. we hypothesized that these motifs contribute to viral pathogenicity. by transiently expressing pedv s proteins with mutations in the motifs, we confirmed that the motif kvhvq is involved in retention of the s proteins in the endoplasmic reticulum (er)-golgi intermediate compartment (ergic). in addition, we showed that the yxxφ motif triggers endocytosis of s proteins. these two motifs synergistically regulate the level of s expressed on the cell surface. to investigate their role in viral pathogenicity, we generated three recombinant pedvs by introducing deletions or a mutation in the two motifs of the infectious clone of pedv pc22a strain (icpc22a): (i) icδ10aa (δyxxφekvhvq), (ii) icδ5aa (δkvhvq), and (iii) icya (y1378a, to an inactivated motif, aevf). infection of vero cells with icδ10aa resulted in larger syncytia and more virions, with reduced numbers of s protein projections on the surface compared with icpc22a. furthermore, we orally inoculated five groups of 5-day-old gnotobiotic piglets with the three mutants, icpc22a, or a mock treatment. mutant icδ10aa caused less severe diarrhea rate and significantly milder intestinal lesions than icpc22a, icδ5aa, and icya. these data suggest that the deletion of both motifs can reduce the virulence of pedv in piglets. importance many coronaviruses (covs) possess conserved motifs yxxφ and/or kxhxx/kkxx in the cytoplasmic tail of the s protein. the kxhxx/kkxx motif has been identified as the er retrieval signal, but the function of the yxxφ motif in the intracellular sorting of cov s proteins remains controversial. in this study, we showed that the yxxφ of pedv s protein is an endocytosis signal. furthermore, using reverse genetics technology, we evaluated its role in pedv pathogenicity in neonatal piglets. our results explain one attenuation mechanism of vero cell-adapted pedv variants lacking functional yxxφ and kvhvq motifs. knowledge from this study may aid in the design of efficacious live attenuated vaccines against pedv, as well as other covs bearing the same motif in their s protein. taxonomically, pedv is a member of the alphacoronavirus genus within the coronaviridae family. the mature pedv virion consists of four structural proteins: spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. as the major glycoprotein on the pedv envelope, s proteins form trimers, which appear as projections on the surface of a virion using an electron microscope, and bind to cellular receptors and mediate virus-host membrane fusion. proteolytic cleavage of s proteins expressed on the cell surface triggers syncytium formation (5, 6) . like those of other coronaviruses (covs), pedv virions assemble at the endoplasmic reticulum (er)-golgi intermediate compartments (ergic) (7) (8) (9) . the amounts of pedv s proteins in the ergic, in other organelles, or on the cell surface are likely regulated by two nearby motifs in its cytoplasmic tail (ct): a tyrosine-based motif, yxx⌽ (x is any residue and ⌽ is a bulky hydrophobic residue: f, m, i, l, or v), and an er retrieval signal (errs), kvhvq (10) (11) (12) (13) , as well as other viral and cellular proteins. the cov errs, either in the dilysine or the dibasic form (kxkxx, kkxx, or kxhxx), is a weak ergic retention signal (14, 15) . it interacts with coatomer complex i (copi), a cellular protein involved in cargo transportation from the golgi to er, and prevents large amounts of the s proteins from being transported to the cell surface through the canonical secretory pathway (16, 17) . in addition, the errs in the s protein of severe acute respiratory syndrome cov (sars-cov) promotes the interaction between s and m proteins in the golgi region (16) . inactivation of the errs in the sars-cov s protein impaired its incorporation into virus-like particles when coexpressed with the m in the cells (15) . for pedv, the amino acid sequence of the errs is kvhvq, which is highly conserved among different genotypes. one study demonstrated that a single amino acid substitution in this motif (kvhvq to kvrvq) weakens the intracellular retention function of the s proteins of the 10th passage of a murine-adapted pedv variant, mk-p10 (18) , resulting in enhanced syncytium formation in vero cells. however, this impaired kvrvq motif does not alter the incorporation of s into the mk-p10 virions (6) . although the yxx⌽ motif is a well-studied, clathrin-dependent endocytosis signal among numerous viral and host cellular transmembrane proteins (19) (20) (21) (22) (23) (24) (25) , its function in cov s proteins has not been fully understood. most s proteins of alphacoronaviruses, such as transmissible gastroenteritis virus (tgev), and gammacoronaviruses, such as infectious bronchitis virus (ibv), contain this motif in their cts (fig. 1a) . a previous study demonstrated that the yxx⌽ motif is responsible for intracellular retention but not endocytosis of tgev s proteins into cells (13) . interestingly, this retention signal could be transformed into a strong endocytosis signal when the first lysine residue of the tgev kxhxx motif was replaced by methionine (yepiekvhvh to yepiemvhvh; change is in bold type and yxx⌽ and kxhxx motifs are underlined) (26) . for ibv, the role of the yxx⌽ motif in s protein intracellular transport is controversial. one group reported that this motif is not an endocytosis signal (11) . the other group demonstrated that the yxx⌽ motif functioned as an endocytosis signal in a chimeric protein composed of the vesicular stomatitis virus (vsv) g protein ectodomain and the ct of the ibv s protein (14) . it has also been demonstrated that a recombinant ibv harboring a mutated yxx⌽ (yttf to ytta) in the ct of the s protein could not be recovered in cell culture, suggesting the essential role of this motif in the ibv life cycle. moreover, since the 3=end of the ibv s gene overlaps with the transcription regulatory sequence (trs) of accessory gene 3, mutagenesis of the ibv errs (kksv to aasv) impaired the synthesis of subgenomic rna 3 (sgrna 3), resulting in decreased expression of e proteins in the ibv-infected cells (12) . previously, we generated an attenuated pedv strain by serially passaging a highly virulent strain, pc22a, in vero cells. at the 120th (p120) and later passages, we identified a premature stop codon in the 3=end of the s gene (27) . this stop codon truncates the s protein by 9 amino acids (aa) (evfekvhvq) . interestingly, at least five pedv variants (fl2013, sm-98, chm2013, avct12, and knu-141112) containing a premature terminated s gene have been reported (table 1) (28) (29) (30) (31) . all these pedv variants are either cell culture attenuated or clinically mild in pigs, except for avct12, whose pathogenicity is unknown. these variants share the same s truncation pattern as pc22a-p120: partially ablated yxx⌽ and completely deleted kvhvq. it is possible that the loss of kvhvq may enhance the surface s protein level and increase its ability to induce syncytia in cell culture. however, the function of the incomplete yxx⌽ motif in viral replication and pathogenicity is unknown. notably, two types of yxx⌽ motif exist in the cts of s proteins among pedv genotypes: classical strains (g1a) and some highly virulent asian strains (g2a) contain a yeaf motif, whereas highly virulent north american strains (g2b), s insertion and deletion (indel) strains (g1b), and some g2a strains harbor a yevf motif. we hypothesized that the yxx⌽ motif of pedv, regardless of its amino acid sequence, is an endocytosis signal and determines viral virulence in pigs. in this study, we evaluated the phenotypes of transiently expressed s mutants containing mutations or deletions in these two motifs in mammalian cells and the virulence of three representative recombinant pedvs in gnotobiotic piglets. motifs yxx⌽ and kvhvq regulate the levels of pedv s proteins on the cell surface. to determine the roles of the two motifs in the intracellular sorting of s proteins, we constructed 12 plasmids bearing a wild-type s gene (of pc22a) or an s gene with different mutations in the cts ( fig. 1b and c) . also, the dispensable 197-aa region (residues 34 to 230) in domain 0 of each s protein was replaced by fluorescent protein mcherry (236 aa), as we demonstrated previously that this 197-aa region does not interfere with the formation of s protein trimers (32) . wt1 and wt2 contain the yevf and yeaf motifs, respectively. using these two wild-type constructs as templates, we generated additional 10 mcherry-tagged s mutants, including mutants bearing point mutations and the ct-truncated variants ( fig. 1 ; table 1 ). the δ10aa mutant lacks both motifs. the δ9aa and hd mutants contain the cts of pedv strains pc22a-p120 and knu-141112, respectively. the δ7aa mutant bears the same ct as four s-truncated pedv variants, fl2013, sm-98, avct12, and chm2013. mk-p10 contains the identical ct to that of the pedv strain mk-p10. to evaluate the difference in functions between yevf and yeaf, three pairs of mutants (ya and ya-a, δ5aa and δ5aa-a, and mk-p10 and mk-p10-v) were generated. to confirm the role of the kvhvq motif in ergic retention, transiently transfected vero cells were fixed with methanol at 12 h posttransfection (hpt) and stained with an antibody against ergic marker ergic-53 ( fig. 2a) . we observed distinct sorting patterns among the 12 recombinant s proteins. quantification of the colocalization between the s protein and ergic signals was indicated as pearson's correlation coefficient (pcc) value (33, 34) , by measuring 20 to 25 individual cells from each sample (fig. 2b) . the higher pcc values indicate the higher levels of colocalization between the two fluorescent signals. for the four s proteins (wt1, wt2, ya, and ya-a) with an intact kvhvq motif in the ct, most intracellular s proteins were concentrated at the ergic region, with significantly higher pcc values than for the other s mutants (p ͻ 0.05). interestingly, only the six s proteins with an intact yxx⌽ motif in their cts (wt1, wt2, δ5aa, δ5aa-a, mk-p10, and mk-p10-v) formed clear puncta in the cells. to determine the steady-state levels of these s proteins, we quantified the fluorescent intensities of the mcherry signals in 50 individual vero cells of each sample at 12 hpt (fig. 3b) . no statistically significant difference (p ͼ 0.05) was observed among samples, indicating that all the recombinant s proteins were expressed at similar levels. next, we measured the levels of s proteins on the cell surface using digital quantitative method and syncytium induction assay. the transfected cells were fixed with 4% formaldehyde without permeabilization, and the surface s proteins were stained with green fluorescence (fig. 3a) . total (both surface and intracellular) s proteins were observed for the red fluorescence. since each s protein produces equal intensity of red fluorescent signal, we quantified the levels of surface s proteins by measuring the ratios of gray values of the green fluorescent signal to mcherry signal and designated it the surface/total ratio. fifty individual cells in each sample were measured and are plotted in fig. 3c . wt1 did not differ statistically from wt2. eight mutants (δ10aa, ya, ya-a, δ9aa, δ7aa, hd, mk-p10, and mk-p10-v) expressed higher levels of s proteins than the two wild-type s proteins on the cell surface, while δ5aa and δ5aa-a expressed significantly lower levels (p ͻ 0.05). because proteolytic cleavage of surface s proteins triggers cell-cell fusion, a syncytium induction assay was performed by culturing the transfected cells in medium containing trypsin. the syncytia induced by the eight mutants (δ10aa, ya, ya-a, δ9aa, δ7aa, hd, mk-p10, and mk-p10-v) contained significantly more nuclei than those induced by the two wild types (wt1 and wt2), while almost no syncytia were induced by the δ5aa and δ5aa-a mutants ( fig. 3d and e). collectively, these data suggest that an intact kvhvq motif (wt1, wt2, ya, and ya-a) retains s proteins in the ergic, and the two motifs synergistically regulate the level of pedv s protein expressed on the cell surface via different mechanisms. the yxx⌽ motif is an endocytosis signal and regulates surface s protein internalization to early endosomes. to determine whether the yxx⌽ motif functions as an endocytosis signal in pedv s protein, we performed an antibody-antigen (gp17-s protein) uptake assay. we evaluated the portions of the gp17-bound surface s proteins (green) being endocytosed after incubation at 37°c. as controls, one set of cells were incubated at 4°c, whereby the cell endocytosis activities ceased. therefore, both the gp17-bound s proteins (green) and the surface s proteins (red), which were labeled after incubation and fixation without permeabilization, were colocalized on the cell surface ( fig. 4) . on the other hand, for the cells being incubated at 37°c, a large portion of the gp17-bound s proteins (green) of wt1, wt2, mk-p10, mk-p10-v, δ5aa and δ5aa-a formed puncta in the cells and were not colocalized with the surface s proteins (red). this was further confirmed when we compared the pcc values between the gp17-bound s protein signal and total surface s protein signal of each s protein at 4°c and 37°c (fig. 5a ). we found that the pcc values of the six s proteins (wt1, wt2, mk-p10, mk-p10-v, δ5aa, and δ5aa-a) at 37°c were significantly lower than those at 4°c, suggesting that partial gp17-bound s proteins of those six samples were internalized from the cell surface after incubation at 37°c. additionally, we probed the total gp17 signals on the cell surface without permeabilization in the gp17-s uptake assay (fig. 5b ). we measured the ratios of surface s (gp17) signal to total s (mcherry) signal in the transfected cells incubated at 4°c and 37°c. wt1, wt2, and the mk-p10, mk-p10-v, δ5aa, and δ5aa-a mutants had significantly lower ratios at 37°c than at 4°c. in contrast, the other six mutants (δ10aa, ya, ya-a, δ9aa, δ7aa, and hd) had similar pcc values (fig. 5a ) and surface/total ratios (fig. 5b ) at 4°c and 37°c. these results indicate that the s mutants without an intact yxx⌽ motif were defective in endocytosis. endocytotic vesicles deliver cell surface proteins to early endosomes in the cytoplasm (35) . to determine whether the internalized s puncta of wt1, wt2, mk-p10, mk-p10-v, δ5aa, and δ5aa-a ( fig. 2a and fig. 4 ) were located in early endosomes, we performed the antibody-antigen (gp17-s protein) uptake assay (fig. 6 ). after performing fixation and permeabilization of cells, we immunostained the gp17-bound s proteins (green) and the early endosome marker rab5 proteins (red). the gp17-bound s protein signals in the cells incubated at 37°c showed a significantly higher percentage 6 ). collectively, these data suggest that an intact yxx⌽ motif in the ct is necessary for endocytosis of the s protein from the cell surface to early endosome. pig anti-s1 antiserum gp17 at 4°c for 10 min. after washing with pbs three times, one set of cells was cultured at 37°c for 30 min to allow endocytosis. another set of cells was kept at 4°c for 30 min as the control group. cells were fixed with 4% formaldehyde without permeabilization. surface s proteins were stained with mouse anti-s2 monoclonal antibody sd129-5 and goat anti-mouse af647-conjugated secondary antibodies (red). then the cells were permeabilized with triton x-100 and stained with goat anti-guinea pig af488-conjugated secondary antibodies (green). nuclei were stained with dapi (blue). cells were randomly selected, and images were taken by using a leica tcs sp6 confocal microscope. scale bar: 10 m. virulence of the recombinant pedv lacking both the yxx⌽ and kvhvq motifs was reduced in neonatal gnotobiotic pigs. to investigate the function of the two motifs in pedv replication and pathogenesis, we generated three recombinant pedvs by introducing the δ10aa, δ5aa, and ya mutations into the ct of the pedv infectious cdna clone of strain pc22a (icpc22a) (32) . we inoculated 5-day-old gnotobiotic piglets with the three mutant pedvs, icpc22a, and phosphate-buffered saline (pbs; mock treatment) to determine the role of the two motifs in viral pathogenicity. clinical signs fig. 4 . to stain the gp17 bound on the cell surface, cells were fixed with 4% formaldehyde without permeabilization. gp17 was probed using af488-conjugated secondary antibodies (green). the fluorescent intensities of surface (af488) to total s (mcherry) ratios were measured in 20 to 25 individual cells. values are plotted in a bar chart, and shown as means ϯ sds. values of the same sample cultured at 4°c and 37°c were analyzed by student's t test. ns, p ͼ 0.05; **, p ͻ 0.01; ***, p ͻ 0.001; ****, p ͻ 0.0001. deletion of yxx and kvhvq from s attenuates pedv journal of virology internalized s proteins colocalized with early endosomes. antibody-s protein uptake assays were performed at 24 hpt for the two wild-type s proteins (wt1 and wt2) and the four mutants, δ5aa, δ5aa-a, mk-p10, and mk-p10-v, that formed puncta as shown in fig. 4 . cells were incubated with guinea pig anti-s1 antiserum gp17 at 4°c for 10 min. after washing with pbs three times, one set of cells were cultured at 37°c for 30 min to allow endocytosis and another set of cells were incubated at 4°c for 30 min. after fixation with methanol, early endosome marker rab5 proteins were stained with rabbit anti-rab5 mab (c8b1) followed by af647-conjugated goat anti-rabbit antibody (red). gp17-bound s proteins were stained with goat anti-guinea pig af488-conjugated secondary antibodies (green 7a ). the villous atrophy in the duodenum, jejunum, and ileum was quantified by measuring villus height to crypt depth (vh/cd) ratios of each piglet (fig. 7d to f). a lower vh/cd value indicates more severe atrophy in the intestines. compared with the virulent icpc22a group, the lesions of icδ10aa-infected pigs were the least severe in all three small intestinal regions (duodenum, jejunum, and ileum). although there were no statistically significant differences in duodenum and ileum, the lesions in the jejunum of icya-infected piglets were significantly milder than those of the icpc22a-and icδ5aa-infected pigs. we did not observe significant differences in clinical signs, fecal viral shedding titers, or vh/cd ratios between the icδ5aa and icpc22a groups. we concluded that the virulence of icδ10aa was milder than that of icpc22a, and the icδ5aa maintained virulence similar to that of icpc22a. these data suggest that deletion of both the yxx⌽ and kvhvq motifs from the s protein significantly impaired the pathogenicity of the highly virulent pedv icpc22a. the yxx⌽ and kvhvq motifs are dispensable for pedv replication, but the lack of either impairs viral replication efficiency in vitro. we measured the one-step (with a multiplicity of infection [moi] of 5) growth kinetics of the viruses in vero cells (fig. 8a) . the three pedv mutants had significantly lower peak titers than icpc22a at 12 hpi. subcellular structures of the four recombinant pedv-infected vero cells at 12 hpi were visualized by transmission electronic microscopy (tem) of cellular sections. like other covs, replication of pedv induces dramatic membrane rearrangements (9, 36). we did not observe differences in the rearranged membrane structures among cells infected with the four recombinant pedvs. however, in the icpc22a-infected cells, golgi vacuoles contained more mature virions, which are smaller particles with denser cores than immature virions (9), than those in the icδ10aa-and icya-infected cells (fig. 8f and g) . the icδ5aa-infected cells contained intermediate amounts of mature virions in the vacuoles. trans-golgi vacuoles containing large numbers of mature virions were observed exclusively in icpc22a-infected cells. this single-membrane structure contains viral particles prior to their egress. collectively, these data suggest that neither the yxx⌽ nor kvhvq motif is essential for pedv replication, but the absence of either motif impairs the generation of infectious pedv viral particles. recombinant pedvs lacking an intact yxx⌽ motif in the ct of the s protein formed larger syncytia than the wild-type virus. we measured multistep growth kinetics (moi ϭ 0.001) of the four recombinant pedv in vero cells (fig. 8b) . we found that the speed of viral replication varied among the four recombinant viruses and the infectious titers gradually decreased after the majority of cells were lysed due to the formation of syncytia. in the growth curve, the peak titers of icδ10aa and icya occurred earlier but were lower than those of icpc22a and icδ5aa. replication of icδ5aa in vero cells showed a peak titer similar to that of icpc22a, but with a delay. we performed plaque assays and measured the kinetics of the plaque formation of the four viruses ( fig. 8c and d) . at 40 hpi, the icδ10aa virus had induced the largest plaques, followed by the icya and then by the parental icpc22a, whereas the icδ5aa had formed the smallest plaques. we stained the surface s proteins of infected cells at an early time point (8 hpi) (fig. 8e) . the fluorescent signals represented s proteins anchoring either on cell membrane or on the egressing virions on the surface. the s proteins of icpc22a-infected cells clustered mainly in a small area on the cell surface, presumably virions clustering at the egress sites, in contrast to the evenly distributed surface s proteins of icδ10aa and icya on the entire cytoplasmic membrane, presumably s proteins anchoring directly on the cell membrane. the surface s proteins of icδ5aainfected cells were distributed in a pattern similar to that of icpc22a but showed less intense staining. these data suggest that the yxx⌽ motif decreases the amount of s proteins on the cell surface in pedv-infected cells. to determine whether the attenuation of icδ10aa in vivo was due to the amount of s proteins incorporated into virions, we visualized the s proteins on the purified virion surface using tem. the culture and purification of the four recombinant pedvs were performed simultaneously to minimize the variations due to sample preparation. we observed virions with or without s protein projections on virions in all four samples (fig. 9a) . the virions without s projections could be those that lack enough s proteins on the surface or whose s projections were lost during sample preparation for tem. the icδ10aa pedv contained the lowest percentage of virions with s protein projections, followed by icya and then by icpc22a and icδ5aa. next, we quantified the number of s projections on the individual virions of each pedv sample. among the four mutants, the icδ10aa pedv contained significantly fewer s projections than icpc22a, icδ5aa, and icya (p ͻ 0.05). these data suggest that the deletion of both the yxx⌽ and kvhvq motifs from the s protein impairs the incorporation of s proteins into pedv virions. previous studies showed that the m protein retained the s protein of sars-cov in the golgi regions when they were coexpressed in the same cell (15, 16) . to characterize whether the s proteins of the four recombinant pedvs can still interact with m, we cloned the pedv m gene into the vector puc19-sgrna. the s and m proteins were coexpressed in vero cells (fig. 10a ) or the s protein alone was expressed. when the s and m proteins were coexpressed in cells, we observed that they colocalized, with equal pcc values ( fig. 10b and c) . we stained the surface s proteins of the cells cotransfected with the s and m plasmids or transfected with the s plasmid alone and measured the ratios of fluorescent intensities of surface s to total s. we found that the expression of m significantly decreased the surface s protein levels in the same cell (fig. 10c) , as evident by the significantly lower surface s to total s ratios in the m and s plasmidcotransfected cells compared with those in the cells transfected with only s plasmid. in this study, we determined that the yxx⌽ and kvhvq motifs at the ct of pedv s proteins are dispensable for pedv replication. however, the deletion of both motifs reduced the viral virulence in vivo and the incorporation of s proteins into virus journal of virology particles in vitro. the yxx⌽ motif and the kvhvq motif function as an endocytosis signal and an er retrieval signal, respectively. yxx⌽ and kvhvq together regulate surface s protein levels and s proteininduced syncytium formation. the number of nuclei in a pedv s protein-induced syncytium, or the size of a syncytium, correlates with the amount of s proteins on the cell surface (6) . collectively, our data support the hypothesis that both motifs are involved in the regulation of pedv s proteins on the cell surface: the kvhvq motif retains the s proteins in the ergic, limiting its transport to the cell surface, whereas the yxx⌽ motif decreases the amount of surface s proteins via endocytosis. the mutants δ10aa, δ9aa, δ7aa, and hd, which lack the complete kvhvq motif and complete/partial yxx⌽ motif, had the highest level of surface s proteins ( fig. 3a and c) , probably due to the lack of both er retrieval and endocytosis. the mutants ya, ya-a, mk-p10, and mk-p10-v had the second highest level of cell surface s proteins. for the ya and ya-a, this is probably due to the inactivation of the endocytosis signal, while their errs remained intact. for mk-p10 and mk-p10-v, it may be due to the impaired errs (kvrvq; r is the mutation that leads to the impaired errs) while their yxx⌽ motif was intact. surprisingly, the mutants δ5aa and δ5aa-a had significantly lower levels of surface s proteins than wt1 and wt2. the lack of the entire kvhvq motif resulted in significantly lower levels of ergic colocalization of δ5aa and δ5aa-a than of wt1 and wt2 (fig. 2) , confirming that the kvhvq motif functions as an errs in pedv as described previously (6, 16) . why, then, did we not observe an increased level of surface s proteins for these two mutants? we hypothesized that the loss of the entire kvhvq motif in the ct of the s protein altered the intracellular sorting of s proteins. one possibility could be that the potency of their yxx⌽ motifs, as the endocytosis signal, was dramatically enhanced due to the lack of the entire errs motif. a similar endocytosis enhancement effect was reported for the yxx⌽ motif of tgev when its kxhxx motif's conformation was changed due to the k-to-m mutation (26) . another possibility could be that the deletion of the entire kvhvq motif may cause other deficiencies in s protein sorting, such as impaired export from the er or altered transportation to other organelles. detailed mechanistic studies regarding the sorting of δ5aa and δ5aa-a s proteins are necessary to answer this question. the endocytosis role of the yxx⌽ motif in pedv s intracellular sorting. the conserved location of the yxx⌽ and kxhxx/kkxx motifs in the ct implies that the s protein sorting mechanisms may be similar among different covs (fig. 1a) . however, previous studies on the yxx⌽ motif of tgev and ibv s proteins did not provide consistent conclusions (11, 13, 14, 26) . one reason could be that the expression efficiencies of vectors varied in those studies. due to the small amount of s proteins expressed on the cell surface, the endocytosed s proteins might be too subtle to be detected in the antibody-antigen uptake assay. in our study, the plasmid puc19-sgrna overexpressed pedv s proteins in vero cells compared to some commercially available vectors (data not shown), probably due to the inclusion of the 5=leader sequence of subgenomic rna (sgrna) and pedv 3=untranslated region (utr) that enhanced the translation. although we determined that both yevf and yeaf are endocytosis signals of pedv s proteins, it is possible that their efficiencies may vary. in the future, more advanced quantitative approaches, such as mechanically induced trapping of molecular interactions (mitomi) (19) , could be used to determine their affinities for binding to cellular factors. additionally, one limitation of this study is that we did not applied approaches to monitor the kinetic trafficking of the endocytosis of s proteins from the cell surface. future studies to address these points are necessary. in this study, we did not detect the colocalization of endocytosed s protein puncta and lysosomal marker lamp1 in wt1-, wt2-, δ5aa-, δ5aa-a, mk-p10-and mk-p10-v-puc19-sgrna transfected cells (data not shown). also, the total s protein levels (mcherry signals) of those mutants did not decrease during up to 2 days' culture. we hypothesize that the internalized pedv s proteins are transported back to the virion assembly sites rather than being degraded in the lysosome. for pedv, this endocytosis may be a strategy to efficiently use the expressed s proteins for the generation of infectious viral particles. this hypothesis may be supported by the quantitative data on s proteins incorporated into virions (fig. 9) , because the icδ5aa pedv with an intact yxx⌽ motif had a number of s projections similar to that of icpc22a. this endocytosis signal yxx⌽ motif may also help pedv evade host immune responses by constantly decreasing the amounts of viral s proteins on the cell surface. previous studies reported that inactivation of the yxx⌽ motif in the envelope proteins of simian immunodeficiency virus (siv) and human immunodeficiency virus (hiv) attenuated the corresponding virus and enhanced the t cell responses in vivo (37, 38) . moreover, host antibodies that have already bound to cell surface s proteins would be endocytosed into the cytoplasm, to evade antibody-dependent cell lysis (39, 40) . another possible function of this yxx⌽ motif in pedv s protein is that it could downregulate the levels of viral receptors on the cell surface, which may be involved in superinfection exclusion. the role of the yxx⌽ and kvhvq motifs in pedv pathogenicity. in the gnotobiotic piglet study, we observed that the virulence of icδ10aa was significantly reduced compared with that of the parental virus, icpc22a. in contrast, no statistically significant differences were observed in the virulence between icpc22a and the icδ5aa mutant, which lacks the kvhvq motif. mutant icya caused significantly milder villous atrophy in the jejunum than icpc22a. although not significant, it caused a delayed onset of diarrhea and lower peak fecal shedding titers than did icpc22a. therefore, icya pedv with an inactivated yxx⌽ motif was slightly attenuated. we investigated why the deletion of both motifs from the s protein (icδ10aa), but not the deletion of errs alone (icδ5aa), significantly attenuates a virulent pedv. tem images showed that icδ5aa-infected vero cells contained significantly higher numbers of mature virions in the golgi vacuoles than those infected with icδ10aa ( fig. 8f and g) . particularly, icδ10aa had a significantly lower percentage of purified virions with s projections on the surface and lower numbers of s projections on individual virions with surface s projections than the other three viruses (fig. 9) . therefore, icδ10aa may generate the highest level of defective virions (without enough s protein projections on the viral surface) among the four pedvs, causing attenuation in vivo. the difference in s incorporation into viral particles among the pedv mutants may be due to three reasons. first, the lack of both sorting signals renders more icδ10aa s proteins to be transported onto the cell surface, decreasing its level in the virus assembly site, ergic. second, the intense and rapid cell-cell fusion induced by surface s proteins in the infected cells causes significant intracellular membrane rearrangements (41) , which may interfere with the icδ10aa budding and egress process. in contrast, icδ5aa does not cause significant syncytium formation. the third reason may be the altered interaction between s and m proteins in the virion assembly site. although some previous studies revealed that mutation of the errs (klhyt) of sars-cov s protein altered the s-m interaction and s incorporation into virions (15, 16) , another study suggested that inactivation of this motif in the s of ibv did not affect the s-m interaction (12) . for pedv, a previous study of mk-p10 also showed that the impaired kvrvq motif did not alter the s incorporation (6) , which is similar to our observation for icδ5aa (fig. 9 ). in our s and m coexpression assay, we found that the four s proteins colocalized with the m proteins in the cytoplasm at the same level, and the coexpression of m protein significantly decreased the transportation of the four s proteins onto the cell surface, implying that those four s mutants may still interact properly with m proteins, although more experiments to evaluate s-m physical interactions are needed. our current data support the first and second possibilities. another reason for the attenuation of icδ10aa pedv in pigs is that mutation of pedv yxx⌽ may also interfere with the synthesis of orf3 sgrna, since the predicted trs of open reading frame 3 (orf3) sgrna overlaps with the nucleotide sequences encoding the tyrosine of the yxx⌽ motif (42) . it was reported that a recombinant ibv bearing mutations in its dilysine motif (kkxx) expressed less e protein, due to the decrease of sgrna 3 synthesis (12) . in this study, the sgrna 3 levels in the icδ10aa-and icya-infected cells were about 3 log 10 and 2 log 10 lower than that in the icpc22a-infected cells, as determined by sgrna 3-specific taqman real-time reverse transcription-qpcr (rt-qpcr) (data not shown). sgrna 3 expresses the accessory protein orf3. we do not know whether the e gene in the sgrna 3 can be translated during pedv infection. therefore, it is highly possible that the orf3 protein level was decreased in the icδ10aa-and icya-infected cells. however, due to the lack of effective antibodies against pedv orf3 and e proteins, we could not confirm these results at the protein level. the decrease of orf3 expression may also contribute to the significant attenuation of icδ10aa and the slight attenuation of icya in piglets because we determined previously that a pedv lacking an intact orf3 gene is partially attenuated in piglets (43) . the role of s ct truncation in vero cell-adapted pedvs and pedv vaccine development. in our study, the deletion of both the yxx⌽ and kvhvq motifs from the s protein could not completely attenuate a highly virulent pedv strain in piglets, as icδ10aa still caused moderate diarrhea in all the piglets (n ϭ 4). therefore, the ablation of the two motifs was probably not the only determinant of the reduced virulence for those attenuated/mild pedv variants (fl2013, sm-98, chm2013, pc22a-p120, and knu-141112). the attenuation of these strains must be the combined consequence of all the mutations in their genomes. we investigated why some vero cell-adapted pedv variants (pc22a-p120, sm-98, and knu-141112) lost function of the yxx⌽ and kvhvq motifs during continuous passaging. we found that the corresponding mcherry-tagged s proteins (δ9aa, δ7aa, and hd) caused larger syncytia than the wild-type s proteins. the loss of the two motifs may facilitate the rapid spread of those pedv variants between cells through enhanced syncytium formation. this idea was supported by a study of the mutant avct12. jengarn et al. (29) demonstrated that recombinant avct12 was rescued in vero cells only if the infectious cdna clone contained the δ7aa mutation in the s ct. we conclude that inactivation of both the yxx⌽ and kvhvq motifs of the s protein could be applied in the rational design of a pedv vaccine since it results in more s proteins being transported onto cell surface in the infected enterocytes, facilitating antigenic recognition by the host immune system, and may not decrease protective immunogenicity because no neutralizing epitope has been identified in the s ct of pedv. we aim to test this hypothesis in future studies. to achieve complete attenuation and to increase the safety of an attenuated vaccine, the inactivation of the two motifs should be combined with mutations in other key genes in the pedv genome. in conclusion, we showed that the yxx⌽ and kvhvq motifs in the ct of pedv s protein regulate the surface s protein level. the deletion of both motifs significantly reduced the virulence of pedv in pigs. our discovery can aid in the rational design of attenuated vaccines for pedv and other covs containing similar motifs in their s proteins. cells, reagents, and antibodies. vero cells (atcc ccl81) were cultured in dulbecco modified eagle medium (dmem; gibco, carlsbad, ca) supplemented with 5% fetal bovine serum (fbs) (atlanta biologicals, flowery branch, ga). the guinea pig polyclonal antibody (pab) gp17 against pedv s1 was generated in our laboratory as described below. pedv s1 subunit (aa 1 to 421) gene of vero cell-adapted pedv pc177 strain at passage level 2 (genbank accession no. km392229) was cloned into pfuse-higg1-fc2 vector (invivogen, san diego, ca), expressed in hek-293t cells, and purified using a protein a affinity column (pierce, waltham, ma). guinea pigs were first immunized with the purified s1 subunit mixed with freund's complete adjuvant via subcutaneous injection, followed by two booster injections of the same dose in freund's incomplete adjuvant with a 2-to 3-week interval between doses. the total igg antibodies in the hyperimmune serum were purified. the production of guinea pig serum was approved by the institutional animal care and use committee (iacuc) of the ohio state university. mouse monoclonal antibodies (mabs) sd129-5 against pedv s2 (residues 747 to 774) (44) and sd17-103 against pedv n proteins were kindly provided by diego diel and eric nelson at south dakota state university, respectively. rabbit hyperimmune serum against pedv m proteins was kindly provided by ying fang at kansas state university. mouse mab against the ergic-53 (oti1a8) was purchased from enzo life science (farmingdale, ny). rabbit mab against rab5 (c8b1) were purchased from cell signaling technology (danvers, ma). alexa fluor 488 (af488)-or af647-conjugated secondary goat antibodies against the igg antibodies of different species were all purchased from life technologies (gibco, carlsbad, ca). icδ5aa (n ϭ 4), icya (n ϭ 4), or pbs (mock treatment; n ϭ 3). postinoculation, we observed clinical signs and collected rectal swabs every 12 h. all the piglets were euthanized at 52 hpi to evaluate histopathological lesions at the acute phase of infection. taqman rt-qpcr for the detection of fecal pedv n rna, plaque assay for the detection of fecal infectious pedv titers, and immunohistochemistry staining of pedv antigens using mouse mab against pedv n protein (sd17-103) were performed as described previously (48, 49) . this animal study was approved by the iacuc of the ohio state university. growth curves of infectious pedvs. the growth curves of pedv replication in vero cells were determined by inoculation of monolayers of cells with each of the four recombinant pedvs with an moi of 0.001 or 5. after 1 h absorption, the inocula were removed and the cell monolayers were washed with pbs three times. maintenance medium containing 10 g/ml of trypsin was added to the cell culture. the total viruses (intracellular and extracellular) in each sample were collected by freezing and thawing the culture at appropriate time points. the infectious titers of viral samples were titrated by plaque assays. plaque assays. plaque assays were performed as described previously (50) . briefly, monolayers of vero cells were inoculated with appropriate dilutions of recombinant pedvs for 1 h. then the inocula were removed and the cells were washed with pbs three times. the cells were overlaid with an agarose-mem (gibco, carlsbad, ca) mixture containing 10 g/ml of trypsin. the diameters of individual plaques were measured by using an ix-70 fluorescence microscope (olympus, tokyo, japan) and its software. tem. transmission electron microscopy (tem) of pedv-infected cell sections was performed as described previously (50) . to visualize the subcellular structure in tem, recombinant pedv-infected vero cells (moi ϭ 5) were cultured in trypsin-free medium for 12 h and fixed with fixative (3% glutaraldehyde, 1% paraformaldehyde in 0.1 m potassium phosphate buffer [ph 7.2]) for 4 h. samples were embedded in agarose, dehydrated, and re-embedded in em bed812 resin (electron microscopy science, hartfield, pa). ultrathin sections of each sample were prepared and stained. to visualize s protein projections on the virions, vero cells were inoculated with the recombinant pedvs (moi ϭ 2) in the presence of trypsin. without freezing and thawing, virions in 500-ml culture supernatants of each recombinant pedv were purified by using peg-8000 (sigma-aldrich, st. louis, mo) precipitation, followed by 30% sucrose-cushion ultracentrifugation (10,000 ϫ g for 2 h), according to a previous protocol (51) . to maintain consistency, the four pedvs were cultured and prepared simultaneously. samples of subcellular section and purified virions were stained with 2% uranyl acetate prior to tem imaging. images were visualized using an h-7500 tem (hitachi, japan). statistical analysis. the statistical analyses were performed using graphpad prism 6.0. the data in fig. 2b , 3b to e, 7d to f, 8g, and 10b are shown in box-whisker plots. the outliers were the values 3/2 greater than the upper quartile or 3/2 smaller than the lower quartile (52) . comparisons of means of groups in these figures and fig. 8a, 8d , 9b, and 9c were analyzed by one-way analysis of variance (anova) followed by tukey's multiple-comparison test. in the fig. 5a , 5b, 6b, and 10c, the two groups were analyzed by student's t test. a p value between each two groups less than 0.05 was considered significantly different. porcine epidemic diarrhea in china updated estimated economic welfare impacts of porcine epidemic diarrhea virus (pedv) assessment of the economic impacts of porcine epidemic diarrhea virus in the united states emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture mutation in 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glycoprotein b regulates cell fusion and skin pathogenesis vzv gb endocytosis and golgi localization are mediated by yxx motifs in its cytoplasmic domain mutation of the dominant endocytosis motif in human immunodeficiency virus type 1 gp41 can complement matrix mutations without increasing env incorporation endocytosis of the nipah virus glycoproteins a lysine-methionine exchange in a coronavirus surface protein transforms a retention motif into an endocytosis signal attenuation of an original us porcine epidemic diarrhea virus strain pc22a via serial cell culture passage identification and pathogenicity of a variant porcine epidemic diarrhea virus field strain with reduced virulence genetic manipulation of porcine epidemic diarrhoea virus recovered from a full-length infectious cdna clone development of the full-length cdna clones of two porcine epidemic diarrhea disease virus isolates with different virulence genetic characteristics, pathogenicity, and immunogenicity associated with 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cell lysis antibody-induced internalization of viral glycoproteins and ge-gi fc receptor activity protect pseudorabies virus-infected monocytes from efficient complement-mediated lysis syncytia formation induced by coronavirus infection is associated with fragmentation and rearrangement of the golgi apparatus pedv leader sequence and junction sites characterization of a pathogenic full-length cdna clone and transmission model for porcine epidemic diarrhea virus strain pc22a the s2 glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes engineering the largest rna virus genome as an infectious bacterial artificial chromosome measurement of colocalization of objects in dual-colour confocal images the gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses experimental infection of a us spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original us pedv infection pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene purification and electron cryomicroscopy of coronavirus particles some implementations of the boxplot complete genome sequence of novel porcine epidemic diarrhea virus strain gd-1 in china sequence of the spike protein of the porcine epidemic diarrhoea virus phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china this study was funded by the national institute of food and agriculture, u.s. department of agriculture, under award number 2015-67015-23067 (q.w., principal investigator [pi]; l.j.s., co-pi).we thank andrea kaszas and xiaohong wang for technical assistance and juliette hanson, ronna wood, megan strother, dennis hartzler, sara tallmadge, and jeff ogg for animal care assistance. we thank chang-won lee and scott kenney for their critical reviews of the manuscript. we are grateful to diego diel and eric nelson at south dakota state university for providing us the mouse mabs against pedv s and n proteins and to ying fang at kansas state university for providing us the rabbit pab against pedv m proteins. key: cord-349249-jwvz1ux2 authors: singh, gagandeep; singh, pankaj; pillatzki, angela; nelson, eric; webb, brett; dillberger-lawson, steven; ramamoorthy, sheela title: a minimally replicative vaccine protects vaccinated piglets against challenge with the porcine epidemic diarrhea virus date: 2019-10-22 journal: front vet sci doi: 10.3389/fvets.2019.00347 sha: doc_id: 349249 cord_uid: jwvz1ux2 porcine epidemic diarrhea virus (pedv), is an economically important enteric coronavirus, with over a 90% mortality rate in neonatal piglets. the virus emerged in the us in 2013, resulting in severe production losses. effective vaccine development against pedv is a challenge. inactivated vaccines are of questionable efficacy. attenuated vaccines, while more effective, require a relatively long lead development time, are associated with safety concerns and are also unable to prevent new field outbreaks. to combine the safety and efficacy advantages of inactivated and attenuated pedv vaccines, respectively, in this study, we tested the hypothesis that subjecting pedv virions to heat treatment at 44°c for 10 min to reversibly unfold structural proteins, followed by exposure to rnase to fragment the genome, would result in a vaccine preparation with intact viral structure/antigenicity but highly diminished replicative abilities. we expected the vaccine to be both safe and effective in a piglet challenge model. following the heat and rnase treatment, pedv virions had an intact electron microscopic ultrastructure and were amplified only in the 3rd passage in vero cells, indicating that diminished replication was achieved in vitro. strong pedv spike-protein specific and virus neutralizing antibody responses were elicited in vaccinated piglets. upon challenge, all vaccinated pigs were protected against fecal viral shedding and intestinal pathology, while the unvaccinated controls were not. the vaccine virus was not detected in the fecal matter of vaccinated pigs prior to challenge; nor did they develop intestinal lesions. thus, the described approach has significant promise in improving current approaches for pedv immunization. porcine epidemic diarrhea virus (pedv) is an enteric coronavirus which causes diarrhea, vomiting, severe dehydration, and death in pigs. neonatal pigs are particularly susceptible, with mortality rates that can be as high as 90-100%. in older pigs, manifestation of the disease is milder but growth and production parameters are affected (1, 2) . classical strains of pedv (g1 strains) were first detected in the uk in 1971, and spread to asia and europe. more recently, highly virulent strains (g2 strains) which emerged in china have spread to other countries, with the first case in the us being recorded in 2013 (1, 3, 4) . it is estimated that the outbreak resulted in the losses of $0.9-1.8 billion and the death of 7 million pigs (5, 6) . the availability of effective vaccines and the practice of stringent biosecurity measures are critical for the prevention of pedv. however, the development of effective vaccines has been complicated by frequent viral evolution and the fact that ped is most severe in immunologically naïve neonates. effective and safe vaccines development was also challenging because active vaccine replication in the gut is required to induce good and lasting mucosal immunity. both attenuated and inactivated pedv vaccines have been routinely used in asian countries for several years. vaccination of sows prior to farrowing induces lactogenic immunity which is transferred to neonatal piglets via colostrum. inactivated vaccines are very safe but have a low duration of immunity and appear to produce a predominantly th2 type immune response (7) . attenuated vaccines, produced by serially passaging field strains between 83 and 100 passages, are more effective against homologous strains but have a long lead development time and have been associated with safety concerns of recombination with field strains (2) . regardless of the type of vaccine used, viremia and transmission of pedv is not prevented in vaccinated animals. outbreaks in vaccinated herds and the periodical emergence of new, highly pathogenic strains are not uncommon in countries were vaccines have been routinely used for many years (2, (7) (8) (9) . in north america, a s-protein based subunit vaccine (iped plus, harris vaccines inc.) and inactivated vaccines produced by zoetis and vido-intervac were conditionally licensed. however, their efficacy has also been questioned by independent studies, as vaccination of pedv naïve sows did not result in strong protection in neonatal piglets (8, 10, 11) . as the strong need for effective pedv vaccines remains unmet, the practice of feeding back minced intestines from infected piglets to sows, in an attempt to induce more effective immunity against pedv, is common in the field (8, 10, 11) . the use of autogenous vaccines, where a custom inactivated vaccine tailored to each herd is prepared using a sample provided from the production unit, is also practiced (8, 12, 13) . both the feedback and autogenous vaccine approaches are, once again, associated with significant safety and efficacy issues but natural or intentional exposure of pigs of all ages to pedv provides stronger homologous and partial heterologous protection (2, 8) . further, vaccination of naïve animals is less effective than vaccination of previously exposed pigs, indicating that current vaccines are less effective than natural infection at priming the immune response but can effectively boost the memory response (14) . it is established that the viral spike protein is a critical protective antigen, as anti-spike protein-specific serum igg levels correlate well with protection against pedv and virus neutralizing responses (15) . however, the s-protein based subunit vaccine (iped plus, harris vaccines inc.) is of questionable efficacy, indicating that other viral components could contribute to protection. based on the above, we hypothesized that development of a process whereby the structural integrity of the virus was maintained but viral replication was highly diminished but not abrogated, would result in a vaccine with the combined advantages of inactivated and attenuated vaccines, namely, high safety and efficacy margins. previously published data shows that the sars coronavirus capsid is metastable and can be reversibly denatured by changes in temperature or ph, with unfolding commencing at 35 • c and complete denaturation occurring at 55 • c (16) . hence, in this study, our vaccine development approach consisted of exposing pedv virions to 44 • c to unfold the capsid, followed by fragmentation or digestion of the genome with rnase to diminish viral replication and subsequent refolding of the capsid at 25 • c. gamma-irradiated pedv virions were used as an inactivated control vaccine (17) . the objective of this study was to evaluate the heat and rnase treated pedv vaccine for its safety, immunogenicity and ability to reduce viremia in a weanling piglet model, with the ultimate goal of developing a process which can potentially reduce lead vaccine development time, is safe and be easily applied to newly emerging strains. porcine epidemic diarrhea virus (pedv) strain pedv co2013 [national veterinary services laboratory (nvsl), ames, ia] was cultured at a multiplicity index (moi) of 0.1 using vero cells in the presence of trypsin as previously described (18, 19) . the stock virus was titrated three times to obtain the mean 50% tissue culture infectious dose [tcid 50 ] using the spearman and karber formula (20) and stored in aliquots at −80 • c until further use. to optimize the temperature, time of incubation, and dose of rnase treatment, the virus stock was resuspended to 1 × 10 5 tcid 50/ /ml in media (ph 7.2). diluted virus culture was exposed to temperatures ranging from 37 to 60 • c for 10 min for unfolding, followed by incubation at 25 • c for 30 min for refolding, and then moved to 4 • c for 1 h, as previously described for the sars coronavirus (16) . cultures were visualized by electron microscopy to ensure structural integrity. a temperature of 44 • c for 10 min was selected for unfolding. similarly, to fragment the genomic rna, varying combinations of concentrations of rnase a (ameresco) and rnase t (thermo scientific) were tested by adding them to the unfolded virus cultures, followed by incubation for 5, 4, 3, or 2 h at 44 • c. treated cultures were then exposed to 25 • c for 30 min for refolding and cooled down on ice for 1 h. the final optimized protocol consisted of exposing the virus culture, resuspended to 10 5 tcid 50 /ml, to 44 • c for 10 min, followed by 0.1 mg/ml of rnase a and 1 µl/ml of rnase t1 (equivalent to 10 units/ml rnase a or 1,000 units/ml of rnase t1), incubation at 44 • c for 4 h, exposure to 25 • c for 30 min and cooling down on ice for 1 h before storage at −80 • c for further testing. the final process was tested 3 times to ensure reproducibility. to prepare the inactivated control vaccine, 1 × 10 5 tcid 50/ /ml of pedv was irradiated in a cesium-137 source gamma (γ) irradiator at time points of 8 h to 24 h at 753 rad/min. an effective dose of 24 h (1,084,320 rad), was used to prepare the irradiated vaccine, after validation as described above. to determine the effect of the treatment on viability, the treated virus and an untreated control were serially passaged 3 times in vero cells as described above. after each passage, flasks were subject to three freeze-thaw cycles. the culture obtained was centrifuged at 10,000 × g for 10 min 4 • c to remove debris. one ml of the supernatant was used to infect vero cell monolayers and also infect 8 well chamber slides (nunc) to visualize viral replication by an indirect immunofluorescence assay (ifa) as described below. visualization of viral replication in treated and untreated cultures was achieved using an indirect immunofluorescence assay (ifa), performed essentially as described previously (18, 19) . cultured and fixed cells were stained with polyclonal swine anti-pedv sera (nvsl) and examined with a fluorescent microscope for green cytoplasmic fluorescence characteristic of rna viral replication. to visualize structure, treated, and untreated viral cultures were negatively stained by standard methods (21) . stained grids were examined with a jeol jem-100cx ii transmission electron microscope (figure 1 ). possible genetic differences between untreated and treated vaccine virions were assessed by deep sequencing. heat and rnase treated and untreated viral particles were purified from infected cells by ultra-centrifugation at 100,000 × g for 2.50 h and re-suspended in pbs. unpackaged rna and dna were removed by a rnase and dnase cocktail containing 20 units of rnase one (promega), 20 units benzonase (novagen), and 14 units of turbo dnase (ambion) incubated in 1x buffer (ambion) for 37 • c for 1.5 h. viral rna was then isolated by using the qiamp viral rna isolation kit (qiagen) according to the manufacturer's protocol. purified viral rna was deep sequenced by a commercial vendor (bgi genomic). the cdna library was prepared using truseq library construction kit (illumina inc., usa) with random hexamer primers. the prepared cdna library was then sequenced using hiseq 4000 pe100 platform (illumina inc., usa) and raw reads (100 bp) were obtained. the resultant sequences reads were analyzed by bgi genomic, philadelphia, pa. the raw reads were filtered out using soapnuke to get "clean reads" by removing the reads with adaptors, reads with more than 5% of unknown bases (n), and low-quality reads (22) . clean reads were mapped to reference pedv genome (genbank: kf267450.1) using hisat (hierarchical indexing for spliced alignment of transcripts) (23) . the genome mapping results further analyzed using the genome analysis toolkit (gatk) to call single nucleotide polymorphism (snp) and indel (insertion and deletion of bases) (24) . only snps with a quality score above the threshold (qpred > 20) and with a snp frequency of over 85% were included in assembling the consensus sequences. the consensus sequences of the treated and untreated samples were compared by alignment with clustal omega (25) to obtain changes which could be attributed to the treatment. detected changes were annotated to include the locations and proteins affected ( table 1) . clean reads were mapped to the reference genome using bowtie2 to detect differentially expressed genes. gene expression levels were calculated with rsem version 1.2.12 (26) . differentially expressed genes were identified by the possiondis, ebseq software for samples without replicates (27) . all animal experimentation was approved by the institutional animal care and use committee (iacuc) of s. dakota state universities (sdsu) (protocol number: 15-013a). no other specific permissions were required for these activities. this study did not involve endangered or protected species. twenty-four, 2 to 3-week-old piglets which were negative for pedv by pcr and serology were divided into 3 groups; group 1-unvaccinated control group (n = 8) (2 ml of pbs intramuscular and oral route each), group 2-rnase and heat treated pedv vaccine group (pedv-vac) group (n = 8) (2 ml of 10 5 tcid 50 /ml, intramuscular and oral route each) and group 3-irradiated pedv vaccine group (n = 8) (2 ml of 10 5 tcid 50 /ml, intramuscular and oral route each). piglets were boosted by the same route and dose at dpv 14 and 28. on dpv 43, small intestine, heart, liver, and spleen were collected 2 piglets from each group (n = 2/group) to assess vaccine safety. the remaining piglets (n = 6/group) were challenged orally with 10 5 tcid 50 /ml of pedv co2013, as previously described (28, 29) . post-challenge, the piglets were observed daily for clinical signs of ped. all piglets were euthanized 1-week post challenge (dpc) or at dpv 49 and three sections of the small intestine (duodenum, jejunum, and ileum) were collected for histopathological (hp) and immunohistochemical (ihc) analysis. serum was collected from all piglets on dpv 0, 14, 28, 43, and 49 to measure binding and neutralizing ab responses. fecal swabs were collected at dpv 7, 21, 38, and 42 from all piglets to measure shedding of the vaccine virus by rt-qpcr. fecal swabs were collected on dpv 45 and 49 (dpc day 3 and 7) from all piglets to measure protection against shedding of the challenge virus by rt-qpcr. spike protein-specific igg responses in pigs were measured in duplicate by an indirect elisa as previously described, using the pedv s antigen or np antigen for capture (18) . the assay format was pre-validated at the animal disease research and diagnostic laboratory (adrdl), sdsu, using serum samples from animals of known serological status. a standardized operating procedure was followed in sample analysis. the results were calculated as sample to positive (s/p) ratios as follows: s/p = optical density (od) of the sample-od of buffer/od of positive control-od of the buffer. 7.83 ± 6.50 (5/6) (p = 0.37) rnase + heat treated pedv/ unchallenged total number of pigs = 8, no. of pigs sacrificed for vaccine safety assessment prior to challenge = 2, no. of pigs sacrificed at day 7 post challenge = 6. % total atrophic enteritis score for the ileum, jejunum, duodenum where 0, negative; 2, mild; 4, moderate; 6, severe; 2, sections with crypt hypertrophy. & total immunohistochemistry (ihc) for the ileum, jejunum, duodenum where 0, negative; 2, positive; ≤10%, 4, positive, 11-50%; 6, positive, >50%. * fecal score at necropsy-formed feces = 0, semi-formed feces = 3, liquid feces = 6. # sum of the microscopic and fecal scores. @ p < 0.05 as determined by the mann-whitney u-test, compared to the unvaccinated group. to assess the neutralizing antibody responses elicited by vaccination, a pre-validated fluorescent focus neutralization (ffn) assay was used as previously described (18), following the standard operating procedures of the adrdl, sdsu. briefly, doubling dilutions of heat inactivated sera were incubated with 100 foci forming units, incubated for 1 h and cultured on vero cell monolayers. plates were stained with a pedv-specific fluorescein-labeled monoclonal antibody (sd6-29) to visualize the end point, which was defined as a 90% reduction of foci compared to the controls. virus shedding through fecal route was assessed by a rt-qpcr performed by the ndsu veterinary diagnostic laboratory, using pre-validated standard operating procedures, and a commercial pcr kit called the swine enteric pcr panel (thermo fisher) following the manufacturer's instructions. each pig was considered a biological replicate (n = 6, as 2 pigs/ group were sacrificed to assess vaccine safety prior to challenge), and each sample was assessed in duplicate. the obtained ct-values were converted to viral copy numbers using a standard curve and log transformed for representation. tissue samples, collected as described above, were fixed in neutral buffered formalin for 48 h, trimmed, processed, and embedded in paraffin. tissues were cut into 5 µm thick sections and stained with hematoxylin and eosin (he) or a pedv n protein-specific monoclonal antibody (sd6-29) for immunohistochemistry (ihc) following the standard operating procedures of the adrdl, sdsu. scores were recorded in a blinded fashion by a board-certified veterinary pathologist. scores to measure atrophic enteritis characteristic of ped were assigned as follows: 0 = negative, 2 = mild, 4 = moderate, 6 = severe. sections with crypt hypertrophy were assigned an additional 2 points. antigen detection in enterocytes by ihc was semi-quantitatively scored based on the following criteria: 0 = negative, 2 = positive, ≤10%, 4 = positive, 11-50%, 6 = positive, >50%. the consistency of fecal matter during necropsy was assigned scores as follows: formed feces = 0, semi-formed feces = 3, liquid feces = 6. total scores were calculated as the mean sum of the histology and fecal scores ( table 1) . significant differences between treatments were assessed by anova and when significant (p < 0.05) post-hoc analysis was used to determine differences between groups. the student's ttest was used for the serology and rt-qpcr data and the mann-whitney u-test for the pathology lesion scores. the mean values of replicates, standard deviation and statistical significance are represented in the figures and tables. to achieve the targeted outcomes of maintaining structural integrity while achieving diminished viral replication, rather than complete inactivation, pedv virus cultures were first exposed to temperatures ranging from 37 to 60 • c for 10 min and visualized by electron microscopy. intact structures were detected at all temperatures tested. however, increasing numbers of misshapen and fragmented virions were detected at 50 • c and above. cultures treated at 37 and 45 • c remained viable as viral replication was visible by immunofluorescence (ifa) in infected vero cells using a pedv-specific antibody, without any amplification by serial passaging. virus was detected after the 1st passage in the cultures treated at 50 • c. virus cultures treated at 55 and 60 • c were not amplified even after four serial passages in vero cells, indicating that complete inactivation occurred at these temperatures. hence a temperature of 44 • c for 10 min was chosen for reversible unfolding of the viral capsid ( figure 1b) without completely inactivating the virus. untreated control virus culture remained structurally intact as expected ( figure 1a) . similarly, while rnase treatment alone did not affect viability, the reduction in viral replication was proportional to the dose and time of exposure to rnase in the heat-treated virions. a dose of 10 units of rnase a and 1,000 units of rnase t with an exposure time of 4 h was chosen as optimal for the final vaccine preparation. while the untreated virus control showed robust replication (figure 2a) , following the heat and rnase treatment protocol, viral replication was detected only in the 3rd passage in vero cells (figure 2b ). for the gamma (γ) irradiated, inactivated control vaccine, typical icosahedral structures were seen in electron microscopy after 23 h of exposure to radiation. however, the corona-like layer containing the protective spike antigens appeared to be damaged (figure 1c) . at this dose of radiation, the virus was not detected by the ifa with a pedv-specific ab at the third serial passage in cell culture ( figure 2c) . hence, a final dose of 24 h (1,084,320 rad) was selected to prepare the inactivated control vaccine. measurement of ab responses against the pedv spike and nucleocapsid proteins (np) by elisa (18) showed that animals vaccinated with the heat and rnase treated virions mounted strong ab responses against the protective pedv spike antigen following the booster vaccinations on dpv 14 and 28 (figures 3a,b) . however, ab responses to non-structural nucleocapsid protein (np) remained low prior to the challenge. in pigs immunized with the irradiated vaccine, ab responses to both viral antigens were low. the mean optical density values for the elisas were significantly different between the groups (figures 3a,b) . to that of the spike protein-specific abs. strong virus neutralizing ab responses, were detected in animals vaccinated with the heat and rnase treated virions but not in the pigs which received the irradiated viral vaccine. the differences between the groups was statistically significant (figure 3c) . the spike protein-specific ab and virus neutralizing ab levels were strongly correlated in the heat and rnase treated pedv vaccinated pigs, with a correlation coefficient of 95.11%. as expected, the unvaccinated control pigs remained sero-negative for the duration of the study. to assess the efficacy of the vaccine in protecting against challenge, shedding of the challenge viral rna in fecal matter was assessed by a pedv-specific rt-qpcr on days 0, 3, and 7 post-challenge. all experimental animals were rt-qpcr negative on day 0 post-challenge (dpc). at dpc 3 and 7, challenge viral rna was not detected in any of the pigs vaccinated with the heat and rnase treated pedv vaccine (figure 4) , while 4 of the 6 pigs administered the irradiated vaccine were positive by rt-qpcr on dpc3. all 6 pigs in the irradiated vaccine group turned positive by dpc7 (figure 4) . as expected, viral rna was detected in the fecal matter of all unvaccinated pigs on both sample collection days with titers increasing between dpc 3 and 7. while the viral rna loads were significantly different between the two vaccine groups at both time points, there were no significant differences between the unvaccinated controls and pigs administered the irradiated vaccine at both the time points tested, indicating that the irradiated vaccine did not provide protection against viral replication and shedding in the host. examination of the intestinal tissue of the experimental animals by histology and immunohistochemistry (ihc) showed that the heat and rnase treated pedv vaccine completely protected vaccinated pigs against the development of microscopic lesions following challenge. characteristic microscopic intestinal lesions of atrophic enteropathy and crypt hyperplasia were detected in the duodenum, jejunum, and ileum of animals in the control groups (figures 5d-g) . viral antigen was also detected in the enterocytes in all three sections using a pedvspecific monoclonal ab-based immunohistochemistry assay (figures 6a-e) . there were no significant differences between the 3 sections, indicating the entire small intestine was affected. the total microscopic score, including the histopathology and immunohistochemistry scores was 4.16 for the unvaccinated animals and 7.33 for the pigs immunized with the irradiated vaccine and 0 for pigs administered the heat and rnase treated vaccine. while the difference between the unvaccinated group and irradiated vaccine group was not statistically significant, the irradiated vaccine appeared to enhance intestinal pathology (table 1) . similarly, the total necropsy scores, a sum of both the fecal and histology scores, were significantly different (p = 0.04) between the two vaccine groups but not between the unvaccinated group and the irradiated vaccine group (p = 0.37) ( table 1 ). no side effects or clinical signs of ped were observed in vaccinated pigs after either the primary or booster vaccines. vaccine viral rna was not detected by rt-qpcr in the fecal matter of any of the vaccinated pigs from both groups at 7 days after the primary vaccination or at 1 week after the boosters. all animals remained pcr negative until the day of challenge. therefore, although the heat and rnase treated pedv virions were detected by amplification after 3 serial passages in vero cells, replication of the vaccine virus in the host appeared to be curtailed by its immune system. in the 2 pigs euthanized from each group prior to challenge, stools were fully formed at necropsy ( table 1) . no microscopic lesions or viral antigen were detected in the small intestine sections, heart, spleen, and liver of the 2 animals necropsied from each group prior to challenge ( table 1) . representative images of the duodenum, jejunum, and ileum are depicted in figure 5 . to identify possible mutations that could explain the highly effective attenuation observed, deep sequencing of heat and rnase treated virions from infected vero cells resulted in a total of 59.42 and 24.44 mb of raw reads were obtained by rna seq for the treated and untreated samples, respectively. clean reads obtained after trimming were 26.94 and 19.53 gb, respectively. the qphred20 values for the clean reads were 96.69 and 98.49 for the untreated and tread samples, respectively, indicating satisfactory quality of the data obtained. as listed in table 2 snps and insertions or deletions (indels) were detected in the polyprotein, spike and envelope proteins ( table 2 , s1 sequence file and supplementary figures 1-3 ) of heat and rnase treated virions, when compared to the untreated virions. in addition, insertions and deletions were detected in the s1 region for the spike protein. the n terminal signal peptide region of the spike protein had a 2 amino acid deletion and one non-synonymous change at position 355, changing the sequence from igen to k-n. a conservative in-frame insertion was detected at position 355 in the s1 region, changing the amino acid sequence from l----at to lkkkgat ( table 2 and supplementary figure 2) . chemical methods for inactivation of viruses have long been in use for vaccine development. while they are rapid and convenient, commonly used inactivation agents may not only affect nucleic acids but also protein structures and hence antigen presentation and vaccine efficacy. gamma irradiation has been traditionally used to inactivate viruses. the mechanisms involved include nucleic acid degradation, destruction of covalent bonds, and release of free radicals (30) . as commercial inactivated vaccines were not available at the time of testing gamma irradiation was selected as the method of choice to prepare an inactivated control vaccine for this study. moreover, similar to the heat and rnase treated vaccine, the virus-like-particulate structure was more likely to be maintained by gamma irradiation, while achieving complete inactivation. gamma irradiation had been previously used for vaccine development with varying success, depending on the pathogen (17) . for example, we have previously demonstrated that a gamma irradiated vaccine against neospora caninum was effective in mice (31) . however, a gamma irradiated, lassa virus vaccine failed to protect vaccinated mice (32) . although both approaches tested in this study targeted nucleic acids and preservation of structure, the protective outcomes varied significantly between the two vaccines tested. it is possible that release of free radicals during the irradiation process could have a deleterious effect on integrity of antigenic structures and antigen presentation in vivo. a more detailed characterization of these parameters will be the focus of future studies. similar results for the gamma irradiated vaccine in this study, it has been shown that a dendritic cell targeted spike protein-based subunit vaccine against pedv exacerbated intestinal pathology in vaccinated pigs, despite stimulating strong cd4 + /cd8 + t cell responses (33) . while characterizing the exact physical interactions involved in the heat and rnase treatment is not within the scope of this study, our finding that exposure of pedv to temperatures below 50 • c did not affect structure was similar to other studies showing that the sars coronavirus structure is metastable and can be reversibly denatured by exposure to varying physical conditions such ph and temperature (16, 34) . although the heat and rnase treated virus culture was amplified after 3 passages in cell culture (figure 2) , the absence its detection by rt-qpcr (figure 4) , or immunohistochemistry (table 1 and figure 5 ) and the lack of strong ab responses to the non-structural np (figure 3) , in vaccinated pigs prior to challenge indicates that active vaccine viral replication was absent in the host or was undetectable by the techniques used. therefore, unlike other attenuated pedv vaccines or vaccination strategies that rely on prior exposure to field strains, it is highly improbable that reversion to virulence or recombination with field strains could occur with the heat and rnase treated vaccine. viral genomes that were identical to the untreated parental virus were not detected by deep sequencing of the heat and rnase treated virus from infected vero cells. insertions and deletions in the spike protein, especially the s1 region, influence pathogenicity, and immunogenicity of pedv. the core neutralizing epitope of the pedv spike protein has been localized to amino acid positions 503-568 (35, 36) . the snps identified in the spike protein of the vaccine virions ( table 2 ) did not map to these residues. while a limitation of the described method is that genetic changes induced by treatment and repair are unpredictable, repair of mutations (37) or complementation in trans of the fragmented genome could have led to detection of a fluorescent signal in the 3rd passage after treatment. indeed, it has been shown that replication deficient genomes with deletions or mutations are produced during serial passaging of foot and mouth disease virus (fmdv) for attenuation. they are not infective by themselves, but when present in the same cell, the mutations in the genomes can complement each other in trans to produce plaques in vitro. when the defective-complementing virus system was used as a vaccine by rodriguez-calvo et al. vaccine virus replication was not detected but strong protection was elicited. this observation can be explained by vaccine virus replication in the host being limited by the requirement of coinfection of the same cell. even if such an unlikely coinfection event were to happen despite active host innate immunity, the recombined progeny viruses were more likely to be highly attenuated than acquire virulence, thus providing an additional vaccine safety barrier in vivo (38) . in vivo, the presence of the host innate immune system was likely able to effectively curtail replication, despite exposure to 10 5 tcid 50 of the heat and rnase treated virus culture. more detailed studies are required to confirm these hypotheses, but they are not within the scope of this manuscript. the importance of spike protein-specific antibodies for protection against pedv is well-established (15) . several studies describing experimental subunit and vectored vaccines or commercial attenuated and inactivated vaccines against pedv establish a strong correlation between spike protein-specific antibodies, virus neutralization titers and protection against infection (9, 29, 37, (39) (40) (41) (42) (43) . similar to these studies, strong spikeprotein specific ab responses and virus neutralizing responses were noted in the pigs immunized with the heat and rnase treated vaccine. a commercial inactivated vaccine was able to reduce challenge viral shedding by 3-4 logs but an attenuated vaccine induced iga responses but did not affect viral shedding (43) . testing of two attenuated pedv strains produced by serial passage in weanling pigs showed that the passaged viruses were attenuated but were not protected against challenge viral shedding or clinical signs (29) . while direct comparisons are not possible due to differences in experimental conditions, unlike the other cited studies, intestinal lesions, or challenge virus was not detected by qpcr in the heat and rnase treated vaccine group in this study. although boosters were incorporated in the study design to minimize risk, it is likely that they were not required to achieve adequate protection as strong spike protein specific antibody responses and virus neutralizing responses were detected after the first dose of the heat and rnase treated vaccine, at dpv 14 (figure 3) . while cell mediated immunity was not assessed due to difficulties with transportation of cells, it is very likely that it was not compromised by the process used as the heat and rnase treated vaccine was very effective in preventing challenge viral replication in vaccinated pigs. while ideal for pedv, studying vaccine efficacy in pregnant sows and neonatal pigs is expensive and procedurally tedious. although clinical signs are less severe in older piglets (28) and virulence can vary between isolates used for challenge (44, 45) , pedv can infect and replicate well in pigs of all ages (14, 46) . hence several researchers have used weanling piglets to screen vaccine candidates for efficacy and safety (9, 13, 29, 43, (47) (48) (49) (50) (51) (52) . this approach can help reduce animal use and cost if the candidates fall short of expectations. several swine bioassay studies in growing piglets have reported that peak pedv replication occurs between dpi 3 and dpi 7 after which viral loads decrease (28, 43, 47, 48) . similar patterns of infectivity were observed in this study, as the uninfected control pigs had a mean fecal viral rna load of 8.35 log copy numbers at dpi 7 (figure 4 ) developed microscopic lesions, but not severe clinical signs. in comparison to the untreated control and irradiated vaccine groups, no fecal viral shedding or intestinal pathology was detected in the pigs immunized with the heat and rnase treated vaccine, indicating that vaccine induced immunity was highly effective against pedv challenge, within the limits of this weanling pig study model. the primary advantages of this innovative approach are safety, efficacy, convenience and a short development time. as the method can be easily adapted to newly evolving strains, provided they are readily cultured, this approach is very relevant to current field immunization practices of feedback exposure and autogenous vaccination. our future goals include testing the heat and rnase treated vaccine in pregnant sows, and improving oral and respiratory mucosal vaccine delivery systems to target improved protection. the datasets generated for this study are available on request to the corresponding author. the animal study was reviewed and approved by institutional animal care and use committee (iacuc) of s. dakota state universities 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