key: cord-012654-m8nlsutd authors: song, zhiquan; xie, ying; guo, zongpei; han, yang; guan, hua; liu, xiaodan; ma, teng; zhou, ping-kun title: genome-wide identification of dna-pkcs-associated rnas by rip-seq date: 2019-07-05 journal: signal transduct target ther doi: 10.1038/s41392-019-0057-6 sha: doc_id: 12654 cord_uid: m8nlsutd nan highest binding potential. motif analysis showed that dna-pkcs preferentially binds the agga sequence, which was in accordance with previous findings (fig. 1c) . 7 then, the docking between dna-pkcs rna-binding sites deduced from the web server pridictor and the rna motif agga was performed on the frodock webserver, and the docking structure was analyzed using pymol software (fig. 1c) . after analysis with a stringent cutoff,~500 rnas were precipitated by dna-pkcs. to categorize the rnas bound by dna-pkcs, we performed kegg analysis. this analysis showed a number of signatures involved in the focal adhesion and receptor-ecm interaction pathways (fig. 1d) . then, the itga3, itga5, itgav, sdc4, and cd44 rnas were selected for validation of the rip-seq results. the rip-qpcr results showed different fold enrichment values for these five rnas, which are involved in cell adhesion (fig. 1e) . regulation of rna alternative splicing is a crucial process in rna-binding proteins function, and aberrant splicing is often associated with various human diseases including cancers; 8 therefore, to discern how dna-pkcs modulates bound rnas, we sought to determine whether dna-pkcs could affect cd44 alternative splicing. specific primers to amplify the cd44 standard sequence and variants were designed, and qpcr was performed to examine the expression of different variants after u2os cells were treated with nu7441 and nu7026, which target dna-pkcs. the results showed that v4, v9, and v10 increased. next, western blotting further verified that after dna-pk inhibition, expression of cd44 variants increased (fig. 1f) . in summary, our findings strongly support a model wherein the dna-pkcs protein controls a variety of biological processes, including alternative splicing, through its rna-binding activity. further work will elucidate the accessory factors of dna-pkcs in regulating alternative splicing and how alternative splicing may contribute to the dna damage response mediated by dna-pkcs. geometry of a complex formed by double strand break repair proteins at a single dna end: recruitment of dna-pkcs induces inward translocation of ku protein autophosphorylation of the dna-dependent protein kinase catalytic subunit is required for rejoining of dna double-strand breaks beyond dna repair: dna-pk function in cancer human ku70/80 interacts directly with htr, the rna component of human telomerase the human telomerase rna component, htr, activates the dna-dependent protein kinase to phosphorylate heterogeneous nuclear ribonucleoprotein a1 long noncoding rna linp1 regulates repair of dna double-strand breaks in triple-negative breast cancer dna-dependent protein kinase (dna-pk) phosphorylates nuclear dna helicase ii/rna helicase a and hnrnp proteins in an rna-dependent manner therapeutic targeting of splicing in cancer 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-283807-4yo27web authors: ashtari, parviz; he, xiaoxiao; wang, kemin; gong, ping title: an efficient method for recovery of target ssdna based on amino-modified silica-coated magnetic nanoparticles date: 2005-09-15 journal: talanta doi: 10.1016/j.talanta.2005.06.043 sha: doc_id: 283807 cord_uid: 4yo27web abstract in this paper, an improved recovery method for target ssdna using amino-modified silica-coated magnetic nanoparticles (asmnps) is reported. this method takes advantages of the amino-modified silica-coated magnetic nanoparticles prepared using water-in-oil microemulsion technique, which employs amino-modified silica as the shell and iron oxide as the core of the magnetic nanoparticles. the nanoparticles have a silica surface with amino groups and can be conjugated with any desired bio-molecules through many existing amino group chemistry. in this research, a linear dna probe was immobilized onto nanoparticles through streptavidin conjugation using covalent bonds. a target ssdna(i) (5′-tmr-cgcatagggcctcgtgatac-3′) has been successfully recovered from a crude sample under a magnet field through their special recognition and hybridization. a designed ssdna fragment of severe acute respiratory syndrome (sars) virus at a much lower concentration than the target ssdna(i) was also recovered with high efficiency and good selectivity. the separation and recovery of an analyte is a fundamental technique in many fields, including chemistry, biology, biomedicine, industry, and environmental control. target dna separation and recovery is important for numerous applications in biotechnology and medicine. these applications include gene transfection, accurate pcr for mutation detection, gene therapy, species identification, and evolutionary analysis [1] . there are two principles for recovery of target dna. gel electrophoresis is a traditional method of dna separation and recovery [2] [3] [4] , in which a strand is cut into many pieces and passed through a porous gel, wherein shorter pieces move faster and farther than the longer ones. from the distribution of the fragments, information about the genetic content can be determined through comparison with the marker dna. to recover the wanted target dna, the further extremely time-consuming experiments must be performed, including recovery of dna from agarose by electroe-lution, recovery of dna from agarose gels with spin column, recovery of dna from agarose gels by phenol extraction, and recovery of dna from low-melting agarose with licl [5] . on the other side, the recovery of trace target nucleic acids in complex chemical and genetic backgrounds (e.g. pathogen detection) can also be realized based on the nucleic acid or peptide nucleic acid affinity-purification methods [6, 7] . however, these experimental methods possess a typical run time of more than 10 h due to the complex separation steps, such as centrifugation, columniation, filtration, and so on. in recent years, the integration of nanotechnology with biology and medicine has utilized functionalized nanoparticles in molecular diagnostics, therapeutics, molecular biology, bioengineering, and bio-separation [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] . all kinds of functionalized nanoparticles have been developed; in particular, substantial progress has been made in magnetic nanospheres and ferrofluids development technologies [18, 19] . due to their size-dependent properties and dimensional similarities to biomacromolecules [20] [21] [22] , these magnetic nanoparticles can be used as contrast agents for in vivo magnetic resonance imaging (mri) [23, 24] , as longcirculating carriers for drug release/delivery, and poten-tially as oligonucleotide separation devices in medicine and biotechnology. magnetic isolation of these bio-molecules is faster than standard liquid chromatography and electrophoresis techniques. target molecules, such as peptides, proteins, and oligonucleotides can be separated and recovered from untreated crude samples without any filtration, and pretreatment steps by magnetic bio-conjugates, i.e. by attaching bio-specific ligands to them. tan and co-workers developed a novel genomagnetic nanocapture for the collection of trace amounts of dna/mrna molecules based on the molecular beacon [25] . in fact, this novel nanocapture is a kind of magnetic nanoparticle, which incorporates dna probes. a weakness of this protocol is that dna probe immobilization was carried out through conjugation of avidin onto the nanoparticles by use of a weak electrostatic interaction. such linking is not stable, which will affect the lifetime of the nanocapture. at the same time, tan et al. reported the collection of dna using a molecular beacon (mbs) probe that is a class of dna probes widely used in chemistry, biology, biotechnology and medical sciences for bio-molecular recognition. however, in most biosensor applications, the sensitivity of the mbs immobilized on a silica surface is usually low due to its steric hindrance on the solid surface [26] . in addition, the sensitivity of the dna probe will directly affect the efficiency of the separation and recovery. as known, the lifetime and the efficiency of separation and recovery are two important factors to the nanocapture. in this paper, we reported an improved method for recovery of target ssdna using amino-modified silica-coated magnetic nanoparticles (asmnps). the amino-modified silicacoated magnetic nanoparticles were prepared through the controlled synchronous hydrolysis of tetraethoxysilane and n-(␤-amimoethyl)-␥-aminopropyltriethoxysilane in a water nanodroplet using water-in-oil microemulsion, and employed iron oxide as the core of the amino-modified silica-coated magnetic nanoparticles. in this protocol, the dna probe was immobilized onto nanoparticles through streptavidin conjugation using covalent bonds. such linking is much more stable than electrostatic interaction. moreover, we selected a linear dna probe as a capture ssdna, which is better than mbs in this method due in part to its ease of design, less steric hindrance, and high immobilization efficiency on the silica surface. a target ssdna(i) (5 -tmr-cgcatagggcctcgtgatac-3 ) was successfully recovered by using this protocol. this method has also been used successfully to recover trace concentrations of target ssdna fragment of sever acute respiratory syndrome (sars) virus with high efficiency and good selectivity. it introduces an effective, selective, and rapid method for recovery of target ssdna of other viruses, which is very important for disease or mutant detection. cyclohexane, n-hexanol, triton x-100, tetraethylorthosilicate (teos), n-(␤-ethylenamine)-␥-propylamine triethoxylsilane (eptes) and fluorscamine were purchased from sigma-aldrich (st. louis, mo). glutaraldehyde (50%) was obtained from amresco chemical company (usa). streptavidin was from promega corporation (madison, wi, usa). all dna were synthesized at bioasia biologic technology co. ltd. (beijing, china). the dna sequences are shown in table 1 . all other chemicals used were analytical grade reagents and used without any further purification. all solutions were prepared with nanopured-deionized water (branstead co., usa). the magnet property of nanoparticles was measured using an alternating gradient magnetometer (agm, micromag tm 2900). the size of nanoparticles was measured with transmission electron microscope (tem) (hitachi-800) and atomic force microscopy (afm) (spm 3800n-400, seiko). all fluorescence measurements were performed by hitachi f-4500 fluorescence spectrophotometer (kyoto, japan). uv-visible measurements were carried out by du-800 spectrophotometer (beckman, england). zeta potential was measured using the malvern zetasizer 3000hs (malvern, england). fluorescent images were taken with a confocal laser scanning microscope (fv500-ix700 olympus, japan). the magnetic nanoparticles coated with amino-modified silica were prepared using the previously reported water-intable 1 sequence of dna probes and targets oil microemulsion [16] . first, suspension of ferrofluid was synthesized by precipitation from appropriate mixture solution of ferrous sulfate and ferric chloride with ammonia. a microemulsion formed by adding 7.2 ml of cyclohexane, 1.8 ml of n-hexanol, 1.8 ml of triton x-100, and adequate aqueous magnetic ferrofluid (fe 3 o 4 ). in the presence of teos and eptes (5:3, v/v), polymerization reaction was initiated by adding 100 l of concentrated nh 4 oh. the reaction was allowed to continue for 24 h to produce magnetic nanoparticles. then, the magnetic nanoparticles were isolated by magnetic separation and washed with ethanol and water for several times to remove all remaining material and surfactant molecules. based on the results of nanoparticle's zeta potential, the proper dispersant was selected (when zeta potential >30 or ≤30 mv, nanoparticles suspension is stable and disperse) to make the nanoparticle suspension. then, the suspension was added dropwise onto the carbon-coated copper membrane and dried at room temperature. the size of aqueous magnetic ferrofluid and amino-modified silica magnetic nanoparticles were measured by a transmission electron microscope. afm measurements were carried out at room temperature on freshly cleaved mica foils. the magnetization properties of both the magnetic core and amino-modified silica magnetic nanoparticles have been measured by using the alternating gradient magnetometer (agm). amino functional groups on amino-modified silica-coated magnetic nanoparticles were confirmed by fluorescent measurement of fluorescamine acetone solution (0.2 mg/ml) before and after incubation with the amino-modified silica-coated magnetic nanoparticle solution. in order to recover the target ssdna, the capture ssdna was first immobilized onto the amino-modified silica-coated magnetic nanoparticles as follows: 10 mg amino-modified silica-coated magnetic nanoparticles was added to 5 ml of glutaraldehyde (2.5 wt.%) and reacted at room temperature for 2 h. then, glutaraldehyde-activated nanoparticles were washed three times with 0.01 m pbs buffer (ph 7.4) and separated under the magnet field. after that, 200 l 1 mg/ml of streptavidin diluted in 0.01 m pbs buffer was added to the nanoparticles suspensions. nanoparticles were washed with 0.01 m pbs buffer solution after being stirred at 4 • c for 24 h. fluorescence spectroscopy of the supernatant was measured to determine the modification efficiency of the streptavidin. then, 1 ml of 4.1 × 10 −6 m biotin-labeled capture ssdna(i) (5 -biotin-aaaaaaaaaagtatcacgaggccctatgcg-3 ) solution was added to the streptavidin derivatived amino-modified silica-coated magnetic nanoparticles and reacted at room temperature for 4 h. then, the nanoparticles were separated from supernatant under the magnet field. the final product was washed, re-suspended in 0.01 m pbs buffer and stored at 4 • c for future use. the modification of biotin-labeled capture ssdna(i) on the nanoparticles was investigated by measuring the absorption of biotin-labeled capture ssdna(i) before and after reaction with the nanoparticles. the mixture was incubated in a 50 • c water bath for 30 min. finally, the magnetic dna bio-conjugate formed between the capture ssdna(i) and its target ssdna(i) was separated from solution under the magnet field and washed three times with 0.01 m pbs buffer. in the control experiment, only streptavidin derivative amino-modified silica-coated magnetic nanoparticles was used to incubate with the target ssdna(i). in this method, the target ssdna is labeled with fluorophore. the recovery efficiency was determined by fluorescent measurement of target ssdna solution before and after hybridization. the fluorescence of the dissociated target ssdna from the magnetic dna bio-conjugate were detected at the same time as follows. first, the separated magnetic dna bio-conjugate was denaturalized to release the bounded target ssdna. it was put in water bath at different temperature (55, 60, 65, 70, 80, 90, and 95 • c) for about 10 min at each temperature. the nanoparticles were separated from supernatant under the magnet field. then, the fluorescence of the supernatant was detected. fluorescence microscopy imaging was also used to detect whether the fluorescence-labeled target ssdna was captured by the capture ssdna-modified magnetic nanoparticles. tem images of both: (a) magnetic core and (b) aminomodified silica-coated magnetic nanoparticles, are shown in fig. 1 . the aqueous ferrofluid (magnetic core) contains regularly shaped magnetite nanoparticles with an average diameter of about 8 nm (fig. 1a) . the nanoparticles did not aggregate even after several weeks based on our experience. when the aqueous ferrofluid was treated with the w/o microemulsion in the presence of a mixture of tetraethoxysilane, n-(␤ethylenamine)-␥-propylamine triethoxylsilane and ammonia (28-30%, w/w), the magnetite core was covered by silica shell and increased the nanoparticle size up to 40 ± 5 nm (fig. 1b) . the prepared nanoparticles appeared to be homogenous with good dispersion. this sample was also examined by afm, and the diameter was in good agreement with the result obtained from tem. both aqueous magnetic ferrofluid and amino-modified silica-coated magnetic nanoparticles have magnetic property. amino functional groups on the aminomodified silica-coated magnetic nanoparticles are important for the further modification of the magnetic nanoparticles. a significant enhancement in fluorescence of the fluorescamine solution after incubation with the amino-modified silicacoated magnetic nanoparticles suspension was observed in the fluorescamine tests. thus, it can be concluded that there are -nh 2 functional groups on the amino-modified silicacoated magnetic nanoparticles. the fluorescence of streptavidin solution at 340 nm decreased from 231.3 (initial solution) to 49.7, after it was reacted with the glutaraldehyde-activated amino-modified silica-coated magnetic nanoparticles for 24 h. these results indicated successful modification of amino-modified silicacoated magnetic nanoparticles with streptavidin. then, this streptavidin-derivatized amino-modified silica-coated magnetic nanoparticles could be used to bind the biotin-labeled capture ssdna(i). the absorption of 4.1 m biotin-labeled capture ssdna(i) solution at 260 nm decreased from 1.1022 to 0.0715 in supernatant after it was incubated with the streptavidin-derivatized amino-modified silica-coated magnetic nanoparticles. the aforementioned results indicated that the bio-molecules have been easily and successfully modified on the nanoparticles using the existing molecular immobilization methods through the amino groups on the nanoparticles. the scheme of recovery of target ssdna based on aminomodified silica-coated magnetic nanoparticles is shown in fig. 2 . strepavidin was first modified onto the aminomodified silica-coated magnetic nanoparticles. the biotinlabeled capture ssdna was then linked with the streptavidinactivated amino-modified silica-coated magnetic nanoparticles through special recognition and binding of streptavidin and biotin. the target ssdna can be recognized fig. 2 . the method scheme for recovery of target ssdna based on amino-modified silica-coated magnetic nanoparticles (asmnps). and hybridized with the capture ssdna to form the dna bio-conjugate, and it can be separated from the solution under the magnet field due to the magnetic characteristics of the amino-modified silica-coated magnetic nanoparticles. however, other mismatches and non-complementary ssdna could not be hybridized completely. capture ssdna(i)-modified amino-modified silicacoated magnetic nanoparticles were used to recover target ssdna(i) in our lab. the fluorescence of the target ssdna(i) solution obviously decreased after it was incubated with the capture ssdna(i)-modified amino-modified silica-coated magnetic nanoparticles, as shown in fig. 3 . meanwhile, the fluorescence intensity of the dissociated target ssdna(i) solution increased gradually when the separated magnetic dna bio-conjugate was denaturalized at a different temperature, as shown in fig. 4 . after denaturalization at 95 • c, for about 10 min, the fluorescence intensity of the dissociated target ssdna was almost the same as that of the initial target ssdna(i) solution (fig. 3) . these results indicate that the target ssdna(i) can be recovered from solution by this method and dissociated from magnetic dna bio-conjugate, which can be used for further research. the fluorescence microscopy imaging of the nanoparticles also showed that the capture ssdna(i)-modified amino-modified silica-coated magnetic nanoparticles displayed intensive fluorescence after it was incubated with the fluorescence-labeled target ssdna(i) and separated under the magnet field. after the separated magnetic dna bio-conjugate was denatured in 95 • c water bath for 10 min and washed for several times, the fluorescence on the separated magnetic dna bio-conjugate disappeared due to the release of the fluorescence-labeled target ssdna(i) from the nanoparticles. in contrast, the control experiment with only streptavidin-modified aminomodified silica-coated magnetic nanoparticles did not show any fluorescence after it was incubated with the fluorescence-labeled target ssdna(i) and separated under the magnet field (fig. 5) . the results demonstrated that the target ssdna(i) could be easily recovered from the solution by using the capture ssdna(i)-modified amino-modified silica-coated magnetic nanoparticles. it became clear that the existence of capture ssdna(i) on the amino-modified silica-coated magnetic nanoparticle was the major cause of the target ssdna(i) recovery. therefore, this method can be widely used to recover different target ssdna when amino-modified silica-coated magnetic nanoparticles is modified with different capture ssdna. a ssdna fragment (3 -cacgaacgtgacgaat-fitc-5 ) of severe acute respiratory syndrome virus was designed as a sample [e.g. target ssdna(ii)] and a trace amount of the sample was recovered from the solution using this method. the 4.1 m biotin-labeled capture ssdna(ii) (5biotin-aaaaaaaaaagtgcttgcactgctta-3 ) was first modified onto the nanoparticles as mentioned above. the target ssdna(ii) was then added to the capture ssdna(ii)modified nanoparticles suspension until the concentration was 6.5 nmol/l. the mixture was incubated in 50 • c water bath for 30 min. then the magnetic dna bio-conjugate was separated from the supernatant under the magnet field. the recovery efficiency was determined by fluorescent measurement of target ssdna(ii) solution before and after hybridization. the selectivity of this method has also been investigated by comparing the separation efficiency of target ssdna(ii) with mismatched ssdna(ii) [one base mismatch ssdna, three bases mismatch ssdna and random ssdna]. recovery efficiency of this method for target ssdna(ii) was shown in fig. 6 . by using capture ssdna(ii), the recovery efficiency for 6.5 nmol/l target ssdna(ii) is 68.17 ± 3.29%, for one base mismatch ssdna is 31.53 ± 2.57%, for three bases mismatch ssdna is 15.33 ± 3.24%. the recovery efficiency for random ssdna is very low. the above results demonstrated the recovery of target ssdna at trace concentrations. good selectivity was confirmed by comparing the recovery efficiency of target ssdna with the mismatched ssdna. in conclusion, we have developed an improved method for recovery of target ssdna based on amino-modified silicacoated magnetic nanoparticles. this bio-separation technology based on magnetic nanoparticles has some unique advantages over the traditional bio-separation technology. it can be done in many laboratories due to the ease on preparation, low sample consumption and simple equipment. this method has been successfully used to recover trace concentrations of a target ssdna fragment of sever acute respiratory syndrome virus with high efficiency and good selectivity. it introduces an effective, selective, and very fast method of recovery for the target ssdna of other viruses, which is very important for disease or mutation detection. scientific and clinical applications of magnetic carriers key: cord-007755-o2r8ktie authors: kokoszka, malgorzata e.; kay, brian k. title: mapping protein–protein interactions with phage-displayed combinatorial peptide libraries and alanine scanning date: 2014-10-20 journal: peptide libraries doi: 10.1007/978-1-4939-2020-4_12 sha: doc_id: 7755 cord_uid: o2r8ktie one avenue for inferring the function of a protein is to learn what proteins it may bind to in the cell. among the various methodologies, one way for doing so is to affinity select peptide ligands from a phage-displayed combinatorial peptide library and then to examine if the proteins that carry such peptide sequences interact with the target protein in the cell. with the protocols described in this chapter, a laboratory with skills in microbiology, molecular biology, and protein biochemistry can readily identify peptides in the library that bind selectively, and with micromolar affinity, to a given target protein on the time scale of 2 months. to illustrate this approach, we use a library of bacteriophage m13 particles, which display 12-mer combinatorial peptides, to affinity select different peptide ligands for two different targets, the sh3 domain of the human lyn protein tyrosine kinase and a segment of the yeast serine/threonine protein kinase cbk1. the binding properties of the selected peptide ligands are then dissected by sequence alignment, kunkel mutagenesis, and alanine scanning. finally, the peptide ligands can be used to predict cellular interacting proteins and serve as the starting point for drug discovery. very often in research projects, there is interest in mapping the protein-protein interactions of a protein of interest as a way of understanding its function in the cell or virus. while a variety of techniques exist for this purpose, such as yeast two-hybrid screening, mass spectrometry, and protein complementation assays, another approach is the use of phage-displayed combinatorial peptide libraries. in such an approach, one takes a purifi ed, recombinant protein and isolates peptide ligands to it through affi nity selection (fig. 1 ) . interestingly, the phage-displayed peptides bind at "hot spots" for molecular interaction and very often share the same primary structure as short regions within cellular interacting proteins. several examples where this approach has proven useful include such targets as protein interacting domains [ 1 , 2 ] . to demonstrate the utility of this approach, we describe its application to two targets, human protein tyrosine kinase, lyn, and the yeast serine/threonine-protein kinase cbk1. lyn was fi rst discovered as a viral oncogene [ 3 ] and later appreciated to be a proto-oncogene in humans. it is a non-receptor protein tyrosine fig. 1 a general workfl ow diagram for isolating and characterizing the peptide ligands to protein domains or fragments using phage display methods. in principle, coding regions of any protein domain of interest can be converted into recombinant dna and expressed as a fusion protein to a partner such as glutathione s-transferase (gst). with a soluble protein in hand, one can perform affi nity selections with phage-displayed combinatorial peptide libraries to identify its peptide ligands. the binding properties of those ligands can be then further characterized through affi nity and specifi city measurements. to assess the importance of each residue in the peptide ligand, a mutagenic analysis known as alanine scanning can be performed on the recombinant dna. with that knowledge, potential interacting partners of the protein of interest can be predicted [ 14 ] . finally, improving affi nity and specifi city of selected ligands through directed evolution may lead to the development of antagonists, which could be used as inhibitors of specifi c cellular interaction for proof-of-principle experiments of drug development kinase, which is a member of the src family of proteins. it has a modular architecture: a src homology 3 (sh3) domain, a src homology 2 (sh2) domain, several linker regions, and a catalytic domain that phosphorylate tyrosines in proteins [ 4 ] . the sh3 domain plays a role in mediating protein-protein interactions and has been described to bind proline-rich motifs in proteins. the cbk1 belongs to a large family of ndr/lats protein kinases, which is conserved across eukaryotes and includes members such as myotonic dystrophy kinase [ 5 ] . cbk1 plays a role in controlling cell separation after cytokinesis, cell integrity, and polarized growth in saccharomyces cerevisiae [ 6 ] . to date, only a few substrates of cbk1 have been reported. one of them, ace2, a transcription factor that is activated by cbk1 via three phosphorylation sites [ 7 ] , is responsible for transcriptional control of enzymes required for septum degradation after cytokinesis [ 6 ] . the other, ssd1, is an rnabinding protein that suppresses translation of certain mrnas and which loses activity when phosphorylated by cbk1 [ 8 ] . we chose these two proteins as targets in parallel affi nity selection experiments to illustrate that the same phage-displayed combinatorial peptide library can yield very different peptide ligands to different targets. the lyn sh3 domain has previously been used in affi nity selection experiments, and so it served as a positive control. the cbk1 protein had not been used previously in affi nity selection of combinatorial peptide libraries. we also show that the kunkel mutagenesis in combination with alanine scanning and elisa are very simple and expedient methods for evaluating the contribution of certain amino acids in the peptide ligands for binding to their targets. prepare using deionized water (dih 2 o). autoclave or fi lter sterilize and store at room temperature, unless indicated otherwise. single-stranded dna samples were isolated with the qiaprep spin m13 purifi cation kit (qiagen). 6. covalently closed, circular double-stranded m13 dna, synthesized via kunkel mutagenesis [ 9 , 10 ] , was purifi ed with qiaquick pcr purifi cation kit (qiagen). 7. concentrations of dna samples were determined using nanodrop nd-1000 uv spectrophotometer (thermo fisher scientifi c, inc.) and measuring the optical absorbance at 260 nm. 8. the following steps: phosphorylation of oligonucleotides, annealing to the template, and synthesis of covalently closed circular double-stranded dna (cccdna) were performed using dna engine dyad ® thermal cycler (bio-rad). in described methods, affi nity selections with phage-displayed combinatorial peptide libraries (figs. 2 and 3 ) were employed to identify peptide ligands that bind to human lyn sh3 domain and yeast cbk1 protein kinase. in general, once peptide ligands have been identifi ed for a protein target, an interacting motif can be deduced via sequence alignment (logo, fig. 4 ) of the primary structures of the displayed binding peptides. however, to assess the functionality of this motif, amino acid replacements of the critical residues are generally necessary. while this is possible through the chemical synthesis of variant peptides, it is more expedient to use mutagenesis techniques, such as alanine scanning (fig. 5 , [ 11 ] ). for the purpose of this selection protocol, the target domain was overexpressed in escherichia coli in the form of a glutathione s-transferase (gst) fusion protein. recombinant protein can be prepared via commercially available pgex vectors (ge healthcare). figure 2 presents a schematic diagram of the selection protocol utilizing glutathione-conjugated magnetic beads. to maintain optimum sterility and eliminate carryover, only fi ltered tips should be used throughout the selection procedure. 1. add 20 μl of glutathione magnetic bead slurry (5 μl of settled beads) into 2 ml eppendorf tube, and wash twice with 600 μl of ice-cold wash buffer #1 (pbs) on magnetic separator. remove the supernatant. 4. remove the supernatant. resuspend in 200 μl of blocking buffer #1. add 50 μg of gst protein to remove potential gst-binding phage clones and 20 library equivalents (e.g., if the library size is 10 10 clones and the titer 10 13 pfu/ml use 20 μl). bring the volume to 600 μl with ice-cold wash buffer #1. incubate at 4 °c for 1 h on the rotating shaker. 5. remove the supernatant. wash three times with 800 μl of icecold wash buffer #2 (pbst). remove supernatant, add 800 μl of ice-cold wash buffer #1, and transfer into a new 2 ml eppendorf tube to eliminate any plastic-bound phage. 6. repeat the washing step two more times with wash buffer #1. remove the supernatant. 7. elute the phage with 200 μl of elution buffer. incubate for 10 min at room temperature, and gently mix the content every couple minutes. 8. place the tube on magnetic separator, and transfer the eluted phage supernatant into a new 1.5 ml eppendorf tube containing 12 μl of neutralization buffer, and mix well. fig. 3c ) revealed a known y/fxfp docking motif to cbk1 [ 17 ] . also, it should be noted that sequence 3 (fig. 3c ) contains the fkfp motif, which is present in ssd1, a known cbk1 substrate [ 8 , 17 ] . our fi ndings clearly indicate that potential binding partners of certain targets can be deduced via selections with phage-displayed peptide libraries. as only three sequences were used for alignment, any additional amino acid preference analysis could be biased. ( c ) further characterization of the isolated y/fxfp motif showed preference to positively charged residues, r and k, at the "x" position. other allowed residues at that position included v, m, t, q, when at least one positively charged residue was observed outside the motif, or c, when a second cysteine was present following the motif. all sequences incorporated in the logo (32 isolates, data not shown) were isolated by screening phage-displayed focused library. the library was synthesized via kunkel mutagenesis (as described in subheading 3. 13. if blue-white screening can be performed, add 45 μl of 100 mm iptg and 80 μl of 2 % x-gal. 14. immediately before plating, add 4 ml of 2 × yt top agar (0.8 %), gently swirl, and pour over prewarmed 2 × yt agar plates. incubate overnight at 37 °c. 7. add anti-m13 hrp-conjugated antibody diluted 1:5,000 in pbst (same per well volume as the target protein), and incubate 30 min to 1 h at room temperature. 8. wash the plates with 200 μl of pbst three more times. 9. add chromogenic substrate solution (subheading 2.1 , item 21 ) (same per well volume as the target protein), and incubate for 10-30 min. 10. quantify the results by measuring the optical absorbance at 405 nm, using a microtiter plate spectrophotometer. all binding phage clones isolated via elisa are amplifi ed and their binding regions subsequently identifi ed by dna sequencing ( see note 7 ). if the selection generates enough unique isolates, the binding motif of the target can be predicted by sequence alignment known as logo plot (fig. 4 ) . with that knowledge, one can attempt to better defi ne the known interactions and to predict new substrates of the target. to further assess the importance of each residue in the motif, mutagenic analysis known as alanine scanning can be performed (fig. 5 , [ 11 ] ). in this method, each residue is replaced, one by one, by alanine (or by glycine if originally occupied by alanine). one way to generate all the variants is to incorporate them into m13 phage genome via kunkel mutagenesis (described in subheading 3.3.1 ) [ 10 , 12 , 13 ] . once the phage-displayed mutant pool is generated, the effect of the substitutions on their binding to the target is evaluated by phage elisa (described in subheading 3.2 ). for the purpose of this protocol, a modifi ed m13 phage vector containing an amber stop codon has to be fi rst obtained [ 9 ] . in the vector constructed by scholle et al., an amber stop codon, tag, has been placed at the n-terminus of the coding region of gene iii, following the signal sequence of protein iii (piii). that modifi cation eliminates the need for generation of uracil-containing single-stranded dna (u-ssdna) template, using e. coli strain cj236 ( dut − ung − ). a mutagenic oligonucleotide, containing both complementary and exogenous dna fragments, is then annealed to the ssdna template, with the exogenous fragment looping over the tag-containing vector region. once the double-stranded dna (dsdna) is synthesized from the ssdna template, it is electroporated into non-suppressor e. coli strain (e.g., ss320). if the tag triplet-containing region has not been replaced by the exogenous fragment, the phage will not be propagated as the minor coat protein, piii, cannot be translated. the wild-type phage can be propagated via the suppressor e. coli strain such as tg1 cells. the amber stop codon is then translated into glutamine. the use of tag-containing template facilitates nearly 100 % recombination effi ciency [ 9 ] . 2. if low quantities of target are available, a small amount can be used to coat the beads (e.g., 10 μg of protein and 10 μl of slurry, respectively). also, decreasing the amount of used target can facilitate isolation of higher affi nity clones. 3. for convenience, target-bound beads can be prepared for the entire selection procedure and stored at 4 °c in the blocking buffer #1 (pbs containing 3 % bsa (w/v)) for up to a week. 4. as long as no contamination is introduced, phage particles can be stored in a culture medium at 4 °c for many years. 5. since the phage titer, recovered after fi nal round of selection, depends on many controllable and uncontrollable factors such as stringency of the selections (i.e., number of washes, amount of target used) or type and structure of the target, from our experience, we recommend to cover a wide dilution range from at least 10 −4 -10 −8 . 6. if fi ltered tips are used for phage transfer, it is convenient to briefl y rinse and remove the tips with multichannel pipette. 7. in order to identify dna sequences of potential "binders," the phage is fi rst amplifi ed by infecting 200 μl of tg1 cells (from a mid-log phase culture) with 10 μl of phage supernatant (or single plaque) and incubated overnight in a shaking incubator (240 rpm), in 5 ml fi nal volume. the pelleted cells are then used for isolation of dsdna that consists of replicative form of phage dna. subsequently, the samples are analyzed via sequencing. remaining phage supernatant can be stored at 4 °c ( see note 4 ). 8 . in order to isolate high quantity of ssdna template, large amount of phage particles has to be fi rst collected. amplifi cation of phage by infecting cells with just a single plaque may result in low quantity of template. thus, we suggest a two-step preparation process, where the peg-precipitated concentrated virions are used to infect the fresh mid-log cells. 9. to remove any polyethylene glycol (peg) residues from the surface of the tube prior to phage reconstitution, it is recommended to fi rst briefl y rinse the side of the tube with pbs, avoiding the phage pellet. usually 1-3 ml of room temperature pbs is used to resuspend the pelleted virions. one of the major advantages of affi nity selection of phage-displayed combinatorial peptide libraries is the potential for rapid discovery of peptide ligands to a target protein. it is relatively straightforward to use homemade or commercially purchased libraries to isolate peptide ligands to a given target and then deduce a binding motif. a peptide with a consensus motif can then be used to study the biology of the target (i.e., predict cellular interacting partner, solve the three-dimensional structure of the peptide and target in a complex, and to inhibit the target in cells). however, when only one or a few peptide ligands are isolated to a target, it is diffi cult to know a priori what residues in the phage-displayed peptide ligand contribute to binding. while one can explore the binding parameters of the peptide sequence through chemical synthesis of peptides with systematic amino acid replacements across the primary structure, we present an alternative, faster, and easier approach involving kunkel mutagenesis, alanine scanning, and elisa. we fi nd that by coupling these three techniques, one can readily determine at fi rst pass many important aspects of the binding interaction. exploring protein -protein interactions using peptide libraries displayed on phage mapping intracellular protein networks the yesrelated cellular gene lyn encodes a possible tyrosine kinase similar to p56lck src family kinases: regulation of their activities, levels and identifi cation of new pathways cbk1p, a protein similar to the human myotonic dystrophy kinase, is essential for normal morphogenesis in saccharomyces cerevisiae the saccharomyces cerevisiae mob2p-cbk1p kinase complex promotes polarized growth and acts with the mitotic exit network to facilitate daughter cell-specifi c localization of ace2p transcription factor the ndr/lats family kinase cbk1 directly controls transcriptional asymmetry cbk1 regulation of the rna-binding protein ssd1 integrates cell fate with translational control effi cient construction of a large collection of phage-displayed combinatorial peptide libraries effi cient site-directed mutagenesis using uracilcontaining dna highresolution epitope mapping of hgh-receptor interactions by alanine-scanning mutagenesis improvements to the kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries rapid and effi cient sitespecifi c mutagenesis without phenotypic selection convergent evolution with combinatorial peptides can we infer peptide recognition specifi city mediated by sh3 domains? distinct ligand preferences of src homology 3 domains from src, yes, abl, cortactin, p53bp2, plcy, crk, and grb2 proteome-wide discovery of evolutionary conserved sequences in disordered regions this work was funded by the chicago biomedical consortium, with support from the searle funds at the chicago community trust. we would like to thank mr. kyle schneider, dr. brian yeh, and dr. eric weiss (nu) for providing the gst-cbk1 dna constructs and purifi ed gst-cbk1 fusion protein. we are grateful to dr. michael kierny and dr. renhua huang (uic) for their helpful comments on this manuscript. key: cord-286684-2xmd3jfo authors: stefanetti, valentina; compagnone, agnese; sordini, chiara; passamonti, fabrizio; rampacci, elisa; moscati, livia; marenzoni, maria luisa title: retrospective biomolecular investigation of coxiella burnetii and leptospira spp. dna in cases of abortion, stillbirth and neonatal mortality in dogs and cats date: 2018-08-20 journal: top companion anim med doi: 10.1053/j.tcam.2018.08.005 sha: doc_id: 286684 cord_uid: 2xmd3jfo abortion and neonatal mortality are events that can occur in breeding bitches and queens. it has been reported that up to 55% and 33% of these cases remain without a known cause, respectively, in canine and feline pregnancies. unusual abortigenic and potentially zoonotic agents, including coxiella burnetii and leptospira spp., may be involved in these cases. c. burnetii is able to cause reproductive disorders in cattle, sheep and goats, and cases of abortion have been observed in dogs and cats. moreover, several outbreaks of c. burnetii infection in humans have been caused by delivering bitches and queens, and some of these animals experienced abortion. leptospira interrogans sensu lato is able to cause abortion or stillbirth in several animal species and its abortigenic role has occasionally been described in bitches and queens. the aim of this study was to search for c. burnetii and leptospira spp. dna in a retrospective series of 103 cases of canine and feline abortion, stillbirth, and neonatal mortality submitted for the identification of possible infectious agents. one hundred and fifty-one specimens were tested using pcr assays and found negative for c. burnetii and leptospira dna. however, in 49 samples (47.6%) other infectious causes of abortion, stillbirth, and neonatal mortality were identified. these results showed that c. burnetii and leptospira spp. are probably not common abortigenic agents or causes of neonatal deaths in dogs. however, given the potential abortigentic and zoonotic role of these agents, surveillance of canine and feline abortion, stillbirth, and neonatal mortality could be advisable for a systematic investigation of these events. abortion, stillbirth, and neonatal mortality are events that can occur in canine and feline reproductive medicine. their reported incidence ranges from 5% to 35%. the etiology of these losses is complex and can be classically divided into infectious and noninfectious causes. among the infectious causes, bacterial diseases are reported as a primary cause of mortality in puppies and kittens during the first week of age. 1 staphylococcus spp., streptococcus spp., escherichia coli, brucella canis, campylobacter spp., and salmonella spp., are described as the most frequently isolated pathogens causing pregnancy losses in bitches and queens and canine and feline neonatal mortality. 2à4 canine distemper virus, canine herpesvirus (chv-1), canine parvovirus (cpv), canine minute virus, feline herpesvirus (fhv-1), feline panleukopenia virus (fpv), and feline infectious peritonitis, caused by a coronavirus, are reported as the most common viral causes of canine and feline pregnancy loss and neonatal mortality. toxoplasma gondii and neospora caninum also are considered as rare causes of abortion and neonatal mortality. 1, 5 nevertheless, a percentage of these cases of abortion, stillbirth, and neonatal mortality remain without an aetiological identification. for example, it has been reported that 33% of feline neonatal mortality 2 and 55% of canine neonatal deaths 3 were attributed to unknown etiology. in these cases, the involvement of unusual infectious pathogens not routinely investigated in canine and feline abortion, stillbirth, and neonatal mortality cannot be excluded. worldwide, c burnetii and leptospira interrogans sensu lato are bacterial agents well known for their potential abortigenic role in many animal species. c. burnetii is the causative agent of an important and underdiagnosed zoonosis with worldwide distribution, q fever. livestock species have traditionally been considered as reservoirs of infection, with microorganisms found in high concentrations in the placenta and reproductive tracts of cattle, sheep, and goats. however, the potential role of other animal reservoirs, including cats and dogs, has been supposed. 6 antibodies against c. burnetii have been detected in the serum of cats worldwide. 7 moreover, it has been hypothesized that cats have been responsible for transmission of c. burnetii to humans in q fever outbreaks due to exposure to parturient queens, in which various degrees of seroprevalence have been detected. 7à10 nagaoka et al. 11 provided the first evidence of active infection, isolating 9 of 29 cases of c. burnetii from vaginal swabs of domestic queens, and c. burnetii dna was amplified through pcr assay from 3 out of 37 uterine tissues from cats with and without a history of reproductive abnormalities. 12 in canine species, the epidemiological role of c. burnetii infection is still debated; however, seropositivity has been reported in dogs, and human outbreaks associated with parturient bitches have been described. 13, 14 c. burnetii dna has also been recently detected in 4 of 54 canine placentas originating from aborting animals in the netherlands. 15 leptospirosis is a worldwide re-emerging disease caused by a gram-negative bacterium of the genus leptospira. there are over 250 pathogenic serovars that are adapted to different wild or domestic animal reservoir hosts, and serovars are further grouped into antigenically related serogroups. infection with pathogenic leptospires can cause a wide range of clinical manifestations from subclinical to severe and potentially lethal disease. 16 however, there are only a few reports of reproductive disorders in dogs related to leptospiral infection; abortion has occurred in dogs after transplacental spread of the serovar buenos aires, 17 and a report suggested that abortion was associated with serovar bratislava infection. 18 some commercial companies that promote diagnostic devices currently suggest including leptospirosis testing in cases of abortion in dogs. in feline species, seroprevalence of leptospira spp. varies from 0% to 35% depending on the geographical area and the diagnostic methods used 16 ; however, despite serological evidence, there is little information about clinical leptospirosis in cats. a recent report described 3 confirmed and naturally infected clinical cases of feline leptospirosis presenting with renal failure. 19 stillbirths, abortions, and retained placenta due to leptospira spp. also have been reported in queens. 20 the aim of this study was to search for c. burnetii and leptospira spp. dna in a retrospective series of cases of canine and feline abortion, stillbirth, and neonatal mortality submitted to a diagnostic laboratory of infectious diseases. considering a normal gestation period of 57-72 days for dogs and 52-74 days for cats, the cases were classified as follows: abortion was defined as fetal loss during the second half of pregnancy and characterized by the expulsion of a dead conceptus or a living one incapable of independent life; stillbirth was defined as puppies or kittens reported as dead at birth; neonatal mortality was divided into early neonatal mortality if puppy or kitten death occurred within 7 days after birth and late neonatal mortality if the death occurred 7-28 days after birth. according to this classification, abortions, stillbirth cases, and dead neonatal dogs and cats were collected between 2008 and 2015 by clinicians specializing in small animal reproduction. the samples were promptly sent to the infectious disease laboratory, department of veterinary medicine (university of perugia) to search for at least one of the most common infectious agents of abortion or neonatal mortality, both bacterial and viral, including chv-1 and cpv for dogs and fpv and fhv-1 for cats. the pathological materials arrived at the laboratory within 24 hours of the event (abortion, stillbirth, or neonatal mortality) and were maintained refrigerated at 4°c until processing. when the veterinarians were not able to send the pathological material within 24 hours, it was stored frozen and sent afterward. while a biomolecular method was used to detect the viral agents (chv-1 and cpv for dogs; fpv and fhv-1 for cats), a standard bacteriological examination was performed. these examinations were not carried out systematically, but only when required by the referring veterinarian and only when the samples were suitable for the test required (for example, only refrigerated samples for the bacteriological examination). for the bacteriological examination, samples were inoculated onto blood agar, mannitol-salt agar, and macconkey's agar plates. the plates were incubated for a minimum of 24 hours at 37°c under aerobic and anaerobic conditions. isolation of a pure or predominant bacterial species from all the specimens originating from the same puppy or kitten, was considered a positive result. the identification of bacteria was performed by examining colony characteristics, gram staining, and use of biochemical tests and commercially available api kits (biomerieux, etoile, france). breed and exposure to risk factors for infectious diseases were not recorded or investigated. dna extraction from placenta (when available) and a pool of lung, liver, and spleen from the canine or feline foetuses or neonates was performed using the genelute mammalian genomic dna miniprep kit (sigma-aldrich, st. louis). the concentration and purity of the extracted dna were quantified using a nanodrop spectrophotometer (nanodrop 2000, thermo fisher scientific, milan, italy). the extracted dna was stored at ¡20°c until use. a pcr protocol described by berri et al. 21 amplifying a fragment of 687 bp of the is1111 insertion sequence was used to detect c. burnetii dna. the previously reported detection limit of the c. burnetii pcr assay was 10 ¡2 dilution of c. burnetii positive control dna (comparable to 12 pg/ml of dna), 12 but considering that the mean number of is1111 copies varies between 7 and 110, 22 no exact limit was defined. a nested pcr protocol for leptospira spp. was developed to increase the sensitivity of a conventional pcr previously described, 23 amplifying a preserved fragment of the 16s rrna gene present in both saprophytic and pathogenic leptospira spp. the first pcr was performed using the previously published primer pair, resulting in a 330 bp product, whereas an internal pair of primers (fn: 5 0 -catg-caagtcaagcggagta-3 0 ; rn: 5 0 -ggctcatctccgagcaataa-3 0 ) was designed using the primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/primer3/), yielding a fragment of 166 bp. serial 10-fold dilutions of the leptospira controls, ranging from 5 ng/ml to 0.05 pg/ml, were carried out to determine the increase in sensitivity of the nested protocol in the biological samples compared with the single protocol. both saprophytic and pathogenic leptospirae, kindly provided by the national reference centre for animal leptospirosis (l. interrogans serovar pomona, type mezzano i; l. interrogans serovar hardjo, type hardjoprajitno; l. interrogans serovar bratislava, type riccio 2; l. kirschneri serovar grippotyphosa, type moskva v; l. biflexa serovar patoc, type patoc), and unrelated bacteria (streptococcus spp.; eschericia coli; klebsiella pneumoniae; pseudomonas aeruginosa; staphylococcus intermedius; proteus vulgaris; enterococcus faecalis), as well as dna from bovine and caprine faeces and clinical specimens from animals with previously diagnosed leptospira infection, were used to test the specificity of the protocol. a total of 25 ml of reaction mixture contained 10 £ buffer, 3 mm mgcl 2 , 200 mm each deoxyribonucleotide triphosphate, 1 mm each primer (sigma-genosys), 0.5 u taq dna polymerase (microtech, italy). a fixed volume of dna was used, 5 ml for the first cycle and 1 ml for the nested protocol. the cycling conditions were as follows: 95°c for 5 minutes, 35 cycles of 94°c for 30 seconds, 62°c for 30 seconds for leptospira and 59°c for 30 seconds for c. burnetii, and 72°c for 45 seconds, and 72°c for 10 minutes. in each set of reactions, a positive control (l. interrogans serovar bratislava, type riccio 2; c. burnetii 9 mile/i/ep1, atcc vr-615), and a negative control (negative sample), as well as a negative reaction mix control (containing the reagents and water instead of dna), were included in each run. a previously published pcr protocol targeting the lipl32 gene of pathogenic leptospira spp. was planned to confirm any case of positivity for leptospira dna. 24 a total of 151 specimens were examined in this study, originating from 103 cases in dogs (n = 94) and cats (n = 9), among which there were 5 abortions (4.8%), 3 stillbirths (2.9%), 24 cases of early neonatal mortality (23.3%), 18 cases of late neonatal mortality (17.5%), and 53 cases in which no age was recorded (51.4%). all the samples tested were negative for leptospira and c. burnetii dnas. detection limits of the leptospira spp. pcr assay were 0.05 ng/ ml in the first cycle and 0.5 pg/ ml in the nested cycle. the nested protocol increased the sensitivity of the test 100 times. considering that the genome of a single leptospira, corresponding to 4.3 £ 10 6 bp in l. interrogans and 3.6 £ 10 6 bp in l. biflexa, weights 5-10 fg, the protocol was able to detect 50-100 bacteria/ml. all the tested leptospira strains used as controls were positive, whereas no amplification was obtained from bacteria other than leptospira spp. or bovine and caprine faeces, confirming the specificity of the test. although not systematically investigated, in 49 cases (47.6%) other likely causes of abortion and neonatal mortality were identified. overall, chv-1 was detected in 7.8% cases (6/77), and one of these showed a simultaneous coinfection with a beta-haemolytic strain of e. coli. fhv-1 was detected in 11% (1/9) and fpv in 9% (1/11) of the cases. bacteriological examination was performed in 62 cases, 38 (61.3%) of which were positive. in particular, e. coli (20/ the role of c. burnettii and leptospira spp. in canine and feline abortion, stillbirth, and neonatal mortality is generally minimally investigated. in this study, no evidence of c. burnetii and leptospira spp. dna was found in cases of canine and feline abortion, stillbirth, and neonatal mortality submitted for the investigation of infectious causes; in fewer than the half of the cases had an infectious agent already been identified as a possible cause. the presence of c. burnetii and leptospira has been previously reported in the same geographical area and time frame in which this study was conducted, although in different animal species; leptospiral dna was found in equine abortion and stillbirth, 25 whereas c. burnetii has been found in sheep in the same area. 26 moreover, diagnoses of cases of leptospirosis in dogs are reported regularly. 27 according to these previous studies, it might be supposed that our negative results could be related to the fact that cases in dogs and cats not exposed to risk factors for leptospira spp. and c. burnetii infections were analysed in this study. indeed, q fever and coxiellosis are traditionally associated with contact with cattle, sheep, and goats, which are considered the main reservoir of infection. in a previous study, which tested canine sera to detect antibodies for c. burnetii, dogs that had contact with ruminants were 10 times more likely to be positive. 28 on the other hand, it has been observed that dogs living in kennels had a higher prevalence of leptospira spp. when compared with client-owned dogs, and that, despite vaccination, risk factors for exposure are relevant for the infection. 29 similarly, in cats, a significantly higher c.burnetii antibody positivity rate was found in stray cats than in pets, 30 and leptospiral antibody prevalence is higher in outdoor cats, as well as in cats living in urban areas and cats that are known hunters. 31 it has been suggested that the active predatory behaviour of some cats increases the chance of infection for both c. burnetii and leptospira spp., due to contact with reservoirs such as rodents. 7, 16 unfortunately, in the current study, a serological investigation to establish the exposure to these pathogens was not performed in the bitches and queens, and no indication of breed or lifestyle was specifically requested upon admission of the specimens. further studies targeting especially populations at risk for these infections should be designed, using animals exposed to water sources or environments contaminated by faeces, urine, or parturition products of animal reservoirs in order to increase the likelihood of finding positive results and determining whether c. burnetii and leptospira spp. are responsible for canine and feline abortion and neonatal losses. overall, it is possible that only a specific subpopulation of dogs and cats, especially pedigree cats and pets, was submitted for diagnostic examination in cases of abortion and neonatal mortality in the current study. a previous retrospective analysis of feline neonatal mortality in the united kingdom suggested that pedigree cat breeders generally investigate the reasons for such deaths 2 and it is possible that infectious diseases associated with environmental exposure are underrepresented in this group of animals. the failure to confirm the presence of c. burnetii and leptospira spp. in this study could also be attributed to the timing of sample collection and to the nonoptimal storage conditions that can occur, according to the specific infection. 32 additionally, in this study placentas were very rarely submitted for analysis because bitches and queens commonly ingest them. examination of the placenta and umbilical cord is recognized as an important means of diagnosing leptospirosis in some species. 33 in addition, there is strong evidence that the placenta represents the critical tissue also for the detection of c. burnetii, because up to 10 9 c. burnetii per g of placental tissue are released in some species at the time of delivery. 34 these results showed that c. burnetii and leptospira spp. are probably not common agents of canine abortion or neonatal deaths, whereas a higher number of feline cases is needed to arrive at conclusions. however, considering that reproductive failure can occur following c. burnetii and leptospira spp. infection, these 2 agents should always be considered in the differential diagnosis in any case of abortion, stillbirth, and neonatal mortality, especially when none of the usually suspected pathogens causing reproductive abnormalities has been identified. 12, 35 moreover, due to the zoonotic potential of these aetiological agents and the close relationship between companion animals and humans, an active surveillance programme encompassing a larger number of canine and feline abortions/stillbirths/neonatal deaths, including a standardized form for recording signals and investigating some risk factors, could be useful to identify these uncommon agents of abortion and neonatal losses and improve the epidemiological knowledge of them. fetal loss in the dog and cat kitten mortality in the united kingdom: a retrospective analysis of 274 histopathological examinations a review of the fading puppy syndrome (also known as fading puppy complex) bacterial and protozoal causes of pregnancy loss in the bitch and queen molecular characterization of canine minute virus associated with neonatal mortality in a litter of jack russell terrier dogs investigating coxiella burnetii infection in a breeding cattery at the centre of a q fever outbreak exposure to parturient cats: a risk factor for acquisition of q fever in maritime canada truckin' pneumoniaàan outbreak of q fever in a truck repair plant probably due to aerosols from clothing contaminated by contact with newborn kittens an outbreak of cat-associated q fever in the united states isolation of coxiella burnetii from the vagina of feline clients at veterinary clinics evaluation of associations among coxiella burnetii and reproductive abnormalities in cats a dog-related outbreak of q fever draft genome sequence of coxiella burnetii dog utad, a strain isolated from a dog-related outbreak of q fever search for possible additional reservoirs for human q fever european consensus statement on leptospirosis in dogs and cats buenos aires, a new leptospira serovar of serogroup djasiman, isolated from an aborted dog fetus in argentina clinical leptospirosis in three cats feline stillbirths associated with mixed salmonella typhimurium and leptospira infection the detection of coxiella burnetii from ovine genital swabs, milk and fecal samples by the use of a single touchdown polymerase chain reaction saint girons i. polymerase chain reaction for detection of leptospira spp. in clinical samples detection of pathogenic leptospira spp. through taqman polymerase chain reaction targeting the lipl32 gene causes of equine abortion, stillbirth and neonatal death in central italy investigation of coxiella burnetii occurrence in dairy sheep flocks by molecular analysis in umbrian area: a pilot study serological surveillance of leptospirosis in italy: two-year national data survey of seroprevalence of q fever in dogs in the southeast of france serological survey of leptospiral infection in kennelled dogs in italy seroprevalence of coxiella burnetii infections among cats in different living environments serologic and urinary pcr survey of leptospirosis in healthy cats and in cats with kidney disease canine and feline abortion diagnostics funisitis associated with leptospiral abortion in an equine placenta q fever: a zoonosis canine leptospirosisàdo we have a problem? the authors would like to thank carlo sanesi for his helpful technical support. we also gratefully acknowledge dr silvia tagliabue and the national reference centre for animal leptospirosis (istituto zooprofilattico sperimentale lombardia ed emilia romagna) for kindly providing the leptospiral serovars used as positive controls. key: cord-009894-iciaa829 authors: scott‐taylor, tim h.; ahluwalia, gurmuk; dawood, magdy; hammond, gregory w. title: detection of enteric adenoviruses with synthetic oligonucleotide probes date: 2005-12-07 journal: j med virol doi: 10.1002/jmv.1890410414 sha: doc_id: 9894 cord_uid: iciaa829 the abilities of hybridization probes to detect all human adenovirus types and to identify enteric adenovirus types were evaluated. the efficiency of hybridization was compared to other tests currently in routine laboratory use on clinical specimens from young children with gastroenteritis, probes were derived from various regions of the adenovirus types 2 and 41 genomes, and were evaluated by hybridization with a series of dna quantities from 1 μg to 10 pg of one adenovirus type from each human subgenus, lambda phage, and hep 2 cells. the sensitivity of hybridization with the hpii probe (92.7%), containing the conserved hexon gene, compared well with em (54.6%), culture and neutralization (45.5%), and enzyme immunoassay (61.8%). the sensitivity of detection of enteric adenovirus isolates by the cloned bg/ii d fragment probe (92.9%) and by a synthetic probe (85.7%), manufactured from type‐specific sequences of the ad41 hexon gene were comparable to ad40/ad41 specific enzyme immunoassay (84.6%). hybridization was found to be a sensitive method of adenovirus detection in comparison to traditional methods of laboratory diagnosis. synthetic oligonucleotides enable specific detection of individual enteric adenovirus types. hybridization had additional advantages over other tests in identifying cases of infection with more than one adenovirus type and in allowing an estimate of the concentration of adenovirus in the specimen. despite the specific detection of adenoviruses in 5 1 7 % of young children with gastroenteritis [christiensen, 19891 , determination of the involvement of adenoviruses in the aetiology of gastroenteritis has been difficult. adenoviruses have been found consistently in the stools of apparently healthy children during surveillance programs [fox et al., 1977; rodriguez et al., 19851. the ubiquity of adenoviruses makes it difficult to establish criteria to define adenoviral agents of gastroenteritis and prevents unequivocal substantiation of adenoviral causation of diarrhoea [madeley, 19831 . the problem is compounded by differences in pathogenicity and cultivability between adenovirus types. the readily grown, lower numbered adenovirus types can be carried asymptomatically [fox et al., 1977; kidd et al., 1982; rodriguez et al., 19851 , while isolates of the enteric adenovirus types 40 and 41 that cause the majority of clinical disease [uhnoo et al., 1984; wigand et al., 19831 have fastidious growth characteristics and tend to escape identification. additionally, enteric adenoviruses present in dual infections are frequently not observed [brown, 19851. adenoviruses of a different type or viruses of most other groups tend to overgrow enteric adenoviruses in culture, even when present at a lower concentration [brown, 19901 , and fastidious adenoviruses are underdiagnosed. adenoviruses are found as the only pathogen present in a high proportion of stools of sick children [christiensen, 19891 and should be included in a diagnostic protocol for pediatric gastroenteritis. an adenovirus test should operate directly on the initial specimen, to avoid difficulties with culture. the capacity of the test to distinguish the enteric adenovirus types, which have a specific association with gastroenteritis, would be advantageous. in this study we examined cloned and synthesized sequences for the detection of all adenovirus types and for the specific identificationof enteric types by hybridization. the sensitivity and specificity of the hybridization probes were evaluated in comparison to other conventional methods of adenovirus detection on clinical specimens from children with gastroenteritis. strain dugan, and ad41 strain tak from each subgenus a to f were obtained from american type culture collection (atcc, 12301 parklawn drive, rockville, md). viruses were cultured, purified on cscl density gradients, and extracted as previously described [scott-taylor and hammond, 19921 . fragments of the adenovirus genome ad41 strain tak were amplified in competent e . coli strain jm109 in plasmid pgem 32 (promega biotech, mississauga, ontario) . ecori fragments a, b, and c were cloned in plasmids p41eac (containing both a and c fragments), p41ea, p41eb, and p41ec. ad41 hexon gene sequences were electroeluted from p41ea after digestion with sali and hindiii. an attempt was also made to isolate the type-specific hexon gene sequences by cloning hind11 fragments of the p41ea plasmid in the blunt-ended smai site in the pgem 32 vector. useful transformants were identified by colony hybridization with the salid fragment of the ad41 genome. the bgliid fragment probe was kindly donated by howard takiff in the form of a n insert in vector pat153 [takiff et al., 19851 . the hpii probe, a hindiii-puuii fragment enclosing the complete sequence of the ad2 hexon gene, was evaluated in previous experiments as containing the most conserved adenovirus sequences, which enable this probe to detect all adenovirus subgenera with uniformity [scott-taylor et al., 19921 . adenovirus dna sequences were analysed with pustell sequence analysis software (ibi, new haven, ct) on genomic sequences recorded by genbank. the suitability of oligonucleotide sequences for use as probes was determined with the program oligo (national bioscience, hamel, mn). to ad41 or ad40 lwood et al., 19891 , supplied by j a n de jong, were used in a blocking immunoassay as a further means of identification of a sample of 5 uncultivable isolates. identification was determined by the ability of the specific antisera to block the binding of the isolates to microtitre wells coated with a capture antibody (ahluwalia et al., in preparation) . the specificity of probes was evaluated with dilutions of dna of one type of adenovirus from each subgenus, lambda phage, and hep 2 cells, spotted a t 10 pg to 100 pg per ml. dna preparations were denatured with the addition of 0.1 volume of 3 m naoh, neutralized after 30 min incubation with 0.1 volume of 3 m ammonium acetate, and equilibrated to physiological salt conditions with the addition of 0.3 volumes of 20x ssc ( i x ssc = 0.15 m nac110.015 m na citrate). then 150 p1 of each dilution was applied by a slot blot apparatus (schleicher and schuell, no. 03431, keene, nh) under very low vacuum to nylon membrane prewetted in 6 x s s c . preliminary investigation demonstrated that the protein extraction of the clarified stool suspension improved the clarity and completeness of hybridized spots, as noted by kidd et al. [19821. stool suspensions in 450 pl aliquots were incubated with 50 pl of 10% sds and 250 pg of proteinase k for 30 min at 37°c before extraction with phenol and chloroform. extracted samples were then boiled and cooled in ice water before 150 pl of sample were applied to prewetted nylon membranes with the slot blot manifold under low vacuum. membranes were washed twice in 6x ssc, air dried, and baked at 80°c for 2 h prior to hybridization. hybridization was carried out a t 68°c as previously described [scott-taylor et al., 1992al with a t least lo7 cpm/ml random prime labelled probe (boehringer mannheim, kit no. 1004 760). the melting temperatures of some hybridizations were lowered by the addition of formamide to the hybridization solution, according to the estimate that 1% formamide lowers the melting temperature by 0.72"c [mcconaughy et al., 19691 . enteric adenovirus ad41 ecori fragments a, b, and c, together comprising 84% of the genome [scott-taylor et al., 19921 , were used to evaluate the specificity of ad41 sequences for the detection of enteric adenoviruses. the ecori fragments, cloned in plasmid vectors, were hybridized with a series of adenovirus dna preparations of each subgroup. the reaction of the dna dilutions with a genomic ad41 dna probe is shown in the first panel of figure 1 as the standard to which other probe reactions were compared. the reaction of plasmid p41ea with the subgroup dnas is shown in panel ii. this large plasmid, containing over 50% of the ad41 genome from 8 to 61 map units, reacted more strongly with the dna between july 1990 and june 1991, 1,071 stool specimens received a t the cadham provincial laboratory were examined for the presence of adenovirus by electron microscopy (em), tissue culture, and enzyme immunoassay (eia). ten percent stool suspensions were made by emulsification of approximately 1 g/10 ml phosphate buffered saline (pbs) containing antibiotics in polypropylene tubes with glass beads over a vortex mixer. suspensions were clarified by centrifugation at 3,020 x g (5,000 rpm in a sorval rt600 centrifuge) for 20 min. em examination was enhanced by ultracentrifugation of clarified suspensions onto formvar-coated grids by means of an airfuge [hammond et al., 19811 . two commercial immunoassay kits were employed for detection of all adenoviruses and for identification of enteric types according to the manufacturer's instructions (cambridge bioscience, worcester, ma). culture was performed by inoculation of 100 p1 of clarified suspension applied to semiconfluent monolayers of 293 cell, primary rmk and hep 2 cell lines. specimens which grew virus in the conventional hep 2 or rmk cells were further tested with neutralizing antisera (obtained from atcc) to the 6 lowest numbered species. non-neutralized virus isolates were identified by restriction analysis. two monoclonal antibodies specific of other subgroups than the whole ad41 genomic probe, detecting lower amounts of heterologous subgroup dna relative to the quantity of ad41 dna detected in the same autoradiographic time interval. various puui fragments were electroeluted from the p41ea plasmid to assess the specificity of isolated central sequences of the ad41 genome as probes. puui fragments b, d, e, and f, extending from map units 26 to 48,48 to 59,17 to 21, and 21 to 26, respectively (fig. 1, re map) , all demonstrate reactions (fig. 1, panels iii, iv, v, and vi) comparable to the parent plasmid. isolation of segments of the ecori a fragment by cleavage with puui did not demarcate any area able to better distinguish between the dna of ad41 and other types. the puui f fragment shown in panel vi may be useful as a subgenus f specific probe. this fragment detected ad41 dna within one log dilution of the reaction with ad41 dna and distinguished between other subgroups by at least 3 log dilutions. puui f fragment probe has an approximately equal reactivity with enteric types and would not detect other adenovirus types unless present a t 1,000 times the concentration of ad41 virions. type-specific probe a number of fragments from different areas of the ad41 genome were examined for their ability to differentiate between enteric adenovirus types. the reactions of these various probes are shown in a succession of panels in figure 2 as tested against both enteric adenovirus dna preparations on membrane spotted with dna of species of each subgroup. in comparison to the whole ad41 dna probe in the uppermost panel, the ecori b and c fragments inserted in pgem 32 vector have a relatively insensitive reaction with the dna of other subgroups (panels ii and iv), corresponding with their position at the nonconserved right-hand end of the genome. the bgzii d fragment, derived from the portion of the ecori b fragment nearer the right terminus of the ad41 genome, hybridized with greater relative intensity with homologous ad41 dna than the parent plasmid. the reaction of the bgzii d fragment with ad40 dna in panel iii of figure 2 is highly equivalent in sensitivity to the homologous dna reaction. the difference in sensitivity for ad41 and ad40 dna, apparently only 2 to 4 fold, is the least of any of the ad41 fragments tested, and the bglii d fragment was the best prospect for use as a n enteric adenovirus specific probe defined. further attempts to distinguish an ad41 specific probe were made by testing small restriction fragments from within the hexon gene that code for the type-specific epitopes of the capsid [roberts et al., 19861 . the reaction of electroeluted sazi d and hind111 i fragment probes (fig. 2 , panels v and vi) do not adequately distinguish ad41 dna from dna of other types for use as specific probes. the reaction of a cloned hind11 hexon fragment probe, called plasmid p41hh in panel vii of figure 2 , varies with the different subgroup dna prep-arations. no ad41 specific probe was isolated by cloning or electroelution of ad41 dna fragments. published ad41 sequences were compared to ad2 and ad40 genes to determine exact sequences unique to ad41. the longest stretches of unique ad41 sequence in the available sequences were found in the hexon gene. the l1 surface epitope sequence [roberts et al., 19861 was divided into 4 sequences of variation of 30 base pairs or more that could serve as diagnostic probes. the most suitable sequence, however, was a fifth unique stretch of 84 nucleotides from residues 1225 to 1308 of the ad41 hexon sequence [toogood and hay, 19881 forming the l2 epitopic loop. this sequence was synthesized as two 40 base oligomers, designated hex5a and hex5b, signifying the fifth unique hexon region, with the following sequences: the sensitivity of the two probes was tested empirically by hybridization with dilutions of adenovirus, a phage, and hep 2 dna in conditions of increasing stringency as shown in a series of panels in figure 3 . hex5a has a greater content of guanine and cytosine residues and could be used in conditions of greater stringency (fig. 3a) . hex5b sequence is unique to ad41. this is reflected in the loss of reactivity with ad40 and nonhomologous dna in reactions carried out above 35°c (fig. 3b ). the hexeib probe was used in evaluation of the hybridization test with clinical samples. electron microscopy, eia, and viral culture of stool specimens were performed routinely through the period of study. adenovirus was detected by a t least one conventional test in 55 specimens of the 1,071 stool samples examined. isolates were identified by neutralization or restriction analysis. all identified enteric isolates had restriction patterns of the ad41a strain [scott-taylor et al., 19901 . the positive samples were spotted in random order among 200 samples on a nylon membrane for hybridization. suspensions used in prior evaluations of 9 adenovirus positive specimens were spotted to determine whether virus in original suspensions had deteriorated. samples 81 and 88 were from a single specimen, spotted twice to ensure reproducibility. the 200 spotted samples consisted, therefore, of 55 unique adenovirus positive specimens, 28 of which were assessed as enteric isolates, 10 duplicates, and 135 adenovirus negative specimens. twelve of the adenovirus negative samples contained rotavirus, 8 grew enteroviruses, and small round virus particles were seen by electron microscopy in 6 and coronavirus in 1 more. the specimens reacting with the various diagnostic table i in the order spotted on the hybridization membrane. a total of 60 of the samples, including 51 of the unique specimens, reacted with the hpii probe in the first panel of figure 4 . the bgeii d fragment and synthetic hex5b probes in the two panels below reacted with 26 and 24 specimens, respectively. the amount of viral dna in the specimen could be estimated from the dilution series of control dna below the specimens. the ad41 genomic dna probe and plasmid p41ec, containing the ad41 ecori c fragment, hybridized with specimens containing adenoviruses other than enteric types (reactions not shown). cloned probes p41ec and bgzii d reacted with negative specimens 122,124, and 166. specimens 27 and 199 reacted strongly with all ad41 dna probes, although types ad2 and ad5 emerged from culture. these specimens were subsequently tested with the subgenus f-specific eia and were found to harbour a conventional type as well as a n enteric adenovirus in concurrent infection. a sample of 5 of the 9 uncultivable specimens not identified to type was tested in the blocking assay. preincubation with the ad41 monoclonal antibody reduced binding to the capture antibody by between 12% and 37%. the ad40 antibody had no effect and for the purposes of calculation of test performance all the uncultivable specimens were presumed to contain ad41. the sensi-tivity and specificity of the diagnostic tests are compared in table 11 . the 92.7% sensitivity of the hpii probe in hybridization, detecting 51 of 55 unique specimens, compares favourably with conventional diagnostic tests. the specificities of the genomic ad41 dna, p41ec, andbgzii d probes were evaluated on the detection of enteric adenovirus types and were reduced by reactions with unrelated adenovirus types or negative specimens. the synthetic dna probe specificity was evaluated for ad41 specimens alone. no false positives were attributed to hpii or hex5b probes. the predictive values of the tests (table 11) indicate that most hybridization probes had greater reliability in reporting a positive or negative test result than the conventional diagnostic methods. hybridization demonstrated a higher sensitivity than the methods of adenovirus detection currently employed. results indicate that hybridization could improve the efficiency of diagnosis of adenovirus infection in gastroenteritis by one and a half times or more over individual methods in routine use. the technique has great flexibility and can utilize cloned or synthetic sequences to diagnose groups or individual types of virus. oligomeric probes can evidently form effective means of diagnosis, and selection of the appropriate shared or unique sequence can enable differentiation of groups or individual adenovirus types according to the degree of specificity required. the type-specific hexon sequences provide a means to differentiate between closely related adenoviruses by hybridization with dna probes or neutralisation with antipeptide sera [toogood et al., 19921 . hybridization had several additional advantages over other diagnostic methods in enabling the detection of dual infections and allowing an estimate of the concentration of viral particles present in the specimen. dual infections are probably not uncommon judging from the numbers of specimens that yield more than one adenovirus type upon careful culture [brandt et al., 1986; brown, 1985; kidd et al., 1982; wigand et al., 19831 , and none of the traditional methods in use are capable of identifying more than one isolate in a specimen. the intense hybridization associated with most enteric adenovirus isolates indicated that these types were excreted in greater concentration than the conventional types. the most reactive of the enteric specimens were defined from the control dilution series as present in excess of 100 ng of viral dna in 150 pl of the 10% stool suspension spotted. since the ad41 genome comprises 34,600 base pairs [scott-taylor and hammond, 19921 and has a molecular weight of approximately 23 x lo6, it can be calculated that there are about 2.5 x 10" molecules of the ad41 genome per microgram of dna. therefore, in the 150 pl of stool suspension, containing 15 mg of stool and 0.1 pg of viral dna, there are more than 2.5 x lo6 genomes, and there are more than 1.7 x 10l1 virus particles per gram in the most reactive stool specimens. this is in close agreement with the previous evaluation that enteric adenoviruses can be excreted in excess of 10l1 particles per gram of stool [takiff et al., 19811 and demonstrates that hybridization can define viral concentration with some reliability. an association between virus concentration and disease is not clear at present. examination of the relationship between viral burden and prognosis with this technique could yield significant results. additionally, evaluation of viral concentration may be helpful in determining a critical level for defining causation of gastroenteritis by certain adenovirus types. the loss of specificity of some hybridization probes was probably due to reaction with plasmid dna from bacterial flora, as several false-positive specimens reacted only with probes amplified in bacteria. the reaction of vector dna with plasmids derived from alimentary flora has previously been observed [huang and deibel, 1988; takiff et al., 19851 . the lack of falsepositive reaction of the hpii probe, however, demonstrates that extensive electroelution can render probes sufficiently free of plasmid dna contamination for use with faecal specimens. it may be advisable to saturate the probe with unlabelled plasmid dna to completely eliminate this source of false results in hybridization. the uncultivable specimens tested with the blocking assay were reduced in binding by less than the 50% critical value that identifies a n adenovirus type by the test methods. the low reduction in binding may signify that either these specimens were grossly denatured or that commercially available ad41 monoclonal antibodies [herrmann et al., 1987; wood et al., 19891 are not able to react efficiently with local variant strains of ad41 specific to manitoba. the prevalent ad41 strain in manitoba was not detected by the first commercial enzyme immunoassay [scott-taylor et al., 19901 marketed with monoclonal antibodies developed to the prototype strain tak of ad41 [herrmann et al., 19871. these observations suggest that dna hybridization tests, less affected by the variation found in circulating strains of adenovirus types, may have more long-term efficacy than highly specific serological detection methods. simultaneous infections with different enteric and respiratory tract viruses selection of nonfastidious adenovirus species in 293 cells inoculated with stool specimens containing adenovirus 40 laboratory identification of adenoviruses associated with gastroenteritis in canada from 1983 to 1986 human viral gastroenteritis the seattle virus watch. vii. observations of adenovirus infections improved detection of viruses by electron microscopy by direct ultracentrifuge preparation of specimens preparation and characterization of monoclonal antibodies to enteric adenovirus types 40 and 41 nucleic acid hybridization for detection of cell culture amplified adenovirus faecal adenoviruses from glasgow babies. studies on culture and identity viruses and diarhoea-problems in proving causation nucleic acid reassociated in formamide fecal adenoviruses from a longitudinal study of families in metro three dimensional structure of the adenovirus major coat protein hexon restriction analysis of the prototype strain of enteric adenovirus type 41 with exonuclease 111 prevalent enteric adenovirus variant not detected by commercial monoclonal antibody enzyme immunoassay conserved sequences of the adenovirus genome for detection of all human adenovirus types by hybridization propagation and in vitro studies of previously non-cultivable enteral adenoviruses in 293 cells detection of enteric adenoviruses by dot-blot hybridization using a molecularly cloned viral dna probe dna sequence of the adenovirus type 41 hexon gene and predicted structure of the protein antipeptide sera define neutralising epitopes on the adenovirus hexon importance of enteric adenovirus 40 and 41 in acute gastroenteritis in infants and young children isolation and identification of enteric adenoviruses evaluation of a commercial monoclonal antibody-based enzyme immunoassay for detection of adenovirus types 40 and 41 in stool specimens key: cord-291749-revhbd0q authors: mongan, arthur elia; tuda, josef sem berth; runtuwene, lucky ronald title: portable sequencer in the fight against infectious disease date: 2019-10-03 journal: j hum genet doi: 10.1038/s10038-019-0675-4 sha: doc_id: 291749 cord_uid: revhbd0q infectious disease is still a major threat in the world today. five decades ago, it was considered soon to be eradicated, but the adaptation of pathogens to environmental pressure, such as antimicrobials, encouraged the emergence and reemergence of infectious disease. the fight with infectious disease starts with prevention, diagnosis, and treatment. diagnosis can be upheld by observing the cause of disease under the microscope or detecting the presence of nucleic acid and proteins of the pathogens. the molecular techniques span from classical polymerase chain reaction (pcr) to sequencing the nucleic acid composition. here, we are reviewing the works have been undertaken to utilize a portable sequencer, minion, in various aspects of infectious disease management. infectious disease has always been intertwined with human history. poliomyelitis was already documented in egyptian papyrus. leprosy, plague, cholera, yellow fever, influenza epidemics, or pandemics were the norm [1] . as the civilization went forward, the development of microscope allowed the visualization of microorganisms for the first time, changing the paradigm of infectious disease [2] . soon, the discovery of pathogenic agents displaced the preexisting theories of the cause of infectious disease. vaccines were developed to prevent contracting or spreading the disease and methods of food sterilization were deemed valuable for disease control. the fight against infectious agents was so efficient that in the 1960s, control and prevention measures had decreased the incidence of many infectious diseases [1] . smallpox and rinderpest were controlled from the world. nevertheless, emerging and reemerging infections are becoming significant worldwide problems. pathogen adaptation seems to hold the key for the emergence or reemergence of infectious disease. the acquisition of drug resistance, for example malaria parasites to chloroquine or the mosquito vector to insecticide, is one of the factors that prevent the eradication of malaria worldwide in 1955-1978 [3] . other example is the human immunodeficiency virus emergence in the past century due to simian immunodeficiency virus coincidental transmission to humans [4] [5] [6] and the 2009 h1n1 pandemic influenza virus that emerged from pigs, is a legacy of influenza a virus causing pandemic in 1918 [7] . the definitive diagnosis of many infectious diseases still relies on direct observation of the causing pathogens. however, at many times this is difficult to achieve, especially for viral diseases because of the size of virus particles. for example, the microscopic examination of acid-fast bacilli remains the main tool for tuberculosis detection, yet the technique sensitivity varies on smearing, staining, and slide reading [8] . parasites such as microfilaria depends on the sampling time for positive observation [9] . therefore, molecular techniques, such as pcr or enzyme-linked immunosorbent assay have been developed to assist in a more reliable diagnosis. they are designed to detect the presence of infecting pathogen molecularly. because it directly measures the nucleic acids or proteins of infecting pathogens, the risk of missing the organism in direct observation may be avoided. traditionally, laboratory test for drug resistance is a long and laborious process. first, culturable microorganisms must be cultured on various growth mediums. the time needed for colonies to form also varies greatly. then the colonies must be subjected to different type of antimicrobials until the susceptibility or resistance can be distinguished. in certain cases, drug resistance analysis is very critical to patients' prognosis, therefore traditional process is impractical. here is the niche for genetic analysis to expedite the drug resistance tests. there are many genetic analyses designed to confirm diagnosis or to test for microbial resistance. pcr can be used to detect the presence of pathogen dna. using multiplex primers targeting dna of many organisms, an investigator can detect the presence of single or multiple infecting agents [10] . further investigation using primers targeting the presence of known mobile genetic elements can decipher the antibiotics resistance that may be present in the clinical sample [11] . pcr can also be used to check for single nucleotide polymorphisms conferring drug resistance. by creating primers matched the substituted nucleotide, the susceptibility or resistance can be determined by the absence or presence of certain bands [12] . combination with real-time pcr using probes specific to mutations will amplify mutated gene and can be observed in real time [13] . sequencing of pcr amplicons or whole pathogen genomic dna can be applied to comprehensively screen for infecting agents and their drug resistance phenotype, if exists, at the same time. sequencing technique has been around since 1975 by the works of frederick sanger, allan maxam, and walter gilbert [14] . since then, the technology has been modified and refined with the automation of the original technique, the inception of next-generation sequencing, and the advent of long-read sequencing. with each iteration, the data throughput is increased, cost is reduced, and physical form of the sequencer is reduced. sequencing ensures detection of dna composition unique to an organism, the presence of antimicrobials-destroying genes, or mutation that can change the function or conformation of proteins related to drug resistance. further application of sequencing using the next-generation platforms encompasses epigenetic profiling as well [15] . next-generation sequencing incorporates many technologies, but mainly they include the incorporation of terminating nucleotides to a dna polymerization process [14] . these termination nucleotides can be paired with excitable compounds that is excited by laser beam; and depending on the compound, the excitation process will emit different wavelengths which can be identified as different nucleotide. a different kind of dna sequencer uses nanopore protein to recognize the nucleotide in question. here, the disturbance in the basal electrical current inside the protein caused by certain nucleotide strands will be recognized as specific patterns and converted into sequences of dna by specific algorithm [16] . the deviation from common sequencing principles allows the new sequencing platform to be miniaturized ( fig. 1 ). this opens new powerful aspects of sequencing that could never been achieved before. the portable sequencer, minion, can be transported to countries or locations where performing sequencing is difficult or transferring samples to other countries or locations is forbidden. for example, minion has been taken to a remote rainforest of tanzania [17] , ecuador [18] , the canadian high arctic [19] , and even the international space station [20] . minion helped the surveillance and sequencing of zika virus in the 2016 brazil outbreak [21] . during the project, it was found that most of the genome were fragmentary. low titer was correlated to sequencing less than 50% coverage, so 'tiling amplification' was used to amplify the whole genome of zika virus. this technique also was employed in the ebola virus (ebov) outbreak in guinea to get a high nucleic acid concentration for sequencing [22] . the portability of minion comes at the expense of sequencing accuracy. despite recent improvements to the chemistry and computational tools, the sequencing errors are still between 5 and 15% [23] . consensus calling using bioinformatics tools can improve accuracy to more than 97% [24, 25] . when consensus calling was applied to simultaneously screen for multiple antimalarials resistance, it was clear that north indonesia, north vietnam, and southeast thailand had different mutation patterns in k13 gene [26] , the gene related to artemisinin resistance, that were in concordance with a major paper describing the artemisinin-resistance related mutations around the mekong region [27] . nevertheless, the mutations conferring resistance to chloroquine were similar in these three regions. this method applies the amplification of multiple genes with pcr. the sequencing depth is high so consensus sequence can be generated with high confidence. targeted sequencing is cost efficient by performing multiplex sequencing. although the running cost of other sequencing platform is cheaper than minion (for example, $67.82 for miseq compared with $71.56 for minion per sample [28] ), minion has significantly lower cost to set up. targeted sequencing with minion is powerful and fast to detect pathogens in clinical samples. bacterial fig. 1 the portable sequencer, minion, is being handled prior to sequencing composition from empyema patient's pleural effusion could be understood within 2 hours after obtaining the dna sample [29] . here, the amplicons of 16s rrna gene were sequenced to identify the bacteria. due to the capability of long-read sequencing, the gene can be sequenced in entirety, increasing the resolution to detect the infecting bacteria species. bioinformatics tools can be used to increase the accuracy of the amplicons and determine the bacteria composition up to species level [30] . the employment of targeted sequencing using 285 and 256 primer pairs enabled the recognition of the causative agent of viral hemorrhagic fever within 10 min of sequencing, and a definitive diagnosis can be procured in less than 3.5 h [31] . other techniques can be used to amplify the gene of interest instead of pcr (see table 1 for an outline of methods employed with minion). loop-amplified isothermal amplification (lamp) is method to amplify a gene segment flanked by two to three pairs of primers in an isothermal condition [32] . further, lamp reagents can be dried to assure their stability upon transportation to the field [33] . applying the lamp technique as the amplification method, one group of researchers performed a genomic epidemiology study of dengue virus (denv) in indonesia and vietnam [34] . up to 141 and 80 denv-positive samples were amplified isothermally and sequenced with minion. the successful detection rate was 79%. serotype could also be determined despite the 74-80% identity. the lamp-minion technique can be applied to other pathogens as well, such as malaria [35, 36] and chikungunya [37] . with the increase of minion sequencing power, wholegenome sequencing of pathogens [38] [39] [40] and human [41] using minion has become common practice in the field or clinical settings. metagenomic sequencing, previously limited to high-throughput short-read sequencers, has gotten more applications with minion [42] . although the ideal approach is to make no presumptions about the infecting pathogens and sequence all the dna materials contained in the environmental or clinical specimens, there are many technical difficulties, such as the low titer of the infecting agents [21, 34] and large host dna background [43] . sequencing the cultured isolates might overcome the problems. however, because only 1-2% of bacteria can be cultured [44] , unculturable bacteria might be overlooked, so are the cases with virus, fungi, or parasite infections. for metagenomic sequencing, there are several methods can be employed to overcome the difficulties. host dna depletion with saponin [45] , negative cpg selection [46] , or physical disruption of bacterial wall [47, 48] may be used to remove or reduce the background dna and to increase sensitivity. filtration, nuclease digestion, and random amplification allow reliable recovery of viral genomes [49] . whole genome amplification by multiple displacement amplification (mda) can also be performed for low dna concentration [50] . this technique uses the high-fidelity phi29 polymerase combined with random hexamer primers to amplify dna in isothermal reaction. specific mda protocols for amplifying plasmodium dna from whole blood [51] and mycobacteria dna from sputum [52] have been published. metagenomic sequencing with minion has been used in many clinical studies. chikungunya virus (chikv), ebov, and hepatitis c virus can be detected from clinical samples after amplification of viral genomes [38] . in a similar experiment, a coinfection of denv and chikv was found from one clinical specimen [39] . pathogens causing lower tract infections can be identified by sequencing bronchial lavage with or without host depletion [45, 53] . this allowed the finding of bacteria composition and drug resistance phenotype in lower respiratory infection, which led to early antibacterial therapy within 6 h [45] . other advantage of minion is the ability to physically sequence long reads. using ultra-long-read sequencing protocol, a read >2 megabases is obtainable with special protocol [54] . this is particularly important for de novo sequencing of clinical samples, which can have a very elaborate gene structures harboring drug resistant genes with high copy number. for example, plasmid-mediated extended spectrum beta-lactamase escherichia coli strains can have large numbers beta-lactamase genes, leading to high-level resistance [55] . the long-read sequencing could assist the investigation of antimicrobial gene location, copy number, and potential transposon-driven rearrangements of e. coli [56, 57] and salmonella typhi [58] isolates that amplified with lamp [34] [35] [36] [37] whole-genome sequencing metagenomic sequencing [38-41, 45-49, 51-53, 59] isolate sequencing [56] [57] [58] advanced techniques rna/cdna sequencing [61] [62] [63] epigenetic sequencing [66, 70, 71] otherwise could not be capture by short reads by assembling the genome de novo. furthermore, field sequencing using minion of lassa virus outbreak in nigeria molded the management response by the government [59] . by confirming the outbreak was caused by the same strains that transmitted from rodents to humans, the government focused the efforts on rodent control and safe food storage. since lassa virus is genetically highly diverse, long-read sequencing is the method of choice. nanopore sequencing is not limited to dna. rna sequencing, either directly or after cdna conversion, has been widely performed with minion. studies in rna virus will gain substantial benefit because most of the high-profile human viral diseases that recently emerged are caused by rna virus [60] ; sequencing the virus in their natural state can open a lot of exciting findings. already, direct sequencing of rna virus and transcripts of dna virus reveals the structural variants of coronavirus [61] , an mrna that accumulates late in infection of herpes simplex virus-1 [62] , and the discovery of novel rna molecules and transcript isoforms of varicella zoster virus [63] . advanced application of minion in the field of infectious disease is the epigenetic sequencing. epigenetics is the study of heritable changes in the gene expression that do not involve changes in genomic dna sequence [64] . in infection, the researches are mainly focused on the changes in host's dna methylome, histone marks, and microrna profiles as a response to pathogen's invasion. relatively new, the number of infection-related epigenetic researches are still low compared with cancer epigenomics [65] . minion has a high potential assisting epigenetic research. due to its principle of directly sequencing nucleic acid molecules, any modifications in the nucleotides are also recorded in the raw signals. therefore, untreated nucleic acid sequenced with minion has enough information to distinguish 5-methylcytosine from cytosine if carefully analyzed [66] . some software have been developed specifically to read these signals and translate them into methylation marks [66, 67] . these developments open many research possibilities by allowing both genome and epigenome to be analyzed from a single sequencing run [68] . for example, 16s ribosomal rna (16s rrna) can be sequenced with rna sequencing. theoretically, the method can identify bacteria species and screen for antibiotics resistance simultaneously. by identifying gain or loss of ribosomal rna base modifications, antibiotic resistance can be inferred [69] . sequencing of e. coli 16s rrna, as well as its dna has been shown to be capable of detecting 7-methyl-guanosine, pseudouridine, 5-methylcytosine, and 6-methyladenine modifications [70, 71] . the progression of this field may have clinical applications in the future. sequencing as a technique may become the gold standard of diagnosis with the advent of portable sequencing. the technology is still evolving but it has shown development in portability, sequencing accuracy, and ease of operation. in portability, new type of flowcell and equipment can enhance sequencing in the field or in the laboratory. additional equipment such as voltrax (an add-on for the flowcell which will automate library preparation) or minit (a gpu computer) may eliminate the need to for extraction kit or laptop computer. many methods have been shown to be successful coupled with minion. if targeted sequencing is needed, existing methods, such as lamp can amplify genes of interest in an isothermal reaction, eliminating the need for thermal cycler. mda can amplify whole genome of pathogens to reach the optimum input dna concentration. the isothermal nature of the enzymes used in lamp and mda is also advantageous for field or clinical settings. the sequencing power is now enough for unbiased metagenomic sequencing. all of these will pave the way for the portable sequencer be used in field or clinical setting to assist in the fight against infectious disease. 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of this license, visit http://creativecommons. org/licenses/by/4.0/. infectious diseases-past, present, and future centers for disease control and prevention. the history of malaria, an ancient disease dating the age of the siv lineages that gave rise to hiv-1 and hiv-2 the evolution of hiv-1 and the origin of aids evolutionary rate shifts suggest speciesspecific adaptation events in hiv-1 and siv the persistent legacy of the 1918 influenza virus improving sensitivity of direct microscopy for detection of acid-fast bacilli in sputum: use of chitin in mucus digestion evaluation of a commercial multiplex pcr assay for detection of pathogen dna in blood from patients with suspected sepsis pcr-based detection of composite transposons and translocatable units from oral metagenomic dna pcr-based detection methods for single-nucleotide polymorphism or mutation: real-time pcr and its substantial contribution toward technological refinement pcr-based methods for mutation detection the sequence of sequencers: the history of sequencing dna profiling dna methylation based on next-generation sequencing approaches: new insights and clinical applications successful test launch for nanopore sequencing on site dna barcoding by nanopore sequencing real-time dna barcoding in a rainforest using nanopore sequencing: opportunities for rapid biodiversity assessments and local capacity building in situ field sequencing and life detection in remote (79â°26â�²n) canadian high arctic permafrost ice wedge microbial communities nanopore dna sequencing and genome assembly on the international space station mobile real-time surveillance of zika virus in brazil real-time, portable genome sequencing for ebola surveillance from squiggle to basepair: computational approaches for improving nanopore sequencing read accuracy a complete bacterial genome assembled de novo using only nanopore sequencing data fast and accurate de novo genome assembly from long uncorrected reads nanopore sequencing of drug-resistanceassociated genes in malaria parasites a worldwide map of plasmodium falciparum k13-propeller polymorphisms targeted sequencing workflows for comprehensive drug resistance profiling of mycobacterium tuberculosis cultures using illumina miseq and nanopore minion: comparison of analytical and diagnostic performance, turnaround time and cost. biorxiv a portable system for rapid bacterial composition analysis using a nanopore-based sequencer and laptop computer nanoampli-seq: a workflow for amplicon sequencing for mixed microbial communities on the nanopore sequencing platform development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics loop-mediated isothermal amplification of dna direct blood dry lamp: a rapid, stable, and easy diagnostic tool for human african trypanosomiasis serotyping dengue virus with isothermal amplification and a portable sequencer a novel diagnostic method for malaria using loopmediated isothermal amplification (lamp) and minion tm nanopore sequencer an innovative diagnostic technology for the codon mutation c580y in kelch13 of plasmodium falciparum with minion nanopore sequencer field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (lamp) assay and minion sequencing rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis assessment of metagenomic nanopore and illumina sequencing for recovering whole genome sequences of chikungunya and dengue viruses directly from clinical samples whole genome sequencing of influenza a and b viruses with the minion sequencer in the clinical setting: a pilot study sequencing of human genomes with nanopore technology the road to metagenomics: from microbiology to dna sequencing technologies and bioinformatics towards precision quantification of contamination in metagenomic sequencing experiments unculturable bacteria-the uncharacterized organisms that cause oral infections rapid diagnosis of lower respiratory infection using nanopore-based clinical metagenomics. biorxiv comparison of microbial dna enrichment tools for metagenomic whole genome sequencing ultra-deep, long-read nanopore sequencing of mock microbial community standards the three peaks challenge and developing extraction methods suitable for long-read, ultra-deep stool metagenomics on the promethion optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples whole-genome multiple displacement amplification from single cells multiple displacement amplification for malaria parasite dna minion nanopore sequencing of multiple displacement amplified mycobacteria dna direct from sputum. biorxiv rapid pathogen identification in bacterial pneumonia using real-time metagenomics loose m whale watching with bulkvis: a graphical viewer for oxford nanopore bulk fast5 files. biorxiv past and present perspectives on beta-lactamases. antimicrob agents chemother minion nanopore sequencing identifies the position and structure of bacterial antibiotic resistance determinants in a multidrug-resistant strain of enteroaggregative escherichia coli complete assembly of escherichia coli sequence yype 131 genomes using long reads demonstrates antibiotic resistance gene variation within diverse plasmid and chromosomal contexts minion nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island metagenomic sequencing at the epicenter of the nigeria 2018 lassa fever outbreak infectious disease direct rna nanopore sequencing of fulllength coron-avirus genomes provides novel insights into structural variants and enables modification analysis direct rna sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus epigenetics of host-pathogen interactions: the road ahead and the road behind detecting dna cytosine methylation using nanopore sequencing mapping dna methylation with high throughput nanopore sequencing nanopore sequencing meets epigenetics what do we know about ribosomal rna methylation in escherichia coli? reading canonical and modified nucleobases in 16s ribosomal rna using nanopore native rna sequencing single-molecule sequencing detection of n6-methyladenine in microbial reference materials key: cord-001090-qg2r691d authors: twin, jimmy; bradshaw, catriona s.; garland, suzanne m.; fairley, christopher k.; fethers, katherine; tabrizi, sepehr n. title: the potential of metatranscriptomics for identifying screening targets for bacterial vaginosis date: 2013-09-27 journal: plos one doi: 10.1371/journal.pone.0076892 sha: doc_id: 1090 cord_uid: qg2r691d background: the ribosomal rna content of a sample collected from a woman with bacterial vaginosis (bv) was analysed to determine the active microbial community, and to identify potential targets for further screening. methodology/principal findings: the sample from the bv patient underwent total rna extraction, followed by physical subtraction of human rrna and whole transcriptome amplification. the metatranscriptome was sequenced using roche 454 titanium chemistry. the bioinformatics pipeline mg-rast and desktop dna analysis platforms were utilised to analyse results. bacteria of the genus prevotella (predominately p. amnii) constituted 36% of the 16s rrna reads, followed by megasphaera (19%), leptotrichia/sneathia (8%) and fusobacterium (8%). comparison of the abundances of several bacteria to quantitative pcr (qpcr) screening of extracted dna revealed comparable relative abundances. this suggests a correlation between what was present and transcriptionally active in this sample: however distinct differences were seen when compared to the microbiome determined by 16s rrna gene amplicon sequencing. to assess the presence of p. amnii in a larger pool of samples, 90 sexually active women were screened using qpcr. this bacterium was found to be strongly associated with bv (p<0.001, or 23.3 (95%ci:2.9–190.7)) among the 90 women. conclusions/significance: this study highlighted the potential of metatranscriptomics as a tool for characterising metabolically active microbiota and identifying targets for further screening. prevotella amnii was chosen as an example target, being the most metabolically active species present in the single patient with bv, and was found to be detected at a high concentration by qpcr in 31% of cohort with bv, with an association with both oral and penile-vaginal sex. the advent of molecular-based screening utilising massively parallel dna sequencing has increased our capability to characterise the microbial ecology of human clinical samples, both rapidly and economically [1] . in addition it has allowed a better understanding of the normal endogenous microbiota. the vaginal microbiota is complex, varying at different stages of reproductive life, as well as during the menstrual cycle. bacterial vaginosis (bv) is a condition affecting the vaginal microbiota where in the childbearing age woman the natural microbiota (typically lactobacillus spp.) is depleted, and replaced by an overgrowth of mixed, primarily anaerobic bacteria [2] . this condition has significant associations with miscarriage, premature birth and pelvic infections, and can increase a woman's risk of acquiring sexually transmitted infections and hiv [3] . no single aetiological agent has been identified yet for bv, and is now generally considered likely to be of polymicrobial aetiology. bv is typically diagnosed using either the nugent scoring method [4] that examines bacterial composition via a gram smear or the amsel criteria [5] that considers factors such as presence of discharge, amine production, presence of clue cells and a vaginal ph greater than 4.5. through microbiome studies, the microbiology of bv has been better characterised. these studies have generally been carried out using pcr-derived 16s rrna gene fragments, using ''universal'' bacterial 16s rrna gene primers [2] . while this approach is able to provide a comprehensive understanding of the bacterial community membership, it is not able to determine which members are transcriptionally active. metatranscriptomics is the analysis of the rna transcripts being expressed by a community at a given point of time. for bacteria, the predominant rna classes are ribosomal (primarily 16s rrna and 23s rrna), which are able to be used as taxonomic identifiers. the metatranscriptomic screening of clinical samples has been described for gut and dental microbiomes [6, 7] , with only one study to date applying this methodology to bv [8] . an important difficulty faced with the application of this technique to clinical samples is the low levels of rna present in many samples and the overabundance of host rna. this requires reduction of host rna and subsequent linear amplification of rna for transcriptomic analysis [9, 10] . this study describes a method to analyse microbial rrna from vaginal samples that is applicable to variety of clinical sample types. it demonstrates how the data gained can provide valuable information for larger qpcr based screening studies. a 26 year old woman presenting with abnormal vaginal discharge, odour, 4/4 amsel criteria and a nugent score of 10 was recruited with written consent from melbourne sexual health centre (mshc), victoria, australia. this patient reported no recent antibiotic use, had no recent male sexual partner, and reported only having one female partner in the prior 3 months and three in the past 12 months. vaginal discharge was collected using ten flocked swabs and immediately rotated and pooled in 4 ml rnalater (life technologies, grand island, usa). the rnalater solution was then stored at 280uc until processing. for the second component of this study, extracted dna (stored at 230uc) was utilised from a randomly selected subset of 90 samples obtained from a cross sectional study of sexually active women attending mshc in 2003/4 [11] . this set of samples comprised 34 women with normal microbiota (nugent 0-3), 20 with intermediate microbiota and 36 with bv . written consent for all de-identified participants and ethical approval for this study was obtained previously from the alfred hospital human research ethics committee. figure 1 provides an overview of the workflow for this study. initially, total rna was extracted from 2 ml rnalater solution [12] . total rna extraction was performed using trizol (life technologies) with use of 1-bromo-3-chloropropane in place of chloroform followed by rna purification using a rneasy column (qiagen, hilden, germany) [13] . any remaining dna was digested using the turbo dna-free enzyme (life technologies). human ribosomal content was subtracted using the mi-crobenrich kit (life technologies) as per manufacturer instructions followed by removal of small rnas with the megaclear kit (life technologies). rna concentration was determined by agilent 2100 bioanalyzer (agilent technologies inc., santa clara, usa). to increase the rna content needed for sequencing, whole transcriptome amplification (wta) and reverse-transcription using the transplexh complete whole transcriptome amplification kit (sigma-aldrich corporation, st. louis, usa) was utilized. this resulted in a cdna concentration of 50 ng/ml (from original rna content of 3.5 ng/ml) with 2 mg submitted to agrf (australian genome research facility ltd., brisbane, australia) for 454 sequencing (flx titanium chemistry; roche/454 life sciences, branford, usa). genomic dna was precipitated from the remaining trizol buffer using 0.3 ml of 100% ethanol per 1 ml of trizol buffer followed by two washes of the dna pellet with 0.1 m trisodium citrate in 10% ethanol [14] . an amplicon-based metagenomic library was generated from the extracted dna using the universal bacterial pcr primers 27f and 338r that target the v1-v2 hypervariable regions of the 16s rrna gene as previously described [15] . both cdna and 16s rrna gene amplicon libraries were subsequently bar-coded using mid tags and sequenced together utilising a quarter region of a 454 sequencing plate. subsequent to sequencing, all raw sequencing data was demultiplexed according to their mid tags and the data obtained from each sample was then imported into genomics workbench version 4.5.1 (clc bio, aarhus, denmark), for removal of wta primers and random primer sequences from each applicable read and data was filtered (low quality score limit of 0.05; maximum 2 ambiguous nucleotides allowed; minimum sequence length of 100 nt). each dataset was then screened for chimeric sequences using uchime [16] , with the trimmed and filtered data submitted to the mg-rast (http://metagenomics.anl.gov) bioinformatics pipeline for analysis (cdna library mg-rast id = 4461586.3; dna amplicon mg-rast id = 4461792.3) [17] . a 90% cut-off was used for database searches within mg-rast as an arbitrary cut-off for genus identities using the rdp database, and a 98% cut-off was used for species level identification [18] . mg-rast generates abundance counts based on the number of unique hits a particular sequence has against a particular database. it is therefore highly likely that a single read may have multiple abundance counts assigned to it if there is an equal relatedness. the identities for each read were sorted using excel 2007 (microsoft corporation, redmond, usa) to eliminate multiple identical hits for individual reads, with manual analysis being carried out using the blast algorithm for discrepant samples. graphical representation of bacterial abundances was achieved using krona charts [19] . functional genes from the cdna library were characterised by seed analysis within mg-rast. reads derived from the cdna library assigned to each major genus (comprising .10% of total population) using mg-rast were also imported into lasergene 8 (dnastar inc., madison, usa) for manual sequence alignment was carried out using a 98% sequence match cut-off. the consensus sequences of alignments were assigned an identity using the blast algorithm with a $98% identity required to assign a species name. all assays were performed on the lc480 real-time instrument (roche diagnostics, mannheim, germany) using 5 ml dna in a 20 ml reaction. the bacteria targeted were atopobium vaginae, leptotrichia/sneathia spp., megasphaera type i [20] , gardnerella vaginalis [21] , and the genera prevotella [22] and lactobacillus [23] . a primer set for prevotella amnii was developed using the forward and reverse primers 27f and 338r [24] with the incorporation of a taqman probe 59-[6fam] aaa gtt ggc cta atg ccc tat g[bhq1]-39, specific to p. amnii, based on 454 sequencing data from this study and all available relevant sequences on the genbank database. the total bacterial content of each sample was determined by the modification of an assay by nikkari et al. (2002) [25] with the incorporation of a taqman probe, 516f (59-[6fam] tgc cag cag ccg cgg taa [bhq1]-39) to calculate relative bacterial abundances and to determine sample adequacy for stored dna samples. relative bacterial abundances were compared using a paired t test or chi square test. the association of p. amnii with bv, defined as a nugent score of 7-10, was made using a chi square test, reported with odds ratios (or) with 95% confidence intervals (ci). all analyses were carried out using stata version 12 (statacorp lp, college station, usa) [26] . in total, 89969 raw sequencing reads were obtained with an ultimate post data filtration of 78285 reads (average length = 268691 bp; gc content = 4965%). overall, this library consisted of 72826 (93%) bacterial reads, 3865 (4.9%) human reads and 34 (0.04%) either plastid, fungal or viral reads (table 1) . of the bacterial reads, 31857 (43.7%) were of the 16s rrna gene and 4349 (6.0%) were non-ribosomal rna (eg. mrna) with the remaining matching other ribosomal regions such as the 23s rrna gene and intergenic regions. of the 16s rrna gene reads, the most dominant genus identified was prevotella (36% of the reads), followed by the genera megasphaera (19%), leptotrichia/ sneathia (8%), fusobacterium (8%), and 9% of the reads matching uncultured members of the fusobacteriales (figure 2 ). of the prevotella reads, 78% formed contigs of $1000 bp in length that matched p. amnii. analysis of assembly free reads of the prevotella spp. reads at the species level (11725 reads with $98% match to the rdp database) showed similar results, however distinction between p. amnii and p. bivia was not possible for the majority of these sequences (data not shown). the major fusobacterium was f. nucleatum, and the majority of megasphaera reads were $98% similar to the currently uncharacterised veillonellaceae bacterium s3pf24 (genbank accession jx104009). the bioinformatics pipeline of mg-rast was able to annotate 2053/4349 of the non-ribosomal rna reads, which seed analysis revealed to consist of genes involved primarily in protein metabolism (20.6%), carbohydrate/lipid utilisation (16.3%) and cluster based subsystems (13%). the most abundant functional gene read identities were primarily represented by genes expressed by the genus prevotella ( table 2) . comparison of cdna to dna from nugent 10 patient all of the predominant bacteria with .1% total abundance identified in the cdna library were also detected using the 16s rrna gene amplicon-based approach on extracted dna. there was a significant difference in the relative abundances given between both libraries for the most dominant taxa, such as the genus lactobacillus (primarily l. iners) that comprised 1.4% of the cdna reads and 23% of the dna amplicon library (p,0.0001). also of particular note were reads pertaining to the genus gardnerella that were present in 7% of the cdna library, but were nearly non-existent in the dna amplicon library with only seven reads detected (0.006%). when comparing the relative abundances of the seven taxa screened for using qpcr to the abundances given for the cdna library, there was no significant difference (p = 0.9093). we found that the proportion of lactobacillus spp. determined by qpcr to be 1.9% (cdna = 1.4%; dna amplicon = 23.1%), and the proportion of prevotella spp. was 48% (cdna = 35%; dna amplicon = 11%) ( table 3) . qpcr screening of sexually active women p. amnii was detected in 11/36 (30.6%) women possessing a nugent score of 7-10, whereby nine had a nugent score of 7 and two with a nugent score of 10. this bacterium was also detected in a single case possessing intermediate microbiota with a nugent table 4 ). the bacterial load of p. amnii in these 12 positive cases was high, averaging 1.41610 9 copies per swab (range = 7.74610 6 to 4.66610 9 ). this bacterium was found to be strongly associated with bv (p,0.001, or 23.3 (95%ci:2.9-190.7)) among the 90 women. when the 90 women were stratified by different sexual exposures the association between bv and p. amnii persisted for different sexual exposures in men. while the associations between bv and p. amnii were not significant among women reporting receptive oral sex with a woman or sex with a woman in the last 3 months, these analyses involved only small numbers (table 4 ). this study explored the active microbial diversity of a vaginal sample taken from a lesbian woman with symptomatic bv to demonstrate the potential of metatranscriptomics for identifying targets for screening studies. prevotella amnii was found to be the most active species present in this sample and was also detected at a high concentration by qpcr in 30.6% of bv cases in sexually active women. although we identified p. amnii as the predominant prevotella species in this sample (78% of prevotella reads; 28.1% of total bacterial population), there may be a possible overestimation in the cdna abundance given, as conserved regions shared by multiple prevotella spp. may be involved through chimeric assembly. this must be kept in mind when comparing to qpcr data (33.1% of prevotella reads; 15.8% of total bacterial population) that would otherwise suggest that this species is more active than others in this sample. this bacteria has only recently been associated with bv in the literature [27] . its closest relative, p. bivia, is highly cited as being associated with this condition, and in vitro studies have shown this bacterium to form a symbiotic relationship with g. vaginalis [28] . these two prevotella spp. share many phenotypic traits and it has been noted that culture-based methodologies may not differentiate between these two species, with full length 16s rrna gene sequencing recommended for species differentiation [29] . the other predominant bacteria in this case were identified through 16s rrna gene as the currently uncharacterised isolate veillonellaceae bacterium s3pf24 (phylogenetically belonging to the genus megasphaera) that was originally isolated from a vaginal environment, and the bacterium fusobacterium nucleatum. f. nucleatum has been described in cases of bv previously [30] . interestingly it has been implicated in amniotic infection and preterm birth in cases where the bacterium was also detected in the patients' oral cavity [31, 32] . f. nucleatum, as well as both prevotella and megasphaera spp. are regularly associated with periodontal disease [33, 34, 35] , and it is of interest to note that the single nugent score 10 participant analysed in this study had a history of recent female-female oral sex. these three bacteria were more abundant than the usual predominant g. vaginalis and a. vaginae in the cdna library, which collectively only comprised 10% [2] . further work is warranted to determine the role of these bacteria, in particular p. amnii, in the pathogenesis of bv. of particular interest is whether their association with oral sex represents a possible route of transmission. although not a focus of this study, mrna reads were also identified in this study that were primarily associated with protein metabolism and carbohydrate/lipid utilisation pathways of the genus prevotella. the low abundance of mrna reads (5.6%) was not surprising, given the low general relative abundance of mrna in relation to total rna in bacterial cells [36] . to focus more upon this class of rna transcripts, an additional enrichment step, such as the removal of bacterial rrna can be employed [37] , in the same manner as human rrna was reduced in this study. a comparison was made between the cdna and dna amplicon libraries to determine what proportion of the microbial community was active, and differences were observed between these libraries. this may suggest that there was a significant difference between what is present and what is transcriptionally active, although these findings are limited by the fact that this is a single sampling point. the broad range amplification of bacterial dna using the 16s rrna gene may have potential bias in the community structure, as has been found in numerous studies [38, 39, 40, 41] . our data suggests that g. vaginalis was underrepresented in the dna amplicon library when compared to the qpcr data from the same dna sample. despite being one of the most utilized primers for microbiome analysis, there has been suggestion in the literature that the forward pcr primer 27f may not reliably amplify this species [42] , and our analysis has demonstrated this similarly through qpcr for g. vaginalis. also, given that l. iners was found to be underrepresented in the cdna library when compared to the dna amplicon library, we applied a genus-specific lactobacillus qpcr assay to the dna sample, as well as one that targeted the genus prevotella that was present at a much higher abundance in the cdna library. we found that there was no statistical difference in the abundances given between qpcr and the cdna library for these genera. based on this limited data, we can suggest misrepresentation of several taxa in the dna amplicon library occurred, and that in this case, the vast majority of the bacteria physically present in this sample were also transcriptionally active. this further highlights the advantage of transcriptomics for such analysis as our data suggest that this analysis gives better representation of bacterial levels across the board. mg-rast for the analysis of metatranscriptomic work can be carried out using a standard desktop or laptop computer. however, this method may lead to misclassification of certain reads. for example, in this study it was found that many gardnerella reads were being classified as the genus bifidobacterium, megasphaera reads as the genera veillonella and dialister, and those of the genera leptrotrichia/sneathia as streptobacillus. therefore care must be taken in the interpretation of results, and manual checking of representative reads is required to ensure an accurate identification of sequencing reads to the genus level. in summary, this study has shown how metatranscriptomics can be utilised as a useful tool to carry out an in-depth examination of the active microbial ecology of an environment, and is capable of generating more taxonomic and potential functional data than an amplicon sequencing-based approach alone, and avoid potential misrepresentation issues caused by pcr primer selection. we have demonstrated that this kind of metatranscriptomic approach can be used to identify targets for screening in larger studies. as this study focussed on a single individual, generalisations based on the data are not possible and transcriptomic analyses on a large cohort is warranted. this being said, our limited findings reinforce that p. amnii may be an important bv-associated bacterium, and should be included in future studies investigating the pathogenesis of bv. its previously known association with periodontal disease, and the association with oral sex in this study, make it interesting to speculate about transmission of pathogenic bacteria between the oral and vaginal cavities, and their role in the development of bv. metagenomic analysis of bacterial infections by means of high-throughput dna sequencing the vaginal microbiome: new information about genital tract flora using molecular based techniques the aetiology of bacterial vaginosis reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation nonspecific vaginitis. diagnostic criteria and microbial and epidemiologic associations effect of periodontal pathogens on the metatranscriptome of a healthy multispecies biofilm model metatranscriptomic approach to analyze the functional human gut microbiota composition of the vaginal microbiota in women of reproductive age -sensitive and specific molecular diagnosis of bacterial vaginosis is possible? representation is faithfully preserved in global cdna amplified exponentially from subpicogram quantities of mrna broadspectrum respiratory tract pathogen identification using resequencing dna microarrays higher-risk behavioral practices associated with bacterial vaginosis compared with vaginal candidiasis simultaneous extraction of high-quality rna and dna from small tissue samples substitution of chloroform by bromochloropropane in the single-step method of rna isolation a reagent for the single-step simultaneous isolation of rna, dna and proteins from cell and tissue samples vaginal microbiome of reproductive-age women uchime improves sensitivity and speed of chimera detection the metagenomics rast server -a public resource for the automatic phylogenetic and functional analysis of metagenomes study of inter-and intra-individual variations in the salivary microbiota interactive metagenomic visualization in a web browser changes in vaginal bacterial concentrations with intravaginal metronidazole therapy for bacterial vaginosis as assessed by quantitative pcr detection of bacterial vaginosis-related organisms by real-time pcr for lactobacilli, gardnerella vaginalis and mycoplasma hominis comparative assessment of human and farm animal faecal microbiota using real-time quantitative pcr diversity of cervicovaginal microbiota associated with female lower genital tract infections inverse association of h 2 o 2 -producing lactobacilli and vaginal escherichia coli colonization in women with recurrent urinary tract infections broadrange bacterial detection and the analysis of unexplained death and critical illness stata statistical software: release 12 bacterial communities in women with bacterial vaginosis: high resolution phylogenetic analyses reveal relationships of microbiota to clinical criteria evidence for a commensal, symbiotic relationship between gardnerella vaginalis and prevotella bivia involving ammonia: potential significance for bacterial vaginosis prevotella amnii sp. nov., isolated from human amniotic fluid molecular analysis of the diversity of vaginal microbiota associated with bacterial vaginosis the origin of fusobacterium nucleatum involved in intra-amniotic infection and preterm birth abnormal bacterial colonisation of the genital tract and subsequent preterm delivery and late miscarriage pattern of distribution of prevotella species/phylotypes associated with healthy gingiva and periodontal disease identification of candidate periodontal pathogens and beneficial species by quantitative 16s clonal analysis the detection of eight putative periodontal pathogens in adult and rapidly progressive periodontitis patients: an institutional study chemical composition of escherichia coli validation of two ribosomal rna removal methods for microbial metatranscriptomics coverage evaluation of universal bacterial primers using the metagenomic datasets capturing greater 16s rrna gene sequence diversity within the domain bacteria improved detection of bifidobacteria with optimised 16s rrna-gene based pyrosequencing selection of primers for optimal taxonomic classification of environmental 16s rrna gene sequences the human vaginal bacterial biota and bacterial vaginosis we would like to thank the staff and students of melbourne sexual health centre and women's centre for infectious diseases for their assistance in this study, which was also supported by the victorian government's operational infrastructure support program. key: cord-292033-zkwiag7a authors: balboni, andrea; bassi, francesca; de arcangeli, stefano; zobba, rosanna; dedola, carla; alberti, alberto; battilani, mara title: molecular analysis of carnivore protoparvovirus detected in white blood cells of naturally infected cats date: 2018-02-05 journal: bmc vet res doi: 10.1186/s12917-018-1356-9 sha: doc_id: 292033 cord_uid: zkwiag7a background: cats are susceptible to feline panleukopenia virus (fpv) and canine parvovirus (cpv) variants 2a, 2b and 2c. detection of fpv and cpv variants in apparently healthy cats and their persistence in white blood cells (wbc) and other tissues when neutralising antibodies are simultaneously present, suggest that parvovirus may persist long-term in the tissues of cats post-infection without causing clinical signs. the aim of this study was to screen a population of 54 cats from sardinia (italy) for the presence of both fpv and cpv dna within buffy coat samples using polymerase chain reaction (pcr). the dna viral load, genetic diversity, phylogeny and antibody titres against parvoviruses were investigated in the positive cats. results: carnivore protoparvovirus 1 dna was detected in nine cats (16.7%). viral dna was reassembled to fpv in four cats and to cpv (cpv-2b and 2c) in four cats; one subject showed an unusually high genetic complexity with mixed infection involving fpv and cpv-2c. antibodies against parvovirus were detected in all subjects which tested positive to dna parvoviruses. conclusions: the identification of fpv and cpv dna in the wbc of asymptomatic cats, despite the presence of specific antibodies against parvoviruses, and the high genetic heterogeneity detected in one sample, confirmed the relevant epidemiological role of cats in parvovirus infection. parvoviruses are non-enveloped single-stranded dna viruses which infect a wide range of mammalian species, including several members of the order carnivora. the carnivore protoparvovirus 1, belonging to genus protoparvovirus, family parvoviridae, subfamily parvovirinae, includes several closely related autonomous viruses causing a range of serious conditions, especially in young animals: feline panleukopenia virus (fpv, the prototype virus of the former carnivore parvovirus), canine parvovirus (cpv), mink enteritis virus (mev), and raccoon parvovirus (rapv) [1] . feline panleukopenia virus has been known to be a cause of disease in cats since the beginning of the twentieth century, although there are other similar parvovirus species affecting cats, such as mev and cpv. natural infections in cats with cpv have been reported but fpv remains the most prevalent parvovirus causing disease in cats [2] [3] [4] . since cats are susceptible to fpv and cpv 2a, 2b, 2c variants, superinfection and co-infection with multiple parvovirus strains associated with high viral genetic heterogeneity can occur with relatively high frequency in feline hosts [3, [5] [6] [7] . parvoviruses commonly cause acute infection with high levels of viral shedding which generally ceases within 1-2 weeks post-infection, after the development of high titres of virus-neutralising antibody [8, 9] . nevertheless, parvoviruses can be detected in faeces forup to 6 weeks after recovery, depending on the sensitivity of the diagnostic method used [10] . cats experimentally infected with fpv shed the virus in both urine and faeces up to day 41-42 post-infection with parvovirus persisting in the lungs and kidneys for more than 50 weeks in cats which have recovered [11] . the detection of fpv and cpv variants in apparently healthy cats suggests that parvovirus infection may be common in some populations of clinically normal cats, and that asymptomatic cats may be able to shed parvovirus for prolonged periods of time [12] [13] [14] . furthermore, the ability of fpv and cpv to persist in the peripheral blood mononuclear cells (pbmc) of cats irrespective of the presence of neutralising antibodies [13] [14] [15] [16] [17] and the presence of parvoviral dna in the bone marrow of healthy cats [18] , suggests that parvovirus may persist long term in the tissues of cats post-infection without causing clinical signs. the aim of this study was to screen a population of 54 cats from sardinia (italy) for the presence of both fpv and cpv dna within buffy coat samples. the dna viral load, genetic diversity, phylogeny and antibody titres against parvoviruses were investigated in the cats testing positive to dna parvoviruses. this was a retrospective study, carried out on stored blood samples taken for routine diagnostic investigations undertaken at the department of veterinary medicine, university of sassari -uniss (sassari, no sardinia, italy). buffy coats from cats sampled between october 2011 and march 2012 were tested for the presence of feline and canine parvovirus dna using real-time polymerase chain reaction (pcr). the partial vp2 gene of the viruses identified was sequenced and used for statistical analyses and phylogenetic comparisons. in addition, the sera of the cats which were positive to dna parvoviruses were tested for the parvovirus antibody using the haemagglutination inhibition (hi) assay. samples of owned or stray cats with different life-style conditions (indoor or outdoor, living alone or in community) were tested to evaluate different exposure to parvoviruses. date of sampling, gender, age, breed, habitat and the clinical symptoms of the 54 cats sampled are reported in table 1 . according to these data, the animals were divided into two groups called a and b. the cats included in groups a were predominantly healthy cats from four multi-cat households. the cats in group b were sampled in the emergency room of the veterinary teaching hospital of the department of veterinary medicine (uniss) and were predominantly stray cats showing different clinical signs (sick cats). twenty-two of the 54 (40.7%) cats were clinically normal; 26/54 (48.1%) showed clinical signs attributable to various diseases; 3/54 (5.6%) showed gastrointestinal signs, such as vomiting and diarrhoea compatible with parvovirus infection; clinical status was unavailable for 3/54 (5.6%) cats. the vaccination status of the cats included in the study was unknown, although it was hypothesised that the stray cats were unvaccinated. anti-coagulated peripheral blood samples in ethylenediaminetetraacetic acid (edta) and coagulated blood for serology were collected from each cat. the blood samples were stored a + 4°c and sera at − 20°c until use. buffy coat-containing mononuclear cells was isolated from 3 ml of edta anti-coagulated peripheral blood samples using histopaque-1077 (sigma aldrich, st. louis, mo, usa). the dna was extracted using the dneasy blood and tissue kit (qiagen, hilden, germany), according to the manufacturer's instructions. the extracted dna was eluted in 100 μl of ultrapure rnasi and dnasi free water, and was stored at − 20°c after analysis. parvovirus screening was carried out using real-time pcr using two conserved primers (a-for and b-rev, table 2 ) targeting a 99 bp fragment of the vp2 gene. quantitative pcr (qpcr) was carried out using sybr premix ex taq ii (takara bio inc., shiga, japan) and the rotor-gene 3000 system (corbett research, mortlake, nsw, australia). the fluorescence signal was acquired on the fam channel (multi-channel machine, source, 470 nm; detector, 510 nm; gain set to 5) with a fluorescence reading taken at the end of each elongation step. each run consisted of an initial incubation in order to activate the hot-start dna polymerase at 95°c for 30 s followed by 40 cycles of denaturation at 95°c for 10 s, annealing at 60°c for 20 s and polymerisation at 72°c for 30 s. during the melt cycle, the temperature was increased by increments of 1°c from 65°c to 95°c. a pcr 4 plasmid (invitrogen, carlsbad, california, usa) containing one copy of the vp2 target sequence was produced as the external standard for the construction of the assay standard curve for quantitative analysis. duplicates of six 10-fold dilutions of the standard plasmid, duplicates of the buffy coat dna extracts of the cats sampled and a no template control were simultaneously analysed. specimens were considered positive if the fluorescence curve in the amplification plot showed an exponential increase, and if a specific melting peak was observed. copies of viral dna were expressed per microlitre of dna extract. amplification and sequencing of the vp2 gene in order to differentiate between fpv and cpv variants 2a, 2b and 2c, a fragment of the vp2 gene coding for critical amino acid residues 297, 300, 305, 323 and 426 affecting the biological and antigenic proprieties was amplified and sequenced for each virus identified using real-time pcr. a hemi-nested pcr assay was developed by using three conserved primers (c-for, d-rev and e-rev, table 2 ) for this purpose. both pcr reactions were carried out using taq dna polymerase (qiagen, hilden, germany) producing dna fragments of 881 bp and 569 bp in length for the first and the second reaction, respectively. the temperature cycling protocol of the first amplification consisted of 94°c for 5 min, 45 cycles with 1 cycle at 94°c for 30 s, at 48°c for 1 min, and at 72°c for 1 min, followed by a final elongation at 72°c for 10 min. in the second amplification, the pcr conditions were 94°c for 5 min, 35 cycles with 1 cycle at 94°c for 30 s, at 49°c for 1 min, and at 72°c for 45 s, followed by a final elongation at 72°c for 10 min. in both pcr reactions, fpv 1033/09 [3] was used as a positive control while ultrapure water was used in each experiment to avoid false positive results. the nucleotide sequences were obtained using both forward and reverse primers. direct sequencing of the pcr products of one virus identified (number 41/2011) showed an unusually high number of ambiguities, suggesting a mixed viral population. therefore, the amplification product was cloned into the pcr 4/topo vector using the topo cloning kit (invitrogen, carlsbad, ca, usa), and was transformed into escherichia coli dh5α-competent cells according to the manufacturer's protocol. ten recombinant clones were sequenced using both forward and reverse primers. the nucleotide sequences obtained were assembled and translated into amino acid sequences using bioedit sequence alignment editor version 7.2.5 [19] . the vp2 assembled nucleotide sequences were aligned with reference sequences of feline and canine parvoviruses available in the genbank (http://www.ncbi.nlm.nih.gov/genbank), including the modified live fpv vaccine strains available, using the clustalw method implemented with bioedit software. a variety of statistical analyses aiming to investigate nucleotide diversity, sequence variability and natural selection were carried out on the sequence data set using dnasp package version 5.10.01 [20] . statistical analysis was carried out on the subpopulations, grouping the sequence data in the fpv, cpv and 41/2011 (all clones of sample 41/2011) clusters. the following parameters were estimated for each cluster: total number of mutations (η), nucleotide diversity (π) and its standard error, total number of synonymous differences (syndif), and total number of non-synonymous differences (nsyndif). mutation frequency (total number of changes/total number of bases sequenced) and the percentage of mutated clones were used as indicators of genetic diversity of the viral population of virus 41/2011. phylogenetic relationships among the viruses detected and the parvovirus reference sequences were evaluated using mega version 7.0.20 [21] . the best-fit model of nucleotide substitution was determined using the find best dna/protein model function implemented in mega, and the tamura 3-parameter model was found to be optimal for all the sequence data (including reference strains). phylogenetic trees were constructed using the maximum likelihood method, and bootstrap values were determined by 1000 replicates to assess the confidence level of each branch pattern. the nucleotide sequences obtained in this study have been submitted to the genbank under accession numbers kt151621 to kt151638. the parvovirus antibody titre was investigated using the haemagglutination inhibition (hi) test in the sera of the cats which tested positive to parvovirus dna by direct molecular diagnosis. a cpv-2b isolate (number 115/2010), recovered from the faeces of a dog with non-fatal enteritis, and isolated by the authors on the crandell rees felinekidney (crfk, cell line was obtained from the biobanking of veterinary resources of the istituto zooprofilattico sperimentale della lombardia e dell'emilia romagna izsler "bruno ubertini" http://www.ibvr.org/services/cellcultures.aspx) cell line, was used as the viral antigen. this strain was used in hi experiments because there are no significant differences in serum titres inhibiting the haemagglutination reaction when cats are infected by fpv or cpv, if cpv is used as an antigen [22] . to calculate 8 haemagglutinating units (hau) of the viral antigen, a haemagglutination (ha) assay was carried out on the cpv-2b isolate following procedures described previously by carmichael et al. [22] . porcine erythrocytes were washed three times in bis-tris buffered saline (btbs, ph 6.2 at 4°c) and suspended into 0.5% (v/v) btbs containing 1% bovine serum albumin. to reveal the haemagglutination of both viruses, the test was conducted at a ph of 6-6.8 and was incubated at 4°c [23, 24] . the hi tests were carried out as previously reported by senda et al. [25] . all sera were tested in duplicate on two different plates; two-fold dilutions of a commercial canine hyperimmune serum raised against canine distemper, canine hepatitis and canine parvovirosis (stagloban, ati, ozzano emilia, bo, italy), was used as positive controls. for all sera, the mean value of the results from the duplicate hi tests was calculated. if the mean value of the result from one sera was included between two successive dilutions, the lower titre was reported. cats with an hi titre ≥1:8 were considered positive and cats with an hi titre ≥1:80 were considered to be protected from infection. the buffy coat dna extracts of 54 cats were tested in duplicate for the presence of parvovirus using a sybr green real-time pcr assay which showed a limit of detection of 1 copy of the vp2 target dna per microlitre of extract. nine table 1) ; no cat with gastrointestinal signs tested positive for parvovirus dna. in the positive samples, the amount of viral dna ranged from orders of magnitude 10 0 to 10 2 copies of dna/μl of extract. a melting curve analysis showed a solitary peak between 81.8°and 82°c for standard plasmid dilutions and between 81°and 82°c for each positive cat sample. the nucleotide sequences of the partial vp2 gene obtained from positive samples were 532 bp in length (177 amino acid codons), and corresponded to residue 295-471 of the fpv reference strain cu-4 (genbank accession number m38246). nucleotide sequences of the vp2 partial gene were also obtained for ten recombinant clones of sample 41/2011, numbered from c01 to c10, respectively. clone c01 was an fpv which showed a change located in residue 312 (lys → arg); clone c03 was an fpv which displayed two coding changes in thr391-to-ala and pro461-to-ser; c04 was a cpv-2c with an amino acid change located at position 437 (gly → arg); c09 was a typical cpv-2c and clone 41/2011-c08 displayed amino acids identical to fpv in residues 297, 300, 305 and 323 while it showed glutamic acid typical of cpv-2c in position 426. the predicted amino acid sequence changes are summarised in table 3 . several synonymous and non-synonymous substitutions were detected by comparing the sequences as reported in table 4 . feline panleukopenia viruses showed a total number of mutations slightly higher than cpv viruses, although synonymous changes predominated in the fpv sequences while non-synonymous changes were prevalent in the cpv viruses. sample 41/2011 showed high genetic complexity generated within the host, and its sequence variability was higher than the variability of the other sequence data analysed as was seen by the values of parameter π. the non-synonymous fraction was greater for sample 41/2011, indicating clear prevalence of the number of non-synonymous mutations in the sample: 10 non-synonymous mutations out of a total of 15 mutations. the proportion of mutated viral clones was 60% for sample 41/2011 and the mutation frequency was on the order of 2.8 × 10 − 3 (table 4) . unrooted phylogenetic trees constructed from alignments of the nucleotide sequences obtained in this study with additional reference sequences showed two main clusters referable to fpv and cpv. the sequence of clone 41/2011-c08 formed a monophyletic branch inside the fpv clade (fig. 1) . nucleotide sequences of the modified live fpv vaccine strains available from genbank were distinguishable from all the fpv and cpv sequences obtained in this study, and direct relationships between them were not evident from the phylogenetic tree. the hi titres obtained are reported in table 5 . all the cats which tested positive for parvovirus dna were also positive for antibodies against parvoviruses; cats 22/2011 and 55/2012 showed a titre higher than 1:80. the present study screened the occurrence of parvovirus dna and the feline host-immune status in a cat population. asymptomatic and symptomatic cats sampled in sardinia (italy) during 2011-2012 were tested for the presence of both fpv and cpv dna in wbc using qpcr, and the partial vp2 gene of the viruses identified was characterised. parvoviruses have commonly been titrated in the faeces of infected animals, insofar as the viruses shed in faeces reflect virus replication; the authors chose to analyse the wbc for the presence of viral dna as, in addition to the intestinal crypt cells, bone marrow and other lymphoid tissues are also a major target for parvoviruses in both dogs and cats [9] . furthermore, since parvovirus can frequently be isolated in infected cats, even in the presence of high virus-neutralising antibodies [15, 17] , antibody titres against parvovirus were established in the sera of positive cats using an hi assay. nine (16.7%) out of a total of 54 cats tested positive for parvovirus dna with viral dna quantities ranging from 10 0 to 10 2 copies/μl of extract, demonstrating that the viral genome was detectable, although at low levels, in the wbc of a relatively large number of cats. all the cats which tested positive for parvovirus dna had table 3 change of amino acids to vp2 partial protein the presence of fpv and cpv dna in the faecal and peripheral blood samples of healthy cats has previously been reported [3, 12, [15] [16] [17] , raising important questions regarding the role of cats in the epidemiology of parvoviruses. in our study, the absence of clinical signs consistent with parvovirosis in the cats which tested positive, together with the low amount of viral dna detected, suggested that the infection was asymptomatic or that residual viral dna remains in the organism after recovery from acute infection. furthermore, the detection of parvoviral dna in wbc reveals the presence of the virus in the bone marrow or in other lymphoid tissues which might reflect chronic or latent infection. the detection of cpv dna in apparently healthy domestic cats confirmed a previous survey in which a high prevalence (37%) of cpv in apparently healthy domestic cats living in rescue shelters was identified [14] . canine parvovirus-like dna was also detected in the tissues of wildlife carnivores which had no fig. 1 unrooted phylogenetic tree. the phylogenetic tree was constructed with the nucleotide sequences of the partial vp2 gene generated in this study and with feline and canine parvovirus reference sequences available on the genbank nucleotide database or analysed by the authors in a previous study (battilani et al., [3] ). bootstrap values greater than 50%, calculated on 1000 replicates, are indicated on the respective branches. the feline and canine parvoviruses identified in italy not detected in this study but included in the phylogenetic analysis are designated by it. in bold: nucleotide sequences generated in this study. underlined: clone 41/2011-c8 clinical signs of active infection and, therefore, it was likely that this virus caused latent or persistent infection not only in domestic cats [26] . animal parvoviruses, such as rodent protoparvovirus and aleutian mink disease parvovirus, have been shown to persist in their host [27, 28] . persistence is a common feature also for human parvovirus b19 (b19v) infection [29] : b19v parvoviral dna has been documented in a wide range of tissues and the bone marrow of asymptomatic adults, although the majority of people harbour parvoviral dna in a form which does not actively replicate [30, 31] . of the nine cats testing positive for parvovirus dna, four showed fpv dna, four cpv dna (three cpv-2b and one cpv-2c), and cat 41/2011 showed unusually high genetic diversity and evidenced dna belonging to two species of parvovirus in the same patient. the pathogenicity of cpv variants for cats is not fully understood. some studies have suggested that cpv had the same pathogenic potential as fpv in cats [3, [32] [33] [34] [35] ; in other studies, clinical signs were not observed in infected animals, with the exception of transient leukopenia [36, 37] . these results led to the speculation that cpv, compared to fpv, can most frequently cause asymptomatic and persistent infection in cats, even if additional studies are clearly needed to fully understand the potential of cats as cpv carriers. in our study, an equivalent prevalence of fpv and cpv was found in the samples examined; this result might be related to the type of cat population sampled, which consisted mainly of healthy cats and cats showing clinical signs not related to parvovirosis. alternatively, it could be due to the biological matrix analysed since parvoviruses have commonly been investigated in the faeces of infected animals; instead, in our survey the wbc were analysed. the rates of variation of the fpv and cpv nucleotide sequences analysed in this study were similar, although genetic diversity in the fpv sequences was generated primarily by synonymous mutations which did not result in amino acid substitutions. this result was congruent with the evolutive behaviour of fpv which, since its emergence in 1920, has not undergone significant changes in antigenic and biological properties. feline panleukopenia virus varied at a slow rate by random genetic drift and it maintained host-specificity [38] . instead, in the cpv sequence data set, non-synonymous mutations were predominant. this finding is compatible with the pattern of evolution observed for cpv. since its emergence in the late 1970s, cpv evolution has been driven by strong positive selection, giving rise to new antigenic variants which have replaced the original type [38] . an unusual genetic complexity was reported for sample 41/2011, with six different viral dnas ascribable to two distinct species of parvovirus, fpv and cpv type 2c. although co-infection by more than one parvovirus species is a rare event, it has already been described in a cat simultaneously infected by fpv and cpv-2a [7] . carnivore protoparvoviruses show an estimated annual substitution rate on the order of 10 − 4 to 10 − 5 whereas the mutation frequency detected in sample 41/2011 was on the order of 2.8 × 10 − 3 , a value which determines the quasispecies distribution in rna virus populations. carnivore parvoviruses are very prone to genetic evolution, showing substitution rates similar to those of rna viruses, with values of approximately 10 − 4 substitutions per site per year. this result, together with the detection of cpv-2c, which has already been reported in multiple infections of high genetic complexity [3, 5, 39] , confirmed that co-infection with different species of parvovirus in feline hosts led to a high genetic variability and to the potential emergence of new viruses [3] . the presence of distinctive mutations between the fpv dna sequences detected and the sequences of modified live fpv vaccine strains available, together with the lack of information regarding the vaccination history of the cats sampled, allowed the authors to exclude the possibility that dna from vaccine strains was detected and did not allow speculation regarding the persistence of modified live vaccine strains in the cats sampled. however, viraemia persisting up to 24 days post vaccination has been reported in dogs vaccinated with modified live canine parvovirus [40] . additional studies are required to investigate the nature and clinical significance of the presence of parvovirus dna in the wbc of healthy cats and the potential of cats as parvovirus carriers. ideally, faeces for the examination of shedding should also be included in the sampling; viral isolation could be useful for testing viral vitality, and reverse transcription-pcr assay targeting international committee on taxonomy of viruses ictv genetic analysis of feline panleukopenia viruses from cats with gastroenteritis genetic complexity and multiple infections with more parvovirus species in naturally infected cats genetic analysis of feline panleukopenia virus full-length vp2 gene in domestic cats between high genetic diversity of the vp2 gene of a canine parvovirus strain detected in a domestic cat within-host genetic diversity of endemic and emerging parvoviruses of dogs and cats co-infection with feline and canine parvovirus in a cat infectious diseases of the dog and the cat infectious diseases of the dog and the cat canine parvovirus post-vaccination shedding: interference with diagnostic assays and correlation with host immune status immune carrier state of feline panleukopenia virus-infected cats antigenic and genomic variabilities among recently prevalent parvoviruses of canine and feline origin in japan feline host range of canine parvovirus: recent emergence of new antigenic types in cats canine parvovirus in asymptomatic feline carriers isolation of feline parvovirus from peripheral blood mononuclear cells of cats in northern vietnam predominance of canine parvovirus (cpv) in unvaccinated cat populations and emergence of new antigenic types of cpvs in cats characterisation of cross-reactivity of virus neutralising antibodies induced by feline panleukopenia virus and canine parvoviruses identification of parvovirus in the bone marrow of eight cats bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt. nucl acids symp ser dnasp v5: a software for comprehensive analysis of dna polymorphism data mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets hemagglutination by canine parvovirus: serologic studies and diagnostic applications canine parvovirus: strain difference in haemaglutination activity and antigenicity characterization of parvoviruses from domestic cats in brazil an improved hemagglutination test for study of canine parvovirus host-specific parvovirus evolution in nature is recapitulated by in vitro adaptation to different carnivore species pathogenesis of aleutian mink disease parvovirus and similarities to b19 infection minute virus of mice: antibody response, viral shedding, and persistence of viral dna in multiple strains of mice persistence of human parvovirus b19 in human tissues evidence for persistence of human parvovirus b19 dna in bone marrow tissue persistence of parvovirus b19 genotypes in asymptomatic persons isolation of canine parvovirus from a cat manifesting clinical signs of feline panleukopenia antigenic type distribution among canine parvoviruses in dogs and cats in germany characterisation of canine parvovirus strains isolated from cats with feline panleukopenia western european epidemiological survey for parvovirus and coronavirus infections in dogs efficacy of feline panleucopenia vaccine to prevent infection with an isolate of cpv2b obtained from a cat pathogenic potential of canine parvovirus types 2a and 2c in domestic cats high rate of viral evolution associated with the emergence of carnivore parvovirus co-infection with multiple variants of canine parvovirus type 2 (cpv-2) long-term viremia and fecal shedding in pups after modified-live canine parvovirus vaccination comparison of polymerase chain reaction with virus isolation and haemagglutination assays for the detection of canine parvoviruses in faecal specimens analysis of canine parvovirus sequences from wolves and dogs isolated in italy none. this research received no grant from any funding agency in the public or commercial sectors. the nucleotide sequences obtained in this study are available at genbank (no. kt151621 -kt151638). sequence alignment are available from the corresponding author (prof. mara battilani) upon request. alignment and phylogeny data were linked to the dryad repository via treebase search id: http://purl.org/phylo/treebase/phylows/study/tb2:s22165. authors' contributions mb and aa designed the study. rz and cd collected the blood samples and the background information on the samples. preparation of buffy coat and dna extraction was performed by aa. the pcr and vp sequencing was carried out by aa, fb and sda under the supervision of mb. ab analysed the sequence data and drafted the manuscript. mb critically revised the manuscript for important intellectual content. all the authors read the manuscript and approved the final version. the study was carried out using stored blood samples which had been collected for clinical and laboratory purposes independent of the study with the agreement of the cat owners who presented their cats to the veterinary teaching hospital (university of sassari). as stored blood samples were used, no separate ethical approval was required for the study. all efforts were made to minimise the discomfort of the animals during sampling. 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.• 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-000010-prsvv6l9 authors: qin, jian; jones, robert c.; ramakrishnan, ramesh title: studying copy number variations using a nanofluidic platform date: 2008-08-18 journal: nucleic acids res doi: 10.1093/nar/gkn518 sha: doc_id: 10 cord_uid: prsvv6l9 copy number variations (cnvs) in the human genome are conventionally detected using high-throughput scanning technologies, such as comparative genomic hybridization and high-density single nucleotide polymorphism (snp) microarrays, or relatively low-throughput techniques, such as quantitative polymerase chain reaction (pcr). all these approaches are limited in resolution and can at best distinguish a twofold (or 50%) difference in copy number. we have developed a new technology to study copy numbers using a platform known as the digital array, a nanofluidic biochip capable of accurately quantitating genes of interest in dna samples. we have evaluated the digital array's performance using a model system, to show that this technology is exquisitely sensitive, capable of differentiating as little as a 15% difference in gene copy number (or between 6 and 7 copies of a target gene). we have also analyzed commercial dna samples for their cyp2d6 copy numbers and confirmed that our results were consistent with those obtained independently using conventional techniques. in a screening experiment with breast cancer and normal dna samples, the erbb2 gene was found to be amplified in about 35% of breast cancer samples. the use of the digital array enables accurate measurement of gene copy numbers and is of significant value in cnv studies. variation in the human genome occurs on multiple levels, from single nucleotide polymorphisms (snps) to duplications or deletions of contiguous blocks of dna sequences (1) (2) (3) (4) (5) . copy number variation (cnv) is an important polymorphism of dna segments across a wide range of sizes and one of the primary sources of variation in the human genome (6) . recently, cnv has been studied extensively because of its close association with large numbers of human disorders (7, 8) . an understanding of this variation is important not only to understand the full spectrum of human genetic variation but also to assess the significance of such variation in disease-association studies. the first human cnv map was constructed from a study of 270 normal individuals with a total of 1447 cnv regions in the whole genome (9) ; more than 15 000 cnvs have been found in the human genome (http://projects. tcag.ca/variation). a recent paper demonstrated the presence of 525 novel insertion sequences across the genomes of eight unrelated individuals, which were not present in the human reference genome, and showed that many of these have different copy numbers (10) . however, the current cnv analysis is mainly dependent upon microarray-based snp and comparative genomic hybridization (cgh) platforms, or dna sequencing, and is therefore subject to low sensitivity and low resolution. these techniques are high throughput but lack the flexibility of analyzing individual genes or sequences of interest. other existing technologies, such as quantitative polymerase chain reaction (pcr), are limited because of their inability to reliably distinguish less than a twofold difference in copy number of a particular gene in dna samples (11) (12) (13) . in this study we demonstrate the use of a unique integrated nanofluidic system, the digital array, in the study of cnvs. the digital array (14, 15) is able to accurately quantitate dna samples based on the fact that single dna molecules are randomly distributed in more than 9000 reaction chambers and then pcr amplified. the concentration of any sequence in a dna sample (copies/ml) can be calculated using the numbers of positive chambers that contain at least one copy of that sequence. in order to ensure that the apparent difference in gene copy numbers in different samples are real, and not distorted by differences in sample amounts, we use the expression 'relative copy number'. the relative copy number of a gene is the number of copies of that gene per haploid genome. it can be easily expressed as the ratio of the copy number of a target gene to the copy number of a single copy reference gene (two copies per cell) in a dna sample, which is always 1 per haploid genome. by using two assays for the two genes (the gene of interest and the reference gene) with two fluorescent dyes on the same digital array, we are able to simultaneously quantitate both genes in the same dna sample. the ratio of the numbers of molecules of these two genes is the relative copy number of the gene of interest in a dna sample. a single copy gene should have a relative copy number of 1. a relative copy number greater than 1 indicates the presence of duplication of the target gene while a number smaller than 1 implies deletion of this gene. our data show that the digital array is able to distinguish less than twofold differences in gene copy number and differentiate between 1, 2, 3, 4, 5, 6 and 7 copies of a gene with great accuracy. it provides a reliable and robust platform to study copy number variations and has great advantages over conventional techniques. the sequence of the rpp30 synthetic construct and the sequences of the primers and probe used to amplify this construct are shown in supplementary the taqman assay for the rnase p gene (vic) was ordered from applied biosystems (foster city, ca). the feasibility of digital pcr has previously been demonstrated by performing pcr on a single dna sample obtained by a serial dilution process (16, 17) . target molecules in a dna sample could be quantitated by counting the number of positive reactions. we utilize the principle of partitioning instead of dilution in order to identify and quantitate individual dna molecules. the fluidigm digital array is a novel nanofluidic biochip where digital pcr reactions can be performed (14, 15) . utilizing nanoscale valves and pumps, the digital array delivers up to 12 mixtures of sample and pcr reagents into 12 individual panels. each panel contains 765 independent 6-nl chambers. this nanofluidic platform utilizes soft lithography and silicone rubber to create nanoscale valves and pumps that can be used in serial or parallel applications. the digital array is composed of a pdms (silicone rubber) integrated fluidic circuit, an integrated heat spreader to ensure rapid heat transfer and temperature uniformity within the array and an sbs-formatted carrier with inputs and pressure accumulator to act as an interface between the user and the pdms chip. there are 12 carrier inputs corresponding to 12 separate sample inputs to the chip. individual samples of a minimum volume of 8 ml each are delivered into 765 6-nl preprogrammed partitioning chambers in the chip by pressure-driven 'blind filling' in the pdms. control lines are primed with control fluid and are pressurized to actuate valves between the reaction chambers. the valves partition individual chambers that are kept closed during the pcr experiment. one of the important applications of the digital array is absolute quantitation (14, 15) . the dna molecules in each mixture are randomly partitioned into the 765 chambers of each panel. the chip is then thermocycled on fluidigm's biomark system and the positive chambers that originally contained one or more molecules will generate fluorescent signals and can be counted by the digital pcr analysis software. since the volumes and dilution factors of the dna samples are known prior to loading into the digital array, the dna concentrations can be accurately calculated. the precision of this test is only dependent upon the sampling randomness and, like any biological experiments, will improve with multiple tests (panels). digital array has been routinely used by us to quantitate dna samples of unknown concentration and, especially, cdna samples whose concentrations of the sequences of interest are hard to determine otherwise. when duplication occurs, multiple copies of a gene might be closely linked on the same chromosome and therefore might not be separated from each other, even on the digital array. as a result, multiple copies might behave as a single molecule and the total number of copies of the gene would be underestimated. when two copies are separated by a large genomic distance, some of them might be separated when dna molecules are fragmented during purification. however, in most cases this would not be sufficient (see table 2 , sample na11994 genomic dna data). specific target amplification (sta) is a good solution to this problem. sta is a simple pcr reaction with primers for both the reference gene and the gene of interest. it is typically performed for a limited number of thermal cycles (five in this study). the copy numbers of both genes are proportionally increased. using this process, multiple copies of the gene of interest will be amplified separately and later randomly partitioned into chambers in the digital array. since the newly generated molecules of both genes reflect the original ratio and they are not linked any more, a digital chip analysis can quantitate the molecules of the two genes and measure their ratio, and therefore the copy number of the gene of interest, very accurately ( figure 3 ). it is very important that the amplification efficiencies of the two pairs of primers be approximately equal in order not to introduce any bias in the ratio of the two gene copy numbers in the limited number of sta thermal cycles, although this is likely to have an insignificant effect on our results since we utilized only five cycles of preamplification. the amplification efficiency of any pair of primers can be easily measured using real-time pcr (18) . sta was performed on a geneamp pcr 9700 system (applied biosystems, foster city, ca) in a 5 ml reaction containing 1 â taqman preamp master mix (applied biosystems, foster city, ca), 225 nm of primers for both rnase p and the target gene and 10-50 ng dna. thermocycling conditions were 958c, 10 min hot start and five cycles of 958c for 15 s and 608c for 2 min. the products were diluted prior to the copy number analysis on the digital array based on their initial concentrations so that there would be about 500-600 rnase p molecules per panel. copy number analysis using the digital array on the biomark system each panel of a digital array contains a total of 4.59 ml (6 nl â 765 chambers) pcr reaction mix. however, 10 ml reaction mixes were normally prepared for each panel, containing 1 â taqman gene expression master mix (applied biosystems, foster city, ca), 1 â rnase p-vic taqman assay, 1 â taqman assay (900 nm primers and 200 nm probe) for the target gene, 1 â sample loading reagent (fluidigm, south san francisco, ca) and dna with about 1100-1300 copies of the rnase p gene. the reaction mix was uniformly partitioned into the 765 reaction chambers of each panel and the digital array was thermocycled on the biomark system (http:// www.fluidigm.com/products/biomark-main.html). thermocycling conditions included a 958c, 10 min hot start followed by 40 cycles of two-step pcr: 15 s at 958c for denaturing and 1 min at 608c for annealing and extension. molecules of the two genes were independently amplified. fam and vic signals of all chambers were recorded at the end of each pcr cycle. after the reaction was completed, digital pcr analysis software (fluidigm, south san francisco, ca) was used to process the data and count the numbers of both fam-positive chambers (target gene) and vic-positive chambers (rnase p) in each panel. there are 765 chambers in each of the 12 panels in a digital array. when single dna molecules are randomly partitioned into these chambers, it is possible that multiple molecules could partition into the same chamber. as a result there could be more molecules in each panel than positive chambers. the true number of molecules per chamber can be estimated using a simple poisson distribution equation as described by sindelka et al. (15) . we have developed a more robust computational algorithm to analyze cnv data obtained from the digital array. this algorithm has been integrated into the digital pcr analysis software and is detailed in (19) . a proof-of-principle spike-in experiment was performed using a synthetic construct to explore the digital array's feasibility as a robust platform for the cnv study. a 65-base oligonucleotide that is identical to a fragment of the human rpp30 was ordered from integrated dna technologies (coralville, ia, usa). rnase p, a single copy gene, is used as reference in this study (20, 21) . both rpp30 synthetic construct and human genomic dna na10860 from the coriell cell repositories (camden, nj, usa) were quantitated using the rpp30 assay on a digital array. different amounts of rpp30 synthetic construct were then spiked into the genomic dna so that mixtures with ratios of rpp30 and rnase p of 1 : 1 (no spike-in), 1 : 1.5, 1 : 2, 1 : 2.5, 1 : 3 and 1 : 3.5 were made, simulating dna samples containing two to seven copies of the rpp30 gene. these spike-in mixtures were analyzed on the digital arrays. five panels were used for each mixture and 400-500 rnase p molecules were present in each panel. the ratios of rpp30/rnase p of all samples were calculated and are plotted against the expected ratios in figure 1 . a good linear relationship can be observed. also shown in figure 2 is an example of a typical digital array experiment. cnvs of the cyp2d6 gene cyp2d6 belongs to the cytochrome p450 system responsible for the metabolism of many commonly prescribed medications (22, 23) . the cyp2d6 gene is highly polymorphic and this can significantly influence the metabolic activity of the enzyme it codes for (debrisoquine 4-hydroxylase) and the therapeutic efficacy of the drugs. therefore, the pharmacogenetic polymorphism information of this gene would be of great clinical importance in therapeutic decision-making (24) (25) (26) (27) . more than 100 alleles of the cyp2d6 gene have been identified (http://www.cypalleles. ki.se/cyp2d6.htm). allele-associated variations in the activity of the cyp2d6 enzyme have been observed and individuals carrying these alleles are classified into poor, intermediate, extensive and ultrarapid metabolizers (28, 29) . genotyping patients would be able to identify those who are at risk of severe toxic responses (poor metabolizer) or in need of more than standard level of drugs (ultra rapid metabolizer). it has been shown that some poor metabolizers and ultra rapid metabolizers are caused by the deletion or duplication of the entire cyp2d6 gene (30, 31) . these large structural changes can be detected using conventional technologies such as southern blot and longrange pcr. however, it is believed that real-time pcr is figure 1 . quantitation of the rpp30 copy number in spike-in samples that contain two to seven copies of the rpp30 molecules per two haploid genomes. the x-axis shows the expected ratio of the numbers of rpp30 molecules to rnase p molecules. the y-axis shows the observed ratios. each value is calculated using five panels of the same sample mix and the error bars represent standard errors. a good linear correlation can be seen with a coefficient of determination (r 2 ) of 0.996. currently the only promising technique that is able to provide information about the exact copy number of the cyp2d6 gene in a routine clinical setting (32) (33) (34) . we used the digital array to measure the cyp2d6 copy numbers of three dna samples from paragondx (morrisville, nc). the cyp2d6 genotypes of these dna samples had been characterized (table 1 ). the samples were sta-treated (see figure 3 and materials and methods section) and the products were analyzed using five panels each on the digital arrays. the relative copy numbers of these three samples are 0, 0.49 and 0.98, respectively, highly consistent with their assumed cyp2d6 diploid copy numbers (0, 1 and 2) based upon their genotypes. we also studied five cell line dna samples from coriell cell repositories (camden, nj). first, we measured their relative copy numbers using genomic dna. the results showed that two of them have a single copy and two have two copies of the cyp2d6 gene per cell ( table 2) . one sample had a relative copy number of about 1.17, equal to a diploid copy number of 2.34. we then sta-treated these five samples and ran the products on digital arrays. the relative copy numbers of the 1-and 2-copy samples remained the same and the fifth sample showed a relative copy number of about 1.5 or a diploid copy number of 3. apparently this sample had a duplication of the cyp2d6 gene on one of the two chromosomes (35) . it has been previously demonstrated (31, 36) that when cyp2d6 duplication occurs, the two copies are separated by 12.1 kb. therefore, the diploid copy number of 2.34 obtained when genomic dna was used is likely the result of dna breakage in this 12.1 kb genomic region in some dna molecules that separated the two cyp2d6 copies. to confirm this, we ran a long range pcr [see (31) cyp2d6 duplication was observed only in the sample with a relative copy number of 1.5 ( figure 4) . erbb2 (also known as her2) is a receptor tyrosine kinase gene overexpressed in up to 30% of invasive breast cancer, resulting in a loss of normal cellular growth control. most of these cases (97%) are caused by the amplification of this gene and the number of extra copies is closely related to the protein expression level (37) (38) (39) (40) . erbb2 amplification is well correlated with an aggressive phenotype characterized by reduced response to chemotherapy, high recurrence rate and short survival time and serves as a significant prognostic predictor for breast cancer patients (37, 41) . trastuzumab (herceptin), an fda-approved monoclonal antibody against the erbb2 protein, has been shown to dramatically increase response rate and extend survival in breast cancer patients with erbb2 amplification. given trastuzumab's proven efficacy and substantial benefit in multiple clinical trials, detection of erbb2 amplification has become critical (42) (43) (44) (45) . there are different methodologies of determining the erbb2 status in breast cancer. immunohistochemistry (ihc) and fluorescence in situ hybridization (fish) are two fda-approved technologies for the detection of erbb2 amplification. the former detects overexpression of the erbb2 receptor on the cell membrane while the latter detects the copy number of the gene itself relative to the chromosome 17 centromere. ihc is less expensive and easy to perform but is prone to a high rate of inaccuracies due to variations in tissue preparation, protein stability, antibody sensitivity and scoring subjectivity. on the other hand, fish is accurate with good clinical correlation but it is expensive, time consuming, and labor intensive and requires very experienced personnel. therefore, suggestions have been made to use a combination of ihc and fish, where ihc is used as a screening procedure followed by a fish confirmation if necessary (46, 47) . we used digital arrays to analyze the erbb2 copy numbers of 40 breast cancer and 8 normal breast tissue dna samples from biochain (hayward, ca). all dna samples were from asian individuals except one normal sample that was from a caucasian. of the 40 breast cancer samples, 3 are adenocarcinoma, 1 is fibroadenoma, 2 are invasive lobular carcinoma, 1 is infiltrative ductal carcinoma and 33 are invasive ductal carcinoma. the samples were sta-treated and, for screening purpose, the products were analyzed using only two panels for each sample on digital arrays. the results are shown in figure 5 . fourteen breast cancer samples (35%) had a diploid erbb2 copy number of more than five while all control samples were below five copies [an absolute number of erbb2 copies greater than 4.0 per cell is considered amplification in fish analysis (47) . here we use five as the threshold]. the copy numbers shown are not all integers due to (i) heterogeneity of the cancer cells and (ii) sampling variations as only two panels were used for each sample. a real-time pcr reaction was also performed on these 48 samples. twenty-four replicates were used for each sample. although the average copy numbers were close to the digital array data, large fluctuations (sds of up to 0.5) were observed in the 24 reactions of each sample. studies on other genes (for example, cyp2d6) showed that real-time pcr does not always produce accurate results (data not shown). genomewide analyses have shown the existence of large numbers of cnvs in the entire human genome with large interindividual diversity (48) (49) (50) (51) (52) (53) . many of these cnvs colocalize with genes involved in a variety of diseases or disease susceptibility and are believed to play some role in pathogenesis (54) (55) (56) (57) . the first mendelian disorder associated with the amplification of a 750 kb dna fragment was reported recently (54) . it appears to only be a the results of both genomic dna and sta products are shown. the ratios of the cyp2d6 gene to the rnase p gene should be close to multiples of 0.5. the genomic ratio of 1.17 for sample na11994 (corresponding to a diploid copy number of 2.34) reflects the partial separation of the duplication alleles in the genomic dna. a ratio of 1.51 (diploid copy number of 3) was obtained when the sample was subjected to sta prior to the digital pcr analysis. question of time before more genetic conditions related to cnv are identified. two standard genomewide scanning methods for cnv detection are array-based cgh and high-density snp genotyping arrays and both were employed in the construction of the first human cnv map (58) . these microarray techniques are able to generate whole-genome cnv data and are important in cnv discovery. their resolution is also improving with the development of new probes. however, since they are both based on hybridization, the detection of copy number changes largely depends on signal-to-noise ratio, which is sensitive to reagent and manufacturing variability. therefore, false positive and false negative results are sometimes inevitable (59) . additionally, the lack of standard reference genomes in the studies using these technologies further complicates the interpretation of the results (60). on many occasions, gene-or locus-specific (other than the whole genome) copy number information is required. this is especially true in the cases of cyp2d6 and erbb2 described above in which therapeutic decision needs to be made based upon the copy numbers of these genes. in addition to other conventional methods (southern blot, long-range pcr and fish), the possibility of using quantitative pcr in the cnv study of these two genes has been previously explored (61) (62) (63) (64) (65) . quantitative pcr is simple and easy to perform. however, since the copy number of the target gene is derived from the ct difference between the target gene and a reference gene, the results are very sensitive to the efficiency of the amplification reaction. even if one compensates for the amplification efficiency, it is considered difficult to obtain a discrimination power of better than twofold (66) . the digital array has the ability to absolutely quantitate any type of dna sample. in a multiplex pcr reaction with two assays, the quantitation of two or more genes/sequences in a single sample becomes possible, effectively eliminating pipetting variations inherently occurring in any quantitation experiment. the accuracy of the results is only subject to the random distribution of the molecules and, like any biological experiments, can improve with the use of multiple replicates for each sample. sta can efficiently separate the linked copies of a gene on the same chromosome when duplication occurs while other methods, such as restriction digestion are also valid (data not shown). we performed three experiments to test the feasibility of the digital array in the cnv study. first we measured the copy numbers of the rpp30 gene of a series of mixtures made of a human genomic dna and a synthetic rpp30 construct. we observed a very good correlation between the results and the expected outcome. we then studied the cyp2d6 copy numbers of some dna samples that were either genotyped elsewhere or characterized by us using conventional techniques. the results were also consistent. lastly, we screened 40 breast cancer samples for the amplification of the erbb2 gene. although the clinical data (other than pathological classification) of these samples were lacking, about 35% of the samples had an increased number of this gene above 5, very close to the erbb2 amplification frequency reported in the literature (67) . in conclusion, this study shows that the digital array provides a new and robust technology to study geneand sequence-specific cnv and is able to detect gene copy numbers with great accuracy. digital arrays provide a much greater discrimination power than quantitative pcr. cnv studies on the digital array are easy to perform, fast and the data obtained is easy to interpret. furthermore, the platform is very flexible and can be tailored to any gene/sequence. it can also serve as an independent measure to verify results from the whole-genome scans using array technologies. the digital array is an excellent cnv platform for both basic research and clinical investigation. supplementary data are available at nar online. funding for open access charge: fluidigm corporation. conflict of interest statement. the authors declare competing financial interests. all are employees of fluidigm corporation. (19) . the essence of snps recent duplication, domain accretion and the dynamic mutation of the human genome detection of large-scale variation in the human genome large-scale copy number polymorphism in the human genome segmental duplications and copy-number variation in the human genome structural variation in the human genome new perspectives for the elucidation of genetic disorders genomic rearrangements and sporadic disease global variation in copy number in the human genome mapping and sequencing of structural variation from eight human genomes novel real-time quantitative pcr test for trisomy 21 digital pcr for the molecular detection of fetal chromosomal aneuploidy two-fold differences are the detection limit for determining transgene copy numbers in plants by real-time pcr high throughput gene expression measurement with real time pcr in a microfluidic dynamic array intracellular expression profiles measured by real-time pcr tomography in the xenopus laevis oocyte nanoliter scale pcr with taqman detection application of real-time quantitative pcr in the analysis of gene expression. dna amplification: current technologies and applications mathematical analysis of copy number variation in a dna sample using digital pcr on a nanofluidic device real-time reverse transcriptionpolymerase chain reaction assay for sars-associated coronavirus structure and transcription of a human gene for h1 rna, the rna component of human rnase p the effect of cytochrome p450 metabolism on drug response, interactions, and adverse effects overview of enzymes of drug metabolism the clinical role of genetic polymorphisms in drug-metabolizing enzymes individualized drug therapy the prevalence and clinical relevance of cytochrome p450 polymorphisms pharmacogenetics and adverse drug reactions cyp2d6 polymorphisms and the impact on tamoxifen therapy clinical implications of cyp2d6 genetic polymorphism during treatment with antipsychotic drugs deletion of the entire cytochrome p450 cyp2d6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism ultrarapid metabolizers of debrisoquine: characterization and pcr-based detection of alleles with duplication of the cyp2d6 gene cyp2d6 genotyping strategy based on gene copy number determination by taqman real-time pcr determination of cytochrome p450 2d6 (cyp2d6) gene copy number by real-time quantitative pcr pharmacogenetic screening of the gene deletion and duplications of cyp2d6 the human debrisoquine 4-hydroxylase (cyp2d) locus: sequence and identification of the polymorphic cyp2d6 gene, a related gene, and a pseudogene ultrarapid drug metabolism: pcr-based detection of cyp2d6 gene duplication human breast cancer: correlation of relapse and survival with amplification of the her-2/neu oncogene studies of the her-2/neu proto-oncogene in human breast and ovarian cancer detection and quantitation of her-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization prognostic and predictive value of her2/neu oncogene in breast cancer erbb2 oncogene in human breast cancer and its clinical significance use of chemotherapy plus a monoclonal antibody against her2 for metastatic breast cancer that overexpresses her2 ongoing adjuvant trials with trastuzumab in breast cancer trastuzumab after adjuvant chemotherapy in her2-positive breast cancer ) 2-year follow-up of trastuzumab after adjuvant chemotherapy in her2-positive breast cancer: a randomised controlled trial prognostic and predictive value of her2/neu oncogene in breast cancer her2 testing: a review of detection methodologies and their clinical performance recent duplication, domain accretion and the dynamic mutation of the human genome detection of large-scale variation in the human genome large-scale copy number polymorphism in the human genome segmental duplications and copy-number variation in the human genome structural variation in the human genome challenges and standards in integrating surveys of structural variation autosomal-dominant microtia linked to five tandem copies of a copy-number-variable region at chromosome 4p16 a comprehensive analysis of common copynumber variations in the human genome new perspectives for the elucidation of genetic disorders genomic rearrangements and sporadic disease global variation in copy number in the human genome methods and strategies for analyzing copy number variation using dna microarrays challenges and standards in integrating surveys of structural variation cyp2d6 genotyping strategy based on gene copy number determination by taqman real-time pcr determination of cytochrome p450 2d6 (cyp2d6) gene copy number by real-time quantitative pcr pharmacogenetic screening of the gene deletion and duplications of cyp2d6 her-2/neu gene copy number quantified by real-time pcr: comparison of gene amplification, heterozygosity, and immunohistochemical status in breast cancer tissue reliability and discriminant validity of her2 gene quantification and chromosome 17 aneusomy analysis by real-time pcr in primary breast cancer digital pcr for the molecular detection of fetal chromosomal aneuploidy human breast cancer: correlation of relapse and survival with amplification of the her-2/neu oncogene the authors would like to thank dr stephen quake for his assistance in the interpretation of the results, as well as his careful reading of this article. key: cord-007613-g4s0v8ra authors: rimstad, espen; reubel, gerhard h.; dean, gregg a.; higgins, joanne; pedersen, niels c. title: cloning, expression and characterization of biologically active feline tumour necrosis factor-α date: 2000-03-10 journal: vet immunol immunopathol doi: 10.1016/0165-2427(94)05345-s sha: doc_id: 7613 cord_uid: g4s0v8ra we report the cloning, expression and characterization of biologically active feline tumour necrosis factor-α (ftnf-α). messenger rna was extracted from feline peritoneal macrophage cultures and used to synthesize cdna for polymerase chain reaction (pcr) amplification. the pcr products were cloned into the plasmid vector pcrii and sequenced, showing 99.3% homology with a published ftnf-α gene sequence. subcloning into the vector pgex-2t and subsequent expression resulted in a 43 kda fusion protein of ftnf-α and glutathione s-transferase (gst). thrombin cleavage of the fusion protein yielded a 17 kda protein. this protein cross-reacted with a monoclonal anti-human tnf-α antibody in western blotting, but not with a polyclonal anti-murine tnf-α serum. recombinant ftnf-α (rftnf-α) and rftnf-α-gst had a cd(50) of 15 ng ml(−1) and 230 ng ml(−1), respectively, in the l929 cytotoxicity assay. cats given rftnf-α-gst intravenously manifested the typical biological effects of tnf-α, including fever, depression, and piloerection. the rftnf-α-gst upregulated il-2 receptor and mhc-ii antigen expression on peripheral blood mononuclear cells stimulated in vitro, but had no effect on tnf-α receptor and mhc-i antigen expression. tumour necrosis factor-alpha (tnf-a ) is a cytokine with multifunctional activity. although its original activity was recognised against tumour cells (carswell et al., 1975) , it is now known to play an important role in immune and inflammatory responses as well as in the pathogenesis of many human and animal diseases (reviewed by jhattelh, 199 1). tnf-a may also play a crucial role in the pathogenesis of human aids (matsuyama et al., 199 1) . tnf-c~ stimulates human immunodeficiency virus (hiv) replication in both established lymphoid and primary t cell cultures (suzuki et al., 1989) . this enhanced replication is mediated through tnf-a inducible nuclear factors like nfkb and the kb enhancer elements of the hiv ltr (osborn et al., 1989) . induction of hiv gene expression is regulated by interactions of dna binding proteins with specific gene sequences (folks et al., 1989; osborn et al., 1989; poli et al., 1990) . the levels of tnf-(w are increased in patients with aids and may upregulate virus replication in an autocrine fashion (poli et al., 1990 ). in addition tnf-a, may play an important role in some clinical manifestations of hiv infection; dramatic improvement in aphthous stomatitis and esophagitis is seen in aids patients treated with a tnf-a inhibitor (thalidomide) (nicolau and west, 1990) . tnf-a! is an important reagent, therefore, for studies of hiv pathogenesis. the nucleotide sequences for human, mouse, sheep, pig, rabbit and cat tnf-a, has previously been reported (pennica et al., 1985; shirai et al., 1985; ito et al., 1986; drews et al., 1990; mcgraw et al., 1990; green and sargan, 1991) . both human and murine recombinant tnf-a proteins have been expressed in different systems, and used in several studies (pennica et al., 1985; shirai et al., 1985) . however, recombinant feline tnf-a (rftnf-cu) protein has not been expressed. the aim of this study was to clone the cdna of feline tnf-cu and to express it in e~/zerichia coli in a biologically active form. our goal is to use rftnf-a to study immunodeficiency virus pathogenesis using the feline immunodeficiency virus (fiv) infection model. fiv infection has been shown to be a valid animal model for hiv studies because similar changes in tnf-a! expression have also been observed in fiv infected cats (lawrence et al., 1992; lehmann et al., 1992; pedersen, 1993) . adult specific pathogen free (spf) cats were obtained from the breeding colony of the feline retrovirus research laboratory, university of california, davis. animals were housed in quarters provided by the animal research service, university of california, davis. peritoneal macrophages were obtained by peritoneal saline lavage from two specific pathogen free cats as previously described (stoddart and scott, 1988; brunner and pedersen, 1989) . cats were inoculated intraperitoneally with 0.75 ml of human diphtheria/pertussis/tetanus vaccine, and the peritoneal cavities lavaged 4 days later. cells were pelleted by centrifugation and resuspended in rpm1 medium with 10% fetal bovine serum (fbs ) and cultured for 8 h. adherent cells were determined to be virtually 100% macrophages by non-specific alpha-naphthyl esterase staining (stoddart and scott, 1988 ) . the macrophage cultures were then stimulated with 100 ng ml-' of e. coli lipopolysaccharide (lps ) (sigma, st. louis, mo) for 2 h, and frozen at -70°c until further use. the 2 h duration of lps stimulation was based upon the kinetics of tnf-a, production after lps stimulation in macrophages from other animal species (green and sargan, 1991 ). messenger rna was extracted both from 4 x 1 o6 unstimulated and 4 x 1 o6 lps stimulated macrophages using an mrna extraction kit (micro-fast trac, invitrogen, san diego, ca); 0.08 pg mrna from each source was used for cdna synthesis. synthesis of cdna was performed at 42°c for 2 x 60 min using oligo-dt primers (cdna-cycle kit, invitrogen). the cdna was then used in a polymerase chain reaction (pcr) with a 100 ~1 reaction mixture consisting of 10 mm tris-hcl (ph 8.3), 50 mm kcl, 1.5 mm mgcl,, 0.0 1% gelatine, 200 pm of each dntp, 30 pmol of each primer, and 2.5 u of pfu dna polymerase. three primers, making up two pairs p 1 /p3 and p2/p3, were constructed from a previously published sequence of feline tnf-a (mcgraw et al., 1990) : p 1, gggatccatgagcactgaaagcatgatccg; p2, ggggatcccagaa-cactcagatcatcttctc; p3, ggctgcagaattcacagggcaat-gatcccaaagta. the primers had restriction sites for bamhi, psti and ecorl in the 5'-ends to make directionally cloning of the pcr products possible. the forward primer p 1 was located at the start of the ff nf-a gene and the forward primer p2 at the assumed start of the coding sequences for the mature tnf-cr protein, while the backward primer p3 was located at the 3'-end of the coding sequences of the gene. the mixtures were overlaid with 30 ~1 mineral oil and heated at 94°c for 5 min and then cycled 35 times at 94°c for 1 min, 55 "c for 1 min, 72°c for 1 min with a final extension at 72°c for 7 min. the pcr products were subjected to electrophoresis on a 1.7% agarose gel using 3 v cm-' for 2 h in 0.5x tbe-buffer ( 1 xtbe = 0.09 m tris-borate, 2 mm edta ) and then stained with ethidium bromide. to assure that stimulated macrophage mrna was the actual source for cdna, cdna derived by reverse transcription of mrna from unstimulated macrophage cultures, as well as feline genomic dna, were extracted and used as targets in separate and parallel pcrs. the amplified fragments generated by both the p 1 /p3 and p2/p3 primers were separately cloned into the plasmid vector pcri1 (ta-cloning kit, invitrogen), and the nucleotide sequences were determined by conventional dideoxy sequencing of both strands. the cloned fragment from the amplification with p2/p3 was digested out with bamhi/ecori, and subcloned directly into the expression vector pgex-2t (pharmacia, uppsala, sweden). the pgex-2t vector has been used previously to express fiv-pl7 and -p24 proteins (reid et al., 199 1) . this expression vector contains an open reading frame encoding glutathione s-transferase (gst), followed by unique restriction endonuclease sites for bamhi, smal and ecorz, followed in turn by termination codons in all three frames. a thrombin cleavage site is constructed into the vector between gst and the protein to be expressed (chang, 1985 ) . the resulting plasmid, designated pgex-ft nf, was used to transform the e. coli strain xll-blue. a 100 ml overnight terrific-broth (t-broth) culture of e. coli containing pgex-ff nf was diluted 1: 10 in t-broth and incubated for 1 h at 37' c. all bacterial cultures contained 50 ,ug ml-' ampicillin and 12.5 ,ug ml-' tetracycline. expression of the recombinant protein was induced by adding isopropyl-p-d-thiogalactopyranoside (iptg) to a concentration of 0.3 mm. the culture was further incubated at 37°c for 5 h and then centrifuged for 10 min at 5oooxg. the supematant was discarded. the bacterial pellet was resuspended in 10 ml ice-cold phosphate-buffered saline (pbs), and sonicated twice for 30 s; samples were kept on ice. triton-x-100 was added to a concentration of 1%, the solution was centrifuged for 5 min at 10 000 xg, and the supernatant collected. one milliliter of a 50% slurry of glutathione-agarose beads was added to the supernatant and gently mixed for 3 min at room temperature, followed by three times washing with pbs. the fusion protein was eluted by adding 1 ml of 50 mm tris-cl (ph 8.0) with 10 mm reduced glutathione. the elution was repeated three times. the purity of the rftnf-cu, the efficiency of the elutions, and its relative molecular size were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). the part of the rfi'nf-cr-gst that bound to the glutathione-agarose beads, was washed with thrombin cleavage buffer (50 mm tris-cl, ph 7.5, 150 mm nacl, 2.5 mm cacl,) and incubated with 1% thrombin for 1 h at room temperature (chang, 1985) . the cleaved protein products were analyzed on sds-page. the concentrations of the purified recombinant proteins were analysed as described earlier (bradford, 1976) . peripheral blood mononuclear cells ( pmbcs) from four different normal donor cats were purified on ficoll-hypaque density gradients, and resuspended in growth medium (rpmi, 10% fbs, 1 ,ug ml-' concanavalin a (cona) ) to a concentration of 1 o6 cells ml-'. tenfold increasing concentrations (0, 1, 10, 100 and 1000 ng ml-' ) of rftnf-a-gst or rfi'nf-a were added to quintriplicate wells and the cells maintained in culture for 48 h before being analyzed for cell surface receptor expression. control wells contained growth medium without cona and no tnf-a. pmbcs from each culture were pelleted by low speed centrifugation, washed twice with pbs containing 2% fbs and 0.1% sodium azide (pbs/fbs/nan, ). the pelleted cells were then resuspended in one of the following reagents and incubated for 15 min: ( 1) 15 ~1 of tissue culture supernatant from mouse hybridoma cell line 9f23 containing antibodies against the alpha subunit of the feline interleukin-2 receptor (il-2r) (kindly provided by dr. k. ohno, tokyo, japan) (ohno et al., 1992) ; (2) 10 ~1 of tissue culture supematant from mouse hybridoma cell line 42.382, which contains antibodies to feline mhc class ii antigen (rideout et al., 1992 ) ; ( 3 ) 15 ~1 of tissue culture supematant from mouse hybridoma w6/32 which contains antibodies against feline mhc class i antigen (pollack et al., 1988) ; (4) 10 pg of rftnf-a. control cultures were left untreated. following incubation, cells were pelleted, and washed twice with pbs/ fbs/nan3. the tnf-a treated cells were incubated for an additional 15 min with 25 ~1 of mouse monoclonal antibody to human tnf-a receptor (biosource international, camarillo, ca), washed twice with pbs/fbs/nan,, and pelleted. cell pellets were then resuspended in 25 ~1 of a 1:25 dilution of goat f(ab' )2 anti-mouse igg-fitc (caltag laboratories, san francisco, ca) and 10 ~1 propidium iodide ( 100 mg ml-' ) were added to each tube and the samples incubated at 37°c for 15 min. samples were then washed twice with pbs/fbs/nan, buffer and 10 000 cells were analyzed immediately by flow immunocytometry using a 488 nm argon laser, standard filter configuration for two color analysis and consort 30 software (facscan, becton-dickinson, san jose, ca). data were analyzed with lysys software. events were gated on forward and log side scatter light characteristics and dead cells were eliminated from analysis based on propidium iodide staining. live cells were evaluated using log green fluorescence and analysis regions were set such that less than 2% of control cells were in the positive analysis region. western blot analysis of rffnf-cr and rftnf-a-gst were performed as described earlier (lutz et al., 1980) . polyclonal rabbit anti-murine tnf-a (genzyme, cambridge, ma) and monoclonal anti-human tnf-a (biosource international, camarillo, ca) were used. two adult new zealand white rabbits were immunised subcutaneously with 100 pg of rftnf-a-gst at weeks 0 and 2, and with the same amount of rffnf-(y on weeks 4, 6, 14, and 20. the first dose of antigen was in freund's complete adjuvant, while subsequent doses were in freund's incomplete adjuvant. major antibody activity was demonstrated against both gst and tnf-a component by western blotting. serial dilutions of recombinant proteins were tested for cytotoxic activity using the murine tibroblast cell line l929 as described (flick and gifford, 1984) . briefly, l929 cells were seeded into flat bottomed 96-well microtiter plates and incubated overnight in eagle's minimum essential medium (mem) supplemented with 5% fbs. medium was then replaced with mem containing 5 ,ug ml-' actinomycin d (sigma, st. louis, mo), samples were tested in quadruplicates of 100 ~1 and incubated for 16 h at 37°c and 5% co*. the cell supernatant was removed thereafter and the monolayer stained with crystal violet for 10 min. the absorbance of washed stained cell monolayers was measured at a wavelength of 595 nm using an automatic plate reader (biorad, hercules, ca). medium and recombinant feline immunodeliciency virus p24-gst (fiv-rp24-gst) were used as a negative control and recombinant mouse tnf-cr (rmtnf-ar, genzyme ) as a positive control. the concentration of ffnf-c~ resulting in 50% of the absorbance of the controls was considered the 50% cytotoxic dose ( cdso). recombinant ff nf-a ( 500 ng ml-' ) was incubated with different dilutions of both preimmune and immune rabbit anti-rltnf-a serum for 30 min at room temperature. the ffnf-cu antiserum mixtures were then tested with l929 cells as described above. the first study involved three groups of adult spf cats, each consisting of two animals. each group was injected i.v. with 25 or 50 pg kg-' of rftnf-cu-gst or 50 pg kg-' of gst dissolved in pbs. the cats were observed for clinical symptoms for a period of 48 h and rectal temperature was measured every 20 min the first 3 h. in a second study, two adult cats were each inoculated intravenously with 50 pg kg-' of rftnf-a-gst dissolved in pbs. clinical symptoms and rectal temperatures were measured 0,2,6, 12,24 and 48 h following treatment. a 700 bp dna fragment was amplified using primer pair p 1 /p3 and cdna from lps stimulated macrophages as target (fig. 1, lane 1) . no pcr product was amplified from cdna produced from mrna of unstimulated macrophages (fig. 1, lane 2 ) . three fragments of 1.7 kb, 400 bp and 250 bp were amplified from genomic dna (fig. 1, lane 3) . primer pair p2/p3 amplified a 500 bp dna fragment from cdna of stimulated macrophages (fig. 1, lane 4) . no pcr products were amplified from cdna derived from the mrna of unstimulated macrophages (fig. 1, lane 5 ) , while an 850 bp fragment was amplified from genomic feline dna (fig. 1, lane 6 ) . the primers pl /p3 of lps-stimulated, unstimulated feline macrophages, and genomic dna as targets, respectively. lanes 4, 5 and 6: rt-pcr using primers p2/p3 of lps-stimulated. unstimulated feline macrophages, and genomic dna as targets, respectively. sizes of the 1.7 kb and 850 bp amplified fragments from genomic feline dna corresponded to the distance between the pi-p3 and p2-p3 primers in the genomit ftnf-cu nucleotide sequence, respectively. similarly the 700 bp and 500 bp bands amplified from cdna from lps-stimulated macrophages corresponded to the estimated respective sizes of mrna for the pre-and mature-proteins of tnfo!. sequence analysis of the p 1 /p3 amplified product from cdna from stimulated macrophages showed a 99.3% homology to previously reported genomic dna sequence of ftnf-a gene, and 98.7% homology on the amino acid level. there were 9 1% and 8 1% sequence homologies between the mrna of ttnf-cu and the respective human and murine tnf-(y genes. the homologies between the deduced amino acid sequence of the mature part of ffnf-a, i.e. between primer pair p2/p3, to those of human and murine tnf-a, were 92% and 78%, respectively. a single band with a molecular size of 43 kda was observed in sds-page gels of the purified fusion protein rftnf-gst expressed by pgex-ffnf (fig. 2, lane 1) . thrombin cleaved the fusion protein into two fragments one with a size of 17 kda, which corresponds to the size of tnf-(u in other species (marmenout et al., 1985; pennica, et al., 1985) (fig. 2, lane 2) , the other was retained on the glutathione agarose beads after thrombin cleavage had thus a size of 26 kda, which corresponded to the size of gst (smith et al., 1986) . the amount of purified rftnf-gst produced was about 4 mg 1-l bacterial culture medium. however, the amount of rftnf-a after thrombin cleavage was only about one-tenth of this. no cross-reactivity was observed between polyclonal rabbit anti-murine tnf-(y antibodies and rffnf-a in western blot. as predicted from sequence homologies and previous studies (lehmann, et al., 1992) monoclonal anti-human tnfcr antibodies reacted specifically to both rffnf-gst and rff nf-(r (fig. 3 ) . l929 mouse tibroblast cells were susceptible to the toxic effects of both rffnfa-gst and rttnf-a (fig. 4) . at concentration below 125 ng ml-' the rffnf-a was significantly more toxic than a similar concentration of rftnf-lu-gst. the cds,, for the l929 cells was estimated to be 230 ng ml-' and 15 ng ml-' for rftnf-cy-gst and the rffnf-a, respectively. the cytotoxic effect of purified rffnf-a on l929 cells was completely neutralised by a 1: 10 dilution, and partially neutralised by a 1: 100 dilution, of rabbit anti rttnf-a! serum. preimmune serum had no inhibitory effect on the cytotoxicity. cats given rff nf-cu-gst, became clinically ill between 15 min and 10 h after treatment. a fever peaked at 4-5 h after treatment and disappeared after 10 h and was the most prominent syndrome (fig. 5) nously. an increase in the rectal temperature was apparent within 2 h in cats treated with rttnf-cu-gst but not with gst alone. depression, immobility caused by malaise, protrusion of the nictitating membranes, piloerection (especially along the dorsum of neck and back and on the tail) and hemoconcentration. clinical signs peaked at 4 h following treatment and had largely disappeared by 10 h. the clinical signs and their severity were similar in cats given either 25 and 50 pg kg-' of the fusion protein, except for one cat that was treated with 50 pg kg-' of rft nf-a-gst and developed moderately severe hypovolemic shock and signs of cerebellar hypoxemia (disorientation, loss of balance ). these signs appeared 4 h following treatment and lasted for about 1 h before spontaneously resolving. no clinical signs of illness were seen in two control cats that were given only the gst portion of the fusion protein (fig. 5 ) , although some variation was evident between the four individual donor cats, rftnf-a induced a dose related increase of ig2r and mhc class ii antigen expression on the cell surface of in vitro stimulated feline pmbcs (fig. 6 ) . at the highest rftnf-cr concentration, this increase was 13-23% for il-2r and 7-30% for mhc class ii antigen expression. recombinant ftnf-a had no stimulatory effect in vitro on mhc class i or tnf-cu receptor expression. biologically active recombinant feline tnf-a was expressed in e. coli. the entire itnf-a gene was cloned from cdna by pcr since the feline genomic tnf-a gene is interrupted by three introns, making it difficult to clone a functional tnf-a gene directly from genomic dna. although genomic dna was not used in the experiment the pcr primers were constructed from a tnf-a sequence that was itself derived from genomic dna . the cdna encoding the functional tnf-a gene was derived from mrna by reverse transcription. the mrna was extracted from peritoneal macrophages that were induced to produce high levels of tnf-a (and specific mrna) by e. coli lps stimulation. this procedure allowed for the ultimate construction of a plasmid containing only the relevant portions of the tnf-(r gene in a continuous linear configuration. the protein expression system used the pgex-2t vector. the gst fusion protein encoded by the plasmid pgex-itnf had a molecular size of 43 kda of which rftnf was making up 17 kda. this is in about the same size range as human and murine tnf-a (marmenout et al., 1985) ) and correlates well with the estimated molecular size ( 17.9 kda) of the 157 amino acid long mature feline tnf-cw. the reduction in the yield of rffnf-(y after the fusion protein had been cleaved with thrombin could have been caused by the lower solubility of the cleaved protein compared with the fusion protein. a possible obstructive effect of the agarose beads on the efficacy of thrombin cleavage could not be ruled out. analysis of the nucleotide sequence of the cloned mrna encoding for the preprotein of feline tnf-cy showed a homology of 99.3% with the coding sequences of a previously sequenced feline tnf-a gene ) and 98.7% homology at the amino acid level (98.8% homology for the mature part). the small nucleotide sequence divergence (0.7%) between these two ftnf-a genes reflects either limited genetic variations between individual cats and/or small errors in reverse transcription/pcr. if the differences were due to errors, however, the errors were not sufficient to alter the biological activity of the protein. the fusion protein and rftnf-a, both appeared to be biologically active. both proteins killed tnf-a-sensitive l929 mouse cells; rftnf-cr-gst had a toxic effect on these cells comparable to rftnf-cx at concentrations above 125 ng ml-', but at levels below 125 ng ml-' rff nf-cu was significantly more toxic than rfinf-(r-gst. this indicated some interference by the gst moiety on the rftnf-cu portion of the molecule. polyclonal rabbit anti-human tnf-a prevented the toxic effect of both rftnf-a-gst and rffnf-a on l929 cells, again demonstrating that both the cleaved and fused fi'nf-a! proteins were biologically and antigenitally intact. the rftnf-cr-gst was also biologically active when injected intravenously into cats. the onset and the character of clinical signs resembled those observed for tnf-(i! in other species (creagan et al., 1988) . since i.v. administration of recombinant gst did not induce any clinical signs, it can be deduced that the in vivo effects were caused by the rff nf-a portion of the fusion protein and not by the gst. as expected, it was shown that rftnf-a-gst upregulated the expression of ig2r and mhc class ii antigens in normal cultures of feline pmbcs. the induction of ig2r and mhc class ii antigen mrna by tnf-a involves the activation of transcriptional factors (maniatis et al., 1987) . tnf-a activates nfkb proteins that can induce expression of genes possessing kb-like enhancer elements in their regulatory regions (lowenthal et al., 1989b) . included in this group are the genes encoding ig2r (lowenthal et al., 1989a) , mhc class ii antigen (pessara and koch, 1990) , and human, feline and simian immunodeliciency viruses (folks et al., 1989; osbom et al., 1989; dewhurst et al., 1990; poli et al., 1990; sparger et al., 1992) . feline tnf-a possessed considerable antigenic cross-reactivity with human, but not with murine tnf-a! in western blotting. the degree of antigenic crossreactivity corresponded with the deduced amino acid sequence of the tnf-cz coding regions of the three species; ffnf-cu showed a genetic homology of 92% and 78% with human and murine tnf-a, respectively. in conclusion, our results demonstrate that rff nf-cz could prove to be a useful reagent for the study and treatment of feline disease. studies of the cytokinevirus interactions in the fiv infection of cats could be useful for human aids research. a rapid and sensitive method for the quantification of microgram quantities of proteins utilising the principle of protein dye binding infection of peritoneal macrophages in vitro and in vivo with feline immunodeficiency virus an endotoxininduced serum factor that causes necrosis of tumours thrombin specificity. requirement for a polar amino acid adjacent to the thrombin cleavage site of a polypeptide substrate a phase i clinical trial of recombinant human tumour necrosis factor sequence analysis and acute pathogenicity of molecularly cloned sivsmm_rbj14 gene sequence of porcine tumour necrosis factor alpha comparison of in vitro cell cytotoxic assays for tumour necrosis factor tumor necrosis factor a induces expression of human immunodeticiency virus in a chronically infected t-cell clone sequence of the cdna encoding ovine tumour necrosis factoralpha: problems with cloning by inverse pcr molecular cloning of the gene encoding rabbit tumour necrosis factor biologic activities and mechanisms of action of tumour necrosis factor-a/cachectin decreased mitogen responsiveness and elevated tumour necrosis factor production in cats shortly after feline immunodeficiency virus infection tumor necrosis factor a levels in cats experimentally infected with feline immunodeticiency virus: effects of immunization and feline leukemia virus infection tumor necrosis factor-alpha activation of the il2 receptor-alpha gene involves the induction of kappa b-specific dna binding proteins tumor necrosis factor a induces proteins that bind specifically to kb-like enhancer elements and regulate interleukin 2 receptor a-chain gene expression in primary human t-lymphocytes humoral immune reactivity to feline leukemia virus and associated antigens in cats naturally infected with feline leukemia virus regulation of inducible and tissue-specific gene expression molecular cloning and expression of human tumour necrosis factor and comparison with mouse tumour necrosis factor i99 1. cytokines and hiv infection: is aids a tumour necrosis factor disease ? gene sequence of feline tumour necrosis factor alpha thalidomide: treatment of severe recurrent aphthous stomatitis in patients with aids production of a monoclonal antibody that defines the alpha subunit of the feline il-2 receptor tumor necrosis factor alpha and interleukin i stimulate the human immunodeficiency virus enhancers by activation of the nuclear factor kb the feline immunodeficiency virus cloning and expression in e. co/i of the cdna for murine tumour necrosis factor tumor necrosis factor a regulates expression of the major histocompatibility complex class ii-associated invariant chain by binding of an nf-kb-like factor to apromotor element tumor necrosis factor a functions in an autocrine manner in the induction of human immunodeficient virus expression the detection ofconven-e. rimstad et al. / veterinary immunology and immunopathology 45 (i 99s) 297-310 tional class i and class ii i-e homologue major histocompatibility complex molecules on feline cells immunodiagnosis of feline immunodeficiency virus infection using recombinant viral p 17 and ~24 persistent upregulation of mhc class ii antigen expression on t lymphocytes from cats experimentally infected with feline immunodeliciency virus cloning and expression in escherichia coli of the gene for human tumour necrosis factor m, 26000 antigen of schistosoma japonicum recognized by resistant wehi 129/j mice is a parasite glutathione s-transferase regulation of gene expression directed by the long terminal repeat of the feline immunodeficiency virus isolation and identification of feline peritoneal macrophages for in vitro studies of coronavirus-macrophage interactions augmentation of in vitro hiv replication in peripheral blood mononuclear cells of aids and arc patients by tumour necrosis factor this work was supported by public health service grants ai-25802-03 and ca-50179-01. key: cord-286877-0h5vgi5c authors: dahiya, shyam s.; saini, mohini; kumar, pankaj; gupta, praveen k. title: immunogenicity of a dna-launched replicon-based canine parvovirus dna vaccine expressing vp2 antigen in dogs date: 2012-10-31 journal: research in veterinary science doi: 10.1016/j.rvsc.2012.01.017 sha: doc_id: 286877 cord_uid: 0h5vgi5c abstract a replicon-based dna vaccine encoding vp2 gene of canine parvovirus (cpv) was developed by cloning cpv-vp2 gene into a replicon-based dna vaccine vector (palpha). the characteristics of a replicon-based dna vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. when the palpha-cpv-vp2 was injected intradermal as dna-launched replicon-based dna vaccine in dogs, it induced cpv-specific humoral and cell mediated immune responses. the virus neutralization antibody and lymphocyte proliferative responses were higher than conventional cpv dna vaccine and commercial cpv vaccine. these results indicated that dna-launched replicon-based cpv dna vaccine was effective in inducing both cpv-specific humoral and cellular immune responses and can be considered as effective alternative to conventional cpv dna vaccine and commercial cpv vaccine. canine parvovirus (cpv) is an extremely virulent and contagious non-enveloped single-stranded dna virus belonging to family parvoviridae and genus parvovirus affecting dogs, wolves, foxes and other canines. cpv, a strain evolved from feline parvovirus occurs as three different mutated forms, namely, cpv-2a, cpv-2b and cpv-2c. the disease caused by this virus is considered as most threatening to puppies between the time of weaning and 6 months of age. in young and adult dogs, it causes a severe acute leukopenia and enteritis leading to death by dehydration and shock in a large proportion of cases (carmichael, 2005) . with severe disease, dogs can die within 48-72 h without treatment. cpv spreads from dog to dog by direct or indirect contact with feces (parrish, 1990) . conventional vaccines against cpv include killed and modified live virus (mlv) vaccines (smith-carr et al., 1997; martella et al., 2005) . the killed vaccine requires high dose of antigen per immunization and adjuvant while, mlv could be excreted post-vaccination and not recommended during pregnancy. furthermore, newborns are generally considered unsuitable vaccine recipients due to passive transfer of maternal antibodies leading to antigen clearances and immaturity of their immune system. to overcome these problems, attempts were made to develop new cpv vaccines including, a recombinant vaccine utilizing a baculovirus expression system and a synthetic peptide vaccine (turiso et al., 1992; casal et al., 1995) . dna vaccination against cpv has also been investigated with several advantages over conventional cpv vaccines including, eliminating the use of adjuvant and effective in presence of maternal derived antibodies (mda) in age at which the animal is supposed to be immune (jiang et al., 1998; tarpey and greenwood, 2001; gupta et al., 2005; patial et al., 2007; patel and heldens, 2009) . although dna immunization has several advantages but there are few limitations, namely, dna vaccination can induce long-term uncontrolled expression of a transgene, possibility of integration into the host genome and possible induction of anti-dna antibodies (macgregor et al., 1998; martin et al., 1999; beger et al., 2002) . further, enhancing dna vaccine immunogenicity remains a challenge in large animals (macgregor et al., 1998; johnson et al., 2000; babiuk et al., 2003) . to increase antigen production and immunogenicity with dna vaccines, a new strategy has been developed to express the target heterologous antigen under the control of replicon from positive-strand rna viruses with the promise of using the ability of these viruses to produce large amounts of viral proteins in infected cells. in addition, exclusive cytoplasmic replication of replicon rna and inability of the replicon rna to escape from the transfected cell makes the vector biologically safe (berglund et al., 1999; leitner et al., 2000a; lundstrom, 2000) . rna replicon-based expression vectors have been developed from representatives of most of the positive-strand rna virus families, namely, togaviridae, flaviviridae and picornaviridae. several members of alphavirus genus of togaviridae family, including, sindbis virus (xiong et al., 1989; herweijer et al., 1995; hariharan et al., 1998; miller et al., 2008; saxena et al., 2008; gupta et al., 2009 ), semliki forest virus (liljestrom and garoff, 1991; berglund et al., 1999; zhao et al., 2009) , venezuelan equine encephalitis virus (davis et al., 1989; lee et al., 2003) and flavivirus genus, including, tickborne encephalitis virus, kunjin virus (anraku et al., 2002 (anraku et al., , 2008 , pestivirus-bvdv and coronavirus-hcov (curtis et al., 2002) have received considerable attention. these demonstrated that rna replicon-based dna vaccines provide higher levels of protective immunity and break immunological tolerance by activating innate dsrna-mediated anti-viral pathways (frolov and schlesinger, 1994; diebold et al., 2009 ) and significant dose-sparing advantages compared with conventional dna vaccines (miller et al., 2008; leitner et al., 2003) . in this preliminary study, the potential of a replicon-based cpv dna vaccine to induce the cpv-specific humoral and cellular immune responses were analyzed in immunized dogs and compared with immune responses induced with conventional cpv dna vaccine and commercial cpv vaccine. madin darby canine kidney (mdck) cell line was used to propagate cpv-2b for use in virus neutralization (vn) test and in preparation of cpv antigen. the cell lines like, baby hamster kidney-21 (bhk-21) and human embryonic kidney-293 (hek-293) were used for in vitro transfection. all cell lines were procured from national center for cell science (nccs), pune, india and grown at 37°c under 5% co 2 in dulbecco's modified minimum essential medium (dmem, hyclone), supplemented with 10% fetal bovine serum (fbs, hyclone) and 50 lg/ml gentamicin. cpv isolate no. natp/2002/b03, used in this study was isolated from a clinical case from india and characterized as cpv type 2b . this virus was used in virus neutralization (vn) test and in preparation of inactivated cpv antigen. the conventional cpv dna vaccine, ptarget-cpv-vp2, encoding vp2 gene of cpv-2b was used in this study . megavac-p inact (inactivated monovalent cpv vaccine, indian immunologicals, india) was used as commercial cpv vaccine. to construct replicon-based cpv dna vaccine (palpha-cpv-vp2), the dna fragment containing full length vp2 gene was isolated by digesting ptarget-cpv-vp2 with nhei and smai restriction endonucleases and ligated into xbai and stui sites of the replicon-based dna vaccine vector, palpha. the vp2 gene insert and orf in recombinant plasmid was confirmed by restriction digestion and dna sequencing. the escherichia coli dh5a transformed with recombinant plasmid palpha-cpv-vp2 was grown in lb broth containing kanamycin (50 lg/ml). the replicon-based cpv dna vaccine contained cmv promoter at 5 0 end, 5 0 utr, non-structural genes (nsp1-4), 26s subgenomic promoter, cpv-vp2 gene, 3'utr and polya signal sequence. the palpha-cpv-vp2 plasmid was isolated using endofree plasmid column (qiagen) and transfected into hek-293 cells using lipofectamine 2000 transfection reagent (invitrogen) following manufacturer's instructions. the cpv-vp2 protein in transfected cells were detected in sds-page and western blot. at 48 h posttransfection the transfected cells were lysed in sds-page sample buffer and separated on 10% sds-page along with protein molecular weight marker (fermentas). the proteins in sds-page were stained using coomassie brilliant blue staining. for western blotting, proteins after sds-page were transferred onto nitrocellulose membrane and probed with anti-cpv polyclonal sera raised in rabbit (with igg elisa titer >6400). the bound antibodies were detected using anti-rabbit secondary antibodies conjugated with alkaline phosphatase (sigma) and visualized with nbt/bcip substrate solution (ameresco). to analyze the self-amplification of cpv-vp2 transcripts, the bhk-21 cells were transfected with palpha-cpv-vp2 plasmid. as non-amplification control, the plasmid palpha-cpv-vp2 with deleted 3 0 utr (palpha-d3 0 utr-cpv-vp2) was used. the recombinant plasmids palpha-cpv-vp2 and palpha-d3'utr-cpv-vp2 were transfected into bhk-21 cells using lipofectamine 2000 reagent (invitrogen) following manufacturer's instructions. at 48 h posttransfection, the total rnas were isolated from transfected and control bhk-21 cells using trizol ls reagent (invitrogen) and treated with dnase i (fermentas) following manufacturers' instructions. the dnasei-treated total rna samples were analyzed for free of dna contamination in pcr using total rna as template and cpv-vp2 gene specific primers. for quantification of cpv-vp2 mrna transcripts, the total rnas were reverse transcribed into cdna using mmlv-reverse transcriptase (fermentas) and oligo dt primer (fermentas). in real-time pcr, cdnas from palpha-cpv-vp2 mrna was kept as test template and cdna from palpha-d3 0 utr-cpv-vp2 mrna was kept as calibrator template and gapdh as internal control. the cdnas were 1:10 diluted and used for quantitative evaluation of replicase activity using platinum sybr green qpcr supermix-udg (invitrogen) kit based on sybr green dye following the manufacturer's protocol. the primers used were either cpv-vp2 gene-specific primers (cpv-f: 5 0 -tac-catggtacagatccag-3 0 ; cpv-r: 5 0 -cctctatatcaccaaagtt a-3 0 ) or gapdh primers (gapdh-f: 5 0 -cctggagaaacctg ccaagt-3 0 ; gapdh-r: 5 0 -gccaaattcattgtcgtacca-3 0 ) . the cq values for all the reactions were recorded and fold difference in palpha-cpv-vp2 gene mrna transcripts in bhk-21 cells in comparison with cells transfected with palpha-d3 0 utr-cpv-vp2 was determined after normalization with the help of gapdh internal control as described by pfaffl (2001) . to analyze the induction of apoptosis, the bhk-21 cells were transfected with either palpha-cpv-vp2 or ptarget-cpv-vp2 or empty vector (palpha) using lipofectamine 2000 reagent (invitrogen). at 48 h post-transfection cells were analyzed for early onset of apoptosis using annexin v-fitc apoptosis detection kit (sigma) following manufacturer's instructions. the transfected bhk-21 cells were probed with annexin v-fitc fluorescent antibody probe which bound to phosphatidylserine translocated outside in apoptotic cells. after counter staining with propidium iodide, the apoptotic cells were seen as green fluorescent cells under fluorescent microscope. similarly, the transfected bhk-21 cells were also analyzed for apoptosis specific chromosomal dna fragmentation using deadend™ fluorometric tunel system (promega) following manufacturer's instructions. the apoptotic cells with fragmented chromosomal dna ends were labeled with rtdt which demonstrated bead-like green fluorescence under fluorescent microscope. the dna vaccines (replicon-based cpv dna vaccine, conventional cpv dna vaccine and empty vector) were isolated using endofree plasmid giga kit (qiagen, cat#12391). for each plasmid preparation, a single colony was picked and inoculated in 5 ml luria bertani (lb) broth with appropriate antibiotic (kanamycin, at 50 lg/ml or ampicillin, at 100 lg/ml) and incubated overnight at 37°c with shaking. five ml of overnight culture was inoculated into 500 ml of lb broth with appropriate antibiotic and grown overnight at 37°c with shaking. bacterial cells were separated by centrifugation and plasmid dna was isolated using endofree plasmid giga kit following manufacturer's instructions. the purity of plasmid dna preparations was checked and concentration was estimated spectrophotometrically. the plasmid dnas used for immunization were ethanol precipitated and resuspended in 150 mm nacl at concentration of 1 mg/ml. all dna preparations were stored at à20°c until used for immunization. a total of 15 healthy mongrel dogs aged between 4 and 8 weeks screened seronegative (with vn titer <1:10) were used in this study. three groups of seronegative dogs (each n = 3) were injected intradermal each with 50 lg of either replicon-based cpv dna vaccine (paplha-cpv-vp2) or conventional cpv dna vaccine (ptar-get-cpv-vp2) or empty vector (palpha) in each ear pinna. one group (n = 3) of dogs was immunized intramuscularly with 1 ml (one dose) of commercial inactivated cpv vaccine, megavac-p inact. one group (n = 3) of dogs received pbs injection and kept as negative control group. all groups of dogs received booster on day 21 post-immunization. for the safety assessment of the vaccines, standard health parameters of immunized dogs which included monitoring of food intake, body weight, general behavior, etc. were recorded. food and water intakes were monitored daily while rectal temperature and physical examination including, body weight, observations of hair coat, salivation, respiration character and rate, eye prominence, and tremors were monitored twice a week. the serum samples were collected from immunized dogs on day 0, 21, 30 and 40 for determination of cpv-specific igg and vn antibody response. to evaluate anti-cpv igg antibody response in immunized dogs, sera from all immunized dogs were analyzed in elisa following the method described earlier using purified inactivated cpv as elisa coating antigen (patial et al., 2007) . for end point titer determination, a positive was scored for any sample with an absorbance more than absorbance from healthy dogs sera with two times the standard deviation. the elisa titers were defined as the reciprocal of the highest serum dilution positive in elisa and presented as gmt ± sem. to determine the vaccine immunogenicity against cpv in the vaccinated group, vn test was performed following the method described earlier by preparing two fold heatinactivated serum dilutions starting from 1:2 to 1:1024 in 96 well microtiter plates in triplicate and mixed with 100 tcid 50 of cpv-2b. the sera from non-vaccinated dogs were also included as control. after neutralization, the mdck cells were mixed and incubated for three days at 37°c. the observation was recorded with positive and negative for cpv-specific cytopathic effect and no cytopathic effect, respectively. the vn antibody titer was calculated as the reciprocal of the highest serum dilution of sera that neutralized 100 tcid 50 of cpv-2b and presented as gmt ± sem. a vn titer 1:20 and above was considered as protective as described earlier (pollock and carmichael, 1982a,b; smith-carr et al., 1997; http://www.veterinarypartner.com). the cpv-specific cmi response in immunized dogs was determined by lymphocyte proliferation test and immunophenotyping of effectors cells in pbmcs from all immunized dogs on day 40 post-immunization. the pbmcs isolated from each dog were stimulated with inactivated cpv antigen in triplicates in 96 well microtiter plates along with positive stimulator (con a or pha) and negative (pbs) control. after 72 h of stimulation at 37°c, the stimulated cells were mixed with mtt and incubated further for 4 h at 37°c. the formazon crystals formed were dissolved in 150 ll dmso and absorbance was recorded at 540 nm with 620 nm as reference wavelength. stimulation indices (si) were calculated as ratio of absorbance of stimulated cells to absorbance of unstimulated cells. for immunophenotyping of effectors (cd4 + and cd8 + ) cells, the pbmcs were stimulated in vitro with inactivated cpv antigen for 48 h. the stimulated cells were stained with fitc, rpe and alexa 647-conjugated cocktail monoclonal antibodies (serotec) specific for cell surface antigens cd3, cd4 and cd8, respectively. unstimulated cells from each immunized dog were also stained. briefly, cocktail of conjugated antibodies was mixed with about 1 â 10 6 cells as per the manufacturer's instructions and incubated at room temperature for 30 min. stained cells were washed twice with pbs-bsa and resuspended in pbs containing 1% paraformaldehyde. the numbers of cd3 + cd4 + and cd3 + cd8 + cells from duplicate samples collected from all dogs were acquired per 10,000 cells per sample using bd facs calibur flowcytometer (bd biosciences) and data acquired was analyzed using bd cellquest program (bd biosciences). fold increase in effectors cell population on stimulation with inactivated purified cpv antigen over unstimulated cells was calculated. the induction of cpv-specific igg elisa, vn titers and lymphoproliferative responses were compared by statistical analysis using the repeated measures two-way anova followed by bonferroni post-tests to compare vaccinated and control dogs. all data were presented as the gmt ± sem. differences between the groups with p < 0.05 were considered statistically significant. the replicon-based cpv dna vaccine plasmid (palpha-cpv-vp2) encoding cpv-vp2 was constructed and evaluated to express cpv-vp2 protein in transfected cells in sds-page and western blot (data not shown). for quantitative evaluation of cpv-vp2 gene transcripts the bhk-21 cells, palpha-cpv-vp2-transfected cells were analyzed using real time pcr and cq values obtained were compared with non-amplifying control (palpha-d3 0 utr-cpv-vp2). the number of cpv-vp2 mrna transcripts in palpha-cpv-vp2-transfected cells was higher (cq = 16) than palpha-d3 0 utr-cpv-vp2-transfected cells (cq = 28). after normalizing with respective gapdh internal controls, the cq values difference was found 14.59. this indicated that the cpv-vp2 transcripts in palpha-cpv-vp2-transfected cells were 2 14.59 -fold or 24,748 times higher than transcripts from palpha-d3 0 utr-cpv-vp2-transfected cells. in palpha-cpv-vp2-transfected and palpha-transfected bhk-21 cells, there were large numbers of cells showing apoptosis specific green fluorescence while no in ptarget-cpv-vp2-transfected and in mock-transfected bhk-21 cells after staining with annexin v-fitc antibody. this indicated induction of apoptosis specific to replication of the rna replicon (fig. 1) . when transfected cells were analyzed using deadend™ fluorometric tunel system, bead-like green fluorescence indicative of dna fragmentation in apoptotic cells or in apoptotic bodies was found in cells transfected with either palpha-cpv-vp2 or with palpha vector (fig. 2) . there was no bead-like green fluorescence in cells transfected with either ptarget-cpv-vp2 or mock-transfected bhk-21 cell control. these results indicated that there was induction of apoptosis with chromosomal dna fragmentation in bhk-21 cells due to replication of the rna replicon (fig. 2) . to assess the immunogenicity of replicon-based cpv dna vaccine, cpv-specific humoral igg immune response in immunized dogs was measured at different intervals and compared with conventional cpv dna vaccine and commercial cpv vaccine immunized dogs. standard health parameters monitored on vaccinated dogs indicated no adverse reaction in immunized dogs, which recommended the vaccine safe for clinical use. cpv-specific serocon-version was observed in all groups of dogs receiving different cpv vaccine. the dogs immunized with either replicon-based cpv dna vaccine or conventional cpv dna vaccine demonstrated igg elisa titer on day 21 post-immunization which boosted after booster immunization. the titer was significantly higher in dogs immunized with replicon-based cpv dna vaccine than conventional cpv dna vaccine (fig. 3) . the empty vector immunized and healthy controls also showed non-significant seroconversion on day 21, 30 and 40 post-immunization. to assess the protective efficacy of different cpv vaccines, sera collected from all immunized and control dogs were analyzed for presence of virus neutralizing (vn) antibody. the dogs immunized with replicon-based cpv dna vaccine and conventional cpv dna vaccine demonstrated vn antibody response on day 21 postimmunization. however, only two out of three dogs immunized with replicon-based cpv dna vaccine crossed the protective status (vn titer p1:20) . the vn titer in all groups of cpv vaccine immunized dogs boosted after booster immunization and crossed the protective status. however, the vn antibody titer was always maximal with replicon-based cpv dna vaccine. there was non-significant seroconversion in dogs immunized with empty vector on day 30 and 40 post-immunization (fig. 4) . the cpv-specific cmi immune response elicited by repliconbased cpv dna vaccine was analyzed in pbmcs after in vitro stimulation with inactivated cpv antigen and compared with those elicited by the conventional cpv dna vaccine and commercial cpv vaccine. the cpv-specific lymphocyte proliferative responses, as shown in fig. 5 , was higher in dogs immunized with either replicon-based cpv dna vaccine or commercial cpv vaccine compared to dogs immunized with conventional cpv dna vaccine. ptarget-cpv-vp2 there was non-significant cpv-specific proliferative response detected in dogs receiving the empty vector or dogs receiving pbs. the proliferative response of pbmcs from all immunized and control dogs with non-specific stimulator (con a or pha) confirmed that the cells were healthy and competent to proliferate. to characterize the cpv-specific proliferation of lymphocytes for the most crucial components of antiviral effectors (cd4 + and cd8 + ) the stimulated lymphocytes were phenotypically analyzed using a panel of different monoclonal antibodies against cd3 + , cd4 + and cd8 + markers and compared with their respective unstimulated controls (fig. 6) . the analysis of the increase in effectors cells population after in vitro stimulation with inactivated cpv antigen indicated cpv-specific sensitization of cd4 + and cd8 + lymphocytes. there was significant increase in number of cd4 + and fig. 3 . cpv-specific serum elisa igg antibody response in immunized dogs. sera from different groups of immunized dogs were collected on day 0, 21, 30 and 40 and analyzed for cpv-specific elisa igg response. elisa titers were presented as gmt ± sem from all immunized dogs in each group. the titers significantly different from controls are marked with ⁄ , ⁄⁄ , ⁄⁄⁄ at p < 0.05, p < 0.01, p < 0.001, respectively. the titers having similar superscript letters differ significantly at p < 0.05. fig. 4 . cpv-specific virus neutralization (vn) antibody response in immunized dogs. sera from different groups of immunized dogs were collected on day 0, 21, 30 and 40 and analyzed for cpv-specific vn antibody response. vn titers were presented as gmt ± sem from all immunized dogs in each group. dotted line represents the protective 1:20 vn titer. the titers significantly different from controls are marked with ⁄ , ⁄⁄⁄ at p < 0.05, p < 0.001, respectively. the titers having similar superscript letter differ significantly at p < 0.001. fig. 5 . proliferative responses of pbmcs from immunized dogs after in vitro stimulation with vaccine antigen and inactivated cpv antigen. the pbmcs from different groups of immunized dogs were isolated and stimulated with different non-specific and specific (antigens) stimulators. after 72 h of stimulation, mtt dye assay was done to determine cpv-specific proliferative response in each group. stimulation index (si) was calculated as ratio of od of antigen stimulated cells over od of unstimulated cells. the cona and pha were taken as non-specific stimulator. data represents mean ± sem of triplicate wells. cd8 + lymphocytes in dogs immunized with replicon-based cpv dna vaccine compared to controls (fig. 7) . to enhance immunogenicity and to improve biosafety of the conventional cpv dna vaccine, in this study, we developed a replicon-based cpv dna vaccine (palpha-cpv-vp2). on transfection, the vaccine plasmid transcribed the self-replicating rna using cmv promoter and host rna polymerase ii inside the nucleus. this transcribed rna is transported to cytoplasm and translated into replicase protein. the expressed replicase protein catalyzed the synthesis of negative-sense transcripts to act as template for further self-amplification of rna transcripts and transcription of cpv-vp2 mrna using 26s subgenomic promoter. sds-page and western blot analysis of the palpha-cpv-vp2-transfected cell lysate confirmed the translation of cpv-vp2 protein (data not shown). we reasoned that the replicon-based cpv dna vaccine fig. 6 . immunophenotyping of lymphocyte effectors population after in vitro stimulation with inactivated cpv antigen. pbmcs from different vaccinated and control dogs were isolated and cultured in vitro with inactivated cpv antigen in duplicate for 48 h. the stimulated and unstimulated pbmcs were stained with anti-dog cd3, cd4 and cd8 antibodies. the numbers of cd3 + cd4 + and cd3 + cd8 + cells were counted by flowcytometry. fig. 7 . fractional increase in effectors cd4 and cd8 population of cells after in vitro stimulation with inactivated cpv antigen. pbmcs from different groups of immunized dogs were stimulated in triplicate with inactivated purified cpv antigen for 48 h and stained with anti-cd3, cd4 and cd8 antibodies. the cd3 + cd4 + and cd3 + cd8 + cells were counted by flow cytometry. fractional increase in effectors cell population on stimulation with inactivated cpv antigen over unstimulated cells was calculated. data represents mean ± sem of triplicate samples collected from each group from two independent experiments. would amplify the cpv-vp2 mrna transcripts utilizing self-replicating ability of replicase and induce apoptosis in transfected cells. to determine the self-replicating ability of replicase-based cpv dna vaccine, cpv-vp2 mrna transcripts were quantified using real-time pcr in palpha-cpv-vp2-transfected bhk-21 and compared with non-replicating control plasmid (palpha-d3 0 utr-cpv-vp2). the deletion of 3 0 utr has made the plasmid non-replicating as the 3 0 utr of positive sense rna virus has been reported as essential element for initiation of anti-sense rna template and replication (richard and charles, 2005; james et al., 2007) . there were over 24 thousand times more cpv-vp2 mrna transcripts in palpha-cpv-vp2-transfected bhk-21 cells compared to respective non-replicating control indicating the self-replicating ability of replicon-based cpv-vp2 dna vaccine. another characteristic of a replicon-based dna vaccine, induction of apoptosis was also analyzed in palpha-cpv-vp2-transfected cells. to detect induction of apoptosis with replicon-based cpv dna vaccine specific to replication of the replicon, apoptotic cell death was analyzed in both palpha-cpv-vp2-and empty vector (palpha)-transfected cells and compared with ptarget-cpv-vp2transfected cells. the induction of apoptosis was detected only in replicon-based cpv dna vaccine and palpha vector as confirmed by annexin v and tunel assays. the induction of apoptosis due to formation of double-stranded rna intermediates produced by alphaviral replicase during rna replication (leitner et al., 2000b; glasgow et al., 1997) and subsequent activation of dsrna-dependent pathways (pkr and 2 0 -5 0 -a synthetase/rnase l) provided a mechanistic explanation for the observation that every cell transfected in vitro with replicon-based dna or rna undergoes apoptosis (leitner et al., 2003) . in addition, induction of apoptosis also confers potential immune-potentiating effects and increased safety for the clinical use of dna vaccines (ljungberg et al., 2007) . after in vitro characterizing replicon-based cpv dna vaccine, immune responses to vaccine construct was evaluated by injecting intradermally in ear pinna in dogs. cpv-specific virus neutralizing (vn) antibody and igg elisa responses were detected in all vaccinated dogs. the vn antibody response was significantly higher in dogs immunized with replicon-based cpv dna vaccine than conventional cpv dna vaccine and commercial cpv vaccine. further, the lymphoproliferative response and cpv-specific priming of cd4 + /cd8 + effectors cells were significantly higher with repliconbased cpv dna vaccine compared to conventional cpv dna vaccine. similar enhanced immune responses with replicon-based vaccines over conventional dna vaccines have been reported earlier for other diseases (zhou et al., 1994; dalemans et al., 1995; hariharan et al., 1998; vignuzzi et al., 2001; xiao et al., 2004; arbele et al., 2005; saxena et al., 2008) . from this preliminary study, it is premature to compare conventional cpv vaccines with replicon-based cpv dna vaccine due to the limited number of dogs per group studied. nonetheless, it is clear from induced antibody and cellular immune responses that some differences are apparent and replicon-based cpv dna vaccine induced significantly higher responses. the replicon-based cpv dna vaccine induced dsrna-mediated apoptotic cell death which makes this vaccine approach effective and biologically safe compared to conventional dna vaccine. further, the response of replicon-based cpv dna vaccine needs to be evaluated in large number of dogs for early immune response, duration of immunity and response in presence of maternally derived antibodies (mda) before it can be considered as effective alternative to commercial cpv vaccine. kunjin virus replicon vaccine vectors induce protective cd8+ t-cell immunity kunjin replicon-based simian immunodeficiency virus gag vaccines humoral and cellular immune response to rna immunization with flavivirus replicons derived from tick-borne encephalitis virus induction of immune responses by dna vaccines in large animals a peptide dna surrogate accelerates autoimmune manifestations and nephritis in lupus-prone mice immunization with recombinant semliki forest virus induces protection against influenza challenge in mice an annotated historical account of canine parvovirus peptide vaccine against canine parvovirus: identification of two neutralizing subsites in the n terminus of vp2 and optimization of the amino acid sequence heterologous gene expression from transmissible gastroenteritis virus replicon particle protection against homologous influenza challenge by genetic immunization with sfv rna encoding flu ha in vitro synthesis of infectious venezuelan equine encephalitis virus rna from a cdna clone: analysis of a viable deletion mutant role of tlr3 in the immunogenicity of replicon plasmid-based vaccines translation of sindbis virus mrna: effects of sequences downstream of the initiating codon death mechanisms in cultured cells infected by semliki forest virus sindbis virus replicon-based dna vaccine encoding rabies virus glycoprotein elicits specific humoral and cellular immune response in dogs cloning of canine parvovirus vp2 gene and its use as dna vaccine in dogs dna immunization against herpes simplex virus: enhanced efficacy using a sindbis virus-based vector a plasmid-based self-amplifying sindbis virus vector efficient replication, and evolution of sindbis virus genomes with non-canonical 3 0 a/u-rich elements (nc3are) in neonatal mice nucleic acid immunization protects dogs against challenge with virulent canine parvovirus plasmid dna encoding influenza virus haemagglutinin induces th1 cells and protection against respiratory infection despite its limited ability to generate antibody responses venezualan equine encephalitis virus-vectored vaccines protect mice against anthrax spore challenge alphavirus-based dna vaccine breaks immunological tolerance by activating innate antiviral pathways enhancement of tumor-specific immune response with plasmid dna replicon vectors dna and rna-based vaccines: principles, progress and prospects a new generation of animal cell expression vectors based on the semliki forest virus replicon increased immunogenicity of a dna-launched venezuelan equine encephalitis virus-based replicon dna vaccine alphavirus vectors: applications for dna vaccine production and gene expression first human trial of a dna-based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response immunogenicity of an intranasally administered modified live canine parvovirus type 2b vaccine in pups with maternally derived antibodies plasmid dna malaria vaccine: the potential for genomic integration after intramuscular injection sindbis virus vectors elicit hemagglutinin-specific humoral and cellular immune responses and offer a dose-sparing strategy for vaccination emergence, natural history and variation of canine, mink and feline parvoviruses review of companion animal viral diseases and immunoprophylaxis virus neutralizing antibody response in mice and dogs with a bicistronic dna vaccine encoding rabies virus glycoprotein and canine parvovirus vp2 a new mathematical model for relative quantification in realtime rt-pcr dog response to inactivated canine parvovirus and feline panleukopenia virus vaccines maternally derived immunity to canine parvovirus infection: transfer, decline, and interference with vaccination isolation of canine parvovirus in crfk cell line requirements at the 3 0 end of the sindbis virus genome for efficient synthesis of minus-strand rna a sindbis virus replicon-based dna vaccine encoding the rabies virus glycoprotein elicits immune responses and complete protection in mice from lethal challenge canine parvovirus. part i. pathogenesis and vaccination canine parvovirus dna vaccination. us patent 6187759b1 recombinant vaccine for canine parvovirus in dogs naked rna immunization with replicons derived from poliovirus and semliki forest virus genomes for the generation of a cytotoxic t cell response against the influenza a virus nucleoprotein comparison of immune responses and protective efficacy of suicidal dna vaccine and conventional dna vaccine encoding glycoprotein c of pseudorabies virus in mice sindbis virus: an efficient, broad host range vector for gene expression in animal cells assessment of the cell-mediated immunity induced by alphavirus replicon vector dna vaccines against classical swine fever in a mouse model selfreplicating semliki forest virus rna as recombinant vaccine we would like to thank the director, indian veterinary research institute, izatnagar for providing facilities for carrying out this key: cord-005377-36io7zsm authors: sidoti, francesca; bergallo, massimiliano; costa, cristina; cavallo, rossana title: alternative molecular tests for virological diagnosis date: 2012-04-09 journal: mol biotechnol doi: 10.1007/s12033-012-9533-8 sha: doc_id: 5377 cord_uid: 36io7zsm several nucleic acid amplification techniques (naats), particularly pcr and real-time pcr, are currently used in the routine clinical laboratories. such approaches have allowed rapid diagnosis with a high degree of sensitivity and specificity. however, conventional pcr methods have several intrinsic disadvantages such as the requirement for temperature cycling apparatus, and sophisticated and costly analytical equipments. therefore, amplification at a constant temperature is an attractive alternative method to avoid these requirements. a new generation of isothermal amplification techniques are gaining a wide popularity as diagnostic tools due to their simple operation, rapid reaction and easy detection. the main isothermal methods reviewed here include loop-mediated isothermal amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. in this review, design criteria, potential of amplification, and application of these alternative molecular tests will be discussed and compared to conventional naats. at present, a wide variety of diagnostic techniques are applied for the detection of viral pathogens. traditional diagnostic methods, like virus isolation and serology, have been the mainstay of the clinical laboratory, especially in the past two decades. in recent years, several previously unknown viral pathogens have been discovered for which classical culture is unrealized or even lacks sensitivity. to overcome the shortcomings of the traditional diagnostic methods, molecular techniques have been developed. several nucleic acid amplification techniques (naats), particularly pcr and real-time pcr, are currently used in the routine clinical laboratories. such approaches have allowed rapid diagnosis with a high degree of sensitivity and specificity. moreover, naats have offered additional advantages over traditional methods by production of easily standardized protocols, thus resulting a potential for automatization with a range of options for real-time detection chemistries. the advent of fully automated systems with faster turnaround times has given clinical laboratories the tools necessary to report out accurate and sensitive results to clinicians. however, all these in vitro nucleic acid amplification methods have several intrinsic disadvantages, such as the requirement for precision thermal cycling between three temperatures during the reaction and an elaborate method for detection of amplified products. moreover, real-time pcr machines are very expensive requiring an instrumentation platform that consists of a thermal cycler, computer, optics for fluorescence excitation, emission collection, data acquisition, and analysis software. in this context, a new generation of isothermal amplification techniques are gaining a wide popularity as diagnostic tools due to their simple operation, rapid reaction, and easy detection. these new techniques do not require thermal cycler and can be performed simply by using a heating block and/or water bath with a low-energy consumption. the main isothermal methods reviewed here include loop-mediated isothermal amplification (lamp), nucleic acid sequence-based amplification (nasba), and helicase-dependent amplification (hda). moreover, in this review, design criteria, potential of amplification, and application of these alternative molecular tests in the detection of viral pathogens will be discussed. lamp represents today a better innovative nucleic acid amplification method which exceeds the classical pcr in its reaction simplicity, accuracy, and higher amplification efficiency. the whole procedure is very rapid and the nucleic acid amplification can be completed in less than 1 h under isothermal conditions. the main advantage of the lamp technique is that it does not require thermocyclers and the amplification can be performed simply with a water bath or heating block necessary to maintain the required temperature. moreover, the design of lamp assay is very simple requiring only the dna polymerase along with dntps, reaction buffer, and two sets of specially primers that can be developed using the free software primer explore (lamp primer designing support software program, net laboratory, japan, http://venus.netlaboratory. com). the addition of reverse transcriptase make it possible to amplify cdna from rna sequences (rt-lamp). lamp is a one-step amplification reaction that amplifies target dna from a few copies to 10 9 -10 10 copies and proceeds at isothermal conditions for 1 h or less depending on the efficiency of the designed primers. lamp employs a dna polymerase with strand displacement activity (bst dna polymerase), along with two internal primers (fip, bip), and two outer primers (f3, b3) which recognize six different sequences in the dna template, by incubating all the reagents in a single tube at a constant temperature, usually 63°c which is optimum for the activity of dna polymerase (fig. 1) . the chemistry of lamp amplification is based on the principle of strand displacement reaction which has been described thoroughly by notomi et al. [1] . in particular, the mechanism of the reaction can be explained in three steps, an initial non-cyclic step, a cyclic amplification step, and an elongation step. an animation that is useful for better understanding of the principle is available at the web site http://loopamp.eiken.co.jp/e/ index.html. the addition of a primer set that anneals at the loop structure in lamp amplicons enhances specificity of the reaction and accelerates further the amplification time [2] . in particular, using these specific primers, named loop-primers (lf, lb), the reaction time is reduced by half, making it a more efficient tool used in the practical applications of lamp. moreover, the employment of reverse transcriptase in addition to dna polymerase allows the synthesis of cdna molecules from rna template. reverse transcriptase is added to the reaction mixture and, after mixing and incubating at a constant temperature between 60 and 65°c, amplification and detection can be carried out in a single step (rt-lamp). as concerns the visualization of amplified product obtained from lamp reaction, several methods may be used. firstly, product is visualized by agarose gel analysis stained with an intercalating agent such as ethidium bromide or sybr green i using a common uv transilluminator. as the product of the lamp is a mixture of different length dna fragments, the gel will show several bands which will appear as a smear. another method, based on real-time turbidity measurement, allows to quantify the amount of dna template formed by lamp amplification. the increase of turbidity in the reaction mixture is directly proportional to the amount of dna synthesized. precisely, the lamp method yields large amounts of pyrophosphate ions in the course of the amplification reaction leading to a white precipitate of insoluble magnesium pyrophosphate in the reaction mixture. since the production of precipitate correlates with the increase of turbidity, real-time monitoring of the lamp reaction kinetics can be achieved by measurement of turbidity using an inexpensive turbidimeter. gene copy number can also be quantified by using a standard curve obtained from different concentrations of gene copy number plotted against time of positivity. finally, a new detection method of amplified products has been developed [3] . this method uses fluorescent intercalating dye, like calcein, the fluorescence of which is quenched by the binding of manganese ions bound by pyrophosphate ions produced in the course of the amplification reaction. the presence of fluorescence indicates the presence of dna template and a simple visual detection can be achieved by using an uv lamp. recently, lamp products have also been detected electrochemically in a microchip [4] . based on these assumptions, it is possible to make a number of considerations. lamp assay is more specific towards the template sequences than classical pcr. this is caused because four primers recognize six separate regions within a target dna and the amplification reaction occurs only when all these six regions are correctly recognized by the primers. furthermore, lamp is more sensitive than conventional dna-based detection systems and its ability to amplify from fewer copies of initial target dna than pcr has been demonstrated [5] [6] [7] [8] . in particular, the lamp assay was found to be 10-to 100-fold more sensitive than pcr with a detection limit of 0.01-10 pfu of virus [9] [10] [11] . the development of lamp assay is very simple and allows the use of cost-effective reaction equipment. the simplicity of this method comes from the facility of designing primers and from the fact that only the dna polymerase along with dntps, reaction buffer, and a common water bath or heating block are necessary for the development of lamp assay. moreover, lamp has higher amplification efficiency compared with the pcr, with dna being amplified 10 9 -10 10 times. this high amplification efficiency is attributed to no time loss of thermal change because of its isothermal reaction. finally, rt-lamp assay demonstrated faster in comparison to conventional rt-pcr (30 min vs 3-4 h), because no additional reverse transcriptase step is required. a survey of the literature shows that the lamp has already been applied to detect many kinds of pathogens including viruses and bacteria [12] [13] [14] . in particular, the lamp method has been developed for most emerging human viral pathogens like west nile, dengue, chikungunya, japanese encephalitis, sars, highly pathogenic avian influenza (hpai) h5n1, and norwalk viruses [9] [10] [11] [15] [16] [17] [18] [19] . rt-lamp assays for rapid detection of several respiratory viruses as influenza a and b virus, measles virus, and mumps virus have also been evaluated [20] [21] [22] [23] [24] . moreover, the usefulness of lamp for amplification of dna viruses was also reported for cytomegalovirus, herpes simplex virus, varicella zoster virus, human herpes virus 6-7, adenovirus, bk virus, and human papilloma virus type-6, 11, 16, 18 [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] . the lamp technology has now been developed into commercially available detection kits and some of them have been adopted as the officially recommended methods for detecting various pathogens. lamp kits for the detection of escherichia coli, mycobacterium, salmonella, legionella, vibrio cholerae, listeria, campylobacter, and criptosporidium have been commercialized [37] [38] [39] [40] . considering the advantages of rapid amplification, and easy detection, the current focus of lamp methodology is towards a simple diagnostic tool to be routinely employed in resource-limited laboratories in developing countries where many fatal tropical diseases are endemic, without requiring sophisticated equipment or skilled personnel. however, the combination of lamp methodology and innovative microchip technologies may facilitate the realization of novel testing systems to be used by both developed and developing countries in the near future. nucleic acid sequence-based amplification (nasba) nasba technology has provided an alternative method to conventional procedures with a broad application for the detection of several nucleic acid targets. in particular, nasba is an isothermal transcription-based amplification method, first described by guatelli et al. [41] , particularly suitable for the detection and quantification of genomic, ribosomal, and messenger rna. nasba offers potential advantages compared to conventional rt-pcr. first of all, it is a continuous, isothermal process that does not require a thermocycler and the optimal annealing temperature for primers does not have to be determined empirically. moreover, because nasba is a method based on the isothermal reaction occurring at a temperature of 41°c, and does not require denaturation, it prevents amplification of dna genome in case of contamination, thus being very selective for rna target amplification. however, the low temperature occurring in the reaction could be representing a risk factor for the specificity of the method. anyhow, the specificity rate is increased by a well-constructed method for detecting amplified products using additional hybridization with target-specific probes. another advantage is that no additional reverse transcriptase step is required, thus saving time and reducing the risk of contamination. the only restriction of nasba method is probably that individual preparation of the chemical reagents mixture is difficult and commercial kits are expensive. the nasba method nasba amplification consists of a repeated process of primer annealing, formation of double-stranded dna molecule containing a t7 promoter site, and t7-rna polymerase mediated transcription of multiple anti-sense copies of rna amplicons (fig. 2) . held at 41°c, the reaction uses two oligonucleotide primers specific to the rna target, p1 (forward primer), p2 (reverse primer), and three enzymes: avian myeloblastosis virus reverse transcriptase (amv-rt) which has also polymerase activity, rnase h, and t7 rna polymerase. during the reaction, a dna intermediate is generated through a process that involves the hybridization of a primer to the rna target. this primer (p1), which contains a t7 rna polymerase promoter sequence, is then extended by amv-rt to form a rna-dna hybrid. the digestion of the rna component of the hybrid by rnase h permits the binding of a second primer (p2) to the remaining dna strand. the second primer is then extended by amv-rt to form the doublestranded dna intermediate, which contains the t7-rna polymerase promoter needed for transcription. finally, the t7 rna polymerase produces numerous rna copies and once transcription is initiated, the resulting single-stranded rna transcripts, which are anti-sense to the original rna, can serve as a template to start a new amplification process. the amplification product of nasba can be detected by liquid or gel-based probe-hybridization assays, electrochemiluminescence, or microfluidic electrochemical detection [42] [43] [44] [45] . recently, real-time assays incorporating amplification and detection in a single step have been reported and applied to a wide range of targets. in particular, quantitative real-time nasba assays using molecular beacons have been developed and utilized for the detection and quantification of several rna target in all published real-time procedures whether for commercially available kits or for in-house diagnostic assays [46, 47] . these realtime nasba assays appear to be rapid (about 1.5 h), specific and sensitive with rna amplification and a targetspecific fluorescent signal achieved simultaneously in one tube with measurements obtained by using a simple fluorometer. real-time nasba methodology seems to be a suitable alternative to other real-time amplification techniques such as rt-pcr without the need for expensive thermocyclers. because nasba amplification involves three separate enzymes with their own kinetic parameters, variability in every measurement is inevitable [48] . weusten et al. [49] were the first to describe a mathematical model for rna amplification of both target and internal calibrator rna in a molecular beacon-based nasba reaction to normalize enzyme efficiency differences between reactions. however, the description of this model did not include all of the essential parameters needed to operate the model. consequently, analysis using this model requires software calibrated to each target and is commercially available for only a few specific targets. on the contrary, in our study an alternative method for normalizing nasba data by using a simple time to positivity (tpp) calculation in the presence of an internal control that reduces the variability between replicates has been described [47] . to date, the role of primers and kcl concentration for nasba optimization has not been considered. nasba is able to specifically amplify target rna by using specific primers in the presence of kcl. initially, the primers' concentration is very high and is not rate limiting; relatively small amounts of primers are consumed in depletion of the initially present pool of rna copies (linear phase of nasba process). at some time point, the primers' concentration do become rate limiting and decline towards zero. at this time point, the dna intermediate levels have reached their peak and rna production proceeds at high speed. from now on the only reaction that can proceed is t7 rna polymerase-mediated formation of rna from the dna intermediate templates. this time interval represents the second phase of nasba process characterized by an exponential kinetics (fig. 2) . in our study, we evidenced for the first time that high concentrations of primers and kcl elongate the linear phase of nasba process by shorting the exponential amplification; whereas, low concentrations of primers and kcl promote the exponential phase [47] . in particular, in our study we used relatively low concentrations of primers and kcl (0.3 lm and 80 mm, respectively) to elongate the exponential phase of nasba process, and accordingly, to minimize the reaction-to-reaction variation. nasba has proven to be a useful technique for the highly sensitive detection of several pathogens in clinical, environmental, and food samples including, in particular, different rna viruses (table 1) . although nasba methods offer a powerful tools for molecular diagnosis, their sensitivity and specificity are limited by several factors. amplification inhibitors and rna integrity are the main cause of concern when preparing clinical specimens for nasba. efficiency of rna extraction methods is determined by the rna recovery rate and nasba inhibitor reduction during rna extraction. many rna commercial extraction methods have been tested for the reduction or removal of nasba inhibitors. in particular, rna extraction originally performed with phenol-chloroform has been widely replaced by the boom method which is suitable for use in nasba and reagents for this are commercially available [72] . however, these methods are time consuming, labor intensive, and susceptible to contamination. lately, complete automatization was introduced performing rna extraction within 20-40 min on high numbers of samples. several studies showed that robotic automated sample preparation and the performance of the automated magnapure and the nuclisens extraction procedures (easymag and minimag) were more table 1 applications of nasba assay in the detection of several rna viruses enterovirus [50] [51] [52] influenza a virus [52, 53] influenza b virus [52, 54] influenza a virus (h1n1v) * [55] influenza a virus (h5n1) [56] respiratory syncytial virus [52, 57] hiv-1 [58] [59] [60] parainfluenza virus type 1 [52] parainfluenza virus type 2 [52] parainfluenza virus type 3 [52] parainfluenza type 4 [52] norovirus [61] metapneumovirus [62] sars coronavirus (sars-cov) [63] chikungunya virus [64] st. louis encephalitis virus [65] dengue virus [42] west nile virus [65] hepatitis a virus [66] hepatitis c virus [67, 68] human rhinovirus [47, 69] measles virus [70] rubella virus [52] rabies virus [71] * h1n1v, h1ni variant consistently than manual techniques [73, 74] . as concerns the development of in-house real-time nasba assays, a commercial kit is available (''nuclisens easyq ò basic kit'' ([biomérieux]) [75] . it contains the necessary reagents for nasba amplification process including amv-rt, rnase h, t7 rna polymerase enzymes in the form of lyophilized spheres, the enzyme diluent that consists of sorbitol in aqueous solution, the reagent lyophilized spheres containing nucleotides, dithiothreitol, mgcl 2 with their diluent (tris/hcl, 45% dmso), and kcl solution. the primers and specific probe are to be synthesized for each target. in particular, the design of primers and probe for nasba can be performed using the ''beacon designer tm '' program developed by premier biosoft international (www. premierbiosoft.com), and the stability of predicted structure beacons can be analysed by using the european mfold server (http://frontend.bioinfo.rpi.edu/applications/ mfold/cgi-bin/dna-form1.cgi). the amplification conditions for real-time nasba are generally constant, and optimization of conditions for each new assay can be simpler than rt-pcr. the concentration of enzymes is standardized and does not differ from assay to assay. the variable factors that have to be optimized are the kcl, primers and probes concentrations. in conclusion, nasba is a simple and rapid alternative method to conventional procedures, and its isothermal nature and specificity for rna versus dna make it an important technique in rna research and diagnostics. hda is an isothermal amplification reaction inspired by the natural mechanism of the dna replication fork. this new technology mimics dna replication in vivo by using a dna helicase to separate two complementary dna strands (dsdna) into each single-stranded templates for primers hybridization and subsequent extension by a dna polymerase. as the dna helicase unwinds double-stranded dna enzymatically, the initial heat denaturation and subsequent thermocycling are not necessary, and the entire hda reaction can be performed at a single uniform temperature. thus, this alternative technique provides a useful tool to amplify dna in vitro under isothermal conditions with a very simple reaction scheme. the amplification scheme of the hda method is shown in fig. 3 . in this method, double-stranded dna is unwound enzymatically by a dna helicase in the presence of chemical energy. the displaced dna strands are stabilized by single-stranded dna (ssdna)-binding proteins (ssbs). in particular, these ssb proteins bind specifically to the single-stranded part of dna in order to prevent reannealing of the complementary ssdna templates and to protect them from degradation. two sequence-specific primers hybridize to the 3 0 -end of each ssdna template, and a dna polymerase extends the primers annealed to the templates to produce a dsdna. the two newly synthesized dsdnas are used as substrates by the dna helicase, entering the next round of the reaction. therefore, a simultaneous chain reaction proceeds resulting in exponential amplification of the selected target sequence. it has been reported that rna target as well as dna was also amplified and detected by hda method followed by reverse transcription step [76, 77] . initially, the hda systems were developed using escherichia coli uvrd helicase and t7 bacteriophage gp4 helicase. these current hda systems will be briefly described in this review with consideration of the processivity and efficiency of dna amplification. hda system using escherichia coli uvrd helicase the first hda system for isothermal dna amplification was developed by using e. coli uvrd dna helicase (*82 kda) along with a dna polymerase, and two accessory proteins (ssbs): t4 gene 32 or rb 49 gene 32 proteins [78, 79] . initially, e. coli uvrd helicase was chosen due to its ability to unwind blunt-end substrates (dsdna) as well as nicked circular dna [80] . this hda system mimics the in vivo dna replication and is able to amplify several hundred base pairs of dna with a detection limit ranging from 10 to 10 3 dna copies in less than 1 h [76, [81] [82] [83] . moreover, to further improve the sensitivity and specificity of dna amplification in the hda reaction a very simple expedient as the use of thermostable uvrd helicase at elevated temperatures (60-65°c) was considered. however, the efficient amplification of long target sequences is not possible, probably due to the low processivity and limited speed of dna synthesis by uvrd helicase. it has been reported that uvrd helicase has a limited speed (20 bp/s) and processivity (less than 100 bp per binding) [84, 85] . the performance of an hda system may be further improved by testing different helicases. a new hda system with high processivity and speed was developed by using the t7 bacteriophage gp4 helicase. hda system using t7 bacteriophage gp4 helicase (t7 bacteriophage replisome) the t7 bacteriophage replisome consists of four proteins necessary for amplification process: t7 gp4 helicase-primase, t7 gp5 dna polymerase, t7 gp2.5 (ssb protein), and the processivity factor e. coli thioredoxin (trx) [86, 87] . the t7 gp4 helicase-primase is an hexameric protein composed by two subunits, the gp4a (*63 kda) with both helicase and primase activities, and the gp4b (*56 kda) with only helicase activity [86, 88, 89] . in the t7 helicasebased hda system, the helicase t7 gp4 unwinds the dsdna at a rate of 300 bp/s with high processivity, whereas the primase domain of t7 gp4 produces the primers [90] . in particular, this hda system has been applied to amplify both long linear and circular ssdna templates, and the primase activity of t7 gp4 allows for whole genomes to be amplified without the need for additional dna primers [91] . as concerns the t7 gp5 dna polymerase activity itself is not processive, whereas together with the processivity factor e. coli thioredoxin (t7 gp5 dna polymerase-e. coli thioredoxin complex), the speed and processivity are enhanced by up to[100 nt/s and [10 kb per binding, respectively [92] . recent progress in understanding the function of helicases has enabled researchers to use a helicase/polymerase pair (helicase/ polymerase fusion complex) which can move in a coordinated way to further improve the speed and the processivity of hda systems, allowing for the amplification of dna fragments up to 2.3 kb compared to the original limit of 400 bp [93] . future experiments will be certainly directed towards improving the performance of hda systems by testing several helicases/polymerases complex, and by optimizing the existing hda systems. applications of hda assay hda assay has been used to detect several viruses in different clinical samples. in particular, tang and colleagues developed an innovative isothermal amplification hda with lateral flow to detect hiv-1 in human plasma, whereas kim and colleagues developed a qualitative hda method for the detection of herpes simplex virus (hsv) types 1 and 2 from genital lesions [94, 95] . moreover, a novel one-tube isothermal reverse transcription-thermophilic hda (rt-thda) system has been developed to detect rna viruses, including enterovirus and ebola virus [76] . thermophilic hda in combination with enzyme-linked immunosorbent assay was also used by gill et al. [82, 96] for the detection of helicobacter pylori. in addition, they also developed a colorimetric method to detect h. pylori by using isothermal 3' step 1 step 2 step 3 step 4 fig. 3 amplification scheme of hda method. (step 1) dna helicase unwinds doublestranded dna. ( step 2) ssb proteins stabilize the displaced dna strands. ( step 3) specific primers hybridize to the ssdna template and are extended by dna polymerase. (step 4) a double-stranded copy of the dna target is produced hda and gold nanoparticle probes. andresen et al. [97] incorporated hda on a microarray for quantitative detection of antibiotic-resistant pathogens neisseria gonorrhoeae and staphylococcus aureus. microfluidic chips have also been developed for hda at 62°c for quantification of sars cdna [98] . a fully integrated microfluidic device for dna extraction and hda at 65°c on samples containing live bacteria has been developed by mahalanabis et al. [99] . this microfluidic device was the first to combine bacterial lysis, nucleic acid extraction, and dna amplification on the same chip. finally, kivlehan et al. [100] reported for the first time the utilization of a quantitative electrochemical method to monitor in real-time the hda of nucleic acids in less than 1 h at a single constant temperature. the principle of detection consists of monitoring a decrease in the electrochemical current response of a reporter probe during the amplification process. the detection strategy is analogous to that of real-time hda assay. however, this innovative electrochemical method offers some advantages compared to conventional realtime assays being potentially more robust, simpler, and less expensive. isothermal hda kits are currently available and commercially developed at biohelix (beverly, ma, usa). in conclusion, it is expected that more useful and simpler isothermal amplification techniques will be invented to be used for the detection of different pathogens. loop-mediated isothermal amplification of dna accelerated reaction by loop mediated isothermal amplification using loop primers loopmediated isothermal amplification (lamp) of gene sequences and simple visual detection of products simple and accurate determination of cyp2d6 gene copy number by a loop-mediated isothermal amplification method and an electrochemical dna chip detection of koi herpesvirus in common carp, cyprinus carpio l., by loop-mediated isothermal amplification detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification sensitive and rapid detection of edwardsiellosis in fish by a loop-mediated isothermal amplification method a loop mediated isothermal amplification (lamp) method for detection of infectious hematopoietic necrosis virus (ihnv) in rainbow trout (oncorhynchus mykiss) development and evaluation of a novel loop mediated isothermal amplification (lamp) method for rapid detection of sars corona virus real-time reverse transcription loop mediated isothermal amplification for rapid detection of west nile virus rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loopmediated isothermal amplification assay application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant staphylococcus aureus (mrsa) in blood cultures lamp-an emerging technology for the detection of water-and food-borne protozoan parasites loop-mediated isothermal amplification (lamp): a rapid, accurate, and cost-effective diagnostic method for infectious diseases rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay rapid diagnosis of h5n1 avian influenza virus infection by newly developed influenza h5 hemagglutinin gene-specific loop-mediated isothermal amplification method rapid detection and quantification of japanese encephalitis virus by real-time reverse transcription loop-mediated isothermal amplification. microbiology and immunology rapid and real-time detection of chikungunya virus by reverse transcription loop mediated isothermal amplification assay development and evaluation of reverse transcription loop mediated isothermal amplification assay for rapid and real-time detection of japanese encephalitis virus a simple method for the detection of measles virus genome by loop-mediated isothermal amplification (lamp) rapid diagnostic method for detection of mumps virus genome by loop-mediated isothermal amplification detection of human influenza a viruses by loop-mediated isothermal amplification rapid detection and typing of influenza a and b by loop-mediated isothermal amplification: comparison with immunochromatography and virus isolation mumps virus reinfection is not a rare event confirmed by reverse transcription loop-mediated isothermal amplification rapid diagnosis of human herpesvirus 6 infection by a novel dna amplification method, loopmediated isothermal amplification rapid detection of varicellazoster virus infection by a loop-mediated isothermal amplification method rapid and sensitive diagnosis of adenoviral keratoconjunctivitis by loop-mediated isothermal amplification (lamp) method detection of human herpesvirus 7 dna by loop-mediated isothermal amplification rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method sensitive and rapid detection of herpes simplex virus and varicella-zoster virus dna by loop-mediated isothermal amplification comparison of loop-mediated isothermal amplification, real-time pcr, and virus isolation for the detection of herpes simplex virus in genital lesions development of the loop-mediated isothermal amplification method for rapid detection of cytomegalovirus dna development of a loop-mediated isothermal amplification assay for rapid detection of bk virus loop-mediated isothermal amplification method for detection of human papillomavirus type 6, 11, 16, and 18 direct detection of human herpesvirus 6 dna in serum by the loop-mediated isothermal amplification method rapid detection of human herpesvirus 8 dna using loop-mediated isothermal amplification rapid and simple detection of legionella species by lamp, a new dna amplification method loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. intracellulare in sputum samples development and preliminary evaluation of a loop-mediated isothermal amplification procedure for sensitive detection of cryptosporidium oocysts in fecal and water samples sensitive and rapid detection of cholera toxin-producing vibrio cholerae using a loop-mediated isothermal amplification isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication detection of dengue viral rna using a nucleic acid sequence-based amplification assay highly sensitive and specific detection of viable escherichia coli in drinking water human pathogenic cryptosporidium species bioanalytical detection method with single oocyst detection capability. analytical and bioanalytical chemistry pmma biosensor for nucleic acids with integrated mixer and electrochemical detection real-time nucleic acid sequencebased amplification is more convenient than real-time pcr for quantification of plasmodium falciparum development of a quantitative real-time nucleic acid sequence-based amplification assay with an internal control using molecular beacon probes for selective and sensitive detection of human rhinovirus serotypes increased precision of microbial rna quantification using nasba with an internal control principles of quantitation of viral loads using nucleic acid sequence-based amplification in combination with homogeneous detection using molecular beacons development and implementation of real time nucleic acid amplification for the detection of enterovirus infections in comparison to rapid culture of various clinical specimens evaluation of lightcycler as a platform for nucleic acid sequence-based amplification (nasba) in real time detection of enterovirus development of multiplex nucleic acid sequence-based amplification for detection of human respiratory tract viruses development and evaluation of a real-time nucleic acid sequence based amplification assay for rapid detection of influenza a dry cotton or flocked respiratory swabs as simple collection technique for the molecular detection of respiratory viruses using real time nasba detection of novel swine origin influenza a virus (h1n1) by real-time nucleic acid sequence-based amplification development and validation of a commercial real time nasba assay for the rapid confirmation of influenza ah5n1 virus in clinical samples clinical evaluation of nuclisens magnetic extraction and nu-clisens analytical specific reagents for the real-time detection of respiratory syncytial virus (rsv) in pediatric respiratory specimens a one-tube quantitative hiv-1 rna nasba nucleic acid amplification assay using electrochemiluminiscent (ecl) labelled probes quantitation of human immunodeficiency virus type 1 rna in different biological compartments single rapid realtime monitored isothermal rna amplification assay for quantification of human immunodeficiency virus type 1 isolates from groups m, n, and o evaluation of the persistence of infectious human norovirus on food surfaces by using real time nucleic acid sequence-based amplification diagnosis of human metapneumovirus infection in immunosuppressed lung transplant recipients and children evaluated for pertussis real time nasba detection of sarsassociated coronavirus and comparison with real-time reverse transcription-pcr evaluation of real time nucleic acid based amplification for detection of chikungunya virus in clinical sample nucleic acid sequencebased amplification assays for rapid detection of west nile and st. louis encephalitis viruses real time nucleic acid sequence-based amplification assay for detection of hepatitis a virus characterization of the quantitative hcv nasba assay evaluation of a new nasba assay for the detection of hepatitis c virus based on the nuclisens basic kit reagents detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-pcr, in children with acute respiratory infections during a winter season a sensitive and robust method for measles rna detection comparative detection of rabies rna by nasba, real-time pcr and conventional pcr rapid and simple method for the purification of nucleic acids comparison of five methods for extraction of legionella pneumophila from respiratory specimens evaluation of nuclisens easymag for automated nucleic acid extraction from various clinical specimens development and evaluation of nucleic acid sequence based amplification (nasba) for diagnosis of enterovirus infections using the nuclisens ò basic kit development of a novel one-tube isothermal reverse transcription thermophilic helicase-dependent amplification platform for rapid rna detection helicase dependent onchip-amplification and its use in multiplex pathogen detection bacteriophage t4 gene 32 protein: modulation of protein-nucleic acid and protein-protein association by structural domains snapshot of the genome of the pseudo-t-even bacteriophage rb49 escherichia coli helicase ii (uvrd) protein can completely unwind fully duplex linear and nicked circular dna characterization of a thermostable uvrd helicase and its participation in helicase-dependent amplification colorimetric detection of helicobacter pylori dna using isothermal helicase-dependent amplification and gold nanoparticle probes improving isothermal dna amplification speed for the rapid detection of mycobacterium tuberculosis an oligomeric form of e. coli uvrd is required for optimal helicase activity helicase-dependent isothermal dna amplification complete nucleotide sequence of bacteriophage t7 dna and the locations of t7 genetic elements bacteriophage t7: minimal requirements for the replication of a duplex dna molecule characterization of the helicase and primase activities of the 63-kda component of the bacteriophage t7 gene 4 protein cloning and expression of gene 4 of bacteriophage t7 and creation and analysis of t7 mutants lacking the 4a primase/ helicase or the 4b helicase dna replication dna-dependent nucleoside 5 0 -triphosphatase activity of the gene 4 protein of bacteriophage t7 escherichia coli thioredoxin confers processivity on the dna polymerase activity of the gene 5 protein of bacteriophage t7 isothermal dna amplification in vitro: the helicase-dependent amplification system nucleic acid assay system for tier ii labs and moderately complex clinics to detect hiv in low-resource settings a rapid and simple isothermal nucleic acid amplification test for detection of herpes simplex virus types 1 and 2 detection of helicobacter pylori by enzyme-linked immunosorbent assay of thermophilic helicase-dependent isothermal dna amplification helicase-dependent amplification: use in onchip amplification and potential for point-of-care diagnostics real-time pcr array chip with capillary-driven sample loading and reactor sealing for point-ofcare applications an integrated disposable device for dna extraction and helicase dependent amplification real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids key: cord-269839-jxqs51o5 authors: bitome-essono, paul-yannick; ollomo, benjamin; arnathau, céline; durand, patrick; mokoudoum, nancy diamella; yacka-mouele, lauriane; okouga, alain-prince; boundenga, larson; mve-ondo, bertrand; obame-nkoghe, judicaël; mbehang-nguema, philippe; njiokou, flobert; makanga, boris; wattier, rémi; ayala, diego; ayala, francisco j; renaud, francois; rougeron, virginie; bretagnolle, francois; prugnolle, franck; paupy, christophe title: tracking zoonotic pathogens using blood-sucking flies as 'flying syringes' date: 2017-03-28 journal: elife doi: 10.7554/elife.22069 sha: doc_id: 269839 cord_uid: jxqs51o5 about 60% of emerging infectious diseases in humans are of zoonotic origin. their increasing number requires the development of new methods for early detection and monitoring of infectious agents in wildlife. here, we investigated whether blood meals from hematophagous flies could be used to identify the infectious agents circulating in wild vertebrates. to this aim, 1230 blood-engorged flies were caught in the forests of gabon. identified blood meals (30%) were from 20 vertebrate species including mammals, birds and reptiles. among them, 9% were infected by different extant malaria parasites among which some belonged to known parasite species, others to new parasite species or to parasite lineages for which only the vector was known. this study demonstrates that using hematophagous flies as ‘flying syringes’ constitutes an interesting approach to investigate blood-borne pathogen diversity in wild vertebrates and could be used as an early detection tool of zoonotic pathogens. doi: http://dx.doi.org/10.7554/elife.22069.001 emerging and re-emerging human infectious diseases have increased in recent years. around onefourth of the 1415 pathogens known to infect humans appeared between 1940 and 2004 and their appearance has gradually increased since 1980 (taylor et al., 2001; woolhouse and gaunt, 2007; jones et al., 2008; daszak et al., 2004) . today, seven new pathogens appear every year and this number should reach 15-20 by 2020 (woolhouse et al., 2008) , mostly due to the growth of human activities that increase contact with novel sources of pathogens and favor their spread worldwide (murray et al., 2015) . emerging threats mainly concern viruses, such as hiv (sharp and hahn, 2011) , sars-cov and mers-cov (de wit et al., 2016) , avian flu (alexander, 2007) and more recently ebola (baize et al., 2014) , chikungunya (burt et al., 2012) and zika (wikan and smith, 2016) . however, disease emergence and re-emergence also concern bacteria (e.g. helicobacter pylori, salmonella sp., etc.) and parasites (e.g. plasmodium knowlesi in south-east asia). sixty per cent of diseases emerging in humans are zoonoses and wildlife plays a key role by providing a zoonotic pool from which previously unknown pathogens may emerge (taylor et al., 2001; woolhouse and gaunt, 2007; jones et al., 2008; daszak et al., 2004) . the case of p. knowlesi in south-east asia is a good example. this parasite emerged in the human population after a transfer from asian macaques. it is now considered as the fifth human malaria agent after plasmodium falciparum, plasmodium vivax, plasmodium malariae and plasmodium ovale (singh and daneshvar, 2013) . such emerging diseases constitute a massive public health issue that requires active monitoring for signs of outbreaks and rapid diagnosis of the involved pathogen. therefore, it is crucial to anticipate and prevent potential epidemic and pandemic outbreaks by developing new methods for the early detection and monitoring of infectious agents in wild animal sources (kuiken et al., 2005; wolfe et al., 2005) . however, in many cases, monitoring is limited or impossible due to our poor knowledge about the ecology of these pathogens (i.e. where, when and how these agents circulate in the wildlife). the case of the ebola virus is quite exemplary. indeed, the exact nature of its reservoir(s) remains uncertain, although thousands of animals have been screened during the last 40 years (e.g. [marí saéz et al., 2015] ). nowadays, pathogen circulation in wild animals is screened using mainly two methods: bushmeat analysis or direct trapping of animals for organ and tissue collection. these methods are pertinent in many cases, but present some weaknesses. bushmeat represents only a fraction of the fauna (the one consumed by humans), whereas animal trapping can be difficult or dangerous. moreover, such manipulation may be harmful for threatened and protected species. as a consequence, several methods were developed in the last years to study pathogen diversity from wild fauna without the need of direct contacts with animals, for example, by using fecal, urine or saliva samples (e.g. [santiago et al., 2002; prugnolle et al., 2010; pesapane et al., 2013; taberlet et al., 2012] ). however, the value of these non-invasive methods remains limited because not all pathogens can be detected and not all reservoirs can be explored by these methods (for instance, it is difficult to collect feces or saliva of reptiles without trapping them). therefore, new non-invasive methods are crucially needed to provide new opportunities for screening a larger range of hosts and pathogens. the use of hematophagous flies as 'flying syringes' may constitute a new approach to track and survey blood-borne pathogens in the wild (calvignac-spencer et al., 2013) . nucleic acids (dna or elife digest about 60% of new infectious diseases in humans come from animals. their increasing number and rapid spread are linked to increasing levels of contact between humans and wildlife, as recently highlighted by the epidemics of zika in brazil or ebola in west africa. to anticipate and prevent similar outbreaks in the future, it would be ideal to develop new methods for the early detection and monitoring of infectious diseases in wild animals. currently, three methods are mainly used to screen wild animals for infectious disease, but these all have limitations. analyses of bushmeat and game meat only investigate those animals that are eaten by humans. testing the organs and tissues of trapped animals can be difficult and harmful for both the humans and animals involved. collecting and examining samples of feces, urine or saliva cannot detect all diseases and can be difficult to do for some species. bitome-essono et al. now demonstrate a new method for assessing the diseases carried by wild animals: using blood-sucking flies as 'flying syringes' to collect their blood. during several weeks of sampling in gabon, central africa, bitome-essono et al. trapped thousands of these flies, about a third of which were engorged with blood. analyses of these blood samples revealed that they had come from 20 different species, including birds, mammals and reptiles. different malaria parasites could also be detected in the blood. although the study performed by bitome-essono et al. only focused on malaria parasites, in the future the technique could be extended to analyze a number of disease-causing microbesincluding viruses, bacteria, protozoa and macroparasites -that are found in the blood of wild animals. rna) of vertebrate hosts or of pathogens in arthropod blood meals are preserved and detectable for several days (calvignac-spencer et al., 2013; kent, 2009; muturi et al., 2011; grubaugh et al., 2015; lee et al., 2015) . for example, hiv was detected 8 days and 10 to 14 days after blood ingestion by bugs and by ticks, respectively (webb et al., 1989; humphery-smith et al., 1993) . recently, the h5n1 flu virus was found viable in mosquitoes (barbazan et al., 2008) , although its transmission by these insects is unproven (sawabe et al., 2006) . grubaugh and colleagues (grubaugh et al., 2015) applied such an idea (that they called 'xenosurveillance') using anohpeles mosquitoes to estimate the diversity of viruses infecting human populations in remote areas. nevertheless, bloodengorged mosquitoes are very difficult to collect in forest and often show strong host preferences (in particular for mammals). arthropods with more generalist blood feeding patterns would be more useful to survey pathogens from a large range of vertebrates (including mammals, birds and reptiles) in these highly complex ecosystems. hematophagous flies (tsetse flies, stomoxids and tabanids) could be good candidates for this purpose since they are usually large diptera (length comprised between 3 and 25 mm) and hematophagous in both sexes, with the exception of male tabanids (mullens, 2002) . they are easy to trap and some studies performed on tsetse flies and stomoxids showed that 20 to 40% of trapped flies are engorged with blood (mavoungou et al., 2008; simo et al., 2012) . these flies feed on a large spectrum of vertebrate hosts, including birds, reptiles and mammals (muturi et al., 2011; clausen et al., 1998; muzari et al., 2010) . the omnipresence of hematophagous flies in certain habitats and their opportunistic blood-feeding behaviour (muturi et al., 2011; muzari et al., 2010; späth, 2000) make of them compelling candidates to obtain blood meals from different vertebrate hosts for pathogen detection. in the present study, we investigated the possibility of using hematophagous flies as 'flying syringes' to explore the diversity of extant malaria parasites (haemosporida) infecting wild vertebrates living in the forests of gabon (central africa). a total of 4099 hematophagous flies were caught in four national parks of gabon during dry and rainy seasons over a cumulated sampling period of 16 weeks ( figure 1a ). among them, six tsetse fly species, six stomoxid species and six tabanid species were identified ( table 1) . among the 4099 caught flies, 1230 (30%) were engorged with blood. these were mostly tsetse flies (n = 1218; 99%), particularly glossina palpalis palpalis (n = 662; 54%) and g. fuscipes fuscipes (n = 214; 18%) specimens. the blood meal origin was successfully identified in 33% and 43% of these flies, respectively (table 1) . overall, the blood meal origin was successfully identified in 428 fly samples (35%) using a pcr system amplifying long fragments of cytb (450 bp) or coi genes (330 bp or 660 bp). specifically, blood meals were from 20 vertebrate species, including 12 families and 8 orders (figure 1b and tables 2 and 3). a trial study using a pcr system amplifying a shorter fragment (150 bp of the gene 16s) to deal with potential dna degradation in the blood meal showed a high gain of sensitivity in the determination of the origin of the blood meal. thus, out of 89 previously unidentified blood meals, the host was identified for 76% (n = 68) of them. the list of newly identified hosts is given in figure 2 . this shows a high gain of sensitivity with the new pcr system. extant malaria parasites were detected in 37 (8.7%) of the 428 identified blood meals (figure 1c , red isolates). phylogenetic analyses revealed that 29.7% of these parasites belonged to plasmodium falciparum (n = 11, figure 1c ; group 1), 8.1% to plasmodium adleri (n = 3, figure 1c ; group 2), and 8.1% to a recently described lineage of parasites infecting wild ungulates (n = 4, figure 1c ; group 3) (boundenga et al., 2016) . for all blood meals, the identified host represented the known natural host (or one of the hosts) of such parasites. sequences of unknown parasite lineages or of parasites for which the hosts were not known were also obtained. for instance, one sequence ( figure 1c ; group 4) detected in a blood meal originating from an ungulate was related to parasites previously :"$6(-,$6/%)%3-@05/-+,#) isolated only from anopheles mosquitoes (boundenga et al., 2016) . one sequence detected in a blood meal originating from a bird was related to bat haemosporida (nycteria), (figure 1c ; group 5). finally, 18 sequences ( figure 1c ; group 6) that were amplified from blood meals originating from ungulates formed an independent and never described lineage related to groups 3 and 4. in addition, 100 additional samples for which identification of the blood meal failed were randomly chosen for malarial parasite screening. this analysis showed that 7% were infected with p. falciparum (n = 4, group 1), p. praefalciparum (n = 1, group 7), malaria parasites of antelopes from group 6 (n = 1) and parasites of tortoises (group 8, n = 1) ( figure 1c , green isolates). for the parasite, the use of a shorter pcr system led to less conclusive results than those obtained for the host identification. out of the 91 blood meals that were negative to plasmodium with a pcr system amplifying a long cytb fragment, only one was found positive with the new system. the positive individual corresponded to a tragelaphus spekii and was infected with a parasite belonging to group 3 ( figure 1c ). in this study, we tested whether hematophagous flies could be used as 'flying syringes' to identify blood-borne pathogens circulating in the wild vertebrate fauna of gabon. our results show that the blood meals of the captured engorged flies can be successfully used to analyze the diversity of extant malaria parasites. despite a limited sampling effort (a total of 4 weeks of sampling for each park), we could screen the diversity of haemosporidian parasites from a large range of vertebrate hosts, including mammals, birds and reptiles. parasites were detected in more than 8% of the analyzed samples. these malaria parasites belonged to already known, but also to never previously table 1 . number and proportion of specimens captured per fly species. the number of engorged specimens and blood meals identified in each fly species are also indicated. mammals artiodactyla table 4 continued on next page concerning the method efficiency, 30% of blood meals were obtained from 4099 hematophagous flies. this result is consistent with previous studies (mavoungou et al., 2008; simo et al., 2012) showing that most hematophagous flies caught using traps are often seeking hosts for a blood meal. other methods using a dip net seem to have a better capture efficiency with more than 40% of engorged flies caught on their resting places (gouteux et al., 1984) . however, this method requires spending a lot of time in the field because of difficulties in finding their resting sites and catching the flies. tsetse flies provided 99% of the collected blood meals (54% by glossina palpalis palpalis) and they are an interesting candidate as 'flying syringes'. indeed, differently from stomoxids and tabanids, both sexes are exclusively hematophagous in tsetse flies. in addition, g. p. palpalis is considered to be an opportunistic species concerning its feeding behaviour, thus explaining the large diversity of blood meals (clausen et al., 1998; simo et al., 2008; weitz, 1963) . conversely, stomoxids and tabanids show sex-specific differences in feeding behaviour and this may partly explain the smaller number of blood meals collected in these two families. in stomoxids, both sexes are unknown_host_819 ky631984 doi: 10.7554/elife.22069.008 hematophagous, but males sometimes feed on nectar (wall and shearer, 1997) . moreover, the digestion of stomoxids starts more rapidly than in the other hematophagous flies (moffatt et al., 1995) . male and female tabanids feed on nectar just after their emergence as adults. only after having been fertilized, females start sucking blood (mullens, 2002) . therefore, engorged stomoxid and tabanid flies are more difficult to capture. additionally, the lack of engorged stomoxids and tabanids could be explained by the fact that we sampled flies only at floor level. indeed, some stomoxid species readily feed on arboreal monkeys that are mostly found higher in the tree layer (mavoungou et al., 2008) . the low rate (35%) of blood meal identifications could be explained by the degradation of host dna during digestion in the fly midgut or by a too small blood quantity in the midgut. the stage of digestion might influence dna degradation and the host identification efficiency. nevertheless, the diversity of hosts we successfully identified, mainly in tsetse fly blood meals, was large, including big terrestrial (elephants) and semi-aquatic mammals (hippopotamus) and also reptiles and birds. as previously noted, the diversity of blood meals can be due to the fly high mobility, their opportunistic feeding behaviour and their frequent feeding. in our study, most blood meals were from terrestrial animals (i.e. that live primarily on the ground) and very few from arboreal species. as mentioned above, this result is potentially biased by the trophic preferences of tsetse flies and by the capture method that excluded canopy levels. previous studies have shown that hematophagous flies sampled in canopies mainly feed on arboreal species (mavoungou et al., 2008) . therefore, changes in trap position could broaden the range of host species analysed. we can also notice the absence of small mammals (e.g., rodents or bats) within the diversity of host vertebrates we identified. this may be explained by the trophic preferences of the flies we sampled which could have a preferential taste for large vertebrates as previously documented for tsetse flies (e.g. [muturi et al., 2011; späth, 2000] ). concerning pathogen detection, we detected extant haemosporidian parasites in 8.65% of the 428 blood meals for which the host origin was successfully identified. moreover, we also detected parasites in blood meals of unknown origin, thus increasing the number of detected parasites. together, these results show that blood meals collected from hematophagous flies are suitable for tracking blood-borne pathogens from wild animals. haemosporidian pathogens ingested by hematophagous flies during their blood meal can remain detectable in the fly digestive tract even after partial digestion of the blood meal. we observed congruence between the identified hosts and the detected pathogens. as expected, p. falciparum was detected in human blood and p. adleri in gorilla blood. haemosporidian lineages are often host-specific or restricted to certain classes of vertebrate hosts. therefore, the unknown host could be inferred from the detected haemosporidian species (figure 1c) . for example, the blood meal from unknown host n˚110 could have originated from a kinixys turtle (kinixys sp.). similarly, the blood meals from the unknown hosts n˚649, 520, 665, 512 and 819 could have originated from humans (homo sapiens). the present study demonstrates the possibility to use hematophagous flies as 'flying syringes' to analyze the diversity of pathogens circulating in wildlife. we think that there is now room for improvement of the tool; for instance, by improving the methods used to identify the blood meals and the pathogens. since dna is likely to be degraded in many blood meals (calvignac-spencer et al., 2013; schnell et al., 2012) , the use of pcr systems targeting fragments of shorter size could potentially improve the performance of detection. a trial study based on 89 previously unidentified blood meals using a pcr system amplifying a shorter fragment (<150 bp) (boessenkool et al., 2012) than the one used in the present study allowed the identification of 76% (n = 68) of the hosts (figure 2) . this represents an important gain of sensitivity. however, these primers are still not ideal for our purpose as they were designed for optimal amplification of mammal dna and often fail to properly amplify the dna of other classes of vertebrates. a similar pcr system targeting the entire range of vertebrates still remains to be developed. for plasmodium, our trial for amplifying a shorter fragment of cytb (<200 bp) using a combination of previously published primers did not increase the sensitivity. indeed, out of 91 samples for which the blood meal was successfully identified but in which no haemosporidian infection was detected with our long cytb pcr system, only one was shown to be positive with the short pcr system. however, it is possible that other pcr systems, more optimized, could indeed improve the sensitivity of plasmodium detection. another direction of improvement could be the use of high-throughput sequencing technologies on pools of blood-engorged flies or amplicons to ease the identification of both hosts and parasites (especially in the case of mixed blood meals or mixed infections). finally, another way to improve the tool could be to use high-throughput multiplexed pathogen detection methods for the simultaneous testing of many samples in rapid succession. with such improvements, this approach of 'xenorsurveillance' could usefully complete recently developed methods based on the analysis of other invertebrates (carrion flies (hoffmann et al., 2016) , mosquitoes [grubaugh et al., 2015] ) and become an innovative way for the concomitant surveillance of many enzootic blood-borne pathogens, such as viruses (chikungunya, zika), bacteria, protozoa and macro-parasites. the use of hematophagous flies as 'flying syringes' could indeed improve public health management by allowing the surveillance and early detection of zoonotic pathogens and thus prevent they spread to humans before they cause massive infections. this tool could also help to better understand the circulation in wildlife of other enzootic viruses, such as chikungunya or zika, especially at the interface between natural/sylvan environments and, consequently, improving our knowledge of their natural history. from a broader perspective, this method could also be useful for people interested in wildlife biodiversity and conservation. indeed, it could help monitoring the wildlife diversity within a specific region as demonstrated with other invertebrate systems (calvignac-spencer et al., 2013; lee et al., 2015; schnell et al., 2012; schubert et al., 2015) . more importantly, it could also allow detecting the emergence of new diseases in wild animals that may threaten their long-term survival. despite the significant scientific advances in the medical field, humans are still unable to predict where, when and how epidemics arise. around 60% of emerging diseases in humans are of zoonotic origin. the progressive reduction of wild habitats will increase the contacts between humans and species that are potential reservoirs of diseases. we propose here a new non-invasive tool that can help identifying pathogens that circulate in wildlife before they spread in humans. the fly sampling was carried out in four wildlife reserves in gabon (figure 1a hematophagous flies were sampled during the rainy and dry seasons between 2012 and 2014. in inp and mdnp, sampling was done during two years following a gradient of human activity from primary forest to villages. in the other parks, flies were sampled during a single year. flies were collected by using vavoua and nzi traps (laveissiere and grebaut, 1990; acapovi et al., 2001; mihok, 2002; gilles et al., 2007) . the vavoua trap, initially developed for the capture of tsetse flies was also successfully used for the capture of stomoxids at la ré union island (laveissiere and grebaut, 1990; gilles et al., 2007) . the nzi trap was more adapted to the capture of glossina pallidipes and tabanids in africa (acapovi et al., 2001; mihok, 2002) . in each park, we placed 24 traps (12 vavoua and 12 nzi) during 2 weeks per climatic season. each trap was activated from 7:00 am to 5:00 pm. freshly collected hematophagous flies were identified using a stereo-microscope and taxonomic procedure. the fly species (tsetse, stomoxids and tabanids) was determined following the determination keys of pollock (1982) , brunhes et al., 1998 , zumpt, 1973 , garros et al. (2004 and oldroyd (1973) , on the basis of their morphological characteristics, such as size, color, wing venation structure and proboscis. after species identification, engorged flies were dissected individually in a drop of dulbecco's phosphate buffered saline solution (1x dpbs) to isolate blood meals from midgut. each hematophagous fly was dissected on a slide using one forceps and one scalpel that were changed each time to avoid contaminations. each blood meal was transferred in a 1.5-ml microtube containing 50 ml of rnalater stabilization solution (qiagen: store at rt tissue collection) to stabilize and protect nucleic acids of vertebrate hosts and pathogens contained in the blood meals. samples were kept at ambient temperature during field session and then frozen at à80˚c until dna extraction. samples were centrifuged at 15,000 rpm at 4˚c for 10 min to remove the rnalater solution. pellets were used to extract dna using the dneasy blood and tissue kit (qiagen) according to the manufacturer's instructions. extracted dna was eluted in 100 ml of buffer ae and stored at à20˚c. the origin of blood meals was determined using the extracted dna to amplify a 450 bp fragment of the cytochrome b (cytb) gene using previously published primers (townzen et al., 2008) . pcr amplifications were performed using a geneamp 9700 thermal cycler (applied biosystems, usa) with 50 ml reaction mixtures containing 4 ml template dna, 10 mm tris-hcl (ph = 9), 50 mm kcl, 3 mm mgcl 2 , 20 pmol each primer (5'cccctcagaatgatatttgtcctca3' and 5'ccatccaacatc tcagcatgatgaaa3'), 200 mm dntp and 1 u taq polymerase. the thermal cycling conditions consisted of 3.5 min at 95˚c, 40 cycles of 30s at 95˚c, 50s at 58˚c, and 40s at 72˚c, followed by 5 min at 72˚c. when cytb amplification failed, a 330 bp and/or a 660 bp fragment of the cytochrome oxydase subunit i (coi) gene was amplified using previously described primers and protocols (townzen et al., 2008) . all pcr-amplified products (10 ml) were run on 1.5% agarose gels in tbe buffer, and positive samples were sent to beckman coulter genomics (france) for sequencing in both directions (forward and reverse) after purification. consensus sequences were compared with existent sequences using the ncbi nucleotide blast search (altschul et al., 1990) to determine the host species. hosts were identified when the amplified and reference sequences showed at least 98% similarity. haemosporidian parasite detection was performed in samples with identified blood meal origin and also in 100 randomly chosen samples for which blood meal origin could not be identified. haemosporidian parasites were detected by pcr amplification of a portion of the cytb gene (~790 bp) using a nested pcr protocol, as previously published (ollomo et al., 2009) . pcr products were checked on 1.5% agarose gels before shipment to eurofins mwg (germany) for sequencing in both directions (reverse and forward) after purification. multiple alignments of haemosporidian sequences were done using muscle (edgar, 2004) . a phylogenetic tree with the haemosporidian sequences obtained in our study and a set of reference sequences was built using maximum likelihood (ml) methods and phylogeny.fr (dereeper et al., 2008 ) (see table 4 for accession numbers). the ml model used for construction of the tree was gtr (general time reversible)+g (gamma distribution)+i (invariable site distribution). several measures were taken to avoid contaminations during our manipulations. extraction of dna was performed at the cirmf (gabon) in a laboratory working on mosquitoes. the room in which extraction was performed was away from the rooms in which dna was amplified in this lab. dna extracts were then sent to france at the ird (montpellier). there, blood meal and plasmodium identification was performed. this lab had never worked before on plasmodium from ungulates or reptiles. amplification of host dna was never or very rarely performed in this lab. when the work was performed, no work on plasmodium has been performed in this lab for almost 4 years. in addition, the laboratory is designed to avoid contaminations. clearly defined and separated areas are devoted for each step of the pcr process: one area is devoted to the preparation of reagents (mix pcr). another room is dedicated to the pre-pcr manipulation (loading of native dna). this step is done under a cabinet to avoid contamination of the sample with dna from the operator. finally, an area is devoted to pcr-amplified dna. in this area, cabinets are used to deposit the first pcr product into the reagents of the second pcr (for nested pcrs). all cabinets are equipped with uv lamps and are always decontaminated with dna-free solutions before and after manipulations. gloves and coats are changed when moving between the areas and plugged tips are used at all steps. blank controls were always incorporated at all steps of the experimental procedure and were always negative. several observations confirm the authenticity of our results: (1) >80% of the hosts that were found have never been manipulated in our lab (hosts that are not humans or non-human primates); (2) the parasite always corresponded to the expected host (antelope parasites were always found in antelopes, human parasites in humans and gorilla parasites in gorillas). contaminations by external dna would have lead to random association of hosts and parasites; (3) a new lineage of parasites was discovered. since dna is likely to be degraded in many of our samples, the use of pcr systems targeting fragments of shorter size might improve performance. to determine if this could be the case with our study system, we performed supplementary analyses using (1) a pcr system targeting a shorter fragment of the vertebrate mitochondrial dna to identify the blood meal origin and (2) a pcr system targeting a shorter fragment of the cytb dna to identify the parasite. for the identification of the host, the pcr system used was the one amplifying a fragment of 150 bp of 16s as described in boessenkool et al. (2012) and using the primers 16smam1 and 16smam2. this pcr system was used on blood meals that failed to be identified using our original pcr system (see the paragraph 'blood meal identification'). a total of 89 blood meals were tested for this trial study. for the parasite, we designed new primer sets to amplify a shorter fragment of the cytb gene of the parasite (~177 bp). this new pcr system was applied to blood meals for which the host was identified but that were negative to plasmodium with our long pcr system (~790 bp, see material and methods above). a total of 91 blood samples were tested. for the first round of amplifications, we used 6 ml of dna template in a 25 ml reaction volume, containing: 12.5 ml of mix pcr (qiagen), 2.5 ml solution q (qiagen), and 4 pmol of each primer (cytb1f ctctattaatttagttaaagcacactt and 454r ccwgtwgcytgcatytatct). cycling conditions were 15 min at 95˚c, 30 s at 94˚c, 90 s at 57˚c, 90 s at 72˚c (40 cycles), and 10 min at 72˚c. for the second round of amplification, we used 1.5 ml of the first pcr template in a 25 ml reaction volume, containing 2.5 ml of 10â buffer, 1.25 mm mgcl 2 , 250 mm of each dntp, 10 pmol of each primer (454f2 waattayccatgyccattraa and plas1rc caccatccactccataattctc), and 0.1 unit taq platinum (invitrogen). cycling conditions for the second round were 5 min at 95˚c, 30 s at 94˚c, 30 s at 50˚c, 90 s at 72˚c (35 cycles), and 10 min at 72˚c. the amplified products (5 ml) were run on 1.5% agarose gels in tae buffer. the pcr-amplified products (177 bp) were used as templates for sequencing. dna sequencing was performed by eurofins mwg. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. abondance relative des tabanidé s dans la ré gion des savanes de cô te d'ivoire. revue d'élevage et de médecine vétérinaire des pays tropicaux an overview of the epidemiology of avian influenza basic local alignment search tool emergence of zaire ebola virus disease in guinea detection of h5n1 avian influenza virus from mosquitoes collected in an infected poultry farm in thailand blocking human contaminant dna during pcr allows amplification of rare mammal species from sedimentary ancient dna haemosporidian parasites of antelopes and other vertebrates from gabon, central africa les glossines ou mouches tsé -tsé . logiciel d'identification et d'enseignement chikungunya: a re-emerging virus an invertebrate stomach's view on vertebrate ecology: certain invertebrates could be used as "vertebrate samplers" and deliver dna-based information on many aspects of vertebrate ecology host preferences of tsetse (diptera: glossinidae) based on bloodmeal identifications conservation medicine and a new agenda for emerging diseases sars and mers: recent insights into emerging coronaviruses phylogeny.fr: robust phylogenetic analysis for the non-specialist muscle: multiple sequence alignment with high accuracy and high throughput un nouveau caractè re morphologique pour distinguer stomoxys calcitrans et s. niger (diptera: muscidae) : comparaison de populations de l'ile de la ré union efficiency of traps for stomoxys calcitrans and stomoxys niger niger on reunion island ecologie des glossines en secteur pré -forestier de cô te d'ivoire. 9. les lieux de repos xenosurveillance: a novel mosquito-based approach for examining the human-pathogen landscape assessing the feasibility of fly based surveillance of wildlife infectious diseases evaluation of mechanical transmission of hiv by the african soft tick, ornithodoros moubata global trends in emerging infectious diseases molecular methods for arthropod bloodmeal identification and applications to ecological and vector-borne disease studies public health. pathogen surveillance in animals mise au point d'un modè le é conomique: le piè ge 'vavoua'. tropical medicine and parasitology: official organ of deutsche tropenmedizinische gesellschaft and of deutsche gesellschaft für technische zusammenarbeit reading mammal diversity from flies: the persistence period of amplifiable mammal mtdna in blowfly guts (chrysomya megacephala) and a new dna mini-barcode target ecology of stomoxyine fulies (diptera: muscidae) in gabon. ii. blood meals analysis a nd epidemiologic consequences the development of a multipurpose trap (the nzi) for tsetse and other biting flies studies on the synthesis and secretion of trypsin in the midgut of stomoxys calcitrans horse flies and deer flies (tabanidae). in: medical and veterinary entomology global biogeography of human infectious diseases tracking the feeding patterns of tsetse flies (glossina genus) by analysis of bloodmeals using mitochondrial cytochromes genes host preferences of tabanid flies based on identification of blood meals by elisa tabanidae. in: insects and other arthropods of medical importance a new malaria agent in african hominids tracking pathogen transmission at the human-wildlife interface: banded mongoose and escherichia coli tsetse biology, systematics and distribution, techniques african great apes are natural hosts of multiple related malaria species, including plasmodium falciparum investigating the zoonotic origin of the west african ebola epidemic sivcpz in wild chimpanzees detection and isolation of highly pathogenic h5n1 avian influenza a viruses from blow flies collected in the vicinity of an infected poultry farm in kyoto screening mammal biodiversity using dna from leeches targeted detection of mammalian species using carrion fly-derived dna origins of hiv and the aids pandemic tsetse fly host preference from sleeping sickness foci in cameroon: epidemiological implications identification of different trypanosome species in the mid-guts of tsetse flies of the malanga (kimpese) sleeping sickness focus of the democratic republic of congo human infections and detection of plasmodium knowlesi feeding patterns of three sympatric tsetse species (glossina spp.) (diptera: glossinidae) in the preforest zone of cô te d'ivoire risk factors for human disease emergence identification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit i and cytochrome b gene sequences veterinary entomology potential for insect transmission of hiv: experimental exposure of cimex hemipterus and toxorhynchites amboinensis to human immunodeficiency virus the feeding habits of glossina zika virus: history of a newly emerging arbovirus bushmeat hunting, deforestation, and prediction of zoonoses emergence ecological origins of novel human pathogens temporal trends in the discovery of human viruses the stomoxynae biting flies of the world authors thank all the reviewers for their constructive and helpfull comments. this study was carried out with a financial support of: 'agence universitaire de la francophonie' (auf), the 'service de cooperation et d'action culturelle' (scac) of french embassy in gabon, the 'institut franç ais' of libreville (if), the 'conseil ré gional de bourgogne' and the 'bonus qualité recherche' (bqr) of université de bourgogne. this work was also funded by institut de recherche pour le dé veloppement (laboratoire mixte international zofac), centre international de recherches mé dicales de franceville (cirmf), as well as the agence nationale de la recherche (anr) programme jeunes chercheuses jeunes chercheurs (jcjc) sciences de la vie, de la santé et des ecosystè mes 7-2012 project origin (anr jcjc svse 7-2012 origin). we thank the agence nationale des parcs nationaux (anpn) and the centre national de la recherche scientifique et technologique (cenarest) of gabon who authorized this study and facilitated the access to national parks. authors also thank eric willaume from the park of la lé ké di for his help. key: cord-284582-xwedgllw authors: korabecna, m.; zinkova, a.; brynychova, i.; chylikova, b.; prikryl, p.; sedova, l.; neuzil, p.; seda, o. title: cell-free dna in plasma as an essential immune system regulator date: 2020-10-15 journal: sci rep doi: 10.1038/s41598-020-74288-2 sha: doc_id: 284582 cord_uid: xwedgllw the cell-free dna (cfdna) is always present in plasma, and it is biomarker of growing interest in prenatal diagnostics as well as in oncology and transplantology for therapy efficiency monitoring. but does this cfdna have a physiological role? here we show that cfdna presence and clearance in plasma of healthy individuals plays an indispensable role in immune system regulation. we exposed thp1 cells to healthy individuals’ plasma with (np) and without (tp) cfdna. in cells treated with np, we found elevated expression of genes whose products maintain immune system homeostasis. exposure of cells to tp triggered an innate immune response (iir), documented particularly by elevated expression of pro-inflammatory interleukin 8. the results of mass spectrometry showed a higher abundance of proteins associated with iir activation due to the regulation of complement cascade in cells cultivated with tp. these expression profiles provide evidence that the presence of cfdna and its clearance in plasma of healthy individuals regulate fundamental mechanisms of the inflammation process and tissue homeostasis. the detailed understanding how neutrophil extracellular traps and their naturally occurring degradation products affect the performance of immune system is of crucial interest for future medical applications. to catch and destroy the infectious microorganisms. these nets are subsequently cleared from the circulation by dnase i and their insufficient clearance can result in occlusion of blood capillaries, leading to impaired microcirculation, enzymatically damaging tissues and further progression of inflammation 15 . the elevated formation of nets was reported in most comorbidities worsening the clinical course of covid-19 16 . the hyperactivated neutrophils and monocytes-macrophages are the usual initiators of the cytokine storm responsible for the most serious consequences of coronavirus sars cov-2 infection 17 . bacteria and protozoa are able to convert the nets using their own enzymes into the dna degradation product such as deoxyadenosine which is toxic for macrophages and causes their apoptosis 18, 19 . despite all these findings and the massive cfdna-based diagnostic technique developments, there are surprisingly few studies focused on the fundamental biological function of cfdna in healthy individuals. one study treated human monocytes either with the plasma of dialyzed patients or healthy individuals, both containing cfdna. only the plasma from dialyzed patients stimulated the production of pro-inflammatory interleukin il 6 in target cells 20 . it was also revealed that the cfdna isolated from individual tumor cell lines or complex tumors injected into animal blood circulation caused tumor transformation, referred to as genometastasis 21 . the biological character and function of unmethylated fetal cfdna was explored and the increased proportion of fetal cfdna in maternal circulation during pregnancy was found 22, 23 .this fetal cfdna stimulated a maternal immune response against the placenta, resulting in the proper timing of labor 22 . the influence of cfdna on human macrophages was tested using isolated cfdna from various blood products with different storage time to emulate the conditions immediately after transfusion. the cells were cultivated in the presence of calf serum bearing its own cfdna. the study found increased expression of genes involved in the innate immune response (iir), including chemokines and their receptors in macrophages, and concluded that cfdna contained in the stored blood products might interfere with the immune system of transfusion recipients 24 . the authors reported the elevated expressions of cxcl8 and ddit3 in experiments, but the results may be affected by the presence of calf serum in all experiments. this raises the following question: what is the fundamental role of cfdna in healthy organisms? we assumed that we could find it by exposing identical cell lines to plasma with and without cfdna and excluding any other factors, such as calf serum or potential damage of cfdna complexes by isolation procedure. we performed all stimulatory experiments using the thp1 cell line as a representative of primary human monocytes 25 to show the fundamental role of cfdna in healthy organisms. the experiments were conducted in duplicates using plasma containing cfdna (np) and the reference one with cfdna removed by dnase (tp) to recognize the effect of plasma cfdna on transcriptome and proteome of monocytes. we used native human plasma samples obtained from healthy volunteers with no animal serum addition to the cultivation medium in order to avoid the presence of uncharacterized animal cfdna and dnases in the experiments. in the discovery phase, we used six plasma samples, searching for differences in transcriptomes related to treatment with np and tp using genechip human gene 2.1 st array strip (thermo fisher scientific). we detected significant differences ( fig. 1a ; supplementary table 1) , which were further validated using single-target quantitative pcr (qpcr) and another ten plasma samples ( fig. 1b-d) . to differentiate between the effects of buffers alone and the effect of dnase turbo treatment, we performed a set of experiments ( supplementary fig. 1 ). the addition of activation buffer containing divalent cations is necessary for the activity of dnase turbo in plasma originally treated with ethylenediaminetetraacetic acid (edta). the addition of this buffer alone led after incubation to the decrease of cfdna in the plasma sample to 57.42% of its original level due to the activation of plasma endogenous dnase i. the incubation of plasma sample with this buffer and dnase turbo resulted in the detection 7.07% of cfdna original amount when measured after its isolation from plasma using qpcr. in this set of stimulation experiments, we tested the effects of the complete procedure allowing the dnase turbo activity in plasma and its subsequent removal but without the addition of dnase turbo itself and compared the results with np and tp samples. all these experiments were performed using a plasma sample obtained from an identical donor. we examined the expression of all validated genes and concluded that the activation of endogenous dnase i was mostly sufficient to produce the basic differences which could be in some cases further strengthened with subsequent dnase turbo treatment ( supplementary fig. 1 ). the procedure serving for the removal of divalent cations was applied during the processing of all treated plasma samples to avoid the elevated concentrations of these ions in samples due to the dnase turbo activation buffer addition. we received the results identical with the results of validation experiments in eight out of eleven examined genes as we explored the stimulatory capacity of only one differentially treated plasma sample. the expressions of didt3 and sesn2 were significantly elevated in cells treated with np samples in validation experiments but decreased in cells treated with np sample of this donor. the ccl24 expression was significantly increased in cells stimulated with tp samples in validation study but elevated when treated with np sample of this individual. the inconsistencies found in the expressions of ddit3, sesn2 and ccl24 may thus reflect the individual variability deserving of further study. we used the validation phase results to perform a direct comparison of signaling pathways activated in cells as a consequence of their treatment with np or tp samples (table 1a , b) using the database reactome. this analysis demonstrated the critical importance of the presence/absence of the intact cfdna for the expression profile of cells and regulatory pathways activation. this fact was also documented by the reactome analysis of results obtained by mass spectrometry used for proteome examination of thp1 cells treated with np or tp (table 1c) . the presented work documented that the cfdna and its clearance in plasma is under physiological conditions indispensable for immune system performance. we demonstrated that monocytes in intact cfdna presence (np) upregulated the central pathways responsible for immune system homeostasis, especially notch signaling 26 | (2020) 10:17478 | https://doi.org/10.1038/s41598-020-74288-2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ and unfolded protein response (table 1a) 27 while the degraded cfdna in plasma (tp) resulted in upregulated cxcl8 expression (table 1b) . this mrna should translate into interleukin 8 (il8), a pivotal protein involved in the direct activation of iir 28 and also regarded as a marker of cellular senescence 29 , but its elevated level was not found in proteomic analysis after our three-hour-long cultivation experiments. nevertheless, we detected the upregulation of proteins contributing to the activation of complement (table 1; supplementary table 2) , confirming the inflammatory state of these cells. the software ingenuity pathways analysis (ipa) was used to explore all obtained data (supplementary table 3 ). the ipa results ( fig. 2a , b) confirmed the findings of reactome analysis (summarized in fig. 2b inset). in thp1 cells cultivated with tp, multiple pathways involved in immune response were detected among significantly changed canonical pathways ( fig. 2a) and overrepresented disease-specific pathways (fig. 2b) . ipa also predicted the accumulation of granulocytes, leucocytes, myeloid cells, phagocytes, and complement activation as the consequence of events signalizing the presence of cells that are confronted mainly with degraded cfdna (fig. 3 ). we developed and tested an experimental workflow which allowed us to compare the effects of cfdna pool in native plasma and cfdna in plasma degraded by endogenous dnase i and additionally with dnase turbo on the thp1 cells. the native plasma samples were fixed by edta as a potent indirect dnase i inhibitor. in the edta treated plasma samples, the activity of the endogenous dnase i is completely stopped 30 . the activation of endogenous dnase i and the subsequent activity of dnase turbo were allowed by addition of an activation buffer containing divalent cations to the edta treated plasma samples. to inactivate these divalent cations, the dnase turbo inactivation procedure was applied according to the protocol provided by the manufacturer for the aqueous solutions of isolated rnas. our results are of course limited by the fact that we are working with such a complex sample as human blood plasma. it is not possible to design the experiments to exclude completely the influence of changing divalent cations concentrations 31,32 during the entire experimental procedure, the presence of cfdna hidden in plasma exosomes 33, 34 or in supramolecular complexes and on cell surfaces 35 . nevertheless, when we quantified the cfdna isolated from np, from the sample exposed to the entire cfdna removal procedure but without addition of dnase turbo and from the tp sample, we found striking gradual decrease in cfdna levels toward the last sample. the changes in expression profiles of selected validated genes were detectable after the decrease of cfdna levels to 69.10% of its original native concentration as the result of endogenous dnase i activity ( supplementary fig. 1 ). therefore we could speculate also about the role of cfdna www.nature.com/scientificreports/ degradation products in the induction of these expression changes. the significance of differences in expression profiles of cells treated with np and tp was statistically proven in validation experiments. we detected namely the differences in immune system regulatory pathways discussed in the next paragraphs. the inflammatory response in mammalian cells is regulated by notch signaling pathways acting through four different notch receptors (notch 1-4) transducing extracellular signals 26 ; the notch 1 is involved in myeloid lineage differentiation leading to monocytes 36 . the constitutive tonic activity of notch signaling pathways was described in non-activated immune cells 26 . we found that the monocytes treated with np expressed hes1 mrna by ≈ 4.63 × more than the ones treated with tp (fig. 1d) . this elevated expression documents the notch pathways' activity in these cells. the pathways leading from toll-like receptors (tlrs) may provide additional signals to the notch ligands 26 . in primary macrophages, it has been shown that the notch target genes, like hes1, can also be induced exclusively by tlr stimulation 37 . in such a model, the presence of cfdna itself could ensure the constitutive tonic notch signaling via tlr9 as a receptor specialized in dna sensing. different types of cellular stress lead to unfolded protein accumulation in the lumen of the endoplasmic reticulum. this accumulation activates signal-transduction cascade known as unfolded protein response (table 1a) . it has been demonstrated that this cascade plays the central role in the modulation of immune system functions regarding both innate and adaptive responses 27 . in our experiments, the expression of sesn2 (sestrin 2) is upregulated in cells treated with np (fig. 1b) . sesn2 is well-known as a stress-inducible protein suppressing inflammasome activation by the induction of mitophagy. sesn2 plays a crucial role in this unique regulatory mechanism of mitophagy activation which is pivotal for the maintenance of immunological homeostasis and protection from sepsis 38 . we found the increased expression of arrdc4 and irf1 genes in cells treated with np (fig. 1b) additionally to the genes involved in the pathways detected by reactome ( table 1 ). the role of arrestin domain-containing 4 (arrdc4) in iir was recognized but only partially understood 39 . this gene is transcribed in monocytes in healthy individuals; after a viral infection, its levels were increased and correlated with concentrations of interleukins in serum. interferon regulatory factor-1 (irf1) is a transcription factor expressed at low levels in immune system cells and induced by different cytokines. it controls the transcription of its target genes in different types of immune cells 40 . cultivation of thp1 cells with tp led to the change of expression pattern. apart from increased cxcl8 expression, we found upregulated expression in mtts1, gpr1, and ccl24 (fig. 1c) . metastasis suppressor-1 (mtss1) was originally identified as a metastasis suppressor in the carcinoma cell line. nowadays, it is known that this multifunctional cytoskeleton scaffold protein regulates the cytoskeleton dynamics and inhibits cell migration 41 . g protein-coupled receptor 183 (gpr183) is a member of the signaling pathway leading to repression of notch signaling 42 . ccl24 codes for eotaxin 2, which is produced by activated monocytes and attracts lymphocytes, basophils, eosinophils, and monocytes to the site of inflammation. it has been reported that eotaxin 2 induced apoptosis of thp1 cells 43 . significantly higher expression of proteins belonging to complement cascade is evident at the proteomic level upon the treatment of cells with tp in comparison with cells treated with np (table 1c ). the soluble complement www.nature.com/scientificreports/ proteins detected in plasma are synthesized mainly in the liver, but their local production by circulating immune cells including monocytes is well described 44 . we documented that the monocytes without contact with physiological cfdna concentrations in plasma activated emergency mechanisms and began to initiate iir. the normal functions of these cells were downregulated (notch signaling), potential migration was inhibited, and the genes for attractants of immune cells (cxcl8 and ccl24) were overexpressed (fig. 2b inset and fig. 3) . previously, we found elevated expression of another key member of the cytokine network, namely tumor necrosis factor-alpha (tnf-α) in tp treated cells 45, 46 . tnf-α functions as a master regulator of inflammation and ensures tissue homeostasis 47 . we reported the statistically significant differences validated in qpcr experiments earlier on two different occasions-in our study in which plasma samples of healthy volunteers were used for stimulation of thp1 cells 45 and in the report studying the stimulatory capacity of plasma samples obtained from patients with celiac disease 46 . under the stringent conditions set by us for the evaluation of genechip experiments, the tnf-α was not reported as differentially expressed, but its expression was higher in cells treated with tp per our previous results. to date, the regulatory mechanisms keeping the cfdna concentrations in plasma at physiological levels are not well understood. natural regulatory mechanisms are balancing the nets production and their clearance. these mechanisms may involve the negative closed feedback loop system, which is widely spread in biology and also used by pharmacologists for drug delivery systems 48 . the failure of the feedback loop mechanism might cause exacerbated dnase i production, resulting in iir. some bacteria are equipped not only with dnases but also with their own 3´-nucleotidases. the activity of these enzymes destructs nets and converts their degradation products into deoxyadenosine which is toxic namely for macrophages and induces their apoptosis 49 . different ectonucleotidases are found on the surfaces of different human cells 50, 51 , their participation in nets degradation and toxic product formation cannot be excluded. our results suggest that the exposition of cells to relatively elevated concentration of cfdna degradation products can evoke and promote the inflammatory state as the consequence of clearance of high cfdna concentrations associated with tissue damage 52 or with the affected clearance of the products of netosis 53 . elevated deregulated netosis is reported in common comorbidities not only in dialyzed patients 54 but it is also typical in most comorbidities characterized as risk factors predisposing to serious complications of sars cov-2 infection 16 . here we provided evidence that the cfdna in human plasma and its clearance represents an essential natural tool for regulation of innate immune response. we used the thp1 cell line as a model of human monocytes to demonstrate that cfdna plays an indispensable role in the immune system homeostasis. the cells treated with the native plasma of healthy volunteers expressed genes whose products maintain immune system homeostasis. however, the cells treated with identical plasma samples with degraded cfdna directly activate iir with elevated production of mrna for interleukin 8 at the transcriptomic level. they also upregulated the complement compounds at the proteomic level. the role of intact and degraded cfdna and their sensing by cells seems to be one of essential aspects of immune system performance; therefore, further studies focusing on this subject are highly topical. it is of utmost importance to understand the mechanisms of cfdna release, the clearance and mechanisms of the homeostasis maintenance, as well as the role of different types of cfdna sequences and their chemical modifications in cfdna mediated regulatory events, in addition to the recognition of all aspects of cfdna presence sensed by the cells. the detailed knowledge of mechanisms involved in immune system regulation by the levels of circulating cfdna may lead to new clinical applications, especially concerning the complete understanding of the pathogenesis of sepsis, covid-19 and therapeutic dnase treatment. monitoring the cfdna level can serve as an actual tool for early well-advanced diagnoses of several diseases such as cancer, sepsis and covid-19, as well as labor timing. this study is the first to show the fundamental role of cfdna and its clearance in plasma of healthy individuals in the regulation of innate immune response, thus warranting further research in this direction. subjects. plasma donors were selected from healthy volunteers who satisfied the following criteria: 1. they were taking no medication 2. they had no chronic illness 3. they had not recovered from an infectious disease in the last two weeks 4. they were in very good physical and psychical status. the subset of samples described in our previous study 45 was utilized for expression arrays experiments. five women and five men aged between 21 and 64 years with age of (35.8 ± 13.8) year (mean ± standard deviation from 10 samples) were selected for validation of quantitative pcr (qpcr) experiments. the ethical committee of the 1st faculty of medicine of charles university and general faculty hospital in prague, czech republic, approved our study. we obtained informed consent from all study participants. all methods were performed per the relevant guidelines and regulations. preparation of plasma samples. whole blood was collected into vacuette tubes containing k3 edta (greiner bio-one). we separated blood plasma using the following steps: centrifugation rate, time and temperature set to 1,800 rpm, 10 min and 4 °c, respectively; then the transfer of plasma into 2 ml low binding collection tubes and centrifugation set to rate of 14,500 rpm for 5 min to remove the residual cells. finally, we transferred the plasma into clean 2 ml dna lobind tubes (eppendorf). the samples were stored at a temperature of − 80 °c. | (2020) 10:17478 | https://doi.org/10.1038/s41598-020-74288-2 www.nature.com/scientificreports/ before cell experiments, the plasma sample thawed and split into two identical aliquots. the first one was used at its native state (np), and the second one was treated with turbo dna-free dnase (thermo fisher scientific) according to the manufacturer's recommendation (tp). each 100 μl of the plasma sample was treated with 1 μl turbo dnase, 7 μl water and 12 μl 10 × turbo dnase buffer. this mix was incubated at 37 °c for 20 min and then turbo dnase was inactivated by adding 12 μl dnase inactivation reagent, mixed and incubated at room temperature for 5 min. dnase inactivation reagent was removed by centrifugation at an acceleration force of 10,000 g for 1.5 min and the supernatant was transferred into the new tube. in order to evaluate the influence of individual protocol steps on the alteration of expression profiles, the following experiments were performed: the plasma sample was handled according to the protocol for dnase turbo treatment but dnase turbo was supplemented by water to recognize the effect achieved by this enzyme. inactivation buffer was added either immediately or after incubation step to recognize the influence of sample incubation at 37 °c with activation buffer. identical experiments were performed with dnase turbo addition. all these plasma samples were used for stimulation experiments with thp1 cells and expressions of all validated genes was determined as described below. for determination of cfdna levels in plasma before and after treatment with turbo dna-free kit components, the qpcr at quantstudio 12 k flex real-time pcr system (applied biosystems, usa) was performed using the cfdna samples isolated by qiaamp circulating nucleic acid kit. powerup sybr green pcr master mix (life technologies, usa) constituted one half of 20 μl reaction, the total cfdna amount was measured using primers for the gene 36b4 45 and the standard curve dilution had range from 5 ng to 0.312 ng per reaction. cell cultivation and stimulation. we used the thp1 cell line for stimulation experiments. after the recovery of cells from a deep-frozen state, we used the protocol published earlier 45, 46 . the cells were stimulated for 3 h in the rpmi 1640 medium (sigma aldrich) containing 10% of a plasma sample. the cells were collected after 3 h of cultivation; after centrifugation, the supernatant was removed, and the cells were preserved in lysis solution (sigma-aldrich) and stored at − 80 °c. the results were multiplied by a factor of 10 3 to increase resolution and presented in arbitrary units (au). statistical analysis was done in graphpad prism 5.00.288 software (graph-pad software). first, we performed the d´agostino-pearson normality test. in the case of parametric data distribution, the t-test was used; the wilcoxon matched-pairs signed-rank test was applied for non-parametric data distribution. the statistical significance was set to level ≤ 0.05 for all comparisons. sample preparation for mass spectrometry. frozen thp-1 cell pellets containing ≈ 500,000 cells were resuspended in 100 μl p phosphate-buffered saline and lysed using buffer composed of 1% sodium dodecyl sulfate (sigma-aldrich) in 50 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer with ph 8.5 (carl-roth) supplemented with 1× complete ultra protease inhibitor cocktail-ethylenediaminetetraacetic acid (roche) and shortly sonicated. mixtures were heated for 5 min at 95 °c and subsequently cooled by placing samples on ice. twenty-five units of benzonase nuclease (sigma-aldrich) was added into each tube, and the tubes were incubated at 37 °c for 30 min to degrade chromatin. the obtained protein mixtures were centrifuged to remove cell debris, and the supernatant was determined by the bca method using the nanodrop uv-vis spectrophotometer (thermo fisher scientific). | (2020) 10:17478 | https://doi.org/10.1038/s41598-020-74288-2 www.nature.com/scientificreports/ proteins in the lysate (25 μg) were reduced in the protein lobind tubes (eppendorf) by the addition of dithiothreitol solution (sigma-aldrich) to a final concentration of 10 mm and incubated for 30 min at 45 °c. alkylation was performed by the addition of iodoacetamide to a final concentration of 40 mm and incubation for 30 min at 25 °c in the absence of ambient light. reactions were quenched by the addition of 1 μl of 1 m dithiothreitol per tube. then the protein solution was acidified by 1% formic acid to reach ph value between 2 and 3. in the same tube, 50 μg of suspension of paramagnetic carboxylate-modified microparticles sera-mag speedbeads 1 μm (ge healthcare) was resuspended in the protein sample solution. acetonitrile was immediately added to obtain fifty percent solution, and sample suspension was mixed and incubated for 20 min at 25 °c. next, the tube was placed into a magnetic stand, and paramagnetic microparticles with captured proteins were washed twice using 1 ml of 70% ethanol for 30 s. finally, microparticles were dried by 200 μl of acetonitrile for 15 s and resuspended in ≈ 50 μl of ≈ 50 mm triethylammonium bicarbonate buffer ph 8.0. proteins were eluted by on-bead digestion after the addition of trypsin/lys-c protease mixture (promega) in enzyme to substrate ratio of 1:35 and overnight incubation at 37 °c 55 . the next day, the obtained peptide eluate was discharged from microparticles, which were again washed twice using 25 μl of 25 mm triethylammonium bicarbonate ph 8.0. the pooled peptide mixture was acidified by 4 μl of 10% trifluoroacetic acid and then desalted using omix c18 pipette tips (agilent) according to the user manual. the clean peptide sample was evaporated on the vacuum concentrator (eppendorf) and stored at − 80 °c in protein lobind tubes. nanolc-ms analysis. nano reversed-phase columns (easy-spray column, 50 cm × 75 µm id, pepmap c18, 2 µm particles, 10 nm pore size) were used for liquid chromatography/mass spectrometry analysis. mobile phase buffer a and b was 0.1% formic acid in water and acetonitrile, respectively. samples were loaded onto the trap column model c18 pepmap100 with 5 μm particle size and dimension of 300 μm × 5 mm (thermo scientific) for 4 min at 17.5 μl min −1 . the loading buffer was composed of water, 2% acetonitrile and 0.1% trifluoroacetic acid. peptides were eluted with the mobile phase b gradient from 4 to 35% b in 120 min. eluting peptide cations were converted to gas-phase ions by electrospray ionization and analyzed on a thermo orbitrap fusion modelq-ot-qit (thermo fisher scientific). survey scans of peptide precursors from 350 to 1400 m z −1 were performed at 120 k resolution at 200 m/z with a 5 × 10 5 ion count target. tandem ms (ms 2 ) was performed by isolation at 1.5 th with the quadrupole, hcd fragmentation with a normalized collision energy of 30, and rapid scan ms 2 analysis in the ion trap. the ms 2 ion count target was set to 10 4 , and the maximum injection time was set to 35 ms. only those precursors with charge states from 2 to 6 were sampled for ms 2 . the dynamic exclusion duration was set to 45 s with a 10 ppm tolerance around the selected precursor and its isotopes. monoisotopic precursor selection was turned on. the instrument was run in top speed mode with 2 s cycles 56 . ms 2 data analysis. all data were analyzed and quantified with the maxquant software (version 1.6.1.0) 57 . the false discovery rate (fdr) was set to 1% for both proteins and peptides, and we specified a minimum peptide length of seven amino acids. the andromeda search engine was used for the ms 2 spectra search against the human as downloaded from current uniprot human database. enzyme specificity was set as c-terminal to arg and lys, also allowing cleavage at proline bonds and a maximum of two missed cleavages. dithiomethylation of cysteine was selected as fixed modification and n-terminal protein acetylation and methionine oxidation as variable modifications. the match between runs feature of maxquant was used to transfer identifications to other lc-ms 2 runs based on their masses and retention time with a maximum deviation of 0.7 min; this was also used in quantification experiments. quantifications were performed with a label-free algorithm in maxquant41 58 . data analysis was performed using perseus 1.6.2.2. software 59 . bioinformatic analysis. the sets of differentially transcribed genes and differentially expressed proteins were analyzed using reactome dababase 60 . the sets were also subjected to ingenuity pathway analysis (qiagen) 61 . studies on the chemical nature of the substance inducing transformation of pneumococcal types: induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type iii comptes rendus des seances de la societe de biologie et de ses filiales modifications transmitted to the offspring, provoked by heterograft in'solanum melongena' a historical and evolutionary perspective on the biological significance of circulating dna and extracellular vesicles characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic lupus erythematosus presence of fetal dna in maternal plasma and serum liquid biopsies: genotyping circulating tumor dna genomics-based non-invasive prenatal testing for detection of fetal chromosomal aneuploidy in pregnant women cell-free dna (cfdna): clinical significance and utility in cancer shaped by emerging technologies validation of a clinical-grade assay to measure donor-derived cell-free dna in solid organ transplant recipients cell-free dna comprises an in vivo nucleosome footprint that informs its tissues-of-origin the virtosome-a novel cytosolic informative entity and intercellular messenger high-resolution profiling of fetal dna clearance from maternal plasma by massively parallel sequencing increase in and clearance of cell-free plasma dna in hemodialysis quantified by real-time pcr deoxyribonuclease is a potential counter regulator of aberrant neutrophil extracellular traps formation after major trauma sars-cov2 may evade innate immune response, causing uncontrolled neutrophil extracellular traps formation and multi-organ failure clinical, molecular and epidemiological characterization of the sars-cov2 virus and the coronavirus disease 2019 (covid-19), a comprehensive literature review staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death cloning, expression and purification of 3'-nucleotidase/nuclease, an enzyme responsible for the leishmania escape from neutrophil extracellular traps apoptotic cell-free dna promotes inflammation in haemodialysis patients origins, structures, and functions of circulating dna in oncology cell-free fetal dna-a trigger for parturition cell-free fetal dna in maternal plasma during physiological single male pregnancies: methodology issues and kinetics cell-free nucleic acids are present in blood products and regulate genes of innate immune response establishment and characterization of a human acute monocytic leukemia cell line (thp-1) role of notch signaling in regulating innate immunity and inflammation in health and disease the unfolded protein response in homeostasis and modulation of mammalian immune cells role of the cxcl8-cxcr1/2 axis in cancer and inflammatory diseases oncogene-induced senescence relayed by an interleukin-dependent inflammatory network edta-mediated inhibition of dnases protects circulating cell-free dna from ex vivo degradation in blood samples modulating macrophage polarization with divalent cations in nanostructured titanium implant surfaces divalent cation signaling in immune cells new evidence that a large proportion of human blood plasma cellfree dna is localized in exosomes mechanisms of nuclear content loading to exosomes generation of blood circulating dnas: the sources, peculiarities of circulation and structure distinct and regulated expression of notch receptors in hematopoietic lineages and during myeloid differentiation integrated regulation of toll-like receptor responses by notch and interferon-gamma pathways sesn2/sestrin2 suppresses sepsis by inducing mitophagy and inhibiting nlrp3 activation in macrophages arrdc4 regulates enterovirus 71-induced innate immune response by promoting k63 polyubiquitination of mda5 through trim65 irf family of transcription factors as regulators of host defense mtss1: a multifunctional protein and its role in cancer invasion and metastasis g protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via notch1 inhibition eotaxin-2 induces monocytic apoptosis in patients who have undergone coronary artery bypass surgery and in thp-1 cells in vitro regulated by thrombomodulin production of complement components by cells of the immune system cell-free dna from human plasma and serum differs in content of telomeric sequences and its ability to promote immune response immunoregulatory properties of cell-free dna in plasma of celiac disease patients-a pilot study checkpoints in tnf-induced cell death: implications in inflammation and cancer advances in bioresponsive closed-loop drug delivery systems infection: microbial nucleases turn immune cells against each other nucleotidase activities in soluble and membrane fractions of three different mammalian cell lines the ectonucleotidases cd 39 and cd 73: novel checkpoint inhibitor targets neutrophil dysfunction, immature granulocytes, and cell-free dna are early biomarkers of sepsis in burn-injured patients: a prospective observational cohort study molecular mechanisms regulating netosis in infection and disease netosis provides the link between activation of neutrophils on hemodialysis membrane and comorbidities in dialyzed patients ultrasensitive proteome analysis using paramagnetic bead technology the one hour yeast proteome maxquant enables high peptide identification rates, individualized ppb-range mass accuracies and proteomewide protein quantification accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed maxlfq the perseus computational platform for comprehensive analysis of (prote) omics data the reactome pathway knowledgebase causal analysis approaches in ingenuity pathway analysis m.k. designed the experiments, interpreted the results and wrote the manuscript; a.z. and i.b. performed preparation of plasma samples, cell cultivation and qpcr experiments; b.c. performed microarray experiments; p.p. conducted proteomic analysis and data interpretation; l.s. and o.s. conducted ipa analysis; p.n. was involved in results interpretation and manuscript writing. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-74288 -2.correspondence and requests for materials should be addressed to m.k.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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 licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence 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 licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-000436-k1hwh640 authors: amidi, maryam; de raad, markus; crommelin, daan j. a.; hennink, wim e.; mastrobattista, enrico title: antigen-expressing immunostimulatory liposomes as a genetically programmable synthetic vaccine date: 2010-10-26 journal: syst synth biol doi: 10.1007/s11693-010-9066-z sha: doc_id: 436 cord_uid: k1hwh640 liposomes are versatile (sub)micron-sized membrane vesicles that can be used for a variety of applications, including drug delivery and in vivo imaging but they also represent excellent models for artificial membranes or cells. several studies have demonstrated that in vitro transcription and translation can take place inside liposomes to obtain compartmentalized production of functional proteins within the liposomes (kita et al. in chembiochem 9(15):2403–2410, 2008; moritani et al.in febs j, 2010; kuruma et al. in methods mol biol 607:161–171, 2010; murtas et al. in biochem biophys res commun 363(1):12–17, 2007; sunami et al. in anal biochem 357(1):128–136, 2006; ishikawa et al. in febs lett 576(3):387–390, 2004; oberholzer et al. in biochem biophys res commun 261(2):238–241, 1999). such a minimal artificial cell-based model is ideal for synthetic biology based applications. in this study, we propose the use of liposomes as artificial microbes for vaccination. these artificial microbes can be genetically programmed to produce specific antigens at will. to show proof-of-concept for this artificial cell-based platform, a bacterial in vitro transcription and translation system together with a gene construct encoding the model antigen β-galactosidase were entrapped inside multilamellar liposomes. vaccination studies in mice showed that such antigen-expressing immunostimulatory liposomes (anexils) elicited higher specific humoral immune responses against the produced antigen (β-galactosidase) than control vaccines (i.e. anexils without genetic input, liposomal β-galactosidase or pdna encoding β-galactosidase). in conclusion, anexils present a new platform for dna-based vaccines which combines antigen production, adjuvanticity and delivery in one system and which offer several advantages over existing vaccine formulations. synthetic biology is a rapidly emerging interdisciplinary research field that aims to construct new biological parts and systems with new functionalities through a process of engineering and standardization. vaccines may also benefit from a synthetic biology-based design. with vaccination the aim is to delude the immune system with an antigenic formulation to make it believe it is dealing with a natural infection, however, without causing illness. at present, the majority of vaccines on the market consist of attenuated or inactivated pathogens. although effective, these systems are poorly defined, suffer from batch-to-batch variation and can only be used for pathogens that can be readily cultivated in the lab at scales that permit vaccine production. moreover, as the ratio of antigenic compounds within the pathogen is more-or-less fixed, there is poor control over the direction against which antigenic compound an immune reaction will be evoked. the bottom-up design of vaccines which consist of well-defined antigenic compounds (e.g. proteins or nucleic acids) offers better control over the evoked immune reaction, however, the design of such vaccines is often empirical and in general yields vaccines that are poorly immunogenic and rely on adjuvants in order to be effective. moreover, most vaccine production schemes are rather time-consuming, and therefore not suitable for rapid response intervention, for example, to prevent the pandemic spread of a new influenza strain in the human population. cell-free synthetic biology may offer new ways to design potent and genetically programmable vaccines (jewett et al. 2008; tsuboi et al. 2008 tsuboi et al. , 2010a zichel et al. 2010; kanter et al. 2007; simpson 2006) . it is based on the in vitro transcription and translation of single or multiple gene constructs in order to obtain a functional part or system. applications of cell-free biology include the production of membrane proteins that are difficult to express in heterologous hosts (henderson et al. 2007; junge et al. 2010; beebe et al. 2010; nozawa et al. 2010; reckel et al. 2010; ishihara et al. 2005; berrier et al. 2004) , high-throughput screening of protein libraries by in vitro compartmentalization (mastrobattista et al. 2005) , generation of artificial cells by encapsulation of these complex biochemical reactions into cell-sized compartments, like liposomes (kita et al. 2008; sunami et al. 2006 sunami et al. , 2010 yamaji et al. 2009; murtas et al. 2007; ishikawa et al. 2004; oberholzer et al. 1999 ). recently, we have shown that protein expression in liposomes can yield microgramquantities of a model antigen (amidi et al. 2010) . in this study, we propose the use of antigen-expressing immunostimulatory liposomes (anexils) as artificial microbes for vaccination (fig. 1) . the potential advantages of such antigen-expressing immunostimulatory liposomes (anexils) over conventional vaccines are numerous: the specificity of the vaccine can be easily altered by simply changing the dna templates without having to change the entire vaccine formulation. moreover, compared to dna vaccines anexils do not exclusively rely on the often inefficient transfection of dna into cells of the vaccine recipient in order to be effective. secondly, compared to vector-based vaccines, anexils will be unaffected by neutralizing antibodies against the vector. thirdly, there is no limit to the number of genes that can be expressed inside anexils. lastly, anexils mimic characteristics of viruses and bacteria without the safety concerns related to the use of attenuated pathogens for vaccination. here, we show that anexils expressing b-galactosidase are well tolerated after i.m. injection and were capable of inducing strong systemic immune responses, which were superior to that of liposomal dna or protein vaccines encoding the same antigen. egg-derived l-a-phosphatidylcholine (epc), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (peg) 5000 (dspe-peg 5000) and 1,2-dioleoyl-sn-glycero-3-([n(5-amino-1-carboxypentyl) iminodi-acetic acid] succinyl) (dogs-nta) were purchased from avanti polar lipids, inc. (alabaster, alabama, usa). luria broth, 2mercaptoethanol, adenosine-5 0 -triphosphate (atp), phosphoenol-pyruvate (pep), cytidine-5 0 -triphosphate (ctp), guanosine-5 0 -triphosphate (gtp), 3 0 -5 0 -cyclic adenosine monophosphate (camp), folinic acid, cholesterol (chol) and b-galactosidase enzyme (400 iu/mg) and each of the 20 encoded amino acids were purchased from sigma (saint louis, mo, usa). the fluorescein di-b-d-galactopyranoside (fdg) was supplied from marker gene technologies (eugene, or, usa). e-coli trna, creatine kinase and creatine phosphate were obtained from roche (basel, switzerland). uridine 5 0 -triphosphate (utp) and t7 polymerase were supplied from fermentas (burlington, ontario, canada). dithiothreitol (dtt), lysogeny broth (lb) and pyruvate kinase (pk) were from flucka (seelze, germany). rabbit polyclonal anti-b-galactosidase igg and cy-5 conjugated goat igg anti-rabbit immunoglobulin was from abcam (cambridge, uk). horseradish peroxidase (hrp)-labeled goat anti-mouse total igg and hrp-labeled rabbit anti-mouse igg1 were purchased from invitrogen (breda, the netherlands). hrp-labeled rat monoclonal anti-mouse igg2a was obtained from abcam (cambridge, the united kingdom). peg 8000 was from promega (madison, wi, usa). all other materials used were of analytical or pharmaceutical grade. preparation of peg-liposomes and ni 2? nta liposomes a mixture of 2.5 lmol of total lipids (epc, chol and dspe-peg 5000 with a molar ratio of epc:chol:peg fig. 1 schematic representation of anexil formulations used in this study 5000 = 1.6:0.9:0.025) or (epc, chol, dogs-nta) with a molar ratio of epc:chol:dogs-nta = 1.55:0.9: 0.025) were dissolved in dichloromethane:diethylether (1:1, v/v) in a round bottom flask. a thin, dry lipid film was obtained after evaporating the solvents using a rotary evaporator under vacuum at 30°c and subsequently dried with nitrogen for 30 min. the lipid film was hydrated in distilled water by shaking using glass beads to form large multilamellar liposomes, further sonicated with a probe sonicator to produce unilamellar liposomes. the liposomes suspensions were divided into 100 ll aliquots in 1.5 ml tubes (6 lm of total lipids/batch), freeze-dried and the obtained lyophilized lipid cakes were stored in a desiccator at room temperature until used. volume-weighted mean diameters and size distributions of the liposomes were determined by single particle optical sensing (accusizer tm 780, santa barbara, california, usa). for cell-free protein expression, e. coli b-galactosidase was used as a model antigen. lacz, encoding e. coli b-galactosidase was cloned into vector pivex2.2em, which allowed t7 promoter-driven expression in prokaryotic cell-free transcription and translation systems. the vector appends a 6-histidine (6-his) coding sequence to the c-terminal end of lacz for efficient purification of the b-galactosidase protein (amidi et al. 2010) . the e.coli tuner tm strain, which is devoid of endogenous b-galactosidase (merck chemicals ltd., nottingham, uk), was used to make s30 bacterial extract as described previously (amidi et al. 2010) . a coupled in vitro transcription/ translation reaction mixture (further referred to as ivtt mix), consisted of 30% (v/v) s30 extract, 175 lg/ml trna, 250 lg/ml creatine kinase, 5.8 mm magnesium acetate, 260u t7 polymerase, and 50% (v/v) lowmolecular-weight mix (lm mix) containing: 110 mm hepes, 3.4 mm dtt, 2.4 mm atp, 1.6 mm ctp, 1.6 mm gtp, 1.6 mm utp, 0.8 m creatine phosphate (cp), 0.65 mm camp, 0.05 mm folinic acid, 0.21 m potassium acetate, 27 mm ammonium acetate, 2 mm each of the 20 amino acids, and 8% (v/v) peg8000, was used for protein synthesis. to initiate protein expression, plasmid dna was added to the ivtt mix at a final concentration of 20 nm and the reaction mixture was incubated for 3 h at 30°c. generation of b-galactosidase-producing anexils for preparation of anexils with b-galactosidase expressed inside liposomes (further referred to as anexil-in), 75 ll of ivtt mixture and pivex2.2em-lacz with a final concentration of 20 nm, was used to rehydrate a batch of 6 lm of peg-lipid cakes in order to form liposomes encapsulating ivtt mix and pdna. the liposomes were incubated on ice for 10 min to complete the rehydration process. to inactivate protein expression outside liposomes, rnase with a final concentration of 10 lg/ml, was added to the liposomal suspension. samples were incubated at 30°c for 3 h to allow protein synthesis to complete. to prepare b-galactosidase-bound onto ni 2? -nta liposomes (further referred to as anexil-on), b-galactosidase was expressed in bulk and subsequently bound onto liposomal bilayers as described below. ninety-ll of ivtt and pivex2.2em-lacz as a pdna template with a final concentration of 20 nm was used for bulk expression of a b-galactosidase with a c-terminal 6his-tag. ivtt mix with dna template was incubated at 30°c for 3 h. after the expression was completed, 75 ll of the reaction mix containing expressed b-galactosidase was added to the nta-lipid cakes (6 lm lipids) and incubated for 10 min at room temperature to form liposomes. the expressed b-galactosidase has a 6-histidine residue which binds to ni 2? ions of ni 2? -nta liposomes. to remove the non attached b-galactosidase, the liposomes were washed three times with 0.5-1.5 ml phosphate buffer saline (pbs, nacl concentration was adjusted to make it isotonic with the ivtt mix) by centrifugation at 8,000-9,0009g for 10 min at 4°c. after washing, the liposomes were re-suspended in 50 ll pbs. an aqueous solution of b-galactosidase protein (40 iu/ml) was prepared in pbs. subsequently, 100 ll of each solution was slowly added to freeze-dried lipids (6 lm) and incubated for 10 min at room temperature until rehydration was completed and liposomes were formed. to remove nonencapsulated b-galactosidase, liposomes were washed three times with pbs by centrifugation at 8,000-9,0009g for 10 min at 4°c and re-suspended in 50 ll pbs. the amount of encapsulated b-galactosidase in peg-liposomes was calculated from total amount of protein entrapped in antigen-expressing immunostimulatory liposomes as a genetically programmable synthetic vaccine 23 liposomes. for some formulations pdna encoding bgalactosidase was co-entrapped with the b-galactosidase enzyme at a concentration of 20 nm. the amount of encapsulated b-galactosidase was measured based its enzymatic activity. quantification and antigenicity of the produced b-galactosidase b-galactosidase activity assay the amount of encapsulated b-galactosidase as well as expressed b-galactosidase in bulk, inside the peg-liposomes or bound onto ni 2? -nta liposomes were determined based on enzymatic activity of b-galactosidase using fluorescence spectroscopy (amidi et al. 2010) . briefly, in this assay, fdg as a substrate was cleaved by b-galactosidase into fluorescein, and two galactose molecules in a twostep reaction. by using high fdg concentrations relative to enzyme concentrations, the concentration of formed bgalactosidase could be directly deduced from the substrate conversion rate as measured by an increase in fluorescein fluorescence in time (excitation/emission wavelengths: 488/ 512 nm). a calibration curve of 0-500 lg/ml (corresponding to 0-200 iu/ml) of commercially available purified e. coli b-galactosidase was used in all experiments. to determine the amount of encapsulated b-galactosidase or formed by ivtt in liposomes, 10 ll of each liposome suspension was diluted in 90 ll cell lytic b buffer (sigma, saint louis, mo, usa) to disrupt the liposomes and release the produced protein. a same treatment was done with a blank liposomal formulation and standards to correct for possible interferences of lipid components and cell lytic b buffer with generated fluorescent signal. subsequently 100 ll of substrate (fdg, 1 mm) was automatically added to each sample and standard and generated fluorescent signal was measured every 0.5 s over a period of 120 s at 25°c using a fluorescent well-plate reader (flu-ostar optima, bmg-labtech, offenburg, germany). to determine the concentration of encapsulated or expressed bgalactosidase, initial substrate conversion slopes (relative light unit (rlu)/s) were determined and compared to those of standards with known concentrations of b-galactosidase (amidi et al. 2010) . western blotting was done to evaluate the antigenicity of the expressed b-galactosidase. liposomes containing b-galactosidase were disrupted by adding 100 ll of cell lytic b buffer to 55 ll of liposome suspension. after adding electrophoresis loading-buffer (60 mm tris-hcl, ph 6.8, with 25% glycerol and 2% sds containing 0.1% bromophenol blue solution, and b-mercaptoethanol), the samples were heated for 5 min at 95°c and electrophoresed at 125 v under reducing conditions, in a 10% sds-polyacrylamide gel (bollag and edelstein 1991) . after gel electrophoresis, antigen bands were transferred to a nitrocellulose membrane by using a dry western blotting system (iblot, invitrogen, frederick, md) in 6 min. after blotting the free sites were blocked with 1% non-fat milk-powder in pbs containing 0.05% tween 20 (pbs-t). next, the membrane was incubated with a solution of goat anti-b-galactosidase polyclonal antibody in pbs-t containing 0.1% non-fat milk powder. the membrane was then washed to remove the unbound antibody and incubated with cy-5-labeled, rabbit igg anti-goat immunoglobulin. the blotted antigen-antibody complexes were visualized by a fluorescence using a typhoon imager (amersham corporation, arlington heigths, il, usa). female balb-c mice, 6-8 weeks old (charles river, netherlands), were housed in groups of 5 and maintained in the animal facility of utrecht university with a 12 h day and night schedule, while food and water were ad libitum. the experiments were approved by the ethical committee for animal experimentation of utrecht university. mice (5 per group) were intramuscularly (i.m.) immunized twice with 2-week intervals. mice received 100 ll of different formulations (table 1 ) divided over two batches of 50 ll which were injected in each of the hind leg muscles. before each immunization and 2 weeks after the last immunization, the mice were bled by cheek puncture and then sacrificed by inhalation of excess co 2 . individual serum samples were separated from blood cells and coagulated proteins by centrifugation for 5 min at 14,0009g and 4°c, and stored at -20°c. b-galactosidase-specific antibody responses were determined by using an enzyme-linked immunosorbent assay (elisa). briefly, elisa plates (high binding capacity, greiner, alphen a/d rijn, the netherlands) were coated overnight at ambient temperature with b-galactosidase (100 ng in 100 ll/well) in coating buffer (0.05 m carbonate/bicarbonate, ph 9.6). to measure antibody responses in mice vaccinated with liposomes containing expressed b-galactosidase, the elisa plates were coated with recombinantly produced b-galactosidase in e. coli strain bl21 (de3) (invitrogen carlsbad, ca) and purified by using an akta purifier equipped with 5 ml his-trap hp columns (ge healthcare, uppsala, sweden). for serum samples of mice vaccinated with control formulations containing b-galactosidase encapsulated in liposomes, the plates were coated with the same commercial source of b-galactosidase that was used for entrapment. after coating, plates were washed and blocked by incubation with 2.5% (w/v) milk powder in coating buffer (200 ll/well) for 1 h at 37°c. subsequently, the plates were washed with pbs containing 0.05% tween, ph 7.4 (pbs/tween). appropriate dilutions of sera of each individual mouse were applied to the plates, serially diluted two-fold in pbs/ tween and incubated for 2 h at 37°c. plates were then washed and incubated either with horseradish peroxidase (hrp)-conjugated goat antibody against mice igg or hrpconjugated rabbit antibody against mice igg1 or hrpconjugated rat antibody against mice igg2a (all diluted 1:5000 in pbs/tween, 100 ll/well) for 1 h at 37°c. thereafter, the plates were washed twice with pbs/tween and once with pbs. specific antibodies were detected by adding 100 ll of 3,3 0 ,5,5 0 -tetra methyl benzidine (tmb, 0.1 mg/ml) in 10 mm sodium acetate ph 5.5 buffer also containing 0.06% (v/v) hydrogen peroxide to each well. after 10 min, the reaction was stopped by adding 50 ll 2 m h 2 so 4 to each well. total igg, igg1 and igg2a antibody titers are expressed as the reciprocal sample dilution corresponding with an a 450 of 0.2 above the background (amidi et al. 2007 ). comparison between mice of different groups with positive titers was made by a oneway anova test. characterization of anexils and liposomes loaded with b-galactosidase and/or pdna cell-free protein synthesis was used to transcribe and translate the lacz gene encoding for e. coli b-galactosidase either in bulk, inside liposomal compartments (anexils-in) or adhered onto the surface of liposomes (anexil-on) (for details of liposome preparation see materials & methods section). the volume-weighted mean diameter of b-galactosidase-loaded liposomes and anexil formulations was approximately 1.5 lm. the dose of b-galactosidase was standardized to 800 mu per formulation (see ''materials and methods''), which corresponds to approximately 2 lg of active b-galactosidase from a commercial source (sigma). the s30 extract used for ivtt was derived from e. coli bl21 tuner tm strain, which is devoid of endogenous b-galactosidase. this is important to avoid interference of endogenous b-galactosidase in the experiments (amidi et al. 2010 ). sds-page and western blot analysis of s30 extract made from e-coli tuner tm strain proved absence of endogenous b-galactosidase in s30 extract (fig. 2) . importantly, western blot analysis confirmed that the expressed b-galactosidase was recognized by polyvalent anti-bgalactosidase antibodies and the antigenicity of b-galactosidase was preserved in ivtt mix (fig. 2) . systemic antibody responses after i.m. immunization in mice to investigate the potency of anexils as alternatives for conventional protein or dna vaccines, we compared systemic humoral responses of mice immunized i.m. with anexil-in, anexil-on, pcmv-lac-z encapsulated in liposomes (further referred to as liposomal dna vaccine), b-galactosidase encapsulated in liposomes (further referred to as liposomal protein vaccines) and b-galactosidase coencapsulated with pdna (pivex-lac-z) in liposomes (further referred to as liposomal protein/dna vaccines). the liposomal dna vaccine was poorly immunogenic and induced very low serum igg titers in only some of the vaccinated animals after single dose i.m. immunization (fig. 3a) . in contrast, anexil-in induced high serum antibody responses after i.m. immunization, which were significantly higher than those achieved after i.m. injection of liposomal protein and liposomal protein/dna vaccines (fig. 3b) (p \ 0.001). single i.m. vaccination with anexil-on could elicit substantially higher serum antibody responses than those elicited by other vaccines (fig. 3a , b) (p \ 0.001). it has been shown that surfacelinked liposomal antigens could enhance presentation of antigens to apcs and induce stronger immune responses (taneichi et al. 2006a; naito et al. 1996; uchida and taneichi 2008) . altogether, these results point to strong immunostimulating effects of anexil vaccines and robust impact of surface antigen presentation on systemic antibody response. since there are usually needs for booster vaccinations in order to induce prolonged and strong immune responses, the effect of a booster immunization on the systemic antibody production was studied in mice. after booster immunizations all group of mice showed higher igg titers but not significantly higher than those after prime immunization (fig. 3) . to exclude that the observed anti-b-galactosidase serum titers were induced by components within the s30 bacterial extract, serum antibody titers of mice injected with anexils without the pdna encoding b-galactosidase were measured. after i.m. immunizations with pdnalacking anexils, mice showed a negligible b-galactosidase specific antibody response (fig. 3b) . this clearly indicates that the antigen specific immune response is exclusively mediated by the genetic input. to gain more insight into the nature and quality of the systemic immune response, igg1 and igg2a subclasses of b-galactosidase-specific antibodies (fig. 4a, b) were determined. after i.m. immunizations with liposomal dna vaccine, there were no detectable levels of igg1 or igg2a in the sera of the vaccinated mice. as compared to liposomal protein and protein/dna vaccines, anexil-in and the anexil-on vaccines showed superior igg1 (p \ 0.05) and igg2a (p \ 0.001) responses and those induced by anexil-on was the highest (fig. 4a, b) . the igg1 and igg2a responses of control and tested formulations largely corresponded to the total igg responses (fig. 4a, b) . remarkably, the anexil-on induced significantly higher igg2a antibody levels (fig. 4a) and consequently notably higher igg2a/igg1 ratios after booster vaccination compared to the other formulations (fig. 4d) . sera were collected 14 days after each immunization. antibody titers are expressed as mean of the responding mice; the bars represent the 95% confidence intervals. the numbers above the columns indicate the number of responders per group. asterisks indicate titers that are significantly (* p \ 0.05; ** p \ 0.001) higher than those of the group immunized (booster vaccination) with liposomal protein/dna vaccines. circles indicate titers that are significantly (°p \ 0.05) higher than those of the group immunized (booster vaccination) with anexil-in the results presented here demonstrate that anexils can be used as a synthetic biology platform to construct vaccines which can be genetically programmed in order to obtain antigen-specificity. anexils mimic characteristics of natural pathogens, without being virulent, and were able to induce strong humoral immune responses. the particle size of anexils is amenable for uptake by antigen presenting cells (foged et al. 2005; tabata et al. 1996; xiang et al. 2006; yoshida and babensee 2006) . the amount of pivex2.2em-lacz-6his encapsulated in liposomes (20 nm) was sufficient to initiate in vitro protein expression in liposomal compartments. b-galactosidase was successfully produced and quantified in liposomes. enzymatic activity was used as a fast and sensitive method for evaluating biological activity and quantification of expressed b-galactosidase in anexils and showed that the produced enzyme has a correct folding and is active in cellfree protein synthesis. western blot analysis showed that antigenicity of the expressed b-galactosidase was preserved. these results indicate that cell-free protein synthesis can efficiently produce antigens avoiding complexity and maintenance of cell viability associated with recombinant and in vivo systems (jewett et al. 2008 ). furthermore, it was demonstrated that the use of anexils can circumvent current problems of low immunogenicity with non-adjuvanted dna vaccines (liu and ulmer 2005; rosenberg et al. 2003; trimble et al. 2009 ). as opposed to i.m. vaccination with liposomal dna vaccine, i.m. immunization with anexils resulted in strong humoral immune responses. this result is in agreement with other studies showing that dna vaccination is incapable of inducing strong antibody responses most probably because of poor transfection of dna in endogenous cells, which leads to low doses of antigens produced (abdulhaqq and weiner 2008; lu 2008 ). moreover, anexils were able to induce significantly higher antibody responses as compared to conventional liposomes encapsulating antigen with or without pdna. these results demonstrate strong immunostimulating effect of anexils as alternative for dna and protein vaccines. factors that might contribute to the adjuvant effect of anexils include efficient delivery of antigen to apcs, possible adjuvant activities of pdna (due to cpg methylation pattern) (klinman et al. 2009 ) and the presence of pathogen-associated molecular patterns (pamps) in the bacterial s30 extract which can activate the innate immune system via pattern recognition receptors. anexil-on with surfaced-linked b-galactosidase was tested in mice as an alternative to anexil-in formulation, in which antigen is entrapped inside vesicles. from the results of the vaccination studies with anexil-on, it can be concluded that antigen presentation on the surface of the liposomes could significantly enhance systemic immune responses as compared to the other tested formulations and even a single immunization was sufficient to strongly stimulate an immune response superior to those achieved after i.m. immunization with conventional liposomal vaccines. several studies have previously shown that surfacelinked liposomal antigens could enhance presentation of antigens to apcs and induce stronger immune responses matsui et al. 2010; ohno et al. 2009; takagi et al. 2009; taneichi et al. 2006b ). the impact of the anexils and other control vaccines on the antibody subtype profile was also investigated. in most of infectious diseases protection against viral or bacterial infections are mainly mediated by neutralizing immunoglobulins that bind to the viral or bacterial antigens (gissmann 2009; ho et al. 2002; montefiori et al. 2007; montefiori and mascola 2009; sattentau 2008; smith 2009 ). in mice igg1 and igg2a antibodies are known to contribute to virus neutralization. igg2a has been reported to contribute in complement activation and antibody-dependent cell-mediated immunity and is more effective than igg1 in protection against viral infections by preventing virus replication (coutelier et al. 1987; hocart et al. 1989 ). on the other hand, the absolute concentration of igg is important for reducing the viral shedding (hocart et al. 1989; hovden et al. 2005) . therefore, induction of a combined igg2a/igg1 (t-helper 1 /t-helper 2 ) response may improve vaccine efficacy especially against viral infections. in this study, i.m. immunizations with all vaccines resulted in a mixed th 1 /th 2 immune response and i.m. administered anexils were able to markedly enhance both the igg1 and the igg2a response after i.m. vaccination as compared to protein and dna liposomal formulations (fig. 4a, b) , which may be advantageous for protection against viral or intracellular bacterial infections. the igg2a/igg1 ratio of anexil-in was the highest among those of other vaccines after prime vaccination (fig. 4c) and that of anexil-on was substantially increased after booster immunization (fig. 4d) . these data suggest that the quality of the immune response to b-galactosidase vaccine is substantially affected by the characteristic of anexil formulations. the work presented here shows for the first time that liposomes can be used as antigen-producing artificial-cells for vaccination. anexils combine antigen-production, delivery and adjuvanticity in one system, making them more potent in inducing antibody responses compared to liposomal dna vaccines as shown here. still, the specificity of anexils is only determined by its genetic input which offers great flexibility in vaccine production and formulation. preliminary data from our lab show that it is possible to store anexils lyophilized. upon hydration with a dna solution, the dna is being transcribed and translated inside the rehydrated liposomes (data not shown). such a universal vaccine platform may solve some of the problems of production lag-time with conventional vaccines (hinman et al. 2006; desroches et al. 2005; ulmer et al. 2006 ). furthermore, it may allow easy production of personalized vaccines for e.g. cancer vaccination, in which the antigens are optimized for each individual (poland et al. 2008) . although not tested here, the injection of bacterial components as essential part of the anexil formulation may pose problems of reactogenicity. s30 extract derived from e. coli is an essential component of anexils and which is likely to contain the pyrogenic endotoxin lps. visual inspection of the mice after i.m. administration of the anexils did not show any discomfort or illness. nevertheless, alternative sources of ivtt mixes that are free of endotoxins should be tested. for example, extracts prepared from gram-positive bacteria, wheat germ or the synthetic purexpress system (shimizu et al. 2001 ) may be substituted for the e. coli-based extract. although we have only used a single antigen to show proof-of-concept of our anexil vaccination platform, mixtures of antigens can be expressed inside anexils by simply mixing pdnas encoding different antigens. although different antigens will have different levels of expression in cell-free expression systems, in general, the expression levels are high enough for the purpose of vaccination, where only microgram quantities of proteins are required. in fact, membrane proteins which are often difficult to produce in bacteria show remarkably high levels of expression in prokaryotic cell-free systems in the presence of liposomes (junge et al. 2010; goren et al. 2009; kuruma et al. 2010; moritani et al. 2010; nozawa et al. 2010; reckel et al. 2010) . besides dna encoding antigens, a variety of genetic inputs can be used in order to control the type of immune response provoked. for example, genetic adjuvants, such as chemokines or cytokines may be coexpressed inside the anexils in order to enhance the influx of immune cells at the site of injection and uptake of anexils by antigen presenting cells. as such anexils form an excellent synthetic biology platform for genetic programming using standard biological parts (canton et al. 2008; knight 2003) . in conclusion, we have shown proof-of-concept for a genetically programmable vaccine based on the in situ production of antigens from dna templates. further immunization studies focusing on co-administering of dnas, encoding biologically relevant antigens and genetic adjuvants (e.g. cytokines) are needed to demonstrate the possibilities and limits of the anexil vaccination platform. the authors declare that they have conflict of interest. open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. dna vaccines: developing new strategies to enhance immune responses diphtheria toxoidcontaining microparticulate powder formulations for pulmonary vaccination: preparation, characterization and evaluation in guinea pigs optimization and quantification of protein synthesis inside liposomes robotic large-scale application of wheat cell-free translation to structural studies including membrane proteins cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent refinement and standardization of synthetic biological parts and devices igg2a restriction of murine antibodies elicited by viral infections americans' responses to the 2004 influenza vaccine shortage particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model hpv vaccines: preclinical development cellfree translation of integral membrane proteins into unilamelar liposomes cell-free analysis of tail-anchor protein targeting to membranes vaccine shortages: history, impact, and prospects for the future risk factors for subsequent cervicovaginal human papillomavirus (hpv) infection and the protective role of antibodies to hpv-16 virus-like particles the immunoglobulin g subclass responses of mice to influenza a virus: the effect of mouse strain, and the neutralizing abilities of individual protein a-purified subclass antibodies two doses of parenterally administered split influenza virus vaccine elicited high serum igg concentrations which effectively limited viral shedding upon challenge in mice expression of g protein coupled receptors in a cell-free translational system using detergents and thioredoxinfusion vectors expression of a cascading genetic network within liposomes an integrated cell-free metabolic platform for protein production and synthetic biology advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins cell-free production of scfv fusion proteins: an efficient approach for personalized lymphoma vaccines antigen-expressing immunostimulatory liposomes as a genetically programmable synthetic vaccine 29 replication of genetic information with self-encoded replicase in liposomes cpg oligonucleotides as adjuvants for vaccines targeting infectious diseases idempotent vector design for standard assembly of biobricks. mit artificial intelligence laboratory efficient induction of cytotoxic t lymphocytes specific for severe acute respiratory syndrome (sars)-associated coronavirus by immunization with surface-linked liposomal peptides derived from a non-structural polyprotein 1a production of multi-subunit complexes on liposome through an e.coli cell-free expression system human clinical trials of plasmid dna vaccines immunogenicity of dna vaccines in humans: it takes two to tango high-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions a ctl-based liposomal vaccine capable of inducing protection against heterosubtypic influenza viruses in hla-a*0201 transgenic mice neutralizing antibodies against hiv-1: can we elicit them with vaccines and how much do we need? antibody-based hiv-1 vaccines: recent developments and future directions direct integration of cell-free-synthesized connexin-43 into liposomes and hemichannel formation protein synthesis in liposomes with a minimal set of enzymes ovalbumin-liposome conjugate induces igg but not ige antibody production production of membrane proteins through the wheat-germ cell-free technology protein expression in liposomes synthetic peptides coupled to the surface of liposomes effectively induce sars coronavirus-specific cytotoxic t lymphocytes and viral clearance in hla-a*0201 transgenic mice personalized vaccines: the emerging field of vaccinomics strategies for the cell-free expression of membrane proteins inability to immunize patients with metastatic melanoma using plasmid dna encoding the gp100 melanoma-melanocyte antigen correlates of antibody-mediated protection against hiv infection cell-free translation reconstituted with purified components cell-free synthetic biology: a bottom-up approach to discovery by design botulism and vaccines for its prevention femtoliter compartment in liposomes for in vitro selection of proteins synthesis of functional proteins within liposomes size effect on systemic and mucosal immune responses induced by oral administration of biodegradable microspheres highly efficient antiviral cd8(?) t-cell induction by peptides coupled to the surfaces of liposomes antigen chemically coupled to the surface of liposomes are cross-presented to cd8 ? t cells and induce potent antitumor immunity induction of differential t-cell epitope by plain-and liposome-coupled antigen a phase i trial of a human papillomavirus dna vaccine for hpv16 ? cervical intraepithelial neoplasia 2/3 wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates the wheat germ cell-free protein synthesis system: a key tool for novel malaria vaccine candidate discovery an efficient approach to the production of vaccines against the malaria parasite clinical application of surface-linked liposomal antigens vaccine manufacturing: challenges and solutions pathogen recognition and development of particulate vaccines: does size matter? protein synthesis in giant liposomes using the in vitro translation system of thermococcus kodakaraensis molecular aspects of microparticle phagocytosis by dendritic cells efficacy of a potential trivalent vaccine based on hc fragments of botulinum toxins a, b, and e produced in a cell-free expression system key: cord-001732-4eyn7pjq authors: riede, o; seifert, k; oswald, d; endmann, a; hock, c; winkler, a; salguero, f j; schroff, m; croft, s l; juhls, c title: preclinical safety and tolerability of a repeatedly administered human leishmaniasis dna vaccine date: 2015-04-30 journal: gene ther doi: 10.1038/gt.2015.35 sha: doc_id: 1732 cord_uid: 4eyn7pjq the leishmaniases are a complex of vector-borne diseases caused by protozoan parasites of the genus leishmania. leishdnavax is a multi-antigen, t-cell epitope-enriched dna vaccine candidate against human leishmaniasis. the vaccine candidate has been proven immunogenic and showed prophylactic efficacy in preclinical studies. here, we describe the safety testing of leishdnavax in naive mice and rats, complemented by the demonstration of tolerability in leishmania-infected mice. biodistribution and persistence were examined following single and repeated intradermal (i.d.) administration to rats. dna vectors were distributed systemically but did not accumulate upon repeated injections. although vector dna was cleared from most other tissues within 60 days after the last injection, it persisted in skin at the site of injection and in draining lymph nodes. evaluation of single-dose and repeated-dose toxicity of the vaccine candidate after i.d. administration to naive, non-infected mice did not reveal any safety concerns. leishdnavax was also well tolerated in leishmania-infected mice. taken together, our results substantiate a favorable safety profile of leishdnavax in both naive and infected animals and thus, support the initiation of clinical trials for both preventive and therapeutic applications of the vaccine. the leishmaniases are a complex of diseases caused by protozoan parasites of the genus leishmania with up to 1.6 million cases reported worldwide annually 1 and~350 million people at risk to develop leishmaniasis. 2 it is estimated that the most severe form, visceral leishmaniasis (vl), causes up to 40 000 deaths per year. 1 measures to control transmission of the parasite were of limited success to date. despite advances in treatment of vl over the last decade, drug toxicity and heat stability, difficult routes of administration and variation in drug efficacy between endemic areas remain issues to be fully solved. a preventive and therapeutic leishmaniasis vaccine prospectively represents the most cost-effective and successful measure to control the leishmaniases worldwide. 3 immunity to the intracellular parasite leishmania is associated with t-cell-mediated immune responses. 4 dna vaccines are particularly able to deliver antigens into the major histocompatibility complex class i processing pathway, thereby inducing cd8 cytotoxic t-cell immune responses, 5, 6 necessary for clearance of leishmania. in addition, cd4 t cells and b cells are activated by dna vaccines. hence, this vaccination technology is a promising approach for developing a leishmaniasis vaccine. 7 an appropriate preclinical safety profile of a vaccine candidate has to be established prior to initiation of clinical phase 1 studies. the us food and drug administration (fda) and the world health organization recommend the evaluation of distribution and persistence of dna vaccines as well as their local reactogenicity and systemic toxicity. 8, 9 results of corresponding studies suggest that dna vaccines are safe, 10, 11 which has been confirmed in several clinical trials. 12 however, immunogenicity of dna vaccines was often modest in humans, necessitating better delivery methods and improved vaccine antigen expression. 12 in addition, safety concerns prompted avoidance of antibiotic resistance genes and other selection markers for plasmid propagation. 13 state-of-the-art plasmids are consequently more efficacious, safer and smaller than classical plasmids. 14 minimalistic immunogenically defined gene expression (midge) vectors represent one of the most rigorous concepts for improvement. 15 midge vectors are small linear double-stranded dna (dsdna) molecules, solely containing sequences required for their function in vivo and no bacterial plasmid backbone sequences as they have been shown to negatively impact transgene expression and immunogenicity. 16, 17 midge-th1 vectors are midge vectors with a short peptide (pkkkrkvedpyc) covalently attached, enhancing the immune responses to encoded antigens. 18, 19 recently, preclinical data on biodistribution and toxicity of midge and midge-th1 vectors have been published, 20, 21 indicating an excellent safety profile after a single administration. leishdnavax is a mixture of five midge-th1 vectors each encoding a different leishmania antigen: kmp11, cpa, cpb, p74 (elongation factor 1-alpha) or tsa. 22 antigen selection and sequence design followed a novel rational approach. in a series of animal experiments it was demonstrated that leishdnavax is immunogenic and effective against challenge with leishmania donovani, the causative agent of vl. 22 the vaccine is aimed to be administered to humans in both preventive and therapeutic settings. here, we present comprehensive preclinical safety and tolerability data for leishdnavax. a repeated-dose toxicity study in naive mice and a biodistribution and persistence study in rats were performed. moreover, the effect of leishdnavax administration on parasite burden and standard tolerability parameters in mouse models of vl were evaluated. in all studies, potential cumulative effects of repeated dosing of leishdnavax were addressed to allow for a better risk-assessment prior to multi-dose clinical trials. leishdnavax is systemically distributed after intradermal administration biodistribution of the vaccine was assessed in rats after intradermal (i.d.) injection of a 120 μg dose. total dna was extracted from tissue samples taken 24 h post injection and midge-th1 vector dna quantified using a quantitative pcr (qpcr) assay. vector dna was found in a varying number of animals per group and in all tissues except for bone marrow (figure 1a ). the vector copy numbers per μg total dna ranged from 4.04 × 10 3 (geometric mean, ovaries, 4/5 animals positive) to 2.55 × 10 9 (geometric mean, skin of injection site, all animals positive). comparable distribution pattern after single or repeated administration next, we tested whether repeated administration alters the distribution pattern or causes accumulation of vector dna in tissues. one hundred and twenty micrograms of leishdnavax were administered to rats i.d. at the same site four times at weekly intervals. vector dna was quantified in total dna extracted from tissue samples collected 24 h after the last injection. distribution patterns and amounts of vector dna were comparable 24 h after single and repeated injection (figures 1a and b) . copy numbers per μg total dna ranged from 4.63 × 10 3 (geometric mean, lung tissue, 5/10 animals positive) to 4.50 × 10 7 (geometric mean, skin of injection site, all animals positive). midge-th1 vectors persist at injection site and in draining lymph nodes persistence of midge-th1 vectors was examined 14 days and 60 days post treatment. fourteen days after four injections leishdnavax was cleared from the majority of organs (figure 1c ). high vector copy numbers per μg total dna were found in skin of injection site (all animals positive, geometric mean: 3.45 × 10 6 copies) and in inguinal lymph nodes (7/10 animals positive, geometric mean: 2.88 × 10 4 copies). sixty days after four injections, midge-th1 vectors were cleared from most tissues but persisted in inguinal lymph nodes (5/10 animals positive, geometric mean: 1.44 × 10 4 copies) and skin of injection site (all animals positive, geometric mean: 6.93 × 10 5 copies) (figure 1d ). to assess toxicity of leishdnavax in naive mice, sterile phosphate-buffered saline (pbs, placebo) and ascending doses of the vaccine were injected either once or five times in weekly intervals (table 1) . leishdnavax was well tolerated at all doses tested. no animal died during the in-life phase, and no change in behavior or external appearance of the animals was noted. comparing placebo-treated to leishdnavax-treated animals, no differences were observed in body weight, food and drinking water consumption, ophthalmological and auditory examinations, urinalysis, organ weights and the neurological screening. no local intolerance reactions were noted after single or repeated vaccine dosing. minor deviations in single animals/groups for few blood parameters (large unstained cells, albumin/globulin ratio, αamylase) were classified as not vaccine-related. macroscopic post mortem examination of organs revealed an enlarged spleen in a single male animal 1 day after single administration of 100 μg leishdnavax, which possibly indicated the activation of the lymphatic system. in all animals the histological findings were within the normal range of variation. no leishdnavax-related morphological lesions were found. no signs of auto-immune reactions were observed, and there were no elevated levels of serum antibodies against dsdna detected. biological activity of the vaccine lot was proven by induction of antigen-specific serum immunoglobulin g (igg) in mice of satellite groups (table 1) . a dose-dependent increase of leishdnavaxspecific igg levels was observed (figure 2 ), in line with data from efficacy studies as previously reported. 22 rats receiving the vaccine during the biodistribution study did not exhibit any vaccine-related local or systemic signs of toxicity, and also necropsy did not reveal any vaccine-related toxicity. leishdnavax is well tolerated in l. donovani-infected mice tolerability of leishdnavax and effects on visceral parasite burden were evaluated in balb/c and c57bl/6 mice. groups of mice infected with l. donovani were injected with one, two or three doses of 100 μg leishdnavax or pbs in weekly intervals. parasite burden was evaluated seven days and 29 days after the last injection. no significant difference in hepatic or splenic parasite burden was found between groups receiving equal numbers of injections of pbs or leishdnavax. in both treatment groups infection with l. donovani followed the known kinetic 23 with distinct organ-specific growth patterns in livers and spleens of infected mice. figure 3 shows the parasite burden as determined in the balb/c and one c57bl/6 experiment. no significant difference in weights between pbs and leishdnavax-treated groups of mice was observed. serum samples from mice, which had received three injections of pbs or leishdnavax, were subjected to analyses of standard biochemical parameters; alanine transaminase, aspartate aminotransferase, urea and creatinine. no significant differences between pbs and leishdnavax-treated groups were found for these parameters in balb/c mice. significant differences were found in levels of creatinine in c57bl/6 mice in two separate experiments. however, neither the time point nor the direction of difference were consistent between experiments ( table 2) . histological analysis of livers and spleens in repeated experiments and the kidney, heart and lung in a single experiment (c57bl/6 mice) revealed similar lesions in all animals (pbs and leishdnavax treated) in terms of location and severity. lesions were consistent with infection and included severe granulomatous hepatitis and splenitis with intralesional parasites, granulomatous/interstitial pneumonia and interstitial nephritis and glomerulonephritis. biodistribution and persistence of dna vaccines are studied to estimate the duration of antigen expression and the risk of vector integration into host genomic dna. 8, 9 we examined the biodistribution of leishdnavax after a single and, in contrast to previously published results, 20,21 also after a series of four injections at weekly intervals. this condensed application scheme is relevant for clinical approaches with: (i) a limited number of injections over a longer period of time as accepted for prophylactic vaccinations and (ii) vaccination regimes requiring more injections in shorter intervals, for example, therapeutic vaccinations. in line with recommendations of the us fda, 8 organ and tissue samples were taken at several time points following injections and vector dna was quantified using quantitative pcr. twenty-four hour after single or repeated injection, midge-th1 vector dna was detected in almost all organs and tissues examined, suggesting that it was distributed systemically, most likely via the lymphatic system and the blood stream. a similar distribution pattern has been described for midge-th1 vectors per day of sampling, five male and five female animals per group were killed. b satellite group to assess biologic activity of the vaccine lot and induction of antibodies against dsdna. figure 2 . immunogenicity of ascending leishdnavax doses in mice. groups of 10 balb/c mice (5 male, 5 female) were immunized i.d. at the tail base five times in weekly intervals with either pbs or leishdnavax (10, 50 or 100 μg per dose). fourteen days after the last immunization leishdnavax-specific antibodies (total igg) in sera were quantified by elisa using plates coated with a mixture of recombinant leishdnavax antigens. ***p ⩽ 0.001 (student's t-test); n.s.: not significant. encoding hepatitis b surface antigen, 21 indicating that distribution of midge-th1 vectors is independent of the encoded protein as previously reported for plasmid dna vectors. 11 systemic distribution at early time points has also been shown for naked plasmid dna upon i.m. and i.d administration. 24, 25 in accordance with other reports, 11, 26, 27 the highest vector amounts were detected in samples taken from the site of injection. consistently, lymph nodes draining the site of injection contained the second highest copy numbers, probably due to skin immune cells taking up dna at the site of injection and migrating to the lymph nodes. at day 14 after the fourth injection, vectors were cleared from nearly all organs except from skin and inguinal lymph nodes. whereas the amount of vector dna in skin samples was~10-fold lower compared with 24 h after the last injection, vector copy numbers in inguinal lymph node samples were reduced 100-fold. this finding is in line with reports on i.d.-administered plasmid dna vaccines, [27] [28] [29] suggesting that plasmids and midge-th1 vectors ( 21 and this report) follow a similar pattern of distribution and persistence. midge-th1 vector dna was not cleared from skin and inguinal lymph nodes within 60 days after four injections. although this 20 this short time to overall clearance was probably related to wider dispersion of dna upon multiple jet injection, the lower dna concentration (five jet injections of 10 μg dna at 1 mg ml − 1 versus one needle injection of 120 μg dna at 4 mg ml − 1 ), as well as the rapid entry into the blood circuit due to the high vascularization and low retention rate of macromolecules in tumor tissue as compared with normal skin tissue. though only a side-by-side comparison would be conclusive, we propose that these parameters strongly affected the time to overall clearance than the dna dose. in fact, the rats in our study received up to 0.6 μg leishdnavax dna per gram body weight, thus a lower relative dna dose than that administered to mice of the referenced study (~1.6 μg tumor necrosis factor alpha midge dna per gram body weight). 20 a persistence of at least 60 days as observed in our study suggests that vector dna had entered long-living cells residing in the skin. whether or not this vector dna is still functioning cannot be concluded from our results, as the quantitative pcr method targeted a short consensus sequence only. notably, midge-th1 vector dna did not accumulate in any tissue after repeated injections. at 60 days after one injection 21 or after four injections of leishdnavax, comparable amounts of midge-th1 vector dna were found, indicating that repeated injections do not enhance persistence. therefore, future evaluations of biodistribution and persistence of midge-th1 vectors in similar settings can be based on a single injection. long-term persistence of vector dna in tissues can be related to integration into host genomic dna and thus, theoretically bears tumorigenic potential. however, published data show that the integration rate of plasmid dna vectors does not exceed the rate of spontaneous mutation events within the host genome. 26, 27, 30 notably, for linear dna vectors with a structure similar to midge vectors a very low level of integration has been described. 31 furthermore and in contrast to closed circular plasmid dna molecules, integration of covalently closed linear dna constructs rather leads to disruption of chromosomes followed by apoptosis of affected cells, hence minimizing the risk of replicating unwanted genetic rearrangements. 32 on the basis of these published results, the risk related to potential integration of midge-th1 vectors into host genomic dna can be considered as low, though it was not assessed in this work. in a repeated-dose toxicity study in naive mice, we tested three ascending doses of leishdnavax. the highest dose of 100 μg corresponds to a human dose of~300 mg dna on a mg kg − 1 body weight base. doses in published clinical trials ranged from 0.1 mg 33 to 4 mg 34 of plasmid dna, so our study design includes a high safety margin. to further extend the safety margin, the vaccine was administered in a condensed schedule of five vaccinations (one more than the estimated maximum number for clinical application) in weekly intervals. importantly, a no observed adverse effect level, that is, the highest dose without significant toxicity, was not reached with the doses tested. no local intolerance reactions related to the vaccine were observed. the results established in naive mice are corroborated by safety data obtained from the biodistribution study. rats received 120 μg leishdnavax, corresponding to a human dose of~24 mg (based on mg kg − 1 body weight). there were no vaccine-related toxic effects observed either. our results further confirm the findings of other investigators reporting excellent preclinical safety profiles of dna vectors. 10, 11, 20, 26, 27, 30 in leishmania-infected and diseased individuals, tolerability of the vaccine may be different than in healthy, naive vaccinees. moreover, owing to the ability of leishmania amastigotes to exploit host igg as virulence factor, 35 there is the theoretical risk of vaccine-related immune-enhancement of infection and pathogenesis as discussed for other infectious diseases. 36, 37 hence demonstration of absence of disease exacerbation was included into the development program of leishdnavax. we evaluated the tolerability of leishdnavax in two different mouse strains, chosen for their inherent differences in cytokine responses to infection with l. donovani. 38 these experiments demonstrate that leishdnavax had no effect on the kinetics of the parasite burden with no vaccine-related enhancement of infection at any of the time points investigated. the kinetic of infection corresponded to the well-documented pattern in mice with a rapid increase in hepatic parasite burden, followed by a decline in parasite numbers and clearance and a slower increase in parasite numbers in the spleen with the establishment of chronic infections. 23 results for all other standard parameters were similar for treated and nontreated mice, demonstrating a good tolerability of the vaccine candidate in infected animals. in summary, we have shown here that leishdnavax, a novel dna vaccine candidate against leishmaniasis is safe and well tolerated in both naive and leishmania-infected mice. after repeated i.d. injections, the vaccine was rapidly cleared from most organs and tissues of rats and persisted in the skin of injection site and its draining lymph nodes only. we conclude that leishdnavax has a favorable safety profile that supports the initiation of human clinical trials. leishdnavax is an equimass mixture of five midge-th1 vectors, dissolved in sterile pbs. each vector encodes one leishmania antigen: kmp11, cpa, cpb, p74 or tsa, respectively. their dna sequences and expression cassettes of midge-th1 vectors have been described previously. 22 midge-th1 vectors were synthesized in a simple and standardized procedure. 18 forty wistar rats (20 per sex, janvier labs, saint-berthevin, france), 8 weeks of age, were allocated to single-or four-dose study groups with 10 animals each (five per sex). animals received needle injections of 120 μg leishdnavax per injection (~5.3 × 10 13 midge-th1 vector molecules, injection volume 30 μl) i.d. at the dorsal base of tail, either once or four times in weekly intervals. animals injected with a single dose were killed 24 h after administration. four-dose groups were either killed 24 h, 14 days or 60 days after the fourth injection. clinical signs were recorded before, 30 min and 4 h after treatment, thereafter once daily. twenty-four hours after injection, a modified irwin test 39 was performed on the single-dose group and on a non-treated control group to assess acute neurological toxicity of the vaccine. throughout the study, mortality was evaluated once daily and body weight once weekly. at killing, animals were anesthetized deeply by isoflurane inhalation and blood was collected from the plexus ophthalmicus. gross pathology was performed and samples of the following tissues were taken: blood, thigh bone marrow, brain, heart, kidneys, liver (lobus quadratus), lymph nodes (lnn. axillares, inguinales, mesenteriales), lung, thigh muscle, ovaries, skin of injection site, spleen and testes. to avoid cross-contamination with vector dna single-use scalpels were applied for each sample. all other equipment was thoroughly decontaminated after each dissection. samples were immediately frozen in liquid nitrogen and stored at − 80°c until processing. quantitative analyses of dna vector amounts were performed at imgm laboratories (martinsried, germany). total dna was extracted from safety and tolerability of a leishmaniasis dna vaccine o riede et al ~20 mg tissue or up to 280 μl blood, respectively, using the dneasy blood&tissue kit (qiagen, hilden, germany) and stored at − 20°c. a taqman-based quantitative realtime-pcr assay was established and characterized using a standard curve of leishdnavax ranging from 10 vector copies per reaction to 1.05 × 10 11 vector copies per reaction with 200 ng genomic dna from the livers of naive animals as background. measurements were carried out with 200 ng of extracted total dna in 384 well plates with a reaction volume of 20 μl on an ab7900ht instrument (life technologies, carlsbad, ca, usa). primers (eurofins genomics, ebersberg, germany) and hydrolysis mgb probe (life technologies) were designed to detect a consensus sequence present on each midge-th1 vector. the forward primer (5′-gtcgtttagtgaaccgtcagatca-3′) anneals to the cmv promoter region, the hydrolysis mgb probe (5′-fam-tttattgcggtagtttatca-nfq-3′) and the reverse primer (5′-gcacg actgcgttagcaatttaa-3′) anneal to the intron. limit of detection and lower limit of quantification of the assay were determined at 525 copies of midge-th1 vector per reaction (that is, 2625 copies per μg total dna). all samples of extracted dna were measured in triplicates. midge-th1 vector copy numbers were calculated according to the leishdnavax standard curve running in parallel on each plate and expressed as copy number per μg total dna. acceptability criteria of measurements included standard curve coefficient of correlation ⩾ 0.95 and detected copy numbers of no-template-controls below the limit of detection. in addition, the s.d. of the quantification cycle values of at least two replicate measurements had to be below 0.5, otherwise the measurement was repeated. assessment of vaccine toxicity. the study was performed in accordance with glp regulations principles and german animal welfare regulations at lpt laboratory of pharmacology and toxicology, hamburg, germany with prior approval by lpt's institutional animal care and use commissary (study no. 26814) and by the competent authority (behörde für gesundheit und verbraucherschutz, amt für verbraucherschutz lebensmittelsicherheit und veterinärwesen, billstraße 80, 20539 hamburg, germany, v 11307-591-00.33). one hundred and forty balb/c mice (70 male, 70 female) were randomized and allocated to study groups. animals were housed individually. at first treatment mice were 44-49 days (males) or 60-65 days (females) of age with a body weight of 18.2-22.5 g (males) or 18.0-20.4 g (females). ten animals each (five per sex) received one i.d. needle injection (volume: 25 μl) into the dorsal tail base with a dose of 10, 50 or 100 μg leishdnavax or the placebo (pbs). they were killed 24 h post injection for evaluation of acute toxic reactions. thirty animals each receiving placebo and 100 μg dose, or 20 animals each receiving 10 μg and 50 μg dose (injection volume: 25 μl) were injected by needle i.d. into the dorsal tail base five times in weekly intervals. animals were killed after a recovery period of 24 h, 14 days or 61 days (placebo and 100 μg dose group only) following the fifth injection (table 1) . animals were observed for clinical signs including injection site reactogenicity before and 30 min and 4 h after each dosing and daily between injections. mortality was evaluated twice daily throughout the study. body weight of animals was recorded at day of group allocation, on the day of first dosing and three times a week thereafter. food consumption was determined weekly and water consumption was recorded daily. neurological screening 40 and assessment of grip strength 41 was conducted on all animals 24 h after first dosing. ophthalmological and auditory examinations were performed pre-dose, at the end of the treatment period and at the end of the recovery period. for urinalysis, mice were placed in funnel cages in groups of five per sex and received 30 ml tap water per kg body weight by gavage. subsequently, urine was collected for 6 h and the following parameters were measured: volume, weight, ph, specific gravity, osmolality, protein, glucose, bilirubin, urobilinogen, ketones, hemoglobin, nitrite, epithelial cells, leukocytes, erythrocytes, organisms, further constituents, crystalluria. at killing, blood was taken for clinical biochemistry (albumin, globulin, bilirubin, cholesterol, triglycerides, creatinine, glucose, protein, urea, calcium, alanine aminotransferase, alkaline phosphatase, α-amylase, aspartate aminotransferase, creatine kinase, glutamate-dehydrogenase, γ-glutamyl-transferase, lactate dehydrogenase) and hematology (hemoglobin content, erythrocytes, leukocytes, differential blood count, reticulocytes, platelets, mean platelet volume, red cell distribution width, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration). gross pathological examination was conducted, organ weights were taken and samples of the following organs and tissues processed for histological examination: adrenal gland, aorta abdominalis, bone (os femoris with joint), bone marrow, brain, cecum, coagulating gland with seminal vesicle, epididymis, eye with optical nerve and harderian gland, exorbital lacrimal gland, gall bladder, heart, skin and subcutaneous tissue of injection site, large intestine, small intestine, kidney and ureter, liver, lung, lymph nodes (mandibular, mesenteric), mammary gland, muscle, sciatic nerve, esophagus, ovary, pancreas, pituitary gland, prostate, salivary glands, spinal cord, spleen, sternum, stomach, testicle, thymus, thyroid, tongue, trachea, urinary bladder, uterus and vagina. histological examination was performed on samples of all animals with the highest expected burden, that is, after receiving five injections of each 100 μg leishdnavax and 24 h of recovery, and the respective placebo group as control. moreover, the immunologically relevant organs thymus, spleen and lymph nodes as well as the skin at the injection site of all animals of all groups were examined histologically. in general, tissue samples were fixed in 7% buffered formalin (eyes in davidson´s solution), embedded in paraffin, sections prepared and routinely stained with haematoxylin-eosin. samples from mice receiving five times a dose of 10 or 50 μg, respectively, were scheduled for histologically examination only in case of vaccine-related findings in the group receiving five times 100 μg, or if gross pathology revealed vaccine-related changes. as there were no respective findings, these samples were not histologically examined. biologic activity of the vaccine lot and assessment of antibodies against dsdna. in all, 100 balb/c mice (50 male, 50 female) were randomized and allocated to satellite study groups of the toxicity study resembling the repeated-dose treatment schedule (table 1) . at first treatment, animals were 44-49 days (males) or 60-65 days (females) of age with a body weight of 17-19.3 g (males) or 17-18.4 (females) . at study termination, blood samples were taken and serum obtained for enzyme-linked immunosorbent assay to test for antibodies against vaccine antigens and for radioimmunoassay to test for antibodies against dsdna. these analyses were performed under non-glp regulations conditions. leishdnavax-specific antibodies were detected performing an enzyme-linked immunosorbent assay as previously described 22 with minor modifications. plates (nunc maxisorp, thermo scientific, roskilde, denmark) were coated per well with 100 μl of 5 μg ml − 1 antigen-mix (kmp11, cpa, cpb, p74, tsa 1:1:1:1:1) in pbs (fisher scientific, paisley, uk). recombinant proteins were obtained from proteogenix, oberhausbergen, france (cpa, cpb, p74, tsa) or were kindly provided by professor c jaffe (kmp11). antigen mix was assembled at lpt. plates were incubated overnight at 4°c, then washed three times with wash buffer (0.05% (v/v) tween in pbs) and subsequently blocked with assay diluent (5% (w/v) bovine serum albumin (sigma aldrich, st louis, usa) in pbs) for 1 h at room temperature. plates were washed three times with wash buffer and 100 μl per well of 1:50 diluted serum samples were added. the plates were incubated for 2 h at room temperature and subsequently washed five times with wash buffer. hundred micolitre per well of anti-mouse igg-hrp (conc.: 11.3 mg ml − 1 ), (sigma aldrich) were added at a 1:5000 dilution and plates were incubated for 2 h at room temperature. after washing five times developer solution containing 3,3',5,5'-tetramethylbenzidine (sigma aldrich) was added and the reaction stopped by adding acid solution when the color developed. absorbance was measured at 450 nm. to assess the induction of an immune response against dsdna a radioimmunoassay was performed at ibl international gmbh (hamburg, germany) using a kit for detection of anti-dsdna antibodies (ibl international). in brief, 25 μl of serum sample was mixed with 150 μl of 125 i-labeled dsdna tracer and incubated at 37°c for 60 min. subsequently, 1 ml of cold ammonium sulfate solution was added to the sample followed by vortexing. tubes were centrifuged (15 min at 1500 g) and supernatant was removed. radioactivity was counted using a gamma counter and the dsdna binding capacity was calculated according to a standard curve. under specific pathogen-free conditions in individually ventilated cages and exposed to 12 h light-12 h dark cycles. standard rodent diet (rm no 1 expanded) and de-ionized water were supplied ad libitum. l. donovani (strain mhom/et/67/hu3) was maintained in rag-1 (b6) ko mice and amastigotes harvested from spleens of infected animals 440 days after infection. infection and treatment schedule. female balb/c and c57bl/6 mice (6-8 weeks of age at the start of experiments) were infected by injecting 2 × 10 7 freshly harvested l. donovani amastigotes intravenously into a tail vein as described. 42 following infection, animals were sorted into groups of three to five mice per cage and two cages assigned to each treatment group. groups of mice received either one, two or three injections of leishdnavax containing 20 μg dna/antigen (corresponding to 100 μg total dna) or pbs, administered in volumes of 25 μl i.d. at the base of the tail using 29g single-use needles (bd microfine plus insulin syringes). the first dose was administered 7 days after infection and repeat doses in 7-day intervals. groups of mice were killed 7 days after having received the last dose of leishdnavax or pbs. in a further experiment in c57bl/6 mice additional groups were killed 29 days after the last of a total of three doses of leishdnavax or pbs. determination of efficacy and tolerability parameters. mouse weights were recorded prior to the first dose of treatments administered and in weekly intervals thereafter. the injection site was monitored following administration of treatments and animals were observed daily by trained staff for the whole duration of the experiments. mice were humanely killed by exsanguination under terminal anesthesia and blood collected by cardiac puncture. livers and spleens were removed and their weight recorded. tissue impression smears were prepared, fixed in 100% methanol and stained in 10% giemsa. parasite burden was determined microscopically and leishman-donovan units calculated by the formula number of parasites per host cell nucleus × organ weight in mg, as described previously. 43 serum was harvested from blood stored overnight at 4°c by centrifugation at 1500 g, 4°c for 15 min and stored at − 80°c. biochemical analysis of standard serum parameters was carried out by laboklin gmbh&co.kg (bad kissingen, germany). for histology, organs were fixed in 10% neutral buffered formalin, embedded in paraffin and routinely stained with hematoxylin and eosin. histological data were evaluated in blinded fashion. data from the repeated-dose toxicity study in naive mice were analyzed using student's t-test for numerical functional tests (p ⩽ 0.01), dunnet's multiple t-test for body weight, food consumption, hematology, clinical biochemistry and organ weights (p ⩽ 0.01) and fisher's exact test for histology (p ⩽ 0.05), respectively. toxicity data of the biodistribution study were evaluated as follows: parameters of the irwin test were analyzed by mann-whitney u-test (p ⩽ 0.05) and normality of body weight data by shapiro-wilks test. in case of normality, an analysis of variance was performed with post hoc dunnett's test for multiple comparisons; otherwise, a non-parametric kruskal-wallis test with post hoc dunnett's test was employed (p ⩽ 0.05). igg levels were statistically analyzed using graphpad prism 5 (graphpad software inc., la jolla, usa). normality of data was tested with shapiro-wilk test prior to applying either two-tailed student's t-test or mann-whitney test to analyze differences between means of two groups (p ⩽ 0.05). to evaluate tolerability of the vaccine in infected mice, comparisons between three or more groups were made by one-way analysis of variance followed by bonferroni's multiple comparison's test with comparison between pbs and vaccine treated groups. comparisons between two groups were made by an unpaired t-test assuming gaussian distribution (graphpad prism 6). a p-value of o0.05 was considered statistically significant. or, do, ae and cj are employees and ms is chairman of the executive board of mologen ag. ch is employee of imgm laboratories gmbh. aw is employee of lpt gmbh & co. kg. mologen ag owns a patent for the midge-th1 vector (pct/ de02/03798p74). these affiliations do not alter the authors' adherence to gene therapy policies on sharing data and materials. fjs declares no conflict of interest. leishmaniasis worldwide and global estimates of its incidence report of a meeting of the who expert committee on the control of leishmaniases case study for a vaccine against leishmaniasis leishmaniasis: complexity at the host-pathogen interface heterologous protection against influenza by injection of dna encoding a viral protein induction of antigen-specific cytotoxic t lymphocytes in humans by a malaria dna vaccine dna vaccines: a rational design against parasitic diseases us food and drug administration. guidance for industry. considerations for plasmid dna vaccines for infectious disease indications who annex 1: guidelines for assuring the quality and non-clinical safety evaluation of dna vaccines toxicological safety evaluation of dna plasmid vaccines against hiv-1, ebola, severe acute respiratory syndrome, or west nile virus is similar despite differing plasmid backbones or gene-inserts biodistribution of dna plasmid vaccines against hiv-1, ebola, severe acute respiratory syndrome, or west nile virus is similar, without integration, despite differing plasmid backbones or gene inserts clinical applications of dna vaccines: current progress cpmp/bwp/3088/99: note for guidance on the quality, preclinical and clinical aspects of gene transfer medicinal products marker-free plasmids for biotechnological applicationsimplications and perspectives dna immunisation with minimalistic expression constructs silencing of episomal transgene expression by plasmid bacterial dna elements in vivo minicircle dna is superior to plasmid dna in eliciting antigen-specific cd8+ t-cell responses priming of immune responses to hepatitis b surface antigen with minimal dna expression constructs modified with a nuclear localization signal peptide effect of different nuclear localization sequences on the immune responses induced by a midge vector encoding bovine herpesvirus-1 glycoprotein d intratumoral dispersion, retention, systemic biodistribution, and clearance of a small-size tumor necrosis factor-alpha-expressing midge vector after nonviral in vivo jetinjection gene transfer combination of midge-th1 dna vaccines with the cationic lipid saint-18: studies on formulation, biodistribution and vector clearance modular multiantigen t cell epitope-enriched dna vaccine against human leishmaniasis differential regulation of the immune response in the spleen and liver of mice infected with leishmania donovani safety of a gm-csf adjuvant-plasmid dna malaria vaccine safety of interleukin-12 gene therapy against cancer: a murine biodistribution and toxicity study biodistribution, persistence and lack of integration of a multigene hiv vaccine delivered by needle-free intradermal injection and electroporation nonclinical biodistribution, integration, and toxicology evaluations of an h5n1 pandemic influenza plasmid dna vaccine formulated with vaxfectin(r) biodistribution and general safety of a naked dna plasmid, gtu-multihiv, in a rat, using a quantitative pcr method poloxamer-formulated plasmid dna-based human cytomegalovirus vaccine: evaluation of plasmid dna biodistribution/persistence and integration immunogenicity, safety, biodistribution and persistence of advax, a prophylactic dna vaccine for hiv-1, delivered by in vivo electroporation construction and characterization of an in-vivo linear covalently closed dna vector production system dna ministrings: highly safe and effective gene delivery vectors phase 1 clinical trials of the safety and immunogenicity of adjuvanted plasmid dna vaccines encoding influenza a virus h5 hemagglutinin phase 1 safety and immunogenicity evaluation of advax, a multigenic, dna-based clade c/b' hiv-1 candidate vaccine intrinsic antibodydependent enhancement of microbial infection in macrophages: disease regulation by immune complexes after a tick bite in a tick-borne encephalitis virus endemic area: current positions about post-exposure treatment traitors of the immune system-enhancing antibodies in hiv infection: their possible implication in hiv vaccine development the capacity to produce ifn-gamma rather than the presence of interleukin-4 determines the resistance and the degree of susceptibility to leishmania donovani infection in mice comprehensive observational assessment: ia. a systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse a neuromuscular screen for use in industrial toxicology a method for the routine assessment of fore-and hindlimb grip strength of rats and mice n-(2-hydroxypropyl)methacrylamide-amphotericin b (hpma-amb) copolymer conjugates as antileishmanial agents regulation of leishmania populations within the host. i. the variable course of leishmania donovani infections in mice the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material we thank the leishdnavax consortium for helpful discussions, mologen ag's production and qc department for manufacturing the vaccine, shantanabha das for providing the leishmania antigen elisa protocol and charles jaffe for providing recombinant kmp11. this work was funded by the european commission as part of the 7th framework programme (#223189). key: cord-000988-79fp75u3 authors: al-siyabi, turkiya; binkhamis, khalifa; wilcox, melanie; wong, sallene; pabbaraju, kanti; tellier, raymond; hatchette, todd f; leblanc, jason j title: a cost effective real-time pcr for the detection of adenovirus from viral swabs date: 2013-06-07 journal: virol j doi: 10.1186/1743-422x-10-184 sha: doc_id: 988 cord_uid: 79fp75u3 compared to traditional testing strategies, nucleic acid amplification tests such as real-time pcr offer many advantages for the detection of human adenoviruses. however, commercial assays are expensive and cost prohibitive for many clinical laboratories. to overcome fiscal challenges, a cost effective strategy was developed using a combination of homogenization and heat treatment with an “in-house” real-time pcr. in 196 swabs submitted for adenovirus detection, this crude extraction method showed performance characteristics equivalent to viral dna obtained from a commercial nucleic acid extraction. in addition, the in-house real-time pcr outperformed traditional testing strategies using virus culture, with sensitivities of 100% and 69.2%, respectively. overall, the combination of homogenization and heat treatment with a sensitive in-house real-time pcr provides accurate results at a cost comparable to viral culture. human adenoviruses (hadv) are ubiquitous dna viruses that cause a wide spectrum of illness [1] . the majority of hadvs cause mild and self-limiting respiratory tract infections, gastroenteritis or conjunctivitis; however, more severe disease can occur such as kerato-conjunctivitis, pneumonitis, and disseminated disease in the immunodeficient host [2] [3] [4] [5] . hadv is increasingly being recognized as a significant viral pathogen, particularly in immunocompromized patients where accurate and timely diagnosis can play an integral part of management [6] [7] [8] [9] [10] [11] [12] . hadv diagnosis can be achieved using virus culture, antigen-based methods (immunofluorescence, enzyme immunoassays or immunochromatography), or nucleic acid amplification tests (naats). for respiratory viruses, naats are well established as the most sensitive methods for detection and have become front-line diagnostic procedures [7, [13] [14] [15] [16] [17] [18] . most commercially available naats are highly multiplexed assays and enable simultaneous detection of several respiratory pathogens; however, their poor performance for detecting hadv emphasizes the need for single target detection [15, 17, 19] . adenovirusspecific naats have been challenged by the diversity of hadv species, which now include more than 60 different types [20, 21] . commercial qualitative and quantitative naats are available for the detection of all hadv species and most types, yet these are cost prohibitive for many laboratories. "in-house" real-time pcr assays are relatively inexpensive alternatives to commercial naats that provide rapid and accurate results [7, 17, 18, [21] [22] [23] [24] [25] [26] . wong and collaborators [18] developed an in-house real-time pcr assay that has been designed for the detection of all hadv species. it has been extensively validated using a variety of clinical specimens [17, 18] . in addition to the pcr reaction itself, extraction of nucleic acids prior to pcr is also a substantial contributor to cost. recently, a crude mechanical lysis using silica glass beads (i.e. homogenization) and heat treatment was shown to recover herpes simplex virus dna from swabs submitted in universal transport media (utm) [27, 28] . while defying the traditional paradigm of specimen processing for molecular testing, homogenization with heat treatment was shown to be a cost effective alternative to nucleic acid extraction. this study evaluated whether the combination of homogenization and heat treatment with an in-house real-time pcr would be a cost effective strategy for the detection of hadv from viral swabs transported in utm. in patients suspected of respiratory or conjunctivitis, flocked nasopharyngeal or ocular swabs, respectively, were submitted for adenovirus detection. swabs were collected by clinicians at the capital health district authority (cdha) and were submitted to the cdha microbiology laboratory (halifax, ns, canada) between april 2010 and march 2012. the swabs were transported in 3 ml of utm (copan diagnostics inc., murrieta, ca) and kept at 4°c for no more than 24 hours prior to processing. viral cultures were performed as part of routine diagnostic testing by experienced technologists. following virus culture, specimens were transferred in aliquots into cryotubes (without any identifiable patient information) and the anonymized specimen tubes were archived at −80°c for retrospective molecular analyses. twentyseven virus culture-positive specimens and 169 virus culture-negative specimens were randomly selected and tested for the presence of hadv using a well established in-house real-time pcr assay [18] following recovery of viral dna was recovered by homogenization with heat treatment or automated nucleic acid extraction. the world medical association (wma) declaration of helsinki is a statement of ethical principles for medical research involving human subjects, including research on identifiable human material and data. since the purpose of this clinical validation was quality improvement of the laboratory detection of adenovirus and relied exclusively on anonymous human biological materials that did not use or generate identifiable patient information, research ethics board (reb) review was not required based on chapter 2, article 2.4 of the tri-council policy statement: ethical conduct for research involving humans (2nd edition). viral cultures were performed as part of routine diagnostic testing by experienced technologists in the cdha microbiology laboratory (halifax, ns, canada). briefly, 500 μl of specimen was inoculated onto cultured a549 cells (atcc ccl-185), incubated at 37°c in a 5% co 2 atmosphere, and monitored daily for the presence of characteristic cytopathic effect (cpe) [29] . if cpe was observed, cells were fixed with acetone and stained using specific fluorescein isothiocyanate (fitc)-labeled monoclonal antibodies in the d 2 ultra dfa reagent kit (diagnostic hybrids, athens, oh). in absence of cpe, cells were fluid changed on day 7 and incubated for an additional 7 days. on day 14, the culture was discontinued and a terminal stain was performed. a549 cells were propagated in nutrient mixture f-12 ham with lglutamine (sigma-aldrich canada ltd., oakville, on) supplemented with 1% fetal calf serum (hyclone, thermo fisher scientific, ottawa, on), 2 μg/ml amphotericin b (sigma-aldrich), 25 μg/ml ampicillin (novapharm ltd, toronto, on), and 1 mg/ml vancomycin (sigma-aldrich). for quantification, 10-fold dilutions of hadv-c, type 6 (strain tonsil 99, atcc vr-6) were inoculated onto 96-well plates in volumes of 100 μl. cells were maintained as described above and after 14 days were subjected to direct immunofluorescence (dfa) to determine the 50% tissue culture infective dose (tcid 50 ). results were expressed as tcid 50 /ml and represent 8 replicates obtained in four independent experiments (n = 32). prior to molecular testing, viral dna was recovered from specimens using either homogenization with heat treatment as previously described [27] , or using a commercial nucleic acid extraction as recommended by the manufacturer. for homogenization, 500 μl of specimen and 0.5 g of various sized acid-washed silica beads: ≤106 μm; 150-212 μm; 719-1180 μm at a ratio of 3:2:1 (sigma-aldrich, oakville, on) were placed on a fastprep-24 homogenizer (mp biomedicals, solon, oh) at 6.5 m/s for 45 s. following a brief centrifugation at 14,000 × g for 1 min, 200 μl of the supernatant was diluted in two volumes of te buffer (10 mm tris-hcl, 1 mm edta, ph 8.0). the homogenate was then heated at 95°c for 15 min, cooled to room temperature, and 5 μl was subjected to adenovirus real-time pcr. automated extractions were performed on 200 μl of specimen using a magna pure total nucleic acid isolation kit (roche diagnostics, mannheim, germany) on a roche magnapure lc instrument. the elution volume was 100 μl. specimens with discordant results during method comparison were subjected to a manual dna extraction using a qiaamp dna blood mini kit (qiagen, toronto, on) with a sample volume of 200 μl. the dna was eluted in 100 μl, and concentrated 10-fold using a qiagen minelute pcr purification kit. plasmid dna, used for the internal control, was purified from a 5 ml overnight culture using a qiaprep spin miniprep kit (qiagen) as recommended by the manufacturer. for molecular typing, amplicon was purified using a qiaquick gel extraction kit (qiagen) with a final elution volume of 50 μl. all nucleic acid extractions were performed using manufacturers' instructions. nucleic acids were used immediately following extraction and aliquots were placed at −80°c for longterm storage. the real-time pcr has been extensively validated using respiratory specimens [18] . to facilitate workflow in the cdha microbiology laboratory (halifax, nova scotia, canada), the in-house assay was optimized for amplification and detection on a roche lightcycler 2.0 platform. real-time pcr was performed as duplex reactions with primers and probes (table 1 ) targeting the adenovirus hexon gene and an exogenous internal control. for adenovirus, two sets of primers and probes were used to span the genetically diverse adenovirus types [17, 18] . primers were synthesized by sigma genosys (oakville, on). probes for adenovirus and the internal control were purchased from biosearch technologies (novato, ca) and tib molbiol llc (adelphia, nj), respectively. the internal control, termed pgfp, is added to each reaction to monitor for the presence of pcr inhibitors. pgfp is a pmk-derived plasmid with a fragment of the gene encoding green fluorescence protein (gfp). the construct was synthesized, assembled, and transformed into escherichia coli k12 by life technologies (burlington, on). the final construct was verified by dna sequencing and restriction endonuclease digestion. e. coli harboring pgfp was inoculated into luria bertani broth supplemented with 50 μg/ml kanamycin. plasmid dna was purified from a 5 ml overnight culture and plasmid dna was quantified by spectrophotometry. ten-fold serial dilutions were used as template for the in-house realtime pcr. an inverse linear relationship (y = −3.3916× + 40.275; r 2 = 0.9982) was generated by plotting crossing points (cp) values against plasmid concentration (data not shown). the linear range spanned cp values ranging from 7 to 37, corresponding to concentrations of 10 0 to 10 9 copies per μl, respectively. for each pcr reaction, approximately 2000 copies were added. real-time pcr assay was performed using the light-cycler dna master hybprobe kit (roche diagnostics) in 20 μl reactions consisting of: 5 μl of template, 1 × lightcycler faststart mix, 3 mm mgcl 2 ; 0.5 units of heat-labile uracil-n-glycosylase [30] ; 5 μl the internal control at 400 copies/μl; 400 nm of each adenovirus primer (adv2f, adv2r, adv4f, adv4r) and 200 nm of probe (adv2pr and adv4pr); and 500 nm of each pgfp primer (fgfp and rgfp) and 300 nm of each probe (gfppr1 and gfppr2) ( table 1 ). amplification and detection were performed using the lightcyler 2.0 instrument under the thermocycling conditions described for the roche hsv-1/2 detection kit: initial activation at 95°c for 10 min, followed by 45 amplification cycles of denaturation at 95°c for 10 s, annealing at 55°c for 15 s, and elongation at 72°c for 15 s. following amplification, melting temperature (tm) analysis was performed by measuring the fluorescent signal during the following cycling profile: 95°c for 0 s, 40°c for 60 s, and 80°c for 0 s with a 0.2°c/s transition. fluorescence was acquired at the annealing stage during amplification and continuously during the melting curve. cp and tm values were determined using software provided by the manufacturer. the 530 nm (adenovirus) and 705 nm (pgfp) channels were analyzed for presence or absence of target. pcr inhibition was suspected by either loss of positivity in the 705 nm channel, or a shift in cp values greater than two standard deviations (cp ≥ 1.0) from the value obtained with the negative control. to resolve discrepant results obtained between the inhouse pcr assay and virus culture, or quantify the adenovirus dna during evaluation of the analytical sensitivity, the adenovirus r-gene kit (argene inc., sherley, ny) was used according to the manufacturer's protocol following a manual dna extraction. this internally controlled quantitative real-time pcr assay targets the hexon gene of adenovirus, and is validated for detection table 1 nucleotide sequences of primers and probes used in this study sequence (5′ to 3′) reference cca gga cgc ctc gga gta [18] adv2r aaa ctt gtt att cag gct gaa gta cgt [18] adv2pr fam-agt ttg ccc gcg cca cca ccg -bhq1 * [18] adv4f gga cag gac gct tcg gag ta [18] adv4r ctt gtt ccc cag act gaa gta ggt [18] adv4pr fam-cag ttc gcc cgy gcm aca g -bhq1 * [ of types 1 to 52 [7] . the kit contains: a ready-to-use premix contains (primers, probe, polymerase, and buffer) needed for amplification, 4 quantification standards (at 50, 500, 5,000, and 50,000 copies/reaction), and a sensitivity-control at 10 copies/reaction. results were expressed as the number of copies per reaction. analytical specificity, limit of detection, and reproducibility the analytical specificity was first determined in silico by performing a basic local alignment search tool (blast) for primers, probes, and entire amplicon sequences using the national center for biotechnology information website (http://www.ncbi.nlm.nih.gov). in addition, high titer nucleic acids were extracted from a panel of microorganisms chosen based on their ability to cause similar diseases or their potential for being found in the clinical specimen as a pathogen or normal flora ( (figure 1 and table 2 ). the analytical sensitivity (or limit of detection, lod) of the homogenization with heat treatment or nucleic acid extraction, in combination with the real-time pcr, was determined using 10-fold serial dilutions (in utm) of a cultured hadv-c type 6. each dilution was simultaneously processed by both extraction methods, and an aliquot immediately inoculated onto a549 cells for virus culture. the lod was defined by probit analysis [31] using triplicate values obtained in four independent experiments by two different operators (n = 24). each virus dilution was expressed as tcid 50 /ml in the original sample. the virus dilutions were also quantified using a commercial real-time pcr and expressed as target copies/ reaction for each assay. intra-and inter-assay reproducibility were calculated for each dilution and expressed as % coefficients of variation (%cv). the performance of each method was compared to a modified gold standard to determine sensitivity, specificity, accuracy and precision. a case was defined by concordant results (positive or negative) between at least two assays. to resolve discrepant results obtained between the in-house real-time pcr assay and virus culture, dna was extracted manually and was subjected to commercial real-time pcr. the 27 virus culture-positive specimens were subjected to pcr targeting the conserved segments surrounding the hypervariable region 7 (hvr7) of the hexon gene terminator chemistry on the applied biosystems 3130 × l dna sequencer. type designation was undertaken by blast analysis, and confirmed by comparison to a database generated from sequences obtained from genbank [32] . sequence analysis and multiple sequence alignments (clustalw analysis) were performed using the seqman and megalign components of lasergene 6 software (dnastar, madison, wi). the phylogenetic tree was inferred using a neighbor-joining (nj) method with bootstrapping analysis for n = 1000. chi-square and two-tailed fisher's exact tests were used to compare proportions in 2-by-2 contingency tables. confidence intervals (99%) for the estimated parameters are computed by a general method based on "constant chisquare boundaries" [33] . agreement between assays was measured using kappa statistics. the statistical package for social sciences (spss) software v.10 was used and p ≤ 0.01 was used to denote a statistically significance. clades are shaded to depict species a to f. analytical specificity, limit of detection, and reproducibility blast searches of primers and probes targeting the adenovirus hexon gene the internal control sequences revealed that these were highly specific targets. in fact, no cross reactions were observed with high-titer nucleic acids extracted from other respiratory viruses or bacteria ( table 2 ). the in-house real-time assay was able to detect serogroups a to f, including a variety of genetically diverse types: 1, 2, 3, 4, 6, 7, 10, 20, 26, 31, and 40 ( figure 1 and table 2 ). as seen in figure 2 , the performance of the inhouse pcr following the homogenization-or nucleic acid extraction-based protocols was equivalent. for each method, overlapping linear relationships were observed (y = −3.7668 × + 44.733; r 2 = 0.9987 compared to y = −3.9058 + 45.313; r 2 = 0.9985, respectively) that spanned eight orders of magnitude with cp values ranging from 14 to 40 (figure 2a ). the intra-and inter-assay reproducibility of the real-time pcr following homogenization and heat treatment ranged from 0.03 to 4.80%, and 1.45 to 3.79%, respectively. similarly, intra-and inter-assay reproducibility of following the nucleic acid extraction protocol ranged from 0.2 to 2.15% and 0.85 to 3.15%. as expected, the highest %cv values observed for both methods were with virus dilutions near the lod. for hadv-c type 6, the lod for virus culture was 0.2 tcid 50 /ml. the in-house real-time pcr was reproducibly positive following nucleic acid extraction or homogenization with viral stock dilutions corresponding to 0.02 tcid 50 /ml (24/24 and 24/24, respectively), and positive pcr reactions were frequently observed using virus dilutions of 0.002 tcid 50 /ml (20/24 and 21/24, respectively). virus stock dilutions were quantified using commercial real-time pcr assay, and the lod for homogenization or nucleic acid extraction-based protocols were shown to be approximately equivalent (figure 2) . with a probability of 95%, the lod for the homogenization-and nucleic acid extraction-based protocols were 12 copies/reaction (log 10 = 1.08) and 18 copies/reaction (log 10 = 1.08), respectively ( figure 2b) . dilutions corresponding to the lod for virus culture were also quantified by real-time pcr and estimated at approximately 380 copies/reaction ( figure 2b ). of the 196 clinical specimens, 157 concordant negative and 27 concordant positive results were obtained when comparing virus culture to the in-house pcr following either of the two extraction methods ( figure 3a and table 3 ). real-time pcr generated 12 additional positive results that were later resolved as true positives using a manual dna extraction and a commercial real-time pcr ( figure 3a ). all 12 pcr-positive culture-negative results were detected following homogenization protocol, whereas 11 were detected following nucleic acid extraction ( figure 3a) . the single discordant result between the molecular assays had a cp value of 37.22, suggesting that it may be attributed to sampling error (poisson distribution) at low concentrations of template [34] . since the internal control also failed to amplify in this sample, the negative result could also be attributed to pcr inhibition. upon repeat processing by automated and manual nucleic acid extractions, positive results were obtained. therefore, the original specimen result was considered a false negative. overall, compared to the modified gold standard, the sensitivity of the inhouse real-time pcr following homogenization with heat treatment or nucleic acid extraction was approximately equivalent at 100% (89.7-100%) and 97.4% (86.4-97.4%), respectively (table 3 ). in contrast, the sensitivity of virus culture was only 69.7% (56.0-69.2%) ( table 3 ). the accuracy of each method was 100% (95.6-100%), 99.5% (95.1-99.5%), and 93.9% (88.6-93.9%), respectively ( table 3) . all assays showed a high degree of specificity and precision (table 3) . when comparing cp values for the positive results obtained with the real-time pcr following both extraction methods, a linear relationship was observed (y = 0.9416 × + 4.5731; r 2 = 0.9756) ( figure 3b ). cp values for homogenization with heat treatment were consistently higher than those obtained using the nucleic acid extraction; however, no significant differences in sensitivity (analytical or clinical) were observed (figure 2 and table 3 ). as expected, virus culture-positive specimens had positive pcr results with low cp values, whereas the virus culture-negative specimens had pcr-positive results with cp values greater than 30 ( figure 3b ). dna extracted from the 39 real-time pcr positive specimens were subjected to a conventional pcr targeting the conserved segments surrounding the hvr7 of the hexon gene [32] . successful sequences were obtained from dna extracted from the 27 specimens that were both virus culture and real-time pcr-positive. a type could be assigned using multiple sequence alignment of sequences derived from genbank, as previously described [32] . individual blast analysis yielded similar results. three serogroups were observed: b (types 3, 7, 14, and 34), c (types 2 and 6), and d (types 8, 10, and 29). the predominant types observed were: 3 (37.0%), 29 (18.5%), 2 (14.8%), and 8 (1.1%). the conventional pcr was unable to amplify the target sequences from dna extracted from the 12 virus culture-negative/real-time pcr-positive specimens. the cp values for these specimens ranged from 30 to 40, suggesting only low quantities of virus were present (figure 3 ). dna sequencing was also used to distinguish the prototypic hadv type 14p (strain de wit) from newly emerged type 14p1. adenovirus type 14p1 has been associated with severe disease in europe and the north america [2] [3] [4] [5] . while the hexon hvr7 sequences obtained in this study share 100% identity with hadv type 14p1, only two mutations (g1341a and g1491a) separate types 14p1 from 14p in this region. to further characterize the a b figure 2 analytical sensitivity of the in-house real-time pcr. prior to amplification, 10-fold serial dilutions of hadv-c type 6 were processed by homogenization and heat treatment (open circles, solid line), or nucleic acid extraction (filled squares, dashed line). in both cases, equivalent results were obtained in respect to: a) the linear range; and b) the lod determined by probit analysis (n = 24). at a probability of 95%, the lod for the homogenization-and nucleic acid extraction-based protocols were 12 copies/reaction (log 10 = 1.08) and 18 copies/reaction (log 10 = 1.08), respectively. the same dilutions used for inoculate virus culture and dfa staining (indicated by open triangles, dotted line) were also quantified and demonstrated a lod of approximately 380 copies/ml (log 10 = 2.58). virus, the fibre knob gene was sequenced with primer pair f14mut and r14mut (table 1) , using reaction conditions, thermocycling parameters, and dna sequencing as described for the molecular typing. compared to wild-type 14p, the fiber knob gene of hadv type 14p1 displays a 6-bp deletion (referred to as the k250-e251 deletion) [4, 35, 36] . the adenovirus type 14 from this study harbored the characteristic 6-bp deletion, consistent with hadv type 14p1 (figure 4) . an exogenous internal control was used in this study which is non-competitive (contains a primer pair that does not target adenovirus). the addition of the internal control and primers and probes to the in-house pcr reaction did affect the analytical sensitivity of the assay (data not shown). since the internal control was added at the level of pcr, both extraction methods could be directly evaluated for the presence of pcr inhibitors. despite a subsequent heat treatment and dilution step, homogenization is a crude method to recover viral dna and may not sufficient to remove or inactivate pcr inhibitors. amplification of the internal control in adenovirusnegative specimens is consistent with a true negative result and not simply attributed to pcr inhibition. pcr inhibition was suspected by either loss of positivity in the 705 nm channel, or a shift in cp values greater than two standard deviations (which corresponds to approximately ±1.0 cp) from the value obtained with the negative control. this value was established previously, where the internal control cp values from 150 consecutive hsv-negative specimens were compared by homogenization and heat treatment or nucleic acid extraction [27] . this cutoff value remains true for the internal control used in this study. since the in-house pcr was performed as a duplex with an internal control added at the level of pcr, the 196 clinical specimens processed following homogenization and heat treatment or nucleic acid extraction could be monitored directly for the presence of potential pcr inhibitors. potential inhibitory substances were observed in two distinct cases: the first was a specimen that had been processed by homogenization with heat treatment, and the second, in a specimen subjected to nucleic acid extraction. in both cases, pcr inhibition was not observed upon repeat processing, suggesting either a processing error had occurred or the pcr inhibitor was labile [37] . therefore, pcr inhibition could not be proven or excluded. as a result, the rate of possible pcr inhibition with either extraction method was equivalent at 0.51% (1/196). at cdha (halifax, ns, canada), the average number of specimens submitted yearly for adenovirus testing is 312 (range 208 to 466 for years 2009 to 2012) and the turnaround time for virus culture can be up to 14 days. a cost analysis was performed that assumed a more practical approach of bi-weekly molecular testing (3-5 specimens with positive, negative and reagent controls). excluding labor, the average cost of a commercial pcr following nucleic acid extraction would range from $45 to $55 (cad) per specimen. in comparison, the inhouse real-time pcr following a nucleic acid extraction would reduce the cost approximately~2-fold ($21.44 to $25.97). replacement of the nucleic acid extraction with the homogenization-base protocol further reduces the cost~2-fold ($8.84 to $10.97), which is comparable to the average cost of virus culture ($9.47 to 11.64). the time require for bi-weekly processing for either molecular methods is~5 h/week, which is far lower than the time required for weekly maintenance and processing of specimens using cell culture and dfa staining. naats like real-time pcr have revolutionized the detection of human pathogens in clinical microbiology laboratories. rapid specimen throughput and excellent performance characteristics make them an appealing alternative to traditional culture methods; however, cost limits their use in many clinical laboratories. both the recovery of nucleic acids using extraction and the pcr reaction itself contribute to the cost. we have shown that combining a crude extraction method like homogenization with heat treatment [27] and an in-house real-time pcr [18] is a cost effective strategy for the detection hadv from swabs submitted in utm. homogenization uses multidirectional motion to disrupt cells through contact with silica beads and the heat treatment [27, 28] . in combination with a subsequent heat treatment to inactivate heat-labile pcr inhibitors, this crude mechanical lysis had been shown to be a cost-effective method to recover viral dna from swabs transported in utm [27] . the performance characteristics of this approach were equivalent to using traditional nucleic acid extraction and both molecular methods far exceeded those obtained with virus culture. replacing the nucleic acid extraction with the homogenization protocol did not affect the analytical (or clinical) sensitivity of the real-time pcr (figure 2 and table 3 ). using dilutions of hadv-c type 6, the lod for the homogenization protocol was approximately 12 copies/reaction, was consistent with previously reported values (22-33 copies/reaction) for hadv types 2 and 4 [18] . this analytical sensitivity is approximately 32-fold more sensitive than the estimated lod for virus culture. furthermore, positive results could be even obtained at 6 copies/reaction with a probability of 87.5% ( figure 2b ). while no significant differences were observed between the molecular assays, both demonstrated a high level of analytical sensitivity. when comparing 196 clinical specimens using a modified gold standard, the in-house pcr following homogenization and heat treatment or nucleic acid extraction demonstrated similar sensitivities of 100% and 97.4%, respectively (table 3 ). this far surpasses the performance of virus culture at 69.2%. the 30% increase in positivity is consistent with the~32-fold increase in analytical sensitivity and is not surprising since similar results were observed when transitioning other viruses from culture to naats [38] [39] [40] [41] . when comparing positive results from the in-house real-time pcr, cp values obtained following the homogenization protocol were consistently higher than those obtained following nucleic acid extraction ( figure 3b ). however, the analytical and clinical sensitivities of each assay were not significantly different ( figure 2 and table 3 ). it should be noted that all virus culture-negative/pcr-positive specimens had cp values greater than 30, corresponding to viral loads that fell below the lod for virus culture ( figure 3b ). the homogenization-or nucleic acid extraction-based protocols both showed excellent analytical specificity, with no cross-reactions from other organisms ( table 2) . both methods were able to detect diverse hadv types spanning all the different species (figure 1 and table 2 ). of the virus culture-positive specimens, the most predominant types detected were 3, 29 and 2, belonging to species b, d and c, respectively. these hadv types are well-recognized causes of acute respiratory tract and ocular infections and are consistent with the distribution reported by others regions in canada [42, 43] . interestingly, a variant of hadv type 14, termed 14p1, has been described as an emerging pathogen associated with outbreaks and sporadic cases of acute respiratory disease in europe and the united states [2] [3] [4] [5] . while most recorded cases were mild infections, severe disease and deaths have occurred. hadv type 14p1 has a characteristic 6-bp deletion (k250-e251) in the fiber knob gene [4, 35, 36] . the adenovirus type 14 from this study was consistent with type 14p1 and harbored these mutations ( figure 4 ). while there has been a number of reports of type 14p1 circulating in the us and europe, this variant has only once been reported in canada [4] . the first adenovirus 14p1 cases in canada were reported from nova scotia's neighboring province, new brunswick, and included one fatality (figure 4 ) [4] . the specimen identified as 14p1 in this study was obtained from a fatal case dating back to same time period as the new brunswick cases. further epidemiological investigations are underway. while severe and fatal cases associated with type 14p1 have been reported, similar outcomes have been reported with many other common hadv types [6, 7, 10, 44] . the most likely culprit of disease severity is the immune status of the host, not the adenovirus type or species. it should be noted that the thermocycling conditions for the adenovirus pcr were modified to allow simultaneous processing of other real-time pcr assays (hsv and vzv) in the cdha microbiology laboratory [18] . simultaneously processing of multiple pcr assays on the same lightcycler instrument allows more efficient batch testing when equipment availability is limited. interestingly, these modifications allowed the detection of hadv type 31 which had previously been problematic on an abi instrument [18] . difference between assays can be attributed to a numerous factors (i.e. instrumentation, kits, etc.); however, the most likely explanation in this case is the annealing temperature. using the original pcr protocol [18] , hadv type 31 could only be detected when the annealing temperature was reduced from 60°c to 57°c [18] . the annealing temperature in this study is 55°c. using conditions described in this study, the detection of hadv type 31 has now been replicated in both collaborating laboratories. a limitation of this study is that the validation of homogenization was only performed using swabs in utm. future experiments will need to examine whether homogenization can be applied to other relevant specimen types (urine, stool, blood and tissue); however, the realtime pcr following a nucleic acid extraction has been shown to be effective for this purpose [18, 21] . secondly, the performance characteristics of homogenization may vary between pcr assays and should not be implemented without proper validation [27] . while homogenization with heat treatment has shown to be effective for the recovery of viral dna from hadv (this study), hsv [27] , and varicella zoster virus, decreased sensitivity was observed for enveloped rna viruses like mumps and influenza viruses ( [24, 45] leblanc, j. unpublished data). homogenization and heat treatment showed performance characteristics equivalent to a commercial nucleic acid extraction for the detection of hadvs. in combination with a sensitive in-house real-time pcr, homogenization with heat treatment generated results far superior than virus culture, and at a comparable cost. by modifying the thermocycling conditions to those used by other assays in the cdha microbiology laboratory, it further streamlined workflow and facilitated transition from virus culture to molecular testing. compared to virus isolation and propagation using culture, molecular testing also further reduces the risk of laboratoryacquired infections [46] . overall, homogenization with heat treatment combined with a sensitive in-house real-time pcr is a cost-effective method for the detection of hadvs. s principles and practice of infectious diseases a communitybased outbreak of severe respiratory illness caused by human adenovirus serotype 14 severe pneumonia due to adenovirus serotype 14: a new respiratory threat? adenovirus serotype 14 infection first reported cases of human adenovirus serotype 14p1 infection a: treatment of adenovirus infections in patients undergoing allogeneic hematopoietic stem cell transplantation comparison of in-house realtime quantitative pcr to the adenovirus r-gene kit for determination of adenovirus load in clinical samples quantification of adenovirus dna in plasma for management of infection in stem cell graft recipients t-cell immunotherapy for adenoviral infections of stem-cell transplant recipients clinical features and treatment of adenovirus infections high levels of adenovirus dna in serum correlate with fatal outcome of adenovirus infection in children after allogeneic stem-cell transplantation invasive adenoviral infections in t-cell -depleted allogeneic hematopoietic stem cell transplantation: high mortality in the era of cidofovir comparison of three multiplex pcr assays for the detection of respiratory viral infections: evaluation of xtag respiratory virus panel fast assay, respifinder 19 assay and respifinder smart 22 assay switching gears for an influenza pandemic: validation of a duplex reverse transcriptase pcr assay for simultaneous detection and confirmatory identification of pandemic (h1n1) 2009 influenza virus comparison of the filmarray respiratory panel and prodesse real-time pcr assays for detection of respiratory pathogens development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay detection of adenoviruses detection of a broad range of human adenoviruses in respiratory tract samples using a sensitive multiplex real-time pcr assay comparison of the luminex xtag respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections members of the adenovirus research community: toward an integrated human adenovirus designation system that utilizes molecular and serological data and serves both clinical and fundamental virology real-time qualitative pcr for 57 human adenovirus types from multiple specimen sources development of a pcrbased assay for detection, quantification, and genotyping of human adenoviruses molecular detection and quantitative analysis of the entire spectrum of human adenoviruses by a two-reaction real-time pcr assay multiplexed, realtime pcr for quantitative detection of human adenovirus pring-akerblom p: rapid and quantitative detection of human adenovirus dna by real-time pcr evaluation of type-specific real-time pcr assays using the lightcycler and j.b.a.i.d.s. for detection of adenoviruses in species hadv-c homogenization with heat treatment: a cost effective alternative to nucleic acid extraction for herpes simplex virus real-time pcr from viral swabs a reliable and inexpensive method of nucleic acid extraction for the pcr-based detection of diverse plant pathogens presumptive identification of common adenovirus serotypes by the development of differential cytopathic effects in the human lung carcinoma (a549) cell culture uracil-dna glycosylase (ung) influences the melting curve profiles of herpes simplex virus (hsv) hybridization probes probit analysis comprehensive detection and serotyping of human adenoviruses by pcr and sequencing logistic regression quantitation of targets for pcr by use of limiting dilution genome sequences of human adenovirus 14 isolates from mild respiratory cases and a fatal pneumonia, isolated during 2006-2007 epidemics in north america genome sequence of the first human adenovirus type 14 isolated in china inhibition and facilitation of nucleic acid amplification a comparison of cell culture versus real-time pcr for the detection of hsv1/2 from routine clinical specimens adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis comparison of a commercial qualitative real-time rt-pcr kit with direct immunofluorescence assay (dfa) and cell culture for detection of influenza a and b in children efficacy of pcr and other diagnostic methods for the detection of respiratory adenoviral infections epidemiology of severe pediatric adenovirus lower respiratory tract infections in manitoba, canada characterization of culture-positive adenovirus serotypes from respiratory specimens in genome type analysis of adenovirus types 3 and 7 isolated during successive outbreaks of lower respiratory tract infections in children detection of mumps virus rna by real-time one-step reverse transcriptase pcr using the lightcycler platform viral agents of human disease: biosafety concerns a cost effective real-time pcr for the detection of adenovirus from viral swabs we would like to thank members of division of microbiology, department of pathology and laboratory medicine at cdha (halifax, nova scotia) for their ongoing support and for funding for this project. in particular, we are indebted to wanda brewer for the propagation and maintenance of a546 cells, and the various technologists responsible for routine virus culture. the authors declare that they have no competing interests.authors' contributions jl conceived the study. jl, th and rt participated in its design and coordination. ta, kb, and jl carried out the molecular testing. mw quantified the adenovirus stocks and established tcid 50 values. ta and kb performed statistical analyses. jl analyzed the dna sequencing results. rt, sw and kp were involved in the phylogenetic analyses and typing of the adenoviruses as well as preparing the specificity panels. all authors were involved in the preparation of the manuscript. all authors have read and approved the final manuscript. key: cord-007724-2nwrhk1d authors: hofmann, martin a.; brian, david a. title: sequencing dna amplified directly from a bacterial colony date: 1993 journal: pcr protocols doi: 10.1385/0-89603-244-2:205 sha: doc_id: 7724 cord_uid: 2nwrhk1d a few hundred bacterial cells obtained by touching a bacterial colony with a sterile toothpick can be used directly in a polymerase chain reaction (pcr) amplification procedure to identify and orient a plasmid insert (1,2). by combining this procedure with one in which asymmetrically amplified dna is used for sequencing (ref. 3 and fig. 3), we have demonstrated that dna amplified from a bacterial colony can be sequenced directly by the dideoxy chain-termination method to yield results as good as those obtained when purified template dna is used for amplification (ref.4 and fig. 2). by end-labeling the primer that is used in limiting amounts during the amplification step and using it for sequencing, an entire insert of 300 nucleotides or less can be sequenced in one step. inserts of larger size can be sequenced by using labeled primers that bind within the amplified single-stranded dna sequence. the procedure is rapid and enables one to obtain sequences from as many as 20 clones in a single day. a few hundred bacterial cells obtained by touching a bacterial colony with a sterile toothpick can be used directly in a polymerase chain reaction (pcr) amplification procedure to identify and orient a plasmid insert (1, 2) . by combining this procedure with one in which asymmetrically amplified dna is used for sequencing (ref. 3 and fig. l) , we have demonstrated that dna amplified from a bacterial colony can be sequenced directly by the dideoxy chain-termination method to yield results as good as those obtained when purified template dna is used for amplification (ref. 4 and fig. 2 ). by end-labeling the primer that is used in limiting amounts during the amplification step and using it for sequencing, an entire insert of 300 nucleotides or less can be sequenced in one step. inserts of larger size can be sequenced by using labeled primers that bind within the amplified single-stranded dna sequence. the procedure is rapid and enables one to obtain sequences from as many as 20 clones in a single day. fig. 1 . the asymmetric pcr reaction. in the procedure described in this chapter, primer 2 is the limiting primer and will be 5' end-lab&d and used in the sequencing reaction for sequencing the single-stranded dna product. in water. approximately 1 mg of oligonucleotide is purified on a naps sephadex g-25 column (pharmacia, piscataway, nj), which has a bed volume of 3 ml. oligonucleotides are quantitated by spectrophotometry (1 aw u = 33 j.tg/ml) and diluted to a final concentration of 10 p&f or 0.1 pm (depending on the use; see below) in water. store at -20°c. 1. prepare a 2oyl end-labeling reaction mix by adding together 1 jtl of 10 pa4 primer 2 (10 pmol), 11 pl of water, 2 pl of 10x kinase buffer, 5 pl of [t-~~p]atp, 1 pl (10 u) of t4 polynucleotide kinase. this makes enough for 10 sequencing reactions. 2. incubate at 37°c for 30 min, then add 30 pl of hz0 to make a total volume of 50 p.l. 3. purify end-labeled primer by passing it through a biospin column that has been equilibrated with water according to manufacturer's instructions. 4. estimate the volume of eluate and use 0.1 vol (x pl used in section 3.3., step 1 below) (1 pmol of radiolabeled primer) in the sequencing reaction below. one microliter of the eluate can be counted to determine the specific activity of the radiolabeled primer. approximately lo6 cpm cerenkov counts/pmol primer is needed. this is essentially as described by innis add 20 pl of pcr mix (from section 3.1., step 6) containing singlestranded dna, mix by pipetting 10 times c reaction tubes, which contain 2.5 pl each of the respective termination mixes and mix by pipetting five times terminate the reactions by adding 4 lrl of formamide stop solution. store at -20°c for sequencing, the termination mix (10 pl) is heated at 100°c for 3 min and 3 pl/lane is loaded onto a dna sequencing gel (see note 2) pj4 each) so that residual amounts would not interfere with dideoxynucleotide chain termination reactions (innis et al., ref. 3). we have learned that when residual amounts do cause a problem (i.e., when short termination products cannot be seen on the sequencing gel), generally as a result of short (~200 nucleotides) inserts in the clone, two approaches can be used to solve this problem: (1) additional cycles of the pcr (50-60 total) can be run to deplete the dntps and (2) the product from the asymmetric reaction direct clone characterization from plaques and colonies by the pulymerase chain reaction rapid one-step characterization of recombinant vectors by direct analysis of transformed escherichra coli colonies dna sequencing of polymerase chain reaction-amplified dna sequencing pcr dna amplified directly from a bacterial colony sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes molecular clonmg: a laboratory manual the 5-prime end of coronavirus minusstrand rnas contain a short poly(u) tract key: cord-008333-1wepke2o authors: weisz, ora a.; machamer, carolyn e. title: chapter 7 use of recombinant vaccinia virus vectors for cell biology date: 2008-02-28 journal: methods cell biol doi: 10.1016/s0091-679x(08)60602-0 sha: doc_id: 8333 cord_uid: 1wepke2o this chapter describes the use of several of the recombinant vaccinia expression systems, focuses on the systems that are most useful for cell biologists, and discusses their advantages and limitations. vaccinia-mediated expression can be used for assessing cellular localization, posttranslational modifications, oligomerization, and transport and turnover rates. the system provides a rapid method for screening mutant proteins for expression and targeting. it is an excellent way of quickly deciding which mutant proteins might be worth further studying using stable expression systems. expression of foreign genes using vaccinia virus is based on recombinant viruses constructed by insertion of complementary dna (cdna) into the nonessential thymidine kinase (tk) gene. both direct and indirect methods of expression are possible. the foreign gene can be inserted into the vaccinia genome by homologous recombination using a plasmid with flanking regions of vaccinia dna. the recombinant virus is selected, expanded, and used to infect cells, which then express high levels of the foreign protein. recombinant vaccinia viruses are generated by subcloning the foreign gene into a plasmid transfer vector so it is flanked by dna from the vaccinia (tk) gene, which is nonessential for growth of the virus in tissue culture. this plasmid is then transfected into vaccinia-infected cells. homologous recombination of the plasmid and the vaccinia genome generates a recombinant virus with an inactive tk gene. use of several of the recombinant vaccinia expression systems pioneered by bernard moss and colleagues. we will focus on the systems that are most useful for cell biologists, and discuss their advantages and limitations. vacciniamediated expression can be used for assessing cellular localization, posttranslational modifications, oligomerization, and transport and turnover rates. the system provides a rapid method for screening mutant proteins for expression and targeting. it is an excellent way of quickly deciding which mutant proteins might be worth further study using stable expression systems. some uses of vaccinia vectors will not be discussed here, including methods for largescale production of proteins. for detailed methods for large-scale production of proteins from cdnas, or other vaccinia methods not discussed here, see earl and moss (1991) , moss er al. (19901, and moss (1991) . vaccinia virus is the best-studied member of the poxviridae, the largest and most complex of the animal viruses. widespread vaccination with vaccinia virus (probably derived from cowpox virus) resulted in the worldwide eradication of smallpox. the double-stranded linear genome of vaccinia virus is nearly 200 kb, and there are over 250 potential genes (goebel et al., 1990; moss, 1991) . the virions are enveloped and are approximately 200 x 300 nm in size, with a characteristic brick shape (fig. 1) . several features of the vaccinia life cycle make it unique as a eukaryotic expression vector, at least 25 kb of dna can be inserted into the vaccinia genome without detrimental effects on viral replication or assembly. vaccinia replicates completely within the cytoplasm of the host cell, and thus imports or directs the synthesis of its own polymerases and transcription factors. in addition, the virus has a wide host range, so most cultured mammalian cell lines are susceptible to infection. the virus is easy to grow and purify in large quantities, and is relatively safe to work with. considering the size of vaccinia virus, the complexity of replication and assembly is not surprising (figs. 1 and 2) . after binding to the host cell, the viral envelope is believed to fuse directly with the plasma membrane (doms et al., 19901 , releasing the core virion into the cytoplasm. little is known about the uncoating steps, but transcription of early genes begins within 1 hr of infection. the virion core contains all the enzymes it needs for transcription of the early genes. transcribed rnas are capped, methylated, and polyadenylated in the cytosol by viral enzymes. dna replication begins with 3-4 hr, and occurs in discrete juxtanuclear regions of the cytosol termed "viral factories" near the golgi complex. after transcription of intermediate and late viral genes (by different viral rna pol ymerases), viral assembly begins. this complex process appears to involve two separate membrane "enwrapping" events, each of which results in a double lipid bilayer surrounding the core particle. the first double membrane is derived from the "intermediate compartment" between the endoplasmic reticulum (er) and golgi (sodeik er al., 1993) . the viral dna is inserted into a crescent-shaped region of the intermediate compartment membrane which then fuses around it, generating intracellular mature virus (imv), formerly called intracellular "naked" virus. the second enwrapping membranes are derived from the trans-golgi network (schmelz et al., 1994) plasma membrane, releasing virions with three membranes into the extracellular space. however, some strains of vaccinia are inefficiently released from cells. approximately 95% of the commonly used wr strain remains cell associated. other strains (e.g., ihd-j) produce substantial amounts of extracellular virus. since both forms of intracellular virus (singly and doubly enwrapped) are infectious, virus is usually purified from cell homogenates rather than from the culture medium. expression of foreign genes using vaccinia virus is based on recombinant viruses constructed by insertion of cdna into the nonessential thymidine kinase (tk) gene. both direct and indirect methods of expression are possible (fig. 3) . the foreign gene can be inserted into the vaccinia genome by homologous recombination using a plasmid with flanking regions of vaccinia dna. ase (vtf7-3) is used to infect cells (fuerst et al., 1986) . the t7 rna polymerase is expressed efficiently in the cytoplasm early after infection. transfection of cells immediately after infection with a vector containing the gene of interest cloned behind a t7 promoter results in rapid and efficient expression of the encoded protein. this system is convenient, since recombinant virus production is unnecessary. commonly used vectors such as pbluescript (stratagene, la jolla, ca) can be used. recombinant vaccinia viruses are generated by subcloning the foreign gene into a plasmid transfer vector so it is flanked by dna from the vaccinia (tk) gene, which is nonessential for growth of the virus in tissue culture. this plasmid is then transfected into vaccinia-infected cells. homologous recombination of the plasmid and the vaccinia genome generates a recombinant virus with an inactive tk gene. a lysate from the infected cells is used to infect a thymidine kinase negative (tk-) cell line, usually human osteosarcoma 143b cells (mackett et ul., 1984) . recombinant viruses are enriched by growing the cells in the presence of bromodeoxyuridine (brdu), since only recombinant viruses (and tk mutants) are able to replicate. if the plasmid transfer vector used for recombination contains the p-galactosidase gene (see subsequent discussion), recombination viruses can be distinguished from tk mutants by color screening using 5-bromo-4-chloro-3-indolyl-@-~-galactoside (x-gal; chakrabarti et ul., 1985) . various vectors for recombination into the vaccinia genome are available earl and moss, 1991; moss, 1992) . the use of an early vaccinia promoter to drive the foreign gene is essential for cell biology applications, since the protein will be expressed before most of the cytopathic effects of the virus infection become evident. the vector we use for production of recombinant virus is pscl iss (fig. 4a ) or its derivative psc65. pscl lss contains the promoter p,., that is transcribed both at early and late times after infection; psc65 contains a synthetic earlyhate promoter. following the promoter is a short polylinker into which the foreign gene is cloned. another vaccinia promoter, p,, (a late promoter), drives expression of the bacterial @-galactosidase gene and allows color screening of plaques to help identify recombinants. both expression cassettes are flanked by portions of the vaccinia tk gene. for subcloning the gene of interest into the recombination vector, several things must be kept in mind. since splicing of the subcloned dna will not occur, cdnas must be used. the 5' and 3' untranslated regions should be kept short. also, it is important to check for a transcription termination sequence that is recognized by the early rna polymerase. the sequence tttttnt (t,nt, where n can be any nucleotide) will cause termination of transcripts about 50 bp downstream (yuen and moss, 1987) . thus, silent mutations that eliminate any t,nt sequences should be introduced by site-directed mutagenesis prior to subcloning. although proteins can still be expressed from recombinant viruses made with pscl iss or psc65 when their cdnas contain t,nt sequences, expression will only occur during the late phase of virus infection (a) pscllss is used for producing vaccinia recombinants. the foreign gene (with its own start and stop codons) is subcloned into the stic i or sol i sites behind the vaccinia p7,1i promoter. (b) par2529 is used for t7 polymerase-mediated expression after infection with vtf7-3 (section 1v.a). other plasmids such as pbluescript (stratagene) can also be used: however, the absence of the t7 polymerase terminator sequence and the presence of the g-c rich region in the bluescript polylinker upstream of the foreign gene can reduce mrna production and consequently decrease protein expression. (>6 hr) when the late rna polymerase (which does not terminate transcripts at t,nt) is active. after the subcloning step is completed, purified plasmid dna should be prepared in cscl gradients or by the qiagen method (qiagen, inc., chatsworth, ca). nonpurified miniprep dna does not work when the ca,(po,), method of transfection is used. work with vaccinia virus should be performed under standard biosafety level 2 (bl-2) conditions, including the use of class i or i1 biological safety cabinets. national institutes of health (nih) and centers for disease control (cdc) guidelines recommend that workers be vaccinated every 3 years, although each institution sets its own requirements for vaccination and physical containment. regulations should be obtained from institutional biosafety offices before initiating projects involving vaccinia virus. we use class i1 biological safety cabinets, autoclave disposable items that have been in contact with virus, and inactivate used solutions containing infectious virus with bleach. a convenient way of inactivating virus-containing solutions is to aspirate them into the reservoir flask in the hood and, immediately afterward (using the same pasteur pipet), aspirate bleach through the line. additional precautions for using large quantities of vaccinia virus (for example, when growing and purifying large stocks) are discussed in section iii,a. 1. production of recombinant viruses 1. hela or cv-i cells are usually used for production of recombinant viruses. they are grown in dulbecco's modified eagle's medium (dmem) containing 5% fetal calf serum (fcs). plate approximately 5 x lo5 cells in a 3.5-cm dish for cells to be about 80% confluent the next day. 2a. rinse the cells once in serum-free dmem, and add 0.25 ml serum-free medium containing 0.05-0.1 plaque-forming units (pfu)/cell of the wr strain of vaccinia virus (we use lo5 pfu total). 2b. some investigators employ a trypsinization step prior to infection, which is required when the virus stock is a cell lysate instead of a purified preparation. add an equal volume of 0.25 mg/ml trypsin to the amount of lysate that will be required and incubated at 37°c for 30 min, with occasional vortexing. this helps dissociate aggregates and releases infectious virus from cell debris. if trypsinization is used, dmem containing 2.5% fcs should be used to dilute and adsorb the virus. fcs inhibits the trypsin. 3. after addition of the virus inoculum, cells are returned to the incubator and rocked every 5-10 min for 45 min (or 2 hr if trypsinized virus in serumcontaining medium is used). toward the end of the infection period, prepare a ca,(po,), precipitate of the dna for transfection. add 5 pg dna to 125 pl 2 x hepes buffer (50 mm hepes, ph 7.1, 0.28 m nacl, 1.5 m m sodium phosphate). add an equal volume of 0.25 m caci, dropwise with continuous vortexing, and allow a precipitate to form by leaving the mixture at ambient temperature for 20-30 min. the precipitate should be very fine, turning the solution slightly opaque. 5. after the adsorption period, remove the virus inoculum and add 2.25 ml dmem/5% fcs to the dish. allow the ph to equilibrate in the co, incubator for 15 min. 6. add the precipitated dna solution dropwise while gently swirling the dish, and return the cells to the incubator. 7. replace dna-containing medium with fresh growth medium the following morning. 8. prepare a cell lysate 2 days after infection by scraping the cells into their medium and centrifuging for 5 min at 650 g at 4°c. 9. resuspend the cell pellet in 0.25 ml complete medium and lyse the cells by freezing (in dry ice/ethanol) and thawing (at 37°c) three times. 10. the lysate is then stored at -80°c until selection and screening is performed. 1. prepare a brdu stock solution (5 mg/ml) in water, sterilize by filtering, and store at -20°c. 2. grow human tk-143b cells in dmem containing 10% fcs and 25 pg/ ml brdu. plate 143b cells in a 6-well tissue culture dish at 5 x lo5 cells/well in 2 ml complete medium and grow to near confluence (usually overnight). 3. thaw an aliquot (100 pl) of transfected cell lysate, add an equal volume of 0.25 mgiml trypsin, and incubate the mixture at 37°c for 30 min. 4. make i0-fold serial dilutions (to of trypsinized lysate in dmem with 2.5% fcs. 5 . aspirate the medium from the cells and infect cells in the wells in duplicate with i ml 10-2-10-4 dilutions for 2 hr with intermittent rocking. toward the end of the infection period, the agarose overlay is prepared. although most of the virus remains cell-associated, an overlay helps insure that a plaque is derived from a single virion. melt a previously autoclaved solution of 2% low melting point agarose (no. 5517; gibco/brl, grand island, n y ) in water, aliquot enough for 1 ml per well into a tube, and equilibrate to 50-55°c. [some lots of difco (detroit, mi) agar work well, but some are toxic to cells andlor plaque formation.] 7. warm 2-fold concentrated dmem containing 10% fcs and 50 pg/ml brdu (1 ml/well) to 37°c. 8. at the end of the infection period, aspirate the inoculum from the cells, mix the agarose and 2 x medium, and overlay each well with 2 ml. after the overlay has solidified at room temperature, return the dishes to the co, incubator. 9. after 2 days, the infected cells will be rounded and dead and can be seen as clear areas (plaques) in the cell lawn. to see these plaques more clearly and to identify recombinants, prepare a second overlay by mixing an equal volume of melted agarose with 2 x dmem containing 0.1 mg/ml neutral red (from a 10 mg/ml stock) and 0.6 mg/ml x-gal (from a 60 mg/ml stock in dimethyl sulfoxide or dimethyl formamide). 10. overlay each well with 2 ml, allow the agarose to solidify, and incubate the dishes overnight. 11. the following morning, pick any blue plaques by inserting a pasteur pipet or a yellow pipet tip through the agarose until it contacts the cell layer; scrape the cell monolayer gently and discharge the agarose plug into a sterile microfuge tube containing 0.4 ml dmem/5% fcs. 12. these plaque isolates should be vortexed vigorously, then frozen and thawed three times, and stored at -80°c until further purification. typically 6 plaques are picked and taken through two additional rounds of plaque purification, as described subsequently, to ascertain that the recombinant virus is clonally derived and devoid of nonrecombinant virus. for each round of plaque purification, a 6-well dish of 143b cells is plated as just described for each isolated plaque. the frozen and thawed cell lysate is diluted serially from lo-' to and duplicate wells of the confluent mono-layers are infected with 1 ml of each dilution of virus for 2 hr. after infection, the wells are overlaid first with brdu-agarose for 2 days, and then with a second overlay containing x-gal, as described. the plaque-purified recombinants can be tested by several different methods (northern or southern blotting, immunofluorescence, dot blotting, or immunoprecipitation) to insure that they contain the gene of interest inserted into the vaccinia tk gene. we commonly use immunofluorescence after 2-3 plaque purifications. a portion of an isolated plaque is used to infect cells on a cover slip, and cells are fixed and stained 1-2 days later (see protocol in section iv,c,3). cells surrounding the plaques should express the foreign protein. for other methods of screening, plaques should be expanded by successively infecting larger and larger numbers of cells. this is usually done by infecting cells in one well of a 12-well dish with half of an isolated plaque; after 2 days a cell lysate is made (as described) and is used to infect cells on a 35-mm dish. before growing a large-scale preparation of the recombinant, we also test that the expressed protein is the correct size by immunoprecipitation from radiolabeled infected cells, followed by electrophoresis in sds polyacrylamide gels (see section iv,c,l). after subcloning into the recombination vector, approximately 3 wk will be required for generating and purifying the vaccinia recombinant. if all goes well, and if cells for successive plaque purifications are plated to be ready the same day plaques are picked, the time required may be somewhat shorter. the recombination frequency should be about 1 in 1000, which is relatively high. however, occasionally only a few blue and many clear plaques are obtained. one problem might be that the vaccinia virus used to infect the cells for the recombination contains a large population of tk-virus. the solution is to plaque purify the wild-type vaccinia, and test several plaques for their ability to grow in 143b cells in the presence of brdu. select a plaque thatfails to grow (i.e., tk+) for future use. another problem might be that expression of the foreign protein is incompatible with vaccinia replication or assembly. we have had this problem with several proteins that accumulate in the intermediate compartment and cis-golgi network. either the recombinant is never obtained, or dna encoding the foreign gene is lost, giving the virus a growth advantage over virus that still contains the foreign gene. in this case, one alternative is to use adouble infection system (moss el al., 1990; earl and moss, 1991) . the cdna encoding the foreign gene is cloned into a recombination vector with the t7 rna promoter instead of the p,,s promoter (ptm-i), and a recombinant virus is produced as described already. since the protein is not expressed, it will not inteifere with recombinant virus production. expression is mediated by co-infecting the cells with another vaccinia virus encoding t7 rna polymerase (vtf7-3). alternatively, the infection/transfection system can be used (section iv,b). 2b. if the virus inoculum is a crude cell lysate, the volume required to give 0.05-0.1 pfu/cell should be trypsinized as described in section ii,c and diluted in medium with 2.5% serum for infection. rock the infected cells every 15 min for 2 hr. 3. at the end of the infection period, add 20 ml dmem/5% fcs to each dish. purify the virus 2-3 days after infection, at which time most or all of the cells should appear rounded, but should remain attached to the dish. although a frozen and thawed preparation of infected cells can be used for expression work, purified virus stocks are recommended. sonication and trypsinization of cell lysates is the only way to disrupt virus aggregates generated by freezing in medium, and must be performed before infecting cells. purified virus is stored at high ph in a buffer lacking salt, which minimizes aggregation. thus trypsinization and sonication are unnecessary when infecting with purified preparations. 1. virus purification should be performed in a laminar flow hood and gloves should be worn. pipets and any glassware used should be soaked in bleach or autoclaved after use to destroy infectious virus. 2 . scrape the infected hela cells into their medium using a rubber policeman; then combine and transfer to 50-ml disposable sterile tubes and centrifuge for 5 min at 200 g (1000 rpm in a tabletop centrifuge) at 4°c. 3. resuspend the pellets in a total of 8 ml of 10 m m tris hcl, ph 9.0, and homogenize (in two batches) on ice in a 7-ml dounce with 40-60 strokes using the tight pestle. centrifuge the homogenate in two 15-ml disposable tubes for 5 min at 200 g at 4°c to remove nuclei. collect the supernatant and save on ice while the pellets are washed. resuspend the pellets in a small volume of 10 m m tris-hc1, ph 9.0, dilute to 10 ml with the same solution, and recentrifuge as in step 4. 6. transfer the supernatants along with those from the previous spin into 50-ml tubes and centrifuge at 650 g (2000 rpm) for 10 min at 4°c to remove any remaining debris. 7. sonicate the supernatants for 2-5 min in a water bath sonicator; then distribute into two sw28 tubes and underlay with an equal volume of 36% (w/ v) sucrose in 10 mm tris-hc1, ph 9.0, to fill the tubes. 8. centrifuge the tubes at 13500 rpm (33000 g) in a sw28 rotor for 80 min at 4°c. 9. after centrifugation, remove the supernatant, treat with bleach, and discard. resuspend the pellets in 2 ml of 1 mm tris-hc1, ph 9.0, per tube (they should be easy to resuspend but can be sonicated if necessary). combine and dispense into small (50-i00 pl) aliquots. 10. purified virus is stored at -80°c. usually, virus purified with this protocol yields 2-8 x 10' pfu/ml. after an aliquot is thawed for use, any remainder can be refrozen at -80°c and used once or twice more without a substantial loss of titer. titering the virus is performed by infecting cells (typically hela, cv-1, or bsc-i) with various dilutions of virus and determining the number of infectious particles per milliliter by counting the number of plaques that form. we usually use hela cells, but plaques formed on bsc-i monolayers are easier to distinguish and quantify. the protocol for titering the virus is basically the same as that described for selection of vaccinia recombinants (section 11,c). however, the purified virus does not require treatment with trypsin prior to plaquing. also, an agarose overlay is not necessary, since most of the virus remains cellassociated and thus does not spread readily through the culture medium. an agarose overlay containing neutral red stain and/or x-gal added to the cells just prior to counting the plaques can aid in visualizing them. grow cells in a 6-well dish until they are nearly confluent (usually 4-5 x lo5 cellslwell plated the day before). 2. to titer the virus, thaw an aliquot of purified virus and dilute serially into serum-free dmem to include a negative control (no virus) and a positive control (purified vaccinia virus of known titer diluted to a concentration designed to give 50-100 plaques). 3. after rinsing the cells in pbs, add 100 p1 serum-free dmem to each well; 4. place the dish in the incubator and rock every 5-10 min. 5. after 30 min, replace the inoculurn with 2 rnl complete medium. 6. after 3 days, aspirate the medium and stain the cells with 0.1% crystal violet in 20% ethanol for 5 min. aspirate the stain and allow the well to dry before counting the plaques. alternatively, the wells can be overlaid with 3 ml agarose containing neutral red and/or x-gal. the overlay procedure is described in section ii,c. count the plaques in each well (where possible) and determine the titer (pfu/ml) based on the dilution of virus used to infect the cells. then add 100 pl diluted virus (use dilutions ranging from to lo-*). as mentioned in the introduction, the t7 rna polymerase method of vaccinia-mediated expression is quite convenient since only one recombinant virus is needed (vtf7-3). in addition, the gene to be expressed can be cloned into commonly used vectors such as pbluescript. however, specially designed vectors containing a slightly extended version of the t7 promoter and the t7 terminator sequence give better expression (fig. 4b ). for very high levels of expression, vectors have been constructed that contain an untranslated leader region from encephalomyocarditis virus as well as the t7 promoter and terminator. the message produced by t7 polymerase is inefficiently capped by the vaccinia capping enzyme, and the leader region from encephalomyocarditis virus allows cap-independent translation (elroy -stein et al., 1989) . however the 5to 10-fold increase in protein expression obtained with this system may not be practical for cell biological studies (see section v,b). the protocols for the direct and the indirect methods of expression are essentially the same, with the exception of an additional transfection step following the infection for the t7 polymerase-mediated system. we discuss both protocols for protein expression together in this section, with additional steps for the t7 system described in the appropriate places. a wide variety of cell lines can be used for expression of the protein of interest. chinese hamster ovary (cho) cells are an exception, since they are nonpermissive for vaccinia infection. in addition, polarized madin-darby canine kidney (mdck) and caco-2 cells are poorly infected. 1. to examine protein expression using metabolic labeling, imrnunoblotting, or immunofluorescence, cells are plated the day before infection to reach 40-70% confluence (approximately 2 x lo5 cells/35-mm dish). larger dishes may be used for some applications; however, expression is usually high enough that this is not required. 2. cells are rinsed once in pbs, and then infected with vtf7-3 or another recombinant virus. the virus (10-20 pfu/cell or lo7 pfu/35-mm dish) is added in 0.3 ml serum-free dmem per dish. 3. after addition of the virus, dishes are returned to the incubator and rocked every 5-10 min for 30 min. if the protein of interest is expressed directly by the recombinant virus, the inoculum is replaced after infection with 2 ml regular growth medium containing serum and step 4 is omitted. note: if expression is mediated via t7 polymerase (vtf7-3), the infection period can be used to prepare the dna for transfection. transfection can be performed using either cationic lipid or calcium phosphate as a carrier. 4a. for lipid-mediated transfection, 2-5 pg plasmid dna (csc1-purified or qiagen-purified works best, but miniprep dna can also be used) is added to 0.75 ml serum-free dmem per 35-mm dish of cells. after mixing, 10 p1 lipofectace (no. 18301; gibco/brl) is added to the side of the tube and immediately vortexed for 5 sec. [lipofectin (no. 18292 ; gibco/brl) also works well, but is more expensive.] the mixture is incubated at ambient temperature for up to 30 min. at the end of the viral infection period, the inoculum is replaced with the dna-transfectace mixture. 4b. for calcium phosphate-mediated transfection, a precipitate containing 5 pg purified plasmid dna (encoding the foreign gene behind the t7 promoter) is prepared and added to cells exactly as described in section ii,b. we no longer use the ca,(po.,), method of transfection because it is much less reproducible than the cationic lipid method. after transfection, dishes are returned to the incubator. the incubation time varies depending on the type of analysis that will be performed (see the next section). metabolic labeling can be initiated as early as 3 hr postinfection. cells should be starved for 15 rnin in medium lacking the amino acid that will be used to radiolabel cells if a short pulse label will be used. because of the high expression level, pulse periods can be short (5 rnin in 100 pci/ml ["s]methionine typically gives an overnight exposure). after the appropriate pulse-chase period, cells are lysed in nonionic detergent. we use a detergent solution containing 50 mm tris-hc1, ph 8.0, 62.5 mm edta, 0.4% deoxycholate, 1% np-40, and 0.04 tiu aprotinidml. other typical lysis buffers contain 1% triton x-100 in trisbuffered saline with protease inhibitors. after spinning out nuclei and debris (1-15 rnin in a microfuge), the protein of interest is immunoprecipitated and electrophoresed as desired. solubilization and immunoprecipitation conditions must be determined empirically for each antibody-antigen combination. 1. we typically lyse cells from a 35-mm dish in 0.5 ml detergent solution (described earlier). all polyclonal antibodies and many monoclonal antibodies seem to work well in this mixture. 2. after 10 min on ice, lysates are transferred to a microfuge tube and nuclei and debris are removed by spinning for 1 min. 3. the supernatants are transferred to a fresh tube, and 10% sds is added to a final concentration of 0.2%. the sds helps reduce background binding. for immunoprecipitation with monoclonal antibodies, we generally omit the sds. 4. antibody is added to the tubes, and they are rotated (2 hr to overnight) at 4°c. we use fixed staphylococcus aureus cells (no. 507861; calbiochem, san diego, ca) to collect the antigen-antibody complexes (20 min at 4°c); protein a-sepharose can also be used. 6. the immunoprecipitates are washed 3 times in ripa buffer (0.15 m nacl, 10 mm tris-hc1, ph 7.4, 1% np-40, 1% deoxycholate, 0.1% sds). 7. the samples are usually eluted directly in sds sample buffer containing reducing agent and are electrophoresed in sds-polyacrylamide gels. alternatively, the samples can be eluted and treated with endo-or exoglycosidases to analyze carbohydrate modifications. our protocol for digestion with endoglycosidase h is given in the legend to fig. 7. 2. immunoblotting i . if the expressed protein is to be detected by immunoblotting, it is advisabie to wait until 6-8 hr postinfection before lysing the cells to allow chemical amounts of protein to accumulate. cells are infected (and transfected if necessary) as described already. 2. after the appropriate incubation time, the medium is removed (and trichloroacetic acid precipitated if the protein is secreted) and the dishes are rinsed once in cold pbs. 3. after removing the last trace of pbs, cells are lysed on the dish by adding 50 p1 laemmli sample buffer containing a reducing agent and swirling with a pipet tip to draw the lysate together (the dna from the lysed nuclei makes a viscous solution). 4. the samples are transferred to microfuge tubes, heated at 100°c for 3 min (or longer if required to fully shear dna), and loaded onto sds-polyacrylamide gels. after electrophoresis, the proteins are transferred to nitrocellulose (or other suitable membrane) and incubated with primary antibodies. 6. proteins are detected using colorimetric or chemiluminescence methods after incubation with an appropriately conjugated second antibody. an example of immunoblotting used to follow the time course of protein expression is shown in fig. 5 . significant levels of expressed proteins can usually be detected by immunofluorescence staining between 4 and 8 hr after infection, depending on the cell type used and the subcellular localization of the protein. proteins that become concentrated in subcellular compartments (e.g., nuclear, golgi, or lysosomal proteins) may be detectable at shorter times after infection than proteins with diffuse localizations (e.g., plasma membrane or cytoplasmic proteins). at long times after infection (>7-8 hr), localization by immunofluorescence becomes fig. 5 time course of vsv g protein expression after vtf7-3-mediated expression. bhk cells were infected with vtf7-3, then transfected as described with 5 pg per dish of par/g, which encodes the g protein of vesicular stomatitis virus (vsv). at each time point, individual dishes were rinsed once with pbs, then solubilized in 50 pl laemmli sample buffer containing 5% p-mercaptoethanol. samples were heated to i00"c for 3 min prior to electrophoresis in a 10% polyacrylamide gel. proteins were transferred to nitrocellulose and incubated overnight with anti-vsv polyclonal antibody after blocking in tris-buffered saline containing 0.05% tween-20 and 5% nonfat dry milk. primary antibody was detected with horseradish-conjugated goat anti-rabbit igg and enhanced chemiluminescence (ecl; amersham, arlington heights, 1l). increasingly difficult as the cells become rounded. the change in cell shape makes it difficult to focus on certain intracellular compartments. if long infection times must be used, confocal microscopy is recommended. 1. when proteins are to be detected by immunofluorescence, cells are plated on glass cover slips in 35-mm dishes the day before infection. for many cell types, plain cover slips can be used (after sterilization by autoclaving). however, some cells attach better if the glass is pretreated by acid washing, or by coating with poly-~-lysine ( i mg/ml) or extracellular matrix components such as fibronectin (1 pg/ml). 2. vaccinia infection and subsequent dna transfections are performed as described in section 1v.b. after the desired incubation period, cells are fixed in formaldehyde or methanol-acetone. methanol-acetone fixation may be required if components of the cytoskeleton are being analyzed, and usually consists of 5-10 min in methanol at -2o"c, followed by 30 sec to 1 min in acetone at -20°c. no further permeabilization is required. note: we find that formaldehyde fixation works well for all the antibodies we use (see subsequent discussion). as for other methods of analysis, optimal antibody dilutions and other parameters should be determined for each protein. as a guideline, polyclonal sera are usually diluted 1 : 200-1 : 1000, monoclonal antibodies from tissue culture supernatant are used straight or diluted up to 1:200, and affinity-purified antibodies or purified iggs are used at 2-10 pgl ml. commercially prepared secondary antibodies conjugated to fluorescein, rhodamine, or texas red are available from many suppliers. we have had excellent success with affinity-purified preparations from jackson immunoresearch (west grove, pa). 4. our protocol includes fixation in 3% paraformaldehyde in pbs for 20-30 5. the fixative is quenched using pbs-gly (pbs with 10 m m glycine). 6. if an intracellular epitope is being labeled, the cells are permeabilized for 4 min with 0.5% triton x-100 in pbs-gly. 7. after rinsing once with pbs-gly, we typically add a 5 rnin incubation in blocking solution (0.25% ovalbumin in pbs-gly. j 8. after removing aggregates by centrifuging antibodies in a microfuge for about 30 sec, both primary and secondary antibodies are diluted into the blocking solution. throughout the staining procedure, it is important that the cover slips never dry out. 9. the cover slips are lifted with forceps, a corner is touched to a kimwipeb to drain as much liquid as possible, and the slip is placed cell-side down onto a drop (50 pl) of primary antibody on a sheet of parafilm and incubated 20 min at room temperature. min at room temperature. 10. the cover slips are then returned (cell-side up) to the dishes, washed for 10 min in several changes of pbs-gly, and incubated in 50 pl fluorochromeconjugated secondary antibody, as described, for 20 min in the dark. 11. after washing in pbs-gly for 20-30 min, the backs of the cover slips are gently wiped and they are inverted onto a small drop of glycerol on a microscope slide. we use glycerol containing 100 mm n-propyl gallate to reduce photobleaching; glycerol containing phenylaminediamine or a commercial "mounting medium" can also be used. 12. the excess mounting medium is aspirated (and the cover slip may be sealed to the slide with nail polish) before viewing in a microscope equipped with epifluorescence and the appropriate barrier filters. an example of indirect immunofluorescence detection of proteins expressed using the t7 polymerase-vaccinia hybrid system is shown in fig. 6 . this chapter has discussed the preparation and use of recombinant vaccinia viruses to express proteins in mammalian cells. this method offers many advantages over other expression systems for the expression of some proteins (see below). however, the limitations of this method should be taken into account before choosing this technique. 1. vaccinia virus-mediated expression is rapid and efficient: experiments can be performed in i day. furthermore, most of the cells in a dish can be infected, resulting in a much higher efficiency of expression than other in transient expression systems. 2 . vaccinia has a wide host range, so most mammalian tissue culture cell lines are susceptible to infection. one exception is the cho cell line, which is not susceptible to infection with the virus. however, cowpox virus does infect cho cells, and a gene that allows this infection has recently been characterized (spehner et al., 1988) . it is possible that construction of a vacciniavirus recombinant containing this cowpox gene will allow productive infection of cho cells. 3. because of the high efficiency of expression, coexpression of two or more proteins is simple using this technique. the expression level of each protein can be varied with the amount of virus or dna used, depending on the method of expression (e.g., zagouras et ul., 1991) . 4. because this expression system operates cytoplasmically, it can be used to express genes from rna viruses that contain cryptic splice sites that are used when the cdna is inserted into a vector that must be transcribed in the nucleus (machamer and rose, 1987) . 5. infection with recombinant vaccinia viruses results in high expression levels in >90% of cells on a dish soon after infection. this method is therefore ideal for subcellular localization of expressed proteins by immunoelectron microscopy (machamer et d . , 1990; krijnse locker et d., 1992) . early times postinfection (<6 hr) should be analyzed to avoid complications from viral cytopathic effects. localization of proteins expressed via the vaccinia-t7 polymerase hybrid system is more difficult since the celluiar morphology can be altered by the dna carriers used for transfection, especially cationic lipid. 6. vaccinia virus-mediated expression can be useful for production of biologically active proteins. because proteins are expressed in mammalian cell lines, glycosylation and other post-translational modifications that may be necessary for activity are preserved. 7. an advantage specific to the t7 polymerase hybrid expression system is that it can be used to screen rapidly for expression of newly cloned cdnas and of mutant proteins generated by site-directed mutagenesis. the same vector can be used for mutagenesis, sequencing, in uitro transcription/translation, and vaccinia virus-mediated expression. in addition, a high percentage of cells is transfected because the dna only needs to reach the cytoplasm and not the nucleus to be expressed. 1. a serious disadvantage of the vaccinia virus system is that the expression level may be so high that it overwhelms cellular translation and translocation machinery. this can result in decreased efficiency of membrane translocation, post-translational modifications, and transport through the secretory pathway. at very high expression levels, newly synthesized proteins that enter the secretory pathway may accumulate in the er. we have observed reduced rates of transport of the vesicular stomatitis virus (vsv) g protein to the cell surface, compared with vsv-infected cells, when the protein is expressed with the vaccinia-t7 hybrid system (fig. 7) . whereas vsv g becomes resistant to endoglycosidase h (as it moves through the golgi complex) with a half-time of <25 min in vsv-infected hela cells, the half-time is considerably slower (-40 min) in vtf7-3-infected and -transfected cells. the difference in transport kinetics may be less dramatic in other cell types. 2. the cytopathic effects of vaccinia infection can be a problem for certain types of analysis. for example, an early effect of the infection is rounding of cells (probably due to changes induced in the cytoskeleton), which can make conventional immunofluorescence difficult to interpret (fig. 6 ) . in addition, host cell protein synthesis is inhibited in infected cells. vaccinia encodes homologs of cellular proteins including superoxide dismutase, epidermal growth factor, and profilin (goebel et al., 1990) , expression of which could affect cell morphology and behavior in unpredictable ways. although viral dna replication and early assembly steps can be blocked (by treating infected cells with hydroxyurea or rifampicin, respectively), early cytopathic effects such as cell rounding are unfortunately not prevented. 3. recombinant vaccinia viruses can be difficult and time-consuming to produce. because they inhibit transcription from early promoters, any t,nt sequences in the gene of interest must be mutagenized prior to subcloning into the recombination vector. even then, some recombinant viruses are impossible to produce, perhaps because expression of the foreign protein is incompatible with vaccinia replication or assembly. a final consideration prior to using the vaccinia virus-mediated protein expression system is the safety of laboratory workers. institutional guidelines for use of vaccinia virus recombinants must be followed. some institutions require a vaccination against smallpox by all vaccinia users once every 3 years (as recommended by the nih and cdc; however, see grist, 1989; wenzel and vtf7-3-infecteditransfected cells. hela cells grown in 35-mm dishes were infected with vtf7-3 followed by lipofectace-mediated transfection with 2 pg par/g per dish, as described in section iv,b, or with vsv (20 pfuicell). at 3.75 hr postinfection. cells were rinsed once in pbs, starved for 15 min in methionine-free dmem. and pulse-labeled for 5 min with 50 pc "s in uioo-labeling mix (amersham) in 0.5 mi methionine-free dmem per dish. cells were chased in growth medium. at the indicated chase times, individual dishes were lysed in detergent solution and immunoprecipitated using a polyclonal anti-vsv antibody, as described in section iv.c.1. samples were eluted in 20 pi 50 m m tris ph 6.8. 1% sds for 3 min at 100°c. eluents were divided in half, and either 10 &i 0.1s m citrate, ph 5.5, or the same amount of buffer containing 0.3 mu endoglycosidase h (endo h) was added. after overnight incubation at 37°c. concentrated laemmli sample buffer with 0-mercaptoethanol was added; the samples were heated to 100°c for 3 min and electrophoresed on laemmli sds-polyacrylamide gels. the percentage of vsv g protein that was resistant to endo h at each time point was quantitated by densitometric scanning of fluorographed gels. nettleman, 1989) . bl-2 restrictions also apply. if available, a virus-only laminar flow hood is recommended to avoid accidental infection of cell lines during routine tissue culture, although we have not had problems with accidental infection when the ultraviolet light is left on for an adequate time following work with infected cells. a small aliquot of vaccinia virus (both wild-type and the recombinant vtf7-3) and vectors for producing recombinant viruses and for t7 polymerase-mediated expression can be obtained by writing to dr. bernard moss (laboratory of viral diseases, niaid, nih, 9000 rockville pike, bldg 4-rm 229, bethesda, md 20892). new users must demonstrate that their facilities meet biosafety requirements, and that they have followed the institutional guidelines regarding vaccination. in addition, a material transfer agreement from the nih must be completed and signed by your institution prior to receipt of these stocks. vaccinia virus expression vector: coexpression of p-galactosidase provides visual screening of recombinant virus plaques fusion of intra-and extracellular forms of vaccinia virus with the cell membrane generation of recombinant vaccinia viruses cap-independent translation of mrna conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia viruslbacteriophage t7 hybrid expression system eukaryotic transient-expressions system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase the complete dna sequence of vaccinia virus cell biology of viruses that assemble along the biosynthetic pathway smallpox vaccination for investigators 0-glycosylation of the coronavirus m protein a specific transmembrane domain of a coronavirus el glycoprotein is required for its retention in the golgi region the el glycoprotein of an avian coronavirus is targeted to the golgi complex general method for production and selection of infectious vaccinia virus recombinants expressing foreign genes vaccinia virus: a tool for research and vaccine development poxvirus expression vectors new mammalian expression vectors assembly of vaccinia virus: the second wrapping cisterna is derived from the trans golgi network recombinant vaccinia virus vectors sodeik assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the golgi stacks a cowpox virus gene required for multiplication in chinese hamster ovary cells smallpox vaccination for investigators using vaccinia recombinants oligonucleotide sequence signaling transcriptional termination of vaccinia virus early genes dissociation and reassociation of oligomeric viral glycoprotein subunits in the endoplasmic reticulum key: cord-005400-50lmj4op authors: ada, gordon title: overview of vaccines and vaccination date: 2005 journal: mol biotechnol doi: 10.1385/mb:29:3:255 sha: doc_id: 5400 cord_uid: 50lmj4op of the 80-plus known infectious agents pathogenic for humans, there are now more than 30 vaccines against 26 mainly viral and bacterial infections and these greatly minimize subsequent disease and prevent death after exposure to those agents. this article describes the nature of the vaccines, from live attenuated agents to subunits, their efficacy and safety, and the kind of the immune responses generated by those vaccines, which are so effective. to date, all licensed vaccines generate especially specific antibodies, which attach to the infectious agent and therefore can very largely prevent infection. these vaccines have been so effective in developed countries in preventing mortality after a subsequent infection that attempts are being made to develop vaccines against many of the remaining infectious agents. many of the latter are difficult to manipulate; they can cause persisting infections or show great antigenic variation. a range of new approaches to improve selected immune responses, such as immunization with dna or chimeric live vectors, viral or bacterial, are under intense scrutiny, as well as genomic analysis of the agent. vaccines are designed as a prophylactic measure to induce a lasting immune response so that on subsequent exposure to the particular infectious agent, the extent of infection is reduced to such an extent that disease does not occur (1). there is also increasing interest in designing vaccines that may be effective as a therapeutic measure, immunotherapy. there are two contrasting types of infectious processes. some organisms, including all viruses and some bacteria, are obligate intracellular infectious agents, as they only replicate inside a susceptible cell. some parasites, such as plasmodia, have intracellular phases as part of their life cycle. in contrast, many bacteria and parasites replicate extracellularly. the immune response required to control the different patterns of infections may therefore differ. in the case of an acute infection, exposure of a naïve individual to a sublethal dose of the infectious agent may cause disease, but the immune response generated will clear the infection within a period of days or weeks. death occurs if the infecting dose is so high that the immune response is qualitatively or quantitatively insufficient to prevent continuing replication of the agent so that the host is overwhelmed. in contrast, many infections persist for months or years if the process of infection by the agent results in the evasion or subversion of what would normally be an effective immune control reaction. most of the vaccines registered for use in developed countries, and discussed briefly in the next section, are designed to prevent acute human infections. 1. one approach, pioneered by edward jenner, is to use a virus that is a natural pathogen in another mammalian host as the basis of a vaccine in humans. examples of this approach are the use of cowpox and parainfluenza viruses in humans, and the turkey human virus in chickens. more recently, the use of avipox viruses such as fowlpox and canarypox, which undergo an abortive infection in humans, is being used in humans as vectors of dna coding for antigens of other infectious agents for which vaccines are not yet available (2). 2. the measles, mumps, rubella, and yellow fever vaccines are typical of the second approach. the wild-type viruses are extensively passaged in tissue culture/animal hosts until an acceptable balance is reached between loss of virulence and retention of immunogenicity in humans. 3. type 2 polio virus is a naturally occurring attenuated strain that has been highly successful. more recently, rotavirus strains of low virulence have been recovered from children's nurseries during epidemics (3). 4 . a fourth approach has been to select mutants that will grow at low temperatures but very poorly above 37°c (4). the cold-adapted strains of influenza virus grow at 25°c and have mutations in four of the internal viral genes (5). such strains were first described in the late 1960s and have since been used successfully in russia, have undergone extensive clinical trials in the united states (6) and the vaccine has now been licensed, but not for young children and the elderly. in contrast to these successes, bacillus calmette-guerin (bcg) for the control of tuberculosis was for many years the only example of a live attenuated bacterial vaccine. although still widely used in the world health organization (who) expanded programme of immunization (epi) for infants, it has given highly variable results in adult human trials. in general, it has proven more difficult to make highly effective attenuated bacterial vaccines, but with increasing examples of antibiotic resistance occurring, there is now a greater effort. a general approach is to selectively delete or inactivate one or more genes (7). salmonella strain ty21a has a faulty galactose metabolism, and strains with other deletions are being made. the most recent addition to this category is a vibrio cholerae vaccine. a new approach is to sequence the bacterial genome to establish the properties and hence the probable location of different proteins in the bacteria, and this has now been done for many different bacteria (8). genetic modification can be useful for complex viruses. thus, 18 open-reading frames, including six genes involved in nucleotide metabolism, have been selectively deleted from the copenhagen strain of vaccinia virus. the product, nyvac, has low virulence but has retained immunogenicity (9). the same approach has been used with simian immunodeficiency virus (siv) but with limited success. it now seems very unlikely that a live, attenuated strain of human immunodeficiency virus (hiv) will ever become available. live, attenuated agent vaccines have the potential to stimulate strong humoral and cell-mediated immune responses that can be highly effective in preventing or clearing a later infection in most recipients. viruses and bacteria can be treated to destroy their infectivity (inactivation) and the product used with varying efficacy as a vaccine ( table 1) . compared to attenuated preparations, inactivated preparations must be given in larger doses and sometimes administered more frequently. the vi-molecular biotechnology volume 29, 2005 ral vaccines are generally effective in preventing disease. the lower efficacy of the influenza viral vaccine is partly due to the difficulty of closely matching the specificity of the virus strains that are circulating when the vaccine finally becomes available. this is due to the continuing antigenic drift that characterizes this virus (10). the only bacterial vaccine of this nature widely used is the whole cell pertussis vaccine, which is quite effective ( table 2) , but has been replaced in some developed counties by the subunit (acellular) vaccine to avoid the adverse effects attributed to the whole cell vaccine (11). inactivated whole vaccines should induce many of the desirable immune responses, particularly infectivity-neutralizing antibodies. generally, they do not induce a class i major histocompatibility complex (mhc)-restricted cytotoxic t-cell response, which is the major response required to clear intracellular infections by viruses, and by some bacteria and parasites. class ii mhc neutralizing antibodies are adequately induced. the generation of antibodies that prevent infections by both intra-and extracellular microorganisms has been regarded as the prime requirement of a vaccine. the epitopes recognized by such antibodies are usually restricted to one of a few proteins or carbohydrate that is exposed at the external surface of the microorganisms. isolation (or synthesis) of such components formed the basis of the first viral and bacterial subunit vaccines. the influenza virus vaccine was composed of the two surface protein antigens, the hemagglutinin and neuraminidase, and the hepatitis b virus vaccine of the surface antigen, hbsag. encapsulated bacteria have a coating of polysaccharides that is not recognized by the immune system of very young children (<2 yr old) and only moderately well by older individuals (mainly an immunoglobulin[ig] m response), therefore polysaccharide vaccines needed to be improved. in 1929, it was found (12) that immunizing with a polysaccharide/protein conjugate gave a much stronger antipolysaccharide response (mainly iggs, because t cells were now involved). much later, we now have three such conjugate vaccines ( table 1 ) that are highly immunogenic and these are saving especially many young lives (13). more will become available. the two bacterial toxoids, tetanus and diphtheria, represent a special situation in which the primary requirement was neutralization of the toxin secreted by the invading bacteria. whereas toxoids have traditionally been made by treatment of the toxins with chemicals, it is now being achieved by genetic manipulation (14) . hbsag exists in the blood of hepatitis b virus (hbv)-infected people and infected blood was the source of antigen for the first vaccines. production of the antigen in dna-transfected yeast cells initiated the era of genetically engineered vaccines (15, 16) . a second genetically engineered subunit preparation from borrelia burgdorferi to control lyme disease later became available in the mid-1990s but was withdrawn from sale in 2002 because of poor sales. all available data concerning the efficacy and safety of candidate vaccines are reviewed by regulatory authorities before registration (17). at that stage, potential safety hazards, which occur at a frequency of about 1/5000 doses, should have . this advice has been accepted by several other developed countries such as australia. after successful vaccination campaigns that greatly reduced disease outbreaks, the low levels of undesirable side effects after vaccination gained some notoriety. the evidence bearing on causality and specific adverse health outcomes following vaccination against some childhood viral and bacterial diseases, mainly in the united states, has been evaluated by an expert committee of the institute of medicine (iom) (22). the possibility of adverse neurological effects was of particular concern, and evidence for these as well as several immunological reactions, such as anaphylaxis and delayed type hypersensitivity, was examined in detail. in the majority of cases, there was insufficient evidence to support a causal relationship, and where the data were more persuasive, the risk was considered to be extraordinarily low. measles has provided an interesting example of vaccine safety. the experience of the who epi shows that the vaccine is very safe (23). although natural measles infection induces a state of immunosuppression, even immunocompromised children rarely show this effect after vaccination (22) . in developing countries, the epi schedule is to give the vaccine at 9 mo of age. this delay is meant to allow a sufficient drop in the level of maternally derived antibody so that the vaccine can take. in some infants, this decay occurs by 6 mo, resulting in many deaths from measles infection in the ensuing 2 to 3 mo, "high-titer" vaccines were therefore developed that could be given at 6 mo of age. trials in several countries showed the apparent safety and efficacy of the new vaccine, but after who authorized its wider usage, some young girls in disadvantaged countries died, leading to the withdrawal of the vaccine (24). one possibility is that the high titer vaccine caused a degree of immunosuppression sufficient to allow infections by other infectious agents. another example is the rotavirus vaccine that was registered for use in the united states in 1998. it was withdrawn in 1999 after administration to 1.5 million children because of an unacceptable level-about one case per 10,000 recipients in some areas-of the condition intussusception (25). this was surprising because the disease itself does not cause this condition. fortunately, other preparations are well advanced as the need for such a vaccine is great especially in developing countries. it is particularly difficult to attribute causality to the onset of diseases that may occur many months after vaccination. when such claims are made, national authorities or who establish expert committees to review the evidence. there have been claims-rarely in the medical literature but often by antivaccination groups-that a vaccine can cause sudden infant death syndrome (sids), multiple sclerosis, autism, asthma, or a specific allergy. there is no sound medical, scientific, or epidemiological evidence to support these claims. for example, at least 11 different investigations have found no evidence that inflammatory bowel disease and autism occur as a result of measles, mumps, and rubella (mmr) vaccination (26, 27) . this claim was apparently made by the antivaccination lobby simply because the increase in austism in the united states late last century coincided with the introduction of a second mmr vaccination. many countries keep yearly records of diseases incidence and the cdc in atlanta have kept records from as early as 1912. table 2 compares the incidence of cases of some common childhood infectious diseases during a major epidemic before the vaccine was licensed, with levels in 1997 and 2002, some years after the introduction of the vaccine. most show a very high level of efficacy. pertussis, the cause of whooping cough, is an exception in showing an increase in cases recently and this may lead to the introduction of an additional dose of the vaccine. the crowning achievement of a vaccination program would be to eradicate some infectious diseases. jenner (28) had proposed that his new vaccination procedure could be used to eradicate smallpox, but a century and a half passed before this suggestion was taken seriously. having achieved control of smallpox infections in their country, the russians in 1954 proposed to the world health assembly (wha) the global eradication of that disease. a 10-yr unfunded, voluntary program was initiated by who. great progress was achieved in developed countries but in 1964, there were 2 million deaths and 15 million disease cases in developing countries. in 1966, a 10-yr funded (usd $300 million) program was begun and despite many difficulties, such as vaccine shortages and wars, the last case was treated in 1977. eradication was declared to the wha in 1980 (28). three senior investigators, f. fenner, d. a. henderson, and i. arita shared the 1988 japan prize for this achievement. would it be possible to repeat this outstanding success with another infectious disease? poliomyelitis and measles were two considered. both ful-filled the necessary requirements but not the desirable properties ( table 3) . the former was chosen as there was already some success in limiting transmission of the disease in developed countries. the global polio eradication initiative was spearheaded by who, rotary international, the united nations children's fund (unicef), and cdc in 1988, and joined later by other groups. there were many difficulties. the infection persisted in some individuals for many months and at one stage, one of the three vaccine virus strains mutated into a virulent form. repeated vaccinations became necessary so that the deadline had to be progressively extended until to 2005. in 2004, the virus was still endemic (less than 1000 cases) in six countries, but especially in nigeria. in kano province, a religious group refused vaccination because of concerns that the vaccine was contaminated with hiv and other factors that could affect the fertility of their children. by the time (mid-2004) such contamination claims were shown to be wrong and the concern abated, infectious cases had occurred in 12 previously infection-free countries in west and central africa. re-vaccination of children under the age of 5 yr in 22 countries in this region will begin in late 2004 and extend into 2005, provided an ongoing funding gap of usd $100 million is overcome (29). the reason for such funding is that for every case of infection found, up to 1 million children in surrounding areas may need to be re-vaccinated. having been at the edge of successful eradication, it would be a disaster if the extra funding was not made available. as of early october 2004, this vaccination program has begun. for 50 yr until the measles vaccine was introduced in 1963, the yearly incidence of infections in the united states was never less than 100,000 cases, with epidemics occurring every 2 to 3 yr. the maximum incidence of reported cases was 894,134 in 1941 ( table 2) . introduction of the measles vaccine in 1963 led to a reduction in incidence of cases by >99.9%. a 3-yr epidemic of 55,000 cases began in 1989 and led to the introduction of a second vaccine dose for mainly chil-molecular biotechnology volume 29, 2005 dren about to go to school. in 2000, the cdc concluded that measles was no longer an endemic disease in the united states. in 2001, it was established that all 132 cases had been brought into the country by visitors from specified countries. in 1994, ministers of health from north and south america met and resolved to eliminate measles from all the western hemisphere by 2000. the pan american health organization (paho) under ciro de quadros contributed greatly with catch-up and follow-up campaigns with the result that for 9 mo in 2003, transmission of measles in the whole of the americas was prevented. in 1996, the paho strategy was successfully extended to seven south african countries (30). although a major difficulty to prevent transmission of measles is the high level of vaccination coverage necessary (95%), the interruption of indigenous measles transmission was also achieved in cuba from 1988, and in england and wales from 1995. some countries such as japan, italy, france, and germany do not consider this a national priority (31). but there is still hope that one day, a global eradication program will be announced: "it is not a dream to imagine a world free of measles by year 2015" (30). this may depend on the final result of the poliomyelitis eradication campaign. and new vaccines vaccines are not currently available for more than 40 infectious agents pathogenic for humans. vaccine development is well advanced for some of these and is in progress for the most of the remainder. table 4 contains a short list of agents for which improved vaccines are desirable, and a longer list of those agents that should have priority in vaccine development plans. although the current measles vaccine is highly effective, a vaccine that could be administered well before 9 mo would save many lives. in the new category, the "terrible trio," hiv, m ycobacterium tuberculosis, and malaria, would be at the top of many lists. they are major killers and despite great efforts over many years, effective vaccines are some way off. fortunately in subtropical developed countries, and with effective drugs, each agent is largely controlled. the advantages of this approach include the fact that the anti-idiotype should mimic both carbohydrate-based and peptide-based epitopes, and the conformation of the epitopes in question. the potential advantages of the former point have disvaccines (12,13) . the use of this technology may be largely restricted to very special situations, such as identifying the nature of the epitope recognized by very rare antibodies that neutralize a wide spectrum of hiv-1 primary isolates (32). the receptors on t cells (lymphocytes) recognize on the surface of the antigen-presenting cell (apc) or infected target cell, a complex composed of a peptide from a protein in the apc or infected cell attached to a mhc antigen. simi-larly, the ig receptors on b cells may recognize a pattern on an antigen often formed by a linear peptide sequence. therefore, it could be advantageous to link such linear sequences to form a particular antigen. the sequences may contain either b-cell epitopes or t-cell determinants, or often both (see subheading 8.2.). sequences containing b-cell epitopes may either be conjugated to carrier proteins, which act as a source of t-helper cell determinants, or linked in different ways to achieve particular tertiary configurations. some of the obvious advantages of this approach are that the final product contains the critical components of the antigen and avoids other sequences that may mimic host antigen sequences, and thus potentially induce an autoimmune response. multiple antigenic peptide systems (maps) are more immunogenic than individual sequences (33), and the immunogenicity of important "cryptic" sequences may sometimes be enhanced by the deletion of other segments (34). new methods of synthesis offer the possibility of more closely mimicking the conformational patterns in the original protein. this approach is likely to be applicable, especially for some bacterial and parasitic vaccines. however, the first peptide-based candidate vaccine, despite giving encouraging results in an early small trial, gave disappointing results in an efficacy trial in malaria endemic regions (35). a preparation composed of polymers of linked peptides from group a streptococcus, which was effective in a mouse model (36), is currently undergoing clinical trials. this is now a well-established procedure. three cell types have been used: (1) prokaryotes (bacteria), (2) lower eukaryotes, mainly yeast; and (3) mammalian cells, either primary cells (e.g., monkey kidney), cell strains (with a finite replicating ability), or cell lines (immortalized cells such as chinese hamster ovary cells [cho]). each has its own advantages. as a general rule, other bacterial proteins should preferably be made in transfected bacterial cells, and human viral anmolecular biotechnology volume 29, 2005 tigens, especially glycoproteins, in mammalian cells, because of the substantial differences in properties, such as post-translational modifications in different cell types. table 5 lists the viruses and bacteria mostly used for this purpose. of the viruses, the greatest experience has been with vaccinia and its derivatives such as the highly attenuated, modified vaccinia virus ankara. these have a wide host range, possess many different promoters, and can accommodate dna coding for up to 10 averagesized proteins. the avipox viruses, canary and fowlpox, undergo abortive infection in mammals, making them very safe to use as vectors (37). adenovirus (38), polioviruses (39), and salmonella (40) are mainly used for delivery by a mucosal route, although vaccinia and bcg have been administered both orally and intranasally. making such chimeric vectors has also been an effective way to evaluate the potential role of different cytokines in immune processes. inserting dna coding for a particular cytokine as well as that for the foreign antigens results in the synthesis and secretion of the cytokine so its maximum effect should be displayed. thus, interleukin (il)-4 and il-12 have been shown to have dominant effects in inducing a humoral or cell-mediated immune response, respectively. incorporation of dna coding for il-4 into the genome of ectrome-lia virus greatly increased the virulence of this virus in otherwise resistant mice, and even if the latter had been immunized to increase resistance before challenge (41). the most fascinating and exciting of the recent approaches to vaccine development has been the injection of plasmids containing the dna coding for antigens of interest, either directly into muscle cells or as dna-coated tiny gold beads into the skin, using a "gene gun" (42,43) . in the latter case, some coated beads bind to the toll-like receptor (tlr) 9 on langerhan's (dendritic) cells, and during passage of the cells to the draining lymph nodes (24 h), the expressed foreign protein is processed. appropriate peptide sequences attach to mhc molecules and the resulting complex is expressed at the cell surface. these complexes are recognized by naïve, immunocompetent t cells in the node and this stimulates activation of the cells. a basically similar process occurs after muscle injection. in lower primates, this procedure primarily induces a type-1 t-cell response, resulting in both an antibody-and cell-mediated immune response, with both cd4 + and cd8 + effector t cells. in many respects, this is similar to the response following an acute infection. one advantage is that the response to dna occurs in the presence of specific antibody to the encoded antigen. our knowledge of the properties of lymphocytes-the cell type of major importance in vaccine development-has increased enormously in recent years (44). the major role of b lymphocytes is the production of antibodies of different isotypes and of course, specificity. the other class of lymphocytes, t cells (so-called because they mature in the thymus), consist of two main types. one, with the surface marker cd4, exists in two subclasses, the th-1 and th-2 cells (h = helper activity). the major role of th-2 cells is to help b cells differentiate, replicate, and in the form of plasma cells, secrete antibodies of a defined specificity, and of different subclasses, igg, ige, and iga. furthermore, there are different subclasses of igg. this is facilitated by the secretion of different cytokines (interleukins), which are listed in table 6 . th-1 cells also have a small but important role in helping b cells produce antibody of various igg subtypes, but the overall pattern of cytokine secretion is markedly different. factors, such as interferon (ifn)-γ, tumor necrosis factor (tnf)-α, and tnf-β, have several functions, such as antiviral activity and up-regulation of components (e.g., mhc antigens on other cells). with macrophages, this can lead to their activation and if infected, greater susceptibility to recognition by t cells. th-1 cells can also mediate (through cytokine secretion) delayed-type hypersensitivity (dth) responses that may have a protective role in some infections. it was recognized that during an infection by a pathogenic microorganism, the immune response may be suppressed to limit immunopathology, and this has recently been shown to be due to a particular cd4 + t cell, now called a regulatory cell (treg). it is not expected that these cells would be activated during vaccination by a live attenuated vaccine. a second type of t cell has the cell-surface marker cd8, and its pattern of cytokine secretion is similar to that of cd4 + th-1 cells, although they generally mediate dth responses rather poorly. as primary effector cells in vivo, cd8 + t cells recognize and lyse cells infected by a virus or by intracellular bacteria and some parasites, hence the name cytotoxic t lymphocytes (ctls). an important aspect of this arm of the immune remolecular biotechnology volume 29, 2005 sponse is that susceptibility of the infected cell to lysis occurs shortly after infection, and many hours before infectious progeny is produced, thus giving a "window of time" for the effector cell to find and destroy the infected cell (45). both cd4 + and cd8 + t cells recognize on the surface of the apc a complex between a mhc molecule and a peptide from the foreign antigen. in the first case, the peptide (average length, 15 amino acids), which is derived from antigen being degraded in the lysosomes, complexes with class ii mhc antigens. in the second case, the peptide (average length, 9 amino acids) is derived from newly synthesized antigen in the cytoplasm, and binds to class i mhc molecules. class ii mhc antigens are expressed mainly on cells that can act as apcs. in contrast, nearly all cells in the body may become infected and express class i mhc molecules. thus, the role of ctls has been described as performing a continuous molecular audit of the body (46). table 6 ascribes particular roles to specific antibody and to the t-cell subsets. some general conclusions regarding adaptive immune responses are: 1. specific antibody is the major mechanism for preventing or greatly limiting an infection; 2. ctls are the major mechanism for controlling and finally clearing most acute intracellular infections (47 during an acute model infection such as murine influenza, the sequence of the appearance in the infected lung of adaptive responses is: first, cd4 + th cells, then cd8 + ctls, and finally antibody-secreting cells (ascs). the ctls are largely responsible for virus clearance and it has been thought that the subsequent decline in ctl activity that occurs shortly after infectious virus can no longer be recovered (50), and this is because of the short half-life of these cells. the two dominant cytokines are il-12, which favors a th-1 response (51), and il-4, which favors a th-2 response. the production of il-4, which results in the enhancement of the antibody response, may curtail the life of the ctls. the finding that if il-4 is "artificially" induced very early after infection, ctl production is substantially suppressed (52), supports this notion. one important point is that a large pool of memory ctls has already been formed by the time that ctl activity usually disappears. these memory cells persist, and are rapidly activated if the host is exposed to the same or a closely related infectious agent at a later time. it is now recognized that, as well as affecting the magnitude and persistence of immune responses to noninfectious preparations, adjuvants can also greatly influence the type of immune response (53). some adjuvants, such as alum and cholera toxin and its b subunit, favor cd4 + th-2 responses. water-in-oil emulsions, such as freund's complete and incomplete adjuvant and lipopolysaccharide, favor a th-1 response. a variety of delivery systems is available for the induction of ctl responses (54). although new technologies are making it more likely that attempts to develop vaccines to an in-creasing number of infectious agents will be successful, many other factors can influence the final outcome. some of these are: 1. the simpler the agent, the greater the chance that important protective antigens will be identified. 2. the occurrence of great antigenic diversity in the pathogen can be a major hurdle, especially in the case of rna viruses because escape mutants (antigenic drift) may readily occur. 3. integration of dna/cdna into the host cell genome is likely to lead to lifelong infection, which is difficult for a vaccine to prevent/overcome. 4. if a sublethal natural infection does not lead to protection from a second infection, it becomes necessary to understand the pathogenesis of the infection and how the normal protective responses (antibody, cell-mediated) are subverted or evaded. 5. the ready availability of an inexpensive animal model that mimics the human disease can be very helpful, but is rare. most early studies are done with mice but it is becoming increasingly common to then work with lower primates before initiating clinical studies. vaccine delivery is a major cost component in vaccination programs. combining vaccines so that three or more can be administered simultaneously results in considerable savings, therefore there are determined efforts to add other vaccines to longtime successful combinations (diphtheria, acellular pertussis, tetanus [dapt] and mmr) such as dapt-hepatitis b-h aemophilus influenzae type b. there must be compatibility and no interference by one component on the other. there is the risk of antigenic competition that occurs at the t-cell level, and the likelihood of such interference is difficult to predict. however, in the case of mixtures of protein/carbohydrate conjugates, using the same carrier protein should decrease this risk. the use of the same vector (e.g., the same poxvirus) in mixtures of chimeric constructs should minimize this difficulty. vaccination with dna should offer the same advantage. some antigens to be used in vaccines, mainly subunits, and many antibodies can be produced in transgenic plants. initially it was thought that simply "eating the fruit of the plant" would achieve satisfactory vaccination (i.e., edible vaccines) and that the technique would be particularly suitable for use in developing countries (55). but the idea has evolved more recently into obtaining licensed products under strict biological regulations and subject to the same safeguards as products produced by conventional techniques (56). in the long run, such products may be less expensive than conventional products, but not by a very large margin. most recently, although 45 different antigens have been produced in this way, there are still substantial regulatory hurdles to be overcome in the use of these products (57). in an effort to limit vaccine administration by needles, progress is being made in inducing immunization by direct application of the antigen, with a strong adjuvant such as cholera toxin, to prewashed skin, using a patch. with this technique, called transcutaneous immunization, the antigen rapidly contacts and is taken up by dendritic (langerhan's) cells in the epidermis and later, on arrival at draining lymph nodes, the processed antigen is presented to t cells (58). using a gene gun is an efficient way of introducing dna coding for different antigens into the epidermis and directly into langerhan's cells and sometimes into the cell's nucleus. tiny gold beads are coated with the dna and this becomes an efficient way to induce an immune response (59). in contrast with the time-honored approach of giving several doses of the same preparation of an antigen to obtain a stronger and longer immune response, the concept arose of priming with one formulation of an antigen and boosting with a different formulation. immunization of naïve volunteers with an hivgp160/ vaccinia virus construct followed by boosting with a recombinant gp160 preparation gave higher anti-gp160 antibody titers compared to using either preparation for both priming and boosting (60). it was then shown that molecular biotechnology volume 29, 2005 mice immunized with a chimeric dna preparation and later boosted with chimeric fowlpox virus both expressing influenza hemagglutinin (ha), gave anti-ha titers up to 50-fold higher than those found after two injections of the same preparation (61). this approach has been vigorously pursued to induce high and persistent ctl responses to hiv-1, siv, ebola virus, m. tuberculosis, and plasmodia antigens with positive results in mice or monkeys (2). two phase 1 clinical trials using preventive aids vaccine candidates, one by an australian consortium supported by the national institutes of health, washington, and the other, an oxford-nairobi-uganda partnership supported by the international aids vaccine initiative, new york, were recently completed. both used chimeric dna followed by a chimeric live poxvirus vector to generate strong cell-mediated immunity (cmi) responses (ctls), but unfortunately they gave disappointing results (62). the reason is not clear but at least temporally, the results cast a shadow over this technology in humans. studies are proceeding using different chimeric live vectors for priming and boosting. whole genome sequencing of complex microbes such as bacteria and parasites is poised to revolutionize the way different components are chosen to form a vaccine (63).this enables the characterization of potential candidate proteins that might be recognized by infectivity-neutralizing antibodies, and which ones may provide important t-cell determinants. in one recent example, mice immunized with with 6 of 108 proteins from steptococcus pneumoniae that had been identified from the dna sequence as having appropriate structural characteristics, were protected from disease when later challenged with this organism (64). infectious agents in his book, guns, germs and steel (65), jared diamond retraces the history of civilization over the past 13,000 yr. (this should be of special interest to those younger than 50 yr old.) because of the exposure, of especially europeans, to domesticated and other animals for most of this period, some of the animal infectious agents had evolved over many years to become essentially human infectious agents. these include smallpox, influenza, tuberculosis, malaria, plague, measles, and cholera. many infected people died, but the population survived and expanded. but when they traveled abroad, some of the group might be infected. beginning with columbus's voyage in 1492, shortly followed by cortes' invasion of the aztec empire, the european invasion of north america, and in the late eighteenth century, the british invasion of australia, the mortality rate of indigenous peoples exposed for the first time to these agents was often as high as 50% and occasionally much higher. within the first 100 yr, the indigenous populations in these regions had decreased by at least 90%, due mainly to the introduced infections. at the beginning of the twentieth century, in well-off countries, families were large because it was expected that several children in one family could die from childhood infections. parents were especially proud if they reached the biblical goal of three score years and ten. but why was the mortality rate so high? the 1996 nobel prize in physiology or medicine was awarded to peter doherty and rolf zinkernagel for research they had done in the early 1970s at the australian national university. based on their experimental findings, they predicted that ctls could distinguish between infected and normal cells because the former expressed at the cell surface a complex between a mhc molecule and a protein (later shown to be a peptide) from the infecting virus (66). thirteen years later, this interpretation was shown to be correct by xray crystallography (67). the human chromosome 6 contains 9 loci, each containing dna coding for a particular mhc antigen. at conception, the fetus receives 9 such genes from each parent giving a total of 18. dna at six loci codes for class i mhc molecules and at the other 12 loci, the dna codes for class ii mhc molecules. yet on a population basis, there are many, sometimes >100 dna molecules coding for different mhc antigen specificities, anyone of which can occupy the same loci in different individuals. thus, individual humans possess only a small sample of the total number of mhc specificities. one result is that it was highly unlikely that all people would have the best protective response to all infections. person a may make a strongly protective response against infection x, but a weakly protective response against infection y. person b may make the opposite responses. the resistance or susceptibility of different inbred strains of mice to different pathogens supports this view. thus, c57bl mice are relatively resistant to ectromelia (mousepox) infection, whereas balbc mice are susceptible, with a high mortality rate. the protection afforded by the vaccination programs, especially death from childhood infections in nonindigenous populations in developed countries, has dramatically changed the previous family pattern. at present, the family size is generally much smaller as parents expect all children to survive, and many adults are living much longer, well into their 80s or 90s. there are many known infectious agents for which vaccines are not yet available, but the biggest danger is emerging infectious diseases. a recent article on this topic lists about a dozen such agents (68). table 7 lists some of these agents where there is substantial evidence for their animal origin, showing them as zoonoses or vector-borne diseases. many induce a high mortality rate in humans. some such as sars (severe acute respiratory syndrome) has spread around the world. there were three influenza virus pandemics in the past century, the 1918 "spanish flu" causing 50 million deaths due to the special properties of the hemagglutinin gene of the virus (69). the appearance of a/h5n1 in hong kong in 1997 and in neighboring countries in 2004, and possibly the first indication (new york times, sept 30, 2004) that it may have recently spread from one person to another, has heightened concern. the maximum opportunity for a pandemic occurs when the bird virus infects a person currently infected with a human strain, allowing the different particles to enter the same cells and while replicating, to exchange genes. the hiv-1, which causes aids, is also a very serious hazard as there have now been 60 million infections worldwide. it jumped from non-human primates about 70 yr ago, probably by the consumption of infected "bushmeat" and this form of transmission has occurred six more times since. in richer countries, it is controlled by a complex mixture of drugs. in the absence of treatment, the average time to death is 10 yr but can be as long as 20 yr, so it is difficult to know the exact death rate, but it is >90%. there is a small group of long-term nonprogressers whose immune system keeps the hiv virus levels very low. in some african countries, which have about a 30% incidence of hiv infection, the average lifespan has dropped from 50 yr to about 30 yr. despite intensive efforts, it has so far not been possible to develop an effective vaccine. other infections listed in table 7 have mortality rates varying from about 9% (sars) to between 50% and 90% (ebola and marburg hemorrhagic fevers). there is a number of infections called reemerging diseases. these are infections that were reasonably localized but have suddenly spread to other regions. although originally confined to one region, one example, west nile virus, is causing epidemics of encephalitis in the united states and russia due to migratory birds, and an abundance of both vector mosquitoes and susceptible local bird species in those two countries. as the population of the world grows and there is increasingly frequent interaction between mankind and wildlife, it is inevitable that new diseases and re-emerging diseases will continue to occur. efforts to rapidly identify, investigate, and control such outbreaks will continue to be coordinated by who, ably assisted by designated authorities in different countries such as the cdc in the united states. although special situations such as hiv/aids can be kept under control in rich countries by a cocktail of drugs, the basic global need is to be able to develop appropriate vaccines. this must continue to be a top priority if we and our descendants are to live fruitful lives vaccines advances in immunology: vaccines and vaccination rotavirus vaccine temperature sensitive mutant vaccines influenza vaccine-live current status of live, attenuated influenza virus in the us regulation of host immune responses by modification of salmonella virulence genes the use of complete genome sequences in vaccine design nyvac, a highly attenuated strain of vaccinia virus cocirculation and divergence of human influenza viruses pertussis vaccine chemoimmunologial studies on conjugated carbohydrate proteins. 11. immunological specificity of synthetic sugar-protein antigen preparation of polysaccharide-conjugate vaccines genetic detoxification of bacterial toxins vaccine perspectives from the vantage of hepatitis b the cellular basis for the lack of antibody responses to hepatitis b vaccine in humans assuring the quality and safety of vaccines. regulatory expectations for licensing and batch release vaccine protocols an epidemiological and clinical evaluation of guillain-barre syndrome reported in association with the administration of swine influenza virus vaccine acute encephalopathy followed by permanent brain injury or death associated with further attenuated measles vaccines vaccines today adverse events associated with childhood vaccination. evidence bearing on causality. institute of medicine indications and contraindications for vaccines used in the expanded programme of immunization increased mortality following high titer measles vaccines: too much of a good thing intussusception among infants given an oral rotavirus vaccine measles, mumps, rubella immunization, autism, and inflammatory bowel disease: an update. communicable disease intelligence 23 mmr vaccine: the continuing saga smallpox and its eradication. world health organization new polio cases confirmed in guinea, mali and sudan. cases reported as kano, nigeria, resumes immunizations is global measles eradication feasible? perspectives for the elimination/eradication of diseases with vaccines crystal structure of a neutralizing human igg against hiv-1: a template for vaccine design synthetic peptide vaccine design: synthesis and properties of a high density multiple antigenic peptide system efficacy trial of a malaria vaccine spf66 in gambian infants towards a vaccine for rheumatic fever: identification of a conserved target epitope on m protein of group a streptococci new multi-determinant strategy for group a streptococcal vaccine designed for the australian aboriginal population live viral vectors: vaccinia virus live viral vectors: construction of a replication-deficient recombinant adenovirus academic development of attenuated salmonella strains that express heterologous antigens expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox molecular medicine; dna vaccines dna vaccination: an update the immune system cytotoxic t cells recognise very early, minor changes in ectromelia-infected target cells unravelling the mysteries of molecular audit: mhc class i restriction the immunology of vaccination the role of antibody in controlling and/or clearing virus infections expression of cytokines by recombinant vaccinia viruses: a model for studying cytokines in virus infections in vivo the immune response to antigens; the immunological principles of vaccination the adjuvant effect of il-12 in a vaccine against leishmaniasis major interleukin 4 mediates down regulation of antiviral cytokine expression and cytotoxic t lymphocyte responses and exacerbates vaccinia virus infection in vivo adjuvant formulations for experimental vaccines comparison of numerous delivery systems for the induction of cytotoxic t lymphocytes by immunization plant biotechnology: new procedures and applications oral vaccines derived from transgenic plants edible vaccines not ready for main course transcutaneous immunization: a human vaccine delivery strategy using a patch the powderjet particle-mediated epidermal delivery of dna vaccines. a new technology augmentation of human immunodeficiency virus type-1 neutralizing antibody by priming with gp160 recombinant vaccinia and boosting with rgp160 in vaccinia-naive adults generation of enhanced immune responses by consecutive immunization with dna and recombinant fowlpox virus hiv dodges one-two punch the use of complete genome sequences in vaccine design use of a whole genome approach to identify vaccine molecules affording protection against streptococcus pneumoniae infection guns, germs and steel. a short history of everybody for the past 13,000 years. vintage a biological role for the major histocompatibiliy antigens the foreign antigen-binding site and t cell recognition regions of class 1 histocompatibility antigens the challenge of emerging and re-emerging infectious diseases enhanced virulence of influenza a viruses with the haemagglutinin of the 1918 pandemic virus key: cord-278250-dwok857k authors: li, heng; li, hongzhe; wang, jingjing; guo, lei; fan, haitao; zheng, huiwen; yang, zening; huang, xing; chu, manman; yang, fengmei; he, zhanlong; li, nan; yang, jinxi; wu, qiongwen; shi, haijing; liu, longding title: the altered gut virome community in rhesus monkeys is correlated with the gut bacterial microbiome and associated metabolites date: 2019-08-19 journal: virol j doi: 10.1186/s12985-019-1211-z sha: doc_id: 278250 cord_uid: dwok857k background: the gut microbiome is closely associated with the health of the host; although the interaction between the bacterial microbiome and the whole virome has rarely been studied, it is likely of medical importance. examination of the interactions between the gut bacterial microbiome and virome of rhesus monkey would significantly contribute to revealing the gut microbiome composition. methods: here, we conducted a metagenomic analysis of the gut microbiome of rhesus monkeys in a longitudinal cohort treated with an antibiotic cocktail, and we documented the interactions between the bacterial microbiome and virome. the depletion of viral populations was confirmed at the species level by real-time pcr. we also detected changes in the gut metabolome by gc-ms and lc-ms. results: a majority of bacteria were depleted after treatment with antibiotics, and the shannon diversity index decreased from 2.95 to 0.22. furthermore, the abundance-based coverage estimator (ace) decreased from 104.47 to 33.84, and the abundance of eukaryotic viruses also changed substantially. in the annotation, 6 families of dna viruses and 1 bacteriophage family were present in the normal monkeys but absent after gut bacterial microbiome depletion. intriguingly, we discovered that changes in the gut bacterial microbiome composition may promote changes in the gut virome composition, and tryptophan, arginine, and quinone may play roles in the interaction between the bacterial microbiome and virome. conclusion: our results indicated that the clearly altered composition of the virome was correlated with depletion in the bacterial community and that metabolites produced by bacteria possibly play important roles in the interaction. electronic supplementary material: the online version of this article (10.1186/s12985-019-1211-z) contains supplementary material, which is available to authorized users. the gut microbiome of an animal consists of bacteria, viruses, fungi and so on. this intricate ecosystem interacts with the adjacent epithelial layer, and the microbes perform metabolic functions, protect against pathogens, and condition the immune system, and through these basic functions, these microbes directly or indirectly affect most of the physiological functions of the host [1, 2] . in recent years, variations in the bacterial community composition have been shown to correlate with infection outcome, inflammatory bowel disease [3] , diabetes [4] , obesity [5] , and depression [6] , and fecal microbiome transplantation has become an effective treatment for refractory clostridium difficile infections and other diseases [7, 8] . the mechanisms of interaction between the gut bacterial microbiome and the host are very complex, and other components also play crucial roles in this process. in addition to bacteria, viruses are also abundant in the gut [9] and have been hypothesized to markedly alter the structure and function of the bacterial community [10] [11] [12] . additionally, chronic viral infection can confer increased resistance against pathogenic challenges [13] . gut virome alteration has been observed in inflammatory diseases such as inflammatory bowel disease and crohn's disease [14] . the recent advent of highthroughput sequencing methods has made it possible to study these communities and their relationships with health and disease in detail [15] . bacterial communities play an essential role in host health, but further research is still warranted to obtain an in-depth understanding of the mechanisms underlying this role. transfer of whole virome communities between humans was documented in fecal microbiome transplantation [16] , and the difference varied more widely between gut viromes than between gut bacterial microbiomes in humans [17] . however, the relationship between the bacterial microbiome and the virome has rarely been studied, despite its likely medical importance. previous research has shown close relationships between single viral species and single bacterial species [18, 19] , and single viral species could trigger shifts in the bacterial microbiome and the virome [20, 21] . at the same time, enteric bacteria were seen to be required for efficient infection by [22, 23] or suppression of [24] viruses, and the richness of the gut bacterial microbiome had an obvious effect on bacteriophage composition [25] ; moreover, the gut virome composition in humans was examined, and bacteriophage diversity was found to be inversely correlated with naturally occurring bacterial diversity in human infants during healthy development [26] . however, few studies have focused on how the whole virome, a diverse community consisting of eukaryotic rna and dna viruses and bacteriophages, interacts with the bacterial microbiome. rhesus monkeys are good mammalian research models that are closely related to humans, and the virome composition of these animals was seen to be affected by simian immunodeficiency virus infection [20] . we hypothesized that there is a close relationship between the whole gut virome and bacterial microbiome, and the bacterial microbiome could be depleted by treatment with an antibiotic cocktail in rhesus monkeys. we then examined the virome composition to detect the direct effects of the bacterial microbiota on the virome. we performed 16s rrna amplicon sequencing of the fecal bacteria and metagenomic analysis of fecal viromes from rhesus monkeys treated with an antibiotic cocktail. our results suggest that a majority of bacteria were depleted after the monkeys were treated with antibiotics and that the composition of the whole virome changed drastically. importantly, alteration of the virome along with shifts in the composition and function of the gut bacterial community and metabolites from gut bacteria may have played an important role in the interaction. the rhesus monkey cohort described in this study was housed at the institute of medical biology, chinese academy of medical sciences (imbcams). an antibiotic cocktail containing ampicillin, streptomycin, kanamycin, metronidazole, and vancomycin was administered orally at a dose of 15 mg/ kg 3 times per day for 2 weeks. three healthy one-year-old rhesus monkeys were treated with antibiotics, and fresh fecal samples were collected one day before treatment with antibiotics and 5, 8, and 9 days after treatment with antibiotics and stored at − 80°c for subsequent analysis. fresh fecal samples from an additional three normal monkeys were collected after the monkeys were treated with antibiotics for 9 days. the bacterial community of each sample was detected by 16s rrna amplicon sequencing, and the virome communities in the samples collected before treatment with antibiotics and in those collected after treatment with antibiotics for 9 days were detected by deep sequencing. because metabolome analysis requires 6 biological duplications, the metabolomes of samples collected before treatment with antibiotics and of those collected after treatment with antibiotics for 8 and 9 days were detected by gc-ms and lc-ms. in our analysis, samples collected before treatment with antibiotics were used as the control group, and samples collected after treatment with antibiotics were used as the experimental group. bacterial 16s rrna amplicon sequencing dna was extracted from fecal samples, and pcr was performed with the barcode primers 338f/806r to obtain amplicons of hypervariable regions v3 and v4 for phylogenetic discrimination analysis [27] . libraries were pooled by using a truseqtm dna sample prep kit and sequenced using an illumina miseq sequencer. sequences were assigned to closed-reference operational taxonomic units (otus) at a 97% identity threshold using bacterial 16s rrna amplicon sequences from the silva 128/16sbacteria database. the otu data were rarefied to the smallest effective sample sizes [28] ; rarefaction is a homogenization method that is used to randomly draw otus to the same quantity based on a minimum value. the α diversity, which includes the abundance-based coverage estimator (ace) and shannon diversity index, was analyzed by mothur (version v.1.30.1), and statistical significance was evaluated by student's t-test. kyoto encyclopedia of genes and genomes (kegg) prediction analysis of the bacterial microbiome [29, 30] we performed a kyoto encyclopedia of genes and genomes (kegg) prediction analysis of the bacterial microbiome using picrust. picrust contains the cluster of orthologous groups of proteins (cog) and kegg ortholog (ko) information corresponding to greengene id numbers. for metagenome prediction, picrust takes an input otu table containing identifiers that match tips from the marker gene with corresponding abundances for each of the otus across one or more samples. first, picrust normalizes the otu table based on 16s rrna amplicon copy number prediction so that the otu abundances accurately reflect the true abundances of the underlying organisms. the metagenome is then predicted by looking up the precalculated genome content for each otu, multiplying the normalized otu abundance by each ko abundance in the genome and summing these ko abundances together per sample. the prediction yields a table of ko abundances for each metagenome sample in the otu table. analysis of similarities (anosim) is a nonparametric test that shows whether the difference between groups is greater than that within groups. the analyses were performed in vegan or qiime in r (version3.2.2) by using the bray-curtis algorithm [31] . fecal samples were suspended in phosphate-buffered saline (pbs) and filtered through a filter with a pore size of 0.45 μm (millipore). the supernatant was enriched by a 30-kda molecular mass filter (ultra-15 30 k, millipore). the concentrate was treated with dnase i (takara) at 37°c for 30 min to eliminate unencapsulated nucleic acids. subsequently, total viral dna was extracted from half of the concentrate using the qiaamp dna stool kit (qiagen), and at the same time, total viral rna was extracted from the other half using the qiaamp viral rna kit (qiagen). the extracted rna was synthesized into double strands using the nebnext rna first strand synthesis module (neb) and the nebnext mrna second strand synthesis module (neb). the dna and double-stranded cdna were amplified by whole-genome amplification (repli-g mini kit, qiagen) and then fragmented into approximately 300-bp fragments by a covaris m220 instrument. then, the fragments were amplified into a pe library by the truseq™ dna sample prep kit and fixed to the chip by bridge pcr using the hiseq 3000/4000 pe cluster kit. the constructs were sequenced on the illumina hiseq platform using hiseq 3000/4000 sbs kits. for virome analysis, we first sheared the adaptor sequences with seqprep and removed reads that were shorter than 50 bp and those that contained n bases with sickle to retain the paired-end reads and single-end reads. we compared these reads to the host (rhesus monkey) genome by bwa and removed the reads belonging to the host. then, we compared all the clean reads with the u.s. national center for biotechnology information (ncbi) nucleotide database to identify the sequences that belonged to viruses and the sequences that did not belong to any known genome, such as those of bacteria, fungi or other known microorganisms. then, contigs were built from these reads. the contigs and all reads that could not be mapped to any known genome in ncbi were compared with the virus protein database in the ncbi nonredundant refseq database (including sequences from swissprot, pir, prf, and pdb and coding sequences (cds) from genbank and refseq) based on amino acid sequences using blastp (blast version 2.2.31+, e-value: 1e-5). these results constituted our virus database and were used to obtain the nonredundant gene catalog by cd-hit. all the reads were compared to our virus database to analyze their richness. the spliced read alignments were predicted by meta-gene, compared to the eggnog database and virulence factors database (vfdb) for cog analysis using blastp (blast version 2.2.31+, e-value: 1e-5) and annotated using vfdb. the abundance results that were similar in 2 or more monkeys were selected, and real-time pcr was used to validate the changes in these viruses (sybr premix ex taq ii, takara). as the template, we used the dna and cdna extracted from fecal samples. the samples that were not detected directly from the dna or cdna were subjected to multiple displacement amplification (mda) (total nucleic acid was amplified by multiple displacement to comprehensively detect both dna and rna viruses [26] ) and then analyzed by real-time pcr. for real-time pcr, we used the ct numbers to show the richness of the virus. viruses that were not detected were not shown. primers were designed to amplify specific regions in the bdellovibrio phage phimh2k (5′-aatcctcaattccagacttcca-3′ (f) and 5′-ccat ttccataagtccgagtg-3′ (r)), bacillus phage b103 (5′-tggcgatgttgatgatgac-3′ (f) and 5′-cttt atttgcgtctgttgtcg-3′ (r)), columbid circovirus (5′-tcaggagacgaaggacacg-3′ (f) and 5′-tggc atcatacatcgggac-3′ (r)), potato virus m (5′-cgct tcgctgctttcg − 3′ (f) and 5′-cggaccattcatac cacca-3′ (r)), marseillevirus marseillevirus (5′-aaagtc ccaagttatcacaagc-3′ (f) and 5′-tttctcgcag cgtcaatg-3′ (r)), simian sapelovirus (5′-ttccatct gctctaaatgctca-3′ (f) and 5′-cagcagttagag cgggtg-3′ (r)), and andean potato mild mosaic virus (5′-aagcccaacatcgttctcc-3′ (f) and 5′-aaga ggatacgggagaaagg-3′ (r)). redundancy analysis (rda) shows the interactions between sample distribution and environmental factors. we used vegan's rda analysis in r with the phylumlevel abundances of the bacterial microbiome as environmental factors. we ran a regression analysis between bacterial microbiome diversity and virome richness with the stats package and plotted the results using the ggplot2 package. the stool samples were suspended in methanol:h 2 o (4:1), ground, ultrasonicated, concentrated and dried so that the metabolome could be analyzed by gc-ms and lc-ms. the derivatized samples were analyzed on an agilent 7890a gas chromatography system coupled to an agilent 5975c msd system (agilent). an hp-5 ms fusedsilica capillary column (30 mm × 0.25 mm × 0.25 μm, agilent) was utilized to separate the derivatives. helium (> 99.999%) was used as the carrier gas at a constant flow rate of 6.0 ml/min through the column. the injector temperature was maintained at 280°c. a volume of 1 μl was injected in splitless mode. the oven temperature was initially 60°c and was then ramped up to 125°c at a rate of 8°c/min, to 190°c at a rate of 10°c/min, to 210°c at a rate of 4°c/min and to 310°c at a rate of 20°c/min; finally, the temperature was held at 310°c for 8.5 min. the temperatures of the ms quadrupole and ion source (electron impact) were set to 150°c and 230°c, respectively. the collision energy was 70 ev. mass data were acquired in full-scan mode (m/z 50-600), and the solvent delay time was set to 5 min. the acquired ms data from gc-ms were analyzed by chromatof software (v 4.34, leco, st joseph, mi). metabolites were qualitatively assessed by the fiehn database, which is linked to chromatof software. briefly, after alignment with the statistic compare component, a csv file was obtained with three-dimensional data sets, including sample information, peak name, retention time, m/z and peak intensities. the resulting data were normalized to the total peak area of each sample in excel 2007 (microsoft, usa) and imported into simca (version 14.0, umetrics, umeå, sweden) to define the 95% confidence interval of the modeled variation. the differential metabolites were selected on the basis of the combination of a statistically significant threshold of variable influence on projection (vip) values obtained from the opls-da model and p values from a two-tailed student's t-test on the normalized peak areas, where metabolites with vip values larger than 1.0 and p values less than 0.05 were included. lc-ms was performed on an ultimate 3000-velos pro system equipped with a binary solvent delivery manager and a sample manager coupled with an ltq orbitrap mass spectrometer equipped with an electrospray interface (thermo fisher scientific); an acquity beh c18 column (100 mm × 2.1 mm i.d., 1.7 μm; waters) was used. the column was maintained at 45°c, and separation was achieved using the following gradient: 5% b-25% b from 0 to 1.5 min, 25% b-100% b from 1.5 to 10.0 min, 100% b from 10.0 to 13.0 min; 100% b-5% b from 13.0 to 13.5 min, and 5% b from 13.5 to 14.5 min at a flow rate of 0.40 ml/min, where b was acetonitrile (0.1% (v/v) formic acid), and a was aqueous formic acid (0.1% (v/v) formic acid). the injection volume was 3.00 μl, and the column temperature was set at 45.0°c. the mass spectrometric data were collected using an ltq orbitrap mass spectrometer equipped with an electrospray ionization (esi) source operating in either positive or negative ion mode. the capillary and source temperatures were set at 350°c, with a desolvation gas flow of 45 l/h. centroid data were collected from 50 to 1000 m/z with a resolution of 30,000. xcms (http://masspec.scripps.edu/ xcms/xcms.php) was used for nonlinear alignment of time domain data and automatic integration and extraction of the peak intensities. default xcms parameter settings were used (major default parameters: profmethod = bin; method = matched filter; step = 0.1) except for full width at half maximum = 8, bandwidth (bw) = 6 and snthresh = 5. variables with < 30% relative standard deviation (rsd) in qc samples were then retained for further multivariate data analysis. the result was a three-dimensional matrix that included retention time and m/z pairs (variable indices), sample names (observations), and normalized ion intensities (variables). the positive and negative data were merged into a combined data set, which was imported into simca-p+ 14.0 software (umetrics, umeå, sweden). the differential metabolites were selected on the basis of a combination of statistically significant vip values obtained from the opls-da model and p values from a two-tailed student's t-test on the normalized peak areas, where metabolites with vip values larger than 1.0 and p values less than 0.05 were included. the differential metabolites were qualitatively assessed using the human metabolome database (http://www.hmdb.ca/) and metlin (https://metlin. scripps.edu/). we performed metagenomic sequencing of fecal samples to detect the bacterial microbiome and virome composition of healthy one-year-old rhesus monkeys housed at the imbcams (fig. 1) . the rhesus monkeys were monitored by blood cell analysis, which is the examination of blood condition and disease by observing the number and distribution of blood cells during the course of antibiotic treatment [32] , and we found no obvious differences between the normal monkeys and those treated with antibiotics (additional file 1: figure s1 ). the composition of the bacterial microbiome was investigated by extracting dna directly from feces for 16s rrna gene amplification. we used the hypervariable regions v3 and v4 to perform phylogenetic discrimination with the barcode primers 338f/806r [27] . in total, 556, 012 amplicon reads (37,067 ± 6872 per sample) were obtained. at the phylum level, the fecal bacterial communities were composed predominantly of high abundances of bacteroidetes (53.1%) and firmicutes (42%) and low levels of proteobacteria (3.82%) (additional file 2: figure s2a ). as expected, the enteric bacterial microbiome was depleted significantly after exposure to antibiotics (fig. 2, 3a ; additional file 2: figure s2 , additional file 3: table s1 , additional file 4: figure s3 ). the levels of firmicutes and bacteroidetes decreased to 7.69% and below 0.01%, respectively; at the same time, the abundance of proteobacteria increased to 92% (additional file 2: figure s2b ). escherichia-shigella were the major constituents of proteobacteria, and other genera belonging to proteobacteria were markedly depleted (additional file 2 : figure s2b , additional file 3: table s1 ). the fecal samples were spread on plates with the antibiotic cocktail, and a large number of colony-forming units (cfus) were observed in the sample from d9 but not in the sample from d0. the 16s rrna amplicons of single clones were sequenced, and we found that these clones belonged to escherichia-shigella (data not shown). as expected, the overall diversity and richness of the bacterial microbiome were depleted both stably and continuously (fig. 3 b, c) . the shannon diversity index showed that the diversity of the bacterial microbiome was significantly decreased after treatment with antibiotics and remained at a low level (fig. 3b , additional file 2: figure s2c ). the richness of the bacterial microbiome was significantly decreased, as measured by the ace (fig. 3c , additional file 2: figure s2d ). in addition, fig. 1 experimental procedure. three rhesus monkeys were treated with an antibiotic cocktail to control their gut bacterial microbiome, and we detected the longitudinal changes in the gut bacterial microbiome at d0, d5 and d9 by 16s rrna amplicon sequencing. then, we extracted nucleic acids from the fecal supernatant at d0 and d9 and scanned the gut viromes of the monkeys. the samples for metabolomics were collected on d0, d8 and d9 and scanned by gc-ms and lc-ms. we comprehensively analyzed the interactions among the gut virome, bacterial microbiome and metabolomes based on the above results there were noticeable differences in bacterial β-diversity between control and experimental animals, as determined using principal component analysis (pca), and the results showed good repeatability within a single group (additional file 2: figure s2f ). together, these data suggest that the richness and diversity of the bacterial microbiome composition were depleted stably and continuously. therefore, we assessed the virome composition in the control and antibiotictreated experimental monkeys on the ninth day. in previous experiments, examination of microbiota genomes from rhesus macaques (macaca mulatta) showed that a majority of the sequences in the fecal samples were mapped to bacterial genomes, while the percentage of sequences mapped to viral genomes was very low [20] . to comprehensively detect both dna and rna viruses, we filtered the fecal samples with a 0.45-μm filter and treated the samples with dnase i, after which, total dna and rna were extracted separately from the same fecal sample. owing to low yield, amplification of the dna and double-stranded cdna by whole-genome amplification (using mda) was necessary. using the miseq 2 × 250 paired-end protocol on the illumina miseq platform, we obtained an average of 54,590,439 ± 14,898, 536 clean reads per mda sample library and generated a total of 88,610 mbp from 12 samples, allowing detailed investigation of the viral populations. to catalog the present genes, we predicted open reading frames (orfs). a total of 478,694 orfs were predicted; the average orf length was 398.1603279 bp, with a maximum of 16,635 bp and a minimum of 100 bp. the fig. 2 the bacterial microbiome was obviously depleted by treatment with antibiotics. heatmap of the out percentage of every genus at 3 timepoints. each point represents 3 biological replicates. the genera that belong to the same phylum are shown in the same color on the left. the total otu number of 3 biological replicates for every genus was more than 10. the color bar represents the log of the percentage, the numbers in the heatmap are the log values of the otu numbers, and the numbers in the bar are the percentages ecological signatures of the intestinal virome have been characterized. the largest percentage of sequences mapped to viromes in the fecal samples belonged to bacteriophages, accounting for over 80% of the sequences (additional file 5: table s2 ). we also identified eukaryotic dna viruses and rna viruses, as well as other viral families that are defined as "unclassified" in the ncbi taxonomy database. in the raw data from the virome metagenomic analysis, rna virus sequences were found in the dna virus results, and dna virus sequences were found in the rna virus results, because the rna virus sequences were wrongly affiliated with dna viral genomes and vice versa. in addition, dna viruses, especially bacteriophages, were the main components of the virome in our sequencing data, and the rna from these viruses may have been extracted together with rna viruses and vice versa. thus, we excluded these data. we observed that the fecal virome composition was noticeably altered after depletion of the bacterial microbiome treated with antibiotics. anosim showed that the distance between groups was greater than that within groups for dna viruses and bacteriophages (additional file 6: figure s4 ). dna viruses, including members of the families poxviridae, iridoviridae, ascoviridae, baculoviridae, marseilleviridae, and mimiviridae and bacteriophages, such as members of the family inoviridae, were present in the normal monkeys but absent after the gut bacterial microbiome was depleted (fig. 4) . dna viruses, including members of the families herpesviridae, nanoviridae, and phycodnaviridae, were present in three biological replicates before antibiotic treatment but in only one biological replicate after the gut bacterial microbiome was depleted (fig. 4) . most of the reads from bacteriophages were noticeably depleted after the gut bacterial microbiome was depleted (additional file 7: figure s5 , additional file 8: table s3 ). rna viruses, including members of the families picornaviridae and tymoviridae, were present in three biological replicates but were present in only one biological replicate after the gut bacterial microbiome was depleted, and rna viruses belonging to nodaviridae were present in two biological replicates but absent after the gut bacterial microbiome was depleted (fig. 4 ). in addition, many kinds of viral groups, including circoviridae, geminiviridae, microviridae, podoviridae, myoviridae, siphoviridae, picornaviridae, and retroviridae, were present regardless of whether the bacterial microbiota was depleted, but the sequencing reads showed that the abundances of these viruses may have decreased with bacterial microbiota depletion (additional file 7: figure s5 ). however, we could not validate this decrease regarding the abundances of viruses at the species level, the results that were similar in 2 or more monkeys were selected, and we used real-time pcr to validate changes in the richness of the viruses. as the figure shows, we analyzed fecal samples that had not been subjected to mda. after the bacterial microbiome was depleted, the results from real-time pcr validated the depletion of 4 viral species: bdellovibrio phage phimh2k, which belongs to the microviridae family of bacteriophages; bacillus phage b103, which belongs to the podoviridae family of bacteriophages; columbid circovirus, which belongs to the circoviridae family of dna viruses; and potato virus m, which belongs to the betaflexiviridae family of rna viruses (fig. 5a) . the samples that were not detected directly from dna or cdna were subjected to mda and then detected by real-time pcr. the depletion of 3 viral species was detected (fig. 5b) : marseillevirus marseillevirus, which belongs to the marseilleviridae family of dna viruses; simian sapelovirus, which belongs to the picornaviridae family of rna viruses; and andean potato mild mosaic virus, which belongs to the tymoviridae family of rna viruses. in addition, mason-pfizer monkey virus, which belongs to the retroviridae family, was detected among the rna viruses; this virus is very dangerous in monkey populations and has been shown to cause an aids-like disease in rhesus macaques [33] . encouragingly, no mason-pfizer monkey virus was detected by real-time pcr. briefly, the fecal virome composition was noticeably altered after depletion of the bacterial microbiome, and the abundances of many dna viruses, bacteriophages and rna viruses in the gut were clearly decreased. in addition, in the metagenomic analysis, we found high numbers of reads from dna viruses and bacteriophages; however, low numbers of reads from rna viruses were found (additional file 7: figure s5 , additional file 8: table s3 ). these results may be due to the limited application of mda in rna viruses. the microbiota structure is the result of dynamic interactions among various community members. we found fig. 4 the composition of the virome changed noticeably after treatment with antibiotics. the presence-absence heatmap shows the virome characterized by metagenomic analysis. due to the presence of low-complexity/repetitive regions in the reads, false-positive virus family taxonomic assignments with fewer than 3 reads were omitted from the analyses [26] a close interaction between the whole virome (dna viruses, rna viruses and bacteriophages) and the bacterial microbiome. we analyzed the effects on the abundance of the virome by rda, taking the richness of bacteria at the phylum level as environmental factors, and found a negative interaction between the abundances of dna viruses and bacteriophages at d9 and the abundances of most bacteria (fig. 6a, b) . however, rna viruses exhibited chaotic interactions due to weak repeatability. next, we conducted linear regression analysis between virome abundance and both the ace and shannon diversity index, and we found positive correlations in bacteriophages (fig. 6c, d) . in addition, the dna and rna viruses showed a positive trend (additional file 9: figure s6 ) but weak confidence levels. overall, these results support our hypothesis that a clear interomic relationship exists between the virome and bacterial microbiome. positive correlations were found between virome abundance and the richness and diversity of the bacterial microbiome. metabolites could inhibit or promote viruses in vivo [34] and in vitro [35] [36] [37] , and the metabolites produced by bacteria play important roles in host physiology [38] . to interrogate the functions associated with the response to depletion in the bacterial microbiome, we performed a kegg prediction analysis of the bacterial microbiome using picrust. we observed significant differences in functional systems along with shifts in the composition of the bacterial microbiome, according to the predictions by picrust. kegg pathways associated with bacterial toxins were downregulated significantly (fig. 7a) , perhaps as a result of antibiotic treatment. the pathway associated with d-arginine metabolism showed a 9-fold decrease; in contrast, the pathways associated with fatty acid metabolism and tryptophan metabolism showed a 2-fold and 2.6-fold increase, respectively. at the same time, the biosynthetic pathway for ubiquinone and other terpenoidquinones showed a 2-fold increase. moreover, the glycosaminoglycan degradation pathway exhibited low diversity and a 1300-fold decrease (fig. 7a) . glycan [39] , glycosaminoglycan [40] , quinone [41] and arginine [42, 43] are well known to support the inhibition of viruses, while tryptophan [44, 45] and fatty acids [36] promote viral survival. interestingly, we detected the metabolites in the fecal samples by metabolome scanning and found that the changes in some metabolite levels were consistent with our prediction based on the normalization of otus in the 16s rrna amplicon sequencing data. because metabolomic analysis requires 6 biological duplications, the metabolomes of 6 samples collected before treatment with antibiotics and those of samples collected after treatment with antibiotics for 8 and 9 days were examined by gc-ms and lc-ms. the metabolomic results exhibited good repeatability (additional file 10: figure s7 ). in gc-ms (fig. 7b) , n-dimethylarginine was not detected in monkeys that were not treated with antibiotics, but the metabolite was present after antibiotic treatment. the levels of n-acetyltryptophan and n-methyltryptophan showed a 3.3-fold and 1.85-fold decrease, respectively, after treatment with antibiotics. in lc-ms (fig. 7c) , we found that the levels of abruquinone b and sulfated dihydromenaquinone-9 exhibited a 30-fold and 5.6fold increase, respectively. regrettably, in the present study, we did not measure the levels of glycosaminoglycan, which plays a very important role in reducing the viral population [40] . as reported by adina howe, yatsunenko t and alejandro reyes, in the same environment and feeding conditions, the composition of the microbiota and virome could remain stable within an individual [17, 46, 47] . however, the gut microbial composition could be influenced by multiple interacting factors, such as diet [46] , antibiotic use [48] , age, geographical setting [47] , and several diseases, including chronic inflammation, obesity and diabetes [4] . in our study, the major reason for depletion of the gut bacterial microbiota was treatment with the antibiotic cocktail. the feeding conditions of the rhesus monkeys were stable in terms of their food and water consumption, and blood samples were monitored routinely, showing that there was no infection during the study period (additional file 1: figure s1 ). the bacterial composition exhibited stable and continuous depletion after treatment with the antibiotic cocktail, and we found that the virome composition changed noticeably and was correlated with the shifts in the bacterial community. moreover, we found that metabolites produced by the gut bacterial microbiome may play a role in the interrelation. in addition, we found that the composition of the rhesus monkey enterovirus group was similar to that of the human enterovirus group [26] , and our results may be beneficial for research on the composition of the human virome. when the bacterial microbiome was depleted, ampicillin could kill most bacteria, including gram-positive and gram-negative bacteria; streptomycin could kill most bacilli; kanamycin could kill most gram-negative bacteria; metronidazole could kill most anaerobic bacteria and parasites; and vancomycin could kill most gram-positive bacteria. of course, the numbers of drug-resistant bacteria are increasing, but we believe that the cocktail of five antibiotics could deplete most of the commensal bacteria in the gut. as expected, the whole gut bacterial microbiome, including gram-positive and gram-negative bacteria (additional file 2: figure s2e ), was depleted after treatment with the antibiotic cocktail, except for escherichia-shigella species belonging to proteobacteria, which were resistant to the cocktail. escherichia harbored the most diverse antibiotic resistance genes, including genes resistant to multidrug treatments, tetracycline, aminoglycoside, macrolide-lincosamidestreptogramin b, β-lactams, and sulfonamides [49] . we maintained the bacteria belonging to escherichia-shigella in plates with the antibiotic cocktail. in the future, we will investigate the specific resistance and antibiotic resistance genes in this bacterium. notably, our study focused on the interaction between virome composition and the bacterial microbiome in rhesus monkeys and may serve as a model for gut microbiota analysis. therefore, we used the administration of 5 distinct antibiotics at high dosages and high frequencies for 2 weeks to deplete the whole gut bacterial microbiome. in our results, the richness and diversity of the bacterial community were depleted. because our study did not involve clinical treatment, the normal dose of antibiotics was not evaluated by our procedure. people are widely prescribed antibiotics each year [50] , and while antibiotics exert very complex effects on the whole bacterial microbiome [48] , the effects of these drugs on the virome are not clear. antibiotics can directly affect viruses but do not exhibit a wide range of roles. minocycline [51] , berberine, abamectin, ivermectin [52] , glycopeptides [53] , and teicoplanin [54] could inhibit the corresponding viruses. in our study design, an antibiotic cocktail that included ampicillin, streptomycin, kanamycin, metronidazole, and vancomycin was administered orally. no study has yet reported that these antibiotics directly affect viruses. based on our results, the richness of these viruses was very low in the gut, and we had to use mda to perform deep sequencing, although the detection by deep sequencing was very sensitive. we first characterized the shift in the gut virome by deep sequencing, and the samples were amplified by mda. mda is used as a general technique in virome research, especially for dna virome detection [26] . to a certain extent, the amplification read-out can also represent the virus quantity. however, mda is not well suited to the detection of rna viruses. the sequence-independent amplification (sia) approach is more appropriate than mda for detecting rna viruses [55] . in the future, we can use this approach to precisely detect rna viruses. in this case, we validated the depletion of the virome composition, including dna viruses, rna viruses and bacteriophages, by real-time pcr. although the number of cycles seemed high, these results were verified via three biological replicates, and the results of the no-template control (ntc) were not detected. in addition, we performed serial dilution of the in vitro transcribed rna of coxsackievirus a16 to generate a standard curve and found that a ct of 39.96 represents 23 genomic copies (data not shown). in our opinion, these viruses are components of the gut microbiome with low richness and may be involved in host physiology. the metabolites produced by gut bacteria play very important roles in host physiology [38] , although the effects of these metabolites on virome composition have rarely been reported. glycan [39] , glycosaminoglycan [40] , quinone [41] and arginine [42, 43] support the inhibition of viruses, while tryptophan [44, 45] and fatty acids [36] promote viral survival. although most metabolites that can inhibit or promote viruses play roles in human viruses, tryptophan could promote the simian immunodeficiency virus in macaques [45] . in addition, most pandemics originating in animals, such as severe acute respiratory syndrome and pandemic influenza, could start to appear because of ecological, behavioral, or socioeconomic changes [56] . many human viruses are zoonotic, and some human viruses, such as human enterovirus 71, can infect animals, especially monkeys [32] . we believe that metabolites play roles in a broad spectrum of viruses and that changes in the metabolites may correlate with depletion of the virome. in our results, the level of quinone, which decreases the abundance of viruses, was increased in the gut metabolome, and the levels of some amino acids that promote the survival of viruses, such as tryptophan, were decreased. importantly, glycosaminoglycan, which can reduce the populations of various viruses, was noticeably increased in the kegg pathways of the bacterial microbiome, but we did not measure glycosaminoglycan levels in the present study. it is very difficult to detect glycosaminoglycan by metabolic scanning because glycosaminoglycan has a very high molecular weight. in the future, glycosaminoglycan levels could be measured by time-offlight mass spectrometry. first, the polysaccharide needs to be dispelled, followed by detection of the monosaccharide to calculate the polysaccharide levels based on the relationships among the monosaccharides in a specific database. however, this process is very complicated, and the database is not sufficiently large at present. by analyzing the relevant data, we found that depletion of bacteria directly promoted changes in the concentrations of some metabolites, which may play important roles in reducing the abundance of dna viruses. our metagenomic-scale characterization of the virome composition after treatment with antibiotics supports the notion that the composition of the virome is noticeably altered in correlation with bacterial community depletion and that metabolites produced by bacteria possibly play important roles in the interaction. the next step will be to investigate the underlying mechanisms in detail. additional file 1: figure s1 . the detection of blood cell analysis during the course of antibiotic treatment. white blood cells (wbc), neutrophilic granulocytes (neut), lymphocytes (lymph), monocytes (mono#), eosinophils (eo#), and basophilic granulocytes (baso#) were counted, and the counts were compared between the monkeys that were treated with antibiotics and ones were not, and there was no obvious difference. (tif 597 kb) additional file 2: figure s2 . the richness and diversity of gut bacterial microbiota were depleted obviously stably and continuously. (a, b) the community analysis of gut bacterial microbiota on phylum level. the phylum was represented by own color.(c, d) the student's t-test of alpha diversity index (the shannon diversity index and the ace estimator) in genus level. 0.01 < p ≤ 0.05 was marked *, 0.001 < p ≤ 0.01 was marked * *, p ≤ 0.001 was marked * * * .(e)the longitudinally reads of otu in grampositive and gram-negative bacteria. (f) the repeatability analysis of 16s rrna amplicon sequencing by pca. (tif 978 kb) additional file 3: table s1 . the reads number on the genus level in the gut bacterial communities in a longitudinal cohort treated with an antibiotic cocktail. (xls 22 kb) gut microbiota modulation and its relationship with obesity using prebiotic fibers and probiotics: a review front microbiol the gut microbiome in health and in disease stress and the gut: pathophysiology, clinical consequences, diagnostic approach and treatment options assessing the human gut microbiota in metabolic diseases an obesity-associated gut microbiome with increased capacity for energy harvest microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders duodenal infusion of donor feces for recurrent clostridium difficile update on fecal microbiota transplantation 2015: indications, methodologies, mechanisms, and outlook the human gut virome: inter-individual variation and dynamic response to diet going viral: next-generation sequencing applied to phage populations in the human gut a composite bacteriophage alters colonization by an intestinal commensal bacterium bacteriophage adhering to mucus provide a non-host-derived immunity herpesvirus latency confers symbiotic protection from bacterial infection the virome in mammalian physiology and disease two distinct metacommunities characterize the gut microbiota in crohn's disease patients transfer of viral communities between human individuals during fecal microbiota transplantation viruses in the faecal microbiota of monozygotic twins and their mothers influenza promotes pneumococcal growth during coinfection by providing host sialylated substrates as a nutrient source protection against streptococcus pneumoniae invasive pathogenesis by a protein-based vaccine is achieved by suppression of nasiopharyngeal bacterial density during influenza a virus coinfection siv infection-mediated changes in gastrointestinal bacterial microbiome and virome are associated with immunodeficiency and prevented by vaccination influenza virus affects intestinal microbiota and secondary salmonella infection in the gut through type i interferons enteric bacteria promote human and mouse norovirus infection of b cells the influence of commensal bacteria on infection with enteric viruses microbiota regulates immune defense against respiratory tract influenza a virus infection gnotobiotic mouse model of phage-bacterial host dynamics in the human gut early life dynamics of the human gut virome and bacterial microbiome in infants berberine blocks the relapse of clostridium difficile infection in c57bl/6 mice after standard vancomycin treatment members of the human gut microbiota involved in recovery from vibrio cholerae infection predictive functional profiling of microbial communities using 16s rrna marker gene sequences impact of prematurity and perinatal antibiotics on the developing intestinal microbiota: a functional inference study skin fungal community and its correlation with bacterial community of urban chinese individuals neonatal rhesus monkey is a potential animal model for studying pathogenesis of ev71 infection simian aids: isolation of a type d retrovirus and transmission of the disease inhibitory effects and related molecular mechanisms of total flavonoids in mosla chinensis maxim against h1n1 influenza virus a synthetic glycosaminoglycan mimetic blocks hsv-1 infection in human iris stromal cells de novo fatty acid biosynthesis contributes significantly to establishment of a bioenergetically favorable environment for vaccinia virus infection the plant-derived naphthoquinone droserone inhibits in vitro measles virus infection gut microbiota regulation of tryptophan metabolism in health and disease glycans controlling virus infections: meeting report on the 1st international symposium on glycovirology schontal broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry inhibitory effects of quinones on rnase h activity associated with hiv-1 reverse transcriptase arginine as a synergistic virucidal agent synergistic virus inactivation effects of arginine 5-hydroxytryptophan, a major product of tryptophan degradation, is essential for optimal replication of human parainfluenza virus distinct patterns of tryptophan maintenance in tissues during kynurenine pathway activation in simian immunodeficiency virus-infected macaques divergent responses of viral and bacterial communities in the gut microbiome to dietary disturbances in mice human gut microbiome viewed across age and geography uncovering effects of antibiotics on the host and microbiota using transkingdom gene networks antibiotic-mediated changes in the fecal microbiome of broiler chickens define the incidence of antibiotic resistance genes. microbiome the use of antibacterials in children: a report of the specialist advisory committee on antimicrobial resistance (sacar) paediatric subgroup minocycline suppresses dengue virus replication by down-regulation of macrophage migration inhibitory factor-induced autophagy discovery of berberine, abamectin and ivermectin as antivirals against chikungunya and other alphaviruses glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) an analogue of the antibiotic teicoplanin prevents flavivirus entry in vitro metagenomic analysis of human diarrhea: viral detection and discovery prediction and prevention of the next pandemic zoonosis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank majorbio for their contributions to the 16s rrna amplicon and virome sequencing projects. additional file 4: figure s3 . the phylogenetic tree on genus level of gut bacterial microbiome. the number in the line represents the genetic distance. every phlym was showed in own color. the bar in the right were caculated according to the number of reads. (tif 966 kb) additional file 5: table s2 . the reads number on the family level in the gut virome communities in a longitudinal cohort treated with an antibiotic cocktail. (xls 2 kb) additional file 6: figure s4 . the anosim analysis of virome groups. the ordinate represents the distance value. r value represents the statistic results, and the closer the r value is to 1, the greater the difference between groups than the difference in the group, and the grouping was reasonable. (tif 495 kb) additional file 7: figure s5 all data generated or analyzed during this study are included in this published article and the additional files. we have deposited the raw sequencing data into ncbi and the number is prjna555120.ethics approval and consent to participate all animal experiments were conducted under prior approval from the animal ethics committee of the institute of medical biology, chinese academy of medical sciences, with permit number dwsp201803006, according to the national guidelines on animal work in china. not applicable. the authors declare that they have no competing interests.author details key: cord-009376-a35a92gh authors: lovatt, archie title: applications of quantitative pcr in the biosafety and genetic stability assessment of biotechnology products date: 2002-01-07 journal: nan doi: 10.1016/s1389-0352(01)00043-5 sha: doc_id: 9376 cord_uid: a35a92gh high throughput screening, increased accuracy and the coupling of real-time quantitative pcr (q-pcr) to robotic set-up systems are beginning to revolutionise biotechnology. applications of q-pcr within biotechnology are discussed with particular emphasis on the following areas of biosafety and genetic stability testing: (a) determination of the biodistribution of gene therapy vectors in animals; (b) quantification of the residual dna in final product therapeutics; (c) detection of viral and bacterial nucleic acid in contaminated cell banks and final products; (d) quantification of the level of virus removal in process validation viral clearance studies; (e) specific detection of retroviral rt activity in vaccines with high sensitivity; and (f) transgene copy number determination for monitoring genetic stability during production. methods employed for q-pcr assay validation as required in ich topic q2a validation of analytical methods: definitions and terminology (1st june 1995) are also reviewed. real-time quantitative polymerase chain reac-ž . tion q-pcr has revolutionised the detection and quantification of nucleic acid and retroviral reverse transcriptase because of fast throughput ž capability morris et al., 1996; lovatt et al., 1999; . nitsche et al., 1999 . the incorporation of this bulk harvest material. during amplification with target-specific oligonucleotide primers, a fluorogenic oligonucleotide probe with both reporter ž . dye fam, vic, tet, joe, or hex and ž . quencher dye tamra attached, anneals specifically to the amplified product between the amplimers. the detection reaction utilises the 5ј exonuclease activity of amplitaq gold tm dna polymerase to cleave the reporter dye from the probe, which results in an increased fluorescence ž . when target is present fig. 1a . a sequence detector instrument calculates a reporter dye value ž . r for each sample during each cycle of amplin fication. the value of r , or the normalised ren porter signal, represents the fluorescence of the reporter dye divided by a passive reference dye. during pcr, r increases as the amplicon copy n number increases until the reaction approaches a plateau. the value ⌬ r represents the norn ž . malised reporter signal r minus the baseline n signal established in the first few cycles of pcr. like r , ⌬ r increases during pcr as the amn n plicon copy number increases until the reaction ž . reaches a plateau fig. 1b . the use of a sequence detector allows measurement of the amplified product in direct proportion to the increase in fluorescence emission continuously during pcr ž . amplification. the threshold cycle c value is t calculated in real time and is defined as the pcr cycle number at which an increase in reporter fluorescence above the baseline signal can first be detected. therefore the cycling point of a given amplification where a significant increase in the fluorescence signal associated with exponential growth of the pcr product is first detected, rep-ž resents the c value of that amplification fig. t . 1b . a linear relationship exists between the c t value and starting quantity of target molecule, allowing the construction of a standard curve. the greater the amount of target copies in the ž .ž reaction, the lower the c value fig. 2 heid et t . al., 1996; livak and perkin elmer, 1998 . there are many advantages of real time q-pcr over conventional end-point pcr. for example, real-time q-pcr is performed in a closed reaction system and eliminates the post-pcr processing of pcr products, thereby increasing throughput and reducing the chances of carryover con-tamination. moreover, the taqman q-pcr system incorporates a chemical system termed am-perase to avoid amplicon contamination. like conventional qualitative pcr assays, q-pcr assays should operate under a strict contamination ž control system i.e. separate air spaces for all . reactions to give increased assurance of re-avoiding cross-contamination. because of the increased throughput of real time q-pcr, a higher assay sensitivity can be achieved with relative ease. multiple replicated reactions can be used to increase the chance of observing a low level virus ž below the limit of detection heid et al., 1996; . livak and perkin elmer, 1998 . in addition, coupling real-time q-pcr to robotic extraction and pcr set-up systems will increase throughput further and minimise human error. c values are also less sensitive than end-point t reactions to the effects of inhibitors. compared to endpoint pcr, real time q-pcr offers streamline assay development, reproducible results and a large dynamic range. more importantly, if the sample contains viral sequences, then the amount of target nucleic acid molecule present is given. this will provide an indication of the level of viral contamination present, which could be useful in deciding what further action to take. if, for example, the pcr-positive sample was from bulk harvest material, further process validation could be used to determine if the sample still gave a posi-ž tive result heid et al., 1996; livak and perkin . elmer, 1998 . primer design for real-time taqman pcr can be performed using the primer express tm software on the abi 5700, 7700, or 7900ht following pe applied biosystems recommendations. one of the first steps in the identification of the nucleic acid target is comparison of the target sequence or amplicon using multiple sequence alignments with dna sequence databanks. this allows an assessment of potential cross-reaction with other related sequences that may produce problems in the q-pcr testing. determination of primer specificity is of utmost importance when performing pathogen detection studies. false positive reactions, although uncommon, can lead to major problems in the release of biotechnology products for clinical trial. therefore, assay specificity studies are critical during validation to ensure confidence in reporting positive results. the q-pcr assay should be able to discriminate between the target and nucleic acids that are likely to be present in test material. for detection of viruses such as human im-ž . munodeficiency virus hiv , and other viruses which show sequence variation, it is extremely important to ensure the primers and probe are targeted to a conserved region of the viral genome. selection of a region with highly conserved sequences facilitates the design of degenerate amplimers and probes, allowing detection of the maximum amount of variants and strains possible ž . shown in fig. 3 for bovinerporcine circovirus . in addition, the stability of the target sequence should be considered, in an attempt to ensure that the amplicon does not reside on an unstable genetic element that may be a hot spot for deletions, such as those that occur in the polyoma ž . virus bendig and folk, 1979; persing, 1993 . to ensure technical reliability, assay optimisa-tion studies are essential, together with validation ž to ich guidelines ich topic q2a, 1995; ich . topic q2b, 1997; cope, 2000 . a minimum of two operators should perform specificity, linearity, detection limit, accuracy and repeatability studies. each operator should determine these assay parameters at least once, using a minimum of five concentrations over the range of the assay. the importance of optimisation and validation is highlighted when working with non-validated rapidly developed q-pcr assays. high background fluorescent signals leading to reduced assay sensitivity and abnormal amplification signals have been observed in assays before assay optimisation and validation, further supporting the need for assay validation. more importantly, a less experienced operator may not be able to distinguish between genuine and abnormal amplification signals during testing with a non-optimised, non-validated assay. primer optimisation is carried out using an array of primer concentrations with a constant amount of probe and target template. because high ⌬ r values reflect increased levels of amplin fication product and efficient reactions, the combination of forward and reverse primer showing the maximum ⌬ r , is selected. probe optimisan tion is performed using the optimised primer conditions, whereby the concentration of probe is varied. the probe concentration showing the lowest c value is chosen as optimal, and reflects t the most efficient reaction conditions. assay linearity, detection limit and precision are assessed using eight replicated reactions over a range of five concentrations. to assess repro-ž 2 . ducibility, the correlation coefficient r , y-inter-cept and slope of the regression line should be calculated using a minimum of two independent operators on the abi 7700, 7900ht, or 5700 systems. the minimum accepted value for the linear correlation coefficient at the ps 0.05 statistical level when using four standards is 0.88 ž . wardlaw, 1987 , and q-pcr assays will normally show values of r 2 ) 0.90. two major complicating factors in establishing the detection limit for a real-time pcr assay ž . using biological material are: 1 the number of target nucleic molecules present in infected cells ( ) and clinical samples, or electron microscopy ž . counted viral stocks, may be variable; 2 there may be no culture methods available for the target organism. therefore, the limit of detection is firstly validated using purified target molecules in the form of recombinant plasmid dna or recombinant rna. the assay limit of detection with 95% confidence, is defined as the lowest amount of target producing a confidence interval that is significantly lower than that obtained from eight negative controls and no template controls. within the group of eight replicates containing target levels at the limit of detection, there should be no failed reactions, and all replicates should produce amplification signals with a c value less t than 40. assay limit of detection is routinely determined as 10᎐100 nucleic acid copies in a ž background of 200 000 cellular genomes for dna . ž targets or 100 ng of cellular rna for rna . targets . ultimate assessment of assay reproducibility requires the positive cut-off point to be determined. the positive cut-off level is the minimum number of target molecules that are detected in 95% of test runs. the positive cut-off point of an assay can be measured over a period of 12 months using different batches of reagents and several different operators. the extraction of viral nucleic acids is widely used in diagnostic and research laboratories. however, the suitability or yield of nucleic acid from extraction procedures can vary depending on the nucleic acid and the biological material ž . kok et al., 2000 . therefore, to ensure efficient extraction of target nucleic acid, the procedure should be qualified for the particular target and biological material to be used. since q-pcr allows the quantification of a nucleic acid target molecule, it can be used to determine the percentage recovery of the viral or cellular dna target achieved during the extraction procedure ž . table 1 . to assess further the performance of the extraction procedures, the extracted test nucleic acid can be spiked with target molecules to determine the percentage of pcr inhibition. the calculated copy number of the test sample is subsequently adjusted, taking into account the amount lost in extraction and the estimated level of inhibition. pcr inhibition is particularly important to assess when analysing many different biological materials that may contain various levels of inhibitory substances. when extracting nucleic acid from cell free samples, especially final products, it is important to include a carrier molecule in the procedure. this allows maximum recovery of low levels of nucleic acid and is critical for ensuring maximum sensitivity of the pcr assay. this is essential when screening vaccines for contaminating host cell dna. as determined by q-pcr, typical extraction recoveries of 22% and greater can be achieved in 78 pg of human cell line dna per 0.5-ml therapeutic dose, using the qiagen dna ž . mini kit table 1 . similar results are obtained when using q-pcr assays for primate, rodent, chicken and porcine cellular dna. inhibitory factors which interfere with pcr are most apparent when performing extraction of viral or plasmid nucleic acid from animal tissues and secretions. substances such as: heparin; urea and haemoglobin; ethanol; and iso-propanol will inhibit pcr and affect extraction efficiency ž . qiagen, 1999 . the use of methods which remove these molecules are of utmost importance when screening tissue during gene and cell therapy biodistribution studies. therefore in these studies, the use of optimised high throughput nucleic acid extraction systems and the examination of the recovery of 10᎐100 copies of target nucleic acid from the highest amount of tissue possible should be determined. recovery in percentage values of a plasmid dna per 100 g of animal ž . tissue are shown table 2 . after cycling is complete, data are analysed in real time and the c values of each replicate t observed and recorded. the standard deviations and confidence limits of c values of the repli-t cates are calculated to detect similarities and significant differences between groups of replicates. firstly, it is not impossible that a positive result may derive from nucleic acid of previously unrecognised origin that shows non-specific interactions, due to sequence homology with the amplification target used in detection. in a valid assay, a positive result for the test sample is considered to have occurred when the following four conditions are met when testing the sample with two independent amplimer sets to the specified target. ž . 1 there are significant amplification signals pre-ž . sent in the test sample reactions. 2 there are no amplification signals present in the sentinel con-ž . trols. 3 there are no amplification signals pre-ž . sent in the negative controls. 4 there are significant amplification signals present in the test article spiked at the limit of detection. detection of the target sequence by pcr in any of the sentinel or negative controls invalidates the assay and is indicative of contamination. in a positive test with confidence, analysis of data results in the c values of the test sample, spiked t test sample and positive control samples being significantly less than the negativersentinel reactions. when the level of target molecules is below the limit of detection, positive reactions can occur in one of three or two of three replicates. because negative replicates produce a c s 40, the t confidence interval of the test sample is not significantly different from the negative control reactions. in this case, a confidence interval that contains positive amplification signals in test sample reactions is produced, but overlaps with the negative controls. the result could therefore be considered 'equivocal with signals detected below the validated assay sensitivity'. increases in sample volume can be analysed, or if appropriate, testing carried out by an independent procedure, such as cell infectivity with q-pcr end point analysis in a valid assay, a negative result for the test sample is considered to have occurred when the following four conditions are all met when testing ž . the sample. 1 there are no amplification signals ž . present in the test sample reactions. 2 there are no amplification signals present in the sentinel ž . controls. 3 there are no amplification signals ž . present in the negative controls. 4 there are amplification signals detected in the all spiked positive control reactions. in a negative test with confidence, analysis of the data should result in the c value of the t sentinel controls, negative controls and the test article being significantly greater than those from the test article spiked at the limit of detection. occasionally, one in three, or two in three replicates of the test sample spiked at the limit of ( ) detection may contain c s 40. if no amplifica-t tions are detected in the test article, the confidence intervals show that the spiked data is not significantly different from the test article and negative control data. in this case, the result would be considered negative below confidence, since the test was not able to detect amplification signals in all of the spiked replicates. the accuracy of the quantitative data for a number of q-pcr assays can be determined using a minimum of eight replicates containing identical amounts of dna or viral rna spread across a range of concentrations. during the analysis, four of the replicates are termed standards and the other four replicates designated unknowns. after generation of the standard curve and quantification of the unknowns, the accuracy of the quantitative software in combination with an assessment of the q-pcr assay was determined. the results show that q-pcr assays are able to quantify accurately the correct amount to within one standard deviation for both dna ž . and rna targets table 3 . in more detail, the ž value x s 1000 on the standard curve which is the actual amount in the enterovirus rna in the . standard , corresponds to a calculated value y s w ž 994 " 97 which is the mean " standard devia-. tion amount calculated in the unknown replicates containing identical amounts of nucleic acid x as the standard . because the sensitivity of the abi 7700 and 7900ht is a two-to fourfold copy number difference, a mean value of y that varies by more than fourfold could be calculated as being different from the actual value of y, if working at the two-to fourfold copy number difference level. therefore, for an actual value ž . x of 1000 copies the acceptable criteria could be a one-to twofold variation on either side of ž the 1000 copies e.g. from 500᎐750 to 1500᎐2000 . copies ; a total of two-to fourfold variation of the ž . actual value x . therefore, the value of y s 994 " 97 would be within the acceptable criteria. any value outside of the acceptable limit could be considered as too highly variable for accurate quantification. this approach may allow one to ž . actual values x are the known amount in the standard ž . reaction. values y are the amounts calculated in the 'standardised unknowns' from the generated standard curve, and are expressed as mean copy number "s.d. assess the performance of the standards and the overall q-pcr assay in terms of quantification accuracy. adventitious agents pose significant risk if present in vaccines and therapeutics. therefore, highly sensitive pcr assays for the detection of these agents are crucial for safety testing. numerous viral nucleic acid detection assays are available, and the available number of fully validated q-pcr pathogen detection assays is increasing at a considerable rate. besides viral-specific nucleic acid detection, one very important use of q-pcr technology in safety testing is the detection of contaminating retroviruses by the identification of the presence of retroviral reverse transcriptase ž . activity lovatt et al., 1999 . the fluorescent product enhanced reverse tran-ž . scriptase f-pert assay, is an extremely sensitive tests for the detection of reverse transcrip-ž . 5 tase rt . f-pert is up to 10 -fold more sensitive than conventional rt assays for detecting the ž presence of retroviruses maudru and peden, . 1997; lovatt et al., 1999 . this increased sensitivity, prompted the center for biologics evalua-ž . tion and research cber to request pert testing on all viral vaccines being investigated for ( ) humans. consequently, f-pert is the first choice for detection of rt in live viral vaccines, gene therapy preparations and the screening of animals and patients for porcine endogenous retro-ž . virus perv in xenotransplantation trials. f-pert is a rt-dependent q-pcr assay and therefore combines the broad specificity of conventional rt assays with the high sensitivity and fast throughput of q-pcr. as with conventional rt assays, f-pert is utilised to detect the rt activity packaged into extracellular retrovirus particles. the assay involves converting rna template to cdna and then amplifying the cdna using product-specific primers. as no exogenous rt activity is added to the reaction, cdna will only be generated if the sample itself contains rt activity. when rt activity is not present, the ž product will not be detected silver et al., 1993; pyra et al., 1994; heneine et al., 1995; arnold et . al., 1998 . one major advantage of f-pert assays over other similar types of assays, is the capability to discriminate between the amplification signals generated by retroviral rt activity and those which are a result of the rt-like activity from dna polymerases. f-pert uses activated calf thymus dna to suppress the rt-like activity from cellular and taq dna polymerases with no ž . reduction in the rt activity table 4 . a linear ž . relationship between threshold cycle c and the t number of mlv retrovirus particles or rt and perv is approximately 1000 virion particles and 1000᎐10 000 molecules of purified amv rt enzyme, however, the assay can detect lower levels of retrovirus with decreased frequency. an overview of the f-pert assay is described in a flow diagram in fig. 4 . the high sensitivity of f-pert has ensured that this q-pcr assay plays a significant role in the evaluation of the retroviral status of vaccines and other biological material from the biotechnology industry, where safety of products is paramount. more specifically, when performed in combination with co-cultivation, induction and infectivity assays, together with electron microscopy ž . em and pathogen-specific pcr detection assays, f-pert assists in providing a detailed risk assessment for the presence of adventitious agents in therapeutics. viruses may contaminate cell lines from a ž . number of sources including: 1 those present in 2 viruses of animal origin in media and tissue ž . culture reagents; and 3 those from operators or other sources that could have contaminated the cells during handling in non-gmp conditions. for these reasons, all cell lines, primary cells, or tissue products used for therapeutic purposes require testing for the presence of a range of viruses. the selection of viruses to be tested depends upon the origin of the cell line and raw material used in manufacture. the development and validation of pcr assays to meet the requirements of regulatory authorities is a key element in the production and marketing of final products. an increasing number of published or validated q-pcr assays are becoming available, and these tests can be utilised for cell line, raw material, or ž . final product testing tables 5 and 6 . testing for the presence of human viruses is required when cells of human origin or products obtained from human blood or tissues are involved. examples include mouse᎐human and human᎐human hybridomas, and cell lines of human origin such as mrc-5, and per.c6 used in the production of adenovirus vectors for therapy. in broad terms, the viruses that have to be screened for in a human cell line are those associated with severe or oncogenic diseases, and those that are likely to infect the cell type, particularly those that might establish latent or abortive replication in cells. animal viruses pose significant risk if the cell line or therapeutic is of animal origin andror if animal products such as foetal calf serum, trypsin, or insulin are used in the manufacturing cultures. all new animal reagents used in manufacture should be screened by appropriate techniques. bovine serum and porcine trypsin are usually screened using an appropriate title 9 code of ž . federal regulation 9cfr assay. however, some caution is required as 9cfr assays using minimal volumes may miss critical viruses. in addition, some viruses are not detected by these assays, therefore pcr should be employed. many viruses are of concern in the manufacturing process and are not the subject of this review. instead, this section focuses on examples of viruses that have recently emerged as agents of concern, namely bovine polyomavirus, bunyavirus and porcine circovirus. recent concern has been expressed about the contamination of bovine products with the bovine ž . polyomavirus bpyv . this virus belongs to a family, some of which are oncogenic in their own host or in heterologous hosts. infection of cattle is widespread, and up to 70% of foetal calf serum batches contain bpyv sequences by pcr and infectious virus can be isolated from some batches ž . schuurman et al., 1991 . the most worrying feature of this virus is that it is probably zoonotic. it replicates in primate cells, and while antibody to the virus is lacking in the general population, 71% of veterinarians, 50% of cattle farmers and ž 40% of abattoir workers are sero-positive parry . and gardner, 1986 . detection of bpyv by quantitative pcr is fully validated to ich guidelines and the assay is capable of detecting 100 viral genome copies on a routine basis. the bunyaviridae family contains over 300 viruses grouped into five genera. unlike most other enveloped viruses, they bud into intracytoplasmic vesicles associated with the golgi apparatus forming virions 80᎐120 nm in diameter. the genome of the virus is segmented into three strands permitting complex reassortants, as occurs with influenza virus. consequently, the taxonomy of these viruses can be complex žlundstrom, 1999; borucki et al., 1999; mahy, . 1998 . within the bunyavirus genus, the most relevant serogroups or genetic complexes are the californian serogroup and the bunyamwera serogroup. within the bunyamwera group, the cache ž . valley virus cvv is a recognised infection of sheep and cattle. cvv can be transmitted congenitally and infection of foetal calf serum is ž possible mclean et al., 1996; blackmore and grimstad, 1998; edwards et al., 1997; chung et . al., 1991 , emphasising that cvv has the potential to be problematic in large scale fermentation culture during manufacturing. cvv is lytic to cell lines including, cho-k1 and vero, and therefore can also be detected by culture-based bio-assays ž . edwards et al., 1997 . since cvv is suspected to cause foetal malformations in cattle the presence of this virus in serum represents a major safety ž . concern edwards et al., 1997 . detection of cvv by quantitative rt-pcr is fully validated to ich guidelines and the assay is capable of detecting 100 viral rna copies on a routine basis. the 95% confidence interval of c values produced by 100 t viral copies per 100 ng of non-infected cellular rna was significantly different from the negative ž . control rna using two operators circoviruses are amongst the smallest mammalian viruses at approximately 17 nm, and like parvoviruses, are highly resistant in the environž . y q ( ) ž . ment. porcine circovirus type 2 pcv2 has a nucleotide sequence homology to bovine cir-ž . covirus bcv and has been shown to be involved in the formations of organ lesions in pigs. the close relationship between these two viruses has led to suggestions that this group of viruses may cross species barriers. in some cases, circoviruses can remain as latent and persist as non-cytopathic ž . infections in vitro allan and ellis, 2000 . techniques currently available for detection of all forms of circovirus are limited, and there is no current test that differentiates between infectious virus and nucleic acids. consequently, specific q-pcr assays are required to detect these viruses. as shown in fig. 3 , multiple sequence alignment of the bovine circovirus and porcine circovirus genomes has identified the q-pcr primers and probe that will detect a range of strains isolated from many global locations. the bovine circovirus and porcine circovirus q-pcr is fully validated to ich guidelines and can routinely detect 100 genome copies. in summary, cvv, bpyv, porcine circovirus and bovine circovirus represent a new threat to biotechnology products and steps need to be incorporated to monitor these viruses. appropriate sub-pool testing of foetal calf serum and the testing of other raw materials should be conducted. since it is possible that effects on cell viability may not be noted, appropriate bulk harvest screening of cultures should be undertaken to exclude the presence of these viruses, thereby avoiding expensive contamination of the downstream purification train. process validation studies are usually performed using a downscale of the manufacturer's purification process. this allows the assessment of the capacity and efficiency of the process to remove and inactivate viruses, dna and other contaminants. all manufacturers producing recombinant proteins from rodent cell lines, e.g. hybridoma and cho, are required to perform murine leukemia virus clearance studies for phase irii clinical trials. generally, processes for phase iii studies require clearance of three or more other ž viruses to be demonstrated federal register, . 1998 . the federal guidelines specify that infectivity should be used, but where this is not technically feasible alternative technologies may be justified. however, an increasing number of manufacturers are using both infectivity and q-pcr to demonstrate viral removal. murine leukemia viruses are endogenous proviruses that are divided into two main groups, ecotropic and non-ecotropic viruses. ecotropic viruses can infect only mouse cells and non-ecotropic viruses are sub-divided into three groups: xenotropic; polytropic; and modified polytropic. xenotropic mlv infect human cells, but not their natural rodent host cells. polytropic mlvs, also ž . termed mink-cell focus mcf forming mlv can infect both rodent and non-rodent cells, and have a broader host range than xenotropic mlv. unlike xenotropic and ecotropic mlv, infectious polytropic mlv do not pre-exist in the mouse germ line, but can arise by recombination between infectious ecotropic mlv and endogenous mlv-related polytropic sequences. in certain laboratory mouse strains, the progeny of several non-ecotropic mlv can recombine with exogenous or endogenous mlv sequences to result in oncogenic variants such as mcf virus ž . tomonaga and coffin, 1998 . proviral xenotropic mlv that are capable of producing infectious virus are present in inbred mice populations, such as balb-c. an endogenous xenotropic mlv, termed bx¨-1 appears to be the ltr donor for in leukemogenic mcf forming mlv. in addition, xenotropic mlv derived from the bx¨-1 locus, first described in balbrc mice are infectious ž . kozak and ruscetti, 1992 . murine hybridoma cells used for pharmaceutical monocolonal antibody production express xenotropic mlv, and regulatory agencies recommend that retrovirus should be inactivated or removed during manufacturing. in addition, mlv is used as a model virus to evaluate the elimination of non-infectious cho cell retrovirus particles. such retroviral clearance studies can use ( ) virus inactivation treatments such as low ph of elution buffers during manufacturing. viral clearance studies in scaled down conditions, involve spiking the load sample with a known amount of infectious virus to allow assessment of retroviral removal by examination of the eluate by infectivity assay or q-pcr. however, when buffers that inactivate virus are used in the chromatography process, the reduction of infectious virus titre in the eluate is due to the combined result of chro-ž matography removal and buffer inactivation lau . et al., 1999 . in these cases, infectivity assays will not provide clearance information, whereas q-pcr resolves this problem. initially, it is essential to qualify the viral clearance q-pcr assay using quantitative results on both the spiked load and unspiked load control. for cell lines other than murine hybrodoma, this ensures the absence of non-specific interactions that obscure the clearance data. for retroviral nucleic acid clearance, it is essential to perform dna and rna copy number quantification on the viral spike and any spiked column elutions. analysis of the x-mlv nucleic acid copy number in the absence of rt enzyme allows an estimation of the percentage of proviral dna targets which can result from the background residual host cell dna of the viral production cell line. by recalculating the rna copy number, this ensures that detection of viral rna genome is achieved and the results take into account any background integrated provirus present in residual production cell genomic dna. the difference between two rna copy numbers obtained from the viral spike and spiked column elutions allows calculation of ž . viral removal fig. 5 . for the column elution samples that already contain xenotropic mlv, during rna extraction a different virus can be spiked as an internal extractionramplification control in order to distinguish it from the mlv already present. the internal control spike should not cross-react with the actual xenotropic mlv to simplify calculations. an unrelated rna virus of known tcid 50 or naked viral rna can be used to control for pcrrextraction interference and viral recovery during extraction. viral removal can be obtained by calculating the mean rna copy number of the load, hold control and eluate from column runs. in addition to x-mlv quantification, q-pcr assays to measure clearance of the endogenous ž . retrovirus from chinese hamster ovary cho ž cells and sv40 are now available shi et al., . 1999a,b; de wit et al., 2000 . taken together these approaches will allow the manufacturer's clearance process to be assessed for the ability to remove logs of viral genomes and thereby pave the way for optimisation of procedures to generate maximum viral clearance. manufacturers of biopharmaceuticals must ensure that final products produced from starting cell substrates contain minimum levels of residual host cell dna. current regulatory guidelines established by the fda and who recommend that products manufactured from continuous cell lines contain less than 100 pg of cellular dna per dose ž . lewis et al., 1999a . the dangers of residual dna include: malignant transformation by activated cellular andror viral oncogenes; aberrant gene expression by insertion of sequences in sensitive control regions of genes; as well as the production of infectious viruses from viral dna. in response to these concerns, a number of q-pcr assays have been designed to detect and ž . quantify residual dna table 8 . the quantification limit of residual dna q-pcr assays are usually within the 5 pg per reaction or 50᎐100 pg ž slot-blot technique using p-radiolabeled probe ge-. nomic dna , hybridisation and autoradiography. . 10᎐20 genome equivalents per ml. this sensitivity is expected when using a target such as the beta actin gene as the target. sensitivity can sometimes be increased if a target with a higher copy number is used, for example a sequence repeat region within the genome. we are developing q-pcr assays for these repeat regions for rodent and primate residual dna to obtain sensitivity in the fg range, however, one advantage of using a gene such as ␤-actin is gene stability, as this target should not undergo copy number changes within a stable cell line. repeat se-ž quences are known to be less stable lewis et al., . 1999b; lobachev et al., 2000 and may undergo significant changes in copy number on the genome, and therefore have the potential to obscure the residual dna data if a shift in target copy number occurs during production. for these reasons, the choice of pcr target is important and the use of repeat regions may not be necessary, when sensitivity to pg levels is required. besides the qualification of final products for batch release, residual dna assays are used to demonstrate clearance of host cell dna during process validation. in addition, since some test samples are most likely to contain detectable levels of residual dna, another appropriate internal control dna that is unrelated to the host cell and test sample is spiked into the sample. the recovery of the internal control is quantified using a pcr reaction specific for the unrelated target dna. it is important that the internal control target and test sample target do not cross-react in terms of sequence specificity. this simplifies dna recovery calculations for each individual test sample. q-pcr for residual dna is far more robust than conventional hybridisation methods. for example, q-pcr is more precise and has a larger dymanic range than conventional hybridisation ž . methods smith et al., 1999 . problems with standard curves can be encountered when performing linear regression with conventional hybridisation autoradiography. this is due to the fact that x-ray films have a threshold for both response and sensitivity, resulting in a non-linear response ž . to the hybridisation signal cornett et al., 1999 . q-pcr on the other hand, combines a highly linear response coupled with sensitivity comparable to hybridisation assays. in addition, turnaround time for the q-pcr technique is more rapid, less labour intensive and can be automated with robotic systems to remove operator error during sample manipulation. q-pcr can still estimate the amount of dna in benzonase treated products, for example, if dna is sheared down to sizes of 50᎐100 bp. q-pcr is essential in determining the efficacy of gene and cell therapeutics. moreover, the biodistribution analysis of such novel therapeutics is critical for assessment of their bio-safety and for meeting the increasingly stringent regulatory guidelines. in this way, regulatory issues concerning infection or the transfer of the gene to normal or distal tissues, as well as the target site, are addressed. in addition, investigations of the possibility of integration or expression in the germ line are performed. if possible, whole organs should ( ) be homogenised, an aliquot extracted and the remaining homogenate archived for further analysis. homogenisation of whole organs gives insurance that patches or localisation of infection in the organ can be detected. if the therapeutic is viral or cellular based, then detection of viral or cellular rna expression in tissues of concern may be indicative of virus or cellular replication in these tissues. therefore, the assay target, conditions and controls used in the biodistribution study are critical for generating the highest quality data to satisfy regulatory authorities. a prestudy will be most important, to assess the best conditions that can be applied in the overall biodistribution assay. problems that can occur during a bio-distribution study are dependant on the complexity of the study design. pre-studies can be widely variable depending on the complexity of the therapeutic vector. however, in simplistic terms, the pre-study can be used to demonstrate that optimal pcr, nucleic acid extraction and maximum assay sensitivity are all addressed. for high quality data, it may also be necessary for the preparation of an inactivated vector to provide the material for administration into the negative control animals. this will help control for the presence of any residual vector nucleic acid that becomes lodged in animal organs and therefore provide information to distinguish between signals that can be generated from the replicating vector and those from the residual nucleic acid. in addition, if the vector is a dna virus, the use of q-rt-pcr with the incorporation of dnase during extraction and a minus rt reaction control will help distinguish between viral expression and residual dna in the animal tissue. the method for inactivation of the viral vector will depend on the virus, and may be either uv or b-propriolactone treatment. the inactivated preparation should then be verified by amplification using cell culture and the replicating virus vector examined by an appropriate detection method, for example cytopathic effect or q-pcr. healthy cells that have been treated with inactivated vector can be passed through several times to dilute any residual inactivated target in the culture. samples of cells taken at each passage time point can be quantified by q-pcr with and without rt enzyme to demonstrate reduction of residual viral nucleic acid and the absence of low level infection. detection limit, linearity and reproducibility should all be determined during assay development and validation. once the reaction conditions are optimal, spiked negative control organs are analysed to determine the lowest amount of target that can be detected in the maximum amount of test sample tissue. the optimal extraction conditions can be applied in the biodistribution study. the animal species used in the biodistribution study is extremely important. the species must be acceptable to the regulatory authorities for use in safety evaluation studies and extensive background data should be available. the chosen animal species should be susceptible to infection. the route of administration can be the intended human route for the vector, however, the intravenous route allows better assessment of the vector biodistribution. both animal sexes should be used in the study and the administrative dose should be calculated using maximum human dose based on equivalent unit per kilogram of body weight. an example of an animal study design to examine vector biodistribution could be as fol-ž lows: group a: high dose 50᎐100 = human maxi-. ž . mum ; group b: low dose 1 = human dose ; and group c: high dose inactivated. each group contains three female and three male animals. at time points 8, 48 and 168 h, one animal is killed and blood, lymph nodes, lung, spleen, brain, kidneys, gonads, heart and liver harvested under carefully controlled conditions to prevent crosscontamination of vector between different organs. organs should be either snap frozen or stored at y80 њc in individual vials for q-pcr analysis. to generate confidence intervals, a minimum of three replicated wells are required, but more ( ) are preferred. this ensures that results are expressed as positive or negative with confidence. the use of only duplicate or single wells for the controls does not allow comparison of groups to be carried out accurately, due to the significant high value of the t-statistic when using one degree of freedom. however, if working to a limited budget, performing the study using duplicate reaction replicates can allow the data to be expressed as the mean copy number within a specified standard deviation. the use of duplicate wells with a mean and standard deviation allows the generation of a 68.2% confidence interval. however, the use of single wells gives no information on well-to-well reproducibility and should only be used for research purposes rather than bio-safety testing, where confidence in results is essential. to produce an accurate set of data covering the dynamic range of the assay a standard curve with a minimum of four or five standards should be included. a minimum of four or five different 10-fold dilutions should in most cases achieve an assessment of the c values over the range of the t assay on every pcr run. these controls are essential since some of the animals dosed with a gene therapy vectors will most likely contain some pcr-positive tissues. if sentinel extractions are not included while handling positive samples, there will be no control for the level of cross-contamination between samples during extraction. the importance of the sentinel extraction is highlighted since the q-pcr reaction sentinel can be negative and the extraction sentinels positive. this control ensures recovery of test animal nucleic acid. this is necessary since any validation of the extraction procedure will have been performed in another time point than the actual test sample assay. this control can be replaced with the pre-extraction spike recovery control mentioned below, if the target is not integrated into the cellular dna. if the integration of vector dna into the host genome is not being assessed, both pre-extraction and post-extraction spike controls to quantify the percent of recovery during extraction and any inhibition during pcr should be performed. even if a pre-study nucleic acid extraction validation already gives this information, the test sample biodistribution assay is performed at a time point other than that of the pre-study. possible variables between those time points are, for example, variation in solution batches, different operators, errors, or problems with extraction reagents. the spike recovery controls performed at the time of testing ensure that these potential problems are detected. ipc, also known as taqman exogenous internal positive control reagents, can be included in all q-pcr master mixes. this control can replace post-extraction spike controls, since pcr inhibition is detected with ipc reagents. ipc is a set of primers and probe targeted to a non-biological synthetic template. in the multiplexed pcr reaction, ipc is detected using a vic-labelled probe and the viralrcell target nucleic acid is detected using a fam-labelled probe. ipc reagents are spiked into the reactions to distinguish true target negatives from pcr inhibition. in addition, data generated from pcr-negative wells in the presence of ipc ensure that they are truly negative for the viral target, and are not due to a failed amplifications, thereby giving extra confidence in the results. in addition to viral detection and quantification, q-pcr is widely used for the detection of changes in gene copy number, mrna expression and plasmid dna levels. q-pcr combined with dna sequencing and restriction mapping are commonly used in genetic stability testing. examples of common uses of q-pcr in research, de-ž . velopment and quality control include: 1 detec-( ) tion of changes in plasmid dna copy number in e. coli and yeast cell banks from development ž . projects to large scale production; 2 detection of changes in transgene copy number of chromoso-ž mal-integrated expression cassettes yeast and . ž . mammalian cell banks ; 3 copy number of trans-ž . gene mrna levels to assess expression and 4 changes in gene expression in response to the therapeutic product. recent approaches to vaccination involve the direct introduction of recombinant plasmid dna ž gene therapy vectors into appropriate tissue ro-. bertson and griffiths, 1998; mahato et al., 1999 . this includes the administration of plasmid dna complexed with cationic lipids for the treatment ž of genetic and acquired diseases gao and haung, . 1995 . since, plasmids are receiving increasing attention for use as vectors in gene therapy preparations, methods to monitor their stability and yield is of fundamental importance during manufacturing. monitoring changes in transgene copy number and levels of mrna expression is critical for the isolation of stable cell lines and in the streamlining of constructs for production. however, production and purification of large amounts of the therapeutic plasmid vector can be hindered by a number of factors, including bacterial host strain, plasmid dna instability and copy number per cell. together with restriction endonuclease mapping, nucleotide sequence analysis and phenotypic testing, routine monitoring of plasmid copy number is of utmost importance in large scale cultures where genetic instability can ž . cause significant problems smith et al., 1996 . conventional procedures for the determination of plasmid dna copy number are laborious, requiring dna extraction, restriction endonuclease digestion, agarose electrophoreseis and ethidium bromide staining. therefore, any high throughput methods which can calculate the copy number of plasmid vector constructs during fermentation are paramount in obtaining maximum efficiency in any downstream process. using q-pcr assays targeted to regions such as: the plasmid origin of replication; the antibiotic resistance gene; the human cmv promoter; or the therapeutic transgene, it is possible to identify specific changes in plasmid copy number of different microbial constructs. by assessing the relationship between cell number and c values, t linearity was demonstrated allowing the direct determination of plasmid dna copy number from culture samples relative to the reference plasmid containing e. coli strain. a fourfold difference in plasmid dna copy number was detected, therefore the procedure can be used to monitor microbial fermentation or optimise production ž . methods tables 9 and 10 . the two methods for quantifying target molecules by real-time q-pcr are termed relative and absolute quantification. relative quantification can use an endogenous control to normalise the amount of plasmid molecules in a sample. the normalised value can assess significant increases or decreases in the copy number of plasmid dna and is independent of conventional measurement of cell number. the incorporation of relative quantification could provide the potential to screen colonies or volumes of culture dia fourfold difference in cell number was detected. confidence interval data show statistical differences. ( ) rectly. the relative quantification will calculate numerical difference between the endogenous chromosomal gene and the number of plasmid molecules, in a microbial sample. in this way, significant differences between plasmid target and endogenous control, are detected to provide information on plasmid dna molecules per cell. using this approach, the assay could be independent of od standardisation and still be able to 600 detect a two-to fourfold difference in copy number. taken together, q-pcr has severe implications in monitoring the genetic stability of plasmid dna in microbial constructs selected for production. q-pcr can be used to measure the copy number over a wide range of different cell numbers or genome equivalents, and is able to assess copy ž . number stability in master cell bank mcb , ž . working cell bank wcb and post production ž . cell bank pcb . significant changes are demonstrated by performing statistical analysis on the c value obtained from the mcb, wcb and t pcb. the copy number for each cell bank is expressed in a confidence interval and if the numerical c values of the confidence intervals t from each cell bank overlap, this demonstrates that the data derived from each stage of the manufacturing process are derived from the same statistical populations. the statistical data at the ps 0.05 level, provides a means of monitoring significant changes in the copy number during the manufacturing process. q-pcr is more accurate than the southern blot technique for the detection of alterations in the gene copy number, since estimation by southern blotting relies on comparison of the banding intensity on an autoradiograph. the southern blot analysis generates useful information about the structural integrity of the integrated construct but provides only an approximate evaluation of the copy number. in terms of stability, an increase in copy number of the transgene in the cell line can occur by chromosomal duplication. if significant increases in copy number are detected in the manufacturing process, fluorescent in situ ž . hybridisation fish analysis can be performed to investigate further the stability of the construct. direct chromosomal duplication, with no associated structural alterations, would be missed by southern analysis, if the integrated expression cassette remains unaltered when duplicated. structural analysis will only detect changes in the expression cassette, such as those caused by deletions, insertions and recombinationrduplication of the integrated construct to another chromosomal location. therefore, rapid screening with q-pcr assays will aid in the detection of any chromosomal duplication, which results in an increase in copy number but no associated structural alterations detectable by southern blot analysis. because of the increasing rate of discovery of previously unrecognised viruses, safety is becoming of the utmost concern in biotechnology manufacturing. in addition, there is additional pressure for fast assay development, optimisation and validation of testing methods. now that validated tests are available, q-pcr is now beginning to revolutionise the production and batch release of products. the high versatility of q-pcr assays coupled with the high reproducibility and fast throughput ability will no doubt assist in decreasing the time for the development, safety testing and final marketing of novel biotechnology products. that they have carried out for the development program, and for significantly contributing towards solving the problems encountered. a quantitative, internally controlled real-time pcr assay for the detection of parvovirus b19 dna porcine circoviruses: a review one step fluorescent probe product enhanced reverse transcriptase assay deletion mutants of polyoma virus defining a nonessential region between the origin of replication and the initiation codon for early proteins rapid quantification and differentiation of human polyomavirus dna in undiluted urine from patients after bone marrow transplantation cache valley and ž . potosi viruses bunyaviridae in white tailed deer ž . odocoileus¨irginianus : experimental infections and antibody prevalence in natural populations bunyavirus superinfection and segment reassortment in transovarially infected mosquitos use of quantitative product enhanced reverse transcriptase assay to monitor retrovirus levels in mab cell-culture and downstream processing cache valley virus infection in texas sheep flocks analytical method validation real time quantitative pcr for retrovirus-like particle quantification in cho cell culture ovine fetal malformations induced by utero inoculation of main drain, san angelo and la crosse viruses cationic liposome-mediated gene transfer development of a fluorogenic ž . polymerase chain reaction assay taqman for the detection and quantification of varcella zoster virus real time quantitative pcr detection of reverse transcriptase by a highly sensitive assay in sera from persons infected with human immunodeficiency virus type 1 sensitive and robust one-tube real-time reverse transcriptase-polymerase chain reaction to quantify siv rna load: comparison of one-versus two-enzyme systems validation of analytical methods. definitions and terminology. the european agency for the evaluation of medicinal products validation of analytical methods. methodolgy. the european agency for the evaluation of medicinal products detection and quantitation of human papillomavirus by using the fluorescent 5ј exonuclease assay development of a tt virus dna quantification system using realtime detection pcr human herpes virus 8 ž hhv-8 in kaposi's sarcoma: lack of association with bcl-2 and p53 expression quantitative analysis of epstein᎐barr virus load by a using real time pcr assay comparison of six nucleic acid extraction methods for the detection of viral dna or rna sequences in four different non-serum specimen types retroviruses in rodents quantitative competitive reverse transcription-pcr as a method to evaluate retrovirus removal during chromatography procedures real-time taqman pcr as a specific and more sensitive ( ) alternative to the branched-chain dna assay for quantitation of simian immunodeficiency virus rna a defined approach to the regulatory assessment of the use of neoplastic cells as substrates for viral vaccine manufacture. a draft. for the cell᎐ substrate ᎐ adventitious agent workingrinterest group palindromic dna and genome stability. further studies quantitation of dnarrna using real time pcr detection inverted alu repeats unstable in yeast are excluded from the human genome high throughput detection of retrovirus-associated reverse transcriptase using an improved fluorescent product enhanced reverse ž transcriptase assay f-pert and its comparison to conventional detection methods pharmaceutical perspectives of nonviral gene therapy zoonoses and haemorrhagic fever elimination of background signals in a modified polymerase chain reaction-based reverse transcriptase assay the role of deer as a possible reservoir host of potosi virus recognised arbovirus in the united states simultaneous screening for hbv dna and hcv rna genomes in blood donations using a novel taqman pcr rapid reverse transcription-pcr detection of hepatitis c virus rna in serum using taqman fluorogenic detection system development of a real-time pcr procedure including an internal control for the measurement of hcmv viral load different real-time pcr formats compared for the quantitative detection of human cytomegalovirus dna human exposure to bovine polyomavirus: a zoonosis? target selection and optimisation of amplification reactions ultra-sensitive retro-v irus detection by a reverse transcriptase assay based on product enhancement critical factors for successful pcr. practical guidelines technical literature who guidelines for assuring the quality of dna vaccines development of a high-throughput quantitative assay for detecting herpes simplex virus dna in clinical samples application of a fluorogenic pcr assay for typing and subtyping of influenza viruses in respiratory samples frequent detection of bovine polyomavirus in commercial batches of calf serum by using the polymerase chain reaction a real time quantitative pcr-based method for the detection and quantification of simian virus 40 real time quantitative pcr as a method to evaluate simian virus 40 removal during pharmaceutical protein purification an rt pcr assay for the enzyme activity of reverse transcriptase capable of detecting single virions fast and accurate method for quantitating e. coli host᎐cell dna contamination in plasmid dna preparations plasma siv rna viral load determination by real time quantification of product generation in reverse transcriptase polymerase chain reaction structure and distribution of endogenous non-ecotropic murine leukemia virus in wild mice simultaneous detection of influenza viruses a and b using real-time quantitative pcr multiplex detection of four pathogenic retroviruses using molecular beacons correlation, regression and line fitting through graph points: standard curves. practical statistics for experimental biologists key: cord-010037-1bpc8g6n authors: wu, hui; ma, zhen; wang, ming‐ming; qin, ai‐li; ran, jin‐hua; wang, xiao‐quan title: a high frequency of allopolyploid speciation in the gymnospermous genus ephedra and its possible association with some biological and ecological features date: 2016-02-16 journal: mol ecol doi: 10.1111/mec.13538 sha: doc_id: 10037 cord_uid: 1bpc8g6n the origin and evolution of polyploids have been studied extensively in angiosperms and ferns but very rarely in gymnosperms. with the exception of three species of conifers, all natural polyploid species of gymnosperms belong to ephedra, in which more than half of the species show polyploid cytotypes. here, we investigated the origin and evolution of polyploids of ephedra distributed in the qinghai–tibetan plateau (qtp) and neighbouring areas. flow cytometry (fcm) was used to measure the ploidy levels of the sampled species that are represented by multiple individuals from different populations, and then, two single‐copy nuclear genes (lfy and ddb2) and two chloroplast dna fragments were used to unravel the possible origins and maternal donors of the polyploids. the results indicate that the studied polyploid species are allopolyploids, and suggest that allotetraploidy is a dominant mode of speciation in ephedra. the high percentage of polyploids in the genus could be related to some of its biological attributes such as vegetative propagation, a relatively high rate of unreduced gamete formation, and a small genome size relative to most other gymnosperms. significant ecological divergences between allotetraploids and their putative progenitors were detected by pcas and anova and tukey's tests, with the exception of e. saxatilis. the overlap of geographical distributions and ecological niches of some diploid species could have provided opportunities for interspecific hybridization and allopolyploid speciation. polyploidy or whole-genome duplication (wgd) has long been recognized as an important process in plant evolution (otto & whitton 2000; soltis et al. 2009 ). at least one round of wgd occurred before the divergence of seed plants (jiao et al. 2011) , and multiple rounds of wgd have been reported in angiosperms (vision et al. 2000; simillion et al. 2002; bowers et al. 2003 ; also see review by leitch & leitch 2012) . polyploidy may have broad-scale effects on genomic repatterning, gene expression and genetic networks due to the inheritance of an additional set of chromosomes, and can produce immediate shifts in morphology, breeding system and ecological tolerances (otto 2007; soltis et al. 2010; fawcett et al. 2013; weiss-schneeweiss et al. 2013) , although some studies found no correlation between polyploidization rate and species richness (wood et al. 2009; mayrose et al. 2011) . while it has been recognized that a polyploid species often has multiple origins (see review by soltis et al. 2014a) , it remains challenging to evaluate the direct effect of polyploidy on evolutionary success of the species (madlung 2013; soltis et al. 2014b ). moreover, although some studies indicate that ecological divergence is an important driver of polyploid speciation, especially allopolyploid speciation (fawcett et al. , 2013 van de peer et al. 2009; ramsey 2011) , it remains unresolved whether diploid and polyploid plants can share broad-scale climatic niches (glennon et al. 2014) . in contrast to the high frequency of polyploids documented in angiosperms, polyploidy is exceedingly rare in gymnosperms excluding ephedra (khoshoo 1959; ahuja 2005; murray 2013; wang & ran 2014) . when excluding three species of conifers, that is the two tetraploids juniperus chinensis 'pfitzeriana' and fitzroya cupressoides and the hexaploid sequoia sempervirens (ahuja 2005) , the remaining natural polyploids of gymnosperms all belong to the genus ephedra, in which 50-65% of species show tetraploid or very rarely octoploid cytotypes (khoshoo 1959; huang et al. 2005) . it is of interest to investigate why and how so many polyploids have evolved in this genus. ephedra comprises about 50 extant species that are mainly shrubs distributed in both temperate and subtropical arid environments in the northern hemisphere and south america (kubitzki 1990; ickert-bond et al. 2009 ), with the basal-most lineages distributed in the mediterranean area (rydin & korall 2009; qin et al. 2013 ). these species originated by radiative speciation in the cenozoic with a crown age of about 30 ma (ickert-bond et al. 2009 ), although the earliest fossil record of the genus is from the early cretaceous (yang & wang 2013) . as a secondary diversification centre, the qinghai-tibetan plateau (qtp) and adjacent regions harbour approximately 16 species of ephedra (fu et al. 1999; yang 2002; yang et al. 2003) , of which nine were reported to be polyploids or have polyploid cytotypes, including e. distachya, e. equisetina, e. gerardiana, e. glauca, e. intermedia, e. likiangensis, e. monosperma, e. saxatilis and e. sinica. all of the 16 species form a monophyletic clade with several other species from northern and western asia and horn of africa (huang & price 2003; rydin & korall 2009) and are generally geographically isolated from the other species of the clade, whereas the nine polyploids are respectively located in three well supported subclades, southern qtp, eastern qtp and northern china (qin et al. 2013) . it was inferred that the subclade divergence occurred in the miocene and was very likely linked to the uplift of the qtp and the asian aridification (qin et al. 2013) . however, the origin of these polyploid species remains unknown. the dna sequence markers, single-/low-copy nuclear genes in particular, are increasingly and successfully used to study allopolyploid speciation in plants, such as in oryza (ge et al. 1999) , paeonia (ferguson & sang 2001) , persicaria (kim et al. 2008) , solanum (cai et al. 2012) , nicotiana and sequoia (yang et al. 2012b) . also, in recent years, the whole-genome or whole-transcriptome analysis has greatly advanced our knowledge of origin and evolution of polyploids in angiosperms (e.g. roulin et al. 2012; page et al. 2013; renny-byfield et al. 2013) . however, genome sequencing of any gymnosperm remains a huge and expensive task, although a draft assembly of the genome has been generated for a couple of conifers, including norway spruce (nystedt et al. 2013) , white spruce (birol et al. 2013 ) and loblolly pine (neale et al. 2014 ). therefore, current phylogenetic analysis using single-/low-copy nuclear genes is still the best approach for exploring the evolution of polyploids in ephedra. the previous inference of several allotetraploids from karyomorphological data (mehra 1946) and the presence of hybridogenic speciation in ephedra (cutler 1939; wendt 1993 ) allow us to hypothesize that allopolyploid speciation might be common in the genus. this study aims to investigate the origin and evolution of polyploids of ephedra distributed in the qtp and neighbouring areas. first, flow cytometry was used to measure ploidy levels of the sampled species, most of which were represented by multiple individuals from different populations. then, two single-copy nuclear genes (lfy and ddb2) and two chloroplast dna (cpdna) fragments (trnt-trnf and trns-trnfm) were used to investigate whether these polyploids are allopolyploids or autopolyploids and which species could be the maternal donors of the allopolyploids. finally, based on a comprehensive analysis of biological attributes, phylogenetic relationships, geographical distributions and ecological factors of the habitats, we discussed the possible correlation between allopolyploid speciation and some biological and ecological features. a total of 48 populations of twelve ephedra species (one with two varieties) distributed in the qtp and adjacent regions were sampled for the measurement of ploidy levels, and the cpdna and nuclear gene analyses (table 1) . young branchlets were collected from the individuals that were at least 50 m apart from each other. the remaining four species that also occur in the qtp and neighbouring areas, namely e. distachya, e. lomatolepis, e. rituensis and e. fedtschenkoae, were not included in the study due to the controversy of their species status (fu et al. 1999; yang 2002; yang et al. 2003) , or a lack of enough population samples. among them, e. fedtschenkoae seems unique in being table 1 the chlorotypes, nuclear gene alleles and ploidy levels detected in the sampled populations of the studied ephedra monoecious, but florin (1933) reported that monoecious individuals are common in ephedra. the other three species are tetraploids based on previous studies (e.g. leitch et al. 2001 ) and our preliminary investigation. therefore, the exclusion of these species should not greatly affect our inference about the origins of other tetraploid species (mostly allotetraploids, see results). information on the geographical variation of cytotypes is critical for studies of origin and evolution of polyploids. to determine the ploidy levels, we initially analysed 10 individuals from each of the three populations kbd, xhy and kbx, and did not find ploidy variation within populations. consequently, we analysed five individuals from each of the other populations with the exception of four populations (sbg, ghz, mgd and mge), from which fewer than five individuals were available. to investigate variation patterns and evolutionary relationships of the maternally inherited cpdna, sequences of two fragments (trnt-trnf and trns-trnfm) were analysed for a total of 740 samples, including 306 individuals sampled in this study and 434 individuals reported in qin et al. (2013) . based on the analyses of ploidy and cpdna variation, we further chose 58 individuals to explore nuclear gene relationships of the 12 species, one with two varieties. also, one individual of the saudi arabian e. foeminea was sampled as outgroup based on the results of previous phylogenetic analyses (ickert-bond et al. 2009; rydin & korall 2009 ). the samples were consistently used in the three analyses (ploidy, cpdna and nuclear genes), with the exception that different sample sizes were used. chromosome numbers were counted for two species, e. equisetina (cultivated in the beijing botanical garden) and e. intermedia. fresh root tips were pretreated with 0.01% colchicine solution for 5-6 h and fixed in a mixture of ethanol/acetic acid (3:1) for 12 h at room temperature. after being macerated in 1n hcl at 60°c for 5-10 min, the materials were stained with 1% carbolfuchsin, and then were squashed and observed under a light microscope. the chromosome number of each species was counted based on at least five cells. the flow cytometry (fcm) has made it convenient to detect the variation of dna ploidy level in large samples from herbaria and silica gel-dried materials (suda & tr avn ı cek 2006; sch€ onswetter et al. 2007; krejcikova et al. 2013; vr ana et al. 2014) . in ephedra, the leaves are reduced to small membranous sheaths. therefore, we used the silica gel-dried young branchlets for the fcm measurement, mainly following the protocol of suda & tr avn ı cek (2006) . approximately 0.3 g silica gel-dried branchlets per individual was chopped with a razor blade in a petri dish containing 1 ml of otto i buffer (0.1 mol/l citric acid monohydrate, 0.5% (v/v) tween-20, ph 2-3). after filtering through a 50-lm nylon mesh and centrifuging at 100 g for 8 min, the pellet was resuspended in 200 ll buffer of a 1:2 mixture of otto i and otto ii (0.4 mol/l na 2 hpo 4 12h 2 o) and stained with 50 lg/ml pi including 50 lg/ml rnase. the fcm measurements were taken using an elite flow cytometer (bd facscalibur, usa). to guarantee the reliability of the measurements, several samples were reanalysed (up to four times) on different days to assess between-run fluctuations, and the results showed that the measurements are very consistent. if the coefficient of variation (cv) of the histogram peak exceeded 5%, the sample was discarded or remeasured. the dna ploidy levels were inferred based on dna contents measured in plants with known chromosome numbers. that is, based on chromosome number counts (see results), the two species of ephedra, e. equisetina (2n = 14, diploid) and e. intermedia (2n = 28, tetraploid), were used as external reference standards, with their dna contents measured and shown in fig. s1 (supporting information). total dna was isolated from silica gel-dried young branchlets by the modified ctab method (rogers & bendich 1985) . two cpdna regions, trnt-trnf and trns-trnfm, were amplified and sequenced following the protocols of qin et al. (2013) . the lfy gene was amplified with the forward primer lfye2f2 (5 0 -gacagttggtg ctttaatagg -3 0 ) located at the second intron and the reverse primer lfye3r1 (5 0 -cctcatctttggcttgtt-tat -3 0 ) at the third exon, and the ddb2 gene was amplified with ddb2s2 at the second exon (5 0 -acag ccaggtgattgttatgag -3 0 ) and ddb2a1 at the fifth exon (5 0 -tctaaggaggtgacccgtctact -3 0 ). the pcrs were conducted in a volume of 25 ml, containing 50-75 ng total dna, 6.25 pmol of each primer, 200 mmol/l of each dntp and 0.75 unit of taq dna polymerase (takara biotech co., china). pcr cycles were as follows: 4 min at 94°c, 36 cycles of 30 s at 94°c, 30 s at 58°c and 1-2 min at 72°c, with a final extension of 10 min at 72°c. the pcr products were purified using a tiangel midi purification kit (tiangen, china) for the nuclear markers and then cloned with the pgem-t easy vector system ii (promega). for each individual, 6-20 clones (6-12 for diploids, and 8-20 for tetraploids and the outgroup) were sequenced using primer t7 or lfy e3r1. the sequencing products were separated on an abi prism 3730xl dna analyzer (applied biosystems). the sequences generated in this study are deposited in genbank under accession numbers kt033384-kt033389 (trns-trnfm), kt033390-kt033395 (trnt-trnf), kt033145-kt033275 (lfy) and kt033276-kt033383 (ddb2). the dna sequences were aligned and manually adjusted in bioedit v.7.0.9 (hall 1999) . haplotype networks were constructed with network 4.6.1.2 (bandelt et al. 1999) , and each indel was treated as a single mutation event. phylogenetic trees of cpdna haplotypes and the two nuclear genes were constructed by maximum parsimony (mp), maximum likelihood (ml) and bayesian inference (bi) methods, using paup*4.0b10 (swofford 2002) , phyml3.0 (guindon et al. 2010 ) and mr-bayes 3.1.2 (ronquist & huelsenbeck 2003) , respectively. the gaps were treated as missing data. the mp analysis used a heuristic search with 1000 random addition sequence replicates, tree-bisectionreconnection (tbr) and multrees on. branch support was evaluated by a bootstrap analysis (felsenstein 1985) of 1000 replicates using the same heuristic search settings, and a 50% majority-rule consensus was used. in the ml analysis, jmodeltest 2 (guindon & gascuel 2003; darriba et al. 2012 ) was used to determine the best-fit nucleotide substitution models under the akaike information criterion (aic), which were gtr+i for cpdna, tvm+g for lfy and tvm+i for ddb2. as a starting point for the ml search, a bionj tree was used (gascuel 1997) . branch support was estimated by bootstrap analysis with 1000 replicates. the bi analysis used the best-fit models determined also by jmodeltest 2, including gtr+g for cpdna [nst = 6, rates = gamma, prset statefreqpr = dirichlet(1,1,1,1)], gtr+g for lfy [nst = 6, rates = gamma, prset statefreqpr = dirichlet(1,1,1,1)] and hky+g for ddb2 [nst = 2, rates = gamma, and prset statefreqpr = dirichlet(1,1,1,1)]. one cold and three incrementally heated markov chain monte carlo (mcmc) chains were run for 1 000 000 generations each, and trees were sampled every 100 generations with the first 300 samples discarded as burn-in. to show explicitly the origin, particularly the putative progenitors, of the allopolyploid species, the program padre (lott et al. 2009a,b) was used to generate a reticulate phylogenetic network of the studied species based on a collection of lfy, ddb2 and cpdna trees under default settings. the input topologies were 50% majority-rule consensus mp trees that were generated from the reduced matrices comprising 13 species (including outgroup) and 19 representative individuals, which included one allele (diploids) or two alleles (polyploids) distributed in different main clades of the two nuclear gene phylogenies. to investigate whether there was an association between speciation and climatic factors, ecological niche divergence among the species was also investigated. a total of 557 georeferenced occurrence records ( hijmans et al. 2005 ) and the mean annual potential evapotranspiration (pet) from cgiar-csi (http://www.cgiar-csi.org; trabucco et al. 2008 ) at a resolution of 30 arc seconds (about 1 km) based on the geocoordinates using arcgis 10.2. the 20 bioclim variables were examined for pairwise pearson correlations, and 10 variables (bio2, 3, 4, 8, 12, 14, 15, 18, 19 and pet, shown in table s2 , supporting information) with correlation coefficients lower than 0.8 were finally selected. to determine the ecological characteristics of all studied species in the qtp and neighbouring areas and investigate whether ecological divergence had driven polyploid speciation, we used two approaches. first, to identify the divergence of ecological niches, a principal component analysis (pca) using 10 bioclim variables implemented in ade4-r package (dray et al. 2007 ) was conducted for all taxa, and allotetraploids vs. their putative progenitors, respectively. then, to identify bioclim variables associated with speciation, a permutational analysis of variance (permanova) was performed to assess the variation of principal components (pc1 and pc2) and each bioclim variable among and within species using the lmperm package (wheeler 2010) . the principal components and bioclim variables with significant differences indicated by the anova were further assessed by the tukey's honestly significant difference (hsd) test for every two taxa using the stats package. the variation of these variables was shown by boxplots. the analyses of pca, anova and tukey's hsd were conducted in r version 3.1.2 (r development core team, 2014). to confirm the chromosome numbers of the two samples used as external standards for the fcm analysis, we observed mitotic cell divisions in root tips by light microscopy and found that the chromosome number was 2n = 14 for e. equisetina and 2n = 28 for e. intermedia. therefore, the fcm fluorescence histograms of the two samples, as shown in fig. s1 (supporting information), were used to represent diploid (2x) and tetraploid (4x) nuclei, respectively. the peak ratio of e. intermedia to e. equisetina is 1.98. for all the ephedra samples (12 species, 48 populations and 248 individuals) analysed by fcm, none of the cv values exceeded 5%. based on the peak positions that indicate the relative dna contents, the fluorescence histograms could be clearly divided into two cytotypes, corresponding to diploids and tetraploids, respectively. the peak ratios of diploids to e. equisetina ranged from 0.95 to 1.08, and those of tetraploids to e. intermedia ranged from 0.94 to 1.07. the fcm measurement indicated that three species (e. gerardiana, e. przewalskii and e. regeliana) harboured both diploid and tetraploid cytotypes, four species (e. equisetina, e. minuta, e. monosperma and e. rhytidosperma) showed only the diploid cytotype, and six taxa (e. likiangensis, e. glauca, e. intermedia, e. saxatilis, e. saxatilis var. mairei and e. sinica) exhibited only the tetraploid cytotype (table 1) . in our survey, two ploidy levels (2n = 14, 28) were found in different populations of e. przewalskii and e. regeliana (table 1) , from which only diploids were previously reported (kong et al. 2001; jiang 2006; wu et al. 2009 ). moreover, for some species which were reported to have two or more cytotypes (table s3 , supporting information), we only found a single cytotype, such as only diploids in e. equisetina and e. monosperma (table 1) . the trnt-trnf and trns-trnfm sequences were obtained from all of the 740 samples, including 306 individuals determined in this study and 434 individuals reported in qin et al. (2013) (table 1 ). the new sequences generated are mainly from populations in northern china. the alignment of the combined two cpdna fragments is 1115 bp in length, including 27 nucleotide substitutions and eight indels that were used to designate 24 haplotypes (h1-h23 for ingroups and h24 for outgroup, see table s1 , supporting information), of which five (h6-8, h14, h15) were newly detected. the distributions of the ingroup haplotypes are shown in fig. 1 . when the sequence of e. foeminea (h24) was used as outgroup, three main lineages (i-iii) were consistently resolved in the network and phylogenetic tree of cpdna haplotypes (figs 2 and s3) . lineage i consisted of 10 haplotypes (h1-h10) that occurred in five species (e. glauca, e. intermedia, e. przewalskii, e. regeliana and e. sinica) mainly distributed in northern china, but only three of them were shared among species, including h1 among e. glauca, e. intermedia, e. przewalskii and e. sinica, h3 between e. przewalskii and e. glauca, and h4 between e. przewalskii and e. regeliana. lineage ii comprised four haplotypes (h12-h15), of which h12 was the most widely distributed, and was shared by six species (e. equisetina, e. likiangensis, e. minuta, e. monosperma, e. rhytidosperma and e. saxatilis var. mairei). lineage iii harboured eight haplotypes (h16-h23) that occurred in two species and a variety (e. gerardiana, e. saxatilis and e. saxatilis var. mairei) from the qtp. the haplotype h11 detected in the population jjs of e. equisetina was not grouped into any of the three lineages. of the 48 ephedra populations analysed, 37 (77%) harboured a single haplotype, 9 (19%) exhibited two haplotypes, and only two had more than two haplotypes ( fig. 1; table 1 ). for each of the nuclear genes lfy and ddb2 that were pcr-amplified and cloned, 1-2 distinct clones (alleles) were obtained from each diploid individual, and 2-4 alleles were detected in each tetraploid individual, with the exception of two tetraploid individuals of e. przewalskii from populations mgf and wb, each of which contained only 1-2 alleles ( table 1 ). the distributions of the alleles of lfy and ddb2 are shown in figs 3 and 4, respectively. the lfy gene sequences (alleles) were 462-791 bp in length, and the sequence alignment contained 886 sites, of which 244 were variable and 167 were parsimonyinformative. the ddb2 gene sequences ranged from 546 to 551 bp, and the sequence alignment contained 554 sites, of which 87 were variable and 52 were parsimony-informative. the generated mp, ml and bi trees of each gene were highly congruent, and the lfy and ddb2 gene trees were also congruent in deep branches (fig. s4 , supporting information). the simplified strict consensus mp trees of the two genes are shown in fig. 5 , both strongly supporting clades a, b, c and d. clade a (a-type) sequences (alleles) were from northern china and northern qtp, clade b (b-type) alleles occurred in southern and eastern qtp, and clade c (c-type) alleles had a very wide distribution (figs 3 and 4) . the clade d (type d) sequences were all from e. rhytidosperma, a species narrowly distributed in the helan mountain. the diploid species or populations contained only one type of sequences, type a (diploid populations of e. przewalskii and e. regeliana), type c (e. equisetina, e. minuta, e. monosperma, and diploid populations of e. gerardiana) or type d (e. rhytidosperma). in contrast, the tetraploid species contained two types of sequences, types a and c (e. glauca, e. intermedia and e. sinica) or types b and c (e. likiangensis, e. saxatilis, e. saxatilis var. mairei, and the tetraploid population of e. gerardiana). however, the tetraploid populations of przewalskii and e. regeliana only had a-type sequences (fig. 5 ). a phylogenetic network generated from the integration of all three gene trees (cpdna, lfy and ddb2) is shown in fig. 6 , from which eight allotetraploid taxa could be inferred, including e. gerardiana, e. glauca, e. intermedia, e. likiangensis, e. regeliana, e. saxatilis, e. saxatilis var. mairei and e. sinica. the diploid progenitors of these allotetraploids were not well resolved, but it seems that all studied diploid ingroup species, with the exception of e. rhytidosperma, could have been involved. for example, the three tetraploids e. glauca, e. intermedia and e. sinica possibly originated from hybridization with diploids most closely related to e. przewalskii in clade a as the maternal parents and diploids of clade c (e. equisetina, e. minuta and e. monosperma) as the paternal parents (see discussion). the diploid e. regeliana does not share chlorotypes with any of the tetraploids, and therefore is not very likely to have acted as a maternal parent in the allotetraploid speciation. more than 10 georeferenced occurrence records were collected for each of the studied species, with the exception of e. glauca, e. minuta and e. rhytidosperma due to their narrow distributions. results of the pcas are shown in figs 7 and 8, with the factor loadings shown in table s2 (supporting information). for all of the 13 taxa, the pca revealed two components that cumulatively explained 60.11% of variation, and the scatter plot showed that these taxa were clearly divided into two groups by pc1 and pc2 (fig. 7) . table 1 . group 1 include four taxa distributed in the south and east of qtp (e. gerardiana, e. likiangensis, e. saxatilis and e. saxatilis var. mairei), which show a distinct ecological niche with higher isothermality (bio3) and precipitation in the warmest quarter (bio18) and a lower temperature seasonality (bio4), and group 2 comprise the remaining nine taxa. although the anovas detected significant differences of 10 bioclim variables among the 13 taxa, the tukey's hsd tests indicated that only two bioclim variables (bio3 and bio4) were significantly differentiated between group 1 and group 2 species (p < 0.01; table s4 and fig. s5a , b, supporting information), with the exception of a nonsignificant difference between group 1 species and e. minuta in bio4. in addition, e. likiangensis and e. saxatilis var. mairei show significant differences from group 2 species in bio12 and bio18 (p < 0.001 for hsd; fig. s5c , d, supporting information). the two taxa occur in a moist climate with higher annual precipitation (bio12) and precipitation of the warmest quarter (bio18). for comparisons between the allotetraploids and their putative progenitors, the pca revealed two components (pc1 and pc2) that collectively explained 55.63-67.74% of variation (table s2 , supporting information). all but one (e. saxatilis) of the allotetraploids show ecological divergence from their putative progenitors (fig. 8) . two divergence patterns were found: (i) the allotetraploids, including e. likiangensis and e. saxatilis var. mairei, occupy separate ecological niches from their putative maternal progenitors; and (ii) the allotetraploids, including e. glauca, e. intermedia and e. sinica, show separate ecological niches from one of their putative paternal progenitors but have partially overlapped ecological niches with their other putative progenitors. for example, the niche of e. glauca is completely different from that of e. minuta, but is slightly overlapped with that of e. monosperma and more overlapped with those of e. equisetina and e. przewalskii (fig. 8a) ; e. intermedia has a much wider niche than e. minuta and a separate niche from e. equisetina, and is clearly differentiated from e. equisetina, e. monosperma and e. przewalskii along pc2 (fig. 8b) . the allotetraploid e. saxatilis has a similar niche with its putative maternal progenitor e. gerardiana (fig. 8d) . table 1. significant ecological divergences between the allotetraploids and their putative progenitors were also detected by the anova and tukey's tests of the two principal components (pc1, pc2) and the 10 bioclim variables (tables 2 and s4) . results of the anova indicate that the mean squares of all comparisons among species are higher than those within species with the exception of the comparison between e. saxatilis and e. gerardiana, and almost all interspecific divergences are significant (p < 0.05; table s4 , supporting information). the tukey's test detected significant niche divergence between the allotetraploids and their putative progenitors in 2-7 bioclim variables ( table 2 ). the divergence between e. glauca and e. minuta and between e. glauca and e. monosperma occurred, respectively, in seven and five bioclim variables, which is consistent with the results of the pca. compared to e. minuta and e. monosperma, e. glauca is higher in mean temperature of the wettest quarter (bio8) and annual potential evapotranspiration (pet), intermediate in annual precipitation (bio12), and lower in precipitation seasonality (bio15) and precipitation of the warmest quarter (bio18) ( fig. s5e -i, supporting information). relative to their putative progenitors, e. intermedia is lower in temperature seasonality (bio4) and bio8 but higher in bio12 and precipitation of the coldest quarter (bio19), with the exception of the comparison between it and e. minuta (fig. s5j -m, supporting information), and e. likiangensis is higher in isothermality (bio3), bio12, bio18 and pet but lower in bio4, with the exception of the comparison between it and e. minuta in bio4 (fig. s5n -r, supporting information). like e. likiangensis, e. saxatilis var. mairei is higher in bio3, bio12 and bio18 except the comparison between it and e. gerardiana in bio3 (fig. s5s -u, supporting information). the allotetraploid e. sinica also shows niche divergence from most of its putative progenitors and tends to occupy a niche with higher bio12, bio15 and pet ( fig. s5v -x, supporting information). all tetraploid species of ephedra from the qtp and adjacent regions originated by allopolyploid speciation. phylogenetic analysis of single-/low-copy nuclear genes can be effective in revealing allopolyploid parental lineages (e.g. ge et al. 1999; ferguson & sang 2001; kim et al. 2008; cai et al. 2012; yang et al. 2012b; kelly et al. 2013) , when an allopolyploid species, particularly of recent origin, has paralogous sequences inherited from its two or more parental species. these paralogues can place an allotetraploid in different parental clades. in the present study, the fcm measurement of the ploidy levels in a number of individuals from different populations indicates that six (46%) of the studied 13 ephedra taxa, including e. glauca, e. intermedia, e. likiangensis, e. saxatilis, e. saxatilis var. mairei and e. sinica, are tetraploids (table 1 ). in both single-copy nuclear gene trees (lfy and ddb2), each of the six taxa contains two types of sequences that are distributed in different major clades a and c or b and c (figs 5 and 6) , strongly suggesting an allopolyploid origin involving diploid parents from these clades. compared to clades a and c, clade b only contains tetraploids. this could be attributed to the extinction or lack of sampling of diploids in this clade. the three tetraploid species e. glauca, e. intermedia and e. sinica exhibit nuclear gene alleles in both clades a and c (fig. 5) . these species are mainly distributed in northern china, and their chlorotypes belong to lineage i which is also confined to northern china (figs 1 and 2) . in particular, all of them share chlorotypes with diploid e. przewalskii rather than with diploid e. regeliana (fig. 1) . therefore, the three tetraploid species possibly originated from hybridization with diploids most closely related to e. przewalskii (in clade a of fig. 5 and lineage i of fig. 2 ) from northern china as the maternal parents and diploids most closely related to the widespread e. equisetina-e. minuta-e. monosperma (in clade c of fig. 5 and lineage ii in fig. 2 ) as the paternal parents (see the reticulate network in fig. 6 ). in contrast, the three tetraploid taxa e. likiangensis, e. saxatilis and e. saxatilis var. mairei exhibit nuclear gene alleles in both clades b and c (fig. 5) . the chlorotypes of e. saxatilis belong to lineage iii (fig. 2) , and are narrowly distributed in southern qtp (fig. 1) , corresponding to the geographical distribution of this species. hence, this species possibly originated by allopolyploidy with a maternal progenitor from southern qtp, very likely the diploid cytotype of e. gerardiana (in lineage iii of fig. 2 and clade c of fig. 5) , and a paternal progenitor from clade b (fig. 6) . however, as mentioned earlier, the ancient diploids in clade b could be currently extinct. the chlorotypes of e. likiangensis belong to lineage ii with a relatively wide distribution (figs 1 and 2) , and thus, its maternal progenitor could be a diploid species in this lineage, such as e. equisetina, e. minuta and e. monosperma (with nuclear gene alleles in clade c), whereas its paternal progenitor should belong to clade b (figs 5 and 6) . the e. saxatilis var. mairei harbours a high frequency of chlorotypes of lineage iii and a much lower frequency of chlorotypes of lineage ii (figs 1 and 2) . therefore, the maternal progenitor of this taxon could be diploid species from the two lineages such as e. gerardiana (2x), e. equisetina, e. minuta and e. monosperma, and its paternal progenitor could be from clade b (figs 5 and 6 ). fig. 6 a reticulate network constructed from the reduced 50% majority-rule consensus mp trees of lfy, ddb2 and cpdna using the program padre. different colours of lines indicate the positions of the species in the cpdna network (lineages i-iii in fig. 2 ) and the alleles in the two nuclear gene phylogenies (clades a-c in fig. 5) : green, lineage i, clade a; blue, lineage ii, clade c; purple, lineage iii, clade c; red, clade b; grey, the lineage of cpdna haplotype h11. bold solid and dashed lines represent putative maternal and paternal progenitors of the tetraploids, respectively. diploids and autotetraploids are in black, and allotetraploids are in colour. letters following species names are population names corresponding to those in table 1. as discussed above, the widespread diploids of e. equisetina-e. minuta-e. monosperma or their progenitors may have played important roles in allopolyploid speciation of ephedra in the qtp and adjacent regions. interestingly, most of the above six tetraploid taxa harbour two or more chlorotypes, even chlorotypes from different main lineages such as in e. saxatilis var. mairei (figs 1 and 2) . this may indicate multiple origins of a tetraploid taxon, divergence of chlorotypes subsequent to polyploidy, or interspecific chloroplast introgression. both diploid and tetraploid cytotypes are present in e. gerardiana, e. przewalskii and e. regeliana (table 1) . the tetraploids of e. przewalskii only have a2-type sequences of the nuclear gene lfy (in clade a, fig. 5 ) as its diploids (figs 3 and 4) , but do not share all chlorotypes with the diploids (table 1; fig. 1 ), although their chlorotypes all belong to lineage i (fig. 2) . in particular, the chlorotype h3 is shared between the tetraploids of e. przewalskii and e. glauca (table 1; fig. 1 ). therefore, the tetraploids of e. przewalskii may be autopolyploids or young allopolyploids with very closely related parental species. in the lfy tree (fig. 5) , the tetraploids of e. regeliana harbour two subtypes of sequences in clade a, including a2 with the diploids and tetraploids of e. przewalskii and the tetraploid e. glauca, and a3 with the diploid e. regeliana and two tetraploid species (e. sinica and e. intermedia). notably, the diploids and tetraploids of e. regeliana do not share chlorotypes (table 1 ; fig. 1 ). thus, the tetraploids of e. regeliana could be allopolyploids that might have originated by hybridization between the diploids of e. regeliana and e. przewalskii (fig. 6) , although an origin by hybridization between an autotetraploid of e. regeliana and a tetraploid of other species cannot be ruled out. in addition, there could be incomplete linage sorting or introgression at the tetraploid level. however, it is unknown why this tetraploid population (xab) only exhibits private chlorotypes. the diploids and tetraploids of e. gerardiana occur in clade c and clade b+ clade c, respectively (fig. 5) , and they do not share chlorotypes (table 1 ; fig. 1 ). this may also suggest an allopolyploid origin or a complicated origin of the tetraploid cytotype like in the tetraploid e. regeliana (fig. 6) , although the two cytotypes of e. gerardiana do not show clear morphological difference. to understand the origin of the tetraploids of this species, more samples need to be studied in the future. polyploid evolution in ephedra and its correlation with some biological and ecological features evolution of polyploids. of the 36 ephedra species that have been cytologically studied, 24 are polyploids or contain polyploid cytotypes (66% of species). although intraspecific polyploidy has been documented in about half of the species, the results of some early cytological studies need to be checked carefully as mentioned earlier, and more population samples are necessary to study the ploidy levels of a species. notably, all polyploids in the 24 species are tetraploids with the exception of an octoploid cytotype reported in e. funerea and e. gerardiana (table s3 , supporting information). according to the available information (table s3 , supporting information), tetraploids are present in about 75% of the old world species and 62.5% of the new world species. our present study found that all tetraploid ephedra species from the qtp and neighbouring areas, excluding the species with both diploid and tetraploid populations, are allotetraploids. therefore, we may conclude that allotetraploidy is a dominant mode of speciation in ephedra, although the origin of other polyploids in this genus, especially from europe and america, needs to be further studied. in fact, all of the remaining three polyploid species from other gymnosperm lineages were also deduced to be allopolyploids by yang et al. (2012b) . the biological features related to polyploid speciation. the high percentage of polyploids in ephedra could be related to some attributes of the genus such as a shrub habit, vegetative propagation, a relatively high rate of unreduced gamete formation, and a relatively low basic chromosome number and small genome size (at diploid level) for a gymnosperm (leitch & leitch 2012 grif (2000) reported that plant taxa with a small dna value per genome have a high percentage of polyploidy and show higher ploidy levels, and wood et al. (2009) found that the generic base count (the minimum number of chromosomes reported in a genus) is negatively associated with polyploid incidence in angiosperms. in particular, the union of unreduced gametes has been considered as the most likely way of polyploid formation in plants (harlan & de wet 1975; de wet 1980; soltis et al. 2010; brownfield & k€ ohler 2011) , and the different rates of unreduced (bretagnolle & thompson 1996; ramsey & schemske 1998 , 2002 ramsey 2006; younis et al. 2014) . according to the karyomorphological study of ephedra based on pollen germination, three of five tetraploid species exhibited unreduced pollen grains that accounted for 2-5% of the total amount (mehra 1946) . also, dimorphism of pollen size in the same herbarium specimen has been reported from seven species of ephedra, including e. alata, e. americana, e. aphylla, e. breana, e. chilensis, e. ochreata, e. torreyana, e. trifurca and e. tweediana (beug 1956; kedves 1987; ickert-bond et al. 2003) . hence, a relatively high rate of unreduced gamete formation in ephedra has very likely contributed to the high frequency of polyploids in the genus. the ecological differentiation associated with polyploid speciation. polyploids possess the potential for evolving into new species with evolutionary novelty, and some previous studies suggest that ecological divergence may play a prominent role in polyploid speciation (brochmann et al. 2004; hijmans et al. 2007; du skov a et al. 2010; mcintyre 2012; theodoridis et al. 2013) . in this study, we also found that ecological divergence was associated with the speciation or divergence of polyploids in ephedra. significant ecological divergences between the allotetraploids and their putative progenitors were detected by the pcas and the anova and tukey's tests, with the exception of e. saxatilis (figs 8 and s5 ). the allotetraploids have separate ecological niches from their putative maternal progenitors, or occupy separate ecological niches from one of their putative paternal progenitors but have partially overlapped ecological niches with their other putative progenitors (figs 8 and s5 ). for example, e. likiangensis and e. saxatilis var. mairei endemic to the qtp inhabit dense grass swards or dwarf-shrub communities and prefer a moister climate with higher annual precipitation and precipitation of the warmest quarter than their putative progenitors e. equisetina, e. minuta and e. monosperma (table s5 ; fig. s5p , q, t, u, supporting information); e. intermedia favours conditions that have lower temperature seasonality and mean temperature of the wettest quarter and favours higher annual precipitation and precipitation of the coldest quarter, compared to its putative maternal progenitor e. przewalskii and paternal progenitors e. equisetina and e. monosperma (table 2 ; figs 8b and s5j-m). the allotetraploid e. sinica has a vast distribution from northwestern china northward to mongolia and russia and eastward up to the gulf of bohai. similarly, the allotetraploid e. intermedia is widely distributed in irano-turanian and central asian floristic regions. both these species widely occur in open areas such as zonal steppe, desert steppe or coarse-textured-skeletal-sandy soils (our unpublished observations; freitag & maierequisetina -*** -*** *** *** *** ns ns *** *** *** e. minuta ns ns ns ns ns ns ns *** ** ** e. monosperma -*** ns *** *** *** ns *** *** *** *** e. przewalskii -*** -** *** *** *** ns ns ns *** ns e. likiangensis e. equisetina *** *** -*** *** ** *** ns *** *** ns * e. minuta * ns -*** ns ns *** ns ns *** ns ** e. monosperma *** *** -*** *** ns *** ns ns *** ns *** e. saxatilis var. mairei e. equisetina *** *** ns *** *** ** *** ns * *** ns e. gerardiana ns ns ns ns ns ns *** ns ns *** ns e. minuta ns ns ns ** ns ns *** ns ns *** ns e. monosperma *** *** ns *** *** ns *** ns ns *** ns e. sinica e. equisetina *** ns ns ns ns ns *** *** *** * *** ** e. minuta ns *** ns ns * *** ns ns ns ns ns ** e. monosperma ** ns ns ns ns *** *** ns *** ns ns *** e. przewalskii * *** ns ns ns ns *** ns *** *** * ns ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; -, p > 0.05 in the anovas. stolte 1994; fu et al. 1999 ). however, their putative progenitors e. equisetina, e. minuta and e. monosperma are usually subordinate components of acrophytia with narrow distributions in dry rocky slopes (table s5 ; fig. s2 , supporting information). it is particularly interesting that most of the allotetraploid taxa we found, including e. intermedia, e. likiangensis, e. saxatilis var. mairei and e. sinica, have adapted to a moister climate with a higher annual precipitation than their putative progenitors ( fig. s5l , p, t, v, supporting information), considering that the genus ephedra generally occurs in dry habitats. the ecological divergence associated with allotetraploidy or its divergence could be associated with new habitats triggered by the fast uplift of the qtp and the asian aridification in the middle to late miocene guo et al. 2002; spicer et al. 2003; dupont-nivet et al. 2007; jiang & ding 2008; royden et al. 2008) , during which the tetraploid ephedra species originated (qin et al. 2013) . such a scenario has been argued for angiosperm polyploids both geographical distributions and ecological niches of some diploid species, such as e. equisetina, e. monosperma and e. regeliana (2x), mostly or partially overlap (figs 7 and s2 ), which could have provided opportunities for the hybridization between these species, giving rise to allotetraploid species by allopolyploid speciation. to investigate whether ecological divergence had driven polyploid speciation of ephedra in the qtp and neighbouring areas, more molecular markers or phylogenomic approaches should be used to resolve the parental species of these polyploids, and the molecular mechanisms underlying the adaptation to a specific ecological factor could be explored in future studies. additional supporting information may be found in the online version of this article. table s1. the cpdna haplotypes detected in the studied ephedra species and their genbank accessions. table s2 . a summary of the principal component analysis. table s3 . the chromosome numbers in ephedra l. table s4 . results of the anova and tukey's hsd tests for the two principal components revealed by the pca and the 10 bioclim variables. table s5 . morphological characteristics, habitat preference and distribution of ephedra species from the qtp and adjacent regions. polyploidy in gymnosperms: revisited evolution of asian monsoons and phased uplift of the himalaya-tibetan plateau since late miocene times median-joining networks for inferring intraspecific phylogenies assembling the 20 gb white spruce (picea glauca) genome from whole-genome shotgun sequencing data unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events an experimental study of ecological differences in winter growth between sympatric diploid and autotetraploid dactylis glomerata polyploidy in arctic plants unreduced gamete formation in plants: mechanisms and prospects single copy nuclear gene analysis of polyploidy in wild potatoes (solanum section petota) ploidy variation in buddleja l. (buddlejaceae) in the sino-himalayan region and its biogeographical implications monograph of the north american species of the genus ephedra 2: more models, new heuristics and parallel computing the ade4 package-ii: two-table and k-table methods. r news tibetan plateau aridification linked to global cooling at the eocene-oligocene transition genome size correlates with growth form, habitat and phylogeny in the andean genus lasiocephalus (asteraceae) plants with double genomes might have had a better chance to survive the cretaceous-tertiary extinction event significance and biological consequences of polyploidization in land plant evolution confidence limits on phylogenies: an approach using the bootstrap speciation through homoploid hybridization between allotetraploids in peonies (paeonia) uber einige neue oder wenig bekannte asiatische ephedra-arten der sektion pseudobaccatae stapf chorology of trees and shrubs in southwest asia and adjacent regions 10 bionj: an improved version of the nj algorithm based on a simple model of sequence data phylogeny of rice genomes with emphasis on origins of allotetraploid species. proceedings of the national academy of sciences of the united states of america evidence for shared broad-scale climatic niches of diploid and polyploid plants some aspects of plant karyology and karyosystematics a simple, fast and accurate method to estimate large phylogenies by maximum-likelihood new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml 3.0 onset of asian desertification by 22 myr ago inferred from loess deposits in china bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/ nt on € o. winge and a prayer: the origins of polyploidy very high resolution interpolated climate surfaces for global land areas geographical and environmental range expansion through polyploidy in wild potatoes (solanum section petota) estimation of the age of extant ephedra using chloroplast rbcl sequence data phylogenetic relationships in ephedra (ephedraceae) inferred from chloroplast and nuclear dna sequences the incidence of polyploidy in natural plant populations: major patterns and evolutionary processes pollen dimorphism in ephedra l. (ephedraceae) a fossil-calibrated relaxed clock for ephedra indicates an oligocene age for the divergence of asian and new world clades and miocene dispersal into south america study on the genetic relationship analysis and determination of main medical composition for ephedra a 20 ma pollen record of east-asian summer monsoon evolution from guyuan ancestral polyploidy in seed plants and angiosperms lm and em studies on pollen grains of recent welwitschia mirabilis hook. and ephedra species reconstructing the complex evolutionary origin of wild allopolyploid tobaccos (nicotiana section suaveolentes). evolution allopolyploid speciation in persicaria (polygonaceae): insights from a low-copy nuclear region a study on karyotypes of two species in ephedra high ploidy diversity and distinct patterns of cytotype distribution in a widespread species of oxalis in the greater cape floristic region the families and genera of vascular plants genome size and its uses: the impact of flow cytometry ecological and genetic factors linked to contrasting genome dynamics in seed plants nuclear dna c-values complete familial representation in gymnosperms uniformity of karyotypes in rheum (polygonaceae), a species-rich genus in the qinghai-tibetan plateau and adjacent regions padre: a package for analyzing and displaying reticulate evolution inferring polyploid phylogenies from multiplylabeled gene trees a karyological study of six species of silene l. (caryophyllaceae) from the hengduan mountains polyploidy and its effect on evolutionary success: old questions revisited with new tools recently formed polyploid plants diversify at lower rates polyploidy associated with altered and broader ecological niches in the claytonia perfoliata (portulacaceae) species complex a study of the karyotypes and the occurrence of diploid male gametophytes in some species of the genus ephedra polyploidy and new chromosome counts in anaphalis (asteraceae: gnaphalieae) from the qinghai-tibet plateau of chromosome numbers and polyploidy in leontopodium (asteraceae: gnaphalieae) from the qinghai-tibet plateau of sw china. caryologia decoding the massive genome of loblolly pine using haploid dna and novel assembly strategies the norway spruce genome sequence and conifer genome evolution the evolutionary consequences of polyploidy polyploid incidence and evolution insights into the evolution of cotton diploids and polyploids from whole-genome re-sequencing phylogeographic evidence for a link of species divergence of ephedra in the qinghai-tibetan plateau and adjacent regions to the miocene asian aridification a language and environment for statistical computing. r foundation for statistical computing unreduced gametes and neopolyploids in natural populations of achillea borealis (asteraceae) polyploidy and ecological adaptation in wild yarrow pathways, mechanisms, and rates of polyploid formation in flowering plants neopolyploidy in flowering plants quaternary range dynamics and polyploid evolution in an arid brushland plant species (melampodium cinereum, asteraceae) diploidization and genome size change in allopolyploids is associated with differential dynamics of low-and high-copy sequences extraction of dna from milligram amounts of fresh, herbarium and mummified plant tissues mrbayes 3: bayesian phylogenetic inference under mixed models the fate of duplicated genes in a polyploid plant genome the geological evolution of the tibetan plateau evolutionary relationships in ephedra (gnetales), with implications for seed plant phylogeny sympatric diploid and hexaploid cytotypes of senecio carniolicus (asteraceae) in the eastern alps are separated along an altitudinal gradient the hidden duplication past of arabidopsis thaliana polyploidy and angiosperm diversification what we still don't know about polyploidy are polyploids really evolutionary dead-ends (again)? a critical reappraisal of the polyploidy revolution then. . .and now: stebbins revisited constant elevation of southern tibet over the past 15 million years reliable dna ploidy determination in dehydrated tissues of vascular plants by dapi flow cytometry-new prospects for plant research phylogenetic analysis using parsimony (and other methods) 4.0 beta. sinauer associates, sunderland divergent and narrower climatic niches characterize polyploid species of european primroses in primula sect climate change mitigation through afforestation reforestation: a global analysis of hydrologic impacts with four case studies the flowering world: a tale of duplications the origins of genomic duplications in arabidopsis flow cytometry in plant research: a success story evolution and biogeography of gymnosperms evolutionary consequences, constraints and potential of polyploidy in plants evolutionary diversifications of plants on the qinghai-tibetan plateau a new variety of ephedra torreyana (ephedraceae) from west texas and chihuahua, with notes on hybridization in the e. torreyana complex origins of polyploids lmperm: permutation tests for linear models. r package version the frequency of polyploid speciation in vascular plants staining and slide-preparing technique of mitotic chromosomes and application in karyotype determination of ephedra systematics and evolution of ephedra l. (ephedraceae) from china the earliest fleshy cone of ephedra from the early cretaceous yixian formation of northeast china a new species of ephedra (ephedraceae) from china great genetic differentiation among populations of meconopsis integrifolia and its implication for plant speciation in the qinghai-tibetan plateau three genome-based phylogeny of cupressaceae s.l.: further evidence for the evolution of gymnosperms and southern hemisphere biogeography exploitation of induced 2n-gametes for plant breeding polyploidy in aconitum subgenus lycoctonum (ranunculaceae) phylogenetic relationships and character evolution of rhodiola (crassulaceae) based on nuclear ribosomal its and plastid trnl-f and psba-trnh sequences designed the study. h.w. and z.m. performed the laboratory work. m.m.w. and a.l.q. contributed plant materials we thank drs. yu-zhi cun and fu-sheng yang, and mr. cai-yuan qiao for their help in sample collection; ms. wan-qing jin for her assistance in dna sequencing. we also thank the royal botanic garden, edinburgh, for providing the sample of ephedra foeminea for dna analysis. we appreciate the subject editor and the three anonymous reviewers for their insightful comments and suggestions on the manuscript. this study was supported by the national natural science foundation of china (grant nos 31330008, 31170197 and 30730010) and the chinese academy of sciences (kjzd-ew-l07 and the cas/ safea international partnership program for creative research teams). key: cord-290550-u8x9drva authors: radford, alan d.; chapman, david; dixon, linda; chantrey, julian; darby, alistair c.; hall, neil title: application of next-generation sequencing technologies in virology date: 2012-09-17 journal: j gen virol doi: 10.1099/vir.0.043182-0 sha: doc_id: 290550 cord_uid: u8x9drva the progress of science is punctuated by the advent of revolutionary technologies that provide new ways and scales to formulate scientific questions and advance knowledge. following on from electron microscopy, cell culture and pcr, next-generation sequencing is one of these methodologies that is now changing the way that we understand viruses, particularly in the areas of genome sequencing, evolution, ecology, discovery and transcriptomics. possibilities for these methodologies are only limited by our scientific imagination and, to some extent, by their cost, which has restricted their use to relatively small numbers of samples. challenges remain, including the storage and analysis of the large amounts of data generated. as the chemistries employed mature, costs will decrease. in addition, improved methods for analysis will become available, opening yet further applications in virology including routine diagnostic work on individuals, and new understanding of the interaction between viral and host transcriptomes. an exciting era of viral exploration has begun, and will set us new challenges to understand the role of newly discovered viral diversity in both disease and health. we need only to look at the size and growth of international nucleotide databases to realize the importance of sequencing to science in general, and virology in particular. in the latest published release of genbank ( # 179; august 2010), there were some 970 million and 43 million bases of viral and phage origin respectively, representing an annual growth of 20-24 % (fig. 1a ) (benson et al., 2011) , a growth rate comfortably above average for the database as a whole. the sequencing effort is being driven almost entirely by human clinical significance, with 17 of the top 20 sequenced viruses causing disease in humans (fig. 1b) . in its entirety, this sequencing effort represents a significant achievement; the information generated has wide-ranging impact on all areas of virology, from diagnosis to pathogenesis, and from vaccine design to viral evolution and ecology. all of this output has only been possible through embracing what were, at their outset, two groundbreaking and revolutionary methodologies. these allowed those interested in understanding viruses first to amplify and then to sequence viral nucleic acids, namely dna amplification by pcr (mullis et al., 1986) and dna sequencing with chain-terminating inhibitors (sanger sequencing or first-generation sequencing) (sanger et al., 1977) . using these technologies, even a fairly basic laboratory could amplify and generate 100-1000 bases of sequence relatively easily in a single day, and by applying first-generation sequencing technology on a large scale in highly specialized sequencing centres, the first large highprofile genomes were published, notably those for homo sapiens, plasmodium falciparum and mycobacterium tuberculosis (cole et al., 1998; gardner et al., 2002; lander et al., 2001) . the main limitations to the use of these technologies are restricted scalability, their cost when applied to large genomes and their frequent reliance on prior and specific template amplification by pcr or in bacterial clones. as such, genome projects were largely restricted to high-profile organisms, model organisms and human pathogens. more commonly, next-generation sequencing; ngs) that have entirely revolutionized our ability to sequence. overnight, it is now possible to generate many millions of bases of sequence and this has opened many new opportunities, making large-scale sequencing projects accessible to us all, whatever our field of biology. in this review, we shall give an overview of these methods and look particularly at their increasing application in virology, specifically in virus discovery, genome sequencing and transcriptomics. there are an increasing number of ngs technologies in the marketplace, all using slightly different methodologies to achieve clonal amplification and sequencing (table 1) . these methodologies are likely to be subject to continual modification, although the basic principles will probably remain the same. instruments 454 sequencing (roche diagnostics) . dsdna in solution is first sheared by nebulization (converted into a fine spray) or amplified by pcr, and fragments of the appropriate size are selected (fig. 2) . these fragments are blunt-ended and dephosphorylated, followed by the ligation of two separate adapters (a and b). the b adapter is biotinylated, allowing subsequent purification using streptavidin-coated beads. subsequently, denaturation releases from the beads only those molecules that contain an a primer on one end and a b primer on the other. these molecules constitute the dna library, which is bound to microbeads through primer hybridization under conditions that favour a single molecule per bead (fig. 3a) . subsequently, an emulsion is formed in a water and oil mixture, thus capturing individual beads and amplification reagents, including primers, one of which is biotinylated, in their own emulsion microreactor (fig. 3b) . thermal cycling then results in the emulsion-based clonal amplification of individual molecules (fig. 3c ). after amplification, the emulsion is disrupted and beads coated with amplified products are enriched using streptavidin-coated beads and magnetic separation. the amplified products are denatured, bound to a sequencing primer and separated into individual wells of a picotitre plate, each well being large enough to accommodate a single bead (fig. 4a) . sequencing then takes place by pyrosequencing, whereby each nucleotide incorporation leads to the release of pyrophosphate (pp i ) (fig. 5b) . this is converted via atp to generate light, the amount of which is proportional to the number of bases incorporated. the strength of this system is its read length of approximately 500 bases, making it particularly suitable for amplicon sequencing and bridging across complex sequences. its achilles' heel is its reliance on pyrosequencing, which creates difficulties for sequencing homopolymer runs, leading to an error rate in individual reads of ¡1 %, mostly attributable to (benson et al., 2011) . (b) the 20 most frequently sequenced viruses appearing on genbank. sequences were identified based on their organism name as described in each submission; prrsv, porcine reproductive and respiratory syndrome virus. these data were compiled using eid2 (anonymous, 2012) . insertions and deletions (gilles et al., 2011; wang et al., 2007) . whilst depth of coverage can usually overcome this, it becomes more problematic when individual read data are used to assess population diversity (see subsection on targeted amplification). solid sequencing (life technologies). this methodology starts in a similar way to that described above, with dna fragmentation (fig. 2) , ligation to beads, and emulsion pcr (fig. 3a-c) . following denaturation of amplified products, the beads are attached to a glass slide for sequencing (fig. 4b) . the density of beads can be extremely high, and determines the ultimate number of reads achieved. unlike all other methodologies, sequencing occurs by hybridization and ligation ( fig. 5f ) with fluorescently labelled 8mers, which are of the general sequence 39-ctnnnzzz-59-label, where n represents a degenerate base, and z a 'universal' modified base with no binding preference (fig. 6 ). specificity of ligation and sequencing comes only from the 39 dinucleotides, of which there are clearly 16 combinations. these 16 sequencing probes are labelled with one of four dyes, leaving any individual dye associated with four primers. sequencing starts by hybridization of a primer and proceeds by the progressive ligation of 8mers, fluorescence detection to identify which sequencing probe pool was incorporated, and cleavage to remove the label and three 59 universal bases. this leaves a 5mer in place on the newly extended strand, from which the process is repeated. this first round of ligation reactions gathers sequence information on positions 1 and 2, 6 and 7, 11 and 12, etc. this process is then repeated with a starting primer that is displaced by 1 nt in the 59 direction (upstream), allowing sequence information to be gathered on positions 2 and 3, 7 and 8, 12 and 13, etc. the process is repeated from starting primers displaced 2, 3 and 4 nt upstream. independent ligation reactions starting from each of these five primers allows the software to recreate the final sequence. it also means that each position in the sequence is interrogated twice, leading to high accuracy. illumina sequencing. following dna fragmentation, adapter ligation and gel purification, adapter-ligated, single-stranded sequences are annealed to a glass plate that is precoated with oligos complementary to the adapters ( fig. 3d-h) . these oligos serve both to capture the template dna and as primers for subsequent amplification. amplification occurs on the slide by a process termed 'bridge amplification', in which each singlestranded molecule binds at both ends to the oligo primers on the slide. successive rounds of pcr result in the generation of tiny islands or clusters of amplified molecules (fig. 3h) , which serve as clones for subsequent sequencing using chain terminators, similar to traditional sanger sequencing. however, unlike the sanger method, illumina uses fluorescently labelled reversible terminators, such that each single base incorporation on each molecule temporarily terminates the reaction (fig. 5g) . a high-resolution (loman et al., 2012) . dapproximate values based on data published on the companies' websites on 9 march 2012. these data are for guidance only and are subject to change; readers interested in the details should consult either the manufacturers or those that are offering the sequencing service. next-generation sequencing digital image is used to determine which nucleotide is incorporated in each dna clonal cluster. after imaging, the terminator is reversed chemically, allowing the template molecule to be extended again in the next round of sequencing. as such, the sequencing reaction proceeds to conclusion on the majority of molecules. helicos sequencing (helicos biosciences). helicos has been the first true single-molecule sequencing technology to enter the marketplace, with no template amplification prior to sequencing. the methodology starts in a similar way to those already described, with template fragmentation and adapter ligation (fig. 2) . this time, a poly(a) adapter is added to the 39 end of the single-stranded dna template, finishing with a single fluorescently labelled datp. this poly(a) tag is used to capture each template molecule onto a flow cell using oligo(dt) probes, at a density of over 100 000 000 cm 22 (fig. 4d) . a laser locates the bound templates, prior to the cleavage of the 39 fluorescent label. a dna polymerase and fluorescently labelled, reversible terminator nucleotides are then added sequentially, with imaging detecting each incorporated base (fig. 5g ). this method has been used to sequence rna directly without prior cdna synthesis (ozsolak et al., 2009) . pacbio sequencing (pacific biosciences). unlike other ngs technologies, in this method it is the dna polymerase that is immobilized on the floor of a microcell. fragmented dsdna is ligated to hairpin adapters to create circular dna ( fig. 3i-m) . these are amplified linearly using primers complementary to hairpin sequence, and then captured by a single molecule of dna polymerase and sequenced in the bottom of a well ('zero mode waveguides') ( fig. 4e ). fluorescently labelled nucleotides (one colour for each base) diffuse into the cell from above. unlike other systems, the fluorescent molecule is phospholinked, meaning that it is cleaved on incorporation (fig. 5e ). as these labelled nucleotides diffuse around the polymerase active site, they generate a small noise signal. however, when the dna polymerase encounters the nucleotide complementary to the next base in the template, it is incorporated into the growing dna chain, and held in place for orders of magnitude longer than the average diffusing nucleotide. this creates a measurable coloured signal that can be differentiated from simple diffusion. following incorporation, the fluorescent label is cleaved (as part of the chemistry of forming the phosphate chain) and diffuses away, allowing the dna polymerase to continue to incorporate multiple bases per second. ion torrent (life technologies). as with other methodologies, the first stage of ion torrent sequencing relies on adding adapters of known sequence to template dsdna by ligation or pcr. these adapters are used to capture the library clonally onto solid particles, and then to amplify the target sequences through emulsion pcr ( fig. 3a -c). the amplified library is then separated, one bead per well, on a high-density array (fig. 4a ). these wells sit on top of an ion-sensitive semiconductor. during dna polymerase-catalysed extension, a hydrogen ion is released as part of the normal chemistry of nucleotide incorporation ( fig. 5c ). this ion is detected by the semiconductor as a small change in ph. like pyrosequencing, which measures released pp i , the extent of the change in ph with ion torrent sequencing is determined by the number of base incorporations, rendering it sensitive to misreading the length of homopolymers. pcr reagents deciding which technology is best is a contentious issue and will ultimately depend on the specific experiment being planned. important factors include the size of the genome being considered, its complexity including g+c content, as well as the depth of coverage and accuracy required. it is therefore most important to take advice from local service providers. that said, some general principles based on current technology performance may help to guide the decision-making process. for those looking to assemble complex genomes de novo, longer read lengths may be appropriate. for those seeking faster turnaround times, then the smaller platforms may offer greater flexibility, depending on the size of the laboratory (loman et al., 2012) . 454 sequencing (roche), whilst currently relatively expensive, still has a niche in amplicon sequencing because of its longer reads. other platforms may be more appropriate for direct rna sequencing (helicos) and very long reads (pacbio). however, as both are single-molecule sequencers, accuracy may become an important issue depending on the experiment. in the authors' experience, the illumina and solid platforms currently offer the best all-round value for money, accuracy and throughput for rna-seq (see subsection on transcriptomics), and those projects requiring high depths of coverage. the amount of sequence generated by each platform may seem excessive for viral genomes that typically range in size from 5 to 350 kb. however, in most cases sequencing occurs on a solid platform, whether a glass or microwell plate. these surfaces can be readily partitioned to allow several samples to be sequenced independently on a single run of the machine. in addition, by the use of distinct adapters that contain unique sequence motifs or tags that are also sequenced during the reaction, libraries of different samples can be mixed and sequenced together, their sequences being partitioned back to their respective samples based on the unique sequence tags in their adapters. this process is usually referred to as barcoding. in addition, many of the companies that manufacture the platforms discussed above are producing smaller machines with such users in mind. one of the crucial features for ultimate sequencing success is how the template nucleic acid is prepared. clearly, the appropriate nucleic acid must be purified, whether it be rna or dna. as obligate intracellular organisms, viral preparations are usually heavily contaminated by host nucleic acid, and it is wise to remove as much of this as is practical in order to ensure as many of the resulting sequence reads are of viral rather than host origin. for dna and rna viruses, the nature of contaminating nucleic acid may vary. in all cases, the best starting material contains little other genetic information, such as cerebrospinal fluid and serum. however, methods are also described for more cellular material (daly et al., 2011) . in many cases, simple viral purification can work: low-speed centrifugation to remove cellular debris, followed by highspeed sedimentation to concentrate packaged viral genomes. rnase and/or dnase treatment can be used to remove unpackaged and unprotected contaminating nucleic acids. in other cases, more specific purification may be followed based on density gradients. where the goal is to sequence one or just a small number of genomes, crude purification is often sufficient, especially where the academic value of the genomes is high. under these circumstances, only a small percentage (1 % or less) of the reads would typically be of viral origin. if the goal is to sequence many hundreds of genomes, and where it is important to achieve full genome coverage, then simple methods of prior virus enrichment, including pcr, are needed. more recently, hybridization capture has been used to enrich viral nucleic acid prior to deep sequencing, allowing whole herpesvirus genomes to be sequenced from clinical samples (depledge et al., 2011) . in each experiment, there is a clear trade-off between sample purity and time/cost for purification, and each project needs assessment on its own values. the informatics requirements of ngs projects are ignored at the researcher's peril. arguably, it is easy to now generate many million bases of sequence; the challenge can be to use it efficiently. a full discussion of informatics is beyond the scope of this article. however, key processes in the pipeline are quality scoring, sequence assembly and annotation. manipulating typical output files, including both individual reads and their alignments into continuous sequences (contigs), can be challenging due to their size. post-sequencing, there are two approaches that can be used in analysis of read data. if a reference genome is available, then the sequences can be mapped directly to this reference using a mapper such as bwa (li & durbin, 2009 ). this provides rapid information about substitutions, insertions, deletions and gene loss, but is not applicable if the aim of the research is to look for novel genes or sequence. under these circumstances, or where a suitable reference genome is not available, individual sequencing reads can be assembled de novo using other software such as velvet (zerbino & birney, 2008) or mira (chevreux et al., 1999) , which use algorithms to find overlapping information between reads, leading to the generation of contigs. this approach allows discovery of novel genes and sequences, as well as small variants (e.g. snps and indels). both the de novo and mapping strategies may not cover the entire genome, due either to insufficient depth of coverage or to genomic repeats that, if the read length is smaller than the repeat, cause gaps in the genome assembly. the size of these gaps can be approximated by comparison with a reference genome or, alternatively, may be crossed using paired-end (or mate-paired) libraries. here, larger fragments of dna are first purified (e.g. 2-8 kb), adapters are added on either end and the molecule is circularized. this brings two regions of the genome, previously separated by several thousands of bases, into close contact, separated by the adapters. these circular dna molecules are fragmented and sequenced in the usual way. this not only allows the original ends of the molecule to be sequenced, but crucially also identifies their relative position and separation in the genome. sequence from paired-end libraries can be built informatically into genome 'scaffolds', showing the order and relative positions of individual contigs, thereby facilitating genome closure. genome gaps can be closed by conventional pcr followed by more traditional sanger sequencing; whether this is necessary depends on the nature of the project. genome sequencing perhaps the most obvious application of these technologies is genome sequencing. although viral genomes are relatively small, their academic value is often extremely high, and, when coupled with barcoding and partitioning, ngs can represent a highly efficient way of sequencing full viral genomes. it is impractical to provide an exhaustive list of the viral genomes published by ngs, and any such list would rapidly go out of date. rather, we provide two examples of this approach, chosen from extremes of the interrogates base positions 1 and 2, 6 and 7, etc. interrogates base positions 2 and 3, 7 and 8, etc. almost at the extreme other end of viral biodiversity from marseillevirus are the influenza viruses, with relatively small, segmented rna genomes. the need for genome sequencing of these viruses has been driven by the emergence of h5n1 (höper et al., 2009) and an h1n1 pandemic, with an urgent need to understand the evolution and molecular epidemiology of these viruses. added sequencing complexity comes from a segmented genome and associated recombination events, meaning that phylogenetic analyses based on partial sequence information can never pick up the full complexity of historical recombination events that often lead to pandemic emergence. clearly, full genome sequences can be obtained from clinical material by segment-specific pcr and more traditional sanger sequencing (smith et al., 2009; vijaykrishna et al., 2011) . however, more recently, methods have been described based on pooling of segment-specific pcrs obtained directly from clinical material (höper et al., 2009 (höper et al., , 2011 or following purification of virus particles from cell culture (lorusso et al., 2011) , followed in both cases by tagging and sequencing. by allowing many full genomes to be sequenced, ngs has effectively removed a major bottleneck to our understanding of the emergence and transmission of these important viruses (baillie et al., 2012) . the remaining challenge is to reduce the time for sequence production to a point where sequence information can be used routinely in real time to aid health protection agencies in the control of outbreaks. in the authors' laboratories, we have now used ngs platforms to obtain de novo sequence from multiple virus families, including poxviridae, parvoviridae, picornaviridae, herpesviridae, asfarviridae (chapman et al., 2011) and rhabdoviridae, underlying the broad applicability of these techniques to this field of virology. cleary, single full genome sequences are incredibly valuable to understanding virus biology, but perhaps of more interest is the ability that ngs provides to sequence and compare multiple full genomes of distinct types, to identify important genetic differences between them (szpara et al., 2010). one of the striking features of viral genomes is their potential for high evolution rates, a feature of short generation times and, for rna viruses in particular, lowfidelity polymerases. this has for many years been enshrined in the concept of the viral quasispecies, whereby many rna viruses are believed to exist at the sample level, not as a single sequence but as a collection of closely related variants (lauring & andino, 2010) . this diversity creates a challenge for those wishing to sequence viruses, such that many of the sequences for rna viruses, particularly those based on pcr without cloning, represent average, majority or consensus sequences for these populations. generally, minority members of the population are ignored, which in most cases is entirely acceptable practice. however, in evolutionary terms, this existing diversity represents a massive reserve on which selection can occur and from which fitter variants can emerge rapidly. these are of concern not only for immune escape, but also in antiviral chemotherapy, where minor population variants harbouring resistance mutations can be rapidly selected for, leading to failure of antiviral therapy. this has led the world health organization (who) to establish a global strategy for the prevention and assessment of human immunodeficiency virus (hiv) drug resistance and, amongst its many roles, the global influenza surveillance and response system has been closely monitoring the evolution of influenza viruses infecting humans, including their susceptibility to antiviral drugs. there is a long history of attempts to characterize the viral quasispecies and to identify clinically significant minor variants within it. generally, this has been done by pcr amplification and direct sequencing, which may only be able to detect mutations that exist at .10-20 % frequency in the population (varghese et al., 2009; wang et al., 2007) , or line probe assays, which, although perhaps slightly more sensitive, can only detect known mutations (lok et al., 2007) . sensitivity can be increased by using limitingdilution pcr, i.e. essentially sequencing multiple individual molecules from each sample; however, this clearly comes at a considerable cost (palmer et al., 2005; wang et al., 2007) . in comparison, it is generally accepted that ngs of amplicons can detect minority variants present in 1-2 % of sequence reads (varghese et al., 2009) , and it is now accepted that ngs detects many more minority mutations than more traditional methods (margeridon-thermet et al., 2009) . for example, in one study of hiv population mutations, ultradeep pyrosequencing detected on average seven times more variants than conventional methods, with all variants present in 3 % of genomes and 57 % of variants present in ,3 % of genomes confirmed by limiting-dilution sequencing . the limitation to this detection threshold is driven both by error rates of the polymerases used and by pyrosequencing errors around homopolymers, and has been estimated by amplifying plasmid sequences (margeridon-thermet et al., 2009; mitsuya et al., 2008; solmone et al., 2009; varghese et al., 2009; wang et al., 2007) . to the authors' knowledge, error rates associated with reverse transcription have not been included, so this represents an important area for future research, particularly as direct rna sequencing becomes available (ozsolak et al., 2009 ). ngs has predominantly been used to monitor population diversity in hiv (eriksson et al., 2008; hoffmann et al., 2007; ji et al., 2010; wang et al., 2007) and hepatitis b virus (homs et al., 2011; margeridon-thermet et al., 2009; solmone et al., 2009 ), but it is now starting to be used for other viruses (geret et al., 2011; görzer et al., 2010; hiraga et al., 2011; tapparel et al., 2011; verbinnen et al., 2010; wellehan et al., 2010) . the methodology is starting to shed new light on other areas of viral pathogenesis where minor population variants may also be highly significant. these include assays for receptor usage (raymond et al., 2011) and minor pathogenic variants (geret et al., 2011) , and studies to understand immune escape in simian immunodeficiency virus (bimber et al., 2009; hughes et al., 2010) . studies using ngs to dissect the dynamics of transmission have shed new light on the differences between resistance acquired by transmission and that which has evolved within the patient (varghese et al., 2009 ). in addition, some studies are now moving away from sequencing of specific genomic regions to estimating the diversity across the whole genome tapparel et al., 2011; willerth et al., 2010) . such an approach is likely to generate new insights into the roles of previously understudied viral genes in pathogenesis. the use of barcoding and multiplexing provides opportunities to increase the cost-effectiveness of this approach (hoffmann et al., 2007; ji et al., 2010) . metagenomics can be defined as the characterization of genetic information directly from samples. it has the benefit of not requiring previous culture, making it particularly attractive when trying to characterize the 'viral metagenome' or 'virome'. the term 'metagenome' first appeared in pubmed in 1998 (handelsman et al., 1998) in relation to classifying unculturable bacteria from soil, when such early studies relied on the use of random bacterial cloning and sequencing (breitbart et al., 2003) . the availability of ngs has allowed these metagenomes to be analysed in unbiased ways at previously unseen resolution, and is providing a wealth of new opportunities in two major areas: viral candidate pathogen discovery and viral ecology. virus candidate pathogen discovery. the threat of newly emerging viruses to important species remains everpresent. in humans, although viruses currently make up a small proportion of known human diseases, they make up the majority of newly identified ones (woolhouse & gaunt, 2007) . the rate of discovery of new virus infections has remained largely unchanged over many decades and suggests that many new viruses remain to be discovered (woolhouse et al., 2008) . in the face of new outbreaks of a disease, there is an imperative to rapidly characterize the infectious agent to understand better the disease, and to allow the development of specific diagnostic tests and control measures. ideal methods are non-specific and have included electron microscopy, virus isolation, cloning, degenerate or consensus pcr, representational difference analysis (rda) and sequence-independent single primer amplification (sispa) (ambrose & clewley, 2006; tang & chiu, 2010) . the most high-profile recent example of this was the sars (severe acute respiratory syndrome) coronavirus. the new respiratory syndrome was first reported by the who on 14 march 2003 (anonymous, 2003) . initially, large-scale screens for known pathogens were carried out to try to identify a common factor in cases using virus isolation, electron microscopy, pcr and antigen-based methods (drosten et al., 2003) . several 'red herrings' were identified, most notably a paramyxovirus. ultimately, random pcr (drosten et al., 2003) or degenerative coronavirus primers (ksiazek et al., 2003) amplified the first sars coronavirus sequences, which were published electronically in the new england journal of medicine on 10 april 2003. on 5 july 2003, the who declared the sars outbreak contained (http://www.who. int/mediacentre/news/releases/2003/pr56/en/). whilst the sars story represented a considerable triumph in global cooperation and pathogen discovery, with only approximately 1 month between syndrome description and pathogen discovery, it is highly likely that any such future discovery projects would be carried out by ngs in a fraction of the time, and here we describe just one of many examples that shows the speed of such approaches. in 2008, an outbreak of unexplained haemorrhagic fever in people was reported in south africa. the index patient was admitted to a clinic on 12 september 2008. over the following 2 weeks, secondary and tertiary cases were reported, with four of the five patients dying. such outbreaks of haemorrhagic fever are highly emotive events, necessitating a rapid response to control both infection and public anxiety. rna extracts from two post-mortem liver biopsies (cases 2 and 3) and one serum sample (case 2) were submitted to ngs. blast analysis of the resulting sequences identified contigs corresponding to about half of the approximately 10 kb genome of a novel arenavirus (briese et al., 2009) . the majority of sequences were obtained from serum rather than tissue, presumably reflecting the higher levels of host dna obtained from the highly cellular tissue samples. such rapid results are highly significant for several reasons. as the method used is not predicated on sequence-specific amplification, it has a high probability of success, providing that sufficient sequencing is done. indeed, for influenza virus it has been shown that ngs has sensitivity on clinical specimens close to that of specific pcr (greninger et al., 2010) . secondly, rapid pathogen discovery is now accessible to all outbreaks, regardless of the profile of the disease, as reported recently for schmallenberg virus of ruminants (hoffmann et al., 2012) . thirdly, it works very quickly, allowing authors to obtain a candidate diagnosis in only 72 h. finally, when used in novel samples, it often finds a wealth of previously unrecognized viruses (day et al., 2010) . this last point highlights a key issue: clearly, the identification of viral sequences in clinical material is far from showing an association with disease (lipkin, 2009 ). in some cases, small case-control studies have sought to compare metagenomes in health and disease, including in cystic fibrosis (willner et al., 2009 ) and chronic fatigue syndrome (sullivan et al., 2011) , to try to determine the significance of any one genome to disease. however, cost will currently limit such an approach to those diseases where a single pathogen is perhaps unlikely. more typically, traditional methods will be employed to seek proof of an association between any newly discovered virus and disease. these methods include classical infection studies to achieve koch's postulates, larger case-control studies using traditional diagnostics methods such as pcr to compare the prevalence of the new viral sequences in health and disease, or the detection of acquired immunity showing a temporal association between infection and disease. even when all this is done, it is likely that ngs technologies will lead to the identification of an expanding class of viruses that historically would have been called 'orphan' viruses, viruses in search of a disease, and commensals (dolgin, 2011 (donaldson et al., 2010; li et al., 2010; reyes et al., 2010) , sewage (cantalupo et al., 2011) , vaccines , plants (notably grapevines) (coetzee et al., 2010) and environmental samples including water (ló pez-bueno et al., 2009; rodriguez-brito et al., 2010) . a recent paper has described the use of blood-feeding mosquitos as a way of sampling a broad range of viral diversity (vector-enabled metagenomics) and of identifying a broad range of animal, plant, insect and bacterial viruses (ng et al., 2011) . a review of ocean viromes suggests that the sea is dominated by rare genes, many of which might be contained within virus-like entities such as gene-transfer agents (kristensen et al., 2010) . the same study predicted a wealth of dna viruses belonging to the eukaryotic ncldvs, and suggested that the rna virome was dominated by picorna-like viruses. although virus candidate pathogen discovery and environmental viral metagenomics can be broadly thought of as separate disciplines, they are highly related, as they both essentially seek to identify correlations between virus populations and sample phenotype. recurring themes often include using samples low in contaminating host nucleic acid, such as faeces (victoria et al., 2009) or serum (towner et al., 2008) , filtration of samples to remove contaminating genetic material, and sequence-independent reverse transcription and pcr amplification of capsidprotected, nuclease-resistant viral nucleic acids. as the wide range of undiscovered viral diversity can now begin to be illuminated, an important challenge is how to identify truly novel sequences when comparing newly generated sequence to published databases. several approaches have been developed to facilitate this, including searching based on amino acid similarity and by the recreation of theoretical ancestral sequences (delwart, 2007) . as with other molecular methods, the sensitivity of ngs, which can detect rare viral transcripts at frequencies lower than 1 in 1 000 000 (moore et al., 2011) , does bring with it considerable potential for sample contamination (schmieder & edwards, 2011) . whilst genomic data are clearly extremely valuable in their own right, other areas of science are turning increasingly to the transcriptome, the measurement of mrna, to achieve new insights into genome expression and how this may be modified in health and disease. historically, oligonucleotide microarrays have been used to quantify gene expression throughout biology, but now these approaches are largely being replaced by ngs of transcribed rna -so called rna-seq (marioni et al., 2008; wilhelm & landry, 2009) . key steps in the process firstly include isolation of rna and removal of host genomic dna. subsequently, rrna is also removed by selection of polyadenylated rna or removal of rrna with antisense oligos. complementary dna synthesis is primed either by oligo(dt) or randomly. on long transcripts, the former can lead to bias sequencing of the 39 end and failure to determine the transcript start site. strand specificity of transcription is lost in routine preparation of double-stranded cdna. however, it can be maintained either by adding different adapters to the 59 and 39 ends of the rna transcript, or by marking one strand for degradation by chemical modification (levin et al., 2010) . analysis of rna-seq data is usually achieved by mapping sequence reads to a reference genome, using software that can map reads over gene splice junctions (trapnell et al., 2009) . statistical analysis of the number of reads per genome region (gene) can be used to quantify relative levels of expression (anders & huber, 2010; robinson et al., 2010) . whilst the use of these technologies is still in its infancy in virology, they are beginning to provide new insight on genome coding capacity and biology of transcription, particularly for large dna viruses. for example, in mimiviruses, rna-seq has identified a new role for palindromic sequences in transcription termination (byrne et al., 2009) , confirmed that all predicted orfs are transcribed, as well as identifying new orfs (legendre et al., 2010 (legendre et al., , 2011 . in poxviruses, rna-seq has been used to revisit temporal trends in gene expression (yang et al., 2011b) , to identify interactions between the host and poxviral transcriptome (yang et al., 2010) and to map transcription start and stop sites precisely (yang et al., 2011a) . in herpesviruses, new insight has been made into the role of viral gene expression in epstein-barr virus latency , and in cytomegalovirus, the significance of non-coding transcripts and splice variants has been characterized, as well as identifying new proteincoding transcripts (gatherer et al., 2011) . the technology is clearly not limited to large dna viruses, and has also been used to identify differences in expression and posttranscriptional modification associated with lentivirus virulence (ertl et al., 2011) . ngs also provides a new way to explore the role of micrornas in virus replication and pathogenesis. these short rna molecules of about 21-22 bases in length function as negative regulators of mrna translation (sayed & abdellatif, 2011) and their role is now being explored in several viral systems, including latency in herpes simplex virus (umbach et al., 2008) and epstein-barr virus (riley et al., 2012) , cytomegalovirus (stark et al., 2012) , transformation in marek's disease virus (burnside et al., 2006) and kaposi's sarcoma-associated herpesvirus (wu et al., 2011) , and grapevine pathology (pantaleo et al., 2010) . of course, there are two sides to a transcriptome for viruses, namely that of the virus and its host. virusinduced perturbations to the host transcriptome are clearly of critical importance to infection outcome. however, methodological limitations have meant that, until recently, the study of pathogenesis has been heavily driven by targeted analysis of specific genes or pathways thought to be critical to disease progression. although this review has concentrated on the use of ngs to study viral sequences directly, it is clear that these approaches provide new opportunities to explore the interaction of replicating viruses with their host, particularly their transcriptome, in a non-targeted, hypothesis-generating mode. recent examples include gene profiling of early and advanced liver disease in chronic hepatitis c virus (hcv)-infected patients (khalid et al., 2011) , the replication of hcv in vitro (woodhouse et al., 2010) , host gene shut-off in kaposi's sarcoma-associated herpesvirus, and the identification of host transcripts refractory to shut-off (clyde & glaunsinger, 2011) . despite the rapid progress that has been made in these technologies over the last 5 years, new developments are still likely. in the last year, there has been a lot of publicity around single-molecule nanopore technologies that aim to allow simple, cheap, single-molecule dna sequencing in devices no larger than a smartphone (eisenstein, 2012) . these technologies work by pulling dna through tiny pores and reading the bases as they pass through as changes in electric current across the pore. this process could remove the need for enzymic processes and imaging completely and could potentially generate very long reads very rapidly. whilst no substantial data have yet been seen, this method could become available in 2013. if the technology delivers, then devices could be available in clinics very rapidly afterward, becoming a key technology in diagnostic medicine. as all of the available sequencing chemistries are becoming established and maturing, read lengths and fidelity should increase, allowing us to explore deeper into viral genome diversity. all of the major platforms are being developed to be easier to use and more cost-effective, and some of the major companies have now released cheaper benchtop machines, with the aim of democratizing this technology and making it more readily available (loman et al., 2012) . the ultimate consequence of such democratization will be to bring ngs sequencing into many more research laboratories, as well as routine diagnostics for human, animal and plant disease, thereby providing new insights into the complex virome in which we all live. in medicine, routine sequencing of the human genome will herald new opportunities for personalized medicine. it perhaps follows that similar opportunities allowing antiviral therapy to be better-matched to individual genome sequences will exist, as already happens to some extent for hiv. improvements in the area of sample preparation have been rapid, including recent developments in genome partitioning (also known as enrichment). this allows targeting of a specific region, sets of genes or indeed entire exomes. technology platforms include either microarrays on solid substrates, free rna or dna oligos that can be separated from other fragments using biotin tags, or high-throughput pcr-based techniques (reviewed by mamanova et al., 2010) . such approaches may be particularly appropriate for viruses as a way of removing contaminating host dna. virus discovery by sequenceindependent genome amplification differential expression analysis for sequence count data acute respiratory syndrome. china, hong kong special administrative region of china, and viet nam enhanced infectious diseases (eid2) database evolutionary dynamics of local pandemic h1n1/2009 influenza virus lineages revealed by whole-genome analysis metagenomics and the molecular identification of novel viruses ultradeep pyrosequencing detects complex patterns of cd8+ t-lymphocyte escape in simian immunodeficiency virus-infected macaques whole-genome characterization of human and simian immunodeficiency virus intrahost diversity by ultradeep pyrosequencing giant marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms metagenomic analyses of an uncultured viral community from human feces genetic detection and characterization of lujo virus, a new hemorrhagic fever-associated arenavirus from southern africa marek's disease virus encodes micrornas that map to meq and the latency-associated transcript the polyadenylation site of mimivirus transcripts obeys a stringent 'hairpin rule raw sewage harbors diverse viral populations genomic analysis of highly virulent georgia 2007/ 1 isolate of african swine fever virus genome sequence assembly using trace signals and additional sequence information deep sequencing reveals direct targets of gammaherpesvirus-induced mrna decay and suggests that multiple mechanisms govern cellular transcript escape deep sequencing analysis of viruses infecting grapevines: virome of a vineyard deciphering the biology of mycobacterium tuberculosis from the complete genome sequence a viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing metagenomic analysis of the turkey gut rna virus community viral metagenomics specific capture and whole-genome sequencing of viruses from clinical samples sequencing reveals suite of commensal and pathogenic viruses metagenomic analysis of the viromes of three north american bat species: viral diversity among different bat species that share a common habitat identification of a novel coronavirus in patients with severe acute respiratory syndrome oxford nanopore announcement sets sequencing sector abuzz viral population estimation using pyrosequencing viral transcriptome analysis of feline immunodeficiency virus infected cells using second generation sequencing technology genome sequence of the human malaria parasite plasmodium falciparum high-resolution human cytomegalovirus transcriptome ló pez-bao feline leukemia virus outbreak in the critically endangered iberian lynx (lynx pardinus): high-throughput sequencing of envelope variable region a and experimental transmission accuracy and quality assessment of 454 gs-flx titanium pyrosequencing deep sequencing reveals highly complex dynamics of human cytomegalovirus genotypes in transplant patients over time a metagenomic analysis of pandemic influenza a molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products rapid emergence of telaprevir resistant hepatitis c virus strain from wild type clone in vivo dna bar coding and pyrosequencing to identify rare hiv drug resistance mutations novel orthobunyavirus in cattle ultra-deep pyrosequencing analysis of the hepatitis b virus precore region and main catalytic motif of the viral polymerase in the same viral genome simple, sensitive, and swift sequencing of complete h5n1 avian influenza virus genomes a comprehensive deep sequencing strategy for full-length genomes of influenza a dynamics of haplotype frequency change in a cd8 + tl epitope of simian immunodeficiency virus hiv drug resistance surveillance using pooled pyrosequencing gene profiling of early and advanced liver disease in chronic hepatitis c patients new dimensions of the virus world discovered through metagenomics a novel coronavirus associated with severe acute respiratory syndrome initial sequencing and analysis of the human genome quasispecies theory and the behavior of rna viruses & other authors (2010). mrna deep sequencing reveals 75 new genes and a complex transcriptional landscape in mimivirus breaking the 1000-gene barrier for mimivirus using ultradeep genome and transcriptome sequencing comprehensive comparative analysis of strand-specific rna sequencing methods fast and accurate short read alignment with burrows-wheeler transform bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses quantitative and qualitative rna-seq-based evaluation of epstein-barr virus transcription in type i latency burkitt's lymphoma cells microbe hunting in the 21st century antiviral drugresistant hbv: standardization of nomenclature and assays and recommendations for management performance comparison of benchtop high-throughput sequencing platforms high diversity of the viral community from an antarctic lake genetic and antigenic characterization of h1 influenza viruses from united states swine from targetenrichment strategies for next-generation sequencing ultra-deep pyrosequencing of hepatitis b virus quasispecies from nucleoside and nucleotide reverse-transcriptase inhibitor (nrti)-treated patients and nrti-naive patients rna-seq: an assessment of technical reproducibility and comparison with gene expression arrays minority human immunodeficiency virus type 1 variants in antiretroviral-naive persons with reverse transcriptase codon 215 revertant mutations the sensitivity of massively parallel sequencing for detecting candidate infectious agents associated with human tissue specific enzymatic amplification of dna in vitro: the polymerase chain reaction broad surveys of dna viral diversity obtained through viral metagenomics of mosquitoes direct rna sequencing multiple, linked human immunodeficiency virus type 1 drug resistance mutations in treatment-experienced patients are missed by standard genotype analysis deep sequencing analysis of viral short rnas from an infected pinot noir grapevine frequency of cxcr4-using viruses in primary hiv-1 infections using ultra-deep pyrosequencing viruses in the faecal microbiota of monozygotic twins and their mothers ebv and human micrornas co-target oncogenic and apoptotic viral and human genes during latency edger: a bioconductor package for differential expression analysis of digital gene expression data viral and microbial community dynamics in four aquatic environments dna sequencing with chain-terminating inhibitors micrornas in development and disease fast identification and removal of sequence contamination from genomic and metagenomic datasets origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic use of massively parallel ultradeep pyrosequencing to characterize the genetic diversity of hepatitis b virus in drug-resistant and drug-naive patients and to detect minor variants in reverse transcriptase and hepatitis b s antigen highresolution profiling and analysis of viral and host small rnas during human cytomegalovirus infection an unbiased metagenomic search for infectious agents using monozygotic twins discordant for chronic fatigue metagenomics for the discovery of novel human viruses rhinovirus genome variation during chronic upper and lower respiratory tract infections newly discovered ebola virus associated with hemorrhagic fever outbreak in uganda tophat: discovering splice junctions with rna-seq micrornas expressed by herpes simplex virus 1 during latent infection regulate viral mrnas minority variants associated with transmitted and acquired hiv-1 nonnucleoside reverse transcriptase inhibitor resistance: implications for the use of second-generation nonnucleoside reverse transcriptase inhibitors tracking the evolution of multiple in vitro hepatitis c virus replicon variants under protease inhibitor selection pressure by 454 deep sequencing metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis viral nucleic acids in liveattenuated vaccines: detection of minority variants and an adventitious virus long-term evolution and transmission dynamics of swine influenza a virus characterization of mutation spectra with ultra-deep pyrosequencing: application to hiv-1 drug resistance characterization of san miguel sea lion virus populations using pyrosequencing-based methods rna-seq-quantitative measurement of expression through massively parallel rna-sequencing development of a low bias method for characterizing viral populations using next generation sequencing technology metagenomic analysis of respiratory tract dna viral communities in cystic fibrosis and non-cystic fibrosis individuals transcriptome sequencing, microarray, and proteomic analyses reveal cellular and metabolic impact of hepatitis c virus infection in vitro ecological origins of novel human pathogens temporal trends in the discovery of human viruses the manipulation of mirna-gene regulatory networks by kshv induces endothelial cell motility simultaneous high-resolution analysis of vaccinia virus and host cell transcriptomes by deep rna sequencing genome-wide analysis of the 59 and 39 ends of vaccinia virus early mrnas delineates regulatory sequences of annotated and anomalous transcripts expression profiling of the intermediate and late stages of poxvirus replication velvet: algorithms for de novo short read assembly using de bruijn graphs key: cord-277665-ac8txr3h authors: grichko, varvara p.; shenderova, olga a. title: 15 nanodiamond designing the bio-platform date: 2006-12-31 journal: ultrananocrystalline diamond doi: 10.1016/b978-081551524-1.50017-2 sha: doc_id: 277665 cord_uid: ac8txr3h publisher summary this chapter discusses various methods of surface modification for the development of functionalized diamond nanoparticles for biomedical applications. to be used in biomedical applications, nanoparticles must be biocompatible, non-toxic, non-detective by immune systems, and should not induce side effects. size control of particles is a prerequisite for biomedical applications. carbon nanostructures span the same length scale as bio-compounds, ranging from subnanometer-size nucleotides to tens and hundreds of nanometer-sized organelles and viruses, and up to micron-sized cell sizes. the chapter also summarizes different approaches to the surface functionalization of nanodiamonds (nd) particles—that is, the key in successful biomedical applications followed by a discussion of modification of diamond surfaces with nucleic acids and proteins. both current and potential applications of diamond films and particles in the area of biosensing are addressed. all major forms of carbon at the nanoscale -fullerenes, nanotubes, and nanodiamond (nd), the last in the forms of both particulate and filmsappear to be valuable materials for biomedical applications (freitas, 1999; . importantly, carbon nanostructures span the same length scale as bio-compounds ( fig. 15.1) , ranging from subnanometer-size nucleotides, to tens and hundreds of nanometer-sized organelles and viruses, and up to micron-sized cell sizes. in the mid 1990s it was discovered that fullerene compounds have biological activity, and their potential as therapeutic agents for the treatment of several diseases was demonstrated. as a result, a private biopharmaceutical company, c sixty inc., has been established with a primary focus on the discovery and development of novel fullerene-based therapeutics. at 7.2 å in diameter, c 60 is similar in size to steroid hormones and peptide alpha-helices, and, thus, fullerene compounds are ideal molecules to serve as ligands for enzymes and receptors (wilson, 2000) . within the last few years, a number of useful fullerene-based therapeutic applications have been developed, including as antiviral agents and anticancer drugs (www.csixty.com) and biosensors for diagnostic applications (anonymous, 2001) ; a protective agent against iron-induced oxidative stress (lin et al., 1999) ; and an in vitro antibacterial agent (da ros et al., 1996) . the exploration of buckytubes in biomedical applications is also underway. multiwall carbon nanotubes have been used for immobilization of proteins, enzymes, and oligonucleotides (lin et al., 2004) . significant progress has been made within the last few years in an effort to overcome some of the fundamental and technical barriers toward bioapplications of carbon nanotubes, especially on issues concerning solubility in water, biocompatibility, modifications of carbon nanotubes with various biological and biologically active compounds, and both design and fabrication of biosensor prototypes (lin et al., 2004) . nanodiamond particles and their derivatives, diamondoids and their derivatives, and ultrananocrystalline diamond (uncd) films all have high potential in biotechnological and biomedical applications. uncd films have been suggested to be the ideal platforms for future biochips and biosensors because of their superior mechanical, thermal, and chemical properties as compared to those of glass, silicon, and gold surfaces (yang et al., 2002) . uncd particles had been probed in the separation of proteins (bondar and puzr, 2004) , fabrication of integrated biochips and sensors (puzyr et al., 2004a) , and even in anticancer applications (dolmatov, 2001) . in general, multifunctional hierarchical structures consisting of nanoparticle derivatives of organic and inorganic molecular entities began to play increasingly important roles in a variety of applications in nanobiotechnology (niemeyer, 2001) , genomics (wengel, 2004) , drug discovery (ozkan, 2004) , and nanomedicine (freitas, 2003) . for example, zero-dimensional nanostructures reported for medical applications include gold and magnetic nanoparticles, semiconductor quantum dots, a wide variety of polymer-based nanoparticles, nanoshells consisting of metals and dielectrics, and many others. nanoparticles act as potential carries for several classes of drugs such as anticancer agents, antihypertensive agents, immunomodulators, and hormones; and macromolecules such as nucleic acids, proteins, peptides, and antibodies. an absence of narrow fractions of nanodiamond particulate on the market hindered their biomedical applications. recently, however, production of narrow fractions of nd (5 nm sized particle suspensions; several fractions within the 40-100 nm size range as well as fractions above 100 nm) has been achieved in research laboratories (chapter 3 of this book and private communications). in combination with the advances in the production of high-purity particles and the fact that uncd particles of detonation origin are relatively inexpensive (compared to fullerenes, pure carbon nanotube (cnt), and gold particles, for example), a fast growth of bioapplications of nd particulate is expected. this chapter will be organized as follows: in the next section different approaches to the surface functionalization of nd particles, that is, the key in successful biomedical applications, will be summarized, followed by a discussion of modification of diamond surfaces with nucleic acids and proteins. after that both current and potential applications of diamond films and particles in the areas of biosensing and medicine will be addressed. the concluding section will summarize results reported on the biocompatibility of nd, the paramount property in the biomedical applications of artificial nanostructures. diamond possesses a number of distinct properties that make it an attractive biotechnological material. although natural diamond is highly hydrophobic, hydrophilic surface groups, important for bioapplications, can be generated on a diamond surface by heating to high temperature in an oxygen atmosphere and using ion bombardment and other rather aggressive treatments. nd particles synthesized by detonation have numerous oxygen-containing chemical groups on the surface and, thus, are intrinsically hydrophilic. the detonation nd particles are 4-5 nm round-shaped monocrystals that form tightly and loosely bond aggregates of about 50 nm and 100-200 nm in diameter, correspondingly. detonation nd possesses a very large specific surface area (300-400 m 2 g −1 ) (dolmatov, 2001) , which is an important factor for bioapplications. detonation nd is likely to have a significant number of unpaired electrons that make it an efficient free radical scavenger and opens up the opportunity for its use in medicine (kulakova et al., 2000; nachalnaya et al., 2000) . in general, the size, purity, and surface chemistry of detonation nd varies considerably from one manufacturer to another. the composition of the explosive mixture, coolant media, chamber size, and consequent purification of detonation soot are all important factors in the manufacture of uncd particles with the desirable physical and chemical properties of their surface (donnet et al., 1997) . the methods of extraction and purification of nd from the detonation product may include ozone treatment, oxidation by different reagents with/without catalysts, treatment with acids, modification of the nd surface in gaseous and liquid media (kulakova, 2004) , selective inhibition of nd oxidation (chiganov, 2004) , and many others. disaggregating of detonation nd can be achieved, for example, by its graphitization in a n 2 atmosphere at 1000°c followed by oxidation in air at 450°c in order to remove the surface graphite layer. in this way, the average size of nd aggregates can be reduced to 50 nm or less with a very high yield (xu and xue, 2004) . dispersion efficiency and stability of colloidal solutions of nd in water is important for nd applications in nanobiotechnology and medicine, and is determined by the electrostatic, hydration, and hydrophobic (the tendency of lypophilic nd to form aggregates in an aqueous solution) interactions among the particles (xu et al., 2005a) . within the last few years a number of new methods have been introduced to control nd solubility in water by biological and chemical modifications of the nd surface. to obtain stable suspensions of well-dispersed nd particles in water the mechanochemical procedure has been developed, which employs a set of different mechanical treatments (high-power sonification or vibration milling techniques) along with the application of surfactant agents (xu et al., 2005b; 2005c) . the mechanochemical modification with anionic surface modifiers increased the zeta potential and lowered the amount of hydroxyl groups and the size of individual particles . the mechanochemical processes were also successfully applied for the preparation of stable highly dispersed nd suspensions in non-polar solvents : an oil suspension of nd particles with an average size of 55 nm was prepared and stored for 6 months without any visible signs of sedimentation. there has been steady progress in the development of chemical methods of nd surface modification as well. a summary of methods of altering the nd surface chemistry with the final purpose of biofunctionalization is illustrated in fig. 15 .2. various organic molecules can be attached to polycrystalline diamond films when under irradiation with uv light. prior to exposure to uv light the hydrogen-terminated diamond surface must be coated with a thin film of the solution containing organic molecules in order to be immobilized. by attaching molecules with specific protecting groups and removing protecting groups after the attachment, it is possible to obtain diamond surfaces functionalized with carboxyl or primary amine groups that may facilitate further steps in chemical modification of diamond surfaces such as dna and protein immobilization (strother et al., 2002) . to increase nd solubility in water and prepare the nd surface for biomolecule attachment, oxidation of the nd surface is frequently required (fig. 15.2) . under mild conditions the diamond surface can be directly functionalized with carboxylic acids by initiating radical reactions (tsubota et al., 2004) . however, the number of carboxylic acid residues introduced on the diamond surface may be low in some cases. indeed, oxidation of nd does not proceed easily under the mild conditions. in an aqueous slurry, nd of 4 nm size may react with ozone, though a very long time is needed in order to functionalize the surface of nd with the ketonic groups (cataldo and koscheev, 2003) . in air nd may suddenly explode 15: designing the bio-platform, grichko and shenderova 533 above a temperature of 450°c. the functionalization of nds at elevated temperature affects both their size and surface chemistry. the weight of nd particles was reported to decline by 11.5% as a result of heating at 900°c in an inert atmosphere (cataldo and koscheev, 2003) . spherical diamond powder with a particle size ranging from 150 to 600 nm can be oxidized in an oxygen atmosphere at 450-610°c (lee et al., 2004) . the oxidized diamond powders were further successfully functionalized by reacting with 4-(trifluoromethyl)benzylamine containing the reactive amino group . under certain conditions the high rate of diamond oxidation may result in an explosive process. ida et al. (2003) have investigated the reactivity of hydrogenated diamond surfaces with peroxide radical initiators such as benzoyl peroxide, lauroyl peroxide, dicumyl peroxide, and di-t-butyl peroxide. with benzoyl peroxide, they detected ir peaks that were then assigned to the aromatic c-h and c=o stretching vibrations. the c=o stretching vibration was observed after the diamond was exposed to lauroyl peroxide. the areas under the peak of spectra determined with fourier transform infrared spectroscopy (ftir) increased with reaction time and amount of reagent. dicumyl peroxide and di-t-butyl peroxide did not react with diamond surfaces. while the yield of reaction of diamond surface with radical species generated from benzoyl peroxide depends on the organic solvent used, the same functional groups were synthesized in toluene, tetrahydrofuran, n,ndimethylformamide, cyclohexane, and hexane (tsubota et al., 2002a) . the hydroxyl groups located on the surface of diamond powders, which was treated either with sulfuric acid alone or with a mixture of sulfuric and nitric acids, can be reacted with metoxy groups of silanecoupling reagents (3-aminopropyltrimethoxysilane, 3-mercaptopropyltrimethoxysilane, or n-octyltrimethoxysilane) with the formation of stable modified diamonds (tsubota et al., 2002b) . the silane-modified nds can be used for surface synthesis of dna and protein immobilization. the substantial increase in the number of surface c-h groups was achieved by the gas treatment of nd powder with h 2 , plasma-ionized hydrogen, n 2 , methane and air, and in vacuum at different temperatures (jiang et al., 1996) . surface decomposition, decarbonylation, and decarboxylation were likely to be the main reactions. the h-terminated nd surfaces can be further reacted photochemically (λ = 254 nm) with longchain ω-unsaturated amines to produce a homogeneous layer of amine groups for consequent dna attachment (yang et al., 2002) . nd purification and functionalization can also be carried out using gas and vapor reactive media ( fig. 15 .2). the chlorinated diamond was pre-pared by sotowa et al. (2004) by irradiating hydrogenated diamond with uv light in the presence of elemental chlorine. the formation of chlorinated diamond was confirmed by diffuse reflectance ftir, which revealed a strong peak corresponding to the c-cl stretching. the researchers then treated the chlorinated diamond surface further with ammonia and found that diamond amination is a temperature-dependent process and results in the formation of nh 4 + ; c=n and nh 2 ; and imines at room temperature, 100°c, and 200°c, respectively. high-temperature treatment of detonation nd with hydrogen, ccl 4 , or nh 3 has been studied recently by spitsyn et al. (2005) . nd, which was annealed in hydrogen flow at 850°c for 5 h, possessed about 1000 cal g −1 lower combustion heat than non-modified nd powder. moreover, the initial dangling bonds density of ∼1.16 × 10 20 spin/cm 3 was reduced 1.5 times after the treatment with hydrogen. after treatment in a ccl 4 /ar mixture at 450°c for 0.5-3 h the hydrophilicity of nd was changed significantly: the atmospheric water vapor readsorbance was at least 20 times lower than in the nd samples treated in pure ar. after treatment in a nh 3 flow at 600°c for 70 min the number of oxygen-containing groups had decreased and atmospheric water vapor readsorbance was four times lower than in the initial nd samples. a number of characterization methods such as chemical analysis, raman, ftir, electron spin resonance (esr), and chromatomass spectrometry confirmed the possibility of extensive modification and controlled functionalization of the nd using gas treatment (in terms of hydrophilic/ hydrophobic or acidic/basic nd termination) (spitsyn et al., 2005) . this prevents agglomeration of hydrophilic and hydrophobic detonation nd in polar and non-polar solvents, correspondingly. fluorination is another efficient method for nd chemical modification that enables a variety of applications in engineering and biological sciences ( fig. 15 .2). treatment of detonation uncd powder (1-2 µm sized particles composed of 3.5-6.5 nm diamond nanocrystals, >97% purity) with a f 2 /h 2 mixture at 150-470°c resulted in the formation of fluorinated nds with 8.6 at.% fluorine . the fluorinated nd material was then used as a precursor for preparation of alkyl-, amino-, and amino acid-functionalized nds that showed an increased solubility in polar solvents and reduced particle agglomeration . application of fluorinated nd has been found in cost-effective synthesis of diamond coatings covalently bonded to glass surfaces (liu et al., 2005) . before that the production of diamond thin films could only be achieved by chemical vapor deposition that requires heating to 1000°c. liu et al. (2005) applied a silane coupling agent, 3-aminopropyltriethoxysilane, to attach fluoro-nd to the glass slide surface, which was preliminary func-tionalized with terminal amino groups. using atomic force microscopy (afm), sem, and x-ray photoelectron spectroscopy (xps) analysis it was established that surface-bonded fluoro-nd particles were closely packed and had an average size of 10-40 nm. the fluoro-nd, which is covalently attached to the surface of the glass slide, can be further modified by chemical substitution of residual fluorine. nanoparticles are valuable platforms in controlled drug delivery and can be administered via most routes to carry various therapeutics, anticancer, antiviral, antibacterial, and antihypertensive agents, immunomodulators, hormones, antibodies, proteins, peptides, and nucleic acids to isolated cells, tissues, and organs (bala et al., 2004; ozkan, 2004) . successful design of highly efficient drug delivery systems may solve many problems faced by present-day medical sciences. in gene therapy, genes are delivered to the cell nucleus allowing cells to produce therapeutic proteins. gene delivery is achieved using either viral or non-viral vectors. viral vectors are genetically engineered adenoviruses, retroviruses, and other viruses that are very efficient when used for gene transfer in vivo. methods of non-viral gene delivery, including nanoparticle carriers, represent a small fraction of all methods used for transfection in vivo. however, they have gradually become as popular as the viral vector-based technology. in the development of non-viral technologies, the main goal is to have a transfection method that is efficient, reproducible, non-toxic, and allows the prolonged gene expression to occur. efficiency of targeted delivery of nanoparticles loaded with biologically active molecules is affected by many factors including particle size, surface charge, and chemistry, and mechanism of target recognition. over the years, a number of natural and synthetic materials have been used to prepare nanoparticles, and their stability, biocompatibility, and biodegradability were investigated (bala et al., 2004) . in some cases nanocarriers protect naked dna from nucleases while allowing dna plasmids to pass the cell membrane and get into the nucleus (kneuer et al., 2000; roy et al., 2005) . to optically monitor intracellular trafficking and gene transfection events roy et al. (2005) first prepared fluorescently labeled organically modified silica nanoparticles for use in non-viral gene delivery and biophotonics applications and then showed that these nanoparticles can serve as a delivery platform with superior efficacy in targeted drug therapy and as the realtime monitoring of drug action. the highly monodispersed stable water suspensions of the organically modified silica nanoparticles, which were labeled with the fluorescent dyes and functionalized by amino groups, were prepared using micelle chemistry. the nanoparticles efficiently bound dna due to positively charged amino groups and protected it from digestion by dnase i. imaging by fluorescence confocal microscopy confirmed that in vitro cells efficiently took up the nanoparticles in the cytoplasm, and the nanoparticles delivered dna to the nucleus. very recently bejjani et al. (2005) reported the breakthrough discovery that polylactic nanoparticles enable in vivo gene transfer and expression with a high efficiency. solid nanoparticles can be used to deliver drugs and biologically active molecules to any body organ including the brain, because they can cross the blood-brain barrier (lockman et al., 2002; koziara et al., 2003) . the nanoparticles that are biodegradable in a controlled way are likely to become the carriers of choice for in vivo non-viral gene delivery. surfacemodified and appropriately labeled nd particles may also serve as an efficient platform for in vivo transfection. however, because of the very low, if any, biodegradability of nd, nd nanoparticles, when applied in vivo, must be of a size small enough to allow their excretion by the kidney or applied cutaneously. immobilization of dna on diamond surfaces via covalent bonding has been explored intensively. for example, ushizawa et al. (2002) first modified diamond powder with particle sizes of 1-2 µm (fig. 15. 3) by oxidation in a heated mixture of sulfuric acid and nitric acid and then converted it in chlorocarbonyl-diamond by reacting with thionyl chloride at 50°c for 1 day. chlorocarbonyl-diamond was then reacted with thymidine in the presence of 4-dimethylaminopyridine. the dna was attached to the 3′-end of diamond-attached thymidine by 5′-end phosphatization. the formation of ester bonds was confirmed by diffuse reflectance ftir spectroscopic analysis. preparation of dna-modified diamond films for use in hybridization has received increased attention. the chemical stability of diamond surfaces is substantially greater than that of gold or silicon surfaces (lu et al., 2004) , and the dna molecules attached to the diamond surfaces are easily accessible to enzymes. nanocrystalline diamond thin films covalently modified with dna oligonucleotides following the photochemical modification of h-terminated surfaces with amine groups provide a very stable and highly selective platform for the surface hybridization reaction (yang et al., 2002) . after linking dna to the amine groups, hybridization reactions with fluorescently tagged complementary and non-complementary oligonucleotides did not reveal any non-specific adsorption, with extremely good selectivity between matched and mismatched sequences (yang et al., 2002) . in a similar manner, hydrogen-terminated diamond substrates were photochemically converted into amine-terminated surfaces following by linking to thiol-terminated dna oligonucleotides reported by knickerbocker et al. (2003) . the dna hybridization on dna-modified polycrystalline diamond is highly specific and when compared to the hybridization on dna-modified surfaces of crystalline silicon shows that the diamond surface exhibits superior chemical stability (knickerbocker et al., 2003) . dna-modified diamond surfaces are particularly suitable for invasive cleavage reactions, in which introduction of target dna to solution results in the specific cleavage of surface-bound probe oligonucleotides, permitting snp (single nucleotide polymorphisms) detection. the sensitivity of the analysis can be improved 100 times by replacing the dna-modified gold surface with a more stable dna-modified diamond surface (lu et al., 2004) . cvd diamond has also found some applications for dna immobilization. using thymidine as a linker molecule, the fragment of the human pku gene was covalently bound to the cvd diamond film by wenmackers et al. (2003) . boron-doped diamond (bdd) thin films with enhanced conductivity offer a substantial advantage for use in dna hybridization analysis. for example, gu et al. (2004) electropolymerized a thin layer of polyaniline/poly(acrylic acid) onto the diamond surface. the carboxylic acid residues in the polymer film enhanced the electron transfer between dna and a bdd surface and acted as the binding sites for dna attachment. both fluorescence microscopy and cyclic voltammograms indicated that the polymer-modified bdd did not show significant non-specific dna adsorption, while providing a stable transduction platform for dna detection by hybridization. the absence of efficient technology for in vivo delivery of oligonucleoticles limits many therapeutic applications. for successful application in vivo, in most cases in vitro delivery platforms should be extensively modified in order to provide targeted drug delivery. the adamantanebased materials have found application in the preparation of carriers for in vivo nucleic acid delivery because they can be modified by using cyclodextrin/adamantane host/guest interactions to provide the particles suitable for systemic application (pun and davis, 2002) . transferrinmodified nanoparticles containing dnazymes (dna enzymes that are rna-cleaving phosphodiester-linked dna-based enzymes, which cleave their target mrna in a gene-specific fashion) for targeting tumors were prepared by using conjugates of adamantane with poly(ethylene glycol) and administered to tumor-bearing nude mice by intraperitoneal bolus and infusion, intravenous bolus, and subcutaneous injection. dnazymes packaged in polyplex formulations were concentrated and retained in tumor tissue, whereas unformulated dnazyme was eliminated from the body within 24 hours after administration . in wound healing therapy, the localized delivery of growth factors is achieved by gene transfer to the wound site. synthetic biocompatible materials prepared with a linear, beta-cyclodextrin-containing polymer and an adamantane-based crosslinking polymer are very suitable for in vivo gene delivery to fibroblasts via the inclusion of adenoviral vectors in the synthetic construct . gene-deleted adenoviral vectors were originally developed as delivery vehicles for use in gene therapy trials and are currently being developed as hiv vaccines and in other medical applications. from a different perspective, in biological materials science and in rapidly emerging angstrom-scale chemical engineering the nucleic acids are thought to find many applications in the fabrication of self-assembling, multi-dimensional materials (wengel, 2004) . nd and diamondoid structures can be valuable candidates for crosslinking of oligonucleatides in carbon-based systems. both selective adsorption of proteins and their immobilization onto surfaces of nd particles (fig. 15.4 ) may be advantageous in nanobiotechnological applications and medicine. fibrinogen is widely accepted as an indicator in a biocompatibility test and material-caused inflammation. the adsorption of human fibrinogen on the surface of chemical-vapor-deposited diamond has been studied by tang et al. (1995) and was the first report on the diamond interaction with choi, 2004.) a protein and its biocompatibility. the cvd diamond was found to be biologically compatible to the same extent as titanium and stainless steel. in 1995 kossovsky et al. demonstrated that surface-modified nd particles with a size ranging from 5 to 300 nm provided both conformational stabilization and a high degree of surface exposure to protein antigens and used them to generate antibodies. recently huang and chang (2004) developed the universal procedure for protein immobilization onto the surface of 5 nm nd particles. it starts with nd particles with strong acids followed by modifying their surface with poly-l-lysine. covalent attachment of proteins is then carried out by activating the amine-terminated nd to react with the heterobifunctional linker ssmcc followed by mixing with the protein. the researchers successfully immobilized both alexa fluor 488 dye and yeast cytochrome c using the free sh group for linkage. more information on the application of protein-modified nd in biosensing and biotechnology can be found in section 15.5. hollow nanoparticles are thought to be the most suitable carriers of proteins and peptides susceptible to degradation. many nanoparticles can be made hollow by heating at high temperature, treating with strong acids, alkali, and organic solvents, or using gentle chemical treatment to encapsulate susceptible organic molecules such as proteins, peptides, and enzymes or others inside the cavity of hollow nanoparticles (sharma et al., 2005) . nd particles produced by detonation of the mixture of trinitrotoluene and cyclomethylenetrinitramine have a tetragonal structure and according to vereshchagin and yurjev (2003) are hollow particles with an inner diameter of 18.94 å and outer diameter of 25.47 å. yurjev et al. (2005) used synchrotron x-ray diffraction to characterize these spherical hollow detonation nds that can be modified even further by grinding in a planetary mill. the surface-modified nds and diamondoids are expected to find vast application in the development of a new generation of protein delivery platforms and antigen carriers because they can be readily modified to both carry and stabilize biologically active molecules, proteins, and enzymes. in addition, these particles are rigid, biocompatible, available in different shapes, and span the subcellular size range (fig. 15.1) . a biosensor (fig. 15 .5) generally comprises biomolecules sensing an analyte, e.g., dna, antibody, receptor, or enzyme, and the electrochemical, optical, calorimetric, or piezoelectric transducer that detects an attach-ment of an analyte to the biorecognition layer (deisingh and thompson, 2004) . the physiochemical properties that determine the analytical characteristics of biosensing devices are surface characteristics of the sensing area and its size, as well as intrinsic properties of the material. in the past few years, significant advances have been made toward the development of both biosensors and biochips for single-cell analysis, detecting of pathogens and toxins (vo-dinh et al., 2001) , and other biological and medical applications. diamond films occupy a special place as an electrode material in biosensors. when made sufficiently electrically conducting by boron doping, thin-film and free-standing diamond electrodes exhibit remarkable chemical resistance to etching, a wide potential window, low background current responses, mechanical stability toward ultrasound-induced interfacial cavitation, a low stickiness in adsorption processes, and a high degree of tunability of the surface properties (reviewed by compton et al., 2003) . tatsuma et al. (2000) have examined direct electron transfer from bdd electrodes to heme peptide and horseradish peroxidase for the application to h 2 o 2 biosensors. diamond electrodes exhibit low sensitivity to interfering agents and may become a capable h 2 o 2 biosensor. nanocrystalline diamond films can be both a platform for biofunctionalization and serve as an electrode in biosensors based on electrochemical reactions. different proteins can be covalently attached to the hydrogen-terminated nanocrystalline diamond films modified with amino groups and remain fully functional. hartl et al. (2004) functionalized nanocrystalline diamond electrodes with catalase and detected a direct electron transfer between the redox centre of the enzyme and the diamond electrode. also, the electrode was found to be sensitive to hydrogen peroxide. hydrogenated diamond can gain surface conductivity after exposure to air and by transfer doping with c 60 resulting in a significant rise in two-dimensional conductivity (strobel et al., 2004) . the fully hydrogen terminated diamond surfaces are not ph sensitive but can gain this sensitivity after a mild surface oxidation by ozone (garrido et al., 2005) . the difference in dna adsorption on the h-and o-terminated diamond surfaces may be useful in the nanofabrication of biosensors . compared to multiwalled carbon nanotube-based electrodes, bdd electrodes exhibit less selective voltammetric responses to the different biomolecules and slower electron-transfer kinetics. bdd electrodes have no intrinsic selective response to l-ascorbic acid, and surface modification by anodic polarization is required to resolve l-ascorbic acid and dopamine (poh et al., 2004) . highly conductive bdd electrodes are especially suited for electrochemical detection of nucleic acids in aqueous solutions. their distinctive features are high reproducibility, small background currents at high positive potentials, and robustness under extreme conditions (prado et al., 2002) . importantly, bdd electrodes can be used in analysis involving a heating step and ultrasonic treatment. well-defined peaks, observed with trna, single-and double-stranded dna, and 2′deoxyguanosine 5′-monophosphate were directly assignable to the electrooxidation of deoxyguanosine monophosphate (prado et al., 2002) . polycrystalline diamond films deposited by microwave plasma cvd were incorporated in the design of four different glucose sensors by troupe et al. (1998) . while a diamond-platinum-glucose oxidase sensor was affected by the presence of electroactive chemicals in blood, usage of bdd as a conducting electrode in place of the platinum provided a strong and repeatable response to glucose. the sensor that was fabricated with the surface-modified diamond allowed attachment of biologically active molecules and electron transfer from glucose oxidase to the electrode. this approach received further development in a study by . however, a 3,3′-diaminobenzidine-electropolymerized carbon nanotube-based electrode outperformed the diamond one in terms of selectivity and sensitivity. in another attempt to develop a nd-based biosensor immobilized polyclonal antibodies against salmonella typhimurium and staphylococcus aureus on nanocrystalline diamond films and evaluated the efficacy of immobilization by enzymelinked immunosorbent assay (elisa). the immobilization of antibodies and attachment of bacteria measured by sem were more efficient on a surface of air plasma-treated diamond than on a surface of diamond treated preliminarily with the hydrogen plasma. the plasma-oxidized surface of nd was more hydrophilic and was terminated with hydroxyl and carbonyl groups. modified nd particles and films have been used in heterogeneous and electrochemical oxidation catalyses and related applications, and in electrochemical analysis (bogatyreva et al., 2004; song et al., 2004; yang et al., 2005) . using the new amperometric biosensors designed with monocrystalline diamond and l-and d-amino acid oxidases, stefan et al. (2004) conducted differential pulse voltammetric assay of l-pipecolic and d-pipecolic acids in serum samples with a low limit of detection. the impedance of the diamond film can be affected by dna hybridization at the interface that induces a field effect in the diamond space-charge layer. by identifying a range of impedances, where the impedance is dominated by the diamond space-charge layer, and measuring the interfacial impedance, it is possible to directly monitor dna hybridization. no dna labeling is required. frequency-dependent interfacial electrical properties of nanocrystalline diamond films that were covalently linked to dna oligonucleotides were changed significantly in the presence of complementary dna oligonucleotides, with only minimal changes due to the presence of non-complementary dna oligonucleotides . the electrolyte-solution-gate field-effect transistors with hterminated polycrystalline diamond surface were shown to be sensitive to cl − and br − and can find application in cystic fibrosis tests . the capability for biomolecular recognition was provided to the highly sensitive field-effect transistor (bio-fet) made of a nanocrystalline diamond thin film by linking human immunoglobulin g to the diamond surface. electrical measurements showed that the bio-fet responded specifically to the anti-igg antibody . puzyr et al. (2004a) have recently developed a prototype nd-based biochip for use in bioluminescent analysis. the biochip incorporates aluminum oxide film-adhesive layer-deposited nd particulate-luciferase, which retained substantial enzymatic activity. it is also worth mentioning here that diamond sensor applications include high-sensitivity detection of charged particles such as protons and pions that are possible due to diamond's high radiation tolerance. a cvd diamond strip detector that can be used for tracking and detection of charged particles has been recently introduced on the market. fast charged particles create charge carriers in the irradiated diamond followed by induction of an electric charge on the strips. similarly, detonation nd and monocrystal cvd diamonds may be used for detecting both intermediate and high-energy heavy ions and in other instrumental analysis (adam et al., 2002; berdermann et al., 2004) . because both carboxylated and oxidized nd exhibit a remarkably high affinity for proteins, proteins in dilute solutions can be easily captured by nds with a size of 100 nm, separated by centrifugation, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms) without any need for pre-separation of the adsorbed proteins from nd (kong et al., 2005) . with the dilute mixed solution of cytochrome c, myoglobin, and albumin that preferentially adsorbs on a hydrophilic surface, the developed method offered significantly higher sensitivity than conventional maldi-tof-ms and had a limit of detection of 100 pm for a 1 ml sample solution. the potential of nd-assisted maldi-tof-ms for use in clinical proteomics was demonstrated with human blood serum analysis (kong et al., 2005) . if necessary, surfaces of nd particles can be modified to decrease non-specific protein adsorption and optimize their use for bioanalysis (lasseter et al., 2004) . application of detonation nds facilitated separation of obelia longissima apoobelin from the recombinant e. coli cell extract and made possible the preparation of purified protein with a yield of up to 38% . within the nd family, to a large extent medical applications have been developed for diamondoids. diamondoids such as adamantane derivatives (single-molecule unit of diamond with the formula c 10 h 16 ) have been used in pharmacology, clinical medicine, and biosensing (freitas, 2003) . until recently the cholinesterase inhibitors were the only available drugs for the treatment of alzheimer's disease, which is one of the leading causes of death for people over 65 years of age. unfortunately, the cholinesterase inhibitors cannot stop the process of neurodegeneration and just symptomatically enhance the cognitive state to some degree (sonkusare et al., 2005) . in alzheimer's disease, neuronal death is caused by glutamate excitotoxicity mediated through the n-methyl-d-aspartate (nmda) receptors making them an excellent target for preventing the disease. excitotoxicity is excessive exposure to the neurotransmitter glutamate or overstimulation of its membrane receptors, leading to neuronal injury or death. activity of the nmda receptor is also essential for normal neuronal function, and neuroprotective agents that fully block nmda receptor activity will have severe side effects (lipton, 2004) . memantine (1-amino adamantane derivative) is the potent nmda-receptor antagonist that blocks excessive nmda receptor activity without disrupting normal activity through its action as an uncompetitive, low-affinity, open-channel blocker. it also has beneficial effects in parkinson's disease, stroke, epilepsy, central nervous system (cns) trauma, amyotrophic lateral sclerosis, drug dependence, chronic pain, depression, glaucoma, and severe neuropathic pain. memantine is available in europe and has been recently approved for treatment of dementia in the usa. at the present time the second-generation memantine derivatives, which take advantage of the additional modulatory sites in the nmda receptor that could also be used for clinical intervention, are under development (lipton, 2004; sonkusare et al., 2005) . many adamantane derivatives possess antibacterial, antiviral, and antifungal activity (wang et al., 1998; el-sherbeny, 2000; orzeszko et al., 2002; el-emam et al., 2004) . for example, 5-(iadamantyl)-2-substituted thio-1,3,4-oxadiazoles and 5-(1-adamantyl)-3substituted aminomethyl-1,3,4-oxadiazoline-2-thiones exhibit substantial antimicrobial activity against gram-positive bacteria and the antiviral activity against hiv-1 significantly reducing viral replication at 2-50 µg ml −1 concentrations (el-emam et al., 2004) . adamantane-based drugs, namely, amantadine and tromantadine, are excreted unaltered in the urine and are not susceptible to hydroxylation (koppel and tenczer, 1985) . in 2005, hodek et al. discovered that adamantane, diamantane, triamantane, 2-isopropenyl-2-methyladamantane, and 3-isopropenyl-3methyldiamantane inhibited cytochromes p450 of subfamily iib by binding to the active site and, thus, could be potent inhibitors for hepatic oxidative drug metabolism in humans. theoretical and experimental values of the dissociation constant of cytochrome p450 complexes with the diamondoids were in good agreement and confirmed the high potency of identified inhibitors. bananins, the antiviral agents that possess a trioxaadamantane moiety attached to a pyridoxal derivative, were shown to be potent inhibitors of the sars (severe acute respiratory syndrome) coronavirus helicase and can prevent replication of animal scv (sars-related coronavirus) (tanner et al., 2005) . recently, the adamantane derivatives have been identified as the potential drugs acting on the p2x(7) receptor, which is involved in signaling in many inflammatory processes (cytokine release, no generation, cytotoxicity, killing of intracellular pathogens) (baraldi et al., 2004; romagnoli et al., 2005) . because nd is not mutagenic or toxic (oral ld50 value for rats is 7 g kg −1 ), can neutralize free radicals, and possesses a very large surface area, it was suggested that detonation nd particles may have some antitumor activity (dolmatov and kostrova, 2000 ; see also chapter 13). indeed, mice with erlich ascetic carcinoma that were given supplements with the nd suspensions were more active and lived almost 40% longer than non-treated animals. in 2001, dolmatov reported the results of a new medical study of the possible oral administration of water suspensions of nds to terminally ill cancer patients. the oral administrations of nd did not result in any side effects and, moreover, were moderately beneficial in some cases. diamond has the outstanding reputation of a chemically inert and uniquely biologically compatible material that has found a number of applications in medicine (freitas, 2003) . diamond is biocompatible in both bulk and particulate forms. in orthopedic surgery the use of a diamond coating on the metallic components reduces generation of macrophages and improves the wearability of devices (santavirta et al., 1999) . in addition, nanocrystalline diamond films show an excellent resistance to bacterial colonization (jakubowski et al., 2004) . the diamond-like carbon coating is also very inert and particularly suitable for use in orthopedic implants. in an early, pioneering study thomson et al. (1991) grew mouse peritoneal macrophages and fibroblasts on tissue culture plates coated with 0.4 µm of an amorphous diamond-like carbon layer and assessed the biocompatibility both biochemically and morphologically. the diamond-like carbon coating caused no adverse effects on cells in the culture. recently diamond-like carbon films have been reexamined and it was reported that they have good biocompatibility and high corrosion resistance (kim et al., 2005) . additional evidence of diamond biocompatibility came from zheng et al. (2005) who fabricated nanocrystalline diamond films (ndfs) on optical glass using microwave plasma assisted cvd and used osteoblast cell cultures and platelet adhesion tests for in vitro evaluation of biocompatibility of ndfs. their results indicated that the diamond films exhibit good tissue compatibility and hemocompatibility, which makes them very suitable for biomedical applications. the excellent chemical inertness and smoothness of ndfs made them a promising material for medical implants, cardiovascular surgery, and coating of artificial heart valves (mitura et al., 1996; 1999) . in 2004 specht et al. were able to demonstrate the ordered growth of mammalian neurons on diamond. to accomplish this the researchers patterned proteins on diamond surfaces by micro-contact printing and cultured mouse cortical neurons on these substrates. the diamond biocompatibility and the suitability of neuron interfacing with the surface make this an interesting approach for implant engineering. the high biocompatibility, corrosion resistance, chemical inertness, low friction coefficient, electrical insulation, and excellent mechanical characteristics of cvd diamond suggested that diamond-coated materials may find numerous applications in medicine (tang et al., 1995) . in biomaterials research, it is known that the biocompatibility of a bulk material is not necessarily the same as the biocompatibility of fine parti-cles of the same material, which may penetrate inside live cells and their organelles (freitas, 2003) . the main threat to cell viability comes from possible mechanical damage to cellular organelles and membranes. foreign intracellular particles with a diameter of 20-200 nm do not damage cells mechanically (lu and rosenzweig, 2000) . detonation particles possess a rounded shape, superior lubricity characteristics, hardness, and wear resistance. the fine diamond particles and diamond-like carbon coatings were always found to be very inert and non-inflammatory (tse and phelps, 1970; hedenborg and klockars, 1989; swan et al., 1990; grill, 2003) . when higson and jones (1984) treated both pig and horse neutrophils with diamond crystals, no induction of peroxide and superoxide generation was observed. the interaction of leucocytes with diamonds with a size of 4-8 µm in 0.2% diamond suspension did not cause any cell damage. no increase in degranulation and production of cell motility factors, and cell death, was observed (swan et al., 1990) . compared particles of diamond, sic, and hydroxyapatite in serum-free cultures of human monocytes and found that all particles were phagocytozed, and monocyte morphology changed except after the ingestion of diamond. it was concluded that diamond particles were inert in a serum-free human monocyte culture, while both sic and hydroxyapatite had a stimulatory effect comparable to that of polymethylmethacrylate. when suspensions of phagocytosable particles of diamond and sic in hyaluronan were introduced into a canal traversing the bone implant in rabbits neither the diamond nor the sic particles caused any decrease in bone formation. it confirmed that particles of diamond and sic are harmless . human blood cells' lack of adherence to the cvd diamond substrates, and blood clotting on diamond, produced a less rough surface than blood clotting on glass (baranauskas et al., 2004) . recent reports on possible nd-induced damage to both white and red human blood cells in vitro (puzyr et al., 2002; 2004b; raised the question about the mechanism promoting these phenomena. prior to use, nds are usually modified to gain high stability in water colloids , and it is not clear whether bacterial contamination, unidentified impurities, defects (shames et al., 2002) , and surfactants were not the factors involved. indeed, in those studies, the total content of nondiamond carbon, non-combustible residue, and volatile compounds reached 16% in some samples (puzyr et al., 2002) . in contrast to these observations, dion et al. (1993) did not notice any in vitro hemolysis when 14% blood solutions were treated with 0.5 g cm −3 diamond powder produced by de beers industrial diamond division, and the early reports on crystalline diamond-induced damage to cells were never confirmed (freitas, 2003) . rodil et al. (2005) reported that the diamond-like coating did not have any toxic effect on human osteoblasts cells in vitro. interestingly, monteiro-riviere et al. (2005) found that carbon nanotubes, which were grown using a microwave plasma enhanced cvd system, accumulated within cytoplasmic vacuoles of the human epidermal keratinocytes and stimulated release of the proinflammatory cytokine interleukin 8. there are no reports on site-specific intracellular accumulation of nd particles by live cells. as was mentioned in the previous section, administration of detonation nd particles to both erlich ascetic carcinoma mice and terminally ill cancer patients did not result in any side effects. moreover, it was beneficial in some cases (dolmatov and kostrova, 2000; dolmatov, 2001) . however, 0.002-0.01% nd suspensions administered orally to white mice over the course of 3 months did cause a substantial increase in leukocyte content in the blood of animals (puzyr et al., 2004c) . the treatment did not affect the weight of the experimental mice though. very unexpectedly, the intravenous administration of 0.3 ml of sterile colloids of 1% modified nds in 10% glucose to rats and dogs did not result in sickness or premature death of the animals (puzyr et al., 2005b) . when 1 ml of 1% nd was administered to a dog followed by the second administration 1 week later, the ecg tests revealed substantial changes in heart activity immediately after each treatment. however, the cardiac condition became normal again within 1 day. the safety and effectiveness of nanosystems and platforms made of nds will fully depend on their compatibility with human organs, tissues, cells, and cellular organelles. extensive thorough research has yet to be done to clearly establish physiological, immunological, and cytological responses of the human body to nd particles and films. in this review, methods of surface modification were discussed for the development of functionalized diamond nanoparticles for biomedical applications. to be used in biomedical applications, nanoparticles must be biocompatible, non-toxic, non-detective by immune systems, and should not induce side effects. size control of particles is a prerequisite for biomedical applications. to meet all these criteria, the diameter of particles should be less than 100 nm and their surfaces should be modified by hydrophilic moieties. such nanoparticles are likely to avoid uptake by the reticuloendothelial system and remain in blood at a high enough concen-tration to reach the target organs, tissues, or cells. the surface of nanoparticles has to be preliminarily modified by functional ligands with a high affinity to a disease site to achieve site-specific delivery. in many cases, nanoparticles may encapsulate and protect therapeutic agents against enzymes and hydrolysis; it is not clear yet how nanodiamond particles can satisfy this requirement. it is obvious from the reviewed literature that both nanodiamond particulates and films have become very popular objects in biomedical research. performance of irradiated cvd diamond micro-strip sensors nanotechnology in biology: the good of small things. the economist benign response to particles of diamond and sic: bone chamber studies of new joint replacement coating materials in rabbits plga nanoparticles in drug delivery: the state of the art agonists and antagonists acting at p2x(7) receptor analysis of the coagulation of human blood cells on diamond surfaces by atomic force microscopy behar-cohen f (20905) nanoparticles for gene delivery to retinal pigment epithelial cells synthetic biocompatible cyclodextrin-based constructs for local gene delivery to improve cutaneous wound healing charged particle detectors made of singlecrystal diamond application of modified nanodiamonds as catalysts of heterogeneous and electrochemical catalyses nanodiamonds for biological investigations applications of nanodiamonds for separation and purification of proteins a study on the action of ozone and on the thermal stability of nanodiamond selective inhibition of the oxidation of nanodiamonds for their cleaning progress in enzyme-based biosensors using optical transducers electroanalysis at diamondlike and doped-diamond electrodes easy access to watersoluble fullerene derivatives via 1,3-dipolar cycloadditions of azomethine ylides to c60 biosensors for the detection of bacteria detonation synthesis ultradispersed diamonds: properties and applications detonation-synthesized nanodiamonds and the possibility to develop a new generation of medicines detonation and shock synthesis of nanodiamonds b synthesis, antimicrobial, and anti-hiv-1 activity of certain 5-(i-adamantyl)-2-substituted thio-1,3,4-oxadiazoles and 5-(1-adamantyl)-3-substituted aminomethyl-1,3,4-oxadiazoline-2-thiones synthesis, antitumor activity, and anti-hiv-1 testing of certain heterocyclic systems containing an adamantane nucleus nanomedicine, volume i: basic capabilities ph sensors based on hydrogenated diamond surfaces diamond-like carbon coatings as biocompatible materials-an overview conductive polymer-modified boron-doped diamond for dna hybridization analysis protein-modified nanocrystalline diamond thin films for biosensor applications quartz-dust-induced production of reactive oxygen metabolites by human-granulocytes oxygen radical production by horse and pig neutrophils induced by a range of crystals structural requirements for inhibitors of cytochromes p4502b: assessment of the enzyme interaction with diamondoids adsorption and immobilization of cytochrome c on nanodiamonds immobilization of antibodies and bacterial binding on nanodiamond and carbon nanotubes for biosensor applications chemical reaction of hydrogenated diamond surface with peroxide radical initiators nanocrystalline diamond surface is resistant to bacterial colonization ftir studies on the spectral changes of the surface functional groups of ultradispersed diamond powder synthesized by explosive detonation after treatment in hydrogen, nitrogen, methane and air at different temperatures electrochemical behavior of diamond-like carbon films for biomedical applications a nonviral dna delivery system based on surface modified silicananoparticles can efficiently transfect cells in vitro dna-modified diamond surfaces highaffinity capture of proteins by diamond nanoparticles for mass spectrometric analysis a revision of the metabolic disposition of amantadine surface-modified diamond nanoparticles as antigen delivery vehicles situ blood-brain barrier transport of nanoparticles surface chemistry of nanodiamonds chemical properties of detonation-synthesized ultradispersed diamond covalently modified silicon and diamond surfaces: resistance to nonspecific protein adsorption and optimization for biosensing oxidation of cvd diamond powders reactions of amines with cvd diamond nanopowders carboxyfullerene prevents iron-induced oxidative stress in rat brain advances toward bioapplications of carbon nanotubes paradigm shift in nmda receptor antagonist drug development: molecular mechanism of uncompetitive inhibition by memantine in the treatment of alzheimer's disease and other neurologic disorders functionalization of nanoscale diamond powder: fluoro-, alkyl-, amino-, and amino acidnanodiamond derivatives fluorinated nanodiamond as a wet chemistry precursor for diamond coatings covalently bonded to glass surface nanoparticle technology for drug delivery across the blood-brain barrier diamond and carbon nanotube glucose sensors based on electropolymerization nanoscale fluorescent sensors for intracellular analysis invasive cleavage reactions on dna-modified diamond surfaces influence of carbon coatings origin on the properties important for biomedical application nanocrystalline diamond coatings multi-walled carbon nanotube interactions with human epidermal keratinocytes special features of structure and physico-mechanical properties of natural diamonds of ukraine nanoparticles, proteins, and nucleic acids: biotechnology meets materials science human monocytes stimulation by particles of hydroxyapatite, silicon carbide and diamond: in vitro studies of new prosthesis coatings synthesis and antimicrobial activity of new adamantane derivatives iii quantum dots and other nanoparticles: what can they offer to drug discovery? biosensing properties of diamond and carbon nanotubes electrochemical analysis of nucleic acids at boron-doped diamond electrodes development of a nonviral gene delivery vehicle for systemic application targeted delivery of rna-cleaving dna enzyme (dnazyme) to tumor tissue by transferrinmodified, cyclodextrin-based particles damaging effect of detonation diamonds on human white and red blood cells in vitro design of a luminescent biochip with nanodiamonds and bacterial luciferase destruction of human blood cells in interaction with detonation nanodiamonds in experiments in vitro dynamics of the selected physiological responses in laboratory mice under the prolonged oral administration of nanodiamond suspentions destruction of human blood cells upon interaction with detonation nanodiamonds in experiments in vitro a possibility of using of intravenous administration of sterile colloids of modified nanodiamonds in vitro cytotoxicity of amorphous carbon films recent progress in the discovery of antagonists acting at p2x(7) receptor optical tracking of organically modified silica nanoparticles as dna carriers: a nonviral, nanomedicine approach for gene delivery studies of host response to orthopedic implants and biomaterials defects and impurities in nanodiamonds: epr, nmr and tem study enzymes in the cavity of hollow silica nanoparticles carbon nanostructures cl-sensitive biosensor used electrolyte-solution-gate diamond fets surface-modified diamond field-effect transistors for enzyme-immobilized biosensors dementia of alzheimer's disease and other neurodegenerative disorders-memantine, a new hope effect of treatment temperature on the amination of chlorinated diamond ordered growth of neurons on diamond purification and modification of nanodiamond new amperometric biosensors based on diamond paste for the assay of l-and d-pipecolic acids in serum samples surface transfer doping of diamond photochemical functionalization of diamond films a comparison of the effects of urate, hydroxyapatite and diamond crystals on polymorphonuclear cells-relationship of mediator release to the surface-area and adsorptive capacity of different particles diamond nanofabrication and characterization for biosensing application biocompatibility of chemical-vapor-deposited diamond the adamantane-derived bananins are potent inhibitors of the helicase activities and replication of sars coronavirus electron transfer from diamond electrodes to heme peptide and peroxidase biocompatibility of diamondlike carbon coating diamond-based glucose sensors polymorphonuclear leukocyte motility in-vitro. 5. release of chemotactic activity following phagocytosis of calcium pyrophosphate crystals, diamond dust, and urate crystals abstraction of hydrogen atoms on diamond surface using benzoyl peroxide as a radical initiator surface reforming of the oxidized diamond surface with silane coupling reagents chemical modification of diamond surface with various carboxylic acids by radical reaction in liquid phase covalent immobilization of dna on diamond and its verification by diffuse reflectance infrared spectroscopy structure of detonation diamond nanoparticles nanosensors and biochips: frontiers in biomolecular diagnostics in vitro and in vivo growth inhibition of cancer cells by adamantylmaleimide derivatives nucleic acid nanotechnology -towards angstrom-scale engineering covalent immobilization of dna on cvd diamond films biological aspects of fullerenes a new method for deaggregation of nanodiamond from explosive detonation: graphitization-oxidation method dispersion and stability of nanodiamond in clean oil effect of sodium oleate adsorption on the colloidal stability and zeta potential of detonation synthesized diamond particles in aqueous solutions mechanochemical dispersion of nanodiamond aggregates in aqueous media influence of surface modification adopting thermal treatments on dispersion of detonation nanodiamond fabrication and characterization of a biologically sensitive field-effect transistor using a nanocrystalline diamond thin film dna-modified nanocrystalline diamond thin-films as stable, biologically active substrates interfacial electrical properties of dna-modified diamond thin films: intrinsic response and hybridization-induced field effects electrically addressable biomolecular functionalization of conductive nanocrystalline diamond thin films structural study of detonation nanodiamonds mpacvd nanocrystalline diamond for biomedical applications. high performance ceram chemical mechanical modification of nanodiamond in an aqueous system key: cord-262353-iips79vo authors: veltkamp, henk-willem; akegawa monteiro, fernanda; sanders, remco; wiegerink, remco; lötters, joost title: disposable dna amplification chips with integrated low-cost heaters † date: 2020-02-25 journal: micromachines (basel) doi: 10.3390/mi11030238 sha: doc_id: 262353 cord_uid: iips79vo fast point-of-use detection of, for example, early-stage zoonoses, e.g., q-fever, bovine tuberculosis, or the covid-19 coronavirus, is beneficial for both humans and animal husbandry as it can save lives and livestock. the latter prevents farmers from going bankrupt after a zoonoses outbreak. this paper describes the development of a fabrication process and the proof-of-principle of a disposable dna amplification chip with an integrated heater. based on the analysis of the milling process, metal adhesion studies, and comsol multiphysics heat transfer simulations, the first batch of chips has been fabricated and successful multiple displacement amplification reactions are performed inside these chips. this research is the first step towards the development of an early-stage zoonoses detection device. tests with real zoonoses and dna specific amplification reactions still need to be done. diseases were and can still be a major problem in the world. examples are outbreaks of zoonoses. one very recent example is the covid-19 coronavirus outbreak in the people's republic of china. zoonoses are also a widespread problem in animal husbandry [1] . this group encompasses diseases which can be transferred between animals (usually vertebrates) and between animals and humans. they are transmitted through zoonotic agents (e.g., bacteria, viruses, fungi, and parasites) [2] [3] [4] . examples of bacterial zoonoses are the infections caused by coxiella burnetii (q-fever), mycobacterium bovis (bovine tuberculosis), and by species of the salmonella (salmonellosis), campylobacter (campylobacteriosis), and escherichia (escherichiasis) genus [2, 5] . these diseases are of potential risk for humans and livestock of farms. poon et al. show that early-stage detection of coronaviruses positively influence the survival chances of patients [6] . an outbreak among the livestock of a farm is often disastrous to the owner of that farm, and for people living in the proximity of that farm [7] . often, more animals of the livestock are infected and the whole livestock is exterminated out of precaution, which could lead to bankruptcy of the farmer. therefore, early-stage detection of this group of diseases, and other diseases as well, is often the key to save lives and livestock. as these diseases are also encountered at remote locations and in developing countries, it is desired that such detection equipment is portable and as cheap as possible. a lab-on-a-chip platform can be used for this early-stage detection. in the early stage of diseases, the agent, and therefore its genetic material, is only present in low concentrations within the infected human or animal, making detection rather difficult. one way to overcome this low concentration is to amplify the genetic material of the agent, i.e., deoxyribonucleic acid (dna) in case of bacteria and dna or ribonucleic acid (rna) in case of viruses, until a certain threshold is reached and detection of the disease is made possible. when this amplification reaction is specific to certain dna or rna sequences, for example, by using polymerase chain reaction (pcr) [6, 8] , helicase-dependent amplification (hda) [9, 10] , or loop-mediated isothermal amplification (lamp) [11] , and when a fluorescent dna or rna binding dye is used, a simple yes-or-no answer for a specific disease can be obtained. in the past, several chip-based dna and rna amplification devices are reported. it goes beyond the scope of this paper to discuss the state-of-the-art of dna amplification chips in-depth. there are several good review papers written on this topic [12] [13] [14] [15] [16] [17] [18] [19] [20] . readers are referred to these for a comprehensive overview of the field. in this paper, the state-of-the-art is divided into several discussion points, i.e., the heating method, the temperature control method, and the substrate material and fabrication technique. these points will be discussed separately. with respect to heat supply, different methods have been employed. almassian et al. give a comprehensive overview of different possible heating methods in their review paper [12] . not all of the mentioned methods are easy to implement in low-cost and portable lab-on-a-chip devices due to their bulkiness or implementation costs. examples of these rather difficult methods are using heating via induction, infrared, or microwave radiation. others are not useful due ot their challenging temperature control, like with heating up the system using exothermic reactions. within the field of dna amplification, different mechanisms of amplification exist. some are based on thermo cycling processes, e.g., pcr, whereas others are isothermal. the use of an isothermal amplification technique puts less requirements on the heaters. isothermal processes are either truly isothermal or consisting of three different temperatures, as they have a thermal denaturation step before and a termination step after the elongation step. the switching between these temperature steps does not have to be as fast as with thermal cycling steps in, for example, pcr amplification reactions. the use of less temperature variations makes it easier to maintain the set temperature as there is less heating an cooling involved. furthermore, it eliminates the use of a continuous flow approach in systems with low thermal conductivities, e.g., polymers. therefore, it is easier to implement within lab-on-a-chip devices [17, 21] . isothermal dna amplification reactions can already be performed by putting the chip on a commercially available hotplate [22, 23] or peltier elements [24, 25] . however, these heating systems are bulky and power-consuming. therefore, they are not useful for portable equipment or operation at remote locations. miniaturizing heaters lowers the bulkiness and power consumption. miniaturized heaters can be integrated as integrated resistive heaters, e.g., as deposited thin-film metal [26] [27] [28] [29] or as laminated cu foil [30] , or as micro-peltier elements [31, 32] . these miniaturized heaters can be implemented directly onto the microfluidic chip [28] or on a different substrate and leter incorporated onto the microfluidic chip [33] [34] [35] [36] . the geometry of such a heater contributes significantly to the uniformity of the heat distribution within the chip [26, 37] . one method to accurately control the temperature is the use of a proportional-integral-derivative (pid) controlled thermostat. these pid controllers are coupled to the electrical heaters and use a thermocouple as feedback-loop to the controller [22] [23] [24] [25] . there are various materials that can be used to fabricate lab-on-a-chip devices for dna or rna amplification. in the past 15 years, more than ten polymers, ceramic materials, and metals have successfully been used to fabricate such devices [15] . the major property playing a role here is the biocompatibility of the material. the surface of the microfluidic structure should not inhibit the amplification reaction. this biocompatibility can be an intrinsic property of the material or the surface can be modified or coated to achieve this [12] [13] [14] [15] [16] [17] [18] [19] [20] . one often used material is polydimethylsiloxane (pdms) [22, 23, 31, 32, 34, 35, 38] , which can be processed using soft lithography [39] . however, this is a fabrication technology used in academia and is not suitable for upscaling to mass production [40] . fabrication methods suitable for mass production are thermoforming/embossing or injection molding [41] . one of the materials which is biocompatible and suitable for both industrial scale fabrication technologies is cyclic olefin copolymer (coc) [42] , which is one of the materials used in the past as well [28, 36, [43] [44] [45] . guckenberger et al. estimates the costs of injection molding of only 50 simple microfluidic devices on $47, but this becomes cheaper when the mass production stage is reached [41] . another benefit of coc is the possibility to shape it using micromilling. this technique is a rapid prototyping technology and therefore very useful within proof-of-concept projects [41] . integrating resistive metal tracks onto a coc substrate have also been done in the past. some papers describe the use of a surface modification step done before metal deposition in order to enhance adhesion between the coc and the metal layer, like a pretreatment with plasma [46] or an organic solvent [47] . other papers describe the direct deposition of metal onto the coc surface [28, 48] . chung et al. specifically, fabricated an amplification chip in coc with integrated au heaters [28] . however, their system required heating from both sides as the used grade of coc has a glass transition temperature (t g ) of 130 • c. this coc could not withstand the required heater temperatures to have enough heat flux into the system. they had to heat up the heater to temperatures above 130 • c, which caused cracking of the heater tracks due to deformation of the coc. with their double-sided heating they ensured that the reaction mixture had the desired pcr temperatures. however, double-sided heating doubles the amount of metal required, increases the amount of fabrication steps, and therefore increases the price per chip. the work presented at the 4th microfluidic handling systems conference and which is extended in this paper aims at the development of a disposable, polymer-based dna amplification lab-on-chip system with integrated resistive heater based on the world health organization (who) sexually transmitted diseases diagnostics initiative (sdi) assured criteria. devices which are assured are (a) affordable, (s) sensitive, (s) specific, (u) user-friendly, (r) robust and rapid, (e) equipment-free, and (d) deliverable to those who need them [20, 49] . the first step towards such a device is the development of the chip itself. this paper focuses on the choice of substrate material, metal deposition method, and type of metal. although, it is mentioned above that pcr and hda are sequence specific, the reaction chosen is the isothermal multiple displacement amplification (mda) [50] . this reaction is more straightforward [51] , as it amplifies any present dna, and is therefore better suitable as a proof-of-principle amplification reaction to show the functioning of the integrated heater and the biocompatibility of the substrate after the fabrication process. the use of an isothermal amplification technique also simplifies the final device and lowers its footprint, as there are no pumps required. in this research, external analysis methods are used which do not contribute to the who-sdi assured criteria due to their bulkiness, costs, and difficulty. however, suggestions and comments on the integration of low-cost detection methods, which are assured, are given in section 5. the proof-of-principle amplification of choice is a mda reaction, which is a non-specific isothermal method of amplification performed around 30 • c [50] . mda is a method of whole genome amplification (wga), as it amplifies all present dna [52] . it is commonly used when the initial amount of dna sample is very low. after the wga is performed, a sequence specific amplification can be done since the quality the amplified dna by mda is very high [53] . the amplification reaction is illustrated below in figure 1 (the contour of the amplified sequence is highlighted in black for clarity). starting with a double stranded dna (dsdna) molecule, a denaturation step at 95 • c is required, giving the random hexamer-primers and the φ29 dna polymerase access to the bases of single stranded dna (ssdna) strands. the hexamers anneal themself to aleatory parts of the ssdna sequence. these hexamers work as initiation sites for the φ29 dna polymerases. after denaturation at 95 • c, the mixture is cooled down to ice temperature and the rest of the reagents are added. the mixture is heated up to~30 • c so the polymerase starts to complete the complementary ssdna sequence, creating again a dsdna strand, eventually it encounters a hexamer from another annealing site. once this happens the polymerase will lift up that hexamer and starts to separate the amplified sequence formed from that annealing site. as the polymerase displaces the formed strand ahead of it, it continues to complete the sequence. the displaced strand becomes a new ssdna strand and therefore, it gives new sites for more primers to attach and initiation sites for the polymerase, continuing the amplification, and thus creating a web of dna strands. finally, the inactivation of the polymerase is done by heating up the system to 65 • c. even though mda is considered an isothermal process, prior to the reaction and to the addition of most reactants, the dsdna and a buffer are heated up to 95 • c to denature the dsdna to ssdna and to give hexamers the initial access to the ssdna. after the amplification reaction, the polymerase has to be inactivated at 65 • c. however, this does not require fast temperature changes, as would be the case with, for example, the temperature cycling in pcr amplifications. this, together with the robustness of the amplification (it is a self-limiting reaction that amplifies all present dna [50] ) makes mda perfectly suitable as proof-of-principle amplification reaction for such devices. in panel (b), denaturation happens at 95 • c. in panel (c), the random hexamer-primers (purple) and φ29 dna polymerase (blue arrow) bind to the initiation sites. in panel (d), the amplification is performed by the polymerase, which binds complementary bases to the ssdna strand. in panel (e), the polymerase encounters another hexamer binded to an initiation site and starts lifting up this hexamer. in panel (f), a hexamer binds to the displaced ssdna strands and the polymerase starts the amplification from this initiation site. for clarity, the amplified dna is bordered with black. the microfluidic structure consists of two chambers, i.e., a reaction chamber and a temperature monitor chamber. in figure 2 , a close-up of the final chip is shown. for clarity reasons, the two microfluidic structures are colored with food coloring dye. the reaction chamber is based on the work of bruijns et al. [36] and its dimensions are chosen in such way that the internal volume of the reaction chamber is the same as the reaction volume of the used illustra genomiphi v2 dna amplification kit (ge healthcare life sciences, eindhoven, the netherlands) together with the evagreen dye solution (biotium, fremont, ca, usa), while maintaining an as low as possible surface-area-to-volume ratio [44] . using solidworks 2018 computer-aided design (cad) software (dassault systemes, vélizy-villacoublay, france), the 3d image of the chip is drawn and with the use of the autodesk hsmworks computer-aided manufacturing (cam) plug-in (autodesk inc., san rafael, ca, usa), this image is transferred into a computer numerical control (cnc) milling code. the total chip size is 3 cm by 3 cm and contains an inlet and outlet of 1.5 mm diameter. the inlet and outlet are of such size that the reaction chamber can be filled using pipette tips. in between the inlet and outlet, a rectangular reaction chamber of 10 mm by 3 mm is located. two trapezoid structures are placed in the tapered channels between the inlet/outlet and the chamber. the function of these trapezoids is twofold: first, they minimize the dead volume between the inlet/outlet and the reaction chamber, locating as much as possible of the reaction mixture inside the chamber. second, they provide support for the chamber closure. a stadium-shaped channel of 1.5 mm wide and 1.0 mm deep is located next to the reaction chamber, in such way that this channel is also covered by the heater. this channel serves as temperature monitor chamber. a thermocouple is inserted in this channel for real-time monitoring of the temperature inside the chip. this way, a more accurate temperature of the reaction mixture inside the chip can be obtained. via a feedback loop, the input potential can be changed when required. in figure 3 , the solidworks design of the chamber-based chip with both chambers is shown. in figure a1 , in appendix a, the technical drawing of the chip can be found. a resistive heater structure will be placed at the bottom side of the chip using shadow masks and a metal deposition method capable of being used for large-scale production. a meandering heater design is chosen, as this minimizes the input power required to heat up the heater. this is evident from equation (1), which is the relation between joule's law, ohm's law, and pouillet's law. here, p is the input power, a cross−section is the cross-sectional area of the resistor, v is the input potential, ρ res,i is the resistivity of resistor material i, and l heater is the length of the resistor. this makes a meandering structure, or any other narrow line structure, a quite often used pattern for heaters or electrodes within micro-electromechanical structures and microfluidics [26, 29, 54, 55] . mda is being done at temperatures of around 30 • c [50] , which is lower than, for example, temperatures required for hda (64 • c) [9] or lamp (65 • c) [11] and the required pcr temperatures of chung et al. (95 • c, 54 • c, and 72 • c) [28] . however, most amplification methods require a dna denaturation step at 95 • c. equation (2) is used to make an estimation of the required heating powers for a coc-h 2 o-coc stack (in the real device, the upper plate is an adhesive pcr foil, but the thermal properties of this foil are unknown). here, r th is defined as the sum of all thermal resistances in series: here, p is the required power, ∆t is the temperature difference, r th is the thermal resistance , a heated is the heated area, h is the convective heat transfer coefficient (being 10 w m −2 k −1 for convection to air [56] ), κ i is the thermal conductivity of substance i, and l i is the thickness of substance i. values for κ i can be found in appendix b. from equation (3), the product r th × a can be defined as the sum of 1 /h and l i/κ i . based on this summation, one can conclude that the convective heat transfer to the air is the most present heat transfer mechanism within the system (begin almost a factor 100 higher than the heat lost in the coc and h 2 o). this is also evident from solving equation (2) for every individual temperature differences within the system and also including convective heat transfer directly from the heater into the air. if a heated area of 7.7 mm by 10.1 mm is assumed, which covers both the reaction chamber and the temperature monitor chamber, and a system consisting of 1 mm coc-0.5 mm h 2 o-0.1 mm coc is assumed, than the heater temperatures and powers in table 1 are required. these are all in the workable range when a coc of a proper grade is chosen (e.g., topas 6017 has a t g of 170 • c). the only side note here is that at higher temperatures, the temperature gradient through the system also becomes larger. this can be eliminated by using double-sided heating, like chung et al. [28] . to determine the optimal heater width and heater spacing in the heated area, a parametric study using comsol multiphysics 5.3a finite element method simulations with the heat transfer in solids (ht) package is done (comsol inc., burlington, ma, usa). the model is designed such that it consists of two parallel rectangles of coc (in the real device, the upper plate is an adhesive pcr foil) with h 2 o in between. the meandering heater are assumed to be lines at the bottom side of the layer stack. this reduces the required complexity of the mesh tremendously, as the heater in the real device will be approximately 100 nm in thickness. the heater temperature is set at a constant temperature of 303.15 k. this makes the heater material independent and the model purely focused on the heat transfer inside the coc-h 2 o-coc stack. all used values and equations are given in appendix b. the layer stack is meshed with an extremely fine mapped mesh consisting of 280.650 elements with average quality of 0.9966. a parametric sweep from 0.3 mm to 2.0 mm, in steps of 0.1 mm, is done for both the heater width (w heater ) and the heater spacing (s heater ), giving 324 combinations. the simulations are solved by using the fully coupled, direct pardiso solver on a custom-build and 40% cpu overclocked simulation computer, containing the equipment listed in table 2 . to validate whether the metal tracks can withstand the required current, a quick analysis is done for the four extreme cases (i.e., w heater of 0.3 mm and 2.0 mm and s heater of 0.3 mm and 2.0 mm). in the same heated area of 7.7 mm by 10.1 mm a 100 nm (t heater ) thick heater track consisting of rectangles is assumed. the total amount of large and smaller interconnecting rectangles for all 4 cases is estimated in table 3 . based on the polynomial approximation equations for the resistivity of au and pt (ρ res,i , where i is either au or pt) which comsol multiphysics 5.3a uses (equations (4) and (5)) and equation (6) an estimation is made for the required input currents and the created current densities (defined as i i/a cross−section , in a m −2 ) when the heater is operated at 129.4 mw to get a temperature of 95 • c. these estimations are also given in table 3 . in which ρ res,i is the resistivity, t is the temperature, i i is the current going through the resistor, p i is the input power, r is the resistance of the resistor, l heater is the length of the resistor, and a cross−section is the cross-sectional area of the resistor defined as width times thickness (w heater × t heater ). the subscript i denotes the material, being au or pt. all these current densities are below the critical current densities for au and pt, which are around 10 10 a m −2 [57] and 10 11 a m −2 [58] , respectively. therefore, any possible combination of heater width and heater spacing will give a resistive that can withstand its operation. coc [42] is chosen as polymeric substrate because of its biocompatibility, optical transparency, physical resistance, chemical resistance, electrical insulation, and price. this copolymer consists of two monomers, an apolar bridged cyclic hydrocarbon (norbornene) monomer and a linear, lesser apolar, linear ethene monomers. injection molded coc plates (10 cm by 10 cm and 1.5 mm thickness) of the grade topas 6017 (see figure 4a ) are obtained via kunststoff-zentrum leipzig (kunststoff-zentrum ggmbh, leipzig, germany). this grade is chosen because of its high norbornene content, giving it a relatively high t g of 170 • c. this minimizes the chance of melting during the milling process and decreases the chance of heater failure due to a deforming substrate during operation of the heater [28] . the microfluidic structure explained in section 2.1 is cnc-milled using a mikron wf 21c milling machine (mikron sa agno, agno, switzerland), as can be seen in figure 4b . milling is a very fast prototyping technique and chosen because of its flexibility [41] . the milling creates a surface roughness, which increases the surface-area-to-volume ratio. this roughness increases the chance of inhibition during the amplification due to the interaction of the used chemicals with the surface [44] . it also causes a considerable loss of optical transparency, which could obstruct the potential use of in situ fluorescence detection in future devices. therefore, a chemical post-treatment with cyclohexane vapor is done (see figure 4c ). such treatment dissolves a thin outer layer of the coc substrate and causes reflowing of the surface roughness due to the surface tension of the material, restoring the optical transparency and reducing the surface roughness [59] . cnc milling and subsequent cyclohexane vapor post-treatment are less suitable for mass production. however, coc has the possibility of being injection molded [42] . the used substrates are made using this method. this is a large-scale production method and could lower the costs of the eventual product and it eliminates the cyclohexane vapor post-treatment, as injection-molded chips would have the same optical transparency as the pristine substrates. guckenberger et al. mention production costs of $ 47 per simple microfluidic device when only 50 pieces are fabricated [41] . this price is expected to drop drastically when large numbers are fabricated. a metal is deposited on the backside of the substrate using two laser-cut metal (mo) shadow masks to outline the shape of the resistive heater (see figure 4d ). mo has a smaller coefficient of thermal expansion than stainless steel, and therefore gives less deformation during the deposition. metals of interest are au or pt, which are commonly used metals to function as resistive heaters [54] . the deposition methods studied are dc magnetron sputtering using a custom-build machine (techno centrum voor onderwijs en onderzoek, university of twente, enschede, the netherlands) and e-beam physical vapor deposition (evaporation) using a balzers bak 600 ce (oerlikon balzers limited, balzers, principality of liechtenstein). both deposition methods are capable of large-scale production, which will lower the production costs in the large-volume production stage. the metal and deposition method will be chosen based on the metal adhesion performances on the coc substrate, which is studied using the scotch tape test [60, 61] , and the resistance versus temperature behavior in the range 20 • c to 100 • c, which is measured in a heraeus t5025 oven (heraeus holding gmbh, hanau, germany), customized with electrical readout and connected to a custom-build national instruments labview program (austin, tx, usa). the chambers with the resistive heater on the backside, are intensively cleaned by rinsing with acetone, milliq di water, ethanol, and isopropanol [45] . each cleaning step was done 3 times and the chips are blow dried using n 2 gas. after drying, the chambers are closed using microseal "b" pcr plate sealing foil from bio-rad (bio-rad inc., hercules, ca, usa), which is cut in the proper size and manually attached on top of the substrate (see figure 4e ). the dna, reactants and buffer solutions from the illustra genomiphi v2 dna amplification kit and an evagreen fluorescence dye are pipetted inside the chip using the inlet aperture, after which the inlet and outlet are closed using the same pcr foil. an input potential is applied on the resistive heater using a keithley 2602 system sourcemeter (cleveland, oh, usa) until they acquire the desired temperature for the amplification. the temperature is real-time monitored by inserting a 162 series rs technics thermocouple k (rs components b.v., haarlem, the netherlands) in the temperature monitor chamber. the thermocouple is read out with a tenma 72-7715 thermometer (premier farnell ltd., leeds, uk). the source and the read-out of the thermocouple are operated using a custom-programmed labview program. the initial potential is based on the heater characterization measurements, but will be adjusted according to the feedback-loop of the thermocouple. detection of the amplification is done ex-situ by using quartz cuvets and an horiba scientific fluoromax+ spectrofluorometer (horiba scientific, piscataway, nj, usa). in figure 5 , the results of heat transfer simulations of two different heater spacings are shown. the heater width for both geometries is 0.3 mm, while the heater spacing in figure 5b ,c are 0.3 mm and 2.0 mm, respectively. in figure 6a -d, tables with the results of the full parametric sweep for different heater widths and heater spacings are shown. figure 6a shows the temperature deviation between the highest and lowest temperature at the top of the chamber, i.e., the second h 2 o and coc interface (∆t top of chamber = t top,max − t top,min ). figure 6b shows the deviation between the highest and lowest temperature inside the chamber, i.e., between the two coc and h 2 o interfaces (∆t across chamber = t bottom,max − t top,min ). figure 6c shows the temperature deviation between the highest and lowest temperature at the bottom of the chamber, i.e., the first coc and h 2 o interface (∆t bottom of chamber = t bot,max − t bot,min ). figure 6d shows the deviation between the set heater temperature of 30 • c and the lowest temperature at the top of the chamber, i.e., the second h 2 o and coc interface (∆t deviation from set t = t heater − t top,min ). as can be seen from the results in figure 6 , a combination of small heater widths and heater spacings will result in smaller temperature differences inside the reaction mixture. this is evident as smaller heater spacings will result in a better coverage of the heated area by heater material. the smaller heater widths will result in a smaller heater cross-sectional area, and thus can be operated at lower powers, as is evident from equation (1). resulting in the fact that a densely packed meander structure with small heater widths and small heater spacings can dissipate more heat into the system. 3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 based on these results and its simplicity, a meandering heater pattern of a heater with a width of 0.3 mm and a spacing of 0.3 mm in between the lines is designed. a side note on the chosen heater width and heater spacing is that according to the simulations, the temperature differences within the chamber are less than ±0.3 • c for the most unfavorable dimensions. this temperature difference is still well-accepted in the temperature window in which the mda reaction takes place (25 • c to 35 • c). however, as pointed out, a smaller cross-sectional area will result in a lower power consumption and therefore these dimensions are chosen. it is known that a meandering heater structure does not give the optimal temperature distribution over the device [26] . therefore, the heater lines are longer than the width of the reaction chamber, and thus also covering the bulk material outside the chamber in order to improve the temperature uniformity inside the reaction mixture. the heater pattern is divided over two shadow masks to minimize the length of the narrow mo tracks in between the meandering structure. this prevents curvature due to intrinsic stresses. see figure 7 for the outlines of both shadow masks, together with the resulting pattern on coc. the use of two shadow masks will give a metal track in which small parts has the double thickness. here, the temperature will be lower. the system is designed such that these thicker parts are outside the reaction chamber and temperature control chamber region. the milling increased the surface roughness of the coc plates also increases the surface area. inhibition of the amplification can be caused by large surface areas as the used chemicals have more surface to have interaction with [44] . the created surface roughness is visualized using a fei sirion high resolution scanning electron microscope (hr-sem) (fei company, hillsboro, or, usa) and measured using a bruker icon dimension afm in tapping mode with bruker tespa-v2 cantilevers (bruker nano surfaces, santa barbara, ca, usa) and gwyddion 2.52 open source freeware [62] . the results are shown in figure 8 . the surface roughness of pristine coc had a r rms of 3.5 nm. this increased two orders of magnitude after milling (r rms of 310.1 nm). with the reported surface treatment [44, 59] we were capable of decreasing the surface roughness to a value even lower than that of pristine coc and the lowest reported in literature (r rms of 0.9 nm). for this grade of coc (topas 6017) it worked the best to do four short exposures of 5 s, with n 2 blow drying after each exposure, instead of one longer exposure, as is more common in other grades of coc [44, 59] . the difference in duration for the cyclohexane vapor post-treatment can be explained by the different ratios of the copolymers present in each grade. as the grade number increase, the ratio changes towards more norbornene monomers and less linear ethene monomers. the norbornene is more apolar due to the bridged cyclic hydrocarbon present in its molecular structure and therefore, will dissolve faster in non-polar solvents, like cyclohexane (vapor). lowering the surface roughness also increased the optical transmittance fivefold. transmittance measurements in the visible range are done using a woollam m-2000ui ellipsometer (j.a. woollam co., lincoln, ne, usa). the results can be seen in figures 9 and 10 . having a high optical transparency in the visible range can be desired when in situ fluorescence detection will be implemented (e.g., evagreen fluorescence dye has an excitation wavelength of 500 nm and emission wavelength of 525 nm [63] ). however, as in situ fluorescence detection is not used yet in this system and can also be done through the transparent pcr plate sealing foil, no further effort is put into optimizing this procedure to get even better optical transmittance. the graph in figure 10 shows the transmittance data of these substrates. to get reliable heaters, four possible options are investigated for their adhesion properties to the coc substrate. the adhesion of au and pt deposited by either evaporation or dc magnetron sputtering is investigated using the scotch tape test [60, 61] before and after temperature cycling up to 100 • c. test patterns consisting of rectangular metal strips of 2 mm by 14 mm are fabricated by depositing 100 nm of metal using a hand-made shadow mask made out of dupont kapton ® hn polyimide film of 0.05 mm thickness (rs components b.v., haarlem, the netherlands). see table 4 for the results of the scotch tape test. normally, heating up a glass or si substrate with thin metal strips while measuring the resistance (r t ) in these metal strips at certain temperature intervals (t) yields directly a linear relation, which can be fitted with r t/r 0 = 1 + α (t − t 0 ) [64] , in which α is the the temperature coefficient of resistance (tcr) value. the thin-film tcr values have to be measured as they differ from the bulk tcr values due to its dependency on layer purity, grain size, and deposition method [65, 66] . belser and hicklin also lists other attributes, such as surface roughness, porosity, and adsorbed materials present in or on the substrate which could influence the tcr value [64] . the bulk tcr values are 0.0034 k −1 and 0.0037 k −1 for au and pt [67] . the tcr characterizations of the metal strips on a coc substrate did not yield trustworthy tcr values at the first cycle. the first temperature cycle can be seen as a kind of thermal annealing, and therefore gives an hysteresis in the graphs, as can be seen in figure a2 in appendix c.1. after this first cycle, the values more or less show the linear behavior. the resulting tcr of this linear part is in agreement with the tcr ranges of belser and hicklin [64] and is given in table 4 . belser and hicklin used for their experiments substrates with coefficients of linear thermal expansion lower than 1.2 × 10 −5 • c −1 [64] . the coefficient of linear thermal expansion for au and pt are 1.42 × 10 −5 k −1 and 0.88 × 10 −5 k −1 , respectively [68] . coc of the grade topas 6017 has a coefficient of linear thermal expansion of 6.0 × 10 −5 k −1 [42] . this mismatch in coefficients of linear thermal expansion can give strain in the metal layers. both au [69] [70] [71] and pt [72] [73] [74] are used as strain-sensitive gauges, and thus are sensitive to strain-induced geometry changes due to thermal expansion. another effect influencing the tcr value of the metal layer is aging. as can be seen in figure a3 in appendix c.2, the tcr value already changes after two weeks storing in ambient conditions. this could be due to adsorbed materials present on the surface [64] . however, in this device, the tcr is not of importance as the metal structure will not be used as temperature sensor. real-time temperature sensing is done using a thermocouple in the temperature monitor chamber. the resistance of the heater structure changes with temperature; thus, the dissipated power changes when a fixed voltage or current is used. however, the results in section 3.4 show a 25 h stability test with a constant input potential and only a ±1.5 • c deviation. the tcr can become more important when other (higher) temperatures are required for the amplification. based on the results in table 4 , the choice of heater material and deposition method to be used in the actual device is au deposited using sputtering. sputtering is an industrial-scale technique that is already being used in, for example, the car mirror and headlight industry [75] . characterization of the actual heat distribution is done using a flir one pro ios thermal camera (flir systems, inc., wilsonville, or, usa). thermal images of the heat distribution are made at the side of the substrate without the resistor, whereas different input powers are used to heat up the heater. au reflects the infrared radiation of the environment directly, therefore an image with the resistor facing the camera would give a heat map of the surrounding and not of the real temperature of the heater. these measurements also gives a better insight of the heat distribution inside the reaction chamber. the images are processed using the flir postprocessing freeware. results of these measurements are shown in figure 11a ,b. the results are in good agreement with the estimations in table 1 . the slight deviation between the values can be explained by the fact that the heated area in the calculations had an assumed value, the thermal camera measurements used 1.5 mm thick coc substrates without a water-filled chamber, the actual resistors have small parts wich have a double thickness due to the two used shadow masks, and rounding of the values used in the calculations. the reliability of the heater is tested by inserting the thermocouple into the temperature control chamber (see figure 3a) . a constant input potential of 4 v is applied using the keithley source and the temperature is measured for 25 h. this exceeds the required operation time at least twelve-fold, meaning that it is a good indication for the reliability of the heater and thermocouple. the results are shown in figure 11c . to perform on-chip amplifications, the resistive heater on the chip is connected to the keithley source using crocodile connections and the thermocouple is inserted in the temperature control chamber and connected to a tenma 72-7715 thermometer (see figure 12 ). first, to determine the temperature window of operation, mda reactions are performed at 25 • c and 30 • c using the illustra genomiphi v2 dna amplification kit and evagreen fluorescence dye. from the literature, we know that this reaction does not work above 35 • c due to degradation of the protein activity in presence of a substrate [44] . in figure 13 , a graph of the fluorescence signal during mda reactions at 25 • c and 30 • c, together with their non template control (ntc) is shown. these reactions are carried out in a conventional bio-rad cfx96 touch real-time pcr machine (bio-rad laboratories, inc., hercules, ca, usa) and the results show that the chosen proof-of-principle dna amplification reaction is temperature dependent to some extent, but that there is a wide range of temperatures at which the amplification can be performed, i.e., 25 • c to 35 • c. this makes the functioning of the integrated resistive heater less critical than the stability shown in figure 11c . data is collected using a flir one pro ios thermal camera. at mda temperature the heat distribution over the reaction chamber area is more uniform than at higher temperatures. (c) duration stability test of the heater. a constant input potential of 4 v is applied and the temperature is measured for 25 h using the type k thermocouple. the slight decrease in temperature between 7 h and 15 h is due to the night. however, it is within the temperature range for mda. mda reactions are also performed inside an eppendorf tube (eppendorf ag, hamburg, germany) and inside the chip, again using the illustra genomiphi v2 dna amplification kit and evagreen fluorescence dye. as heat supply the water bath of an ika rotary evaporator rv 8v (ika-werke, staufen im breisgau, germany) is used. this water bath is according to its specification stable within a range of the set temperature ±0.1 • c. the chip and an eppendorf tube are loaded with the reaction mixture containing the dna sample and the evagreen dye solution. here, the eppendorf tube is serving as a control to show that the fabrication steps of the chips are not inhibiting the mda reaction. the inlet and outlet of the chip are sealed with the microseal "b" pcr plate sealing foil. the closed chip and tube are heated up in a separate water bath to 95 • c and kept at that temperature for 3 min to denaturate the dsdna. subsequently, the chip and tube are cooled down by placing it in an ice bath for 5 min after which the rest of the reagents are added. the complete mixtures are according to table a3 in appendix d. after closing the chip and tube again, they are placed in the water bath of the rotary evaporater and left there for 90 min, after which the reaction is terminated at 65 • c. the mda is also performed inside the chip, but with the integrated au resistive heater serving as heat source. the set up shown schematically in figure 12 . the same procedure is followed as with the water bath heated test. denaturation is done in a separate water bath. the heater is driven by an input potential of 3.2 v to get to a temperature of 30 • c and at the end of the reaction, the system is heated up to 65 • c by applying a potential of 9.2 v in order to terminate the amplification. in figure 14 the logged temperature during the amplification is shown. after the amplifications, the reaction mixtures are pipetted out of the chips and tubes and into 1 ml quartz cuvettes containing 55 µl milliq di water (merck millipore, burlington, ma, usa). fluorescence measurements are done in a horiba scientific fluoromax+ spectrofluorometer to verify each amplification. the mixture is excitated at a wavelength of 500 nm and the emission spectrum is measured at wavelengths from 510 nm to 550 nm (bounded evagreen dye has a peak at 525 nm [63] ). the measured spectra are normalized by subtracting the background signal of a mixture containing only the reaction buffer, the sample buffer, evagreen, and dna. no enzyme was added to this mixture, therefore no amplification could take place. see figure 15 for the results obtained in the eppendorf tube and chips. figure a4 in appendix e shows the background signal which is subtracted from all measurements. as can be seen in figure 15 , the spectra of the amplification performed inside the chip, and by applying heat with the water bath as well as with the integrated au-resistive heater, show the same trend as the amplification performed in the eppendorf and heated by water bath. there is an order of magnitude difference in the fluorescence signal. however, the fluorescence intensity cannot be used as a value to quantify the amount of dna. evagreen is a bis-intercalating cyanine fluorescence dye consisting of two monomeric dna-binding dyes which are linked by a flexible spacer. these two dna-binding dyes bind each in between two base pairs, which make them simple and fast, but also nonuniform and non-specific [44, 63] . however, with this dye, a simple yes-or-no answer can be obtained if the amplification took place, as can be seen in figure 15 . the aim of this study was to fabricate biocompatible, low-cost, and disposable chips with integrated heater, which should be able to perform dna amplification, and possible in situ fluorescence detection in the near future. in this case there is no interest in quantification of the dna, but only in amplification of dna until the detection threshold is reached. as proof-of-principle the mda reaction and ex-situ fluorescence measurements were used. with the proposed fabrication process, low-cost and biocompatible chips (figure 12b ) were fabricated. the integrated resistive heaters on the chips were characterized and showed a temperature stability of ±2 • c over a time period of 25 h, which is at least twelve-fold longer than the required operating times for dna amplification reactions [6, [8] [9] [10] [11] . the main cause of this period of lowered temperature was due to the fact that the measurement was run overnight. with the proof-of-principle device, successful dna amplifications using mda inside a disposable polymeric chip were achieved. the heat for the reaction was applied using the integrated low-cost au-resistive heater. the device was operated at a suitable temperature for mda reactions and the amplified dna was measured using evagreen fluorescence dye and an ex situ spectrofluorometer. a distinct peak is visible in the reaction mixtures which is absent in the ntc mixtures. the operating temperature for mda reactions is around 30 • c, which is comparable with a nice summer day. using amplification reactions which such low reaction temperatures could encounter problems at warmer locations. however, as mda is not sequence specific, this reaction will not be integrated in the final protocols. mda was only used as proof-of-principle reaction to show the biocompatibility of the device and functioning of the integrated heater. sequence specific amplifications, e.g., hda and lamp, are performed at higher temperatures, as will be discussed in section 5. this makes the system less sensitive to the hot summer days. the device in its current state is not fully conform the who-sdi assured criteria [49] as it still relies on the use of (expensive) external equipment. however, the first steps are made to an assured device. future steps which will make the device fully assured are given in the next section (section 5). future steps, which will result in a device for early-stage detection of, for example, zoonoses, include studies on the optimization of this device for sequence specific dna amplifications (e.g., primer design and reaction optimization), i.e., hda or lamp. hda utilizes dna helicase (an enzyme also used in vitro during dna replication) to separate the dsdna instead of thermal denaturation. after separation, ssdna binding proteins hybridize on the ssdna strands for stabilization, ensuring that the next primer will have time to bind to the ssdna stripe and a dna polymerase will extend the primers with the complementary bases. this method is a truly isothermal technique in which the separation of the dsdna can be performed at the same temperature as the amplification reaction, i.e., 64 • c [9] . lamp is more similar to mda in the way it also uses heat to denature the dsdna. after denaturation, a set of four primers (six can be used as well to achieve better selectivity) and a dna polymerase is used at isothermal conditions (65 • c) to amplify the dna [11] . when used in combination with reverse transcriptase, lamp becomes a rna amplification method, which could be used for rna-containing viruses [11] , like virus-based zoonoses diseases as the corona viruses [22] . despite not being a truly isothermal technique, lamp offers the possibility to use turbidity as detection method [76] . such a detection method would simplify the required equipment even further as a decrease in transmitted light through the chip can be used as detection method. different amplification techniques require different temperatures. based on table 1 one can conclude that a higher temperature would also give a larger temperature gradient within the system. this can be disadvantageous for amplification reactions, as optimal denaturation temperatures are in the range 92 • c to 94 • c [77] . the denaturation in this research was done in a separate water bath, so this temperature gradient was circumvented. however, when on-chip denaturation and/or another amplification technique will be used, a second step will be the optimization of the heater in order to create better temperature uniformity within the system. this can be done by using different heater geometries [26, 37] or using double-sided heating [28] . the third step that has to be optimized in the sample collection and work-up procedure. one has to think of what kind of samples to collect in order to have the biggest chance of having the agent of the disease present in that sample (i.e., blood, mucus, saliva, etc.). such crude samples contain full cells, with the dna present within. there are different approaches to perform cell lysis in order to extract the dna [78] . various components of bodily fluids, and reagents and products of the lysis are well-known to inhibit the amplification reaction [79] . however, mda [80] and hda [9] could also be performed on crude samples. a fourth step in the near-future is the development of a first prototype with all hardware integrated in a single piece of equipment. such a device in its pure essence will consist of a battery to power the heater and detection, a chip holder to firmly keep the chip in its place, a thermocouple for real-time monitoring of the temperature, and a led light and a photodiode. the lamp and photodiode could both be used for fluorescence measurements and turbidity measurements. the authors declare no conflict of interest. the following abbreviations are used in this manuscript: figure a1 . technical drawing of the dna amplification chip. all dimensions are in mm. the total chip size is 3 mm by 3 mm. values for the density (ρ coc = 1020 kg m −3 ), specific heat (c coc , see table a2 ), and thermal conductivity (κ coc,23 • c = 0.17 w m −1 k −1 and κ coc,320 • c = 0.24 w m −1 k −1 , linear fit in between these point) of topas 6017 coc are obtained via topas advanced polymers (topas advanced polymers, farmington hills, mi, usa). the heat capacity at constant pressure (c p,coc ) is calculated assuming a homogeneous body of mass m, via equation (a5), where comsol interpolated linearly in between the points. convective heat loss to the air is also taken into account with equation (a6), which is often used in simulations [56] . h = 10 w m −2 k −1 (a6) the two subsections below show two effects on the tcr measurements, i.e., the effect of thermal annealing during the first temperature cycle and the effect of aging. the graph below is an example of the measured thermal annealing of a metal layer on coc. here, only the graph for evaporated au is shown, but all except evaporated pt show this behavior. evaporated pt had some contaminants in the metal track. this is most probably caused by the shadow mask. the layer analyzed in figure a2 is stored for two weeks in ambient conditions and the tcr is analyzed again. from the graph it is visible that the aging has some effect on the tcr of the metal layer. as deposited two weeks old legend figure a3 . the effect of two weeks aging in ambient conditions on a 100 nm au layer. the tcr of the as deposited layer is 0.001 61 k −1 and the tcr of the two week old layer is 0.002 24 k −1 . figure a4 . the background signal of the mixture in table a4 . zoonotic diseases and phytochemical medicines for microbial infection in veterinary science: current state and future perspective review: the important bacterial zoonoses in "one health" concept. front. public health et al bacterial zoonoses transmitted by household pets: state-of-the-art and future perspectives for targeted research and policy actions a comprehensive review of common bacterial, parasitic and viral zoonoses at the human-anmial interface in egypt extended-spectrum β-lactamase-producing and ampc-producing escherichia coli from livestock and companion animals, and their putative impact on public health: a global perspective early diagnosis of sars coronavirus infection by real time rt-pcr epidemic q fever in humans in the netherlands enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia helicase-dependent isothermal dna amplification helicase-dependent amplification of nucleic acids loop-mediated isothermal amplification of dna portable nucleic acid thermocyclers lamp-on-a-chip: revising microfluidic platforms for loop-mediated dna amplification microfluidic devices for forensic dna analysis: a review microfluidic dna amplification-a review extraction, amplification and detection of dna in microfluidic chip-based assays miniaturized isothermal nucleic acid amplification, a review forensic dna analysis on microfluidic devices: a review flow-through polymerase chain reactions in chip thermocyclers miniaturized nucleic acid amplification systems for rapid and point-of-care diagnostics: a review isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review three-dimensional on-chip continuous-flow polymerase chain reaction employing a single heater recent progress in lab-on-a-chip technology and its potential application to clinical diagnoses exploring the limits of ultrafast polymerase chain reaction using liquid for thermal heat exchange: a proof of principle highly-integrated lab-on-chip system for point-of-care multiparameter analysis thechnological journey towards reliable microheater development for mems gas sensors: a review thermal modeling and characterization of a thin-film heater on glass substrate for lab-on-chip applications comparison of different metal film thicknesses of cyclic olefin copolymer-substrate polymerase chain-reaction chips with single-side and double-side heaters experimental study of heat-treated thin film ti/pt heater and temperature sensor properties on a si microfluidic platform all-plastic, low-power, disposable, continuous-flow pcr chip with integrated microheaters for rapid dna amplification thermal management in microfluidics using micro-peltier junctions fast thermo-pneumatic actuation of a thin pdms membrane using a micro peltier-element for microfluidic applications bulk-micromachined submicroliter-volume pcr chip with very rapid thermal response and low power consumption a micro circulating pcr chip using a suction-type membrane for fluidic transport thermal modeling and design analysis of a continuous flow microfluidic chip on-chip real-time monitoring of multiple displacement amplification of dna a comprehensive methodology for design and development of an integrated microheater for on-chip dna amplification loop-mediated isothermal amplification integrated on microfluidic chips for point-of-care quantitative detection of pathogens soft lithography comparing microfluidic performance of three-dimensional (3d) printing platforms micromilling: a method for ultra-rapid prototyping of plastic microfulidic devices cyclic olefin copolymer (coc) an integrated disposable device for dna extraction and helicase dependent amplification microfluidic devices for presumptive forensic tests cyclic olefin copolymer microfluidic devices for forensic applications surface modification of cycloolefinic copolymers for optimization of the adhesion to metals organic pre-etch treatment for metal plating of cyclic olefin polymers. u.s. patent ush1807h cyclic olefin copolymer plasma millireactors instrument-free nucleic acid amplification assays for global health settings multiple displacement amplification to create a long-lasting source of dna for genetic studies whole genome amplification in preimplantation genetic diagnosis the future is now: single-cell genomics of bacteria and archaea sequence quality is maintained after multiple displacement amplification of non-invasively obtained macaque semen dna a review of heating and temperature control in microfluidic systems: techniques and applications a versatile technology platform for microfluidic handling systems, part i: fabrication and functionalization modeling natural and forced convection in comsol multiphysics electromigration in thin gold films power regulation and electromigration in platinum microwires reduction of surface roughness for optical quality microfluidic devices in pmma and coc improved adhesion of thin conformal organic films to metal surfaces tough bonding of metallic layers to hydrocarbon surfaces by depositing ag films characterization of evagreen and the implication of its physicochemical properties for qpcr applications temperature coefficients of resistance of metallic films in the temperature range 25 • c to 600 • c electrical and structural properties of thin gold films obtained by vacuum evaporation and sputtering characterization of evaporated and sputtered thin au layers on poly(ethylene terephtalate) handbook of modern sensors: physics, designs, and applications thermal and physical properties of pure metals longitudinal and transverse strain sensitivity of gold film thin gold film strain gauges structural engineering of gold thin films with channel cracks for ultrasensitive strain sensing diapragm-type sputtered platinum thin film strain gauge pressure transducer stress relaxation study of sputtered platinum thin films at near room temperature using an ultrasensitive strain gauge simultaneous measurement of strain and temperature with two resistive strain gauges made from different materials better aluminium mirrors by integrating plasma pretreatment, sputtering, and plasma polymerization for large-scale car headlight production detection of loop-mediated isothermal amplification reation by turbidity derived from magnesium pyrophosphate formation rapid cycle dna amplification: time and temperature optimization a review on macroscale and microscale cell lysis methods inhibition and facilitation of nucleic acid amplification comprehensive human genome amplification using multiple displacement amplification water-heat capacity (specific heat) this article is an open access article distributed under the terms and conditions of the creative commons attribution the reaction mixture for the on-chip mda reaction consisted of the following. all fluorescence measurements in the horiba scientific fluoromax+ spectrofluorometer are normalized by subtracting the background signal. this background signal is measured using the mixture in table a4 . key: cord-270082-byxd4o4m authors: doheny, kimberly floy; sorger, peter k.; hyman, anthony a.; tugendreich, stuart; spencer, forrest; hieter, philip title: identification of essential components of the s. cerevisiae kinetochore date: 1993-05-21 journal: cell doi: 10.1016/0092-8674(93)90255-o sha: doc_id: 270082 cord_uid: byxd4o4m abstract we have designed and utilized two in vivo assays of kinetochore integrity in s. cerevisiae. one assay detects relaxation of a transcription block formed at centromeres; the other detects an increase in the mitotic stability of a dicentric test chromosome. ctf13-30 and ctf14-42 were identified as putative kinetochore mutants by both assays. ctf14 is identical to ndc10 cbf2 , a recently identified essential gene that encodes a 110 kd kinetochore component. ctf13 is an essential gene that encodes a predicted 478 amino acid protein with no homology to known proteins. ctf13 mutants missegregate chromosomes at permissive temperature and transiently arrest at nonpermissive temperature as large-budded cells with a g2 dna content and a short spindle. antibodies recognizing epitope-tagged ctf13 protein decrease the electrophoretic mobility of a cen dna-protein complex formed in vitro. together, the genetic and biochemical data indicate that ctf13 is an essential kinetochore protein. the term chromosome cycle describes a fundamental aspect of the cell division cycle in which each of the chromosomal dna molecules first is replicated and then undergoes a series of morphological changes and complex movements to ensure its faithful distribution at mitosis. the gene products responsible for execution of the chromosome cycle include structural components, such as those that assemble into the kinetochore, and regulatory components, such as those that establish checkpoints monitoring the proper completion of ordered events within the cell cycle. saccharomyces cerevisiae offers two major advantages as an experimental organism in which to study the chromosome cycle. first, it is possible to combine classical §present address: european molecular biology laboratory, meyerhoff strasse 1, heidelberg 6900-de, federal republic of germany. lipresent address: center for medical genetics, department of medicine, johns hopkins school of medicine, baltimore, maryland 21205. genetics (isolation and phenotypic analysis of mutants) with recombinant genetics (manipulation of cloned dna segments by recombinant dna methods and subsequent reintroduction into the yeast host). second, all of the cisacting dna sequence elements required for chromosome maintenance are cloned and well characterized, including functional centromere dna, chromosomal origins of dna replication, and telomere dna (reviewed by newlon, 1988) . one structure clearly essential to chromosome distribution is the kinetochore (centromere dna and associated proteins), providing the site of attachment of spindle microtubules. the kinetochore is a relatively simple structure in s. cerevisiae in comparison with the large and complex trilaminar structures seen in higher eukaryotes (rieder, 1982; pluta et al., 1990) . in electron microscopic studies of s. cerevisiae chromosomes, one microtubule is seen to interact with each chromatin molecule, but astructurally differentiated kinetochore is not visible (peterson and ris, 1976) . the kinetochore of s. cerevisiae is composed of an approximately 160-220 bp nuclease-resistant core that is centered around the centromere (cfn dna) sequence and flanked by an ordered array of nucleosomes (bloom et al., 1964; funk et al., 1989) . the cen dna sequence requirements for s. cerevisiae have been rigorously and extensively characterized through mutational analysis (reviewed by carbon and clarke, 1990) . approximately 125 bp is sufficient for centromere function (cottarel et al., 1989) . comparison of centromeres from different chromosomes reveals that they consist of three centromere dna sequence elements (cdei, cdeii, and cdeiii) (fitzgerald-hayes et al., 1962; hieter et al., 1985) . cdei (8 bp) and cdeiii (25 bp) exhibit dyad symmetry and are separated by cdeii, a 76-86 bp sequence of over 90% at content. deletions of cdei and cdell reveal that they are important but not essential for chromosome segregation, while single-nucleotide point mutations in cdeiii can completely destroy centromere function. although a great deal is known about cen dna sequence determinants in s. cerevisiae, little is known about the proteins required for kinetochore activity or its regulation within the cell cycle. biochemical purification of kinetochore proteins through sequence-specific affinity purification with cen dna (ng and carbon, 1967; lechner and carbon, 1991) has proven to be difficult, apparently owing to the low abundance of the kinetochore proteins and the requirement of accessory factors for binding in vitro. to date, only one cen dna-binding protein, cpfl (also known as cpl or cbfl), has been extensively characterized (baker and masison, 1990; mellor et al., 1990; cai and davis, 1990) . cpfl is a member of the helix-loophelix family of dna-binding proteins and binds as a homodimer to cdei. a null mutation in cpfl results in only a io-fold decrease in chromosome segregation, indicating that it is important but not essential for kinetochore function. lechner and carbon (1991) have described the isolathe amino-terminal actin orf is represented by the stippled boxes, separated by a line representing the actin intron. the in-frame lacz coding sequence is shown as a hatched box. cen dna is indicated by open boxes, with roman numerals i, ii, and iii indicating cdei, cdeii, and cdeiii, respectively. transcription initiation from gal70 is indicated by the solid arrow, and the length and number of transcripts by the length and width of the dashed arrow. (a) control experiments (cen dna mutation in cis). cen dna transcriptional blocks (wild-type and cdeiii-1% mutant) were tested in a wild-type strain. q-galactosidase activity levels were normalized to 100% with a strain containing the reporter with no cen transcriptional block (top). (6) proposed relaxation of the wild-type cen transcriptional block due to mutation of a kinetochore protein component. perier and carbon (1992) recently described a reporter with cen dna within the gal7 promoter. this situation presumably sets up a competition for binding between kinetochore proteins and transcription initiation factors. the reporter used here is different in that it assays for the relief of a transcriptional block caused by a cen placed downstream of the gal10 promoter. tion of a multicomponent protein-cen dna complex, cbf3, which is defined as an in vitro activity that can bind cen dna sequences in a cdeiii sequence-specific manner. three major protein species of 110 kd, 84 kd, and 58 kd apparent molecular weight are present in approximately equimolaramounts in the most highly purified preparations, although many substoichiometric species are also present (lechner and carbon, 1991) . the cbf3 preparation has recently been shown to exhibit a minus end mechanochemical motor activity in vitro, observed as translocation of a latex bead covalently attached to cen dna along polymerized microtubules (hyman et al., 1992) . classical genetic approaches have also been undertaken to identify s. cerevisiae genes required for chromosome transmission, some of which are expected to encode kinetochore components. several mutant collections have been isolated with the primary criterion of chromosome missegregation, including the ctf(chromosome transmis-sion fidelity; spencer et al., 1990) , ch/(chromosome loss; kouprina et al., 1993), tin (chromosome instability; hoyt et al., 1990) , mcm (minichromosome maintenance; maine et al., 1984) , and m/f (mitotic fidelity; meek+wagner et al., 1988) mutants. these mutants could have defects in any of the many components necessary for the chromosome cycle to proceed with high fidelity. secondary criteria can be applied to identify those mutants defective in a particular structure or process. for example, sensitivity to benomyl (a microtubule-destabilizing drug) was used as a secondary screen for the tin collection to identify mutants involved in microtubule function. this recently resulted in the identification of cln8 and klpl (cin9) (hoyt et al., 1992; saunders and hoyt, 1992; roof et al., 1992) two kinesin-related proteins that are involved in mitotic spindle function. we have designed two secondary screens in order to identify kinetochore mutants. one assay monitors transcriptional readthrough of a centromere, and the other monitors the stability of a test dicentric chromosome. these in vivo assays of the integrity of a test kinetochore were used to screen the cff mutant collection. the cff collection consists of 138 independent mutants that exhibit increased loss of a nonessential chromosome. this collection represents approximately 50 genes whose products are required for high fidelity chromosome transmission in the mitotic cell cycle (spencer et al., 1990) . two mutations, cff13-30 and ctf14-42, tested positive in both secondary screens. we found that ctf14 was identical to ndcloi cbf2, recently shown to encode a 110 kd kinetochore protein (goh and kilmartin, 1993; jiang et al., 1993) . through a combination of genetic and biochemical approaches, we have shown that ctf13 is a previously unidentified essential protein that is a component of the s. cerevisiae kinetochore. readthrough assay and secondary screen of the ctf collection when transcription from a strong promoter is initiated toward a cen dna sequence, the mitotic segregational function of the centromere is destroyed (hill and bloom, 1987) without disruption of its 180-220 bp nuclease protected region (bloom et al., 1984; hill and bloom, 1987) , indicating that at least some of the kinetochore complex remains intact. furthermore, it has been observed that the majority of transcripts terminate at the border of the cen sequence (p. phillipsen, personal communication). these observations suggest that the cen dna-protein complex is responsible for this transcriptional block. in the reporter plasmid used to test this hypothesis (figure l) , the gal70 promoter initiates transcription of an actin-laczfusion gene. a wild-type ceng(185 bp) inserted in the actin intron allowed only 1% of the j3-galactosidase levels seen when no cen was present ( figure 1 ). the structurally dicentric reporter plasmid was maintained in a functionally monocentric state by keeping transformed strains on medium containing galactose. transcription initiated from the gal70 promoter inactivates the segrega tional function of the test cen (hill and bloom, 1987) . to test the hypothesis that a cfn dna-protein complex was responsible for the transcriptional block, we replaced the wild-type ceng sequence with a cen6 sequence containing a single-nucleotide point mutation (cdeiii-15c) in the central element of the palindrome of cdeiii (ccg). this transversion from g to c causes a 250-fold increase in the rate of mitotic missegregation of a chromosome fragment (hegemann et al., 1988; jehn et al., 1991) . similar central element mutations have been shown by in vivo footprinting to result in decreased methylation protection of cen dna (densmore et al., 1991) . the cdeiii-15c cen mutant inserted in the actin intron allowed approximately 20% of the 8-galactosidase levels seen when no cfn was present ( figure 1 ). thus, a cen dna mutation affecting kinetochore integrity caused an increase in transcriptional readthrough that was detectable by increased levels of 8-galactosidase activity. this increase in @galactosidase activity could also be detected as blue colony color when strains were grown on solid medium containing x-gal (see experimental procedures), providing a sensitive visual assay for rapid screening of the ctf collection. we proposed that the transcriptional block provided by the full-length wild-type cfn6 might be relaxed in cff strains with mutant kinetochore proteins. the reporter minichromosomes used in the control experiments would very likely be present in highly variable copy number in these ctf strains, owing to increased rates of nondisjunction and/or loss. this could result in the appearance of false positives and false negatives. to maintain the reporter in single copy, we integrated it into the cff strains. two independent transformants of each cff strain, containing an integrated wild-type cen reporter, were plated on medium containing x-gal and monitored for the appearance of blue colony color (see experimental procedures). of 34 cff mutants screened (see experimental procedures for list), 7 were identified as putative kinetochore mutants because they produced an intermediate level of blue colony color between the levels of the ctf+ strains carrying the wild-type and mutant cen reporters. five of these mutant strains (designated "s" followed by an isolate number) are members of complementation groups, ~10 (ctf7) s9 (ctf8), s30 (ctfl3), ~42 (ctfl4), and s61 (ctfl7) and two contain independent mutations, ~26 and s58 (table 1 ). quantitative measurement of p-galactosidase activity levels in protein extracts from these strains verify the identifi-cation of a relaxed transcriptional block by the colony color assay (table 2) . dicentric chromosome stabilization assay and secondary screen the second assay we developed to screen for kinetochore mutants among the cffcollection is based upon the behavior of dicentric chromosomes as they undergo mitotic segregation. if a chromosome has two centromeres, kinetochores on the same chromatid may become attached to opposite poles of the mitotic spindle ( figure 2a ). when this occurs, the dna molecule usually breaks, and the dicentric chromosome is rapidly lost or is rearranged to a stable form (mann and davis, 1983; haber and thorburn, 1984) . a kinetochore mutant might assemble kinetochores that have a weakened attachment of chromosomal dna to microtubules. this could lead to microtubule detachment before chromatid breakage ( figure 28 ) resulting in stabilization of the dicentric chromosome. the artificial chromosome fragment present in the c?f strains was an appropriate substrate for the construction of a dicentric test chromosome. the chromosome fragment, a nonessential disome, possesses all the sequences required for proper chromosome segregation. its stability can be visually monitored by the degree of colony color sectoring (see figure 38 ) (spencer et al., 1990; shero et al., 1991) , and selective pressure for rearrangement to a stable form is absent because the chromosome fragment is not essential for viability. the gal&en constructs developed in the transcriptional readthrough assay allow control of the mitotic activity of a centromere by the choice of carbon source in the medium. we constructed a vector that would direct integration of these test conditional centromeres to the /eubd 7 locus present approximately 23 kb from the centromere on the chromosome fragment (see experimental procedures). in control experiments, we examined the stability of dicentric chromosome fragments containing either a nearly wild-type (acdei) or a highly defective (cdeiii-15c) secondary conditional cfn ( figures 3a and 38 ). we predicted that upon activation assays were performed on strains grown at 30%. cent, wild-type ceng. b s16(ctf9) was not identified as a putative kinetochore mutant by the transcriptional readthrough assay and serves as a negative control. d s42 (ctf14) is inviable at 30%. in (b), the arrowhead indicates release of the microtubule attachment to the chromosome, allowing the dicentric chromatid to proceed intact to the spindle pole. the hypothesis that a kinetochore mutant might result in thestabilization of a linear dicentric chromosome is based on a previous study of the behavior of dicentric minichromosomes (koshland et al., 1987) . a circular minichromosome carrying a single wild-type centromere is quite stable in s. cerevisiae (maintained in 98%-990/o of the population under selection), whereas a minichromosome with two wild-type centromeres is highly unstable (maintained in only 6% of the population). however, when two identical partially defective centromeres (which by themselves allowed maintenance of minichromosomes in 91% of the population) were placed on the same minichromosome, the plasmid was not destabilized to the same degree (maintained in 49% of the population). in this case, defective kinetochore function was due to mutation of the cea! dna sequence. by analogy, it is possible that kinetochore dysfunction due to a defective or aberrant protein will also result in stabilization of a test dicentric chromosome. of the secondary cen, the dicentric chromosome fragment containing a strong secondary cen would be highly unstable, resulting in a frequent sectoring phenotype, and the dicentric chromosome fragment containing one wild-type and one defective cfnwould be relatively stable, resulting in fewer sectors per colony ( figure 3b ). the actual sectoring phenotypes that resulted, shown in figure 4 , were consistent with our hypothesis, indicating that the dicentric stability assay was a feasible screen for kinetochore mutants among the cff collection ( figure 3c ). to screen the cff mutants for stabilization of a dicentric chromosome, the conditional nearly wild-type cen, acdei, was integrated into the chromosome fragment present in each strain. the strains were maintained on galactose to induce the gal.70 promoter and inactivate the conditional cen (see experimental procedures and figure 3a ). two transformants of each cff strain were streaked onto medium containing dextrose to activate the dicentric state by repressing transcription from the gal70 promoter. the stability of the dicentric chromosome fragment in the crf strains was visually assessed and compared with the stability of the dicentric chromosome frag-ment in the ctf+ strain. if the dicentric chromosome fragment was as unstable as in the wild-type background ( figure 3b ), the cti strain was scored negative in this assay. with 27 cff mutants tested (see experimental procedures for list), 2 exhibited a reduction in sectoring frequency relative to the wild-type control and were thus identified as putative kinetochore mutants: ~30 (ctfl3), and ~42 (ctfl4) (see table 2 ). the sectoring phenotypes exhibited by ~30 (ctfl3) and a representative mutant that was scored negative, ~16 (ctfg), are shown in figure 4 . the sectoring phenotype of ~42 (ctfl4) carrying the dicentric chromosome fragment is similar to that seen for ~30 (ctfl3). the sectoring phenotypes of these mutants are similar to that seen with a test dicentric chromosome carrying a weak secondary cen. two &strains, ~30 (ctfl3) and ~42 (ctfl4), were scored as putative kinetochore mutants in both secondary screens. the ctf74-42 mutation identifies a recently characterized kinetochore component, ndcloicbf2 (see below). we therefore explored further whether the cff13-30 and active on dextrose medium (the test chromosome behaves as a dicentric). (b) the stability of the test dicentric chromosome fragment can be monitored visually. a trna suppressor gene (sup1 1) present on the chromosome fragment partially suppresses the accumulation of red pigment caused by the ade2-101 ochre mutation in our strain backgrounds. if the chromosome fragment is present, the strain is white; if it is lost, the strain is red. thus, the numberof red sectors that develop during the growth of a colony founded by a haploid cell containing the chromosome fragment (white) is indicative of the rate of loss of the chromosome fragment in the strain. the lines within the circles represent the presence of such red sectors in a white colony. the sectoring phenotypes pictured are those predicted and observed (see figure 4 ) when nearly wild-type (cen*v) and highly defective (cen-) cen the centromere originally present on the chromosome fragment is fully wild type (cenw). labels at the left indicate the type and number of cen dnas present on the test chromosome fragment. labels across the top indicate the relevant genotype of the pictured strain. sl6 (ctfg) was scored negative; its sectoring phenotype with the test dicentric chromosome fragment (column 2, row 2) was the same as that seen in the ctf+ background (column 1, row 2). s3g (ctfl3) was scored positive; its sectoring phenotype with the test dicentric chromosome fragment (column 3, row 2) was not as severe as that seen in the ctf+ background (column 1, row 2) and looked similar to that seen with the test dicentric chromosome carrying the weak secondary cen (column 1, row 3). mutation had identified a gene encoding a kinetochore component. molecular cloning cff73-30 is completely deficient for growth at 37%, and this temperature sensitivity was shown to cosegregate with its moderate sectoring phenotype at 25%. ctf73 was cloned by complementation of lethality at 37% (spencer et al., 1988) . a 2.2 kb sau3a fragment that complemented the temperature sensitivity of ctf73-30 was shown to correspond to ctf73 by the directing of an integration event in a heterozygous diploid. this event introduced a prototrophic marker at the genomic site of the cloned dna segment and deleted approximately half of the 2.2 kb genomic sequence (almost the entire ctf73 open reading frame [orf]; see experimental procedures and figure 58 ). when the diploid transformants were dissected, it was found that viability segregated 2:2 (see experimental procedures). we concluded that the cloned dna encodes wild-type ctf73 and that ctf73 is an essential gene in s. cerevisiae. ctf 73 was localized to the right arm of chromosome xiii using both physical and genetic mapping methods ( figure 5a ). from the mapping data, we concluded that ctf73 is a previously unidentified gene in s. cerevisiae. the nucleotide sequence of the 2.2 kb ctf73 clone contains a 1.4 kb orf that encodes for a protein of 478 amino acids with a predicted molecular weight of 58 kd ( figures 5b and 8 ). the ctf73 protein shows no significant overall homology at the amino acid level to entries in gen-bank, genpept, gpupdate, swissprot, pir, embl, and emblupdate data bases as of january 1993. the homology searches were performed on the national center for biotechnology information blast network (altschul et al., 1990) . the ctf7 3 protein contains a short acidic serinerich region (amino acids 200-230) that is approximately 40% identical to the first acidic block found in a mammalian centromere-associated protein, cenp-b (pluta et al., 1992) . the significance of this small region of similarity is unclear, and there are no other significant homologies found outside this area. interestingly, there is a possible cdc28 phosphorylation site in the ctf73 protein (sspss, amino acids 224-228) (figure 8 ). analysis lechner and carbon (1991) have described a multiprotein complex (cbf3) present in nuclear extracts of s. cerevisiae cells that binds in vitro to a 350 bp fragment of cen dna. dna footprinting reveals that cbf3 interacts with the cdeiii sequence element. using a modification of the methods of lechner and carbon (1991) we were able to detect the binding of cbf3 complexes present in wholecell extracts to an 88 bp dna probe that spans cdeiii but lacks cdei and cdeii. to determine whether ctf13 is a component of the cdeiii-binding complex, the ctf13 orf was fused to peptide epitopes against which antibodies had been preby southern hybridization to an etectrophoretic karyotype (spencer et al., 1966; gerring et al., 1990a) . ctf73 was positionally mapped by the method of chromosome fragmentation (gerring et al., 199oa) and was localized to the right arm of chromosome xiii, 475 kb from the right arm telomere and 445 kb from the left arm telomere (see experimental procedures). this physical location was verified by using the 2.2 kb saum fragment to probe a set of filters containing contiguous overlapping 1 clones that cover 66% of the yeast genome (l. riles and m. olson, unpublished data). ctf73 was localized to overlapping clones 4199 and 6643, placing it on an -15 kb segment of dna located on the right arm of chromosome xiii between adh3 and i/v2 the temperature-sensitive cff7530 mutation was meiotically mapped and found to be located 34 cm proximal to the cin4 locus, which agrees well with the physical mapping data (see of ctf13 (see experimental procedures). in the first construct, an 11 amino acid epitope derived from the ha1 protein of influenza virus (field et al., 1988) was inserted in frame into the amino terminus of ctf13 ( figure 5c ). in the second construct, two tandem copies of the el epitope (pluta et al., 1992) , derived from the carboxy-terminal 25 amino acids of an avian coronavirus glycoprotein (machamer and rose, 1987) were placed in frame at the amino terminus of the ctf13 orf under the transcriptional control of the gal7 promoter ( figure 5c ). both epitope-tagged ctf13 derivatives were able to rescue viability in a ctfl3 al::his3 null strain. extracts were prepared from cells carrying either wildtype or epitope-tagged ctf13, reacted with 32p-labeled cdeiii dna, and complexes were resolved on a nondenaturing gel (figure 7) . a single band corresponding to a cdeiii-protein complex was observed (figure 7 , lanes 1, 7, and 10); no complex was observed with a nonfunctional cdeiii variant (data not shown). the addition of antiepitope antibodies to binding reactions containing extracts from cti73 mutant strains rescued by the respective ctfl3-epitope fusion protein resulted in the appearance of a complex with significantly decreased electrophoretic mobility (figure 7, lanes 2-4 and 11 ). this supershift is dna-protein complexes formed with yp-labeled cdeiii probe and whole-cell extracts were analyzed on a nondenaturing acrylamide gel. antibodies were added to preformed complexes, and samples were incubated for 20 min at room temperature before gel analysis. unbound probe was run off the bottom of the gel. lanes 1-6, extracts from cff73d7::h/s3 null cells carrying an ha epitope-ctf13 fusion (see figure 5c ) and incubated with antibodies at various dilutions; lanes 7-9, extracts from cff13dl::h/s3 null cells carrying a ctf13 plasmid reacted with the indicated antibody (controls for lanes l-6); lanes 10-12, extracts from ctf73-30 cells carrying an el epitope-ctf13 fusion (see figure 5c ) and incubated with indicated antibodies (control is lane 6). ha indicates the addition of 12ca5 monoclonal antibody, which is directed against the ha epitope; el indicates the addition of a polyclonal serum directed against the el epitope; peptide indicates the addition of ha peptide to 1 mm prior to the addition of 12ca5 antibody. clearly antibody specific, because antibodies directed against the el epitope did not recognize the ha-ctf13 fusion protein (figure 7, compare lanes 6 and 4) and antibodies directed against the ha epitope did not recognize the el -ctf13 fusion protein (figure 7 , compare lanes 12 and 11). as expected, the supershift was also shown to require the presence of el-ctf13 (figure 7 , compare lanes 6 and 11) or ha-ctf13 (figure 7 , compare lanes 8 and 9 with lanes 3 and 4) and to be competed with ha peptide (figure 7, compare lanes 5 and 3) . these results show that the supershifted band shown in lanes 2-4 and 11 of figure 7 is composed of a complex containing proteins, dna, and antibody. these results demonstrate that ctf13 is present in the protein complex that binds to the essential cdeiii region of s. cerevisiae cfn dna in vitro. because all of the cdeiiiprotein complex formed in our reactions were able to be supershifted by antibodies directed against epitope-tagged ctf13, the stoichiometry of ctf13 and dna in the complexes must be at least 1 to 1. we conclude from these data that ctf13 is a major component of the yeast kinetochore, which, probably in combination with other proteins, interacts with cdeiii. phenotypic analysis the cff73-30 mutant allowed transcriptional readthrough of a test cen and stabilized a test dicentric chromosome fragment. further phenotypic analysis of this mutant revealed defects consistent with defective kinetochore function. the colony color assay for chromosome fragment stability can be used to monitor the rates of chromosome fragment loss and nondisjunction events in diploids. these rates were measured for a ctf13-30 homozygous diploid and its wild-type parent at permissive temperature (25oc). the cti73-30 homozygous diploid exhibited an approximately 50-fold elevation both in the rates of nondisjunction and in loss of the chromosome fragment (table 3a) . the rates of mitotic missegregation and recombination of a suitably marked endogenous chromosome ill were also measured. the mitotic missegregation rate of chromosome ill was elevated 1 o-fold in the ctf73-30 homozygous diploid, while the mitotic recombination rate was only elevated 4-fold (table 38) . we conclude that the cti73-30 mutation confers mitotic segregation and recombination phenotypes consistent with a role in the segregational machinery. cti73-30 causes cells to arrest at the g2/m phase of the cell cycle when shifted to the nonpermissive temperature. flow cytometric analysis of dna content per cell revealed an accumulation of cells with a g2 dna content during log phase growth at the permissive temperature and a single peak of g2 content dna after arrest at the nonpermissive temperature ( figure 8a ). quantitation of cell and nuclear morphology at the permissive temperature also indicated an accumulation of cells with a g2 content; 13% of cells were large budded with the nucleus at the neck in a cff73-30 background, while only 2% of wild-type cells had this morphology ( figure 8c ). cff73-30 is a cdc-like mutation that arrests with a cell morphology indicative of the g2/m preanaphase portion of the cell cycle. after 3 hr at nonpermissive temperature (38x), approximately 80% of cff73-30 homozygous diploid cells had arrested as large-budded cells with an undivided nucleus positioned at or near the neck between the mother and daughter cells. the mitotic spindle was very short in virtually every cell; a medium or long (anaphase b-like) spindle is never seen (figure 86, upper panels) . the cdc arrest is leaky in cff73-30 at 38oc, and the uniform cell morphology decays with time (see figures 88 and 8c ). the mitotic spindle phenotype also becomes less uniform with the appearance of misaligned and aberrantlooking spindles. interestingly, after 5 hr at the nonpermissive temperature, a "cut"-like phenotype is observed in approximately 10% of the population (though present in only 2% of the population at the 2 hr time point). this morphology is reminiscent of the phenotype of schizosaccharomyces pombe cut mutants (hirano et al., 1986) as well as of the phenotype observed in topoisomerase ii mutants of s. cerevisiae (holm et al., 1985) . we define this cut-like cell morphology as a very narrow-necked, large-budded cell in which the nucleus straddling the neck has a pinched appearance (see figure 8b , lower panel). these data demonstrate that the cff73-30 mutation results in a defect revealed in the g2/m phase of the cell cycle, consistent with a defect in kinetochore function. the ctistrain s42 (ctfl4) was also identified by both secondary screens as a putative kinetochore mutant. a clone that complemented the temperature sensitivity of clf74-42 was obtained and mapped to chromosome vii essentially as described for clf73-30 (data not shown). nuclear division cycle 70 (ndcio), recently identified by goh and kil-cells is slightly tighter (a single g2 peak) when incubated at 36% for 3 hr (data not shown). shown above the columns. the numbers shown represent the percentage of total cells scored; small-budded cells with a single nucleus were quantitated (percentage is 100 minus the sum indicated) but are not shown. at 25°c 1500 cells were scored; 200 cells were scored for each time point at 36%. martin (1993) as an essential gene involved in chromosome segregation in s. cerevisiae, is identical to cbf2, a gene recently identified by jiang et al. (1993) that encodes the 110 kd subunit of the cbf3 complex (lechner and carbon, 1991) . multiple internal restriction fragments from the nix70 clone were found to comigrate with fragments from the cti74-42 complementing clone. moreover, the temperature-sensitive mutation cff74-42 failed to complement the temperature sensitivity of n&70-7. we conclude that the cff74-42 mutation is present at the ndc70/ cbf2 locus. thus, the only two cff mutants identified by both secondary screens as putative kinetochore mutants have now been shown to be defective in essential kinetochore components. although the cen dna sequence elements from budding yeast have been cloned for over 10 years (clarke and carbon, 1980) , identification of the genes encoding proteinsessentialforcentromerefunction hasprovendifficult. we describe a genetic approach using two independent in vivo genetic assays to screen an existing large reference set of mitotic segregation mutants (the cffcollection; spencer et al., 1990) for altered kinetochore integrity. in combination, these assays identified two mutant strains, ~30 (ctf 13) and ~42 (ctfl4), as putative kinetochore mutants. biochemical and further phenotypic analysis indicated that the ctf73 gene product was indeed an essential structural component of the kinetochore, and the ctf74 gene product was shown to be identical to ndc70kbf2, a recently characterized essential kinetochore component (goh and kilmartin, 1993; jiang et al., 1993) . mutants theoretically, the transcriptional readthrough assay might result in both false negatives (e.g., kinetochore protein mutations that fail to relieve a transcription block) and false positives(e.g., mutationsthat affect transcriptional regulation or chromatin structure). similarly, a dicentric chromosome could be stabilized by mutants affecting dna metabolism or spindle integrity and assembly. in light of these caveats, we used these assays to screen a set of mutants previously shown to have defects in mitotic chromosome segregation. it is not known how efficient either of these secondary assays would be in a primary screen. our experience suggests that kinetochore mutants can be recognized by the combined phenotype of transcriptional readthrough and dicentric chromosome stabilization. in theory, the degree to which the integrity of the kinetochore must be compromised to result in either of these phenotypes could be quite different. in a simplified view, mutations affecting the interaction of the kinetochore complex with the cen dna should be detected by both assays, while mutations affecting kinetochore to microtubule interactions may only be detected by the dicentric stabilization assay. however, we note that kinetochores participate in several distinct processes in vivo, including microtubule capture, congresaion to the metaphase plate, and poleward migration. in addition, a viable kinetochore mutation would most likely be a leaky mutation, which might exhibit complex consequences following the primary defect. thus, in reality, it is quite possible that some of the mutations identified by only one of these secondary screens indeed disrupt kinetochore integrity but perhaps lead to more subtle alterations than cff73-30 or cff74-42. it is clear that these two screens, whether used alone or in combination, have the potential to aid in the identification of additional regulatory and structural components of the s. cerevisiae kinetochore. they may also be adaptable to other organisms. ctf13 is an esserttial kinetochore compomnt we have presented a combination of in vivo and in vitro evidence demonstrating that the ctf13 protein is a cornponent of the s. cerevisiae kinetochore. in vivo, tf& ctf73-30 mutation confers relaxation of a transcriptional mock mediated by the kinetochore and stabilizes a test dioentric chromosome fragment. in vitro, we demonstrate otat the ctf13 protein is a component of the cen dna-@rot&n complex and, specifically, that it interacts withcdelll. the predicted molecular mass of 56 kd for the ctf13 protein is the approximate size seen on a western blot (data not shown). therefore, the ctf13 protein seemed to be a very good candidate for the 58 kd subunit of the cbf3 complex (lechner and carbon, 1991) , and in fact, the predicted amino-terminal amino acid sequence of the ctf13 protein was found to be identical to tryptic peptide sequence obtained from the purified 58 kd protein component of the cbf3 complex (j. lechner, personal communication). the ctf13 protein appears to be limiting for cdeiiiprotein complex formation in vitro. when extracts derived from a strain overproducing ctf13 are used in the band shift assays (see figure 7 , lanes lo-12), the amount of cdeiii-protein complex formed is increased relative to the amount seen with extracts from nonoverproducing cells (see figure 7 , lanes l-9). also, a cti73 heterozygous diploid strain (cti7347::h/%/ctf13) exhibits a mild but detectable sectoring phenotype. this indicates that the amount of ctf13 protein produced by one copy of the ctf73 locus is not sufficient to keep the fidelity of chromosome segregation at a wild-type level. these observations suggest that ctf13 may be limiting for kinetochore function in vivo. phenotypic analysis of the temperature-sensitive cff73-30 mutation is consistent with a kinetochore defect. cti73-30 causes an increase in the mitotic rate of chromosome missegregation and results in a terminal phenotype indicative of a defect in the g2/m phase of the cell cycle. it has been previously proposed that missegregation mutants will fall into two broad groups: those affecting the pathways of dna metabolism and those affecting the mitotic segregational machinery. a mutation affecting dna metabolism was expected to cause increased rates of both chromosome loss and mitotic recombination, while a mitotic segregation mutant was expected to cause only an increase in chromosomal loss events. phenotypic analysis of a known dna metabolic mutant, dna polymerase a (c&77; hartwell and smith, 1985) , and a known spindle mutant, b-tubulin (r&2; huffaker et al., 1988), supported this model. examination of these phenotypes for cff73-30 strains revealed a significant increase in the rate of chromosomal missegregation with only a very slight elevation in the rate of mitotic recombination (table 38 ). in addition, we now have the ability to distinguish between loss (1:o) and nondisjunction (2:0) missegregation events, and we find that the rates of both of these events are significantly elevated in.& ctf73-30 background (table 3a) . thus a known kinetochore mutation, cti73-30, has been shown to result in phenotypes consistent with previous expectation, and we can perhaps extend this expectation to include a predicted increase in the rates of both chromosomal loss and nondisjunction in mitotic segregation mutants. the ctf73.30 kinetochore defect may be recognized by a cell cycle checkpoint critical events in the cell cycle are temporally ordered and coordinated by a series of dependency pathways in which late events are dependent on the successful completion of earlier events. these dependencies can result from a substrate-product mechanism or from extrinsic control by a monitoring function termed cell cycle checkpoint control (hartwell and weinert, 1989) . checkpoints are responsible for a subset of observed cell cycle arrests or delays. cell cycle arrests or delays associated with kinetochore defects have been reported in several systems. in animal cells, a delay in the initiation of anaphase is correlated with the failure of chromosomes to achieve bipolar attachment to the spindle (rieder and alexander, 1989; zirkle, 1970) and a metaphase arrest is observed with kinetochore disruption by injection of anti-centromere antibodies (bernat et al., 1990) . in yeast, one abberant kinetochore on a single chromosome can cause a mitotic delay (spencer and hieter, 1992) . ctf73-30 strains exhibit a g2/m phase accumulation in logarithmic cultures at permissive temperature and a preanaphase arrest morphology at nonpermissive temperature. cell morphology and dna content do not critically distinguish g2 and m phases in yeast. however, at permissive temperature, crf73-30 strains exhibit a detectable increase in hl kinase activity relative to ctf73 controls, and at nonpermissive temperature, hl kinase activity levels in cff73-30 strains are equivalent to nocodazole-arrested strains (data not shown). thus, hl kinase activity measurements suggest that the cti73-30 mutation causes an accumulation in m phase. it is tempting to speculate that this cell cycle alteration is similar to those described above and that these are a result of checkpoint control exerted in the presence of defective kinetochores. checkpoints are defined by two experimental criteria: first, identification of mutations or conditions that allow bypass of an arrest or delay (resulting in the accumulation of errors) and second, an observed error correction when cell cycle delay is reintroduced experimentally. alternatively, defective substrate-product conversion that becomes rate limiting for progress may also result in cell cycle delay. these alternatives have not been distinguished for the delays seen associated with kinetochore defects. conditional mutations in kinetochore proteins will provide important tools for exploring the relationship be-tween kinetochore structure and cell cycle progression. examination of the terminal phenotype of cti73-30 mutants raises several interesting questions. the fact that the ctf73-30 defect does not lead to a permanent and uniform arrest morphology may simply be a result of the presence of a small amount of active ctf13 protein that eventually allows completion of mitosis, or mitosis may never be completed but cytokinesis may still eventually be attempted in some cells. consistent with the latter possibility, we have observed the accumulation of cells with a cut-like phenotype: 10% of all cells exhibit this phenotype after 5 hr at the nonpermissive temperature. bernat et al. (1990) describe a similar cut-like phenotype after injection of mammalian cells with anti-centromere antibodies and propose that it is a result of the cells' eventual attempt to undergo cytokinesis after prolonged mitotic arrest. because it is not known whether this subset of the cff73-30 population is still capable of dividing, it is unclear whether cytokinesis has trapped nuclei in these cells or whether they are caught undergoing nuclear transits at the time of fixation (palmer et al., 1989) . the terminal phenotype of ~7773-30 is quite different from the terminal phenotype of the other described temperature-sensitive kinetochore mutant, n&70-7 (goh and kilmartin, 1993). n&70-7 mutants exhibit detatchment of the chromosomes from one spindle pole and progression through the cell cycle in the absence of chromosome segregation (most cells produce one aploid daughter and one daughter of increased ploidy). if there is checkpoint control exerted in response to events at the kinetochore, the ndclo-7 defect is not recognized. perhaps this is because the ndc70 protein itself is involved in the recognition and/ or signaling of a dysfunctional complex, or alternatively, checkpoint control may be disabled by complete disruption of kinetochore structure. future experiments addressing the relationships of kinetochore proteins to the control of progression through mitosis should help define important molecular determinants of the temporal order of events in chromosome segregation. will the molecular dissection of the s. cerevisiae kinetochore aid the understanding of kinetochore function in more complex eukaryotes? at this time, analysis of the dna sequence and protein component requirements of the kinetochore is significantly more advanced in s. cerevisiae than in any other eukaryotic organism, although there is great speculation about the relevance of these studies to the understanding of the much larger and morphologically more elaborate kinetochores present in other eukaryotes. while there may be a need for additional components to ensure fidelity in more complex eukaryotes, we think it is probable that the basic mechanisms of the segregational process, including those involved in centromere function, will have been conserved through evolution. a repeat subunit model for the centromere-kinetochore complex has recently been proposed by zinkowski et al. (1991) . this model describes the kinetochore as organized in multiple small repeat units that fold together into a contiguous plate-like structure when condensed at metaphase. zinkowski et al. propose that each unit is capable of microtubule binding and segregational function. in this context, the s. cerevisiae kinetochore, which binds a single microtubule, could represent the simplest ancestral unit of the eukaryotic kinetochore (fitzgerald-hayes et al., 1982; koshland et al., 1987) . identification and characterization of the s. cerevisiae kinetochore components will facilitate the definition of the activities necessary for the completion of proper mitotic segregation in this organism and may well provide substrates for the identification of kinetochore components in other eukaryotic organisms. yeast strains and media the c/f and wild-type parental strains containing chromosome fragments that can be monitored by a visual assay have been previously described (spencer et al., 1990; shero et al., 1991) . the et/collection of 136 originally isolated mutants can be represented in 18 complementation groups and 41 single isolates. all cff mutant isolates that are members of a complementation group retain the original isolate number as an allele number (e.g., 930 contains cff/3-30). one member of each complementation group (the isolate with the most severe sectoring phenotype) and 19 single isolates (those that were his3-) were tested in the two kinetochore screens. media for yeast growth and sporulation were as described (rose et al., 1990 ) except that where sectoring was examined, adenine was added to 6 us/ml to minimal (sd) medium to enhance the development of red pigment in ada2-101 strains. x-gal plates were made as described for synthetic complete (sc) medium (rose et al., 1990) except for the addition of 0.1 m napol(ph 6.6) and 40 pg/ml x-gal (5-bromo4chloro-5indolyl-6-d-galactopyranoside) (use a 20 mg/ml stock in dimethylformamide). all yeast transformations were done by the method of ito et al. (1963) . readthrough assay the reporter construct schematically pictured in figure 1 was modified from pgab (u. vijayraghavan and j. abelson, unpublished data) obtained from r. parker. (pgab is a modification of pyahb2 [vijayraghavan et al., 19661, a cen-arsplasmid containing an actin-/acz fusion gene that has been used extensively to study splicing in s. cerevisiae [vijayraghavan et al., 1966; cellini et al., 19861 mann et al., 1988) . the dna sequence and orientation of the test cen6 sequences in pkf16 were.verified by sequencing (sanger et al., 1977; hattori and sakaki, 1966) ,the resulting plasmids, pkf19 and pkf44, contain wild-type and mutant (cdeiii-15c) cen8 sequences, respectively, in the prientatipn placing cdei 5'to cdeiii. in control experiments, pkfi 6, pkf19, and pkf44 were transformed into the wild-type strain yph102 tiai$ ura3-52 /ys2-801 ada2-101 his3-a200 /eu2-a 1, and p-galactosidase assays were done on independent ura+ transformants (see figure 1 ). the structurally dicentric plasmids (all ycpbo derivatives) were maintained in a functionally monocentric state by keeping transformed strains on medium containing galactose as the carbon source causing transcriptional inactivation of the test centromeres (hill and bloom, 1967) . 6galactosidase assays were performed essentially as described (rose et al., 1990) following the protocol for assay of crude extracts, except that cells were grown in scgal-his liquid medium or scraped off scgal-his plates. the values for optical density at 420 nm were zeroed to an isogenic yeast strain that did not contain a reporter plasmid. for screening of the ctfcollection, the reporter was integrated into chromosome xv as follows: the gal&io-actin-test ceng-/acz fragment described above was inserted into a genomic xhol site immediately 3' to the his3 gene contained on a pbr322-based plasmid, psz62-xbal (mccleod et al., 1966) kindly provided by j. broach. the resulting bamhl fragment containing his3 and the test reporter fragment was transformed into yph276 selecting for replacement of the his3a200 locus on chromosme xv. independent his+ transformants were picked and analyzed by southern blotting toverify insertion of the reporter construct into chromosome xv at the his3 locus. pkf71 contains the wild-type cen6 reporter, and pkf72 contains the mutant cdeiii (15c) cen6 reporter inserted into the his3 bamhl fragment. yph977 and yph976 contain the pkf71-and pkf72derived bamhl fragments, respectively, and were maintained in medium containing galactose. strains were tested for the production of blue color on medium containing the chromogenic substrate of 6galactosidase, x-gal. yph977 colonies appear white (this progresses to a very faint blue color after several days), while yph978 colonies develop a deep blue color. the reporter containing the wild-type cen6 (pkf71) was inserted into chromosome xv in each of the cff mutants as follows. each cff strain was made competent for transformation in sc medium containing 2% galactose and transformed with the bamhl fragment of pkf71. transformants were selected, and the colony was purified on scgal-his plates at 25oc. two independent transformants of each cff strain tested were then plated at a low density (-200 two p679 and two pkf77 transformants of each cff strain tested were streaked onto synthetic complete dextrose plates containing a limiting amount of adenine. the switch to dextrose as a carbon source causes the gal70 promoter to be turned off, resulting in activation of the second conditional centromere and a functionally dicentric chromosome fragment. sectoring phenotypes were directly compared with those of a yph276 p679 or pkf77 transformant streaked onto the same plate. the &strains tested with the stabilization of a dicentric assay were: 659 (ctf2), ~50 (ctf4), ~31 (ctf5), ~53 (ctf6), ~10 (ctfi'), s9 (ctf6), ~16 (ctf9), ~67 (ctfll), ~16 (ctfl2), s30 (ctf13), ~42 (ctfl4), yph960 mata his3-a200 ade2-101 lys2-801 leu2-al cdl&124 cfvll (radld.yptf 275) ura3 sup1 1, ~61 (ctflir), yph961 mata ~6~3-52 lys2-801 adeb 101 his3-a200 trpl-a 1 leula 1 ctf18-160 cflll (cen3.l. yph278) ura3 supil, s3, s4, s12, s17, ~20, ~22, ~41, s.47, ~55, ~56, ~62, ~63, and ~64. ~31 (ctf5) and ~20 were unscorable in this screen because the chromosome fragment present in the p679 derivative strains was extremely unstable. true positives were verified in multiple independent transformants. one source of false positives was transformants containing two chromosome fragments, only one of which carried the conditional secondary centromere. these false positives were easily identified by the demonstration that sectored colonies were his+. for ~30 (ctft3), the phenotype of stabilization of the test dicentric chromosome was shown to be due to a mutation in the ctf73 gene product by transformation of a pkf76 derivative of ~30 with pkf1 i, a prs3lcbased (sikorski and hieter, 1969) plasmid carrying the ctff3 locus. the presence of the wild-type ctf73 gene product resulted in destabilization of the dicentric chromosome fragment back to the level seen in the wild-type parent, yph276. characterization of ctfl3 the 2.2 kb sau3a subclone that rescues the temperature sensitivity of s30 (ctfl3) was inserted into the polylinker of prs314 (sikorski and hieter, 1969) , resulting in pkf1 i, deletion derivatives of the pkft 1 insert were made using existing restriction sites (see figure 5) . a bglll to polylinker deletion, as well as a clal to polylinker deletion, was unable to rescue the temperature sensitivity of ~30 (ctfl3). the dna sequence encoding the entire orf was determined using a set of unidirectional deletions (henikoff, 1967) by standard methods (sanger et at., 1977; hattori and sakaki, 1966) . the sequence of the second strand of the orf was obtained using synthetic oligonucleotides as primers. the ctf13 clone was shown to correspond to the cff73-30 locus, and ctf73 was shown to be an essential gene in s. cerevisiae by using the ctfl3 clone to direct an integration event (sikorski and hieter, 1969) that replaced a majority of the ctf73 orf with vector and his3 sequences. the integration vector, pkf93, was constructed by inserting the -600 bp bglll (polylinker)-bglll fragment and the -200 bp clal-ecori (polylinker) fragment from pkfl1 (see figure 58 ) into the bamhl site and clal-ecori sites of prs303 (sikorski and hieter, 1969) respectively. pkf93 was linearized with ecorl and transformed into a cff73-30btf13 heterozygous diploid strain, yph974, selecting for his+ transformants. integration of pkf93 should delete the ctfl3 orf from amino acid 57 to amino acid 467 (see figure 58 ). approximately half of the his+ diploid transformants obtained exhibited the cff73-30 sectoring phenotype, indicating that the cff73-30 locus was being targeted by pkf93. the integration of pkf93 and deletion of ctf73 sequences was confirmed by southern analysis (data not shown). two sectoring diploid isolates (ctfl3 locus deleted) and two nonsectoring diploid isolates (cff1530 locus deleted) were sporulated, and tetrads were dissected. viability segregated 22 in all 31 tetrads dissected, and all viable spores were his-. all viable spores resulting from the sectoring diploids were temperature sensitive, and all of the viable spores resulting from the nonsectoring diploids were not temperature sensitive. ctf73 was physically mapped by the previously described method of chromosome fragmentation (gerring et al., 199oa) , using the 2.2 kb ctf13 fragment. the sizes of the resulting stable chromosome fragments were determined by orthogonal field-alteration gel electrophoresis (ofage) analysis (carle and olson, 1964) . and assignment of ctf13 to an arm of chromosome xiii was accomplished by hybridization of a left arm telomere-adjacent probe, tub3, to a southern blot of the ofage gel. tub3 was obtained from p. schatz, and the probe used was a 1.2 kb hindlll fragment, radioactively labeled with 'p (feinberg and vogelstein, 1964) . tub3 hybridized to the 445 kb proximal fragment. to obtain a meiotic map position, a diploid strain was constructed that was heterozygous for cff73 and cin4 (~61530/+, +/chc:ura3). the meiotic distance was calculated from the following data by using the formula of perkins: cff73cin4 34 cm (parental ditypel nonparental ditypeitetratype = 42/2/56). ctfl3 was placed proximal to cin4 by probing the ctf73 chromosome fragmentation ofage blots with a 2 kb sacl-kpnl c/n4 fragment obtained from a. hoyt. c/n4 hybridized to the 475 kb distal ctf73 chromosome fragment, placing ctf73 proximal to cin4. analysis the plasmid containing the el tag fused to ctf13, pkf60, was constructed from the base plasmid p414geul (j. kroll, unpublished data). p414geul has a 460 bp gal7 promoter fragment cloned into the kpnl site and two tandem copies of the el tag sequence, described by pluta et al. (1992) , inserted in frame into the apal and xhol sites of prs414 (sikorski and hieter, 1969) . the gal7 promoter directs transcription from its own atg toward the polylinker. an ecorl fragment containing the entire 2.2 kb insert of pkfl1 was cloned into the ecorl site of p414geul in the appropriate transcriptional orientation. the 5' -600 bp of the ctf73containing fragment (up to the bglll site; see figure 58 ) were removed and replaced with an -200 bp polymerase chain reaction product containing sequences from the atg of ctf13 to the bglll site. this allowed the in-frame fusion of the tandem el tags to ctf73 under the transcriptional control of gal7 (see figure 5c ). pkf60 was transformed into yph972 and shown to rescue the temperature sensitivity caused by the ctf73-30 mutation on both galactose-and dextrosecontaining media. the plasmid containing the ha epitope fused to ctf13, psfl97a, was constructed by using a synthetic oligonucleotide to fuse the ha epitope and linker sequences to the amino terminus of ctf13 (see figure 5c ). the fusion protein and -200 bp of 3'noncoding sequence from the ctfl3 locus were cloned into prs315 (sikorski and hieter, 1969) downstream of a 625 bp fragment of 5' flanking dna that is presumed to include the ctf73 promoter. psf197a was transformed into yph975. transformants were streaked onto medium containing 5-fluoroorotic acid to select against the ctfl3-ura3 plasmid (boeke et al., 1967) and it was shown that psf197a would rescue viability in the resulting ctf73al::h/s3 strain. the preparation and analysis of cbf3-dna complexes was performed using a modification of procedures previously described by lechner and carbon (1991) . cells in log phase were harvested by centrifugation, frozen in liquid nitrogen, and mechanically disrupted by fragmentation with a liquid nitrogen-cooled mortar and pestle in 30 mm sodium phosphate (ph 7.0). 60 mm ftglycerophosphate, 1 m kci, 6 mm egta, 6 mm edta, 6 mm naf, 10% glycerol, 1 mm phenylmethytsulfonyi fluoride, and 10 &ml (each) leupeptin, pepstatin, and chymostatin. whole-ceil extract (40 ug) was incubated for 30 min at room temperature with 20 fmol of "p-labeled dna probe, 5 ug of salmon sperm dna, 5 pg of poly(dl-dc), and 10 pg of bovine serum albumin in 30 nl of 10 mm hepes (ph 6.0) 1 mm naf, 6 mm mgci,, 10% glycerol, and kci at a final concentration of 125 mm. the 66 bp dna probe was derived from cen3 and spans the core region of cdeiii, from 5 bp to the left of cdeiii to 59 bp to the right of cdeiii. binding reactions were electrophoresed on 4% polyacrylamide gels as described (ng and carbon, 1967) and visualized by autoradiography. basic local alignment search tool isolation of the gene encoding the saccharomyces cerevisiae centromere-binding protein cpi chromatin conformation of yeast centromeres 5-fluoroerotic acid as a selective agent in yeast molecular genetics yeast centromere binding protein of the helix-loop-helix protein family, is required for chromosome stability and methionine prototrophy we thank c. connelly for significant contributions to this work. we would like to thank ft. parker for sending pgab, j. kroll for allowing us to use p414geu1, and a. pluta for kindly providing us with poly clonal el antibodies.we would like to acknowledge s. holloway, r. sikorski, and n. kouprina for helpful theoretical discussions and h. varmus and t. mitch&on for support during this project. we are also grateful to j. kilmartin, j. carbon, and j. lechner for communicating results prior to publication.we thank d. koshland, w. earnshaw, and t. kelly for critical reading of the manuscript.k. f. d. is a student in the predoctoral training program in human genetics at johns hopkins (national institute of general medical sciences grant p32gm07614). s. t. is supported by the national institutes of health departmental training grant 5t32ca09139. p. k. s. and a. a. h. are biomedical scholars of the lucille p. markey charitable trust. this work was supported by a national institutes of health grant (ca16519) to p. h. and an american cancer society grant (cd-509) to f. s. carbon, j., and clarke, l. (1990) . centromere structure and function in budding and fission yeast. new biologist 2, 10-19. carle, g. f., and olson, m. (1964) . separation of chromosomal dna molecules from yeast by orthogonal-field-alteration gel electrophoresis. nucl. acids res. 12, 5647-5665.cellini, a., parker, r., mcmahon, j., guthrie, c., and rossi, j. (1966) . activation of a tactaac box in the saccharomyces cerevisiae actin intron. mol. cell. biol. 6, 1571 -1576 . clarke, l., and carbon, j. (1960 . isolation of a yeast centromere and construction of functional small circular chromosomes. nature 287, 504-509.cottarel, g., shero, j., hieter, p., and hegemann, j. (1969) . a 125base-pair cen8 dna fragment is sufficient for complete meiotic and mitotic centromere functions in saccharomyces cerevisiae. mol. cell. biol. 9, 3342-3349. densmore, l., payne, w., and fitzgerald-hayes, m. (1991) . in vivo genomic footprint of a yeast centromere. mol. cell. biol. 77, 154-165. feinberg, a. p., and vogelstein, b. (1964) . a technique for radiolabeling dna restriction endonuclease fragments to high specific activity. anal. biochem. 732, 6-13. field, j., nikawa, j., broek, d., macdonald, b., rogers, l., wilson, i., lerner, r., and wigler, m. (1966) . purification of a ras-responsive adenylyl cyclase complex from saccharomyces cerevisiae by use of an epitope addition method. mol. cell. biol. 8, 2159 -2165 . fitzgerald-hayes, m.. clarke, l., and carbon, j. (1962 . nucleotide sequence comparisons and functional analysis of yeast centromere dnas. cell 29,235-244. funk, m., hegemann, j., and philippsen, p. (1969) . mellor, j., jiang, w., funk, m., rathjen, j., barnes, c., hiz, t., hegemann, j., and philippsen, p. (1990) . cpfl, a yeast protein which functions in centromeres and promoters. embo j. 9, 4017-4028. mullis, k. b., and faloona, f. a. (1987) . specific synthesis of dna in vitro via a polymerase catalysed chain reaction. meth. enzymol. 755, 335-350. newlon, c. (1988) . yeast chromosome replication and segregation. microbial. rev. 52, 588-801. ng, r., and carbon, j. (1987) . mutational and in vitro protein-binding studies on centromere dna from saccharomyces cerevisiae. mol. cell, biol. 7, 4522-4534.palmer, r., koval, m., and koshland, d. (1989) . the dynamics of chromosome movement in the budding yeast saccharomyces cerevisiae. j. cell biol. 109, 3355-3388. perier, f., and carbon, j. (1992) . a colony color assay for saccharomyces cefevisiae mutants defective in kinetochore structure and function. genetics 132, 39-51.peterson, j., and ris, h. (1978) . electron microscope study of the spindle and chromosome movement in the yeast s. cerevisiae. j. cell sci. 22, 219-242.pluta, a. f., cooke, c. a., and earnshaw, w. c. (1990) . structure of the human centromere at metaphase. trends biochem. sci. 75, 161-185.pfuta, a. f., saitoh, n., goldberg, i., and earnshaw, w. c. (1992). identification of a subdomain of cenp-b that is necessary and sufficient for focalization to the human centromere.j. cell biol. 716,1081-1093. rieder, c. l. (1982) . the formation, structure and composition of the mammalian kinetochore fiber. int. rev. cytol. 79, l-88. rieder, c. l., and alexander, s. p. (1989) . the attachment of chromosomes to the mitotic spindle and the production of aneuploidy in newt lung cells. in mechanisms of chromosome distribution and aneuploidy, m. resnick and b. vig, eds. (new york: liss), pp. 185-194. roof, d. m., meluh, p. b., and rose m. d. (1992) . kinesin-related proteins required for assembly of the mitotic spindle. j. cell biol. 118, 95-108. rose, m. d., winston, f., and hieter, p. (1990) . methods in yeast genetics (cold spring harbor, new york: cold spring harbor laboratory pressj. sanger, f., nicklen, s., and coulson, a. r. (1977) . dna sequencing with chain-terminating inhibitors. proc. natl. acad. sci. usa 74,5483-5487. saunders, w. s., and hoyt, m. a. (1992) the accession number for the ctf73 sequence reported in this paper is l10083. key: cord-000049-rl7sdzd7 authors: lee, david; la mura, maurizio; allnutt, theo r; powell, wayne title: detection of genetically modified organisms (gmos) using isothermal amplification of target dna sequences date: 2009-02-02 journal: bmc biotechnol doi: 10.1186/1472-6750-9-7 sha: doc_id: 49 cord_uid: rl7sdzd7 background: the most common method of gmo detection is based upon the amplification of gmo-specific dna amplicons using the polymerase chain reaction (pcr). here we have applied the loop-mediated isothermal amplification (lamp) method to amplify gmo-related dna sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions. results: we have tested the specificity and sensitivity of the technique for use in gmo studies. results show that detection of 0.01% gmo in equivalent background dna was possible and dilutions of template suggest that detection from single copies of the template may be possible using lamp. conclusion: this work shows that gmo detection can be carried out using lamp for routine screening as well as for specific events detection. moreover, the sensitivity and ability to amplify targets, even with a high background of dna, here demonstrated, highlights the advantages of this isothermal amplification when applied for gmo detection. the ability to detect the presence of gmo is pivotal for consumers to be able to exercise their lifestyle choice of whether to consume, or not, food containing gmos. though the detection and quantification of gmo proteins using immunoassay has been reported [1] , denaturation of the protein during processing makes the method unsuitable for gmo testing and quantification of food. the durability of dna makes it a better substrate for testing and its amplification by pcr is the method of choice, as recommended by the ec (2004/787), for detection and quantification of gmo in samples. an alternative dna amplification method was described by notomi and coworkers [2] called 'loop mediated isothermal amplification' (lamp). the lamp assay relies on the design of a set of primers that generate stem looped (hairpin) structures during the early stage of dna synthesis. the hairpin structures form because two of the primers used contain, at their 5' end, a reverse complement of a sequence that is present in the target further downstream of the initial binding site. displacement primers help the formation of these hairpins at the ends of the dna strands and once formed, these structures can be copied into a series of dna fragments containing multiple units of the target sequence under isothermal conditions utilizing the displacement properties of bst polymerase (see [3] ). although lamp was first described using a set of four primers, enhanced sensitivity was reported using an additional pair of loop primers [4] . as the reactions are performed at a single temperature, lamp assays can be performed very quickly since there are no separate denaturation, annealing and extension steps, and as such, reactions do not require thermalcyclers. here we assess the lamp protocol for the detection of gmos using primers that target event-specific sequences for transgenic ms8 and rf3 oilseed rape (brassica napus l.) and generic gmo sequences such as the cauliflower mosaic virus 35s promoter (p-35s) and the promoter and terminator for the nopaline synthase gene (p-nos and t-nos, respectively) from agrobacterium spp. the lamp technique relies on the design of an interrelated set of primers. the orientation and positions are important for self-priming through stem-looped products that drive and perpetuate the reaction. the osr events ms8 and rf3 arise from the insertion of two closely related transgenes from the plasmids, pthw107 and pthw118, respectively [5] . the former encodes the barnase gene that give rise to male sterility, which is replaced in the latter by the barstar gene which restores fertility: both also have the selectable marker bar which confers tolerance to the herbicide glufosinate ammonium. though the left border of the rf3 insert has undergone rearrangement in the form of duplication and inversion [5] , the right borders of both events are relatively intact (our data [6] which agree with the two sequences in the database). even though the insertions of the transgenes have different breakpoints from the plasmids, they are very close so it was possible to design assays for the rf3 and ms8 events utilizing a common set of primers within the transgenes ( figure 1 ). when used in conjunction with primers for the plant sequence at the border of each event, they are able to detect each event ( figure 2 ). since they have 50% of primers in common, it was important to determine whether there was any cross reaction between the assays. specificity of the two assays was tested using plasmid dna for each event. no cross-reaction between the two targets was observed ( figure 2 ). the sensitivity of the lamp reaction was assessed in two ways: copy number detection and background in which 10 copies of the target could be detected. copy number detection was measured by serial dilutions of known amounts of dna containing the target sequences, either as genomic or plasmid dna ( figure 3 ). reactions fail in both assays at dna molecule number of less than 1 which is consistent with the stochastic probability of a target being present [7] . we note that sometimes non-specific amplification can also take place, especially where the target dna is absent (see figure 3 ) and there is low amounts of dna present in the reaction (cf figures 3a and 4) . these do not form the specific banding patterns representing the different multimeric lamp products that are characteristic for each assay and thus can be easily distinguished on a gel. alternative banding patterns have been observed, also for low template reactions; analyses of these products show that they are formed by interactions of the primers used [8] , to form lamp equivalents of primer-dimers. two factors seem to be important: in the absence of target, low background dna may aid the formation of non-specific products that go on to be amplified; and freeze-thaw repetitions may induce damage to the primers to permit the formation of 'primer intermediates' which can then be amplified. since lamp is capable of non-specific amplification, techniques that rely on the detection of by-products of dna synthesis, e.g., the use of magnesium pyrophosphate precipitation [9] or the use of sybr green dye [10] may not be able to distinguish between real and right border sequences of ms8 and rf3 figure 1 right border sequences of ms8 and rf3. sequences of the plant (above) and transgene (below) at the right border junctions for ms8 and rf3. highlighted sequences are the targets of the lamp primers. the plant sequences are those shown in table 1 : dark blue bases highlighted are the outer displacement primers, yellow and green sequences are the 5' and 3' ends of the lamp primers respectively, and light blue sequences depict the loop primers. the sensitivity of the lamp assay and its suitability for practical gmo detection was tested using assays for commonly-used sequence motifs, the camv 35s promoter (p-35s) and the agrobacterium promoter and terminator for the nopaline synthase gene (p-nos and t-nos, respectively). these sequences are commonly used in constructs used to create approved gm events (see [11] ). roundup ready™ soya construct contains both p-35s and t-nos so provided convenient template for testing the assays. we used a sample where the copy number of the gm has been well characterized and thus control the number of template in each reaction. lamp sensitivity was assessed by the detection of ten roundup ready™ gmo targets in a background of 100 ng of genomic oilseed rape dna (figure 3 ). since the c value of both species is approximately 1 pg [12] , this background dna represents a gmo level of 0.01% for both tnos and p-35s assays. osr dna was used because we did not have any soya dna free from roundup ready™. dna extracted from our 0% crm was shown to contain 0.002% gmo [13] . the use of 100 ng of this sample would be equivalent to adding 200 copies of the transgene sequence, considerably more than the experimental input of 10 copies. we believe the use of osr dna to be a valid substitute since none of the primer sequences for either assay will be present in non-transgenic soya or oilseed rape. we have tested the upper limits of dna that lamp reactions can tolerate and found that up to 200 ng dna in a 20 μl reaction, positive detection is reproducible. above this dna level, reactions become unreliable (data not shown). we have found that denaturation of template was a prerequisite step prior to amplification, unlike results found by nagamine and co-workers [14] . this can be explained by the fact that we do not use a base pair destabiliser, such as betaine, in the reaction buffer. since we are detecting down to near single copies of templates, our results suggest no benefit to the sensitivity of the assay by their inclusion. the consistent amplification within all dilutions showed that lamp is an 'all or nothing' reaction, with little of the tailing off effect that is often observed in pcrs with diluting templates. this makes it easy to identify positive reactions. together with specificity and the speed at which reactions can be performed, lamp is an excellent method for diagnostics [8, 10, 15] . the use of camv 35s promoter sequence in lamp has previously been reported as a screening method [16] . here we demonstrate the sensitivity and reliability of the lamp method for gmo detection, both with generic and gmo-specific assays. the ability to perform reactions in a simple heated block or water bath without the need for thermal cycling makes testing using lamp more accessible. that lamp is able to detect very small amounts of target and do that even in high amounts of background dna makes it ideal for gmo detection. gmo testing can be performed in steps: routine screening for the presence of gmos using generic assays such as for 35s promoters and t-nos; and if required, identification of specific events can be performed using event-specific assays. equally, direct screening using event-specific assays is also feasible. the levels of sensitivity are orders of magnitude below the permissible threshold for gmo in food and feed (ec regulation 1830/2003), ensuring the detection of the presence of gmos at acceptable levels and the reliable detection of any presence of unauthorised gm events, for which at present there is no legally acceptable lower limit (according to ec regulations). conventional oilseed rape (osr) seed, variety 'hearty' was a gift from christine lewis, niab. the sample was originally purchased from monsanto uk ltd (cambridge, uk). dna was extracted by grinding 2 g seed with 10 ml extraction buffer [0.5 m nacl, 0.1 m edta ph 8 and 1% (w/v) sds] in a mortar with a pestle. the sample was emulsified with 5 ml of chloroform:isoamyl alcohol (24:1) and poured into a 20 ml falcon polypropylene tube. after centrifugation at 1000 g for 5 mins, the aqueous phase was transferred to a new tube and nucleic acids dna from the oilseed rape ms8/rf3 was extracted from seedlings from a selfed ms8/rf3 plant [17] using dneasy plant dna extraction kit (qiagen, crawley, uk). the parent plant was genotyped to be ms8ms8/rf3rf3 using real-time pcr (data not shown). the sample was quantified using picogreen fluorescence (molecular probes inc., invitrogen). dna containing roundup ready™ soya was extracted from soya roundup ready™ gmo reference material (fluka biochemika, sigma-aldrich, dorset, uk) and the gmo concentration of the sample has been accurately determined by dilutions of template combined with statistical analysis [13] . the plasmid pgreenii 0049 was a gift from mark smedley and wendy harwood of the john innes centre. details of the plasmid can be found at the website: http:// www.pgreen.ac.uk/jit/pgreen0000_fr.htm sensitivity assessment of lamp detection figure 3 sensitivity assessment of lamp detection. a. sensitivity of lamp using genomic target. dna from ms8/rf3 sample (16 ng.μl -1 ) was serially diluted and amplified by lamp, in triplicate, using primers to assay for the rf3 junction. the numbers in parenthesis represent the approximate copy numbers of the target assuming that the sample represents rf3 in a hemizygous state (determined using rt-pcr data not shown) for the transgene and using 1 pg as the genome size for oilseed rape. c is the no dna control and m represents molecular size markers. the smear (*) shows an example of non-specific amplification. b. sensitivity of lamp using plasmid target. serial dilutions of the plasmid pgreenii were amplified using primers for the pnos target. numbers represent the calculated copy numbers of the plasmid derived from the dna value. c is the no dna control and m represents molecular sized markers. plasmids containing each event were constructed to test the specificity of the ms8 and rf3 assays separately. the junction at the right borders of the transgenes were amplified by pcr from the ms8/rf3 dna sample using the displacement (outer) primers of the lamp reactions, ms8-rf3 displr (b3c) separately with ms8 displf (f3) and rf3 displf (f3), to amplify the ms8 and rf3 junctions, respectively (see figure 1 and table 1 ). the fragments were cloned into pgem-t vector (promega, southampton, uk) and transformed into dh5α. clones containing the correct inserts were confirmed by sequencing. target sequences for p-35s, p-nos and t-nos were chosen based upon common identity between different plasmids in the embl database containing the promoters and terminator. the sequences of the targets and positions of the primers are provided (see additional file 1). primers for each target segment have tm's of 50-52°c (calculated using the 2 × at, 4 × gc formula), except for the f2 and b2 regions (3' of the lamp primers), where the tm was 54-56°c. primer sequences are listed in table 1 . for lamp reactions, primers were purchased from sigmagenosys (table 1) . reactions were performed in 20 μl containing 1 × bst pol buffer (neb, ipswich, uk) with 0.4 mm each dntp with the appropriate primers listed in table 1 : displacement primers were each used at a concentration of 0.2 μm; loop primers at 0.4 μm and lamp primers at 0.8 μm in the reactions. enough reaction reagents, without template and enzyme, were mixed together and split into two. template (1 μl) was added to 9 μl of the mix and the samples denatured at 95°c for 2 mins and then cooled to 4°c. bst pol was added to the remaining reaction mix to a concentration of 3.2 u.μl -1 , mixed thoroughly, and 10 μl was added to each reaction. reactions were incubated at 55°c for 2 hours, followed by 80°c for 10 mins to inactivate the enzyme and stored at 4°c until analysed. aliquots of the reactions (5 μl) were run on 1.5% (w/v) agarose gels, containing ethidium bromide validation of an immunoassay for detection and quantitation of a genetically modified soybean in food and food fractions using reference materials: interlaboratory study loop-mediated isothermal amplification of dna loop animation eiken genome site accelerated reaction by loopmediated isothermal amplification using loop primers report on the molecular characterisation of the genetic map of event ms8 × rf3 plasmid standards for real time pcr and gm enforcement testing the limits of gmo detection loop-mediated isothermal amplification for detection of african trypanosomes detection of loop mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. intracellulare in sputum samples genetically modified (gm) crops: molecular and regulatory details nuclear dna content of some important plant species. table i (nuclear dna content of a number of important plant species as determined by flow cytometry) quantitation using informative zeros (quiz): application for gmo detection and quantification without recourse to certified reference material loopmediated isothermal amplification reactions using a nondenatured template development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus real-time loop-mediated isothermal amplification for the camv-35s promoter as a screening method for genetically modified organisms crop-to-crop gene flow using farm scale sites of oilseed rape (brassica napus) in the uk andy greenland and giacomo morreale are thanked for their comments. this work was carried out with the financial support of the co-extra project: gm and non-gm supply chains, their co-existence and traceability (contract 007158), funded by the european commission through the sixth framework programme under the food quality and safety priority. dr olga gandelman (lumora ltd, ely, uk) is thanked for help in primer design. mlm is the recipient of a ministero dell' universita' e della ricerca award from university of naples federico ii. dl, as project leader, coordinated the work and wrote the manuscript. mlm performed lab work. ta provided data and material for the experiments. wp, as group leader provided focus and direction. all authors contributed to discussions, read and approved the final manuscript. sequences of lamp targets. the sequences of the different genetic elements used to design the lamp assays are shown together with a genebank accession number that contains the sequence. the sequences targeted by the lamp primers are coloured. click here for file [http://www.biomedcentral.com/content/supplementary/1472-6750-9-7-s1.doc] sequence ( key: cord-003674-3ajyr5e4 authors: nagao, konomu; makino, ryohei; apego, francis victor; mekata, hirohisa; yamazaki, wataru title: development of a fluorescent loop-mediated isothermal amplification assay for rapid and simple diagnosis of bovine leukemia virus infection date: 2019-03-27 journal: j vet med sci doi: 10.1292/jvms.19-0009 sha: doc_id: 3674 cord_uid: 3ajyr5e4 bovine leukemia virus (blv) causes enzootic bovine leukosis (ebl), a condition that threatens the sustainability of the livestock industry. a fluorescent loop-mediated isothermal amplification (flamp) assay targeting blv env sequences was developed and used to evaluate 100 bovine blood samples. compared with a conventional real-time pcr (rpcr) assay, the flamp assay achieved 87.3% (62/71) sensitivity and 100% (29/29) specificity. the rpcr assay took 65 min, while the flamp assay took 8 min to 30 min from the beginning of dna amplification to final judgement with a comparable limit of detection. the flamp is a potential tool for the rapid and simple diagnosis of blv infection to supplement elisa testing and can be used by local laboratories and slaughterhouses without special equipment. doi: 10 .1292/jvms.19-0009 [5, 14] . fluorescent (flamp) reagents are commercially available, and this real-time amplification approach allows extremely rapid and accurate diagnosis through the use of an improved chain replacement enzyme and annealing analysis compared with tlamp [4, 6, 17] . here we used 100 bovine blood samples obtained from farms in the kagoshima, miyazaki and oita prefectures in japan to develop an flamp assay that we compared with a published real-time pcr assay. from march to june 2017, blood samples of 20 brown-swiss, 85 holstein and 304 japanese black cattle were collected from five farms in kagoshima, miyazaki and oita prefectures, japan. an evacuated tube containing edta was used for blood collection, all samples were centrifuged at 1,500 × g for 5 min, and the plasma was used for the anti-blv antibody-elisa (blv elisa test; jnc co., ltd., tokyo, japan) according to manufacturer's instructions, including with previously described modifications [8, 9] . genomic dna extraction was performed according to our previous study [9] . briefly, among 409 blood samples, 80 seropositive and 20 seronegative samples were randomly chosen. the buffy coat was collected from the centrifuged edta-treated blood sample described above, and genomic dna was extracted using a wizard genomic dna purification kit (promega, fitchburg, wi, u.s.a.) in accordance with the manufacturer's instructions. using a nanodrop 8000 spectrophotometer (thermo fisher scientific, waltham, ma, u.s.a.), the dna concentration was measured and adjusted to 20 ng/µl using distilled water. the genomic dna of fetal lamb kidney cells infected with blv (flk-blv) [21] was extracted in the same manner described above for the comparative determination of the lod. genomic dnas were used immediately, otherwise they were stored at −20 or −80°c. we used primer explorer v5 software (fujitsu system solutions ltd., tokyo, japan) to design a new primer set based on blv env sequences. we used clustal omega for multiple alignment of 180 blv complete env sequences (https://www.ebi.ac.uk/tools/ msa/clustalo/), as well as jalview to identify conserved env nucleotide sequences in an alignment of 401 blv partial env gene sequences [22] . details of the primers are shown in table 1 . the predicted specificities of the six primers were determined using the basic local alignment search tool (blast) (https://blast.ncbi.nlm.nih.gov/blast.cgi). the flamp assay was performed using a genie iii (optigene, horsham, u.k.). amplification was performed at 67°c for 30 min, followed by inactivation of enzymatic activity at 98°c for 2 min, and cooling to 80°c for annealing analysis with ramping at 0.05°c/sec. the 25-µl flamp reaction comprised 15 µl of an isothermal master mix (iso-002, optigene), 0.4 µl each of fip and bip primers (100 pmol/µl), 0.2 µl each of lf and lb primers (100 pmol/µl), 0.05 µl each of f3 and b3 primers (100 pmol/µl), 3.7 µl of nuclease-free water, and 5 µl of the dna template. all lamp primers were produced using column-grade purification methods by hokkaido system science (sapporo, japan). when the fluorescence intensity reached 20,000 within a 30-min amplification, and the annealing temperature (ta) value ranged between 89.0 and 92.0°c, the results were interpreted as positive. time of positivity (tp) was automatically calculated by the genie iii. rpcr was performed using a lightcycler 96 (roche molecular systems, pleasanton, ca, u.s.a.) to detect pol sequences according to rola-łuszczak, et al. [16] and the oie terrestrial manual [23] . reactions were modified using shorter times for denaturation and annealing according to the instructions provided with the cycleave pcr reaction mixture (takara bio inc., otsu, japan). briefly, 20-µl rpcr reactions comprised 10 µl of 2x cycleave pcr reaction mixture (takara bio), 0.08 µl each of primers (100 pmol/µl, hokkaido system science, 0.04 µl of probe (100 pmol/µl, hokkaido system science), 4.8 µl of nucleasefree water, and 5 µl of the dna template. the cycling conditions were as follows: one cycle at 95°c for 10 sec, 50 cycles each at 94°c for 5 sec, and 60°c for 30 sec. each amplification was performed in duplicate, and an average c t (threshold cycle) value was automatically calculated. details of the primers and probes are shown in table 1 . based on the sequence of lc164083, a partial sequence of pol for rpcr encompassing primer target sequences was synthesized and cloned into the pex-k4j1 vector by eurofins genomics, co., ltd (tokyo, japan). the amplified sequence was used to calculate the proviral loads in clinical samples as well as to prepare serial 10-fold serial dilutions of the dna templates for lod determination. further, the tlamp assay targeting the ltr region was performed to compare the lod according to a published method [5] . two dna templates were used for further comparisons of different genotypes of the flk strain (genotype 1) and a blv-genomic dna obtained from a naturally blv-infected cow in miyazaki (ja366, genotype 3). ten-fold serial dilutions in distilled water were prepared, and the flamp, tlamp and rpcr assays were performed as described above. when a sample was positive or negative in duplicate analyses, the result was interpreted as a positive in all three assays. we retrieved 401 blv complete and partial env sequences from genbank to design a blv-specific lamp primer set. the locations and nucleotide variations of the lamp primers representing 180 complete env sequences are shown in fig. 1 . although lamp primers were derived from the conserved region of env, to maintain amplification speed and accuracy, mixed bases were incorporated into the fip and b3 primers because of the variability of the target sequences that introduce mismatches at the crucial regions between target sequences and primers (table 1 and fig. 1) . in a preliminary test, we attempted to design a lamp primer set using well-conserved pol sequences. the pol gene was not amplified by any lamp primer set that we designed using primer explorer v5 software. this may be explained by the the low gc content of pol. therefore, we chose the conserved sequence in env to design the specific lamp primer set. to determine primer specificities, each of the six primers representing eight distinct regions of env were analysed using blast to query genbank. there were no matches between the first six bases of the eight primer-binding regions with non-blv sequences. (table 1) . further, 20 samples did not yield c t values using the blv-specific real-time pcr (rpcr) assay, and their values were significantly below the threshold of baseline absorbance in the anti-blv antibody-elisa. these samples were all blv-negative in the flamp assay. further, false-positive signals were not detected (fig. 2, table 2 ). we used 100 bovine clinical blood samples, comprising 80 elisa-positive and 20 elisa-negative samples, to evaluate the performance of the blv specific flamp assay. the results were compared with those of the rpcr assay [16, 23] . as shown in table 2 and fig. 2 , among the 80 elisa-positive samples, 62 and 71 were positive and 18 and 9 were negative in the flamp and rpcr assays, respectively. the 20 elisa-negative samples were negative in both assays. compared with the elisa results, the flamp assay achieved 77.5% (62/80) sensitivity and 100% (20/20) specificity and the rpcr assay showed 88.8% (71/80) sensitivity and 100% (20/20) specificity. compared with a conventional real-time pcr (rpcr) assay, the flamp assay achieved 87.3% (62/71) sensitivity and 100% (29/29) specificity. the rpcr assay required 65 min from the beginning of reaction to the final extension step. in contrast, the flamp assay yielded 62 positive results between 7 min, 15 sec and 28 min (mean tp 10 min 8 sec ± sd 3 min 23 sec). among the 13-samples that were low proviral loads (2-39 copies per 100 ng dna), corresponding to weak rpcr positives (c t range, 35.37-39.00), the flamp assay generated four true-positives and nine false-negatives ( table 2 , fig. 2 , and table s1 ). the flamp assay did not detect blv sequences in 29 samples comprising nine rpcr-negative/elisa-positive and 20 rpcr-and elisa-negative samples ( table 2) . a comparison between flamp and rpcr data sets is shown in fig. 2 . the rpcr assay yielded 71 positives with 2-25,883 proviral loads (copies per 100 ng dna), corresponding to c t values ranging from 24.94 to 39.00 (mean c t , 29.74 ± sd 3.86). in contrast to the elisa results, the remaining nine samples were negative in the rpcr assay. a 10 −5 -dilution of the blv reference strain flk (genotype 1) returned a "weak" c t value (39.32) in one of two duplicate samples ( table 3 ). the lods of the flamp assay were comparable, or 1-log less sensitive, compared with those of the rpcr and tlamp assays ( table 3) . the difference in the lod values may explain why nine samples were flamp-negative but rpcr-positive. the flamp assay was remarkably rapid. two reports describe the development of a lamp assay for blv based on the ltr region [5] and its application to routine surveys [14] . however, these studies used a tlamp assay, requiring amplification times (22-60 min) [5, 14] that are significantly longer compared with those reported here (8-30 min). in our present study, the tlamp and flamp assays took 16-34 min and 8-25 min to amplify target dnas, respectively (table 3) . further, the tlamp assay is unable to confirm the specificity of the amplified product as judged by annealing temperatures [4, 6, 17] . for this purpose, the tlamp assay requires an open reaction tube for electrophoresis, which may be susceptible to contamination. a diagnostic system based on single genomic targets risks a false-negative diagnosis [20] . for this reason, the development of a fluorescent-based assay using a distinct target gene with high specificity should be useful as a safeguard. therefore, the flamp provides a potential tool for the rapid and simple diagnosis of blv infection to supplement elisa testing and can be used by local laboratories and slaughterhouses without special equipment. the worldwide spread of blv emphasizes the requirement for early diagnosis and control of disease to minimize economic losses, as well as to ensure animal welfare. the flamp assay was applied to rapid and simple diagnosis of human and veterinary infectious diseases [4, 6, 17] . specific amplification using the lamp assay occurs at a constant temperature, minimizing reliance on expensive equipment [2, 24] . consequently, these assays may facilitate the development of an inexpensive test. for example, the flamp takes 8-30 min from the beginning of amplification and <10 min for enzyme inactivation and annealing to final judgment. the application of the lamp assay for rapid screening of clinical samples would save time and costs, enabling detection of blv-positive cases during the early phase of infection because of its lower or equivalent lods compared with those of conventional pcr and nested pcr assays [5, 14] . further, the flamp assay can be used at farms, slaughterhouses, and wholesale markets in combination with direct dna detection techniques of clinical samples [4, 17] , which would enhance the utility of flamp when performed using a portable real-time detector such as the genie iii [4, 6] . as ideal quantitative tools, digital lamp assays have been developed for clinically important infectious diseases [7, 18] . in the future, application of the blv-specific lamp primers described here to the lamp assay should facilitate more precise and rapid quantification of blv as a countermeasure in countries with endemic blv infections. the determination of the precise blv proviral loads of individual cattle within a herd and the isolation of cattle with high blv proviral loads is essential to develop an effective strategy to control the transmission of blv [8] [9] [10] [11] . in conclusion, the flamp assay serves as a simple and rapid tool that has the potential for effectively controlling of blv infection. provirus variants of the bovine leukemia virus and their relation to the serological status of naturally infected cattle direct blood dry lamp: a rapid, stable, and easy diagnostic tool for human african trypanosomiasis development of a bovine leukemia virus polymerase gene-based real-time polymerase chain reaction and comparison with an envelope gene-based assay evaluation of two lyophilized molecular assays to rapidly detect foot-and-mouth disease virus directly from clinical samples in field settings development of loop-mediated isothermal amplification method for diagnosis of bovine leukemia virus infection development and evaluation of reverse transcription-loop-mediated isothermal amplification (rt-lamp) assay coupled with a portable device for rapid diagnosis of ebola virus disease in guinea digital quantification of dna via isothermal amplification on a self-driven microfluidic chip featuring hydrophilic film-coated polydimethylsiloxane evaluation of the natural perinatal transmission of bovine leukaemia virus horizontal transmission and phylogenetic analysis of bovine leukemia virus in two districts of miyazaki diagnosing bovine leukemia virus infection the recent prevalence of bovine leukemia virus (blv) infection among japanese cattle loop-mediated isothermal amplification of dna eradication of enzootic bovine leukosis from finland simple and rapid method for routine screening of bovine leukemia virus by loop-mediated isothermal amplification assay rapid and real-time detection technologies for emerging viruses of biomedical importance development of an improved real time pcr for the detection of bovine leukaemia provirus nucleic acid and its use in the clarification of inconclusive serological test results improving detection accuracy and time for campylobacter jejuni and campylobacter coli in naturally infected live and slaughtered chicken broilers using real-time fluorescent lamp approach phenotypic antibiotic susceptibility testing using digital lamp quantification in clinical samples pathobiology of bovine leukemia virus development of fluorescent reverse transcription loop-mediated isothermal amplification (rt-lamp) using quenching probes for the detection of the middle east respiratory syndrome coronavirus replicationof bovine leukemia virus in mono-layercell cultures jalview version 2-a multiple sequence alignment editor and analysis workbench world organization for animal health (oie) development and evaluation of multiplex rt-lamp assays for rapid and sensitive detection of foot-and-mouth disease virus conflict of interest. we declare that there are no conflicts regarding the subject matter of the manuscript.acknowledgments. this research was supported by grants from jsps kakenhi grant number jp18k05535, and training program for asian veterinarians from the japan racing horse health association. we thank junko sasaki and mari yamamoto for technical assistance. key: cord-001677-p6ikd8ns authors: hansra, satyender; pujhari, sujit; zakhartchouk, alexander n title: exploration of new sites in adenovirus hexon for foreign peptides insertion date: 2015-05-29 journal: open virol j doi: 10.2174/1874357901509010001 sha: doc_id: 1677 cord_uid: p6ikd8ns adenoviral vectors are now being explored as vaccine carriers to prevent infectious diseases in humans and animals. there are two strategies aimed at the expression of a vaccine antigen by adenoviral vectors. the first includes an insertion of the foreign gene expression cassette into the e1 region. the second strategy is antigen incorporation into the viral capsid proteins. to extend this methodology, we have searched for new sites at the human adenovirus serotype 5 major capsid protein hexon for a vaccine antigen insertion. to this end, we utilized sites in the hexon hypervariable region (hvr) 7, 8 and 9 to display a 15-mer peptide containing the main neutralizing epitope of porcine reproductive and respiratory syndrome virus. however, we could not rescue the viruses with the insertions of the peptide into hvr 8 and 9, consistent with the viruses being unable to tolerate insertions at these sites. in contrast, the virus with the insertion of the peptide in hvr 7 was viable growing well in cell culture and the inserted peptide was exposed on the virion surface. human adenovirus type 5 (had5) vector has been shown to be an excellent vaccine delivery system in humans and animals [1, 2] . the replication-defective (e1-deleted) and replication-competent (e3-deleted) had5 have both been used as vaccine delivery vectors. for biosafety concerns, a replication-defective had5 is considered to be more suitable for vaccination. replication-defective vectors may also have deletion in the e3 region to increase the amount of foreign dna that can be inserted. conventional strategy for the expression of a vaccine antigen by adenoviral vector includes insertion of the foreign gene expression cassette into the e1 region. this antigen is expressed as transgene after the infection of permissive cells with recombinant adenovirus. apparent drawbacks of the conventional transgene expression of antigen include an inability of the had5-based vectors to produce a potent humoral response against certain antigens and, in some cases, the inability of the vector to express a foreign gene [3] . to overcome such hurdles, the "antigen capsid-incorporation" strategy was developed [4] . incorporating an immunogenic peptide into the had5 capsid offers several potential advantages. importantly, the processing of capsid-incorporated antigen via the exogenous pathway could result in a strong humoral response, similar to that generated by native had5 capsid proteins, and this strategy may also be useful in boosting antigen-specific antibody immune responses. adenoviral capsid consists of the hexon, penton base and fiber [5] . antigenic epitopes could be incorporated into these proteins as well as into the minor capsid protein ix [6] . importantly, hexon is the most abundant of the capsid proteins, accounting for more than 83% of the capsid protein [7] . furthermore, hexon is shown to be a vaccine adjuvant [8] . early analysis of the hexon protein sequences revealed that, in addition to the conserved regions, there were 7 discrete hypervariable regions (hvrs) [9] . in a subsequent study, they are reclassified as regions 1 to 9 [7] . these hvrs do not appear to be involved in the binding of any other viral proteins, and the loops at the top of the hvrs are the most amenable to modifications. it was also shown that short heterologous peptides can be incorporated within the hvrs of hexon without affecting the viability of the virus [10, 11] . incorporating antigens into hexon is applicable towards vaccination for several pathogens including the poliovirus, pseudomonas, b. anthracis, influenza, malaria and hiv [12] [13] [14] [15] [16] [17] . in selected studies, protective immune responses in a mouse model are documented [12, 16] . in had5, hvr 1 through 6, and 8 have been used for the incorporation of foreign peptides [10, 18] . in this study, we explored different sites in hvrs 7, 8 and 9 of had5 hexon for the insertion of a peptide corresponding to the porcine reproductive and respiratory syndrome virus (prrsv) main neutralizing epitope b [19] of the gp5 protein. our results provide potentially applicable information for adenovirusvectored vaccine development involving such hexon modifications, particularly in hvr 7. in order to access new sites in had5 hexon, tailored for the incorporation of foreign peptide sequences, we genetically inserted a 15 amino acid (aa) residue peptide into hvr7, hvr8 or hvr9 region of the had5 hexon. the peptide consisted of embedding a 9 aa residue sequence containing the prrsv major neutralizing epitope flanked by the lgs spacer sequences (fig. 1) . fig. (1) . sites in the hvrs that were modified with a peptide containing the prrsv major neutralizing epitope b. the arrows mark the position where the peptide was inserted. the numbers show the positions of the amino acid residues in had5 hexon. peptide sequence corresponding to the prrsv epitope is underlined. insertion of the dna encoding peptide into the had5 hexon gene was achieved by pcr followed by bacteriumbased homologous recombination. the linearized dna of the plasmid containing the right portion of the had5 genome with modified hexon and the 1878 bp deletion in the e3 region was co-transfected into human embryo kidney (hek) 293 cells together with a plasmid containing the left portion of the had5 genome with an e1-deletion. homologous recombination in hek 293 cells led to generation of the recombinant virus, termed ad5hvr7epb. multiple attempts to rescue the other two recombinant viruses (ad5hvr8epb and ad5hvr9epb) were not successful. this inability to rescue recombinant viruses with the foreign peptide insertion into hvr8 and hvr9 of hexon suggested that the insertion interferes with the formation of viral particles. interestingly, in a previous study [10] , hexon-modified virus with incorporation of the his 6 peptide into hvr8 was reported to be viable. our result with hvr8 is contradictory, evidently due to the differences in the peptides sequences and their lengths. further, while the two studies are made with changes within hvr8, the specific locations of the incorporations differ as well. notably our study uses a foreign sequence inserted between asn437 and gly438 ( fig. 1) , while wu et al. [10] placed his 6 between pro431 and trp439 substituting for 7 aa residues of hexon. to corroborate the insertion of the peptide encoding dna into the hexon gene of had5, the viral genome fragment was amplified using pcr from the viral dna extracted from ad5hvr7epb-infected cells, and the pcr product was digested with avrii since the peptide encoding dna contained the single avrii site. predictably, a 1053 bp pcr fragment was amplified from ad5hvr7epb dna (fig. 2 , lane 1), and its site specific cleavage by avrii provided two fragments: 650 bp and 403 bp (fig. 2, lane 2) . the results of this analysis suggested that recombinant ad5hvr7epb contained the epitope b encoding sequence in the hvr7 of hexon. the growth kinetics of the recombinant virus ad5hvr7epb was analyzed in hek 293 cells. cultures of the cells were infected with ad5hvr7epb or ad5-empty (e1-deleted and partially e3-deleted had5 with unmodified hexon), and the cells were harvested at 12, 24, 36, 48, 72 and 96 hr post infection intervals. virus in each sample was released by freeze-thawing and titered on monolayers of hek 293 cells. fig. (3) shows the insertion of the peptide in hvr7, resulting in similar virus yields to unmodified virus with maximal titers ranging from 2 x 10 11 to 10 12 tcid 50 per ml in a culture of 2 x 10 6 cells. we also determined the viral particle/infectious particle (vp/ip) ratio for cscl gradient purified preparations of the control virus ad5-empty, as well as the hexon-modified virus ad5hvr7epb. a 2.7 fold increase was observed in the vp/ip ratio of ad5hvr7epb preparations as compared to the unmodified control ad5-empty ( table 2) . a similar observation is reported for hexon-modified had5 with peptide incorporation into hvr2 or 5 [11] . a typical vp/ip ratio of unmodified adenoviruses ranges from 10 to 44 [20] . we next examined whether prrsv epitope was presented on the surface of the virion, using an elisa binding assay. different amounts of cscl gradient purified viruses (ad5hvr7epb and ad5-empty) were immobilized on the wells of 96-well plates and incubated with peptide antisera. the binding of antibodies to the virions was detected with alkaline phosphatase (ap)-conjugated secondary antibody, followed by ap color reaction hvr7: ……. 420 development. evidently, the anti-peptide antibodies bind well to adhvr7epb virions and not to ad5-empty (fig. 4) . this suggests that the prrsv epitope inserted into hvr7 of hexon is exposed on the virion surface. while a previous study showed that epitopes were exposed on the virion surface when incorporated in hvr1, hvr2 and hvr5, and not exposed in hvr3 and hvr6, as well as poorly exposed when a peptide was placed in hvr8 [10] . in the current study, we find a new site in hvr7 for incorporation of foreign peptide into had5 hexon, and determine that the peptide was also exposed on the virion surface making it readily accessible for antibody binding and be potentially useful for vaccination. . (4) . the prrsv epitope incorporated in hvr7 of hexon is accessible in the context of an intact virion. in the assay, varying amounts of purified viruses were immobilized in the wells of elisa plate and incubated with anti-peptide antibody. the binding was detected with an ap-conjugated secondary antibody. values are expressed as the mean and standard deviation. * indicates a p value of < 0.05. herein, we report a novel adenovirus vector (ad5hvr7epb) with the insertion of the prrsv main neutralizing epitope b in 3 different hvr regions of the major capsid protein hexon. while the insertion of the peptide into hvrs 8 and 9 were not tolerated by the adenovirus, the 9-mer epitope placed within hvr7 is determined to be exposed on the virion surface. plasmid puc-ad5-hex was used as a template for pcr and as a cloning vector. this plasmid contained a 5.2 kb dna fragment of had5 genome encoding a portion of the hexon gene. to insert the dna sequence encoding the main neutralizing epitope b of prrsv in hvr7 and obtain the dna fragment for cloning, four consequent pcrs were performed using phusion high-fidelity dna polymerase (neb). first, a 1 kb fragment was amplified using primers bl7f1 and nhe-r, and puc-ad5-hex as a template. second, a 1 kb fragment was amplified using primers blf2 and nhe-r, and the product of pcr 1 as a template. third, a 690 bp fragment was amplified using primers bl7r and nde-f, and puc-ad5-hex as a template. fourth, a 1.7 kb fragment was amplified using primers nde-f and nhe-r, and the mixture of the products of pcr 2 and 3 from above as a template. the final 1.7 kb pcr product was digested with ndei and nhei and cloned into ndei and nhei sites of puc-ad5-hex, creating puc-ad5hex-hvr7epb. similar strategy was used to construct puc-ad5hex-hvr8epb and puc-ad5hex-hvr9epb. specific sequences of the primers are presented in table 1 . the plasmid ph5r-hexhvr7epb was generated by homologous dna recombination in e. coli bj5183 [21] between a 2.6 kb dna fragment excised by kpni and ndei from puc-ad5hex-hvr7epb and bamhi-linearized ph5r [22] . similar strategy was used to construct ph5r-hexhvr8epb and ph5r-hexhvr9epb. fig. (1) shows the sites of the insertion of the peptide in the had5 hexon protein sequence. human embryonic kidney (hek) 293 cells were transfected with paci-digested dnas of the plasmids ph5r-hexhvr7epb, ph5r-hexhvr8epb or ph5r-hexhvr9epb and paci-digested ph5-l [22] using lipofectamine-2000 (invitrogen). homologous recombination in the transfected cells led to generation of the recombinant adenovirus, and the virus was detected by developing cytopathic effect (cpe) in the cell monolayer. if cpe was not detected by the 14th day after transfection, the cells and the cell culture media were collected and freeze-thawed. this material was used to infect a fresh monolayer of hek 293 cells. the virus was amplified by passaging in hek 293 cells and purified by cscl gradient centrifugation [23] . the concentration of virus particles (vp) in purified virus preparations was determined by measuring the optical density at 260 nm as previously described [24] , whereas numbers of infectious particles (ip) were determined by the tissue culture infective dose 50 (tcid 50 ) method [25] . hek 293 cells grown in wells of 6-well plates were infected with ad5-empty or ad5hvr7epb viruses at a multiplicity of infection (m.o.i.) of 5. infected cells were harvested at the indicated time intervals post-infection. cells were lysed in the growth medium by freezing-thawing three times. the titres of infectious viral progeny were determined by tcid 50 method in monolayers of hek 293 cells. hek 293 cells grown in t75 flasks were infected with the virus ad5hvr7epb. after 48 hours post infection, when full cpe was observed, the cells and cell culture media were harvested followed by 3 cycles of freezing and thawing. cell debris was removed by centrifugation at 1,500 rpm for 5 min, and the supernatant (275 µl) was used for viral dna isolation. briefly, the supernatant was incubated with 10 µl of dnase i (10 mg/ml) at 37 0 c for 30 min, followed by addition of 6 µl of 0.5 m ethylenediaminetetraacetic acid (edta) (ph 8.0), 7.5 µl of 20 % sodium dodecyl sulfate (sds), 1.5 µl of proteinase k (20 mg/ml) and incubation at 50 0 c for 1 h. total dna was further extracted using a geneclean spin kit (mp biomedicals) according to the manufacturer's instructions. the isolated dna was subjected to pcr using fermentas (2x) pcr master mix and primers (5'-atcatgcagctgggagagtc-3' and 5'-cattgcccagcaacattgag-3') that were designed to anneal in the sites flanking the hexon hvrs. amplification product was digested by avrii and size-separated by electrophoresis in 1 % agarose gel. the peptide, containing amino acid residues of the prrsv neutralizing epitope b (shlqliynl), was synthesized by genscript inc. the peptide was conjugated to keyhole limpet hemocyanin (klh) or bovine serum albumin (bsa) as carrier molecules. two new zealand white rabbits were immunized with the conjugated peptide (500 µg/rabbit) emulsified with freund's complete adjuvant (fca) followed by two injections