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Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, UK
Correspondence
Venugopal K. Nair
venu.gopal{at}bbsrc.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Witter et al., 1970). As a member of the genus Mardivirus (Fauqet et al., 2005), HVT is antigenically related to Marek's disease virus (MDV), the causative agent of the highly contagious neoplastic Marek's disease (MD) in chickens. The antigenic relatedness between HVT and MDV has been exploited in inducing protection against MD (Okazaki et al., 1970) and successful field trials with the HVT FC126 strain confirmed its usefulness as an effective vaccine against MD (Purchase & Okazaki, 1971). Widespread use of HVT vaccine in the early 1970s has dramatically reduced losses from MD (Witter, 1998). However, the increasing virulence of MDV strains has necessitated the introduction of new generations of MD vaccine strains, such as SB-1 (Schat & Calnek, 1978) and CVI988 (Rispens et al., 1972), to induce protection against very virulent MDV pathotypes. Nevertheless, HVT-based vaccines are still being used widely for the control of MD (Bublot & Sharma, 2004), particularly with other strains to exploit the synergistic protective effects of the combined vaccines (Witter & Lee, 1984).
Because of the strict cell-associated nature of MDV, the vaccines derived from serotypes 1 and 2 are available only as ‘wet’ vaccines that comprise suspensions of viable, infected cells. HVT vaccine was also originally developed as a ‘cell-associated’ wet vaccine. However, because of the distinct ability of HVT to produce cell-free virus in infected tissue culture (Witter et al., 1970), HVT vaccine is also available in a cell-free ‘dry’ lyophilized form that is used widely in countries where the cold storage necessary for the viability of wet vaccines is a problem. HVT is also used as a vector for generating recombinant vaccines and HVT expressing protective genes of avian pathogens such as Newcastle disease virus, Infectious bursal disease virus, infectious laryngotracheitis virus, Avian leukosis virus and Eimeria have been developed (Bublot & Sharma, 2004).
The complete genome sequence of the FC126 strain of HVT has recently been determined (Afonso et al., 2001; Kingham et al., 2001). The 160 kbp-long genome is estimated to contain at least 99 functional genes, many of which have homologues in MDV and other herpesviruses. HVT lacks 16 genes that are present in MDV, but has 13 genes for which there are no MDV homologues, including vNr-13, the first Bcl-2 homologue found in an alphaherpesvirus (Afonso et al., 2001). A detailed understanding of the functions of the HVT-encoded genes is very important to elucidate the unique biological features of HVT as well as to develop HVT-based vectors with enhanced efficacy and versatility.
Analyses of gene functions by manipulation of herpesvirus genomes have been revolutionized by the development of bacterial artificial chromosome (BAC) technology and BAC clones containing full-length genomes of several herpesviruses including MDV have been constructed (reviewed by Zelnik, 2003). In this paper, we describe the construction of full-length HVT BAC (pHVT) clones and show that infectious viruses recovered from these clones are indistinguishable from the wild-type HVT (WTHVT) in plaque morphology. We also show that pHVT-derived viruses, in spite of the differences in growth kinetics in vitro, were as effective as WTHVT in inducing protection against virulent MDV. This is the first report on the construction of BAC clones of HVT and will provide the opportunity for rapid manipulation of the viral genome to identify the molecular determinants associated with the unique features of HVT and for development of more efficient and versatile recombinant vaccine vectors.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was used as the challenge virus in protection studies. All viruses were propagated in primary chicken embryo fibroblasts (CEFs) prepared from 10-day-old specific-pathogen-free embryos.
Construction of HVT BAC.
Construction of the BAC clone was carried out by insertion of the bacterial miniF plasmid into the US region of HVT, essentially using the method described for the construction of MDV BAC (Schumacher et al., 2000; Petherbridge et al., 2003, 2004). For the construction of the transfer vector with homologous sequences to regions of the HVT genome, 2·0 kbp and 2·7 kbp fragments flanking the HVT US2 gene were amplified by PCR using appropriate primers (Table 1). The PCR products amplified from total DNA extracted from HVT-infected cells were digested with appropriate restriction enzymes (EcoRI, PacI and BamHI) and cloned into the pGEM-T vector (Promega) linearized with EcoRI and BamHI to create the pHVTDS vector. The 7·3 kbp PacI fragment containing the miniF plasmid released from the plasmid pHA1 (kindly provided by Dr M. Messerle, Ludwig-Maximilians-Universität, Munich, Germany) was cloned into the pHVTDS vector to generate the pHVTDS–pHA1 recombination transfer vector. BAC clones of HVT were generated following the procedures described previously (Petherbridge et al., 2003, 2004) and by Schumacher et al. (2000). Briefly, primary CEF cultures were co-transfected with approximately 2 µg FC126 genomic DNA and 10 µg pHVTDS–pHA1 DNA. Genomic DNA extracted from these CEF cultures after four rounds of selection in medium containing mycophenolic acid, xanthine and hypoxanthine were electroporated into Escherichia coli DH10B cells. Chloramphenicol-resistant colonies containing high molecular mass DNA were tested for infectivity after transfection into CEFs using lipofectamine (Invitrogen).
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) and glycoprotein B (gB) using standard methods (Sambrook & Russell, 2001). The sequences of the primers used to synthesize the DIG-labelled probes are shown in Table 1
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Construction of gB deletion mutant and revertant of pHVT3.
Replacement of the UL27 gene encoding gB with the KanR gene was carried out by a procedure of homologous recombination using the products of 
phage genes exo, 
and 
encoded by the temperature-sensitive plasmid pKD46 under the control of an L-arabinose-inducible promoter (Datsenko & Wanner, 2000). Briefly, electrocompetent cells were prepared from E. coli strain DH10B (harbouring the pHVT3 and the pKD46 plasmids) grown at 30 °C in LB medium containing ampicillin (100 µg ml–1), chloramphenicol (30 µg ml–1) and 10 mM L-arabinose to an optical density (OD600) of 0·6. The KanR cassette (nt 1923–2738), amplified by PCR from the pACYC177 plasmid (NEB) using the 
gB Kan sense and gB Kan antisense primers (Table 1), which contained an additional 50 nt of homology with the region flanking the HVT UL27 gene to allow recombination, was electroporated into the competent cells. BAC DNA was prepared from kanamycin- and chloramphenicol-resistant colonies using a Qiagen plasmid Maxi kit according to the manufacturer's instructions and examined by restriction digestion for the presence of HVT genomic sequences.
We also constructed an identical gB deletion mutant of pHVT3 by Red mutagenesis (Yu et al., 2000; Lee et al., 2001) using EL250 cells (kindly provided by Dr N. Copeland, NCI Frederick, MD, USA) by inserting a KanR cassette flanked by FRT sites, amplified using oligonucleotides gB Kan_FRT sense and gB Kan_FRT antisense (Table 1) from the plasmid pKD13 (Datsenko & Wanner, 2000). This gB mutant, after excision of the KanR cassette by the expression of FLPe recombinase using 0·2 % arabinose, was used for the construction of the revertant virus. For this, a 3·0 kbp fragment of HVT gB (Tulman et al., 2000) with 350–450 bp homology at each end was amplified by PCR and cloned into the pCR2.1Topo vector (Invitrogen). A spectinomycin cassette from the pCR8 vector (Invitrogen) with flanking FRT sites was inserted at the end of the gB coding region. Finally, a 4·5 kbp PCR product containing the whole gB ORF with flanking homologous sequence and FRT-spectinomycin cassette was amplified from this vector and electroporated into EL250 competent cells containing pHVT-3gB to induce recombination. BAC DNA from revertant clones resistant to spectinomycin and chloramphenicol was transfected into primary CEFs using lipofectamine.
Comparison of the rates of in vitro growth of the WTHVT strain and pHVT viruses
Plaque assay.
The rates of in vitro growth of the viruses were studied on CEFs by counting the p.f.u. at various time points. Briefly, 100 p.f.u. of each of the viruses was inoculated onto 60 mm tissue-culture dishes seeded with 2x106 CEFs and incubated at 38·5 °C, 5 % CO2. At 0, 12, 24, 48, 72, 96 and 120 h after inoculation, the infected cells were trypsinized and serial 10-fold dilutions were added in triplicate onto the six-well plates of CEFs. The titres of the virus at each time point were calculated after 4 days from the number of p.f.u. from each of the dilutions. The fold increase in the number of plaques compared with those at the zero time point was plotted against hours post-infection for each of the three viruses.
Assay of HVT genome copy numbers by quantitative PCR (qPCR).
DNA was prepared by phenol extraction (Sambrook & Russell, 2001) from infected CEFs harvested at 0, 12, 24, 48, 72, 96 and 120 h after inoculation. HVT genome copies per 10 000 CEFs were quantified using real-time PCR, taking the mean value for duplicate wells for each test sample. The method used was essentially as described previously, using a duplex PCR to detect both the virus gene and the host ovotransferrin gene (Baigent et al., 2005), with the following modifications. The primers and probe used were specific for the HVT gene sORF1 as published previously by Islam et al. (2004). The standard curve for the SORF1 reaction, for calibration of HVT genome copy number, was prepared using a dilution series of pHVT BAC3 DNA. The fold increase in HVT genome copies per 10 000 cells compared with those at the zero time point was plotted against hours post-infection for each of the three viruses.
The ability of the viruses to produce cell-free virus from infected CEFs was also compared. For this, tissue-culture supernatants were collected 4 days after infection from CEFs infected with 100 p.f.u. WTHVT or pHVT virus stocks. After centrifugation at 450 g for 5 min to remove any cells, 10-fold dilutions were added to duplicate wells of fresh CEFs in a six-well plate. Tenfold dilutions of cell lysates obtained after four rapid freeze–thaw cycles were also titrated similarly on duplicate wells of CEFs. The virus titres were determined from infected cells 4 days after titration.
Immunological detection of HVT plaques.
HVT plaques were detected by immunohistochemical staining with a polyclonal antiserum collected from birds infected with the HPRS-16 strain of MDV. CEF monolayers were fixed in acetone–methanol, blocked for 1 h with 5 % newborn calf serum in PBS and incubated with a 1 : 1000 dilution of the antiserum at room temperature for 1 h. After washing the cell sheets three times in PBS containing 0·1 % Tween 20, the cells were left at room temperature for 1 h with a 1 : 500 dilution of anti-chicken–horseradish peroxidase conjugate (Sigma). After further washing, the cells were incubated for 1 h at room temperature in a developing solution of 3-amino-9-ethylcarbazole (Sigma), diluted to a final concentration of 0·2 mg ml–1 in 0·1 M sodium acetate (pH 4·8) with 0·015 % hydrogen peroxide, and the mean virus titres were calculated from the number of plaques. HVT plaques were also detected by immunofluorescence staining with the above chicken antiserum and anti-chicken–fluorescein isothiocyanate conjugates (Sigma).
In vivo protection studies.
Protection experiments were carried out in 1-day-old specific-pathogen-free Rhode Island Red chicks, maintained at the Poultry Production Unit of the Institute for Animal Health. All experiments were carried out in separate rooms of the experimental animal house as per UK Home Office guidelines. Chicks were randomly divided into groups of 15 and vaccinated intramuscularly with a total of 5000 p.f.u. of either WTHVT, pHVT3 or pHVT4 in two doses at 1 and 7 days of age. A further group was inoculated with non-infected CEFs as a negative control. At 13 days of age these birds were then infected intra-abdominally with 1000 p.f.u. RB-1B virus. The birds were inspected regularly and all the birds that died during the experiment or were killed at the end of the trial were evaluated for gross and histological lesions by necropsy. Cumulative survival rates were used to assess the protective efficacy of each vaccine virus.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). BamHI–EcoRI digestion of the DNA isolated from pHVT clones 3 and 4 showed identical restriction patterns, indicating that the two clones were indistinguishable from each other at the molecular level (Fig. 2). The pattern and size of the restriction fragments were also identical to the expected digestion pattern predicted from the full-length genome sequence of HVT (GenBank accession nos AF282130 and AF291866), indicating that the pHVT BAC clones contained the full complement of the HVT genome.
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) and plaque titration (Fig. 3b) showed that pHVT3 virus replicated at a higher rate than WTHVT and pHVT4. The growth rate measured by virus plaques peaked at 96 h, whereas the genome copy numbers remained high even at 120 hours post-infection. Since HVT is capable of producing cell-free virus in tissue culture, we also examined whether pHVT could produce cell-free virus in infected CEFs. Titration of the culture supernatant and cell lysates from infected CEFs showed that the two pHVT clones were capable of producing cell-free virus, although the levels were lower than those of WTHVT (Fig. 3c).
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gB) by using a one-step mutagenesis procedure to replace the gB gene with a KanR gene by homologous recombination and selecting for chloramphenicol- and kanamycin-resistant colonies. BamHI–EcoRI digestion of the DNA extracted from one of these clones showed a very similar restriction pattern to that of the donor pHVT3 BAC, indicating that the molecular integrity of the genome was not affected by the mutation. The appearance of an extra band of 12·5 kb and the loss of two bands of 7·2 and 7·0 kb (Fig. 2), resulting from the loss of an EcoRI site, is in agreement with those calculated after insertion of the KanR gene in the gB coding sequence.
Southern blotting hybridization of the BamHI digest of the DNA from the pHVT3, pHVT4 and pHVT3gB clones with the gpt probe identified a single 14·0 kb band, which corresponded exactly to that predicted after insertion of the pHA1 sequences into the HVT genome (Fig. 4a). A single band of identical size was also detected in the digests of DNA prepared from the virus stocks reconstituted from the pHVT clones. The specificity of the probe was confirmed by the absence of signal from the DNA of WTHVT-infected cells. From these results, it was concluded that the miniF plasmid was stable and correctly inserted in the US2 region of the pHVT clones.
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gB clone. For this, the gpt probe was stripped off the membrane, which was then hybridized with a DIG-labelled gB probe generated by PCR using specific primers (Table 1). As expected, a single band of the predicted size of about 25 kb was detected in the DNA from the two pHVT BAC clones as well as in the virus stocks reconstituted from these clones. A similar-size band was also detected in the DNA samples from WTHVT-infected cells. However, no signals were obtained on pHVT3gB DNA (Fig. 4b), confirming the deletion of gB from this clone. In order to check the effect of the deletion of gB on virus replication, DNA prepared from pHVT3 and pHVT3gB clones was transfected onto fresh CEFs and incubated at 37 °C for 3–4 days. HVT plaques, the specificity of which was confirmed by positive immunofluorescence staining with a polyclonal anti-MDV serum, could readily be detected in cells transfected with pHVT3 DNA from 48 hours post-infection (not shown). Compared with this, no plaques were visible in the cells transfected with pHVT3gB DNA. However, single cells that expressed HVT proteins were demonstrated by immunofluorescence, indicating that, in the absence of gB, HVT is unable to spread from cell to cell. This was also demonstrated using a second pHVT3gB mutant prepared with a KanR cassette flanked by FRT sites. To confirm that this defect in cell-to-cell spread was due to the deletion of gB, we constructed a revertant virus in which the deleted region was replaced with the UL27 gene by recombination. Transfection of the DNA from the revertant BAC clone produced virus plaques (data not shown).
Reconstituted HVT from pHVT clones protects against virulent MDV challenge
Since HVT is used as a highly efficient vaccine against oncogenic MDV strains, we compared the protective ability of the virus stocks reconstituted from the two pHVT clones with that of WTHVT in an experimental MDV-challenge model. The protective efficacies of the vaccine viruses were determined by the cumulative survival rates and gross/histological lesions in vaccinated and unvaccinated chickens experimentally challenged with the highly virulent RB-1B strain of MDV. Evidence of MD could be observed in the unvaccinated control birds from about 4 weeks after infection with the RB-1B strain and nearly 80 % of the birds developed MD during the 60-day experimental period (Fig. 5). Post-mortem examination of these birds showed evidence of lymphoid tumours in several visceral organs. Compared with this, none of the birds vaccinated with WTHVT or either of the two pHVT-derived viruses showed any evidence of the disease. Tissues from all birds collected at the end of the experiment were also examined for evidence of any histological lesions. These studies further showed that vaccination with either WTHVT or the pHVT-derived viruses were effective even against the induction of microscopic lesions, although one bird from the group vaccinated with WTHVT showed histological lesions of MD (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), HVT vaccines are still being used widely either alone or in combination with other vaccines. Despite its widespread use, little is known about the functions of HVT genes that are associated with the immune responses that control MDV infection. Recent studies using microarray analysis have shown that HVT infection of CEFs has a wide range of effects on cellular homeostasis (Karaca et al., 2004).
HVT is antigenically related to MDV and induces a persistent viraemic infection with similar infection kinetics (Holland et al., 1998). However, there are several features that are unique to HVT. For example, (i) HVT is distinct from MDV in its ability to produce cell-free virus in culture, a property made use of in the production of freeze-dried cell-free vaccines, (ii) HVT appears to replicate less efficiently in the skin than does MDV, a phenotype that is thought to be associated with the relatively infrequent transmission of HVT among chickens (Cho & Kenzy, 1975) and (iii) unlike MDV, HVT has the ability to replicate in embryonic tissues (Sharma, 1987), a phenomenon of great significance since the widespread practice of in ovo vaccination for the control of MD. A fundamental understanding of the functions of HVT genes and the molecular basis for the unique features of HVT would be useful in improving the immune responses induced by HVT.
The availability of the complete genome sequence of the FC126 strain of HVT (Afonso et al., 2001; Kingham et al., 2001) has enabled comparisons between the HVT and MDV genomes to examine the molecular bases for some of the distinct biological features of HVT. For example, the absence of several important MDV-specific genes such as meq and vCXC chemokine or the presence of distinct HVT-specific genes such as the Bcl-2 homologue vNr-13 in the HVT genome might account for some of the distinct features, such as the non-pathogenic phenotype (Afonso et al., 2001). Although such genomic comparisons are valuable, delineation and precise mapping of the functional determinants would require the application of reverse genetic tools for the rapid manipulation of the genomes. The manipulation of large herpesvirus genomes has been facilitated by the development and widespread application of BAC technology (Brune et al., 2000). BAC clones of various strains of MDV have been constructed and used for examining gene function. As a first step in identifying the molecular determinants associated with the unique features of HVT, we have generated BAC (pHVT) clones of the FC126 strain. Infectivity of the pHVT clones was confirmed by the ability to reconstitute the virus from the transfected DNA. The viruses rescued from the two pHVT clones were indistinguishable from the parental virus in plaque morphology (Fig. 1). However, there were differences in the replication rates between the BAC and wild-type viruses (Fig. 3). Virus derived from the pHVT3 clone showed a much higher replication rate compared with WTHVT. On the other hand, pHVT4 virus replicated at a slower rate than WTHVT. These results show that the BAC clones represent the individual genomes present in the WTHVT pool that may differ in biological characteristics. Titration of the culture supernatant and cell lysates from infected CEFs showed that the pHVT clones produced cell-free virus. However, the titres of cell-free viruses were much lower than those produced by WTHVT. Although the reasons for this defect are not clear, it may be related to the loss of US2. We are currently trying to restore US2/SORF3 in the BAC clones to examine this. The pHVT-derived viruses could be passaged several times in vitro, demonstrating that the pHVT clones are stable and could be useful as seed stocks for production of vaccines.
Since HVT is primarily used as a vaccine against MDV, we compared the immunogenicity of viruses derived from the two pHVT clones with that of the FC126 strain of WTHVT. In the virulent RB-1B-challenge infection model, viruses derived from the two pHVT clones induced 100 % protection, similar to WTHVT. In comparison, nearly 80 % of the unvaccinated birds developed MD during the 60-day experimental period, demonstrating that the viruses derived from the pHVT clones are equally effective as WTHVT in inducing protective immune responses. Birds vaccinated with pHVT were completely protected even against the development of microscopic lesions, further proving induction of strong protective responses against MD.
We also examined the ease with which the pHVT clones could be subjected to mutagenesis techniques. For this, we chose to construct a gB deletion mutant of HVT using the one-step mutagenesis protocol described by Datsenko & Wanner (2000). Using this approach we were able to manipulate the HVT genome and construct the gB deletion mutant pHVT3gB. The deletion of gB from the pHVT3gB clone was confirmed by restriction digestion and Southern blotting (Figs 2 and 4). Under the same transfection conditions in which the pHVT3 DNA produced viral plaques on CEFs, pHVT3gB DNA was unable to produce any viral plaques. However, single cells expressing HVT proteins could be detected by immunofluorescence staining of cells using polyclonal antiserum from MDV-infected birds, indicating that the deletion of gB interfered with the cell-to-cell spread of HVT. This defect in pHVT3gB was rescued using a revertant virus in which UL27 was restored. These results further confirm the essential nature of gB for replication of herpesviruses (Pereira, 1994; Schumacher et al., 2000). HVT encodes other glycoproteins present in MDV, including gB, gC, gD, gE, gH, gI, gK, gL, gM and gN (Kingham et al., 2001). Previous studies have shown that the requirement of glycoprotein complexes for MDV replication is distinct from that of other herpesviruses (Osterrieder & Vautherot, 2004). Although most alphaherpesviruses can grow in cell culture in the absence of various glycoproteins, deletion of gE, gI, gM or gN blocks MDV replication. This differential requirement of glycoproteins is suggested to be attributable to the strict cell-associated nature of MDV (Tischer et al., 2002). As HVT can produce cell-free virus in culture, it is possible that the requirements of glycoprotein complexes of HVT are distinct from those of MDV. The availability of the BAC clones provides the opportunity to compare the requirements of individual glycoproteins for HVT replication.
HVT is also used as a vector for expression of heterologous antigens. Recombinant HVT expressing antigens from various avian pathogens has been constructed using conventional recombination approaches (Bublot & Sharma, 2004). The construction of the pHVT clones will considerably speed up the development of new, highly immunogenic, multivalent recombinant HVT containing multiple antigens or cytokines.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Baigent, S. J., Petherbridge, L. J., Howes, K., Smith, L. P., Currie, R. J. & Nair, V. K. (2005). Absolute quantitation of Marek's disease virus genome copy number in chicken feather and lymphocyte samples using real-time PCR. J Virol Methods 123, 53–64.[CrossRef][Medline]
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Received 5 September 2005;
accepted 8 December 2005.
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) or without (
) microscopic lesions.