Imperfect Vaccination Can Enhance the Transmission of Highly Virulent Pathogens.

Could some vaccines drive the evolution of more virulent pathogens? Conventional wisdom is that natural selection will remove highly lethal pathogens if host death greatly reduces transmission. Vaccines that keep hosts alive but still allow transmission could thus allow very virulent strains to circulate in a population. Here we show experimentally that immunization of chickens against Marek's disease virus enhances the fitness of more virulent strains, making it possible for hyperpathogenic strains to transmit. Immunity elicited by direct vaccination or by maternal vaccination prolongs host survival but does not prevent infection, viral replication or transmission, thus extending the infectious periods of strains otherwise too lethal to persist. Our data show that anti-disease vaccines that do not prevent transmission can create conditions that promote the emergence of pathogen strains that cause more severe disease in unvaccinated hosts.

Influenza A virus PB1-F2 protein prolongs viral shedding in chickens lengthening the transmission window.

Avian influenza is a significant economic burden on the poultry industry in geographical regions where it is enzootic. It also poses a public health concern when avian influenza subtypes infect humans, often with high mortality. Understanding viral genetic factors which positively contribute to influenza A virus (IAV) fitness - infectivity, spread and pathogenesis - is of great importance both for human and livestock health. PB1-F2 is a small accessory protein encoded by IAV and in mammalian hosts has been implicated in a wide range of functions that contribute to increased pathogenesis. In the avian host, the protein has been understudied despite high-level full-length conservation in avian IAV isolates, which is in contrast to the truncations of the PB1-F2 length frequently found in mammalian host isolates. Here we report that the presence of a full-length PB1-F2 protein, from a low pathogenicity H9N2 avian influenza virus, prolongs infectious virus shedding from directly inoculated chickens, thereby enhancing transmission of the virus by lengthening the transmission window to contact birds. As well as extending transmission, the presence of a full-length PB1-F2 suppresses pathogenicity evidenced by an increased minimum lethal dose in embryonated chicken eggs and increasing survival in directly infected birds when compared to a virus lacking an ORF for PB1-F2. We propose that there is a positive pressure to maintain a full-length functional PB1-F2 protein upon infection of avian hosts as it contributes to the effective transmission of IAV in the field.

Prototype endogenous avian retroviruses of the genus Gallus.

Ancient endogenous retroviruses (ERVs), designated endogenous avian retrovirus (EAVs), are present in all Gallus spp. including the chicken, and resemble the modern avian sarcoma and leukosis viruses (ASLVs). The EAVs comprise several distinct retroviruses, including EAV-0, EAV-E51 and EAV-HP, as well as a putative member previously named the avian retrotransposon of chickens (ART-CH). Thus far, only the EAV-HP elements have been well characterized. Here, we determined sequences of representative EAV-0 and EAV-E51 proviruses by cloning and data mining of the 2011 assembly of the Gallus gallus genome. Although the EAV-0 elements are primarily deleted in the env region, we identified two complete EAV-0 env genes within the G. gallus genome and prototype elements sharing identity with an EAV-E51-related clone previously designated EAV-E33. Prototype EAV-0, EAV-E51 and EAV-E33 gag, pol and env gene sequences used for phylogenetic analysis of deduced proteins showed that the EAVs formed three distinct clades, with EAV-0 sharing the last common ancestor with the ASLVs. The EAV-E51 clade showed the greatest level of divergence compared with other EAVs or ASLVs, suggesting that these ERVs represented exogenous retroviruses that evolved and integrated into the germline over a long period of time. Moreover, the degree of divergence between the chicken and red jungle fowl EAV-E51 sequences suggested that they were more ancient than the other EAVs and may have diverged through mutations that accumulated post-integration. Finally, we showed that the ART-CH elements were chimeric defective ERVs comprising portions of EAV-E51 and EAV-HP rather than authentic retrotransposons.

Relationship between levels of very virulent MDV in poultry dust and in feather tips from vaccinated chickens.

To assess the effect of various vaccine strains on replication and shedding of virulent Marek's disease virus from experimentally infected chickens, quantitative PCR (q-PCR) methods were developed to accurately quantify viral DNA in infected chickens and in the environment in which they were housed. Four groups of 10 chickens, kept in poultry isolators, were vaccinated at 1 day old with one of four vaccines covering each of the three vaccine serotypes, then challenged with very virulent MDV strain Md5 at 8 days of age. At regular time-points, feather tips were collected from each chicken and poultry dust was collected from the air-extract prefilter of each isolator. DNA was extracted from feather and dust samples and subjected to real-time q-PCR, targeting the U(S)2 gene of MDV-1, in order to measure Md5 level per 10(4) feather tip cells or per microgram of dust. Accuracy of DNA extraction from dust and real-time q-PCR were validated by comparing either q-PCR cycle threshold values or the calculated MDV genome level; for use in q-PCR, DNA was extracted from serial dilutions of MDV-infected dust diluted with noninfected dust, or DNA from MDV-infected dust was diluted with DNA from noninfected dust. The results confirmed the accuracy and sensitivity of dust DNA extraction and subsequent q-PCR and showed that differences in virus levels between dust samples truly reflect differences in shedding. Vaccination delayed both replication of Md5 in feather tips and shedding of Md5. First detection of Md5 in feather tips always preceded or coincided with first detection in dust in each group. pCVI988 and HVT+SB-1 were the most efficient vaccines in reducing both replication and shedding of Md5. There was close correlation between mean virus level in feathers of each group and mean virus level in the dust shed by that group. This relationship was similar in each of the vaccinated groups, demonstrating that measurement of the virus in dust can be used to monitor accurately both the infection status of the chickens and environmental contamination by MDV.

A virus-encoded telomerase RNA promotes malignant T cell lymphomagenesis.

Telomerase is a ribonucleoprotein complex consisting of two essential core components: a reverse transcriptase and an RNA subunit (telomerase RNA [TR]). Dysregulation of telomerase has been associated with cell immortalization and oncogenesis. Marek's disease herpesvirus (MDV) induces a malignant T cell lymphoma in chickens and harbors in its genome two identical copies of a viral TR (vTR) with 88% sequence identity to chicken TR. MDV mutants lacking both copies of vTR were significantly impaired in their ability to induce T cell lymphomas, although lytic replication in vivo was unaffected. Tumor incidences were reduced by >60% in chickens infected with vTR- viruses compared with animals inoculated with MDV harboring at least one intact copy of vTR. Lymphomas in animals infected with the vTR- viruses were also significantly smaller in size and less disseminated. Constitutive expression of vTR in the chicken fibroblast cell line DF-1 resulted in a phenotype consistent with transformation as indicated by morphological alteration, enhanced anchorage-independent cell growth, cell growth beyond saturation density, and increased expression levels of integrin alpha v. We concluded that vTR plays a critical role in MDV-induced T cell lymphomagenesis. Furthermore, our results provide the first description of tumor-promoting effects of TR in a natural virus-host infection model.

Rapid, Sensitive, and Species-Specific Detection of Conventional and Recombinant Herpesvirus of Turkeys Vaccines Using Loop-Mediated Isothermal Amplification Coupled With a Lateral Flow Device Readout.

Marek's disease, an economically important disease of chickens caused by virulent serotype 1 strains of the Mardivirus Marek's disease virus (MDV-1), is effectively controlled in the field by live attenuated vaccine viruses including herpesvirus of turkeys (HVT)-both conventional HVT (strain FC126) and, in recent years, recombinant HVT viruses carrying foreign genes from other avian viruses to protect against both Marek's disease and other avian viral diseases. Testing to monitor and confirm successful vaccination is important, but any such test must differentiate HVT from MDV-1 and MDV-2, as vaccination does not prevent infection with these serotypes. End-point and real-time PCR tests are widely used to detect and differentiate HVT, MDV-1 and MDV-2 but require expensive specialist laboratory equipment and trained operators. Here, we developed and validated two tube-based loop-mediated isothermal amplification tests coupled with detection by lateral flow device readout (LAMP-LFD): an HVT-specific test to detect both conventional and recombinant HVT strains, and a second test using novel LAMP primers to specifically detect the Vaxxitek® recombinant HVT. Specificity was confirmed using DNA extracted from virus-infected cultured cells, and limit of detection was determined using plasmid DNA carrying either the HVT or Vaxxitek® genome. The LAMP-LFD tests accurately detected all HVT vaccines, or Vaxxitek® only, in crude DNA as well as purified DNA extracted from field samples of organs, feathers, or poultry house dust that were confirmed positive for HVT by real-time PCR. These LAMP-LFD tests have potential for specific, rapid, simple, and inexpensive detection of HVT vaccines in the field.

Industry-Wide Surveillance of Marek's Disease Virus on Commercial Poultry Farms.

Marek's disease virus is a herpesvirus of chickens that costs the worldwide poultry industry more than US$1 billion annually. Two generations of Marek's disease vaccines have shown reduced efficacy over the last half century due to evolution of the virus. Understanding where the virus is present may give insight into whether continued reductions in efficacy are likely. We conducted a 3-yr surveillance study to assess the prevalence of Marek's disease virus on commercial poultry farms, determine the effect of various factors on virus prevalence, and document virus dynamics in broiler chicken houses over short (weeks) and long (years) timescales. We extracted DNA from dust samples collected from commercial chicken and egg production facilities in Pennsylvania, USA. Quantitative PCR was used to assess wild-type virus detectability and concentration. Using data from 1018 dust samples with Bayesian generalized linear mixed effects models, we determined the factors that correlated with virus prevalence across farms. Maximum likelihood and autocorrelation function estimation on 3727 additional dust samples were used to document and characterize virus concentrations within houses over time. Overall, wild-type virus was detectable at least once on 36 of 104 farms at rates that varied substantially between farms. Virus was detected in one of three broiler-breeder operations (companies), four of five broiler operations, and three of five egg layer operations. Marek's disease virus detectability differed by production type, bird age, day of the year, operation (company), farm, house, flock, and sample. Operation (company) was the most important factor, accounting for between 12% and 63.4% of the variation in virus detectability. Within individual houses, virus concentration often dropped below detectable levels and reemerged later. These data characterize Marek's disease virus dynamics, which are potentially important to the evolution of the virus.

Vaccinal control of Marek's disease: current challenges, and future strategies to maximize protection.

Marek's disease is an economically important lymphoid neoplasm of chickens, caused by oncogenic strains of Marek's disease herpesvirus. The disease can be successfully controlled by vaccination with attenuated or non-pathogenic MDV strains. However, vaccine failures do occur as field strains continue to evolve towards pathotypes of greater virulence, and this evolution is likely to be driven by the vaccines themselves. Two general strategies can be considered to improve protection by vaccination. Firstly by the development of novel vaccines, and secondly by maximizing the potential of existing vaccines. This second goal requires investigation of optimal timing and vaccine delivery route, and optimal vaccination regimes for different breeds of chick. Accurate quantitation of Marek's disease vaccine virus in vaccinated chicks will contribute significantly to our understanding of vaccinal protection. We recently developed a real-time polymerase chain reaction (PCR) assay for quantitation of CVI988 vaccine virus in the feather tips, a rich source of viral DNA which can easily be sampled in a non-invasive manner. This PCR test is now used commercially to confirm the successful vaccination of chicks. We have also used the PCR to examine various aspects of vaccination in experimental chicks and commercial chicks with a view to determining how vaccine level in feathers correlates with protection against challenge, and for identifying optimal timing and vaccine delivery route, and optimal vaccination regimes for different breeds of chick. In this article we review some aspects of the current vaccinal control of Marek's disease, before highlighting some of the problems associated with current vaccines and vaccination strategies, and the challenges for the future. We go on to discuss the development and use of our real-time PCR feather test, its current applications and potential opportunities in Marek's disease vaccine research.

Real-time PCR for differential quantification of CVI988 vaccine virus and virulent strains of Marek's disease virus.

CVI988/Rispens vaccine, the 'gold standard' vaccine against Marek's disease in poultry, is not easily distinguishable from virulent strains of Marek's disease herpesvirus (MDV). Accurate differential measurement of CVI988 and virulent MDV is commercially important to confirm successful vaccination, to diagnose Marek's disease, and to investigate causes of vaccine failure. A real-time quantitative PCR assay to distinguish CVI988 and virulent MDV based on a consistent single nucleotide polymorphism in the pp38 gene, was developed, optimised and validated using common primers to amplify both viruses, but differential detection of PCR products using two short probes specific for either CVI988 or virulent MDV. Both probes showed perfect specificity for three commercial preparations of CVI988 and 12 virulent MDV strains. Validation against BAC-sequence-specific and US2-sequence-specific q-PCR, on spleen samples from experimental chickens co-infected with BAC-cloned pCVI988 and wild-type virulent MDV, demonstrated that CVI988 and virulent MDV could be quantified very accurately. The assay was then used to follow kinetics of replication of commercial CVI988 and virulent MDV in feather tips and blood of vaccinated and challenged experimental chickens. The assay is a great improvement in enabling accurate differential quantification of CVI988 and virulent MDV over a biologically relevant range of virus levels.

Absolute quantitation of Marek's disease virus genome copy number in chicken feather and lymphocyte samples using real-time PCR.

A real-time PCR method was developed, optimised and validated, to enable quantitation of Marek's disease virus genomes as copy number per million host cells. The duplex PCR measured the virus meq gene and host ovotransferrin gene in a single reaction enabling correction for differences in amount of sample DNA added. A bacterial artificial chromosome (BAC) clone of the virus genome, and a plasmid (pGEM-T-ovo) bearing a fragment of the chicken ovotransferrin gene, were used to quantify virus and host genomes respectively. This sensitive and reproducible assay was established initially using chicken lymphocyte DNA, then adapted for feather tip DNA by inclusion of bovine serum albumin in the reaction to overcome inhibition by melanin. The principal advantages are: (1) determination of absolute virus genome copy number enabling meaningful comparison between samples; (2) expression of copy number per million cells, allowing direct correlation with plaque assays; (3) using BAC-cloned whole virus genome as a standard potentially enables any virus gene to be used as the PCR target. This is the first report of quantitation of MDV genomes in feather tips, and application of this assay could significantly further our understanding of pathogenesis, spread, diagnosis, genetic resistance and vaccinal control of Marek's disease.

Correlation of Marek's disease herpesvirus vaccine virus genome load in feather tips with protection, using an experimental challenge model.

We previously developed a real-time polymerase chain reaction (PCR) assay for absolute quantitation of serotype 1 Marek's disease virus in feather tips of chickens, and this has been used clinically to monitor a flock's response following vaccination with CVI988, an attenuated serotype 1 strain. The level of vaccine virus in feather tips associated with protection against challenge by virulent virus is not known. Here, we used an experimental challenge model, in which one dose of vaccine gives over 90% protection against mortality, to investigate correlation between the CVI988 level in feathers and protection. One-day-old chickens were vaccinated with 1, 0.1 or 0.01 commercial dose of CVI988 vaccine, and were then challenged with a virulent strain (RB-1B) 14, 21 or 28 days later. Replication of CVI988 virus was followed in each bird by real-time PCR analysis of feather DNA samples. Since the PCR does not differentiate between CVI988 and RB-1B, samples were taken only prior to challenge to ensure that the virus being measured was CVI988. Administration of one dose of vaccine ensured a uniform, rapid and high replication amongst birds, while replication following administration of the 0.1 or 0.01 dose was very variable. However, given time, a low early level of vaccine virus eventually replicated to high levels in some birds. Both the dose of vaccine virus administered and the level of vaccine virus in feather tips at 13 days post vaccination showed significant correlation with protection against challenge. A level of CVI988 vaccine virus of 132 genome copies/10000 feather tip cells was calculated to be the level required for 90% protection in this experimental model. The potential of this assay, and its limitations for monitoring protection in the field, are discussed.

Horizontal transmission of Marek's disease virus requires US2, the UL13 protein kinase, and gC.

Marek's disease virus (MDV) causes a general malaise in chickens that is mostly characterized by the development of lymphoblastoid tumors in multiple organs. The use of bacterial artificial chromosomes (BACs) for cloning and manipulation of the MDV genome has facilitated characterization of specific genes and genomic regions. The development of most MDV BACs, including pRB-1B-5, derived from a very virulent MDV strain, involved replacement of the US2 gene with mini-F vector sequences. However, when reconstituted viruses based on pRB-1B were used in pathogenicity studies, it was discovered that contact chickens housed together with experimentally infected chickens did not contract Marek's disease (MD), indicating a lack of horizontal transmission. Staining of feather follicle epithelial cells in the skins of infected chickens showed that virus was present but was unable to be released and/or infect susceptible chickens. Restoration of US2 and removal of mini-F sequences within viral RB-1B did not alter this characteristic, although in vivo viremia levels were increased significantly. Sequence analyses of pRB-1B revealed that the UL13, UL44, and US6 genes encoding the UL13 serine/threonine protein kinase, glycoprotein C (gC), and gD, respectively, harbored frameshift mutations. These mutations were repaired individually, or in combination, using two-step Red mutagenesis. Reconstituted viruses were tested for replication, MD incidence, and their abilities to horizontally spread to contact chickens. The experiments clearly showed that US2, UL13, and gC in combination are essential for horizontal transmission of MDV and that none of the genes alone is able to restore this phenotype.

Herpesvirus of turkey reconstituted from bacterial artificial chromosome clones induces protection against Marek's disease.

Herpesvirus of turkey (HVT) is an alphaherpesvirus that is widely used as a live vaccine against Marek's disease because of its antigenic relationship with Marek's disease virus (MDV). In spite of a similar genome structure, HVT has several unique genes, the functions of which are not completely understood. As a first step in carrying out detailed analysis of the functions of the HVT genes, a full-length infectious bacterial artificial chromosome (BAC) clone of HVT was constructed. DNA from two independent BAC clones, upon transfection into chicken embryo fibroblasts, produced plaques similar to those produced by the wild-type virus. Viruses derived from the BAC clones were stable during in vitro passage, but showed differences in in vitro growth kinetics compared with the wild-type virus. Using a one-step mutagenesis protocol to delete the essential glycoprotein B gene from the HVT genome, followed by construction of the revertant virus, BAC clones of HVT were shown to be amenable to standard mutagenesis techniques. In spite of the difference in in vitro growth, viruses from both clones induced 100 % protection against infection by the virulent MDV strain RB-1B, indicating that the BAC-derived viruses could be used as vaccines with efficacies similar to that of the parental virus. The construction of HVT BAC is a major step in understanding the functions of HVT genes by exploiting the power of BAC technology. Furthermore, the availability of the BAC clones enables use of HVT as a vector for expressing foreign genes.

A robust system for RNA interference in the chicken using a modified microRNA operon.

RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo.

Replication kinetics of Marek's disease vaccine virus in feathers and lymphoid tissues using PCR and virus isolation.

CVI988 (Rispens), an avirulent strain of Marek's disease virus, is the most widely used vaccine against Marek's disease. The kinetics of replication of CVI988 was examined in tissues of chickens vaccinated at either 1 day or 14 days of age and sampled regularly up to 28 days post-vaccination. Age at vaccination had no significant effect on the kinetics of CVI988 virus replication. During the cytolytic phase of infection (1-7 days), virus levels peaked in the spleen, bursa and thymus with very close correlation among these organs. Virus load in peripheral blood lagged behind and did not reach high levels. Significant numbers of virus genomes were detected in the feather tips only after 7 days, but subsequently rose to levels almost 10(3)-fold greater than in the other tissues. This is the first accurate quantitative data for kinetics of CVI988 replication in a variety of tissues. There was good correlation between data from virus isolation and PCR, with real-time PCR being the preferred method for rapid, accurate and sensitive quantification of virus. Feathers were ideal for non-invasive sampling to detect and measure CVI988 in live chickens and, from 10 days onwards, virus load in feather tips was predictive of virus load in lymphoid tissues where immune responses will occur. The potential for real-time PCR analysis of feather samples for further investigation of the mechanism of vaccinal protection, and to assist optimization of vaccination regimes, is discussed.

Attenuation of Marek's disease virus by deletion of open reading frame RLORF4 but not RLORF5a.

Marek's disease (MD) in chickens is caused by the alphaherpesvirus MD virus (MDV) and is characterized by the development of lymphoblastoid tumors in multiple organs. The recent identification and cloning of RLORF4 and the finding that four of six attenuated strains of MDV contained deletions within RLORF4 suggested that it is involved in the attenuation process of MDV. To assess the role of RLORF4 in MD pathogenesis, its coding sequence was deleted in the pRB-1B bacterial artificial chromosome clone. Additionally, RLORF5a was deleted separately to examine its importance for oncogenesis. The sizes of plaques produced by MDV reconstituted from pRB-1BdeltaRLORF5a (rRB-1BdeltaRLORF5a) were similar to those produced by the parental pRB-1B virus (rRB-1B). In contrast, virus reconstituted from pRB-1BDeltaRLORF4 (rRB-1BdeltaRLORF4) produced significantly larger plaques. Replication of the latter virus in cultured cells was higher than that of rRB-1B or rRB-1BdeltaRLORF5a using quantitative PCR (qPCR) assays. In vivo, both deletion mutants and rRB-1B replicated at comparable levels at 4, 7, and 10 days postinoculation (p.i.), as determined by virus isolation and qPCR assays. At 14 days p.i., the number of PFU of virus isolated from chickens infected with rRB-1BdeltaRLORF4 was comparable to that from chickens infected with highly attenuated RB-1B and significantly lower than that from rRB-1B-infected birds. The number of tumors and kinetics of tumor production in chickens infected with rRB-1BdeltaRLORF5a were similar to those of P2a chickens infected with rRB-1B. In stark contrast, none of the chickens inoculated with rRB-1BdeltaRLORF4 died up to 13 weeks p.i.; however, two chickens had tumors at the termination of the experiment. The data indicate that RLORF4 is involved in attenuation of MDV, although the function of RLORF4 is still unknown.

vLIP, a viral lipase homologue, is a virulence factor of Marek's disease virus.

The genome of Marek's disease virus (MDV) has been predicted to encode a secreted glycoprotein, vLIP, which bears significant homology to the alpha/beta hydrolase fold of pancreatic lipases. Here it is demonstrated that MDV vLIP mRNA is produced via splicing and that vLIP is a late gene, due to its sensitivity to inhibition of DNA replication. While vLIP was found to conserve several residues essential to hydrolase activity, an unfavorable asparagine substitution is present at the lipase catalytic triad acid position. Consistent with structural predictions, purified recombinant vLIP did not show detectable activity on traditional phospholipid or triacylglyceride substrates. Two different vLIP mutant viruses, one bearing a 173-amino-acid deletion in the lipase homologous domain, the other having an alanine point mutant at the serine nucleophile position, caused a significantly lower incidence of Marek's disease in chickens and resulted in enhanced survival relative to two independently produced vLIP revertants or parental virus. These data provide the first evidence that vLIP enhances the replication and pathogenic potential of MDV. Furthermore, while vLIP may not serve as a traditional lipase enzyme, the data indicate that the serine nucleophile position is nonetheless essential in vivo for the viral functions of vLIP. Therefore, it is suggested that this particular example of lipase homology may represent the repurposing of an alpha/beta hydrolase fold toward a nonenzymatic role, possibly in lipid bonding.

Monocytosis is associated with the onset of leukocyte and viral infiltration of the brain in chickens infected with the very virulent Marek's disease virus strain C12/130.

Marek's disease (MDV) virus is mainly known for the induction of visceral lymphomas and lymphoid infiltration of peripheral nerves. Recently, additional tropism for the central nervous system has been recognised as a distinct feature of disease induced by very virulent MDV isolates. During the analysis of changes in the peripheral blood leukocyte subpopulations in chickens infected with either a virulent (HPRS-16) or a very virulent (C12/130) strain of MDV, we observed a marked monocytosis in chickens infected with C12/130. Perivascular cuffing in brain and mononuclear cell infiltration into the meninges of chickens infected with C12/130 were associated with the appearance of the monocytosis from 6-10 days post-infection. Our results show that a peripheral blood monocytosis may be a contributory factor in establishing or accelerating the severity of mononuclear infiltration into the meninges and perivascular spaces in the brain during infection by very virulent C12/130 strain of MDV.

Assessing the roles of endogenous retrovirus EAV-HP in avian leukosis virus subgroup J emergence and tolerance.

Avian leukosis virus (ALV) subgroup J is thought to have emerged through a recombination event between an unknown exogenous ALV and the endogenous retrovirus elements designated EAV-HP. All EAV-HP elements identified to date in the chicken genome show large deletions, including that of the entire pol gene. Here we report the identification of four segregating chicken EAV-HP proviruses with complete pol genes, one of which shows exceptionally high sequence identity and a close phylogenetic relationship with ALV-J with respect to the env gene. Embryonic expression of EAV-HP env has been suggested as a factor associated with immunological tolerance induction in a proportion of ALV-J-infected meat-type chickens. In support of this, env gene transcripts expressed from two of the four newly identified EAV-HP proviruses were demonstrated in chicken embryos. However, when ALV-J-infected outbred meat-type chickens were assessed, the presence of intact EAV-HP proviruses failed to directly correlate with ALV-J tolerance. This association was further examined using F(2) progeny of two inbred lines of layer chicken that differed in EAV-HP status and immunological responses to ALV-J. Immunological tolerance developed in a small proportion of F(2) progeny birds, reflecting the expected phenotypic ratio for inheritance of a double-recessive genotype; however, the status of tolerance did not show any direct correlation with the presence of the intact EAV-HP sequence. Nevertheless, identification of an intact chicken EAV-HP locus showing a uniquely close relationship to the ALV-J prototype clone HPRS-103 in the env region provides the strongest evidence of its contribution to the emergence of ALV-J by recombination.

Infection of macrophages by a lymphotropic herpesvirus: a new tropism for Marek's disease virus.

Marek's disease virus (MDV) is classified as an oncogenic lymphotropic herpesvirus of chickens. MDV productively and cytolytically infects B, alphabetaT and gammadeltaT lymphocytes and latently infects T-helper lymphocytes. The aims of this study were to identify whether MDV infects macrophages in vivo and, if so, whether quantitative differences in macrophage infection are associated with MDV strain virulence. Chickens were infected with either virulent MDV (HPRS-16) or 'hypervirulent' MDV (C12/130). Flow cytometry with monoclonal antibodies recognizing MDV pp38 antigen and leukocyte antigens was used to identify MDV lytically infected cells. Macrophages from HPRS-16- and C12/130-infected chickens were pp38(+). It is demonstrated that macrophages are pp38(+) because they are infected and not because they have phagocytosed MDV antigens, as assessed by confocal microscopy using antibodies recognizing MDV antigens of the three herpesvirus kinetic classes: infected cell protein 4 (ICP4, immediate early), pp38 (early) and glycoprotein B (gB, late). Spleen macrophages from MDV-infected chickens were ICP4(+), pp38(+) and gB(+), and ICP4 had nuclear localization denoting infection. Finally, MDV pp38(+) macrophages had high inherent death rates, confirming cytolytic MDV infection, although production of virus particles has not been detected yet. These results have two fundamental implications for understanding MDV pathogenesis: (i) MDV evolved to perturb innate, in addition to acquired, immunity and (ii) macrophages are excellent candidates for transporting MDV to primary lymphoid organs during the earliest stages of pathogenesis.