The alphaherpesvirus conserved pUS10 is important for natural infection and its expression is regulated by the conserved Herpesviridae protein kinase (CHPK).

Conserved Herpesviridae protein kinases (CHPK) are conserved among all members of the Herpesviridae. Herpesviruses lacking CHPK propagate in cell culture at varying degrees, depending on the virus and cell culture system. CHPK is dispensable for Marek's disease herpesvirus (MDV) replication in cell culture and experimental infection in chickens; however, CHPK-particularly its kinase activity-is essential for horizontal transmission in chickens, also known as natural infection. To address the importance of CHPK during natural infection in chickens, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics of samples collected from live chickens. Comparing modification of viral proteins in feather follicle epithelial (FFE) cells infected with wildtype or a CHPK-null virus, we identified the US10 protein (pUS10) as a potential target for CHPK in vivo. When expression of pUS10 was evaluated in cell culture and in FFE skin cells during in vivo infection, pUS10 was severely reduced or abrogated in cells infected with CHPK mutant or CHPK-null viruses, respectively, indicating a potential role for pUS10 in transmission. To test this hypothesis, US10 was deleted from the MDV genome, and the reconstituted virus was tested for replication, horizontal transmission, and disease induction. Our results showed that removal of US10 had no effect on the ability of MDV to transmit in experimentally infected chickens, but disease induction in naturally infected chickens was significantly reduced. These results show CHPK is necessary for pUS10 expression both in cell culture and in the host, and pUS10 is important for disease induction during natural infection.

Viral proteogenomic and expression profiling during productive replication of a skin-tropic herpesvirus in the natural host.

Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little is known about the viral genes that mediate transmission, mostly due to a lack of natural virus-host model systems. Marek's disease is a devastating herpesviral disease of chickens caused by Marek's disease virus (MDV) and an excellent natural model to study skin-tropic herpesviruses and transmission. Like varicella zoster virus that causes chicken pox in humans, the only site where infectious cell-free MD virions are efficiently produced is in epithelial skin cells, a requirement for host-to-host transmission. Here, we enriched for heavily infected feather follicle epithelial skin cells of live chickens to measure both viral transcription and protein expression using combined short- and long-read RNA sequencing and LC/MS-MS bottom-up proteomics. Enrichment produced a previously unseen breadth and depth of viral peptide sequencing. We confirmed protein translation for 84 viral genes at high confidence (1% FDR) and correlated relative protein abundance with RNA expression levels. Using a proteogenomic approach, we confirmed translation of most well-characterized spliced viral transcripts and identified a novel, abundant isoform of the 14 kDa transcript family via IsoSeq transcripts, short-read intron-spanning sequencing reads, and a high-quality junction-spanning peptide identification. We identified peptides representing alternative start codon usage in several genes and putative novel microORFs at the 5' ends of two core herpesviral genes, pUL47 and ICP4, along with strong evidence of independent transcription and translation of the capsid scaffold protein pUL26.5. Using a natural animal host model system to examine viral gene expression provides a robust, efficient, and meaningful way of validating results gathered from cell culture systems.

ICTV Virus Taxonomy Profile: Herpesviridae 2021.

Members of the family Herpesviridae have enveloped, spherical virions with characteristic complex structures consisting of symmetrical and non-symmetrical components. The linear, double-stranded DNA genomes of 125-241 kbp contain 70-170 genes, of which 43 have been inherited from an ancestral herpesvirus. In general, herpesviruses have coevolved with and are highly adapted to their hosts, which comprise many mammalian, avian and reptilian species. Following primary infection, they are able to establish lifelong latent infection, during which there is limited viral gene expression. Severe disease is usually observed only in the foetus, the very young, the immunocompromised or following infection of an alternative host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Herpesviridae, which is available at ictv.global/report/herpesviridae.

Expression of the Conserved Herpesvirus Protein Kinase (CHPK) of Marek's Disease Alphaherpesvirus in the Skin Reveals a Mechanistic Importance for CHPK during Interindividual Spread in Chickens.

The Herpesviridae encode many conserved genes, including the conserved herpesvirus protein kinase (CHPK) that has multifunctional properties. In most cases, herpesviruses lacking CHPK can propagate in cell culture to various degrees, depending on the virus and cell culture system. However, in the natural animal model system of Marek's disease alphaherpesvirus (MDV) in chickens, CHPK is absolutely required for interindividual spread from chicken to chicken. The lack of biological reagents for chicken and MDV has limited our understanding of this important gene during interindividual spread. Here, we engineered epitope-tagged proteins in the context of virus infection in order to detect CHPK in the host. Using immunofluorescence assays and Western blotting during infection in cell culture and in chickens, we determined that the invariant lysine 170 (K170) of MDV CHPK is required for interindividual spread and autophosphorylation of CHPK and that mutation to methionine (M170) results in instability of the CHPK protein. Using these newly generated viruses allowed us to examine the expression of CHPK in infected chickens, and these results showed that mutant CHPK localization and late viral protein expression were severely affected in feather follicles wherein MDV is shed, providing important information on the requirement of CHPK for interindividual spread.IMPORTANCE Marek's disease in chickens is caused by Gallid alphaherpesvirus 2, better known as Marek's disease alphaherpesvirus (MDV). Current vaccines only reduce tumor formation but do not block interindividual spread from chicken to chicken. Understanding MDV interindividual spread provides important information for the development of potential therapies to protect against Marek's disease while also providing a reliable natural host in order to study herpesvirus replication and pathogenesis in animals. Here, we studied the conserved Herpesviridae protein kinase (CHPK) in cell culture and during infection in chickens. We determined that MDV CHPK is not required for cell-to-cell spread, for disease induction, and for oncogenicity. However, it is required for interindividual spread, and mutation of the invariant lysine (K170) results in stability issues and aberrant expression in chickens. This study is important because it addresses the critical role CHPK orthologs play in the natural host.

Exocytosis of Progeny Infectious Varicella-Zoster Virus Particles via a Mannose-6-Phosphate Receptor Pathway without Xenophagy following Secondary Envelopment.

The literature on the egress of different herpesviruses after secondary envelopment is contradictory. In this report, we investigated varicella-zoster virus (VZV) egress in a cell line from a child with Pompe disease, a glycogen storage disease caused by a defect in the enzyme required for glycogen digestion. In Pompe cells, both the late autophagy pathway and the mannose-6-phosphate receptor (M6PR) pathway are interrupted. We have postulated that intact autophagic flux is required for higher recoveries of VZV infectivity. To test that hypothesis, we infected Pompe cells and then assessed the VZV infectious cycle. We discovered that the infectious cycle in Pompe cells was remarkably different from that of either fibroblasts or melanoma cells. No large late endosomes filled with VZV particles were observed in Pompe cells; only individual viral particles in small vacuoles were seen. The distribution of the M6PR pathway (trans-Golgi network to late endosomes) was constrained in infected Pompe cells. When cells were analyzed with two different anti-M6PR antibodies, extensive colocalization of the major VZV glycoprotein gE (known to contain M6P residues) and the M6P receptor (M6PR) was documented in the viral highways at the surfaces of non-Pompe cells after maximum-intensity projection of confocal z-stacks, but neither gE nor the M6PR was seen in abundance at the surfaces of infected Pompe cells. Taken together, our results suggested that (i) Pompe cells lack a VZV trafficking pathway within M6PR-positive large endosomes and (ii) most infectious VZV particles in conventional cell substrates are transported via large M6PR-positive vacuoles without degradative xenophagy to the plasma membrane.IMPORTANCE The long-term goal of this research has been to determine why VZV, when grown in cultured cells, invariably is more cell associated and has a lower titer than other alphaherpesviruses, such as herpes simplex virus 1 (HSV1) or pseudorabies virus (PRV). Data from both HSV1 and PRV laboratories have identified a Rab6 secretory pathway for the transport of single enveloped viral particles from the trans-Golgi network within small vacuoles to the plasma membrane. In contrast, after secondary envelopment in fibroblasts or melanoma cells, multiple infectious VZV particles accumulated within large M6PR-positive late endosomes that were not degraded en route to the plasma membrane. We propose that this M6PR pathway is most utilized in VZV infection and least utilized in HSV1 infection, with PRV's usage being closer to HSV1's usage. Supportive data from other VZV, PRV, and HSV1 laboratories about evidence for two egress pathways are included.

The Herpesviridae Conserved Multifunctional Infected-Cell Protein 27 (ICP27) Is Important but Not Required for Replication and Oncogenicity of Marek's Disease Alphaherpesvirus.

The Herpesviridae conserved infected-cell protein 27 (ICP27) is essential for cell culture-based replication of most herpesviruses studied. For members of the Alphaherpesvirinae, ICP27 regulates the expression of many viral genes, including expression of pUL44 (gC), pUL47 (VP13/14), and pUL48 (VP16). These three viral proteins are dysregulated during Marek's disease alphaherpesvirus (MDV) replication in cell culture. MDV replicates in a highly cell-associated manner in cell culture, producing little to no infectious virus. In contrast, infectious cell-free MDV is produced in specialized feather follicle epithelial (FFE) cells of infected chickens, in which these three genes are abundantly expressed. This led us to hypothesize that MDV ICP27, encoded by gene UL54, is a defining factor for the dysregulation of gC, pUL47, and pUL48 and, ultimately, ineffective virus production in cell culture. To address ICP27's role in MDV replication, we generated recombinant MDV with ICP27 deleted (vΔ54). Interestingly, vΔ54 replicated, but plaque sizes were significantly reduced compared to those of parental viruses. The reduced cell-to-cell spread was due to ICP27 since plaque sizes were restored in rescued viruses, as well as when vΔ54 was propagated in cells expressing ICP27 in trans In chickens, vΔ54 replicated, induced disease, and was oncogenic but was unable to transmit from chicken to chicken. To our knowledge, this is the first report showing that the Herpesviridae conserved ICP27 protein is dispensable for replication and disease induction in its natural host.IMPORTANCE Marek's disease (MD) is a devastating oncogenic disease that affects the poultry industry and is caused by MD alphaherpesvirus (MDV). Current vaccines block induction of disease but do not block chicken-to-chicken transmission. There is a knowledge gap in our understanding of how MDV spreads from chicken to chicken. We studied the Herpesviridae conserved ICP27 regulatory protein in cell culture and during MDV infection in chickens. We determined that MDV ICP27 is important but not required for replication in both cell culture and chickens. In addition, MDV ICP27 was not required for disease induction or oncogenicity but was required for chicken-to-chicken transmission. This study is important because it addresses the role of ICP27 during infection in the natural host and provides important information for the development of therapies to protect chickens against MD.

Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways.

UNLABELLED: Varicella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (∼100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome. IMPORTANCE: VZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an intersection of viral exocytosis and autophagy pathways. Specifically, both LC3-II and Rab11 proteins copurified with some infectious VZV particles. The results suggested that a subpopulation of VZV particles were carried to the cell surface in single-walled vesicles with attributes of an amphisome, an organelle formed from the fusion of an endosome and an autophagosome. Our results also addressed the interpretation of autophagy/xenophagy results with mutated herpes simplex virus lacking its ICP34.5 neurovirulence gene (HSVΔ34.5). The VZV genome lacks an ICP34.5 ortholog, yet we found no evidence of VZV particles housed in a double-membraned autophagosome. In other words, xenophagy, a degradative process documented after infection with HSVΔ34.5, was not observed in VZV-infected cells.

Interindividual Spread of Herpesviruses.

Interindividual spread of herpesviruses is essential for the virus life cycle and maintenance in host populations. For most herpesviruses, the virus-host relationship is close, having coevolved over millions of years resulting in comparatively high species specificity. The mechanisms governing interindividual spread or horizontal transmission are very complex, involving conserved herpesviral and cellular proteins during the attachment, entry, replication, and egress processes of infection. Also likely, specific herpesviruses have evolved unique viral and cellular interactions during cospeciation that are dependent on their relationship. Multiple steps are required for interindividual spread including virus assembly in infected cells; release into the environment, followed by virus attachment; and entry into new hosts. Should any of these steps be compromised, transmission is rendered impossible. This review will focus mainly on the natural virus-host model of Marek's disease virus (MDV) in chickens in order to delineate important steps during interindividual spread.

Temporal Dynamics of Purinergic Receptor Expression in the Lungs of Marek's Disease (MD) Virus-Infected Chickens Resistant or Susceptible to MD.

Marek's disease virus (MDV) is an economic concern for the poultry industry due to its poorly understood pathophysiology. Purinergic receptors (PRs) are potential therapeutic targets for viral infections, including herpesviruses, prompting our investigation into their role in MDV pathogenesis. The current study is part of an experimental series analyzing the expression of PRs during MDV infection. To address the early or short-acting P2 PR responses during natural MDV infection, we performed an "exposure" experiment where age-matched chickens were exposed to experimentally infected shedders to initiate natural infection. In addition, select non-PR regulatory gene responses were measured. Two groups of naïve contact chickens (n = 5/breed/time point) from MD-resistant (White Leghorns: WL) and -susceptible (Pure Columbian) chicken lines were housed separately with experimentally infected PC (×PC) and WL (×WL) chickens for 6 or 24 h. Whole lung lavage cells (WLLC) were collected, RNA was extracted, and RT-qPCR assays were used to measure specific PR responses. In addition, other potentially important markers in pathophysiology were measured. Our study revealed that WL chickens exhibited higher P1 PR expression during natural infection. WL chickens also showed higher expression of P1A3 and P2X3 at 6 and 24 h when exposed to PC-infected chickens. P2X5 and P2Y1 showed higher expression at 6 h, while P2Y5 showed higher expression at 6 and 24 h; regardless of the chicken line, PC chickens exhibited higher expression of P2X2, P2Y8, P2Y10, P2Y13, and P2Y14 when exposed to either group of infected chickens. In addition, MDV infection altered the expression of DDX5 in both WL and PC groups exposed to PC-infected birds only. However, irrespective of the source of exposure, BCL2 and ANGPTL4 showed higher expression in both WL and PC. The expression of STAT1A and STAT5A was influenced by time and breed, with major changes observed in STAT5A. CAT and SOD1 expression significantly increased in both WL and PC birds, regardless of the source of infection. GPX1 and GPX2 expression also increased in both WL and PC, although overall lower expression was observed in PC chickens at 24 h compared to 6 h. Our data suggest systemic changes in the host during early infection, indicated by the altered expression of PRs, DDX5, BCL2, ANGPTL4, and other regulatory genes during early MDV infection. The relative expression of these responses in PC and WL chickens suggests they may play a key role in their response to natural MDV infection in the lungs and long-term pathogenesis and survival.

mRNA Splicing of UL44 and Secretion of Alphaherpesvirinae Glycoprotein C (gC) Is Conserved among the Mardiviruses.

Marek's disease (MD), caused by gallid alphaherpesvirus 2 (GaAHV2) or Marek's disease herpesvirus (MDV), is a devastating disease in chickens characterized by the development of lymphomas throughout the body. Vaccine strains used against MD include gallid alphaherpesvirus 3 (GaAHV3), a non-oncogenic chicken alphaherpesvirus homologous to MDV, and homologous meleagrid alphaherpesvirus 1 (MeAHV1) or turkey herpesvirus (HVT). Previous work has shown most of the MDV gC produced during in vitro passage is secreted into the media of infected cells although the predicted protein contains a transmembrane domain. We formerly identified two alternatively spliced gC mRNAs that are secreted during MDV replication in vitro, termed gC104 and gC145 based on the size of the intron removed for each UL44 (gC) transcript. Since gC is conserved within the Alphaherpesvirinae subfamily, we hypothesized GaAHV3 (strain 301B/1) and HVT also secrete gC due to mRNA splicing. To address this, we collected media from 301B/1- and HVT-infected cell cultures and used Western blot analyses and determined that both 301B/1 and HVT produced secreted gC. Next, we extracted RNAs from 301B/1- and HVT-infected cell cultures and chicken feather follicle epithelial (FFE) skin cells. RT-PCR analyses confirmed one splicing variant for 301B/1 gC (gC104) and two variants for HVT gC (gC104 and gC145). Interestingly, the splicing between all three viruses was remarkably conserved. Further analysis of predicted and validated mRNA splicing donor, branch point (BP), and acceptor sites suggested single nucleotide polymorphisms (SNPs) within the 301B/1 UL44 transcript sequence resulted in no gC145 being produced. However, modification of the 301B/1 gC145 donor, BP, and acceptor sites to the MDV UL44 sequences did not result in gC145 mRNA splice variant, suggesting mRNA splicing is more complex than originally hypothesized. In all, our results show that mRNA splicing of avian herpesviruses is conserved and this information may be important in developing the next generation of MD vaccines or therapies to block transmission.

Oncogenic Animal Herpesviruses.

Oncogenic viruses play a pivotal role in oncology due to their unique role in unraveling the complexities of cancer development. Understanding the role viruses play in specific cancers is important to provide basic insights into the transformation process, which will help identify potential cellular targets for treatment. This review discusses the diverse role of animal herpesviruses in initiating and promoting various forms of cancer. We will summarize the mechanisms that underlie the development of animal herpesvirus-induced cancer that may provide a basis for developing potential therapeutic interventions or preventative strategies in the future.

Marek's disease virus expresses multiple UL44 (gC) variants through mRNA splicing that are all required for efficient horizontal transmission.

Marek's disease (MD) is a devastating oncogenic viral disease of chickens caused by Gallid herpesvirus 2, or MD virus (MDV). MDV glycoprotein C (gC) is encoded by the alphaherpesvirus UL44 homolog and is essential for the horizontal transmission of MDV (K. W. Jarosinski and N. Osterrieder, J. Virol. 84:7911-7916, 2010). Alphaherpesvirus gC proteins are type 1 membrane proteins and are generally anchored in cellular membranes and the virion envelope by a short transmembrane domain. However, the majority of MDV gC is secreted in vitro, although secondary-structure analyses predict a carboxy-terminal transmembrane domain. In this report, two alternative mRNA splice variants were identified by reverse transcription (RT)-PCR analyses, and the encoded proteins were predicted to specify premature stop codons that would lead to gC proteins that lack the transmembrane domain. Based on the size of the intron removed for each UL44 (gC) transcript, they were termed gC104 and gC145. Recombinant MDV viruses were generated in which only full-length viral gC (vgCfull), gC104 (vgC104), or gC145 (vgC145) was expressed. Predictably, gCfull was expressed predominantly as a membrane-associated protein, while both gC104 and gC145 were secreted, suggesting that the dominant gC variants expressed in vitro are the spliced variants. In experimentally infected chickens, the expression of each of the gC variants individually did not alter replication or disease induction. However, horizontal transmission was reduced compared to that of wild-type or revertant viruses when the expression of only a single gC was allowed, indicating that all three forms of gC are required for the efficient transmission of MDV in chickens.

Fluorescently tagged pUL47 of Marek's disease virus reveals differential tissue expression of the tegument protein in vivo.

Marek's disease virus (MDV), a lymphotropic alphaherpesvirus, causes Marek's disease (MD) in chickens. MD is characterized by neurological signs, chronic wasting, and T cell lymphomas that predominate in the visceral organs. MDV replicates in a highly cell-associated manner in vitro and in vivo, with infectious virus particles being released only from feather follicle epithelial (FFE) cells in the skin. Virus produced and shed from FFE cells allows transmission of MDV from infected to naïve chickens, but the mechanisms or roles of differential virus gene expression have remained elusive. Here, we generated recombinant MDV in which we fused enhanced green fluorescent protein (EGFP) to the C terminus of the tegument protein pUL47 (vUL47-EGFP) or pUL49 (vUL49-EGFP). While vUL49-EGFP was highly attenuated in vitro and in vivo, vUL47-EGFP showed unaltered pathogenic potential and stable production of pUL47-EGFP, which facilitated direct analysis of pUL47 expression in cells and tissues. Our studies revealed that pUL47-EGFP is expressed at low levels and localizes to the nucleus during lytic replication in vitro and in lymphocytes in the spleen in vivo, while it is undetectable in tumors. In contrast, pUL47-EGFP is highly abundant and localizes predominantly in the cytoplasm in FFE cells in the skin, where MDV is shed into the environment. We concluded that differential expression and localization of MDV pUL47-EGFP tegument protein is potentially important for the unique cell-associated nature of MDV in vitro and in lymphocytes in vivo, as well as production of free virus in FFE cells.

Herpesvirus telomerase RNA (vTR) with a mutated template sequence abrogates herpesvirus-induced lymphomagenesis.

Telomerase reverse transcriptase (TERT) and telomerase RNA (TR) represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR) on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV) as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5) by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1) that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2) that this strategy could be used to generate novel vaccine candidates against virus-induced lymphoma.

Importance of differential expression of Marek's disease virus gene pp38 for the pathogenesis of Marek's disease.

The role of pp38 in the pathogenesis of Marek's disease (MD) has not been fully elucidated. Previously, we reported the presence of two splice variants (Spl A and Spl B) for pp38. We also reported that the wild-type pp38 (WT), as well as the mutated pp38 (MUT), altered the oxidative phosphorylation pathway in infected cells. To determine whether the different forms of pp38 are important for the pathogenesis of MD, we generated RB-1B-based bacterial artificial chromosome (BAC) clones expressing pp38MUT, pp38Sp1 A, and pp38Spl B. Infectious viruses were recovered from these BAC clones in chick kidney cells (CKC). The Spl A and Spl B viruses had significantly smaller plaque sizes and replicated to a lesser degree in CKC than the WT and MUT viruses. Two in vivo experiments were conducted by inoculating 7-day-old P2a chicks with 1000 plaque-forming units of each virus. In the first experiment, chicks were sacrificed at 4, 8, 11, and 15 days postinfection (PI). WT and MUT viruses had similar viremia levels using virus isolation and quantitative real-time PCR (qPCR) assays, whereas Spl A and Spl B viruses had significantly lower viremia levels than WT and MUT viruses. In the second experiment, we showed that tumor development and MD mortality were similar in the WT- and MUT-infected chickens, with all birds MD positive at 5 wk PI. In contrast, chickens infected with Spl B and Spl A had a significantly lower MD incidence at 11 wk PI, when the experiment was terminated.

A deletion in the glycoprotein L (gL) gene of U.S. Marek's disease virus (MDV) field strains is insufficient to confer increased pathogenicity to the bacterial artificial chromosome (BAC)-based strain, RB-1B.

Marek's disease (MD) is a highly transmissible, herpesvirus-associated malignancy of chickens and turkeys caused by Marek's disease virus (MDV). MD is currently controlled through the use of nonsterilizing vaccines composed of antigenically related, apathogenic herpesviruses Mardivirus 2 (MDV-2), Meleagrid herpesvirus 1 (herpesvirus of turkeys, HVT), or attenuated MDV-1 strain CVI988 (Rispens). Since the mid-1960s, field strains of MDV have increased in virulence, due, in part, to the widespread use of vaccines since the early 1970s. One mutation that we have identified common to very virulent field strains (vv and vv+MDVs) since the 1990s has been a mutation in the UL1 gene, encoding glycoprotein L (gL). This mutation, a 12-nucleotide (nt) deletion in the signal peptide of gL, has been associated with increased virulence and decreased vaccine protection in the context of challenge with a vv+MDV, strain TK. To determine whether this mutation alone was sufficient to confer increased virulence, we introduced this mutation into the transmission-competent pRB-1B bacterial artificial chromosome (BAC) using two-step, Red-mediated recombination. The resulting mutant, pRB-1BgLdelta, was tested for changes in replication in cell culture using multistep growth curves, plaque size analysis, viral burst analysis, and the ability to compete with the parental virus when co-transfected at different ratios and sequentially passaged. In addition, we examined this mutant for changes in pathogenicity in inoculated and contact-exposed unvaccinated and vaccinated chickens. Our data show minor differences in plaque sizes in cell culture, but no discernible changes in the infection of specific-pathogen-free (SPF) leghorn chickens. We therefore conclude that although this mutation is indeed common to MDV field strains isolated in the eastern United States, it is insufficient to confer increased virulence or loss of vaccine protection previously observed for a vv+MDV strain having this mutation.

Purinergic signaling during Marek's disease in chickens.

Purinergic receptors (PRs) have been reported as potential therapeutic targets for many viral infections including herpesviruses, which urges the investigation into their role in Marek's disease (MD), a herpesvirus induced cancer in chickens that is an important pathogen for the poultry industry. MD is caused by MD virus (MDV) that has a similar viral life cycle as human varicella zoster virus in that it is shed from infected epithelial skin cells and enters the host through the respiratory route. In this report, PR responses during natural MDV infection and disease progression was examined in MD-resistant white Leghorns (WL) and MD-susceptible Pure Columbian (PC) chickens during natural infection. Whole lung lavage cells (WLLC) and liver tissue samples were collected from chickens infected but showing no clinical signs of MD (Infected) or presenting with clinical disease (Diseased). RNA was extracted followed by RT-qPCR analysis with gene specific primers against members of the P1, P2X, and P2Y PR families. Differential expression (p < 0.05) was observed in breed and disease conditions. Some PRs showed tissue specific expression (P1A1, P2X1, and P2X6 in WLLC) whereas others responded to MDV infection only in MD-susceptible (PC) chickens (P1A2A, P2X1, P2X5, P2X7). P2Y PRs had differential expression in both chicken lines in response to MDV infection and MD progression. This study is the first to our knowledge to examine PR responses during MDV infection and disease progression. These results suggest PR signaling may an important area of research for MDV replication and MD.

Herpesvirus telomerase RNA(vTR)-dependent lymphoma formation does not require interaction of vTR with telomerase reverse transcriptase (TERT).

Telomerase is a ribonucleoprotein complex involved in the maintenance of telomeres, a protective structure at the distal ends of chromosomes. The enzyme complex contains two main components, telomerase reverse transcriptase (TERT), the catalytic subunit, and telomerase RNA (TR), which serves as a template for the addition of telomeric repeats (TTAGGG)(n). Marek's disease virus (MDV), an oncogenic herpesvirus inducing fatal lymphoma in chickens, encodes a TR homologue, viral TR (vTR), which significantly contributes to MDV-induced lymphomagenesis. As recent studies have suggested that TRs possess functions independently of telomerase activity, we investigated if the tumor-promoting properties of MDV vTR are dependent on formation of a functional telomerase complex. The P6.1 stem-loop of TR is known to mediate TR-TERT complex formation and we show here that interaction of vTR with TERT and, consequently, telomerase activity was efficiently abrogated by the disruption of the vTR P6.1 stem-loop (P6.1mut). Recombinant MDV carrying the P6.1mut stem-loop mutation were generated and tested for their behavior in the natural host in vivo. In contrast to viruses lacking vTR, all animals infected with the P6.1mut viruses developed MDV-induced lymphomas, but onset of tumor formation was significantly delayed. P6.1mut viruses induced enhanced metastasis, indicating functionality of non-complexed vTR in tumor dissemination. We discovered that RPL22, a cellular factor involved in T-cell development and virus-induced transformation, directly interacts with wild-type and mutant vTR and is, consequently, relocalized to the nucleoplasm. Our study provides the first evidence that expression of TR, in this case encoded by a herpesvirus, is pro-oncogenic in the absence of telomerase activity.

Marek's disease virus late protein expression in feather follicle epithelial cells as early as 8 days postinfection.

Marek's disease virus (MDV) or Gallid herpesvirus 2 (GaHV-2) is a lymphotropic alphaherpesvirus and causes Marek's disease. Former studies have demonstrated that MDV is spread from chicken to chicken about 2 wk postexposure as infectious dander shed from infected chickens. More recent reports, using highly sensitive quantitative PCR analyses of dander from infected chickens, suggested that MDV replicates and is shed from the chicken much earlier (5-7 days). However, detection of viral DNA in chicken dander does not indicate whether fully infectious virus is present. To determine if viral replication is present in the skin of infected chickens at these early times, expression of a late viral protein indicative of fully productive virus replication was evaluated using fluorescent microscopy. To do this, highly virulent and attenuated recombinant (r)MDV was generated that abundantly expresses the monomeric red fluorescent protein fused to the late UL47 (VP13/14) protein in feather follicle epithelial cells. Detection of viral DNA could be detected in the skin of infected chickens as early as 6 days postinfection (p.i.), consistent with previous reports detecting viral DNA in dander shed from infected chickens. Replication of virulent rMDV was evident in the feather follicles as early as 8 days p.i., while attenuated rMDV replication in the feather follicles was delayed 1-2 days. Former studies, using less sensitive techniques, suggested viral protein expression to occur about 10-12 days p.i. Undoubtedly differences in time of detection can partly be explained by multiple factors including the pathotype of virus, the route of infection, and the age and genetic line of the infected chickens used in different studies. In summary, though viral DNA can be detected as early as 6 days p.i., late viral protein expression, indicative of infectious virus production, occurs 2-3 days after DNA detection, but earlier than previously thought.

Coinfection in the host can result in functional complementation between live vaccines and virulent virus.

One of the greatest achievements of the last century is the development of vaccines against viral diseases. Vaccines are essential for battling infectious diseases and many different formulations are available, including live attenuated vaccines. However, the use of live attenuated vaccines has the potential for adverse effects, including reversion of pathogenicity, recombination, and functional complementation in the host. Marek's disease is a serious disease in poultry controlled by live attenuated vaccines that has resulted in increased virulence over the decades. Recombination between circulating field viruses or vaccines is a proposed mechanism for the increase in virulence, however, complementation between vaccines and field strains has not been demonstrated in chickens. Here, we describe functional complementation of vaccines with virulent virus to functionally complement transmission and spread in the host. Using the natural virus-host model of Marek's disease in chickens, our results show dual infection of target cells in chickens with vaccine and virulent virus providing the opportunity for recombination or complementation to transpire. Interestingly, our controlled results showed no evidence of recombination between vaccine and virulent virus, but functional complementation occurred in two independent experiments providing proof for complementation during natural infection in vaccinated individuals. These results suggest complementation as a potential mechanism for vaccine-mediated viral evolution and the potential for complementation should be taken into consideration when developing novel vaccines.