Impact of viral telomeric repeat sequences on herpesvirus vector vaccine integration and persistence.

Marek's disease virus (MDV) vaccines were the first vaccines that protected against cancer. The avirulent turkey herpesvirus (HVT) was widely employed and protected billions of chickens from a deadly MDV infection. It is also among the most common vaccine vectors providing protection against a plethora of pathogens. HVT establishes latency in T-cells, allowing the vaccine virus to persist in the host for life. Intriguingly, the HVT genome contains telomeric repeat arrays (TMRs) at both ends; however, their role in the HVT life cycle remains elusive. We have previously shown that similar TMRs in the MDV genome facilitate its integration into host telomeres, which ensures efficient maintenance of the virus genome during latency and tumorigenesis. In this study, we investigated the role of the TMRs in HVT genome integration, latency, and reactivation in vitro and in vivo. Additionally, we examined HVT infection of feather follicles. We generated an HVT mutant lacking both TMRs (vΔTMR) that efficiently replicated in cell culture. We could demonstrate that wild type HVT integrates at the ends of chromosomes containing the telomeres in T-cells, while integration was severely impaired in the absence of the TMRs. To assess the role of TMRs in vivo, we infected one-day-old chickens with HVT or vΔTMR. vΔTMR loads were significantly reduced in the blood and hardly any virus was transported to the feather follicle epithelium where the virus is commonly shed. Strikingly, latency in the spleen and reactivation of the virus were severely impaired in the absence of the TMRs, indicating that the TMRs are crucial for the establishment of latency and reactivation of HVT. Our findings revealed that the TMRs facilitate integration of the HVT genome into host chromosomes, which ensures efficient persistence in the host, reactivation, and transport of the virus to the skin.

The dominantly expressed class II molecule from a resistant MHC haplotype presents only a few Marek's disease virus peptides by using an unprecedented binding motif.

Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek's disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek's disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek's disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek's disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans.

Avian oncogenic herpesvirus antagonizes the cGAS-STING DNA-sensing pathway to mediate immune evasion.

The cellular DNA sensor cGMP-AMP synthase (cGAS) detects cytosolic viral DNA via the stimulator of interferon genes (STING) to initiate innate antiviral response. Herpesviruses are known to target key immune signaling pathways to persist in an immune-competent host. Marek's disease virus (MDV), a highly pathogenic and oncogenic herpesvirus of chickens, can antagonize host innate immune responses to achieve persistent infection. With a functional screen, we identified five MDV proteins that blocked beta interferon (IFN-ß) induction downstream of the cGAS-STING pathway. Specifically, the MDV major oncoprotein Meq impeded the recruitment of TANK-binding kinase 1 and IFN regulatory factor 7 (IRF7) to the STING complex, thereby inhibiting IRF7 activation and IFN-ß induction. Meq overexpression markedly reduced antiviral responses stimulated by cytosolic DNA, whereas knockdown of Meq heightened MDV-triggered induction of IFN-ß and downstream antiviral genes. Moreover, Meq-deficient MDV induced more IFN-ß production than wild-type MDV. Meq-deficient MDV also triggered a more robust CD8+ T cell response than wild-type MDV. As such, the Meq-deficient MDV was highly attenuated in replication and lymphoma induction compared to wild-type MDV. Taken together, these results revealed that MDV evades the cGAS-STING DNA sensing pathway, which underpins the efficient replication and oncogenesis. These findings improve our understanding of the virus-host interaction in MDV-induced lymphoma and may contribute to the development of novel vaccines against MDV infection.

Efficacy of a turkey herpesvirus double construct vaccine (HVT-ND-IBD) against challenge with different strains of Newcastle disease, infectious bursal disease and Marek's disease viruses.

A double construct vaccine of turkey herpesvirus (HVT) was prepared that contains the fusion (F) gene from Newcastle disease virus (NDV) and the viral protein 2 (VP2) gene from infectious bursal disease virus (IBDV). Safety of the vaccine (HVT-ND-IBD) was confirmed and efficacy was evaluated after subcutaneous (SC) vaccination at 1 day of age or the in ovo route of vaccination. Challenges were performed with velogenic NDV strains (Texas GB and Herts Weybridge 33/56), with different strains of IBDV (classical strain STC; very virulent strain CS89 and variant E strain) and with Marek's disease virus (MDV) strain RB1B. Vaccination with HVT-ND-IBD induced a high level of protection against these challenges. Vaccination with HVT is often combined with Rispens CVI988 vaccine and live ND vaccines for higher and earlier, MD and ND protection, respectively. HVT-ND-IBD vaccination in combination with these vaccines showed MD protection as early as 4 days post vaccination and ND protection as early as 2 weeks post vaccination. The long protection as seen with HVT vaccination was confirmed by demonstrating protection against NDV up to 60 weeks. Finally, to evaluate the performance of the vaccine in commercial birds with maternally-derived antibodies, two field trials were performed, using in ovo vaccination in broilers and SC vaccination in combination with Rispens CVI988 vaccine in layer-type birds. The efficacy was confirmed for all components by challenges. These results demonstrate that HVT-ND-IBD is a safe and highly efficacious vaccine for simultaneous control of ND, IBD and MD. RESEARCH HIGHLIGHTS A double construct HVT vaccine with the NDV F and the IBDV VP2 genes was prepared. The vaccine protects against three important diseases: MDV, NDV and IBDV. In ovo and sub-cutaneous vaccination was evaluated in the field in commercial chickens.

Revisiting cellular immune response to oncogenic Marek's disease virus: the rising of avian T-cell immunity.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes deadly T-cell lymphomas and serves as a natural virus-induced tumor model in chickens. Although Marek's disease (MD) is well controlled by current vaccines, the evolution of MDV field viruses towards increasing virulence is concerning as a better vaccine to combat very virulent plus MDV is still lacking. Our understanding of molecular and cellular immunity to MDV and its immunopathogenesis has significantly improved, but those findings about cellular immunity to MDV are largely out-of-date, hampering the development of more effective vaccines against MD. T-cell-mediated cellular immunity was thought to be of paramount importance against MDV. However, MDV also infects macrophages, B cells and T cells, leading to immunosuppression and T-cell lymphoma. Additionally, there is limited information about how uninfected immune cells respond to MDV infection or vaccination, specifically, the mechanisms by which T cells are activated and recognize MDV antigens and how the function and properties of activated T cells correlate with immune protection against MDV or MD tumor. The current review revisits the roles of each immune cell subset and its effector mechanisms in the host immune response to MDV infection or vaccination from the point of view of comparative immunology. We particularly emphasize areas of research requiring further investigation and provide useful information for rational design and development of novel MDV vaccines.

Unraveling the role of B cells in the pathogenesis of an oncogenic avian herpesvirus.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes immunosuppression, paralysis, and deadly lymphomas in chickens. In infected animals, B cells are efficiently infected and are thought to amplify the virus and transfer it to T cells. MDV subsequently establishes latency in T cells and transforms CD4+ T cells, resulting in fatal lymphomas. Despite many years of research, the exact role of the different B and T cell subsets in MDV pathogenesis remains poorly understood, mostly due to the lack of reverse genetics in chickens. Recently, Ig heavy chain J gene segment knockout (JH-KO) chickens lacking mature and peripheral B cells have been generated. To determine the role of these B cells in MDV pathogenesis, we infected JH-KO chickens with the very virulent MDV RB1B strain. Surprisingly, viral load in the blood of infected animals was not altered in the absence of B cells. More importantly, disease and tumor incidence in JH-KO chickens was comparable to wild-type animals, suggesting that both mature and peripheral B cells are dispensable for MDV pathogenesis. Intriguingly, MDV efficiently replicated in the bursa of Fabricius in JH-KO animals, while spread of the virus to the spleen and thymus was delayed. In the absence of B cells, MDV readily infected CD4+ and CD8+ T cells, allowing efficient virus replication in the lymphoid organs and transformation of T cells. Taken together, our data change the dogma of the central role of B cells, and thereby provide important insights into MDV pathogenesis.

Vaccinal efficacy of molecularly cloned Gallid alphaherpesvirus 3 strain 301B/1 against very virulent Marek's disease virus challenge.

Marek's disease virus (MDV), a causative agent of Marek's disease, has evolved its virulence partly because the current control strategies fail to provide sterilizing immunity. Gallid alphaherpesvirus 3 (GaHV-3) and turkey herpesvirus have been developed as bivalent vaccines to improve upon the level of protection elicited by single formulations. Since the in vitro passage of vaccines can result in attenuation, a GaHV-3 strain 301B/1 was cloned as a bacterial artificial chromosome (BAC) by inserting the mini-F replicon into the virus genome. A fully infectious virus, v301B-BAC, was reconstituted from the 301B/1 BAC clone and had similar growth kinetics comparable to that of the parental 301B/1 virus with strong reactivity against anti-301B/1 chicken sera. Protective efficacies of v301B-BAC, parental 301B/1, and SB-1 vaccine were evaluated against a very virulent MDV Md5 challenge. Clinical signs were significantly lower in the v301B-BAC vaccinated groups (24-25 %), parental 301B/1 (29 %) compare to that of non-vaccinated control (100%) and the removal of BAC sequences from v301B-BAC genome further reduced this to 17 %. The protective indices of v301B-BACs (75-76 %) were comparable with those of both the 301B/1 and the SB-1 vaccine (71%). Removal of the mini-F replicon resulted in a reconstituted virus with a protective index of 83 %. The shedding of challenge virus was notably lower in the v301B-BAC, and v301B-delBAC vaccinated groups. Overall, the protective efficacy of the 301B-BAC-derived vaccine virus against a very virulent MDV challenge was comparable to that of the parental 301B/1 virus as well as the SB-1 vaccine virus.

Attenuation of a very virulent Marek's disease herpesvirus (MDV) by codon pair bias deoptimization.

Codon pair bias deoptimization (CPBD) has enabled highly efficient and rapid attenuation of RNA viruses. The technique relies on recoding of viral genes by increasing the number of codon pairs that are statistically underrepresented in protein coding genes of the viral host without changing the amino acid sequence of the encoded proteins. Utilization of naturally underrepresented codon pairs reduces protein production of recoded genes and directly causes virus attenuation. As a result, the mutant virus is antigenically identical with the parental virus, but virulence is reduced or absent. Our goal was to determine if a virus with a large double-stranded DNA genome, highly oncogenic Marek's disease virus (MDV), can be attenuated by CPBD. We recoded UL30 that encodes the catalytic subunit of the viral DNA polymerase to minimize (deoptimization), maximize (optimization), or preserve (randomization) the level of overrepresented codon pairs of the MDV host, the chicken. A fully codon pair-deoptimized UL30 mutant could not be recovered in cell culture. The sequence of UL30 was divided into three segments of equal length and we generated a series of mutants with different segments of the UL30 recoded. The codon pair-deoptimized genes, in which two segments of UL30 had been recoded, showed reduced rates of protein production. In cultured cells, the corresponding viruses formed smaller plaques and grew to lower titers compared with parental virus. In contrast, codon pair-optimized and -randomized viruses replicated in vitro with kinetics that were similar to those of the parental virus. Animals that were infected with the partially codon pair-deoptimized virus showed delayed progression of disease and lower mortality rates than codon pair-optimized and parental viruses. These results demonstrate that CPBD of a herpesvirus gene causes attenuation of the recoded virus and that CPBD may be an applicable strategy for attenuation of other large DNA viruses.

Detection of p53 mutation and serum monitoring alert caused by Marek's disease virus in poultry.

BACKGROUND: Marek's disease (MD) is a chicken neoplastic disease, which brings huge economic losses to the global poultry industry. The wild type p53, a tumor suppressor gene, plays a key role in blocking cell cycle, promoting apoptosis, and maintaining the stability of the genome. However, the mutant p53 losses its tumor inhibitory role and become an oncogene when a mutation has happened. RESULTS: The mutation rate of p53 was 60% in the experimentally and naturally infected chickens. The mutations included point-mutations and deletions, and mostly located in the DNA-binding domain. The mutated p53 was expressed in various tumor tissues in an infected chicken. The mutant P53 proteins were notably accumulated in the cytoplasm due to the loss in the function of nuclear localization. Unlike the study on human cancer, the concentrations of P53 in the serums of MD infected chicken were significantly lower than the control group. CONCLUSIONS: The p53 mutations were apparent in the development of MD. P53 and P53 antibody level in serum could be a useful marker in the diagnosis and surveillance of MD.

A double recombinant herpes virus of turkeys for the protection of chickens against Newcastle, infectious laryngotracheitis and Marek's diseases.

A double recombinant strain of herpes virus of turkeys (HVT) was constructed that contains the fusion (F) gene from Newcastle disease virus (NDV) and the gD plus gI genes from infectious laryngotracheitis virus (ILTV) inserted into a non-essential region of the HVT genome. Expression of the F protein was controlled by a human cytomegalovirus promoter, whereas expression of gD plus gI was driven by an ILTV promoter. The double recombinant vaccine virus (HVT-NDV-ILT) was fully stable genetically and phenotypically following extended passage in cell culture and infection of chickens. Safety of the vaccine virus was confirmed by overdose and backpassage studies in specific-pathogen-free chickens. Chickens vaccinated with a single dose of HVT-NDV-ILT administered by the in ovo route were highly protected from challenge with the velogenic NDV (GB Texas), ILTV (LT 96-3) and Marek's disease virus (GA 5) strains (97%, 94% and 97%, respectively). Similarly, chickens vaccinated with a single dose by subcutaneous (SC) route at 1 day of age were highly protected from challenge with the same three viruses (100%, 100%, and 88%, respectively). The protection level of a single dose given by in ovo or SC route against challenge with a virulent Marek's disease virus strain demonstrates that insertion of multiple genes from two different pathogens within the HVT genome had no adverse effect on the capacity of HVT to protect against Marek's disease. These results demonstrate that HVT-NDV-ILT is a safe and efficacious vaccine for simultaneous control of NDV, ILTV and Marek's diseases.

Suspension culture of Marek's disease virus and evaluation of its immunological effects.

Marek's disease virus (MDV) is a cell-associated α-herpesvirus of chickens. It is difficult to grow MDV in suspension culture. Therefore, MDV vaccines are currently produced using adherent primary chicken embryo fibroblasts, and on a large scale this is labour-intensive and costly. In this study, the CVI988 strain of MDV was inoculated into chicken fibroblast cell line UMNSAH/DF-1 (DF-1) cultured by microcarrier suspension for the proliferation experiment. Moreover, the effects of culture conditions, such as inoculation method, multiplicity of infection (MOI), microcarrier concentration, and pH value, on the proliferation of MDV were investigated. The results demonstrated that the maximum viral load of 64.76 ± 2.64 × 106 PFU/flask in a working volume of 100 ml could be obtained using synchronous cell seeding and inoculation method at an MOI of 0.02 and a microcarrier concentration of 5 g/l at pH 7.2. At the same time, the CVI988/DF-1 vaccines prepared by the microcarrier culture process and the traditional adherent cell culture process (CVI988/Rispens) were compared through bird experiments. We found a protective rate of 94.4% using the CVI988/DF-1 vaccine with specific pathogen-free chickens that was equivalent to that of the commercial vaccine CVI988/Rispens (protection rate of 94.1%). In this study, the MDV CVI988/DF-1 vaccine prepared by the microcarrier suspension culture of DF-1 cells could provide effective immune protection for specific pathogen-free chickens, providing a reference for the prevention and control of MD and further development of a large-scale bioreactor for producing the MD vaccine.

PFOS mediates immunomodulation in an avian cell line that can be mitigated via a virus infection.

BACKGROUND: Per- and polyfluoroalkyl substances (PFASs) are environmentally persistent and bioaccumulative chemicals. Immunomodulation is among the most concerning of toxic effects linked with PFAS exposure in mammalian models. However, no studies had yet shown this to be true in birds. Thus, we designed and conducted the first study to determine if PFASs could cause immunomodulation in birds. Secondly, we wanted to determine the effects on an avian host when exposed not only to immunomodulating chemicals, but also to a viral challenge. The aim, to determine if PFAS mediated immunmodulation functionally affects a pathogen challenge for a host. As innate immune system signalling pathways initiate crucial responses against a pathogen challenge, and are lesser studied than their adaptive counterparts, we focused on these pathways. To provide the first information on this, an in vitro experiment was designed and performed using chicken embryo fibroblasts exposed to perfluorooctane sulfonate (PFOS) (22 ppm) and immune markers characterised before and after being infected with gallid herpesvirus-2 (GaHV-2). RESULTS: The expression of two pro-inflammatory cytokines, namely interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α), the nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB), and the anti-inflammatory cytokine interleukin 4 (IL-4) were investigated in various scenarios. These results showed that exposure to PFOS decreased immune gene expression in chicken fibroblasts from 36 h post-exposure. Next, it was shown that this decrease could be mitigated by infection with gallid herpesvirus-2, which increased gene expression back to the baseline/control levels. CONCLUSIONS: Not only is this the first study to provide the expected evidence that PFOS has immunomodulatory potential in birds, it also provides unexpected data that virus infections can mitigate this negative effect. Thereby, further research, including in vivo and in situ studies, on the impact of PFOS on host-virus interactions is now warranted, as it has been overlooked and might contribute to our understanding of recent disease outbreaks in wildlife. The mechanisms by which gallid herpesvirus mitigates immunomodulation were beyond the scope of this study, but are now of interest for future study.

Differential attenuation of Marek's disease virus-induced tumours and late-Marek's disease virus-induced immunosuppression.

Marek's disease virus (MDV) is a herpesvirus that induces lymphoma and a variety of non-neoplastic syndromes in chickens. Furthermore, very virulent plus (vv+) MDVs induce a form of immunosuppression (late-MDV-IS) that might involve both neoplastic and non-neoplastic mechanisms. The objective of this study was to evaluate whether the attenuation of MDV-induced tumours and late-MDV-IS occurs simultaneously or can be dissociated. The immunosuppressive ability of three viruses derived from vv+ MDV strain 686 (wild-type 686, the somewhat attenuated molecular clone 686-BAC, and the nononcogenic molecular clone lacking the two copies of the oncogene meq 686-BACΔMEQ) was evaluated. Late-MDV-IS was evaluated indirectly by assessing the negative effect of MDV strains on the protection conferred by infectious laryngotracheitis (ILT) vaccines. Our results showed that the ability to induce late-MDV-IS was attenuated before the ability to induce tumours. Strain 686 induced both tumours and late-MDV-IS, 686-BAC induced tumours but did not induce late-MDV-IS and 686-BACΔMEQ did not induce either tumours or late-MDV-IS. Further comparison of strains 686 and 686-BAC revealed that strain 686 reduced the humoral immune responses to ILTV (1132 vs 2167) more severely, showed higher levels of meq transcripts (2.1E+09 vs 4.98E+8) and higher expression of MDV microRNAs (mdv1-miR-M4-5p and mdv1-miR-M2-3p) in the spleen, and further reduced the percentage of CD45+-MHC-I+splenocytes (13 vs32 %) compared to molecular clone 686-BAC. This study suggests that the immunosuppressive ability of MDV might follow a continuous spectrum and only the most virulent MDVs can overcome a certain threshold level and induce clinical MDV-IS in the ILT model.

Turkey herpesvirus with an insertion in the UL3-4 region displays an appropriate balance between growth activity and antibody-eliciting capacity and is suitable for the establishment of a recombinant vaccine.

We constructed turkey herpesvirus (HVT) vector vaccines in which the VP2 gene of infectious bursal disease virus (IBDV) was inserted into the HVT genome in the following regions: UL3-4, UL22-23, UL45-46, and US10-SORF3. We then evaluated the relationship between the gene insertion site and the capacity of the virus to elicit antibodies. rHVT/IBD (US10) showed good growth activity in vitro, with growth comparable to that of the parent HVT. On the other hand, rHVT/IBD (UL3-4), rHVT/IBD (UL22-23), and rHVT/IBD (UL45-46) exhibited decreased growth activity in chicken embryo fibroblast (CEF) cells compared to the parent HVT. However, the rHVT/IBD (US10) elicited lower levels of virus-neutralizing (VN) antibodies compared to the other constructs. rHVT/IBD (UL3-4) and rHVT/IBD (UL45-46) appeared to be similar in their ability to elicit VN antibodies. Based on the results of in vitro and in vivo assays, rHVT/IBD (UL3-4) was selected for further testing. In a challenge assay, rHVT/IBD (UL3-4) protected chickens from challenge with virulent Marek's disease virus serotype 1 and IBDV. In conclusion, the site of gene insertion may have a strong effect on the growth of the vector virus in vitro and its antibody-eliciting capacity. Insertions in the UL3-4 region permitted a balance between growth activity and VN-antibody-eliciting capacity, and this region might therefore be an appropriate insertion site for IBDV VP2.

Characterization of a Gallid herpesvirus 2 strain with novel reticuloendotheliosis virus long terminal repeat inserts.

A bacterial artificial chromosome clone, designated LCY, was constructed from a Gallid herpesvirus 2 (GaHV-2) isolate from a GaHV-2 and reticuloendotheliosis virus co-infected clinical sample. The LCY GaHV-2 insert was sequenced and found to consist of 175,319 nucleotides. LCY GaHV-2 open reading frames (ORFs) had a high sequence identity to those of reference strains. The major difference was that two REV long terminal repeats (LTRs), in the same direction, were inserted at the internal repeat short (IRs)/unique short (Us) and Us/terminal repeat short (TRs) junctions. In addition, the a-like sequence and UL36 were different from other strains. Phylogenetic analysis revealed that LCY was closely related to pandemic strains in China. A pathogenicity study and a vaccination-challenge test were performed on LCY and the reference strain, GA. The results showed that LCY induced gross Marek's disease (MD) lesions and mortality in 71.4 and 7.1% of chickens, respectively, which are lower rates than those observed for the reference strain GA (85.7 and 35.7%). The commercially available CVI988 vaccine provided complete protection against LCY and GA (100%). These results showed that the isolate exhibited lower pathogenicity in SPF chickens. This study revealed that a novel pattern of LTR inserts was found in the strain LCY and that the strain was of low virulence. The present work expands the available genetic information for GaHV-2 and will be useful for the control of MD in China.

Differential amplification of Marek's disease CVI988 vaccine and of wild-type isolates from organs of commercial chickens using single or duplexed probes in real-time PCR.

The differentiation of Marek's disease virus (MDV)-infected and vaccinated animal (DIVA) test, based on the MDV pp38 gene was described by Baigent et al. [(2016). Real-time PCR for differential quantification of CVI988 vaccine and virulent MDV strains. Journal of Virological Methods, 233, 23-36], using similar primers and alternate probes for virulent MDV-1 and the vaccine CVI988 virus. We explored the assay's applicability for commercial vaccines and commercial chickens, as the above-mentioned study employed tissue-cultured MDV strains and tissues from experimental trials. DNA of visceral organs and feathers of vaccinated or naturally infected chickens was used. Further, the applicability of the DIVA assay was evaluated using single or duplexed probes for the two viruses in the same amplification tube. Due to the high viral content in the commercial vaccines and in the clinical cases of MDV-1 infected commercial chickens, their examination by the MDV-1 DIVA real-time PCR was performed in one step. However, for the feather DNAs of commercially vaccinated birds, a step of pre-amplification was required. The MDV-1 DIVA real-time PCR performed as single probe in separate tubes using the Vir3 probe was very sensitive for virulent MDV-1 strains, but not very specific, as it also gave a clear signal with CVI988 vaccine virus. In contrast, the CVI vaccine probe was specific for CVI988, and did not recognize the MDV-1 strains. When both probes were present in one tube, the CVI probe showed a greater sensitivity for CV1988, while the Vir3 probe showed a much better specificity for virulent MDV-1.

Marek's disease in chickens: a review with focus on immunology.

Marek's disease (MD), caused by Marek's disease virus (MDV), is a commercially important neoplastic disease of poultry which is only controlled by mass vaccination. Importantly, vaccines that can provide sterile immunity and inhibit virus transmission are lacking; such that vaccines are only capable of preventing neuropathy, oncogenic disease and immunosuppression, but are unable to prevent MDV transmission or infection, leading to emergence of increasingly virulent pathotypes. Hence, to address these issues, developing more efficacious vaccines that induce sterile immunity have become one of the important research goals for avian immunologists today. MDV shares very close genomic functional and structural characteristics to most mammalian herpes viruses such as herpes simplex virus (HSV). MD also provides an excellent T cell lymphoma model for gaining insights into other herpesvirus-induced oncogenesis in mammals and birds. For these reasons, we need to develop an in-depth knowledge and understanding of the host-viral interaction and host immunity against MD. Similarly, the underlying genetic variation within different chicken lines has a major impact on the outcome of infection. In this review article, we aim to investigate the pathogenesis of MDV infection, host immunity to MD and discuss areas of research that need to be further explored.

Activation of Toll-like receptor 3 inhibits Marek's disease virus infection in chicken embryo fibroblast cells.

Toll-like receptor 3 (TLR3) is a critical component of the innate immune system against viral infection and controls the activation of adaptive immunity. The role of TLR3 in Marek's disease virus (MDV) infection is not clear. In this study, we found that the abundance of TLR3 mRNA was significantly higher in chicken embryo fibroblast cells (CEF) infected with MDV than in a control group. Activated TLR3 signaling via TLR3 ligand stimulation inhibited replication of the RB1B strain of MDV in CEF cells. In contrast, CEF cells transfected with TLR3 siRNA promoted RB1B infection and replication. However, treatment with other TLR ligands, whether stimulatory (LPS, imiquimod and CpG) or inhibitory (TLR2/4 inhibitor and/or MyD88 inhibitor), had little effect on RB1B infection and replication. In addition, we found that the expression trend of TLR3 mRNA in RB1B-infected CEF cells was similar to that of mdv1-mir-M4-5p (a functional ortholog of oncogenic miR-155 encoded by MDV). Inconsistent with this, the TLR3 protein level was sharply reduced in RB1B-infected CEF cells at 96 hpi, while there was an at least 200-fold increase in miR-M4-5p at the same time point. Additionally, CEF cells transfected with an mdv1-mir-M4-5p mimic promoted RB1B infection and replication, while an mdv1-mir-M4-5p inhibitor inhibited RB1B infection and replication. Similar results were observed in CEF cells transfected with a gga-miR-155 mimic or inhibitor. These findings suggest that TLR3 and MDV-encoded miRNAs might be involved in MDV infection.

Early infection with Marek's disease virus can jeopardize protection conferred by laryngotracheitis vaccines: a method to study MDV-induced immunosuppression.

Marek's disease virus (MDV) is a herpesvirus that induces lymphomas and immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is divided into two phases: early-MDV-IS occurring mainly in chickens lacking maternal antibodies (MAb) against MDV and associated with lymphoid organ atrophy; and late-MDV-IS occurring once MDV enters latency and during tumour development. Our objectives were to document the impact of late-MDV-IS on commercial poultry (meat-type chickens bearing MAb against MDV and that were vaccinated or unvaccinated against MD) and to optimize a model to study late-MDV-IS under laboratory conditions. The impact of late-MDV-IS was evaluated by assessing the effect of early infection (day of age) with a very virulent plus MDV (vv+MDV) on the efficacy of chicken-embryo-origin (CEO) infectious laryngotracheitis (ILT) virus vaccine against ILT challenge. The CEO ILT vaccine was administered in water at 14 days of age and ILT virus (ILTV) challenge was done intratracheally at 30 days of age. Development of ILT was monitored by daily evaluation of clinical signs, development of gross and histological lesions in trachea, and quantification of ILTV transcripts in trachea. Infection with vv+MDV strain 648A resulted in total abrogation of protection conferred by the CEO vaccine against ILTV challenge even in chickens vaccinated at 1 day of age with either HVT, HVT+SB-1, or CVI988. Chickens exposed to vv+MDV prior to vaccination with CEO ILTV vaccine had similar (P < 0.05) clinical scores, gross lesions, histopathologic lesion scores, and load of ILTV transcripts in trachea after ILTV challenge, as chickens that were not vaccinated with CEO ILTV vaccine.

Protection provided by Rispens CVI988 vaccine against Marek's disease virus isolates of different pathotypes and early prediction of vaccine take and MD outcome.

We tested the level of protection provided by the Rispens CVI988 (Rispens) vaccine against challenge with a virulent Marek's disease virus (MDV) pathotype (vMDV) and a very virulent pathotype (vvMDV) and the accuracy of a range of predictive measures of Marek's disease (MD) incidence and vaccine take. Commercial layer chicks (n = 236) were vaccinated (or not) with 4000 plaque-forming units (pfu) of Rispens vaccine at hatch and challenged (or not) with 500 pfu of each challenge virus five days post vaccination. The vvMDV pathotype FT158 induced higher MD incidence (65%) and mortality (33%) when compared with the vMDV pathotype MPF57 (39% and 8%, respectively). The protective index provided by the Rispens vaccine against FT158 (61%) did not differ significantly from that against MPF57 (66%). This provides additional evidence that protection provided by the Rispens vaccine is not influenced by pathotype determined in studies using vaccines of other Mardivirus species. The challenge viruses did not differ in MDV or Rispens viral load in spleen at 14 dpc (days post challenge) determined by specific quantitative polymerase chain reaction test. MDV load in peripheral blood leucocytes at 7 and 14 dpc, splenocytes at 14 dpc, feather cells at 14 and 21 dpc and isolator dust at 21 dpc were significant early indicators of subsequent MD incidence to 56 dpc. These are potentially useful as the sampling can be carried out well before the onset of MD and some measures are non-invasive. The Rispens viral load in both invasive and non-invasive samples was more useful as a measure of vaccine take.