Regulation of Wnt/ß-catenin signaling by Marek's disease virus in vitro and in vivo.

Marek's disease virus (MDV) infection causes immunosuppression in the host, ultimately inducing tumor formation and causing significant economic losses to the poultry industry. While the abnormal activation of the Wnt/ß-catenin signaling pathway is closely associated with the occurrence and development of tumors. However, the relationship between MDV and the Wnt/ß-catenin pathway remains unclear. In this study, we found that the MDV RB1B strain, but not the MDV vaccine strain CVI988, activated the Wnt/ß-catenin signaling pathway by increasing the phosphorylation level of GSK-3ß in chicken embryo fibroblast (CEF). In vivo infection experiments in SPF chickens also confirmed that the RB1B strain activated the Wnt/ß-catenin signaling pathway, while the CVI988 strain did not lead to its activation. Moreover, unlike the Meq protein encoded by the CVI988 strain, the Meq protein encoded by the RB1B strain specifically activated the Wnt/ß-catenin signaling pathway in CEF cells. The findings from these studies extend our understanding of the regulation of Wnt/ß-catenin signaling by MDV, which make a new contribution to understanding the virus-host interactions of MDV.

A quadruplex real-time PCR assay combined with a conventional PCR for the differential detection of Marek's disease virus vaccines and field strains.

To evaluate the effect of the vaccine and differentiate vaccine from virulent MDV, a new quadruplex real-time PCR assay based on TaqMan probes was developed to differentiate and accurately quantify HVT, CVI988 and virulent MDV-1. The results showed that the limit of detection (LOD) of the new assay was 10 copies with correlation coefficients >0.994 of CVI988, HVT and virulent MDV DNA molecules without cross-reactivity with other avian disease viruses. The intra-assay and inter-assay coefficients of variation (CVs) of Ct values for the new assay were less than 3%. Analysis of replication kinetics of CVI988 and virulent MDV of collected feathers between 7 and 60 days post-infection (dpi) showed MD5 had no significant effect on the genomic load of CVI988 (p > 0.05), while vaccination with CVI988 could significantly reduce the viral load of MD5 (p < 0.05). Combined with meq gene PCR, this method can effectively identify virulent MDV infections in immunized chickens. These results demonstrated that this assay could distinguish between the vaccine and virulent MDV strains and had the advantages of being reliable, sensitive and specific to confirm the immunization status and monitor the circulation of virulent MDV strains.

Effect of Insertion and Deletion in the Meq Protein Encoded by Highly Oncogenic Marek's Disease Virus on Transactivation Activity and Virulence.

Marek's disease virus (MDV) causes malignant lymphoma in chickens (Marek's disease, MD). Although MD is currently controlled by vaccination, MDV strains have continuously increased in virulence over the recent decades. Polymorphisms in Meq, an MDV-encoded oncoprotein that serves as a transcription factor, have been associated with the enhanced virulence of the virus. In addition, insertions and deletions in Meq have been observed in MDV strains of higher virulence, but their contribution to said virulence remains elusive. In this study, we investigated the contribution of an insertion (L-Meq) and a deletion in the Meq gene (S-Meq) to its functions and MDV pathogenicity. Reporter assays revealed that both insertion and deletion enhanced the transactivation potential of Meq. Additionally, we generated RB-1B-based recombinant MDVs (rMDVs) encoding each Meq isoform and analyzed their pathogenic potential. rMDV encoding L-Meq indueced the highest mortality and tumor incidence in infected animals, whereas the rMDV encoding S-Meq exhibited the lowest pathogenicity. Thus, insertion enhanced the transactivation activity of Meq and MDV pathogenicity, whereas deletion reduced pathogenicity despite having increased transactivation activity. These data suggest that other functions of Meq affect MDV virulence. These data improve our understanding of the mechanisms underlying the evolution of MDV virulence.

Comparison of three filter paper-based devices for safety and stability of viral sample collection in poultry.

General diagnosis of poultry viruses primarily relies on detection of viruses in samples, but many farms are located in remote areas requiring logistic transportation. Filter paper cards are a useful technology that offer an alternative for collecting and preserving samples without hazardous exposure. The goal of this study was to compare three filter papers: the Flinders Technology Associates filter (FTA®) card, dried blood spot (DBS) card and qualitative filter paper (FP) grade 2 to collect poultry samples. In particular, we have used Newcastle disease virus (NDV) to evaluate safety and a Marek's disease virus (MDV) attenuated vaccine (CVI988) to evaluate stability of viral DNA. This experiment was divided into two parts. The first part was to determine the DNA stability and detection limit of CVI988 in samples collected in different paper supports after four storage times (3, 7, 14 and 30 days post spot). The second part was to determine the safety of papers by evaluating the viral inactivation efficacy using NDV as a representative virus. Results showed that all papers could preserve CVI988 DNA at all times, with a detection limit of 0.5 PFU/5 µl for FTA® and DBS cards, and 5 PFU/5 µl for FP. Our results showed that the NDV remained viable and infectious on the DBS card and FP, while no viable virus was detected on the FTA® card, suggesting that the FTA® card was safest to use. Therefore, the use of the DBS card and FP for infectious sample collection should be discouraged and reconsidered. RESEARCH HIGHLIGHTS The detection limits of the FTA® card, DBS card and FP for CVI988 detection were 0.5, 0.5 and 5 PFU/5 µl, respectively. All three filter papers could preserve viral DNA for at least 30 days of post spot. The DBS card and FP are not suitable for collecting NDV samples, which is one of the major economical threats for the poultry industry worldwide.

Pervasive Differential Splicing in Marek's Disease Virus can Discriminate CVI-988 Vaccine Strain from RB-1B Very Virulent Strain in Chicken Embryonic Fibroblasts.

Marek's disease is a major scourge challenging poultry health worldwide. It is caused by the highly contagious Marek's disease virus (MDV), an alphaherpesvirus. Here, we showed that, similar to other members of its Herpesviridae family, MDV also presents a complex landscape of splicing events, most of which are uncharacterised and/or not annotated. Quite strikingly, and although the biological relevance of this fact is unknown, we found that a number of viral splicing isoforms are strain-specific, despite the close sequence similarity of the strains considered: very virulent RB-1B and vaccine CVI-988. We validated our findings by devising an assay that discriminated infections caused by the two strains in chicken embryonic fibroblasts on the basis of the presence of some RNA species. To our knowledge, this study is the first to accomplish such a result, emphasizing how relevant a comprehensive picture of the viral transcriptome is to fully understand viral pathogenesis.

Marek's Disease Virus Infection of Natural Killer Cells.

Natural killer (NK) cells are key players in the innate immune response. They kill virus-infected cells and are crucial for the induction of adaptive immune responses. Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that causes deadly T cell lymphomas in chickens. Host resistance to MDV is associated with differences in NK cell responses; however, the exact role of NK cells in the control of MDV remains unknown. In this study, we assessed if MDV can infect NK cells and alter their activation. Surprisingly, we could demonstrate that primary chicken NK cells are very efficiently infected with very virulent RB-1B MDV and the live-attenuated CVI988 vaccine. Flow cytometry analysis revealed that both RB-1B and CVI988 enhance NK cell degranulation and increase interferon gamma (IFNγ) production in vitro. In addition, we could show that the MDV Eco Q-encoded oncogene (meq) contributes to the induction of NK cell activation using meq knockout viruses. Taken together, our data revealed for the first time that NK cells are efficiently infectable with MDV and that this oncogenic alphaherpesvirus enhances NK cell degranulation and increased IFNγ production in vitro.

A Common Live-Attenuated Avian Herpesvirus Vaccine Expresses a Very Potent Oncogene.

Vaccines play a crucial role in the protection of animals and humans from deadly pathogens. The first vaccine that also protected against cancer was developed against the highly oncogenic herpesvirus Marek's disease virus (MDV). MDV infects chickens and causes severe immunosuppression, neurological signs, and fatal lymphomas, a process that requires the viral oncogene, meq The most frequently used Marek's disease vaccine is the live-attenuated CVI988/Rispens (CVI) strain, which efficiently protects chickens and prevents tumorigenesis. Intriguingly, CVI expresses at least two isoforms of meq; however, it remains unknown to what extent these isoforms contribute to virus attenuation. In this study, we individually examined the contribution of the two CVI-meq isoforms to the attenuation of the vaccine. We inserted the respective isoforms into a very virulent MDV (strain RB-1B), thereby replacing its original meq gene. Surprisingly, we could demonstrate that the longer isoform of meq strongly enhanced virus-induced pathogenesis and tumorigenesis, indicating that other mutations in the CVI genome contribute to virus attenuation. On the contrary, the shorter isoform completely abrogated pathogenesis, demonstrating that changes in the meq gene can indeed play a key role in virus attenuation. Taken together, our study provides important evidence on attenuation of one of the most frequently used veterinary vaccines worldwide.IMPORTANCE Marek's disease virus (MDV) is one of several oncogenic herpesviruses and causes fatal lymphomas in chickens. The current "gold standard" vaccine is the live-attenuated MDV strain CVI988/Rispens (CVI), which is widely used and efficiently prevents tumor formation. Intriguingly, CVI expresses two predominant isoforms of the major MDV oncogene meq: one variant with a regular size of meq (Smeq) and one long isoform (Lmeq) harboring an insertion of 180 bp in the transactivation domain. In our study, we could break the long-standing assumption that the Lmeq isoform is an indicator for virus attenuation. Using recombinant viruses that express the different CVI-meq isoforms, we could demonstrate that both isoforms drastically differ in their abilities to promote pathogenesis and tumor formation in infected chickens.

Study of Efficacy and Replication of Recombinant Vector Vaccines by Using Turkey Herpesvirus Combined with Other Marek's Disease Vaccines.

Several recombinant turkey herpesviruses (rHVTs) have been developed within the past decades, and they are now used commercially worldwide. In broiler chickens, rHVTs are usually administered alone, but in long-living birds they are used in combination with Marek's disease (MD) vaccines of other serotypes (i.e., CVI988). The objectives of this work were to 1) evaluate protection against MD conferred by HVT and two rHVTs when combined with CVI988 and 2) optimize the use of rHVT in combination with CVI988 to maximize replication of rHVT without compromising MD protection. Various vaccine protocols, all using rHVT or HVT at the recommended dose (RD), were evaluated. Protocols evaluated included in ovo vaccination with HVT+CVI988 or rHVT+CVI988 (using either the double dose [DD] or the RD of CVI988), day of age vaccination of rHVT+CVI988 at DD, and revaccination protocols using rHVT in ovo followed by CVI988 at DD at day of age. Our results show that, when combined with CVI988, HVT and rHVTs confer a similar level of protection against MD (>90%) regardless of whether CVI988 was used at RD or at DD. However, the combination of rHVT with CVI988 at DD resulted in reduced replication rates of rHVT (60%-76% vs. 95%-100%). Our results show that such a negative effect could be avoided without jeopardizing MD protection by administering CVI988 at RD (if combined in ovo with rHVT) or administered rHVT first in ovo followed by CVI988 at DD at day of age.

Estudio de la eficacia y replicación de vacunas con vectores recombinantes mediante el uso del virus del herpes del pavo combinado con otras vacunas contra la enfermedad de Marek. Varios herpesvirus de pavo recombinantes (rHVT) se han desarrollado en las últimas décadas y ahora se utilizan comercialmente en todo el mundo. En pollos de engorde, los rHVT generalmente se administran solos, pero en aves de vida larga se usan en combinación con vacunas contra la enfermedad de Marek (MD) de otros serotipos (especialmente, CVI988). Los objetivos de este trabajo fueron 1) evaluar la protección contra la enfermedad de Marek conferida por herpesvirus de pavo (HVT9 y por dos rHVT cuando se combinan con la cepa CVI988 y 2) optimizar el uso de rHVT en combinación con la cepa CVI988 para maximizar la replicación de rHVT sin comprometer la protección contra la enfermedad de Marek. Se evaluaron varios protocolos de vacunas, todos con rHVT o con HVT a la dosis recomendada (RD). Los protocolos evaluados incluyeron la vacunación in ovo con HVT + CVI988 o rHVT + CVI988 (usando la dosis doble o la dosis recomendada de la cepa CVI988), la vacunación al día de la edad con rHVT + CVI988 con dosis doble, y los protocolos de revacunación usando rHVT seguido por la cepa CVI988 con dosis doble al día de edad. Los resultados muestran que cuando se combinan con CVI988, HVT y rHVT confieren un nivel de protección similar contra la enfermedad de Marek (> 90%) independientemente de que la cepa CVI988 se haya usado a la dosis recomendada o con dosis doble. Sin embargo, la combinación de rHVT con la cepa CVI988 con doble dosis produjo una reducción en las tasas de replicación de rHVT (60% ­76% vs. 95% ­100%). Estos resultados muestran que dicho efecto negativo podría evitarse sin poner en peligro la protección contra la enfermedad de Marek administrando la cepa CVI988 a la dosis recomendada (si se combina in ovo con rHVT) o administrando rHVT primero in ovo, seguido de CVI988 con dosis doble al día de la edad.

The Transcriptional Landscape of Marek's Disease Virus in Primary Chicken B Cells Reveals Novel Splice Variants and Genes.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that infects chickens and poses a serious threat to poultry health. In infected animals, MDV efficiently replicates in B cells in various lymphoid organs. Despite many years of research, the viral transcriptome in primary target cells of MDV remained unknown. In this study, we uncovered the transcriptional landscape of the very virulent RB1B strain and the attenuated CVI988/Rispens vaccine strain in primary chicken B cells using high-throughput RNA-sequencing. Our data confirmed the expression of known genes, but also identified a novel spliced MDV gene in the unique short region of the genome. Furthermore, de novo transcriptome assembly revealed extensive splicing of viral genes resulting in coding and non-coding RNA transcripts. A novel splicing isoform of MDV UL15 could also be confirmed by mass spectrometry and RT-PCR. In addition, we could demonstrate that the associated transcriptional motifs are highly conserved and closely resembled those of the host transcriptional machinery. Taken together, our data allow a comprehensive re-annotation of the MDV genome with novel genes and splice variants that could be targeted in further research on MDV replication and tumorigenesis.

Application of CRISPR/Cas9 Gene Editing System on MDV-1 Genome for the Study of Gene Function.

Marek's disease virus (MDV) is a member of alphaherpesviruses associated with Marek's disease, a highly contagious neoplastic disease in chickens. Complete sequencing of the viral genome and recombineering techniques using infectious bacterial artificial chromosome (BAC) clones of Marek's disease virus genome have identified major genes that are associated with pathogenicity. Recent advances in CRISPR/Cas9-based gene editing have given opportunities for precise editing of the viral genome for identifying pathogenic determinants. Here we describe the application of CRISPR/Cas9 gene editing approaches to delete the Meq and pp38 genes from the CVI988 vaccine strain of MDV. This powerful technology will speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis.

Marek's disease vaccine activates chicken macrophages.

To provide insights into the role of innate immune responses in vaccine-mediated protection, we investigated the effect of Marek's disease (MD) vaccine, CVI988/Rispens, on the expression patterns of selected genes associated with activation of macrophages in MD-resistant and MD-susceptible chicken lines. Upregulation of interferon γ, interleukin (IL)-1ß, IL-8, and IL-12 at different days post-inoculation (dpi) revealed activation of macrophages in both chicken lines. A strong immune response was induced in cecal tonsils of the susceptible line at 5 dpi. The highest transcriptional activities were observed in spleen tissues of the resistant line at 3 dpi. No increase in the population of CD3⁺ T cells was observed in duodenum of vaccinated birds at 5 dpi indicating a lack of involvement of the adaptive immune system in the transcriptional profiling of the tested genes. There was, however, an increase in the number of macrophages in the duodenum of vaccinated birds. The CVI988/Rispens antigen was detected in the duodenum and cecal tonsils of the susceptible line at 5 dpi but not in the resistant line. This study sheds light on the role of macrophages in vaccine-mediated protection against MD and on the possible development of new recombinant vaccines with enhanced innate immune system activation properties.

Marek's disease vaccine activates chicken macrophages

To provide insights into the role of innate immune responses in vaccine-mediated protection, we investigated the effect of Marek's disease (MD) vaccine, CVI988/Rispens, on the expression patterns of selected genes associated with activation of macrophages in MD-resistant and MD-susceptible chicken lines. Upregulation of interferon γ, interleukin (IL)-1β, IL-8, and IL-12 at different days post-inoculation (dpi) revealed activation of macrophages in both chicken lines. A strong immune response was induced in cecal tonsils of the susceptible line at 5 dpi. The highest transcriptional activities were observed in spleen tissues of the resistant line at 3 dpi. No increase in the population of CD3³ T cells was observed in duodenum of vaccinated birds at 5 dpi indicating a lack of involvement of the adaptive immune system in the transcriptional profiling of the tested genes. There was, however, an increase in the number of macrophages in the duodenum of vaccinated birds. The CVI988/Rispens antigen was detected in the duodenum and cecal tonsils of the susceptible line at 5 dpi but not in the resistant line. This study sheds light on the role of macrophages in vaccine-mediated protection against MD and on the possible development of new recombinant vaccines with enhanced innate immune system activation properties.

Early Immune Responses to Marek's Disease Vaccines.

Marek's disease virus (MDV), a highly cell-associated lymphotropic α-herpesvirus, is the causative agent of Marek's disease (MD) in domestic chickens. MDV replicates in chicken cells and establishes a latent infection within CD4+ T cells. Although MD vaccines have been in use for several decades, the exact mechanism of vaccine-induced protection is unclear. It is believed that the innate immune system plays a role in vaccine-induced immunity against pathogenic strains of MDV. To shed light on the possible function of the innate immunity in vaccine-mediated protection, we investigated the effect of vaccination, Rispens/CVI988, on the activation of cellular components of the innate immune system by analyzing the expression pattern of select immune-related genes in the cecal tonsils (CT) and duodenum of two MD-susceptible and MD-resistant chicken lines at 3, 5, and 10 days postvaccination (dpv). The differential expression patterns of the tested genes within the CT and duodenum of vaccinated birds revealed the activation of the innate immune system in both the susceptible and resistant lines. Stronger innate immune response was induced within the CT of the vaccinated birds of the susceptible line at 5 dpv. Upregulation of some of the tested genes at 10 dpv was likely due to the activation and response of the adaptive immune system to vaccination. Immunohistochemical analysis showed no increase in the number of CD3+ T cells in the CT and duodenum of the vaccinated birds of either line at 5 dpv. There was, however, an increase in the macrophage populations within the duodenum of the vaccinated birds of both the susceptible and resistant lines at 5 dpv. The vaccine strain antigen was detected in the CT and duodenum of the susceptible line, but not the resistant line at 5 dpv.

History of the First-Generation Marek's Disease Vaccines: The Science and Little-Known Facts.

Shortly after the isolation of Marek's disease (MD) herpesvirus (MDV) in the late 1960s vaccines were developed in England, the United States, and The Netherlands. Biggs and associates at the Houghton Poultry Research Station (HPRS) in England attenuated HPRS-16, the first cell-culture-isolated MDV strain, by passaging HPRS-16 in chick kidney cells. Although HPRS-16/Att was the first commercially available vaccine, it never became widely used and was soon replaced by the FC126 strain of herpesvirus of turkeys (HVT) vaccine developed by Witter and associates at the Regional Poultry Research Laboratory (now Avian Disease and Oncology Laboratory [ADOL]) in East Lansing, MI. Ironically, Kawamura et al. isolated a herpesvirus from kidney cell cultures from turkeys in 1969 but never realized its potential as a vaccine against MD. Rispens of the Central Veterinary Institute (CVI) developed the third vaccine. His associate, Maas, had found commercial flocks of chickens with MDV antibodies but without MD. Subsequently, Rispens isolated a very low pathogenic strain from hen number 988 from his MD antibody-positive flock, which was free of avian leukosis virus and clinical MD. This isolate became the CVI-988 vaccine used mostly in The Netherlands. During the late 1970s, HVT was no longer fully protective against some new emerging field strains. The addition of SB-1, isolated by Schat and Calnek, to HVT improved protection against the emerging very virulent strains. In the 1990s CVI-988 became the worldwide vaccine gold standard. This review will present data from published papers and personal communications providing additional information about the exciting 15-yr period after the isolation of MDV to the development of the different vaccines.

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.

Effects of Rispens CVI988 vaccination followed by challenge with Marek's disease viruses of differing virulence on the replication kinetics and shedding of the vaccine and challenge viruses.

Vaccination with "imperfect" vaccines that prevent disease but not infection is strongly implicated in the observed increased virulence of Marek's disease virus (MDV) over the past six decades. The current "gold standard" vaccine, Rispens CVI988 (Rispens), has maintained efficacy despite use for five decades, raising the question of whether it too favours higher virulence MDVs. To investigate this, we studied the kinetics of Rispens CVI988 (Rispens) and two MDV strains of different virulence in 236 commercial ISA Brown chickens vaccinated with Rispens at hatch and challenged with vMDV isolate MPF57 or vvMDV isolate FT158 on day five. Each treatment was replicated in two isolators and from 7 to 56 days post infection (dpi) peripheral blood leucocytes (PBL), feather and dust samples were collected and subjected to differential quantitative PCR (qPCR). Rispens vaccination significantly reduced challenge MDV viral load in a sample-dependant manner with evidence of a differentially greater inhibitory effect on the less virulent MDV. Similarly, challenge with the more virulent MDV reduced the Rispens viral load in PBL. Rispens virus load displayed a distinctive pattern of viral load that was similar in PBL and feathers, but different in dust. The clear effects of vaccination and challenge evident in PBL and feather samples were less clearly reflected in dust samples. The data are consistent with the Rispens vaccine reducing replication of lesser virulent MDVs to a greater extent like the HVT vaccine. Likely reasons for the persistent efficacy of Rispens vaccine are discussed.

Detection of specific UL49 sequences of Marek's disease virus CVI988/Rispens strain using loop-mediated isothermal amplification.

Marek's disease (MD) is a tumoral disease of chickens that can be controlled by vaccines based on non-pathogenic strains of turkey herpesvirus (HVT), SB-1 strain belonging to serotype 2, or the attenuated CVI988/Rispens strain belonging to serotype 1 of Marek's disease virus (MDV). Currently, the 'gold standard' in MD prophylaxis is the Rispens strain-based vaccine which protects against very virulent MDV and disease onset. Previous studies have shown that loop-mediated isothermal amplification (LAMP) is a rapid alternative to polymerase chain reaction (PCR) for detection and differentiation of HVT, SB-1 and virulent MDV strains. The aim of this study was to develop and evaluate a novel LAMP assay for the detection of the UL49 Rispens-specific region. This assay was validated using material from infected chicken embryo fibroblasts (CEFs) and tissue samples from vaccinated chickens. The analytical sensitivity of the assay was 10-times higher than PCR and reliably amplified 0.1 log10 TCID50/ml. The MDV Rispens was also detected at 18h after infection of CEFs. The results showed LAMP to be selective and a sensitive method to detect Rispens as early as 3 d.p.v. in all internal organs of chickens. Furthermore, the method was also capable to detect Rispens in 5 out of 26 chickens originating from different flocks. A mismatch amplification mutation assay (MAMA-PCR) confirmed the presence of Rispens strain in all LAMP-positive chickens. This is the first report of the specific visual detection of Rispens in vitro and in vivo using LAMP. The method may be useful for monitoring of successful chicken vaccination as well as in vitro studies in infected cell cultures.

Replication kinetics and shedding of very virulent Marek's disease virus and vaccinal Rispens/CVI988 virus during single and mixed infections varying in order and interval between infections.

Vaccination is thought to contribute to an evolution in virulence of the Marek's disease virus (MDV) as vaccines prevent disease but not infection. We investigated the effects of co-infections at various intervals between Rispens/CVI988 vaccine virus (Rispens) and very virulent MDV (vvMDV) on the replication and shedding of each virus. The experiment used 600 ISA Brown layer chickens in 24 isolators with all treatments replicated in two isolators. Chickens were vaccinated with Rispens and/or challenged with the vvMDV isolate 02LAR on days 0, 5, or 10 post hatching providing vaccination to challenge intervals (VCI) of -10, -5, 0, 5 or 10 days with the negative values indicating challenge prior to vaccination. Peripheral blood lymphocytes (PBL), feathers and isolator exhaust dust were sampled between 7 and 56 days post infection (dpi) and subjected to quantitative real-time polymerase chain reaction (qPCR) to differentiate the two viruses. Overall Rispens significantly reduced the viral load of vvMDV in PBL and feather cells and shedding in dust. Similarly vvMDV significantly reduced the viral load of Rispens in PBL and feather cells but not in dust. VCI significantly influenced these relationships having strong positive and negative associations with load of vvMDV and Rispens respectively. Differences between the two viruses and their effects on each other were greatest in PBL and feathers, and least in dust. This study expands our understanding of the interaction between pathogenic and vaccinal viruses following vaccination with imperfect vaccines and has implications for selection of appropriate samples to test for vaccination success.