Comparison of Marek's Disease Virus Challenge Strains and Bird Types for Vaccine Licensing.

Marek's disease virus (MDV) is an important poultry pathogen that is controlled through widespread vaccination with avirulent and attenuated strains. However, continued evolution of field viruses to higher virulence has required ongoing improvement of available vaccine strains, and these vaccine strains offer an attractive platform for designing recombinant vector vaccines with cross-protection against MDV and additional pathogens. Recent reports of failures in vaccine licensing trials of positive controls to reach appropriately high levels of Marek's disease incidence prompted us to evaluate possible combinations of outbred specific-pathogen-free layer lines and alternative virulent challenge strains that could provide more consistent models for serotype 3 vectored vaccine development. Choice of layer line and virulent MDV challenge strain each contributed to the ability of a challenge model to reach 80% virulence in unvaccinated positive control groups in the majority of trials, without overwhelming serotype 3 vectored vaccine protection in vaccinated groups. Conversely, reducing challenge virus dose by a factor of four, or vaccine dose by half, had no consistent effect across these models. Although MDV strain 617A had the most potential as an alternative to strains that are currently approved for licensing trials, no combination of layer line and challenge virus consistently met the goals for a successful challenge model in all study replicates, indicating that high variability is an inherent difficulty in MDV challenge studies, at least when outbred birds are used.

Artículo regular­Comparación de las cepas de desafío del virus de la enfermedad de Marek y los tipos de aves para la obtención de licencias de vacunas. El virus de la enfermedad de Marek (MDV) es un patógeno importante en la avicultura que se controla mediante la vacunación generalizada con cepas avirulentas y atenuadas. Sin embargo, la evolución continua de los virus de campo hacia una mayor virulencia ha requerido una mejora continua de las cepas vacunales disponibles y estas cepas vacunales ofrecen una plataforma atractiva para diseñar vacunas con vectores recombinantes que induzcan protección cruzada contra el virus de la enfermedad de Marek y patógenos adicionales. Los reportes recientes de fallas en los controles positivos para alcanzar niveles apropiadamente altos de incidencia de la enfermedad de Marek en los ensayos para obtener la licencia de vacunas llevaron a evaluar posibles combinaciones de líneas de postura híbridas libres de patógenos específicos y cepas de desafío virulentas alternativas que podrían proporcionar modelos más consistentes para el desarrollo de vacunas con vectores de serotipo 3. Tanto la elección de la línea de postura como de la cepa de desafío virulenta de Marek contribuyeron a obtener un modelo de desafío con capacidad para alcanzar el 80% de virulencia en grupos controles positivo no vacunados en la mayoría de los ensayos, sin una protección abrumadora de la vacuna con vector de serotipo 3 en los grupos vacunados. Por el contrario, la reducción de la dosis del virus de desafío en un factor de cuatro, o la dosis de vacuna a la mitad, no tuvieron un efecto constante en estos modelos. Aunque la cepa 617A de Marek mostró el mayor potencial como alternativa a las cepas que actualmente están aprobadas para ensayos de licenciar vacunas, ninguna combinación de línea de postura y virus de desafío cumplió consistentemente los objetivos de un modelo de desafío exitoso en todas las réplicas del estudio, lo que indica que la alta variabilidad es una dificultad inherente en los estudios de desafío para la enfermedad de Marek, al menos cuando se utilizan aves híbridas.

Further observations on serotype 2 Marek's disease virus-induced enhancement of spontaneous avian leukosis virus-like bursal lymphomas in ALVA6 transgenic chickens.

Breeders of the 2009 generation of Avian Disease and Oncology Laboratory transgenic chicken line ALVA6, known to be resistant to infection with subgroups A and E avian leukosis virus (ALV), were vaccinated at hatch with a trivalent Marek's disease (MD) vaccine containing serotypes 1, 2, and 3 Marek's disease virus (MDV) and were maintained under pathogen-free conditions from the day of hatch until 75 weeks of age. Spontaneous ALV-like bursal lymphomas, also termed lymphoid leukosis (LL)-like lymphomas, were detected in 7% of the ALVA6 breeders. There was no evidence of infection with exogenous and endogenous ALV as determined by virus isolation tests of plasma and tumour tissue homogenates. For the next three generations, serotype 2 MDV was eliminated from the trivalent MD vaccine used. Results show, for the first time, that removal of serotype 2 MDV from MD vaccines eliminated spontaneous LL-like lymphomas within 50 to 72 weeks of age for at least three consecutive generations. Two experiments were also conducted to determine the influence of in ovo vaccination with serotype 2 MD vaccines on enhancement of spontaneous LL-like lymphomas in ALVA6 chickens. Chickens from the 2012 generation were each inoculated in ovo or at hatch with 5000 plaque-forming units of serotype 2 MDV. Results indicate that by 50 weeks of age the incidence of spontaneous LL-like lymphomas in chickens inoculated in ovo with serotype 2 MDV was comparable with that in chickens inoculated with virus at hatch, suggesting that the augmentation effect of serotype 2 MDV is independent of age of vaccination.

Influence of vaccination with CVI988/Rispens on load and replication of a very virulent Marek's disease virus strain in feathers of chickens.

Several highly efficacious vaccines are currently available for control of Marek's disease, a lymphoproliferative disease in chickens. However, these vaccines are unable to prevent infection with Marek's disease virus (MDV) in vaccinated birds. This leads to shedding of virulent MDV from feather follicle epithelium and skin epithelial cells of vaccinated and infected chickens. The objective of the present study was to study the interactions between a vaccine strain (CVI988/Rispens) and a very virulent strain of MDV (RB1B) in feathers. We examined genome load and replication of CVI988 and MDV-RB1B strains at various time points post infection. Moreover, we evaluated cytokine expression in feathers as indicators of immunity generated in response to vaccines against MDV. Analysis of feathers collected between 4 and 21 days post infection (d.p.i.) revealed a steady level of CVI988 genome load in the presence or absence of RB1B. Infection with MDV resulted in a significant increase in RB1B genome load peaking at 14 d.p.i. Importantly, vaccination with CVI988 resulted in a significant reduction in accumulation of MDV-RB1B in feathers. RB1B genome accumulation in feather tips was associated with increased expression of interferon-α at 14 d.p.i. and interferon-Sγ at earlier time points, 4 and 7 d.p.i. compared with 10 and 14 d.p.i. Interleukin-10 and interleukin-6 were up-regulated at 14 d.p.i. in the infected groups. This study expands our understanding of the dynamics of replication of vaccine and virulent MDV strains in the feathers and illuminates mechanisms associated with immunity to Marek's disease.

A toll-like receptor 3 ligand enhances protective effects of vaccination against Marek's disease virus and hinders tumor development in chickens.

Marek's disease (MD) is caused by Marek's disease virus (MDV). Various vaccines including herpesvirus of turkeys (HVT) have been used to control this disease. However, HVT is not able to completely protect against very virulent strains of MDV. The objective of this study was to determine whether a vaccination protocol consisting of HVT and a Toll-like receptor (TLR) ligand could enhance protective efficacy of vaccination against MD. Hence, chickens were immunized with HVT and subsequently treated with synthetic double-stranded RNA polyriboinosinic polyribocytidylic [poly(I:C)], a TLR3 ligand, before or after being infected with a very virulent strain of MDV. Among the groups that were HVT-vaccinated and challenged with MDV, the lowest incidence of tumors was observed in the group that received poly(I:C) before and after MDV infection. Moreover, the groups that received a single poly(I:C) treatment either before or after MDV infection were better protected against MD tumors compared to the group that only received HVT. No association was observed between viral load, as determined by MDV genome copy number, and the reduction in tumor formation. Overall, the results presented here indicate that poly(I:C) treatment, especially when it is administered prior to and after HVT vaccination, enhances the efficacy of HVT vaccine and improves protection against MDV.

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.

Oncogenic Marek's disease viruses lacking the 132 base pair repeats can still be attenuated by serial in vitro cell culture passages.

Marek's disease virus (MDV) can be attenuated by serially passing the virus in cell culture. During cell culture passage, two copies of a 132 bp repeat are expanded to over 30 copies. We deleted the two copies of the 132 bp repeat region in a pathogenic MDV and demonstrated that the virus was still pathogenic. The pattern and frequency of tumors in the parental and mutant virus were the same. Early virus replication, and the appearance of persistent neurological disease were also similar between the parental and deleted virus. Nevertheless, wild-type MDV and the deletion virus could be attenuated by serial in vitro cell culture passages. Based upon analyzing the passage 40 viruses, attenuation of the MDV lacking the 132 bp repeats appears to occur in a manner that is analogous to the process occurring wild-type MDV attenuation. Whatever process is involved in the cell culture attenuation of MDV, the mechanism does not involve the 132 bp repeat region.

Characterization of LORF11, a unique gene common to the three Marek's disease virus serotypes.

The unique open reading frame 11 (LORF11) of Marek's disease virus (MDV) is present in all three serotypes of MDV and is located in the unique long region of the MDV genome. In the serotype 1 Md5 genome, LORF11 comprises 2711 nucleotides and encodes a predicted protein of 903 amino acids. In order to study the biological function of LORF11 we deleted it from the MDV cosmid A6 by using the RecA-assisted restriction endonuclease cleavage method. The recombinant cosmid, A6DeltaLORF11, was transfected into duck embryo fibroblasts (DEF) in conjunction with parental SN5, P89, SN16, and B40 cosmid clones. Recombinant rMd5DeltaLORF11 plaques were evident at 12-13 days after transfection. Polymerase chain reaction amplification of DEF cells infected with rMd5DeltaLORF11 viruses confirmed the deletion of a 2.57-kb fragment resulting in a 296-bp fragment. Three rMd5DeltaLORF11 mutants were generated and their biological functions were studied in vitro and in vivo. In vitro growth characteristics of rMd5DeltaLORF11 viruses were similar to those of parental rMd5, indicating that LORF11 is not essential for replication in vitro. In vivo studies of rMd5DeltaLORF11 mutants showed that they were impaired in viral replication in the lymphoid organs and had 100x lower viremia than chickens infected with the parental rMd5 virus. Furthermore, rMd5-infected chickens horizontally transmitted the virus to contact controls whereas no horizontal transmission occurred in rMd5DeltaLORF11-infected chickens. Three independent deletion mutants were tested and showed the same phenotypes, so it is unlikely that the observed phenotype is because of any random mutation in the genome. Therefore the LORF11 gene of MDV is essential for normal virus replication in chickens and deletion of LORF11 renders an attenuated virus.

Characterization and localization of the unique Marek's disease virus type 2 ORF873 gene product.

Studies on Marek's disease virus (MDV)-unique genes are important for understanding the biological nature of the virus. Based on complete DNA sequence analyses of the MDV genomes, the MDV genomes contain presumably at least five MDV-unique genes, which are commonly conserved among the three MDV serotypes. A recombinant baculovirus that contains the MDV serotype 2 (MDV2)-unique gene, ORF873, under the polyhedrin promoter was constructed and designated rAcORF873. Polyclonal and monoclonal antibodies, which recognize the recombinant MDV2 ORF873 protein in Spodoptera frugiperda clone 9 (Sf9) cells infected with rAcORF873, were prepared by immunizing mice with a recombinant fusion protein expressed in Escherichia coli. Immunoblot analyses with the antibodies revealed a major protein band with a molecular mass of 108-kDa in both MDV2-infected chick embryo fibroblasts (CEF) and rAcORF873-infected Sf9 cells. By indirect immunofluorescence analyses using monoclonal antibody, the authentic ORF873 protein was localized in the cytoplasm of MDV2-infected CEF cells. The monoclonal and polyclonal sera, which were generated in the present study and reacted effectively to MDV2 ORF873 protein, are considered to be useful reagents for further studying the role(s) of the ORF873 protein in MDV2 infection.

Resistance to Marek's disease virus in White Leghorn chickens: effects of avian leukosis virus infection genotype, reciprocal mating, and major histocompatibility complex.

Genetic improvement for resistance to Marek's Disease (MD) in chickens continues to be of interest to the poultry industry. The aims of this study were to identify effects of the MHC on the molecular level and of avian leukosis virus (ALV) resistance status on MD mortality in two noninbred White Leghorn chicken lines that differ in B blood group type. Previously, within each of the chicken lines, sublines had been selected for resistance or susceptibility to ALV infection with Subgroups A and B. In this study, F2 offspring, obtained by crossing the two ALV-resistant or the two ALV-susceptible sublines, were tested for MD mortality after contact exposure at 1 d of age. Reciprocal matings were made in the grandparental generation. The MD mortality percentages, in an observation period of 17 wk, of F2 offspring from two hatches were 82.63 and 92.35%, respectively. Survival analysis (Cox model) was applied to assess the risk of dying from MD. No differences in MD mortality risk profiles were found between ALV-resistant and ALV-susceptible F2 offspring. Within ALV-susceptible F2 offspring, however, a reciprocal mating effect was observed in both hatches. The MHC Class I, II, and IV restriction fragment length polymorphism (RFLP) analyses were carried out on birds of the first hatch. Although two of 11 MHC class IV RFLP bands displayed a significant effect, in general, a strong association of MHC and MD mortality was not detectable.