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.

Phylogeny and recombination history of gallid herpesvirus 2 (Marek's disease virus) genomes.

Phylogenetic analyses based on concatenated amino acid sequences from orthologous loci from eight genomes of alpha herpesviruses infecting birds provided strong support for the following hypotheses: (1) gallid HV3 is a sister taxon to gallid HV2 but gallid HV1 is not closely related to the other two chicken herpesviruses; (2) meleagrid HV1 is closer to both gallid HV2 and gallid HV3 than is gallid HV1; (3) within gallid HV2, the virulent GA genome forms an outgroup to both the avirulent CVI988 genome and the highly virulent Md5 and Md11 genomes. Analysis of the pattern of synonymous nucleotide substitution between orthologous genes shared by four complete genomes of gallid HV2 showed strong evidence of past events of homologous recombination that homogenized certain loci between genomes. Eight of these loci represented cases of loci homogenized between the CVI988, on the one hand, and the Md5 and Md11 genomes, on the other hand. Two others represented loci where the GA genome was homogenized with those of Md5 and Md11. The two loci (UL49.5 and RLORF12) that were homogenized among the virulent genomes GA, Md5, and Md11 are candidates for contributing to viral virulence.

Absolute quantification using real-time polymerase chain reaction of Marek's disease virus serotype 2 in field dust samples, feather tips and spleens.

Methods for Taqman quantitative real-time PCR (qPCR) assays to detect the three serotypes of Marek's disease virus (MDV) are available, and absolute quantification has been developed for MDV serotype 1 and serotype 3. The development of a method for absolute quantification of Marek's disease virus serotype 2 (MDV2) is described in this paper. Using plasmid DNA, the lower detection limit of the MDV2 assay was determined to be 10 copies. Three independent assay runs showed highly reproducible Ct values and calculated copy numbers, with mean intra- and inter-assay coefficients of variation of less than 3% for Ct and less than 21.5% for calculated copy number. Absolute quantification of MDV2 was performed successfully on dust samples collected from poultry farms across Australia, material from infectious spleens and feather tips from chickens vaccinated with an attenuated strain of MDV2. Thus, it is now possible to use qPCR assays for absolute quantification of all three serotypes of MDV in a sample.