Robert F. Silva

Rescue of a pathogenic Marek's disease virus with overlapping cosmid DNAs: Use of a pp38 mutant to validate the technology for the study of gene function

Mareks disease virus (MDV) genetics has lagged behind that of other herpesviruses because of the lack of tools for the introduction of site-specific mutations into the genome of highly cell-associated oncogenic strains. Overlapping cosmid clones have been successfully used for the introduction of mutations in other highly cell-associated herpesviruses. Here we describe the development of overlapping cosmid DNA clones from a very virulent oncogenic strain of MDV. Transfection of these cosmid clones into MDV-susceptible cells resulted in the generation of a recombinant MDV (rMd5) with biological properties similar to the parental strain. To demonstrate the applicability of this technology for elucidation of gene function of MDV, we have generated a mutant virus lacking an MDV unique phosphoprotein, pp38, which has previously been associated with the maintenance of transformation in MDV-induced tumor cell lines. Inoculation of Mareks disease-susceptible birds with the pp38 deletion mutant virus (rMd5Δpp38) revealed that pp38 is involved in early cytolytic infection in lymphocytes but not in the induction of tumors. This powerful technology will speed the characterization of MDV gene function, leading to a better understanding of the molecular mechanisms of MDV pathogenesis. In addition, because Mareks disease is a major oncogenic system, the knowledge obtained from these studies may shed light on the oncogenic mechanisms of other herpesviruses.

Monoclonal antibody-mediated immunoprecipitation of proteins from cell, infected with marek's disease virus or turkey herpesvirus

The major immunogenic viral proteins of Mareks disease virus (MDV) and turkey herpesvirus (HVT) share antigenic determinants. The polyacrylamide gel electrophoresis pattern of five viral polypeptides immunoprecipitated with homologous convalescent chicken plasma was identical with the pattern obtained after immunoprecipitation with heterologous convalescent chicken plasma. A panel of monoclonal antibodies was used to identify MDV and HVT polypeptides. Sixteen monoclonal antibodies were positive in an immunofluorescence assay. However, only eight monoclonal antibodies immunoprecipitated a total of seven distinct viral proteins from MDV- and HVT-infected cells. Six of these monoclonals immunoprecipitated multiple viral proteins. None of the monoclonal antibodies recognized the common A antigen of MDV or HVT. Monoclonal antibodies immunoprecipitated three glycoproteins (100,000, 60,000, and 49,000 Da) that comigrate in polyacrylamide gels with three of the five common polypeptides obtained with the convalescent chicken plasma. In addition, a 79,000-Da protein was common to all MDV- and HVT-infected cells. Competition immunoprecipitation and peptide mapping by limited proteolysis confirmed that the three glycoproteins and the 79,000-Da protein contain MDV-HVT common epitopes. MDV-specific antigenic determinants were detected on the three remaining viral proteins (41,000, 38,000, and 24,000 Da).

Genetic mechanisms of antigenic variation in infectious bursal disease virus: analysis of a naturally occurring variant virus.

The major immunogenic protein VP2 from a pathogenic field isolate (variant A virus) of infectious bursal disease virus (IBDV) was cloned and sequenced to examine antigenic variations. The VP2 open reading frame consists of 1509 nucleotides and codes for a 503 amino acid protein. Overall, the VP2 amino acid sequence of the variant A virus shares 98.6% identity with VP2 genes from other published IBDV strains. However, within the central region of VP2 (amino acids 222–334) lies a highly divergent area that we have termed thevariable domain. Relative to five other IBDV isolates, a total of six amino acid changes occur within the variable domain of the variant A virus. At positions 284–288, a substitution of isoleucine to threonine, a decrease in the number of Chou and Fasman β turns, and a switch from a hydrophilic to a hydrophobic region are found only in the variant A virus. Together these changes predict a decrease in antigenicity as determined by calculation of potential antigenic sites. This suggests that only minor changes within VP2 contributed to the emergence of a variant virus that can cause disease in immunized birds.

Differentiation of pathogenic and non-pathogenic serotype 1 Marek's disease viruses (MDVs) by the polymerase chain reaction amplification of the tandem direct repeats within the MDV genome.

There are no simple, direct methods to reliably distinguish oncogenic serotype 1 Mareks disease viruses (MDVs) from their attenuated variants. The present study was an attempt to apply polymerase chain reaction (PCR) to develop a rapid and sensitive assay for the presence of the MDV genome. PCR oligos were chosen to flank the 132-base-pair tandem direct repeats in the serotype 1 MDV genome. The PCR reaction was specific for serotype 1 MDVs, amplifying fragments corresponding to one to three copies of the tandem repeats present in Md11/8, JM/102W, and GA viruses. A high-molecular-weight DNA smear was observed when the DNA from an attenuated Md11/100 was PCR-amplified. Use of the PCR technique allowed the detection of two copies of the 132-base-pair repeat in the DNA extracted from MDV-induced lymphomas removed from two chickens. No DNA was amplified from the DNA extracted from lymphomas induced by either an avian leukosis virus (RAV-1) or reticuloendotheliosis virus (chick syncytial virus).

Recombinant Marek's disease virus (MDV) lacking the Meq oncogene confers protection against challenge with a very virulent plus strain of MDV

Mareks disease virus (MDV) encodes a basic leucine-zipper protein, Meq, that shares homology with the Jun/Fos family of transcriptional factors. Conclusive evidence that Meq is an oncogene of MDV came from recent studies of a Meq-null virus, rMd5 Delta Meq. This virus replicated well in vitro, but was non-oncogenic in vivo. Further characterization of this virus in vivo indicated that the meq gene is dispensable for cytolytic infection since it replicated well in the lymphoid organs and feather follicular epithelium. Since rMd5 Delta Meq virus was apathogenic for chickens, we set out to investigate whether this virus could be a good candidate vaccine. Vaccine efficacy experiments conducted in Avian Disease and Oncology Laboratory (ADOL) 15I(5)x 7(1) chickens vaccinated with rMd5 Delta Meq virus or an ADOL preparation of CVI988/Rispens indicated that the Meq-null virus provided protection superior to CVI988/Rispens, the most efficacious vaccine presently available, following challenge with a very virulent (rMd5) and a very virulent plus (648A) MDV strains.

Development of a Polymerase Chain Reaction to Differentiate Avian Leukosis Virus (ALV) Subgroups: Detection of an ALV Contaminant in Commercial Marek's Disease Vaccines

Abstract Avian leukosis viruses (ALVs) are common in many poultry flocks and can be detected using an enzyme-linked immunosorbent assay or any other test designed to identify p27, the group-specific antigen located in gag. However, endogenous retroviruses expressing p27 are often present and can be confused with exogenous ALVs. A more specific and informative assay involves targeting the variable envelope glycoprotein gene (gp85) that is the basis for dividing ALVs into their different subgroups. We designed polymerase chain reaction (PCR) primers that would specifically detect and amplify viruses from each of the six ALV subgroups: A, B, C, D, E, and J. Subgroup B and D envelopes are related, and our B-specific primers also amplified subgroup D viruses. We also designed a set of common primers to amplify any ALV subgroup virus. To demonstrate the usefulness of these primers, we obtained from the Center for Veterinary Biologics in Iowa culture supernatant from chicken embryo fibroblasts infected with an ALV that was found to be a contaminant in two commercial Mareks disease vaccines. Using our PCR primers, we demonstrate that the contaminant was a subgroup A ALV. We cloned and sequenced a portion of the envelope gene and confirmed that the ALV was a subgroup A virus. Unlike typical subgroup A viruses, the contaminant ALV grew very slowly in cell culture. We also cloned and sequenced a portion of the long terminal repeat (LTR) from the contaminant virus. The LTR was found to be similar to those LTRs found in endogenous ALVs (subgroup E) and very dissimilar to LTRs normally found in subgroup A viruses. The E-like LTR probably explains why the contaminant grew so poorly in cell culture.

The pp38 Gene of Marek's Disease Virus (MDV) Is Necessary for Cytolytic Infection of B Cells and Maintenance of the Transformed State but Not for Cytolytic Infection of the Feather Follicle Epithelium and Horizontal Spread of MDV

ABSTRACT Mareks disease virus has a unique phosphoprotein, pp38, which is suspected to play an important role in Mareks disease pathogenesis. The objective of the present study was to utilize a mutant virus lacking the pp38 gene (rMd5Δpp38) to better characterize the biological function of pp38. This work shows that the pp38 gene is necessary to establish cytolytic infection in B cells but not in feather follicle epithelium, to produce an adequate level of latently infected T cells, and to maintain the transformed status in vivo.

An outbreak of lymphomas in commercial broiler breeder chickens vaccinated with a fowlpox vaccine contaminated with reticuloendotheliosis virus.

Gross and microscopic examinations of affected tissues from chickens of two commercial broiler breeder flocks aged 27 and 31 weeks revealed lesions of visceral lymphomas with bursal involvement in some chickens. Reticuloendotheliosis virus (REV), but not avian leukosis virus (ALV), was isolated from blood of affected chickens. Furthermore, DNA extracted from tumours tested positive for REV, but not for ALV or Mareks disease virus by polymerase chain reaction (PCR) test. Attempts to determine the source of REV infection included testing a commercial fowlpox (FP) vaccine used to immunize flocks at 7 days of age. Chicken-embryo fibroblasts inoculated with the FP vaccine tested positive for REV by PCR and immunofluorescent tests. REV was also isolated from plasma of pathogen-free chickens experimentally inoculated with FP vaccine at hatch; two of eight (25%) inoculated chickens developed lymphomas by 34 weeks of age. Antigenic characterization of REV isolated from commercial broiler breeder chickens and from FP vaccine, using monoclonal antibodies, revealed that both isolates belong to subtype 3 of REV. The data represent the first report of an outbreak of REV-induced lymphomas in commercial chickens. The data also indicate that the source of REV infection is an REV-contaminated commercial FP vaccine.

New Serotype 2 and Attenuated Serotype 1 Marek's Disease Vaccine Viruses: Selected Biological and Molecular Characteristics

Two new Mareks disease vaccine viruses, Md11/75C/R2 (serotype 1) and 301B/1 (serotype 2), were evaluated in chickens with maternal antibodies (ab+) or without maternal antibodies (ab-). Strain Md11/75C/R2 was mildly pathogenic in ab--chickens, but this pathogenicity was markedly reduced in ab+ chickens. Md11/75C/R2 spread less by contact and replicated better, both in vivo and in vitro, than CVI988/C, another serotype 1 vaccine virus. Strain 301B/1 was similar to SB-1, another serotype 2 vaccine virus: both were nonpathogenic for ab--chickens, spread readily by contact, and replicated well in vivo. In vitro, 301B/1 grew more rapidly and produced larger plaques than SB-1. Notable characteristics of strain CVI988/C included absence of pathogenicity, poor replicative ability, and the absence of one epitope detected by a common serotype-1-specific monoclonal antibody. All four viruses could be distinguished from each other by restriction enzyme analysis of viral DNA. We conclude that Md11/75C/R2, although exceptionally protective, may require further attenuation. On the other hand, 301B/1, which in other studies induced higher levels of protection than SB-1, is nonpathogenic and may be considered for use as a commercial vaccine.

Load of Challenge Marek's Disease Virus DNA in Blood as a Criterion for Early Diagnosis of Marek's Disease Tumors

Abstract Outbreaks of Mareks disease (MD) in vaccinated flocks still occur sporadically and lead to economic losses. Unfortunately, adequate methods to predict MD outbreaks are lacking. In the present study, we have evaluated whether high load of challenge MD virus (MDV) DNA in peripheral blood could aid in the early diagnosis of MD and in monitoring efficacy of vaccines against MD. One experiment was conducted to simulate field conditions by combining various vaccines (turkey herpesvirus [HVT] and HVT + MDV serotype 2 [SB1]) and challenge viruses (GA, Md5, and 648A). Vaccine efficacy among our experimental groups ranged from 13.3% to 94.2%. Each chicken was sampled three times during the length of the experiment (3, 5, and 15 wk postchallenge [wpc]), and gross lesions were evaluated in chickens that died and at termination of the experiment. DNA was extracted from whole blood and buffy coats from each sample, and the load of challenge MDV DNA and HVT DNA were quantified by real-time polymerase chain reaction. Chickens that developed MD by the end of the experiment had higher load of challenge MDV DNA (threshold cycle [Ct] glyceraldehyde-3-phosphate dehydrogenase [GAPDH]/Ct glycoprotein B [gB] ratios of 1.0, 1.04, and 1.05 at 3, 5, and 15 wpc, respectively) than those that did not develop MD (Ct GAPDH/Ct gB ratios of 0.7, 0.69, and 0.46 at 3, 5, and 15 wpc, respectively). However, load of HVT DNA in blood was not correlated with the development of tumors (Ct GAPDH/Ct HVT ratios from 0.04 to 0.10 in both groups). Vaccinated groups with >75% protection had statistically significant less challenge DNA virus (Ct GAPDH/Ct gB ratios of 0.76, 0.70, and 0.45 at 3, 5, and 15 wpc, respectively) than less protected groups (Ct GAPDH/Ct gB ratios of 0.92, 0.97, and 0.85 at 3, 5, and 15 wpc, respectively). No differences in the load of HVT DNA could be found between protected and nonprotected groups at any time point of the study (Ct GAPDH/Ct HVT from 0.05 to 0.09 in both groups). Our results showed that load of challenge MDV DNA but not load of HVT DNA in blood can be used as criterion for early diagnosis of MD.