Keyvan Nazerian

Nucleotide and predicted amino acid sequences of Marek's disease virus homologues of herpes simplex virus major tegument proteins

The DNA sequence of an 8.4 kbp BamHI-EcoRI fragment of Mareks disease virus (MDV) strain GA was determined. Three of the predicted polypeptides are homologous to UL47, UL48 and UL49 encoding the major tegument proteins of herpes simplex virus type 1 (HSV-1), and four are homologous to HSV-1 UL45, UL46, UL49.5 and UL50. These seven genes are found in the long unique region of the MDV genome and are collinear with homologues in HSV-1 and varicella-zoster virus (VZV). Northern blot analysis revealed different transcriptional patterns from those of HSV-1 and VZV. MDV homologues of UL49.5, UL49 and UL47 lack a poly(A) signal immediately downstream of their coding regions. Amino acid conservation between MDV and HSV-1, and between MDV and VZV is as high as that between HSV-1 and VZV. The MDV homologue of UL48 shows 60% similarity to its HSV-1 counterpart. Amino acid sequence comparison reveals that the MDV homologue of UL48 lacks an acidic carboxyl terminus. This homologue, like the VZV homologue of UL48, may be involved in the trans-activation of immediate early genes and may function as an important component of the structural proteins.

Intracellular State of Marek's Disease Virus DNA in Two Tumour-derived Chicken Cell Lines

SummarynThe intracellular state of Mareks disease virus (MDV) DNA was investigated in two permanent chicken cell lines: HPRS-1 and MSB-1. The HPRS-1 line was established from an ovarian lymphoma of a chicken with Mareks disease and is a virus-non-productive line, while the MSB-1 line originates from another animal with a Mareks disease splenic lymphoma and is a low producer line. By repeated isopycnic centrifugation in CsCl, MDV DNA in the HPRS-1 line showed properties of integrated DNA, whereas in cells of the productive MSB-1 line both integrated and free virus DNA appeared to be present. Under denaturing conditions (0.1 m-NaOH) the virus DNA remained associated with the cellular DNA as revealed by equilibrium centrifugation in CsCl and hybridization of the DNA in each single fraction with 32P-labelled complementary RNA transcribed from the DNA of the GA strain of MDV. Shearing of HPRS-1 DNA to a mol. wt. of about 8 × 106 released only part of the virus DNA to the density of free virus DNA, while a large proportion of MDV DNA could still be localized at the density of cellular DNA. Sedimentation velocity experiments with HPRS-1 and MSB-1 DNA originally fractionated on CsCl gradients revealed integrated virus DNA sequences in both cell lines and an additional peak of virus DNA at the position of free linear MDV DNA in the MSB-1 line. No fast-sedimenting virus DNA molecules with properties of covalently closed circular structures could be detected. Further evidence for the presence of integrated virus DNA sequences in MDV-transformed cells was provided by the Hirt (1967) precipitation procedure.

Identification and functional analysis of the fowlpox virus homolog of the vaccinia virus p37K major envelope antigen gene.

A fowlpox virus (FPV) gene with homology to the vaccinia virus p37K major envelope antigen gene was identified and sequenced. The predicted product has a molecular weight of 43,018 Da (p43K). The FPV p43K gene has 37.5% identity with its vaccinia counterpart and higher homology with a molluscum contagiosum virus gene (42.6% identity). Based on upstream sequences, p43K appears to be regulated as a late gene. Recombinant FPV were generated in which a large portion of p43K was replaced by the Escherichia coli lacZ gene. These recombinants failed to produce visible plaques under standard conditions. After prolonged incubation the microplaques developed into small macroscopic plaques. Plaques were purified on the basis of lacZ expression. Single-cycle growth curves comparing the p43K-deleted recombinant (designated fJd43Z) with parental FPV showed that the two viruses produce identical amounts of intracellular virions, but that fJd43Z released 20-fold fewer infectious particles into the medium. CsCl gradient centrifugation of [3H]thymidine-labeled virus was employed to examine differences in the production of physical particles. The two viruses produced equivalent levels of intracellular virions, but fJd43Z failed to produce detectable levels of released particles. FPV p43K is therefore involved in the release of virions from infected cells.

Insertional inactivation of a fowlpox virus homologue of the vaccinia virus F12L gene inhibits the release of enveloped virions.

Insertion of the Escherichia coli lacZ gene into a ClaI restriction enzyme site of a 5.7 kb HindIII fragment of the fowlpox virus (FPV) genome resulted in the generation of stable recombinants. These recombinants produced plaques that were significantly smaller than those produced by parental FPV or by FPV recombinants containing the lacZ gene at other non-essential sites. Insertion of foreign DNA into the ClaI site disrupts a previously unidentified open reading frame (ORF) which potentially encodes a 74K polypeptide. The predicted amino acid sequence of this FPV ORF has 24% identity with the F12L ORF of vaccinia virus, the function of which is not currently known. Production of intracellular FPV was similar in cells infected with recombinant or parental viruses, but the number of infectious extracellular virions released into the medium by the recombinant was about 20% of that released by the parental virus. Likewise, the release of FPV particles, which were labelled in vivo with [3H]thymidine, was significantly lower in recombinant FPV-infected cells. These results suggest that the FPV homologue of the vaccinia virus F12L ORF is involved in the envelopment or release of infectious extracellular virions.

Structural Proteins of Two Different Plaque-Size Phenotypes of Fowlpox Virus

Structural polypeptides of two plaque-purified variant isolates of fowlpox virus differing in plaque morphology and size were examined by Coomassie blue-staining and immunoblot analysis of purified virions. A total of 30 structural polypeptides were observed, ranging in molecular weight from 14,100 to 122,600. A late polypeptide of 36,400 molecular weight was quite prominent in the small-plaque clone but absent in the large-plaque clone. Two other polypeptides, of 33,700 and 34,800 molecular weight, were present in virions from large-plaque virus and cell lysates of both clones but were absent in the small-plaque virions. These differences were observed whether the viruses were grown in chorioallantoic membrane or in chicken embryo fibroblast cultures. No difference was observed between the growth curves of the two virus clones. Differences observed in the polypeptides of the two viruses may be due to changes in the less conserved regions of viral DNA and may be used for differentiation of virus isolates.

Structural Polypeptides of Type II Avian Adenoviruses Analyzed by Monoclonal and Polyclonal Antibodies

Polypeptides of hemorrhagic enteritis virus (HEV) of turkeys and marble spleen disease virus (MSDV) of pheasants were analyzed by immune precipitation and immunoblot assays. A total of 11 polypeptides ranging in molecular weight from 14,000 to 97,000 were detected in lysates of HEV-infected turkey cells analyzed by immunoblot assay using a polyclonal antibody against HEV. Identical patterns were observed with preparations of MSDV. Five monoclonal antibodies (MAbs) against HEV were chosen based on their virus neutralization activity and used for identification of neutralizing epitopes of these two viruses. Three MAbs precipitated a single 97,000-molecular-weight hexon polypeptide in an immune precipitation assay.

Structural analysis of unstable intermediate and stable forms of recombinant fowlpox virus.

The stability and structure of the products of recombination in a fowlpox virus (FPV) system using the thymidine kinase (TK) gene as the insertion site were examined. A 4.6 kb chimeric DNA fragment from the pUV1 expression vector, containing the bacterial lacZ gene and the vaccinia virus P7.5 promoter, was ligated into the XbaI site of the FPV TK gene. The resulting vector, pFTKlacZb, was transfected into chicken embryo fibroblast cultures infected with FPV at an m.o.i. of 0.1. Recombinants were screened for the expression of beta-galactosidase. Five recombinants were isolated and plaque-purified to 80 to 90% for expression of beta-glucosidase. Serial cell culture passage of the recombinants led to the gradual reappearance of the non-recombinant parental phenotype. Southern hybridization analysis of EcoRI fragments from all five recombinants indicated that a single cross-over homologous recombination had occurred between either the 5 or the 3 end fragments of the TK gene, generating unstable intermediate recombinants incorporating the entire pFTKlacZb vector. Secondary intermolecular or intramolecular recombination of intergenic repetitive sequences within the intermediate recombinants appears to have resulted in frequent regeneration of the parental genotype and an infrequent generation of more stable recombinants. A method was developed to select stable recombinants by passage of the intermediate recombinants in chicken embryo fibroblast cultures treated with 5-bromo-2-deoxyuridine.

A double-antibody enzyme-linked immunosorbent assay for the detection of turkey hemorrhagic enteritis virus antibody and antigen.

A highly sensitive and specific double-antibody enzyme-linked immunosorbent assay (ELISA) is described for the detection of antigen and antibody of turkey hemorrhagic enteritis virus (HEV). The assay utilizes a virus-neutralizing monoclonal antibody (MAb) to capture the antigen and turkey antiserum against HEV as the second antibody. Microtiter plates were first coated with a dilution of 1:3000 of the MAb (300 ng immunoglobulin/well) and are used for detection of both antigen and antibody. For antibody detection, MAb-coated plates were treated with an appropriate dilution of a cell-culture-propagated HEV antigen and then reacted with the test turkey serum. For detection of HEV antigen, MAb-coated plates were treated with appropriate dilutions of test antigens and then reacted with purified anti-HEV turkey immunoglobulins. The assay for HEV antibody detection was more sensitive and specific than previously described single-antibody ELISAs. Using the double-antibody ELISA, it was found that the spleen of HEV-infected turkeys harbors very high levels of antigen. Traces of HEV antigen are present in some other organs. Infectivity assay for HEV is found to be about two orders of magnitude more sensitive than the ELISA for detection of virus.

A Recombinant Fowlpox Virus Expressing the Envelope Antigen of Subgroup A Avian Leukosis/Sarcoma Virus

A recombinant fowlpox virus (FPV) was constructed by inserting cloned sequences from Schmidt-Ruppin subgroup A avian sarcoma virus coding for the viral envelope (env) antigen into a nonessential region of FPV DNA downstream from a synthetic promoter. Sera from chickens hyperimmunized with the recombinant FPV neutralized the infectivity of the homologous subgroup A virus (RCASBP/AP) but only weakly neutralized the infectivity of Rous sarcoma virus, another subgroup A avian leukosis virus. Similarly, vaccination of 1-day-old chicks with this recombinant FPV protected against infection with RCASBP/AP virus but not against infection with another subgroup A Rous-associated virus (RAV-1). These results show that such a recombinant FPV can be used to protect chickens against avian leukosis virus and confirm previous observations that a type-specific antigenic variability existed within the subgroup A avian leukosis/sarcoma virus group.

Mutational analysis of the proteolytic cleavage site of glycoprotein B (gB) of Marek's disease virus.

The Mareks disease virus (MDV) glycoprotein B (gB) precursor, gp100, is proteolytically cleaved into two disulfide-linked subunits, gp60 and gp49. In the gB homologs of most other herpesviruses, a tetrapeptide, Arg-Xaa-Arg-Arg, is immediately upstream from the predicted cleavage site. We have investigated the specificity of the proteolytic cleavage in gp100 by introducing mutations within its predicted cleavage site (Arg-Leu-Arg-Arg) and expressed these mutants in recombinant fowlpox virus (FPV). The results show that all three Arg residues at the predicted cleavage site play an important role in the specific proteolytic cleavage of gp100. Furthermore, we demonstrated that the cleavage of gp100 is not necessary for transport of gB to the cell surface.