HVT-infected Cells Research Articles

Novel Insights into the Roles of Bcl-2 Homolog Nr-13 (vNr-13) Encoded by Herpesvirus of Turkeys in the Virus Replication Cycle, Mitochondrial Networks, and Apoptosis Inhibition.

The Bcl-2 (B cell lymphoma 2)-related protein Nr-13 plays a major role in the regulation of cell death in developing avian B cells. With over 65% sequence similarity to the chicken Nr-13, herpesvirus of turkeys (HVT) vNr-13, encoded by the HVT079 and HVT096 genes, is the first known alphaherpesvirus-encoded Bcl-2 homolog. HVT-infected cells were reported to be relatively more resistant to serum starvation, suggested that vNr-13 could be involved in protecting the cells. Here, we describe CRISPR/Cas9-based editing of exon 1 of the HVT079 and HVT096 genes from the HVT genome to generate the mutant HVT-ΔvNr-13 to gain insights into its functional roles. Overall, wild-type HVT and HVT-ΔvNr-13 showed similar growth kinetics; however, at early time points, HVT-ΔvNr-13 showed 1.3- to 1.7-fold-lower growth of cell-associated virus and 3- to 6.2-fold-lower growth of cell-free virus. In transfected cells, HVT vNr-13 showed a mainly diffuse cytoplasmic distribution with faint nuclear staining. Further, vNr-13 localized to the mitochondria and endoplasmic reticulum (ER) and disrupted mitochondrial network morphology in the transfected cells. In the wild-type HVT-infected cells, vNr-13 expression appeared to be directly involved in the disruption of the mitochondrial network, as the mitochondrial network morphology was substantially restored in the HVT-ΔvNr-13-infected cells. IncuCyte S3 real-time apoptosis monitoring demonstrated that vNr-13 is unequivocally involved in the apoptosis inhibition, and it is associated with an increase of PFU, especially under serum-free conditions in the later stages of the viral replication cycle. Furthermore, HVT blocks apoptosis in infected cells but activates apoptosis in noninfected bystander cells.IMPORTANCE B cell lymphoma 2 (Bcl-2) family proteins play important roles in regulating apoptosis during homeostasis, tissue development, and infectious diseases. Several viruses encode homologs of cellular Bcl-2-proteins (vBcl-2) to inhibit apoptosis, which enable them to replicate and persist in the infected cells and to evade/modulate the immune response of the host. Herpesvirus of turkeys (HVT) is a nonpathogenic alphaherpesvirus of turkeys and chickens that is widely used as a live vaccine against Marek's disease and as recombinant vaccine viral vectors for protecting against multiple avian diseases. Identical copies of the HVT genes HVT079 and HVT096 encode the Bcl-2 homolog vNr-13. While previous studies have identified the potential ability of vNr-13 in inhibiting apoptosis induced by serum deprivation, there have been no detailed investigations on the functions of vNr-13. Using CRISPR/Cas9-based ablation of the vNr-13 gene, we demonstrated the roles of HVT vNr-13 in early stages of the viral replication cycle, mitochondrial morphology disruption, and apoptosis inhibition in later stages of viral replication.

Identification of the Cross-Reactive Antigens between Marek's Disease Virus and Pseudorabies Virus by Monoclonal Antibodies

Among the 33 monoclonal antibodies (MAbs) against pseudorabies virus (PRV) examined, three MAbs (24-17, 74-26, and 8) were found to react with cells infected with Marek's disease virus (MDV)-related viruses by immunofluorescence test. Two of the MAbs (24-17 and 74-26) reacted with the nuclei of cells infected with MDV serotype 1 (MDV1), MDV serotype 2 (MDV2), and herpesvirus of turkeys (HVT), whereas MAb 8 reacted with the cytoplasm of MDV2- and HVT-infected cells. However, none of the MAbs against MDV1, MDV2, and HVT that were examined reacted with PRV-infected cells. None of these three MAbs against PRV reactive with MDV-related viruses cross-reacted with the cells infected with other herpesviruses, such as herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, Epstein-Barr virus, or human herpesvirus 6. Southern-blot hybridization under stringent or less-stringent conditions showed that no significant DNA homology was detected between PRV DNA and MDV DNA.

Identification of Virus-specific Polypeptides by Monoclonal Antibodies against Serotype 2 Marek's Disease Virus

A total of 41 antibody-secreting hybridoma cells against the HPRS24 strain of Marek's disease virus (MDV) type 2 (MDV2) have been isolated. Of these monoclonal antibodies (MAbs), 24 were found by immunofluorescence tests to react specifically with MDV2-infected cells, but not MDV type 1 (MDV1)- or herpesvirus of turkeys (HVT)-infected cells, while eight reacted with MDV1- or MDV2-infected cells and nine with MDV1-, MDV2- or HVT-infected cells. By using these MAbs, seven classes of MDV type-specific or cross-reactive polypeptides were characterized by immunoprecipitation followed by SDS-PAGE. Among them, a 28K/32K glycoprotein differed from the previously identified gA and gB. The 28K/32K glycoprotein was found on the surface of MDV2-infected cells and in the cytoplasm by an immunofluorescence test with MAbs. In addition, a cross-reactive polypeptide of 25K/29K was also detected in MDV1-infected cells with MAbs reactive with the 28K/32K glycoprotein of MDV2.

Antigen A gene of Marek's disease virus (MDV) detects two mRNA species (3.7 and 2.1 kb) in MDV- and HVT-infected cells.

The gene of glycoprotein A of Marek's disease virus (MDV) detected to mRNA species (3.7 and 2.1 kb) by Northern blot hybridization. These two mRNA species were also detected in RNA extracted from herpesvirus of turkeys (HVT)-infected chick embryo fibroblasts (CEF) and from the MSB-1 cell line.

Partial Protection against Marek's Disease in Chickens Immunized with Glycoproteins gB Purified from Turkey-Herpesvirus-Infected Cells by Affinity Chromatography Coupled with Monoclonal Antibodies

Glycoproteins gB of Marek's disease virus (MDV) and herpesvirus of turkeys (HVT) related to virus neutralization were purified from HVT-infected cells by affinity chromatography. Immunization of chickens with purified glycoproteins gB resulted in partial protection against MD. Neutralizing antibodies were detected in chickens immunized with HVT-gB.

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

The major immunogenic viral proteins of Marek's 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 immunofiuorescence 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).

Most virus-specific polypeptides in cells productively infected with Marek's disease virus or herpesvirus of turkeys possess cross-reactive determinants.

Most virus-specific polypeptides in cells productively infected with Marek's disease virus (MDV) or herpesvirus of turkeys (HVT) possess cross-reactive antigenic determinants, although the two viruses have very little DNA homology. The cross-reactivity appeared to be more evident when [35S]methionine-labelled polypeptides were immunoprecipitated than when the [3H]glucosamine-labelled polypeptides were immunoprecipitated, suggesting that the glycoproteins of MDV and HVT may be less related in structure than other proteins. The major cross-reactive glycoproteins excreted into the culture mediums of MDV- and HVT-infected cells had molecular weights of 64 000 (gp64) and 56 000 (gp56) respectively.

Dose-Dependent Inhibition of Virus Rescue from Lymphocytes Latently Infected with Turkey Herpesvirus or Marek's Disease Virus

The number of plaque-forming units (PFU) of turkey herpesvirus (HVT) isolated per 10(6) latently infected splenic lymphocytes was determined by co-cultivation on permissive monolayer cultures in 35-mm-diameter Petri dishes. Doses of 1 x 10(6) spleen cells or less per culture gave uniform dose-related titers, whereas doses of 8 x 10(6) cells often yielded less than 1-2% of the expected number of PFU. Intermediate doses gave proportionally reduced virus yields. This dose-dependent inhibition was observed with spleen cells from birds within a week after infection and became more marked with time. A similar phenomenon occurred with a non-oncogenic Marek's disease virus (MDV) isolate (SB-1) but not with oncogenic MDV isolates (CU-2, JM-10, GA-5), except in genetically resistant birds. High numbers of uninfected spleen cells mixed with low numbers of HVT-infected cells during assay reduced titers only slightly. Immunosuppression by combined neonatal thymectomy and cyclophosphamide treatment before HVT infection prevented the inhibition, but embryonal bursectomy had no effect.

Metabolic characteristics of cells infected with a herpesvirus of turkeys.

Chicken embryo fibroblasts infected with the nononcogenic herpesvirus of turkeys (HVT) displayed an increased rate of glucose uptake, a pronounced alteration of the pH of the medium, and an increased production of lactic acid when compared to mock-infected cultures. Objective estimates of cytopathology (quantifiable neutral red uptake and cell protein determination) showed that cell deterioration was a slow process in HVT-infected cells when compared to infection by herpes simplex virus. Experiments with irradiated host cells demonstrated that HVT required functional cell DNA for replication. The inactivation of the necessary host cell function displayed multihit kinetics. In agreement with data on other herpesviruses, HVT damaged by UV light could be photoreactivated in chick cells. The results indicate that HVT shares biologic properties in common with other herpes and transforming viruses.

Studies of Marek's disease herpesvirus and Turkey herpesvirus specific common antigen which stimulates the production of neutralizing antibodies

An agar-gel precipitation antigen prepared from the skin (feather-Ag) of chicken infected with JM strain of Marek's disease herpesvirus (MDHV) and cell extracts of cultures infected with either Type 2 plaque producing agent (PPA) of MDHV or turkey herpesvirus (HVT) (Type 2-Ag, HVT-Ag) had 1 precipitation line in common. Hyperimmune sera to the common antigen (common-Ag) neutralized to a greater extent by homologous antiserum. Absorption of HVT antiserum or hyperimmune serum to Type 2 PPA with common-Ag reduced their neutralizing activity. This reduction was almost complete with homologous antigen but less complete with heterologous antigen. The location of antigen in Type 2 PPA and HVT infected cells was determined using hyperimmune serum to the common-Ag in fluorescent antibody tests. Antigen mainly occurred in the cytoplasm of cells corresponding to the rounded refractile cells in the plaques, whereas unfixed cells showed antigen on the cytoplasmic membrane. The common-Ag associated with MDHV or HVT infections might be a virus structural component which is associated with the envelope.

Alterations of the immunological specificity of plasma membranes from cells infected with Marek's disease and turkey herpes viruses.

SUMMARY Highly purified plasma membranes were isolated from chicken embryo fibroblasts infected with Marek’s disease virus (MDV) or turkey herpes virus (HVT). The purification was monitored by the incorporation of fucose and marker enzymes specific for plasma membranes. Polyacrylamide gel electrophoresis showed that the membrane preparations contained two new virus-induced proteins. When reacted in the double immunodiffusion test solubilized plasma membranes from MDV-infected cells formed two specific precipitation bands with Marek’s disease immunoglobulins. Antisera prepared against plasma membranes from MDV- or HVT-infected cells neutralized extracellular infectious HVT. After incubation of plasma membranes from MDV-infected cells with Marek’s disease antibodies the buoyant density of the membranes increased from 1.05 g/ml to 1.08 g/ml due to the binding of immunoglobulins to the virus-induced membrane proteins. The Marek’s disease mortality of chickens twice vaccinated with a plasma membrane preparation from HVT-infected cells was reduced by 94%. From the results it was concluded that the plasma membranes acquire new immunologic specificities after infection with MDV or HVT.

Vaccination against Marek's disease: immunizing effect of purified turkey herpes virus and cellular membranes from infected cells.

One-day-old chickens susceptible to Marek's disease were vaccinated with experimental vaccines prepared from purified turkey herpes virus (HVT), inactivated HVT preparations or a membrane fraction isolated from HTV-infected chicken embryo fibroblasts, respectively. Purified HVT was found to be as effective in immunization against Marek's disease as cell-associated virus. The specific mortality of chickens twice vaccinated with cellular membranes from HVT-infected cells was reduced by 94%. These membranes also carried virus-specific antigens as shown by immunodiffusion tests.