Pathogenetical characterization of isolate MHV-60 of mouse herpesvirus strain 68.

Infection of mice with mouse herpesvirus strain 68 (MHV-68) is an excellent small animal model of gammaherpesvirus pathogenesis in a natural host. We carried out comparative studies on MHV-60, another isolate of MHV-68. The acute infection of BALB/c mice inoculated intranasally (i.n.) with MHV-60 as well as its impact on tumor development were investigated. During the acute phase of infection the lungs were the main tissues infected. Our results show that MHV-60 has similar pathological features like other 4 isolates so far examined, namely MHV-72, MHV-78, MHV-Sumava inclusive of MHV-68. Nevertheless, MHV-60 differed from other isolates in following features: (i) the acute phase of infection was established very soon and lasted 10 days post infection (p.i.) in contrast to 14-28 days p.i. in the abovementioned isolates with a peak on days 3-5 p.i. The virus could also be recovered from the spleen, thymus and kidneys but not in other investigated organs. A lymphoproliferative response was associated with splenomegaly. At this time an increase in the number of leukocytes and appearance of atypical leukocytes in peripheral blood were observed. (ii) the infection was localized in the lungs and spleen, while in other isolates it was detected in a much broader scale of organs, and (iii) the acute phase of infection was accompanied by a massive splenomegaly, which was characteristic for the chronic phase of infection. Despite the fact that after clearance of the acute infection the virus was hardly detected, the tumor formation was later observed in 22% of infected mice as compared to 5% in control non-infected mice.

Antigenic relationship between five isolates of murine gammaherpesvirus analysed with monoclonal antibodies.

A panel of six monoclonal antibodies (mAbs) specific for murine gammaherpesvirus (MHV) was used for analysis of the antigenic relationship between five MHV-isolates (MHV 68, MHV 72, MHV 76, MHV 78, MHV S). Two mAbs raised against MHV 72 and four raised against MHV S were used in the study. Antibody-virus interactions were tested using immunochemical (ELISA, Western blot, immunofluorescence) and biological (virus-neutralization) assays. Immunoanalysis by ELISA showed a close antigenic relationship between the five viruses, nevertheless, some antigenic individuality of the isolate MHV S was observed. This isolate originated from a geographically distinct area in Czechia relative to the other four isolates from Slovakia. In Western blot analysis, antibodies to MHV 72 recognized viral antigens with the relative molecular mass about 116,000. Of four mAbs against MHV S, only two reacted with denatured viral antigen in Western blot and showed specificity for the 50-55,000 protein. These findings suggested that both isolates, besides of minor antigenic variability, could differ also in immunodominant proteins. Mabs to MHV S exhibited much stronger virus-neutralizing potency than mAbs to MHV 72, indicating thus that the 50-55,000 antigen might be more relevant for the infectivity of MHV-virus. Immunofluorescence with mAbs allowed specific localization of antigens in virus-infected VERO cells.

Replication and persistence of murine herpesvirus 72 in lymphatic system and peripheral blood mononuclear cells of Balb/C mice.

After intranasal inoculation of murine herpesvirus 72 (MHV-72) to Balb/c mice the virus persisted in adherent lung mononuclear cells (AMC). Infectious virus was occasionally detected during a period of eight months after infection in lymphatic organs (thymus, spleen, lymph nodes), bone marrow, alveolar and peritoneal AMC, and lymphocytes and macrophages of peripheral blood by indirect immunofluorescence and cocultivation with permissive VERO cells. Mouse B-cell lines NS0 and SP2/0 were permissive for MHV-72 infection when inoculated in vitro. Virus-neutralizing antibodies have been detected during one year period in the sera of infected mice, and their levels correlated with activation of the latent MHV-72.

Interferon-neutralizing or enhancing activities in hybridoma cell fluids after in vitro immunization.

Several hybridomas supernatants capable of interferon beta (IFN-beta) or "IFN epsilon" ("IFN-eps") neutralizing or enhancing activities were obtained after in vitro immunization of BALB/c and C57 mice spleen cells and their fusion with Sp2/0 plasmacytoma cells. Besides rather low anti-IFN-beta or "eps" antibody secretion several cloned hybridoma fluids contained a factor potentiating anti-viral activity of the both IFNs. It is speculated that this activity is due to production by some hybridomas of another lymphokine.

Some physicochemical properties of murine herpes virus.

The effect of incubation temperature variation and various pH values on the stability of murine herpesvirus isolate-76 (MHV-76) was investigated. The virus survival in rabbit embryo fibroblasts cells (REF) has been determined. At room temperature (23 degrees C) 50% of the virus became inactivated during 12 days and at 37 degrees C its half life was 9 days. MHV-76 was completely inactivated at 50 degrees C in 1 hr or at 41 degrees C in 7 hrs. MHV-76 retains its maximal infectivity at the pH range between 6-9 regardless of the duration of treatment. The pH range 3, 4, 5 and 10, 11, 12 caused either complete or more or less expressed inactivation of the virus. A complete inactivation MHV-76 was also achieved after treatment with ethyl ether, chloroform and 2M urea.

Polypeptides synthesized in rabbit cells infected with murine herpesvirus (MHV): a comparison of proteins specified by various MHV strains.

Polypeptides synthesized in rabbit embryo fibroblasts (REF) infected with six isolates of murine herpesvirus (MHV) coming from different localities in CSFR were compared by electrophoresis (SDS-PAGE). Altogether 28 virus-coded polypeptides with apparent molecular weights ranging from 240 kD to 16 kD were identified. While isolates MHV-60, MHV-72, and MHV-76 had identical polypeptide profiles, the isolates MHV-68 and MHV-78 lacked a polypeptide with apparent molecular weight of 46 kD, the rest of polypeptides being identical as well. The polypeptide profile of MHV Sumava A.f. was most different from that of other MHV isolates. This result is in agreement with some different biological properties of this isolate. At a high multiplicity of infection (40 TCID50 per cell) first polypeptides with apparent molecular weights 125 kD and 84 kD were detected by 5-9 hr post-infection (p.i.). All other proteins appeared 14-18 p.i.

Comparative polypeptide analysis of human, murine and strigis herpesviruses with murine cytomegalovirus by polyacrylamide gel electrophoresis.

The polypeptide composition of five purified murine herpesvirus (MHV) strains grown in a stable line of rabbit embryo fibroblasts (REF) was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and compared with herpes simplex virus type 1 (HSV-1). About 24 structural polypeptides of molecular mass ranging from 275,000 to 25,000 were identified in MHV and HSV-1. The polypeptide profiles of MHV and HSV-1, showed a close similarity. The polypeptides of MHV were further compared with those of HSV-1, HSV-2, herpes virus strigis (HVS) and murine cytomegalovirus (MCMV). Differences were found between herpesviruses of different origin and MCMV. SDS-PAGE analysis of the six strains of MCMV labelled with 14C-amino acid hydrolysate also revealed differences in electrophor eticprofiles of MHV and MCMV proteins, what was confirmed by densitometric scanning of HSV-1, MHV and MCMV.

Antigenic relatedness of alphaherpesviruses isolated from free-living rodents.

Complement fixation and virus neutralization tests confirmed that alphaherpesviruses isolated from free-living Apodemus flavicollis and Clethrionomys glareolus rodents form an antigenically identical or very close group. Immunofluorescence showed that antigen assembly and distribution within the infected cell resembles that of members of the Alphaherpesvirinae subfamily. Radioimmunoassay revealed close antigenic relatedness between five rodent herpesvirus isolates. Moreover it suggested a possible relatedness of these viruses to some virus species isolated from humans and animals.

Electron microscopy of rabbit embryo fibroblasts infected with herpesvirus isolates from Clethrionomys glareolus and Apodemus flavicollis.

Electron microscopy of rabbit embryo fibroblasts infected with five herpesvirus isolates from murine rodents (Clethrionomys glareolus and Apodemus flavicollis) revealed morphological changes characterized by the formation of intranuclear vacuoles, in which envelopment of nucleocapsids took place. The herpesvirus nucleocapsids were formed in the vicinity of intranuclear granular inclusions. Margination of chromatin, disaggregation of nucleoli and, in some isolates, the occurrence of tubular structures with a diameter of 33--35 nm characterized the ultrastructural changes in the nuclei. In the cytoplasm, membraneous vacuoles, containing numerous naked nucleocapsids and, in some isolates, large electron-dense bodies, containing non-enveloped nucleocapsids, were formed. Extracellular spaces contained either enveloped virions which, besides nucleocapsids, contained in their envelope a part of the dense material from intracytoplasmic dense bodies, or viral envelope-like structures without nucleocapsids partially filled with dense material from intracytoplasmic dense bodies. Ultrastructural development of these isolates in REF resembled that of freshly isolated herpes simplex type 1 viruses but some morphological changes resembled those characteristic of cytomegaloviruses.

Morphological, cytological and biological observations on viruses isolated from patients with subacute thyroiditis de Quervain.

New data on viruses isolated from patients with subacute thyroiditis de Quervain are reported. Characteristic morphological, cytological, some physico-chemical and biological features of the isolated viruses are described. A possible role of these viruses in human and animal health disorders is discussed. The isolated viruses remain unclassified so far.