Ecology of the murine alphaherpesvirus and its isolation from lungs of rodents in cell culture.

Together 381 sera of murine rodents (Apodemus flavicollis, Clethrionomys glareolus, Microtus arvalis and Mus musculus) trapped in different localities of Czechoslovakia were examined for the presence of antibodies to a murine alphaherpesvirus (MHV) and to murine cytomegalovirus (CMV). Positivity of rodent sera to the two MHV and one murine CMV strains varied from none to 12.5% and from none up to 11.3%, respectively, depending on the locality under study. From the lungs of A. flavicollis showing serum antibodies to MHV, a further murine herpes virus strain was isolated in rabbit embryo fibroblasts (REF). The latter MHV reached a titre of 10(10) TCID50/ml in the 13th passage, causing typical cytopathic effect from the 2nd passage on.

Distribution of mouse cytomegalovirus in organs of white mice experimentally infected by natural route.

One, 10, 21-day-old and adult mice were inoculated by peroral and/or intranasal routes with mouse cytomegalovirus (MCMV). In animals surviving generalized infection, the virus could be demonstrated in salivary glands up to 123 days postinfection (p.i.). In mouse females which had eaten their infected and diseased offspring, the virus was detectable in salivary glands up to day 121, p.i. On day 16 p.i., the virus was present in salivary glands, lungs and kidneys of mice of different age groups, but no virus was recovered from their Gasserian ganglia. These results were compared with those obtained after infection with murine alpha herpesvirus.

Experimental pathogenesis of murine herpesvirus in newborn mice.

Newborn white mice were susceptible to peroral (p.o.) infection with murine alphaherpesvirus isolated from free-living Clethrionomys glareolus. Death occurred within 6-8 days in animals infected with the higher virus dose of 4.8 log10 TCID50. Clinical symptoms also occurred in some animals infected with lower doses, while others developed inapparent infection as judged by presence of humoral antibodies at 60 days post-infection (p.i.). The virus was detected in the lungs, blood, liver, spleen, kidneys, heart muscle, brain and urinary bladder of sick animals. Necrotising pneumonia accompanied the replication of the virus in the epithelial cells of alveolar ducts and alveolar lining as confirmed by immunofluorescence and histological examination. Latent infection of Gasserian ganglia in the survivors was not necessarily related to the administered dose of infectious virus. Two of mother females, which had eaten their diseased offspring, became inapparently infected as proved by reisolation of the virus from trigeminal ganglion explants and by detection of specific antibodies at 60 days p.i.

Growth characteristics of herpesviruses isolated from free living small rodents.

Sixteen cell cultures of different origin (bird, rodents, carnivores, pigs, monkey, man) reproduced five strains of herpesviruses isolated from small free living rodents Apodemus flavicollis and Clethrionomys glareolus revealing cytopathic changes typical for members of the family Herpesviridae. The virus titres in different cell cultures ranged from 10(2) to 10(7) TCD50 per ml. The growth curves of two isolates originating from both animal species resembled to those obtained with human herpesviruses type 1 and 2, pseudorabies virus and guinea pig herpes-like virus (Hsiung-Kaplow) in identical cell cultures. Mouse cytomegalovirus was grown practically in mouse embryo fibroblasts only. The five isolates from free living small rodents were classified according to cytopathic changes and growth characteristics in different cell cultures as members of the subfamily Alphaherpesvirinae.

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.

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.

Pathogenesis of acute and persistent murine herpesvirus infection in mice.

Outbred laboratory mice were inoculated at the age of 5, 10 and 21 days by oral and/or intranasal routes with 2 different (a lethal and a nonlethal) doses of the murine herpesvirus isolate 68 (MHV-68). Severe exudative pneumonia with haematogenous dissemination of the virus to liver, heart muscle, and kidneys developed in the 5-day-old as well as in a part of the 10-day-old mice. Virus antigen was found by immunofluorescence (IF) in the alveolar lining of lungs, in heart muscle fibres, in spleen and thymic lymphocytes, in the tubular epithelium cells of kidneys, in the neurons of Gasserian ganglia and in the intima of large pulmonary vessels. Electron microscopy confirmed the transfer of virus particles through the capillary endothelium of the damaged alveolar septa. The surviving progeny and the mothers of animals, which had not succumbed to the lethal virus dose, were kept for 141-169 days when lungs and Gasserian ganglia were examined for virus presence. MHV-68 was recovered both by direct examination of the tissue homogenates as well as by the explantation technique. The results are suggestive for a dynamic persistence of MHV-68 rather than for static latency.

Purification of murine alpha-herpesvirus and some properties of its DNA.

Strain Sumava Af of murine alpha-herpesvirus (MHV) banded in density gradient at the boundary between 10-20% and 20-30% Ficoll solution. In electron microscope, the partially purified virus visualized by negative staining exhibited intact or disrupted envelope structures and capsid particles of about 110 nm in outer diameter. The DNA extracted from partially purified virus and further purified in CsCl density gradient had the Tm-value of 79.0 degrees C. The molecular weight of about 90 million daltons was calculated for viral DNA according to its migration in agarose gel.

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.

Radioimmunoassay of influenza A virus haemagglutinin. II. Antigenic cross-reactions of influenza A (H3 subtype) viruses as determined by radioimmunoassay and haemagglutination inhibition tests.

Individual rabbits differed greatly in their antibody response to the "strain-specific" and "cross-reactive" antigenic determinants on the haemagglutinin (HA) subunit of influenza virus recombinant MRC11 (H3N2) and influenza virus Dunedin (H3N2), after immunization with whole virus or bromelain-released haemagglutinin (B-HA). Consequently, diverse cross-reactions between htese viruses and A/Hong Kong/68 virus were found in the haemagglutination inhibition (HI) test as well as in homologous radioimmunoassay (125I-B-HA from MRC11:anti MRC11 serum, and 125I-B-HA from Dunedin: anti Dunedin serum) when sera from different animals were employed. Radioimmunoassay (RIA), over and above to the HI test, was able to differentiate clearly the respective HAs also with antisera reacting to the same HI titre with both corresponding influenza virus strains. Thus it appeared that antigenic differences could be identified with higher sensitivity by homologous RIA than by the HI test and that multiple antigenic determinants were reactive on the 125I-B-HA in the RIA procedure employed. MRC11 and A/HK/68 viruses were also compared by heterologous RIA (125I-B-HA from MRC11: anti A/HK/68 serum). It was found that preferentially antigenic determinants with a high degree of cross-reactivity could be studied in the heterologous system.

Relative role of B and T lymphocytes in pathogenesis of a murine herpes virus.

Pathogenesis of a murine herpes virus was investigated in inbred strains (BALB/c, CBA, AKR and C57BL/10) of mice. After intranasal inhalation, virus was found to replicate primarily in the lungs, followed by haematogenous spread to the target organs (adrenal glands and ganglia). AKR (H-2k) were found to be most susceptible to virus infection while CBA (H-2k) mice appeared to be relatively resistant. Infection of B-cell depleted BALB/c mice resulted in detection of lower lung virus titres in B-cell depleted animals as compared to normal intact mice. Moreover, 3 of 12 normal mice in untreated group died of virus infection while deaths did not occur in the B-cell depleted group. Results of T-cell subset depletion experiments in BALB/c mice revealed maximum mortality in the group depleted of both Lyt-2+ and L3T4+ subpopulations. Infectious virus titres were also higher in lungs of T-cell depleted animals.

Impairment of calcium ATPases by high glucose and potential pharmacological protection.

The review deals with impairment of Ca(2+)-ATPases by high glucose or its derivatives in vitro, as well as in human diabetes and experimental animal models. Acute increases in glucose level strongly correlate with oxidative stress. Dysfunction of Ca(2+)-ATPases in diabetic and in some cases even in nondiabetic conditions may result in nitration of and in irreversible modification of cysteine-674. Nonenyzmatic protein glycation might lead to alteration of Ca(2+)-ATPase structure and function contributing to Ca(2+) imbalance and thus may be involved in development of chronic complications of diabetes. The susceptibility to glycation is probably due to the relatively high percentage of lysine and arginine residues at the ATP binding and phosphorylation domains. Reversible glycation may develop into irreversible modifications (advanced glycation end products, AGEs). Sites of SERCA AGEs are depicted in this review. Finally, several mechanisms of prevention of Ca(2+)-pump glycation, and their advantages and disadvantages are discussed.