Contribution to the problem of infection of humans with a murine gammaherpesvirus.
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Biomedical subjects
J Mistríková
Publications and source records attributed to J Mistríková.
Establishment and characterization of a tumor cell line derived from a mouse infected with murine gammaherpesvirus 78.
A tumor cell line, NB-78, was derived from a lymphoma from a BALB/c mouse infected with Murine gammaherpesvirus 78 (MHV-78). Cultures of the cells of this line underwent till now more than 100 passages, displaying an epitheloid transformed morphology and a diploid complement of 40 chromosomes. Viral antigen was detected in 12% of cells by immunofluorescence (IF) test. Reactivation of latent MHV-78 was proved by detecting infectious virus in culture medium only at passages 4345. The presence of viral M1, M2, M3, and M4 gene sequences in the genome of the cells was demonstrated by PCR. NB-78 is the first continuous cell line, which originates from a tumor of a MHV-78-infected host, harbors viral genome or at least its several genes, and produces infectious virus only rarely upon reactivation. It can be assumed that this cell line is primarily associated with MHV-78 and will serve as an invaluable tool for studying the MHV-78 latency.
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Leukemia-like syndrome in Balb/c mice infected with the lymphotropic gamma herpesvirus MHV-Sumava: an analogy to EBV infection.
Based on our previous observation that primary infection with the murine gamma herpesvirus (MHV) isolate Sumava (MHV-SU) undergoes a lymphoproliferative phase resembling to Epstein-Barr virus (EBV) induced infectious mononucleosis (IM), we evaluated white blood cell (WBC) counts at late stages following MHV-SU infection. In consequence of intranasal inoculation with MHV-SU a leukemia-like syndrome in Balb/c mice developed. The syndrome in question was accompanied with significant splenomegaly; in the peripheral blood leukocytosis (from 8 x 10(4) to 5 x 10(5) leukocytes/microl) and a high percentage of atypical lymphocytes (60-80%) was found. Presented results are bringing further evidence for lymphoproliferative effect of MHV and point at analogic course of MHV-SU and EBV infections.
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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.
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Immunophenotyping of leukocytes in peripheral blood of BALB/c mice infected with mouse herpesvirus isolate 72.
We characterized leukocytes in peripheral blood of BALB/c mice infected with mouse herpesvirus isolate 72 (MHV-72) representing an isolate of mouse herpesvirus strain 68 (MHV-68, species Murid herpesvirus 4, genus Rhadinovirus, subfamily Gammaherpesvirinae, family Herpesviridae) (van Regenmortel et al., 2000). In acute infection (up to day 30 post infection (p.i.)) the number of CD8+ T cells increased, reaching a maximum at day 11 p.i. This increase correlated with that of CD4+ T, activated CD 19+ B and natural killer (NK) cells. At day 30 p.i. the numbers of CD4+, CD8+, CD14+ and CD19+ cells decreased to normal values. A similar increase in the number of these cells was observed at day 730 p. i. In the course of persistent infection (after day 30 p.i.) some of the mice developed a leukemia-like syndrome characterized by an increase in the number of leukocytes and appearance of atypical, blastic immature forms of leukocytes. The latter forms of leukocytes were characteristic by an increased amount of argyrophilic proteins. These results show further similarities between MHV-72 (another isolate of MHV-68) and EBV infections and justify the use of MHV-68 or MHV-72 as an appropriate mouse model for the study of EBV infection of humans.
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Pathogenetic characterization of a mouse herpesvirus isolate Sumava.
BALB/c mice inoculated intranasally (i.n.) with the mouse herpesvirus isolate Sumava (MHV-Sumava) did not show apparent symptoms of illness. However, they showed an increase in the number of leukocytes and appearance of atypical leukocytes in peripheral blood. The infiltration of spleen with atypical cells resulted in splenomegaly. In the course of the infection the virus persisted in lungs, spleen, thymus, bone marrow, mammary glands, peritoneal macrophages and liver. We regard MHV-Sumava as one of the eight isolates of MHV-68 (a virus and species Murid herpesvirus 4, genus Rhadinovirus, subfamily Gammaherpesvirinae, family Herpesviridae (van Regenmortel et al., 2000)) so far obtained. MHV-Sumava differs from MHV-68, MHV-72 and MHV-76 in some virological and pathogenetical features.
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Increased neoplasm development due to immunosuppressive treatment with FK-506 in BALB/C mice persistently infected with the mouse herpesvirus (MHV-72).
BALB/c mice were infected with the lymphotropic mouse gammaherpesvirus (MHV-72). At late (7-12 months) post-infection intervals the latent virus was detected in the cells of lymphatic system (peripheral blood, lymphocytes and macrophages, thymocytes, lymph nodes, bone marrow, and peritoneal macrophages,) and in the spleen, lungs, liver, and kidney by cocultivation as well as by explantation. The MHV-72 infected mice, in which latency had been established, were treated with the immunosuppressive (IS) drug FK-506 (2 mg/kg/mouse for 30 days). This treatment increased the probability of virus reactivation by over two-fold. During the post-treatment observation period of 19 months, the incidence of lymphomas and the development of MHV-related lymphoproliferative and hemoblastic disorders raised to nearly five-fold in the drug treated mice as compared to untreated animals.
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Detection of abnormal lymphocytes in the blood of Balb/c mice infected with murine gammaherpesvirus strain 72: the analogy with Epstein-Barr virus infection.
We have followed the effect of murine gammaherpesvirus strain 72 (MHV-72) infection and immunosuppression on the differential white blood cell count of Balb/c mice. In both the acute and chronic phase of infection, abnormal lymphocytes resembling human B lymphocytes infected with Epstein-Barr virus (EBV) were detected. Immunosuppression had no significant effect on the haematological changes during the infection. Some of mice, which had developed tumours as a consequence of infection, showed splenomegaly, lymphadenopathy, leukocytosis and high percentage of immature blastic forms of leukocytes.
Acute Disease↗
Effect of immunosuppression on Balb/c mice infected with murine herpesvirus.
Balb/c mice were infected with murine herpesvirus (MHV-72) and subjected to immunosuppression (IS) with the antibiotic FK506 either during the acute or chronic phase of infection. Attempts to detect virus in various organs of immunosuppressed and non-immunosuppressed mice at different time interval were made. In the mice immunosuppressed on days 3-23 p.i. of the acute phase of infection virus was detected in various tested organs and tissues at 2.0 times higher rate than those of control mice. At later intervals of the acute phase of infection (56-84 days p.i.) virus was still recovered in bone marrow and lymph nodes peritoneal cells of immunosuppressed mice, but not in those of control animals. In the chronically infected mice immunosuppressed on days 290-320 p.i., the virus was detected in lungs, thymus, bone marrow, spleen and peritoneal cells at 3.5 times higher rate than in those of control mice.
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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.
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Further isolation of murine herpesviruses from small mammals in southwestern Slovakia.
A total of 69 small mammals of 6 species were collected in localities Marcelová and Kopác (southwestern Slovakia) and investigated. Two strains of murine herpesvirus (MHV) have been isolated and reisolated from Apodemus flavicollis. Both virus strains killed suckling mice after i.c. and i.p. inoculation. Adult mice were killed 4-7 days after i.c. inoculation. Cross antigenic reactions among 14C aminoacid hydrolysate labelled MHV-72 infected Vero cells and mouse immune sera against the two new isolates and rabbit immune sera raised against purified MHV strains No. 72 and No. 76 were done. Profiles of immunoprecipitated proteins are almost identical. All used immune sera crossreacted with major proteins of MHV-72 strain. We conclude that the two virus isolates from Apodemus flavicollis are new strains of MHV.
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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.
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[Classification of herpes viruses isolated from wild rodents].
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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.
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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.
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Isolation of five strains of herpesviruses from two species of free living small rodents.
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Animals↗
A murine gammaherpesvirus.
In 1976, within a project on isolation of herpesviruses from small rodents in former Czechoslovakia, the mouse herpesvirus strain 68 (MHV-68) was isolated (Blaskovic et al., 1980). This virus was accepted by The International Committee on Taxonomy of Viruses (ICTV) as a new, so far unassigned species (member) of the Gammaherpesvirinae subfamily of the Herpesviridae family (Murphy et al., 1995). Besides MHV-68, four more isolates (MHV-60, MHV-72, MHV-76, and MHV-78) similar to MHV-68 were obtained in that field experiment in Slovakia. Later, three more isolates (MHV-Sumava from Bohemia and MHV-4556 and MHV-5682 from Slovakia) were obtained in other field experiments. All these isolates are in some properties similar but in others different from each other. Nevertheless, as their comparative genome sequence analysis is not yet available, we propose at present to regard all the abovementioned isolates as different isolates of the same virus and strain, i.e. MHV-68. It is not excluded that a more detailed characterization of these isolates in the future will lead to proposals of designating some of these isolates as new strains of the virus of concern. This review summarizes the up to date knowledge of various biological and physico-chemical properties of MHV-68. At least three isolates, MHV-68, MHV-72 and MHV-Sumava seem to be involved in malignant neoplasm development in mice. It should be stressed that the pathogenesis of the induced lymphoproliferative disease in mice is similar to that caused by Epstein-Barr virus (EBV) in humans.
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