Murid herpesvirus 4 (MuHV-4, prototype strain MHV-68) as an important model in global research of human oncogenic gammaherpesviruses.

The aim of this work was to give an overview of murid herpesvirus 4 (MuHV-4) (synonyms: murine gammaherpesvirus 68, mouse herpesvirus strain 68), the first model for the study of human and animal oncogenic gammaherpesviruses. Based on our results confirming similarity of murine gammaherpesvirus 68 (MHV-68) to another gammaherpesvirus, human oncogenic Epstein-Barr virus (EBV), we were able to interpret some processes observed in the course of MHV-68 infection in analogy to EBV infection. In particular, that were the processes occurring during MHV-68-induced persistent infection in mice accompanied by tumor formation and leukemia following immunosuppression. Since EBV is a highly species specific virus, infecting humans only, these processes cannot be experimentally examined at the molecular, cellular, and tissue levels in natural host. However, they can be investigated in BALB/c mice infected with MHV-68, which is nowadays generally accepted model mainly thanks to experimental results achieved by our research team. The important mouse model MHV-68 is a prototype strain of MuHV-4 species and is classified as a member of the order Herpesvirales, family Herpesviridae, subfamily Gammaherpesvirinae and genus Rhadinovirus. During 40 years since its isolation from wild rodents, the virus was distributed into many virological laboratories in Europe (such as England, Slovakia, Germany, Italy, Portugal, Belgium, Denmark, Spain, Switzerland, Hungary, Russia, Sweden), USA, Canada, China, Korea, Japan and Australia. Global research of this virus, which has become an irreplaceable animal model, has expanded our understanding of the pathogenesis and immunology of human and animal gammaherpesvirus infections as well as the gammaherpesvirus-associated oncogenesis. No less important fact is that MHV-68 provides an excellent model to explore methods for controlling gammaherpesvirus infections through vaccination and chemotherapy. Keywords: MHV-68; EBV; KSHV; immunology; pathogenesis; oncogenesis; genome.

Recombinant luciferase-expressing murine gammaherpesvirus 68 as a tool for rapid antiviral screening.

Murine gammaherpesvirus 68 (MHV-68) provides a valuable tool to screen novel therapeutic strategies against oncogenic gammaherpesviruses. The development and characterization of antiviral agents usually depend on appropriate screening assays. The aim of this study was to develop rapid and sensitive method for testing antiviral compounds against gammaherpesviruses. For this purpose, a recombinant MHV-68 expressing firefly luciferase (MHV-68/LUC) was constructed. The conditions for MHV-68/LUC infection in Vero cells suitable for novel antiviral screening assay in 96-well plate format were then optimized. The sensitivity of MHV-68/LUC to acyclovir (ACV) and ganciclovir (GCV) was measured by the optimized luciferase activity reduction assay. The 50% inhibition concentration (IC50) values for ACV and GCV were comparable to those determined by conventional plaque reduction assay. Therefore, the luciferase activity reduction assay can efficiently replace the plaque reduction assay. The great advantages of novel assay are represented by the significant reduction in assay time and rapid and objective measurement of the assay. In order to evaluate whether the luciferase activity reduction assay could be used as a screening system for novel antivirals, newly synthesized quinolone/quinoline derivatives were tested for their effects on the replication of MHV-68/LUC in vitro. The compound 2-(1-(b-D-Xylopyranosyl)-1,2,3-triazol-4-yl)-3,4-dibenzyloxy-quinoline showed significant antiviral activity and its IC50 against MHV-68/LUC was estimated to be 1,76 µg/ml. However, this compound was not suitable for in vivo testing due to its narrow selectivity index (SI = 11). Keywords: MHV-68; antiviral screening; luciferase; quinolone/quinoline derivatives.

The effect of Isoprinosine treatment on persistent infection of Balb/c mice infected with murine gammaherpesvirus 68.

We demonstrated the positive effect of Isoprinosine treatment on persistent infection of Balb/c mice with murine gammaherpesvirus 68 (MHV-68). Increased number of leukocytes, increased percentage of neutrophils, elevated levels of virus-neutralizing (VN) antibodies, reduced number of atypical lymphocytes and reduced virus titers were detected in the examined organs after a 14-day treatment. The positive effect of Isoprinosine therapy vanished after 120-150 days. After this interval, we demonstrated lower numbers of leukocytes, lower levels of VN antibodies and an increased number of atypical lymphoid monocytes in the Isoprinosine-treated group. Immunological parameters correlated with increased titers of virus in all investigated organs. Evidence of immunostimulation was demonstrated by lower incidence of tumor formation (7.5%) in the group of MHV-infected and Isoprinosine-treated mice in comparison to group without Isoprinosine treatment (17.5%). The presented results showed that Isoprinosine therapy had a positive impact on persistent infection of mice with MHV-68, but this effect was time-limited. The improvement of the investigated parameters lasted for five months only. Our presented results confirmed that each treatment with Isoprinosine should be repeated and must be long-term in some chronic infections.

The influence of dual infection with herpes and influenza viruses on the differential blood cell count of mice.

Based on our previous results, which confirmed the role of latent gammaherpesvirus infection in alteration of immune homeostasis, we studied the influence of simultaneous infection with gammaherpes and influenza viruses on selected parameters of innate immunity, particularly on the subpopulations of peripheral blood cell leukocytes. The aim was to analyze changes of differential blood cell count of BALB/c mice persistently infected with murine gammaherpesvirus 68 (MHV-68) and subsequently co-infected with influenza A virus (IAV), in comparison to mice infected with MHV-68 or with IAV only. Our results showed that ongoing gammaherpesvirus latency in mice caused a decreased number of leukocytes after acute infection with IAV in comparison to a single acute IAV infection. However, increased proportion of neutrophils was measured in peripheral blood of IAV- infected and co-infected mice. Dual infection had no effect on the proportion of monocytes or basophilic and eosinophilic granulocytes. The number of atypical lymphocytes, usually accompanying the persistent infection with MHV-68, decreased in co-infected mice as a consequence of the acute infection with IAV. Persistent infection with gammaherpesvirus may thus modulate the host immune response to influenza A virus and the acute IAV infection can influence the immune homeostasis established by latent MHV-68 infection.

Sequence variability among murine herpesvirus isolates shows possible effect of long-term in vitro passaging on their genome.

No abstract Keywords: murine herpesvirus 68; virus isolates; sequence analysis; restriction fragment length polymorphism.

Simultaneous infection with gammaherpes and influenza viruses enhances the host immune defense.

We have studied the impact of simultaneous infection of mice with murine gammaherpesvirus (MHV) and influenza A virus (IAV) on the immune response and pathogenesis of both infections. After a persistent MHV-68 herpesviral infection had been established, the same mice were super-infected with IAV. Individual parameters of MHV infection (viral DNA detection in organs and blood) and numbers of leukocytes in lungs and spleens were determined. With regard to the assumed reactivation of MHV-68 (mainly in lungs, spleen, thymus and peritoneal exudate cells) we focused our attention on the detection of transcripts, typical either for lytic infection (ORF50) and/or for latency (ORF73). Herpesviral DNA was detected in above mentioned organs in several intervals during the acute phase of IAV co-infection, but the expression of monitored transcripts was lower, i.e. it has decreased. Though the reason for such limited expression during acute influenza superinfection remains unclear, it is unambiguous that lower MHV-68 expression was detected in lungs and peritoneal exudate cells (PECs) from 3rd to 10th day after co-infection with IAV. Furthermore, our study showed that the ongoing gammaherpesvirus latency in co-infected mice affected the number of cytotoxic T-lymphocytes and neutrophils during the acute IAV infection and lowered their deviations from that of non-infected mice. Therefore, we suppose that co-infection with herpes and influenza viruses could be mutually beneficial for the host by promoting its defense against both viruses.

Possible role of different animal species in maintenance and spread of murine gammaherpesvirus 68 in the nature.

Murine gammaherpesvirus 68 (MHV-68), isolated from a bank vole (Clethrionomys glareolus) in Slovakia in 1976 is a natural pathogen of wild murid rodents. This review is focused to biological properties of this pathogen, the mode of its maintenance in murid rodents as reservoir animals, mechanisms of its spread to other animals in the same biotope as well as to livestock and household animals. Potential role of ticks as vectors and the possibility of infection of humans with this virus are considered as well. All the above evidence of the virus infection of various hosts is based on serological or molecular analytical data. The presented knowledge indicates important epizootologic consequences, namely harboring and permanent maintenance of the virus in murid rodents as reservoir animals with a real possibility of spread to other animals in the same biotope. These relationships imply a cross-species virus transmission with potential serious consequences for the infected animals or humans.

Biological and pathogenetic characterization of different isolates of murine gammaherpesvirus 68 (MHV-68) in the context of study of human oncogenic gammaherpesviruses.

Study of murine gammaherpesvirus 68 (MHV-68), which was discovered in 1980 in Slovakia, has led to many important findings regarding gammaherpesviral properties in general. Nowadays, it is considered to be a universal model used for detailed studies to determine pathogenetic, immunological and molecular aspects of oncogenesis in analogy to Epstein-Barr virus (EBV) and Kaposi΄s sarcoma-associated virus (KSHV). The objective of this work is to characterize biological and pathogenetic properties of the virus with an emphasis on our prior results concerning ecology, epidemiology, viral persistence in peritoneal macrophages, detection of malign and benign lymphoproliferations accompanied by the presence of atypical lymphocytes in blood during IM-like and leukemia-like syndromes. We are trying to elucidate the role of virus-specific genes in virulence, pathogenicity and murine gammaherpesvirus oncogenesis by comparison of molecular-biological, pathogenetic and oncogenic potential of MHV-68 isolates and deletion mutant MHV-76 and therefore help to understand the analogical processes that occur in EBV infected patients.

Vertical transmission of murine gammaherpesvirus 68 in mice.

The mouse infected with murine gammaherpesvirus 68 (MHV-68) is accepted animal model for investigation of pathogenesis, oncogenesis, immunology and molecular biology of gammaherpesviruses in their natural host. However, little is known about the host range, epidemiology and pathogenesis of this natural pathogen of free-living murid rodents. Therefore we addressed the question of transplacental transmission of MHV-68 from pregnant Balb/c mice chronically infected with the virus to their fetuses and shedding of the virus by breast milk from chronically infected mothers to their offspring. The mothers were positive to infectious virus and viral antigen in various organs but mainly in the spleen and peritoneal *macrophages. Virus-neutralizing serum antibodies, and leukocyte count with a proportion of atypical lymphocytes were increasing to elevated values with the time of infection. The chronic infection resulted in premature termination of pregnancy or reduced number and size of newborns due to their retarded development. Out of 10 infected pregnant mice just one died and another developed a tumor. All these results confirm the vertical transmission of MHV-68 in mice, teratogenicity of the virus and the virus shedding by breast milk.

Establishment of cell lines latently infected with non-oncogenic murine gammaherpesvirus 76.

Murine gammaherpesviruses 68 (MHV-68) and 78 (MHV-78), both inducing tumors in mice and a latent infection in cells in vitro, serve as models for study of human oncogenic gammaherpesviruses, namely Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this work, we succeeded in establishing a latent infection of HeLa and CGL1 cell lines with non-oncogenic murine gammaherpesvirus 76 (MHV-76), which differs from MHV-68 and MHV-78 besides by oncogenicity also by deletion of M1-M4 genes and eight tRNA-like sequences. Viral latency in these cell lines, λHeLa and λCGL1, was demonstrated by the presence of viral DNA, suppression of viral latency-associated ORF73 gene and appearance of low amounts of infectious virus following treatment with phorbol 12-myristate 13-acetate (PMA). Both latently infected cell lines showed irregular presence of viral antigen originating apparently from spontaneous reactivation. The growth of latently infected cells in culture was similar to that of non-infected ones. The latently-infected λHeLa cells did not induce tumors in mice following subcutaneous inoculation. These results (i) confirm that MHV-76 is the only non-oncogenic murine gammaherpesvirus of all the so far tested ones, (ii) suggest that some of the genes deleted in MHV-76 might be responsible for the oncogenicity of murine gammaherpesviruses, (iii) confirm that viral ORF73 is one of major latency-associated genes that is suppressed during virus reactivation, and (iv) present MHV-76 as another murine gammaherpesvirus useful as a model for study of gammaherpesvirus pathogenesis, oncogenicity, latency and reactivation.

Tumors induced by Murine herpesvirus 60 or by cell line NB-78 derived from a tumor induced by Murine herpesvirus 78 show presence of the inducing viruses.

Murine herpesviruses 60 and 78 (MHV-60, MHV-78), closely related to Mouse herpesvirus strain 68 (MHV-68), are oncogenic lymphotropic gammaherpesviruses, which may serve as models for study of human oncogenic gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this work, we attempted to detect an analog of the MHV-68 ORF73 gene in tumors induced in mice either directly by MHV-60 or indirectly by MHV-78 via inoculation of NB-78 cells derived from a tumor induced by MHV-78. Besides the ORF73 gene, viral antigen and infectious virus were searched for. We succeeded in inducing lymphomas in mice by NB-78 cells and thus confirmed their transformed character. Importantly, we showed that the tumors induced by either MHV-60 or NB-78 cells were positive for the ORF73 gene, viral antigen and infectious virus. These results confirmed the generally accepted hypothesis about the connection between reactivation of latent gammaherpesviruses and malignant tumorigenesis.

Sequence analysis of the regions flanking terminal repeats of the genome of umava isolate of murine gammaherpesvirus.

The umava isolate of murine gammaherpesvirus (MHV-umava) slightly differs from Murine gammaherpesvirus 68 (MHV-68) and two other isolates of murine gammaherpesvirus (MHV), MHV-76 and MHV-72 in some biological properties. To identify the region(s) in the MHV-umava genome responsible for this phenomenon, we compared the sequences flanking terminal repeats (TRs) of the MHV-umava genome with those of MHV-68, MHV-76 and MHV-72. Restriction and sequence analyses revealed in MHV-umava as compared to MHV-68 approximately 9.3 kbp deletion at the left end of the genome and approximately 1.5 kbp deletion at the right end of the genome. While the approximately 9.3 kbp deletion was similar to that in MHV-76, the approximately 1.5 kbp deletion was unique for MHV-umava.

Murine gammaherpesvirus 68 serum antibodies in general human population.

Previous studies using ELISA and virus neutralization test (VNT) have proved the presence of Murine gammaherpesvirus 68 (MHV-68) serum antibodies in sera of laboratory staff working with MHV-68, as well as in the general population. In this study, we investigated the incidence of serum antibodies to MHV-68 and to human herpesviruses presumably antigenically similar to MHV-68, as Herpes simplex virus 1 (HSV-1), Human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV), in general population using ELISA, VNT, and immunofluorescence assay (IFA). We also searched for the possible detection of false-positive reaction of MHV-68 antibodies due to cross-reactions between MHV-68 and the antibodies to the herpesviruses mentioned above. We found 16% of positive sera for MHV-68 antibodies by ELISA (titers of 1,600-102,400) and 4.5% by VNT and IFA (titers of 8-32). Tested human sera, either positive or negative for MHV-68 antibodies, were positive for antibodies to HSV-1, HCMV, and EBV (71/69%, 69/65%, and 66/49%, respectively). We concluded that the false-positivity of the sera for MHV-68 antibodies detected by ELISA was due to the nonspecific cross-reactions with antibodies to antigenically similar EBV.

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.

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.

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.