Murine cytomegalovirus containing a mutation at open reading frame M37 is severely attenuated in growth and virulence in vivo

A pool of murine cytomegalovirus (MCMV) mutants was generated by using a Tn3-based transposon mutagenesis procedure. One of the mutants, RvM37, which contained the transposon sequence at open reading frame M37, was characterized both in tissue culture and in immunocompetent BALB/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M37 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M37 region, the viral mutant was severely attenuated in growth in both BALB/c and SCID mice after intraperitoneal infection. Specifically, titers of the Smith strain and rescued virus in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice at 21 days postinfection were about 5 x 10(5), 2 x 10(5), 5 x 10(4), 5 x 10(3), and 1 x 10(4) PFU/ml of organ homogenate, respectively; in contrast, titers of RvM37 in these organs were less than 10(2) PFU/ml of organ homogenate. Moreover, the virulence of the mutant virus appeared to be significantly attenuated because none of the SCID mice infected with RvM37 had died by 120 days postinfection, while all animals infected with the wild-type and rescued viruses had died by 26 days postinfection. Our results suggest that M37 probably encodes a virulence factor and is required for MCMV virulence in SCID mice and for optimal viral growth in vivo.     

Latent murine cytomegalovirus DNA in splenic stromal cells of mice.

Latency is an integral feature of the pathogenesis of cytomegalovirus infection and disease. Using in situ hybridization, we detected viral DNA in the splenic stroma of mice with acute murine cytomegalovirus (MCMV) infection but could not detect latent infection. By using enzymatic amplification of a 700-bp region of exon 4 of immediate-early gene 1 of MCMV, viral DNA was consistently detected in whole spleens of latently infected mice. MCMV DNA was detected in 16 of 23 stromal cell fractions from latently infected animals, in only 2 of 13 residual nonstromal cell fractions, and in none of 9 additional lymphocyte or macrophage-enriched nonstromal cell preparations. We conclude that MCMV DNA is maintained predominantly, and possibly exclusively, in stromal cells in the spleens of latently infected mice.     

Murine cytomegalovirus with a transposon insertional mutation at open reading frame m155 is deficient in growth and virulence in mice

A pool of murine cytomegalovirus (MCMV) mutants was previously generated by using a Tn3-based transposon mutagenesis approach (X. Zhan, M. Lee, J. Xiao, and F. Liu, J. Virol. 74:7411-7421, 2000). In this study, one of the MCMV mutants, Rvm155, which contained the transposon insertion in open reading frame m155, was characterized in vitro for its replication in tissue culture and in vivo for its growth and virulence in immunodeficient SCID mice. Compared to the wild-type strain and a rescued virus that restored the m155 region, the mutant is significantly deficient in growth in many organs of the infected animals. At 21 days postinfection the titers of Rvm155 in the salivary glands, lungs, spleens, livers, and kidneys of the intraperitoneally infected SCID mice were lower than the titers of the wild-type virus and the rescued virus by 50-, 1,000-, 500-, 100-, and 500-fold, respectively. Moreover, the viral mutant was attenuated in killing the SCID mice, as none of the SCID mice that were intraperitoneally infected with Rvm155 died until 38 days postinfection while all the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results provide the first direct evidence that a disruption of m155 expression leads to attenuation of viral virulence and growth in animals. Moreover, these results suggest that m155 is a viral determinant for optimal MCMV growth and virulence in vivo.     

Murine cytomegalovirus open reading frame M27 plays an important role in growth and virulence in mice

Using a Tn3-based transposon mutagenesis approach, we have generated a pool of murine cytomegalovirus (MCMV) mutants. In this study, one of the mutants, RvM27, which contained the transposon sequence at open reading frame M27, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. Our results suggest that the M27 carboxyl-terminal sequence is dispensable for viral replication in vitro. Compared to the wild-type strain and a rescued virus that restored the M27 region, RvM27 was attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. Specifically, the titers of RvM27 in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice at 21 days postinfection were 50- to 500-fold lower than those of the wild-type virus and the rescued virus. Moreover, the virulence of the mutant virus appeared to be attenuated, because no deaths occurred among SCID mice infected with RvM27 for up to 37 days postinfection, while all the animals infected with the wild-type and rescued viruses died within 27 days postinfection. Our observations provide the first direct evidence to suggest that a disruption of M27 expression results in reduced viral growth and attenuated viral virulence in vivo in infected animals. Moreover, these results suggest that M27 is a viral determinant required for optimal MCMV growth and virulence in vivo and provide insight into the functions of the M27 homologues found in other animal and human CMVs as well as in other betaherpesviruses.     

In vitro and in vivo characterization of a murine cytomegalovirus with a mutation at open reading frame m166

We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the mutants, Rvm166, which contained the transposon sequence at open reading frame m166, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. The viral mutant replicated as well as the wild-type Smith strain in vitro in NIH 3T3 cells, whereas the transposon insertion precluded the expression of >65% of the m166 open reading frame. Compared to the wild-type strain and a rescued virus that restored the m166 region, the viral mutant was significantly attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. At 21 days postinfection, the titers of the viral mutant in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice were lower than the titers of the Smith strain and the rescued virus by about 30000-, 10000-, 1000-, 300-, and 800-fold, respectively. Moreover, the virulence of the mutant virus appears to be severely attenuated because no death was found in SCID mice infected with the viral mutant up to 90 days postinfection, whereas all of the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results suggest that m166 probably encodes a virulence factor and is required for MCMV virulence in killing SCID mice and for optimal viral growth in vivo.     

In vitro and in vivo characterization of a murine cytomegalovirus with a transposon insertional mutation at open reading frame M43

We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the MCMV mutants, RvM43, which contained the transposon inserted in open reading frame M43, was characterized. Our results provide the first direct evidence to suggest that M43 is not essential for viral replication in vitro in NIH 3T3 cells. Moreover, RvM43 exhibited a titer similar to that of the wild-type virus in the lungs, livers, spleens, and kidneys of both BALB/c and SCID mice and was as virulent as the wild-type virus in killing SCID mice that had been intraperitoneally infected with the viruses. In contrast, titers of the mutant virus in the salivary glands of the infected animals at 21 days postinfection were significantly (100 to 1,000-fold) lower than those of the wild-type virus and a rescued virus that restored the M43 region and its expression. Thus, M43 appears to be not essential for viral growth in vivo in the lungs, livers, spleens, and kidneys of infected animals and is also dispensable for virulence in killing SCID mice. Moreover, our results suggest that M43 is an MCMV determinant for growth in the salivary glands. Studies of viral genes required for replication in the salivary glands are important in understanding the mechanism of viral tropism for the salivary glands and shedding in saliva, which is believed to be one of the major routes of CMV transmission among healthy human populations.     

Murine cytomegalovirus with a transposon insertional mutation at open reading frame M35 is defective in growth in vivo

We had previously constructed a pool of murine cytomegalovirus (MCMV) mutants that contained a Tn3-based transposon sequence randomly inserted in the viral genome. In the study reported here, one of the mutants, RvM35, which contains the transposon insertion at open reading frame M35, was characterized both in vitro in tissue cultures and in immunocompetent Balb/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M35 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M35 region, the viral mutant was attenuated in growth in both the intraperitoneally infected Balb/c and SCID mice. At 21 days postinfection, the titers of the mutant in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice were lower than the titers of the wild-type Smith strain and the rescued virus by 50,000-, 100-, 10-, 100-, and 50-fold, respectively. Moreover, the growth of RvM35 is severely attenuated in the salivary glands. The virulence of the mutant virus also appears to be attenuated, because no death was observed in SCID mice infected with RvM35 until 35 days postinfection, while all the animals infected with the wild-type and rescued viruses died 27 days postinfection. Our results suggest that M35 is important for MCMV virulence in killing SCID mice and is required for optimal viral growth in vivo, including in the salivary glands.     

Studies of latent cytomegalovirus infection: the macrophage as a virus-harboring cell

During chronic infection of mice with mouse cytomegalovirus (MCMV), the virus was isolated from various tissues by cocultivation with allogeneic mouse embryonic fibroblasts (MEF). Infectious virus was recovered from over 15% of the pancreases, salivary glands, kidneys, lacrimal glands, and spleens. When activated macrophages were obtained by intraperitoneal injection of peptone into mice infected 3 months earlier, they harbored MCMV. Macrophages or lymphocytes were infected with MCMV in vitro and injected into normal mice intravenously. The peritoneal cavities of these mice were then stimulated by peptone injection 3 months after the transfer, and peritoneal or splenic macrophages and lymphocytes were cocultured with allogeneic MEF. MCMV was recovered from the peritoneal and splenic macrophages and not from the lymphocytes.     

The effect of biological response modifiers on chronic and latent murine cytomegalovirus infections

Host-mediated antiviral effect of 2 biological response modifiers (BRM), OK-432, and PS-K, against murine cytomegalovirus (MCMV) was evaluated in chronically or latently infected mice. In the early stage of chronic MCMV infection, the BRM-induced resistance was evidenced by decrease in infectious viruses replicated in the salivary glands and by augmented cytotoxic activity of the spleen cells against YAC-1 cells and MCMV-infected mouse embryonic fibroblasts (MEF). In the late stage of chronic MCMV infection, the BRM treatment did not eliminate MCMV from the mice, but did prevent exacerbation of MCMV infection in the salivary glands induced by administration of cyclophosphamide (CY). In mice latently infected by MCMV, BRM treatment suppressed CY-induced reactivation of MCMV in the salivary glands. It was suggested that the antiviral effect of BRM against MCMV in chronically or latently infected mice was based on activation of natural killer (NK) cells and cytotoxic T lymphocytes (CTL).     

Construction and characterization of murine cytomegaloviruses that contain transposon insertions at open reading frames m09 and M83

A transposon derived from Escherichia coli Tn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants, including two recombinant viruses that contained the transposon sequence within open reading frames m09 and M83. Our studies provide the first direct evidence to suggest that m09 is not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and in both BALB/c-Byj and CB17 severe combined immunodeficient (SCID) mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo. Moreover, the virus that contained the insertion mutation in m09 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of both the BALB/c and SCID mice and was as virulent as the wild-type virus in killing the SCID mice when these animals were intraperitoneally infected with these viruses. These results suggest that m09 is dispensable for viral growth in these organs and that the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo. In contrast, the virus that contained the insertion mutation in M83 exhibited a titer of at least 60-fold lower than that of the wild-type virus in the organs of the SCID mice and was attenuated in killing the SCID mice. These results demonstrate the utility of using the Tn3-based system as a mutagenesis approach for studying the function of MCMV genes in both immunocompetent and immunodeficient animals.     

Murine cytomegalovirus gene amplification and culture after submaxillary salivary gland biopsy.

An important target tissue for murine cytomegalovirus (CMV) infection is the submaxillary salivary gland. Submaxillary salivary gland biopsy specimens from BALB/c mice latently infected with murine CMV were examined for murine CMV DNA by in vitro enzymatic amplification using the polymerase chain reaction preceding oligonucleotide hybridization. The amplified sequence was a 152-base pair segment from within the immediate early gene of murine CMV. Biopsy and whole gland specimens from acutely infected BALB/c mice and latently infected, immunosuppressed BALB/c mice were compared for active murine CMV infection. After acute infection with murine CMV, virus was recovered in all cultures of both biopsy and whole salivary gland specimens but from none of the latently infected animals. Reactivated virus was detected by culture of both biopsy (90%) and whole salivary gland specimens (100%) from latently infected mice that received antithymocyte serum. Viral nucleic acid was detected in 90% of biopsy specimens from latently infected animals. Hence, active murine CMV infection can be detected in biopsy specimens from mice with acute and reactivated infection and murine CMV DNA can be amplified and detected in salivary gland biopsy specimens from latently infected animals. Biopsy of this or other target tissues can be useful for obtaining tissue for viral studies where the survival of the animal is important and it is useful to distinguish latent from acute or reactivated infection.     

Persistent humoral immune responses in the CNS limit recovery of reactivated murine cytomegalovirus

Background

Experimental infection of the mouse brain with murine CMV (MCMV) elicits neuroimmune responses that terminate acute infection while simultaneously preventing extensive bystander damage. Previous studies have determined that CD8⁺ T lymphocytes are required to restrict acute, productive MCMV infection within the central nervous system (CNS). In this study, we investigated the contribution of humoral immune responses in control of MCMV brain infection.

Methodology/Principal Findings

Utilizing our MCMV brain infection model, we investigated B-lymphocyte-lineage cells and assessed their role in controlling the recovery of reactivated virus from latently infected brain tissue. Brain infiltrating leukocytes were first phenotyped using markers indicative of B-lymphocytes and plasma cells. Results obtained during these studies showed a steady increase in the recruitment of B-lymphocyte-lineage cells into the brain throughout the time-course of viral infection. Further, MCMV-specific antibody secreting cells (ASC) were detected within the infiltrating leukocyte population using an ELISPOT assay. Immunohistochemical studies of brain sections revealed co-localization of CD138⁺ cells with either IgG or IgM. Additional immunohistochemical staining for MCMV early antigen 1 (E1, m112–113), a reported marker of viral latency in neurons, confirmed its expression in the brain during latent infection. Finally, using B-cell deficient (Jh^−/−) mice we demonstrated that B-lymphocytes control recovery of reactivated virus from latently-infected brain tissue. A significantly higher rate of reactivated virus was recovered from the brains of Jh^−/− mice when compared to Wt animals.

Conclusion

Taken together, these results demonstrate that MCMV infection triggers accumulation and persistence of B-lymphocyte-lineage cells within the brain, which produce antibodies and play a significant role in controlling reactivated virus.     

Interaction of herpes simplex virus type 2 with a rat glioma cell line

The interaction between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and two neural cell lines, mouse neuroblastoma (N1E-115) and rat glioma (C6-BU-1), was investigated. N1E-115 cells were permissive to both types of HSV. In C6-BU-1 cells, on the other hand, all the HSV-1 strains tested so far showed persistent infection, and the infectious virus of HSV-2 strains disappeared spontaneously. The HSV-2-infected C6-BU-1 cells were positive for HSV-2-specific DNA sequences, virus-specific RNA, HSV-2-specific antigens and thymidine kinase activity, when no infectious virus was detected. The HSV-2 was reactivated from those C6-BU-1 cells by superinfection with murine cytomegalovirus (MCMV), but not with UV-irradiated MCMV or human cytomegalovirus. The reactivated HSV-2 was identical to the parental virus, when examined by restriction endonuclease cleavage analysis.     

Lipopolysaccharide, tumor necrosis factor alpha, or interleukin-1beta triggers reactivation of latent cytomegalovirus in immunocompetent mice

We have previously shown that cytomegalovirus (CMV) can reactivate in lungs of nonimmunosuppressed patients during critical illness. Our recent work has shown that polymicrobial bacterial sepsis can trigger reactivation of latent murine CMV (MCMV). We hypothesize that MCMV reactivation following bacterial sepsis may be caused by inflammatory mediators. To test this hypothesis, BALB/c mice latently infected with Smith strain MCMV received sublethal intraperitoneal doses of lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), or saline. Lung tissue homogenates were evaluated for viral reactivation 3 weeks after mediator injection. Because LPS is known to signal via Toll-like receptor 4 (TLR-4) in mice, further studies blocking this signaling mechanism were performed using monoclonal MTS510. Finally, mice were tested with intravenous TNF-alpha to determine whether this would cause reactivation. All mice receiving sublethal intraperitoneal doses of LPS, TNF-alpha, or IL-1beta had pulmonary reactivation of latent MCMV 3 weeks following injection, and LPS caused MCMV reactivation with kinetics similar to those for sepsis. When TLR-4 signaling was blocked, exogenous LPS did not reactivate latent MCMV. Intravenous TNF-alpha administration at near-lethal doses did not reactivate MCMV. Exogenous intraperitoneal LPS, TNF-alpha, and IL-1beta are all capable of reactivating CMV from latency in lungs of previously healthy mice. LPS reactivation of MCMV appears dependent on TLR-4 signaling. Interestingly, intravenous TNF-alpha did not trigger reactivation, suggesting possible mechanistic differences that are discussed. We conclude that inflammatory disease states besides sepsis may be capable of reactivating CMV from latency.     

Lack of Interleukin-6 (IL-6) Enhances Susceptibility to Infection but Does Not Alter Latency or Reactivation of Herpes Simplex Virus Type 1 in IL-6 Knockout Mice

The ability of the pleotropic, proinflammatory cytokine interleukin-6 (IL-6) to affect the replication, latency, and reactivation of herpes simplex virus type 1 (HSV-1) in cell culture and in IL-6 knockout (KO) mice was studied. In initial studies, we found no effect of exogenous IL-6, monoclonal antibodies to IL-6, or monoclonal antibody to the IL-6 coreceptor, gp130, on HSV-1 replication in vitro by plaque assay or reactivation ex vivo by explant cocultivation of latently infected murine trigeminal ganglia (TG). Compared with the wild-type (WT) mice, the IL-6 KO mice were less able to survive an ocular challenge with 10(5) PFU of HSV-1 (McKrae) (40% survival of WT and 7% survival KO mice; P = 0.01). There was a sixfold higher 50% lethal dose of HSV-1 in WT than IL-6 KO mice (1.7 x 10(4) and 2.7 x 10(3) PFU, respectively). No differences were observed in titers of virus recovered from the eyes, TG, or brains or in the rates of virus reactivation by explant cocultivation of TG from latently infected WT or KO mice. Exposure of latently infected mice to UV light resulted in comparable rates of reactivation and in the proportions of WT and KO animals experiencing reactivation. Moreover, quantitative PCR assays showed nearly identical numbers of HSV-1 genomes in latently infected WT and IL-6 KO mice. These studies indicate that while IL-6 plays a role in the protection of mice from lethal HSV infection, it does not substantively influence HSV replication, spread to the nervous system, establishment of latency, or reactivation.     

Pathogenesis of murine cytomegalovirus infection in natural killer cell-depleted mice.

The effect of natural killer (NK) cells on the course of acute and persistent murine cytomegalovirus (MCMV) infection was examined by selectively depleting NK cell activity by inoculation of mice with antibody to asialo GM1, a neutral glycosphingolipid present at high concentrations on NK cells. The dose of MCMV required to cause 50% mortality or morbidity in control C57BL/6 mice dropped 4- and greater than 11-fold, respectively, in mice first treated with anti-asialo GM1. NK cell-depleted mice had higher (up to 1,000-fold) virus titers in their lungs, spleens, and livers at days 3, 5, 7, and 9 postinfection. Spleens and livers of control mice were virus-free by day 7 postinfection, and their lungs showed no signs of active infection at any time. In contrast, MCMV had disseminated to the lungs of NK cell-depleted mice by day 5, and these mice still had moderate levels of virus in their lungs, spleens, and livers at day 9. Markedly severe pathological changes were noted in the livers and spleens of NK cell-depleted, MCMV-infected mice. These included ballooning degeneration of hepatocytes and spleen necrosis. MCMV-infected, NK cell-depleted mice had severe spleen leukopenia, and their spleen leukocytes exhibited a significantly lower (up to 13-fold) response to the T cell mitogen concanavalin A when compared with those of uninfected and MCMV-infected controls. It appeared that NK cells exerted their most potent antiviral effect early in the infection, in a pattern correlating with interferon production and NK cell activation; treatment with anti-asialo GM1 later in infection had no effect on virus titers. The relative effect of NK cell depletion on MCMV pathogenesis depended on the injection route of the virus. NK cell depletion greatly augmented MCMV synthesis and pathogenesis in mice inoculated either intravenously or intraperitoneally but had no effect on the course of disease after intranasal inoculation, at any time point examined. One month after intraperitoneal inoculation of virus, NK cell depletion resulted in a six- to eightfold increase in salivary gland virus titers in persistently infected mice, suggesting that NK cells may be important in controlling virus synthesis in the salivary gland during persistent infection. This treatment did not, however, induce dissemination of virus to other organs. These data support the hypothesis that NK cells limit the severity, extent, and duration of acute MCMV infection and that they may also be involved in regulating the persistent infection.     

Mucosal and Parenteral Vaccination against Acute and Latent Murine Cytomegalovirus (MCMV) Infection by Using an Attenuated MCMV Mutant

We used a live attenuated murine cytomegalovirus (MCMV) mutant to analyze mechanisms of vaccination against acute and latent CMV infection. We selected MCMV mutant RV7 as a vaccine candidate since this virus grows well in tissue culture but is profoundly attenuated for growth in normal and severe combined immunodeficient (SCID) mice (V. J. Cavanaugh et al., J. Virol. 70:1365–1374, 1996). BALB/c mice were immunized twice (0 and 14 days) subcutaneously (s.c.) with tissue culture-passaged RV7 and then challenged with salivary gland-passaged wild-type MCMV (sgMCMV) intraperitoneally (i.p.) on day 28. RV7 vaccination protected mice against challenge with 10⁵ PFU of sgMCMV, a dose that killed 100% of mock-vaccinated mice. RV7 vaccination reduced MCMV replication 100- to 500-fold in the spleen between 1 and 8 days after challenge. We used the capacity to control replication of MCMV in the spleen 4 days after challenge as a surrogate for protection. Protection was antigen specific and required both live RV7 and antigen-specific lymphocytes. Interestingly, RV7 was effective when administered s.c., i.p., perorally, intranasally, and intragastrically, demonstrating that attenuated CMV applied to mucosal surfaces can elicit protection against parenteral virus challenge. B cells and immunoglobulin G were not essential for RV7-induced immunity since B-cell-deficient mice were effectively vaccinated by RV7. CD8 T cells, but not CD4 T cells, were critical for RV7-induced protection. Depletion of CD8 T cells by passive transfer of monoclonal anti-CD8 (but not anti-CD4) antibody abrogated RV7-mediated protection, and RV7 vaccination was less efficient in CD8 T-cell-deficient mice with a targeted mutation in the β2-microglobulin gene. Although gamma interferon is important for innate resistance to MCMV, it was not essential for RV7 vaccination since gamma interferon receptor-deficient mice were protected by RV7 vaccination. Establishment of and/or reactivation from latency by sgMCMV was decreased by RV7 vaccination, as measured by diminished reactivation of MCMV from splenic explants. We found no evidence for establishment of splenic latency by RV7 after s.c. vaccination. We conclude that RV7 administered through both systemic and mucosal routes is an effective vaccine against MCMV infection. It may be possible to design human CMV vaccines with similar properties.