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.     

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.     

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 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.     

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.     

Latency, without persistence, of murine cytomegalovirus in the spleen and kidney.

It is not known if murine cytomegalovirus (MCMV) establishes a state of molecular latency independent of low-level persistent infection. The presence of low levels of infectious MCMV distinguishes persistence from molecular latency. Thus, the distinction between persistence and latency has depended on the sensitivity of plaque assays for detecting low levels of infectious virus in tissue of previously infected mice. To determine whether MCMV establishes molecular latency or remains persistent, we developed two assays for detecting low levels of MCMV in tissue. Using prolonged in vitro culture of virus with either mouse embryonic fibroblasts or the murine 3T12 fibroblast cell line, we reproducibly detected a single PFU of MCMV. Inclusion of undiluted sonicated tissue in this assay decreased sensitivity by up to 100-fold. However, sensitivity was improved to 1 PFU of MCMV when sonicated tissue was appropriately diluted. Severe combined immunodeficient (SCID) mice were also used to detect MCMV in sonicated tissue. Infection of SCID mice with a single PFU of MCMV killed two of eight SCID mice, and the 50% lethal dose of MCMV in SCID mice was 2 to 3 PFU. Applying these two methods, we detected infectious virus in 0 of 34 spleens, 1 of 34 kidneys, and 0 of 37 salivary glands from latently infected mice. Spleens and kidneys assessed for persistent virus contained MCMV DNA by PCR and reactivated after 10 to 50 days in explant cultures. Latently infected kidney cells reactivated after adoptive transfer to SCID mice. Quantitation of the MCMV genome by PCR showed that latently infected spleens without detectable infectious MCMV contained about 3,000,000 copies of the MCMV genome. These results demonstrate that MCMV latency in spleen and kidney exists in the absence of low-level persistent infection. Use of assays with defined sensitivity for detection of MCMV in tissue provides a basis for evaluation of cytomegalovirus gene expression in the spleen and kidney during molecular latency.     

Ocular avirulence of a herpes simplex virus type 1 strain is associated with heightened sensitivity to alpha/beta interferon.

BALB/c mice infected on the scarified cornea with herpes simplex virus type 1 strain 35 [HSV-1(35)] rarely developed ocular disease even at challenge doses as high as 10(7) PFU per eye. In contrast, HSV-1(RE) consistently induced stromal keratitis at an inoculum of 2 x 10(4) PFU. The goal of this study was to determine the reason for the difference in virulence between the two HSV strains. Both HSV-1 strains replicated to similar titers in excised corneal "buttons." However, after in vivo infection of the cornea, the growth of strain 35 was evident only during the first 24 h postinfection, whereas the replication of strain RE persisted for at least 4 days. In vitro tests revealed that HSV-1(35) was greater than 10 times more sensitive to alpha/beta interferon (IFN-alpha/beta) than HSV-1(RE). Both strains induced comparable serum levels of IFN after intraperitoneal inoculation. The kinetics of HSV-1(35) clearance from the eye was markedly altered by treatment with rabbit anti-IFN-alpha/beta. Virus titers exceeding 10(4) PFU per eye could be demonstrated 4 to 5 days postinfection in mice given a single inoculation of antiserum 1 h after infection. Furthermore, anti-IFN treatment in 3-week-old mice infected with HSV-1(35) led to the development of clinically apparent corneal disease which subsequently progressed to stromal keratitis in the majority of recipients. These results indicate that the striking difference in the capacity of HSV-1(35) and HSV-1(RE) to induce corneal disease was related to the inherently greater sensitivity of strain 35 to IFN-alpha/beta produced by the host in response to infection.     

An orally bioavailable antipoxvirus compound (ST-246) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus Challenge

ST-246 is a low-molecular-weight compound (molecular weight = 376), that is potent (concentration that inhibited virus replication by 50% = 0.010 microM), selective (concentration of compound that inhibited cell viability by 50% = >40 microM), and active against multiple orthopoxviruses, including vaccinia, monkeypox, camelpox, cowpox, ectromelia (mousepox), and variola viruses. Cowpox virus variants selected in cell culture for resistance to ST-246 were found to have a single amino acid change in the V061 gene. Reengineering this change back into the wild-type cowpox virus genome conferred resistance to ST-246, suggesting that V061 is the target of ST-246 antiviral activity. The cowpox virus V061 gene is homologous to vaccinia virus F13L, which encodes a major envelope protein (p37) required for production of extracellular virus. In cell culture, ST-246 inhibited plaque formation and virus-induced cytopathic effects. In single-cycle growth assays, ST-246 reduced extracellular virus formation by 10 fold relative to untreated controls, while having little effect on the production of intracellular virus. In vivo oral administration of ST-246 protected BALB/c mice from lethal infection, following intranasal inoculation with 10x 50% lethal dose (LD(50)) of vaccinia virus strain IHD-J. ST-246-treated mice that survived infection acquired protective immunity and were resistant to subsequent challenge with a lethal dose (10x LD(50)) of vaccinia virus. Orally administered ST-246 also protected A/NCr mice from lethal infection, following intranasal inoculation with 40,000x LD(50) of ectromelia virus. Infectious virus titers at day 8 postinfection in liver, spleen, and lung from ST-246-treated animals were below the limits of detection (<10 PFU/ml). In contrast, mean virus titers in liver, spleen, and lung tissues from placebo-treated mice were 6.2 x 10(7), 5.2 x 10(7), and 1.8 x 10(5) PFU/ml, respectively. Finally, oral administration of ST-246 inhibited vaccinia virus-induced tail lesions in Naval Medical Research Institute mice inoculated via the tail vein. Taken together, these results validate F13L as an antiviral target and demonstrate that an inhibitor of extracellular virus formation can protect mice from orthopoxvirus-induced disease.     

Genetic mapping of reovirus virulence and organ tropism in severe combined immunodeficient mice: organ-specific virulence genes.

We used reovirus reassortant genetics and severe combined immunodeficient (SCID) mice to define viral genes important for organ tropism and virulence in the absence of antigen-specific immunity. Adult SCID mice infected with reovirus serotype 1 strain Lang (T1L) died after 20 +/- 6 days, while infection with serotype 3 strain Dearing (T3D) was lethal after 77 +/- 22 days. One hundred forty-five adult SCID mice were infected with T1L, T3D, and 25 different T1L x T3D reassortant reoviruses, and gene segments associated with the increased virulence of T1L were identified. Gene segments S1, L2, M1, and L1 accounted for > 90% of the genetically determined increase in T1L virulence. Gene segment M1 was independently important for virulence, with S1, L2, and L1 alone or in combination also playing a role. T1L grew to higher titers in multiple organs and caused more severe hepatitis than T3D. Seventy adult SCID mice, T1L, T3D, and 15 T1L x T3D reassortant viruses were used to map genetic determinants of viral titers in the brain, intestines, and liver, as well as the severity of hepatitis. Different sets of gene segments were important for determining viral titers in different organs. Gene segments L1 (encoding a core protein) and L2 (encoding the core spike of the virion) were important in all of the organs analyzed. The M1 gene segment (encoding a core protein), but not the S1 gene segment, was a critical determinant of reovirus titer in the liver and severity of hepatitis. The S1 gene segment (encoding the viral cell attachment protein and a nonstructural protein), but not the M1 gene segment, was a critical determinant of titers in intestines and brains. These studies demonstrate that viral growth in different organs is dependent on different subsets of the genes important for virulence. The virion-associated protein products of the four gene segments (L1, L2, M1, and S1) important for virulence and organ tropism in SCID mice likely form a structural unit, the reovirus vertex. Organs (the brain and intestines versus the liver) differ in properties that determine which virulence genes, and thus which parts of this structural unit, are important.     

Detection of murine cytomegalovirus DNA in circulating leukocytes harvested during acute infection of mice.

We used virus assay and in situ hybridization with a cloned fragment of the murine cytomegalovirus (MCMV) genome to study MCMV infection of circulating leukocytes harvested from 3-week-old BALB/c, C57BL/6, and C3H mice infected with MCMV intraperitoneally. Infectious virus or MCMV DNA was detected in leukocytes on days 1 through 21 of infection in BALB/c mice and on days 3 through 7 in C57BL/6 mice. On days 5 and 7, MCMV DNA or infectious virus was detected in the leukocytes of 17 (94%) of 18 BALB/c mice and 10 (59%) of 17 C57BL/6 mice. In both strains infection peaked on days 5 and 7, when as many as 0.01 to 0.1% of the circulating leukocytes contained MCMV DNA. In C3H mice, however, infectious virus was rarely recovered from leukocyte fractions and MCMV DNA was detected in the circulating leukocytes of only one animal. Circulating leukocytes may have an important role in the dissemination of CMV infections in susceptible hosts.     

The murine gammaherpesvirus 68 M2 gene is required for efficient reactivation from latently infected B cells

Murine gammaherpesvirus 68 (gammaHV68) infection of mice provides a tractable small-animal model system for assessing the requirements for the establishment and maintenance of gammaherpesvirus latency within the lymphoid compartment. The M2 gene product of gammaHV68 is a latency-associated antigen with no discernible homology to any known proteins. Here we focus on the requirement for the M2 gene in splenic B-cell latency. Our analyses showed the following. (i) Low-dose (100 PFU) inoculation administered via the intranasal route resulted in a failure to establish splenic B-cell latency at day 16 postinfection. (ii) Increasing the inoculation dose to 4 x 10(5) PFU administered via the intranasal route partially restored the establishment of B-cell latency at day 16, but no virus reactivation was detected upon explant into tissue cultures. (iii) Although previous data failed to detect a phenotype of the M2 mutant upon high-dose intraperitoneal inoculation, decreasing the inoculation dose to 100 PFU administered intraperitoneally revealed a splenic B-cell latency phenotype at day 16 that was very similar to the phenotype observed upon high-dose intranasal inoculation. (iv) After low-dose intraperitoneal inoculation, fractionated B-cell populations showed that the M2 mutant virus was able to establish latency in surface immunoglobulin D-negative (sIgD(-)) B cells; by 6 months postinfection, equivalent frequencies of M2 mutant and marker rescue viral genome-positive sIgD(-) B cells were detected. (v) Like the marker rescue virus, the M2 mutant virus also established latency in splenic naive B cells upon low-dose intraperitoneal inoculation, but there was a significant lag in the decay of this latently infected reservoir compared to that seen with the marker rescue virus. (vi) After low-dose intranasal inoculation, by day 42 postinfection, latency was observed in the spleen, although at a frequency significantly lower than that in the marker rescue virus-infected mice; by 3 months postinfection, nearly equivalent levels of viral genome-positive cells were observed in the spleens of marker rescue virus- and M2 mutant virus-infected mice, and these cells were exclusively sIgD(-) B cells. Taken together, these data convincingly demonstrate a role for the M2 gene product in reactivation from splenic B cells and also suggest that disruption of the M2 gene leads to dose- and route-specific defects in the efficient establishment of splenic B-cell latency.     

Murine viruses in an island population of introduced house mice and endemic short-tailed mice in Western Australia

House mice (Mus domesticus) were recently introduced to Thevenard Island, off the northwest coast of Western Australia. This island is also habitat for an endangered native rodent, the short-tailed mouse (Leggadina lakedownensis). Concerns have been raised that house mice may pose a threat to L. lakedownensis both through competition and as a source of infection. To assess the threat to L. lakedownensis posed by viral pathogens from M. domesticus, a serological survey was conducted from 1994 to 1996 of both species for evidence of infection with 14 common murine viruses (mouse hepatitis virus, murine cytomegalovirus, lymphocytic choriomeningitis virus, ectromelia virus, mouse adenovirus strains FL and K87, minute virus of mice, mouse parvovirus, reovirus type 3, Sendai virus, Theiler's mouse encephalomyelitis virus, polyoma virus, pneumonia virus of mice, and encephalomyocarditis virus) and Mycoplasma pulmonis. Despite previous evidence that populations of free-living M. domesticus from various locations on the Australian mainland were infected with up to eight viruses, M. domesticus on Thevenard Island were seropositive only to murine cytomegalovirus (MCMV). Antibodies to MCMV were detected in this species at all times of sampling, although seroprevalence varied. Infectious MCMV could be isolated in culture of salivary gland homogenates from seropositive mice. In contrast, L. lakedownensis on Thevenard Island showed no serological evidence of infection with MCMV, any of the other murine viruses, or M. Pulmonis, and no virus could be isolated in culture from salivary gland homogenates. Although MCMV replicated to high titers in experimentally infected inbred BALB/c laboratory mice as expected, it did not replicate in the target organs of experimentally inoculated L. lakedownensis, indicating that the strict host specificity of MCMV may prevent its infection of L. lakedownensis. These results suggest that native mice on Thevenard Island are not at risk of MCMV infection from introduced house mice, and raise interesting questions about the possible selective survival of MCMV in small isolated populations of M. domesticus.     

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.     

Attenuated, replication-competent herpes simplex virus type 1 mutant G207: safety evaluation in mice

Herpes simplex virus type 1 (HSV-1) mutants that are attenuated for neurovirulence are being used for the treatment of cancer. We have examined the safety of G207, a multimutated replication-competent HSV-1 vector, in mice. BALB/c mice inoculated intracerebrally or intracerebroventricularly with 10(7) PFU of G207 survived for over 20 weeks with no apparent symptoms of disease. In contrast, over 80% of animals inoculated intracerebrally with 1.5 x 10(3) PFU of HSV-1 wild-type strain KOS and 50% of animals inoculated intracerebroventricularly with 10(4) PFU of wild-type strain F died within 10 days. Similarly, after intrahepatic inoculation of G207 (3 x 10(7) PFU) all animals survived for over 10 weeks, whereas no animals survived for even 1 week after inoculation with 10(6) PFU of KOS. After intracerebroventricular inoculation, LacZ expression was initially observed in the cells lining the ventricles and subarachnoid space; expression decreased until almost absent within 5 days postinfection, with no apparent loss of ependymal cells. G207 DNA could be detected by PCR in the brains of mice 8 weeks after intracerebral inoculation; however, no infectious virus could be detected after 2 days. As a model for latent HSV in the brain, we used survivors of an intracerebral inoculation of HSV-1 KOS at the 50% lethal dose. Inoculation of a high dose of G207 at the same stereotactic coordinates did not result in reactivation of detectable infectious virus or symptoms of disease. We conclude that G207 is safe at or above doses that were efficacious in mouse tumor studies.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus in vivo.

Our data show that 1 X 10(7) to 1.5 X 10(7) lymphocytic choriomeningitis virus-specific, H-2-restricted cloned cytotoxic T lymphocytes (CTL) administered intravenously into acutely infected mice totally cleared virus from the spleens (10(4) to 10(5) PFU per spleen reduced to less than 50 PFU per spleen) by 24 h. This activity was genetically restricted in that cloned CTL could reduce titers of infectious virus in syngeneic C57BL/6 mice but not allogeneic BALB/c mice. Dose-response analysis indicated that at least 3 X 10(6) to 5 X 10(6) cloned CTL injected intravenously were needed to reduce significant amounts of infectious virus in the spleens. No infectious virus could be recovered from the spleens for at least 4 days after injection of cloned CTL. Hence, CTL play a major role in elimination of infectious virus from spleens during lymphocytic choriomeningitis virus infection. Our results also indicate that cloned CTL propagated in vitro for long periods of time can mediate a biologically relevant effect in vivo. These cells should be of considerable value in defining the precise manner in which CTL bring about control of viral infection, analyzing lymphocyte trafficking, and the potential use of cloned CTL in immunotherapy against viral disease.     

Herpes simplex virus type 2 UL24 gene is a virulence determinant in murine and guinea pig disease models

A herpes simplex virus type 2 (HSV-2) UL24 beta-glucuronidase (UL24-betagluc) insertion mutant was derived from HSV-2 strain 186 via standard marker transfer techniques. Cell monolayers infected with UL24-betagluc yielded cytopathic effect with syncytium formation. UL24-betagluc replicated to wild-type viral titers in three different cell lines. UL24-betagluc was not virulent after intravaginal inoculation of BALB/c mice in that all inoculated animals survived doses up to 400 times the 50% lethal dose (LD50) of the parental virus. Furthermore, few UL24-betagluc-inoculated mice developed any vaginal lesions. Intravaginal inoculation of guinea pigs with UL24-betagluc at a dose equivalent to the LD50 of parental virus (approximately 5 x 10(3) PFU) was not lethal (10/10 animals survived). Although genital lesions developed in some UL24-betagluc-inoculated guinea pigs, both the overall number of lesions and the severity of disease were far less than that observed for animals infected with parental strain 186.