Occult cytomegalovirus in vivarium-housed mice may influence transplant allograft acceptance.

We have recently shown that latent murine cytomegalovirus (MCMV) can influence murine transplant allograft acceptance. During these studies we became aware that vivarium-housed control mice can acquire occult MCMV infection. The purpose of this investigation was to confirm occult MCMV transmission and determine the timing, vehicle, and possible consequences of transmission. Mice arriving from a commercial vendor were negative for MCMV both by commercial serologic testing and by our nested PCR. Mice housed in our vivarium became positive for MCMV DNA 30-60 days after arrival, but remained negative for MCMV by commercial serologic testing. To confirm MCMV we sequenced PCR products for several genes and showed >99% homology to MCMV. Further sequence analyses show that the occult MCMV is similar to a laboratory strain of MCMV, but the vehicle of transmission remains unclear. Control tissues from historical experiments with unexplained graft losses were evaluated for occult MCMV, and mice with unexplained allograft losses showed significantly higher incidence of occult MCMV than did allograft acceptors. Deliberate infection with very low titer MCMV confirmed that viral transmission can occur without measurable virus specific antibody or T-cell responses. These data suggest that vivarium-housed mice can develop occult MCMV that is missed by currently available commercial serologic testing, and that these infections may influence transplant allograft acceptance.

Immunopathogenesis and immune modulation of Venezuelan equine encephalitis virus-induced disease in the mouse.

The course of Venezuelan equine encephalitis (VEE) disease in immunodeficient and immunologically normal mice was compared to define the role of the immune system in this disease process. Immunocompetent mice infected with VEE exhibited a biphasic illness characterized by an early self-limiting lymphoid phase and a fatal CNS phase. The lymphoid phase of the illness was characterized by extensive viral replication within spleen, thymus, Peyer's patches, and lymph nodes, was accompanied by a high-titered serum viremia, and resolved with the production of VEE-specific IgM class antibody at 72 h postinfection (p.i.). Immunocompetent animals survived an average of 6.8 +/- 1.2 days before succumbing to fulminant encephalitis. In contrast, SCID mice infected with VEE showed a persistent replication of virus throughout all organs tested beginning at 24 h p.i. VEE-infected SCID mice exhibited a severe spongiform encephalopathy with 100% mortality and an average survival time of 8.9 +/- 0.9 days. These studies indicated that the characteristic organ tropism of VEE in the mouse is due in large part to an early anti-viral state, the establishment of which is dependent upon the presence of an intact immune system. Finally, the CNS pathology in a VEE-infected mouse had a significant immunologic component. However, in contrast to other neurovirulent alphaviruses, VEE was directly cytopathic for the cells of the CNS, even in the absence of an immune response.

Immune responses and cytokine induction in the development of severe hepatitis during acute infections with murine cytomegalovirus.

Salivary gland-derived murine cytomegalovirus (SGV) infections of mice have been widely used as models of human cytomegalovirus infections and in the study of CMV biology. Still, many aspects of SGV pathogenesis are not clearly defined. Fatal and non-fatal SGV infections were investigated to characterize pathogenetic correlates of mortality and to assess the role of the immune response in disease progression. Suppression of immune responses was observed in both lethal and sublethal infections. Depletion of immune cell populations in spleen, however, correlated with severe CMV-induced hepatitis and mortality. In addition, T cell depletion studies indicated a requirement for this immune cell subset in control of liver damage and survival of infected mice. Examination of cytokine responses revealed a previously undescribed shock-like syndrome in lethally-infected mice characterized by high levels of tumor necrosis factor alpha and interferon gamma. Furthermore, the sites of tumor necrosis factor alpha gene induction did not strictly correlate with either viral load or the sites of tissue damage during infection. Taken together, these findings define the pathogenetic progression of disease as it relates to disease outcome and suggests that organ-specific differences in cytokine induction play a significant role in the late stages of acute lethal MCMV infections.

TNFalpha, interferon, and stress response induction as a function of age-related susceptibility to fatal Sindbis virus infection of mice.

The age-related acquisition of resistance to fatal Sindbis virus infection was examined using a molecularly cloned laboratory strain of the AR339 isolate designated TRSB. TRSB caused 100% mortality in mice up to 5 days of age. Resistance to fatal infection developed abruptly between 5 and 9 days of age. Lethal Sindbis virus infection of mice inoculated at 4 days of age was characterized by high levels of virus replication, induction of high levels of interferon-alpha/beta and TNF-alpha and severe thymic involution indicative of a systemic stress response. These changes correlated with predominantly noninflammatory lesions. In contrast, TRSB infection of older mice was characterized by survival, more limited virus replication, reduced cytokine induction, and the development of inflammatory responses leading to encephalitis, myositis, and myocarditis. Previous studies utilized infections of neonatal mice with TRSB and an attenuated mutant of TRSB to compare fatal and nonfatal Sindbis infection (Trgovcich et al., 1996. Virology 224, 73-83). The experiments reported here utilize mouse age at the time of infection to create conditions for examination of fatal and nonfatal TRSB infections. Both experiments suggest that fatal infection is associated with a shock-like syndrome and little or no inflammatory pathology, while survival is correlated with greatly reduced cytokine levels and inflammatory lesions.

Hierarchical and redundant lymphocyte subset control precludes cytomegalovirus replication during latent infection.

Reactivation from latent cytomegalovirus (CMV) infection is often associated with conditions of immunosuppression and can result in fatal disease. Whether the maintenance of systemic CMV latency is mainly governed by factors of the infected cell or by immune control functions is unknown. Likewise, the putative immune control mechanisms which could prevent the induction and spread of recurrent CMV infection are not clearly identified. We took advantage of latently infected B cell-deficient mice and a sensitive method for virus detection to study CMV reactivation after ablation of lymphocyte subsets. A crucial role of both T lymphocytes and natural killer (NK) cells was demonstrated. Within 5 d after depletion of lymphocytes, productive infection occurred in 50% of mice, and 14 d later 100% of mice exhibited recurrent infection. A hierarchy of immune control functions of CD8(+), NK, and CD4(+) cells was established. Reactivation was rare if only one of the lymphocyte subsets was depleted, but was evident after removal of a further subset, indicating a functional redundancy of control mechanisms. The salivary glands were identified as the site of most rapid virus shedding, followed by the detection of recurrent virus in the lungs, and eventually in the spleen. Our findings document a previously unknown propensity of latent CMV genomes to enter productive infection immediately and with a high frequency after immune cell depletion. The data indicate that only the sustained cellular immune control prevents CMV replication and restricts the viral genome to a systemic state of latency.

Sindbis virus infection of neonatal mice results in a severe stress response.

Neonatal mice were infected with virus derived from a molecular clone of a laboratory strain of Sindbis virus, TRSB. The resulting acute fatal infection was typified by few if any of the classic hallmarks of encephalitis, very high levels of interferon-alpha/beta (IFNalphabeta), and lesions in the thymus and hematopoietic tissues usually associated with a severe stress response. Infection with an attenuated mutant of TRSB, which harbors a single amino acid change in the E2 surface glycoprotein (TRSBr114), was characterized by encephalitis, reduced mortality, low levels of IFNalphabeta, and no thymic pathology (J. Trgovcich, J. F. Aronson, and R. E. Johnston, 1996, Virology 224, 73-83). Here we report that infection of neonatal mice with TRSB, but not TRSBr114, resulted in induction of high levels of tumor necrosis factor-alpha as well as high and sustained levels of adrenalcorticotropin-releasing hormone and corticosterone. This syndrome of potentially toxic cytokine and stress hormone induction correlates with lethal Sindbis virus infection and constitutes a previously unrecognized aspect of Sindbis virus pathogenesis in mice.

Fatal Sindbis virus infection of neonatal mice in the absence of encephalitis.

A comparative pathogenesis study was performed in neonatal mice using a molecularly cloned laboratory variant of Sindbis strain AR339, designated TRSB, and a single-site attenuated mutant of TRSB derived by site-directed mutagenesis of the E2 glycoprotein from Ser to Arg at residue 114 (TRSBr114). TRSB caused 100% mortality with an average survival time of 3.0 +/- 0.7 days, whereas mice inoculated with TRSBr114 exhibited an attenuated disease course with 46% mortality and an extended average survival time of 7.5 +/- 3.4 days for those animals that died. Reduced virulence of TRSBr114 was characterized by delayed appearance of detectable virus, relative to TRSB, and by lower peak virus titers in both sera and brains of infected mice. TRSB infection induced very high peak serum titers of interferon alpha/beta (215,000 units/ml compared to 2100 units/ml for TRSBr114). In situ hybridization analysis demonstrated replication of TRSB in brain, but only minimal histopathological changes and no evidence of encephalitis were observed. However, extensive extraneural lesions and viral replication were found in skin, connective tissue, and muscle. Moreover, dramatic involution of the thymus and loss of hematopoietic tissues were observed in the absence of virus replication at these sites, suggesting the involvement of a systemic physiological stress response in TRSB infection. TRSBr114 infection did not cause thymic lesions. Otherwise, the attenuated mutant demonstrated a similar pattern of tissue and organ involvement, but lesions and positive in situ hybridization signal were much more limited in scope and intensity compared to TRSB. TRSBr114-infected mice developed myositis and encephalomyelitis approximately 6 days postinfection. Therefore, TRSB-infected animals may succumb to an early syndrome associated with the stress response, preventing their survival for a time sufficient for the development of encephalitis. Alternatively, a systemic stress response, as evidenced by thymic involution, may result in immunosuppression, thus contributing to the absence of encephalitis. In any event, the attenuating mutation in the E2 glycoprotein significantly altered the course of Sindbis-induced disease by limiting virus replication and associated damage early in infection. Mutant-infected animals survived beyond Day 4 and progressed to a classical encephalomyelitis from which about half recovered.