Activation of cytomegalovirus in pig-to-primate organ xenotransplantation

Xenotransplantation of porcine organs carries the risk of reactivation of latent virus in donor and recipient tissues as well as transmission of viruses between species. We have investigated the activation of baboon cytomegalovirus (BCMV) and porcine CMV (PCMV) in a pig-to-primate model of xenotransplantation. Tissues originating from a series of six swine-to-baboon composite thymokidney xenotransplants were investigated. Four immunosuppressed baboons died (survival range, 7 to 27 days) with the graft in situ. Increases in BCMV DNA copy numbers occurred in three (75%) of these baboons and was thought to be responsible for pneumonitis and the death of one animal. In two baboons, disseminated intravascular coagulation was successfully treated by graftectomy and discontinuation of immunosuppression. PCMV was upregulated in five of six xenografts (83%). PCMV infection was associated with ureteric necrosis in one xenograft. Although significantly increased in native tissues, low levels of BCMV and PCMV were also detected in tissues other than that of the native viral host species. The cross-species presence of CMV did not appear to cause clinical or histological signs of invasive disease. Thus, viral infections with clinical disease were restricted to tissues of the native species of each virus. Intensive immune suppression currently required for xenotransplantation results in a significant risk of reactivation of latent infections by BCMV and PCMV. It is not yet known whether viral DNA detected across species lines represents cellular microchimerism, ongoing viral infection, or uptake of free virus. The observation of graft injury by PCMV demonstrates that CMV will be an important pathogen in immunosuppressed xenograft recipients. Strategies must be developed to exclude CMV from porcine organ donors.     

Leukotriene B4 protects latently infected mice against murine cytomegalovirus reactivation following allogeneic transplantation

Human CMV is often associated with transplant rejection and opportunistic infections such as pneumonia in immunosuppressed patients. Current anti-CMV therapies, although effective, show relatively high toxicity, which seriously limits their long-term use. In this study, we provide evidence that leukotriene B(4) (LTB(4)) plays an important role in the fight against murine CMV (MCMV) infection in vivo. Intravenous administration of 50 and 500 ng/kg/day of LTB(4) to mice infected with a lethal dose of MCMV significantly increases their survival (50 and 70%, respectively), compared with the placebo-treated group (10% of survival). In mice infected with a sublethal dose of MCMV and treated daily with 50 ng/kg/day of LTB(4), the salivary gland viral loads were found to be reduced by 66% compared with the control group. Furthermore, using an allogeneic bone marrow transplantation mouse model, the frequency of MCMV reactivation from latently infected mice was much lower (38%) in LTB(4) (500 ng/kg)-treated mice than in the placebo-treated group (78%). Finally, in experiments using 5-lipoxygenase-deficient mice, MCMV viral loads in salivary glands were found to be higher in animals unable to produce leukotrienes than in the control groups, supporting a role of endogenous 5-lipoxygenase products, possibly LTB(4), in host defense against CMV infection.     

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.     

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.     

Murine cytomegalovirus interference with antigen presentation has little effect on the size or the effector memory phenotype of the CD8 T cell response

As with most herpesviruses, CMVs encode viral genes that inhibit Ag presentation to CD8 T cells (VIPRs). VIPR function has been assumed to be essential for CMV to establish its characteristic lifetime infection of its host. We compared infection of C57BL/6 mice with wild-type murine CMV (MCMV) and a virus lacking each of MCMV's three known VIPRs: m4, m6, and m152. During acute infection, there was very little difference between the two viruses with respect to the kinetics of viral replication and clearance, or in the size and kinetics of the virus-specific CD8 T cell response. During chronic infection, a large, effector memory, virus-specific CD8 T cell population (CD8(low)CD62L(-)CD11c(+)NKG2A(+)) was maintained in both infections; the size and phenotype of the CD8 T cell response to both viruses was remarkably similar. The characteristic effector memory phenotype of the CD8 T cells suggested that both wild-type and Deltam4+m6+m152 virus continued to present Ag to CD8 T cells during the chronic phase of infection. During the chronic phase of infection, MCMV cannot be isolated from immunocompetent mice. However, upon immunosuppression, both Deltam4+m6+m152 and wild-type virus could be reactivated from mice infected for 6 wk. Thus, restoring the ability of CD8 T cells to detect MCMV had little apparent effect on the course of MCMV infection and on the CD8 T cell response to it. These results challenge the notion that VIPR function is necessary for CMV persistence in the host.     

Reactivation of latent cytomegalovirus infection in mouse brain cells detected after transfer to brain slice cultures

Cytomegalovirus (CMV) is the most significant infectious cause of brain disorders in humans involving the developing brain. It is hypothesized that the brain disorders occur after recurrent reactivation of the latent infection in some kinds of cells in the brains. In order to test this hypothesis, we examined the reactivation of latent murine CMV (MCMV) infection in the mouse brain by transfer to brain slice culture. We infected neonatal and young adult mice intracerebrally with recombinant MCMV in which the lacZ gene was inserted into a late gene. The brains were removed 6 months after infection and used to prepare brain slices that were then cultured for up to 4 weeks. Reactivation of latent infection in the brains was detected by beta-galactosidase (beta-Gal) staining to assess beta-galactosidase expression. Viral replication was also confirmed by the plaque assay. Reactivation was observed in about 75% of the mice infected during the neonatal period 6 months after infection. Unexpectedly, reactivation was also observed in 75% of mice infected as young adults, although the infection ratio in the brain slices was significantly lower than that in neonatally infected mice. Beta-Gal-positive cells were observed in marginal regions of the brains or immature neural cells in the ventricular walls. Immunohistochemical staining showed that the beta-Gal-positive reactivated cells were neural stem or progenitor cells. These results suggest that brain disorders may occur long after infection by reactivation of latent infection in the immature neural cells in the brain.     

Cellular Localization of Latent Murine Cytomegalovirus

Herpesviruses typically establish latent infection in their hosts. The cell(s) responsible for harboring latent virus, in most cases, is not known. Using immunofluorescence and PCR-in situ hybridization (PISH), a technique which combines the sensitivity of PCR with the localization and specificity of in situ hybridization, we provide the first direct evidence that endothelial cells are a major site of murine cytomegalovirus (MCMV) DNA in latently infected animals. These findings are consistent with existing knowledge of the biological behavior of CMV, in particular the transmission of latent CMV by solid organ and bone marrow transplantation, in both human and animal models. In addition, we have localized MCMV DNA in the lung alveolar macrophage and in bone marrow cells. Our findings confirm that bone marrow-derived hematopoietic cells are a site of CMV latency and further suggest that bone marrow may be a reservoir of infected progeny capable of migrating into the circulation and establishing latency in various tissues. These findings provide clearly needed insight into the site of latent infection which is central to an understanding of the mechanisms of reactivation.     

Sustained CD8+ T cell memory inflation after infection with a single-cycle cytomegalovirus

Cytomegalovirus (CMV) is a β-herpesvirus that establishes a lifelong latent or persistent infection. A hallmark of chronic CMV infection is the lifelong persistence of large numbers of virus-specific CD8+ effector/effector memory T cells, a phenomenon called "memory inflation". How the virus continuously stimulates these T cells without being eradicated remains an enigma. The prevailing view is that CMV establishes a low grade "smoldering" infection characterized by tiny bursts of productive infection which are rapidly extinguished, leaving no detectable virus but replenishing the latent pool and leaving the immune system in a highly charged state. However, since abortive reactivation with limited viral gene expression is known to occur commonly, we investigated the necessity for virus reproduction in maintaining the inflationary T cell pool. We inhibited viral replication or spread in vivo using two different mutants of murine CMV (MCMV). First, famcyclovir blocked the replication of MCMV encoding the HSV Thymidine Kinase gene, but had no impact on the CD8+ T cell memory inflation once the infection was established. Second, MCMV that lacks the essential glycoprotein L, and thus is completely unable to spread from cell to cell, also drove memory inflation if the virus was administered systemically. Our data suggest that CMV which cannot spread from the cells it initially infects can repeatedly generate viral antigens to drive memory inflation without suffering eradication of the latent genome pool.     

Control of Murine Cytomegalovirus Infection by γδ T Cells

Infections with cytomegalovirus (CMV) can cause severe disease in immunosuppressed patients and infected newborns. Innate as well as cellular and humoral adaptive immune effector functions contribute to the control of CMV in immunocompetent individuals. None of the innate or adaptive immune functions are essential for virus control, however. Expansion of γδ T cells has been observed during human CMV (HCMV) infection in the fetus and in transplant patients with HCMV reactivation but the protective function of γδ T cells under these conditions remains unclear. Here we show for murine CMV (MCMV) infections that mice that lack CD8 and CD4 αβ-T cells as well as B lymphocytes can control a MCMV infection that is lethal in RAG-1^-/- mice lacking any T- and B-cells. γδ T cells, isolated from infected mice can kill MCMV infected target cells in vitro and, importantly, provide long-term protection in infected RAG-1^-/- mice after adoptive transfer. γδ T cells in MCMV infected hosts undergo a prominent and long-lasting phenotypic change most compatible with the view that the majority of the γδ T cell population persists in an effector/memory state even after resolution of the acute phase of the infection. A clonotypically focused Vγ1 and Vγ2 repertoire was observed at later stages of the infection in the organs where MCMV persists. These findings add γδ T cells as yet another protective component to the anti-CMV immune response. Our data provide clear evidence that γδ T cells can provide an effective control mechanism of acute CMV infections, particularly when conventional adaptive immune mechanisms are insufficient or absent, like in transplant patient or in the developing immune system in utero. The findings have implications in the stem cell transplant setting, as antigen recognition by γδ T cells is not MHC-restricted and dual reactivity against CMV and tumors has been described.     

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.     

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.     

The interplay between host and viral factors in shaping the outcome of cytomegalovirus infection

Cytomegalovirus (CMV) remains a major human pathogen causing significant morbidity and mortality in immunosuppressed or immunoimmature individuals. Although significant advances have been made in dissecting out certain features of the host response to human CMV (HCMV) infection, the strict species specificity of CMVs means that most aspects of antiviral immunity are best assessed in animal models. The mouse model of murine CMV (MCMV) infection is an important tool for analysis of in vivo features of host-virus interactions and responses to antiviral drugs that are difficult to assess in humans. Important studies of the contribution of host resistance genes to infection outcome, interplays between innate and adaptive host immune responses, the contribution of virus immune evasion genes and genetic variation in these genes to the establishment of persistence and in vivo studies of resistance to antiviral drugs have benefited from the well-developed MCMV model. In this review, we discuss recent advances in the immunobiology of host-CMV interactions that provide intriguing insights into the complex interplay between host and virus that ultimately facilitates viral persistence. We also discuss recent studies of genetic responses to antiviral therapy, particularly changes in DNA polymerase and protein kinase genes of MCMV and HCMV.