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.     

Suppression of RIP3-dependent Necroptosis by Human Cytomegalovirus

Necroptosis is an alternate programmed cell death pathway that is unleashed by caspase-8 compromise and mediated by receptor-interacting protein kinase 3 (RIP3). Murine cytomegalovirus (CMV) and herpes simplex virus (HSV) encode caspase-8 inhibitors that prevent apoptosis together with competitors of RIP homotypic interaction motif (RHIM)-dependent signal transduction to interrupt the necroptosis. Here, we show that pro-necrotic murine CMV M45 mutant virus drives virus-induced necroptosis during nonproductive infection of RIP3-expressing human fibroblasts, whereas WT virus does not. Thus, M45-encoded RHIM competitor, viral inhibitor of RIP activation, sustains viability of human cells like it is known to function in infected mouse cells. Importantly, human CMV is shown to block necroptosis induced by either TNF or M45 mutant murine CMV in RIP3-expressing human cells. Human CMV blocks TNF-induced necroptosis after RIP3 activation and phosphorylation of the mixed lineage kinase domain-like (MLKL) pseudokinase. An early, IE1-regulated viral gene product acts on a necroptosis step that follows MLKL phosphorylation prior to membrane leakage. This suppression strategy is distinct from RHIM signaling competition by murine CMV or HSV and interrupts an execution process that has not yet been fully elaborated.     

Endothelial cells and CMV dissemination

Using the murine CMV animal model and the well-established model of Cre-lox-P-mediated green-fluorescence tagging of endothelial cell (EC)-derived mouse CMV to quantify the role of infected ECs in transplantation-associated CMV dissemination (in mice expressing Cre recombinase under the control of either the Tie2 or the Tek promoter selectively expressed in vascular EC-Tie-Cre and Tek-Cre mice), it was shown that EC-derived virus contributed to 50% of the total viral load during primary infection, and there was no preference for dissemination of EC-derived viruses over viruses produced by other cell types. In addition, during secondary viremia, there was only a negligible contribution of EC-derived virus to dissemination to other organs. These results are novel in the methodology employed and are somewhat interesting. However, the data are limited to the mouse model with a short-term follow-up, and the immunodeficient host has not yet been studied. In humans, these conclusions must be taken with caution. First, in primary infection occurring through natural routes, epithelial cells are infected first, then ECs, unless primary infection occurs through blood transfusion, in which case endothelial vascular cells may become infected first. In both cases, the virus transport occurs through the intervention of leukocytes, namely monocytes and polymorphonuclear leukocytes. As monocytes differentiate to macrophages, they become highly susceptible to human CMV replication inside organ tissues, while polymorphonuclear leukocytes are active in virus capturing from infected endothelial vascular cells and transporting to distant sites.     

Neutrality of the canonical NF-kappaB-dependent pathway for human and murine cytomegalovirus transcription and replication in vitro

Cytomegalovirus (CMV) is known to rapidly induce activation of nuclear factor kappaB (NF-kappaB) after infection of fibroblast and macrophage cells. NF-kappaB response elements are present in the enhancer region of the CMV major immediate-early promoter (MIEP), and activity of the MIEP is strongly upregulated by NF-kappaB in transient-transfection assays. Here we investigate whether the NF-kappaB-dependent pathway is required for initiating or potentiating human and murine CMV replication in vitro. We show that expression of a dominant negative mutant of the inhibitor of NF-kappaB-alpha (IkappaBalphaM) does not alter the replication kinetics of human or mouse CMV in cultured cells. In addition, mouse embryo fibroblasts genetically deficient for p65/RelA actually showed elevated levels of MCMV replication. Mutation of all NF-kappaB response elements within the enhancer of the MIEP in a recombinant mouse CMV containing the human MIEP (hMCMV-ES), which we have previously shown to replicate in murine fibroblasts with kinetics equivalent to that of wild-type mouse CMV, did not negatively affect replication in fibroblasts. Taken together, these data show that, for CMV replication in cultured fibroblasts activation of the canonical NF-kappaB pathway and binding of NF-kappaB to the MIEP are dispensable, and in the case of p65 may even interfere, thus uncovering a previously unrecognized level of complexity in the host regulatory network governing MIE gene expression in the context of a viral infection.     

Growth retardation and microcephaly induced in mice by placental infection with murine cytomegalovirus

BACKGROUND: The placenta is regarded as a site of congenital cytomegalovirus (CMV) infection. The placental infection of fetuses with murine CMV (MCMV) was investigated in a mouse model. METHODS: The placentas and fetuses were examined using the polymerase chain reaction (PCR) and Southern blotting for viral DNA and immunostaining for viral antigen. Since the transplacental infection rarely occurs, the placentas were directly injected with MCMV at day 12.5 of gestation; the embryos were then allowed to develop until day 18.5 of gestation. RESULTS: Formation of infected foci at day 18. 5 of gestation was found in more than 60% of the injected placentas. Infection of about 50% of the embryos occurred from the infected placentas. The frequency of infection in the brain was 27%, which was the same as that in the liver and higher than that in the lungs. In the brains, infected cells were often observed in the ventricular zone of the cerebrum and sometimes in the cortical plate and the hippocampus. Developmental retardation with microcephaly was observed in about 25% of offspring exposed to infection in utero. CONCLUSIONS: These results suggest that formation of infected foci in the placenta is important for embryonic congenital infection, and that the cerebral ventricular zone is one of the most susceptible sites for CMV infection in the embryonic stage.     

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.     

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.     

Cytomegalovirus-induced embryopathology: mouse submandibular salivary gland epithelial-mesenchymal ontogeny as a model

Background  

Human studies suggest, and mouse models clearly demonstrate, that cytomegalovirus (CMV) is dysmorphic to early organ and tissue development. CMV has a particular tropism for embryonic salivary gland and other head mesenchyme. CMV has evolved to co-opt cell signaling networks so to optimize replication and survival, to the detriment of infected tissues. It has been postulated that mesenchymal infection is the critical step in disrupting organogenesis. If so, organogenesis dependent on epithelial-mesenchymal interactions would be particularly vulnerable. In this study, we chose to model the vulnerability by investigating the cell and molecular pathogenesis of CMV infected mouse embryonic submandibular salivary glands (SMGs).     

Bone marrow failure by cytomegalovirus is associated with an in vivo deficiency in the expression of essential stromal hemopoietin genes.

Bone marrow (BM) failure associated with cytomegalovirus (CMV) infection is a feared complication after clinical BM transplantation. Experiments in long-term BM cultures have indicated that BM stromal cells (BMSC) are targets of productive CMV infection, but an in situ infection of BM stroma remained to be documented, and the pathomechanism is open to question. Here we describe a murine in vivo model of lethal CMV aplastic anemia (CMV-AA). The reconstitution of hematopoietic progenitor cells expressing stem cell factor (SCF) receptor was found to be defective in CMV-AA. While murine CMV replication in permissive parenchymal tissues is cytolytic, the hematopoietic cord was found to be a site of very limited virus production with foci of reticular BMSC expressing the intranuclear viral IE1 protein, but with only a few BMSC positive for viral genome in the in situ hybridization. XX-XY BM chimeras were established in order to quantitate Y-chromosome-tagged BMSC by a PCR specific for the male-sex-determining gene Tdy. This approach revealed that murine CMV infection is not associated with a significant loss of BMSC. Despite the physical integrity of the stromal network, the functional integrity of the stroma was impaired. While housekeeping genes were expressed normally in BMSC of infected mice, the expression of genes encoding the essential hemopoietins SCF, granulocyte colony-stimulating factor, and interleukin-6 was markedly reduced. In conclusion, the mechanism of BM failure is not a stromal lesion but an insufficient stromal function. These findings explain CMV-AA as a manifestation of multiple hemopoietin deficiency.     

Lungs are a major organ site of cytomegalovirus latency and recurrence.

Recurrence of infectious virus from the latent viral genomes is the initiating event in the pathogenesis of cytomegalovirus (CMV) disease during states of immunodeficiency. Interstitial pneumonia is a frequent manifestation of posttransplantation CMV disease, in particular after bone marrow transplantation and heart and lung transplantations. Recurrence can occur within the transplant derived from a latent infected donor as well as within latently infected organs of the transplant recipient. The reason for a predilection of the lungs as a site of CMV pathology is so far unknown. In a murine model of CMV latency, the lungs were identified as an authentic site of latent infection, since the viral genome remained detectable in lung tissue even after it was cleared to an undetectable level in blood and bone marrow. A comparison between the lungs and the spleen, the previously most thoroughly investigated site of murine CMV latency, revealed a 10-fold-higher burden of latent viral genome for the lungs. Most important, the organ-specific risk of in vivo recurrence was found to correlate with the organ-specific viral genomic load. This new finding thus characterizes the lungs as a high-risk organ for CMV recurrence, and this fact may explain in part why interstitial pneumonia is a frequent manifestation of recurrent CMV infection.     

An in vitro mouse model of congenital cytomegalovirus‐induced pathogenesis of the inner ear cochlea

Congenital human cytomegalovirus (CMV) infection is the leading nongenetic etiology of sensorineural hearing loss (SNHL) at birth and prelingual SNHL not expressed at birth. The paucity of temporal bone autopsy specimens from infants with congenital CMV infection has hindered the critical correlation of histopathology with pathogenesis. Here, we present an in vitro embryonic mouse model of CMV‐infected cochleas that mimics the human sites of viral infection and associated pathology. There is a striking dysplasia/hyperplasia in mouse CMV‐infected cochlear epithelium and mesenchyme, including organ of Corti hair and supporting cells and stria vascularis. This is concomitant with significant dysregulation of p19, p21, p27, and Pcna gene expression, as well as proliferating cell nuclear antigen (PCNA) protein expression. Other pathologies similar to those arising from known deafness gene mutations include downregulation of KCNQ1 protein expression in the stria vascularis, as well as hypoplastic and dysmorphic melanocytes. Thus, this model provides a relevant and reliable platform within which the detailed cell and molecular biology of CMV‐induced deafness may be studied. Birth Defects Research (Part A), 2013. © 2012 Wiley Periodicals, Inc.     

Functional interaction of nuclear domain 10 and its components with cytomegalovirus after infections: cross-species host cells versus native cells

Species-specificity is one of the major characteristics of cytomegaloviruses (CMVs) and is the primary reason for the lack of a mouse model for the direct infection of human CMV (HCMV). It has been determined that CMV cross-species infections are blocked at the post-entry level by intrinsic cellular defense mechanisms, but few details are known. It is important to explore how CMVs interact with the subnuclear structure of the cross-species host cell. In our present study, we discovered that nuclear domain 10 (ND10) of human cells was not disrupted by murine CMV (MCMV) and that the ND10 of mouse cells was not disrupted by HCMV, although the ND10-disrupting protein, immediate-early protein 1 (IE1), also colocalized with ND10 in cross-species infections. In addition, we found that the UL131-repaired HCMV strain AD169 (vDW215-BADrUL131) can infect mouse cells to produce immediate-early (IE) and early (E) proteins but that neither DNA replication nor viral particles were detectable in mouse cells. Unrepaired AD169 can express IE1 only in mouse cells. In both HCMV-infected mouse cells and MCMV-infected human cells, the knocking-down of ND10 components (PML, Daxx, and SP100) resulted in significantly increased viral-protein production. Our observations provide evidence to support our hypothesis that ND10 and ND10 components might be important defensive factors against the CMV cross-species infection.     

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.     

Dendritic cell programming by cytomegalovirus stunts naive T cell responses via the PD-L1/PD-1 pathway

Early during infection, CMV targets dendritic cells (DC) and alters their functions. Herein we show that CMV-infected DC maintain the ability to present both virus-derived and exogenous Ags, but that they actively induce tolerance or anergy in Ag-specific T cells. CMV accomplishes this by selectively maintaining high-level expression of the negative costimulatory molecule programmed death ligand-1 (PD-L1), while commensurately down-regulating positive costimulatory molecules and MHC on the DC surface. Consequently, CD4 and CD8 T cells activated by these infected DC have a stunted phenotype, characterized by poor proliferation, effector function. and recall responses. Blocking PD-L1, but not PD-L2, during direct priming of naive T cells by infected DC significantly restores Ag-specific T cell functions. Using systems where direct and cross-priming of T cells can be distinguished revealed that PD-L1/PD-1 signaling contributes only when naive T cells are primed directly by infected DC, and not upon cross-presentation of viral Ags by uninfected DC. These data suggest that murine CMV programs infected DC during acute infection to inhibit early host adaptive antiviral responses by tipping the balance between negative and positive cosignals.     

Glucocortiocoid Treatment of MCMV Infected Newborn Mice Attenuates CNS Inflammation and Limits Deficits in Cerebellar Development

Infection of the developing fetus with human cytomegalovirus (HCMV) is a major cause of central nervous system disease in infants and children; however, mechanism(s) of disease associated with this intrauterine infection remain poorly understood. Utilizing a mouse model of HCMV infection of the developing CNS, we have shown that peripheral inoculation of newborn mice with murine CMV (MCMV) results in CNS infection and developmental abnormalities that recapitulate key features of the human infection. In this model, animals exhibit decreased granule neuron precursor cell (GNPC) proliferation and altered morphogenesis of the cerebellar cortex. Deficits in cerebellar cortical development are symmetric and global even though infection of the CNS results in a non-necrotizing encephalitis characterized by widely scattered foci of virus-infected cells with mononuclear cell infiltrates. These findings suggested that inflammation induced by MCMV infection could underlie deficits in CNS development. We investigated the contribution of host inflammatory responses to abnormal cerebellar development by modulating inflammatory responses in infected mice with glucocorticoids. Treatment of infected animals with glucocorticoids decreased activation of CNS mononuclear cells and expression of inflammatory cytokines (TNF-α, IFN-β and IFNγ) in the CNS while minimally impacting CNS virus replication. Glucocorticoid treatment also limited morphogenic abnormalities and normalized the expression of developmentally regulated genes within the cerebellum. Importantly, GNPC proliferation deficits were normalized in MCMV infected mice following glucocorticoid treatment. Our findings argue that host inflammatory responses to MCMV infection contribute to deficits in CNS development in MCMV infected mice and suggest that similar mechanisms of disease could be responsible for the abnormal CNS development in human infants infected in-utero with HCMV.     

Kinetics of ocular and systemic antigen-specific T-cell responses elicited during murine cytomegalovirus retinitis

Cytomegalovirus (CMV) reactivation in the retina of immunocompromized patients is a cause of significant morbidity as it can lead to blindness. The adaptive immune response is critical in controlling murine CMV (MCMV) infection in MCMV-susceptible mouse strains. CD8(+) T cells limit systemic viral replication in the acute phase of infection and are essential to contain latent virus. In this study, we provide the first evaluation of the kinetics of anti-viral T-cell responses after subretinal infection with MCMV. The acute response was characterized by a rapid expansion phase, with infiltration of CD8(+) T cells into the infected retina, followed by a contraction phase. MCMV-specific T cells displayed biphasic kinetics with a first peak at day 12 and contraction by day 18 followed by sustained recruitment of these cells into the retina at later time points post-infection. MCMV-specific CD8(+) T cells were also observed in the draining cervical lymph nodes and the spleen. Presentation of viral epitopes and activation of CD8(+) T cells was widespread and could be detected in the spleen and the draining lymph nodes, but not in the retina or iris. Moreover, after intraocular infection, antigen-specific cytotoxic activity was detectable and exhibited kinetics equivalent to those observed after intraperitoneal infection with the same viral dose. These data provide novel insights of how and where immune responses are initiated when viral antigen is present in the subretinal space.