Smith Strain Research Articles

Late-rising CD4 T cells resolve mouse cytomegalovirus persistent replication in the salivary gland.

Conventional antiviral memory CD4 T cells typically arise during the first two weeks of acute infection. Unlike most viruses, cytomegalovirus (CMV) exhibits an extended persistent replication phase followed by lifelong latency accompanied with some gene expression. We show that during mouse CMV (MCMV) infection, CD4 T cells recognizing an epitope derived from the viral M09 protein only develop after conventional memory T cells have already peaked and contracted. Ablating these CD4 T cells by mutating the M09 genomic epitope in the MCMV Smith strain, or inducing them by introducing the epitope into the K181 strain, resulted in delayed or enhanced control of viral persistence, respectively. These cells were shown to be unique compared to their conventional memory counterparts; producing higher IFNγ and IL-2 and lower IL-10 levels. RNAseq analyses revealed them to express distinct subsets of effector genes as compared to classical CD4 T cells. Additionally, when M09 cells were induced by epitope vaccination they significantly enhanced protection when compared to conventional CD4 T cells alone. These data show that late-rising CD4 T cells are a unique memory subset with excellent protective capacities that display a development program strongly differing from the majority of memory T cells.

Construction of a mouse model of Posner-Schlossman syndrome by anterior chamber infection with cytomegalovirus

Accumulated clinical evidence has shown that Posner-Schlossman syndrome (PSS) is most likely the result of recurrent human cytomegalovirus (HCMV) infection in the anterior chamber (AC). Establishing an animal model is necessary to investigate the pathogenesis of PSS. In this study, we constructed a mouse model of (PSS) by injecting murine cytomegalovirus (MCMV) into the AC of BALB/c mice. Twenty-five BALB/c mice were divided into 5 groups. Smith strain MCMV expressing enhanced green fluorescent protein (EGFP) was passaged with mouse embryonic fibroblast (MEF). Right eyes in the 4 experiment groups received AC injection of 1 μL of virus solution with concentrations of 103,104,105,106 pfu/mL respectively, and the control group received only PBS. PSS-like signs (mutton-fat keratic precipitates (KP), pupil dilation, IOP elevation and corneal edema) were recorded 0–28 days post-injection (DPI). Sections of eyeballs from another 9 mice harvested on 0,10 and 28 DPI were examined to locate KP and the fluorescence signal of the virus. Reversible PSS-like signs except KP were observed in 20% and 60% mice of 104 and 105 groups while no PSS-like signs in the control and 103 group; 80% in the 106 group with partially unreversible signs till 28DPI. Much More fluorescent signals of virus in the iris and KP were found on 10DPI than 28 DPI, while no fluorescent signals and KP on 0DPI. The extent of PSS-like signs (pupil dilation, IOP elevation and corneal edema) was virus concentration-dependent (Spearman correlation coefficient, r = 0.830, = 0.475, = 0.662, p < 0.0001, <0.05, <0.001, respectively, n = 25). Success rate of PSS model (mice with PSS-like signs) was also virus concentration-dependent (Chi-square trend test, χ2 = 6.828, df = 1, p < 0.01, n = 25). Our results indicate that AC injection of 1 μL MEF passaged MCMV (Smith strain) of 104–106 pfu/mL in BALB/c mice can be used to construct a mouse model of PSS. MCMV can infect iris tissue and replicate in it and then establish latency. This might account for the recurrent and self-limited nature of PSS.

MicroRNA‑1929‑3p participates in murine cytomegalovirus‑induced hypertensive vascular remodeling through Ednra/NLRP3 inflammasome activation.

MicroRNAs (miRNAs) play an important role in the development of vascular remodeling in essential hypertension (EH) by mediating the effects of human cytomegalovirus (HCMV) on the vascular system. Therefore, the aim of the present study was to investigate the effects of murine cytomegalovirus (MCMV) infection on blood pressure and vascular function in mice, in order to elucidate the role of miR-1929-3p in this process. For model development, 7-month-old C57BL/6J mice were infected with the Smith strain of MCMV, and MCMV DNA, IgG and IgM were detected. Subsequently, blood pressure was measured via the carotid artery, and the morphological changes of the aorta were evaluated by hematoxylin and eosin and Masson's trichrome staining. miR-1929-3p transfection was performed using an adeno-associated virus packaged vector and the changes in vascular structure were then observed. The levels of nitric oxide (NO) and endothelial NO synthase were also assessed with colorimetry. Vascular remodeling and expression of NLRP3 inflammasome pathway-related proteins were detected by immunohistochemistry and western blotting. Endothelin-1 (ET-1), interleukin (IL)-1β and IL-18 were assayed by ELISA. The results revealed that MCMV infection increased the blood pressure, promoted vascular remodeling, caused endothelial cell injury, and downregulated miR-1929-3p. However, these effects were alleviated by miR-1929-3p overexpression, which downregulated endothelin A receptor (Ednra) and NLRP3 inflammasome, as well as endothelial injury- and vascular remodeling-related genes. Taken together, the findings of the present study indicated that overexpression of miR-1929-3p may improve MCMV-induced vascular remodeling, possibly via the deactivation of the NLRP3 inflammasome by ET-1/Ednra.

The Role of Amino Acid Substitution in HepT Toward Menaquinone Isoprenoid Chain Length Definition and Lysocin E Sensitivity in Staphylococcus aureus.

ObjectivesStaphylococcus aureus Smith strain is a historical strain widely used for research purposes in animal infection models for testing the therapeutic activity of antimicrobial agents. We found that it displayed higher sensitivity toward lysocin E, a menaquinone (MK) targeting antibiotic, compared to other S. aureus strains. Therefore, we further explored the mechanism of this hypersensitivity.MethodsMK production was analyzed by high-performance liquid chromatography and mass spectrometric analysis. S. aureus Smith genome sequence was completed using a hybrid assembly approach, and the MK biosynthetic genes were compared with other S. aureus strains. The hepT gene was cloned and introduced into S. aureus RN4220 strain using phage mediated recombination, and lysocin E sensitivity was analyzed by the measurement of colony-forming units.ResultsWe found that Smith strain produced MKs with the length of the side chain ranging between 8 and 10, as opposed to other S. aureus strains that produce MKs 7–9. We revealed that Smith strain possessed the classical pathway for MK biosynthesis like the other S. aureus. HepT, a polyprenyl diphosphate synthase involved in chain elongation of isoprenoid, in Smith strain harbored a Q25P substitution. Introduction of hepT from Smith to RN4220 led to the production of MK-10 and an increased sensitivity toward lysocin E.ConclusionWe found that HepT was responsible for the definition of isoprenoid chain length of MKs and antibiotic sensitivity.

Complete Genome Sequence of the Plant Pathogen Ralstonia solanacearum Strain CIAT-078, Isolated in Colombia, Obtained Using Oxford Nanopore Technology.

Moko is one of the main diseases affecting banana and plantain in Colombia. Here, we report the genome sequence of the causal agent, the bacterium Ralstonia solanacearum (Smith) strain CIAT-078, collected in 2004 from affected plantains in central-west Colombia. The assembled genome was obtained using Oxford Nanopore Technology.

Platelet-endothelial associations may promote cytomegalovirus replication in the salivary gland in mice

Platelet decline is a feature of many acute viral infections, including cytomegalovirus (CMV) infection in humans and mice. Platelet sequestration in association with other cells, including endothelium and circulating leukocytes, can contribute to this decline and influence the immune response to and pathogenesis of viral infection. We sought to determine if platelet-endothelial associations (PEAs) contribute to platelet decline during acute murine CMV (mCMV) infection, and if these associations affect viral load and production. Male BALB/c mice were infected with mCMV (Smith strain), euthanized at timepoints throughout acute infection and compared to uninfected controls. An increase in PEA formation was confirmed in the salivary gland at all post-inoculation timepoints using immunohistochemistry for CD41+ platelets co-localizing with CD34+ vessels. Platelet depletion did not change amount of viral DNA or timecourse of infection, as measured by qPCR. However, platelet depletion reduced viral titer of mCMV in the salivary glands while undepleted controls demonstrated robust replication in the tissue by plaque assay. Thus, platelet associations with endothelium may enhance the ability of mCMV to replicate within the salivary gland. Further work is needed to determine the mechanisms behind this effect and if pharmacologic inhibition of PEAs may reduce CMV production in acutely infected patients.

Apoptosis of Murine T-Cell Lymphoma EL4 Cells Induced by Murine Cytomegalovirus and Its Mechanism

To detect whether the murine T-cell lymphoma cell line EL4 could be infected by murine cytomegalovirus (MCMV), and to observe the morphological changes and apoptosis of El4 cells before and after infection. EL4 cells were infected with MCMV smith strain with 1, 10 and 100 multiplicity of infection (MOI) respectively. The morphology of the cells was observed by light microscopy and Wright's-Giemsa staining. The survival rate was calculated by trypan blue staining. RT-PCR-based assay was used to detect the copy number of MCMV-DNA in the infected ELA cells. Flow cytometry was used to detect apoptosis. RT-qPCR was used to detect the mRNA expression levels of P53, P21, cFlip and Caspase 8. The protein expression levels of Caspase8, P53, BAX, BCL-2 and Cleaved Caspase3 proteins were detected by Western blot. After Wright-Giemsa staining, it was found that the infected EL4 cells displayed larger volume, irregular nuclei and the folded twist under light microscopy. Compared with the normal control group, the survival rate of EL4 cells decreased, and the apoptosis rate statistically significantly (P<0.05) increased with the increasing MOI and the infected time in each group. While, the level of apoptosis protein P53, BAX/BCL-2, Cleaved-caspase3 and Caspase8 were up-regulated. And the survival rate, apoptosis rate and the apoptosis protein level of infected EL4 cells with MOI=10 were the most obvious at the 5thday. Compared with MCMV infection group (MOI=60), the content of MCMV DNA in EL4 cells was decreased in MCMV+GGV group ［MOI=60, GCV 25 (g/ml)］, and the cell apoptosis rate and apoptosis protein expression of P53, Caspase8, BAX/BCL-2 were decreased (P<0.05). Murine T-cell lymphoma cell line EL4 can be infected by MCMV and displayes an obvious apoptosis phenomenon. MCMV may up-regulate the expression levels of apoptosis protein P53, BAX-BCL-2, Cleaved-caspase3 and Caspase8 in EL4 cells. The drug ganciclovir reduces the copy mumber of MCMV DNA in infected EL4 cells and inhibited the killing effect of MCMV on EL4 cells.

Determination of suitable reference genes for RT-qPCR analysis of murine Cytomegalovirus in vivo and in vitro

Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to evaluate gene expression. Reliable qPCR results are highly dependent on accurate normalization using suitable reference genes. We investigated expression of commonly used reference genes during murine Cytomegalovirus (mCMV) infection and latency to determine those genes least perturbed by infection. Following mCMV infection in BALB/c mice, lung, salivary gland, liver, spleen and kidney were evaluated. Liver sinusoidal endothelial cells and NIH-3T3 cells were also evaluated. RT-qPCR was performed during acute and latent mCMV infection for 11 commonly used reference genes with comparisons made to uninfected samples. Normfinder, BestKeeper, GeNorm and the comparative delta CT method produced comparable analyses that were combined in RefFinder to generate an overall ranking. Ppia, B2m and Gapdh are the most stable reference genes for in vitro infection studies. For in vivo studies the most suitable reference genes were highly tissue and cell type dependent. Comparing infected and uninfected groups revealed viral influence on transcription of some genes. We provide reference gene guidelines for investigations of gene expression for mCMV Smith strain infection of Balb/cJ mice or NIH-3T3 cells. These results also suggest careful consideration of reference genes for different host tissues evaluated.

Studies on the resistance of poplar hybrids to Aplanobacterium populi (Smith) strain Ride

Studies on the resistance of poplar hybrids to Aplanobacterium populi (Smith) strain Ride

Latent viral infection alters the immune architecture of the lung (VIR5P.1030)

Abstract Human cytomegalovirus (HCMV) is ubiquitous virus infecting most humans and developing persistent latent infections. HCMV reactivation occurs in ~33% of immune-competent patients during critical illness associated with doubled risk of mortality. In many persistent infections Myeloid derived suppressor cells (MDSC) cause T cell dysfunction, creating an immunosuppressive microenvironment. We hypothesized that latent CMV infection induces MDSC, perhaps contributing the impairment of host resolution of infection and thus latency. BALB/c mice were infected with 1×106 PFU of Smith strain murine CMV and lung mononuclear cells (LMNC) were cultured ex vivo or stained for surface markers and analyzed by ELISA or FACS. During latency, we observed a change in frequency (million/gm of lung tissue) of cell subsets such as marked reduction of CD4+T and B cells, increased CD8+T cells and significant increase in NK cells compared to naïve mice. Interestingly, the percentage of MDSC was unchanged at 2 weeks, but was significantly reduced at12 weeks post infection (p.i.). Both IL-6 and TNF-α were elevated in unstimulated or LPS, ConA stimulated LMNC at 2 and 12 weeks p.i. conferring the risk of lung pathology under critical illness. Our findings suggest that during the transition from acute to latent infection, persistent pro-inflammatory lung immune environment is associated with reduced MDSC. Longitudinal studies are underway to explore the role of MDSC in lungs of latently infected mice.

Abstract 4815: Cytomegalovirus enhances glioblastoma via PDGF-B/STAT3 pathway activation

Abstract Recently several groups have demonstrated cytomegalovirus (CMV) protein and DNA in a large majority of glioblastomas (GBMs), though the exact role of CMV in tumors remains controversial. Although CMV is capable of activating several oncogenic pathways, it has never been shown to be a transforming virus. Because of this, we hypothesize that CMV infection can modify the rate of gliomagenesis in the context of genetic mutations known to predispose to tumor formation. To test this, we used the Smith strain mouse CMV (MCMV) to infect Mut3 (GFAP-cre; Nf1loxP/+; Trp53-/+) mice that develop normally but eventually develop spontaneous gliomas at an adult age After intraperitoneal viral inoculation, mice exhibited multisystemic infection throughout the body, including the brain. Mut3 mice infected perinatally (103 plaque forming units) developed gliomas significantly sooner than mock-infected Mut3 mice. MCMV-infected mice developed GBMs (WHO grade IV) at a higher rate than less severe tumors, whereas, mock-infected mice developed less severe gliomas (WHO grade III). Since MCMV preferentially infected the neural stem cell (NSC) niche, an area shown to generate gliomas, we interrogated changes in signaling in this population via microarray of cultured NSCs in MCMV- versus Mock-infected mice. Ingenuity Pathway Analysis identified a PDGF-B network as the highest scoring differentially regulated network between MCMV-and Mock-infected neurospheres. In vitro infection of mouse tumorspheres and human GBM tumorspheres with CMV validated the observed upregulation of PDGF-B, a molecule shown to cause gliomas de novo. Additionally, in vitro infection of human glioma stem cells with HCMV (Towne strain) or exogenous PDGF-B activated STAT3, a key regulator of gliomas. In mice infected with MCMV, PDGF-B is upregulated in CA2/3 neurons and STAT3 is activated in the subventricular zone and dentate gyrus suggesting a paracrine effect of MCMV-induced PDGF-B. Also, infected human brain tumor stem cells demonstrated an increase in proliferation via flow cytometry. Taken together, our data suggest that CMV in gliomas may accelerate GBM progression by activating PDGF-B/STAT3 signaling in the NSC population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4815. doi:1538-7445.AM2012-4815

Virus Progeny of Murine Cytomegalovirus Bacterial Artificial Chromosome pSM3fr Show Reduced Growth in Salivary Glands due to a Fixed Mutation of MCK-2

Murine cytomegalovirus (MCMV) Smith strain has been cloned as a bacterial artificial chromosome (BAC) named pSM3fr and used for analysis of virus gene functions in vitro and in vivo. When sequencing the complete BAC genome, we identified a frameshift mutation within the open reading frame (ORF) encoding MCMV chemokine homologue MCK-2. This mutation would result in a truncated MCK-2 protein. When mice were infected with pSM3fr-derived virus, we observed reduced virus production in salivary glands, which could be reverted by repair of the frameshift mutation. When looking for the source of the mutation, we consistently found that virus stocks of cell culture-passaged MCMV Smith strain are mixtures of viruses with or without the MCK-2 mutation. We conclude that the MCK-2 mutation in the pSM3fr BAC is the result of clonal selection during the BAC cloning procedure.

Latent Cytomegalovirus Infection Exacerbates Experimental Colitis

Inflammatory bowel disease (IBD) severity is positively correlated with cytomegalovirus (CMV) infection. This may reflect CMV triggering and/or exacerbating flares of IBD and/or IBD or immunosuppressive drugs administered to patients with IBD increasing susceptibility to CMV infection. Herein, we performed studies in mice to investigate these possibilities. Mice administered murine CMV (MCMV) developed signs of acute viral infection (malaise and weight loss) and had MCMV loads that were readily detected in numerous organs including the intestine. By 4 weeks, these mice manifested a "latent" infection in which MCMV levels were low but detectable by PCR. Such MCMV infection did not induce acute colitis in either wild-type mice or IL-10(-/-) mice, which are highly prone to developing colitis. However, underlying MCMV infection in an acute or latent state exacerbated the severity of colitis induced by dextran sodium sulfate (DSS). Such potentiation of DSS colitis by latent MCMV infection seemed to occur without viral reactivation. Whereas initial MCMV infection induced acute alterations in serum and intestinal cytokines, such cytokine levels returned to baseline before administration of DSS. However, the initial infection resulted in lasting elevation of antibodies to commensal bacterial antigens, suggesting that MCMV infection may have potentiated colitis via priming of the intestinal immune response to gut microbiota. Thus, underlying CMV infection can alter mucosal immunity, potentially increasing the tendency of CMV-infected hosts to develop colitis.

Allogeneic Stimulation Causes Transcriptional Reactivation of Latent Murine Cytomegalovirus

Cytomegalovirus (CMV) reactivation is a well-described complication of transplantation that may be caused by allogeneic stimulation, immunosuppression, or both. These studies were performed to determine if allogeneic stimulation alone is sufficient to reactivate latent CMV. BALB/c mice latently infected with Smith strain murine CMV (MCMV) received allograft (n = 8), allograft plus cortisol (n = 5), or isograft (n = 4) skin. All allograft recipients rejected their grafts within 9 to 12 days of transplantation. Three weeks after grafting, recipients were evaluated for MCMV reactivation, and all allograft recipients (8/8) showed MCMV reactivation, while no isografts had reactivation (0/4). Surprisingly, cortisol therapy blocked MCMV reactivation (0/5). These data suggested that allogeneic stimulation alone can trigger systemic reactivation of latent CMV. Although immunosuppression is thought to contribute to reactivation, certain agents that impair NF-κB activation may actually reduce reactivation.

Mutations in the temperature‐sensitive murine cytomegalovirus (MCMV) mutants tsm5 and tsm30: A study of genes involved in immune evasion, DNA packaging and processing, and DNA replication

A murine cytomegalovirus (MCMV) temperature-sensitive (ts) mutant, tsm5, of the K181 (Birmingham) strain, showed approximately 10-fold and approximately 10,000-fold reductions in yields at the permissive (33 degrees C) and non-permissive temperature (40 degrees C), respectively. It did not replicate to detectable levels in any tissue of 1-week-old Balb/c mice for up to 21 days following i.p. inoculation with 4 x 10(3) pfu although it did replicate, albeit with considerably delayed kinetics, in SCID mice. tsm5 expressed all kinetic classes of transcript (immediate-early, early and late) both in vitro at the non-permissive temperature and in vivo. To identify mutations contributing to this phenotype, chimaeric viruses produced from overlapping cosmids generated from tsm5 and the Smith strain of MCMV were examined. A virus, Smith/tsm5DGIK, comprising the central conserved region of the tsm5 genome, was not attenuated at 33 or 37 degrees C but was ts at 40 degrees C, although not to the same extent as tsm5. In contrast to tsm5, this chimaeric virus replicated to similar levels as parental viruses in adult BALB/c mice. These results suggested that genes contributing to reduced replication at 33 degrees C and lack of replication in vivo are located at the ends of the tsm5 genome while those contributing to the ts phenotype are located in the central conserved region of the genome. Sequencing of some immune evasion genes known to be located at the 3' or 5' ends of the MCMV genome showed that no mutations were present in ORFs m04, m06, M33, M37, m38.5, m144, m152, or m157 although mutations were found in M27 (A658S) and M36Ex1 (V54I). tsm5 made few capsids at 40 degrees C and these lacked DNA. DNA synthesis was significantly reduced in tsm5-infected cells at 40 degrees C although DNA cleavage occurred with close to wt efficiency. Sequencing of the herpesvirus conserved cis-acting elements, pac1 and pac2, and genes involved in DNA packaging and cleavage located in the central core region of the genome identified few point mutations. Two were identified that alter the encoded protein in tsm5 ORFs M98 (P324S) and M56 (G439R). Furthermore, a point mutation (C890Y) was identified in M70, the primase. Another mutant, tsm30, which is also defective in DNA packaging and processing, has a point mutation in M52 (D494N). Thus, a number of mutations have been identified in tsm5 that suggests that it is defective in genes involved in immune evasion, DNA replication and DNA encapsidation.

Lipopolysaccharide, Tumor Necrosis Factor Alpha, or Interleukin-1β 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.

Transfusion‐transmitted cytomegalovirus (CMV) infections in a murine model: characterization of CMV‐infected donor mice

Donor and recipient mechanisms that modulate the incidence and severity of transfusion-transmitted cytomegalovirus (TT-CMV) are unclear. The kinetics of murine CMV (MCMV) infection in the peripheral blood of donor mice were investigated to determine the utility of this model for studying TT-CMV. BALB/cByJ mice, experimentally infected with Smith strain MCMV, were killed at serial time points up to 28 days after infection. Peritoneal exudate cells (PECs), peripheral blood white blood cells (WBCs), plasma, and marrow were tested for MCMV DNA with quantitative polymerase chain reaction (PCR), replication-competent virus with quantitative culture, and transcription of viral genes with reverse transcription (RT)-PCR targeted at the immediate-early 1 (ie1) gene. PECs, macrophages infected by MCMV shortly after intraperitoneal inoculation, demonstrated high mean levels of MCMV DNA (10(5)-10(7) genome equivalents [geqs]/10(5) PECs), virus production (10(1)-10(4) infectious virions/10(5) PECs), and ie1 gene transcription, demonstrating productive infection. In contrast, while MCMV loads averaged 10(4) to 10(6) geqs per 10(5) peripheral WBCs, all WBC samples were uniformly negative for MCMV ie1 expression by RT-PCR and for culturable virus, consistent with latent MCMV infection. Plasma and marrow showed lower viral loads than WBCs and PECs and were all negative by culture and RT-PCR analysis. Following experimental MCMV infection, murine peripheral blood WBCs appear to be latently infected with virus (MCMV DNA-positive; MCMV RNA-negative; MCMV culture-negative), similar to the latently infected human monocytes in peripheral blood of CMV-seropositive donors. These donor kinetics suggest that the experimental MCMV system can be used to effectively model the mechanisms of TT-CMV infections in humans.

Allogeneic stimulation, not immunosuppression, causes cytomegalovirus reactivation after transplantation

Introduction: For many years, the cause of cytomegalovirus (CMV) reactivation after transplantation has been unclear. While many have hypothesized that requisite immuno-suppression is a cause of CMV reactivation post transplant, recent work has suggested that allogeneic stimulation alone may cause reactivation of CMV. Because of inherent ethical limitations, this has been difficult to prove in-vivo. To date, therefore, allogeneic stimulation as a trigger for CMV reactivation has only been proven in-vitro by mixing peripheral blood mononuclear cells from naı̈ve and latently infected donors (Soderberg-Naucler et al., M.I. Guedes et al.). We hypothesized that allogeneic stimulation may lead to systemic in-vivo reactivation of latent CMV, and further, we sought to define the role of glucocorticoids in this process. Methods: We utilized well-characterized models of murine CMV infection/reactivation and murine skin transplantation. BALB/c mice latently infected with Smith strain MCMV underwent either allogeneic skin transplant (n=9), syngeneic skin transplant (n=4), allogeneic skin transplant + cortisol (100 mg/kg qod), or cortisol alone (100 mg/kg qod). Briefly, square full-thickness skin grafts (approximately 8×10 mm) from either C57BL6 mice (allogeneic) or BALB/c (syngeneic) donors were placed on graft beds prepared on the recipient’s flank. Three weeks after transplantation, all animals were evaluated for CMV reactivation using RT-PCR for CMV glycoprotein B, and antibody (Ab) responses to alloantigens or MCMV using flow cytometry. Results: All allogeneic skin recipients rejected their grafts within 7-10 days of transplantation, and all isografts survived the duration of the experiment. Following graft rejection, 8/9 allogeneic skin recipients had MCMV reactivation in salivary glands, and 3/4 tested had reactivation in lung tissues. In contrast, 0/4 isograft recipients showed reactivation. Interestingly, cortisol treated allogeneic skin recipients showed no evidence of CMV reactivation (0/5), and as expected latently infected mice treated with cortisol alone had no reactivation (0/5). All allogeneic skin recipients were positive for allo-reactive Ab and all animals were positive for CMV reactive Ab. Conclusions: Allogeneic skin transplantation with subsequent rejection is capable of causing systemic reactivation of latent MCMV. Because our previous work has shown that inflammatory mediators can trigger CMV reactivation in-vivo, we suspect that reactivation by allogeneic skin transplant is caused by release of inflammatory mediators. The fact that glucocorticoids block allogeneic stimulated CMV reactivation supports this hypothesis. This model will allow further dissection of the components of allogeneic transplant mediated CMV rejection.

The cytomegalovirus m155 gene product subverts natural killer cell antiviral protection by disruption of H60-NKG2D interactions.

Natural killer (NK) cells are an important early mediator of host immunity to murine cytomegalovirus (MCMV) infection. However, MCMV has evolved mechanisms to elude recognition and clearance by NK cells. We have identified an MCMV immune evasion protein that impairs NKG2D-mediated NK cell antiviral activity. Infection of BALB/c 3T3 cells with the Smith strain of MCMV resulted in strong down-regulation of H60, a high affinity ligand for NKG2D, from the surface of virus-infected cells. The MCMV m155 protein specifically down-regulated H60 without affecting expression of the other known NKG2D ligands, RAE-1 and MULT-1. Treatment with the proteasome inhibitors lactacystin or epoxomicin reversed m155 down-regulation of H60. An MCMV mutant virus lacking m155 was severely attenuated in BALB/c mice; however, treatment with neutralizing anti-NKG2D monoclonal antibody or with NK-depleting anti-asialo GM1 antisera restored virulence of the mutant virus. Thus, down-regulation of H60 by m155 is a powerful mechanism of inhibiting NKG2D-mediated antiviral function.

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.