PFU Of Murine Cytomegalovirus Research Articles

Infection of Retinal Neurons during Murine Cytomegalovirus Retinitis

Previous results suggest that retinal neurons are infected early during murine cytomegalovirus (MCMV) infection of the inner retina. The purposes of this study were to identify which retinal neurons are infected and to determine the routes by which MCMV spreads in the retina. Immunosuppressed (IS) BALB/c mice were inoculated with 5 x 10(3) PFU of MCMV (k181) through the supraciliary route. Injected eyes were collected at several times after inoculation, sectioned, and examined by electron microscopy and by staining for retinal cell antigens and for MCMV early (EA) or late (LA) antigen. MCMV-infected cells were observed in the choroid and RPE by day 3 after infection (PI) and in the inner retina beginning at day 5 PI. At this time, many horizontal and bipolar cells were MCMV-antigen-positive but only rare MCMV-infected amacrine cells (glycine positive or gamma-aminobutyric acid [GABA] positive) or MCMV-infected ganglion cells (NF positive) were observed in the inner retina. At day 10 PI, most virus-infected cells were glial fibrillary acidic protein (GFAP)- and GABA-positive glia. Virions were observed by electron microscopy in the choroid, RPE, and inner nuclear layer of the retina. Although virions were observed in the endothelium of the retinal vessels and the nearby retinal cells, the endothelial cell lining of the retinal vessels remained intact. Both apoptotic cells and necrotic cells were seen in the inner retina. In the inner retina, horizontal and bipolar cells were the early (< or = day 7 PI) targets of MCMV infection. Virus spread from the RPE and the photoreceptor layer to the inner retina through infected Muller cells and within the inner retina horizontally through infected horizontal cells.

Dose and duration-dependence of ganciclovir treatment against murine cytomegalovirus infection in severe combined immunodeficient mice

The present study investigates the full dose–response curve and treatment duration dependence of ganciclovir (GCV) against murine cytomegalovirus (MCMV) infection in severe combined immunodeficiency (SCID) mice. Animals inoculated intraperitoneally with 6.3×10 3 pfu of MCMV per mouse developed typical wasting syndrome rapidly and died around day 12 post-inoculation. Once-daily treatment with subcutaneous GCV for 5 days dose dependently delayed MCMV-induced wasting syndrome and mortality at a dose range of 1–80 mg/kg per day, whereas a dose of 160 mg/kg per day induced reversible side-effects. The effect of GCV treatment on mean death day (MDD) was significantly correlated to reductions of viral titers in the lung ( r=0.969, P<0.05). Treatment duration dependence was examined at the dose of GCV at 80 mg/kg per day for 1, 5, 8 and 12 days. The protective duration, over vehicle-treated mice, was constantly 3–4 days plus the duration of GCV treatment, as evidenced by the delay of viral replication, wasting syndrome and death. At a sub-optimally effective dose of 10 mg/kg per day of GCV, maximum protection was achieved with a 8-day treatment regimen. Prolongation of this treatment to 12 days failed to further delay mean death day and wasting syndrome that started on day 10, indicative of insufficient suppression of viral replication. Treatment with a single dose of GCV failed to show a complete dose–response curve since only minimal protective effects were observed at the dose of 80 mg/kg while side-effects were associated with the dose of 160 mg/kg. The treatment duration dependence and requirement for sufficient dosage of GCV against CMV infection observed in the current model are consistent with clinical observations. It also suggests that 5–8 days treatment duration may be a good balance considering the opportunity for identifying active compounds and speeding up the turnaround time in drug evaluations.

Immunosuppression induces transcription of murine cytomegalovirus glycoprotein H in the eye and at non-ocular sites.

In these studies, DNA PCR was used to identify sites of murine cytomegalovirus (MCMV) latency after inoculation of virus into the supraciliary space of the eye. Reverse transcription (RT) PCR for an immediate early gene and a late gene was used to identify putative sites of virus reactivation after methylprednisolone (steroid)-induced immunosuppression. Ten weeks after inoculation of 5 x 10(2) PFU of MCMV, BALB/c mice were immunosuppressed by intramuscular injection of steroid. Control mice were infected but not immunosuppressed. Two weeks after initiation of immunosuppression, mice were sacrificed. DNA and RNA extracted from homogenized tissues were amplified for immediate early gene 1 (IE1) and late gene, glycoprotein H (gH), DNA and mRNA by PCR and RT-PCR, respectively. Replicating virus was detected in homogenized ocular and non-ocular tissues by plaque assay. In the latently infected PBS-treated control group, viral DNA was detected in the inoculated eye and in several non-ocular tissues; IE1 mRNA was found in most of the DNA-positive tissues, while gH mRNA was amplified only in a few of the MCMV DNA-positive tissues from a single mouse. After immunosuppression, viral DNA and IE1 mRNA were detected at a higher frequency in various tissues of steroid-treated mice. gH mRNA was detected in a significantly higher number of the inoculated eyes, salivary glands and other non-ocular tissues of steroid-treated mice. After immunosuppression, low titers of infectious virus were recovered from the salivary glands of steroid-treated mice, but infectious virus was not recovered from the inoculated eye of either steroid-treated of non-immunosuppressed mice. The DNA PCR results suggest that after inoculation of 5 x 10(2) PFU of MCMV into the supraciliary space of euthymic BALB/c mice, virus becomes latent in the inoculated eye, salivary gland and other extraocular tissues. The RT-PCR results suggest that latent MCMV can be reactivated in multiple tissues by immunosuppression.

Induction of apoptosis of T cells by infecting mice with murine cytomegalovirus.

Cytomegalovirus (CMV) is associated with several lymphocyte dysfunctions, but the precise mechanisms of the dysfunctions are still unclear. To elucidate the mechanisms, a cell cycle-DNA content analysis was performed on splenic T cells of murine CMV (MCMV)-infected BALB/c mice. T cells from mice infected with 3 x 10(3) PFU of MCMV contained a higher percentage of hypodiploid nuclei after 12 or 24 h of culture than those from naive mice. T cells from infected mice also contained a larger amount of fragmented DNA. Taken together, these results suggested that infection with MCMV induced the apoptotic cell death of T cells. This induction of apoptosis accounted for the dysfunction of lymphocytes, at least partially. Flow cytometric analysis showed that T cells as well as B cells from MCMV-infected mice expressed an augmented level of Fas antigen, an apoptosis-associated cell surface molecule, which might be the cause of the apoptosis of cells. T cells from MCMV-infected C57BL/6-lpr/lpr mice with mutations at the lpr/fas locus, however, also showed a substantial level of apoptosis, which was reproducibly lower than that seen in C57BL/6 mice. Therefore, it was suggested that the Fas-mediated pathway contributed to but was not sufficient for the induction of apoptosis and that mechanisms other than the Fas-associated pathway were also involved in the induction of apoptosis.

Retinitis in euthymic mice following inoculation of murine cytomegalovirus (MCMV) via the supraciliary route.

Cytomegalovirus (CMV) retinitis is the most frequent infectious ocular complication of the acquired immune deficiency syndrome (AIDS). Currently, there are few animal models to study the virologic and immunologic factors which contribute to the pathogenesis of CMV retinitis. In these experiments, 1-2 X 10(4) PFU of murine cytomegalovirus (MCMV) was inoculated into the supraciliary space of BALB/c mice. Within three days of inoculation, moderate iridocyclitis was observed which progressed to necrosis of the ciliary body by day 14. Approximately 60% of the mice developed typical retinitis characterized by virus-infected cytomegalic cells in the retina and retinal pigment epithelium, focal retinal infiltrates, transition zones between uninvolved and involved retina, and optic neuritis. The remaining animals exhibited atypical retinitis characterized by non-specific retinal inflammation in the absence of obvious viral infection. This murine model of CMV retinitis shares some features with retinitis observed in AIDS patients with CMV retinitis and may be useful to evaluate the efficacy of immunologic and/or pharmacologic treatment strategies for CMV retinitis.

The protective effects of hyperimmune anti-murine cytomegalovirus antiserum against lethal viral challenge: The case for passive-active immunization

The administration of 0.2 ml of hyperimmune anti-mouse cytomegalovirus (CMV) antiserum intraperitoneally (ip) or intravenously provided complete protection against lethal challenge (10 5.8 PFU ip) with murine CMV. Antiserum protection was complete when the antiserum was administered as long as 24 hr after viral challenge. The administration of antiserum had little effect on the titers of virus in the organs of these animals. Ammonium sulfate-treated antiserum provided similar complete protection. Animals rechallenged with 10 6–10 6.5 PFU of murine CMV 1 month after initial challenge, at a time when the administrated antiserum was no longer detectable, all survived. We conclude that hyperimmune antiserum can provide significant protection against otherwise lethal murine CMV infection, that the protecting material lies within the immunoglobulin fraction, and that long-term immunity results from the combined exposure to virus and antiserum. Such passive-active protection could be useful in protecting against human CMV infection.