Production- and Purification-Relevant Properties of Human and Murine Cytomegalovirus.

There is a large unmet need for a prophylactic vaccine against human cytomegalovirus (HCMV) to combat the ubiquitous infection that is ongoing with this pathogen. A vaccination against HCMV could protect immunocompromised patients and prevent birth defects caused by congenital HCMV infections. Moreover, cytomegalovirus (CMV) has a number of features that make it a very interesting vector platform for gene therapy. In both cases, preparation of a highly purified virus is a prerequisite for safe and effective application. Murine CMV (MCMV) is by far the most studied model for HCMV infections with regard to the principles that govern the immune surveillance of CMVs. Knowledge transfer from MCMV and mice to HCMV and humans could be facilitated by better understanding and characterization of the biological and biophysical properties of both viruses. We carried out a detailed investigation of HCMV and MCMV growth kinetics as well as stability under the influence of clarification and different storage conditions. Further, we investigated the possibilities to concentrate and purify both viruses by ultracentrifugation and ion-exchange chromatography. Defective enveloped particles were not separately analyzed; however, the behavior of exosomes was examined during all experiments. The effectiveness of procedures was monitored using CCID50 assay, Nanoparticle tracking analysis, ELISA for host cell proteins, and quantitative PCR for host cell DNA. MCMV generally proved to be more robust in handling. Despite its greater sensitivity, HCMV was efficiently (100% recovery) purified and concentrated by anion-exchange chromatography using QA monolithic support. The majority of the host genomic DNA as well as most of the host cell proteins were removed by this procedure.

NUDT15 polymorphism influences the metabolism and therapeutic effects of acyclovir and ganciclovir.

Nucleobase and nucleoside analogs (NNA) are widely used as anti-viral and anti-cancer agents, and NNA phosphorylation is essential for the activity of this class of drugs. Recently, diphosphatase NUDT15 was linked to thiopurine metabolism with NUDT15 polymorphism associated with drug toxicity in patients. Profiling NNA drugs, we identify acyclovir (ACV) and ganciclovir (GCV) as two new NNAs metabolized by NUDT15. NUDT15 hydrolyzes ACV and GCV triphosphate metabolites, reducing their effects against cytomegalovirus (CMV) in vitro. Loss of NUDT15 potentiates cytotoxicity of ACV and GCV in host cells. In hematopoietic stem cell transplant patients, the risk of CMV viremia following ACV prophylaxis is associated with NUDT15 genotype (P = 0.015). Donor NUDT15 deficiency is linked to graft failure in patients receiving CMV-seropositive stem cells (P = 0.047). In conclusion, NUDT15 is an important metabolizing enzyme for ACV and GCV, and NUDT15 variation contributes to inter-patient variability in their therapeutic effects.

Atypical cytomegalovirus retinal disease in pyroptosis-deficient mice with murine acquired immunodeficiency syndrome.

Pyroptosis is a caspase-dependent programmed cell death pathway that initiates and sustains inflammation through release of pro-inflammatory cytokines interleukin (IL)-1ß and IL-18 following formation of gasdermin D (GSDMD)-mediated membrane pores. To determine the possible pathogenic contributions of pyroptosis toward development of full-thickness retinal necrosis during AIDS-related human cytomegalovirus retinitis, we performed a series of studies using an established model of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS). Initial investigations demonstrated significant transcription and translation of key pyroptosis-associated genes within the ocular compartments of MCMV-infected eyes of mice with MAIDS. Subsequent investigations compared MCMV-infected eyes of groups of wildtype MAIDS mice with MCMV-infected eyes of groups of caspase-1-/- MAIDS mice, GSDMD-/- MAIDS mice, or IL-18-/- MAIDS mice to explore a possible contribution of pyroptosis towards the pathogenesis of MAIDS-related MCMV retinitis. Histopathologic analysis revealed typical full-thickness retinal necrosis in 100% of MCMV-infected eyes of wildtype MAIDS mice. In sharp contrast, none (0%) of MCMV-infected eyes of MAIDS mice that were deficient in either caspase-1, GSDMD, or IL-18 developed full-thickness retinal necrosis but instead exhibited an atypical pattern of retinal disease characterized by thickening and proliferation of the retinal pigmented epithelium layer with relative sparing of the neurosensory retina. Surprisingly, MCMV-infected eyes of all groups of deficient MAIDS mice harbored equivalent intraocular amounts of infectious virus as seen in MCMV-infected eyes of groups of wildtype MAIDS mice despite failure to develop full-thickness retinal necrosis. We conclude that pyroptosis plays a significant role in the development of full-thickness retinal necrosis during the pathogenesis of MAIDS-related MCMV retinitis. This observation may extend to the pathogenesis of AIDS-related HCMV retinitis and other AIDS-related opportunistic virus infections.

Airborne Aerosolized Mouse Cytomegalovirus From Common Otolaryngology Procedures: Implications for COVID-19 Infection.

OBJECTIVES: To determine whether common otolaryngology procedures generate viable aerosolized virus through a murine cytomegalovirus (mCMV) model for infection. STUDY DESIGN: mCMV model of infection. SETTING: University of Utah laboratory. METHODS: Three-day-old BALB/c mice were inoculated with mCMV or saline. Five days later, each mouse underwent drilling, microdebrider, coblation, and electrocautery procedures. Particle size distribution and PM2.5 (particulate matter <2.5 µm) concentration were determined with a scanning mobility particle sizer and an aerosol particle sizer in the range of 15 nm to 32 µm. Aerosolized samples from these procedures were collected with an Aerosol Devices BioSpot sampler for viral titer based on polymerase chain reaction and for viable virus through viral culture. RESULTS: As compared with the background aerosol concentrations, coblation and electrocautery showed statistically significant increases in airborne aerosols (Tukey-adjusted P value <.040), while microdebrider and drilling at 30,000 rpm did not (.870 < Tukey-adjusted P value < .930). We identified viral DNA in samples from coblation and drilling procedures, although we did not identify viable viruses in aerosol samples from any of the 4 procedures. CONCLUSION: Coblation and electrocautery procedures generate >100-fold increases in aerosol concentrations over background; only coblation and drilling produce aerosolized viral DNA. The high concentration of aerosols from coblation and electrocautery suggests the need for appropriate safeguards against particle exposure to health care workers. The presence of viral DNA from drilling and coblation procedures warrants the need for appropriate protection against droplet and aerosol exposure.

The herpesviral antagonist m152 reveals differential activation of STING-dependent IRF and NF-κB signaling and STING's dual role during MCMV infection.

Cytomegaloviruses (CMVs) are master manipulators of the host immune response. Here, we reveal that the murine CMV (MCMV) protein m152 specifically targets the type I interferon (IFN) response by binding to stimulator of interferon genes (STING), thereby delaying its trafficking to the Golgi compartment from where STING initiates type I IFN signaling. Infection with an MCMV lacking m152 induced elevated type I IFN responses and this leads to reduced viral transcript levels both in vitro and in vivo This effect is ameliorated in the absence of STING Interestingly, while m152 inhibits STING-mediated IRF signaling, it did not affect STING-mediated NF-κB signaling. Analysis of how m152 targets STING translocation reveals that STING activates NF-κB signaling already from the ER prior to its trafficking to the Golgi. Strikingly, this response is important to promote early MCMV replication. Our results show that MCMV has evolved a mechanism to specifically antagonize the STING-mediated antiviral IFN response, while preserving its pro-viral NF-κB response, providing an advantage in the establishment of an infection.

Laboratory diagnostics of murine blood for detection of mouse cytomegalovirus (MCMV)-induced hepatitis.

Mouse models are important and versatile tools to study mechanisms and novel therapies of human disease in vivo. Both, the number and the complexity of murine models are constantly increasing and modification of genes of interest as well as any exogenous challenge may lead to unanticipated biological effects. Laboratory diagnostics of blood samples provide a comprehensive and rapid screening for multiple organ function and are fundamental to detect human disease. Here, we adapt an array of laboratory medicine-based tests commonly used in humans to establish a platform for standardized, multi-parametric, and quality-controlled diagnostics of murine blood samples. We determined sex-dependent reference intervals of 51 commonly used laboratory medicine tests for samples obtained from the C57BL/6J mouse strain. As a proof of principle, we applied these diagnostic tests in a mouse cytomegalovirus (MCMV) infection model to screen for organ damage. Consistent with histopathological findings, plasma concentrations of liver-specific enzymes were elevated, supporting the diagnosis of a virus-induced hepatitis. Plasma activities of aminotransferases correlated with viral loads in livers at various days after MCMV infection and discriminated infected from non-infected animals. This study provides murine blood reference intervals of common laboratory medicine parameters and illustrates the use of these tests for diagnosis of infectious disease in experimental animals.

Nutrition of Preterm Infants and Raw Breast Milk-Acquired Cytomegalovirus Infection: French National Audit of Clinical Practices and Diagnostic Approach.

Raw breast milk is the optimal nutrition for infants, but it is also the primary cause of acquired cytomegalovirus (CMV) infection. Thus, many countries have chosen to contraindicate to feed raw breast milk preterm infants from CMV-positive mothers before a corrected age of 32 weeks or under a weight of 1500 g. French national recommendations have not been updated since 2005. An audit of the French practices regarding the nutrition with raw breast milk in preterm infants was carried out using a questionnaire sent to all neonatal care units. Diagnosed postnatal milk-acquired CMV infections have been analysed using hospitalisation reports. Seventy-five percent of the neonatal units responded: 24% complied with the French recommendations, 20% contraindicated raw breast milk to all infants before 32 weeks regardless of the mothers' CMV-status, whereas 25% fed all preterm infants unconditionally with raw breast milk. Thirty-five cases of infants with milk-acquired CMV infections have been reported. The diagnosis was undeniable for five patients. In France, a high heterogeneity marks medical practices concerning the use of raw breast milk and the diagnostic approach for breast milk-acquired CMV infection is often incomplete. In this context, updated national recommendations and monitored CMV infections are urgently needed.

Suppressor of Cytokine Signaling 1 (SOCS1) and SOCS3 Are Stimulated within the Eye during Experimental Murine Cytomegalovirus Retinitis in Mice with Retrovirus-Induced Immunosuppression.

AIDS-related human cytomegalovirus retinitis remains the leading cause of blindness among untreated HIV/AIDS patients worldwide. To study mechanisms of this disease, we used a clinically relevant animal model of murine cytomegalovirus (MCMV) retinitis with retrovirus-induced murine AIDS (MAIDS) that mimics the progression of AIDS in humans. We found in this model that MCMV infection significantly stimulates ocular suppressor of cytokine signaling 1 (SOCS1) and SOCS3, host proteins which hinder immune-related signaling by cytokines, including antiviral type I and type II interferons. The present study demonstrates that in the absence of retinal disease, systemic MCMV infection of mice without MAIDS, but not in mice with MAIDS, leads to mild stimulation of splenic SOCS1 mRNA. In sharp contrast, when MCMV is directly inoculated into the eyes of retinitis-susceptible MAIDS mice, high levels of intraocular SOCS1 and SOCS3 mRNA and protein are produced which are associated with significant intraocular upregulation of gamma interferon (IFN-γ) and interleukin-6 (IL-6) mRNA expression. We also show that infiltrating macrophages, granulocytes, and resident retinal cells are sources of intraocular SOCS1 and SOCS3 protein production during development of MAIDS-related MCMV retinitis, and SOCS1 and SOCS3 mRNA transcripts are detected in retinal areas histologically characteristic of MCMV retinitis. Furthermore, SOCS1 and SOCS3 are found in both MCMV-infected cells and uninfected cells, suggesting that these SOCS proteins are stimulated via a bystander mechanism during MCMV retinitis. Taken together, our findings suggest a role for MCMV-related stimulation of SOCS1 and SOCS3 in the progression of retinal disease during ocular, but not systemic, MCMV infection.IMPORTANCE Cytomegalovirus infection frequently causes blindness in untreated HIV/AIDS patients. This virus manipulates host cells to dysregulate immune functions and drive disease. Here, we use an animal model of this disease to demonstrate that cytomegalovirus infection within eyes during retinitis causes massive upregulation of immunosuppressive host proteins called SOCS. As viral overexpression of SOCS proteins exacerbates infection with other viruses, they may also enhance cytomegalovirus infection. Alternatively, the immunosuppressive effect of SOCS proteins may be protective against immunopathology during cytomegalovirus retinitis, and in such a case SOCS mimetics or overexpression treatment strategies might be used to combat this disease. The results of this work therefore provide crucial basic knowledge that contributes to our understanding of the mechanisms of AIDS-related cytomegalovirus retinitis and, together with future studies, may contribute to the development of novel therapeutic targets that could improve the treatment or management of this sight-threatening disease.

Depletion of the Receptor-Interacting Protein Kinase 3 (RIP3) Decreases Photoreceptor Cell Death During the Early Stages of Ocular Murine Cytomegalovirus Infection.

Purpose: The purpose of this study was to determine if the receptor-interacting protein kinase 3 (RIP3) plays a significant role in innate immune responses and death of bystander retinal neurons during murine cytomegalovirus (MCMV) retinal infection, by comparing the innate immune response and cell death in RIP3-depleted mice (Rip3-/-) and Rip3+/+ control mice. Methods: Rip3-/- and Rip3+/+ mice were immunosuppressed (IS) and inoculated with MCMV via the supraciliary route. Virus-injected and mock-injected control eyes were removed at days 4, 7, and 10 post infection (p.i.) and markers of innate immunity and cell death were analyzed. Results: Compared to Rip3+/+ mice, significantly more MCMV was recovered and more MCMV-infected RPE cells were observed in injected eyes of Rip3-/- mice at days 4 and 7 p.i. In contrast, fewer TUNEL-stained photoreceptors were observed in Rip3-/- eyes than in Rip3+/+ eyes at these times. Electron microscopy showed that significantly more apoptotic photoreceptor cells were present in Rip3+/+ mice than in Rip3-/- mice. Immunohistochemistry showed that the majority of TUNEL-stained photoreceptors died via mitochondrial flavoprotein apoptosis-inducing factor (AIF)-mediated, caspase 3-independent apoptosis. The majority of RIP3-expressing cells in infected eyes were RPE cells, microglia/macrophages, and glia, whereas retinal neurons contained much lower amounts of RIP3. Western blots showed significantly higher levels of activated nuclear factor-κB and caspase 1 were present in Rip3+/+ eyes compared to Rip3-/- eyes. Conclusions: Our results suggest that RIP3 enhances innate immune responses against ocular MCMV infection via activation of the inflammasome and nuclear factor-κB, which also leads to inflammation and death of bystander cells by multiple pathways including apoptosis and necroptosis.

Glycolytic requirement for NK cell cytotoxicity and cytomegalovirus control.

NK cell activation has been shown to be metabolically regulated in vitro; however, the role of metabolism during in vivo NK cell responses to infection is unknown. We examined the role of glycolysis in NK cell function during murine cytomegalovirus (MCMV) infection and the ability of IL-15 to prime NK cells during CMV infection. The glucose metabolism inhibitor 2-deoxy-ᴅ-glucose (2DG) impaired both mouse and human NK cell cytotoxicity following priming in vitro. Similarly, MCMV-infected mice treated with 2DG had impaired clearance of NK-specific targets in vivo, which was associated with higher viral burden and susceptibility to infection on the C57BL/6 background. IL-15 priming is known to alter NK cell metabolism and metabolic requirements for activation. Treatment with the IL-15 superagonist ALT-803 rescued mice from otherwise lethal infection in an NK-dependent manner. Consistent with this, treatment of a patient with ALT-803 for recurrent CMV reactivation after hematopoietic cell transplant was associated with clearance of viremia. These studies demonstrate that NK cell-mediated control of viral infection requires glucose metabolism and that IL-15 treatment in vivo can reduce this requirement and may be effective as an antiviral therapy.

Reduced frequency of murine cytomegalovirus retinitis in C57BL/6 mice correlates with low levels of suppressor of cytokine signaling (SOCS)1 and SOCS3 expression within the eye during corticosteroid-induced immunosuppression.

AIDS-related human cytomegalovirus retinitis remains a leading cause of blindness worldwide. We compared two C57BL/6 mouse models of experimental murine cytomegalovirus (MCMV) retinitis for intraocular expression of suppressors of cytokine signaling (SOCS)1 and SOCS3, host proteins that are inducible negative feedback regulators of cytokine signaling. These mouse models differed in method of immune suppression, one by retrovirus-induced immune suppression (MAIDS) and the other by corticosteroid-induced immune suppression. Following subretinal injection of MCMV to induce retinitis, intraocular SOCS1 and SOCS3 were only mildly stimulated, and often without significance, within MCMV-infected eyes during the progression of MCMV retinitis in corticosteroid-immunosuppressed mice, contrary to MCMV-infected eyes of mice with MAIDS that showed significant high stimulation of SOCS1 and SOCS3 expression in agreement with previous findings. Frequency and severity of retinitis as well as amounts of intraocular infectious MCMV in corticosteroid-immunosuppressed mice were also unexpectedly lower than values previously reported for MAIDS animals during MCMV retinitis. These data reveal a major difference between two mouse models of experimental MCMV retinitis and suggest a possible link between the amplitude of SOCS1 and SOCS3 stimulation and severity of disease in these models.

Sequence variability among murine herpesvirus isolates shows possible effect of long-term in vitro passaging on their genome.

No abstract Keywords: murine herpesvirus 68; virus isolates; sequence analysis; restriction fragment length polymorphism.

Broncholaveolar lavage to detect cytomegalovirus infection, latency, and reactivation in immune competent hosts.

Roughly 1/3rd of immune competent patients will reactivate latent cytomegalovirus (CMV) during critical illness. There are no standard methods to detect reactivation, and some investigators have postulated that presence of DNA in BAL fluid is indicative of viral replication. To test this hypothesis, we used a murine model that allows inclusion of matched healthy controls which is not possible in human studies. BALB/c mice infected with Smith-murine CMV or PBS (mock) had BAL evaluated 7, 14, or 21 days after acute infections, during latency, or during bacterial sepsis. Plaque assay, PCR, and rtPCR were performed on BALs and concomitantly obtained lung tissue. BAL cellular compositions, including tetramer evaluation of CMV-specific T cells were evaluated by flow cytometry. CMV DNA were detected in BAL at all time-points during acute infection, becoming undetectable in all mice during latency, then were detected again during bacterial sepsis, peaking 3 weeks after onset. mCMV specific T-cells were most numerous in BAL after acute viral infections, decreasing to low levels during latency, then fluctuating during bacterial sepsis. Specifically, mCMV-specific T-cells contracted at sepsis onset, expanding 2-4 weeks post-sepsis, presumably in response to increased viral loads at that time point. Altogether, our results support the use of BAL PCR for the diagnosis of CMV replication in immune competent hosts. Additionally, we demonstrate dynamic changes in CMV-specific T cells that occur in BAL during CMV infection and during sepsis induced viral reactivation. J. Med. Virol. 88:1408-1416, 2016. © 2016 Wiley Periodicals, Inc.

Rat cytomegalovirus (RCMV) English isolate and a newly identified Berlin isolate share similarities with but are separate as an anciently diverged clade from Mouse CMV and the Maastricht isolate of RCMV.

The genome of the rat cytomegalovirus (RCMV) English isolate (MuHV-8) differs significantly from the RCMV Maastricht isolate (MuHV-2) and other cytomegaloviruses (CMVs) in its size, base composition and genomic content. Analysis of the RCMV-Berlin isolate, MuHV-8, revealed that the two MuHV-8 isolates are highly similar in genome size and content, indicating that the smaller genome size (202 946 bp) compared to other known CMVs was not the result of an accidental deletion during passage in tissue culture. Surprisingly, the proteins encoded in MuHV-8 shared more overall similarity with their orthologues from mouse CMV (MuHV-1) compared to their orthologues in rat CMV (MuHV-2). Phylogenetic analyses of conserved viral genes showed that the two MuHV-8 isolates are from the same species and represent a unique clade that is distinct from other rodent CMVs.

Murine cytomegalovirus is not restricted to the house mouse Mus musculus domesticus: prevalence and genetic diversity in the European house mouse hybrid zone.

UNLABELLED: Murine cytomegalovirus (MCMV) is a betaherpesvirus of the house mouse, Mus musculus domesticus. It is a common infectious agent of wild mice and a highly studied pathogen of the laboratory mouse. Betaherpesviruses are specific to their hosts, and it is not known if other Mus taxa carry MCMV or if it is restricted to M. m. domesticus. We sampled mice over a 145-km transect of Bavaria-Bohemia crossing a hybrid zone between M. m. domesticus and Mus musculus musculus in order to investigate the occurrence of MCMV in two Mus subspecies and to test the limits of the specificity of the virus for its host. We hypothesized that if the two subspecies carry MCMV and if the virus is highly specific to its host, divergent MCMV lineages would have codiverged with their hosts and would have a geographical distribution constrained by the host genetic background. A total of 520 mice were tested by enzyme-linked immunosorbent assay (ELISA) and/or nested PCR targeting the M94 gene. Seropositive and PCR-positive individuals were found in both Mus subspecies. Seroprevalence was high, at 79.4%, but viral DNA was detected in only 41.7% of mice. Sequencing revealed 20 haplotypes clustering in 3 clades that match the host genetic structure in the hybrid zone, showing 1 and 2 MCMV lineages in M. m. domesticus and M. m. musculus, respectively. The estimated time to the most recent common ancestor (1.1 million years ago [Mya]) of the MCMVs matches that of their hosts. In conclusion, MCMV has coevolved with these hosts, suggesting that its diversity in nature may be underappreciated, since other members of the subgenus Mus likely carry different MCMVs. IMPORTANCE: Murine cytomegalovirus (MCMV) is a betaherpesvirus of the house mouse, Mus musculus domesticus, an important lab model for human cytomegalovirus (HCMV) infection. The majority of lab studies are based on only two strains of MCMVs isolated from M. m. domesticus, Smith and K181, the latter derived from repeated passage of Smith in mouse submaxillary glands. The presence of MCMV in other members of the Mus subgenus had not even been investigated. By screening mouse samples collected in the European house mouse hybrid zone between M. m. domesticus and M. m. musculus, we show that MCMV is not restricted to the M. m. domesticus subspecies and that MCMVs likely codiverged with their Mus hosts. Thus, the diversity of MCMV in nature may be seriously underappreciated, since other members of the subgenus Mus likely carry their own MCMV lineages.

Resolving the titer of murine cytomegalovirus by plaque assay using the M2-10B4 cell line and a low viscosity overlay.

BACKGROUND: Murine cytomegalovirus (MCMV) is increasingly used as an infectious model to investigate host-pathogen interactions in mice. Detailed methods have been published for using primary murine embryonic fibroblasts (MEFs) for preparing stocks and determining viral titers of MCMV. For determining the titer of MCMV by plaque assay, these methods rely on a high viscosity media that restricts viral spreading through the supernatant of the culture, but is also usually too viscous to pipet. Moreover, MEFs must be repeatedly generated and can vary widely from batch-to-batch in purity, proliferation rates, and the development of senescence. In contrast, the M2-10B4 bone marrow stromal cell line (ATCC # CRL-1972), which is also permissive for MCMV, has been reported to produce high-titer stocks of MCMV and has the considerable advantages of growing rapidly and consistently. However, detailed methods using these cells have not been published. METHODS: We modified existing protocols to use M2-10B4 cells for measuring MCMV titers by plaque assay. RESULTS: We found that MCMV plaques could be easily resolved on monolayers of M2-10B4 cells. Moreover, plaques formed normally even when cultures of M2-10B4 cells were less than 50% confluent on the day of infection, as long as we also used a reduced viscosity overlay. CONCLUSIONS: Overall, our protocol enabled us to use a consistent cell line to assess viral titers, rather than repeatedly producing primary MEFs. It also allowed us to start the assay with 4-fold fewer cells than would be required to generate a confluent monolayer, reducing the lead-time prior to the start of the assay. Finally, the reduced viscosity CMC could be handled by pipet and did not need to be pre-mixed with media, thus increasing its shelf-life and ease-of-use. We describe our results here, along with detailed protocols for the use of the M2-10B4 cell lines to determine the titer and grow stocks of MCMV.

Rapid and sequential quantitation of salivary gland-associated mouse cytomegalovirus in oral lavage.

Cytomegalovirus (CMV) establishes a persistent infection in the salivary glands and transmits to other hosts. Mouse cytomegalovirus (MCMV) is a well-characterized model for studying the mechanisms of host responses against CMV. The viral load in salivary glands has been measured traditionally because it has been considered to reflect the consequence of anti-virus responses by T cells and natural killer (NK) cells. However, the standard plaque assay is cumbersome and it is impossible to monitor sequentially the viral load in same host. Hence, the goal of this study was to develop a real-time quantitative PCR (qPCR)-based procedure to measure the viral load in oral lavage. This report demonstrates that the viral load in oral lavage correlates well with viral titers in the salivary glands. This method allows sequential quantitation of viral loads without sacrificing mice and provides a technique that will facilitate kinetic studies of anti-viral immunity mediated by the innate and adaptive immune systems.

Molecular investigation of the 7.2 kb RNA of murine cytomegalovirus.

BACKGROUND: HCMV encodes a stable 5 kb RNA of unknown function that is conserved across cytomegalovirus species. In vivo studies of the MCMV orthologue, a 7.2 kb RNA, demonstrated that viruses that do not express the RNA fail to establish efficient persistent replication in the salivary glands of mice. To gain further insight into the function and properties of this conserved locus, we characterized the MCMV intron in finer detail. METHODS: We performed multiple analyses to evaluate transcript expression kinetics, identify transcript termini and promoter elements. The half-lives of intron locus RNAs were quantified by measuring RNA levels following actinomycin D treatment in a qRT-PCR-based assay. We also constructed a series of recombinant viruses to evaluate protein coding potential in the locus and test the role of putative promoter elements. These recombinant viruses were tested in both in vitro and in vivo assays. RESULTS: We show that the 7.2 kb RNA is expressed with late kinetics during productive infection of mouse fibroblasts. The termini of the precursor RNA that is processed to produce the intron were identified and we demonstrate that the m106 open reading frame, which resides on the spliced mRNA derived from precursor processing, can be translated during infection. Mapping the 5' end of the primary transcript revealed minimal promoter elements located upstream that contribute to transcript expression. Analysis of recombinant viruses with deletions in the putative promoter elements, however, revealed these elements exert only minor effects on intron expression and viral persistence in vivo. Low transcriptional output by the putative promoter element(s) is compensated by the long half-life of the 7.2 kb RNA of approximately 28.8 hours. Detailed analysis of viral spread prior to the establishment of persistence also showed that the intron is not likely required for efficient spread to the salivary gland, but rather enhances persistent replication in this tissue site. CONCLUSIONS: This data provides a comprehensive transcriptional analysis of the MCMV 7.2 kb intron locus. Our studies indicate that the 7.2 kb RNA is an extremely long-lived RNA, a feature which is likely to be important in its role promoting viral persistence in the salivary gland.

Establishment of rat brain endothelial cells susceptible to rat cytomegalovirus ALL-03 infection.

Endothelial cells have been implicated as key cells in promoting the pathogenesis and spread of cytomegalovirus (CMV) infection. This study describes the isolation and culture of rat brain endothelial cells (RBEC) and further evaluates the infectious potential of a Malaysian rat CMV (RCMV ALL-03) in these cultured cells. Brain tissues were mechanically fragmented, exposed to enzymatic digestion, purified by gradient density centrifugation, and cultured in vitro. Morphological characteristics and expression of von Willebrand factor (factor VIII-related antigen) verified the cells were of endothelial origin. RBEC were found to be permissive to the virus by cytopathic effects with detectable plaques formed within 7 d of infection. This was confirmed by electron microscopy examination which proved the existence of the viral particles in the infected cells. The susceptibility of the virus to these target cells under the experimental conditions described in this report provides a platform for developing a cell-culture-based experimental model for studies of RCMV pathogenesis and allows stimulation of further studies on host cell responses imposed by congenital viral infections.

[A murine model with murine cytomegalovirus infection resulting in colon inflammation after allogeneic skin transplantation].

UNLABELLED: To provide a reliable animal model for study of human CMV disease in gastrointestinal track, we tried to infect with murine cytomegalovirus (MCMV) in mice that were received allogenetic skin transplantation under immunosuppression. (1) Skin transplantation was performed between 18 donor C57BL/6 mice and 72 recipient BALB/c mice. (2) All recipient mice were then given Cyclosporine at 12 mg/kg daily for 2 weeks by intraperitoneal injection. Mice were randomly divided into 3 groups. Two experimental groups were received MCMV-infected mouse embryonic fibroblasts (MEF) at 10(4) PFU and 10(5) PFU respectively, and the control group received MEF only. We observed any possibly pathophysiological behavior changes and recorded the changes in body weight. The mice were sacrificed at 5d, 9d, 14d, 21d post infection and colon tissue was collected for analysis. RESULTS: Mice infected with MCMV at 10(5) PFU group showed anorexia, lethargy and degression in locomotor activity. This group of mice showed significant decrease in body weight than that of other groups. Colon tissues were collected 14 days after infection. Histological examination revealed that the mucous layer became thinner in the proximal colon and increased number of lymphoid follicles in distal colon in infected animals. The changes in the mucosal structure was most prominent in the group 10(5) PFU MCMV. Viral DNA was present in the colon by in situ hybridization for IE1 gene, and viral gB transcript was positive by RT-PCR. One of the viral major proteins, pp65, was widely distributed in the colon by immunohistochemistry. These data demonstrated that MCMV established infection in colon of the mice after allogenetic skin transplantation. Electron microscopy showed that there were herpes virus particles in the colon tissue. CONCLUSION: Infection with MCMV in mouse after allogenetic skin transplantation by nasal cavity inoculation resulted in the pathological changes in colon tissue similar to that of inflammation in human colon. The small animal model of colon inflammation may provide a platform for further study of pathogenesis as well as medical intervention of HCMV involved inflammation of human bowel.