Suppression of rat cytomegalovirus replication by antibodies against gamma interferon.

The role of gamma interferon (IFN-gamma) in the resolution of rat cytomegalovirus (RCMV) infection was investigated. In the spleen, IFN-gamma-producing cells reached maximum numbers on day 7 after infection. Prophylactic treatment with high doses of recombinant rat IFN-gamma exerted antiviral activity in fibroblasts and protected immunosuppressed rats against a lethal RCMV challenge. Remarkably, in immunocompetent rats, neutralization of endogenous IFN-gamma activity significantly reduced the numbers of RCMV antigen-expressing cells in the spleen, the predominant site of viral replication. Moreover, protection of radiation-immunosuppressed infected rats by transferred immune T cells was enhanced by coinjection of IFN-gamma neutralizing antibodies. The observations were paralleled by in vitro findings: low concentrations of IFN-gamma enhanced viral replication in both macrophages and fibroblasts. These data suggest that IFN-gamma can play different and even opposite roles in the regulation of RCMV replication in vivo; T lymphocytes may contribute to the progression of RCMV infection by secreting IFN-gamma.     

Dissemination of rat cytomegalovirus through infected granulocytes and monocytes in vitro and in vivo

The role of leukocytes in the in vivo dissemination of cytomegalovirus was studied in this experiment. Rat cytomegalovirus (RCMV) could be transferred to rat granulocytes and monocytes by cocultivation with RCMV-infected fibroblasts in vitro. Intravenous injection of purified infected granulocytes or monocytes resulted in a systemic infection in rats, indicating that our model is a powerful tool to gain further insight into CMV dissemination and the development of new antivirals.     

Tumor necrosis factor alpha promotes replication and pathogenicity of rat cytomegalovirus.

We investigated the role of tumor necrosis factor alpha (TNF-alpha) in the pathogenesis of rat cytomegalovirus (RCMV) infection. TNF-alpha levels found in the sera of radiation-immunosuppressed rats in the course of infection (> 350 pg/ml) correlated with the development of RCMV disease. Administration of anti-TNF-alpha antibodies strongly reduced the severity of pneumonia and led to a reduction in virus titers. In immunocompetent rats, anti-TNF-alpha antibodies also significantly suppressed viral replication. Conversely, administration of TNF-alpha augmented RCMV replication and aggravated the disease signs. In vitro, TNF-alpha enhanced RCMV replication in the macrophage, whereas a reduction of viral replication was observed in fibroblasts, indicating that the effect on viral replication is cell type specific. Besides activation of viral replication and exacerbation of RCMV disease, TNF-alpha also favored lymphoid and hematopoietic tissue reconstitution after irradiation, which may contribute to antiviral resistance and survival. This finding demonstrates the protean nature of TNF-alpha, with both beneficial and adverse effects for the host. Our results suggest that TNF-alpha plays an important role in modulating the pathogenesis of RCMV infection.     

The Role of Interferon in Influenza Virus Tissue Tropism

We have studied the pathogenesis of influenza virus infection in mice that are unable to respond to type I or II interferons due to a targeted disruption of the STAT1 gene. STAT1−/− animals are 100-fold more sensitive to lethal infection with influenza A/WSN/33 virus than are their wild-type (WT) counterparts. Virus replicated only in the lungs of WT animals following intranasal (i.n.) virus inoculation, while STAT1−/− mice developed a fulminant systemic influenza virus infection following either i.n. or intraperitoneal inoculation. We investigated the mechanism underlying this altered virus tropism by comparing levels of virus replication in fibroblast cell lines and murine embryonic fibroblasts derived from WT mice, STAT−/− mice, and mice lacking gamma interferon (IFNγ−/− mice) or the IFN-α receptor (IFNαR−/− mice). Influenza A/WSN/33 virus replicates to high titers in STAT1−/− or IFNαR−/− fibroblasts, while cells derived from WT or IFNγ−/− animals are resistant to influenza virus infection. Immunofluorescence studies using WT fibroblast cell lines demonstrated that only a small subpopulation of WT cells can be infected and that in the few infected WT cells, virus replication is aborted at an early, nuclear phase. In all organs examined except the lung, influenza A WSN/33 virus infection is apparently prevented by an intact type I interferon response. Our results demonstrate that type I interferon plays an important role in determining the pathogenicity and tissue restriction of influenza A/WSN/33 virus in vivo and in vitro.     

Complete DNA sequence of the rat cytomegalovirus genome

We have determined the complete genome sequence of the Maastricht strain of rat cytomegalovirus (RCMV). The RCMV genome has a length of 229,896 bp and is arranged as a single unique sequence flanked by 504-bp terminal direct repeats. RCMV was found to have counterparts of all but one of the open reading frames (ORFs) that are conserved between murine CMV (MCMV) and human CMV (HCMV). Like HCMV, RCMV lacks homologs of the genes belonging to the MCMV m02 glycoprotein gene family. However, RCMV contains 15 ORFs with homology to members of the MCMV m145 glycoprotein gene family. Four ORFs are predicted to encode homologs of host proteins; R33 and R78 both putatively encode G protein-coupled receptors, whereas r144 and r131 encode homologs of major histocompatibility class I heavy chains and CC chemokines, respectively. An intriguing feature of the RCMV genome is the presence of an ORF, r127, with similarity to the rep gene of parvoviruses as well as ORF U94 of human herpesvirus 6A (HHV-6A) and HHV-6B. Counterparts of these ORFs have not been found in the other sequenced herpesviruses.     

Replication of Murine Cytomegalovirus in Differentiated Macrophages as a Determinant of Viral Pathogenesis

Blood monocytes or tissue macrophages play a pivotal role in the pathogenesis of murine cytomegalovirus (MCMV) infection, providing functions beneficial to both the virus and the host. In vitro and in vivo studies have indicated that differentiated macrophages support MCMV replication, are target cells for MCMV infection within tissues, and harbor latent MCMV DNA. However, this cell type presumably initiates early, antiviral immune responses as well. In addressing this paradoxical role of macrophages, we provide evidence that the proficiency of MCMV replication in macrophages positively correlates with virulence in vivo. An MCMV mutant from which the open reading frames M139, M140, and M141 had been deleted (RV10) was defective in its ability to replicate in macrophages in vitro and was highly attenuated for growth in vivo. However, depletion of splenic macrophages significantly enhanced, rather than deterred, replication of both wild-type (WT) virus and RV10 in the spleen. The ability of RV10 to replicate in intact or macrophage-depleted spleens was independent of cytokine production, as this mutant virus was a poor inducer of cytokines compared to WT virus in both intact organs and macrophage-depleted organs. Macrophages were, however, a major contributor to the production of tumor necrosis factor alpha and gamma interferon in response to WT virus infection. Thus, the data indicate that tissue macrophages serve a net protective role and may function as "filters" in protecting other highly permissive cell types from MCMV infection. The magnitude of virus replication in tissue macrophages may dictate the amount of virus accessible to the other cells. Concomitantly, infection of this cell type initiates the production of antiviral immune responses to guarantee efficient clearance of acute MCMV infection.     

Cytomegalovirus-mediated upregulation of chemokine expression correlates with the acceleration of chronic rejection in rat heart transplants

Cytomegalovirus (CMV) infections have been shown to dramatically affect solid organ transplant graft survival in both human and animal models. Recently, it was demonstrated that rat CMV (RCMV) infection accelerates the development of transplant vascular sclerosis (TVS) in both rat heart and small bowel graft transplants. However, the mechanisms involved in this process are still unclear. In the present study, we determined the kinetics of RCMV-accelerated TVS in a rat heart transplant model. Acute RCMV infection enhances the development of TVS in rat heart allografts, and this process is initiated between 21 and 24 days posttransplantation. The virus is consistently detected in the heart grafts from day 7 until day 35 posttransplantation but is rarely found at the time of graft rejection (day 45 posttransplantation). Grafts from RCMV-infected recipients had upregulation of chemokine expression compared to uninfected controls, and the timing of this increased expression paralleled that of RCMV-accelerated neointimal formation. In addition, graft vessels from RCMV-infected grafts demonstrate the increased infiltration of T cells and macrophages during periods of highest chemokine expression. These results suggest that CMV-induced acceleration of TVS involves the increased graft vascular infiltration of inflammatory cells through enhanced chemokine expression.     

Cytomegalovirus aggravates intimal hyperplasia in rats by stimulating smooth muscle cell proliferation

Epidemiological and animal studies suggest a role for cytomegalovirus (CMV) in restenosis. Previously, we demonstrated that proliferating smooth muscle cells (SMCs) in the injured arterial wall are particularly susceptible to CMV-induced effects. Therefore, we hypothesised that, depending on the time point of infection after vascular injury, CMV infection may affect cell proliferation either in the media or in the neointima, thereby aggravating the process of restenosis. In the present study, we focused on the individual layers of the arterial wall by evaluating, besides the neointima-to-media ratio, the medial and neointimal area and cellularity in the rat femoral artery. Vascular injury was photochemically induced in rat femoral arteries. Immediately or 14 days thereafter, rats were infected with rat CMV (RCMV) or mock infected. The presence of RCMV in the vascular wall was determined at 3, 5, 14 and 35 days after infection by quantitative real-time PCR. When rats were infected immediately after injury, a significant increase was seen only in the medial but not in the neointimal cross-sectional area. On the other hand, when rats were infected 14 days after the initial injury, a significant increase was only seen in the neointimal area, thereby confirming our hypothesis that RCMV infection primary affects proliferating SMCs. As the mean number of SMCs per microm2 in both cell layers was unchanged despite an increase in cross-sectional area, this implies that RCMV stimulated SMC proliferation. Furthermore, these vascular effects were observed without the virus being abundantly present in the vascular wall, suggesting that inflammatory and immune-mediated responses to RCMV infection are more important in aggravating the response to vascular injury than the virus itself.     

Pathogenesis and vertical transmission of a transplacental rat cytomegalovirus

Background

Cytomegalovirus (CMV) congenital infection is the major viral cause of well-documented birth defects in human. Because CMV is species-specific, the main obstacle to developing animal models for congenital infection is the difference in placental architecture, which preludes virus transmission across the placenta. The rat placenta, resembling histologically to that of human, could therefore facilitate the study of CMV congenital infection in human.

Results

In this report, we present clear evidences of the transplacental property of a new rat CMV (RCMV), namely ALL-03, which had been isolated from placenta and uterus of the house rat. Our study signifies the detection of infectious virus, virus particles, viral protein and DNA as well as immune response to demonstrate a natural model of acute CMV infection including the immunocompetent and immunocompromised host associated with or without pregnancy. It is characterized by a full range of CMV related clinical signs; lesions and anatomical virus distribution to uterus, placenta, embryo, fetus, neonate, lung, kidney, spleen, liver and salivary gland of the infected rats in addition to the virus-specific seroconversion. The preference of the virus for different organs mimics the situation in immunocompromised man. Most interestingly, the placenta was observed to be involved in the maternofetal infection and hence confirmed the hypothesis that the RCMV strain ALL-03 is capable to cross the placenta and infect the offsprings congenitally.

Conclusion

The maternal viremia leading to uterine infection which subsequently infecting to the fetus through the placenta is the most likely phenomenon of CMV vertical transmission in our study.     

Large deletion mutations involving the first pyrimidine-rich tract of the 5' nontranslated RNA of human hepatitis A virus define two adjacent domains associated with distinct replication phenotypes.

The 5' nontranslated RNA (5'NTR) of the HM175 strain of human hepatitis A virus contains several pyrimidine-rich regions, the largest and most 5' of which (pY1) is an almost pure polypyrimidine tract located between nucleotides (nt) 99 and 138, which includes five tandem repeats of the sequence motif (U)UUCC(C). Previous modeling of the RNA secondary structure suggested that this region was likely to be single-stranded, but repetitive RNase V1 cleavage sites within these (U)UUCC(C) motifs indicated that pY1 possesses an ordered structure. To assess the role of this domain in replication of the virus, a series of large deletion mutations were created which involved the pY1 domain of an infectious cDNA clone. Deletion of 44 nt between nt 96 and 139, including the entire pY1 domain, did not reduce the capacity of the virus to replicate in BS-C-1 or FRhK-4 cells, as assessed by the size of replication foci in radioimmunofocus assays or by virus yields under one-step growth conditions. In contrast, viable virus could not be recovered from transfected RNAs in which the deletion was extended in a 5' direction by an additional 3 nt (delta 93-134), most likely because of the destabilization of a predicted stem-loop structure upstream of pY1. Deletion mutations extending in a 3' fashion to nt 140, 141, or 144 resulted in moderately (delta 96-140 and delta 96-141) or strongly (delta 99-144, delta 116-144, and delta 131-144) temperature-sensitive replication phenotypes. Although deletion of the pY1 domain did not by itself affect the replication phenotype of virus, the additional deletion of sequence elements within the pY1 domain (nt 99 to 130) substantially enhanced the temperature-sensitive phenotype of delta 131-144 virus. These data suggest that the (U)UUCC(C) motifs within the pY1 domain are conserved among wild-type viruses in order to serve a function required during infection in vivo but not in cell culture. In contrast, the single-stranded region located immediately downstream of pY1 (nt 140 to 144) is essential for efficient replication in cultured cells at physiological temperature. Viruses with deletion mutations involving nt 140 to 144 and viruses with large pY1 deletions but normal replication phenotypes in cell culture may have attenuation properties which could be exploited for vaccine development.     

Assessing the in vitro fitness of an oseltamivir-resistant seasonal A/H1N1 influenza strain using a mathematical model

In 2007, the A/Brisbane/59/2007 (H1N1) seasonal influenza virus strain acquired the oseltamivir-resistance mutation H275Y in its neuraminidase (NA) gene. Although previous studies had demonstrated that this mutation impaired the replication capacity of the influenza virus in vitro and in vivo, the A/Brisbane/59/2007 H275Y oseltamivir-resistant mutant completely out-competed the wild-type (WT) strain and was, in the 2008-2009 influenza season, the primary A/H1N1 circulating strain. Using a combination of plaque and viral yield assays, and a simple mathematical model, approximate values were extracted for two basic viral kinetics parameters of the in vitro infection. In the ST6GalI-MDCK cell line, the latent infection period (i.e., the time for a newly infected cell to start releasing virions) was found to be 1-3 h for the WT strain and more than 7 h for the H275Y mutant. The infecting time (i.e., the time for a single infectious cell to cause the infection of another one) was between 30 and 80 min for the WT, and less than 5 min for the H275Y mutant. Single-cycle viral yield experiments have provided qualitative confirmation of these findings. These results, though preliminary, suggest that the increased fitness success of the A/Brisbane/59/2007 H275Y mutant may be due to increased infectivity compensating for an impaired or delayed viral release, and are consistent with recent evidence for the mechanistic origins of fitness reduction and recovery in NA expression. The method applied here can reconcile seemingly contradictory results from the plaque and yield assays as two complementary views of replication kinetics, with both required to fully capture a strain's fitness.     

Identification and characterization of a spliced C-type lectin-like gene encoded by rat cytomegalovirus

The English isolate of rat cytomegalovirus (RCMV) encodes a 20-kDa protein with a C-type lectin-like domain that is expressed in the delayed-early and late phases of the viral replication cycle. Genomic sequence analysis of the restriction fragment KpnR of RCMV revealed significant homology to several C-type lectin-containing molecules implicated in natural killer (NK) and T-cell interactions, as well as genes from four poxviruses and African swine fever virus. The gene is spliced into five exons and shows a splicing pattern with exon boundaries similar to those observed in the human differentiation antigen CD69. The cap site of the gene was mapped by RNase protection, 5' rapid amplification of cDNA ends, and primer extension experiments. This analysis demonstrated that the core promoter of the RCMV lectin-like gene contains a GATA rather than a TATA box. Splicing patterns were confirmed with isolates from an infected-cell cDNA library. A unique aspect of the protein is that its translation is not initiated by the canonical methionine but rather by alanine. To study its role in virus replication and pathogenesis, a recombinant virus was constructed in which the gene is interrupted. Replication in tissue culture was similar to that of wild-type virus.     

Susceptibility of peritoneal macrophages to rat cytomegalovirus infection

Abstract Peritoneal macrophages from Lewis (Lew) and Brown Norway (BN) rats did not support rat cytomegalovirus (RCMV) replication. Intraperitoneal (i.p.) inoculation of virus into the rats resulted in a rapid clearance of virus from the peritoneal lavage fluid and an uptake of virus in the macrophages. The virus did not persist in the peritoneal macrophages of the rats.     

Neutrophils promote mononuclear cell infiltration during viral-induced encephalitis

Neutrophils are the first infiltrating cell population to appear within the CNS during infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV). To determine whether neutrophils play a role in limiting acute JHMV infection, mice were depleted of neutrophils. Infection of neutropenic animals resulted in increased levels of virus replication and mortality compared with control mice. Furthermore, neutropenia resulted in significantly reduced mononuclear leukocyte infiltration possibly due to reduced loss of blood brain barrier integrity during acute JHMV infection. These data suggest that infiltrating neutrophils are crucial for limiting virus replication during acute JHMV infection, contribute to the loss of blood brain barrier integrity and play a role in shaping adaptive immunity within the CNS.     

Seoul virus infection increases aggressive behaviour in male Norway rats

In natural populations of rodents, males are more likely to engage in aggression and be infected with hantaviruses than females. Whether the relationship between hantavirus infection and aggression is due to host- or parasite-mediated mechanisms is unknown. The aim of this study was to determine whether hantavirus infection causes an increase in aggression in male rats and whether these behavioural changes are due to infection of the central nervous system or peripheral tissues. Male laboratory rats were infected with Seoul virus and tested for aggression in a resident-intruder paradigm 15 and 30 days postinoculation (p.i.). Males tested 30 days p.i. (i.e. during the persistent phase of infection) spent more time engaged in aggression than either uninfected males or males tested during the acute phase of infection (i.e. 15 days p.i.). Males that engaged in aggression for a longer duration had more virus present in lung, kidney and testis than males that spent less time engaged in aggression. Infected males shed virus in saliva, faeces, and urine; virus shedding, however, was not correlated with aggression and neither wounding nor transmission of virus to intruder males occurred during behavioural tests. Infection with Seoul virus did not alter either testosterone or corticosterone concentrations. Seoul virus antigens were not detected in the brains of infected rats. These data suggest that hantavirus infection leads to elevated aggression in infected males and may be a by-product of increased virus replication in peripheral tissues.