Persistent Seoul virus infection in Lewis rats.

Mechanistic studies of hantavirus persistence in rodent reservoirs have been limited by the lack of a versatile animal model. This report describes findings from experimental infection of inbred Lewis rats with Seoul virus strain 80-39. Rats inoculated with virus intraperitoneally at 6 days of age became persistently infected without clinical signs. Tissues from Seoul virus-inoculated 6-day-old rats were assessed at 6, 9, and 12 weeks post-inoculation for viral RNA by RT-PCR and in situ hybridization (ISH) and for infectious virus by inoculation of Vero E6 cells. Virus was isolated from lung and kidney of infected rats at 6 weeks and viral RNA was detected in lung, kidney, pancreas, salivary gland, brain, spleen, liver and skin at 6, 9 and 12 weeks. Rats inoculated with Seoul virus intraperitoneally at 10 or 21 days of age became infected without clinical signs but had low to undetectable levels of viral RNA in tissues at 6 weeks post-inoculation. ISH identified vascular smooth muscle and endothelial cells as common sites of persistent infection. Cultured rat smooth muscle cells and to a lesser extent cultured endothelial cells also were susceptible to Seoul virus infection. Pancreatic infection resulted in insulitis with associated hyperglycemia. These studies demonstrate that infant Lewis rats are uniformly susceptible to asymptomatic persistent Seoul virus infection. Additionally, they offer opportunities for correlative in vivo and in vitro study of Seoul virus interactions in host cell types that support persistent infection.

Molecular mechanisms involved in GnRH analogue-related apoptosis for uterine leiomyomas.

GnRH agonist therapy is known to reduce uterine leiomyoma volume, although the molecular mechanisms responsible for this effect remain poorly understood. In this study, we have investigated the molecular mechanisms involved in the anti-proliferative effect of a GnRH agonist, leuprolide acetate (LA), in uterine leiomyomas obtained from six patients treated with LA for 3 months before surgery (group B), compared with tumours from six untreated patients (group A). To this end, we have evaluated the expression and the activity of molecules involved in the regulation of cell survival and proliferation. In group B, the total activity of PI3K was reduced by 60% compared with control samples. Furthermore, LA caused a reduction of PKB activation of approximately 50%, measured as serine 473 phosphorylation. In parallel with PKB reduction in LA samples, we observed a 60% reduction in the phosphorylation of its substrate BAD. While Bcl-xL/BAD association was not significantly modified in LA-treated leiomyomas, BAD/14.3.3 interaction was reduced, due to a 50% decreased 14.3.3 expression. In addition, LA was able to reduce the expression of the antiapoptotic proteins FLIP and PED/PEA15 by 70 and 50% respectively, compared with control samples. We next evaluated the activation of MAP kinases in leiomyomas. Activation of p42 and p44 MAP kinase isoforms was increased by 30% in group B. However, the phosphorylation of the transcription factor Elk1 was not increased in a similar fashion in LA-treated leiomyomas compared with group A. Thus, these data suggest that LA reduction of leiomyoma volume is mediated at least in part by a decreased activation of the PI3K/PKB survival pathway and by the suppression of antiapoptotic factors.

Prevalence of rat virus infection in progeny of acutely or persistently infected pregnant rats.

Infant rats are susceptible to persistent rat virus (RV) infection, but risk of persistent infection after prenatal exposure to virus is unclear. We examined this aspect of RV infection in the progeny of dams inoculated with virus during or prior to pregnancy. Sprague-Dawley (SD) dams were infected during pregnancy (gestation day 9) by oronasal inoculation with 10(5) TCID50 of the UMass strain of RV. SD rats were infected prior to pregnancy by oronasal inoculation of two-day-old females with 10(2) TCID50 of RV-UMass, which induced persistent infection. They were mated to non-immune males after reaching sexual maturity. Rats were assessed for RV infection by virus isolation, in situ hybridization, contact transmission, or serologic testing. The progeny of dams inoculated with virus during gestation had high prevalence of infection through postpartum week 9 (9 of 12 rats were virus positive at week 3, and 7 of 10 were virus positive at week 9). Additionally, 2 of 10 rats were virus positive at least through postpartum week 15. The progeny from persistently infected, seropositive dams had no evidence of infection and did not transmit infection to contact sentinels. However, 12 dams were virus positive at necropsy and 9 had transmitted infection to their breeding partners. These results indicate that prenatal infection in non-immune dams can lead to RV persistence in their progeny. By contrast, the progeny of persistently infected dams are protected from infection, presumably by maternal antibody, although their dams can transmit infection to their breeding partners.

Persistent rat virus infection in smooth muscle of euthymic and athymic rats.

Rat virus (RV) infection can cause disease or disrupt responses that rely on cell proliferation. Therefore, persistent infection has the potential to amplify RV interference with research. As a step toward determining underlying mechanisms of persistence, we compared acute and persistent RV infections in infant euthymic and athymic rats inoculated oronasally with the University of Massachusetts strain of RV. Rats were assessed by virus isolation, in situ hybridization, and serology. Selected tissues also were analyzed by Southern blotting or immunohistochemistry. Virus was widely disseminated during acute infection in rats of both phenotypes, whereas vascular smooth muscle cells (SMC) were the primary targets during persistent infection. The prevalence of virus-positive cells remained moderate to high in athymic rats through 8 weeks but decreased in euthymic rats by 2 weeks, coincident with seroconversion and perivascular infiltration of mononuclear cells. Virus-positive pneumocytes and renal tubular epithelial cells also were detected through 8 weeks, implying that kidney and lung excrete virus during persistent infection. Viral mRNA was detected in SMC of both phenotypes through 8 weeks, indicating that persistent infection includes virus replication. However, only half of the SMC containing viral mRNA at 4 weeks stained for proliferating cell nuclear antigen, a protein expressed in cycling cells. The results demonstrate that vasculotropism is a significant feature of persistent infection, that virus replication continues during persistent infection, and that host immunity reduces, but does not eliminate, infection.

Humoral immunity and protection of mice challenged with homotypic or heterotypic parvovirus.

BACKGROUND AND OBJECTIVES: Two serotypes of autonomously replicating parvoviruses infect laboratory mice. Genome regions coding for the nonstructural proteins of minute virus of mice [MVM] and mouse parvovirus [MPV] are almost identical, whereas capsid-coding sequences are divergent. We addressed these questions: Does humoral immunity confer protection from acute infection after challenge with homotypic or heterotypic parvovirus, and if it confers protection against acute MPV infection, does it also protect against persistent MPV infection? METHODS: Infant mice without maternal antibody or antibody to MVM or MPV and young adult mice given normal mouse serum or antibody to MVM or MPV were challenged with homotypic or heterotypic virus. In situ hybridization with target tissues was the indicator of infection. RESULTS: Humoral immunity failed to confer protection against acute heterotypic parvovirus infection. In passive transfer studies, MPV DNA was observed occasionally in lymph nodes, intestine, or the spleen of MPV-challenged mice given homotypic antibody and kept for 6 or 28 days. Variable proportions of mice given MPV antibody and homotypic challenge had viral DNA in lymphoid tissues 56 days after virus inoculation. CONCLUSION: A mouse or colony that has sustained infection with MVM or MPV is probably fully susceptible to infection with the heterotypic virus.

Mouse parvovirus infection potentiates allogeneic skin graft rejection and induces syngeneic graft rejection.

BACKGROUND: The recently identified autonomous mouse parvovirus designated mouse parvovirus-1 (MPV-1) persists in adult BALB/c mice for at least 9 weeks, infects lymphoid tissues, interferes with the ability of cloned T cells to proliferate, and exhibits immunomodulatory properties. As a consequence of these findings, the present studies were undertaken to characterize further the inmunomodulatory effects of MPV-1 on T cell-mediated immune responses in vivo and in vitro. METHODS: To evaluate the effect of MPV-1 infection on CD8+ T cell-mediated responses, BALB/c-H2dm2 mice were infected after transplantation of allogeneic BALB/c skin. RESULTS: MPV-1 potentiated the rejection of allogeneic skin grafts. This potentiation was not a result of virus infecting the cellular or vascular component of the graft as determined by in situ hybridization, but was mediated by T cells. However, the proliferative capacity of alloantigen-reactive lymphocytes from graft-sensitized infected mice was diminished. MPV-1 also induced the rejection of syngeneic skin grafts, and T cells from these infected graft-sensitized mice lysed syngeneic P815 target cells. CONCLUSIONS: These results suggest that MPV-1 infection of skin-grafted mice may disrupt normal mechanisms of peripheral tolerance and provide a unique model to study virus-induced autoimmunity.

Rat parvovirus type 1: the prototype for a new rodent parvovirus serogroup.

A newly recognized parvovirus of laboratory rats, designated rat parvovirus type 1a (RPV-1a), was found to be antigenically distinct. It was cloned, sequenced, and compared with the University of Massachusetts strain of rat virus (RV-UMass) and other autonomous parvoviruses. RPV-1a VP1 identity with these viruses never exceeded 69%, thus explaining its antigenic divergence. In addition, RPV-1a had reduced amino acid identity in NS coding regions (82%), reflecting phylogenetic divergence from other rodent parvoviruses. RPV-1a infection in rats had a predilection for endothelium and lymphoid tissues as previously reported for RV. Infectious RPV-1a was isolated 3 weeks after inoculation of infant rats, suggesting that it, like RV, may result in persistent infection. In contrast to RV, RPV-1a was enterotropic, a characteristic previously associated with parvovirus infections of mice rather than rats. RPV-1a also differed from RV in that infection was nonpathogenic for infant rats under conditions where RV infection causes high morbidity and mortality. Thus, RPV-1a is the prototype virus of an antigenically, genetically, and biologically distinct rodent parvovirus serogroup.

Mouse parvovirus infection potentiates rejection of tumor allografts and modulates T cell effector functions.

Lymphocytotropic mouse parvoviruses can perturb immune responses. For example the recently identified mouse parvovirus designated MPV-1 persistently infects lymphoid tissues and interferes with the ability of cloned T cells to proliferate. As a consequence of these findings the present studies were undertaken to characterize further the immunomodulatory effects of MPV-1 on T cell-mediated immune responses in vivo and in vitro. To evaluate the effect of MPV-1 on CD8+ T cell-mediated responses sarcoma I (SaI) cells, devoid of class II major histocompatibility (MHC) antigens, were administered to MPV-1-infected adult BALB/c mice. MPV-1 infection accelerated tumor allograft rejection. Immunofluorescence staining and in situ hybridization studies of tumors suggested that direct infection of the tumor cells was not responsible for accelerated rejection. Furthermore, compared with uninfected mice, T cells from infected mice that had rejected SaI tumors had a diminished cytolytic capacity. Taken together these results suggest that MPV-1 may induce "bystander help." To examine the in vivo effect of MPV-1 on CD4+ T cell mediated responses adult mice were primed with ovalbumin (OVA) and infected with MPV-1. Spleen and popliteal lymph node cells from OVA-primed mice 3 or 7 days after MPV-1 inoculation had reduced proliferation responses, whereas the proliferative capacity of mesenteric lymph node cells from these mice was increased. Similarly, MPV-1 reduced cytokine-induced proliferation of allospecific CD8+ cloned L3 T cells and OVA-reactive CD4+ T cells without effecting cell viability. Since parvoviruses are widespread among laboratory rodents, these findings emphasize the importance of identifying and excluding parvovirus infections in mice used for transplantation studies and in cultures of mouse T lymphocytes.

A nonlethal rat parvovirus infection suppresses rat T lymphocyte effector functions.

Inoculation of the UMass strain of rat virus (RV-UMass) into adult immunocompetent rats results in a prolonged subclinical infection that is resolved in 4 to 8 wk. Co-labeling studies, using in situ hybridization (ISH) and immunohistochemistry (IHC), confirmed that RV-UMass was lymphocytotropic and capable of infecting CD4+ and CD8+ T cells as well as B cells. ISH studies also revealed that virus replication was restricted in unstimulated cells but was productive in concanavalin A-stimulated lymphocytes. A corollary of productive infection of lymphocytes was the suppression of lymphocyte functions. Although RV-UMass did not appear to induce phenotypic changes during the course of infection, cells from infected rats had diminished proliferation and cytolytic responses. Both peripheral and mesenteric lymph node cells exhibited only partial recovery of their proliferative and cytolytic capacities one month after infection. Furthermore, RV-UMass-infected tissue culture maintained alloreactive CD4+ T cells in vitro, and a nonlethal infection of this T cell line inhibited Ag- and IL-2-induced proliferation. Because parvoviruses are widespread among laboratory rodents, these findings emphasize the importance of identifying and excluding parvovirus infection in rodents and in cultures of rat T lymphocytes.

Persistent rat virus infection in juvenile athymic rats and its modulation by immune serum.

In contrast to euthymic juvenile rats, which develop acute, self-limiting infection with rat virus (RV), RV infection of juvenile athymic rats was persistent for up to 12 weeks as demonstrated by recovery of infective virus, transmission to cagemates, and detection of viral DNA in the lungs. Administration of RV antiserum at the time of virus inoculation prevented persistent infection in five of six rats. Among rats given RV antiserum 1 week after virus, the interval at which euthymic rats begin to seroconvert, RV was not detected 1 week later but was recovered from four of six rats 3 weeks later. Results of these studies confirm that T-cell deficiency facilitates persistent RV infection and indicate that antibody provides significant protection from persistent infection only if it is present at the time of virus inoculation. The results support the concept that factors which prevent persistent infection in euthymic rats act early after virus inoculation and may include cellular immunity.

Environmental stability and transmission of rat virus.

The environmental stability and transmission of a field isolate of rat virus was tested under conditions resembling those that may be encountered during the housing of laboratory rats. The rat virus kept in physiologic salt solutions at room temperature remained infective for at least 5 weeks. Similar virus preparations remained infective after drying on a plastic surface for 3 to 5 weeks, depending on initial virus concentration. Varying the protein concentration in the medium had no significant effect on stability. Bedding from cages housing infected litters induced seroconversions in sentinel rats for at least 5 weeks after storage of rat virus at room temperature. Infection was transmitted between rats housed in open cages in a Trexler isolator but not between rats housed in microisolator cages connected by tunnels partitioned by wire screens with a mesh size of 1.67 mm. The results indicate that rat virus can remain infective after prolonged exposure to an ambient environment and suggest that infection is more readily transmitted by animal-to-animal contact or by fomites than by aerosolization of exhaled virus.

Development and optimization of plaque assays for rat coronaviruses.

Plaque assays under Sephadex or agarose overlays are described for rat coronaviruses (RCVs) grown in L2 mouse fibroblasts. A plaque assay using Sephadex was simple; however, viable plaques could not be collected for propagation, and fixation was necessary before evaluation. Plaque formation under agarose was optimized using diethylaminoethyl-dextran (DEAE-D) in the pre-treatment and absorption media and trypsin added to the absorption media and agarose overlay. The use of DEAE-D alone, trypsin alone or trypsin combined with DEAE-D significantly increased plaque numbers and visibility. Plaque numbers were highest when pre-treatment media contained DEAE-D, absorption media contained DEAE-D and trypsin, and the agarose overlay contained trypsin. The assay was useful for plaque isolation and quantification of sialodacryoadenitis virus (SDA), Parker's rat coronavirus (PRCV) and other coronavirus isolates from rats and its specificity was demonstrated by plaque-reduction neutralization testing. These methods will facilitate production of cloned virus stocks for study of RCV biology and virus quantification for in vitro and in vivo studies of RCVs.

Characterization of acute rat parvovirus infection by in situ hybridization.

In situ hybridization and virus titration were used to characterize early stages of rat virus (RV) infection of rat pups after oronasal inoculation. Results suggest that virus enters through the lung and that early viremia leads rapidly to pantropic infection. Cells derived from all three germ layers were infected with RV, but those of endodermal and mesodermal origin were the predominant targets. Infection of vascular endothelium was widespread and was associated with hemorrhage and infarction in the brain. Convalescence from acute infection was accompanied by mononuclear cell infiltrates at sites containing RV DNA. Viral DNA was also detected in endothelium, fibroblasts and smooth muscle myofibers four weeks after inoculation. Further examination of these cells as potential sites of persistent infection is warranted.

In vivo studies with an "orphan" parvovirus of mice.

A virus antigenically related to, but distinct from, minute virus of mice was assessed for infectivity in neonatal and weanling random-bred mice and was equally infectious for both age groups. The virus, designated a mouse "orphan" parvovirus (OPV), was also localized in tissues of experimentally infected random-bred, inbred, and immunodeficient mice by in situ hybridization. Hybridization signal was seen in exocrine and endocrine pancreas, abdominal lymph nodes, mesentery, intestine, and sporadically in other tissues of Sencar, C3H, and DBA mice inoculated as infants. In adult BALB/c severe combined immunodeficient (scid) mice, signal was seen in lung, liver, spleen, lymph nodes, and intestine but not in pancreas. Transmission of OPV by Sencar mice inoculated as infants was intermittent, whereas transmission by Sencar mice inoculated as weanlings was consistent during the first 2 weeks both by direct contact and by exposure to soiled bedding. The longest duration of transmission was 6 weeks among mice inoculated as infants. The results implicate a role for urinary, fecal, and perhaps respiratory excretion of virus, depending on host genotype and route of virus exposure. They also suggest that evaluation of pancreatic and immune function during acute infection is warranted.

Transmission of sialodacryoadenitis virus (SDAV) from infected rats to rats and mice through handling, close contact, and soiled bedding.

Thirty mice and six rats were exposed through handling, soiled bedding, or close contact to rats previously inoculated with sialodacryoadenitis virus (SDAV). All exposed rats developed coronaviral antibody without clinical signs or lesions of SDAV infection. Exposed mice had no lesions or clinical signs of coronavirus infection. Mice exposed by handling or by soiled bedding did not develop coronavirus antibody. Two of 10 mice exposed to SDAV-inoculated rats by close contact were coronavirus seropositive when tested 3 weeks postexposure. SDAV-inoculated rats and mice developed coronavirus lesions and antibody. These results suggest that rat-to-rat transmission of SDAV is likely via fomites or handling; however, rat-to-mouse transmission is unlikely when animals are housed and husbanded using modern techniques. Results also suggest that coronavirus antibody in mice is due to exposure to mouse coronavirus and not to rat coronaviruses.

Susceptibility of rodent cell lines to rat coronaviruses and differential enhancement by trypsin or DEAE-dextran.

Cell lines of rodent origin were tested for susceptibility to infection with rat coronavirus (RCV), including sialodacryoadenitis virus (SDAV) and Parker's rat coronavirus (PRCV). LBC rat mammary adenocarcinoma cells were susceptible only if the cells were treated with diethylaminoethyl-dextran (DEAE-D). A recent report that RCVs grow well in L2 mouse fibroblast cells was confirmed and expanded. RCV infection of L2 cells was substantially enhanced by treatment of cells with trypsin but not by treatment with DEAE-D. Primary isolation of SDAV from experimentally infected rats was accomplished using trypsin-treated L2 cells. One of 13 additional cell lines tested (rat urinary bladder epithelium, RBL-02) supported growth of RCVs, and growth was slightly enhanced by DEAE-D, but not by trypsin. These refinements of in vitro growth conditions for RCVs should facilitate further studies of their basic biology and improve options for primary isolation.

Persistent rat parvovirus infection in individually housed rats.

The duration of infection with rat virus (RV), an autonomous rodent parvovirus, was examined at multiple intervals over 6 months in rats inoculated by the oronasal route at 2 days of age or 4 weeks of age and individually housed after weaning to prevent cross-infection. Infectious virus was recovered by explant culture from 32 of 80 rats inoculated as pups and was detected as late as 6 months after inoculation. Rats inoculated as juveniles developed acute infection, but virus was not detected beyond 7 weeks after inoculation. Tissues from rats in both age groups were surveyed for RV DNA by Southern blotting using a double-stranded DNA probe made from a 1700 bp cloned fragment of RV spanning map units 0.19-0.52. Band patterns representative of acute infection (juvenile rats) were consistent with the replicating form of RV DNA, whereas patterns representative of persistent infection (rats inoculated as pups) were suggestive of defective or non-productive viral replication.

A comparison of the sensitivity and specificity of sialodacryoadenitis virus, Parker's rat coronavirus, and mouse hepatitis virus-infected cells as a source of antigen for the detection of antibody to rat coronaviruses.

Sialodacryoadenitis virus (SDAV) and Parker's rat coronavirus (PRC) are two recognized viral strains which cause spontaneous disease in the laboratory rat. Currently there is no recognized practical procedure which will accurately differentiate infections with these strains. Using SDAV- and PRC-infected L-2 cells as the source of antigen, and sera from rats collected post inoculation with either of these viral strains, the indirect fluorescent antibody (IFA) procedure was used to determine whether antibody titers could be used to differentiate infections from the homologous and heterologous virus. There was no detectable difference in the sensitivity or specificity of these systems in detecting antibody to the homologous or heterologous virus. Thus there was no evidence that SDAV- and PRC-infected cells would serve to differentiate antibody to the homologous virus using the IFA technique. In addition, antibody titers were similar when mouse hepatitis virus (MHV)-infected cells were used as the source of antigen for the IFA technique. However, using MHV or SDAV-infected cells as the source of antigen, there was a significant difference in antibody titers to the homologous virus detected using the immunoenzyme technique.

Modulation of lethal and persistent rat parvovirus infection by antibody.

Two day-old athymic (rnu/rnu) and euthymic (rnu/+) rat pups nursing immune or non-immune dams were inoculated oronasally with the Yale strain of rat virus (RV-Y). All athymic and euthymic pups (57/57) from immune dams remained clinically normal, whereas 51 of 66 athymic and euthymic pups from non-immune dams died within 30 days. Infectious RV was detected by explant culture in 12 of 15 surviving pups of both genotypes from non-immune dams 30 days after inoculation, but in none of the 57 surviving pups from immune dams. RV-Y DNA was detected by Southern blotting in kidneys of surviving athymic pups from non-immune dams but was not detected in pups from immune dams. Euthymic pups from immune dams appeared not to produce endogenous antibody to RV after virus challenge, whereas euthymic pups from non-immune dams produced high-titered RV immune serum. Pups of both genotypes given immune serum prior to or with RV were fully protected from disease and persistent infection, whereas pups given immune serum 24 hours after RV were partially protected. These studies show that RV antibody offers significant protection against lethal and persistent RV infection.