Diane J. Gaertner

Persistent rat parvovirus infection in individually housed rats

SummaryThe 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.

The pathogenesis of rat virus infection in infant and juvenile rats after oronasal inoculation

SummaryThe pathogenesis of rat virus (RV) infection was studied in random-bred Sprague-Dawley rats after oronasal inoculation of a recent RV isolate designated RV-Yale (RV-Y). RV-Y was pathogenic for rats inoculated as infants (2 days) whereas rats inoculated as juveniles (4 weeks) had asymptomatic infection and no lesions. Rats inoculated as infants developed pantropic infection accompanied by hepatic necrosis, granuloprival cerebellar hypoplasia and hemorrhagic encephalopathy. Virological and serological studies showed that virus could persist in inoculated rats for at least 35 days and for at least 28 days after seroconversion was first detected. Immuno-histochemical results indicated that RV-Y infects tissues conducive to virus excretion including kidney and lung. RV-Y also was found in genital tissues of some rats. Athymic juvenile rats inoculated intraperitoneally with RV-Y had a poor humoral immune response and harbored infectious virus for at least 3 weeks, whereas infection in euthymic control rats was detected for 1 week. These studies indicate that RV-Y can persists in the presence of humoral immunity and suggest that transmission of infection could occur for a substantial period after seroconversion. They also suggest that immunodeficient rats have increased susceptibility to persistent infection.

Persistence of rat virus in seropositive rats as detected by explant culture

SummaryRat virus (RV) was detected by explant culture for up to 14 weeks in rats inoculated as infants and for up to 7 weeks in rats inoculated as juveniles, although both groups were seropositve by 3 weeks post-inoculation.

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.

Persistence of rat parvovirus in athymic rats

SummaryEuthymic (SD or outbred rnu/+) and athymic (rnu/rnu) rats were inoculated oronasally or intraperitoneally with the RV-Y strain of rat virus when they were 2 days or 4 weeks old. Clinical signs of infection in athymic infants were similar to those in euthymic infants, but significantly more athymic infants died. Some infants developed anemia and thrombocytopenia. After inoculation of infants, RV-Y was detected in surviving euthymic rats for 7 weeks and in surviving athymic rats for at least 10 weeks. After oronasal inoculation of 4 week-old rats no clinical illness was observed. RV-Y persisted less than 6 weeks in juvenile euthymic rats but at least 12 weeks in athymic juvenile rats. Intraperitoneal inoculation of juveniles resulted in infection for at least 6 weeks. The antibody response of athymic rats to RV-Y was significantly reduced compared to that of euthymic rats. These studies indicate that T cell deficiency increases the severity and duration of RV infection and imply that T cells are required for the full expression of resistance to RV infection. They also suggest that RV-Y induced anemia could serve as a model for human parvovirus-induced anemia.

Modulation of lethal and persistent rat parvovirus infection by antibody

SummaryTwo 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.

Efficient induction of persistent and prenatal parvovirus infection in rats.

Parvoviruses are prevalent and disruptive infectious agents of laboratory rats. Risks to rat-based research from infection are increased by the persistence of virus in immune rats and by prenatal transmission of infection. The mechanisms leading to viral persistence and prenatal infection are poorly understood and have been difficult to study for lack of reliable and humane induction methods. We report here protocols for inducing persistent and prenatal infection without causing clinical disease using the UMass strain of rat virus (RV), a common rat parvovirus. Infant rats inoculated by the oronasal route at 6 days of age had greater than 90% prevalence of persistent infection. RV-UMass also induced intrauterine infection in pregnant rats inoculated by the oronasal route. Inoculation of dams at gestation day 9 frequently caused severe disease in the fetuses whereas inoculation at gestation day 12 caused primarily asymptomatic fetal infection that persisted post partum RV-UMass infection facilitates study of parvoviralhost interactions that are relevant to laboratory rats and which also may improve understanding of persistent and prenatal human parvovirus infection.

Development and optimization of plaque assays for rat coronaviruses.

Abstract 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), Parkers 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.

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

SummaryCell lines of rodent origin were tested for susceptibility to infection with rat coronavirus (RCV), including sialodacryoadenitis virus (SDAV) and Parkers 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.

Optimization of in vitro growth conditions for enterotropic murine coronavirus strains

Abstract Enterotropic mouse hepatitis virus (MHV) strains have been difficult to grow in cell culture. In an attempt to develop an efficient in vitro cultivation system for enterotropic MHV strains (MHV-RI and MHV-Y), 8 murine cell lines were inoculated with MHV-RI- or MHV-Y-infected infant mouse intestinal homogenates and screened for the production of cytopathic effects. MHV-RI and MHV-Y consistently produced cytopathic effects only in J774A.1 cells. Both strains produced titers of 106 TCID50/ml in subsequent passages in J774.1 cells. MHV strains -1, -3, -A59, -JHM, -S and -DVIM also produced high-titer viral stocks in J774A.1 cells. Therefore J774A.1 cells are the first cells found that support the replication of these 8 enterotropic and respiratory MHV strains. After passage in J774A.1 cells, MHV-RI and MHV-Y could infect previously non-susceptible cell lines (17C1-1, CMT-93, N18 and NCTC 1469), though cytopathic effects were often negligible with MHV-RI. MHV-Y, but not MHV-RI, grew in L2(Percy) cells. Using L2(Percy) cells, an agarose overlay and Giemsa staining, MHV-Y could be quantified by plaque assay. Infant mouse bioassays, plaque assays and cell culture infections were compared for their sensitivity in detecting MHV-Y in infected intestinal homogenates and cell supernatants.