A cell culture immunofluorescence (CCIF) assay was optimized for detection of porcine pararotavirus (group C rotavirus) in intestinal contents. The greatest viral infectivity was observed when MAi04 cells (5 days after subculturing) were rinsed and refed in serum-free medium before inoculation, pancreatin was added to the inocula, and the inocula were centrifuged onto the cells. Gentamicin treatment of pararotavirus samples to reduce bacterial contamination also reduced the viral infectivity of these samples for MA104 cells. An indirect CCIF assay was used to determine the prevalence of pararotavirus and rotavirus antibodies in pig sera. In pigs from four herds, pararotavirus antibodies were detected in 100% (68 of 68) of adults and 59% (24 of 41) of weanling pigs, while 86% (24 of 28) of nursing pigs from 12 herds had pararotavirus antibodies. The physicochemical properties of pararotavirus were examined and compared with those of group A rotaviruses by using the CCIF assay to quantitate in vitro changes in viral infectivity. Pararotavirus was inactivated (-99% reduction in titer) by heating to 56°C for 30 min, was slightly labile at pH 3 (16 to 34% reduction in titer), and was stable at pH 5 (0 to 17% reduction in titer) and in ether (3 to 19% reduction in titer). One group A rotavirus (Gottfried strain) was stable at 56°C (0% reduction in titer), whereas the OSU strain of group A rotavirus was inactivated at this temperature (99% reduction in titer).

The Cowden strain of porcine group C rotavirus (pararotavirus) was adapted to serial passage in a continuous monkey kidney cell line (MA104). Key factors in its successful adaptation included use of virus passaged in primary porcine kidney celis as the initial inoculum, use of roller tubes, and addition of pancreatin to the maintenance medium. A cell culture immunofluorescence test was used to quantitate the virus at each passage level, since a possible cytopathic effect was obscured by the effects of pancreatin. The virus titers dropped after initial passage into MA104 cells but increased thereafter, with peak titers evident after 16 passages (107 immunofluorescence U/ml). Immune electron microscopy and genome electropherotyping were used to identify group C rotàvirus particles and confirm group C rotavirus double-stranded RNA gel migration patterns, respectively, from infected cell culture supernatants. The electropherotype of the cell culturepropagated group C rotavirus was identical to that of the gut virulent virus from which it was derived. The cell culture-passaged group C rotavirus also retained its infectivity-for gnotobiotic pigs. No group A rotavirus was detected in the intestinal contents of the pigs or in cell culture fluids from group C rotavirus-inoculated monolayers with the two former techniques or the cell culture immunofluorescence test. This is the first verified report of serial propagation of a non-group A rotavirus in a continuous cell line.

A porcine group C rotavirus was adapted to serial propagation in roller tube cultures of primary porcine kidney cells with high concentrations of pancreatin. Infected cells were identified by immunofluorescence staining of cell monolayers. Only group C rotavirus particles were observed in culture supernatants by immune electron microscopy. Group C rotaviruses, also known as pararotaviruses (PaRV), are morphologically identical to but antigenically distinct from group A rotaviruses (19). PaRV were first detected in swine in 1980 (18) and subsequently verified as a cause of gastroenteritis in swine (5). Rotaviruses with electropherotypes similar to those of PaRV have been identified in humans (2, 7, 8, 15, 16), and several of these have subsequently been verified as serogroup C rotaviruses on the basis of antigenic similarities to the Cowden strain of porcine group C rotavirus (6). Further studies of PaRV have been hindered by the inability to serially propagate these viruses in cell cultures (19). In contrast, most group A rotavirus isolates can be routinely propagated in cell cultures by using proteolytic enzymes or roller tube culture techniques or both (4, 11, 13, 21, 24). Most attempts to serially propagate atypical rotaviruses in cell cultures by similar techniques have failed (19). Only one isolate of avian group D rotavirus has been successfully serially propagated in primary cell cultures of chicken embryo liver cells (14). Conditions for the adaptation of porcine PaRV to serial propagation in primary porcine kidney (PPK) cell cultures are described in this paper.