Cell Culture Immunofluorescence Research Articles

Detection of porcine respiratory coronavirus and transmissible gastroenteritis virus by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) for the detection of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) was developed. A bovine TGEV-specific polyclonal antibody was purified by affinity chromatography with the TRIO Bioprocessing System and was used as the capture antibody, at a concentration of 1.5 μg/well. The F5.39 monoclonal antibody was obtained by the fusion of spleen lymphocytes from TGEV immunized mice with NS-1 myeloma cells. This mAb was used as a second antibody for the ELISA to detect TGEV in field cases, 53 intestinal samples were taken from pigs exhibiting clinical signs of transmissible gastroenteritis. All the positive samples detected by the ELISA were confirmed as positive by immunofluorescence or cell culture immunofluorescence. To study the ability of this ELISA to detect PRCV in nasal swabs and lung samples, 20 seven-day-old piglets were inoculated with a Quebec strain of PRCV. The ELISA was able to detect PRCV in both kinds of samples.

Characterization of the neutralizing epitopes of VP7 of the Gottfried strain of porcine rotavirus

The neutralization epitopes of the outer capsid protein VP7 of a porcine group A rotavirus were studied by using neutralizing monoclonal antibodies (N-MAbs). Six N-MAbs which were specific for the VP7 protein of the Gottfried strain of porcine rotavirus (serotype G4) were used for analyzing the antigenic sites of VP7. Three different approaches were used for this analysis: testing the serological reactivity of each N-MAb against different G serotypes of human and animal rotaviruses, analyzing N-MAb-resistant viral antigenic variants, and performing a nucleotide sequence analysis of the VP7 gene of each of the viral antigenic variants generated. From the serological analyses, three different reactivity patterns were recognized by plaque reduction virus neutralization and cell culture immunofluorescence tests. A single MAb (RG36H9) reacted with animal rotavirus serotypes G3 and G4 but not with human serotypes G3 and G4. The MAb 57/8 (D. A. Benfield, E. A. Nelson, and Y. Hoshino, p. 111, in Abstr. VIIth Internat. Congr. Virol., 1987, and E. R. Mackow, R. D. Shaw, S. M. Matsui, P. T. Vo, D. A. Benfield, and H. B. Greenberg, Virology 165:511-517, 1988) reacted with animal and human rotavirus serotypes G3 and G4 and also with human serotype G9 and bovine serotype G6. The other four MAbs reacted only with the porcine rotavirus serotype G4. The epitope defined by MAb 57/8 and the epitope defined by the other five MAbs appeared to be partially overlapping or close to each other, as identified by viral antigenic variant analysis. However, data from nucleotide and deduced amino acid sequence analyses of the VP7 of each of the viral antigenic variants showed that these two epitopes constituted a large, single neutralization domain.

Production and characterization of monoclonal antibodies to porcine group C rotaviruses cross-reactive with group A rotaviruses

Five monoclonal antibodies (MAbs) to porcine group (gp) C rotaviruses (Cowden and Ah strains) reactive with both gp A and C rotaviruses in cell culture immunofluorescence (CCIF) tests were produced and characterized. These MAbs reacted with three strains of gp A and two strains of gp C rotaviruses in a CCIF test and were classified into two groups based on their CCIF titers. The MAbs also reacted to various degrees with cell-culture-propagated porcine gp C rotavirus (Cowden) and bovine gp A rotavirus (NCDV) in an enzyme-linked immunosorbent assay by using the MAbs as capture antibodies. Fecal samples containing human, bovine, and porcine strains of gp A and C rotaviruses were positive when tested using one of the MAbs in this assay. The MAbs recognized VP6 of gp A rotavirus and the VP6 counterpart (41-kDa protein) of gp C rotavirus in a Western blot assay. Results of competitive binding assays on four MAbs indicated that gp A and gp C rotaviruses share three overlapping epitopes within a single antigenic domain. These results suggest that gp A and C rotaviruses share a common antigen located on the VP6 protein, which is recognized by certain MAbs in various serologic assays.

Prevalence of bovine group A rotavirus shedding among dairy calves in Ohio

Summary Fecal samples were collected from 450 neonatal calves ranging from 1 to 30 days old, between May, 1988 and May, 1989 to estimate the prevalence of bovine group A rotavirus in a stratified random sample of Ohio daily herds. Calves were from 47 dairy herds chosen to be representative of Ohio herds. Bovine group A rotavirus was detected in fecal samples by a cell culture immunofluorescence test (ccif) and elisa. Of 450 samples tested, 46 (10%) were positive by ccif and 67 (15%) were positive by elisa. The agreement beyond chance between the 2 assays was good (kappa = 0.65). The overall prevalence rate of rotavirus shedding was 16.4% (74/450). Forty-three percent (29/67) of the samples positive by elisa were subgroup 1, none were subgroup 2, and the remaining 57% (38/67) could not be assigned to either subgroups 1 or 2. Thirty herds (62.5%) had at least 1 group A rotavirus-positive calf (mean number of samples per positive herd = 12.4), and 17 herds (37.5%) had no rotavirus-positive calves (mean number of samples per negative herd = 6.0). A live oral rotacoronavirus vaccine was used in neonatal calves of only 1 herd and 3 of 17 (17.6%) calves from this herd were positive for group A rotavirus. The percentage of the rotavirus-positive fecal samples from all calves (n = 450) when stratified by fecal consistency was as follows: 28.3% (13/46) had liquid feces; 25.6% (10/39) had semiliquid feces; 23.4% (22/94) had pasty feces; and 10.7% (29/271) had firm feces. Of the rotavirus-positive calves (n = 74), 17.6% (13/74) had liquid feces; 13.5% (10/74) had semiliquid feces; 29.7% (22/74) had pasty feces; and 39.2% (29/74) had firm feces. The average age of calves shedding rotavirus was 14 days (range, 1 to 30 days). Double-stranded (ds) rna extracted from 36 samples positive by 1 or both tests was examined by polyacrylamide gel electrophoresis. All samples positive by this technique (30/36) had long dsrna migration patterns, typical of group A rotaviruses, including samples from calves in the herd in which the oral vaccine was used. Moreover, the electrophoretic migration pattern of group A rotavirus dsrna in these vaccinated calves differed from that of the rotavirus vaccine strain, suggesting the rotavirus strain circulating in this herd was not the vaccine strain. All samples negative by ccif or elisa that had volumes > 5 ml (n = 323) were also subjected to dsrna extraction and polyacrylamide gel electrophoresis for detection of additional group A or nongroup A rotaviruses; none of them were positive by this technique.

Production and Characterization of Monoclonal Antibodies against an Avian Group A Rotavirus

Fifteen monoclonal antibodies (MAbs) against an avian group A rotavirus were cloned and characterized. Eight of the 15 MAbs had neutralizing activity (N-MAbs). Five of the N-MAbs (1G1, 5B8, 4E2, 3G1, 2E3) were VP4-specific by radioimmunoprecipitation assay (RIPA), and two N-MAbs (2D11, 6E8) were possibly VP7-specific (faint bands by RIPA). One N-MAb (4H12) of undefined protein specificity cross-reacted with serotype 3 simian rotaviruses. The other seven N-MAbs did not cross-react with any of the eight distinct serotypes of human and mammalian rotaviruses tested. Of the seven non-neutralizing MAbs, three were VP6-specific (3H10, 4B12, 5F6), two were VP8-specific (6C9, 1D1), one was VP4-specific (4E9), and one was of undefined protein specificity (1B11). Four non-neutralizing MAbs recognized only avian group A rotavirus in cell-culture immunofluorescence tests (6C9, 1D1, 4E9 and 5F6), whereas two MAbs (3H10 and 4B12) cross-reacted with all human and animal rotaviruses tested. The MAb 1B11 did not recognize any human rotavirus serotypes but cross-reacted with all nonhuman animal rotavirus serotypes. The MAbs produced in this study should be useful for the detection and further characterization of avian group A rotaviruses.

Detection of phocid distemper virus RNA in seal tissues using slot hybridization and the polymerase chain reaction amplification assay: genetic evidence that the virus is distinct from canine distemper virus

Slot hybridization and the polymerase chain reaction (PCR) after reverse transcription (RT) were used to detect RNA extracted from tissues of seals after naturally occurring disease and experimental infection with phocid distemper virus (PDV). A phosphoprotein (P) gene-specific cDNA served as a probe for both slot hybridization and the identification of PCR-generated fragments by Southern blotting. As primers for the PCR assay PDV P gene-derived oligonucleotides were used. Hybridization, PCR and partial nucleic acid sequence analysis clearly demonstrated that PDV is a distinct virus (most closely related to canine distemper virus) within the morbillivirus group. PCR, when combined with Southern blot hybridization, was clearly superior to slot hybridization and more sensitive than cell culture isolation and immunofluorescence assays for the detection of virus in tissues. Considerable amounts of viral RNA could be demonstrated in the lungs and spleens. In experimentally infected animals a large quantity of virus-specific RNA was additionally found in colon samples. Using RT-PCR in combination with Southern blotting. PDV could be demonstrated in buffy coat cells using a simple and fast cell lysis procedure.

Comparison of Directigen FLU-A with viral isolation and direct immunofluorescence for the rapid detection and identification of influenza A virus

Directigen FLU-A, an enzyme immunoassay membrane test, was compared prospectively to isolation in cell culture and direct immunofluorescence (IF) for the detection of influenza A virus. One hundred ninety specimens were evaluated by Directigen FLU-A and cell culture; 184 of these specimens were also tested by direct IF. The sensitivity of Directigen FLU-A compared to isolation in cell culture and direct IF was 100%. The specificities of Directigen FLU-A compared to isolation and direct IF were identical, 91.6%. Fourteen specimens that were positive by Directigen FLU-A did not yield virus in culture; two of the specimens, however, were positive by direct IF, and four other specimens were not specimens of choice for the test. A positive Directigen result had positive predictive values of 62.6 and 75.0% compared to isolation and direct IF, respectively; a positive Directigen result with an intensity reading of 2+ or greater, however, had positive predictive values of 85 and 100% compared to isolation and direct IF, respectively. In all comparisons, the negative predictive value was 100%. There was no evidence that cross-reactivity occurred with non-influenza A antigens. Directigen FLU-A should serve as a convenient screening test for influenza A and as a rapid test supported by isolation in cell culture during an influenza outbreak.

Effect of malnutrition on extraintestinal spread of rotavirus and development of hepatitis in mice

The effects of malnutrition on the extramucosal spread and severity of the clinical disease were examined in suckling BALB/c mice infected with heterologous rhesus rotavirus (RRV). Control animals were inoculated with homologous murine rotavirus (MRV). Following oral infection with RRV, 100% of malnourished and 90% of normally nourished mice exhibited watery diarrhea within 48 hours. The clinical symptoms appeared more severe and of longer duration in the malnourished group. At 7 to 14 days post-inoculation, 30 of 79 (38%) malnourished and 19 of 92 (21%) well nourished mice developed icterus, clay-colored feces and dark yellow urine. The mortality rates were 9% and 0%, respectively. Light microscopy revealed an acute hepatitis with foci of necrosis. Rotavirus was detected in liver tissue by cell culture infectivity, immunofluorescence assay and electron microscopy. Homologous MRV infection resulted in diarrhea in all animals and with increased severity in the malnourished group. Significantly, however, none of the control animals developed hepatitis and no virus could be detected in the liver. These results suggest that malnutrition may exert significant influence on the severity of rotavirus infection and on the extent of extramucosal spread of RRV. Since several heterologous rotavirus strains are currently being tested in human vaccine trials, the implications of our observations suggest that special attention should be paid to malnourished infants and that screening of hepatic function should be included in vaccine protocols.

Etiologic, Clinical, and Pathologic Analysis of 31 Fatal Cases of Acute Respiratory Tract Infection in Argentinian Children Under 5 Years of Age

During a 3-year survey of 805 children with acute lower respiratory tract infection (ALRI) who were admitted to three hospitals in Buenos Aires, 31 fatal cases were recorded--a fatality rate of 3.8%. Of the 31 children who died, 77% were less than 1 year of age, 48% were boys, 58% were malnourished, 29% had previous respiratory disease, and 22% had previous congenital disease. All children who died had clinical diagnoses of pneumonia (71%) or bronchiolitis (29%). Autopsies were performed in 14 of the cases. Viral etiology was determined by both cell culture and indirect immunofluorescence (IIF) assay of either nasopharyngeal aspirates (NPA) or lung tissue and bacterial etiology was determined by isolation of organisms from blood, lung tissue, and/or pleural fluid. NPA was examined for Bordetella pertussis by IIF. Pathogens were identified in 65% of fatal cases. Seven cases were bacterial; seven cases were viral; and six cases resulted from mixed infections. Lung tissue yielded positive etiologic results in 10 of 13 cases. Histopathologic examination performed on specimens from the 14 autopsied children revealed necrotizing bronchiolitis with intranuclear inclusions (n = 5) and multifocal pneumonia (n = 9).

Infection of nonlymphoid cells by human immunodeficiency virus type 1 or type 2

Human epithelial cells (L132) derived from embryonic lung and human lung fibroblasts (MRC5) were infected by human immunodeficiency virus type 1 (HIV-1) or type 2 (HIV-2). Surface CD4 protein was detected on these cells, and recombinant soluble CD4 (sCD4) blocked infection, indicating that HIV infection was mediated by the cell surface CD4 protein. In contrast, infection of human primary chondrocyte cells (C23), synovial cells (HSA), and foreskin fibroblasts (F13) was apparently independent of cell CD4-mediated mechanisms. Surface CD4 protein could not be detected on these cells, and sCD4 did not block the infection. F13 cells could be infected only by HIV-2, not by HIV-1, under our experimental conditions. In cells of mesenchymal orgin, viral production could be detected only after cocultivation with the human T-lymphoid H9 cells but not by conventional viral assays, including reverse transcriptase and p24 antigen assays in cell culture supernatant and immunofluorescence of host cells. Our DNA transfection studies indicated that this lack of detectable viral production was not due to the inefficient use of the HIV long terminal repeat or the Tat protein in these cells. These mesenchymal and epithelial cells were susceptible to HIV infection but differed in mechanism of virus entry compared with hematopoietic cells such as T lymphocytes. These observations may provide insights into clinical syndromes such as lung dysfunction in HIV-infected newborns and connective tissue disorders in HIV-infected adults.

Comparison of directigen RSV with viral isolation and direct immunofluorescence for the identification of respiratory syncytial virus

An enzyme immunoassay membrane test (Directigen RSV) for the detection of respiratory syncytial virus in clinical specimens was compared prospectively with isolation in cell culture and direct immunofluorescence (IF). A total of 315 nasopharyngeal wash specimens from pediatric patients were examined. Directigen RSV was 86.1% sensitive and 91.3% specific for specimens positive by isolation in cell culture and/or IF, with 88.6% agreement. The false-positive rate was 16%; 2 of 20 specimens giving false-positive reactions by Directigen RSV were true-positives by blocking assay. Twenty-seven specimens (8.5%) whose results were initially uninterpretable by Directigen RSV due to filtration difficulties were diluted and upon retesting produced acceptable results. Sixty-three viral isolates and/or IF identifications of virus antigens representing seven virus groups other than respiratory syncytial virus were also found; cross-reactions between Directigen RSV and other viruses were not observed. Directigen RSV will be useful as an immediate procedure and in facilities lacking a comprehensive virology laboratory.

Extramucosal spread and development of hepatitis in immunodeficient and normal mice infected with rhesus rotavirus

The pathogenic profiles of two heterologous animal rotaviruses, rhesus rotavirus strain MMU 18006 and bovine rotavirus strain WC3, were evaluated in mice with severe combined immunodeficiency (SCID mice) and normal BALB/c mice. Control animals were inoculated with homologous murine strain EDIM 5099 or a tissue culture-adapted murine rotavirus. Heterologous infection with rhesus rotavirus resulted in hepatitis in 84% of SCID and 21% of BALB/c mice, with mortality rates of 27 and 0%, respectively. Surviving SCID animals developed chronic liver disease, while symptoms in BALB/c mice resolved in 2 to 4 weeks after onset. Histopathologic examination revealed a diffuse hepatitis with focal areas of parenchymal necrosis. Rotavirus was detected in liver tissue from 100% of 29 SCID and 85% (11 of 13) BALB/c animals tested by cell culture infectivity, immunofluorescence, or electron microscopy. No extramucosal spread of virus or hepatitis was observed after infection with heterologous bovine strain WC3 or homologous murine rotaviruses. This finding of a novel rotavirus-induced disease manifestation suggests altered tissue tropism in a heterologous host for a group of viruses previously shown to replicate exclusively in the gut mucosa. The implications of our observations suggest that in human vaccine trials utilizing heterologous rotavirus strains, special attention should be paid to children with immunodeficiency disorders, and screening for hepatic function should be included in vaccine protocols.

Detection of Aujeszky's disease virus in nasal cells of fattening pigs by immunoassay

Both conventional and specific pathogen free pigs were inoculated intranasally with a strain of Aujeszky's disease virus (ADV). Nasal cells were collected daily by swab, aspiration or wash. The nasal cells were examined for ADV by isolation on cell culture, direct or indirect immunofluorescence and immunoperoxidase staining by monoclonal antibodies. The infected pigs were studied for nasal shedding of infected cells until 30 days after infection. The study was also extended to naturally infected farm pigs. Swabbing, washing and aspiration proved effective methods of collecting between 10(5) and 10(8) pavement or columnar epithelial cells and non-epithelial cells. Macrophages and polymorphonuclear leucocytes were also identified. Infected nasal cells were detected by immunofluorescence and immunoperoxidase from one to 21 days after infection. The viral antigen was detected in both epithelial and non-epithelial cells, the fluorescence was nuclear and, or, 'cytoplasmic', in the latter case only the cell membrane was stained. ADV antigens were detected in nasal cavity cells in pigs infected with a virulent and a hypovirulent strain. Nasal swabs proved effective in confirming infection both by virus isolation and immunological assay, and the latter was shown to be a useful experimental tool for the rapid diagnosis of Aujeszky's disease virus infection in fattening pigs suffering from acute respiratory distress.

Bacampicillin to treat non-gonococcal urethritis in men: pilot study.

Of 84 men with non-gonococcal urethritis (NGU), 24 yielded Chlamydia trachomatis by either cell culture or MicroTrak immunofluorescence test. All 84 were treated with bacampicillin 800 mg twice a day for seven days. Five (one chlamydia positive) defaulted from follow up 10 to 14 days after the start of treatment. Of the 23 chlamydia positive patients who attended follow up, 22 became chlamydia negative; 14 of the 23 patients also became asymptomatic and had normal urethral smears. Of the 56 chlamydia negative patients who attended follow up, 21 were cleared of their urethritis. Two patients reported side effects; one drowsiness and one mild diffuse alopecia. Bacampicillin may therefore be a safe and effective alternative to tetracycline or erythromycin in treating chlamydial urethritis in men.

Comparison of DNA-spot hybridization, cell culture and direct immunofluorescence staining for the diagnosis of avian chlamydiae

DNA-spot hybridization, cell culture and direct immunofluoresence staining were compared for the detection of avian Chlamydia psittaci strains in cell culture dilutions and in routine samples submitted for diagnosis. With dilutions of infected cell culture material, growth in BGM cells was by far the most sensitive technique, detecting 0.01 infected cells (20 elementary bodies) ml −1. DNA-spot hybridization and direct immunofluorescence staining were of approximately equal sensitivity, both detecting 16 infected cells (3.2×10 4 elementary bodies) per ml −1. When 27 avian liver and spleen samples were assayed, all 3 tests performed similarly (13 positive and 12 negative by all 3 tests). This suggests that in most avian samples presented for diagnosis, sufficient numbers of chlamydiae are present to allow any of the test to the be used. Thus, the direct immunofluorescence staining method is currently the test of choice for routine diagnosis since it is available in kit form, is relatively simple and quick to perform, and like DNA-spot hybridization, detects non-viable as well as viable organisms. However, if low levels of chlamydiae are to be effectively detected, such as in carrier birds or birds with recently acquired infections, then cell culture should be used.

Serial propagation of porcine group C rotavirus (pararotavirus) in a continuous cell line and characterization of the passaged virus.

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 cells 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 (10(7) immunofluorescence U/ml). Immune electron microscopy and genome electropherotyping were used to identify group C rotavirus particles and confirm group C rotavirus double-stranded RNA gel migration patterns, respectively, from infected cell culture supernatants. The electropherotype of the cell culture-propagated 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.

Serial propagation of porcine enteric calicivirus-like virus in primary porcine kidney cell cultures.

A porcine enteric calicivirus-like virus was adapted to serial propagation in primary porcine kidney cell cultures. Attempts to propagate this virus in primary porcine kidney cells in the presence of trypsin or pancreatin or without medium supplementation were unsuccessful. A low-pH medium (pH 6.8) was also ineffective in virus propagation. Successful serial propagation of the virus required the presence of an intestinal-content preparation, derived from uninfected gnotobiotic pigs, in the cell culture medium. The best results were obtained with six-well plate cultures which were centrifuged after virus inoculation. Infected cells were detected by immunofluorescent staining of cell monolayers or detached cells which were harvested by centrifugation. Infected cells were first detected at passage 4 (1% infected cells), and infectivity increased with successive passages, with as many as 80% of the cells infected by passage 16. Extensive cytopathic effects were observed in inoculated cell cultures, but not in uninoculated control cell cultures, at each passage level after passage 13. The infected cells became separated, rounded, and detached, forming holes in the cell monolayer. Only virus particles exhibiting the six-pointed star appearance or stain-filled, cup-shaped depressions characteristic of caliciviruses were detected in inoculated cell culture supernatants by immune electron microscopy. Attempts to determine the titer of the virus by a cell culture immunofluorescence assay or plaque assay were unsuccessful.

Physicochemical characterization of porcine pararotavirus and detection of virus and viral antibodies using cell culture immunofluorescence

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 MA104 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 (greater than or equal to 99% reduction in titer) by heating to 56 degrees 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 either (3 to 19% reduction in titer). One group A rotavirus (Gottfried strain) was stable at 56 degrees C (0% reduction in titer), whereas the OSU strain of group A rotavirus was inactivated at this temperature (99% reduction in titer).

Isolation and serotyping of porcine rotaviruses and antigenic comparison with other rotaviruses

Seven rotavirus strains were isolated in cell cultures from the intestinal contents of piglets with diarrhea. MA104 cells with pancreatin in the cell culture medium was the host system of choice for virus isolation and replication. A cell culture immunofluorescence test in which MA104 cells were used in microtiter plates was very effective for detecting and assaying rotaviruses. A plaque reduction neutralization test, cross-protection studies in gnotobiotic pigs, and electrophoresis of rotaviral double-stranded RNA were used for comparing viruses. Three strains produced plaques on initial isolation attempts, replicated well in cell cultures, and were antigenically very similar. We suggest that these three strains be considered porcine rotavirus serotype 1, with The Ohio State University (OSU) strain serving as the prototype. The OSU strain was distinct from bovine, simian, canine, and human (Wa and M) rotaviruses by plaque reduction neutralization. Four strains did not produce plaques on initial isolation attempts, were difficult to adapt to cell cultures, and were related to each other but were distinct from the serotype 1 strains. We suggest that the Gottfried (G) strain be tentatively considered as a prototype for porcine rotavirus serotype 2. The G strain was antigenically closely related to canine and simian rotaviruses and less so to human M rotavirus (human rotavirus serotype 3). Canine, simian, and human M rotaviruses were closely related. All seven porcine rotavirus strains caused diarrhea in gnotobiotic pigs. Cell-cultured vaccines of the OSU and G strains caused only mild or no diarrhea in gnotobiotic pigs, and protection occurred when such pigs were challenged with homologous, bur not heterologous, virulent viruses. A survey indicated that 94% of 274 porcine serum samples and 100% of 75 herds were serologically positive to the porcine OSU rotavirus.

Rotavirus serotypes by serum neutralisation.

A neutralisation test using cell culture and indirect immunofluorescence was applied to isolates of rotavirus from 55 patients with gastroenteritis, in order to determine serotypes. Frequent cross-reactions were observed, but subsequent statistical analysis confirmed the existence of at least three distinct serotypes. Some results, not sufficient for analysis, suggest that at least five serotypes probably exist. It is suggested that two or more viral polypeptides might be involved in neutralisation, one of which might be common to another serotype(s). This would explain the frequent cross-reactions detected by this neutralisation test. Evidence suggesting that some strains may be hybrids is also presented.