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.

Immune response of pregnant cows to bovine rotavirus immunization.

Fifteen pregnant Holstein cows were freely assigned to 3 experimental groups (5 cows in each group). Cows in group I were inoculated IM and intramammarily (IMm) with Ohio Agricultural Research and Development Center (OARDC) tissue culture-propagated modified-live Nebraska calf diarrhea bovine rotavirus with added adjuvant (OARDC vaccine-immunized cows). Group II cows were given IM injections of a commercial modified-live rotavirus-coronavirus vaccine (commercial vaccine-immunized cows), and the remaining 5 cows were noninoculated controls (group III). Rotavirus antibody in colostrum and milk was mainly associated with immunoglobulin (Ig)G1, and less so with IgG2, IgA, and IgM, as analyzed by the enzyme-linked immunosorbent assay (ELISA), using monospecific anti-bovine IgG1, IgG2, IgM, and IgA sera. In serum, the rotavirus antibody was distributed almost equally between IgG1 and IgG2. The same relationships appeared in both immunized and nonvaccinated cows. All OARDC vaccine-injected cows had virus-neutralization (VN) and ELISA IgG1 rotavirus antibody titers in serum and mammary secretions at significantly increased levels (at least 100-fold; P less than 0.05) compared with the titers in groups II (commercial vaccine-immunized cows) and III (controls). Serum, colostrum, and milk antibody titers from these latter 2 groups did not differ statistically. The ELISA IgG2, IgA, and IgM rotavirus antibody titers also were significantly greater in mammary secretions from OARDC vaccine-immunized cows than in groups II and III cows. There was a high correlation between ELISA IgG1 and VN rotavirus antibody titers for all samples tested (r = 0.97, P less than 0.001), but ELISA IgG1 antibody titers were consistently higher than VN titers. The ELISA IgG1 and VN antibody titers of milk samples collected from cows 30 days after parturition were higher from the OARDC vaccine-immunized cows (ELISA IgG1, geometric mean titer (GMT) = 3,511; VN GMT = 1,689) than were titers from the group II cows (ELISA IgG1 GMT = 39; VN GMT = 33) or group III cows (ELISA IgG1 GMT = 21; VN GMT = 19). These results indicate that IM plus IMm immunization of pregnant cows, using modified-live bovine rotavirus with added adjuvant, may significantly enhance serum, colostrum, and milk rotavirus antibody titers, whereas IM vaccinal inoculation of pregnant cows with a commercial modified-live rotavirus-coronavirus vaccine may not.

Passive immunity to transmissible gastroenteritis virus: intramammary viral inoculation of sows.

Sows were injected intramammarily with live-attenuated TGE virus, an enteric coronavirus--one sow during pregnancy and three sows during lactation. All sows were TGE antibody seronegative prior to inoculation except for one naturally infected sow inoculated during lactation. The animal injected during pregnancy had primarily IgG TGE antibodies in milk from all glands. By contrast, sows injected during lactation had IgA and IgM initially, and later IgA and IgG TGE antibodies in milk from injected and noninjected glands. The seropositive sow had elevated IgA TGE antibody titers in milk after IMm injection. Both seronegative sows inoculated intramammarily during lactation shed TGE virus in milk from injected glands, and their nursing piglets developed mild diarrhea and shed virus in their feces at three to nine DPE of the sows. Milk from IMm injected glands generally had higher TGE antibody titers than milk from noninjected glands. These results suggest that TGE virus replicates in lactating mammary gland tissue, thereby stimulating IgA immunocytes, leading to secretion of IgA antibodies in milk. Whether the intramammary route presents a natural route of enteric virus exposure in lactating animals (by way of infected nursing piglets), leading to IgA-antibody secretion in milk, requires further investigation.

Porcine pararotavirus: detection, differentiation from rotavirus, and pathogenesis in gnotobiotic pigs.

Some characteristics of a newly recognized porcine enteric virus are described. Tentatively, the virus was referred to as porcine pararotavirus (PaRV) because it resembled rotaviruses in respect to size, morphology, and tropism for villous enterocytes of the small intestine. However, it was antigenically distinct from porcine, human, and bovine rotaviruses and reoviruses 1, 2, and 3, and the electrophoretic migration pattern of PaRV double-stranded RNA was distinct from the electrophoretic migration patterns of the rotaviral and reoviral genomes. By passage in gnotobiotic pigs, PaRV was isolated from two suckling diarrheic pigs originating from two herds. After oral exposure of gnotobiotic pigs, villous enterocytes of the small intestines became infected as judged by immunofluorescence, resulting in villous atrophy and diarrhea. Mortality was high when gnotobiotic pigs less than 5 days old were infected. The C strain of this virus was serially passed 10 times in gnotobiotic pigs, and electron microscopy, immunofluorescence, and serological tests indicated no extraneous agents. The virus was serially passed five times in cell cultures which contained pancreatin in the medium, but replication was negligible or absent, as the number of immunofluorescent cells decreased with each passage. Since rotaviral infections are frequently diagnosed by direct electron microscopy of fecal specimens, the presence of other morphologically similar viruses, such as PaRV, should be considered. The use of immune electron microscopy is suggested as a means of helping recognize this situation.

Rapid, simple method of preparing rotaviral double-stranded ribonucleic acid for analysis by polyacrylamide gel electrophoresis.

A procedure for extracting rotaviral double-stranded ribonucleic acid (RNA) directly from fecal and intestinal specimens collected from calves and pigs is described. This procedure provides a rapid, simple, reproducible method of obtaining rotaviral double-stranded RNA preparations suitable for electrophoretic analysis in polyacrylamide-agarose composite gels. The rotaviral genome electrophoretic migration pattern produced by double-stranded RNA extracted directly from a specimen by this procedure was qualitatively identical to the electrophoretic migration pattern obtained with double-stranded RNA extracted from purified rotavirus derived from the same specimen. Direct extraction of specimens containing porcine rotavirus-like virus by this procedure gave preparations that had electrophoretic migration patterns similar, but not identical, to the characteristic electrophoretic migration pattern of the rotaviral genome. Sufficient rotaviral double-stranded RNA could be extracted from 6 ml of fecal or intestinal specimen by this procedure to permit 15 or more electrophoretic assays.

Rotavirus-like, calicivirus-like, and 23-nm virus-like particles associated with diarrhea in young pigs.

Virus particles morphologically similar to caliciviruses and rotaviruses were detected by electron microscopy (EM) in the intestinal contents of a 27-day-old diarrheic nursing pig. A third small spherical 23-nm virus-like particle was also observed. Calicivirus-like particles averaged 33 nm in diameter. Similar to rotaviruses, rotavirus-like particles were present as single-capsid 55-nm forms or double-capsid 70-nm particles. Most gnotobiotic pigs orally exposed to samples containing these three viruses developed diarrhea and villous atrophy of the small intestine, and all shed the three viruses in their intestinal contents. Attempts to propagate these viruses in cell culture were unsuccessful. The antigenic relationship of the rotavirus-like particles to known rotaviruses was explored by immune EM and immunofluorescent staining. By these techniques, the rotavirus-like particles did not cross-react with antisera to porcine, bovine, or human rotaviruses or to reovirus type 3. Antisera from gnotobiotic pigs exposed to all three viruses had enzyme-linked immunosorbent assay and virus neutralization titers of <4 against porcine rotavirus. Previous infection of gnotobiotic pigs with the mixture containing rotavirus-like particles failed to protect them against a subsequent challenge with porcine rotavirus. The antigenic relationship of the calicivirus-like particles to known caliciviruses was investigated by immune EM and virus neutralization. By these tests, the calicivirus-like particles did not react with antisera against feline calicivirus strain 255 or M-8. In a study conducted at Plum Island Animal Disease Center, antiserum against the three combined agents did not specifically neutralize any serotype of swine vesicular exanthema virus.

Porcine rotaviral infection of cell culture: effects of certain enzymes.

Porcine rotaviral infectivity for continuous porcine kidney (PK-15) cells was enhanced by incorporation of pancreatic endopeptidases into the cell culture maintenance medium. Marked enhancement of infectivity was induced by trypsin, whereas elestase and alpha-chymotrypsin enhanced infectivity to a lesser extent. Bacterial protease also induced some enhancement of porcine rotaviral infectivity. A synergistic enhancement of porcine rotaviral infectivity was noticed with trypsin and alpha-chymotrypsin combined. Porcine rotaviral infectivity was not affected by incorporation of alpha-amylase, alkaline phosphatase, beta-galactosidase, carboxypeptidase-A, deoxyribonuclease, enterokinase, lipase, or ribonuclease into the maintenance medium.

Human rotavirus type 2: cultivation in vitro.

A strain of type 2 human rotavirus (Wa) was grown to relatively high titer through 14 passages in primary cultures of African green monkey kidney (AGMK) cells. This passage series was initiated with virus that had been passaged 11 times serially in newborn gnotobiotic piglets. In contrast, virus present in the stool of patient Wa as well as virus from the first, second, or third passage in piglets could not be propagated successfully in African green monkey kidney cells. Prior to each passage in cell culture, the virus was treated with trypsin and the inoculated cultures were centrifuged at low speed. Cultivation of a type 2 human rotavirus should aid attempts to characterize this virus and to develop a means of immunoprophylaxis for a serious diarrheal disease of human infants.

Concurrent porcine rotaviral and transmissible gastroenteritis viral infections in a three-day-old conventional pig.

A 3-day-old suckling pig with diarrhea was necropsied, and immunofluorescent microscopic examination of the small intestinal mucosa, together with immune electron microscopic examination of the large intestinal contents, provided a presumptive diagnosis of a concurrent infection with transmissible gastroenteritis (TGE) virus and porcine rotavirus. Immunofluorescent microscopic, immune electron microscopic, and serologic data obtained from gnotobiotic pigs experimentally inoculated with the large intestinal contents of the suckling pig confirmed this diagnosis. Two gnotobiotic pigs, convalescent from previous TGE viral infections, became infected with porcine rotavirus only. However, another gnotobiotic pig, convalescent from a previous porcine rotaviral infection, became infected with TGE virus only, following inoculation with the large intestinal contents of the suckling pig.

Rotaviral diarrhea in pigs: brief review.

Rotavirus is a name given to a group of viruses that have similar characteristics and are generally capable of causing diarrhea in the young. Infection of pigs with porcine rotavirus is common and widespread and can result in diarrhea, especially in 1- to 4-week-old pigs. This virus is frequently associated with a diarrheal syndrome popularity known as "white scours," "milk scours," or "3-week-old scours." Pigs less than 1 week old are infrequently infected, presumably because of adequate passive immunity. The infection resembles enzootic transmissible gastroenteritis. Diagnosis can be made by immunofluorescent staining of mucosal scrappings from the small intestines.

Intrafetal inoculation of swine with transmissible gastroenteritis virus.

Fetuses in 3 sows were inoculated (intramuscularly) with transmissible gastroenteritis (TGE) virus on 95th, 77th, and 74th days of the gestation. At 15, 14, and 37 days later (or days when pigs were obtained by hysterectomy), there was evidence of intestinal localization of virus, with villous atrophy and subsequent repair. All intrafetal-inoculated pigs became serologic-positive for TGE. A noninoculated pig shown to be seropositive for TGE at 15 days of age (after hysterectomy) was resistant to challenge exposure with virulent TGE virus given on the 32nd day, in contrast to 3 seronegative littermates that developed typical disease when challenge exposed.

Morphogenesis of porcine rotavirus in porcine kidney cell cultures and intestinal epithelial cells.

The morphogenesis of porcine rotavirus was similar in vitro in porcine kidney (PK) cell cultures and in vivo in porcine epithelial cells as examined by electron microscopy. Infected cells contained cytoplasmic, non-membrane-bound viroplasm and accumulations of virus particles within cisternae of the rough endoplasmic reticulum (RER). Three types of virus particles were noted: double-shelled or complete particles which averaged 77 nm in diam.; single-shelled or naked particles which ranged from 50 to 55 nm in diam.; and electron-dense nucleoids, or cores, 31 to 38 nm in diam. Virus particles acquired outer shells by budding through either matrices of granular, electron-dense viroplasm or membranes of distended RER. Accumulation of numerous single-shelled particles was observed only in PK cell cultures containing a high percentage of infected cells. In these cells, virus release occurred through disruption of the plasma membrane. Tubules, similar in diameter to the single-shelled particles, were observed in the nuclei of a few infected PK cells.

Rotavirus as a cause of diarrhea in pigs.

A rotavirus (reovirus-like agent) was associated with diarrheal diseases occurring in 1- to 4-week-old suckling pigs in 8 herds and in weaned pigs in 2 herds. Transmissible gastroenteritis virus was also detected in 2 of these herds, as was enteropathogenic Escherichia coli in 5 herds. Morbidity was generally greater than 80% in pigs of the affected age group within these herds, and mortality from diarrhea ranged from 7 to 20%. The disease due to rotavirus in suckling pigs appeared similar to the syndrome commonly referred to as milk scours, white scours, or 3-week scours. Diarrhea and villous atrophy, resembling that seen in transmissible gastroenteritis, occurred in naturally infected pigs and in gnotobiotic pigs experimentally infected with rotavirus. Diagnosis was accomplished by immune electron microscopy of intestinal contents and by immunofluorescent staining of enterocytes. A massive infection of enterocytes with rotavirus was demonstrated by immunofluorescence, which helps explain the pathogenesis of this disease. The apparent rarity of clinical rotaviral infections in suckling pigs greater than 7 days old is probably due to the acquisition of passive immunity from immune sows.

Pathogenesis of porcine rotaviral infection in experimentally inoculated gnotobiotic pigs.

Porcine rotavirus was shown to infect gnotobiotic pigs and induce an acute enteric disease clinically characterized by diarrhea, anorexia, depression, and occasional vomition. Onset of clinical signs correlated closely with the appearance of lesions within the small intestinal mucosa, and recovery from infection was associated with the regeneration of normal, functional villous epithelium. Villous atrophy, especially in the caudal two-thirds of the small intestine, was the consistent lesion observed in pigs with clinical signs of rotaviral infection. Villi were often short, blunt, and covered with cuboidal epithelial cells. Immunofluorescent microscopy methods demonstrated that the principal site of rotaviral replication was the villous columnar epithelial cells in the small intestine.

Cell culture propagation of porcine rotavirus (reovirus-like agent).

Two isolates of porcine rotavirus (reovirus-like agent) were isolated and passaged in primary procine kidney cell cultures. Viral infectivity for cells was monitored by immunofluorescence because viral cytopathic effect was moderate. Successful passage of virus in cell culture required that viral suspensions obtained from infected cell cultures be treated with pancreatin prior to inoculation onto cell monolayers. Porcine rotavirus passage in cell culture also was accomplished, using trypsin treatments in lieu of pancreatin treatments. Porcine rotavirus passaged 10 times in cell culture infected gnotobiotic pigs and caused diarrhea. Gnotobiotic pigs that recovered from this infection were resistant to challenge exposure with porcine rotavirus but were susceptible to challenge exposure with transmissible gastroenteritis virus. As determined by immunofluorescent cross reactions, porcine rotavirus was found to be antigenically related to the human and bovine rotaviruses but not to reovirus type 3 or to transmissible gastroenteritis virus.

Immunoglobulin classes of antibodies in milk of swine after intranasal exposure to pseudorabies virus or transmissible gastroenteritis virus.

Experiments were conducted to evaluate whether infection of the respiratory tract of pregnant swine with pseudorabies (Pr) virus would induce the secretion of immunoglobulin A (IgA) antibodies in their milk as was observed after enteric infection with transmissible gastroenteritis (TGE) virus. The immune response of sows to Pr virus inoculated intranasally and to TGE virus inoculated orally/intranasally or via a natural infection was studied. Emphasis was placed upon titers and Ig classes of Pr and TGE virus-neutralizing antibodies in colostrum and milk. All animals exposed to Pr virus (alone or in combination with TGE virus) developed Pr-neutralizing antibody titers in both serum and milk. Pr antibody titers were generally higher in colostrum than in serum, but the opposite was true in milk compared with serum, with milk titers declining markedly during lactation. In contrast, TGE antibody titers in milk from experimentally or naturally infected sows usually remained higher than the corresponding serum titers and persisted at relatively constant levels throughout lactation. Gel filtration studies of milk indicated that the antibody activity against Pr virus was associated almost entirely with IgG fractions, with small amounts of antibody detectable in IgM fractions in colostrum from two of nine sows. By comparison, TGE antibodies were primarily of the IgA class, with varying but lesser amounts of antibody associated with the IgG class. Such results suggest that viral infection of the intestinal tract of the sow, but not the upper respiratory tract, stimulates the secretion of IgA antibodies in the milk.