Studies on rotavirus homologous and heterologous active immunity in infant mice.

Homologous and heterologous active immunity was studied in mice with mammalian group A rotaviruses. One day old mice were vaccinated with one of the following rotaviruses: bovine B641 (serotype 6), bovine B223 (untyped), simian SA11 (serotype 3) and murine EDIM (untyped). At 10 days of age they were challenged with EDIM virulent virus or SA11 virus. All the vaccines induced a serological antibody response in the mice but only the homologous immune response was protective.

Isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves.

Small round viruses (SRV) were isolated from the faeces of diarrhoeic calves from three farms. All three SRV preparations caused diarrhoea experimentally in gnotobiotic calves. Each preparation contained viral particles of two morphological types, "astrovirus-like" and "calicivirus-like", and from one preparation the two particle types were separated from each other. The calicivirus-like agent ("Newbury agent") was 33 nm in diameter, and caused diarrhoea in gnotobiotic calves with villous atrophy and D-xylose malabsorption. This virus did not infect cell cultures. The astrovirus-like agent did not cause diarrhoea in two gnotobiotic calves; however, it infected cell cultures (primary calf kidney) and the infected cells immunofluoresced with convalescent gnotobiotic-calf antiserum. The astrovirus-like agents in the three preparations were antigenically related. Experiments in calves showed that there was a degree of cross-protection between the three SRV preparations, as judged by the presence or absence of diarrhoea, but that at least three unrelated pathogens were present.

Serial studies of virus multiplication and intestinal damage in gnotobiotic piglets infected with rotavirus.

A serial study of rotavirus infection in gnotobiotic piglets is described. They were infected when 7 days old and the course of infection was followed for 21 days, by immunofluorescence and histological examinations of the intestinal epithelium and by titration of the virus content of the gut lumen. There appeared to be two stages of recovery of the intestinal wall, the earlier stage involving non-immune mechanisms and the later invloving antibody.

The isolation of reovirus-like agents (rota-viruses) from acute gastroenteritis of piglets.

Isolations of reovirus-like agents (rotaviruses) were made from nine of 23 outbreaks of piglet diarrhoea on different farms and from both weaned and unweaned piglets. The viruses were shown to be morphologically and anti-genically similar to the rotaviruses of children and calves. Gnotobiotig piglets given intranasal inoculations of five different isolates developed acute gastroenteritis, and the virus was re-isolated from the faeces or intestinal contents. The piglet virus was not adapted to replicate in cell culture. We conclude that the pig rotavirus is commonly associated with outbreaks of gastroenteritis and is probably an important aetiological factor in this disease.

Transmission of human rotaviruses to gnotobiotic piglets.

Faecal filtrates containing rotavirus particles, from children with acute infectious diarrhoea, were inoculated intranasally into gnotobiotic piglets. The piglets developed no symptoms, but birus was readily found by electron microscopy in their faeces during three serial passages. Among 11 piglets tested 3 weeks after inoculation of virus, all had developed fluorescent antibodies against tissue-culture-adapted calf rotavirus but only two had neutralising antibody. Growth of human rotavirus did not occur in either normal or "nude", thymus-deficient suckling mice.

A major rearrangement of the VP6 gene of a strain of rotavirus provides replication advantage.

During coinfection of BSC-1 cells with bovine rotavirus B223 and human rotavirus 69M and subsequent serial passages at low multiplicity of infection (0.1 m.o.i.), a reassortant virus (BMR) with a rearranged VP6 gene became the predominant strain. At passage 24 virus extracted from 50 of 51 plaques (98%) contained the rearranged gene 6, which had been first observed in passage 19. The analyses of the clones obtained from passages before the appearance of the rearranged VP6 gene (passage 15) and after (passage 20) indicated that the B223 VP6 gene was the origin of the rearranged VP6 gene. To test whether the rearranged VP6 gene was responsible for the selection advantage observed, reassortant C11 was generated with BMR and WA rotavirus, containing the rearranged VP6 gene and the other 10 genes from WA. Coinfection of WA rotavirus and reassortant C11 and subsequent serial passages at low m.o.i. resulted in 100% of virus from clones extracted at passage 18 being identical to reassortant C11; demonstrating that the rearranged VP6 gene was once again selected over the normal VP6 gene. The selection advantage of the rearranged VP6 gene could not be explained by comparison of the growth curves of the viruses, as there was no significant difference between the growth cycles of rotavirus B223 and reassortant BMR, nor between rotavirus Wa and reassortant C11. However, the plaque and electropherotype analysis at passage 1 of Wa and C11 coinfection revealed that 85% of the progeny viruses contained the rearranged gene 6. These data show that the gene 6 rearrangement resulted in selection of the relevant reassortant, possibly by suppression of competitive strains, and may indicate a new mechanism for the evolution of rotavirus.

A longitudinal study of rotavirus antibody titers in swine in a closed specific pathogen-free herd.

In a newly established closed specific pathogen-free (SPF) swine herd, gilt/sow suckling and weaned pig rotavirus specific antibody titers were followed for three lactations by enzyme-linked immunosorbent assay (ELISA) to gain insight into the dynamics of herd antibody titers to group A rotavirus. Among gilts/sows, serum antirotavirus IgG titers increased during each lactation with a subsequent drop in titer between farrowings. Serum antirotavirus IgM titers declined during each lactation and with subsequent parity. Serum antirotavirus IgA titers remained constant during lactations and among parities. In colostrum and milk, antirotavirus IgA antibody was abundant. Differences in titer were not noticed between gilts and second litter sows but third litter sows had significantly higher titers than the first two groups. Antirotavirus IgG was high in colostrum but nearly nonexistent in milk. This titer did not vary significantly within or among parities. There was a linear regression in the titers of baby pig serum antirotavirus IgG from the post colostral sample through to seven weeks old, after which titer began to increase. No difference in baby pig serum antirotavirus IgG was noted among the three litters. Serum antirotavirus IgA and IgM were undetectable in baby pig sera after 2-3 weeks of age. Coproantibody to rotavirus was sporadically present in pig feces for 2-3 weeks after birth with highest titers in the IgA fraction. We conclude that although it is probable that age resistance of pigs to rotavirus diarrhea occurs, humoral immunity as measured by ELISA rotavirus antibody titers may not be intimately involved in virus clearance since in our studies baby pigs passively received large amounts of antibody but still excreted pathogenic virus. The finding of increasing levels of serum antirotavirus IgG in gilt/sow serum suggest that exposure to antigen of dams occur without significant increases in antirotavirus IgG titers in either colostrum, milk, or baby pig serum.

Seroepidemiology of Breda virus in cattle using ELISA.

Two direct blocking enzyme linked immunosorbent assays (ELISA) for the detection of antibodies to Breda virus in sera of cattle were compared. An ELISA with consecutive addition of antigen and test serum to an antibody-coated plate gave higher positive: negative absorbence ratios than an ELISA in which antigen and test serum were added simultaneously. Sera collected from breeding and fattening herds in The Netherlands (n = 1313) and the F.R.G. (n = 716) were tested, and antibodies to Breda virus were demonstrated in 94% of adult cattle. Ninety percent of newborn calves had high levels of maternal antibodies, which waned until the age of 3 months. Active seroconversion occurred between 7 and 24 months in most animals.

Cultivation and partial characterization of bovine astrovirus.

Bovine astrovirus serotype 2 (US2) was adapted to primary neonatal kidney cell (NBK) cultures by the addition of 50 micrograms ml-1 of trypsin in the medium. Infectious virus was released from the cells within 7 days post-infection in early passages and within 3 days in later passages. In the absence of trypsin, neither passage of infected cells nor release of infectious virus occurred. The virus was shown to be similar to the fecal astrovirus by a neutralization test and by ultrastructural studies of infected cells. Primary embryo bovine kidney (EBK) and NBK cell cultures supported infection with both fecal and tissue culture adapted (TCA) astrovirus. The time-related development of infection, as studied by immunofluorescence, was similar for both fecal and TCA astrovirus and for both cell culture types. The first indication of viral infection and expression of viral antigens occurred at 7 h post-infection and was characterized by the appearance of a diffuse faint immunofluorescence (IF) of the cytoplasm. Soon after, two or three brilliant IF granules were observed in the nucleus, which appeared to involve the nucleoli. Subsequently, dense granular IF was seen in the perinuclear region of the cytoplasm, which later extended to involve all the cytoplasmic area. In both EBK and NBK cultures infected with either fecal or tissue culture adapted astrovirus, only a minority of cells became infected, even when the multiplicity of infection exceeded one. Occasionally 10-20% of cells were infected, but in most cultures the proportion did not exceed 2% and in NBK cultures, from 3/9 calves, no infected cells were observed. The virus did not infect bovine cell lines. Infectivity of the virus was not removed by treatment with chloroform, and iododeoxyuridine and actinomycin D when added to the medium, did not block replication. Masses of virions were observed by electron microscopy in discrete areas in the cytoplasm, with similar distributions as the viral antigen foci as seen by IF. The mean diameter of the virions was 34 nm. In conclusion, bovine astrovirus lacks both essential lipids and an envelope, probably has an RNA genome, may have a nuclear phase of replication involving the nucleoli which is not blocked by DNA inhibitors, and has a selective cell tropism.

In vitro studies on the use of clay, clay minerals and charcoal to adsorb bovine rotavirus and bovine coronavirus.

Rotaviruses are the leading cause and coronaviruses are the major contributors of acute gastroenteritis in the young of various mammalian and avian species. Despite numerous trials and decades of research, vaccines have limited efficacy particularly for calves. As an alternative method of controlling infection, we have investigated broad spectrum antiviral agents that are not discriminatory among various viruses. This report involves testing a variety of adsorbent agents including charcoal, clay, and clay minerals to adsorb rotavirus and coronavirus in vitro. Results revealed that all the adsorbent agents had good to excellent capability of adsorbing rotavirus and excellent capability of adsorbing coronavirus. Percent adsorptions ranged from 78.74% to 99.89% for rotavirus and 99.99% for coronavirus; while sand (negative control) was < 0.01%. A high affinity binding was present as determined by a low percent desorption (0.06-3.09%). However, the adsorbent bound virus complex retained, and may have actually enhanced, infectivity.

The shedding of group A rotavirus antigen in a newly established closed specific pathogen-free swine herd.

A longitudinal study was undertaken in a newly established specific pathogen-free (SPF) swine herd to determine the dynamics of rotavirus antigen shedding in a closed swine facility. Pregnant SPF gilts which populated the herd, and their offspring, were monitored weekly for three consecutive lactations. Fecal samples were assayed for the presence of group-specific viral antigen by a solid phase immunoassay (ELISA). Results indicate that in the week prior to farrow, 35% of samples from gilts/sows contained rotavirus antigen. During nursing, 37% of the gilts'/sows' fecal samples also contained virus antigen. Over the course of three farrowings, every gilt/sow in the herd excreted virus antigen. Virus antigen was present in 25% of the samples tested from nursing pigs and in 70% of the samples tested from pigs in the postnursing period; 95% of the litters excreted virus antigen either while nursing or postweaning. Seasonal incidence in virus antigen excretion was noted with proportionally more suckling pigs virus antigen-positive in summer and proportionally more sows/gilts positive during winter. Diarrhea occurred only rarely in the sampled population. Although piglets shed rotavirus subclinically, ELISA positive feces from piglets of each lactation caused severe disease when fed to neonatal gnotobiotic pigs. Electropherotyping of these passaged viruses indicated minor variation in RNA banding patterns over time.

Immunodominant neutralizing antigens depend on the virus strain during a primary immune response in calves to bovine rotaviruses.

Sera obtained from gnotobiotic calves (GC antisera) infected with bovine rotavirus strain NCDV or B223 from a previous study (Woode et al., 1987), which have different G (G6 and G10 respectively) and P serotypes, were compared for their neutralization (NT) properties to a number of human and animal rotaviruses (representing G serotype 1-6, 8-10). Two distinct patterns of neutralization were identified from these GC antisera. Of all the serotypes tested, NCDV GC antisera neutralized only B641 to a relatively high titer compared with the homologous titer, implying a narrow pattern of NT response. Analysis with reassortants indicated that the response was primarily to VP4. In contrast, B223 GC antisera neutralized most of the G serotypes tested to titers within 3-7 fold of the homologous titer, demonstrating a broad pattern of NT response. In the earlier study B223 was shown to induce a heterotypic protection against bovine rotavirus B641 (G serotype 6), and the serologic data obtained from this study indicates that a B223 vaccine might provide broad protection against several different serotypes of human and animal rotaviruses.

Studies with an unclassified virus isolated from diarrheic calves.

A transmissible agent (Breda agent) was isolated from a calf with diarrhea and shown to be infectious by inoculation orally into gnotobiotic and conventionally reared calves. The "Breda" agent had the morphology of a virus and possessed a hemagglutinin. Antigenic studies showed the virus to be antigenically different from bovine coronavirus, parainfluenza 3 virus, bovine rotavirus, bovine parvovirus and bovine pestivirus (BVD). Attempts to culture the virus in cell or organ cultures or in embryonated eggs, were unsuccessful. The virus was either spherical or kidney shaped, with 7-9 nm peplomers on the surface. A few particles possessed coronavirus processes of 17-20 nm, but these were arranged irregularly and were thought to be tissue debris. Three out of eight experimental calves developed severe diarrhea and the lesions in the small and large intestines were similar to those reported for coronavirus. The virus replicated in the jejunal and ileal regions of the small intestine and in the spiral colon, as judged by immunofluorescence. The virus multiplied in all experimental calves and was excreted in the feces; excretion correlating with the onset of diarrhea or a change in the appearance of the feces. There was little or no malabsorption measured by the uptake of D-xylose and the fact that infection of both the crypt and villus epithelial cells was observed, suggests that the pathogenesis may be different from rotavirus and coronavirus. Fourteen of forty seven calves in the outbreak were infected with the virus, virus was not identified in other farm outbreaks of the disease.

An in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronavirus infections.

Crude theaflavin was extracted from black tea and then fractionated by HPLC into five components (initial peaks (IP), TF1, TF2A, TF2B, and TF3). The crude extract and the various fractions of theaflavin were collected and tested, individually and in combination, for antirotaviral activity. The mean effective concentration (EC50) was calculated and compared. Activity varied from the most active being the uncharacterized theaflavin-like initial peaks (IP) with an EC50 of 0.125 microgram/ml to the least active being theaflavin-3 monogallate (TF2A) with an EC50 of 251.39 micrograms/ ml. The combination of TF1 + TF2A + TF2B + TF3 was more active than the sum of the activities of these four fractions individually, indicating synergism among the peaks. Only the crude extract was assayed for activity against coronavirus; the EC50 was 34.7 micrograms/ml.

The aetiology of diarrhoea in pigs weaned at two days of age.

When pigs are weaned at two days of age large numbers of Excherichia coli appear in the anterior gut and the incidence of diarrhoea rises. The two phenomena do not appear to be directly related because the strains of E coli isolated are not serotypes previously found to be associated with neonatal pig scouring. Representative strains of the non-enteropathogenic serotypes did not produce enterotoxin and did not adhere to small intestine brush borders. Moreover when antibiotics were fed to eliminate E coli from the gut, the pigs still scoured. Rotavirus was detected in the gut contents and gut epithelium of scouring pigs and a bacteria-free filtrate of gut contents produced diarrhoea when administered to germ-free pigs. It is suggested that rotavirus may be one of the causes of the scouring seen shortly after weaning pigs at two days of age.

Breda virus (Toroviridae) infection and systemic antibody response in sentinel calves.

Enzyme-linked immunosorbent assays were established to detect Breda virus antigen in feces and homologous antibodies of the IgG1, IgM, and IgA isotypes in serum. With the aid of solid-phase immune-electron microscopy, torovirions in fecal material were observed. The course of natural infection was studied in 10 sentinel calves that had been obtained from different farms, and housed together at 1 week of age. They were separated from other cattle until the age of 10 months. Up to the age of 4 months, all calves regularly excreted Breda virus in the feces. Irrespective of the existence of IgG1 isotype maternal antibodies, all calves had early IgM responses in serum, but lack of IgA seroconversion. In 7 calves, antibody titer decreased below detection, whereas 3 calves had an isotype switch, resulting in persistent IgG1 titer. After introduction into the dairy herd at 10 months of age, all calves had diarrhea, and shedding of Breda virus was observed in 8 of them. Seroconversion for all antibody isotypes was observed, indicating lack of mucosal memory. In contrast, coronavirus infection in the presence of maternal antibodies led to isotype switch in all calves but one, and a memory response was observed after introduction into the dairy herd.

Surface proteins of Breda virus.

The serotypes 1 and 2 of Breda virus from feces of experimentally infected gnotobiotic calves were studied with respect to their sedimentation and density properties in sucrose gradients and their structural polypeptides; Berne virus, the proposed prototype of the new family Toroviridae, was included for comparison. After Breda-1 virus had been stored at 4 C for a prolonged period, it showed a heterogeneous sedimentation behavior (480 to 520 Svedberg units [S]) and density (1.18 to 1.21 g/ml) indicative of its poor state of preservation. In contrast, freshly prepared Breda-2 virus sedimented at 350 S and showed a buoyant density of 1.18 g/ml; these values compare well with those of Berne virus (400 S and 1.16 g/ml, respectively). Efficient purification of the Breda viruses could be achieved by a 2-step method, involving pelleting by ultracentrifugation followed by isokinetic and isopyknic sucrose gradient centrifugation. Radioiodinated purified virus showed polypeptides with apparent molecular weights of 105,000, 85,000 37,000, and 20,000; another labeled protein of 65,000 D is of doubtful virus specificity. Mouse immune serum raised against Breda-2 virus recognized the polypeptides of the homologous virus and the 2 highest molecular weight proteins of Breda 1 virus in radioimmune precipitation. The same serum inhibited hemagglutination of the heterologous serotype to a low, but significant, degree and efficiently neutralized the infectivity of Berne virus. These observations are taken as indications that the 105,000- and 85,000-D polypeptides represent surface structures of torovirions, probably peplomeric proteins.

Myoelectric activity of the small intestine in enterotoxin-induced diarrhea of calves.

Electrodes were surgically implanted at 15-cm intervals in the jejunum and ileum of 4 healthy neonatal calves so that myoelectric activity could be recorded on 2 consecutive days. On the first day, each calf received a control treatment, and myoelectric activity was recorded for 340 minutes. Phase I was recorded for a mean of 175.8 +/- 22.8 minutes (51.5%), phase II for 124 +/- 27.4 minutes (36.5%), and phase III for 40.3 +/- 6 minutes (11.9%). On the second day, each calf was treated with approximately 200 micrograms of heat-stable enterotoxin (STa) of Escherichia coli orally. All calves developed diarrhea after the administration of STa. Phase I was recorded for a mean of 92.5 +/- 42.3 minutes (27.2%), phase II for 227.3 +/- 52.5 minutes (66.9%), and phase III for 20.3 +/- 11.4 minutes (6.0%). Increase in phase II and decrease in phases I and III after STa administration were significant (P less than 0.05). Duration of the migrating myoelectric complex was longer after STa administration (median, 64 minutes), compared with the control treatment (median, 54 minutes). Minute rhythms, recorded on the day of toxin administration, ranged from 49 to 153 minutes. There was no difference between the number of migrating action potential complexes on the control days (range, 1 to 10), compared with those on treatment days (range, 1 to 14). These findings are suggestive that enterotoxin-induced diarrhea of calves is accompanied by increased total spiking activity and minute rhythms in the distal portion of the jejunum and ileum.

Isolation of a rotavirus from a newborn dog with diarrhea.

A rotavirus was isolated from a newborn dog that died after having clinical signs of diarrhea. Virus particles with rotaviral morphologic features were observed by transmission electron microscopy in the intestinal homogenate collected at necropsy. Cytopathic effects were observed, and rotaviral antigens were detected by indirect immunofluorescence in MA-104 monolayer cultures (a fetal rhesus macaque kidney cell) inoculated with intestinal homogenate. This rotavirus isolate, designated LSU 79C-36, may be a specific canine rotavirus or a rotavirus from another species.

Effects of microbial and host variables on the interaction of rotavirus and Escherichia coli infections in gnotobiotic calves.

Naturally occurring mixed infections with Escherichia coli and rotavirus have been associated with fatal diarrhea of calves about 1 week old. Experiments were designed to reproduce this syndrome in gnotobiotic calves. Clinical, microbiological, and pathologic data were used to assess severity of disease and mechanisms of the interaction between the 2 infections. An initial study involved 5- to 8-day-old gnotobiotic calves inoculated with a strain of enterotoxigenic E coli (ETEC) and a strain of rotavirus. Calves were observed for 2 days after they were inoculated; fatal diarrhea was not produced. In later studies, variables were tested to identify those that might contribute to fatal diarrhea. Variables which did not result in fatal or severe diarrhea or which did not cause disease that was more severe in dually inoculated calves than that in monoinoculated calves were increasing feed to 2 times base line, increasing dose of ETEC to 10 times base line, inoculating calves when they were 2 days old, using a strain of E coli that causes colisepticemia, and using a different strain of rotavirus. When the observation period was extended from 2 days to 6 days after calves were inoculated, severe, watery, fatal diarrhea occurred in 6 of 12 calves by 32 to 72 hours after dual inoculation was given. Fatal diarrhea was associated with intensive colonization by the ETEC in the caudal half of the small intestine. Microscopic lesions were similar between dually inoculated and rotavirus-monoinoculated calves, except there was more severe atrophy of ileal villi of dually inoculated calves.(ABSTRACT TRUNCATED AT 250 WORDS)