Calf Rotavirus Research Articles

Identification of a neutralization-specific antigen of a calf rotavirus.

Monospecific polyclonal antisera were raised in guinea-pigs against the calf rotavirus polypeptides VP1, VP2, VP3 + 4, VP4.2, VP6, VP7.1, VP7.2 and VP10. All of the antisera gave a similar pattern of cytoplasmic immunofluorescence in rotavirus-infected cells, but spots of fluorescence of varying intensity with different sera were seen over the nucleus. Immune precipitation, using Staphylococcus aureus to collect immune complexes, showed that VP2 was precipitated by antiserum to VP2 (alpha-VP2) and VP6 by alpha-VP6, alpha-VP7.1 and alpha-VP7.2 both precipitated the same range of proteins from infected cells (VP7, VP7.1 and VP7.2) or from virions (VP7.1 and VP7.2). VP10, either from virions or infected cells, was not precipitated by alpha-VP10. The only antiserum which efficiently neutralized infectivity was alpha-VP7.2. There were low levels of neutralization with alpha-VP10 (but the results varied from experiment to experiment) and traces with alpha-VP6. alpha-VP7.1 and the other antisera did not neutralize even though alpha-VP7.1 agglutinated double-shelled particles as seen in immune electron microscopy to a greater extent than alpha-VP7.2. Both VP7.1 and VP7.2 were shown to be glycoproteins by tunicamycin treatment of infected cells. Core particles only were agglutinated by alpha-VP10. All the evidence leads us to conclude that there were major neutralizing antigenic determinants present on VP7.2, a minor component of the outer shell of the virion.

Purification and characterization of bovine rotavirus cores.

Using the chaotropic effect generated by a high concentration of CaCl2, we converted calf rotavirus particles into cores of 40 nm in diameter. These cores were purified by rate zonal centrifugation in sucrose gradients and by isopycnic gradients. They had a sedimentation coefficient of 280S +/- 20S and a density of 1.44 g/ml in CsCl. When analyzed by polyacrylamide gel electrophoresis, they contained three polypeptides (VP125, VP89, and VP78). The major internal polypeptide of the virion (VP39) was recovered in a purified and soluble form in the top fractions of the sucrose gradients. From this stepwise degradation, it appears that VP39 is the most external polypeptide of dense particles. In contrast to reovirus cores, calf rotavirus cores did not exhibit transcriptase activity. Purified VP39 also did not exhibit transcriptase activity when tested after being mixed with purified rotavirus genome RNA as a template. Transcriptase activity was partially recovered when ionic conditions were adjusted to permit the reassociation of VP39 with the cores.

The differentiation of calf rotaviruses by plaque morphology and serum neutralization.

SUMMARY The plaques formed by two calf rotavirus isolates differed in size and appearance. One isolate was more sensitive to homologous than to heterologous antiserum while the other was equally sensitive to both antisera.

The molecular biology of rotaviruses II. Identification of the protein-coding assignments of calf rotavirus genome RNA species

The technique of in vitro translation of dimethyl sulfoxide denatured double-stranded (ds)RNA has been applied to the rotavirus system. Translation of an unfractionated mixture of genome RNA segments from calf rotavirus in a nuclease-treated rabbit reticulocyte system allowed the primary gene products of the viral genome to be defined. Polyacrylamide gel electrophoresis was used to resolve all 11 genome segments and then each was individually isolated and translated, to identify the primary gene product of each genome segment. RNA segments 1, 2, 3, 6, 7, and 11 were shown to code for structural proteins of the inner virus shell, segment 9 coded for the one completely nonstructural protein, and segments 4, 5, 8, and 10 coded for proteins or the precursors of proteins that were either completely or mainly outer shell structural polypeptides.

Preparation and characterization of antisera to electrophoretically purified SA11 virus polypeptides.

Antisera to SA11 virus proteins were prepared by immunizing rabbits with individual polypeptides separated by polyacrylamide gel electrophoresis under reducing or nonreducing conditions; the resulting antisera were characterized by four immunological methods. Results of complement fixation tests with double-shelled rotavirus particles and sera raised against reduced or unreduced proteins of the outer shell of the virus suggested the presence of common antigenic determinants in the outer capsid layers of SA11 and the Northern Ireland strain of calf rotavirus. In this test, antisera to outer shell polypeptides gp34 (O2) and gp25 (O4) cross-reacted with calf rotavirions, whereas those to p62 (O1) and p26 (O3) reacted only with the homologous virus. Antisera to the reduced outer shell proteins of the virus did not neutralize viral infectivity, nor did they possess hemagglutination inhibition activity. Evidence suggesting the presence of type-specific antigenic determinant(s) in the major inner protein p42 (I4) of SA11 virus, capable of inducing neutralizing antibody, is presented and discussed. Antisera produced against unreduced gp34 and p26 polypeptides of the virus contained type-specific neutralizing antibodies. Polypeptide gp34 was also capable of inducing hemagglutination inhibiting antibody. All of the antisera to unreduced polypeptides had agglutinating activity against double-shelled particles of homologous and heterologous rotaviruses.

Characterization of RNA polymerase products of Nebraska calf diarrhea virus and SA11 rotavirus.

The endogenous RNA polymerase of the calf rotavirus was shown to synthesize single-stranded RNA transcripts of one polarity which were identical in size to the denatured parental double-stranded RNA segments. The transcripts were notable in their absence of polyadenylate sequences. The polymerase activity associated with the cores of calf rotavirus was minimally altered by the reaction, since calf rotavirus cores could be reused after purification, whereas the RNA polymerase products appeared to be released from the virion.

Isolation of rotaviruses from turkeys and chickens: Demonstration of distinct serotypes and RNA electropherotypes

Six isolates of rotavirus were made from turkeys and two from chickens. Three of these required trypsin treatment for isolation and serial passage in cell cultures. The remainder were isolated without trypsin treatment. Most of these viruses were isolated in chick embryo liver cell cultures from the faeces of birds aged under 1 week. In six of the eight instances, rotavirus isolation was associated with enteric disturbance, characterised by signs such as diarrhoea, poor or abnormal appetite, abnormally fluid or gaseous intestinal contents or increased mortality. Cross immunofluorescence tests showed that while avian and mammalian rotaviruses shared a common group antigen, the avian viruses were more closely related to each other than to the Nebraska calf rotavirus isolate. On the basis of serum neutralisation tests seven of the eight avian rotaviruses were grouped into three serotypes, with two turkey isolates (Ty1 and Ty3) and a chicken (Ch1) virus being the prototype strains. The remaining virus, Ty2, was intermediate in type between Ty1 and Ch1. Analysis of the RNA of these viruses by polyacrylamide gel electrophoresis showed that they could also be grouped into a number of electropherotypes. The isolates which were serologically distinct were also electrophoretically distinct. Similarly the five isolates which belonged to the Ty3 sero-type were electrophoretically identical. Analysis of the serological and electrophoretic differences suggested that RNA segment 5 may code for a type-specific antigen.

Epizootic diarrhoea of adult cattle associated with a coronavirus-like agent

An epizootic of acute diarrhoea of adult cattle occured in Japan during the winter of 1976 to 1977. A majority of adult cattle clinically diagnosed as having the disease, showed a significant rises in antibody titres to bovine coronavirus, whereas only a smal; minority showed serological evidence of recent infection with calf rotavirus, bovine adenovirus type 7, parainfluenza virus type 3, or bovine viral diarrhoea-mucosal disease virus. A coronavirus-like agent was detected by electron-microscopy in faecal material from a cow with diarrhoea, and was subsequently isolated in primary bovine kidney cell cultures.

Polyacrylamide gel electrophoresis of avian rotavirus RNA.

The RNAs of 4 avian rotavirus isolates were analysed by polyacrylamide gel electrophoresis. One of these isolates was made from chickens, the other three were from turkeys. The RNAs were separated into 8 or 9 bands consisting of 11 segments, ranging in molecular weight from 0.24 X 10(6) to 2.09 X 10(6). In contrast to calf rotavirus, segments 10 and 11 of avian rotavirus RNA had very similar or identical electrophoretic mobilities. This may be a unique feature of avian rotavirus RNA. Co-electrophoresis experiments showed that the RNAs of 2 of the turkey isolates were electrophoretically identical. However, differences existed between these 2 isolates, the chicken isolate and the remaining turkey isolate with respect to segments 5, 6, the 7, 8, 9 complex and 10. Segment 5 of the chicken isolate was sometimes resolved into 2 components, and was further anomalous in that it was absent in some (analyses).

Passive immunity in calf rotavirus infections: maternal vaccination increases and prolongs immunoglobulin G1 antibody secretion in milk.

Ten heifers were inoculated on two occasions with an inactivated preparation of tissue culture-grown calf rotavirus, and a further ten heifers received a placebo vaccine. Serum anti-rotavirus antibody titers were significantly increased throughout pregnancy in the vaccinated group. After calving, the mean neutralizing antibody titer of colostral whey in control cows was 100, associated with immunoglobulins A and G1. No antibody was detected in the milk of these cows after the 4th day postpartum. The colostral whey from the vaccinated cows had a mean antibody titer of 20,452; 28 days after calving, the mean milk antibody titer was 320, associated mainly with immunoglobulin G1. Calves were challenged with a large oral inoculum of calf rotavirus at the 7th day of age. There was significant lengthening of the incubation and prepatent periods in calves born to vaccinated dams, but rotavirus-associated diarrhea of equal severity occurred in both groups. Evidence is presented which suggests that rotavirus antibody in milk can protect against a smaller challenge dose. Maternal immunization against rotavirus may be a practical proposition.

GEL ELECTROPHORESIS OF ROTAVIRUS RNA DERIVED FROM SIX DIFFERENT ANIMAL SPECIES

Rotavirus RNA prepared from calf, pig, mouse, deer, foal and dog-adapted human isolates was compared using polyacrylamide gel electrophoresis. Reproducible differences in the RNA migration patterns were found between all isolates. There were 11 clearly resolved segments in the pig, mouse and foal samples. The calf rotavirus RNA and deer rotavirus RNA separated into 9 bands and 10 bands, respectively. The dog-adapted human virus migrated in 12 bands, and this probably results from the complex passage history of the original human rotavirus isolate.

Rotavirus in diarrhoeic calves in Iran

Rotavirus in diarrhoeic calves in Iran

Rotavirus Polypeptides

Rotavirus infected monkey kidney cells (LLC-MK2) have been labelled with 35S-methionine in the presence of actinomycin D. The cells have been lysed with SDS and the polypeptides separated by discontinuous polyacrylamide gel electrophoresis. Rotavirus polypeptides began to appear 4 to 5 h p.i.; incorporation was maximum at 8 h, but all the polypeptides were still being made 15 to 18 h p.i. Tissue culture adapted calf rotavirus particles were labelled with 35S-methionine and the polypeptides compared with cell associated rotavirus polypeptides. There were four inner coat, four outer coat and three non-structural polypeptides. Several of the outer coat polypeptides have altered mol. wt. on maturation. The polypeptides of rotavirus from seven species (human, pig, calf, lamb, mouse, foal and rabbit) have been compared and their mol. wt. calculated. The polypeptides fell into the same relative groupings for each virus, but there were variations in the mol. wt. of most comparable polypeptides. The polypeptides of tissue culture adapted and non-adapted calf rotavirus from the same original isolate varied only in one of the non-structural polypeptides.

Location of type-specific antigens in calf rotaviruses.

Rotavirus antisera with and without neutralizing activity to calf rotavirus were compared for their ability to agglutinate calf rotavirus particles with and without an outer capsid layer. Particles without the outer capsid layer were agglutinated by antisera with immunofluorescent antibody activity and by antisera with immunofluorescent antibody and neutralizing activity. Particles with the outer capsid layer were agglutinated only by antisera with neutralizing activity. The neutralization test appears to be type specific, and it was concluded that type-specific antigens are associated with the outer capsid layer of rotaviruses. Since particles with the outer capsid layer were not agglutinated by antisera lacking neutralizing activity but possessing immunofluorescent antibody activity, it was concluded that in intact particles the group-specific antigen is masked by the outer capsid layer.

ISOLATION OF ROTAVIRUS FROM FOALS WITH DIARRHOEA

SummaryA rotavirus, morphologically similar to other known rotaviruses, was demonstrated in the faeces of 5 foals with diarrhoea on two properties. Four of these 5 samples produced specific intracytoplasmic fluorescence in cell culture when reacted with calf rotavirus antiserum conjugate. Sixteen affected foals from both properties were depressed, did not suckle and became recumbent. Most had a watery diarrhoea which lasted for 3 days and resulted in some dehydration and loss of body condition. Sick foals were separated from their mothers following the onset of diarrhoea and given fluid therapy and antibiotics, but despite these measures 4 of 12 affected foals on one property died. Acute and convalescent sera were collected from 6 foals, and in each case a rise in the titres of complement fixing and neutralising antibodies was demonstrated. Faecal filtrates containing foal rotaviruses were fed to gnotobiotic piglets and the effects compared with those of other rotaviruses. Viral isolates from both properties produced an asymptomatic infection in piglets. This contrasts with the other rotaviruses isolated in Australia that have been shown to cause diarrhoea in gnotobiotic piglets.

Calf rotavirus: Detection of outer capsid glycoproteins by lectins

Calf rotavirus: Detection of outer capsid glycoproteins by lectins

Serological relationships between rotaviruses from different species as studied by complement fixation and neutralization.

Human, piglet, mouse, foal, lamb, calf and rabbit rotaviruses all infected, but could not readily be subcultured in LLC MK2 cells. Cells infected with mouse and calf rotaviruses reacted by indirect immunofluorescence (FA) with convalescent serum from children, piglets, mice, foals, lambs, calves or rabbits, taken after rotavirus infection. Human, calf, piglet, mouse and foal rotaviruses reacted with human, calf, mouse, foal and lamb convalescent serum by complement fixation (CF). It was not possible to distinguish between different rotaviruses by CF or FA. Neutralization tests, however, detected species-specific rotavirus antigens. Any virus was neutralized by a much higher dilution of homologous species convalescent serum than by any heterologous serum. With the exception of the mouse virus there was very little cross reaction. However, in sera with a very high neutralizing titre for the homologous virus the titre was proportionately raised against heterologous virus. It is, therefore, now possible to type to species an unknown rotavirus by a neutralization test in LLC MK2 cells using convalescent serum from each species.

Further biochemical characterization, including the detection of surface glycoproteins, of human, calf, and simian rotaviruses

Polyacrylamide gel electrophoretic analysis of purified preparation of the simian rotavirus SA-11 indicated eight polypeptide components that migrated in a manner remarkably similar to those of the previously characterized human and calf rotaviruses. Analyses of preparations of single-shelled and double-shelled particles of human, calf, and simian an rotaviruses have also permitted assignment of the polypeptides to the inner or outer shells. The major components of the outer shells of each virus have been identified as glycoproteins, and the importance of this in terms of host cell specificity is discussed. Sensitivities of the various rotaviruses to acid, proteases, and glycosidases were also investigated.

Demonstration of Size Variation of RNA Segments Between Different Isolates of Calf Rotavirus

Polyacrylamide gel electrophosesis of RNA extracted from wild rotavirus isolates and cell culture adapted virus revealed a significant variation in the molecular weight of individual RNA segments. The major differences were observed between wild isolates on the one hand and the adapted strain on the other hand. The slight variations that were observed between different wild isolates were found regularly and appeared to be related to the origin of the samples.

Experimental rotavirus infection in lambs

Inoculation of lamb rotavirus to gnotobiotic lambs produced a disease characterized by diarrhoea and anorexia. Rotavirus was excreted in the faeces of the lambs for several days. One-day-old lambs were more susceptible than 12-day-old lambs. A serological response to infection was detected in all lambs by complement fixation and immunofluorescence techniques, using antigens prepared from calf rotavirus. Lamb rotavirus antiserum did not neutralize calf rotavirus, indicating the distinct nature of the lamb rotavirus. No evidence of latent infections was detected by treating the lambs with an immunosuppressant four months after initial recovery.