Prevalence of antibodies to four bovine rotavirus strains in different age groups of cattle

Neutralizing antibody titers to four bovine rotavirus strains, representing three serotypes, were measured in 160 sera from cattle of different age groups. Age-specific seroprevalence analysis revealed serotype 6, represented by bovine rotavirus (BRV) NCDV, as the predominant rotavirus serotype infecting German cattle and serotype 10, represented by BRV V1005, as the least prominent. Infections with serotype 8, represented by BRV 678, occurred with intermediate frequency. Antibodies of young calves distinguished between NCDV and UK virus, two serotype 6 BRV strains differing in VP4 antigen.     

Neonatal calf diarrhea induced by rotavirus

This presentation summarizes the results of a comprehensive study on rotaviruses isolated in Italy from calves and rabbits affected by neonatal diarrhea. The results clearly indicated that rotavirus infection is widespread and supported the evidence for an etiologic role of these viruses in neonatal diarrhea. The evidence of differences in virulence among bovine rotaviruses appeared also to be confirmed. Conventionally reared calves were fully susceptible to the experimental infection induced by three rotaviruses originating from heterologous hosts, i.e. monkeys, pigs and rabbits, respectively. When rotavirus strains of bovine, simian, porcine and rabbit origin were compared by cross neutralization tests, it was found the simian and porcine strains were indistinguishable and both appeared to relate antigenically to the bovine strain. On the other hand, a reciprocal antigenic correlation was found between bovine and rabbit isolates. Finally, it was proven that feeding newborn calves with colostrum of their dams, previously vaccinated with an inactivated rotavirus vaccine, could prevent the neonatal diarrhea from occurring.     

A study on neonatal calf diarrhea induced by rotavirus

This review summarizes the results of a study on rotaviruses isolated from calves affected by neonatal diarrhea.

The results indicated that rotavirus infection is widespread and supported the evidence for an etiologic role of these viruses in neonatal diarrhea. Differences in virulence among bovine rotaviruses appeared also to be confirmed.

Conventionally reared calves were fully susceptible to the experimental infection induced by rotaviruses originating from heterologous hosts, i.e. monkeys, pigs and rabbits.

When rotavirus strains of bovine, simian and rabbit origin were compared by cross neutralization tests, it was found the simian and porcine strains were indistinguishable and both appeared to relate antigenically to the bovine strain.

Finally, it was proven that feeding newborn calves with colostrum and first milk of their dams, previously vaccinated with an inactivated adjuvanted rotavirus vaccine, could prevent the neonatal diarrhea from occurring.     

Distinct serotypes of porcine rotavirus associated with diarrhea in suckling piglets in southern Quebec.

Cytopathic rotavirus strains were isolated in cell cultures from the intestinal contents of diarrheic piglets on Quebec pig farms where repeated outbreaks of enteritis occurred. All the isolates shared the common group antigens of rotaviruses as revealed by immunofluorescence and counterimmunoelectrophoresis. A hemagglutinating activity was demonstrated with human group O, porcine and guinea pig erythrocytes. At least one of the isolates was clearly distinguished from the American prototype of porcine rotavirus (strain OSU) by neutralization and hemagglutination inhibition tests; a third serotype was also suspected. By polyacrylamide gel electrophoresis of RNA, it was not possible to differentiate these isolates.     

Development of specific nucleic acid probes for the differentiation of porcine rotavirus serotypes

A dot blot hybridization assay is described for the detection and differentiation of porcine rotavirus serotypes. Recombinant complementary DNA (cDNA) representing gene 9 (the gene encoding the neutralization antigens in VP7 glycoprotein) from OSU (porcine rotavirus serotype 1) and Gottfried (porcine rotavirus serotype 2) strains were used to determine the optimal hybridization conditions which allow specific detection of group A porcine rotaviruses. Probes were prepared by excision of the inserts from the recombinant plasmids and radiolabeling of cDNA with 32P by the random primer extension method. Probes were hybridized at various stringencies with viral RNA from different rotavirus serotypes bound to nylon membranes. Hybridization at low stringency (26% base pair mismatch for stable hybrid formation) had high sensitivity but low specificity. Hybridization at high stringency (16% base pair mismatch for stable hybrid formation) produced high specificity but decreased the sensitivity observed at low stringency. Probes were specific for rotavirus at both stringencies and did not hybridize with nucleic acids from other porcine viruses. Subgenomic gene 9 fragments were then tested to provide more specific probes. A 322 bp fragment from OSU gene 9 between nucleotides 382 and 704 and a 266 bp fragment from Gottfried gene 9 between nucleotides 230 and 496 were found to be specific as hybridization probes. These studies demonstrated the feasibility of the dot blot hybridization assay using subgenomic fragments of gene 9 to detect and differentiate serotypes of porcine rotavirus. Additional studies are warranted to further evaluate the sensitivity and the capability of these probes to detect porcine field isolates of the same serotype.     

Detection and differentiation of bovine group A rotavirus serotypes using polymerase chain reaction-generated probes to the VP7 gene.

Dot and Northern blot hybridization assays were developed to detect and differentiate group A bovine rotavirus serotypes using radiolabeled serotype 6 (Nebraska calf diarrhea virus [NCDV] and United Kingdom [UK] strains) or serotype 10 (Crocker [Cr] strain) VP7 gene probes. Partial length VP7-specific cDNA encompassing areas of major sequence diversity were generated by the polymerase chain reaction (PCR) using either cloned VP7 genes (NCDV and UK strains) or reverse transcribed mRNA (Cr strain) as templates. Radiolabeled probes prepared from the PCR-generated cDNA were tested at various stringency conditions to optimize the hybridization assays. At high stringency conditions (52 C, 50% formamide, 5 x standard saline citrate), the NCDV, UK, and Cr probes serotypically differentiated bovine rotavirus isolates in RNA samples prepared from cell culture propagated viruses or in fecal specimens from infected gnotobiotic calves. The sensitivity and specificity of NCDV and Cr VP7 probes were characterized in dot blot hybridization assays, and the probes were estimated to detect at least 1 ng of viral RNA. The serotyping results obtained using VP7 probes were similar to those obtained using serologic assays. Further development of these assays may provide a useful means for the rapid detection and differentiation of bovine rotavirus serotypes in fecal samples from calves in the field.     

Identification of rotaviruses of different origins by the plaque-reduction test

The plaque assay for the simian rotavirus SA11 was shown to be applicable to the economically important calf and porcine rotaviruses. In addition, neutralization of viral infectivity with specific antiserum before assay (plaque-reduction test) was found to be a useful test to identify the species of origin of rotaviruses.     

Diversity and zoonotic potential of rotaviruses in swine and cattle across Europe

Group A rotaviruses can infect both humans and animals. Individual rotavirus strains can occasionally cross species barriers and might hereby contribute to the emergence of new genotypes in heterologous hosts. The incidence and impact of zoonotic rotavirus are not well defined, and one reason for this is a lack of data about strains circulating in suspected reservoir animal hosts. In this study we report the incidence, genetic diversity, and molecular epidemiology of rotaviruses detected in domestic cattle and swine in 6 European countries. From 2003 to 2007, 1101 and more than 2000 faecal specimens were collected from swine and cattle, both healthy and diarrhoeic, and tested for rotaviruses. Viruses from positive stools were genotyped and a subset of strains was characterized by nucleotide sequencing and phylogenetic analysis of the VP7 (G) and VP4 (P) genes. Rotaviruses were detected in 43% of bovine samples and in 14% of porcine samples. In cattle, 10 different combinations of G and P types were identified and the most common strains were G6P[11] and G6P[5]. In swine, the number of identified G-P combinations was higher (n=21), however, no single combination was predominant across Europe. Newly described genotype specificities, P[27] and P[32], were identified in swine. When compared at the nucleotide sequence level, the identified porcine rotavirus strains and contemporary human strains grouped together phylogenetically, whereas bovine rotavirus strains formed separate clades. These data demonstrate large genetic diversity of porcine and bovine rotavirus strains across Europe, and suggest that livestock herds may serve as potential reservoirs for human infections.     

An experimental study of Bungowannah virus infection in weaner aged pigs

Animal-to-human interspecies transmission is one of the evolutionary mechanisms driving rotavirus strain diversity in humans. Although quite a few studies emanating from Africa revealed evidence of bovine-to-human rotavirus interspecies transmission, whole genome data of African bovine rotavirus strains are not yet available. To gain insight into the complete genome constellation of African bovine rotaviruses, the full genomes of three bovine rotavirus strains were extracted from stool samples collected from calves, amplified using a sequence-independent procedure, followed by 454(?) pyrosequencing. Strains RVA/Cow-wt/ZAF/1603/2007/G6P[5] and RVA/Cow-wt/ZAF/1605/2007/G6P[5] were both genotyped as G6-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3 and were probably two variants of the same rotavirus due to their close nucleotide sequence similarity. The genotype constellation of strain RVA/Cow-wt/ZAF/1604/2007/G8P[1] was G8-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The genetic relationships and phylogenetic analyses suggested that these three bovine rotavirus strains may have emerged through multiple reassortment events between bovine, giraffe and antelope rotaviruses. Due to the close relatedness of genome segments 1 (encoding VP1), 7 (NSP2), 9 (VP7) and 10 (NSP4) of strain RVA/Cow-wt/ZAF/1604/2007/G8P[1] to those of the corresponding segments of human rotaviruses, RVA strain 1604 may represent bovine strains that were transmitted to humans and possibly reassorted with human rotaviruses previously. The complete nucleotide sequences of the bovine rotavirus strains reported in this study represent the first whole genome data of bovine rotaviruses from Africa.     

Detection and Genetic Evolution of Diarrhea-related Viruses in Chongqing Beef Cattle

In order to investigate the pathogen prevalence of bovine viral diarrhea in Chongqing, this study carried out an investigation of bovine viral diarrhea virus (BVDV), bovine coronavirus (BCV), bovine rotavirus (BRV) and bovine astrovirus (BAstV) on a total of 81 diarrhea samples of beef cattle which were collected from Chongqing by RT-PCR. After the PCR products were sequenced, phylogenetic analysis was performed with Mega 6.0 software. From 81 samples, the positive rate of BRV,BAstV and BVDV were 66.7%,8.6% and 7.4%, respectively,BCV was not detected.Phylogenetic analysis showed that 5 strains of BRV sequences were clustered into a small branch, which had significant genetic distance with other VP6 sequences in GenBank; 5 strains of BVDV, Chinese and Denmark strains were clustered into one branch,genetic relationship were close; 5 strains of BAstV clustered into a branch with Hongkong strain, but there was still an obvious genetic distance. The result showed that calves under the age of half year were the main group of beef cattle with diarrhea in Chongqing. BRV was an important cause of diarrhea in Chongqing, the genetic diversity of BRV,BAstV and BVDV could be reference to further concern.     

重庆肉牛腹泻相关病毒的检测及遗传进化分析

为了解重庆市肉牛病毒性腹泻的病原流行情况,本研究对重庆市8个肉牛养殖场的81份腹泻粪便样本中牛病毒性腹泻-黏膜病病毒（bovine viral diarrhea virus,BVDV）、牛冠状病毒（bovine coronavirus,BCV）、牛轮状病毒（bovine rotavirus,BRV）和牛星状病毒（bovine astrovirus,BAstV）4种致腹泻病毒进行了RT-PCR检测,对PCR产物进行测序,用Mega 6.0软件进行系统发育分析。结果显示,BRV、BAstV和BVDV检出率分别为66.7%、8.6%和7.4%,BCV未检出。遗传进化分析结果表明,测序的5个BRV单独聚为一小支,与GenBank中其他VP6序列有明显的遗传距离;BVDV与中国株和丹麦株聚为一支,遗传关系最近;5个BAstV单独聚为一支,与中国香港株遗传关系最近,但仍有明显的遗传距离。本试验结果表明,重庆地区肉牛腹泻主要发生在6月龄以下犊牛,BRV是该地区肉牛腹泻的重要原因,BRV、BAstV和BVDV 3种病毒的遗传多样性值得进一步关注。     

Antigenic Relationships of Equine Herpesvirus Strains Demonstrated by the Plaque Reduction and Neutralization Kinetics Test

The antigenic relationships among 50 strains of equine herpesvirus (EHV) were studied by neutralization tests using antisera prepared in rabbits against four EHV reference strains: types 2 and 3, cytomegalo-like virus 82-A, and our leukocyte isolant H-40. No distinctive antigenic differences among reference strains were demonstrated in reciprocal neutralization tests but each antiserum neutralized its homologous virus more rapidly than any heterologous strain. Forty-six EHV strains isolated from peripheral blood leukocytes of apparently healthy horses were antigenically indistinguishable from each other and from the four reference strains. Their high degree of antigenic relatedness suggests that these viruses are isolants of a single, widely distributed serotype of which type 2 (LK) strain is a typical representative.     

Hybridization probes for the detection and differentiation of two serotypes of porcine rotavirus

A dot hybridization assay was developed to detect and differentiate two serotypes of porcine rotavirus by hybridization of specific probes to rotavirus RNA dotted onto nylon membranes. The assay was conducted using radiolabeled probes prepared from cDNA clones of OSU (serotype 5) and Gottfried (serotype 4) rotavirus gene 9 segments. The probes were prepared by excision of full length cDNA copies of gene 9 inserts from recombinant plasmids followed by nick translation and 32P-dCTP labeling. Rotavirus RNA samples used in the assay were prepared from tissue culture, and intestinal contents from gnotobiotic and conventional pigs. Optimal conditions for hybridization consisted of 52 degrees C, 50% formamide, and 5 x SSC. Hybridization studies with the OSU and Gottfried gene 9 probes have shown them to be specific with only low levels of cross-reactivity with heterologous rotavirus preparations. Sensitivity studies have demonstrated the ability of the probes to detect at least 2 ng of rotavirus RNA. Further development and refinement of this technique may provide a useful tool for the detection and differentiation of porcine rotavirus serotypes.     

Phylogenetic analysis of porcine rotavirus in Argentina: increasing diversity of G4 strains and evidence of interspecies transmission

Group A rotaviruses are one of the most frequently detected viral agents associated with neonatal diarrhea in piglets. In order to characterize rotavirus (RV) strains circulating in Argentinean swine, four porcine production farms located in Buenos Aires were studied. RV strains genotyped as P[6]G4, P[6]G8 and P[1]G6 were found in piglets under 30 days of age, without diarrhea. Phylogenetic and sequence analysis of the VP7 gene from G4 strains available in databases, reveals five porcine new lineages (III-VII) and three sublineages (VIIa-VIIc). The G4 porcine Argentinean strains were grouped with a porcine RV strain isolated in Brazil and another RV strain isolated from a child with diarrhea in Mexico, constituting an American lineage (VII). On the other hand, porcine G6 and G8 were closely related to RV's circulating in Argentinean cattle and South-American camelids, respectively. The fact that G4 porcine lineages were epidemiologically related to human strains, and G6 and G8 Argentinean porcine strains were found related to bovine and South-American camelids, respectively, suggests that pigs might play a crucial role as reservoir and generator of newly adapted emerging RV strains for human and other species.     

Relative prevalence of typical and atypical strains among rotaviruses from diarrheic pigs in conventional swine herds

Polyacrylamide gel electrophoresis was conducted on genomic RNA extracted from rotaviruses detected in diarrheic pigs from conventional swine herds. Ninety samples contained sufficient virus for RNA band visualization and genome classification. Genome profiles were characteristic of typical group A rotaviruses in 67.8% of the 90 samples, of group B rotaviruses in 10.0%, and of group C rotaviruses in 11.1%. In 11.1% of the samples, the presence of more than 11 bands suggested concurrent infection with more than 1 strain of rotavirus. In infections among nursing pigs, 76.4% were group A rotaviruses, 7.4% were group B, 7.4% were group C, and 8.8% were coinfections. In infections among weaned pigs, 40.9% were group A, 18.2% were group B, 22.7% were group C, and 18.2% were coinfections. Coelectrophoresis with prototype OSU and Gottfried strains revealed a great diversity in electropherotype among field strains of rotavirus.     

Antiviral activity of Alpinia katsumadai extracts against rotaviruses

In vitro anti-rotavirus activity of Alpinia katsumadai (AK) extracts were evaluated against bovine G8P[7] and porcine G5P[7] rotaviruses in two different assay strategies, a mixed treatment assay and a post treatment assay. In the mixed treatment assay, six AK extracts [AK-1 (EtOH extract), AK-3 (H(2)O layer), AK-5 (40% methanol fraction), and AK-9-11 (H(2)O extract, polysaccharide fraction, supernatant fraction)] exhibited inhibitory activities against G5P[7] rotavirus with the EC(50) values ranging from 0.7±0.4 to 33.7±6.5 μg/mL. Extracts AK-1, AK-3, and AK-5 inhibited rotavirus infection against G8P[7] rotavirus, the with EC(50) values of 8.4±2.2 μg/mL, 6.5±0.8 μg/mL and 8.4±5.0 μg/mL, respectively. By hemagglutination inhibition (HI) assay, six AK extracts completely inhibited viral adsorption onto human RBCs in both strains of rotaviruses at less than 11 μg/mL. However, in the post treatment assay, there was no anti activity shown against both strains of rotaviruses. As a result, six AK extracts were attributed mainly to having a strong interaction with hemagglutinin protein on the outer surface of rotavirus, resulting to blockage of viral adsorption.     

Longitudinal study of bovine rotavirus group A in newborn calves from vaccinated and unvaccinated dairy herds

Reports of rotavirus excretion in calves usually result from cross-sectional studies, and in face of the conflicting results regarding protection of calves born to vaccinated dams against diarrhea, the aim of the present study was to evaluate rotavirus excretion in dairy calves born to vaccinated or unvaccinated dams, to identify the genotypes of bovine rotavirus group A (RVA) strains isolated from these animals as well as to investigate characteristics of the disease in naturally occurring circumstances throughout the first month of life. Five hundred fifty-two fecal samples were taken from 56 calves, 28 from each farm and, in the vaccinated herd, 11/281 samples (3.91%) taken from six different calves tested positive for RVA while in the unvaccinated herd, 3/271 samples (1.11%) taken from 3 different calves tested positive. The genotyping of the VP7 genes showed 91.2% nucleotide sequence identity to G6 genotype (NCDV strain), and for the VP4 gene, strains from the vaccinated herd were 96.6% related to B223 strain, while strains from the unvaccinated herd were 88% related to P[5] genotype (UK strain). Genotypes found in this study were G6P[11] in the vaccinated herd and G6P[5] in the unvaccinated herd. All calves infected with rotavirus presented an episode of diarrhea in the first month of life, and the discrepancy between the genotypes found in the commercial vaccine (G6P[1] and G10P[11]) and the rotavirus strains circulating in both vaccinated and unvaccinated herds show the importance of keeping constant surveillance in order to avoid potential causes of vaccination failure.