Determination of bovine rotavirus G serotype by polymerase chain reaction

A total of 113 diarrheic samples comprising of 68 buffalo calves and 45 cow calves were screened by RNA-PAGE for the detection of presence of rotavirus. RNA-PAGE analysis of these samples revealed 11 (9.73%) was found positive for rotavirus. Out of 68 faecal samples of buffalo calves tested for viral gastroenteritis, 8 (11.76%) were found positive for rotavirus. Similarly, out of 45 faecal samples of cattle calves tested for viral gastroenteritis, 3 (6.66%) was found positive for rotavirus. Rotavirus-positive samples represented long electropherotype. All RNA-PAGE-positive faecal samples for rotavirus subjected to RT-PCR for VP7 gene, ten samples yielded a specific product of 1,013 bp of VP7 gene. All the PCR-positive samples of the present study were subjected to genotyping with primers for G6, G8 and G10 genotype. All positive samples showed G10 genotype. This indicates that G10 may be predominant genotype among bovine calves in Mumbai region in India.     

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.     

Detection and analysis of bovine rotavirus strains circulating in Australian calves during 2004 and 2005

Bovine rotavirus (BRV) has been detected in both dairy and beef cattle herds worldwide. Stool samples collected from calves in the Gippsland region of Victoria, Australia were screened to determine the presence of BRV. A total of 100 faecal samples were collected from calves with and without diarrhoea across three farms during 2004 and 2005. Group A BRV was detected in 26% of faecal samples (22 from diarrheic calves and four from asymptomatic calves). Genotyping analysis of rotavirus positive samples indicated that G6P[5] was the most prevalent genotype (38.5%) followed by G6P[5 + 11] (15.4%). G10P[11] and G6 + G10P[5] were each detected at a rate of 7.7%, and G6 + G10P[11] was found in a single sample (3.8%). Seven samples (26.9%) could not be G and/or P typed. Thirty percent of the BRV positive samples were mixed infections, indicating that individual calves were co-infected with more than one strain of rotavirus. The G6P[5] strains exhibited high VP7 identity (>97% amino acid identity) with B-60, a G6 strain identified in Victorian calves during 1988. A G10P[11] isolate was closely related (>97% amino acid identity in VP7 and VP4 proteins) to a Victorian G10P[11] strain (B-11) also identified during 1988. This study demonstrates that BRV is a contributing pathogen to diarrhoeal disease in Victorian calves, with sequence analysis suggesting long-term conservation of the VP7 protein over a 16-year period.     

Genetic heterogeneity of wild-type G4P[6] porcine rotavirus strains detected in a diarrhea outbreak in a regularly vaccinated pig herd

Group A rotavirus (RV-A) with short electropherotype was identified by ss-PAGE in a neonatal diarrhea outbreak at a Brazilian pig farm where the sows were regularly vaccinated with a commercial vaccine containing OSU (G5P[7]) and Gottfried (G4P[6]) porcine RV-A (PoRV-A) strains. The ss-PAGE positive stool samples (n=20) were characterized as P[6] genotype by multiplex-nested-RT-PCR assay. The nucleotide analysis of the VP4 gene (VP8*) state that the viruses clustered in P[6] lineages that are also shared by RV-A strains identified in human hosts. Nucleotide analysis of the VP7 gene identified different lineages in G4 including a new lineage tentatively designated IX. The immunological pressure induced by commercial vaccine with a rotavirus containing a G4P[6] genotype of porcine origin (Gottfried strain) might have allowed the selection of PoRV-A strains with characteristics found in RV-A strains isolated of human hosts, such as P[6]-Ie and If, and promoted the selection or emergence of RV-A strains with a new lineage of the G4 genotype. The characterization of PoRV-A strains with unusual genotypes described in this study highlight the importance of surveys on the relationship between human and animal rotavirus strains.     

Use of polymerase chain reaction for the differentiation of Group A bovine rotavirus G6, G8, and G10 genotypes in the North Island of New Zealand

AIM: To determine the presence of Group A rotavirus G6, G8, and G10 genotypes in calves in the North Island of New Zealand. METHODS: Faecal samples from 730 calves (<6 weeks old) with diarrhoea were collected during 2006 and 2007 from seven regions in the North Island of New Zealand. The samples were screened for the presence of Group A rotavirus antigen, using a commercial ELISA. Forty-one samples from different farms were randomly selected out of the 385 ELISA-positive samples and tested using PCR for the presence of G6, G8, and G10 genotypes of rotavirus. RESULTS: The PCR analysis of 41 antigen-positive field samples revealed that 37 (90%) contained genotype G6, three (7%) genotype G10, one sample (2%) had both G6 and G10 genotypes, and none contained genotype G8. CONCLUSIONS: Rotavirus genotype G6 was the predominant genotype found in this preliminary study and was present in all seven regions studied. Genotype G10 was also found in some regions of the North Island, whereas genotype G8 was not found in any sample. CLINICAL RELEVANCE: This is the first report on rotavirus G genotypes present in calves in the North Island of New Zealand.     

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.     

Molecular characterization of unusual bovine rotavirus A strains having high genetic relatedness with human rotavirus: evidence for zooanthroponotic transmission

We report here the genomic characterization of two rare rotavirus A (RVAs) G1P[11] and G9P[X] strains detected in cattle calves from two different geographical locations in India during routine rotavirus surveillance. These strains possessed unusual G types (VP7 gene) on a bovine/artiodactyl genotype constellation, G1‐P[11]‐I2‐Rx‐Cx‐Mx‐Ax‐N2‐T6‐E2‐H3 (HR‐B91) and G9‐P[X]‐I2‐Rx‐Cx‐Mx‐Ax‐N2‐T6‐E2‐H3 (WB‐H2). This is the first report on molecular characterization of G9 in cattle, and second report on G1 in cattle. The VP7 gene of HR‐B91 occupied lineage IIc within G1 while that of WB‐H2 occupied IIIb within G9 genotype. The latter was found to be very diverse from other RVA strains of G9 genotype, and this may emerge as a new genotype in due course. The study provides evidence of zooanthroponotic transmission of human G1 and G9 RVA genes to calves. Of note, the G9 genotype was found to serve as the ancestral genotype for G1. Phylogenetic analysis of remaining gene segments revealed close relatedness to artiodactyl or artiodactyl‐like human RVA strains. The findings of this study highlight the potential role of interspecies transmission and reassortment events in generating the rare rotavirus strains.     

Molecular characterization of unusual bovine group A rotavirus G8P[14] strains identified in western India: emergence of P[14] genotype

A total of 78 fecal specimens were collected from both apparently healthy (n=71) and diarrheic (n=7) cattle from an organized farm in Pune, western India in December 2007-January 2008. Three specimens tested positive for group A rotavirus (RV) by antigen capture ELISA were subjected to RT-PCR for amplification of entire coding regions of three structural (VP4, VP6 and VP7) and one nonstructural (NSP4) genes. All three strains were genotyped as G8P[14]. Phylogenetic analysis of the VP7 and VP4 genes showed clustering of the VP7 gene with G8 strains of bovine origin and VP4 gene with P[14] strains of human origin. The identification of VP6 and NSP4 genes to have I2 (subgroup I) and E2 (genotype A) specificity, respectively of bovine and human origin indicated independent segregation of genes in bovine RV strains. This study indicates circulation of a rare RV genotype, G8P[14] in western India. To our knowledge, this is the second report on RV G8[14] isolated from bovine species after bovine group A RV strain, SUN9 from Japan.     

Molecular epidemiology or bovine rotaviruses from the Charolais area.

Molecular epidemiology of bovine rotavirus from the Charolais area (France). Faecal samples from 164 diarrhoeic calves under 60 days of age were collected from the Charolais area of France during winter of 1998. The samples were tested by an enzyme-linked immunosorbent assay (ELISA) to detect the presence of rotavirus antigen. Of 164 dairy calves tested, 45.1% were positive for rotavirus antigen. The presence of rotavirus was confirmed by electrophoresis of genomic segments. Genomic segment 9 coding for the surface glycoprotein VP7 was amplified by RT-PCR using amplimeres corresponding to a conserved sequence located at the 5' and 3' ends. Nucleotides of the region 29 to 320-560 (average 427) was determined by the Taq dye deoxynucleotide cycle sequencing method. By comparison to the 175 sequences of gene 9 previously published, sequence analysis demonstrated that all of the isolates from the present study belong to the genotype G6. This result confirms previously published data indicating the prevalence of rotavirus G6 in bovine, and suggests that a monovalent vaccine based on G6 antigen would be sufficient to elicit a good protection.     

G and P genotype profiles of rotavirus A field strains circulating in beef and dairy cattle herds in Brazil, 2006–2015

The aim of this retrospective study was to use RT-PCR and nucleotide sequencing analysis to determine the G (VP7 gene) and P (VP4 gene) genotypes of 155 Brazilian bovine rotavirus A (RVA) wild-type strains detected in diarrheic calves from all Brazilian geographical regions from 2006 to 2015. The RVA strains evaluated belonged to the G6, G10, P[5], and P[11] genotypes. The G6P[5] genotype was prevalent (65.5%; P < 0.05) in beef, and the G10P[11] (38.4%) and G6P[11] (30.8%) genotypes were more prevalent in dairy cattle herds. The Midwest was the region with the highest number of genotyped RVA strains, where the genotypes G6, P[5], and P[11] were identified. Genotype combination G6-IV/P[5]-IX, prevalent in beef herds, and G6-III/P[11]-III or G10-IV/P[11]-III, prevalent in dairy herds, were detected. In addition, for the first time in Brazil, we detected the P[5] and P[11] genotype RVA strains that belong to lineage II and VII, respectively.     

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.     

Prevalence of serotypes G6 and G10 group A rotaviruses in dairy calves in Quebec.

Fecal samples from diarrheic and nondiarrheic dairy calves (1 to 3 weeks old) from 12 regions of Quebec, collected between 1992 and 1994, were screened for group A bovine rotavirus (BRV) using a combination of 2 VP6-specific monoclonal antibodies (MAbs) in an enzyme-linked immunosorbent assay (ELISA). The overall prevalence of BRV infection was 26.4% (107/405). In diarrheic calves, BRV infection reached 74.3% (55/74), but only 15.7% (52/331) in nondiarrheic calves. BRV-positive samples were serotyped by enzyme-linked immunosorbent assay (ELISA) using G6 and G10 specific MAbs. The analysis of 107 field samples revealed that, in diarrheic calves, 34.5% (19/55) were G6, 27.2% (15/55) were G10, 9% (5/55) were G6 and G10 positive, and 29.9% (16/55) were G6 and G10 negative. In nondiarrheic calves, 19.2% (10/52) were G6, 19.2% (10/52) were G10, 7.6% (4/52) were G6 and G10 positive, and 53.6% (28/52) were G6 and G10 negative. Rotavirus dsRNA was extracted from BRV-positive samples and examined by polyacrilamide gel electrophoresis (PAGE). Of 107 samples tested, 74 (69.1%) were positive, and all the samples demonstrated a typical group A rotavirus migration pattern.     

Recurrent rotavirus diarrhoea outbreaks in a stud farm, in Italy

A total of 47 stool samples were collected at the same stud farm from young foals with rotavirus diarrhoea and from their stud mares. Illness involved foals during three consecutive winter seasons. Infection in the farm appeared firstly in January-February 2008. After vanishing in the warm seasons, cases reappeared in March 2009 and 2010. Determination of the rotavirus G- and P-types was carried out using nested RT-PCR in samples collected in 2009 and 2010. A total of 19 of 47 samples resulted positive for rotavirus. The G type was determined in 19/47 samples, whereas the P genotype was determined in 17/47 samples. All equine strains presented a G14 VP7 in combination with a P[12] VP4, suggesting persistence of the same viral strain in the stud farm, during at least two consecutive winter periods. Sequence analysis of the genes encoding the outer capsid rotavirus proteins VP7 and VP4 revealed that the virus had a close relationship between strains recently isolated in the rest of Europe.     

Molecular characterization of bovine rotavirus circulating in beef and dairy herds in Argentina during a 10-year period (1994-2003)

Group A bovine rotavirus (BRV) is one of the main causes of neonatal calf diarrhea. The present study reports the incidence of rotavirus diarrhea and the genotypes of BRV strains circulating in beef and dairy herds from Argentina, during a 10-year period (1994-2003). Group A BRV was detected in 62.5% (250/400) of the total studied cases of diarrhea. Positive cases were analyzed by heminested multiplex RT-PCR for P and G genotypes identification. Sixty percent of them were typed as P[5]G6, 4.4% P[11]G10, 4.4% P[11]G6 and 2.4% P[5]G10. Additionally, 9.2% of the cases were initially typed as G8 combined with P[5] or P[11], but sequence analysis revealed they belonged to genotype G6, lineage Hun4-like. Partial typing was assessed in 12.0% of the cases. One of the partially typed samples was closely related to genotype G15. BRV was detected in 71% and 58% of the outbreaks registered in beef and dairy farms, respectively. A clear differential distribution of G/P types was found according to the herd type. P[5]G6 was the prevalent strain in beef herds, while P[11] was the prevalent P-type in dairy herds (71%), associated in similar proportions with G6 and G10, These findings indicate that BRV genotypes included in the current commercially available rotavirus vaccines (G6, G10, P[5] and P[11]) should protect calves from most Argentinean field strains. Nevertheless, continuous surveillance is necessary to detect the emergence of new variants.     

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.     

Isolation and molecular characterisation of equine rotaviruses from Germany

A total of 26 rotavirus positive faecal samples of diarrhoeal foals, and 8 equine rotavirus isolates were examined. Viral RNA patterns were generated, G typing was performed by PCR, and a P[12]-specific DNA probe was developed for P typing. Furthermore, five equine rotavirus isolates were sequenced in the genomic regions coding for VP7 and part of VP4. Rotaviruses of genotype G3 P[12] were found in 22 faecal samples and G14 P[12] type could be found in 4 faecal samples. These findings confirm that in Germany G3 P[12] is the predominating type of equine rotaviruses.     

Detection and characterisation of bovine rotavirus in Ireland from 2006–2008

Background

Worldwide, Group A bovine rotavirus (RVA boRV) is one of the main causes of neonatal calf diarrhoea. Currently, limited epidemiological and sequence data exists on the RVA disease in bovines in Southern Ireland only. The aim of the study was to generate epidemiological and sequence data of RVA boRV distributed over a wide geographical area in Ireland.

Findings

272 stool samples were obtained from symptomatic calves and analysed to identify the prevalent G and P genotypes. Viral type combinations including G6P[5], G6P[11] and G10P[11] genotype were the most frequently identified. The G6P[5] combination was predominant throughtout the study, accounting for 70% (n = 191). Sequence analysis of the VP7 gene revealed that Irish G6 strains fell within Lineage IV, similiar to previous reports in Ireland.

Conclusion

The detection of unusual G and P combinations may have an impact on rotavirus control programmes and current vaccines may need to incorporate new strains, as the current vaccine available may not offer protection against all of these circulating types.