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.     

我国部分地区牛轮状病毒的病原学调查及一株G10P[11]型牛轮状病毒的分离鉴定

应用轮状病毒RNA电泳方法,对内蒙古、黑龙江、新疆、北京、安徽等地共90份犊牛腹泻粪便样品进行检测.在内蒙古和新疆采集的粪样中共检出11份轮状病毒阳性样品,平均阳性率为12%(11/90).阳性样品经RT-PCR分型鉴定,新疆2份和内蒙古5份样品属于G10型,新疆另一牛场的4份样品属于G6型.对所有阳性样品进行病毒分离,获得1株能在MA104细胞上稳定传代的病毒株,经鉴定该分离株属于G10P[11]型轮状病毒,命名为HM26.G10型牛轮状病毒的分离在国内尚属首次.     

Genetic diversity and novel combinations of G4P[19] and G9P[19] porcine rotavirus strains in Thailand

Several epidemiological studies reported the detection of rotavirus strains bearing unusual combinations of genetic background of human and porcine rotaviruses. This observation supports the hypothesis of interspecies transmission of rotaviruses in humans and pigs. The aims of this study were to investigate the genotypes and molecular characteristics of rotaviruses in piglets with diarrhea in several farms from two provinces in Thailand. A total of 207 fecal specimens collected from diarrheic piglets were screened for the presence of groups A, B, and C rotaviruses. Group A rotaviruses were detected in 41 out of 207 (19.8%) fecal specimens tested. A wide variety of G-P combination rotavirus strains were detected in this study. The G4P[6] was identified as the most prevalent genotype (39.0%), followed by G4P[23] (12.2%), G3P[23] (7.3%), G4P[19] (7.3%), G3P[6] (4.9%), G3P[13] (4.9%), G3P[19] (4.9%), G9P[13] (4.9%), G9P[19] (4.9%), G5P[6], and G5P[13] each of 2.4%. Furthermore, G5 and G9 in combinations with P-nontypeable strains were also found at each consisting of 2.4% (n = 1) of the collection. It was interesting to note that among diversified porcine rotavirus strains, novel combinations of G4P[19] and G9P[19] strains were detected for the first time in this study. Nucleotide sequences of VP4 and VP7 of these strains were closely related to human rotaviruses reported previously. The data implies that these porcine rotaviruses were probably generated in nature from the reassortment between the viruses of human and porcine origin. This study provides valuable epidemiological information and molecular characteristics of porcine rotaviruses circulating in piglets with diarrhea in northern Thailand.     

Novel group A rotavirus G8 P[1] as primary cause of an ovine diarrheic syndrome outbreak in weaned lambs

Rotavirus is a worldwide major cause of diarrhea outbreaks in neonatal ruminants. An outbreak of ovine diarrheic syndrome (ODS) in 50-75 days-old lambs (weaned lambs) is described. Fecal immunochromatography and intestinal immunohistochemistry for rotavirus group A were performed. In addition, semi-nested multiplex RT-PCR for G and P rotavirus genotyping in combination with sequencing were performed, to support the diagnosis and identify the viral strain. A novel ovine rotavirus group A G8 P[1] strain was determined as the main cause of the ODS observed, whereas other pathogens were ruled out.     

Reassortment among bovine, porcine and human rotavirus strains results in G8P[7] and G6P[7] strains isolated from cattle in South Korea

Group A rotaviruses (GARVs) cause severe acute gastroenteritis in children and young animals. Although zoonotic infections with bovine-like G6 and G8 GARVs have been reported in many countries, there is little evidence for reassortment between bovine GARVs and GARVs from heterologous species. The finding of bovine GARVs with the G6 and G8 genotypes in combination with the typical porcine P[7] prompted us to characterize all 11 genes of 30 bovine GARVs isolated from clinically infected calves. By the comparison of the full-length ORF of VP7 and NSP1-5, and the partial VP1-4 and VP6 nucleotide sequences between the 30 Korean and other known strains, three different genome constellations were found. Twenty seven strains showed the G8-P[7]-I5-R1-C1-M2-A1-N1-T1-E1-H1 genotypes, a single strain possessed the G6-P[7]-I2-R2-C1-M2-A1-N2-T1-E2-H1 genotype constellation and 2 strains the G6-P[7]-I2-R2-C2-M2-A3-N2-T6-E2-H3 genotype constellation. The complete genome of a single reference strains for each of these three genotype constellations (KJ25, KJ9-1 and KJ19-2) was determined and analyzed. A detailed phylogenetic analysis revealed a complicated picture, with several reassortments among bovine-like, porcine-like and human-like GARV strains, resulting in several different reassortant strains successfully infecting cattle.     

Prevalence of rotavirus A infection and the detection of type G3P[11] strain in ruminants in Yobe state,Nigeria

Rotaviruses have a worldwide distribution and the infection is associated with diarrhea in young of ruminants as well as children. However, limited data exist on its prevalence and types in Yobe state, Nigeria. Detection of rotavirus A and types in ruminant population in Yobe state was the aim of the study. A total of 470 diarrheic fecal samples were collected and tested for rotavirus and types using serology and molecular techniques respectively. A prevalence rate of 2.98% (14/470) was found in the three species with specific rates of 2.9% (6/202), 3.8% (6/158), and 1.8% (2/110) in goat, sheep, and cattle respectively. The prevalence rates of 3.6% (12/331), 1.2% (1/84), and 1.8% (1/55) were for those aged < 1–3, 4–6, and 7–9 months old, respectively, while 4.9% (9/185) and 1.7% (5/285) were in males and females respectively. Rotavirus genes VP7 and VP4 were detected in 2 (14.3%) out of the 14 ELISA-positive samples while deduced amino acid sequences of the major variable regions revealed the genes to belong to types G3P[11] strain. Significant association was found between the infection and sex (P < 0.05) unlike in the species and age groups of the ruminants. The circulation of rotavirus virus in ruminants and type G3P[11] in cattle has been confirmed in the study. Hence, there is a need for continuous surveillance, awareness campaign, and assessment of the economic losses and public health implications of rotavirus infection in Nigeria.

     

一株牦牛源G6P[11]型牛轮状病毒的分离鉴定及部分基因的序列分析

本研究的目的是分离、鉴定牦牛源牛轮状病毒(bovine rotavirus, BRV)。将经RT-PCR检测BRV阳性的牦牛腹泻粪便样本接种MA-104细胞,进行病毒分离和鉴定,并对其VP4、VP6和VP7完整基因进行测序,分析其分子特征。结果显示:病毒盲传3代后出现细胞病变,至第7代后出现细胞病变的时间稳定,经蚀斑纯化后测得病毒滴度为10^8.39TCID₅₀·mL^-1。分离株经RT-PCR、间接免疫荧光和电镜观察证实为BRV,命名为HY-1株;扩增HY-1株VP4、VP6和VP7完整基因序列,分析表明HY-1株为G6P[11]I2型;系统发育树显示,HY-1株VP4、VP6和VP7节段分别与中国牛源DQ-75株和印度牛源M-1和RUBV319株遗传演化关系最近,可能为重配毒株。与国内的G6型和P[11]型BRV毒株相比,HY-1株的VP4和VP7蛋白重要氨基酸位点变化较大。成功分离得到一株牦牛源BRV,基因型为G6P[11]型,为我国首次报道。     

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.     

Identification of a G2-like porcine rotavirus bearing a novel VP4 type, P[32]

A porcine group A rotavirus (GARV) strain, 61/07/Ire, was isolated from a 4–5 week asymptomatic piglet, during an epidemiological survey of porcine herds in Southern Ireland, in 2007. The nucleotide (nt) and amino acid (aa) sequence of the full-length VP4 protein of the PoRV strain 61/07/Ire was determined. Based on the entire VP4 open reading frame (nt), strain 61/07/Ire displayed ≤ 76.5% identity to representatives of the established 31 P-types, a value far lower than the percentage identity cutoff value (80%) established by the Rotavirus Classification Working Group (RCWG) to define a novel P genotype. Strain 61/07/Ire revealed low aa identity, ranging from 57.1% to 83.6%, to the cognate sequences of representatives of the various P genotypes. The aa identity was lower in the VP8* trypsin-cleavage fragment of the VP4, which encompasses the VP4 hypervariable region, ranging from 36.9% to 75.3%. Sequence analyses of the VP7, VP6, and NSP4 genes revealed that the GARV strain 61/07/Ire possessed a G2-like VP7, an E9 NSP4 genotype and an I5 VP6 genotype. Altogether, these results indicate that the GARV strain 61/07/Ire should be considered as a prototype of a new VP4 genotype, P[32], and provide further evidence for the vast heterogeneity of group A rotaviruses.     

Detection of a mammalian-like group A rotavirus in diarrhoeic chicken

The present investigation describes detection of a mammalian-like electropherogroup A rotavirus in chicken with diarrhoea. This also records the first detection of a rotavirus in an avian species from India. During the investigation 75 diarrhoeic faecal samples collected from adult chicken were screened for the presence of group A rotavirus antigen by sandwich ELISA. All three samples positive for rotavirus antigen revealed 11 bands of RNA in polyacrylamide gel electrophoresis (PAGE). In contrast to avian group A rotavirus, segment 5 was found to migrate closer to 6 as is the case with mammalian group A rotaviruses. Segments 7, 8 and 9 were found to migrate as a tight triplet, which is characteristic of group A rotavirus.     

Detection of rotavirus antigen in diarrhoeic and non-diarrhoeic piglets in Nigeria

Faecal samples from 96 piglets with acute gastroenteritis and 41 samples from non-diarrhoeic piglets were investigated for porcine rotavirus (PRV) antigens by enzyme-linked immunosorbent assay (ELISA). PRV was detected in 43 of 96 (44.8%) in the diarrhoeic group whilst none of the samples in the non-diarrhoeic group was positive for PRV (P less than 0.01). Age distribution of infection showed a higher infection rate of 30.2% in piglets aged 1-3 weeks, which was significantly higher (P less than 0.05) than in piglets aged 4-6 weeks, where the infection rate was 14.6 per cent. No PRV antigen was detected in diarrhoeic piglets above 6 weeks of age. Titration of 10 randomly selected ELISA-positive samples showed titres ranging from 512 to greater than or equal to 4.096.     

Detection and genetic characterization of porcine group A rotaviruses in asymptomatic pigs in smallholder farms in East Africa: Predominance of P[8] genotype resembling human strains

Viral enteritis is a serious problem accounting for deaths in neonatal animals and humans worldwide. The absence of surveillance programs and diagnostic laboratory facilities have resulted in a lack of data on rotavirus associated diarrheas in pigs in East Africa. Here we describe the incidence of group A rotavirus (RVA) infections in asymptomatic young pigs in East Africa. Of the 446 samples examined, 26.2% (117/446) were positive for RVA. More nursing piglets (78.7%) shed RVA than weaned (32.9%) and grower (5.8%) pigs. RVA incidence was higher in pigs that were either housed_free-range (77.8%) or tethered_free-range (29.0%) than those that were free-range or housed or housed-tethered pigs. The farms with larger herd size (>10 pigs) had higher RVA prevalence (56.5%) than farms with smaller herd size (24.1–29.7%). This study revealed that age, management system and pig density significantly (p < 0.01) influenced the incidence of RVA infections, with housed_free-range management system and larger herd size showing higher risks for RVA infection. Partial (811–1604 nt region) sequence of the VP4 gene of selected positive samples revealed that different genotypes (P[6], P[8] and P[13]) are circulating in the study area with P[8] being predominant. The P[6] strain shared nucleotide (nt) and amino acid (aa) sequence identity of 84.4–91.3% and 95.1–96.9%, respectively, with known porcine and human P[6] strains. The P[8] strains shared high nt and aa sequence identity with known human P[8] strains ranging from 95.6–100% to 92–100%, respectively. The P[13] strains shared nt and aa sequence identity of 83.6–91.7% and 89.3–96.4%, respectively, only with known porcine P[13] strains. No P[8] strains yielded RNA of sufficient quality/quantity for full genome sequencing. However analysis of the full genome constellation of the P[6], two P[13] and one untypeable strains revealed that the P[6] strain (Ke-003-5) genome constellation was G26-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1, P[13] strains (Ug-049 and Ug-453) had G5-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1 while the untypeable strain (Ug-218) had G5-P[?]-I5-R1-C1-M1-A8-N1-T1-E1-H? In conclusion, P[6] and P[8] genotypes detected were genetically closely related to human strains suggesting the possibility of interspecies transmission. Further studies are required to determine the role of RVA in swine enteric disease burden and to determine the genetic/antigenic heterogeneity of the circulating strains for development of accurate diagnostic tools and to implement appropriate prophylaxis programs.     

Lapine rotaviruses of the genotype P[22] are widespread in Italian rabbitries

An epidemiological survey was carried out to investigate the distribution of the VP7 and VP4 specificities of lapine rotaviruses (LRVs) in rabbitries from different geographical regions of Italy. Almost all the strains were characterized as P[22],G3, confirming the presence of the newly-recognized rotavirus P[22] VP4 allele in Italian rabbits. Only one P[14],G3 LRV strain was identified and two samples contained a mixed (P[14] + [22],G3) rotavirus infection. All the LRV strains analyzed exhibited a genogroup I VP6 specificity and a long dsRNA electropherotype. However, one of the P[14],G3 strains possessed a super-short pattern. Altogether, these data highlight the epidemiological relevance of the P[22] LRVs in Italian rabbitries.     

Frequency of group A rotavirus with mixed G and P genotypes in bovines: predominance of G3 genotype and its emergence in combination with G8/G10 types

The present study describes the genotypic distribution of rotaviruses (RVs) in an Indian bovine population with unexpectedly higher proportions of G3 alone or in combination of G8/G10. PCR-genotyping confirmed that 39.4% (13/33) of the prevalent RVs were the G3 type while 60.6% (20/33) were dual G3G10 or G3G8 types. P typing revealed that 93.9% (31/33) of the samples were P[11] while 6.1% (2/33) possessed a dual P[1]P[11] type. Sequence analysis of the VP7 gene from G3 strains viz. B-46, 0970, and BR-133 showed that these strains had sequence identities of 90.5% to 100% with other bovine G3 strains. The highest identity (98.9% to 100%) was observed with RUBV3 bovine G3 strains from eastern India. The G3 strains (B-46, 0970, and BR-133) showed 97.5% to 98.8% sequence homologies with the Indian equine RV strain Erv-80. Phylogenetic analysis demonstrated that G3 strains clustered with bovine RUBV3 and J-63, and equine Erv-80 G3. Overall, these results confirmed that the incidence of infection by RVs with the G3 genotype and mixed genotypes in the bovine population was higher than previously predicted. This finding reinforces the importance of constantly monitoring circulating viral strains with the G3 genotype in future surveillance studies.     

Antibody response in vaccinated pregnant mares to recent G3BP[12] and G14P[12] equine rotaviruses

Background

Both the G3P[12] and the G14P[12] type of equine group A rotavirus (RVA) have recently become predominant in many countries, including Japan. G3 types are classified further into G3A and G3B. The G3A viruses have been circulating in Europe, Australia, and Argentina, and the G3B viruses have been circulating in Japan. However, only an inactivated vaccine containing a single G3BP[12] strain is commercially available in Japan. To assess the efficacy of the current vaccine against recently circulating equine RVA strains, we examined antibody responses in pregnant mares to recent G3BP[12] and G14P[12] strains by virus neutralization test.

Findings

After vaccination in five pregnant mares, the geometric mean serum titers of virus-neutralizing antibody to recent G3BP[12] strains increased 5.3- to 7.0-fold and were similar to that against homologous vaccine strain. Moreover, antibody titers to recent G14P[12] strains were also increased 3.0- to 3.5-fold.

Conclusions

These results suggest that inoculation of mares with the current vaccine should provide foals with virus-neutralizing antibodies against not only the G3BP[12] but also the G14P[12] RVA strain via the colostrum.     

A rotavirus in lambs with diarrhoea.

A reovirus-like agent was identified from an outbreak of enteritis in young lambs. From its morphology and immunological relationship with calf rotavirus, it was concluded that it was a rotavirus which infects lambs.