Complex evolutionary patterns of two rare human G3P[9] rotavirus strains possessing a feline/canine-like H6 genotype on an AU-1-like genotype constellation

The group A rotavirus (RVA) G3P[9] is a rare VP7–VP4 genotype combination, detected occasionally in humans and cats. Other than the prototype G3P[9] strain, RVA/Human- tc/JPN/AU-l/1982/G3P3[9], the whole genomes of only two human G3P[9] RVA strains and two feline G3P[9] RVA strains have been analyzed so far, revealing complex evolutionary patterns, distinct from that of AU-1. We report here the whole genomic analyses of two human G3P[9] RVA strains, RVA/Human-tc/CHN/L621/2006/G3P[9] and RVA/Human-wt/CHN/E2451/2011/G3P[9], detected in patients with diarrhea in China. Strains L621 and E2451 possessed a H6 NSP5 genotype on an AU-1-like genotype constellation, not reported previously. However, not all the genes of L621 and E2451 were closely related to those of AU-1, or to each other, revealing different evolutionary patterns among the AU-1-like RVAs. The VP7, VP4, VP6 and NSP4 genes of E2451 and L621 were found to cluster together with human G3P[9] RVA strains believed to be of possible feline/canine origin, and feline or raccoon dog RVA strains. The VP1, VP3, NSP2 and NSP5 genes of E2451 and L621 formed distinct clusters in genotypes typically found in feline/canine RVA strains or RVA strains from other host species which are believed to be of feline/canine RVA origin. The VP2 genes of E2451 and L621, and NSP3 gene of L621 clustered among RVA strains from different host species which are believed to have a complete or partial feline/canine RVA origin. The NSP1 genes of E2451 and L621, and NSP3 gene of E2451 clustered with AU-1 and several other strains possessing a complete or partial feline RVA strain BA222-05-like genotype constellation. Taken together, these observations suggest that nearly all the eleven gene segments of G3P[9] RVA strains L621 and E2451 might have originated from feline/canine RVAs, and that reassortments may have occurred among these feline/canine RVA strains, before being transmitted to humans.     

Analysis of complete genome sequences of G9P[19] rotavirus strains from human and piglet with diarrhea provides evidence for whole-genome interspecies transmission of nonreassorted porcine rotavirus

Whole genomes of G9P[19] human (RVA/Human-wt/THA/CMH-S070-13/2013/G9P[19]) and porcine (RVA/Pig-wt/THA/CMP-015-12/2012/G9P[19]) rotaviruses concurrently detected in the same geographical area in northern Thailand were sequenced and analyzed for their genetic relationships using bioinformatic tools. The complete genome sequence of human rotavirus RVA/Human-wt/THA/CMH-S070-13/2013/G9P[19] was most closely related to those of porcine rotavirus RVA/Pig-wt/THA/CMP-015-12/2012/G9P[19] and to those of porcine-like human and porcine rotaviruses reference strains than to those of human rotavirus reference strains. The genotype constellation of G9P[19] detected in human and piglet were identical and displayed as the G9-P[19]-I5-R1-C1-M1-A8-N1-T1-E1-H1 genotypes with the nucleotide sequence identities of VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4, and NSP5 at 99.0%, 99.5%, 93.2%, 97.7%, 97.7%, 85.6%, 89.5%, 93.2%, 92.9%, 94.0%, and 98.1%, respectively. The findings indicate that human rotavirus strain RVA/Human-wt/THA/CMH-S070-13/2013/G9P[19] containing the genome segments of porcine genetic backbone is most likely a human rotavirus of porcine origin. Our data provide an evidence of interspecies transmission and whole-genome transmission of nonreassorted G9P[19] porcine RVA to human occurring in nature in northern Thailand.     

The origin of two rare human P[10] rotavirus strains

The Group A rotavirus (RVA) P[10] is a rare genotype of the RVA VP4 gene. To date, the whole genome sequence of only a single P[10] RVA strain, RVA/Human-tc/IDN/69M/1980/G8P4[10], has been determined, revealing a DS-1-like genotype constellation. Whole genomic analyses of P[10] RVA strains with other VP7 genotypes are essential to obtain conclusive data on the origin and genetic diversity of the P10] RVAs. In the present study, the whole genome of a human G4P[10] RVA strain, RVA/Human-tc/IDN/57M/1980/G4P[10], was analyzed. Strain 57M exhibited an unusual G4-P[10]-I1-R1-C1-M1-A1-N1-T2-E1-H2 genotype constellation, and was found to originate from intergenogroup reassortment events involving acquisition of RVA strain 69M-like VP4, NSP3 and NSP5 genes by a co-circulating Wa-like human G4 RVA strain. Although the reference P[10] strain, 69M, exhibits a DS-1-like genotype constellation, the exact origin of this RVA remains to be elucidated. By detailed phylogenetic analyses, we found that the VP1-VP3, VP6, NSP2 and NSP4 genes of 69M originated from artiodactyl and/or artiodactyl-like human P[14] strains, whilst its NSP1, NSP3 and NSP5 genes were more related to those of typical human DS-1-like strains than those of other RVAs. On the other hand, the origin of the VP4 gene of 69M could not be established. Nevertheless, these observations clearly indicated that strain 69M might have originated from reassortment events involving at least the artiodactyl or artiodactyl-like human RVAs and the typical human DS-1-like strains. The present study provided rare evidence for intergenogroup reassortment events involving co-circulating typical human Wa-like RVAs and unusual RVAs of the DS-1-like genogroup, and revealed the presence of artiodactyl-like genes in a human P[10] strain, highlighting the complex evolutionary patterns of the P[10] RVAs.     

Genetic characterization of a novel G3P[14] rotavirus strain causing gastroenteritis in 12 year old Australian child

A genotype G3P[14] rotavirus strain was identified in a 12 year old child presenting to the Emergency Department of the Royal Children’s Hospital, Melbourne, with gastroenteritis. G3P[14] strains have been previously identified in rabbits in Japan, China, the USA and Italy and a single lapine-like strain from a child in Belgium.Full genome sequence analysis of RVA/Human-wt/AUS/RCH272/2012/G3P[14] (RCH272) revealed that the strain contained the novel genome constellation G3-P[14]-I2-R3-C3-M3-A9-N2-T6-E2-H3. The genome was genetically divergent to previously characterized lapine viruses and the genes were distantly related to a range of human bovine-like strains and animal strains of bovine, bat and canine/feline characteristics. The VP4, VP6, NSP2, NSP3, NSP4 and NSP5 genes of RCH272 clustered within bovine lineages in the phylogenetic analysis and shared moderate genetic similarity with an Australian bovine-like human strain RVA/Human-tc/AUS/MG6/1993/G6P[14]. Bayesian coalescent analysis suggested these genes of RCH272 and RVA/Human-tc/AUS/MG6/1993/G6P[14] were derived from a population of relatively homogenous bovine-like ancestral strains circulating between 1943 and 1989. The VP7, VP1, VP2 and NSP1 genes shared moderate genetic similarity with the Chinese strain RVA/Bat-tc/CHN/MSLH14/2011/G3P[3] and the VP3 gene clustered within a lineage comprised of canine and feline strains.This strain may represent the direct transmission from an unknown host species or be derived via multiple reassortment events between strains originating from various species. The patient lived in a household containing domesticated cats and dogs and in close proximity to a colony of Gray-headed Flying-foxes. However, without screening numerous animal populations it is not possible to determine the origins of this strain.     

Whole-genomic analysis of G3P[23], G9P[23] and G3P[13] rotavirus strains isolated from piglets with diarrhea in Thailand, 2006–2008

Group A rotavirus (RVA) is the most common cause of severe acute viral gastroenteritis in humans and animals worldwide. This study characterized the whole genome sequences of porcine RVAs, 2 G3P[23] strains (CMP40/08 and CMP48/08), 1 G9P[23] strain (CMP45/08), and 1 G3P[13] strain (CMP29/08). These strains were collected from diarrheic piglets less than 7 weeks of age in 4 pig farms in Chiang Mai, Thailand, in 2008. The VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes of CMP40/08 and CMP48/08 strains were assigned as G3-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1 genotypes based on their nucleotide sequences and phylogenetic analyses. The CMP29/08 strain was different from the CMP40/08 and CMP48/08 strains only in the VP4 gene, since it was assigned as P[13] genotype. Furthermore, the VP7 gene of the CMP45/08 strain was classified as genotype G9, and the NSP3 gene as T7 genotype. The finding of this study supports the porcine-origin of T7 genotype, although the NSP3 gene of this strain was similar to the bovine UK strain at the highest nucleotide sequence identity of 92.6%. Whole genome sequence analysis of the porcine RVAs indicated that multiple inter-genotypic and intra-genotypic reassortment events had occurred among the porcine RVAs circulating in this studied area. Interestingly, the VP7 gene of the CMP45/08 strain, and the VP1, NSP2, and NSP4 genes of all four porcine RVAs strains described in this study revealed much similarity to those of two porcine-like human RVA strains (RVA/Human-tc/THA/Mc323/1989/G9P[19] and RVA/Human-tc/THA/Mc345/1989/G9P[19]) detected in Thailand in 1989. The present study provided important information on the evolution of porcine RVA.     

Full genomic analysis of rabbit rotavirus G3P[14] strain N5 in China: Identification of a novel VP6 genotype

Group A rotaviruses (RVAs) are major pathogens associated with acute gastroenteritis in young children and in a wide variety of domestic animals. The full-length genome of a rabbit RVA strain, RVA/Rabbit-tc/CHN/N5/1992/G3P[14], showed a G3-P[14]-I17-R3-C3-M3-A9-N1-T1-E3-H2 genomic configuration. A novel VP6 genotype, I17, was confirmed by the Rotavirus Classification Working Group. Phylogenetic analyses revealed that strain N5 possessed VP1–3, VP7, NSP1–2 and NSP4 genes closely related to those of the simian strain TUCH, NSP3 and NSP5 genes closely related to the human strains Wa and 69M, and a VP4 gene closely related to the rabbit strain 30/96 and sheep strain OVR762. The RRV and TUCH shared their ancestry with canine/feline RVAs and showed a close relationship to the human T152/feline-like RVAs. Comparison with the genotypes of the simian strains TUCH and RRV, canine strains A79-10, CU-1, K9, feline strains Cat2 and Cat97, and human strains T152 and 69M showed that RVA/Rabbit-tc/CHN/N5/1992/G3P[14] was possibly of feline/canine origin, or was a multiple reassortment involving canine, feline and human rotaviruses. The sequencing and phylogenetic analyses of rotavirus genomes is critical to the elucidation of the patterns of virus evolution.     

Whole genomic analyses of asymptomatic human G1P[6], G2P[6] and G3P[6] rotavirus strains reveal intergenogroup reassortment events and genome segments of artiodactyl origin

Although P[6] group A rotaviruses (RVA) cause diarrhoea in humans, they have been also associated with endemics of predominantly asymptomatic neonatal infections. Interestingly, strains representing the endemic and asymptomatic P[6] RVAs were found to possess one of the four common human VP7 serotypes (G1–G4), and exhibited little antigenic/genetic differences with the VP4 proteins/VP4 encoding genome segments of P[6] RVAs recovered from diarrhoeic children, raising interest on their complete genetic constellations. In the present study, we report the overall genetic makeup and possible origin of three such asymptomatic human P[6] RVA strains, RVA/Human-tc/VEN/M37/1982/G1P2A[6], RVA/Human-tc/SWE/1076/1983/G2P2A[6] and RVA/Human-tc/AUS/McN13/1980/G3P2A[6]. G1P[6] strain M37 exhibited an unusual genotype constellation (G1-P[6]-R1-C1-M1-A1-N1-T2-E1-H1), not reported previously, and was found to originate from possible intergenogroup reassortment events involving acquisition of a DS-1-like NSP3 encoding genome segment by a human Wa-like RVA strain. On the other hand, G2P[6] strain 1076 exhibited a DS-1-like genotype constellation, and was found to possess several genome segments (those encoding VP1, VP3, VP6 and NSP4) of possible artiodactyl (ruminants) origin on a human RVA genetic backbone. The whole genome of G3P[6] strain McN13 was closely related to that of asymptomatic human Wa-like G3P[6] strain RV3, and both strains shared unique amino acid changes, which might have contributed to their attenuation. Taken together, the present study provided insights into the origin and complex genetic diversity of P[6] RVAs possessing the common human VP7 genotypes. This is the first report on the whole genomic analysis of a G1P[6] RVA strain.     

Characterization of human rotaviruses circulating in Iraq in 2008: Atypical G8 and high prevalence of P[6] strains

Fecal samples from 976 children with gastroenteritis were collected and analyzed for group A rotavirus (RVA), in three different cities in Iraq between January 2008 and December 2008. RVA antigen was detected in 394 (40%) of the samples, and 98 samples were available for further genotype analyses using multiplex RT-PCR and sequence analyses for untypeable strains. The G/P-genotype combination was determined for 69 samples, and 19, 2 and 8 samples remained P-untypeable, G-untypeable and G/P-untypeable (UT), respectively. The most prevalent genotype was G2 (40%, 39/98) most often associated with P[6]. G1 was the second most common genotype (16%, 16/98) mainly associated with P[8] and P[UT]. G3, G4 and G9 were detected at a lower prevalence (3%, 11%, 3%, respectively), mainly associated with P[6]. Surprisingly, five G8P[6], and seven G12 RVA strains in combination with P[6] and P[8] were also detected for the first time in Iraq. Overall, a striking high prevalence of 47% of the analyzed samples possessed the P[6] genotype (65% of the P-typed RVA strains). Atypical genotype combinations such as G1P[4], G1P[6], G2P[8] or strains with mixed G-types were detected sporadically. The detection of unusual G8P[6] RVA strains prompted us to further analyze the NSP2, NSP3, NSP4 and NSP5 gene segments of three selected G8P[6] strains, resulting in their designation to the N2, T2, E2 and H2 genotypes, respectively. The VP7, VP4, NSP2, NSP3 and NSP5 gene segments clustered closely with common human RVA strains, whereas the NSP4 gene sequences were found to cluster with animal derived RVA strains, suggesting a potential reassortment event. The high prevalence of RVA strains with the G8, G12 and P[6] genotypes in combination with a DS-1-like genotype constellation in Iraq, needs to be monitored closely as these RVA strains might challenge the effectiveness of current RVA vaccines.     

Whole genomic analysis reveals the porcine origin of human G9P[19] rotavirus strains Mc323 and Mc345

The group A rotavirus (RVA) P[19] is a rare P-genotype of the RVA VP4 gene, reported so far in humans and pigs. Whole genomic analyses of P[19] strains are essential to study their origin and evolutionary patterns. To date, all the 11 genes of only two P[19] strains, RVA/Human-wt/IND/RMC321/1990/G9P[19] and RVA/Human-wt/IND/mani-97/2006/G9P[19], have been analyzed, providing evidence for their porcine origin. In the present study, the whole genomes of the first reported human P[19] strains, RVA/Human-tc/THA/Mc323/1989/G9P[19] and RVA/Human-tc/THA/Mc345/1989/G9P[19], were analyzed. Strains Mc323 and Mc345 exhibited a G9-P[19]-I5-R1-C1-M1-A8-N1-T1-E1-H1 genotype constellation. With the exception of the NSP5 gene, both the strains were closely related to each other. Most of the genes of Mc323 (VP2-4, VP6-7, NSP1-4 genes) and Mc345 (VP2-4, VP6-7 and NSP1-5 genes) appeared to be of porcine origin, whilst the exact origin of VP1 and NSP5 genes of Mc323 and VP1 gene of Mc345 could not be ascertained. Therefore, strains Mc323 and Mc345 were found to have a porcine RVA genetic backbone, and are likely of porcine origin. Taken together, our observations corroborated the hypothesis that P[19] strains might be derived from porcine RVAs, providing important insights into the origin of P[19] strains, and on interspecies transmission of RVAs.     

Prevalence and genome characterization of porcine rotavirus A in southern Mozambique

Rotavirus A (RVA) is an important pathogen causing gastroenteritis in many species, including humans and pigs. The objective of this study was to determine the prevalence of RVA in pigs from smallholdings and commercial farms in southern Mozambique and characterize the complete genomes of selected strains. RVA was detected at a rate of 11.8% (n = 288), of which 7.6% was detected at commercial farms and 4.2% at smallholdings. The whole genomes of eight rotavirus strains were determined using an Illumina MiSeq platform. Seven displayed a G9P[13] and one a G4P[6] genotype combination, all with a typical porcine backbone (I1/5-R1-C1-M1-A1/8-N1-T1/7-E1-H1). Phylogenetic analysis indicated that the seven G9P[13] strains were in fact one strain that circulated on a commercial pig farm. The genome segments of this strain clustered with diverse segments of human and porcine RVA strains from various Asian countries. Analysis of the G4P[6] strain revealed four distinct genome segments (VP2, VP4, VP6 and VP7) and five genome segments closely related to South African porcine rotavirus strains (NSP1, NSP3, NSP4, NSP5 and VP1). These results suggest that both the G4P[6] and the G9P[13] strains possibly emerged through multiple reassortment events. The presence of these strains on the commercial farms and smallholdings calls for a more in-depth surveillance of rotavirus in Mozambique.     

Multiple reassortment and interspecies transmission events contribute to the diversity of feline, canine and feline/canine-like human group A rotavirus strains

RNA–RNA hybridization assays and complete genome sequence analyses have shown that feline rotavirus (FRV) and canine rotavirus (CRV) strains display at least two distinct genotype constellations (genogroups), represented by the FRV strain RVA/Cat-tc/AUS/Cat97/1984/G3P[3] and the human rotavirus (HRV) strain RVA/Human-tc/JPN/AU-1/1982/G3P3[9], respectively. G3P[3] and G3P[9] strains have been detected sporadically in humans. The complete genomes of two CRV strains (RVA/Dog-tc/ITA/RV198-95/1995/G3P[3] and RVA/Dog-tc/ITA/RV52-96/1996/G3P[3]) and an unusual HRV strain (RVA/Human-tc/ITA/PA260-97/1997/G3P[3]) were determined to further elucidate the complex relationships among FRV, CRV and HRV strains. The CRV strains RV198-95 and RV52-96 were shown to possess a Cat97-like genotype constellation. However, 3 and 5 genes of RV198-95 and RV52-96, respectively, were found in distinct subclusters of the same genotypes, suggesting the occurrence of reassortment events among strains belonging to this FRV/CRV/HRV genogroup. Detailed phylogenetic analyses of the HRV strain PA260-97 showed that (i) 8 genome segments (VP3, VP4, VP6, VP7 and NSP2-5) clustered closely with RV198-95 and/or RV52-96; (ii) 2 genome segments (VP1 and VP2) were more closely related to HRV AU-1; and (iii) 1 genome segment (NSP1) was distantly related to any other established NSP1 genotypes and was ratified as a new NSP1 genotype, A15. These findings suggest that the human strain PA260-97 has a history of zoonotic transmission and is likely a reassortant among FRV/CRV strains from the Cat97 and AU-1-like genogroups. In addition, a potential third BA222-05-like genogroup of FRV and HRV strains should be recognized, consisting of rotavirus strains with a stable genetic genotype constellation of genes also partially related to bovine rotavirus (BRV) and bovine-like rotaviruses. The detailed phylogenetic analysis indicated that three major genotype constellations exist among FRV, CRV and feline/canine-like HRV strains, and that reassortment and interspecies transmission events contribute significantly to their wide genetic diversity.     

Changes in the distribution of lineage constellations of G2P[4] Rotavirus A strains detected in Japan over 32 years (1980–2011)

Rotavirus A (RVA) is a leading cause of acute gastroenteritis in young children worldwide. Most human RVA strains are classified into three major genotype constellations: Wa-like, DS-1-like and AU-1-like. The evolution of G2P[4] strains possessing the DS-1-like genetic background was described in a few recent studies. However, the strains analyzed in these studies were almost exclusively the ones detected after 2000. In recognition of the scarcity of G2P[4] strains detected before 2000 for which whole genome information was available, this study was undertaken to characterize 19 Japanese G2P[4] strains detected between 1983 and 1990 (14 strains) and between 2001 and 2011 (5 strains), and to compare them with 131 G2P[4] strains from across the world. The Japanese strains along with the strains elsewhere in the world underwent stepwise changes from lineage I to IVa in 5 genes (the VP7, VP4, VP2, NSP1 and NSP5 genes) and from lineage I to V in 6 genes (the VP6, VP1, VP3, NSP2, NSP3 and NSP4 genes). Furthermore, G2P[4] strains detected after 2004 appeared to have undergone further intragenotype reassortment, resulting in the emergence of lineage V in the VP7 gene, and VI and VII in the VP3 and NSP4 genes. The time of the most recent common ancestor (tMRCA) for the emergent lineages VI and VII was estimated to be around the early 2000s. However, the year when the ancestor of the emergent lineages diverged from that of the rest of the lineages in the respective genes preceded the tMRCA 80–90 years. The origin of the emergent lineages is likely to be human RVA strains possessing genotypes other than G2P[4], and not RVA strains of an animal origin. In conclusion, stepwise changes in lineages imparted new genomic constellations to G2P[4] strains, which appears to have contributed to their successful spread across the globe, most notably since 2004.     

Multispecies reassortant bovine rotavirus strain carries a novel simian G3-like VP7 genotype

Rotavirus-A (RVAs), are the major cause of severe gastroenteritis in the young of mammals and birds. RVA strains possessing G6, G8, and G10 genotypes in combination with P[1] or P[11] have been commonly detected in cattle. During a routine surveillance for enteric viruses in a bovine population on North-Western temperate Himalayan region of India, an uncommon bovine RVA strain, designated as RVA/Cow-wt/IND/M1/09/2009 was detected in a diarrhoeic crossbred calf. The examination of nearly complete genome sequence of this RVA strain revealed an unusual G-P combination (G3P[11]) on a typical bovine RVA genotype backbone (I2-R2-C2-M2-A11-N2-T6-E2-H3). The VP7 gene of M1/09 isolate displayed a maximum nucleotide sequence identity of 73.8% with simian strain (RVA/Simian-tc/USA/RRV/1975/G3P[3]). The VP4 and NSP5 genes clustered with an Indian pig strain, RVA/Pig-wt/IND/AM-P66/2012/G10P[11] (99.6%), and a caprine strain, RVA/Goat-tc/BGD/GO34/1999/G6P[1] (98.9%) from Bangladesh, respectively, whilst the, VP6, NSP1, NSP3 and NSP4 genes were identical or nearly identical to Indian bovine strains (RVA/Cow-wt/IND/B-72/2008/G10P[X], RVA/Cow-wt/IND/B85/2010/GXP[X], and RVA/Cow-wt/IND/C91/2011/G6P[X]). The remaining four genes (VP1, VP2, VP3 and NSP2) were more closely related to RVA/Human-wt/ITA/PAI11/1996/G2P[4] (93.5%), RVA/Sheep-wt/CHN/LLR/1985/G10P[15] (88.8%), RVA/Human-tc/SWE/1076/1983/G2P2A[6] (93.2%) and RVA/Human-wt/AUS/CK20003/2000/G2P[4] (91.2%), respectively. Altogether, these findings are suggestive of multiple independent interspecies transmission and reassortment events between co-circulating bovine, porcine, ovine and human rotaviruses. The complete genome sequence information is necessary to establish the evolutionary relationship, interspecies transmission and ecological features of animal RVAs from different geographical regions.     

Genomic characterization of G3P[6], G4P[6] and G4P[8] human rotaviruses from Wuhan,China: Evidence for interspecies transmission and reassortment events

We report here the whole genomic analyses of two G4P[6] (RVA/Human-wt/CHN/E931/2008/G4P[6], RVA/Human-wt/CHN/R1954/2013/G4P[6]), one G3P[6] (RVA/Human-wt/CHN/R946/2006/G3P[6]) and one G4P[8] (RVA/Human-wt/CHN/E2484/2011/G4P[8]) group A rotavirus (RVA) strains detected in sporadic cases of diarrhea in humans in the city of Wuhan, China. All the four strains displayed a Wa-like genotype constellation. Strains E931 and R1954 shared a G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1 constellation, whilst the 11 gene segments of strains R946 and E2484 were assigned to G3-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 and G4-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 genotypes, respectively. Phylogenetically, the VP7 gene of R946, NSP3 gene of E931, and 10 of 11 gene segments of E2484 (except for VP7 gene) belonged to lineages of human RVAs. On the other hand, based on available data, it was difficult to ascertain porcine or human origin of VP3 genes of strains E931 and R946, and NSP2 genes of strains R946 and R1954. The remaining genes of E2484, E931, R946 and R1954 were close to those of porcine RVAs from China, and/or porcine-like human RVAs. Taken together, our observations suggested that strain R1954 might have been derived from porcine RVAs, whilst strains R946 and E931 might be reassortants possessing human RVA-like gene segments on a porcine RVA genetic backbone. Strain E2484 might be derived from reassortment events involving acquisition of a porcine-like VP7 gene by a Wa-like human RVA strain. The present study provided important insights into zoonotic transmission and complex reassortment events involving human and porcine RVAs, reiterating the significance of whole-genomic analysis of RVA strains.     

Whole genome analysis of multiple rotavirus strains from a single stool specimen using sequence-independent amplification and 454? pyrosequencing reveals evidence of intergenotype genome segment recombination

Infection of a single host cell with two or more different rotavirus strains creates conditions favourable for evolutionary mechanisms like reassortment and recombination that can generate novel strains. Despite numerous reports describing mixed rotavirus infections, whole genome characterisation of rotavirus strains in a mixed infection case has not been reported. Double-stranded RNA, exhibiting a long electropherotype pattern only, was extracted from a single human stool specimen (RVA/Human-wt/ZAF/2371WC/2008/G9P[8]). Both short and long electropherotype profiles were however detected in the sequence-independent amplified cDNA derived from the dsRNA, suggesting infection with more than one rotavirus strain. 454? pyrosequencing of the amplified cDNA revealed co-infection of at least four strains. Both genotype 1 (Wa-like) and genotype 2 (DS-1-like) were assigned to the consensus sequences obtained from the nine genome segments encoding NSP1-NSP5, VP1-VP3 and VP6. Genotypes assigned to the genome segments encoding VP4 were P[4] (DS-1-like), P[6] (ST3-like) and P[8] (Wa-like) genotypes. Since four distinct genotypes [G2 (DS-1-like), G8, G9 (Wa-like) and G12] were assigned to the four consensus nucleotide sequences obtained for genome segment 9 (VP7), it was concluded that at least four distinct rotaviruses were present in the stool. Intergenotype genome recombination events were observed in genome segments encoding NSP2, NSP4 and VP6. The close similarities of some of the genome segments encoding NSP2, VP6 and VP7 to artiodactyl rotaviruses suggest that some of the infecting strains shared common ancestry with animal strains, or that interspecies transmission occurred previously. The sequence-independent genome amplification technology coupled with 454? pyrosequencing used in this study enabled the characterisation of the whole genomes of multiple rotavirus strains in a single stool specimen that was previously assigned single genotypes, i.e. G9P[8], by sequence-dependent RT-PCR.     

Identification of circulating porcine–human reassortant G4P[6] rotavirus from children with acute diarrhea in China by whole genome analyses

P[6] group A rotavirus (RVA) strains identified in four stool specimens collected from children with acute diarrhea in Guangxi Province, southern China in 2010, with unknown G type were further analyzed by full genomic analysis. It was revealed by whole genome sequencing that 11 genomic cognate gene segments of these P[6] RVA strains shared almost 100% nucleotide identities and all exhibited an identical G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1 genotype constellation. Phylogenetic analyses of VP7, VP1-VP4, NSP1, NSP2, NSP4 and NSP5 genes revealed that these Guangxi G4P[6] RVA strains were closely related to porcine and porcine-like human RVAs, while VP6 and NSP3 were closely related to those of common human RVAs. Interestingly, the four infants from whom these specimens were collected had come from different villages and/or towns. They had not contacted with each other and had had acute diarrhea before admitted into the same hospital. The genomic analyses and the clinical data revealed that these four Guangxi G4P[6] RVA strains from China were reassortants possessing VP6 and NSP3 gene segments of human origin yet all other nine gene segments of porcine origin. It is the first report on porcine–human reassortant G4P[6] RVA with identical genome configuration circulating in children.     

Whole genome analysis provides evidence for porcine-to-simian interspecies transmission of rotavirus-A

We report here whole genome analysis of a porcine rotavirus-A (RVA) strain RVA/Pig-wt/KNA/ET8B/2015/G5P[13] detected in a diarrheic piglet, and nearly whole genome (except for VP4 gene) analysis of a simian RVA strain RVA/Simian-wt/KNA/08979/2015/G5P[X] detected in a non-diarrheic African green monkey (AGM) on the island of St. Kitts, Caribbean region. Strain ET8B exhibited a G5-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1 genotype constellation that was identical to those of Brazilian porcine RVA G5P[13] strains RVA/Pig-wt/BRA/ROTA01/2013/G5P[13] and RVA/Pig-wt/BRA/ROTA07/2013/G5P[13], the only porcine G5P[13] RVAs that have been analyzed for the whole genome so far. Phylogenetically, all the 11 gene segments of ET8B were closely related to those of porcine and porcine-like human RVAs within the respective genotypes. Although the porcine G5P[13] RVAs exhibited identical genotype constellations, ET8B did not appear to share common evolutionary pathways with the Brazilian porcine G5P[13] RVAs. Interestingly, the VP2, VP3, VP6, VP7, and NSP1-NSP5 genes of simian RVA strain 08979 were closely related to those of porcine and porcine-like human RVA strains, exhibiting 99%–100% nucleotide sequence identities to cognate genes of co-circulating porcine RVA strain ET8B. On the other hand, the VP1 of 08979 appeared to be genetically divergent from porcine and human RVAs within the R1 genotype, and its exact origin could not be ascertained. Taken together, these observations suggested that simian strain 08979 might have been derived from interspecies transmission events involving transmission of ET8B-like RVAs from pigs to AGMs. In St. Kitts, AGMs often stray from the wild into livestock farms. Therefore, it may be possible that the AGM acquired the infection from a pig farm on the island. To our knowledge, this is the first report on detection of porcine-like RVAs in monkeys. Also, the present study is the first to report whole genomic analysis of a porcine RVA strain from the Caribbean region.     

A decade of G3P[8] and G9P[8] rotaviruses in Brazil: Epidemiology and evolutionary analyses

This study aims to estimate the frequency of group A rotaviruses (RVA) infection with genotypes G3P[8] and G9P[8] in children that suffered from diarrheal disease (DD) between 2001 and 2011 in different Brazilian regions. In addition, the genetic diversity of G3P[8] and G9P[8] RVA strains recovered from vaccinated and non-vaccinated children was assessed. Laboratory-based RVA surveillance included 15,115 cases of DD, and RVA was detected by enzyme immune-assay and/or polyacrylamide gel electrophoresis in 3357 (22%) samples. RVA was genotyped by the semi-nested RT-PCR and among RVA-positive samples, 100 (2.9%) were G3 (63 G3P[8], 32 G3P not typed [NT], and 5 G3P[6]) and 378 (16.2%) were G9 (318 G9P[8], 59 G9P[NT], and 1 G9P[6]). From the G3 and G9 positive samples, 16 and 12, respectively, were obtained from children aged 4–48 months vaccinated with the monovalent vaccine (Rotarix®, RV1). Phylogenetic analyses of the VP7 and VP8¹ encoding genes were performed for 26 G3P[8] and 48 G9P[8] strains. VP8¹ phylogenetic analysis revealed that all strains analyzed belonged to P[8] lineage III, whereas RV1 belongs to P[8]-I lineage. VP7 analysis revealed that all G3 and G9 strains belonged to G3-lineage III and G9-lineage III. The comparison of the VP7 and VP8¹ antigenic epitopes regions of Brazilian strains with RV1 strain revealed several amino acid changes. However, no particular differences among Brazilian strains detected before and after vaccine introduction were observed, or among strains detected from vaccinated and non-vaccinated children. Complete genome characterization of four G3P[8] and seven G9P[8] strains revealed a typical conserved human Wa-like genomic constellation. Changes in the genetic diversity of G3P[8] and G9P[8] RVA detected from 2001 to 2011 in Brazil seemed not be related to RV1 introduction in Brazil.     

Distinct evolutionary origins of G12P[8] and G12P[9] group A rotavirus strains circulating in Brazil

G12 group A rotavirus (RVA) are currently recognized as a globally emerging genotype and have been described in combination with several P-types. In Brazil, G12 RVA strains have been described in the Southern (2003) and Northern (2008–2010) regions, in combination with the P[9] and P[6] genotype, respectively. To date, few complete genomes of G12 RVA strains have been described (none from Brazilian strains), considering G12P[9] genotype just one strain, RVA/Human-tc/THA/T152/1998/G12P[9], has their 11 gene segments characterized. This study aims to determine the genomic constellation of G12P[9] and G12P[8] RVA strains detected in Brazil between 2006 and 2011. Therefore, the eleven gene segments of five Brazilian G12 RVA strains were amplified and sequenced, and the genotype of each gene segment was assigned using phylogenetic analysis. Complete genome analyses of G12 RVA strain circulating between 2006 and 2011 in Brazil revealed a conserved Wa-like genomic constellation for three G12P[8] RVA strains; whereas the two G12P[9] strains possessed distinct reassorted AU-1-like genomic constellations, closely related to the reference strain RVA/Human-tc/THA/T152/1998/G12P[9] in most genes. The results obtained in the current study suggest that G12P[9] (AU-1-like) and G12P[8] (Wa-like) strains detected in different regions of Brazil do not share a common origin. Moreover, while Brazilian G12P[8] RVA strains showed a complete Wa-like human constellation, both G12P[9] strains possessed an NSP1 gene of bovine origin (NSP1), and RVA/Human-wt/BRA/PE18974/2010/G12P[9] also possessed a VP3 gene of canine/feline origin.