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.     

Molecular characterization of rotavirus strains detected during a clinical trial of a human rotavirus vaccine in Blantyre, Malawi

The human, G1P[8] rotavirus vaccine (Rotarix?) significantly reduced severe rotavirus gastroenteritis episodes in a clinical trial in South Africa and Malawi, but vaccine efficacy was lower in Malawi (49.5%) than reported in South Africa (76.9%) and elsewhere. The aim of this study was to examine the molecular relationships of circulating wild-type rotaviruses detected during the clinical trial in Malawi to RIX4414 (the strain contained in Rotarix?) and to common human rotavirus strains. Of 88 rotavirus-positive, diarrhoeal stool specimens, 43 rotaviruses exhibited identifiable RNA migration patterns when examined by polyacrylamide gel electrophoresis. The genes encoding VP7, VP4, VP6 and NSP4 of 5 representative strains possessing genotypes G12P[6], G1P[8], G9P[8], and G8P[4] were sequenced. While their VP7 (G) and VP4 (P) genotype designations were confirmed, the VP6 (I) and NSP4 (E) genotypes were either I1E1 or I2E2, indicating that they were of human rotavirus origin. RNA-RNA hybridization using 21 culture-adapted strains showed that Malawian rotaviruses had a genomic RNA constellation common to either the Wa-like or the DS-1 like human rotaviruses. Overall, the Malawi strains appear similar in their genetic make-up to rotaviruses described in countries where vaccine efficacy is greater, suggesting that the lower efficacy in Malawi is unlikely to be explained by the diversity of circulating strains.     

Novel NSP1 genotype characterised in an African camel G8P[11] rotavirus strain

Animal–human interspecies transmission is thought to play a significant role in influencing rotavirus strain diversity in humans. Proving this concept requires a better understanding of the complete genetic constellation of rotaviruses circulating in various animal species. However, very few whole genomes of animal rotaviruses, especially in developing countries, are available. In this study, complete genetic configuration of the first African camel rotavirus strain (RVA/Camel-wt/SDN/MRC-DPRU447/2002/G8P[11]) was assigned a unique G8-P[11]-I2-R2-C2-M2-A18-N2-T6-E2-H3 genotype constellation that has not been reported in other ruminants. It contained a novel NSP1 genotype (genotype A18). The evolutionary dynamics of the genome segments of strain MRC-DPRU447 were rather complex compared to those found in other camelids. Its genome segments 1, 3, 7–10 were closely related (>93% nucleotide identity) to those of human–animal reassortant strains like RVA/Human-tc/ITA/PA169/1988/G6P[14] and RVA/Human-wt/HUN/Hun5/1997/G6P[14], segments 4, 6 and 11 shared common ancestry (>95% nucleotide identity) with bovine rotaviruses like strains RVA/Cow-wt/CHN/DQ-75/2008/G10P[11] and RVA/Cow-wt/KOR/KJ19-2/XXXX/G6P[7], whereas segment 2 was closely related (94% nucleotide identity) to guanaco rotavirus strain RVA/Guanaco-wt/ARG/Rio_Negro/1998/G8P[1]. Its genetic backbone consisted of DS-1-like, AU-1-like, artiodactyl-like and a novel A18 genotype. This suggests that strain MRC-DPRU447 potentially emerged through multiple reassortment events between several mammalian rotaviruses of at least two genogroups or simply strain MRC-DPRU447 display a unique progenitor genotypes. Close relationship between some of the genome segments of strain MRC-DPRU447 to human rotaviruses suggests previous occurrence of reassortment processes combined with interspecies transmission between humans and camels. The whole genome data for strain MRC-DPRU447 adds to the much needed animal rotavirus data from Africa which is limited at the moment.     

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.     

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.     

Sequence analysis of the whole genomes of five African human G9 rotavirus strains

The G9 rotaviruses are amongst the most common global rotavirus strains causing severe childhood diarrhoea. However, the whole genomes of only a few G9 rotaviruses have been fully sequenced and characterised of which only one G9P[6] and one G9P[8] are from Africa. We determined the consensus sequence of the whole genomes of five African human group A G9 rotavirus strains, four G9P[8] strains and one G9P[6] strain collected in Cameroon (central Africa), Kenya (eastern Africa), South Africa and Zimbabwe (southern Africa) in 1999, 2009 and 2010. Strain RVA/Human-wt/ZWE/MRC-DPRU1723/2009/G9P[8] from Zimbabwe, RVA/Human-wt/ZAF/MRC-DPRU4677/2010/G9P[8] from South Africa, RVA/Human-wt/CMR/1424/2009/G9P[8] from Cameroon and RVA/Human-wt/KEN/MRC-DPRU2427/2010/G9P[8] from Kenya were on a Wa-like genetic backbone and were genotyped as G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Strain RVA/Human-wt/ZAF/MRC-DPRU9317/1999/G9P[6] from South Africa was genotyped as G9-P[6]-I2-R2-C2-M2-A2-N1-T2-E2-H2. Rotavirus A strain MRC-DPRU9317 is the second G9 strain to be reported on a DS-1-like genetic backbone, the other being RVA/Human-wt/ZAF/GR10924/1999/G9P[6]. MRC-DPRU9317 was found to be a reassortant between DS-1-like (I2, R2, C2, M2, A2, T2, E2 and H2) and Wa-like (N1) genome segments. All the genome segments of the five strains grouped strictly according to their genotype Wa- or DS-1-like clusters. Within their respective genotypes, the genome segments of the three G9 study strains from southern Africa clustered most closely with rotaviruses from the same geographical origin and with those with the same G and P types. The highest nucleotide identity of genome segments of the study strains from eastern and central Africa regions on a Wa-like backbone was not limited to rotaviruses with G9P[8] genotypes only, they were also closely related to G12P[6], G8P[8], G1P[8] and G11P[25] rotaviruses, indicating a close inter-genotype relationship between the G9 and other rotavirus genotypes. Rotavirus strain MRC-DPRU9317 is the first G9P[6] to be characterised on a DS-1-like genetic backbone with a reassortant segment 8 (NSP2) and fourth G9P[6] to be fully sequenced globally.     

Repeated circulation over 6 years of intergenogroup mono-reassortant G2P[4] rotavirus strains with genotype N1 of the NSP2 gene

Human rotavirus A, a major cause of acute diarrhoea in infants and young children worldwide, contains the genome of 11 segments of double-stranded RNA, which is grouped into three major genotype constellations: the Wa genogroup (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1), the DS-1 genogroup (G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2), and the AU-1 genogroup (G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H3). Recently, a G2P[4] strain detected in Kenya in 1982 was reported to be a nonstructural protein 2 (NSP2) gene mono-reassortant between the Wa and DS-1 genogroups. While NSP2 induces heterotypic antibody responses in children, thereby being the likely target of immune selective pressure, it was not known how frequently these NSP2 mono-reassortants circulated among children. In a previous 10 year epidemiological study, out of 100 G2P[4] rotavirus specimens that were typed into 22 distinct electropherotypes (i.e., strains), RNA-RNA hybridization identified that 12 strains were mono-reassortants involving either the NSP2 or the NSP3 gene. The aim of this study was therefore to determine the nucleotide sequences of all 11 genes of the representative mono-reassortant strain (AU605) and the sequences of the reassorted genes of the other mono-reassortant strains. The genome constellation of AU605 was G2-P[4]-I2-R2-C2-M2-A2-N1-T2-E2-H2, indicating that it was a mono-reassortant strain carrying a Wa-like NSP2 genotype on the DS-1 genogroup background. The reassorted gene of the other 11 strains also had the genotype N1 in the NSP2 gene. Given that electrophoretically-identical rotaviruses represent a single strain, the 12 NSP2 mono-reassortants detected in six rotavirus seasons accounted for 36% (36 of 100) of G2P[4] rotavirus specimens in the 10 year collection. The circulation of NSP2 mono-reassortant strains was observed in epidemic seasons when the G2P[4] genotype was not dominant. Taken together with their repeated occurrence at higher-than-expected frequencies, the identification of NSP2 mono-reassortant G2P[4] strains suggests that such genome constellation may also be viable in nature, and that they circulated less rarely than generally believed.     

Identification of a multi-reassortant G12P[9] rotavirus with novel VP1, VP2, VP3 and NSP2 genotypes in a child with acute gastroenteritis

The G12 rotavirus genotype is globally emerging to cause severe gastroenteritis in children. Common G12 rotaviruses have either a Wa-like or DS-1-like genome constellation, while some G12 strains may have unusual genome composition. In this study, we determined the full-genome sequence of a G12P[9] strain (ME848/12) detected in a child hospitalized with acute gastroenteritis in Italy in 2012. Strain ME848/12 showed a complex genetic constellation (G12-P[9]-I17-R12-C12-M11-A12-N12-T7-E6-H2), likely derived from multiple reassortment events, with the VP1, VP2, VP3 and NSP2 genes being established as novel genotypes R12, C12, M11 and N12, respectively. Gathering sequence data on human and animal rotaviruses is important to trace the complex evolutionary history of atypical RVAs.     

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 constellation of the first human G8 rotavirus strain detected in Japan

Human G8 Rotavirus A (RVA) strains are commonly detected in Africa but are rarely detected in Japan and elsewhere in the world. In this study, the whole genome sequence of the first human G8 RVA strain designated AU109 isolated in a child with acute gastroenteritis in 1994 was determined in order to understand how the strain was generated including the host species origin of its genes. The genotype constellation of AU109 was G8-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analyses of the 11 genome segments revealed that its VP7 and VP1 genes were closely related to those of a Hungarian human G8P[14] RVA strain and these genes shared the most recent common ancestors in 1988 and 1982, respectively. AU109 possessed an NSP2 gene closely related to those of Chinese sheep and goat RVA strains. The remaining eight genome segments were closely related to Japanese human G2P[4] strains which circulated around 1985–1990. Bayesian evolutionary analyses revealed that the NSP2 gene of AU109 and those of the Chinese sheep and goat RVA strains diverged from a common ancestor around 1937. In conclusion, AU109 was generated through genetic reassortment event where Japanese DS-1-like G2P[4] strains circulating around 1985–1990 obtained the VP7, VP1 and NSP2 genes from unknown ruminant G8 RVA strains. These observations highlight the need for comprehensive examination of the whole genomes of RVA strains of less explored host species.     

Detection of rare reassortant G5P[6] rotavirus,Bulgaria

During the ongoing rotavirus strain surveillance program conducted in Bulgaria, an unusual human rotavirus A (RVA) strain, RVA/Human/BG/BG620/2008/G5P[6], was identified among 2200 genotyped Bulgarian RVAs. This strain showed the following genomic configuration: G5–P[6]–I1–R1–C1–M1–A8–N1–T1–E1–H1. Phylogenetic analysis of the genes encoding the neutralization proteins and backbone genes identified a probable mixture of RVA genes of human and porcine origin. The VP1, VP6 and NSP2 genes were more closely related to typical human rotavirus strains. The remaining eight genes were either closely related to typical porcine and unusual human–porcine reassortant rotavirus strains or were equally distant from reference human and porcine strains. This study is the first to report an unusual rotavirus isolate with G5P[6] genotype in Europe which has most likely emerged from zoonotic transmission. The absence of porcine rotavirus sequence data from this area did not permit to assess if the suspected ancestral zoonotic porcine strain already had human rotavirus genes in its genome when transmitted from pig to human, or, the transmission was coupled or followed by gene reassortment event(s). Because our strain shared no neutralization antigens with rotavirus vaccines used for routine immunization in children, attention is needed to monitor if this G–P combination will be able to emerge in human populations. A better understanding of the ecology of rotavirus zoonoses requires simultaneous monitoring of rotavirus strains in humans and animals.     

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.     

Whole genome characterisation of a porcine-like human reassortant G26P[19] Rotavirus A strain detected in a child hospitalised for diarrhoea in Nepal, 2007

A rare G26 Rotavirus A strain RVA/Human-wt/NPL/07N1760/2007/G26P[19] was detected in a child hospitalised for acute diarrhoea in Kathmandu, Nepal. The complete genome of 07N1760 was determined in order to explore its evolutionary history as well as examine its relationship to a Vietnamese strain RVA/Human-wt/VNM/30378/2009/G26P[19], the only G26 strain whose complete genotype constellation is known. The genotype constellation of 07N1760 was G26-P[19]-I12-R1-C1-M1-A8-N1-T1-E1-H1, a unique constellation identical to that of the Vietnamese 30378 except the VP6 gene. Phylogenetic analysis revealed that both strains were unrelated at the lineage level despite their similar genotype constellation. The I12 VP6 gene of 07N1760 was highly divergent from the six currently deposited I12 sequences in the GenBank. Except for its NSP2 gene, the remaining genes of 07N1760 shared lineages with porcine and porcine-like human RVA genes. The NSP2 gene belonged to a human RVA N1 lineage which was distinct from typical porcine and porcine-like human lineages. In conclusion, the Nepali G26P[19] strain 07N1760 was a porcine RVA strain which derived an NSP2 gene from a human Wa-like RVA strain by intra-genotype reassortment probably after transmission to the human host.     

Analysis of rotavirus species diversity and evolution including the newly determined full-length genome sequences of rotavirus F and G

Rotaviruses are a leading cause of viral acute gastroenteritis in humans and animals. Eight different rotavirus species (A–H) have been defined based on antigenicity and nucleotide sequence identities of the VP6 gene. Here, the first complete genome sequences of rotavirus F (strain 03V0568) and G (strain 03V0567) with lengths of 18,341 and 18,186 bp, respectively, are described. Both viruses have open reading frames for rotavirus proteins VP1 to VP7 and NSP1 to NSP5 located at the 11 genome segments. Nucleotide sequence identities to other rotaviruses ranged between 29.8% (NSP1 gene) and 61.7% (VP1 gene) for rotavirus F and between 29.3% (NSP1-2 gene) and 65.9% (NSP2 gene) for rotavirus G, thus confirming their classification as separate virus species. Encoded proteins revealed remarkable sequence differences among the rotavirus species. In contrast, the non-coding 5′-terminal sequences of the genome segments are highly conserved among all rotavirus species. Different 3′-terminal consensus sequences are found between rotavirus A/D/F, rotavirus C and rotavirus B/G/H. Phylogenetic analyses indicated a separation of rotaviruses in two major clades consisting of rotavirus A/C/D/F and rotavirus B/G/H. Within these clades, rotavirus F mainly clustered with rotavirus D and rotavirus G with rotavirus B. In addition, differentiation among mammalian and avian rotavirus A strains, host-specific evolution of rotavirus B and C as well as an ancient reassortment event between avian rotavirus A and D are indicated by the phylogenetic data. These results underline the high diversity of rotaviruses as a result of a complex evolutionary history.     

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.     

Molecular characterization of two rare human G8P[14] rotavirus strains,detected in Italy in 2012

Since 2007, the Italian Rotavirus Surveillance Program (RotaNet-Italy) has monitored the diversity and distribution of genotypes identified in children hospitalized with rotavirus acute gastroenteritis.We report the genomic characterization of two rare human G8P[14] rotavirus strains, identified in two children hospitalized with acute gastroenteritis in the southern Italian region of Apulia during rotavirus strain surveillance in 2012.Both strains showed a G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3 genomic constellation (DS-1-like genomic background). Phylogenetic analysis of each genome segment revealed a mixed configuration of genes of animal and zoonotic human origin, indicating that genetic reassortment events generated these unusual human strains. Eight out of 11 genes (VP1, VP2, VP3, VP6, VP7, NSP3, NSP4 and NSP5) of the Italian G8P[14] strains exhibited close identity with a Spanish sheep strain, whereas the remaining genes (VP4, NSP1 and NSP2) were more closely related to human strains. The amino acid sequences of the antigenic regions of outer capsid proteins VP4 and VP7 were compared with vaccine and field strains, showing high conservation between the amino acid sequences of Apulia G8P[14] strains and human and animal strains bearing G8 and/or P[14] proteins, and revealing many substitutions with respect to the RotaTeq™ and Rotarix™ vaccine strains. Conversely, the amino acid analysis of the four antigenic sites of VP6 revealed a high degree of conservation between the two Apulia strains and the human and animal strains analyzed.These results reinforce the potential role of interspecies transmission and reassortment in generating novel rotavirus strains that might not be fully contrasted by current vaccines.     

Phylogenetic analysis of rotavirus A NSP2 gene sequences and evidence of intragenic recombination

The rotavirus non-structural protein NSP2 is one of the earliest and most abundant viral proteins produced during infection. This protein has multiple essential roles in the replication cycle involving RNA binding, viroplasm formation, helicase and can hydrolyse the γ-phosphate of RNA and NTPs acting as an RTPase and NTPase. In studying sequences from rotavirus strains isolated in Australia between 1984 and 2009, the NSP2 gene was seen to be highly conserved and clustered with defined NSP2 genotypes N1 and N2 according to the full genome based rotavirus classification system. Phylogenetic analysis indicated that NSP2 gene sequences isolated from Australian rotavirus strains formed four distinct lineages. Temporal variation was observed in several clusters during the 26 year period, with lineage D identified throughout the entire study period and lineage A only detected since 1999. Phylogenetic analysis and dendrograms identified NSP2 genes that exhibited reassortment between different virus VP7 genotypes, as well as a sequence from a human strain that grouped closely with the NSP2 genes of bovine rotavirus strains. This study also identified a sequence that fell between lineages and exhibited evidence of recombination, the first time that intergenic recombination has been detected in a NSP2 gene sequence. This study increases the understanding of the evolution mechanisms of NSP2 in view of improved vaccine design.     

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.     

Reassortant group A rotavirus from straw-colored fruit bat (Eidolon helvum)

Bats are known reservoirs of viral zoonoses. We report genetic characterization of a bat rotavirus (Bat/KE4852/07) detected in the feces of a straw-colored fruit bat (Eidolon helvum). Six bat rotavirus genes (viral protein [VP] 2, VP6, VP7, nonstructural protein [NSP] 2, NSP3, and NSP5) shared ancestry with other mammalian rotaviruses but were distantly related. The VP4 gene was nearly identical to that of human P[6] rotavirus strains, and the NSP4 gene was closely related to those of previously described mammalian rotaviruses, including human strains. Analysis of partial sequence of the VP1 gene indicated that it was distinct from cognate genes of other rotaviruses. No sequences were obtained for the VP3 and NSP1 genes of the bat rotavirus. This rotavirus was designated G25-P[6]-I15-R8(provisional)-C8-Mx-Ax-N8-T11-E2-H10. Results suggest that several reassortment events have occurred between human, animal, and bat rotaviruses. Several additional rotavirus strains were detected in bats.