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.     

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.     

Whole genome sequencing reveals genetic heterogeneity of G3P[8] rotaviruses circulating in Italy

After a sporadic detection in 1990s, G3P[8] rotaviruses emerged as a predominant genotype during recent years in many areas worldwide, including parts of Italy. The present study describes the molecular epidemiology and evolution of G3P[8] rotaviruses detected in Italian children with gastroenteritis during two survey periods (2004–2005 and 2008–2013). Whole genome of selected G3P[8] strains was determined and antigenic differences between these strains and rotavirus vaccine strains were analyzed. Among 819 (271 in 2004–2005 and 548 in 2008–2013) rotaviruses genotyped during the survey periods, the number of G3P[8] rotavirus markedly varied over the years (0/83 in 2004, 30/188 in 2005 and 0/96 in 2008, 6/88 in 2009, 4/97 in 2010, 0/83 in 2011, 9/82 in 2012, 56/102 cases in 2013). The genotypes of the 11 gene segments of 15 selected strains were assigned to G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1; thus all strains belonged to the Wa genogroup. Phylogenetic analysis of the Italian G3P[8] strains showed a peculiar picture of segregation with a 2012 lineage for VP1-VP3, NSP1, NSP2, NSP4 and NSP5 genes and a 2013 lineage for VP6, NSP1 and NSP3 genes, with a 1.3–20.2% nucleotide difference from the oldest Italian G3P[8] strains. The genetic variability of the Italian G3P[8] observed in comparison with sequences of rotaviruses available in GenBank suggested a process of selection acting on a global scale, rather than the emergence of local strains, as several lineages were already circulating globally. Compared with the vaccine strains, the Italian G3P[8] rotaviruses segregated in different lineages (5–5.3% and 7.2–11.4% nucleotide differences in the VP7 and VP4, respectively) with some mismatches in the putative neutralizing epitopes of VP7 and VP4 antigens. The accumulation of point mutations and amino acid differences between vaccine strains and currently circulating rotaviruses might generate, over the years, vaccine-resistant variants.     

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.     

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.     

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 sequences of Japanese porcine species C rotaviruses reveal a high diversity of genotypes of individual genes and will contribute to a comprehensive,generally accepted classification system

Porcine rotavirus C (RVC) is distributed throughout the world and is thought to be a pathogenic agent of diarrhea in piglets. Although, the VP7, VP4, and VP6 gene sequences of Japanese porcine RVCs are currently available, there is no whole-genome sequence data of Japanese RVC. Furthermore, only one to three sequences are available for porcine RVC VP1–VP3 and NSP1–NSP3 genes. Therefore, we determined nearly full-length whole-genome sequences of nine Japanese porcine RVCs from seven piglets with diarrhea and two healthy pigs and compared them with published RVC sequences from a database. The VP7 genes of two Japanese RVCs from healthy pigs were highly divergent from other known RVC strains and were provisionally classified as G12 and G13 based on the 86% nucleotide identity cut-off value. Pairwise sequence identity calculations and phylogenetic analyses revealed that candidate novel genotypes of porcine Japanese RVC were identified in the NSP1, NSP2 and NSP3 encoding genes, respectively. Furthermore, VP3 of Japanese porcine RVCs was shown to be closely related to human RVCs, suggesting a gene reassortment event between porcine and human RVCs and past interspecies transmission. The present study demonstrated that porcine RVCs show greater genetic diversity among strains than human and bovine RVCs.     

Evidence of discordant genetic linkage in the VP4, VP6, VP7 and NSP4 encoding genes of rotavirus strains from adolescent and adult patients with acute gastroenteritis

The vast diversity within rotavirus strains circulating in the developing countries continues to be a major challenge for the efficacy of currently used preset rotavirus vaccines. The sequence analysis and phylogeny of multiple genes of rotavirus strains enable identification of reassortant strains and their human or animal origin. The objective of this study was to monitor the genetic linkage between the rotavirus VP4(P), VP6(I), VP7(G) and NSP4(E) encoding genes. The G, P, I and E genotypes of a total of 80 rotavirus strains isolated from adolescent and adult cases of acute gastroenteritis at the two time points [1993–1996 (n = 67) and 2004–2007 (n = 13)] were determined by nucleotide sequencing and phylogenetic analysis. The rotavirus strains from the 1990s and 2000s revealed common combinations of genotypes (G1–P[8]–I1–E1, G2–P[4]–I2–E2, G3–P[8]–I1–E1 and G4–P[8]–I1–E1) in 47.8% and 30.8%, unusual combinations of the same genotypes (G2–P[8]–I2–E2, G9–P[6]–I1–E1, G9–P[6]–I1–E2, G9–P[6]–I2–E1 and G4–P[4]–I1–E2, G1–P[4]–I2–E1, G9–P[4]–I1–E1) in 7.5% and 23% and mixed infections of different G and P genotypes in 31.3% and 46.2%, respectively. Discordance in the association of I with E, G with I and E and P with I and E genotypes was found to be contributed respectively by 23.8–38.5%, 40.3–69.8% and 49.3–61.5% of the rotavirus strains at the two time points.The data suggest relatively high occurrence of intergenogroup reassortment in circulating rotavirus strains emphasizing the need for continuous surveillance and whole genome sequence based characterization of rotavirus strains for better understanding of their evolution and ecology.     

Diversity in VP3, NSP3, and NSP4 of rotavirus B detected from Japanese cattle

Bovine rotavirus B (RVB) is an etiological agent of diarrhea mostly in adult cattle. Currently, a few sequences of viral protein (VP)1, 2, 4, 6, and 7 and nonstructural protein (NSP)1, 2, and 5 of bovine RVB are available in the DDBJ/EMBL/GenBank databases, and none have been reported for VP3, NSP3, and NSP4. In order to fill this gap in the genetic characterization of bovine RVB strains, we used a metagenomics approach and sequenced and analyzed the complete coding sequences (CDS) of VP3, NSP3, and NSP4 genes, as well as the partial or complete CDS of other genes of RVBs detected from Japanese cattle. VP3, NSP3, and NSP4 of bovine RVBs shared low nucleotide sequence identities (63.3–64.9% for VP3, 65.9–68.2% for NSP3, and 52.6–56.2% for NSP4) with those of murine, human, and porcine RVBs, suggesting that bovine RVBs belong to a novel genotype. Furthermore, significantly low amino acid sequence identities were observed for NSP4 (36.1–39.3%) between bovine RVBs and the RVBs of other species. In contrast, hydrophobic plot analysis of NSP4 revealed profiles similar to those of RVBs of other species and rotavirus A (RVA) strains. Phylogenetic analyses of all gene segments revealed that bovine RVB strains formed a cluster that branched distantly from other RVBs. These results suggest that bovine RVBs have evolved independently from other RVBs but in a similar manner to other rotaviruses. These findings provide insights into the evolution and diversity of RVB strains.     

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 genome sequencing and phylogenetic analysis of a zoonotic human G8P[14] rotavirus strain

The full-length genome of a rare human G8P[14] rotavirus strain, BP1062/04, identified during a surveillance study in Hungary was determined and analyzed. This strain showed a G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3 genomic constellation. Phylogenetic analysis of each genome segment revealed common origins with selected animal and zoonotic human strains. The closest relatedness was seen with suspect zoonotic Hungarian G6P[14] strains in the NSP1 and NSP3 gene phylogeny, with ovine strains in the VP1, VP2, NSP4 gene phylogeny, and with bovine strains in the NSP5 gene phylogeny. The outer capsid VP7 and VP4 genes could not be derived from cognate genes of any known human or animal G8P[14] strains. The remaining genes, NSP2, VP3 and VP6, gave no definite clues to the host origin, although each was clearly different from true human strains. Altogether, our findings suggest that strain BP1062/04 represents an example of a direct zoonotic transmission event.     

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.     

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.     

A rare G1P[6] super-short human rotavirus strain carrying an H2 genotype on the genetic background of a porcine rotavirus

Rotavirus strains with a rearranged 11th genome segment may show super-short RNA electropherotypes. Examples from human strains were limited to seven strains, 69M, 57M, B37, Mc345, AU19, B4106 and BE2001, which have a variety of G and P genotypes. AU19 is a rare G1P[6] human rotavirus strain detected in a Japanese infant with severe acute gastroenteritis. This study was undertaken to better understand the origin of AU19 by determining the genotype constellation of AU19. Upon nearly-full genome sequencing, AU19 had a G1-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H2 genotype constellation. Possession of I5 and A8 genotypes is indicative of its porcine rotavirus origin, whereas possession of H2 genotype is indicative of its DS-1 like human rotavirus origin. At the phylogenetic lineage level for the genome segments that share the genotype between porcine and human rotaviruses, the VP1-4, VP7, NSP3-4 genes were most closely related to those of porcine rotaviruses, but the origin of the NSP2 gene was inconclusive. As to the NSP5 gene, the lineage containing AU19 and the other three super-short human strains, 69M, 57M and B37, carrying the H2 genotype (H2b) clustered with the lineage to which DS-1- like short strains belonged (H2a) albeit with an insignificant bootstrap support. Taken all these observations together, AU19 was likely to emerge as a consequence of interspecies transmission of a porcine rotavirus to a child coupled with the acquisition of a rare H2b genotype by genetic reassortment probably from a co-circulating human strain. The addition of the AU19 NSP5 sequence to much homogeneous H2b genotypes shared by previous super-short rotavirus strains made the genetic diversity of H2b genotypes as diverse as that of the H2a genotype, lending support to the hypothesis that super-short strains carrying H2b genotype have long been circulating unnoticed in the human population.     

Genotypic linkages of VP4, VP6, VP7, NSP4, NSP5 genes of rotaviruses circulating among children with acute gastroenteritis in Thailand

Rotavirus is the main cause of acute viral gastroenteritis in infants and young children worldwide. Surveillance of group A rotavirus has been conducted in Chiang Mai, Thailand since 1987 up to 2004 and those studies revealed that group A rotavirus was responsible for about 20-61% of diarrheal diseases in hospitalized cases. In this study, we reported the continuing surveillance of group A rotavirus in 2005 and found that group A rotavirus was detected in 43 out of 147 (29.3%) stool samples. Five different G and P genotype combinations were detected, G1P[8] (27 strains), G2P[4] (12 strains), G9P[8] (2 strains), G3P[8] (1 strain), and G3P[10] (1 strain). In addition, analysis of their genotypic linkages of G (VP7), P (VP4), I (VP6), E (NSP4), and H (NSP5) genotypes demonstrated that the rotaviruses circulating in Chiang Mai, Thailand carried 3 unique linkage patterns. The G1P[8], G3P[8], and G9P[8] strains carried their VP6, NSP4, NSP5 genotypes of I1, E1, H1, respectively. The G2P[4] strains were linked with I2, E2, H2 genotypes, while an uncommon G3P[10] genotype carried unique genotypes of I8, E3 and H6. These findings provide the overall picture of genotypic linkage data of rotavirus strains circulating in Chiang Mai, Thailand.     

Detection of the first G6P[14] human rotavirus strain from a child with diarrhea in Egypt

We report the first detection of a G6P[14] rotavirus strain in Egypt from the stool of a child participating in a hospital-based diarrhea surveillance study conducted throughout the year 2004. Rotavirus infection was initially detected using a rotavirus group A VP6 enzyme immunoassay; the P (VP4) and G (VP7) genotypes of the strain were identified by RT-PCR. We sequenced the VP7 gene and the VP8* portion of the VP4 gene and the strain displayed the strongest identity to the VP7 [>94% nucleotides (nt), >97% amino acids (aa)] and VP4 (>93% nt, >98% aa) sequences of PA169, a novel G6P[14] strain first isolated from a child in Italy during the winter of 1987. Additional sequencing and analysis of the other remaining structural (VP1–VP3, VP6) and non-structural (NSP1–NSP5) proteins support this animal-to-human reassortment theory. According to the full genome classification system, the G6P[14] strain (EGY3399) was assigned to G6-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3 genotypes. The greatest similarity of EGY3399 NSP4 and NSP5 gene sequences were to those of ovine and simian origin, respectively. Coupled with other observations, our results suggest G6P[14] isolates rarely cause severe diarrhea in Egyptian children, and support other studies that indicate animal rotavirus contribute to the genetic diversity of rotavirus detected from humans through interspecies transmission and single or multiple segments reassortment.     

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.     

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.     

Molecular characterization of genotype G6 human rotavirus strains detected in Italy from 1986 to 2009

Group A human rotavirus (HRV) strains with a bovine-like (G6) major outer capsid protein VP7 were first detected in Palermo, Italy, in the late 1980s, and subsequently worldwide. During a 25-year rotavirus surveillance period, additional HRV G6 strains, associated with either a P[9] or P[14] VP4 genotype, have been detected sporadically, but repeatedly, in Palermo. Whether these G6 HRVs were transmitted to humans directly from an animal reservoir or could have circulated at low prevalence in susceptible individuals is uncertain. Upon sequence analyses of the VP7, VP4, VP6, NSP4 and NSP5 gene segments, all the Italian HRV strains displayed a conserved genotype constellation, G6-P[9]/[14]-I2-E2-H3. Intra-genotypic lineages and/or sub-lineages were observed among the various HRV strains, with some lineage/sublineage combinations being retained over time. Interestingly, two epidemiologically unrelated G6P[9] viruses, collected in the same rotavirus season, were found to have a clonal origin. In conclusion, our results indicate not only diverse origin of animal derived G6 HRVs in Palermo but also suggest human-to-human transmission of certain strains.     

昆明地区22株人轮状病毒VP7及NSP4基因分析

目的研究昆明地区轮状病毒（RV）不同VP7血清型及NSP4基因变异与腹泻的流行及症状严重程度的关系。方法对2002年和2003年分离于昆明地区的RV，用PCR分型法对四种主要的VP7血清型进行分型，并对从150份RV腹泻标本VP7血清型为G1、G3、（34型RV株中挑出的14份一般腹泻株和8份重症腹泻株的NSP4基因序列进行了分析，与来自GenBank Database的4株人RV（Wa、KUN、AU-1、Hochi）和3株动物RV（EW、OSU、SA11）以及中国不同地区流行株NSP4的基因变异情况进行了比较。结果2002年昆明地区RV流行株以G1型为主，2003年RV腹泻株以G3型为主；昆明地区RV流行株间的氨基酸同源性高达98．9％～99．4％，22株流行株全都属于Wa组；NSP4变异与地域及VP7血清型无关；NSP4基因变异与RV腹泻临床症状严重程度不相关（P〉0．05）。结论不同年份相同季节不同地域流行的RVVP7血清型变异较大，而NSP4基因的相对保守性及其免疫原性使其有可能成为发展疫苗的候选基因。