Rare AU-1-like G3P[9] human rotaviruses with a Kun-like NSP4 gene detected in children with diarrhea in Italy

Three G3P[9] rotaviruses, detected in children hospitalized with gastroenteritis in Palermo, Italy, were found to be genetically related to strains of either human or feline origin in the VP7, VP4, and VP6 genes. In contrast, in the NSP4 gene the viruses resembled G2P[4] human strains, suggesting a reassortment between AU-1-like and Kun-like strains.     

Molecular characterization of rare G3P[9] rotavirus strains isolated from children hospitalized with acute gastroenteritis

In 2004, an epidemiological survey of human rotavirus infection in Chiang Mai, Thailand detected two uncommon human rotavirus strains (CMH120/04 and CMH134/04) bearing AU-1-like G3P[9] genotypes in 1 year old children hospitalized with acute gastroenteritis. The CMH120/04 and CMH134/04 rotavirus strains were characterized by molecular analyses of their VP6, VP7, VP8*, and NSP4 gene segments as well as the determination of RNA patterns by polyacrylamide gel electrophoresis (PAGE). Analysis of the VP8* gene revealed a high level of amino acid sequence identities with those of P[9] rotavirus reference strains, ranging from 94.9% to 98.3%. The highest identities were shared with the human rotavirus AU-1 strain at 97.8% and 98.3% for CMH120/04 and CMH134/04 strains, respectively. Analysis of the VP7 gene sequence revealed the highest identities with G3 human rotavirus strain KC814 at 96.6% and 96.2% for CMH120/04 and CMH134/04 strains, respectively. Based on the analyses of VP7 and VP8* genes, CMH120/04 and CMH134/04 belonged to G3P[9] genotypes. In addition, analyses of VP6 and NSP4 sequences revealed a VP6 subgroup (SG) I, with NSP4 genetic group C specificities. Moreover, both strains displayed a long RNA electrophoretic pattern. The finding of uncommon G3P[9] rotaviruses in pediatric patients provided additional evidence of the genetic/antigenic diversities of human group A rotaviruses in the Chiang Mai area of Thailand.     

Isolation from diarrheal and asymptomatic kittens of three rotavirus strains that belong to the AU-1 genogroup of human rotaviruses.

A survey of 143 stool specimens collected during a 17-month period in Japan from diarrheal and asymptomatic kittens identified three rotavirus strains that were similar in their genomic RNA constellation to human rotavirus AU-1 (G3P3[9]), lending strong support to the view that rotaviruses belonging to the same genogroup are circulating in both the human and the feline population.     

Isolation,serologic and molecular characterization of the first G3 caprine rotavirus

Summary.  We isolated a rotavirus in cell culture, named the GRV strain, from a stool specimen of a Korean goat with diarrhea, and performed an in-depth characterization. At various passage levels in cell culture, the GRV strain retained its pathogenicity for goat kids, thereby for the first time establishing that a caprine rotavirus can cause diarrhea in goat kids. The GRV strain grew to a high titer and agglutinated group O human erythrocytes. The GRV VP7 protein was 96% identical with the RRV (simian rotavirus) and R2 (lapine rotavirus) VP7 proteins, and slightly less similar to the SA11 (simian rotavirus) and HCR3 (feline/canine-like human rotavirus) VP7 proteins. The GRV VP4 protein was 93% identical with the RRV VP4 (P[3]) and 90% identical with the SA11 VP4 (P[2]). However, phylogenetic analysis including more VP4 sequences from representative P[3] strains unambiguously placed the GRV VP4 in the cluster of P[3] VP4s. A high level of two-way cross neutralization with RRV substantiated that GRV was a G3P5[3] strain, thus identifying GRV as the first caprine rotavirus with such a phenotype. The GRV NSP4 sequence belonged to the AU-1 allele, as does the RRV NSP4 sequence. Genetic analysis by RNA–RNA hybridization revealed that the overall genomic RNA constellation of the GRV strain was unique among mammalian rotavirus genogroups and that it was almost equally related to, yet distant from, simian rotavirus RRV, feline/canine rotavirus FRV64 (or CU-1), feline/human rotavirus FRV-1 (or AU-1), and lapine rotavirus R2. The availability of the GRV strain will further expand our limited knowledge of caprine rotaviruses. Present address: Department of Veterinary Pathobiology, Texas Veterinary Medical Center, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4467 U.S.A. Received September 26, 2002; accepted November 28, 2002     

Characterization of nontypeable rotavirus strains from the United States: identification of a new rotavirus reassortant (P2A[6],G12) and rare P3[9] strains related to bovine rotaviruses

Among 1316 rotavirus specimens collected during strain surveillance in the United States from 1996 to 1999, most strains (95%) belonged to the common types (G1 to G4 and G9), while 5% were mixed infections of common serotypes, rare strains, or not completely typeable. In this report, 2 rare (P[9],G3) and 2 partially typeable (P[6],G?; P[9],G?) strains from that study were further characterized. The P[6] strain was virtually indistinguishable by hybridization analysis in 10 of its 11 gene segments with recently isolated P2A[6],G9 strains (e.g., U.S.1205) from the United States, but had a distinct VP7 gene homologous (94.7% a.a. and 90.2% nt) to the cognate gene from P1B[4],G12 reference strain L26. Thus, this serotype P2A[6],G12 strain represents a previously unrecognized reassortant. Three P3[9] strains were homologous (97.8-98.2% aa) in the VP8 region of VP4 to the P3[9],G3 feline-like reference strain AU-1, but had a high level of genome homology to Italian bovine-like, P3[9],G3 and P3[9],G6 rotavirus strains. Two of the U.S. P3[9] strains were confirmed to be type G3 (97.2-98.2% VP7 aa homology with reference G3 strain AU-1), while the other was most similar to Italian bovine-like strain PA151 (P3[9],G6), sharing 99.0% a.a. homology in VP7. Cross-neutralization studies confirmed all serotype assignments and represented the first detection of these rotavirus serotypes in the United States. The NSP4 genes of all U.S. P3[9] strains and rotavirus PA151 were most closely related to the bovine and equine branch within the DS-1 lineage, consistent with an animal origin. These results demonstrate that rare strains with P and G serotypes distinct from those of experimental rotavirus vaccines circulate in the United States, making it important to understand whether current vaccine candidates protect against these strains.     

Genomic characterization of a novel group A lamb rotavirus isolated in Zaragoza, Spain

An ovine rotavirus (OVR) strain, 762, was isolated from a 30-day-old lamb affected with severe gastroenteritis, in Zaragoza, Spain, and the VP4, VP7, VP6, NSP4, and NSP5/NSP6 genes were subsequently characterized molecularly. Strain OVR762 was classified as a P[14] rotavirus, as the VP4 and VP8* trypsin-cleavage product of the VP4 protein revealed the highest amino acid (aa) identity (94% and 97%, respectively) with that of the P11[14] human rotavirus (HRV) strain PA169, isolated in Italy. Analysis of the VP7 gene product revealed that OVR762 possessed G8 serotype specificity, a type common in ruminants, with the highest degree of aa identity (95-98%) shared with serotype G8 HRV, bovine rotavirus, and guanaco (Lama guanicoe) rotavirus strains. Moreover, strain OVR762 displayed a bovine-like NSP4 (genotype E2) and NSP5/NSP6 (genotype H3), and a VP6 genotype I2, as well as a long electropherotype pattern. This is the first report of a lamb rotavirus with P[14] and G8 specificities, providing additional evidence for the wide genetic and antigenic diversity of group A rotaviruses.     

Molecular analysis of the VP7, VP4, VP6, NSP4, and NSP5/6 genes of a buffalo rotavirus strain: identification of the rare P[3] rhesus rotavirus-like VP4 gene allele

We report the detection and molecular characterization of a rotavirus strain, 10733, isolated from the feces of a buffalo calf affected with diarrhea in Italy. Strain 10733 was classified as a P[3] rotavirus, as the VP8* trypsin cleavage product of the VP4 protein revealed a high amino acid identity (96.2%) with that of rhesus rotavirus strain RRV (P5B[3]), used as the recipient virus in the human-simian reassortant vaccine. Analysis of the VP7 gene product revealed that strain 10733 possessed G6 serotype specificity, a type common in ruminants, with an amino acid identity to G6 rotavirus strains ranging from 88 to 98%, to Venezuelan bovine strain BRV033, and Hungarian human strain Hun4. Phylogenetic analysis based on the VP7 gene of G6 rotaviruses identified at least four lineages and an apparent linkage between each lineage and the VP4 specificity, suggesting the occurrence of repeated interspecies transmissions and genetic reassortment events between ruminant and human rotaviruses. Moreover, strain 10733 displayed a bovine-like NSP4 and NSP5/6 and a subgroup I VP6 specificity, as well as a long electropherotype pattern. The detection of the rare P[3] genotype in ruminants provides additional evidence for the wide genetic and antigenic diversity of group A rotaviruses.     

Serological and genomic characterization of human rotaviruses detected in China

A total of 1,385 stool specimens were collected from children with diarrhea at two hospitals in Wuhan, Hubei Province, China, in 1994 and 1995, and screened for rotavirus by polyacrylamide gel electrophoresis of viral RNA. Group A rotavirus was detected with high frequency; 56.5% (87/154) and 40.8% (502/1,231) of the specimens collected in 1994 and 1995, respectively, were positive for rotavirus. Assignment of G serotype and P type (VP4 genotype) of group A rotavirus by ELISA with monoclonal antibodies and/or PCR, respectively, showed that strains of G2-P[4] and G1-P[8] specificity were predominant in 1994 and in 1995, respectively. In contrast, a single strain was found to have a P[9] type specificity, and no G4 strain was detected. Unusual combinations of RNA pattern-subgroup-G serotype-P type, such as long pattern-subgroup I-G1-P[8], short pattern-subgroup II-G3-P[4] and short pattern-subgroup I-G1-P[4], were detected in four specimens. Nucleotide sequences of the VP8* and/or NSP5 genes from two Chinese P[8] strains 470 and 582 and one Chinese P[9] strain 512 as well as five Japanese P[9] strains (K8, AU1, M318, O264, and O265) were determined and compared with the published sequences of the corresponding gene. In the phylogenetic tree of VP8* sequences of P[9] strains, which formed two clusters each having strain K8 or AU-1 as the representative strain, the Chinese P[9] strain was found in the cluster represented by AU-1, although it was most distantly related to other strains. While NSP5 sequences of human strains with P[9] specificity were related to simian and bovine strains, that of Chinese P[8] strains was most closely related to those of porcine strains. A single group C rotavirus (No. 208) was detected. Nucleotide sequences of its VP4, VP6, VP7, and NSP4 genes were very similar to those of group C human rotaviruses detected worldwide. J. Med. Virol. 55:168–176, 1998. © 1998 Wiley-Liss, Inc.     

Relationships among porcine and human P[6] rotaviruses: evidence that the different human P[6] lineages have originated from multiple interspecies transmission events

Porcine rotavirus strains (PoRVs) bearing human-like VP4 P[6] gene alleles were identified. Genetic characterization with either PCR genotyping or sequence analysis allowed to determine the VP7 specificity of the PoRVs as G3, G4, G5 and G9, and the VP6 as genogroup I, that is predictive of a subgroup I specificity. Sequence analysis of the VP8* trypsin-cleavage product of VP4 allowed PoRVs to be characterized further into genetic lineages within the P[6] genotype. Unexpectedly, the strains displayed significantly higher similarity (up to 94.6% and 92.5% at aa and nt level, respectively) to human M37-like P[6] strains (lineage I), serologically classifiable as P2A, or to the atypical Hungarian P[6] human strains (HRVs), designated as lineage V (up to 97.0% aa and 96.1% nt), than to the porcine P[6] strain Gottfried, lineage II (<85.1% aa and 82.2 nt), which is serologically classified as P2B. Interestingly, no P[6] PoRV resembling the original prototype porcine strain, Gottfried, was detected, while Japanase P[6] PoRV clustered with the atypical Japanase G1 human strain AU19. By analysis of the 10th and 11th genome segments, all the strains revealed a NSP4B genogroup (Wa-like) and a NSP5/6 gene of porcine origin. These findings strongly suggest interspecies transmission of rotavirus strains and/or genes, and may indicate the occurrence of at least 3 separate rotavirus transmission events between pigs and humans, providing convincing evidence that evolution of human rotaviruses is tightly intermingled with the evolution of animal rotaviruses.     

Interspecies sharing of two distinct nonstructural protein 1 alleles among human and animal rotaviruses as revealed by dot blot hybridization.

The distribution of the nonstructural protein 1 (NSP1) alleles from human strain AU-1 and canine strain K9 among rotaviruses of human, feline, canine, bovine, and simian origin was studied by a dot blot hybridization assay. Human and feline strains belonging to the AU-1 genogroup had the same NSP1 allele, while canine and feline strains belonging to the canine-feline genogroup shared another NSP1 allele. This canine-feline NSP1 allele had a significant level of homology with the NSP1 of rhesus rotavirus strain MMU18006.     

Characterization of human rotavirus strains with G12 and P[9] detected in Japan

Two G12 human rotavirus strains, CP727 and CP1030, were isolated from the respective diarrheic stools of an infant and an adult in Japan. VP7 gene sequences of strains CP727 and CP1030 showed high identity with that of the G12 prototype strain L26, and with those of G12 strains reported recently from Thailand, the United States, and India. VP4 gene sequences of strains CP727 and CP1030 showed the highest identity with those of P[9] rotaviruses. In Northern blot hybridization, strains CP727 and CP1030 were found to be closely related to strain AU-1 (G3P[9]); nine RNA segments hybridized to each other. Moreover, all segments each of the two Japanese G12 strains hybridized to those of the Thai G12 strain T152. These results suggest that Japanese G12 strains detected in this study are reassortants between a L26-like strain and a strain in the AU-1 genogroup. A similar reassortant was found in the Thai G12 strain T152.     

Molecular characterization of a rare G3P[3] human rotavirus reassortant strain reveals evidence for multiple human-animal interspecies transmissions

An unusual strain of human rotavirus G3P[3] (CMH222), bearing simian-like VP7 and caprine-like VP4 genes, was isolated from a 2-year-old child patient during the epidemiological survey of rotavirus in Chiang Mai, Thailand in 2000-2001. The rotavirus strain was characterized by molecular analysis of its VP4, VP6, VP7, and NSP4 gene segments. The VP4 sequence of CMH222 shared the greatest homology with those of caprine P[3] (GRV strain) at 90.6% nucleotide and 96.4% amino acid sequence identities. Interestingly, the VP7 sequence revealed highest identity with those of simian G3 rotavirus (RRV strain) at 88% nucleotide and 98.1% amino acid sequence identities. In contrast, percent sequence identities of both the VP4 and VP7 genes were lower when compared with those of human rotavirus G3P[3] reference strains (Ro1845 and HCR3). Analyses of VP6 and NSP4 sequences showed a close relationship with simian VP6 SG I and caprine NSP4 genotype C, respectively. Phylogenetic analysis of VP4, VP6, VP7, and NSP4 genes of CMH222 revealed a common evolutionary lineage with simian and caprine rotavirus strains. These findings strongly suggest multiple interspecies transmission events of rotavirus strains among caprine, simian, and human in nature and provide convincing evidence that evolution of human rotaviruses is tightly intermingled with the evolution of animal rotaviruses.     

Evidence for independent segregation of the VP6- and NSP4- encoding genes in porcine group A rotavirus G6P[13] strains

Summary. Molecular characterization of two porcine group A rotavirus strains (HP113 and HP140), detected from eastern India, revealed a VP7 closely related to those of human G6P[14] strains, VP4 with a borderline P[13] genotype, and VP6 related to bovine and human SGI strains rather than porcine SGI and/or SGII group A rotaviruses. Both strains had NSP4 and NSP5 of porcine origin. Therefore, to our knowledge, the present study is the first report of detection of group A rotavirus strains with G6P[13] genotype specificities and provides evidence for independent segregation of the VP6- and NSP4-encoding genes in porcine group A rotaviruses.     

Prevalence of human rotavirus genotypes in Wuhan, China, during 2008-2011: changing trend of predominant genotypes and emergence of strains with the P[8]b subtype of the VP4 gene

Hospital-based surveillance of rotavirus genotypes was conducted in Wuhan, China, between March 2008 and May 2011. The detection rates of group A rotavirus were 24.6% (458/1859) and 12.1% (96/795) in children and adults, respectively, with diarrhea. Among the 554 positive specimens, the most frequent genotype was G3P[8] (57.9%), followed by G1P[8] (29.4%). Compared with previous studies in Wuhan (2000-2008), the relative frequency of G3P[8] has been decreasing year by year, while the predominant genotype G3 shifted to G1 in 2011. In the present study, a rare P[8]b subtype of the VP4 gene (OP354-like P[8]) was identified in nine strains. Full-length sequences of VP7, VP4, VP6 and NSP4 genes of two G9P[8]b strains (RVA/Human-wt/CHN/E1545/2009/G9P[8]b and RVA/Human-wt/CHN/Z1108/2008/G9P[8]b) were determined for phylogenetic analysis. The four genes of these strains were closely related to one another, and the G9-VP7 genes of these strains belonged to lineage III, which contains globally spreading G9 rotaviruses. The full-length sequence of VP4 gene segments of the P[8]b strains in Wuhan clustered with those of P[8]b strains in Vietnam, Russia and Belgium, while they were distinct from those of the OP354 strain from Malawi and Bangladeshi strains. The VP6 and NSP4 genes of two P[8]b strains belonged to the I1 and E1 genotype, respectively, and clustered with those of strains belonging to Wa-like human rotaviruses from various Asian countries. These findings indicate the changing epidemiologic trend of rotavirus genotypes in Wuhan, i.e., the shift of the predominant type from G3 to G1 and the emergence of P[8]b strains genetically related to those distributed in other Asian countries.     

Full genomic analysis of a porcine–bovine reassortant G4P[6] rotavirus strain R479 isolated from an infant in China

During the 2004 surveillance of rotaviruses in Wuhan, China, a G4P[6] rotavirus strain R479 was isolated from a stool specimen collected from a 2‐year‐old child with diarrhea. The strain R479 had an uncommon subgroup specificity I + II, and analysis of the VP6 gene suggested that it was related to porcine rotaviruses. In the present study, full‐length nucleotide sequences of all the RNA segments of R479 were determined and analyzed phylogenetically to identify the origin of individual RNA segments. According to the rotavirus genotyping system based on 11 RNA segments, the genotype of R479 was expressed as G4‐P[6]‐I5‐R1‐C1‐M1‐A1‐N1‐T7‐E1‐H1. This genotype includes the porcine‐like VP6 genotype (I5) and bovine‐like NSP3 genotype (T7). Phylogenetic analysis revealed that R479 genes encoding VP1, VP2, VP3, VP6, VP7, VP8*, NSP1, NSP4, and NSP5 were more closely related to those of porcine rotaviruses than human or other animal rotaviruses. In contrast, it was remarkable that the NSP3 gene of R479 was genetically closely related to only a bovine rotavirus strain UK. The NSP2 gene of R479 was also unique and clustered with only the G5P[8] human strain IAL28 and G3P[24] simian strain TUCH. These results suggested that R479 may be a reassortant virus having the NSP3 gene from a bovine rotavirus in the genetic background of a porcine rotavirus, with an NSP2 gene related to the porcine‐human reassortant strain IAL28. To our knowledge, R479 is the first porcine–bovine reassortant rotavirus isolated from a human. J. Med. Virol. 82:1094–1102, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular characterization of a rare G9P[23] porcine rotavirus isolate from China

The fifth most important G genotype, G9 rotavirus, is recognized as an emerging genotype that is spreading around the world. Sequence analysis was completed of a rare group A rotavirus, strain G9P[23], that was designated rotavirus A pig/China/NMTL/2008/G9P[23] and abbreviated as NMTL. It was isolated from a piglet with diarrhea in China. Nucleotide sequence analysis revealed that the VP7 gene clustered within the G9 lineage VId. The VP4 gene clustered within the rare P[23] genotype. NMTL is the first porcine G9 stain reported in China. Thus, to further characterize the evolutionary diversity of the NMTL strain, all gene segments were used to draw a phylogenetic tree. Based on the new classification system of rotaviruses, the NMTL sequence revealed a G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1 genotype with close similarity to human Wa-like and porcine strains. The results showed that (i) NSP2 and NSP4 genes of NMTL exhibited higher genetic relatedness to human group A rotaviruses than to porcine strains, (ii) the VP2 and VP4 genes clustered with porcine and porcine-like human strains, and (iii) VP1 genes clustered apart from the Wa-like human and porcine clusters. In view of rotavirus evolution, this report provides additional evidence to support the notion that the human and porcine rotavirus genomes might be related.     

G8 rotavirus strains isolated in the Democratic Republic of Congo belong to the DS-1-like genogroup

Several G8P[6] and G8P[8] rotavirus strains were isolated from hospitalized patients in the Democratic Republic of Congo in 2003. To investigate their overall genomic relatedness and to determine to which genogroup they belonged, the complete genomes of strains DRC88 (G8P[8]) and DRC86 (G8P[6]) were determined. Genomic comparison of these two African G8 strains revealed that 10 out of their 11 gene segments, except for VP4, were nearly identical (>98.9% identical at the nucleotide level), suggesting that this rare G8P[8] rotavirus strain originated recently from a reassortment between a common G8P[6] strain and a strain with a P[8] specificity. A very close evolutionary relationship between 9 out of the 11 gene segments of DRC88 and DRC86 and rotavirus strains belonging to the DS-1-like (G2P[4]) "genogroup" was found, and several possible reassortment events preceding the occurrence of G8P[8] and G8P[6] human rotaviruses were hypothesized. Since the genes of G2P[4] rotavirus strains are very well adapted to infect humans, the acquirement of a new VP7 (G8) gene, and especially the replacement of P[6] (believed to be of animal origin) by P[8] (most common in human rotaviruses), might make DRC88-like rotaviruses very well equipped to become a predominant human rotavirus strain and an important pathogen on the African continent and the rest of the world. These findings have important implications for rotavirus vaccine development and highlight that typing of new rotavirus strains by merely sequencing their VP7 and VP4 genes provides us with only the tip of the iceberg regarding rotavirus diversity.     

Serologic and genomic characterization of a G12 human rotavirus in Thailand

The G and P type specificity of the human rotavirus strain T-152 (G12P[9]) isolated in Thailand was serologically confirmed with G12-specific monoclonal antibodies prepared in this study by using a reference G12 strain, L26, as an immunizing antigen and a P[9]-specific monoclonal antibody, respectively. The genomic relationship of strain T-152 with representative human rotavirus strains was examined by means of Northern blot analysis. The results showed that T152 is closely related to strain AU-1 (G3P[9]). Gene 5 (NSP1 gene) of T152, which did not hybridize with those of any other strains examined, was characterized by sequence determination. The T152 NSP1 gene is 1,652 nucleotides in length, encodes 493 amino acids, and exhibits low identity to those of representative human and animal rotaviruses.     

Rotaviruses belonging to the AU-1 genogroup recovered from Israeli infants with diarrhea

Summary The genetic and antigenic diversity of group A rotaviruses recovered from Israeli infants has been expanded recently by the inclusion of three unusual human rotavirus strains. Two rotavirus strains (Ro-5829 and Ro-5960) were shown to be the first viruses outside Japan that resembled the AU-1 genogroup of feline like human rotaviruses in their overall genomic constellation and in the restriction pattern of their polymerase chain reaction amplified gene 4 following digestion with EcoRI. Another strain (Ro-5193) turned out to be an intergenogroup reassortant between viruses belonging to the AU-1 and the bovine genogroups and resembled in that respect similar viruses isolated from infants in Italy.