Full genomic analysis of a human rotavirus G1P[8] strain isolated in South Korea

A rotavirus G1P[8] strain C1‐81 was isolated from a 5‐month‐old female infant admitted to hospital with fever and severe diarrhea in Incheon, South Korea. To investigate its full genomic relatedness and its group, the full genome of strain C1‐81 was determined. Based on a full genome classification system, C1‐81 was shown to possess the typical Wa‐like genotype constellation: G1‐P[8]‐I1‐R1‐C1‐M1‐A1‐N1‐T1‐E1‐H1. On the basis of sequence similarities, the strain was shown to be the closest related strain to contemporary human rotavirus strains with recent strains isolated in Asia. This C1‐81 strain showed the highest degree of nucleic acid similarity (98.8% and 97%) to G1 B4633‐03 and P[8] (Thai‐1604 and Dhaka8‐02), respectively. This is the first report that group A rotavirus was analyzed with G1P[8] in South Korea. The study of the complete genome of the virus will help understanding of the evolution of rotavirus. J. Med. Virol. 85:157–170, 2012. © 2012 Wiley Periodicals, Inc.     

Full genomic analysis of human rotavirus strain TB-Chen isolated in China

A G2P[4]/NSP4[A] rotavirus strain TB-Chen was isolated from a 2-year-old patient hospitalized with acute gastroenteritis in Kunming, China. The strain TB-Chen was demonstrated having group A-specific antigenicity, a “short” (subgroup II) electropherotype. To investigate its overall genomic relatedness and to determine which group it belonged, the complete genome of strain TB-Chen was determined. Genomic comparison based on amino acid sequence identity and phylogenetic analysis revealed that all 11 gene segments of strain TB-Chen were highly identical (> 91.80%) with the representative G2P[4]/NSP4[A] human strains DS-1, S2, NR1 and IS2, suggesting that this rotavirus strain was derived from human host. Besides, almost all the available representative rotavirus gene segments among group A were analyzed and identified within 15 G-types, 28 P-types, and 6 NSP4 genotypes. This is the first report of group A rotavirus genomic analyses in China and the findings have important implications for rotavirus vaccine development.     

Molecular characterization of a porcine Group A rotavirus strain with G12 genotype specificity

Summary. A porcine Group A rotavirus strain (RU172) was detected and molecularly characterized during a surveillance study conducted for rotavirus infection in a pig farm located in a suburban area of Kolkata City, India. The G12 genotype specificity of RU172 was revealed by PCR-based genotyping assays following addition of a G12 type-specific primer (designed in our laboratory to pick up G12 isolates from field samples) and was confirmed by sequence analysis of the VP7-encoding gene. The RU172 strain exhibited maximum VP7 identities of 93.6% to 94.5% with human G12 strains at the deduced amino acid level. In spite of its G12 genotype nature, RU172 appeared to be distinct from human G12 rotaviruses and, on phylogenetic analysis, formed a separate lineage with human G12 strains. Among the other gene segments analyzed, RU172 belonged to NSP4 genotype B, had a NSP5 and VP6 of porcine origin, and shared maximum VP4 identities with porcine P[7] rotaviruses (94.3%–95.4% at the deduced amino acid level). Therefore, to the best of our knowledge, this is the first report of detection of an animal rotavirus strain with G12 genotype specificity. Detection of strains like RU172 provides vital insights into the genomic diversity of Group A rotaviruses of man and animals.     

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.     

Full genomic analysis of a human group A rotavirus G9P[6] strain from Eastern India provides evidence for porcine-to-human interspecies transmission

Deduced amino acid sequence and phylogenetic analyses of a group A rotavirus G9P[6] strain (designated as mcs/13-07), detected from a 3-year-old child in Eastern India, revealed a VP8* closely related to porcine P[6] strains (P[6] sublineage 1D), and the VP7 clustered with G9 lineage-III strains. To our knowledge, this is the first report of human P[6] strain clustering in sublineage Id. Thus, to further characterize the evolutionary diversity of strain mcs/13-07, all gene segments were analyzed. VP6 and NSP4 exhibited genetic relatedness to Wa-like human subgroup II strains, while VP1-3, NSP1-3 and NSP5 were closely related to porcine strains. Based on the new classification system of rotaviruses, mcs/13-07 revealed a G9–P[6]–I1–R1–C1–M1–A8–N1–T1–E1–H1 genotype with close similarity to human Wa-like and porcine Gottfried strains. Therefore, considering the porcine-like or porcine origin of multiple gene segments, it might be tempting to assume that strain mcs/13-07 represents a rare instance of whole-virus transmission from pig to human, after which the virus evolved with time. Alternatively, it is possible that strain mcs/13-07 resulted from multiple reassortment events involving human subgroup II and porcine P[6] strains. Nevertheless, detection of strain mcs/13-07 provides further evidence for complex interspecies transmission events, which are frequent in developing 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.     

Identification of a novel VP4 genotype carried by a serotype G5 porcine rotavirus strain

Rotavirus genome segment 4, encoding the spike outer capsid VP4 protein, of a porcine rotavirus (PoRV) strain, 134/04-15, identified in Italy was sequenced, and the predicted amino acid (aa) sequence was compared to those of all known VP4 (P) genotypes. The aa sequence of the full-length VP4 protein of the PoRV strain 134/04-15 showed aa identity values ranging from 59.7% (bovine strain KK3, P8[11]) to 86.09% (porcine strain A46, P[13]) with those of the remaining 25 P genotypes. Moreover, aa sequence analysis of the corresponding VP8* trypsin cleavage fragment revealed that the PoRV strain 134/04-15 shared low identity, ranging from 37.52% (bovine strain 993/83, P[17]) to 73.6% (porcine strain MDR-13, P[13]), with those of the remaining 25 P genotypes. Phylogenetic relationships showed that the VP4 of the PoRV strain 134/04-15 shares a common evolutionary origin with porcine P[13] and lapine P[22] rotavirus strains. Additional sequence analyses of the VP7, VP6, and NSP4 genes of the PoRV strain 134/04-15 revealed the highest VP7 aa identity (95.9%) to G5 porcine strains, a porcine-like VP6 within VP6 genogroup I, and a Wa-like (genotype B) NSP4, respectively. Altogether, these results indicate that the PoRV strain 134/04-15 should be considered as prototype of a new VP4 genotype, P[26], and provide further evidence for the vast genetic and antigenic diversity of group A rotaviruses.     

Full genomic analysis of a G8P[1] rotavirus strain isolated from an asymptomatic infant in Kenya provides evidence for an artiodactyl-to-human interspecies transmission event

Group A rotavirus (GAR) G8P[1] strains, found sometimes in cattle, have been reported rarely from humans. Therefore, analysis of the full genomes of human G8P[1] strains are of significance in the context of studies on interspecies transmission of rotaviruses. However, to date, only partial-length nucleotide sequences are available for the 11 genes of a single human G8P[1] strain, while the partial sequences of two other strains have been reported. The present study reports the first complete genome sequence of a human G8P[1] strain, B12, detected from an asymptomatic infant in Kenya in 1987. By nucleotide sequence identities and phylogenetic analyses, the full-length nucleotide sequences of VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes of strain B12 were assigned to G8-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3 genotypes, respectively. Each of the 11 genes of strain B12 appeared to be more related to cognate genes of artiodactyl (ruminant and/or camelid) and/or artiodactyl-derived human GAR strains than those of most other rotaviruses. Strain B12 exhibited low levels of genetic relatedness to canonical human GAR strains, such as Wa and DS-1, ruling out the possibility of its origin from reassortment events between artiodactyl-like human and true human strains. These observations suggest that strain B12 might have been directly transmitted from artiodactyls to humans. Unhygienic conditions and close proximity of humans to livestock at the sampling site might have facilitated this rare event. This is the first report on a full genomic analysis of a rotavirus strain from Kenya. To our knowledge, strain B12 might be the oldest G8 strain characterized molecularly from the Africa continent.     

A porcine G9 rotavirus strain shares neutralization and VP7 phylogenetic sequence lineage 3 characteristics with contemporary human G9 rotavirus strains

Of five globally important VP7 (G) serotypes (G1-4 and 9) of group A rotaviruses (the single most important etiologic agents of infantile diarrhea worldwide), G9 continues to attract considerable attention because of its unique natural history. Serotype G9 rotavirus was isolated from a child with diarrhea first in the United States in 1983 and subsequently in Japan in 1985. Curiously, soon after their detection, G9 rotaviruses were not detected for about a decade in both countries and then reemerged in both countries in the mid-1990s. Unexpectedly, however, such reemerged G9 strains were distinct genetically and molecularly from those isolated in the 1980s. Thus, the origin of the reemerged G9 viruses remains an enigma. Sequence analysis has demonstrated that the G9 rotavirus VP7 gene belongs to one of at least three phylogenetic lineages: lineage 1 (strains isolated in the 1980s in the United States and Japan), lineage 2 (strains first isolated in 1986 and exclusively in India thus far), and lineage 3 (strains that emerged/reemerged in the mid-1990s). Currently, lineage 3 G9 viruses are the most frequently detected G9 strains globally. We characterized a porcine rotavirus (A2 strain) isolated in the United States that was known to belong to the P[7] genotype but had not been serotyped by neutralization. The A2 strain was found to bear serotype G9 and P9 specificities as well as NSP4 [B] and subgroup I characteristics. By VP7-specific neutralization, the porcine G9 strain was more closely related to lineage 3 viruses than to lineage 1 or 2 viruses. Furthermore, by sequence analysis, the A2 VP7 was shown to belong to lineage 3 G9. These findings raise intriguing questions regarding possible explanations for the emergence of variations among the G9 strains.     

A novel type of VP4 carried by a porcine rotavirus strain

The gene encoding the VP8* trypsin-cleavage product of the VP4 protein of porcine rotavirus strain A34 was sequenced, and the predicted amino acid (aa) sequence was compared to the homologous region of all known P genotypes. The aa sequence of the VP8* of strain A34 shared low identity, ranging from 39% (bovine strain B223, P8[11]) to 76% (human strain 69M, P4[10]), with the homologous sequences of representative strains of the remaining 21 P genotypes. Phylogenetic relationships showed that the VP8* of strain A34 shares a common evolutionary lineage with those of human 69M (P4[10]) and equine H-2 (P4[12]) strains. Hyperimmune sera raised to strain A34 and to a genetic reassortant strain containing the VP4 gene from strain A34, both with high homologous neutralization titer via VP4, failed to neutralize strains representative of 15 different P genotypes. These results indicate that strain A34 should be considered as prototype of a new P genotype and serotype (P14[23]) and provide further evidence for the vast genetic and antigenic diversity of group A rotaviruses.     

Sequence analysis of three non structural proteins of a porcine group C (Cowden strain) rotavirus

Summary Sequences of three gene products of a group C (Cowden strain) rotavirus are presented and compared with the sequences of the corresponding group A (SA 11) proteins. The degree of similarity for gene 7, 9, and 10 is respectively 34%, 58%, and 45%. Comparison of these 2 viruses allowed to identify several regions well conserved. In the protein coded by Cowden segment 7 (NS 53) only a short cystein and histidine rich region, presenting the zinc finger consensus motif, is common to group A (segment 5) and group C sequences. Conversely the protein coded by segment 9 (NS 35) presented marked homology with group A NS 35. The protein coded by segment 10 (NS 26) is serine rich and presents an accumulation of charged residues near the carboxy terminus, like group A counterpart. This genomic segment presented a single large open reading frame, that contrasts with the group A counterpart for which a second out of phase ORF is used in rotavirus infected MA 104 cells.     

Minimal infective dose of the OSU strain of porcine rotavirus

Summary Infection of piglets, defined as the development of clinical symptoms with or without the excretion of viral particles in stools, was induced by as few as 90 rotavirus particles equivalent to 0.006 CD50% (cytopathic dose 50%) and 0.04mpniu (most probable number of infections units).     

Detection and full genomic analysis of G6P[9] human rotavirus in Japan

A rare genotype G6P[9] was identified in two human group A rotavirus strains designated as KF14 and KF17, that were detected in stool specimens from children with diarrhea in Japan. VP7 gene sequences of these two strains were identical and genetically closely related to G6 human rotavirus strains reported in European countries and the United States. To our knowledge, this is the first report of detection of a G6 human rotavirus in Japan. For further genetic analysis to elucidate the origin of the G6 rotavirus, nearly full-length sequences of all 11 RNA segments were determined for the KF17 strain. The complete genomic constellation of KF17 was determined as G6-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3, a novel genotype constellation for human rotavirus. Phylogenetic analysis indicated that VP6, VP1-3, and NSP2 genes of KF17 clustered with bovine-like G6 human strains and some animal strains into sub-lineages distinct from those of common DS-1-like G2 human rotaviruses. On the other hand, KF17 genes encoding VP4, NSP1, and NSP3-5 showed high sequence identities to the human G3P[9] strain AU-1, and clustered with AU-1 and some feline strains within the same lineage. These findings suggested that the G6P[9] human rotavirus detected in Japan may have occurred through reassortment among uncommon bovine-like human rotaviruses and human/feline AU-1-like rotaviruses.     

Identification of bovine and porcine rotavirus G types by PCR.

A new seminested PCR typing assay has been extended to identify the important veterinary rotavirus serotypes G5, G6, G10, and G11, as well as the rare human serotype G8. The specificity of the method was evaluated with 30 standard laboratory strains of the G1 to G6 and G8 to G11 types. Rotavirus strain types G6 and G8, not previously recognized in pigs, were identified in field specimens of porcine origin.     

Phylogenetic analysis of novel G12 rotaviruses in the United States: A molecular search for the origin of a new strain

Rotavirus serotype G12 was initially identified in the Philippines in 1987 and was not described again until it reemerged more than 13 years later. G12 strains were first detected in the United States in 2002 and have recently assumed a worldwide distribution. The high similarity between the sequence of the major outer capsid VP7 gene of human G12 strains and the single porcine G12 isolate raised the prospect that human strains may have arisen through reassortment with porcine strains or, alternatively, that the porcine strain originally came from humans. We sequenced portions of the remaining 10 segments of two human G12 strains (G12P[8] and G12P[6]) and a currently circulating common strain (G1P[8]) identified during the 2005–2006 surveillance season and compared the sequences with those of strains available through GenBank. By comparison, the three strains were all Wa‐like and not porcine‐like. A newly outlined classification system proposed genotypes for each gene segment based on nucleotide similarity. Using this approach, gene segments VP1–3, VP6 and NSP1–5 grouped within the same genotype, indicating that the three strains analyzed were closely related. These results suggest that the novel G12P[8] strain could have been formed by the solitary introduction of a VP7 gene into a globally common rotavirus strain, G1P[8]. Classifying rotavirus strains based only on VP7 (G) and VP4 (P) genotype potentially underestimates diversity and sequence analysis of the other segments is required to assess the complete genetic relationships between strains. J. Med. Virol. 81:736–746, 2009 © 2009 Wiley‐Liss, Inc.