Genetic characterization of group C rotavirus isolated from a child hospitalized with acute gastroenteritis in Chiang Mai,Thailand

During an epidemiological survey of human rotavirus infection in Chiang Mai, Thailand, from 2002 to 2004, in which 263 stool specimens tested, one isolate of group C rotavirus was detected from a two-year-old child admitted to hospital with acute gastroenteritis. The human group C rotavirus, named CMH004/03, was characterized further by molecular analyses of its VP4, VP6, and VP7 gene segments as well as determination of RNA pattern by polyacrylamide gel electrophoresis (PAGE). Molecular characterization of VP4, VP6, and VP7 genes by sequence analyses showed high levels of sequence identities with those of human group C rotavirus reference strains isolated worldwide at 95.2% to 99.4% on nucleotide and 97.5% to 100% on amino acid levels. In contrast, the CMH004/03 strain exhibited far lesser nucleotide and amino acid sequence identities at 67.7% to 84.1% and 68.7% to 91.3%, respectively, when compared with those of porcine and bovine group C rotaviruses. Phylogenetic analyses of VP4, VP6, and VP7 genes clearly confirmed that the CMH004/03 strain clustered in a monophyletic branch with other human group C rotavirus reference strains and distantly related to the clusters of animal group C rotavirus strains. In addition, the RNA electrophoretic migration pattern of CMH004/03 showed a typical pattern (4-3-2-2) of group C rotavirus. To our knowledge, this study is the second report of group C rotavirus infection in pediatric patients in Thailand after it was reported for the first time about two decades ago.     

Detection of human group C rotaviruses in Nigeria and sequence analysis of their genes encoding VP4, VP6, and VP7 proteins.

In a survey on the etiology of acute gastroenteritis in infants and young children in Nigeria, group C human rotaviruses were detected in two of 112 rotavirus positive stool specimens collected between 1999 and 2000. The VP7, VP6, and VP4 genes of the two Nigerian human group C rotavirus strains (Jajeri and Moduganari) were sequenced in this study. Comparative sequence analysis with other published human group C rotaviruses showed that the genes encoding the three structural proteins were remarkably conserved in primary structure with few mutations. The VP4 and VP7 genes from the two Nigerian strains were related more closely to each other than to those of other published strains, and formed a separate cluster on the phylogenetic tree. In contrast, it was of note that VP6 gene of strain Moduganari was related more closely to the Brazilian strain Belem than to the other Nigerian strain Jajeri. This is the first report of identification of human group C rotavirus in Nigeria and constitutes the first sequence data of human group C rotaviruses in the African continent.     

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.     

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 characterization of unusual human G5P[6] rotaviruses identified in China.

BACKGROUND: We found an unusual human rotavirus, LL36755 of G5P[6] genotype, in a stool sample collected in Lulong County, Hebei Province, China. This is the first detection of rotavirus serotype G5 in Asia. OBJECTIVES: To identify and characterize G5 rotaviruses in 988 stool samples collected from children under 5 years old with acute gastroenteritis. STUDY DESIGN: We analyzed 459 rotavirus-positive samples with RT-PCR using G5 genotype-specific primers. The G5 strains were sequenced. RESULTS: Two additional G5-positive samples (LL3354 and LL4260) were identified. VP7, VP4, VP6 and NSP4 genes of LL3354, LL4260 and LL36755 strains were sequenced. The VP4 sequences formed a group with porcine P[6] strains. The VP6 sequences of strains LL3354 and LL36755 were phylogenetically close to the major clusters of SGI and SGII rotaviruses, respectively. The deduced VP6 protein of strain LL4260 had characteristics of both SGI and SGII strains, but best fit with a cluster of atypical SGI viruses. In addition, based on NSP4 sequences, the three G5 strains belonged to genogroup B and were closest to human strain Wa. CONCLUSION: These results indicate a dynamic interaction of human and porcine rotaviruses and suggest that reassortment could result in the stable introduction and successful spread of porcine gene alleles into human rotaviruses.     

Genetic heterogeneity in the VP7 of group C rotaviruses

Evidence for a possible zoonotic role of group C rotaviruses (GCRVs) has been recently provided. To gain information on the genetic relationships between human and animal GCRVs, we sequenced the VP7 gene of 10 porcine strains detected during a large surveillance study from different outbreaks of gastroenteritis in piglets. Four GCRV strains were genetically related to the prototype GCRV porcine Cowden strain. A completely new VP7 genotype included 4 strains (344/04-7-like) that shared 92.5% to 97.0% aa identity to each other, but <83% to human GCRVs and <79% to other porcine and bovine GCRVs. A unique 4-aa insertion (SSSV or SSTI), within a variable region at the carboxy-terminus of VP7, represented a distinctive feature for these 4 unique strains. An additional strain, 134/04-18, was clearly different from all human and animal GCRVs (<85% aa identity) and likely accounts for a distinct VP7 genotype. The VP7 of a unique strain, 42/05-21, shared similar ranges of aa sequence identities with porcine and human strains (88.0-90.7% to porcine GCRVs and 85.2-88.2% to human GCRVs). Plotting the VP7 gene of strain 42/05-21 against the VP7 of human and porcine strains revealed discontinuous evolution rates throughout the VP7 molecule, suggesting different mutational pressure or a remote intragenic recombination event. These findings provide the need for future epidemiological surveys and warrant studies to investigate the pathogenic potential of these novel GCRVs in pigs.     

Sequence analysis of VP4 and VP7 genes of nontypeable strains identifies a new pair of outer capsid proteins representing novel P and G genotypes in bovine rotaviruses

During a limited epidemiological study, the serotype specificities of several isolates of bovine rotavirus, exhibiting identical electropherotypes, from a single cattle farm near Bangalore, India, could not be determined using a panel of serotyping monoclonal antibodies (MAbs) specific for G serotypes 1-6 and 10. To determine the genotypes of these isolates, the nucleotide sequences of the genes encoding the outer capsid proteins VP4 and VP7 of two representative isolates, Hg18 and Hg23, were determined. The corresponding gene sequences from the two isolates were identical, indicating that these isolates represented a single strain of bovine rotavirus. Comparison of the VP4 nucleotide (nt) and the deduced amino acid (aa) sequences with those of several human and animal rotavirus strains representing all of the currently recognized 20 different VP4 (P) genotypes revealed low nt and aa sequence identities of 61.0 to 74.2% and 57.9 to 78.2% for VP4. The percentages of amino acid homology for the VP8* and VP5* regions of VP4 were 37.7 to 67.9 and 68.1 to 84.2%, respectively. The nt and aa sequences of the VP7 gene were also distinct from those of human and animal strains belonging to the previously established 14 VP7(G) serotypes (65.9 to 75.5% nt and 59.5 to 77.6% aa identities). These findings suggest the classification of the VP4 and VP7 genes of the bovine isolates represented by Hg18 as new P and G genotypes and provide further evidence for the vast genetic/antigenic diversity of group A rotaviruses.     

Detection of a porcine rotavirus strain with VP4, VP7 and NSP4 genes of different animal origins

A new rotavirus strain, sh0902, was detected in diarrheic piglets on a farm in Shanghai, China, and its genotype was characterized as G1P[7]. Analysis of the VP4, VP7 and NSP4 genes demonstrated VP4 homology to bovine and swine rotavirus strains; the nucleotide (nt) and amino acid (aa) identities were 99.7% and 99.5%, respectively. The VP7 gene was highly homologous to that of a giant panda rotavirus strain, with 98.5% similarity at the nt level and 99% similarity at the aa level. The nucleotide sequence of the NSP4 gene displayed high homology to human rotavirus strain R479, with 99.7% identity at the nt level and 99.3% identity at the aa level. This is the first report of an unusual porcine rotavirus strain with VP4, VP7 and NSP4 genes that are highly homologous to bovine, swine, giant panda and human strains isolated at geographically distant sites (South Korea, China and India). Our data indicate that rotaviruses have circulated among humans and animals and undergone genome reassortment.     

Phylogenetic analyses of human rotavirus in central Taiwan in 1996, 2001 and 2002.

BACKGROUND: Rotavirus epidemiology information is required for gastroenteritis disease control and prevention. Information gathered about the serotype distribution of rotaviruses isolated in Taiwan is of crucial significance, before a licensed rotavirus vaccine is introduced. OBJECTIVES: The purpose of the present study is to investigate the epidemiological diversity of rotaviruses in Taiwan. STUDY DESIGN: A total of 51 stool samples taken from cases of acute gastroenteritis were collected from three teaching hospitals in central Taiwan in 1996, 2001 and 2002. The samples were subjected to RT-PCR tests of VP7 gene of the human rotavirus group A, B, C. RESULTS: A total of 16 stool samples were detected positive by RT-PCR and 10 were sequence analyzed and classified into G1, G3, and G9 types. Compared with other HRV strains: the sequences of CS96-40 of G1 are similar to MVD9816 (identity rate 97.15% and 96.09%, respectively, from Uruguay); the sequences of CS02-01 of G3 are similar to 98-B31 (identity rate 98.93% and 98.72%, respectively, from Japan); the sequences of CS01-05, CS01-06, CS01-07, CS01-09, CS01-13, CS02-02, CS02-03, CS02-04 are very similar to other established G9 rotaviruses sequences (identity rate 96.85-99.88%), especially between CS02-04 and SP2737 (from Japan) with an identity rate of 99.88% and 100% nucleotide and amino acid, respectively. Except for CS01-06 strain, it is VR3, but not VR5, VR7 or VR8, that found to be the most frequent mutated amino acid regions of VP7 in these strains. CONCLUSIONS: Our findings are the first to document the high prevalence of G9 HRV strains in Taiwan, and suggest the re-emergence of G3 strains in central Taiwan since 1991. Epidemiological surveys carried out in this study suggest genotype shifts from type G1 before 1996, to G9 in 2001 and 2002 and the re-emergence of G3 type in 2002.     

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.     

Molecular characterization of a human group C rotavirus detected first in Turkey

The present study was done to find out the prevalence of group B and C rotavirus infections in children with diarrhea presented at two major hospitals in Ankara, Turkey. Group B rotavirus was not found in any samples. One of 122 samples was positive for group C rotavirus. Phylogenetic analysis of genes for nonstructural protein NSP4, and structural proteins VP4, VP6, and VP7 confirmed the human origin of this strain. Similar to other human group C rotaviruses, one N-glycosylation site was predicted at amino acid residue 67 on the VP7 of strain GUP188. The genes of strain GUP188 were closely related to those of human group C rotavirus strain from the UK (Bristol) for NSP4, China (208 and Wu82) for VP4 and VP6, and from Colombia (Javeriana) for VP7, indicating that the Turkish group C rotavirus was unique and can serve as an additional reference strain for the molecular epidemiology of group C rotaviruses.     

Whole genome sequencing of lamb rotavirus and comparative analysis with other mammalian rotaviruses

Rotavirus (RV) epidemiological surveys with molecular analysis of various strains are required for gastroenteritis control and prevention. The lamb rotavirus strain NT, isolated from a diarrhea lamb in China, is considered as a promising vaccine strain. The whole genome of the lamb-NT strain was determined by sequence analysis. Sequence identity and phylogenetic analysis defined the lamb-NT strain as group A, genotype G10P[15]/NSP4[A]/SG1 strain. Comparative genomic analysis of the lamb-NT strain and 17 reference strains reveals that gene reassortments between rotaviruses circulating in different species occurred. Alignment of protein sequences of the genes shows some variations in the important functional regions of VP3 and VP4. These variations are related to host range restriction, virulence, and other potential characters of rotaviruses. Besides, this study also makes a significant foundation for the study of genetic classification, epidemiology, and antigenic diversity of rotaviruses on the molecular level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Sequence analysis of Vietnamese P[6] rotavirus strains suggests evidence of interspecies transmission

Nucleotide and amino acid sequences of the VP8* gene of five Vietnamese P[6] rotavirus strains detected from hospitalized patients with acute gastroenteritis were analyzed and compared with other human and porcine P[6] rotaviruses. It is of interest that these strains had greatest identity with two Italian porcine rotavirus strains, 134/04-10 and 134/04-11. To our knowledge, these five Vietnamese rotaviruses are the rare P[6] rotavirus strains belonging to lineage I that cluster into sublineage Ic with porcine rotaviruses, and not into sublineage Ia, as other human P[6] rotaviruses have done so far. Sequence analysis of the VP7 gene of these P[6] rotavirus strains was also performed. The results showed that the Vietnamese G9P[6] strain had high similarity with other human G9 rotaviruses, confirming a human-animal reassortant virus, whereas other three G4P[6] strains had best identity with porcine G4 rotavirus strains, suggesting interspecies transmission of rotavirus between porcine and humans. This result provides the important data on molecular characteristics of Vietnamese rotaviruses, and highlights interspecies transmission events of rotaviruses in Vietnam as well as in Asia.     

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.     

Molecular characterization of novel G5 bovine rotavirus strains

Group A rotaviruses are a major cause of acute gastroenteritis in young children as well as many domestic animals. The rotavirus genome is composed of 11 segments of double-stranded RNA and can undergo genetic reassortment during mixed infections, leading to progeny viruses with novel or atypical phenotypes. The aim of this study was to determine if the bovine group A rotavirus strains KJ44 and KJ75, isolated from clinically infected calves, share genetic features with viruses obtained from heterologous species. All 11 genes sequences of the KJ44 and KJ75 strains were sequenced and analyzed. The KJ44 VP4 had 91.7% to 96.3% deduced amino acid identity to the bovine related P[1] strain, whereas the KJ75 strain was most closely related to the bovine related P[5] strain (91.9% to 96.9% amino acid identity). Both KJ44 and KJ75 strains also contained the bovine related VP3 gene. The remaining 9 segments were closely related to porcine group A rotaviruses. The KJ44 and KJ75 strains showed high amino acid identity to the G5 rotaviruses, sharing 90.4% to 99.0% identity. In addition, these strains belonged to the NSP4 genotype B, which is typical of porcine rotaviruses and subgroup I, with the closest relationship to the porcine JL-94 strain. These results strongly suggest that bovine rotavirus strains with the G5 genotype occur in nature as a novel G genotype in cattle as a result of a natural reassortment between bovine and porcine strains.     

Rotavirus genotypes in Slovenia: unexpected detection of G8P[8] and G12P[8] genotypes

A rotavirus surveillance study was undertaken in Slovenia from December 2005 to March 2006. Stool samples from 114 children hospitalized with acute viral gastroenteritis were collected from two main Slovenian hospitals. These confirmed rotavirus-positive samples were selected for a rotavirus G and P genotype prevalence study. Six untypable strains of genotype G were further analyzed with sequencing of the VP7, VP8*, and NSP4 genes. The findings of the study were that the G1 genotype was the most prevalent, found in 72 samples (63.2%), followed by G9 in 26 samples (22.8%), G4 in 10 samples (8.8%), and G3 in 2 samples (1.7%). All G genotypes were combined with the P[8] genotype specificity. After sequence analysis, one G8 and two G12 genotypes were also characterized. In a VP7-based phylogenetic analysis, the G8P[8] strain (SI-885/06) was more closely related to the Cody I801 bovine strain than to other human strains. Both G12 strains (SI-264/06 and SI-403/06) were shown to belong to the Se585 G12 cluster. In the VP8* phylogenetic tree, all analyzed strains except one, belonged to the P[8] lineage II and shared high identity in amino acid sequence. All characterized strains were clustered into the NSP4 genotype B. The molecular characterization of this G8 strain supports the theory of interspecies transmission of rotaviruses and animal-human genome reassortment. This is the first report on rotavirus G12 detection in Slovenia.