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.     

Emerging G9 rotavirus strains in the northwest of China

Although G9 rotaviruses have become one of the important rotavirus genotypes worldwide, they have been uncommon in China. Recently, we reported G9 rotaviruses as a highly prevalent genotype in Xinjiang, the northwest part of China [Yang, X., Matthijnssens, J., Sun, H., Muhamaiti, J., Zhang, B., Nahar, S., Van Ranst, M., Rahman, M., 2008. Temporal changes of rotavirus strain distribution in a northwest city of China, 1996-2005. Int. J. Infect. Dis., June (Epub ahead of print)]. Here we report the genetic variations of the Xinjiang-G9 rotaviruses isolated between 1999 and 2005. Sequence analysis of the VP7 genes of Xinjiang-G9 strains indicated that they were more closely related to the contemporary global G9 strains than to the prototype Chinese G9 strains. However, their VP4 genes were most similar to those from the locally circulating G1P[8], G2P[4], G3P[6] and G3P[8] strains. This indicates that reassortment rather than antigenic drift might be the preferred evolutionary mechanism for the emergence of the G9 rotaviruses in Xinjiang. These findings will be of major significance for understanding the emergence of newly introduced rotavirus strains.     

Diversity of human rotavirus G9 among children in Turkey

Between September 2004 and December 2005 a prospective study was conducted to understand the epidemiology of rotavirus infection among children with diarrhea attending two hospitals in Ankara, Turkey. Rotavirus was detected in 39.7% of the 322 stool samples and affected mainly children in the age group of 6-23 months. More than 70% and 39% of these cases occurred in children <2 and <1 year of age, respectively. In the temperate climate of Ankara rotavirus infection was prevalent throughout the year. Serotype G1P[8] was dominant followed by G9P[8]. In 38 samples a total of 5 electropherotypes were detected. All G9P[8] were of long electropherotype except one of short electropherotype. A proportion of G1 and G9 strains were in combination with P[6], P[4] or P nontypable. Mixed serotypes were responsible for 2.4% of the infections. A phylogenetic tree constructed with the deduced amino acid sequences of the VP7 gene showed that 16 Turkish G9 strains clustered with rotaviruses of lineage III. One G9 strain formed a new lineage, lineage IV with the Sri Lankan G9 rotaviruses. In the phylogenetic tree of the VP8* gene, the Turkish G9P[6] rotaviruses clustered with human strains of lineage Ia. Increased diversity of the G/P type combination and the presence of infection throughout the year in Turkey was a situation similar to developing countries. The occurrence of rotavirus infection at later age and low level of mixed infections in Turkey represented the situation of developed countries. This study suggests that diverse G9 rotaviruses are emerging in Turkey.     

Characterization of genotype G8 strains from Malawi, Kenya, and South Africa

Reviews of the global distribution of rotavirus genotypes have revealed the continuous circulation of G8 strains in Africa, often responsible for more cases of rotavirus disease than the more common G1-G4 rotavirus strains. During the study, genotype G8 strains from Malawi, Kenya, and South Africa were detected and the VP7 and VP4 genes of selected specimens were sequenced. Results indicated that G8 strains appeared to reassort frequently and were associated with P[6], P[4], and P[8] specificity. Phylogenetic analysis suggested that G8 strains occurred in a North/South African phylogenetic divide. In addition, G8 strains appear to be able to infect non-human primates and strains with close phylogenetic relationships were detected in the same year on two continents. Any rotavirus vaccine introduced into African environments will need to demonstrate protective efficacy against unusual genotype combinations, new serotypes, and animal strains. Therefore, continuous monitoring of rotavirus strains in human and animal populations in Africa is a necessity.     

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.     

Changing pattern of rotavirus G genotype distribution in Chiang Mai, Thailand from 2002 to 2004: decline of G9 and reemergence of G1 and G2

Group A rotaviruses are the most common cause of acute viral diarrhea in humans and animals throughout the world. Previous surveillance studies of group A rotaviruses in Thailand indicated that the dominant types of rotaviruses were changing from time to time. During 2000 and 2001, the G9 rotavirus emerged as the most prevalent genotype, with an exceptionally high frequency (91.6%) in Chiang Mai, Thailand. In the year 2002-2004, group A rotavirus was detected in 98 out of 263 (37.3%) fecal specimens collected from children hospitalized with diarrhea. Of these, 40 (40.8%) were G9P[8], 33 (33.7%) were G1P[8], 23 (23.5%) were G2P[4], and 2 (2.0%) were G3P[9]. The G9P[8] was found to be the most predominant strain in 2002, but the prevalence rate abruptly decreased during the period 2003-2004. In addition, G2P[4] reemerged in the epidemic season of 2003, whereas G1P[8] became the most predominant strain in the following year (2004). Phylogenetic analysis of the VP7 genes revealed that G1, G2, and G9 rotavirus strains clustered together with recently circulating strains, which were isolated from different regional settings in Thailand. In conclusion, the study demonstrated a decrease of incidence of G9P[8] and reemergence of G1P[8] and G2P[4] rotaviruses in Chiang Mai, Thailand during the period 2002-2004. These data imply that the distribution of group A rotavirus genotypes circulating in Chiang Mai, Thailand, changes over time.     

Detection of G3P[3] and G3P[9] rotavirus strains in American Indian children with evidence of gene reassortment between human and animal rotaviruses

The distribution and evolution of human rotavirus strains is important for vaccine development and effectiveness. In settings where rotavirus vaccine coverage is high, vaccine pressure could select for replacement of common strains (similar to those included in rotavirus vaccines) with uncommon strains, some of which could be generated by reassortment between human and animal rotaviruses. Between 2002 and 2004, a phase-III rotavirus vaccine clinical trial was conducted among American Indian children of the Navajo and White Mountain Apache tribes, which are known to be at high risk for rotavirus diarrhea. We evaluated the rotavirus strains collected from study participants who received placebo during the trial to determine the distribution of rotavirus genotypes and to detect emerging strains that contribute to disease and could influence rotavirus vaccine effectiveness. Three uncommon strains of human rotavirus, two G3P[3] and one G3P[9] strains were detected in stools of children aged 3 to 6 months of age. Segments of all 11 rotavirus genes were sequenced and genotyped by comparison of cognate gene fragments with reference strains. The G3P[3] strains had similar genotypes to each other and to reference dog and cat strains. The G3P[9] strain had similar genotypes to cow, cat and dog reference strains. Genetic analyses of these three strains support the known diversity generating mechanisms of rotavirus.     

Dual infection due to simian G3--human reassortant and human G9 strains of rotavirus in a child and subsequent spread of serotype G9, leading to diarrhea among grandparents

Cultivation of rotavirus from day 1 and 3 fecal specimens of a child yielded simian SA11-human reassortant, G3P[8] and AU32 like G9P[8] rotavirus strains, respectively. Diarrhea developed in the grandfather by sheer hospital visits, and in the grandmother, after wiping the vomit of the grandfather. AU32 like G9 strains were isolated from the grandparents also. Rotavirus specific IgM developed in all the three patients. A fourfold rise in G9 neutralizing antibodies was observed in the child and grandmother. The child's mother had asymptomatic rotavirus infection. The study highlights the potential of G9 serotype to spread from children to adults.     

Full genomic analyses of human rotavirus G4P[4], G4P[6], G9P[19] and G10P[6] strains from North-eastern India: evidence for interspecies transmission and complex reassortment events

In hospitalized patients with acute gastroenteritis in Manipur, India, four rotavirus strains were found to possess VP7 and/or VP4 genes with porcine or bovine characteristics. Considering the animal-like nature of these strains, the remaining eight gene segments were analysed to decipher their exact origin. Analyses of full genome of these strains exhibited their origin from porcine/bovine rotaviruses. This study suggests single or multiple events of reassortment involving multiple gene segments of more than one host type among the strains and emphasizes the significance of complete genetic characterization of unusual strains in regions with high incidence and mortality rates.     

Characterization of incompletely typed rotavirus strains from Guinea-Bissau: identification of G8 and G9 types and a high frequency of mixed infections

Among 167 rotavirus specimens collected from young children in a suburban area of Bissau, Guinea-Bissau, from 1996 to 1998, most identifiable strains belonged to the uncommon P[6], G2 type and approximately 50% remained incompletely typed. In the present study, 76 such strains were further characterized. Due to interprimer interaction during the standard multiplex PCR approach, modifications of this procedure were implemented. The modified analyses revealed a high frequency of G2, G8, and G9 genotypes, often combined with P[4] and/or P[6]. The Guinean G8 and G9 strains were 97 and 98%, respectively, identical to other African G8 and G9 strains. Multiple G and/or P types were identified at a high frequency (59%), including two previously undescribed mixed infections, P[4]P[6], G2G8 and P[4]P[6], G2G9. These mixed infections most likely represent naturally occurring reassortance of rotavirus strains. Detection of such strains among the previously incompletely typed strains indicates a potential underestimation of mixed infections, if only a standard multiplex PCR procedure is followed. Furthermore cross-priming of the G3 primer with the G8 primer binding site and silent mutations at the P[4] and P[6] primer binding sites were detected. These findings highlight the need for regular evaluation of the multiplex primer PCR method and typing primers. The high frequency of uncommon as well as reassortant rotavirus strains in countries where rotavirus is an important cause of child mortality underscores the need for extensive strain surveillance as a basis to develop appropriate rotavirus vaccine candidates.     

Characterization of genotype P[9]G12 rotavirus strains from argentina: High similarity with Japanese and Korean G12 strains

The circulation of the unusual P[9]G12 strains was previously reported in suburban Buenos Aires, Argentina and in Far Eastern Asian countries. To examine genetic relationships of these strains the genes coding VP7, VP4, and NSP1 from two Argentine, one Japanese and one Korean P[9]G12 isolates were sequenced and their overall genome relatedness was determined by liquid hybridization. In addition, liquid hybridization was used to compare this group of strains to the previous G12 isolates L26 and Se585, and prototype Wa, DS‐1, and AU‐1 strains. The genomes of the Argentinean, Japanese and Korean strains were virtually indistinguishable by hybridization assays, suggesting very high sequence relatedness for all 11 segments. Hybridization assays also demonstrated that these four strains belong to the AU‐1 genogroup and that their genetic relationship with rotaviruses L26 and Se585 is limited to the VP7 gene. The VP7, VP4, and NSP1 genes of the Argentinean, Japanese and Korean strains were highly homologous to each other and to Thai strain T152 (～99% identity). These results together with the report of a similar strain detected during 2003 in Brazil are consistent with a recent importation and dissemination of the G12 strains from Far Eastern countries into South America. Increasing reports from several regions of the world demonstrating a variety of different G12 reassortant strains suggests that routine surveillance for this serotype should be conducted to determine its potential for global emergence. J. Med. Virol. 81:371–381, 2009. © 2008 Wiley‐Liss, Inc.     

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.     

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.     

Phylogenetic analysis of rotaviruses with predominant G3 and emerging G9 genotypes from adults and children in Wuhan,China

Prevalence and phylogenetic relatedness of rotaviruses causing diarrheal diseases in children and adults were analyzed in Wuhan, China. During a period between June 2006 and February 2008, group A rotavirus was identified in 24.9% (280/1126) and 7.6% (83/1088) of specimens taken from children and adults, respectively. G3P[8] was the most frequent genotype in both children (66.3%) and adults (62.7%), followed by G1P[8] (20.3% and 26.2%, respectively). G9 was detected in specimens from six children (2.0%) and seven adults (5.6%). The VP7 genes of G3P[8] rotaviruses from children and adults showed extremely high sequence identities to each other (98.9–100%) and also to those of G3 viruses isolated in Wuhan in 2003–2004. In the phylogenetic analysis of the VP7 gene, the G3P[8] rotaviruses in Wuhan were clustered into a single lineage with some G3 viruses, which had been referred to as “the new variant G3” rotaviruses, reported recently in East Asia and Southeast Asia. Similar to G3P[8] rotaviruses, extremely high sequence identities between children and adults were observed for VP7 genes of G1 and G9 rotaviruses. The G9 viruses were clustered in the lineage of globally spreading strains, while G1 viruses were genetically close to those reported previously in China and Japan. These findings indicated the persistence of the variant G3 rotaviruses and spread of G9 rotaviruses derived from the global G9 lineage in Wuhan, and suggested that the rotaviruses were circulating among children and adults, irrelevant to the G types. J. Med. Virol. 81:382–389, 2009. © 2008 Wiley‐Liss, Inc.     

Genomic characterization of human rotavirus G8 strains from the African rotavirus network: Relationship to animal rotaviruses

Global rotavirus surveillance has led to the detection of many unusual human rotavirus (HRV) genotypes. During 1996–2004 surveillance within the African Rotavirus Network (ARN), six P[8],G8 and two P[6],G8 human rotavirus strains were identified. Gene fragments (RT‐PCR amplicons) of all 11‐gene segments of these G8 strains were sequenced in order to elucidate their genetic and evolutionary relationships. Phylogenetic and sequence analyses of each gene segment revealed high similarities (88–100% nt and 91–100% aa) for all segments except for gene 4 encoding VP4 proteins P[8] and P[6]. For most strains, almost all of the genes of the ARN strains other than neutralizing antigens are related to typical human strains of Wa genogroup. The VP7, NSP2, and NSP5 genes were closely related to cognate genes of animal strains (83–99% and 97–99% aa identity). This study suggests that the ARN G8 strains might have arisen through VP7 or VP4 gene reassortment events since most of the other gene segments resemble those of common human rotaviruses. However, VP7, NSP2 (likely), and NSP5 (likely) genes are derived potentially from animals consistent with a zoonotic introduction. Although these findings help elucidate rotavirus evolution, sequence studies of cognate animal rotavirus genes are needed to conclusively determine the specific origin of those genes relative to both human and animal rotavirus strains. J. Med. Virol. 81:937–951, 2009. © 2009 Wiley‐Liss, Inc.