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.     

Anticipating rotavirus vaccines in Brazil: detection and molecular characterization of emerging rotavirus serotypes G8 and G9 among children with diarrhoea in Recife, Brazil

In 2006, Brazil will initiate universal immunization of its 4-million infants with a live attenuated serotype G1P[8] human rotavirus vaccine. In anticipation of the national immunization program, this study was undertaken to characterize rotavirus strains circulating among children in Recife, one of the largest cities in the northeast region of Brazil. Group A rotaviruses were detected in 102 (35%) of 290 faecal specimens collected from children under 5 years of age who presented with acute diarrhoea during a 1-year period between May 2004 and April 2005. In addition to the globally common G1P[8] serotype that accounted for 49% of strains, emerging rotavirus serotypes G8P[6] and G9P[8] represented 2% and 29% of strains, respectively. Following cell culture adaptation, RNA-RNA hybridization demonstrated that two Brazilian G8P[6] rotavirus strains shared a high level of genomic RNA homology with Malawian G8P[6] strains, and a Brazilian G9P[8] strain was related most closely to a G9P[8] strain from India. The results suggest that certain rotavirus strains have a much wider global circulation than generally appreciated. Continued global spread of such strains might challenge the efficacy of current rotavirus vaccines.     

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.     

Genotype diversity of group A rotavirus strains in children with acute diarrhea in urban Burkina Faso, 2008-2010

In this study, the diversity of G and P genotypes of rotavirus strains in Burkinabe children were examined. Between November 2008 and February 2010, 447 stool samples were collected from children <5 years of age with acute diarrhea visiting hospital in Ouagadougou. Group A rotavirus was previously detected in 151/447 (33.8%) of the samples tested by an immunochromatographic test and these samples were now tested further for rotavirus G and P genotypes by RT-PCR. Of these, the rotavirus type genes were amplified by RT-PCR for 140/151 (92.7%) samples and G and P genotypes were successfully determined for 81 (57.9%) and 130 (92.9%) samples, respectively. The most prevalent G genotypes were G1, 34/140 (24.3%), and G9, 21/140 (15%), while the predominant P genotypes were P[6], 56/140 (40%), and P[8], 54/140 (38.6%). Among the single infections, 63/140 (45%), the predominant G/P combinations were: G1P[8] (33%), G9P[8] (29%), and G2P[6] (14%). The unusual strains G1P[9] (3%), G12P[6] (3%), G10P[6] (2%), and G2P[8] (2%) were also detected. In a high number of strains 61/140 (43.6%), the G genotype could not be determined and mixed infections were determined in 17/140 (12.1%) of strains identified. This study highlights the high diversity and presence of unusual rotavirus strains in children in Burkina Faso.     

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 human rotavirus serotype G9 isolated in Japan and Thailand from 1995 to 1997.

Serotyping of human rotavirus was conducted in 396 Japanese and 100 Thai rotavirus-positive fecal specimens collected from 1995 to 1997. Serotype G9 was found to be the third most common serotype with frequency of 16.2% in Thailand from 1996 to 1997. It was also detected in Japan with a low frequency (0.7%) in this year. The genetic analyses of VP4 and NSP4 genes of these G9 strains showed that 1 strain from Japan possessed P[8] genotype and NSP4 Wa-group with long electropherotype (e-type). In contrast, 5 strains from Thailand belonged to P[6] and 1 strain belonged to P[4]. All of the Thai strains were in the NSP4 KUN-group with a short e-type. Sequence analysis of their VP7 gene revealed that there was the highest homology among fecal G9 strains (> 96.3%, amino acid identity) and a relatively high degree of homology to standard viruses, F45 from Japan (95.4-96.3%, amino acid identity) and 116E from India (92-92.3%, amino acid identity). However, immunological analysis using G9 specific monoclonal antibodies (Mabs) against VP7 protein showed that the G9 strains isolated from the two countries had different antigenic specificity. It was confirmed further by intraserotypical phylogenetic analysis of VP7 amino acid. These results indicated that the prevalence of G9 rotavirus in 1996-1997 in Thailand was relative to the continuing recent emergence of it on a worldwide basis, while the Japanese G9 strain isolated in this survey was identified to have progenitors common to the F45 strain that was prevalent in 1985 in Japan. Phylogenetic analysis of VP7 amino acid of G1-14 prototype rotavirus showed that the G9 strains were most closely related to the equine G14 rotavirus FI23 strain but G3 strains, interserotypically. These findings suggest that G9 rotaviruses might be divided into two or more subtypes.     

Genotypes of rotavirus strains circulating in Amman, Jordan, in 2006/07 and their significance for the potential effectiveness of future rotavirus vaccination

The prevalence, seasonality and genotypes of rotavirus circulating in Jordan were determined to provide data useful for the implementation of a rotavirus vaccine in the national childhood vaccination program. During 2006/07, rotavirus was detected in 35% of hospitalized children, and 88% of the cases occurred in children aged between 6 and 23 months. Rotavirus infection persisted throughout the year and peaked in winter. Seven genotypes were identified by RT-PCR and sequencing: G1P[8], G9P[8], G2P[4], G1P[4], G3P[8], G4P[8] and G9P[6], which represented 69%, 8.8%, 2.4%, 2.0%, 1.2%, 0.4% and 0.4% of the rotavirus strains, respectively. Thirteen percent and 2.8% of the total strains were partially typed or untypeable, respectively. Eighty-eight percent and 12% of the rotavirus strains possessed a long and short electropherotype, respectively. As more than 90% of the rotavirus strains circulating in Jordan possessed the G1 or P[8]-genotype, it is concluded that the implementation of the rotavirus vaccine in the framework of the national childhood vaccination program of Jordan would most likely be very effective. Continuous monitoring of the currently circulating genotypes in Jordan should be encouraged.     

Dominating prevalence of P[8],G1 and P[8],G9 rotavirus strains among children admitted to hospital between 2000 and 2003 in Budapest, Hungary

Group A rotaviruses are the main cause of acute dehydrating diarrhea in children, responsible for high mortality in developing countries and a significant socio-economic burden associated with treating the disease in developed countries. Two rotavirus vaccine candidates predicated on either homotypic or heterotypic protection have undergone clinical trials recently and await licensure for routine use. In anticipation of a future vaccination campaign in Hungary, the diversity of rotaviruses collected from Budapest between 2000 and 2003 were analyzed by polyacrylamide gel electrophoresis (PAGE) of the viral genome and by serotyping and genotyping of the outer capsid genes, VP7 and VP4. Among 2,763 rotavirus positive specimens available for analysis, we were able to determine the electropherotype of 2,227, and, of these, 1,517 (68.1%) were subjected to G typing and 1,173 (52.7%) were subjected to P typing. We successfully G typed 1,481 (97.6%) and P typed 1,130 (96.3%) strains, respectively. A total of six G types (G1, 50.2%; G2, 2.2%; G3, 1.7%; G4, 5.8%; G6, 0.6%; and G9, 34.4%) and four P types (P[4], 3.0%; P[6], 0.7%; P[8], 89.9%; and P[9], 1.7%) were identified in nine individual combinations (P[8],G1; P[4],G2; P[8],G3; P[8],G4; P[8],G9; P[6],G4; P[4],G1; P[9],G3; and P[9],G6). The prevalence of VP7 and VP4 specificities varied from year to year. In this regard, a shift in serotype predominance from G1 in 2000-2001 (61.8%) and 2001-2002 (69.7%) to G9 in 2002-2003 (51.3%) was an intriguing observation that has been reported recently in some other countries, as well. The emergence of serotype G9 rotaviruses in Hungary and other parts of the world may have implications for future vaccine development and use, particularly, if current vaccine candidates cannot confer adequate homotypic or heterotypic protection against these strains.     

Molecular epidemiology of rotavirus diarrhea among children in Saudi Arabia: first detection of G9 and G12 strains

In anticipation of rotavirus vaccine introduction in Saudi Arabia, this study was undertaken to determine the distribution of the G and P genotypes of rotaviruses in order to examine whether there was any emerging serotype or unusual strain circulating in children in Saudi Arabia. Of 984 stool specimens collected between 17 April 2004 and 16 April 2005, rotavirus was detected by an enzyme-linked immunosorbent assay in 187 (19%) diarrheal children less than 5 years of age. Of these, 160 (86%) were classified into G and P genotypes as follows: G1P[8] (44%), G2P[4] (20%), G9P[8] (11%), G12P[8] (4%), and G3P[8] (4%). RNA polyacrylamide gel electrophoresis identified 94 (50%) specimens as long RNA patterns, 30 (16%) specimens as short RNA patterns, and 1 mixed infection. Only a single long RNA electropherotype was identified for seven specimens containing G12P[8] rotavirus. RNA-RNA hybridization demonstrated that the G12P[8] strains were similar in their genomic constellation to locally cocirculating strains and to a Nepalese G12P[8] strain. The Saudi Arabian G12 VP7 gene had a 99% nucleotide sequence identity with Nepalese and Indian G12 VP7 genes and belonged to the third lineage. This study is the first to describe the distribution of rotavirus G and P types and also the first to identify G9P[8] and G12P[8] strains in the country.     

Evidence of rotavirus AU32 like G9 strains from nontypeable fecal specimens of Indian children hospitalized during 1993-1994

Serotyping of 432 rotavirus positive fecal specimens collected from hospitalized children during 1990-1997 was carried out at National Institute of Virology (NIV), Pune, India, using monoclonal antibodies (MAbs) directed against VP7 determinant of serotypes G1-G4, G6, G8, and G10. However, significant number of specimens, that is, 47.92% remained nontypeable. The aim of the present study was to culture adapt the nontypeable specimens and to characterize them further. Since the fecal specimens were not tested by MAb to G9 serotype, which has emerged as an important serotype infecting humans recently, presence of G9 serotype was expected in nontypeable specimens. Therefore, we selected specimens from those children, who showed higher neutralizing antibody (NAb) titer in their convalescent serum samples to G9 serotype than their mothers. Out of six isolates having long electropherotype, five isolates showed subgroup II, and one showed subgroup I, II. The isolates were confirmed as G9 by MAb based ELISA, neutralization assay, and PCR. The G9 specific nested PCR products of four isolates showed 96-99% identities to AU32 G9 strain reported from Japan. P type of four isolates was determined as P8. Besides isolates, four additional nontypeable fecal specimens were confirmed as G9 by MAb based ELISA. Thus, 10 (28.57%) out of 35 nontypeable specimens were identified as rotavirus serotype G9. The results indicate that serotype G9 may represent significant proportion of specimens, which were previously nontypeable.     

Molecular characterization of rotavirus strains from children with diarrhea in Italy, 2007-2009

The surveillance network RotaNet-Italia was established in 2007 in order to investigate the diversity of co-circulating rotavirus strains in Italy, and to provide a baseline for future assessment of possible effects of vaccine implementation in selecting novel versus common rotavirus strains. A total of 2,645 rotavirus strains from pediatric patients with acute diarrhea were collected over three consecutive seasons from September 2006 through August 2009, and partially characterized by standardized multiplex RT-PCR. Most of strains (89.1%) belonged to genotypes G1-G4, and G9, associated with either P[8] or P[4], commonly found in humans worldwide. However, in at least 2.0% of cases, viruses exhibited either a G or P type typical of animal viral strains, suggesting gene reassortment events between rotaviruses of different origin. Mixed infections with two or more rotavirus strains were observed frequently (7.6% of patients), and depended on the frequencies of co-circulating rotaviruses of one particular genotype. The numbers and genotypes of likely natural reassortants of common genotype rotaviruses were found to be correlated with the observed numbers and genotypes of mixed infections. Large variation in the relative frequency of different rotavirus genotypes was observed between different seasons and/or areas of Italy, suggesting independent evolution or differential introduction of viral strains with respect to both time and space.     

Characterization of neutralization specificities of outer capsid spike protein VP4 of selected murine,lapine, and human rotavirus strains

Neutralization specificities of outer capsid spike protein VP4 of murine rotavirus strains EW (P?[16],G3) and EHP (P?[20],G3) and lapine rotavirus strains Ala (P?[14],G3), C11 (P?[14],G3), and R2 (P?[14],G3) as well as human rotavirus strains PA169 (P?[14],G6) and HAL1166 (P?[14],G8) were determined by two-way cross-neutralization. This was done by generating and characterizing (i) three murine x human, three lapine x human, and two human x human single gene substitution reassortant rotaviruses, each of which bore identical human rotavirus DS-1 strain VP7 (G2), and (ii) guinea pig hyperimmune antiserum raised against each reassortant. Reference rotavirus strains employed in the study represented 10 established VP4 (P) serotypes, including 1A[8], 1B[4], 2A[6], 3[9], 4[10], 5A[2], 5B[2], 5B[3], 6[1], 7[5], 8[11], 9[7], and 10[16] as well as a P serotype unknown P[18]. Murine rotavirus strains EW and EB were demonstrated to share the same P serotype (P10[16]) distinct from (i) 9 established P serotypes, (ii) lapine and human rotavirus strains bearing the P[14] genotype, and (iii) an equine rotavirus strain bearing the P[18] genotype. Both lapine (Ala, C11, and R2) and human (PA169 and HAL1166) rotaviruses were shown to belong to the same VP4 serotype, which represented a distinct new P serotype (P11[14]). P serotype 13[20] was assigned to murine rotavirus EHP strain VP4, which was shown to be distinct from all the P serotypes/genotypes examined in the present study.     

Changing patterns of rotavirus genotypes in ghana: emergence of human rotavirus G9 as a major cause of diarrhea in children

Genotyping of human rotaviruses was performed on 312 rotavirus-positive samples collected from 2,205 young children with diarrhea in the Upper East District of Ghana, a rural community. Of the 271 (86.9%) rotavirus strains that could be VP7 (G) or VP4 (P) characterized, 73 (26.9%) were of G9 specificity. The predominant G9 genotype was G9P[8], which constituted 79.5% of all G9 strains detected, followed by G9P[6] (12.3%), G9P[10] (2.7%), and G9P[4] (1.3%). G9 strains with mixed P types constituted 2.7% of all G9 strains found in the study. All the G9P[8] strains had a long RNA electrophoretic pattern with VP6 subgroup II specificity. Four G9 isolates, GH1319, GH1416, GH3550, and GH3574, which were selected based on the abundance of stool material and were representative of the three electropherotypes observed, were cloned and sequenced. The Ghanaian isolates shared more than 98% sequence nucleotide homology with other G9 strains from the United States (US1205), Malawi (MW69), Brazil (R160), Japan (95H115), and Nigeria (Bulumkutu). However, they showed only 95% nucleotide homology with the Thai G9 strain Mc345. Phylogenetic analysis of the nucleic acid sequence revealed the existence of at least three clusters, with Ghanaian strains forming one cluster, Nigerian and Brazilian strains forming a second cluster, and U.S., Malawian, and Japanese strains forming a third.