G9 Rotaviruses Research Articles

Typing of human rotaviruses: Nucleotide mismatches between the VP7 gene and primer are associated with genotyping failure

BackgroundRotavirus genotyping is performed by using reverse transcription PCR with type-specific-primers. Because the high rotavirus mutation rate generates an extensive genomic variation, different G-type-specific primer sets are applied in different geographical locations. In Bangladesh, a significant proportion (36.9%) of the rotavirus strains isolated in 2002 could not be G-typed using the routinely used primer set. To investigate the reason why the strains were untypeable, nucleotide sequencing of the VP7 genes was performed.ResultsFour nucleotide substitutions at the G1 primer-binding site of the VP7 gene of Bangladeshi G1 rotaviruses rendered a major proportion of circulating strains untypeable using the routine primer set. Using an alternative primer set, we could identify G1 rotaviruses as the most prevalent genotype (44.8%), followed by G9 (21.7%), G2 (15.0%) and G4 (13.8%).ConclusionBecause of the natural variation in the rotaviral gene sequences, close monitoring of rotavirus genotyping methods is important.

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.

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.

Molecular characterization of the first isolation of rotavirus in guanacos (Lama guanicoe).

Rotaviruses (RVs) are a major cause of neonatal diarrhoea in humans and numerous animal species. We report G-type, P-type and phylogenetic analysis of two RV strains isolated from newborn guanacos (Lama guanicoe) with acute diarrhoea in Argentina. Isolates were group A G8 RV. Phylogenetic analysis showed a close relationship to other G8 bovine RV from Japan, U.S.A. and Switzerland. P-types were P[1] and an unusual P[14], related to human and goat P[14] strains. This is the first report of a P[14]G8 strain in our country and the first study on molecular epidemiology of rotavirus found in a new species.

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

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. The purpose of the present study is to investigate the epidemiological diversity of rotaviruses in Taiwan. 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. 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. 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 epidemiology of human P[8],G9 rotaviruses in Hungary between 1998 and 2001.

Increasing numbers of studies have documented the widespread distribution of human G9 rotaviruses and demonstrated that these strains may represent a fifth epidemiologically important G serotype. Serotype G9 strains were identified in Hungary for the first time in the 1997-1998 rotavirus season. Contrary to numerous surveys that reported several unexpected P-G combinations among recent G9 isolates (e.g. genotypes P[4], P[6] and P[19]), all Hungarian strains characterized to date possess the globally most common P-type, P[8], which was found among the first G9 isolates that were identified during the 1980s in the USA (WI61) and Japan (F45). To study the genetic variability within Hungarian G9 strains, RNA profile analysis and nucleotide sequencing were performed on a subset of samples that were collected between 1998 and 2001. These strains could be classified into four major RNA profiles, of which two were characteristic for epidemiologically major and two for epidemiologically minor G9 strains. Phylogenetic analysis demonstrated substantial sequence differences between the VP7 gene of Hungarian G9 strains and early strains that were isolated in the USA (WI61), Japan (F45) and India (116E) and a few recently identified isolates, e.g. from China (97'SZ37) and the USA (OM67) (< 90 % nucleotide sequence similarity). In contrast, the VP7 genes of Hungarian G9 strains were related very closely to the vast majority of G9 strains that were isolated in a variety of countries over the last several years (> 96 % nucleotide sequence similarity). With respect to the VP4 gene, Hungarian G9 rotaviruses fell into two of the major genetic lineages of genotype P[8], one corresponding to the epidemic strains (lineage II; P-like) and the other for two unique strains (lineage I; Wa-like), suggesting independent introduction of distinct P[8],G9 strains into Hungary or genetic reassortment between locally circulating P[8] strains and descendants of G9 isolates that were imported into the country at an earlier time. The unexpected heterogeneity found for G9 VP7 genes from several countries suggests that genetic variation among these strains has not yet been fully explored.

Rotavirus genotyping: keeping up with an evolving population of human rotaviruses

The use of molecular methods for rotavirus characterisation provides not only increased sensitivity for typing, but also allows accurate and more complete characterisation of strains, and the identification of putative reassortant strains. However, due to the constant accumulation of point mutations through genetic drift, and to the emergence of novel genotypes, possibly zoonotic transmission and subsequent reassortment, the reagents and methods used require close monitoring and updating. Methods and oligonucleotide primers are described to overcome failures to type G9, G10 and P[11] rotavirus strains, and cross-reactivity identified between G10 and G3 rotaviruses.

Antigenic and genetic characterization of serotype G2 human rotavirus strains from South Africa from 1984 to 1998.

Within South Africa, cyclic peaks of serotype G2P[4] rotavirus infection have been observed and these strains were prevalent in some locations. To examine the cyclic phenomenon of serotype G2 rotaviruses, historical stool collections from South Africa spanning 15 years were screened for G2 strains. Subgroup (VP6) ELISA, polyacrylamide gel electrophoresis (PAGE), and P genotyping were performed on 43 G2 strains to investigate the associated DS-1 genogroup characteristics. Antigenic variation of the gene encoding the major neutralization glycoprotein (VP7) was also investigated using G2-specific monoclonal antibodies. In addition, the VP7 gene of 14 serotype G2 strains was sequenced to examine genetic variation. Serotype G2 strains from South Africa displayed a 10 year cyclic pattern with major epidemics occurring in 1987 and 1997. Serotype G2 strains were also found co-dominant with G(1) strains in 1984, 1990, and 1993. The G2 strains from the major epidemics appeared to have emerged from community strains in a manner similar to that suggested for G(1) strains The serotype G2 strains displayed subgroup I specificity and short electropherotypes characteristic of DS-1 genogroup rotavirus strains but appeared to differ in the VP4 gene. Genetic analyses revealed three major serotype G2 lineages, i.e., strains isolated prior to 1987, strains isolated between 1988 and 1994, and strains isolated from 1995. The use of monoclonal antibodies and PCR primers designed against older G2 strains has resulted in the failure to serotype G2 strains circulating currently.

Nucleotide variation in the VP7 gene affects PCR genotyping of G9 rotaviruses identified in Italy.

A modified (aFT9m) and a degenerate (aFT9d) version of the rotavirus G9-specific primer (aFT9) allowed strains that were previously untypable, because of point mutations accumulating at the primer binding site, to be G typed by reverse transcription-polymerase chain reaction. The strains were collected during 2001-2002 in Italy in hospitals of the Apulia region, from children affected by severe rotavirus-associated enteritis. Using a wide selection of G9 rotaviruses detected worldwide, sequencing of the G9 untypable strains, sequence comparison, and phylogenetic analysis showed that the Italian strains have strong genetic similarity (< or =99.4%) to G9 rotaviruses identified recently in many parts of the world and different from the old G9 strains identified during the 1980s (less than 90%). Genetic variation of G9 rotaviruses explains the constraints encountered in the typing assays and presumably accounts, together with genetic reassortment events, for the emergence on a global scale of the G9 serotype.

Detection of the emerging rotavirus G9 serotype at high frequency in Italy.

Group A human rotavirus strains belonging to the unusual serotype G9 were detected at high frequency in stool specimens from infected children with acute diarrhea in Bari, Italy, during a 15-month survey from March 2001 to June 2002. This may signify a local reemergence of the G9 rotaviruses detected in Italy in the early and mid-1990s or may be related to the global emergence of G9 rotaviruses in recent years.

Viral agents of acute gastroenteritis in German children: prevalence and molecular diversity.

Acute gastroenteritis is a major source of morbidity and mortality among young children in developed and developing countries. Enteropathogenic viruses are regarded as particularly relevant causative agents. Between February 2001 and January 2002, fecal specimens were obtained from German children admitted to hospital with acute gastroenteritis and examined for rotaviruses, Noroviruses, enteric adenoviruses, and astroviruses using (RT-)PCR methods. Of the 59% (129/217) samples positive for > or =1 viral agent, 79% (102/129) carried rotavirus, whereas Norovirus was detected in 35% (45/129), enteric adenovirus in 14% (18/129), and astrovirus in 4% (5/129). Thirty-eight specimens contained at least two enteropathogenic viruses, with the majority of coinfections attributable to rotavirus/Norovirus dual infections. Sequence analysis revealed a cocirculation of G1, G3, G4, and G9 type rotavirus with G1 being the most common and G9 the second most common rotavirus G-type. Emergence of G9 rotaviruses in Germany may have implications for future vaccine development. A variety of Norovirus genotypes, most belonging to GGII, were found. Apart from subgenus F, adenovirus related genetically to subgenera A-C were detected. All astroviruses belonged to genotype 1. This is the first study concerning German children admitted to hospital that assesses the relative importance of these viruses by nested (RT-) PCR methods.

Virus diversity in a winter epidemic of acute diarrhea in France.

In France, an epidemic peak of acute diarrhea is observed each winter. Previous results suggested a viral etiology for these winter epidemics. We investigated the role of enteric viruses in acute diarrhea and their molecular diversity. One hundred sixty-one patients with acute diarrhea and 45 healthy patients (controls) from the general population were given a standardized questionnaire between December 1998 and May 1999. Stool specimens were screened for group A and C rotaviruses, human caliciviruses, astroviruses, and adenovirus types 40 and 41 by reverse transcription-PCR and/or enzyme immunoassay. Virologic analysis was positive for 63 cases (39%). Caliciviruses and group A rotaviruses were the most frequent (19 and 17% of cases, respectively). Two control stool specimens were found positive for group A rotavirus, and one was found positive for astrovirus. Molecular characterization of the strains disclosed a cocirculation of P[8],G1, P[8],G4, and P[4],G2 rotaviruses; type 1, 2, 3, 4, and 8 astroviruses; and Sapporo-like and Norwalk-like human caliciviruses. These four types of viruses accounted for an attributable risk of acute diarrhea of 34.7% for the general population, under the assumption of a causal role of these viruses.

Detection and characterization of rotaviruses in hospitalized neonates in Blantyre, Malawi.

In five separate fecal collections spanning three years, group A rotaviruses were detected by enzyme-linked immunosorbent assay in 35 (25%) of 142 specimens obtained from nondiarrheic, hospitalized neonates in Blantyre, Malawi. Molecular characterization of each strain identified, for the first time in neonates, a short electropherotype, genotype P[6], G8 strain type, similar to the dominant, cocirculating community strain detected in symptomatic infants in Blantyre. Partial sequence analysis of the VP4 and NSP4 genes of neonatal and community strains failed to identify changes which could explain the differences in clinical outcome. Neonatal serotype G8 rotaviruses should be considered as potential rotavirus vaccine candidates for use in Malawi.

Identification and molecular characterization of a bovine G3 rotavirus which causes age-independent diarrhea in cattle.

G3 rotaviruses have been reported rarely in cattle, and none have been characterized. We report the first genomic characterization of a bovine G3 rotavirus, CP-1, which had been biologically characterized in vivo and shown to cause age-independent diarrhea. CP-1 was a G3 rotavirus as its VP7 had 92 to 96% deduced amino acid identity to those of G3 rotaviruses. However, initially, CP-1 was identified as a G10 rotavirus by RT-PCR even though the CP-1 VP7 had only 81 to 85% deduced amino acid identity to those of G10 rotaviruses. Rotavirus CP-1 was of P[5] specificity, a type common in cattle, and had a bovine NSP1 and NSP4. These results added another animal species to those in which G3 rotaviruses have been found, characterized a bovine rotavirus which caused age-independent diarrhea in calves, and raised the possibility that bovine G3 rotaviruses may be misdiagnosed as G10 rotaviruses.

Bovine rotavirus G and P types and sequence analysis of the VP7 gene of two G8 bovine rotaviruses from Japan

A total of 1481 fecal specimens were collected from diarrheic calves under 1 month of age on 29 dairy and beef farms in 11 prefectures in Japan during the period from 1987 to 2000. Those calves and their dams were not vaccinated against rotavirus. One hundred and forty-two bovine rotaviruses were isolated on MA-104 cell cultures and detected by latex agglutination test. They were classified into 18 G6P[1] (11.2%), 53 G6P[5] (37.3%), 15 G6P[11] (10.6%), 12 G10P[5] (8.5%), 42 G10P[11] (29.6%) and 1 G8P[11] (0.7%) by reverse transcription–polymerase chain reaction. One serotype G8 virus was untypable for the P genotype suggesting a new type of bovine origin. The least common G8 serotype viruses were isolated from the samples of farms from Niigata and Tokushima prefectures. The VP7 gene sequences of the two isolates exhibited a high degree of homology as well as previously reported G8 viruses with 93.3–98.8% identity of deduced amino acids. A phylogenetic analysis of the VP7 gene of the two G8 viruses and 13 previously reported G8 viruses by the neighbor-joining method indicated that the two newly isolated G8 rotaviruses had a common origin and they were assigned to a new disparate cluster.

Rotavirus cross-species pathogenicity: molecular characterization of a bovine rotavirus pathogenic for pigs.

Rotaviruses which cause disease in heterologous animal species have been reported but the molecular basis of cross-species infectivity and disease is not established. We report the molecular characterization of a cloned rotavirus, PP-1, which was originally obtained from cattle and which had been biologically characterized in vivo in two target animal species, gnotobiotic pigs and calves. In pigs, PP-1 caused severe clinical disease but in experimental calves it replicated subclinically. PP-1 was characterized as a G3 reassortant with a porcine VP4 and NSP4 but a bovine NSP1. The PP-1 VP4 had 96 to 97% deduced amino acid identity to P[7] porcine rotaviruses and P[7] specificity was confirmed with VP4-specific monoclonal antibodies. Sequence analysis of the PP-1 NSP1 showed 94 to 99.6% deduced amino acid identity to bovine rotaviruses but the NSP4 protein had 94 to 98% identity to the NSP4 genotype B porcine rotaviruses. G-typing PCR initially classified PP-1 as a G10 rotavirus but sequence analysis revealed 92 to 96% identity of the PP-1 VP7 with porcine, simian, and human G3 rotaviruses. These results, combined with the in vivo properties of PP-1 in the two target species, supported the concept that species-specific VP4 and NSP4, but not NSP1, are required to induce rotavirus disease, at least in calves and pigs. The results illustrate experimentally that rotaviruses circulating in one animal species can pose a risk to another by the emergence of a pathogenic reassortant rotavirus under appropriate conditions.

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.

Expanding Global Distribution of Rotavirus Serotype G9: Detection in Libya, Kenya, and Cuba

Serotype G9 may be the fifth most common human rotavirus serotype, after serotypes G1 to G4. In three cross-sectional studies of childhood diarrhea, we have detected serotype G9 rotaviruses for the first time in Libya, Kenya, and Cuba. Serotype G9 constituted 27% of all rotaviruses identified, emphasizing the reemergence of serotype G9 and suggesting that future human rotavirus vaccines will need to protect against disease caused by this serotype.

Characterisation and phylogenetic analysis of the VP7 proteins of serotype G6 and G8 human rotaviruses.

Serotype G6 and G8 rotaviruses are rarely found in man and may have originated in animals. Human serotype G6 and G8 rotaviruses, isolated from hospitalised children at various locations in Australia, were characterised. Deduced amino acid sequences of the major neutralising antigen, V7, showed significant identity to the cognate proteins of prototype human and bovine G6 and G8 viruses, respectively, and the strains reacted with G6 and G8 serotype-specific neutralising monoclonal antibodies, respectively, in an enzyme immunoassay. The VP4 type was determined as P[14] for all strains tested. Phylogenetic analysis of these and other human and bovine VP7 sequences suggested that a single inter-species transmission event, possibly from cattle, may have led to the emergence of G6 viruses in man. In contrast, the exchange of genes between human and bovine G8 viruses may have occurred onmore than one occasion, or these genes may have originated in a different host.

Rotavirus strain diversity in Blantyre, Malawi, from 1997 to 1999.

In a 2-year study of viral gastroenteritis in children in Blantyre, Malawi, the diversity of rotavirus strains was investigated by using electropherotyping, reverse transcription-PCR amplification of the VP7 and VP4 genes (G and P genotyping), and nucleotide sequencing. Of 414 rotavirus strains characterized, the following strain types were identified: P[8], G1 (n = 111; 26.8%); P[6], G8 (n = 110; 26.6%); P[8], G3 (n = 93; 22.5%); P[4], G8 (n = 31; 7.5%); P[8], G4 (n = 21; 5.1%); P[6], G3 (n = 12; 2.9%); P[6], G1 (n = 7; 1.7%); P[6], G9 (n = 3; 0.7%); P[6], G4 (n = 3; 0.7%); P[4], G3 (n = 1; 0.2%); and mixed (n = 15; 3.6%). While all strains could be assigned a G type, seven strains (1.7%) remained P nontypeable. The majority of serotype G8 strains and all serotype G9 strains had short electropherotype profiles. All remaining typeable strains had long electropherotypes. Divergent serotype G1 rotaviruses, which contained multiple base substitutions in the 9T-1 primer binding site, were commonly identified in the second year of surveillance. Serotype G2 was not identified. Overall, G8 was the most frequently identified VP7 serotype (n = 144; 34.8%) and P[8] was the most frequently detected VP4 genotype (n = 227; 54.8%). Partial sequence analysis of the VP4 gene of genotype P[8] rotaviruses identified three distinct clusters, which predominantly (but not exclusively) comprised strains belonging to a distinct VP7 serotype (G1, G3, or G4). As a result of mutations in the 1T-1 primer binding site, strains belonging to each cluster required a separate primer for efficient typing. One cluster, represented by P[8], G4 strain OP354, was highly divergent from the established Wa and F45 VP4 P[8] lineages. As is the case for some other countries, the diversity of rotaviruses in Malawi implies that rotavirus vaccines in development will need to protect against a wider panel of serotypes than originally envisioned.