VP7 gene polymorphism of serotype G9 rotavirus strains and its impact on G genotype determination by PCR

Rotaviruses are the single most important etiologic agents of severe diarrhea of infants and young children worldwide. Surveillance of rotavirus serotypes/genotypes (both VP7[G] and VP4[P]) is in progress globally in which polymerase chain reaction (PCR) has been the assay of choice. We investigated polymorphism of the VP7 gene of serotype G9 rotavirus strains and its impact on the determination of VP7 gene genotype by PCR assay. By VP7 gene sequence analysis, we and others have previously shown that the G9 rotavirus strains belong to one of three VP7 gene lineages. By PCR assay using three different sets of commonly used primers specific for G1-4, 8 and 9, 23 Brazilian G9 strains and 5 well-characterized prototype G9 strains which collectively represented all three VP7 gene lineages were typed as: (i) G3; (ii) G4; (iii) G9; (iv) G3 and G9; or (v) G9 and G4 depending on a primer pool employed. This phenomenon appeared to be due to: (i) a VP7 gene lineage-specific polymorphism, more specifically mutation(s) in the primer binding region of the VP7 gene of G9 strain; and (ii) the magnitude of difference in nucleotide homology at respective primer binding site between homotypic (G9) and heterotypic (G3 or G4) primers present in a primer pool employed.     

Diverse origin of P[19] rotaviruses in children with acute diarrhea in Taiwan: Detection of novel lineages of the G3, G5, and G9 VP7 genes

We previously reported the detection of genotype P[19] rotavirus strains from children hospitalized with acute dehydrating diarrhea during a 5-year surveillance period in Taiwan. The characterization of five P[19] strains (0.4% of all typed), including three G3P[19], a novel G5P[19], and a unique G9P[19] genotype is described in this study. Phylogenetic analysis of the VP4, VP7, VP6, and NSP4 genes was performed, which demonstrated novel lineages for respective genotypes of the VP4 and the VP7 genes. The sequence similarities of the P[19] VP4 gene among Taiwanese human strains was higher (nt, 91.5-96.2%; aa, 93.7-97.6%) than to other P[19] strains (nt, 83.5-86.6%; aa, 89.4-94.1%) from different regions of the world. The VP7 gene of the three G3P[19] Taiwanese strains shared up to 93.4% nt and 97.5% aa identity to each other but had lower similarity to reference strain sequences available in GenBank (nt, <90.1%; aa, <95.6%). Similarly, the VP7 gene of the novel G5P[19] strain was only moderately related to the VP7 gene of reference G5 strains (nt, 82.2-87.3%; aa, 87.0-93.1%), while the VP7 gene of the single G9P[19] strain was genetically distinct from other known human and animal G9 rotavirus strains (nt, ≤ 92.0%; aa, ≤ 95.7%). Together, these findings suggest that the Taiwanese P[19] strains originated by independent interspecies transmission events. Synchronized surveillance of human and animal rotaviruses in Taiwan should identify possible hosts of these uncommon human rotavirus strains.     

Nigerian rotavirus serotype G8 could not be typedby PCR due to nucleotide mutation at the 3′ endof the primer binding site

Summary.  A rotavirus strain HMG89 from Nigeria with short electrophoretic pattern was typed G3 by PCR. A cDNA clone from the PCR product which hybridised in Northern blots to RNA segment 9 of the homologous Nigerian rotavirus strain HMG89 and laboratory reference strain 69M but not to other mammalian group A rotaviruses was sequenced. The VP7 gene 9 sequence is 1060 nucleotides long with two base deletions at positions 1034–1035. Sequence analysis of the primer (aAT8) used in the previous PCR serotyping assay revealed a mutation in one of the three nucleotide bases at the 3′ end of the primer binding site accounting for our inability to serotype G8 strains in our samples. These findings demonstrate that PCR analysis can, albeit infrequently, lead to error in typing of rotaviruses due to small numbers of mutations in the primer binding region. Received August 20, 1996 Accepted April 21, 1997     

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.     

Characterisation of rotavirus G9 strains isolated in the UK between 1995 and 1998

G9P[6] and G9P[8] rotavirus strains were identified during 1995/96 through the molecular epidemiological surveillance of rotavirus strains circulating in the UK between 1995 and 1998. An increase in the incidence and spread of sporadic infections with rotavirus genotype G9P[8] across the UK was detected in the two following seasons. Partial sequencing of the VP7 gene showed that all the UK strains shared a high degree of homology and were related very closely to G9 strains from the US and from symptomatic infections in India (> or =96% homology). The UK strains were related more distantly to the apathogenic Indian strain 116E (85-87.8% homology). Phylogenetic analysis revealed clustering of the UK strains into 3 different lineages (I to III) and into two sub-lineages within lineage I. There were correlations between VP7 sequence clustering, the P type and the geographical origin of the G9 strains. Partial sequencing of the VP4 gene showed high degree of homology (>98%) among all the P[6] strains, and the sequences obtained from the P[8] strains clustered into 2 of the 3 global lineages described for P[8] strains associated with other G types. These data suggest that G9 strains may be a recent importation into the UK, and that G9P[8] strains may have emerged through reassortment in humans between G9P[6] strains introduced recently and the more prevalent cocirculating G1, G3 and G4 strains that normally carry VP4 genes of P[8] type.     

Genetic variation in the VP7 gene of rotavirus G1P[8] strains isolated in Vietnam, 1998-2009

Group A rotavirus genotype G1P[8] is the most common strain affecting humans around the world over the past few decades. In this study, we examined genetic variation in the VP7 gene of rotavirus G1P[8] strains, detected in children of four major cities of Vietnam during three different rotavirus seasons: 1998-1999, 2007-2008 and 2008-2009 in order to assess the evolution of the virus over 11 years. Fecal samples (n=73) from children hospitalized for gastroenteritis caused by G1P[8] rotavirus were analyzed by DNA sequencing of gene 9 encoding the VP7 capsid protein. Phylogenetic analyses indicated that VP7 gene of the G1 strains from 1999 contained a lineage I, while rotaviruses from 2009 clustered in lineage II. Both of these lineages were found co-circulating in 2007-2008 season. While different sublineages of lineage I and II co-circulated in the 1998-1999 and 2007-2008 seasons, almost all strains in 2009 belonged to sub-lineage II-C. In the analysis using selected 10 strains, the VP4 genes of these 2 VP7-G1 lineages were all grouped in F45-like cluster. Deduced amino acid analyses indicated that there were thirteen amino acid substitutions between strains of two lineages. Of those, two were found in antigenic regions A and C, implying possible antigenic differences between these two lineages. The G1P[8] strains in Vietnam are very genetically diverse and dynamic, implying the frequent monitoring on evolution of rotavirus will be important to assess efficacy of rotavirus vaccine in Vietnam.     

Analysis of human rotavirus G1P[8] strains by RFLP reveals higher genetic drift in the VP7 than the VP4 gene during a 4-year period in Mexico

Several studies have demonstrated that rotaviruses of the G1P[8] genotype are among the most important worldwide. Sequence analysis of G1P[8] strains has revealed high genetic variability of VP4 and VP7 genes. The aim of this study was to investigate by restriction fragment length polymorphism (RFLP) analysis the genetic variability of the VP7 and VP4 genes within rotaviruses of the G1P[8] genotype. A total of 60 rotavirus-positive fecal samples genotyped as G1P[8], were collected from children with acute diarrhea under 5 years of age, between October 1995 and October 1998. The VP7 and VP4 genes were amplified by RT/PCR, using the Beg9/End9 primer pair and the Con3 and Con2 primers, respectively. VP7 amplicons were digested with three restriction enzymes Hae III, Taq I and Rsa I in separate reactions and VP4 amplicons were digested similarly with endonucleases Hinf I, Sau96 I and Rsa I. Analysis of the digested VP7 and VP4 amplicons showed a higher genetic drift for the VP7 gene (18 RFLPs) compared to the VP4 gene (9 RFLPs). The combination of profiles for both VP7 and VP4 amplicons, showed 27 different patterns, none of them similar to the Wa-1 strain. Furthermore, RFLP analysis of these G1P[8] strains, clearly differentiated the viruses into two main clusters, both of them sharing the same restriction pattern for the VP4 gene, and a different one for the VP7 gene.     

Presence of three P types (VP4 serotypes) and two G types (VP7 serotypes) among bovine rotavirus strains

Summary Cross neutralization tests with a panel of rotavirus strains representing previously described nine VP7 (G) serotypes revealed that bovine rotavirus strain KK-3, a prototype Japanese bovine serotype 2, belonged to a new serotype (G10), confirming and extending the recent report of Snodgrass et al. [J. Clin. Microbiol. 28: 504–507 (1990)] which showed that hyperimmune serum to the KK-3 strain neutralized the B223 strain, a proposed type strain of G10. Further antigenic characteristics of the KK-3 strain, as well as the 0510 strain (a G6 strain isolated in Japan), were examined in terms of their VP4 (P) specificity. For the characterization of P types, we employed genetic reassortants that possess VP4 gene for UK and VP7 gene for D (G1), VP4 gene for NCDV and VP7 gene for SA11 (G3), or VP4 gene for SA11 and VP7 gene for NCDV (G6) in the plaque reduction neutralization assay with hyperimmune sera against these two Japanese strains and the prototype bovine rotavirus NCDV strain. While the 0510 strain had UK-like P and NCDV-like G types, the KK-3 strain had a distinct set of P and G types. Thus, at least three P types (NCDV-, UK-, and KK-3-like) and two G types (G6 and G10) are present among bovine rotavirus strains.     

Molecular characterization of serotype G9 rotaviruses circulating in South Korea between 2005 and 2010

A total of 18 rotavirus G9 strains in South Korea were collected during five rotavirus seasons between 2005 and 2010. The relationship between these strains was examined by analyzing the genetic variation of two major structural genes, VP7 and VP4. All the rotavirus isolates were of the G9P[8] genotype. The VP7 phylogenetic analysis demonstrated that all of the G9 rotaviruses circulating in South Korea belonged to lineage IIId and were within three single clusters. The amino acid comparison of the antigenic regions of the VP7 gene suggests possible common progenitors of these strains. Phylogenetic analysis of P[8] VP4 genotypes indicated three lineages, P[8]‐2, P[8]‐3, and P[8]‐4, with P[8]‐3 being the most common. The results of this study provide information on the genetic relatedness of rotavirus G9 strains circulating in South Korea over recent years and can be utilized for the development of effective vaccines and the identification of reference strains for future efficacy studies. J. Med. Virol. 85:171–178, 2012. © 2012 Wiley Periodicals, Inc.     

Acrylamide concentration affects the relative position of VP7 gene of serotype G2 strains as determined by polyacrylamide gel electrophoresis

BACKGROUND: During the course of development and characterization of various rotavirus reassortants, we found that the relative position of the gene encoding neutralization and protective antigen VP7 of certain rotavirus strains in a PAGE gel was influenced by the concentration of acrylamide. OBJECTIVES: We investigated systematically various factors that affected the relative position of the rotavirus VP7 gene in a PAGE gel. STUDY DESIGN: We analyzed dsRNAs of selected rotavirus strains bearing G1, G2, G3 or G9 specificity by PAGE at varying concentrations of acrylamide. RESULTS: We demonstrated that the relative position of the VP7 gene of three G2 strains varied depending upon the concentration of acrylamide in a PAGE gel, which occurred not only in a homologous G2 virus gene background but also in a heterologous G3 virus gene background; and the VP7 gene bearing G1, G3, G4 or G9 specificity did not display this phenomenon when the PAGE running conditions were varied. CONCLUSIONS: The concentration of acrylamide in a PAGE gel was the major factor that influenced the relative position of the VP7 gene of G2 rotavirus strains (i.e., VP7 gene coding assignment by PAGE).     

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.     

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.     

Brazilian P[8],G1, P[8],G5, P[8],G9, and P[4],G2 rotavirus strains: nucleotide sequence and phylogenetic analysis

Rotavirus epidemiological surveys with molecular analysis of strains are required for gastroenteritis control and prevention. Twenty-nine human rotavirus strains detected in Rio de Janeiro, Brazil, from 1986 to 2004 were characterized as P[8],G1, P[8],G5, P[8],G9, and P[4],G2 genotypes. The VP7 genes were sequenced and phylogenetic analysis was performed. Strains of genotype G1 revealed two distinct lineages, G1-3 and G1-4; strains of genotype G2 grouped in lineage G2-1; G5 strains clustered with other Brazilians G5 strains and G9 strains were closely related to each other in lineage G9-3, distinct from the original G9 strains detected in 1980s. The VP4 genes were analyzed and P[8] strains fell into two major genetic lineages, P[8]-2 and P[8]-3. Our findings document an intragenotype diversity represented by lineages and sublineages within rotavirus circulating in Rio de Janeiro from 1986 to 2004, before application of a vaccine (Rotarix) in Brazil. This report emphasizes the importance of continuing monitor genotypes to verify if uncommon strains or newly strains are emerging to be specifically addressed in future vaccine trials.     

Association of the G4 rotavirus genotype with gastroenteritis in adults

Rotavirus is the most common etiological cause of acute viral gastroenteritis in infants and young children worldwide, yet its role in the adult population is less well understood. We have recently identified rotavirus as the causative agent of severe diarrhea in adults, specifically in two gastroenteritis outbreaks in separate care for the elderly homes. Strain typing has shown the continued presence of P[8]G1, the emergence of P[8]G9, and the reemergence of P[8]G4. A total of 26 community cases and 6 outbreak cases of rotavirus infection, positive via a molecular screening assay, were subsequently amplified using VP4 and VP7 specific primers (Con2/Con3 and 1A/1B primer sets, respectively). The age range of patients investigated was from <1 year to 89 years. The resulting PCR products were cloned into TOPO10 PCR IV vector and sequenced to give the P- and G-type accordingly. All sequence data were subjected to BLAST analysis. Three different rotavirus types P[8]G1, P[8]G4, and P[8]G9 were identified. Types P[8]G1 and P[8]G9 were identified as circulating within the community, whereas the third type P[8]G4 was identified only in an elderly care outbreak. The identification of G9 rotaviruses supports evidence of emergence of the genotype on a global scale.     

VP7 Gene of human rotavirus A genotype G5: Phylogenetic analysis reveals the existence of three different lineages worldwide

Group A rotavirus (RV-A) genotype G5, which is common in pigs, was also detected in children with severe diarrhea in Brazil, Argentina, Paraguay, Cameroon, China, Thailand, and Vietnam. To evaluate the evolutionary relationship among RV-A G5 strains, the VP7 and VP4 genes of 28 Brazilian RV-A G5 human strains, sampled between 1986 and 2005, were sequenced and compared with other RV-A G5 strains currently circulating worldwide in animals and humans. The phylogenetic analysis of RV-A G5 VP7 gene strains demonstrates the existence of three main lineages: (a) Lineage I: Brazilian strains grouped with three porcine strains from Thailand; (b) Lineage II: porcine, bovine, and equine strains from different regions; (c) Lineage III: human strains isolated in Asia and Africa, and two porcine strains from Argentina. The VP8* (*non-typable) subunit of VP4 gene sequencing showed that all P[8] strains fell into three major genetic lineages: P[8]-1; P[8]-2; and P[8]-3. These results showed that the RV-A G5 strains circulating in humans are the result of two independent zoonotic transmission events, most likely from pigs.