Amino Acid Substitution within the VP7 Protein of G2 Rotavirus Strains Associated with Failure To Serotype

Abstract

Rotavirus strains collected in the United Kingdom during the 1995-1996 season and genotyped as G2 by reverse transcription-PCR failed to serotype in enzyme-linked immunosorbent assays using three different G2-specific monoclonal antibodies. The deduced amino acid sequences of the antigenic regions A (amino acids 87 to 101), B (amino acids 142 to 152), and C (amino acids 208 to 221) of VP7 revealed that a substitution at position 96 (Asp3Asn) correlated with the change in ability to serotype these G2 strains.Rotaviruses are triple-layered particles of the Reoviridae family that contain 11 segments of double-stranded RNA. The outer layer is composed of VP7 and VP4 proteins, encoded by gene segments 9, 7, or 8 (depending on the strain) and 4, respectively (8). These two proteins elicit neutralizing antibody responses and form the basis for the dual classification system of rotaviruses into G and P types, the designations being derived from glycoprotein (VP7) and protease-sensitive protein (VP4), respectively (8). Comparative sequence analyses of the deduced VP7 amino acid sequences of different animal and human rotavirus serotypes have identified six serotype-specific variable regions (VR) between amino acids (aa) 39 and 50, aa 87 and 101, aa 120 and 130, aa 143 and 152, aa 208 and 221, and aa 233 and 242, and these have been designated VR4 to VR9 (9, 11). VR5, VR7, and VR8 correspond to the antigenic regions A, B, and C, respectively, which have been confirmed as major rotavirus neutralization sites by mapping of neutralization escape mutants (6,7,16,17).Serotyping using G type-specific monoclonal antibodies (MAbs) has been applied widely in rotavirus epidemiological studies. However, the results of many studies have been incomplete due to the limited availability of MAbs specific for types other than G1 to G4, the relatively low sensitivity of the method due mainly to the requirement of intact virus particles, or to the existence of monotypes or antibody escape mutants within the different G types (2-4). Monotypes within G1, G2, G3, and G4 rotaviruses react with different degrees of affinity against different panels of G-specific MAbs (21) .Previously we reported that rotavirus strains collected in the United Kingdom during the 1995-1996 season and genotyped by reverse transcription-PCR as G2 failed to serotype in enzyme-linked immunosorbent assays (ELISAs) using G2-specific MAbs (13). Complementary DNAs of the VP7 genes of a subset of these strains were partially sequenced and compared to corresponding sequences of a subset of successfully serotyped G2 strains collected during 1990 and 1991 in order to identify amino acid substitutions at the VP7 antigenic sites that may be responsible for the failure to react with different G2-specific MAbs.G-serotyping ELISAs and genotyping reverse transcriptionPCRs were performed as previously described (1, 10, 13) using 10% rotavirus-positive fecal suspensions in balanced salt solution. G-serotyping ELISAs (13) Twenty-one G2 rotavirus strains isolated in the United Kingdom-...

Identification of Rotavirus Strains Causing Diarrhoea in Children under Five Years of Age in Yogyakarta, Indonesian

“…One is accumulation of point mutation (genetic drift), which is due to the error-prone nature of RNA-dependent RNA polymerase (RdRp) enzyme (36). Such mutations could be introduced at the primer binding sites and result in typing failure (8,15). Another important mechanism of rotavirus evolution is genetic shift due to reassortment of the segmented RNA genome.…”

“…Rotavirus RNA was analysed to determine the VP7 (G-type) and VP4 (P-type) genotypes using reverse transcriptase polymerase chain reaction (RT-PCR) as previously described (13)(14)(15). For G typing, the primers 9con1L (13) and VP7R (15) were used during the first round of RT-PCR (20 cycles) to amplify the 905-bp of the VP7 gene segment. Then, 9con1L was used in the second round of PCR (20 cycles) with the type-specific primers 9T1 (G1), 9T-2 (G2), 9T-3P (G3), 9T4 (G4) and 9T-9B (G9) (Table 1) (13).…”

“…For G typing, the consensus primers VP7F and VP7R were used in the first round of RT-PCR (30 cycles) to generate the 881-bp of the VP7 gene segment (15). VP7F was used in the second round of PCR (30 cycles) with the type-specific primers aBT1 (G1), aCT-2 (G2), G3 (G3), aDT4 (G4) and G9 (G9) (Table 1) (16).…”

Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network

“…Therefore, failure to type is most likely to be associated with technical issues and/or low viral load, although occasionally they may represent usual genotypes, particularly in those associated with an animal rotavirus G-or P-types [21][22][23][24][25][26]. The failure to identify G-types associated with P [6], P [9], P [10], P [11] and P [14] may be associated with the presence of unusual G-types for which G genotype-specific primers were not included in the study protocol.…”

Group A rotavirus genotypes and the ongoing Brazilian experience: a review

“…Many epidemiological studies have used subgrouping enzyme immunoassays (EIAs). Based upon these analyses, most human isolates fall into SGII (Arista et al 1990, Gómara et al 2001, while most animal isolates fall into SGI (Tang et al 1997). …”