Comparison of enzyme immunoassay and reverse transcriptase PCR for identification of serotype G9 rotaviruses

Abstract

While only four globally important rotavirus G serotypes (1 to 4) have been documented, many studies suggest that serotype G9 viruses may be widely distributed and more important than previously recognized. We have evaluated 10 serotype G9 rotavirus-neutralizing monoclonal antibodies (MAbs) directed to VP7, which bound by direct enzyme immunoassay (EIA) to P1A[8], G9 rotaviruses F45, WI61, and AU32, for their ability to recognize the New Delhi G9 rotavirus 116E. Only one MAb (MAb F45:1) bound to P[11], G9 virus 116E to a high titer by EIA. This MAb was incorporated into an indirect EIA for G serotyping, which was validated with prototype cultivable human rotaviruses of G types 1 to 4 and 9. The EIA was compared with genotyping by reverse transcriptase PCR (RT-PCR) under code for the determination of the G types of rotaviruses obtained from neonates in New Delhi, India. The sensitivities of RT-PCR and EIA (after two additional freeze-thaw cycles) for the typing of G9 rotaviruses were 91 and 86%, respectively, for 24 culture-adapted rotavirus strains. The untypeable culture-adapted rotavirus samples also were unreactive with VP7 group antigen-reactive MAb 60. After two additional freeze-thaw cycles, only 26 of 42 (62%) of stools containing rotavirus typed as G9 by RT-PCR were positive for G9 rotavirus by EIA. Stools containing rotavirus untypeable by EIA contained significantly less MAb 60-reactive VP7 antigen (P = 0. 0001) than the stools containing typeable rotavirus. Thus, RT-PCR genotyping was the more sensitive method for determination of G9 type, but a serotype was readily determined in rotavirus samples containing MAb 60-reactive VP7 antigen by an EIA that incorporates MAb F45:1.

FIG. 1

Relation between levels of typing MAb and MAb 60 reactivities with 25 cultivable human rotaviruses of G serotypes 1 to 4 and 9. The OD₄₅₀ was obtained by the serotyping EIA. The sigmoidal curve of best fit shown was determined by regression analysis (r² = 0.89).

FIG. 2

Relation of level of MAb 60 reactivity to serotyping MAb reactivity in the EIA. cc, cell culture-adapted rotavirus; stool, rotavirus-positive stool sample; NT, not typeable by G-serotyping EIA; T, typeable by G-serotyping EIA. Horizontal bars indicate median values for each group. The positive-negative cutoff for MAb 60 reactivity by EIA is shown with a dashed line.