VP7 and VP4 genotyping of human group A rotavirus in Buenos Aires, Argentina

Abstract

Specific and sensitive tests for the detection and typing of group A rotavirus strains are needed for a more comprehensive knowledge of the epidemiology of rotaviral infection. In this study 500 stool specimens taken from 1996 to 1998 from children with acute diarrhea in Buenos Aires were examined. Group A rotavirus was unequivocally demonstrated in 62% of the samples tested by enzyme-linked immunosorbent assay (ELISA) for detection of VP6 antigen, polyacrylamide gel electrophoresis of double-stranded RNA, and reverse transcription-PCR (RT-PCR) for amplification of the VP7:G (1, 062 bp) and VP4:P (876 bp) genes. Only five positive specimens were found by RT-PCR but not by ELISA. G and P typing was carried out by nested amplification of variable sequences of the VP7 and the VP4 genes with six G- and five P-type-specific primers (multiplex PCR). Results obtained by this method showed the prevalence of the following G and P types: G1, 39%; G2, 43%; G4, 4%; P[8], 16%; P[4], 71%. Unexpectedly, the G-P type combination most frequently found was G2P[4] (43%) rather than G1P[8] (12%), which is the most commonly found worldwide. Unusual strains of the type G1P[4] accounted for 14% of the total, while mixed infections with more than one type were found in 10% of the samples. Detection of fecal rotavirus-specific immunoglobulin M (IgM) and IgA antibodies in consecutive samples of two patients taken at daily intervals demonstrated that high levels of IgM and IgA antibodies were detected on day 1 after the onset of disease and that the samples remained positive for about 10 days, after which virus shedding was no longer observed. Multiplex PCR offers a sensitive and specific alternative to determine the prevalence of group A rotavirus G and P types and to identify the emergence of uncommon strains, whereas detection of fecal IgM and IgA antibodies represents a useful supplement to virus detection for the diagnosis of current or recently acquired infections.

FIG. 1

PAGE analysis of human rotavirus dsRNA. PAGE analysis of the dsRNA genome extracted from stool suspensions taken from children with acute diarrhea is shown. All samples (lanes 1, 2, 3, and 6) exhibited the typical 4-2-3-2 pattern of group A rotavirus. Lane 4, bovine rotavirus strain UK; lane 5, extraction procedure applied to a pool of stool suspensions taken from healthy children. Numbers denote positions of dsRNA segments.

FIG. 2

Amplification of the VP7 and VP4 genes by RT-PCR. Amplification products corresponding to the VP7 (lanes 2, 3, and 4) and VP4 genes (lanes 5, 6, and 7) in three different patient samples are shown. Lane 1, negative control; lane M, molecular weight markers (100-bp ladder). The sizes of amplified bands are indicated.

FIG. 3

Typing of human group A rotavirus VP7 and VP4 genes by multiplex PCR. (A) Amplification products of the VP7 genes in stool samples taken from eight different patients with acute diarrhea. Lanes 3 and 8, type G1; lanes 2, 4, 5, 6, and 7, type G2; lane 1, coinfection with both G1 and G2 types. (B) Amplification products of the VP4 genes in stool samples taken from nine different patients with acute diarrhea. Lanes 2, 3, and 8, type P[8]; lanes 4, 5, 7, and 10, type P[4]; lanes 6 and 9, coinfection with both P[8] and P[4] types. Negative controls consisted of multiplex PCR applied to the VP7 gene (panel A, lane 9) and to the VP4 gene (panel B, lane 1) of bovine group A rotavirus strain UK. Lane M, molecular weight markers (100-bp ladder). Sizes of amplified bands are indicated.

FIG. 4

Detection of virus and coproantibodies in consecutive samples. (A) Consecutive samples from two patients taken at daily intervals and tested for rotavirus antigens (●) and rotavirus IgM (▵) and IgA (▴) antibodies. —, RT-PCR-positive samples. The arrow indicates illness onset. (B) Amplification products obtained by RT-PCR with VP4-specific primers (876 bp) in consecutive samples. Lane 0, disease onset; lanes 1 to 5, 1 to 5 days after onset, respectively; lane M, molecular weight markers (100-bp ladder).