Application of a reverse transcription-PCR for identification and differentiation of Aichi virus, a new member of the Picornavirus family associated with gastroenteritis in humans

Abstract

Aichi viruses isolated in Vero cells from seven patients in five gastroenteritis outbreaks in Japan, five Japanese returning from Southeast Asian countries, and five local children in Pakistan with gastroenteritis were examined for differentiation based on their reactivities with a monoclonal antibody to a standard strain (A846/88) and a reverse transcription-PCR (RT-PCR) of three genomic regions. The RNA sequences were determined for 519 bases of these 17 isolates at the putative junction between the C terminus of 3C and the N terminus of 3D. The analyses revealed an approximately 90% homology between these isolates, which were then divided into two groups: group 1 (genotype A) included six isolates from four outbreaks and one isolate from a traveler and group 2 (genotype B) included one isolate from the other outbreak, four isolates from returning travelers, and all of the isolates from the Pakistani children. Based on the isolate sequences, a primer pair and a biotin-labeled probe were designed for amplification and detection of 223 bases at the 3C-3D junction of Aichi virus RNA in fecal specimens. The Aichi virus RNA was detected in 54 (55%) of 99 fecal specimens from the patients in 12 (32%) of 37 outbreaks of gastroenteritis in Japan. Of the 12 outbreaks, 11 were suspected to be due to genotype A. These results indicated that RT-PCR can be a useful tool to detect Aichi virus in stool samples and that a sequence analysis of PCR products can be employed to identify the prevalent strain in each incident.

FIG. 1

Ethidium bromide-stained agarose gel of Aichi virus (A846/88 strain) RT-PCR products. Lane 1, 100-bp DNA ladder from GIBCO BRL; lanes 2 to 4, PCR with Aichi virus primer sets A (lane 2), B (lane 3), and C (lane 4). The numbers at right indicate the sizes of the RT-PCR products in base pairs.

FIG. 2

Dendrogram of predicted genetic relationships among 17 isolates of Aichi virus by comparison of 519 bases at the putative junction between the C terminus of 3C and the N terminus of 3D.

FIG. 3

Sequence of Aichi virus (A846/88) amplified with primer set C and partial alignment of nucleic acid sequences of isolates in the C terminus of the 3C region. Two genotypic groups (A and B) were defined by the boxed sequences. The position of the cleavage site between the 3C and 3D regions is indicated. The primer pairs C and C94b-245K are underlined, and a biotin-labeled probe (AiPrb2) is double underlined.

FIG. 4

Detection of Aichi virus by RT-PCR with primers C94b-264k (A) and identification by Southern blot hybridization with probe AiPrb2 (B). Serial dilutions (from 10⁻⁶ to 10⁻¹⁰) of Aichi virus (50 mg/ml) were analyzed by agarose gel electrophoresis and stained with ethidium bromide. M, markers; N, negative control.