Complete nucleotide sequence and genetic organization of Aichi virus, a distinct member of the Picornaviridae associated with acute gastroenteritis in humans

Abstract

The complete nucleotide sequence of a novel enteric virus, Aichi virus, associated with nonbacterial acute gastroenteritis in humans was determined. The Aichi virus genome proved to be a single-stranded positive-sense RNA molecule with 8,251 bases excluding a poly(A) tail; it contains a large open reading frame with 7,302 nucleotides that encodes a potential polyprotein precursor of 2,433 amino acids. The genome contains a 5' nontranslated region (NTR) with 712 bases and a 3' NTR with 240 bases followed by a poly(A) tail. The structure of the genome, VPg-5' NTR-leader protein-structural proteins-nonstructural proteins-3' NTR-poly(A), was found to be typical of a picornavirus. The VP0-VP3 and VP3-VP1 cleavage sites were determined to be Q-H and Q-T, respectively, by N-terminal amino acid sequence analyses using purified virion proteins. Possible cleavage sites, Q-G, Q-A, and Q-S, which cleave P2 and P3 polyproteins were found to be similar to those of picornaviruses. A dendrogram based on 3Dpol proteins indicated that Aichi virus is genetically distinct from the known six genera of picornaviruses including entero-, rhino-, cardio-, aphtho-, and hepatovirus and echovirus 22. Considering this together with other properties of the virus (T. Yamashita, S. Kobayashi, K. Sakae, S. Nakata, S. Chiba, Y. Ishihara, and S. Isomura, J. Infect. Dis. 164:954-957, 1991), we propose that Aichi virus be regarded as a new genus of the family Picornaviridae.

FIG. 1

Comparison of conserved sequence elements in the IRES of picornaviruses. A short stretch including the Y_n-X_m-ATG motif and the initiator methionine, according to Jang et al. (16), is shown. Y_n, pyrimidine-rich tract; X_m, nonconserved sequence; ATG, initiator methionine.

FIG. 2

Electron micrograph (A) and SDS-PAGE analysis (B) of Aichi virus full particle (F) and empty particle (E). The intact virions and the empty particles were purified by banding at 100,000 × g for 22 h in CsCl with an initial density of 1.36g/ml followed by 5 to 30% (wt/vol) sucrose density gradient centrifugation at 100,000 × g for 100 min (40). The proteins were analyzed by SDS–12% PAGE, and the bands were visualized by silver staining. For N-terminal sequence analysis, the protein band was transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Bedford, Mass.) and analyzed by an Applied Biosystems model 476A automated protein sequencer.

FIG. 3

Genome organization of Aichi virus and comparison of the structure among picornaviruses. The genome organizations have been shown according to the L434 system (29). P1 represents viral structural proteins. P2 and P3 represent nonstructural proteins. P1 proteins of hepatovirus and E22 produce 6- and 12-aa leaders, respectively (5 and 11 aa without the initiator methionine).

FIG. 4

Relationships between Aichi virus and other picornaviruses based on amino acid differences of 3D^pol proteins. The dendrogram was generated by evolutionary distances computed by UPGMA.