Picornavirus salivirus/klassevirus in children with diarrhea, China.
Subject: Children (Health aspects)
Diarrhea (Health aspects)
Authors: Shan, Tongling
Wang, Chunmei
Cui, Li
Yu, Ying
Delwart, Eric
Zhao, Wei
Zhu, Caixia
Lan, Daoliang
Dai, Xiuqiang
Hua, Xiuguo
Pub Date: 08/01/2010
Publication: Name: Emerging Infectious Diseases Publisher: U.S. National Center for Infectious Diseases Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2010 U.S. National Center for Infectious Diseases ISSN: 1080-6040
Issue: Date: August, 2010 Source Volume: 16 Source Issue: 8
Product: Product Code: E121920 Children
Accession Number: 237533365
Full Text: Diarrhea causes [approximately equal to]2 million deaths each year (1), primarily among young children in developing countries (1,2). The causative agents for ?40% of cases remain unknown (2-4).

Studies have documented an association between Aichi viruses and gastroenteritis (5,6). Recent studies have documented human infections with the salivirus/klassevirus-related Aichi virus (7-9) that were associated with diarrhea (9). The previously unknown picornavirus klassevirus has recently been recently detected in fecal samples from persons with diarrhea in the United States and Australia and in sewage in Spain (7,8). Closely related saliviruses have been identified in fecal samples from persons in Nigeria, Tunisia, and Nepal and have been statistically associated with diarrhea in Nepal (9).

Klassevirus/salivirus is genomically organized similar to other picornaviruses and most closely related to Aichi virus in the genus Kobuvirus (5-7). The family Picornaviridae is highly diverse and contains small, nonenveloped viruses with a single-stranded positive-sense RNA genome that encodes a single polyprotein; it consists of 12 genera and 2 possibly new genera (7), a subset of which can infect and cause disease in humans.

To our knowledge, there have been no reports of infection with this virus in the People's Republic of China. Therefore, to extend these initial findings, we tested for this newly characterized virus in fecal samples from children with diarrhea in China and sequenced the nearly full genome of 1 isolate, SH1.

The Study

During April 2008-March 2009, a total of 216 fecal samples were collected from children, 2-6 years of age, who were hospitalized with diarrhea in Shanghai Children's Hospital, China. A total of 96 children, 3-5 years of age, from 2 childcare centers in Shanghai City were included as healthy controls.

Samples were suspended to 10% (wt/vol) in phosphate-buffered saline (0.01 M, pH 7.4), and total RNA was extracted from 200 [micro]L of the suspension by using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). Viral RNA was dissolved in 30 [micro]L RNase-free water and stored at -80[degrees]C.

To understand the possible association between salivirus/klassevirus and diarrhea, we conducted prevalence studies by using nested reverse transcription-PCR (RTPCR). We used a nested set of PCR primers (SAL-L1, 5'-CCCTGCAACCATTACGCTTA-3'; SAL-R1, 5'-CA CACCAACCTTACCCCACC-3'; SAL-L2, 5'-ATTGAGT GGTGCAT(C)GTGTTG-3'; SAL-R2, 5'-ACAAGCCGG AAGACGACTAC-3') to amplify a 414-bp fragment located in the 5' untranslated region (UTR). The expected size DNA bands were excised from an agarose gel, purified with the AxyPrep DNA gel extraction kit (Axygen, Union City, CA, USA), cloned into pMD-18T vector (TaKaRa, Shiga, Japan), and sequenced on an Applied Biosystems 3730 DNA Analyzer (Invitrogen). Of 216 samples, 9 (4.2%) were positive for the newly described picornavirus; ages of the children were 2 years (n = 1), 3 years (n = 3), 4 years (n = 2), 5 years (n = 1), and 6 years (n = 2). Sequence analysis, based on the 414-bp sequences, showed that these 9 sequences shared 98.3%-99.8% identity with each other, suggesting that they could be considered members of the same virus species. The sequences shared 94.7%-97.3% sequence identities with GenBank isolates nos. GQ253930 (klassevirus 1, Australia), GQ184145 (human klassevirus 1, USA), and GQ179640 (salivirus, Nigeria). The 9 salivirus/klassevirus-positive samples were further investigated for Aichi virus, parechovirus, norovirus, sapovirus, rotavirus, astrovirus, and cosavirus by using RT-PCR with the primers previously described (10-13). Results indicated that 1 sample, for which the 518-bp-specific fragment was sequenced, was also positive for human parechovirus. No salivirus/klassevirus was detected in samples from the 96 healthy controls. The Fisher exact test showed a significant (p = 0.03) association between salivirus/klassevirus detection and diarrhea.

The complete genomic sequence of strain SH1 was then determined by using 10 sets of specific oligo1These authors contributed equally to this article. nucleotide primers designed on the complete genome of GQ184145, GQ253930, and GQ179640. The nearly full-genome genome of this virus strain was 7,798 nt and contained an open reading frame (ORF) with a length of 7,107 nt, encoding a putative polyprotein precursor of 2,369 aa. This ORF is preceded by a 5? UTR at least 624 nt long and followed by a 3? UTR at least 67 nt long. Phylogenetic analysis using the more variable P1 region of SH1 and 45 representative picornaviruses (including 3 salivirus/klassevirus strain) confirmed the close relationship of this strain with strains from other continents; it was most closely related to a human klassevirus from the United States (GQ184145) (Figure).


The nearly full genome of SH1 has been submitted to GenBank under accession no. GU245894. The 9 partial 414-bp sequences of salivirus/klassevirus are deposited in GenBank under accession nos. GU376738-GU376746.


Our finding of salivirus/klassevirus in fecal samples of children with diarrhea in China is consistent with Li et al.'s report of this virus' association with diarrhea (9). This finding, plus the identity with the Nigeria reference strain, support widespread distribution of this newly characterized virus species and its association with diarrhea.

Dr Shan works at Shanghai Jiao Tong University and is interested in discovering novel viruses from biological samples by using metagenomic methods.


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Tongling Shan, (1) Chunmei Wang, (1) Li Cui, Ying Yu, Eric Delwart, Wei Zhao, Caixia Zhu, Daoliang Lan, Xiuqiang Dai, and Xiuguo Hua

Author affiliations: Shanghai Jiao Tong University, Shanghai, People's Republic of China (T. Shan, C. Wang, L. Cui, Y. Yu, W. Zhao, C. Zhu, D. Lan, X. Dai, X. Hua); Huazhong Agricultural University, Wuhan, People's Republic of China (C. Wang); Blood Systems Research Institute, San Francisco, CA, USA (E. Delwart); and University of California, San Francisco (E. Delwart)

Address for correspondence: Xiuguo Hua, Shanghai Jiaotong University, Shanghai 200240, People's Republic of China; email: hxg@sjtu.edu.cn

DOI: 10.3201/eid1608.100087
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