Dispatches.indd

Picornavirus

Salivirus/Klassevirus

in Children with

Diarrhea, China

Tongling Shan,

Chunmei Wang,

Li Cui, Ying Yu,

Eric Delwart, Wei Zhao, Caixia Zhu, Daoliang Lan,

Xiuqiang Dai, and Xiuguo Hua

To learn more about salivirus/klassevirus, we tested

feces of children with diarrhea in China during 2008–2009.

We isolated the virus from 9/216 diarrhea samples and 0/96

control samples. The nearly full polyprotein of 1 isolate, SH1,

showed 95% identity with a salivirus from Nigeria, indicating

widespread distribution and association with diarrhea.

iarrhea causes ≈2 million deaths each year (1), pri-

marily among young children in developing countries

(1,2). The causative agents for ≈40% of cases remain un-

known (2–4).

Studies have documented an association between Aichi

viruses and gastroenteritis (5,6). Recent studies have docu-

mented 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 per-

sons with diarrhea in the United States and Australia and

in sewage in Spain (7,8). Closely related saliviruses have

been identiﬁ ed 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 Picorna-

viridae 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 in-

fection with this virus in the People’s Republic of China.

Therefore, to extend these initial ﬁ ndings, we tested for this

newly characterized virus in fecal samples from children

with diarrhea in China and sequenced the nearly full ge-

nome 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 phos-

phate-buffered saline (0.01 M, pH 7.4), and total RNA was

extracted from 200 μL of the suspension by using TRIZOL

reagent (Invitrogen, Carlsbad, CA, USA). Viral RNA was

dissolved in 30 μL RNase-free water and stored at –80°C.

To understand the possible association between sali-

virus/klassevirus and diarrhea, we conducted prevalence

studies by using nested reverse transcription–PCR (RT-

PCR). 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 lo-

cated in the 5′ untranslated region (UTR). The expected

size DNA bands were excised from an agarose gel, puriﬁ ed

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 salivi-

rus/klassevirus–positive samples were further investigated

for Aichi virus, parechovirus, norovirus, sapovirus, rotavi-

rus, astrovirus, and cosavirus by using RT-PCR with the

primers previously described (10–13). Results indicated

that 1 sample, for which the 518-bp–speciﬁ c 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 signiﬁ cant

(p = 0.03) association between salivirus/klassevirus detec-

tion and diarrhea.

The complete genomic sequence of strain SH1

was then determined by using 10 sets of speciﬁ c oligo-

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 8, August 2010 1303

These authors contributed equally to this article.

Author afﬁ liations: Shanghai Jiao Tong University, Shanghai, Peo-

ple’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 Re-

search Institute, San Francisco, CA, USA (E. Delwart); and Univer-

sity of California, San Francisco (E. Delwart)

DOI: 10.3201/eid1608.100087

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) conﬁ rmed the close relation-

ship 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.

Conclusions

Our ﬁ nding 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

ﬁ nding, 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 in-

terested in discovering novel viruses from biological samples by

using metagenomic methods.

Reference

1. World Health Organization. World Health Report 2004. Geneva:

The Organization; 2004 [cited 2010 Jun 18]. http://www.who.int/

whr/2004/annex/topic/en/annex_2_en.pdf

2. Kosek M, Bern C, Guerrant RL. The global burden of diarrhoeal dis-

ease, as estimated from studies published between 1992 and 2000.

Bull World Health Organ. 2003;81:197–204.

3. Finkbeiner SR, Allred AF, Tarr PI, Klein EJ, Kirkwood CD, Wang

D. Metagenomic analysis of human diarrhea: viral detection and

discovery. PLoS Pathog. 2008;4:e1000011. DOI: 10.1371/journal.

ppat.1000011

4. Denno DM, Klein EJ, Young VB, Fox JG, Wang D, Tarr PI. Explain-

ing unexplained diarrhea and associating risks and infections. Anim

Health Res Rev. 2007;1:69–80. DOI: 10.1017/S1466252307001302

5. Ambert-Balay K, Lorrot M, Bon F, Giraudon H, Kaplon J, Wolfer

M, et al. Prevalence and genetic diversity of Aichi virus strains in

stool samples from community and hospitalized patients. J Clin Mi-

crobiol. 2008;46:1252–8. DOI: 10.1128/JCM.02140-07

6. Pham NT, Khamrin P, Nguyen TA, Kanti DS, Phan TG, Okitsu S,

et al. Isolation and molecular characterization of Aichi viruses from

fecal specimens collected in Japan, Bangladesh, Thailand, and Viet-

nam. J Clin Microbiol. 2007;45:2287–8. DOI: 10.1128/JCM.00525-

7. Holtz LR, Finkbeiner SR, Zhao G, Kirkwood CD, Girones R, Pipas

JM, et al. Klassevirus 1, a previously undescribed member of the

family Picornaviridae, is globally widespread. Virol J. 2009;6:86.

DOI: 10.1186/1743-422X-6-86

8. Greninger AL, Runckel C, Chiu CY, Haggerty T, Parsonnet J, Ganem

D, et al. The complete genome of klassevirus—a novel picornavirus

in pediatric stool. 2009. Virol J. 2009;6:82. DOI: 10.1186/1743-422-

X-6-82

9. Li L, Victoria J, Kapoor A, Blinkova O, Wang CL, Babrzadeh F,

et al. A novel picornavirus associated with gastroenteritis. J Virol.

2009;22:12002–6. DOI: 10.1128/JVI.01241-09

DISPATCHES

1304 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 8, August 2010

Figure. Phylogenetic analysis of the more variable P1 region of the

salivirus/klassevirus isolated from fecal samples of 9 (4.2%) of 216

children with diarrhea in the People’s Republic of China, April 2008–

March 2009, and 45 representative strains. Phylogenetic tree was

constructed by the neighbor-joining method with 1,000 bootstrap

replicates by using MEGA4.0 software (www.megasoftware.

net). Bootstrap values are indicated at each branching point. The

isolate SH1 is marked with a triangle. Scale bar indicates estimated

phylogenetic divergence.

Picornavirus Salivirus/Klassevirus

10. Sdiri-Loulizi K, Gharbi-Kheliﬁ H, de Rougemont A, Chouchane S,

Sakly N, Ambert-Balay K, et al. Acute infantile gastroenteritis as-

sociated with human enteric viruses in Tunisia. J Clin Microbiol.

2008;46:1349–55. DOI: 10.1128/JCM.02438-07

11. Li CS, Chan PK, Tang JW. Prevalence of diarrhea viruses in hospi-

talized children in Hong Kong in 2008. J Med Virol. 2009;11:1903–

11. DOI: 10.1002/jmv.21611

12. Holtz LR, Finkbeiner SR, Kirkwood CD, Wang D. Identiﬁ cation of

a novel picornavirus related to cosaviruses in a child with acute diar-

rhea. Virol J. 2008;5:159. DOI: 10.1186/1743-422X-5-159

13. Shan TL, Guo W, Cui L, Shang XG, Dai XQ, Yuan CL, et al. The

ﬁ rst detection of human parechovirus infections in China. J Clin Vi-

rol. 2009;4:371–2. DOI: 10.1016/j.jcv.2009.05.033

Address for correspondence: Xiuguo Hua, Shanghai Jiaotong

University, Shanghai 200240, People’s Republic of China; email: hxg@

sjtu.edu.cn

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 8, August 2010 1305