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Human cosavirus infections in children in China
Journal of Clinical Virology 48 (2010) 228–229
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Letter to the Editor Human cosavirus infections in children in China To the Editor, New picornaviruses, provisionally named human cosaviruses (HCoSVs), consisting of four species making up a new genus in the Picornaviridae family were recently characterized. HCoSV was detected in the stools of children with acute flaccid paralysis as well as healthy children in Pakistan and Afghanistan.1,2 A fifth species of human cosavirus (HCoSV-E1) was identified in a stool sample from an infant with acute diarrhea in Australia.3 Human cosavirus was also detected in stool of a 64-year-old woman in Scotland as well as in raw sewage from cities in the United States,1,4 indicating that cosavirus is geographically widespread. The pathogenicity of HCoSV is not currently known. From November 2008 to December 2009, stool specimens were collected from 188 hospitalized children with diarrhea and 60 healthy controls in Shanghai, People’s Republic of China. The ages of children ranged from 1 month to 8 years. Viral nucleic acid was extracted with TRIzol reagent from fecal specimens suspended in 6 volumes of phosphate-buffered saline (pH 7.2),5 according to the manufacture’s instructions (Invitrogen, USA). Screening for HCoSV was done by nested reverse transcription-PCR (RT-PCR) with primers targeting the 5 UTR and RdRp region as described by Kapoor et al.,1 which generated a 316 bp and 428 bp amplicons respectively. All positive PCR amplicons were purified by the AxyPrep DNA gel extraction kit (Axygen, USA), cloned into pMD18T vector (TaKaRa, Japan), and were verified by sequencing. By confirming sequences of the 5 UTR and RdRp region, 6 (3.2%) specimens from the 188 hospitalized children and 1 (1.6%) from the 60 healthy children were found to be positive for HCoSV. Additionally, the sequences were submitted to GenBank with the strain names SH-1–8 (accession nos.: GU968209–GU968216). When compared with available cosavirus strains in GenBank on the basis of the sequences of 5 UTR, the majority of positive samples (5 of 7) could be classified within cosavirus species A, indicating that HCoSV-A may be the dominant species in circulation in China. In addition, sequences that identified in the other two positive samples did not cluster with known cosavirus species. Using a combination of mass sequencing, RT-PCR and genome walking, nearly full genome of SH-1 was then determined which is 7262 nt, encoding a putative polyprotein of 2125 aa. Base compositions of the CDS in strain SH-1 were found to be A, 28%, C, 22.6%, G, 20.8%, and U, 28.6%. The structural protein of this strain contains putative VP4, VP2, VP3 and VP1 proteins with lengths of 68 aa, 266 aa, 232 aa and 294 aa, respectively. Regarding the nonstructural proteins, lengths of 30 aa, 121 aa, and 321 aa are predicted for 2A, 2B, and 2C, and of 108 aa (19 aa, 203 aa, 463 aa) for 3A (3B, 3C, 3D). Phylogenetic trees were constructed on the basis of the nearly full genome sequences of the strain SH-1 (Fig. 1, panel A), the capsid protein gene (Fig. 1, panel B), and those sequences avail1386-6532/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.jcv.2010.03.024
Fig. 1. Phylogenetic tree constructed by the neighbor-joining method with 1000 bootstrap replicates using MEGA4.0 software. Bootstrap values are indicated at each branching point. The isolate of SH-1 identified in this study is marked with a red triangle. (A) Sequence similarity detection and phylogenetic relationships of the nearly full genome isolated in this study with other picornaviridae genera. (B) Phylogenetic analysis of the putative amino acid sequence of P1 region between SH-1 and 7 representative strains. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Letter to the Editor / Journal of Clinical Virology 48 (2010) 228–229
able in GenBank. Both phylogenetic trees confirmed that SH-1 belongs to species HCOSV-A. Sequence alignment showed that P1 region of SH-1 and the other HCoSV-A strains available in GenBank (FJ438902–FJ438905) share 66.8%, 67.7%, 69%, 74.4% nucleotide identities and amino acid identity of 67.5%, 68.1%, 70.9%, 78.7%, respectively. These results will provide useful information for further study of HCoSV in China. Conflict of interest We declare that we have no conflict of interests. We had no financial support for this work. References 1. Kapoor A, Victoria J, Simmonds P, Slikas E, Chieochansin T, Naeem A, et al. A highly prevalent and genetically diversified Picornaviridae genus in South Asian children. Proc Natl Acad Sci USA 2008;105(51):20482. 2. Victoria J, Kapoor A, Li L, Blinkova O, Slikas B, Wang C, et al. Metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis. J Virol 2009;83(9):4642. 3. Holtz L, Finkbeiner S, Kirkwood C, Wang D. Identification of a novel picornavirus related to cosaviruses in a child with acute diarrhea. Virol J 2008;5(1):159. 4. Blinkova O, Rosario K, Li L, Kapoor A, Slikas B, Bernardin F, et al. Frequent detection of highly diverse variants of cardiovirus, cosavirus, bocavirus, and circovirus in sewage samples collected in the United States. J Clin Microbiol 2009;47(November (11)):3507–13. 5. 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(February (2)):e1000011.
229
X.Q. Dai Zoonosis and Comparative Medicine Group, Shanghai Jiao Tong University, PR China X.G. Hua ∗ Zoonosis and Comparative Medicine Group, Shanghai Jiao Tong University, PR China T.L. Shan Zoonosis and Comparative Medicine Group, Shanghai Jiao Tong University, PR China a
Eric Delwart a,b Blood Systems Research Institute, San Francisco, CA, USA b University of California, San Francisco, CA, USA
W. Zhao Zoonosis and Comparative Medicine Group, Shanghai Jiao Tong University, PR China ∗ Corresponding
author. Tel.: +86 21 34206149; fax: +86 21 34206263. E-mail address: [email protected] (X.G. Hua) 25 March 2010
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Letter to the Editor Human cosavirus infections in children in China To the Editor, New picornaviruses, provisionally named human cosaviruses (HCoSVs), consisting of four species making up a new genus in the Picornaviridae family were recently characterized. HCoSV was detected in the stools of children with acute flaccid paralysis as well as healthy children in Pakistan and Afghanistan.1,2 A fifth species of human cosavirus (HCoSV-E1) was identified in a stool sample from an infant with acute diarrhea in Australia.3 Human cosavirus was also detected in stool of a 64-year-old woman in Scotland as well as in raw sewage from cities in the United States,1,4 indicating that cosavirus is geographically widespread. The pathogenicity of HCoSV is not currently known. From November 2008 to December 2009, stool specimens were collected from 188 hospitalized children with diarrhea and 60 healthy controls in Shanghai, People’s Republic of China. The ages of children ranged from 1 month to 8 years. Viral nucleic acid was extracted with TRIzol reagent from fecal specimens suspended in 6 volumes of phosphate-buffered saline (pH 7.2),5 according to the manufacture’s instructions (Invitrogen, USA). Screening for HCoSV was done by nested reverse transcription-PCR (RT-PCR) with primers targeting the 5 UTR and RdRp region as described by Kapoor et al.,1 which generated a 316 bp and 428 bp amplicons respectively. All positive PCR amplicons were purified by the AxyPrep DNA gel extraction kit (Axygen, USA), cloned into pMD18T vector (TaKaRa, Japan), and were verified by sequencing. By confirming sequences of the 5 UTR and RdRp region, 6 (3.2%) specimens from the 188 hospitalized children and 1 (1.6%) from the 60 healthy children were found to be positive for HCoSV. Additionally, the sequences were submitted to GenBank with the strain names SH-1–8 (accession nos.: GU968209–GU968216). When compared with available cosavirus strains in GenBank on the basis of the sequences of 5 UTR, the majority of positive samples (5 of 7) could be classified within cosavirus species A, indicating that HCoSV-A may be the dominant species in circulation in China. In addition, sequences that identified in the other two positive samples did not cluster with known cosavirus species. Using a combination of mass sequencing, RT-PCR and genome walking, nearly full genome of SH-1 was then determined which is 7262 nt, encoding a putative polyprotein of 2125 aa. Base compositions of the CDS in strain SH-1 were found to be A, 28%, C, 22.6%, G, 20.8%, and U, 28.6%. The structural protein of this strain contains putative VP4, VP2, VP3 and VP1 proteins with lengths of 68 aa, 266 aa, 232 aa and 294 aa, respectively. Regarding the nonstructural proteins, lengths of 30 aa, 121 aa, and 321 aa are predicted for 2A, 2B, and 2C, and of 108 aa (19 aa, 203 aa, 463 aa) for 3A (3B, 3C, 3D). Phylogenetic trees were constructed on the basis of the nearly full genome sequences of the strain SH-1 (Fig. 1, panel A), the capsid protein gene (Fig. 1, panel B), and those sequences avail1386-6532/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.jcv.2010.03.024
Fig. 1. Phylogenetic tree constructed by the neighbor-joining method with 1000 bootstrap replicates using MEGA4.0 software. Bootstrap values are indicated at each branching point. The isolate of SH-1 identified in this study is marked with a red triangle. (A) Sequence similarity detection and phylogenetic relationships of the nearly full genome isolated in this study with other picornaviridae genera. (B) Phylogenetic analysis of the putative amino acid sequence of P1 region between SH-1 and 7 representative strains. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Letter to the Editor / Journal of Clinical Virology 48 (2010) 228–229
able in GenBank. Both phylogenetic trees confirmed that SH-1 belongs to species HCOSV-A. Sequence alignment showed that P1 region of SH-1 and the other HCoSV-A strains available in GenBank (FJ438902–FJ438905) share 66.8%, 67.7%, 69%, 74.4% nucleotide identities and amino acid identity of 67.5%, 68.1%, 70.9%, 78.7%, respectively. These results will provide useful information for further study of HCoSV in China. Conflict of interest We declare that we have no conflict of interests. We had no financial support for this work. References 1. Kapoor A, Victoria J, Simmonds P, Slikas E, Chieochansin T, Naeem A, et al. A highly prevalent and genetically diversified Picornaviridae genus in South Asian children. Proc Natl Acad Sci USA 2008;105(51):20482. 2. Victoria J, Kapoor A, Li L, Blinkova O, Slikas B, Wang C, et al. Metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis. J Virol 2009;83(9):4642. 3. Holtz L, Finkbeiner S, Kirkwood C, Wang D. Identification of a novel picornavirus related to cosaviruses in a child with acute diarrhea. Virol J 2008;5(1):159. 4. Blinkova O, Rosario K, Li L, Kapoor A, Slikas B, Bernardin F, et al. Frequent detection of highly diverse variants of cardiovirus, cosavirus, bocavirus, and circovirus in sewage samples collected in the United States. J Clin Microbiol 2009;47(November (11)):3507–13. 5. 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(February (2)):e1000011.
229
X.Q. Dai Zoonosis and Comparative Medicine Group, Shanghai Jiao Tong University, PR China X.G. Hua ∗ Zoonosis and Comparative Medicine Group, Shanghai Jiao Tong University, PR China T.L. Shan Zoonosis and Comparative Medicine Group, Shanghai Jiao Tong University, PR China a
Eric Delwart a,b Blood Systems Research Institute, San Francisco, CA, USA b University of California, San Francisco, CA, USA
W. Zhao Zoonosis and Comparative Medicine Group, Shanghai Jiao Tong University, PR China ∗ Corresponding
author. Tel.: +86 21 34206149; fax: +86 21 34206263. E-mail address: [email protected] (X.G. Hua) 25 March 2010