GENOME ANNOUNCEMENT
Complete Genome Sequence of a Novel Picornavirus, Canine Picornavirus, Discovered in Dogs Patrick C. Y. Woo,a,b,c,d Susanna K. P. Lau,a,b,c,d Garnet K. Y. Choi,a Cyril C. Y. Yip,a Yi Huang,a Hoi-Wah Tsoi,d and Kwok-Yung Yuena,b,c,d Department of Microbiology,a State Key Laboratory of Emerging Infectious Diseases,b Research Centre of Infection and Immunology,c Carol Yu Centre of Infection,d The University of Hong Kong, Hong Kong
We discovered a novel canine picornavirus in fecal, nasopharyngeal, and urine samples from dogs. The coding potential of its genome (5=-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3Cpro-3Dpol-3=, where 3Cpro is 3C protease and 3Dpol is 3D polymerase) is similar to those of other picornaviruses, with putative P1, P2, and P3 sharing 54% to 58%, 60%, and 64% to 67% amino acid identities with bat picornavirus groups 1, 2, and 3.
P
icornaviruses are positive-sense, singled-stranded RNA viruses found in humans and a wide variety of animals (3, 5). Based on genotypic and serological characterization, picornaviruses are currently divided into 12 genera. In the last few years, there has been a surge in the number of novel picornaviruses being discovered and in the number of their genomes being sequenced, including those of three avian picornaviruses, three bat picornaviruses, and one feline picornavirus discovered in our previous studies carried out in Hong Kong (1, 2, 7). We also recently reported the discovery of a novel picornavirus-like virus, canine picodicistrovirus, in the picornavirus-like superfamily, with two functional internal ribosomal entry sites (IRES), in dogs (6). During the process of this molecular epidemiology study of picornaviruses in dogs, we discovered a novel picornavirus in fecal, nasopharyngeal, and urine samples from dogs (6). We proposed that this virus be named canine picornavirus (CanPV). The complete genome of CanPV was amplified and sequenced with an EZ1 virus minikit (Qiagen, Germany), using RNA extracted from the fecal swab of a dog positive for CanPV as a template. RNA was converted to cDNA by a combined randompriming and oligo(dT)-priming strategy. The genome was sequenced by genome walking using degenerate primers and additional primers designed from the results of each round of sequencing (1, 2, 4, 6–9). DNA sequencing was performed using an ABI Prism 3700 DNA analyzer (Applied Biosystems, USA). The 5= end of the viral genome was confirmed by rapid amplification of cDNA ends (RACE) using a SMARTer RACE cDNA amplification kit (Clontech, USA). Sequences were assembled and manually edited to produce the final genome sequence (1, 2, 4, 6–9). The genome size of CanPV is 7,948 bases, with a G⫹C content of 41.0% after exclusion of the polyadenylated tract. The genome organization is similar to that of other picornaviruses, with the characteristic gene order 5=-VP4-VP2-VP3-VP1-2A-2B-2C-3A3B-3Cpro-3Dpol-3= (3Cpro and 3Dpol are 3C protease and 3D polymerase, respectively). Both the 5= (668 bases) and 3= (143 bases) ends of the genome contain untranslated regions (UTRs). The genome contains a single open reading frame of 7,137 bases, which encodes a polyprotein precursor of 2,378 amino acids. In the 5= UTR, the conserved sequence Y6-X54-AUG and a GNRA sequence, a motif conserved among picornavirus IRES, are present. The IRES of CanPV conforms to the structure of type I IRES. A leader protein (L) is present, but it does not possess the charac-
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teristic catalytic amino acid residues with proteolytic activity. The 2A of CanPV possesses the characteristic catalytic amino acid residues with trypsin-like proteolytic activity, and its 2C possesses the GXXGXGKS motif for nucleoside triphosphate (NTP) binding and the DDLXQ motif for putative helicase activity. The 3Cpro of CanPV contains the catalytic triad His-Asp-Cys. Like 3Dpol, it contains the conserved KDE(LI)R, GG(LMN)PSG, YGDD, and FLKR motifs. Phylogenetically, CanPV is most closely related to bat picornavirus groups 1, 2, and 3, which we reported previously, with 54% to 58%, 60%, and 64% to 67% amino acid identities between the P1, P2, and P3 of CanPV and those of the three bat picornaviruses, suggesting that they may form a novel genus in Picornaviridae. Nucleotide sequence accession number. The complete genome sequence of CanPV (strain 325F) has been deposited in GenBank under accession no. JN831356. ACKNOWLEDGMENTS We thank Alan Chi-Kong Wong, Siu-Fai Leung, Chik-Chuen Lay, Thomas Sit, K. F. Chan, Michelle L. Yeung, Byung Mo Hwang, Suet Yee Ng, Patrick I. T. Lau, and Steven D. Benton of the HKSAR Department of Agriculture, Fisheries, and Conservation (AFCD) for facilitation and support. We also thank the members of the Animal Management Centres of AFCD. We are grateful for the generous support of Carol Yu, Richard Yu, Hui Hoy, and Hui Ming with the genomic sequencing platform. This work was partly supported by a Research Grant Council grant (HKU 783611 M); the University Development Fund and Strategic Research Theme Fund of, the University of Hong Kong; the Tung Wah Group of Hospitals Fund for Research in Infectious Diseases; the HKSAR Research Fund for the Control of Infectious Diseases of the Health, Welfare and Food Bureau; the Providence Foundation Limited, in memory of the late Lui Hac Minh; and the Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Disease of the HKSAR Department of Health.
Received 28 December 2011 Accepted 3 January 2012 Address correspondence to Kwok-Yung Yuen,
[email protected]. P. C. Y. Woo and S. K. P. Lau contributed equally to this article. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.07228-11
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Genome Announcement
REFERENCES 1. Lau SK, et al. 2011. Complete genome analysis of three novel picornaviruses from diverse bat species. J. Virol. 85:8819 – 8828. 2. Lau SK, et al. 2012. Identification of a novel feline picornavirus from the domestic cat. J. Virol. 86:395– 405. 3. Lau SK, et al. 2009. Clinical and molecular epidemiology of human rhinovirus C in children and adults in Hong Kong reveals a possible distinct human rhinovirus C subgroup. J. Infect. Dis. 200:1096 –1103. 4. Lau SK, et al. 2007. Clinical features and complete genome characterization of a distinct human rhinovirus (HRV) genetic cluster, probably representing a previously undetected HRV species, HRV-C, associated with acute respiratory illness in children. J. Clin. Microbiol. 45:3655–3664. 5. Whitton JL, Cornell CT, Feuer R. 2005. Host and virus determinants of picornavirus pathogenesis and tropism. Nat. Rev. Microbiol. 3:765–776.
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6. Woo PC, et al. 2012. Natural occurrence and characterization of two internal ribosome entry site elements in a novel virus, canine picodicistrovirus, in the picornavirus-like superfamily. J. Virol. 86:2797–2808. 7. Woo PC, et al. 2010. Comparative analysis of six genome sequences of three novel picornaviruses, turdiviruses 1, 2 and 3, in dead wild birds, and proposal of two novel genera, Orthoturdivirus and Paraturdivirus, in the family Picornaviridae. J. Gen. Virol. 91:2433–2448. 8. Yip CC, Lau SK, Woo PC, Chan KH, Yuen KY. 2011. Complete genome sequence of a coxsackievirus A22 strain in Hong Kong reveals a natural intratypic recombination event. J. Virol. 85:12098 –12099. 9. Yip CC, et al. 2010. Emergence of enterovirus 71 “double-recombinant” strains belonging to a novel genotype D originating from southern China: first evidence for combination of intratypic and intertypic recombination events in EV71. Arch. Virol. 155:1413–1424.
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