Abstract
We discovered a novel otarine picobirnavirus in fecal samples of California sea lions. Its genome contains a large segment with two open reading frames (ORFs), ORF1 encoding a putative protein of 163 amino acids with unknown function and ORF2 encoding capsid protein, and a small segment with one ORF encoding RNA-dependent RNA polymerase.
GENOME ANNOUNCEMENT
Picobirnaviruses (PBVs) are small nonenveloped bisegmented double-stranded RNA viruses found in humans and a wide variety of mammals and birds. Since its first discovery in fecal samples of humans and rats in 1988 (8, 9), PBVs have been reported in other mammals and birds (1, 2, 5, 6, 7) and environmental water samples (3). Although >400 sequences of PBVs are available in the GenBank database, there is only one complete genome sequence of human PBV (10). During a molecular epidemiology study for PBVs in mammals, we discovered a novel PBV in fecal samples of California sea lions (Zalophus californianus) in Hong Kong. We proposed that this virus be named otarine PBV (Ot-PBV), and the complete genome of this virus (GI/PBV/California sea lion/Hong Kong/HKG-PF080915/2012) was sequenced. This represents the first complete genome sequence of PBV in marine mammals.
The complete genome of Ot-PBV was amplified and sequenced using published strategies for double-stranded RNA viruses (4), using RNA extracted from the fecal swab of a California sea lion positive for Ot-PBV as the template with the EZ1 virus minikit (Qiagen, Germany). The adaptor primer, with a 3′ NH2 blocking group, was ligated to the 3′ terminus of the viral RNA and subjected to reverse transcription using a complementary primer. After RNA hydrolysis, reannealing, and end filling, single-primer amplification of viral genomic segments was performed using complementary primer. The PCR products were gel purified and cloned into pCR-Blunt II-TOPO vector by using a Zero Blunt TOPO PCR cloning kit (Invitrogen). The clones were sequenced using an ABI Prism 3700 DNA analyzer (Applied Biosystems). Sequences were assembled and manually edited to produce the final genome sequence.
The genome of Ot-PBV is 4,035 bases long, with a large segment (segment 1) and a small segment (segment 2). Segment 1 is 2,347 bases long with a G+C content of 42.8%. The 5′ noncoding region (88 bases) is AU rich (G+C content of 40.9%), whereas the 3′ noncoding region (28 bases) has a G+C content of 71.4%. It contains two open reading frames (ORFs), ORF1 and ORF2. ORF1 (nucleotides [nt] 89 to 577) encodes a putative 18.6-kDa protein of 163 amino acids with unknown function. ORF2 (nt 592 to 2319) encodes a putative 64.0-kDa capsid protein of 576 amino acids. It has 28.0% and 23.2% amino acid identities with genogroup I human PBV strains Hy005102 (NC_007027) and lapine PBV R5-9 (AJ244022), respectively. Segment 2 is 1,688 bases long with a G+C content of 47.45%. The 5′ noncoding region (46 bases) is also AU rich (G+C content of 28.3%), whereas the 3′ noncoding region (43 bases) has a G+C content of 46.5%. It contains one ORF, which encodes the 61.0-kDa RNA-dependent RNA polymerase of 532 amino acids. It has 62.7% and 64.7% amino acid identities with genogroup I human PBV strains Hy005102 (NC_007027) and 1_CHN_97 (AF246939), respectively, and 56.9% amino acid identities with bovine PBV (GQ221268), and 27.2% and 24.5% amino acid identities with genogroup II human PBV strains GPBV6G2 (AB517738) and 4GA-91 (AF246940), respectively.
Nucleotide sequence accession numbers.
The complete genome of Ot-PBV strain PF080915 has been sequenced and submitted to GenBank under accession no. JQ776551 and JQ776552.
ACKNOWLEDGMENTS
We are grateful for the generous financial support of Carol Yu, Richard Yu, Hui Hoy, and Hui Ming in the genomic sequencing platform. This work is partly supported by the Research Grant Council Grant, University Development Fund and Strategic Research Theme Fund, The University of Hong Kong; The Tung Wah Group of Hospitals Fund for Research in Infectious Diseases; the Hong Kong Special Administrative Region (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 Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Disease for the HKSAR Department of Health.
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