Arch Virol DOI 10.1007/s00705-014-2087-2

ANNOTATED SEQUENCE RECORD

Complete sequencing and phylogenetic analysis of porcine kobuvirus in domestic pigs in Northwest China Enli Wang • Bin Yang • Wei Liu • Jixing Liu Xiaojun Ma • Xi Lan



Received: 5 March 2014 / Accepted: 9 April 2014 Ó Springer-Verlag Wien 2014

Abstract Porcine kobuvirus, a member of the genus Kobuvirus that is associated with diarrhea, has been reported in many countries. We determined the complete genome sequence and investigated the genetic evolution of the kobuvirus strain swKoV CH441, which was detected in the highland of Gansu province in Northwest China. The viral genome is 8149 nucleotides (nt) long, including a 29-nt poly(A) tail of the 30 end, and is 90 nt shorter in the 2B coding region than those of other kobuvirus strains whose sequences are available in the GenBank database. Phylogenetic analysis showed that swKoV CH441was most closely related to porcine kobuvirus CH/HNXX-4 but more distantly related to other strains, including the strains GS-1/2012/CH and GS-2/ 2012/CH, which were detected in Gansu province, indicating that porcine kobuvirus may have geographic and host differences in evolution.

[11], goats [9], cats [4] and ferrets [17]. More recently, they have been detected in swine samples from various countries, including Thailand [7], Korea [13], Japan [8], China [20], Brazil [2, 16], the Netherlands [2], Italia [5], Kenya and Uganda [1]. A typical porcine kobuvirus virion is 30 nm in diameter, and its genome is 8210 nt in length and contains a single open reading frame (ORF) encoding a protein of 2488 amino acids (aa). The genome organization is typical for picornaviruses: a 50 UTR, a leader (L) protein, structural proteins P1 (VP0, VP3, and VP1), non-structural proteins P2 (2A, 2B, 2C) and P3 (3A, 3B, 3C, 3D), a 30 UTR and a poly (A) tail. In this study, we sequenced the genome of swKoV CH441 and compared it with other porcine kobuviruses to study their genetic diversity.

Specimens and primers Introduction Kobuviruses are one of the important causes of acute viral gastroenteritis in both human and animals, especially in young animals. Kobuviruses have also been isolated from humans [18], sheep [15], bats [10], canyon mice [14], dogs

E. Wang  B. Yang  W. Liu  J. Liu  X. Lan (&) State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Grazing Animal Diseases, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China e-mail: [email protected] E. Wang  X. Ma College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China

A total of 85 porcine fecal specimens were collected from pig farms in Wuwei, Tianshui, Jingtai, and Jiayuguan of Gansu province. Based on the sequences of S-1HUN, K-30-HUN, WB-1-HUN and SH-W-CHN from GenBank, 10 sets of primers were designed to cover the genome. The 50 and 30 ends of the genome were amplified using a SMART RACE cDNA Amplification Kit (Clontech).

Sequence and phylogenetic analysis The genome of swKoV CH441 is 8120 nt long, with a 90-nt deletion in its 2B coding region when compared with strains from Hungary and part of China. It consists of

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20.41 % A, 20.85 % G, 27.09 % U and 31.65 % C ribonucleotides. The 50 UTR is 577 nt long, and the 30 UTR is 166 nt long. Between these is a single 7377-nt open reading frame encoding a potential 2458-aa polyprotein precursor, which is 30 aa shorter than in other strains. In a recent study, the strains CH/HNXX-4, GS-1/2012/CH and GS-2/ 2012/CH, which were identified in Sus scrofa in Gansu province, had the same deletion in the 2B coding region [3, 6]. Interestingly, the 30 UTR was 166 nt long in the swKoV CH441 strain but only 126 nt long in CH/HNXX-4. We therefore compared the whole genome of strain swKoV

Table 1 Kobuvirus strains with sequences available in the GenBank database that were used in this study Host

Strain

Accession number

Country

Sus scrofa

S-1-HUN

EU787450

Hungary

Sus scrofa

K-30-HUN

GQ249161

Hungary

Sus scrofa

WB-1-HUN

JX177612

Hungary

Sus scrofa

GS-1/2012/CH

KC424639

China

Sus scrofa

GS-2/2012/CH

KC424640

China

Sus scrofa

K-4/2012/CH

KC424638

China

Sus scrofa Pig

WUH1 Y-1-CHI

JQ692069 GU292559

China China

Pig

XX

KC204684

China

Pig

SH-W-CHN

JN630514

China

Pig

SwKoV CH441

KF539763

China

Piglet

CH/HNXX-4

JX401523

China

Cattle

U-1

AB084788

Japan

Human

Aichi virus

AB040749

Japan

Fig. 1 Phylogenetic analysis of the complete genomes of members of the genus Kobuvirus using the neighborjoining method in Mega 5.0 software with 1000 bootstrap replicates. The tree includes strains previously (black filled diamond) and newly (black filled circle) identified in Gansu province as wells as others obtained from GenBank

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CH441 with those of other kobuviruses deposited in GenBank (Table 1). The swKoV CH441 strain shared a high level of nucleotide sequence identity with the strains available in GenBank, ranging from 88.3 to 90.8 %. The amino acid similarity ranged from 94.3 to 94.8 %. The VP1 region is the most variable structural protein gene in kobuviruses [12]. The structural capsid protein VP1 is exposed on the surface of the virus particles, contains an immunodominant epitope, and is the most prone to mutation [19], which plays an important role in the molecular epidemiology and molecular immunology of kobuviruses. In this study, The VP1 region showed 81.4–90.4 % nucleotide sequence identity and 88.4–98.3 % amino acid sequence similarity. In addition, as seen from the phylogenic tree of the complete genome sequence (Fig. 1), strains in China and in Hungary are in different branches. The sample used in this study was collected in the Loess Plateau area, while the other strains were detected in plain areas (Hebei, Jilin, Shanghai, Anhui, Hubei). The strains GS-1/2012 and GS-2/ 2012, which were identified in Sus scrofa, were detected in Gansu province; however, they were in a separate branch from the strain swKoV CH441 in this study. Therefore, we concluded that host, geographic and environmental factors may play roles in evolution process.

Nucleotide sequence accession number The complete sequence of porcine kobuvirus strain swKoV CH441 was submitted to GenBank under the accession number KF539763.

Porcine kobuvirus from pigs in Northwest China

References 1. Amimo JO, Okoth E, Junga JO, Ogara WO, Njahira MN, Wang Q, Vlasova AN, Saif LJ, Djikeng A (2013) Molecular detection and genetic characterization of kobuviruses and astroviruses in asymptomatic local pigs in East Africa. Arch Virol. doi:10.1007/ s00705-013-1942-x 2. Barry AF, Ribeiro J, Alfieri AF, van der Poel WH, Alfieri AA (2011) First detection of kobuvirus in farm animals in Brazil and The Netherlands. Infect Genet Evol 11:1811–1814 3. Cao W, Zheng H, Zhang K, Jin Y, Lv L, Yang F, Liu X (2012) Complete genome sequence of the Porcine Kobuvirus variant CH/HNXX-4/2012. J Virol 86:11947 4. Chung J-Y, Kim S-H, Kim Y-H, Lee M-H, Lee K-K, Oem J-K (2013) Detection and genetic characterization of feline kobuviruses. Virus Genes 47:559–562 5. Di Profio F, Ceci C, Di Felice E, Marsilio F, Di Martino B (2013) Molecular detection of porcine kobuviruses in Italian swine. Res Veterinary Sci 95:782–785 6. Fan S, Sun H, Ying Y, Gao X, Wang Z, Yu Y, Li Y, Wang T, Yu Z, Yang S (2013) Identification and characterization of Porcine Kobuvirus variant isolated from suckling piglet in Gansu Province, China. Viruses 5:2548–2560 7. Khamrin P, Maneekarn N, Kongkaew A, Kongkaew S, Okitsu S, Ushijima H (2009) Porcine kobuvirus in piglets, Thailand. Emerg Infect Dis 15:2075–2076 8. Khamrin P, Maneekarn N, Hidaka S, Kishikawa S, Ushijima K, Okitsu S, Ushijima H (2010) Molecular detection of kobuvirus sequences in stool samples collected from healthy pigs in Japan. Infect Genet Evol 10:950–954 9. Lee M-H, Jeoung H-Y, Lim J-A, Song J-Y, Song D-S, An D-J (2012) Kobuvirus in South Korean black goats. Virus Genes 45:186–189 10. Li L, Victoria JG, Wang C, Jones M, Fellers GM, Kunz TH, Delwart E (2010) Bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses. J Virol 84:6955–6965

11. Li L, Pesavento PA, Shan T, Leutenegger CM, Wang C, Delwart E (2011) Viruses in diarrhoeic dogs include novel kobuviruses and sapoviruses. J General Virol 92:2534–2541 12. Okitsu S, Khamrin P, Thongprachum A, Hidaka S, Kongkaew S, Kongkaew A, Maneekarn N, Mizuguchi M, Hayakawa S, Ushijima H (2012) Sequence analysis of porcine kobuvirus VP1 region detected in pigs in Japan and Thailand. Virus Genes 44:253–257 13. Park S-J, Kim H-K, Moon H-J, Song D-S, Rho S-M, Han J-Y, Nguyen V-G, Park B-K (2010) Molecular detection of porcine kobuviruses in pigs in Korea and their association with diarrhea. Arch Virol 155:1803–1811 14. Phan TG, Kapusinszky B, Wang C, Rose RK, Lipton HL, Delwart EL (2011) The fecal viral flora of wild rodents. PLoS Pathogens 7:e1002218 ´ , Pankovics P, Egyed L (2010) Kobuvirus in 15. Reuter G, Boros A domestic sheep, Hungary. Emerg Infect Dis 16:869 16. Ribeiro J, de Arruda Leme R, Alfieri AF, Alfieri AA (2013) High frequency of Aichivirus C (porcine kobuvirus) infection in piglets from different geographic regions of Brazil. Trop Anim Health Prod 45:1757–1762 17. Smits SL, Raj VS, Oduber MD, Schapendonk CM, Bodewes R, Provacia L, Stittelaar KJ, Osterhaus AD, Haagmans BL (2013) Metagenomic analysis of the ferret fecal viral flora. PloS One 8:e71595 18. Yamashita T, Kobayashi S, Sakac K, Nakata S, Chiba S, Ishihara Y, Isomura S (1991) Isolation of cytopathic small round viruses with BS-Cl cells from patients with gastroenteritis. J Infect Dis 164:954–957 19. Yamashita T, Ito M, Kabashima Y, Tsuzuki H, Fujiura A, Sakae K (2003) Isolation and characterization of a new species of kobuvirus associated with cattle. J General Virol 84:3069–3077 20. J-M Yu, Jin M, Zhang Q, Li H-Y, Li D-D, Xu Z-Q, Li J-S, Cui S-X, Yang S-H, Liu N (2009) Candidate porcine kobuvirus, China. Emerg Infect Dis 15:823

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Complete sequencing and phylogenetic analysis of porcine kobuvirus in domestic pigs in Northwest China.

Porcine kobuvirus, a member of the genus Kobuvirus that is associated with diarrhea, has been reported in many countries. We determined the complete g...
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