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
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