Virus Genes DOI 10.1007/s11262-015-1282-x
Isolation, complete genome sequencing, and phylogenetic analysis of the first Chuzan virus in China Fang Wang1 • Jun Lin2 • Jitao Chang1 • Yingying Cao2 • Shaomin Qin2 Jianmin Wu2 • Li Yu1
•
Received: 10 October 2015 / Accepted: 19 December 2015 Springer Science+Business Media New York 2016
Abstract A Chuzan virus (CHUV), defined as GX871 here, was isolated from blood from a sentinel cattle firstly in China, and its full-length genome was sequenced in this study. The GX871 genome included 10 segments and 18914 bp, one base fewer than the CHUV prototype strain K-47 due to a one-base deletion in the 50 non-coding region of segment 8. A frameshift mutation was detected in a short coding region (1010–1026 nt) corresponding to the VP1 protein; this frameshift resulted in a five-amino acid mutation from 336CVLSY340 to 336YGAKL340. In addition,
there were a one-base deletion at 1713 nt and a one-base insertion at 1682 nt in the 30 non-coding region of segment 5. Based on phylogenetic analysis of the deduced VP2 amino acid sequences, Palyam serogroup viruses were classified into three groups. The Chinese CHUV isolate GX871 was categorized into the same group as CHUV prototype strain K-47. The phylogenetic tree was divided into three clusters according to the geographical distribution of the partial nucleotide sequences of VP7, and this arrangement might define the geographical gene pool of CHUV.
Edited by Juergen A Richt.
Keywords Chuzan virus Isolation in China Complete genome Phylogenetic analysis
Fang Wang and Jun Lin have contributed equally to this work. & Jianmin Wu [email protected] & Li Yu [email protected] Fang Wang [email protected] Jun Lin [email protected] Jitao Chang [email protected] Yingying Cao [email protected] Shaomin Qin [email protected] 1
Division of Livestock Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People’s Republic of China
2
Guangxi Veterinary Research Institute, Nanning, People’s Republic of China
The Chuzan virus (CHUV), a member of the Palyam serogroup within the genus Orbivirus of the family Reoviridae [1], was first demonstrated to be the causative agent of bovine disease among the Palyam serogroup [2]. CHUV is responsible for many reproductive disorders, including abortion, stillbirth, and congenital malformation, which result in considerable economic loss in the cattle industry [2, 3]. Similar to other viruses of the Palyam serogroup, CHUV consists of 10 double-stranded (ds) RNA segments in which segments 1, 2, 3, 4, 6, 7, and 9 encode the structural proteins VP1, VP2, VP3, VP4, VP5, VP7, and VP6 [4, 5], respectively, segments 5 and 8 encode the nonstructural proteins NS1 and NS2 [4], respectively, and the smallest segment 10 encodes two related non-structural proteins, NS3 and NS3A [4]. To date, CHUV has been found in countries near China, such as Korea [6, 7] and Japan [1, 8]. Despite the presence of Culicoides spp., which had been reported as possible vectors for CHUV transmission, no CHUV isolate has been
123
Virus Genes Table 1 Features of Chinese CHUV isolate GX871 genes and their encoded proteins Genome segment
Length (bp)
50 /30 non-coding region
50 /30 non-coding region
Encoded protein
ORF length (aa)
Predicted molecular weight (Da)
1
3930
GTTAAA/ACTTAC
22/23
VP1
1295
149113
2
3055
GTTAAA/ACTTAC
14/35
VP2
1002
117422
3
2774
GTTAAA/ACTTAC
13/49
VP3
904
103866
4
1967
GTTAAA/ACTCAC
9/38
VP4
640
74631
5
1764
GTTAAA/ACTCAC
35/94
NS1
545
64138
6
1610
GTTAAA/GCTTAC
20/27
VP5
521
59018
7 8
1151 1058
GTAAAA/GCTCAC GTAAAA/ACTCAC
18/89 20/39
VP7 NS2
348 333
37994 37164
9
877
GTTAAA/ACATAC
19/42
VP6
272
29713
10
728
GTTAAA/ACTTAC
18/77
NS3/NS3a
211/200
23606/22328
Total
18914
6071
696665
reported in China. To investigate the status of arthropodborne disease in China, a pilot study was performed in 2013. This study aimed to isolate viruses from whole-blood samples collected from sentinel animals in different areas of China. Consequently, the first CHUV strain was isolated, and its complete genome sequence was determined for phylogenetic analysis. To perform virus isolation, whole-blood samples were collected from sentinel cattle in herds scattered across different locations throughout the country. The C6/36 cell line derived from the mosquito (ATCC CRL-1660) was used for primary inoculation. After the first passage, the infected C6/36 cells were used to inoculate baby hamster kidney (BHK21) cells for five blind passages. Samples that exhibited typical cytopathological changes within 5 days after infection were further tested using reverse transcription-PCR-based sequencing. The method of rapid identification of unknown viruses was described previously [9]. Briefly, the culture medium of the virus-infected BHK-21 cells was filtered through a 0.22-lm filter (Millipore) and treated with DNase and RNase. Viral cDNA synthesis was performed via reaction of the extracted viral RNA with the primer K-8N (GAC CAT CTA GCG ACC TCC ACN NNN NNN N). Subsequently, PCR amplification of the extension products was performed using the primer K (GAC CAT CTA GCG ACC TCC AC). The obtained sequences were analyzed for their homology to nucleotide sequences in the GenBank database using a BLASTn search. Consequently, one CHUV isolate (subsequently defined as GX871) was identified. The infected animal was a 12-month-old native cattle reared in the city of Mashan (108–115E, 23.6–25N) in the Guangxi Zhuang Autonomous Region of China in 2013. Neither illness nor disease was observed in the population of these sentinel cattle.
123
To elucidate the molecular characteristics of the CHUV GX871, its complete genome was sequenced according to the full-length amplification of cDNAs (FLAC) technique as described previously [10–12]. The genome was sequenced twice using two separate extracted RNA sample sets, resulting in coverage of a minimum of four reads per nucleotide. The Lasergene Package was used to assemble the generated sequences. The complete genome sequence of CHUV GX871 is available from GenBank (accession number KT887180 * KT887189). The sequence data were analyzed and aligned using the alignment programs BioEdit Sequence Alignment Editor (version 6.0), Mega (version 6.0) and Clustal X. Phylogenetic trees were generated using neighbor-joining (NJ), maximum likelihood (ML) and Bayesian algorithms. The lengths of the CHUV genome segments and their coding capacities are summarized in Table 1. As a result of a one-base deletion in the 50 non-coding region of segment 8, the full-length genome sequence of this virus is 18,914 bp, which is one base fewer than the CHUV prototype strain K-47 [4, 5]. The consensus terminal sequence of the GX871 genome is 50 GU (U/A) AAA… (A/G) C (U/ A) (U/C) AC 30 ; this finding is in agreement with the consensus sequence of Palyam serogroup virus genomes [4, 5]. These motifs in the consensus terminal sequence may serve as important signals for recognition, sorting, and packaging of the RNA segments during virus maturation. RNA segments 2, 3, 6, and 7 encode the major capsid proteins VP2, VP3, VP5, and VP7, respectively [5]. In the virion, the inner capsid (core), consisting of two major proteins (VP3 and VP7) and three minor proteins (VP1, VP4 and VP6), is surrounded by an outer capsid composed of VP2 and VP5, which are responsible for viral neutralization and antigenic variability [13]. Based on comparison
Virus Genes
Fig. 1 Phylogenetic tree based on amino acid sequences of VP2 (a) and the partial nucleotide sequence of VP7 (b) of CHUV. Trees were constructed using the neighbor-joining method and numbers
above the internal nodes indicate the bootstrap values obtained with 1000 replications. BTV-1 and AHSV were used as the outgroup to root the tree (a). The black diamonds indicate Chinese isolate GX871
with the nucleotide and deduced amino acid sequences of VP2 of 6 CHUV isolates, VP2 of Chinese isolate GX871 showed 95.9–98.1 % and 97.6–99 % identities, respectively, except for VP2 of CHUV ON 91-5, which showed 53.2 and 41.5 % identities to VP2 of CHUV GX871, respectively. Among the compared sequences, GX871 VP2 displayed the highest identity (98.1 and 99 %) to the nucleotide and deduced amino acid sequences of K-47 VP2. Phylogenetic analysis of the VP2 protein revealed that these viruses could be divided into three major clusters (Fig. 1a). One cluster contained five Japanese CHUV isolates (K-47, 31, FO 88-2, FO 90-8, and ON-3/E/98) and the Chinese isolate GX871. Four D‘Aguilar virus isolates (KY115, ON-1/E/00, KSB-29/E/01 and B8112), CHUV ON 91-5, and Nyabira virus 792/76 formed another cluster. Finally, four Bunyip Creek virus isolates formed a distinct cluster. Segment 6 of CHUV encodes the outer-coat protein VP5, which is 521 amino acids in length (Table 1). The nucleotide and deduced amino acid sequences of GX871 VP5 showed 96.0 and 99.6 % identities to CHUV K-47 VP5, respectively. That CHUV VP3 and VP7 form core capsids is presumably directly relevant to the intra- and inter-molecular interactions involved in virus assembly [5]. The deduced amino acid sequences of VP3 and VP7 in GX871 showed identities of 99.9 and 100 % to VP3 and VP7 of CHUV K-47, respectively. To determine the relationships between
the Chinese isolates and other Palyam serogroup viruses, a partial 224-bp DNA fragment corresponding to nucleotide positions 261–484 of segment 7 was compared. A high degree of sequence identity, from 97.3 to 100 %, was obtained among the Chinese, Japanese, and Taiwanese isolates. These Asian isolates showed relatively lower identities (from 84.8 to 92.0 %) to Australian and African isolates. Phylogenetic analysis of VP7 revealed that Palyam serogroup viruses were divided into three clusters according to the region from which the viruses were isolated [14]. These clusters consisted of Japanese and Taiwanese isolates, Australian isolates, and African isolates (Fig. 1b). All viruses isolated in Japan were categorized into the same cluster together with GX871 and two Taiwanese isolates. These results suggested that the VP7 protein of Palyam serogroup viruses was conserved among the viruses in the epidemic area even if the serotypes were different. Thus, isolate GX871 and the Taiwanese and Japanese isolates of Palyam serogroup viruses appeared to have been derived from a common genetic pool. CHUV genome segments 1, 4, 5, 8, 9, and 10 encode VP1, VP4, NS1, NS2, VP6, and NS3, respectively. The VP1 protein appears to be highly conserved among Orbiviruses [5]. Chinese isolate GX871 showed high identity with the Japanese isolate K-47. The amino acid sequences of VP1, VP4, VP6, NS1, NS2, and NS3 of isolate GX871 showed 98.1, 97.5, 97.1, 98.2, 98.5, and 96.2 % identities
123
Virus Genes
to the respective proteins of CHUV K-47. However, some unique differences in the genome of Chinese isolate GX871 were observed. Based on comparisons of GX871 with other Palyam serogroup viruses, a frameshift mutation was detected in nt 1010–1026 within the coding region of the VP1 protein of GX871; this frameshift produced a fiveamino acid mutation from 336CVLSY340 to 336YGAKL340. Similarly, there were a one-base deletion at 1713 nt and a one-base insertion at 1682 nt in the 30 non-coding region of segment 5. In addition, a one-base deletion was detected in the 50 non-coding region of segment 8. To our knowledge, this is the first report of a CHUV isolated in China. It is important to understand the genetic and antigenic variations of the CHUV isolates circulating in China, which is near Japan and Korea in East Asia and to develop molecular diagnostic tools and a more effective vaccine for the monitoring and prevention of CHUV infection. The genome sequences of CHUV isolates provide evidence of their functional and phylogenetic relationships to other members of the genus Orbivirus. The establishment a reverse-genetics system is in progress to define the molecular determinants of the virulence of the Chinese CHUV isolate GX871. Acknowledgments This study was supported by Special Fund for Agro-scientific Research in the Public Interest of China (No. 201303035).
123
References 1. Y. Miura, Y. Goto, M. Kubo, Y. Kono, Am. J. Vet. Res. 49, 2022–2025 (1988) 2. Y. Miura, Y. Goto, M. Kubo, Y. Kono, Nihon Juigaku Zasshi 50, 632–637 (1988) 3. Y. Miura, M. Kubo, Y. Goto, Y. Kono, Nihon Juigaku Zasshi 52, 689–694 (1990) 4. M. Yamakawa, M. Kubo, S. Furuuchi, J. Gen. Virol. 80(Pt 4), 937–941 (1999) 5. M. Yamakawa, S. Furuuchi, Y. Minobe, J. Gen. Virol. 80(Pt 1), 205–208 (1999) 6. Y.H. Kim, J.K. Oem, E.Y. Lee, K.K. Lee, S.H. Kim, M.H. Lee, S.C. Park, J. Vet. Med. Sci. 77, 247–250 (2015) 7. S.I. Lim, C.H. Kweon, D.S. Tark, S.H. Kim, D.K. Yang, J. Vet. Sci. 8, 45–49 (2007) 8. E.R. Jusa, Y. Inaba, K. Kadoi, H. Kurogi, E. Fonseca, R.E. Shope, Aust. Vet. J. 71, 57 (1994) 9. J.G. Victoria, A. Kapoor, K. Dupuis, D.P. Schnurr, E.L. Delwart, PLoS Pathog. 4, e1000163 (2008) 10. S. Maan, S. Rao, N.S. Maan, S.J. Anthony, H. Attoui, A.R. Samuel, P.P. Mertens, J. Virol. Meth. 143, 132–139 (2007) 11. S. Anbalagan, E. Cooper, P. Klumper, R.R. Simonson, B.M. Hause, J. Gen. Virol. 95, 434–441 (2014) 12. A.C. Potgieter, N.A. Page, J. Liebenberg, I.M. Wright, O. Landt, A.A. van Dijk, J. Gen. Virol. 90, 1423–1432 (2009) 13. P.P. Mertens, S. Pedley, J. Cowley, J.N. Burroughs, A.H. Corteyn, M.H. Jeggo, D.M. Jennings, B.M. Gorman, Virology 170, 561–565 (1989) 14. S. Ohashi, Y. Matsumori, T. Yanase, M. Yamakawa, T. Kato, T. Tsuda, J. Clin. Microbiol. 42, 4610–4614 (2004)
Isolation, complete genome sequencing, and phylogenetic analysis of the first Chuzan virus in China Fang Wang1 • Jun Lin2 • Jitao Chang1 • Yingying Cao2 • Shaomin Qin2 Jianmin Wu2 • Li Yu1
•
Received: 10 October 2015 / Accepted: 19 December 2015 Springer Science+Business Media New York 2016
Abstract A Chuzan virus (CHUV), defined as GX871 here, was isolated from blood from a sentinel cattle firstly in China, and its full-length genome was sequenced in this study. The GX871 genome included 10 segments and 18914 bp, one base fewer than the CHUV prototype strain K-47 due to a one-base deletion in the 50 non-coding region of segment 8. A frameshift mutation was detected in a short coding region (1010–1026 nt) corresponding to the VP1 protein; this frameshift resulted in a five-amino acid mutation from 336CVLSY340 to 336YGAKL340. In addition,
there were a one-base deletion at 1713 nt and a one-base insertion at 1682 nt in the 30 non-coding region of segment 5. Based on phylogenetic analysis of the deduced VP2 amino acid sequences, Palyam serogroup viruses were classified into three groups. The Chinese CHUV isolate GX871 was categorized into the same group as CHUV prototype strain K-47. The phylogenetic tree was divided into three clusters according to the geographical distribution of the partial nucleotide sequences of VP7, and this arrangement might define the geographical gene pool of CHUV.
Edited by Juergen A Richt.
Keywords Chuzan virus Isolation in China Complete genome Phylogenetic analysis
Fang Wang and Jun Lin have contributed equally to this work. & Jianmin Wu [email protected] & Li Yu [email protected] Fang Wang [email protected] Jun Lin [email protected] Jitao Chang [email protected] Yingying Cao [email protected] Shaomin Qin [email protected] 1
Division of Livestock Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People’s Republic of China
2
Guangxi Veterinary Research Institute, Nanning, People’s Republic of China
The Chuzan virus (CHUV), a member of the Palyam serogroup within the genus Orbivirus of the family Reoviridae [1], was first demonstrated to be the causative agent of bovine disease among the Palyam serogroup [2]. CHUV is responsible for many reproductive disorders, including abortion, stillbirth, and congenital malformation, which result in considerable economic loss in the cattle industry [2, 3]. Similar to other viruses of the Palyam serogroup, CHUV consists of 10 double-stranded (ds) RNA segments in which segments 1, 2, 3, 4, 6, 7, and 9 encode the structural proteins VP1, VP2, VP3, VP4, VP5, VP7, and VP6 [4, 5], respectively, segments 5 and 8 encode the nonstructural proteins NS1 and NS2 [4], respectively, and the smallest segment 10 encodes two related non-structural proteins, NS3 and NS3A [4]. To date, CHUV has been found in countries near China, such as Korea [6, 7] and Japan [1, 8]. Despite the presence of Culicoides spp., which had been reported as possible vectors for CHUV transmission, no CHUV isolate has been
123
Virus Genes Table 1 Features of Chinese CHUV isolate GX871 genes and their encoded proteins Genome segment
Length (bp)
50 /30 non-coding region
50 /30 non-coding region
Encoded protein
ORF length (aa)
Predicted molecular weight (Da)
1
3930
GTTAAA/ACTTAC
22/23
VP1
1295
149113
2
3055
GTTAAA/ACTTAC
14/35
VP2
1002
117422
3
2774
GTTAAA/ACTTAC
13/49
VP3
904
103866
4
1967
GTTAAA/ACTCAC
9/38
VP4
640
74631
5
1764
GTTAAA/ACTCAC
35/94
NS1
545
64138
6
1610
GTTAAA/GCTTAC
20/27
VP5
521
59018
7 8
1151 1058
GTAAAA/GCTCAC GTAAAA/ACTCAC
18/89 20/39
VP7 NS2
348 333
37994 37164
9
877
GTTAAA/ACATAC
19/42
VP6
272
29713
10
728
GTTAAA/ACTTAC
18/77
NS3/NS3a
211/200
23606/22328
Total
18914
6071
696665
reported in China. To investigate the status of arthropodborne disease in China, a pilot study was performed in 2013. This study aimed to isolate viruses from whole-blood samples collected from sentinel animals in different areas of China. Consequently, the first CHUV strain was isolated, and its complete genome sequence was determined for phylogenetic analysis. To perform virus isolation, whole-blood samples were collected from sentinel cattle in herds scattered across different locations throughout the country. The C6/36 cell line derived from the mosquito (ATCC CRL-1660) was used for primary inoculation. After the first passage, the infected C6/36 cells were used to inoculate baby hamster kidney (BHK21) cells for five blind passages. Samples that exhibited typical cytopathological changes within 5 days after infection were further tested using reverse transcription-PCR-based sequencing. The method of rapid identification of unknown viruses was described previously [9]. Briefly, the culture medium of the virus-infected BHK-21 cells was filtered through a 0.22-lm filter (Millipore) and treated with DNase and RNase. Viral cDNA synthesis was performed via reaction of the extracted viral RNA with the primer K-8N (GAC CAT CTA GCG ACC TCC ACN NNN NNN N). Subsequently, PCR amplification of the extension products was performed using the primer K (GAC CAT CTA GCG ACC TCC AC). The obtained sequences were analyzed for their homology to nucleotide sequences in the GenBank database using a BLASTn search. Consequently, one CHUV isolate (subsequently defined as GX871) was identified. The infected animal was a 12-month-old native cattle reared in the city of Mashan (108–115E, 23.6–25N) in the Guangxi Zhuang Autonomous Region of China in 2013. Neither illness nor disease was observed in the population of these sentinel cattle.
123
To elucidate the molecular characteristics of the CHUV GX871, its complete genome was sequenced according to the full-length amplification of cDNAs (FLAC) technique as described previously [10–12]. The genome was sequenced twice using two separate extracted RNA sample sets, resulting in coverage of a minimum of four reads per nucleotide. The Lasergene Package was used to assemble the generated sequences. The complete genome sequence of CHUV GX871 is available from GenBank (accession number KT887180 * KT887189). The sequence data were analyzed and aligned using the alignment programs BioEdit Sequence Alignment Editor (version 6.0), Mega (version 6.0) and Clustal X. Phylogenetic trees were generated using neighbor-joining (NJ), maximum likelihood (ML) and Bayesian algorithms. The lengths of the CHUV genome segments and their coding capacities are summarized in Table 1. As a result of a one-base deletion in the 50 non-coding region of segment 8, the full-length genome sequence of this virus is 18,914 bp, which is one base fewer than the CHUV prototype strain K-47 [4, 5]. The consensus terminal sequence of the GX871 genome is 50 GU (U/A) AAA… (A/G) C (U/ A) (U/C) AC 30 ; this finding is in agreement with the consensus sequence of Palyam serogroup virus genomes [4, 5]. These motifs in the consensus terminal sequence may serve as important signals for recognition, sorting, and packaging of the RNA segments during virus maturation. RNA segments 2, 3, 6, and 7 encode the major capsid proteins VP2, VP3, VP5, and VP7, respectively [5]. In the virion, the inner capsid (core), consisting of two major proteins (VP3 and VP7) and three minor proteins (VP1, VP4 and VP6), is surrounded by an outer capsid composed of VP2 and VP5, which are responsible for viral neutralization and antigenic variability [13]. Based on comparison
Virus Genes
Fig. 1 Phylogenetic tree based on amino acid sequences of VP2 (a) and the partial nucleotide sequence of VP7 (b) of CHUV. Trees were constructed using the neighbor-joining method and numbers
above the internal nodes indicate the bootstrap values obtained with 1000 replications. BTV-1 and AHSV were used as the outgroup to root the tree (a). The black diamonds indicate Chinese isolate GX871
with the nucleotide and deduced amino acid sequences of VP2 of 6 CHUV isolates, VP2 of Chinese isolate GX871 showed 95.9–98.1 % and 97.6–99 % identities, respectively, except for VP2 of CHUV ON 91-5, which showed 53.2 and 41.5 % identities to VP2 of CHUV GX871, respectively. Among the compared sequences, GX871 VP2 displayed the highest identity (98.1 and 99 %) to the nucleotide and deduced amino acid sequences of K-47 VP2. Phylogenetic analysis of the VP2 protein revealed that these viruses could be divided into three major clusters (Fig. 1a). One cluster contained five Japanese CHUV isolates (K-47, 31, FO 88-2, FO 90-8, and ON-3/E/98) and the Chinese isolate GX871. Four D‘Aguilar virus isolates (KY115, ON-1/E/00, KSB-29/E/01 and B8112), CHUV ON 91-5, and Nyabira virus 792/76 formed another cluster. Finally, four Bunyip Creek virus isolates formed a distinct cluster. Segment 6 of CHUV encodes the outer-coat protein VP5, which is 521 amino acids in length (Table 1). The nucleotide and deduced amino acid sequences of GX871 VP5 showed 96.0 and 99.6 % identities to CHUV K-47 VP5, respectively. That CHUV VP3 and VP7 form core capsids is presumably directly relevant to the intra- and inter-molecular interactions involved in virus assembly [5]. The deduced amino acid sequences of VP3 and VP7 in GX871 showed identities of 99.9 and 100 % to VP3 and VP7 of CHUV K-47, respectively. To determine the relationships between
the Chinese isolates and other Palyam serogroup viruses, a partial 224-bp DNA fragment corresponding to nucleotide positions 261–484 of segment 7 was compared. A high degree of sequence identity, from 97.3 to 100 %, was obtained among the Chinese, Japanese, and Taiwanese isolates. These Asian isolates showed relatively lower identities (from 84.8 to 92.0 %) to Australian and African isolates. Phylogenetic analysis of VP7 revealed that Palyam serogroup viruses were divided into three clusters according to the region from which the viruses were isolated [14]. These clusters consisted of Japanese and Taiwanese isolates, Australian isolates, and African isolates (Fig. 1b). All viruses isolated in Japan were categorized into the same cluster together with GX871 and two Taiwanese isolates. These results suggested that the VP7 protein of Palyam serogroup viruses was conserved among the viruses in the epidemic area even if the serotypes were different. Thus, isolate GX871 and the Taiwanese and Japanese isolates of Palyam serogroup viruses appeared to have been derived from a common genetic pool. CHUV genome segments 1, 4, 5, 8, 9, and 10 encode VP1, VP4, NS1, NS2, VP6, and NS3, respectively. The VP1 protein appears to be highly conserved among Orbiviruses [5]. Chinese isolate GX871 showed high identity with the Japanese isolate K-47. The amino acid sequences of VP1, VP4, VP6, NS1, NS2, and NS3 of isolate GX871 showed 98.1, 97.5, 97.1, 98.2, 98.5, and 96.2 % identities
123
Virus Genes
to the respective proteins of CHUV K-47. However, some unique differences in the genome of Chinese isolate GX871 were observed. Based on comparisons of GX871 with other Palyam serogroup viruses, a frameshift mutation was detected in nt 1010–1026 within the coding region of the VP1 protein of GX871; this frameshift produced a fiveamino acid mutation from 336CVLSY340 to 336YGAKL340. Similarly, there were a one-base deletion at 1713 nt and a one-base insertion at 1682 nt in the 30 non-coding region of segment 5. In addition, a one-base deletion was detected in the 50 non-coding region of segment 8. To our knowledge, this is the first report of a CHUV isolated in China. It is important to understand the genetic and antigenic variations of the CHUV isolates circulating in China, which is near Japan and Korea in East Asia and to develop molecular diagnostic tools and a more effective vaccine for the monitoring and prevention of CHUV infection. The genome sequences of CHUV isolates provide evidence of their functional and phylogenetic relationships to other members of the genus Orbivirus. The establishment a reverse-genetics system is in progress to define the molecular determinants of the virulence of the Chinese CHUV isolate GX871. Acknowledgments This study was supported by Special Fund for Agro-scientific Research in the Public Interest of China (No. 201303035).
123
References 1. Y. Miura, Y. Goto, M. Kubo, Y. Kono, Am. J. Vet. Res. 49, 2022–2025 (1988) 2. Y. Miura, Y. Goto, M. Kubo, Y. Kono, Nihon Juigaku Zasshi 50, 632–637 (1988) 3. Y. Miura, M. Kubo, Y. Goto, Y. Kono, Nihon Juigaku Zasshi 52, 689–694 (1990) 4. M. Yamakawa, M. Kubo, S. Furuuchi, J. Gen. Virol. 80(Pt 4), 937–941 (1999) 5. M. Yamakawa, S. Furuuchi, Y. Minobe, J. Gen. Virol. 80(Pt 1), 205–208 (1999) 6. Y.H. Kim, J.K. Oem, E.Y. Lee, K.K. Lee, S.H. Kim, M.H. Lee, S.C. Park, J. Vet. Med. Sci. 77, 247–250 (2015) 7. S.I. Lim, C.H. Kweon, D.S. Tark, S.H. Kim, D.K. Yang, J. Vet. Sci. 8, 45–49 (2007) 8. E.R. Jusa, Y. Inaba, K. Kadoi, H. Kurogi, E. Fonseca, R.E. Shope, Aust. Vet. J. 71, 57 (1994) 9. J.G. Victoria, A. Kapoor, K. Dupuis, D.P. Schnurr, E.L. Delwart, PLoS Pathog. 4, e1000163 (2008) 10. S. Maan, S. Rao, N.S. Maan, S.J. Anthony, H. Attoui, A.R. Samuel, P.P. Mertens, J. Virol. Meth. 143, 132–139 (2007) 11. S. Anbalagan, E. Cooper, P. Klumper, R.R. Simonson, B.M. Hause, J. Gen. Virol. 95, 434–441 (2014) 12. A.C. Potgieter, N.A. Page, J. Liebenberg, I.M. Wright, O. Landt, A.A. van Dijk, J. Gen. Virol. 90, 1423–1432 (2009) 13. P.P. Mertens, S. Pedley, J. Cowley, J.N. Burroughs, A.H. Corteyn, M.H. Jeggo, D.M. Jennings, B.M. Gorman, Virology 170, 561–565 (1989) 14. S. Ohashi, Y. Matsumori, T. Yanase, M. Yamakawa, T. Kato, T. Tsuda, J. Clin. Microbiol. 42, 4610–4614 (2004)
