Journal List > J Bacteriol Virol > v.45(4) > 1034191

Lee, Yang, Kim, Jo, Choi, Park, Choi, and Cho: Recharacterization of Morphological and Genetic Feature of Getah Virus Isolated from South Korea

Abstract

Three QIAG93 strains, QIAG9301, QIAG9302 and QIAG9303 that have been identified as Getah virus (GETV) are analyzed in this study. The morphological features of three virus isolates were observed by using electron microscopy, suggesting that the QIAG9301, QIAG9302 and QIAG9303 isolate can be classified as tentative member of Alphavirus species in the Semliki Forest complex. The full length of the structural polyprotein gene of each QIAG93 isolate (QIAG9301, QIAG9302 and QIAG9303) was determined that are identical in size, comprising 3759 nucleotides that encoded 1253 amino acids. The sequence analysis of the structural polyprotein gene, including the C, E3, E1, 6K and E2 domain, showed that each QIAG93 isolate shares >98.9% sequence identity. The phylogenetic analysis and evolutionary distance (ED) estimation based on the structural polyprotein gene sequence showed that the QIAG9301 isolate is closely related to GETV South Korea strain (99.9% sequence identity and ED value 0.001) and Chinese GETV YN0540 strain (99.3% sequence identity ED value 0.007) than other Alphavirus species analyzed in this study. Both QIAG9032 and QIAG9303 isolate exhibited genetically close relationship with Mongolian GETV LEIV17741MPR strain (at least 99.3% sequence identity and mean ED value 0.0065). Therefore, our findings will be valuable for molecular epidemiological analyses of GETV in Korea and contribute to a further study on pathogenicity of three QIAG93 isolates in animals.

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Figure 1.
Electron microscopy of QIAG93 isolates. QIAG9301 was used as a representative of the three isolates. Virus-like particles in specimens prepared by direct (A) or sucrose gradient centrifugation (B). General morphological characteristics of viruses and cytopathic changes in Vero cells after virus infection for 72 h (C, D and E). Mock-infected cells were used for comparison (F). Inset C, black solid arrows indicate virus-like particles.
jbv-45-328f1.tif
Figure 2.
Amplification of the structural polyprotein gene of the GETV QIAG93 isolate with a specific primer set (see Table 1). Lanes 1 to 5, PCR products amplified using the respective GE1, GE2, GE3, GE4, and GE5 primer sets. Lane M, 100 bp DNA ladder. Use of the GE1 to GE5 primer sets resulted in amplification of products with sizes of 990, 1,030, 990, 880 and 490 bp, respectively.
jbv-45-328f2.tif
Figure 3.
Phylogenetic relationships between QIAG93 isolates and other Alphaviruses within the Semliki Forest antigenic complex. Phylogenies were assessed on the structural polyprotein gene using neighbor-joining methods. A bootstrap method with 1,000 replicates was applied in this analysis to evaluate the reliability of the inferred phylogenetic tree. In the neighbor-joining tree, the monophyletic sister groups that had bootstrap values > 70% were chosen. The GETV strains were classified into three sister groups rooted at GETV MM2021.
jbv-45-328f3.tif
Table 1.
Oligonucleotide primers used for RT-PCR of GETV, designed from the complete genome sequences of GETV South Korea (GenBank Accession No. AY702913)
Primer Sequence (5′ to 3′) Position Length (bp)
GE1F GAT GTC CAC GCT GTC TAA GAG 7401~7421 990
GE1R CGC GGG CTC TGA GCA TGG GA 8371~8390
GE2F TGG CCA TTG TCC TGG GAG GG 8221~8240 1030
GE2R GGG ACA AAC GAG GAG GTG TAC 9230~9250
GE3F CGT GGG CAC CAC CAG TAG CG 9143~9162 990
GE3R GGT GAT GGC ACG ACT GTC TTG 10124~10144
GE4F CTC CCC GAT GAC CCT ACA GC 10043~10062 880
GE4R TCG CGA TCC CGC CGA AGT CC 10901~10920
GE5F GGA CAT CCC GGA CAC CGC GT 10811~10830 490
GE5R CAA GCC TCC CGG AAT GCG GC 11281~11300
Table 2.
Detailed information of the Alphaviruses analyzed in the present study
Taxonomy Isolates Geographic origin Host GeneBank accession
GETV QIAG9301 South Korea Swine KR081238
GETV QIAG9302 South Korea Swine KR081239
GETV QIAG9303 South Korea Swine KR081240
GETV South Korea South Korea Swine AY702913
GETV HB0234 China Mosquito EU015062
GETV M1 China Mosquito EU015061
GETV YN0540 China Mosquito EU015063
GETV Kochi/01/2005 Japan Swine AB859822
GETV LEIV 16275 Mag Russia Mosquito EF631998
GETV LEIV 17741 MPR Mongolia Mosquito EF631999
GETV MM2021 Malaysia Unknown AF339484
SAGV Unknown Japan Unknown AB032553
RRV M1 China Unknown EF011023
CHIKV 0611aTw Singapore Human FJ807896
Table 3.
Nucleotide sequence comparison (above the diagonal) and estimation of evolutionary distance (below the diagonal) based on comparisons of the structural polyprotein gene of QIAG93 isolates with that of other Alphaviruses belonging to the Semliki Forest complex
Virus strains 1 2 3 4 5 6 7 8 9 10 11 12 13 14
1. GETV QIAG9301   99 98.9 99.9 99.1 98.4 99.3 98.2 97.9 99.1 95.4 97.4 98.5 62.8
2. GETV QIAG9302 0.010   99.7 99 98.6 98.7 98.8 98.5 98.2 99.4 95.5 97.6 98.8 62.7
3. GETV QIAG9303 0.011 0.003   98.9 98.5 98.5 98.7 98.4 98.1 99.3 95.4 97.5 98.6 62.6
4. GETV South Korea 0.001 0.010 0.011   99.2 98.4 99.4 98.2 98 99.2 95.4 97.4 98.5 62.9
5. GETV HB0234 0.009 0.014 0.016 0.008   98 99 97.8 97.6 98.7 95.1 97 98 62.8
6. GETV M1 0.017 0.013 0.015 0.016 0.021   98.1 97.8 98.2 98.7 95.7 97.6 99.8 62.8
7. GETV YN0540 0.007 0.012 0.013 0.006 0.010 0.018   98 97.7 99 95.2 97.2 98.2 62.9
8. GETV Kochi/01/2005 0.018 0.015 0.016 0.018 0.023 0.022 0.020   97.4 98.7 94.9 97 97.9 62.7
9. GETV LEVI16275Mag 0.021 0.018 0.019 0.021 0.025 0.018 0.023 0.027   98.3 95.5 97.6 98.3 62.6
10. GETV LEIV17741MPR 0.009 0.006 0.007 0.008 0.013 0.013 0.011 0.013 0.017   95.6 97.7 98.8 62.8
11. GETV MM2021 0.048 0.047 0.048 0.048 0.052 0.045 0.051 0.054 0.047 0.046   95.6 95.8 62.4
12. SAGV 0.027 0.024 0.025 0.027 0.031 0.025 0.028 0.031 0.025 0.024 0.045   97.6 62.6
13. RRV Alpahavirus M1 0.016 0.013 0.014 0.016 0.020 0.002 0.018 0.022 0.017 0.012 0.044 0.024   62.9
14. CHIKV 0611aTw 0.751 0.755 0.760 0.751 0.753 0.754 0.753 0.758 0.762 0.754 0.769 0.763 0.751  
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