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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.
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.
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.
Table 1.
Oligonucleotide primers used for RT-PCR of GETV, designed from the complete genome sequences of GETV South Korea (GenBank Accession No. AY702913)
Table 2.
Detailed information of the Alphaviruses analyzed in the present study
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
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