3.1 Complete nucleotide sequence of Korean ECV5
The Korean ECV 5 genome was sequenced (GenBank accession number HM775882) and its amino acid sequence was deduced. The genome is 7,430 nt in length, excluding the poly(A) tail. The 5′NCR contains 738 nt, followed by an ORF that encodes a viral polyprotein consisting of 2,196 codons, between a start codon (AUG) at position 739 and a stop codon (UGA) at position 7,326. The 3′NCR is 104 nt in length.
3.2 Genome comparison between Korean ECV5 and the Noyce strain
The Korean ECV 5 isolate genome was divided into five regions (5′NCR, P1, P2, P3, and 3′NCR) and aligned with the Noyce strain using Megalign (DNASTAR). The P1 region (85.3%) had the highest level of nucleotide identity, followed by the P3 region (84.8%), the 3′NCR (84.5%), the 5′NCR (81.8%), and the P2 region (80.0%). The P3 region (98.0%) had the highest level of amino acid identity, followed by the P1 region (97.7%), and the P2 region (96.9%).
Most of the cleavage sites were identical between Korean ECV 5 and the Noyce strain. The only exception was the cleavage site between VP1 and 2A, which was TY/GA in the Noyce strain, but TR/GA in the Korean ECV 5 isolate.
3.3 Antiviral activity of Korea ECV5
No signs of cytotoxicity were observed in Vero cells treated with any of the five antiviral drugs at a CC50 value >100 μg/mL. Amantadine and ribavirin exhibited antiviral activity against the Korean ECV 5 strain, though azidothymidine, acyclovir, and lamivudine did not. The amantadine possessed an IC50 value of 1 ± 0.42 μg/mL and a TI value of 100, while the ribavirin possessed an IC50 value of 22 ± 1.36 μg/mL and a TI value of 4.55.
4. Discussion
The ECV 5 is a rare enterovirus strain as causative aseptic meningitis and its sequence information is not common in the previous publication, including genbank database. This report describes the first complete nucleotide sequence for an ECV 5 isolated in Korea. Previous work has shown that sequence identity between different ECV serotypes is relatively high for the 5′NCR sequence, moderate for the P2 and P3 regions, and lowest for the P1 region [21]. This was not the pattern that emerged when we compared the Korean ECV 5 isolate and the Noyce strain; rather, the 5′NCR and P2 regions had relatively low nucleotide sequence identities (<81.8%), while the identities of the P1 and P3 regions were relatively high (>84.5%). Amino acid sequences for the protein coding regions had much higher sequence identities (>96.9%). Cleavage site variations have often been reported for VP2/VP3, VP3/VP1, and VP1/2A. Thus, we were not surprised to find that, though conservation was observed at most sites, there had been a substitution (TY/GA→TR/GA) at VP1/2A. There was about 20% genetic difference between prototype and the current widespread strain, and its difference was found at the cleavage site. Therefore, development and screening of antiviral drugs have to be focused on the object of the current epidemic strain.
Of the five antiviral drugs tested here (azidothymidine, acyclovir, amantadine, lamivudine, and ribavirin), only amantadine (IC50: 1 μg/mL) and ribavirin (IC50: 22 μg/mL) had antiviral activity against Korean ECV 5, with amantadine showing stronger effects than ribavirin. Therefore, the amantadine and ribavirin could be applied to patients infected with ECV 5. It was reported that the amantadine could suppress the IRES mediated translation, and ribavirin is a nucleoside analogue with broad-spectrum antiviral activity by decreasing viral replication in EV71.
In conclusion, this manuscript is the first report of the complete nucleotide sequence of the Korean ECV strain, as well as the first examination of its response to various antiviral drugs. This data should be useful in preventing future outbreaks of ECV5 and in treating patients infected with the strain. Accordingly, it is necessary to test more of the same kind of antiviral drugs and various enterovirus serotypes in future studies.