Molecular characterization and antiviral activity test of common drugs against echovirus 18 isolated in Korea

The Korean ECV 18 strain was isolated from a stool sample of a male patient with aseptic meningitis who had been hospitalized to the Department of Pediatrics at Soonchunhyang University Cheonan Hospital in April 2005. The pretreated specimen was inoculated into Vero cells and incubated at 37°C in an atmosphere of 5% CO₂ until the appearance of cytopathic effects (CPE). The identification of the Korean isolate was verified by a Basic Local Alignment Search Tool search of VP1 sequences at the National Center for Biotechnology Information website http://​www.​ncbi.​nlm.​nih.​gov/​. The VP1 sequences of Korean isolate had the highest nucleotide similarity with ECV 18 serotype strains [3].

The complete nucleotide sequence of the Korean ECV 18 strain was determined using a primer walking strategy; the sequences of the genome termini were determined by random amplification of cDNA ends (RACE) system (Invitrogen, Carlsbad, CA, USA). Polymerase chain reaction (PCR) products were purified using a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA). The purified DNA was added to a reaction mixture containing 2 μL of terminator reaction mix (ABI Prism BigDye Terminator Cycle Sequencing Kit; Applied Biosystems, Foster, CA, USA) and 2 pmole of each primer. Sequencing involved an initial denaturation at 96°C for 1 min and 25 cycles consisting of 96°C for 10 sec, 50°C for 5 sec, and 60°C for 4 min in a Gene Amp PCR system 2700 (Applied Biosystems). The products were purified by precipitation with 100% cold ethanol and 3 M sodium-acetate (pH 5.8), then loaded on an automated 3100 Genetic Analyzer (Applied Biosystems). Nucleotide sequences of ECV 18 Korean isolates were constructed to contig and were compared with the reference Metcalf strain (Accession no. AF317694). The Metcalf nucleotide sequence was obtained from the GenBank database [27]. Sequence analyses were performed using computer software included in the Lasergene package (DNASTAR, Madison, WI, USA).

Assays of antiviral activity and cytotoxicity involved the modified sulforhodamine B (SRB) assay [28‐30]. Ribavirin was purchased from DUCHEFA (Haarlen, Netherlands). Azidothymidine, acyclovir, amantadine, lamivudine, and sulforhodamine B (SRB) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

One day before infection, Vero cells were seeded (2 × 10⁴ cells/well) in wells of a 96-well plate. The next day, the medium was removed from each well and the adherent cells were washed with 1 × phosphate buffered saline (PBS). Infectivity of the virus stock was determined by the SRB assay [28‐30] and was determined as infectivity of the ECV 18 by SRB ID₅₀ (50% infective dose). Subsequently, 0.09 mL of diluted virus suspension of ECV 18 containing CCID₅₀ (50% cell culture infective dose) of the virus stock to produce an appropriate CPE within 2 days after infection and 0.01 mL of medium containing an appropriate concentration of the compounds were added. The antiviral activity of each agent was determined with a 10-fold diluted concentration ranging from 0.1-100 μg/mL. Three wells were used as ECV 18 controls (virus-infected non-drug-treated cells), while three wells were used as cell controls (non-infected, non-drug-treated cells). The culture plates were incubated at 37°C in 5% CO₂ for 2 days. After washing once with 1 × PBS, 100 μL of cold (-20^oC), 70% acetone was added to each well and left for 30 min at -20^oC. The acetone (70%) was discarded and each 96-well plate was dried in an oven for 30 min. 100 μL of 0.4% (w/v) SRB in 1% acetic acid was added to each well and left at room temperature for 30 min. Unbound SRB was removed and the plates were washed five times with 1% acetic acid before oven-drying prior to being left in the drying oven for one day. Bound SRB was solubilized with 100 μL of 10 mM unbuffered Tris-base solution and each plate was left on a table for 30 min. The absorbance was read at 540 nm using a VERSAmax microplate reader (Molecular Devices, Palo Alto, CA, USA) with a reference absorbance at 620 nm. To calculate the IC₅₀ (50% inhibitory concentration) values, the results were transformed to percentage of controls and the IC₅₀ values were graphically obtained from the dose-response curves. The percent protection achieved by the test compound in ECV 18-infected cells was calculated by the following formula:

where (OD_t)_{ECV 18} is the optical density measured with a given concentration of the test compound in ECV 18-infected cells, (OD_c)_{ECV 18} is the optical density measured for the control untreated ECV 18-infected cells, and (OD_c)_mock is the optical density measured for control untreated mock-infected cells. The therapeutic index was defined as 50% cytotoxic concentration (CC₅₀)/IC₅₀.

Vero cells were seeded into wells of a 96-well culture plate at a concentration of 2 × 10⁴ cells/well. The next day, the medium was discarded and the medium in each well was replaced with medium containing the serially-diluted drugs, and the cells were incubated for another 48 hours. The culture medium was removed and the adherent cells were washed with 1 × PBS. The next step assessed antiviral activity as described above. To calculate the CC₅₀ values, the results were transformed to percentage of controls and the CC₅₀ values were graphically obtained from the dose-response curves.

The genome of the Korean ECV 18 isolate was sequenced (GenBank accession no. HM777023) and its amino acid sequence was deduced. The genome was 7,413 nt in length, excluding the poly(A) tail. The 5' UTR contained 743 nt, followed by an ORF that encoded a viral polyprotein consisting of 2,189 codons, between a start codon (AUG) at position 744 and a stop codon (UGA) at position 7,310. The 3' UTR was 103 nt in length.

The Korean ECV 18 isolate genome was divided into five regions (5' UTR, P1, P2, P3, and 3' UTR) and aligned with the Metcalf strain using Megalign. The 3' UTR region had the highest level of nucleotide identity (85.3%), followed by the P3 region (82.2%), 5' UTR (82.0%), P2 (81.2%), and the P1 region (79.7%). The P2 region displayed the highest level of amino acid identity (96.9%), followed by the P3 region (96.8%), and the P1 region (95.2%) (Table 1). Most of the cleavage sites were identical between the Korean ECV 18 isolate and the Metcalf strain. The only exception was the cleavage site between 2B and 2C, which was RQ/NN in the Metcalf strain and RQ/NS in the Korean ECV 18 isolate (Table 2).

No signs of cytotoxicity were observed in Vero cells treated with any of the five antiviral agents at a CC₅₀ value >100 μg/mL. Amantadine and ribavirin exhibited antiviral activity against the Korean ECV 18 strain, while azidothymidine, acyclovir, and lamivudine did not. Amantadine displayed an IC₅₀ value of 4.97 ± 0.77 μg/mL and a TI value of 20.12, while ribavirin displayed an IC₅₀ value of 7.63 ± 0.87 μg/mL and a TI value of 13.11 (Table 3 and Figure 1).