Genetic diversity of a Korean echovirus 5 isolate. Part 2

At present, some 40 antiviral drugs have been formally licensed for use in humans, mostly for treatment of human immunodeficiency virus, hepatitis B virus, and herpesvirus infections. The number of antiviral drugs that have been licensed for use in treating highly-pathogenic RNA virus infections is very limited; the current list of approved drugs includes anti-influenza medications, M2 channel inhibitors (amantadine and rimantadine), and neuramidase inhibitors (oseltamivir and zanaminir). Ribavirin is licensed for the treatment of respiratory syncytial virus and hepatitis C virus infections. Pleconaril, an antiviral drug developed in 1996 for treatment of diseases associated with picornavirus infections, can be used in treatments against enterovirus and rhinovirus infections. However, the treatment of this drug was extremely limited and reported to pleconaril-resistant viruses.

In this study, the molecular biological characteristics and genetic diversity of Korean ECV 5 that do not exist the using antiviral agents for it but widespread currently were analyzed through the complete nucleotide sequencing and compared with ECV 5 prototype strain (Noyce). We also selected 5 kinds of antiviral drugs (azidothymidine, acyclovir, amantadine, lamivudine, and ribavirin) that were used as inhibitors of other viruses and examined for anti-viral activity for Korean ECV 5.

2. Materials and methods

2.1 Virus isolation and identification

The Korean ECV 5 strain was isolated from cerebrospinal fluid sampled from a male patient with aseptic meningitis who had been admitted to the Department of Pediatrics at the Soonchunhyang University Cheonan Hospital, Korea, in June 2006. The sample was inoculated into Vero cells and then incubated at 37°C with 5% CO2 until the appearance of cytopathic effects (CPE) took place. The identification of the Korean isolate was verified by a Basic Local Alignment Search Tool (BLAST) search in VP1 sequences; that is, the VP1 sequences of Korean isolate had the highest nucleotide similarity with ECV5 serotype strains.
Online pharmacy Mexico
2.2 Nucleotide sequencing and sequence analysis

The complete nucleotide sequence of the Korean ECV 5 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). The PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Hamburg, Germany). The purified DNA was added to a reaction mixture containing 2 μL of Big Dye terminator reaction mix (ABI Prism BigDye Terminator Cycle Sequencing Kit; Applied Biosystems, Foster, CA, USA) and 2 pmoles of each primer. Sequencing reactions were subjected to 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 enterovirus isolates were constructed to contig and were compared with a reference, the Noyce strain, which was originally identified in the USA in 1954, and the nucleotide sequence of which was obtained from Genbank databases. Sequence analyses were performed using computer software included in the Lasergene package (DNASTAR, Inc., Madison, WI, USA).