- Short report
- Open Access
Genetic diversity of a Korean echovirus 5 isolate and response of the strain to five antiviral drugs
© Park et al; licensee BioMed Central Ltd. 2011
- Received: 17 October 2010
- Accepted: 24 February 2011
- Published: 24 February 2011
An outbreak of echovirus 5 (ECV 5) occurred in Korea in 2006, marking the first time this virus had been identified in the country since enterovirus surveillance began in 1993. Using a sample isolated from a young male patient with aseptic meningitis, we performed sequencing of the Korean ECV 5 strain and compared it with a prototype strain (Noyce). At the nucleotide level, the P1 region (85.3%) had the highest identity value; at the amino acid level, the P3 region (98.0%) had the highest identity value. The two strains shared all cleavage sites, with the exception of the VP1/2A site, which was TY/GA in the Noyce strain but TR/GA in the Korean ECV 5 isolate. In Vero cells infected with the Korean ECV 5 isolate, no cytotoxicity was observed in the presence of azidothymidine, acyclovir, amantadine, lamivudine, or ribavirin, when the drugs were administered at a CC50 value >100 μg/mL. Of the five drugs, only amantadine (IC50: 1 ± 0.42 μg/mL, TI: 100) and ribavirin (IC50: 22 ± 1.36 μg/mL, TI: 4.55) had any antiviral activity against the Korean ECV 5 isolate.
- Antiviral Activity
- Vero Cell
Human enteroviruses (HEV) are RNA viruses from the Picornaviridae family. The 80 immunologically-distinct serotypes that are known to cause infections in humans can be grouped as follows: polioviruses (PV), echoviruses (ECV), coxsackieviruses A (CVA), coxsackieviruses B (CVB), and enterovirus (EV) types 68-71. These viruses are also classified genetically into five species (HEV-A to HEV-D and PVs). HEV-B group containing ECV 5 are CBV 1 to 6, CVA9, ECV 1 to ECV 7, ECV 9, ECV 11 to ECV 21, ECV 24 to ECV 27, ECV 29 to 33, EV 69, EV 73 [1–5].
ECV cause the same types of infections in humans as the CVB group, but are given a distinct classification primarily because they lack pathogenicity in newborn mice . There are, however, strains of ECV that are pathogenic in mice . ECV 5 infections have been associated with a wide variety of neurological and exanthematic diseases. The prototype strain of ECV 5 was isolated from a patient with aseptic meningitis and was later grouped as the fifth enterovirus serotype . An outbreak of aseptic meningitis caused by ECV 5 occurred in Korea in 2006, marking the first time that ECV 5 had been identified in the country since enterovirus surveillance began in 1993 .
The ECV 5 genome contains approximately 7,500 nucleotide-long single-stranded RNA molecules with polarity and carries a small viral peptide (VPg) covalently attached to its 5' end. The 5' untranslated region (UTR) of the RNA is approximately 700 nt in length and is unusually long compared with the homologous region of cellular mRNA. The internal ribosomal entry site (IRES) was discovered in the 5' UTR of the HEVs. In these viruses, the IRES can fold to be a functional secondary RNA structure and drive translation initiation . The coding region encompasses a single open reading frame that encodes a polyprotein divided into three sub-regions, P1, P2, and P3. The P1 region encodes the genetic information of four structural proteins, VP4, VP2, VP3, and VP1. The non-structural proteins are encoded in the P2 (2A-2C) and P3 (3A-3D) regions. A short 3' UTR of approximately 100 nt separates the coding region from the poly (A) tail [6, 11].
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 [12, 13]. 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 [15, 16].
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.
2.1 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 .
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 (Accession no. AF083069) 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).
2.3 Antiviral drugs and antiviral activity assays
Assays of antiviral activity and cytotoxicity were evaluated by the modified sulforhodamine B (SRB) assay previously described and recently reported by Choi et al [18–20]. Ribavirin was purchased from DUCHEFA (Netherlands); azidothymidine, acyclovir, amantadine, and lamivudine were purchased from Sigma-Aldrich (St. Louis, MO, USA), as was SRB.
where (ODt)ECV is the optical density measured with a given concentration of the test compound in ECV-infected cells; (ODc)ECV is the optical density measured for the control untreated ECV-infected cells; and (ODc)mock is the optical density measured for control untreated mock-infected cells. The therapeutic index was defined as 50% cytotoxic concentration (CC50)/IC50.
2.4 Cytotoxicity assay
Vero cells were seeded into a 96-well culture plate at a concentration of 2 × 104 cells per well. Next day, the medium was removed and the 96-well plates were replaced with media containing the serially diluted compounds, and the cells were further incubated for 48 hrs. The culture medium was removed and washed with 1 × PBS. The next step was conducted by antiviral activity assay 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.
3.1 Complete nucleotide sequence of Korean ECV5
Genome components of the Korean echovirus 5 (ECV 5) isolate and comparison of predicted N-terminal cleavage sites for the Korean ECV 5 isolate and the Noyce strain
Amino acid sequence
Predicted N-terminal cleavage sites
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 (Table 1).
3.3 Antiviral activity of Korea ECV5
Efficacy of antiviral drugs against Korean echovirus 5
1 ± 0.42
22 ± 1.36
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 . 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 [21, 22]. 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 [23, 24].
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.
This study was supported by an intramural research fund from the National Institute of Health, South Korea.
- Huttunen P, Santti J, Pulli T, Hyypiä T: The major echovirus group is genetically coherent and related to coxsackie B viruses. J Gen Virol 1996, 77: 715-725. 10.1099/0022-1317-77-4-715View ArticlePubMedGoogle Scholar
- Mayo M, Pringle CR: Virus taxonomy. J Gen Virol 1998, 79: 649-657.View ArticlePubMedGoogle Scholar
- Oberste MS, Maher K, Kilpatrick DR, Pallansch MA: Molecular evolution of the human enteroviruses: Correlation of serotype with VP1 sequence and application to picornavirus classification. J Virol 1999, 73: 1941-1948.PubMed CentralPubMedGoogle Scholar
- King AMQ, Brown F, Christian P, Hovi T, Hyypiä T, Knowles NJ, Lemon SM, Minor PD, Palmenberg AC, Skern T, Picronaviridae StanwayG: Virus taxonomy Seventh report of the International Committee on Taxonomy of Viruses. Edited by: Van Regenmortel MHV, Fauquet CM, Bishop DHL, Carstens EB, Estes MK, Lemon SM, Maniloff J, Mayo MA, McGeoch DJ, Pringle CR, Wickner RB. Academic Press, San Diego, Calif; 2000:657-678.Google Scholar
- Stanway G, Brown F, Christian P, Hovi T, Hyypiä T, King AMQ, Knowles NJ, Lemon SM, Minor PD, Pallansch MA, Palmenberg AC, Skern T: Family Picornaviridae. In Virus Taxonomy Eighth Report of the International Committee on Taxonomy of Viruses. Edited by: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. Elsevier Academic Press, London; 2005:757-778.Google Scholar
- Melnick JL: Enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses. In Fields Virology. 3rd edition. Edited by: Fields BN, Knipe DM, Howley PM, Channock RM, Melnick JL, Monath TP, Roizman B, Straus SE. Philadelphia: Lippincott-Raven Publishers; 1996:655-712.Google Scholar
- Eggers HJ, Sabin AB: Factors determining pathogenicity of variants of ECHO 9 virus for newborn mice. J Exp Med 1959, 110: 951-967. 10.1084/jem.110.6.951PubMed CentralView ArticlePubMedGoogle Scholar
- Melnick JL: Tissue culture techniques and their application to original isolation, growth, and assay of poliomyelitis and orphan viruses. Ann NY Acad Sci 1955, 61: 754-772. 10.1111/j.1749-6632.1955.tb42532.xView ArticlePubMedGoogle Scholar
- Baek K, Park K, Jung E, Chung E, Park J, Choi H, Baek S, Jee Y, Cheon D, Ahn G: Molecular and epidemiological characterization of enteroviruses isolated in Chungnam, Korea from 2005 to 2006. J Microbiol Biotechnol 2009, 19: 1055-1064. 10.4014/jmb.0810.584View ArticlePubMedGoogle Scholar
- Martínez-Salas E: The impact of RNA structure on picornavirus IRES activity. Trends Microbiol 2008,16(5):230-237.View ArticlePubMedGoogle Scholar
- Hyypiä T, Hovi T, Knowles NJ, Stanway G: Classification of enteroviruses based on molecular and biological properties. J Gen Virol 1997, 78: 1-11.View ArticlePubMedGoogle Scholar
- De Clercq E: Antiviral drugs in current clinical use. J Clin Virol 2004, 30: 115-133. 10.1016/j.jcv.2004.02.009View ArticlePubMedGoogle Scholar
- Leyssen P, De Clercq E, Neyts J: Molecular strategies to inhibit the replication of RNA viruses. Antiviral Res 2008, 78: 9-25. 10.1016/j.antiviral.2008.01.004View ArticlePubMedGoogle Scholar
- Berg AK, Olsson A, Korsgren O, Frisk G: Antiviral treatment of Coxsackie B virus infection in human pancreatic islets. Antiviral Res 2007, 74: 65-71. 10.1016/j.antiviral.2006.12.001View ArticlePubMedGoogle Scholar
- Pevear DC, Tull TM, Seipel ME, Groarke JM: Activity of pleconaril against enterovirus. Antimicrob Agents Chemother 1999, 43: 2109-2115.PubMed CentralPubMedGoogle Scholar
- Pevear DC, Hayden FG, Demenczuk TM, Barone LR, McKinlay MA, Collett MS: Relationship of pleconaril susceptibility and clinical outcomes in treatment of common colds caused by rhinoviruses. Antimicrob Agents Chemother 2005, 49: 4492-4499. 10.1128/AAC.49.11.4492-4499.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Lindberg AM, Johansson S, Andersson A: Echovirus 5: infectious transcripts and complete nucleotide sequence from uncloned cDNA. Virus Res 1999, 59: 75-87. 10.1016/S0168-1702(98)00127-0View ArticlePubMedGoogle Scholar
- Lin ZX, Hoult JR, Raman A: Sulphorhodamine B assay for measuring proliferation of a pigmented melanocyte cell line and its application to the evaluation of crude drugs used in the treatment of vitiligo. J Ethnopharmacol 1999,66(2):141-150. 10.1016/S0378-8741(98)00199-8View ArticlePubMedGoogle Scholar
- Choi HJ, Kim JH, Lee CH, Ahn YJ, Song JH, Baek SH: Antiviral activity of quercetin 7-rhamnoside against porcine epidemic diarrhea virus. Antiviral Res 2009, 81: 77-81. 10.1016/j.antiviral.2008.10.002View ArticlePubMedGoogle Scholar
- Choi HJ, Song JH, Park KS, Kwon DH: Inhibitory effects of quercetin 3-rhamnoside on influenza A virus replication. Eur J Pharm Sci 2009, 37: 329-333. 10.1016/j.ejps.2009.03.002View ArticlePubMedGoogle Scholar
- Lindberg AM, Andersson P, Savolainen C, Mulders MN, Hovi T: Evolution of the genome of Human enterovirus B: incongruence between phylogenies of the VP1 and 3CD regions indicates frequent recombination within the species. J Gen Virol 2003, 84: 1223-1235. 10.1099/vir.0.18971-0View ArticlePubMedGoogle Scholar
- Andersson P, Edman K, Lindberg AM: Molecular analysis of the echovirus 18 prototype: evidence of interserotypic recombination with echovirus 9. Virus Res 2002, 85: 71-83. 10.1016/S0168-1702(02)00019-9View ArticlePubMedGoogle Scholar
- Chen YJ, Zeng SJ, Hsu JT, Horng JT, Yang HM, Shih SR, Chu YT, Wu TY: Amantadine as a regulator of internal ribosome entry site. Acta Pharmacol Sin 2008,29(11):1327-1333. 10.1111/j.1745-7254.2008.00876.xView ArticlePubMedGoogle Scholar
- Li ZH, Li CM, Ling P, Shen FH, Chen SH, Liu CC, Yu CK, Chen SH: Ribavirin reduces mortality in enterovirus 71-infected mice by decreasing viral replication. J Infect Dis 2008,197(6):854-857. 10.1086/527326View ArticlePubMedGoogle Scholar
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