J Bacteriol Virol.  2008 Sep;38(3):127-137. 10.4167/jbv.2008.38.3.127.

Amantadine and Zanamivir Resistance of Influenza A/H3N2 Viruses Isolated in Korea, 2002/03~2003/04

• Kim KA
• Lee JY
• Kim W
• Kim Y
• Park YK
• Kang C

• KMID: 1483971
• DOI: http://doi.org/10.4167/jbv.2008.38.3.127

Abstract

To investigate the emergence and prevalence of antiviral resistance, we analyzed influenza A/H3N2 viruses isolated in Korea during 2002/03 to 2003/04 season by genetic and phenotypic assay. For the genetic analysis to the amantadine, an M2 protein inhibitor, the M gene was amplified by RT-PCR and regions corresponding to the amino acid at positions 27, 30, and 31 were amplified by nested PCR with size of 154 bp, 95 bp, and 153 bp fragments, respectively. A total of 3 of 31 (9.7%) viruses were found to be mutated by restriction fragment length polymorphism (RFLP) with Sca I and sequence analysis, showing the single amino acid change (Ser to Asn) at position 31. Also it was observed that their growths in Madin-Darby Canine Kidney (MDCK) cells were unaffected by amantadine (up to 1 microgram/ml) in both plaque assay and WST-1 assay, confirming that these viruses were resistant against amantadine. We also examined the resistant pattern against zanamivir, a neuraminidase inhibitor, for 15 Korean influenza A/H3N2 viruses isolated in 2002~2003 season. Sequence analysis showed that there were no genetic changes of NA genes including R292K, K274Y, R152K, and E119V which were related to resistance against the neuraminidase inhibitor. In the NA inhibition assay to zanamivir, Korean isolates were found to be sensitive, ranging from 0.17 nM to 1.77 nM in 50% inhibitory concentration (IC(50)). These results suggest that monitoring for the antiviral resistance should be intensified and maintained to provide guideline for prophylaxis and treatment of influenza in Korea.

Keyword

Influenza virus; Amantadine; Zanamivir; Resistance

MeSH Terms

Amantadine
Influenza, Human
Kidney
Korea
Neuraminidase
Orthomyxoviridae
Polymerase Chain Reaction
Polymorphism, Restriction Fragment Length
Prevalence
Seasons
Sequence Analysis
Zanamivir
Amantadine
Neuraminidase
Zanamivir

Figure

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  Figure 1. Amplification of M2 gene of influenza A/H3N2 viruses by RT-PCR M, 50 bp size marker; lanes 1 to 3, position 27 (154 bp); lanes 4 to 6, position 30 (95 bp); lanes 7 to 9, position 31 (153 bp) of the M2 gene.

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  Figure 2. PCR-RFLP analysis by Sca I for the detection of amantadine-resistant virus M, 50 bp size marker; lanes 1 to 3, amantadine-sensitive viruses without substitution at position 31; lanes 4 to 6, amantadine-resistant strains with substitution at position 31. In the lanes 1 to 3, the fragments 37 bp were not clearly visualized due to the very small size.

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  Figure 3. Confirmation of amino acid substitution at position 31 in the M2 gene by sequence analysis.

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  Figure 4. Effect of amantadine on plaque formation of the sensitive strain (A) and resistant strain (B) of influenza A/H3N2 viruses. The concentrations of inhibitor in upper row were 0.01, 0.1, and 1 μg/ml (left to right) and those in the low row were no drug and 0.001 μg/ml (left to right).

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  Figure 5. Effect of amantadine on viral growth in MDCK cells in the WST-1 assay.

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  Figure 6. Neuraminidase inhibition by zanamivir of influenza viruses.

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