台灣北部2000-2004年流行性感冒病毒(A/H3N2)神經胺酸酶之基因變化

Abstract

Influenza virus is a member of Orthomyxoviridae, and infection of influenza virus can cause severe morbidity and mortality in the elderly and children. Influenza A viruses are enveloped and have two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Both of HA and NA undergo antigenic shift and antigenic drift. Antibody to HA is the most important determinant of immunity because it can neutralize the infectivity of influenza virus. Although anti-NA antibodies do not neutralize virus infectivity, they appear to modify the disease and reduce both pulmonary virus titer and the extent of lung lesions. Therefore, antigenic variability of the NA protein should also be considered when analyzing the epidemic impact of influenza virus and predicting newly emerging viruses. However, limited information is available concerning the molecular change of the influenza NA genes. Analysis of NA gene is particularly important since the use of influenza NA inhibitors that target the highly conserved catalytic site of the enzyme. In order to understand the variation of NA gene of influenza A (H3N2) virus in northern Taiwan, 43 strains of clinical isolates in Taipei during 2000-2004 were collected for this study. The result indicated that the amino acid variation rate of NA was about 0.5% per year. As compared with the A/Moscow/10/99 vaccine strain, amino acid changes within at least one of the seven NA antigenic determinants (I-VII) were found in approximate half of the isolates (20/43) and the most common changes were at position 332, 401, 431 and 432. Only one amino acid change (D151G) was observed in the catalytic site of NA. All isolates contained the seven conserved asparagine-linked glycosylation sites found in the NA of the progenitor A/Hong Kong/8/68 strain. In addition, most strains (38/43) had the new glycosylation sites at positions 93 and 329. To understand whether there is gene reassortment recently, we also analyzed the evolutionary relationship of these isolates. It appears that no HA/NA reassortment was found. Variation of plaque size was observed in the plaque assay. After purification of virus, a small-plaque virus strain (NA-) was obtained and a 586 nucleotide deletion (303-888nt) of NA gene was found. Although the deficiency of NA enzyme activity, it still can grow in MDCK cell. However, the virus yield was 45-fold less than wild-type when low MOI (10-5) was used. The receptor-binding ability of the defective virus was low but no compensatory substitutions in the HA gene were found. TamifluTM, a kind of anti-influenza drug, did not influence on NA- virus in vitro. Thus, our results suggest that NA activity may not be essential for influenza A virus growth.