Generation and characterization of variants of NWS/G70C influenza virus after in vitro passage in 4-amino-Neu5Ac2en and 4-guanidino-Neu5Ac2en
Generation and characterization of variants of NWS/G70C influenza virus after in vitro passage in 4-amino-Neu5Ac2en and 4-guanidino-Neu5Ac2en
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DOI：
10.1128/aac.40.1.40
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发表时间：
1996-01-01
影响因子：
4.9
通讯作者：
Penn, CR
中科院分区：
文献类型：
--
作者：
McKimmBreschkin, JL;Blick, TJ;Penn, CR
化合物4-amino-Neu 5Ac 2 en（5-乙酰氨基-2，6-脱水-4-氨基-3，4，5-乳糖-壬-2-烯酸）和4-胍基-Neu 5Ac 2 en（5-乙酰氨基-2，6-脱水-4-胍基-3，4，5-三脱氧-D-甘油-D-半乳糖-壬-2-烯酸），其选择性地抑制流感病毒神经氨酸酶，已经在体外测试了它们产生耐药变体的能力，通过有限稀释传代，在每种药物中培养NWS/G70 C病毒（H1 N9）。在任一化合物中传代5或6次后，出现了对细胞培养物中抑制剂敏感性降低的病毒。变异病毒在空斑试验、液体培养和血凝洗脱试验中的敏感性降低了1，000倍。此外，在所有三种测定中均观察到对两种化合物的交叉耐药性。一些分离株表现出药物依赖性，在空斑测定中空斑的大小和数量均增加，并且在存在抑制剂的情况下液体培养物中病毒产量增加。在体外未检测到神经氨酸酶活性的显著差异，并且未发现神经氨酸酶保守位点的序列变化。然而，检测到血凝素中保守氨基酸的变化，这些氨基酸与血凝素受体结合位点Thr-155或受体结合口袋的左边缘瓦尔-223和Arg-229相关。因此，可以预期这些位点的突变会影响血凝素结合的亲和力或特异性。因此，导致弱结合血凝素的补偿突变似乎通过允许病毒从细胞中释放而较少依赖神经氨酸酶来规避神经氨酸酶的抑制。
The compounds 4-amino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-amino-3,4,5 lacto-non-2-enoic acid) and 4-guanidino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-guanidino-3,4,5-trideoxy-D-glycerol-D-galacto-non-2-enoic acid), which selectively inhibit the influenza virus neuraminidase, have been tested in vitro for their ability to generate drug-resistant variants, NWS/G70C virus (H1N9) was cultured in each drug by limiting-dilution passaging. After five or six passages in either compound, there emerged viruses which had a reduced sensitivity to the inhibitors in cell culture, Variant viruses were up to 1,000-fold less sensitive in plaque assays, liquid culture, and a hemagglutination-elution assay. In addition, cross-resistance to both compounds was seen in all three assays, Some isolates demonstrated drug dependence with an increase in both size and number of plaques in a plaque assay and an increase in virus yield in liquid culture in the presence of inhibitors, No significant difference in neuraminidase enzyme activity was detected in vitro, and no sequence changes in the conserved sites of the neuraminidase were found, However, changes in conserved amino acids in the hemagglutinin were detected, These amino acids were associated with either the hemagglutinin receptor binding site, Thr-155, or the left edge of the receptor binding pocket, Val-223 and Arg-229, Hence, mutations at these sites could be expected to effect the affinity or specificity of the hemagglutinin binding. Compensating mutations resulting in a weakly binding hemagglutinin thus seem to be circumventing the inhibition of the neuraminidase by allowing the virus to be released from cells with less dependence on the neuraminidase.