Mohr Peter G, Deng Yi-Mo, McKimm-Breschkin Jennifer L
CSIRO Australian Animal Health Laboratory, Portarlington Road, Geelong, VIC, 3219, Australia.
WHO Collaborating Centre for Reference and Research on Influenza, 792 Elizabeth Street, Melbourne, VIC, 3000, Australia.
Virol J. 2015 Apr 22;12:67. doi: 10.1186/s12985-015-0295-3.
The neuraminidases (NAs) of MDCK passaged human influenza A(H3N2) strains isolated since 2005 are reported to have dual functions of cleavage of sialic acid and receptor binding. NA agglutination of red blood cells (RBCs) can be inhibited by neuraminidase inhibitors (NAIs), thus distinguishing it from haemagglutinin (HA) binding. We wanted to know if viruses prior to 2005 can demonstrate this property.
Pairs of influenza A(H3N2) isolates ranging from 1993-2008 passaged in parallel only in eggs or in MDCK cells were tested for inhibition of haemagglutination by various NAIs.
Only viruses isolated since 1994 and cultured in MDCK cells bound chicken RBCs solely through their NA. NAI inhibition of agglutination of turkey RBCs was seen for some, but not all of these same MDCK grown viruses. Efficacy of inhibition of enzyme activity and haemagglutination differed between NAIs. For many viruses lower concentrations of oseltamivir could inhibit agglutination compared to zanamivir, although they could both inhibit enzyme activity at comparable concentrations. An E119V mutation reduced sensitivity to oseltamivir and 4-aminoDANA for both the enzyme assay and inhibition of agglutination. Sequence analysis of the NAs and HAs of some paired viruses revealed mutations in the haemagglutinin of all egg passaged viruses. For many of the paired egg and MDCK cultured viruses we found no differences in their NA sequences by Sanger sequencing. However, deep sequencing of MDCK grown isolates revealed low levels of variant populations with mutations at either D151 or T148 in the NA, suggesting mutations at either site may be able to confer this property.
The NA active site of MDCK cultured human influenza A(H3N2) viruses isolated since 1994 can express dual enzyme and receptor binding functions. Binding correlated with either D151 or T148 mutations. The catalytic and receptor binding sites do not appear to be structurally identical since relative concentrations of the NAIs to inhibit enzyme activity and agglutination differ.
据报道，自2005年以来分离出的经MDCK传代的甲型H3N2人流感病毒的神经氨酸酶（NA）具有唾液酸裂解和受体结合的双重功能。神经氨酸酶抑制剂（NAI）可抑制红细胞（RBC）的NA凝集，从而将其与血凝素（HA）结合区分开来。我们想了解2005年之前的病毒是否也具有这种特性。
对1993年至2008年期间仅在鸡胚或MDCK细胞中平行传代的甲型H3N2流感病毒分离株进行检测，以评估各种NAI对血凝的抑制作用。
仅1994年以来分离并在MDCK细胞中培养的病毒通过其NA单独结合鸡红细胞。部分（但并非全部）在MDCK中培养的这些病毒可观察到NAI对火鸡红细胞凝集的抑制作用。不同NAI对酶活性和血凝的抑制效果有所不同。对于许多病毒，与扎那米韦相比，较低浓度的奥司他韦即可抑制凝集，尽管它们在相当的浓度下均可抑制酶活性。E119V突变降低了对奥司他韦和4-氨基DANA的酶活性检测及凝集抑制的敏感性。对部分配对病毒的NA和HA进行序列分析发现，所有经鸡胚传代的病毒的血凝素均有突变。对于许多配对的鸡胚培养和MDCK培养病毒，通过桑格测序我们未发现其NA序列存在差异。然而，对在MDCK中培养的分离株进行深度测序发现，NA中D151或T148位点存在低水平的变异群体，提示这两个位点的突变可能赋予了这种特性。
自1994年以来分离出的经MDCK培养的甲型H3N2人流感病毒的NA活性位点可表达双重酶和受体结合功能。结合与D151或T148突变相关。由于抑制酶活性和凝集所需NAI的相对浓度不同，催化位点和受体结合位点在结构上似乎并不相同。
