H5N1亚型禽流感病毒NS第263—277位核苷酸缺失提高病毒对鸡的致病力

为研究2000年以来绝大多数H5N1亚型禽流感病毒分离株在非结构基因的第263—277位发生15个碱基缺失现象的生物学意义，构建H5N1A／D／SD／04株HA、NA、NS的全基因表达／转录载体，以及NS的删除突变载体（m248），A/D/YZ/04株的NS基因表达／转录载体（848）和其补加15个核苷酸的NS突变载体（m848）。构建的载体分别与编码WSN（H1N1）内部基因载体进行组合转染，拯救获得4个具不同NS的重组的H5N1亚型流感病毒：RWSN-848和RWSN—m248在263-277位缺失15个碱基。RWSN-m848和RWSN-248则在相同位置不发生缺失。4个重组病毒的平均鸡胚繁殖效价（HA）、鸡胚的平均死亡时间（MDT）和鸡胚半数感染量（EID50）均无显著差异；但RWSN-848和RWSN-m248对6周龄SPF鸡的致病力明显高于RWSN—m848和RWSN-248。结果说明H5N1的NS基因在263～277位核苷酸发生缺失后，不影响重组H5N1在鸡胚中的繁殖性能，但提高了病毒对鸡的致病力。

The deletion of nucleotides of NS gene from 263 to 277 of H5Nt increases viral virulence in chicken

Since 2000, more and more NS fragment of H5N1 subtype influenza A virus had a unique deletion of nuleotides from 263 to 277. In order to investigate the biological significance of the mutation, four recombinants, RWSN-848, RWSN-m848, RWSN-248 and RWSN-m248, were generated via eight-plasmid reverse genetic system. These reassortants had the same inner gene and outer gene derived from A/WSN/33(H1N1) and A/D/SD/04(H5N1), respectively. But their NS genes were different. RWSN-m248 and RWSN-848 were isogenic with RWSN-248 and RWSN-m848 respectively, except for the NS gene, the formers encoded a mutant NS with a deletion from 263 - 277 nucleotides. All of the four recombinants could grow well in embryonated chicken eggs and had the similar viral titer, EID50 and MDT. Infections were done with the same Multiplicity of infection (MOI, 0.001), and the viruses in supernatants from infected cells were titrated at 12, 24, 36, and 48 h post-infection by plaque assay in fresh MDCK. The results show that four viruses grown well and had the same viral title in Vero cell, a non-IFN-responsive cell system. But in MDCK and COS-1, the cell lines can produce IFN, RWSN-248 and RWSN-m848 had a higher viral titer (two fold) than RWSN-m248 and RWSN-848. It revealed that the deletion of nucleotides of NS from 263 - 277 of H5N1 influences decreases viral growth ability in IFN producing cell line. IVPI were done for the virulence test of reassorted H5N1 viruses. Ten six-week-old SPF White Leghorn chickens were inoculated intravenously with 0.2mL of a 1:10 dilution of allantoic fluid containing each of the four recombinant viruses. The chickens were daily monitored for disease signs and death for 10 days post-inoculation. Finally, IVPI was counted for each virus. RWSN-m248 caused 4 chickens died in 10 days and its index was 1.63. RWSN-248 only caused one chicken died and its index was 0.45. RWSN848 caused 3 chickens died in 10 days and its index was 0.81. RWSN-m848 caused one of the ten chickens died and its' index was only 0.175. The results revealed that the deletion of nucleotides of NS gene from 263 to 277 sites increases H5N1 pathogenesis in chicken.