A/H6N1亚型禽流感病毒致病性评估及与2009 A/H1N1亚型流感病毒在哺乳动物体内的遗传兼容性

Evaluation of the pathogenicity of A/H6N1 avian influenza virus and its genetic compatibility with 2009 A/H1N1 pandemic influenza virus in mammalian models

目的探讨人、禽流感病毒在哺乳动物体内的遗传兼容性,为下一步研究H6亚型禽流感病毒重配和致病性变异的分子机制奠定基础。方法野鸭源A/H6N1亚型禽流感病毒A/Mallard/SanJiang/275/2007以101EID50～106EID50的攻毒剂量经鼻内途径感染小鼠,通过临床症状观察、病毒滴定和病理切片观察进行病毒学和组织学两方面检测对小鼠的致病性;同时,将此病毒与2009年A/H1N1流感病毒A/Changchun/01/2009(H1N1)混合感染豚鼠,分析两株病毒在哺乳动物体内的遗传兼容性。每天采集豚鼠鼻洗液并用噬斑纯化技术获得重配病毒,对获得的重配病毒进行全基因组序列的测定。结果 H6N1亚型禽流感病毒能直接感染小鼠,但对小鼠不致死。106EID50的攻毒剂量可有效感染小鼠,攻毒后第5天,小鼠表现出被毛较粗乱、活动减少、体重下降、呼吸急促的临床症状,但至攻毒后第10天开始康复,而对照组(MOCK)小鼠在14 d的观察期内无明显临床症状。病毒滴定结果表明,该病毒主要在小鼠肺脏和鼻甲骨中复制,病毒滴度可达104.5EID50/mL。病理学观察发现感染小鼠肺泡壁增厚,有大量炎性细胞浸润,纤维蛋白渗出并伴有轻微出血;在A/H6N1和A/H1N1混合感染豚鼠的重配实验中,经过三轮噬斑纯化从豚鼠鼻洗液中分离到6株重配病毒,说明A/H6N1亚型禽流感病毒与A/H1N1亚型流感病毒具有很好的遗传兼容性,能在豚鼠体内能发生重配。结论野鸭源A/H6N1亚型流感病毒无需适应就能够感染哺乳动物;该病毒与A/H1N1流感病毒具有很好的遗传兼容性,在哺乳动物体内能够发生基因重配,产生新的重配病毒,其公共卫生意义应引起高度关注。

Objective A/H6N1 avian influenza virus has become an epidemic virus in poultry in China, posing a significant threat to public health. In order to evaluate the capacity of pathogenesis and reassortment of H6N1 virus in mam- malians, we first evaluated the pathogenicity of H6N1 virus to mice, and then evaluated the compatibility between H6N1 vi- rus and 2009 A/HIN1 pandemic viruse. Methods For the mouse infection experiment, the mice were inoculated intrana- sally with 101EID^0 -106EIDs0 A/H6N1 AIV （A/Mallard/SanJiang/275/2007 ,which was isolated from wild ducks）, and virological and histological data were collected by clinical sign observation, virus titration, and histopathology with HE stai- ning. For the reassorment experiment in guinea pigs, the animals were inoculated with mixed A/H6N1 and A/HI N1 influ- enza viruses （A/Changchun/01/2009）. The nasal washes of guinea pigs were collected every day, and reassortment viru- ses were obtained by plaque purification and identified by sequence analysis. Results In the mouse infection experiment, the mice were directly infected with H6N1 AIV virus, but were not lethal. The group of mice inoculated with 106 EID50 showed body weight loss, decreased activity, dorsal hair disordered, and tachypnea at day 15 after infection （DPI15）. How- ever, the clinical symptoms disappeared at DPI10. The virus titration results showed that the A/H6N1 virus mainly replicated in the lung, trachea and turbinate of mice, and the virus titers were up to 104＂5/mL. Using HE staining, the pathological ex- amination found that the lungs of the 106 EIDs0 group showed pathological changes, including alveolar wall thickening, infiltra- tion of inflammatory cells, fibrin exudation and slight bleeding. In the reassorment experiment of guinea pigs, six reassortant virus strains were recovered with different gene segments derived from H6N1 and H1N1 viruses after three-time plaque purifi- cation. The results indicated that A/H6N1 and A/H1N1 influenza virus have high genetic compatibility in mammalian mod els. Conclusions A/H6N1 AIV can infect mammals directly without adaption and has a high genetic compatibility in guinea pigs with the H1N1 AIV. Therefore, attention should be paid on its significant threat to public health.