【作者】 朱雯迎；

【导师】 崔言顺；

【作者基本信息】 山东农业大学 ， 预防兽医， 2011， 硕士

【摘要】 禽流感是由A型流感病毒引起的禽类传染病,临床表现为呼吸道感染,产蛋量下降,头颈水肿和腹泻。H9亚型禽流感为低致病力禽流感,能引起少量死亡或不死亡,表现为生长障碍和产蛋量下降。禽流感病毒变异频繁,传播速度快,给疫情的防控造成巨大的困难,使世界养禽业遭受巨大损失。研究并掌握禽流感的生物学特性和遗传进化规律,对有效的防控禽流感疫情有重要意义。为了掌握我国H9亚型禽流感的变异情况和流行规律,本研究从生物学特性和分子水平上对2009年从山东不同地区的规模化肉鸡场中分离到6株H9N2亚型流感病毒进行生物学特性、免疫相关性、分子遗传规律、对哺乳动物的致病性和对鸡的免疫保护性等方面的研究。通过血凝特性的测定、鸡胚致病力的测定等方法对6株禽流感H9N2亚型毒株的致病力进行分析和比较。结果表明:本试验分离到的2009年我国山东地区的这6株禽流感H9N2亚型毒株属于低致病性禽流感病毒,其HA效价在2⁶～2⁹之间,EID₅₀在10^-7.75/0.1mL¹0^-9.25/0.1mL之间,TCID₅₀在10^-8.00/0.1mL¹0^-8.50/0.1mL之间,6株H9N2禽流感病毒的EID₅₀和TCID₅₀数值相差不大,这说明2009年分离的H9流感病毒在鸡胚和哺乳动物细胞上的适应性比较接近。用有限稀释法对这6株病毒进行纯化,制备单因子血清,进行双交叉HI试验,从而分析它们的抗原相关性。不同时期和不同地区分离株的抗原性有所差异但没有发生根本性变化,试验选用的6株病毒的抗原相关性在0.91¹.02之间,同一病毒的单因子血清对该病毒的HI效价较高;6株病毒之间的差异性不大。从分子水平上分别研究了6株AIV H9N2分离株各基因片段的基因类型。对6株H9N2病毒的8个基因片段（PB2,PB1,PA,HA,NP,NA,M和NS）分别进行系统进化分析,作出系统进化树。HA基因进化树中,6株病毒存在同一个进化位置,都属于CK/SH/Y2/2008-like谱系,NA,NP,M,NS,PB1,PA基因进化树和HA基因进化树相同。然而,在PB2基因进化树中,6株病毒可以分为两个不同的进化分支,其中CK/SD/SG2/2009、CK/SD/WL/2009属于CK/SH/Y2/2008-like谱系,另外四株病毒CK/SD/PD/2009、CK/SD/HY/2009、CK/SD/SG1/2009、CK/SD/FX/2009却属于CK/SH/F/98-like谱系。因此6株病毒可以分为两个基因型:CK/SD/SG2/2009、CK/SD/WL/2009属于一个基因型,类似于CK/SH/Y2/2008-like谱系;CK/SD/PD/2009、CK/SD/HY/2009、CK/SD/SG1/2009、CK/SD/FX/2009是CK/SH/Y2/2008-like和CK/SH/F/98-like重组而来。为阐明H9N2流感病毒对哺乳动物致病性,分别以106EID50剂量攻毒6周龄BABL/c雌性小鼠,6株病毒都能在小鼠肺脏中复制,而且两株病毒能在攻毒7天后引起小鼠死亡。该试验结果具有重要公共卫生意义。以CK/SD/SG2/2009和某公司的鸡新城疫禽流感二联灭活疫苗（以下简称:新流疫苗）分别免疫28天的SPF试验鸡,用本试验分离株CK/SD/SG2/2009攻毒,两种疫苗都有保护性,但不同的部分分离的病毒滴度不同。本研究中我国山东分离到的H9N2亚型禽流感病毒不存在明显的抗原性差异,但是疫苗也不能产生完全的保护,这也可能和疫苗不能诱导完全的免疫保护有关。因此,合理的选择抗原针对性强的疫苗株非常重要,最好是选择和当前流行毒株抗原性一致的病毒株作为疫苗株。同时要制定合理的免疫程序,这样可以提高免疫后的抗体水平,更好的发挥疫苗的免疫保护作用。更多还原

【Abstract】 Avian influenza is caused by influenza virus type A. The clinical manifestations are respiratory infection, decline in egg production and alvi profluvium. H9 subtype of avian influenza is low-pathogenicity avian influenza. It can cause decline in egg production, growth retardation and a small quantity death. The Mutation frequency of the Avian influenza virus is frequently and the speed of propagation is quick, so it is very difficult to prevent the dissemination of the Avian influenza virus. There are important significance to explore the genetic mutations of the hemagglutinin gene and the neuraminidase gene and the regularity of molecular epidemiology of H9 subtype avian influenza for prevention and curing the Avian influenza. In order to explore the genetic mutations of the hemagglutinin gene and the neuraminidase gene and the law of molecular epidemiology of H9 subtype avian influenza in China, Six influenza viruses were isolated from broilers in different parts of Shandong province in 2009,and explored their virulence, immune reference and the genetic mutations. The investigation includes five experiments.Through the determination of hemagglutination properties, infected chicken embryo, the virulence was analyzed and compared. The results show that the virulences of there virus were weak and there were small disparity in antigenicity. The HA potency is between 2⁶ and 2⁹, the HI potency for Standard antigen is between 10log2 and 11log2. The EID₅₀ is between 10^-7.75/0.1mL and 10^-9.25/0.1mL. The EID₅₀ is between 10^-8.00/0.1mL¹0^-8.50/0.1mL. There are not quite different between EID₅₀ and TCID₅₀ of the six H9N2 avian influenza virus. This explains the H9 influenza virus which separate in 2009 are adaptability closer in chicken embryos and mammalian cells.Elected the six strains of virus which representing different regions and different periods and depurated these virues by Microdilution, maked manufacture single-factor serum and maked double cross-hemagglutination inhibition test. The correlation of the double cross-hemagglutination inhibition test is between 0.91 and 1.02. It is little different between the viruses.The whole cDNA fragments of the six influenza viruses isolated from broilers in different parts of Shandong province in 2009 were amplified by reverse transcription polymerase chain reaction, with primers specific to each gens. After cloning and sequencing, gene sequences were analyzed on homology and heredity evolution of molecule. HA gene phylogenetic tree, Six viruses exist in the same evolutionary position, all belong to CK/SH/Y2/2008-like lineage. the NA, NP, M, NS, PB1, PA are same with the HA gene phylogenetic tree with the phylogenetic tree. However, in the PB2 phylogenetic tree, Six viruses can be divided into two distinct evolutionary branches. CK/SD/SG2/2009 and CK/SD/WL/2009 are belong to CK/SH/Y2/2008-like lineage, the other four strains CK/SD/PD/2009, CK/SD/HY/2009, CK / SD/SG1/2009, CK/SD/FX/2009 are belong to CK/SH/F/98-like lineages. Therefore, six genotypes of the virus can be divided into two: CK/SD/SG2/2009, CK/SD/WL/2009 belong to a genotype, similar to CK/SH/Y2/2008-like pedigree; CK/SD/PD/2009, CK/SD/HY/2009, CK / SD/SG1/2009, CK/SD/FX/2009 are belone to the genotype whiche is reassortments of CK/SH/Y2/2008-like and CK/SH/F/98-like.To study the pathogenicity to mammals of six viruses, six-week-old-female BABL/c rats were inoculated with 10⁶EID₅₀ dosage of six viruses （in 50μL）, respectively. The results was that all the six viruses can replicate in the lungs of rats, and two of six viruses can cause rats to die at 8 days past inoculation（DPI）, which indicates that the virulence of H9N2 to rats have been increased. Therefore this study posed significance in human public health.Experimental chickens were immunized with the vaccine CK/SD/SG2/2009 and new vaccine, with CK/SD/SG2/2009 attack drugs. Two vaccines are protective. In this study, the six strains of H9N2 avian influenza viruses isolated from broilers in Shandong province are no significant difference in antigenicity, but the vaccine can not produce complete protection, and vaccine may not induce protective immunity completely relevant. Therefore, a reasonable choice of antigens targeted for vaccine strains is very important, it is best to choice the current strains in the same strain of the virus antigen as a vaccine strains. At the same time immune to the development of rational procedures, this can increase the antibody levels after immunization, better play the role of vaccine protection.更多还原