【作者】 徐大伟；

【导师】 李云章； 李泽君；

【作者基本信息】 内蒙古农业大学 ， 临床兽医学， 2012， 博士

【摘要】 H9N2亚型禽流感病毒在全世界流行广泛,危害巨大。通常该亚型禽流感病毒为低致病性,感染禽类后不会引起大批死亡。但最近发现感染该亚型病毒的禽类死亡率很高,应用多聚酶链式反应(PCR)方法诊断,发现感染本病禽类脏器中同时存在H9N2亚型禽流感病毒和新发现的鸭坦布苏病毒。基于此问题,本研究分别对H9N2亚型禽流感病毒和鸭坦布苏病毒的生物学特性进行了深入研究,并对预防这两种病毒的疫苗进行了初步探讨研究。为掌握我国H9N2亚型禽流感病毒流行株的生物学特性,本研究选取2003-2010年分离到的27株病毒进行了系统深入的研究。遗传演化分析发现我国H9N2亚型禽流感病毒的遗传演化复杂,部分毒株PA和NS基因与H9N2亚型禽流感病毒参考序列的同源性均不高,表明这些基因可能来源于其它亚型的流感毒株,序列分析发现H9N2亚型禽流感病毒近期分离株的基因片段主要起源于Ck/Shanghai/F/98(H9N2)株。交叉血凝抑制实验及病毒中和实验表明近年来我国H9N2亚型禽流感病毒的抗原性发生了明显的改变。对BALB/c小鼠致病性实验说明我国H9N2亚型禽流感病毒对小鼠的致病能力呈现下降趋势,尤其是2006年后的病毒分离株在小鼠体内的复制能力明显低于早期分离株。挑取处在不同进化分支的4株H9N2亚型禽流感病毒对4周龄SPF鸡进行了鼻腔接种致病性实验,发现这些毒株主要在感染鸡的气管和肺脏中增殖,但不出现明显症状。根据上述抗原性分析结果,从H9N2亚型禽流感病毒分离株中筛选出一株病毒Ck/Shanghai/441/09(H9N2)(简称Ck441)进行了初步的疫苗(免疫学)研究。Ck441病毒尿囊液灭活后制备灭活(毒株)疫苗,免疫4周龄SPF鸡,免疫3周后,用Ck441和一株2011年H9N2亚型禽流感病毒分离株进行攻毒实验,发现免疫组鸡均不发病、不排毒,而对照组鸡100%排毒,表明Ck441是一株理想的疫苗候选株。为提高病毒株的在细胞上的增殖能力,把Ck441的HA和NA插入到pBD载体SapI酶切位点之间,分别拯救获得了4株重组病毒,通过鸡胚和细胞增殖实验,发现441HANA/PR8-mutNS在鸡胚和细胞上增殖滴度都很高,将该株病毒经鸡胚和细胞扩增、灭活后制备灭活疫苗并免疫SPF鸡,都能够达到100%的保护率。鸭坦布苏病毒是2010年4月以来引起鸭产蛋量下降、生长阻滞甚至死亡的新发鸭传染性疾病的病原,为获得此病毒对哺乳动物和鸡的致病信息,将鸭坦布苏病毒奉贤分离株(FX2010)人工感染BALB/c小鼠和SPF鸡。经肌肉和腹腔注射接种小鼠,各组织脏器均没检测到病毒；经鼻腔接种小鼠,FX2010病毒可在BALB/c小鼠的肺脏中复制增殖；脑内接种,BALB/c小鼠出现明显的临床症状,被毛逆立,体重下降,在小鼠的脑和肺脏中均可分离到病毒,随着接种病毒量的增加,小鼠体重下降更加明显；经8d后,脑内病毒含量仍然很高,而肺脏中的病毒含量明显降低。FX2010经肌肉和鼻腔接种2周龄SPF鸡,经鼻腔接种鸡体重增长明显减缓,肌肉接种体重变化与对照组无明显差异。病毒分离发现,经鼻腔和肌肉接毒的鸡在多个脏器均可分离到病毒；FX2010病毒经鼻腔接SPF产蛋鸡,实验组鸡排出绿色粪便,产蛋量呈现一过性下降,部分脏器能够检测到病毒。在感染鸡的整个实验过程中,未见鸡严重发病或死亡,表明该病毒对鸡呈现低致病性。为初步研制防控鸭坦布苏病毒病的DNA疫苗,将鸭坦布苏病毒E基因和密码子优化后E基因分别插入到真核表达载体pCAGGS,通过IFA和Western blot实验,证明两种重组质粒在真核细胞内都能表达具有免疫原性的E蛋白,而优化后的重组质粒较野生型的表达量更高。重组疫苗(毒株)质粒免疫小鼠后,用间接ELISA检测两种DNA疫苗免疫小鼠的血清抗体,发现密码子优化后E基因的质粒激发的抗体效价高于未优化质粒,该试验为DNA疫苗防治鸭坦布苏病毒病提供了初步研究数据。总之,本研究阐明了H9N2亚型禽流感病毒和鸭坦布苏病毒的部分生物学特性,初步研制了H9N2亚型禽流感的灭活疫苗和鸭坦布苏病毒病的DNA疫苗质粒。这些研究为H9N2亚型禽流感病毒病和鸭坦布苏病毒病的防治提供坚实的理论依据。更多还原

【Abstract】 H9N2avian influenza virus (AIV) was widely spread worldwide, and caused great losses to poultry industry. Normally, H9N2AIV was low pathogenicity to poultry, and can’t cause death, but in the last few years, we found H9N2AIV was involved in the epidemic with poultry death. Using polymerse chain reaction (PCR), both H9N2AIV and Tembusu virus were identified in the lungs of dead ducks. So in this study we focused on biological characteristics of H9N2AIV and Tembusu viruses separately. In addition, we developed primarily the vaccines against the diseases caused by these two viruses.In order to know the biological characteristics of H9N2AIV,27H9N2AIVs isolated during2003to2010were selected. The genetic evolution analysis reveal the viruses posed multiple genotypes and diversity of the biological properties, PA and NS genes may recombine from other influenza virus subtypes, at the same time, we also found Ck/Shanghai/F/98(H9N2) is prevalent gene donator. The cross HI test and neutralization test indicated that the antigenic drift happened in the H9N2AIVs circulated in China. In the mouse experiments, the H9N2AIVs show a decreasing trend of pathogenicity on mice. After inoculated intranasally four-week-old SPF chicken with H9N2viruses, the viruses mainly amplified in tracheas and lungs.Based on the antigenic analysis result, Ck/Shanghai/441/09(H9N2)(abbr. Ck441) was selected as candidate strain for vaccine developement. Four-week-old SPF chickens were injected intramuscularly with inactivated vaccine, and then challenged with Ck441and a recent H9N2AIV isolates. the vaccinated chickens didn’t shed H9N2AIVs after challenge, while100%negative control chickens shed the viruses. In order to obtain high titer of H9N2virus in cell culture, the HA and NA genes of Ck441were inserted into pBD vector, and several reassortant viruses were rescure based on12plasmids system.441HANA/PR8-mutNS was selected as vaccine candidate for its high titers in both embryonated eggs and MDCK cell (HA-2) culture. The inactive vaccine based on the allantoic fluid or the cell culture supernatants containing441HANA/PR8-mutNS provided complete protection against H9N2AIV challenge.Duck Tembusu virus was a newly emerged virus which caused egg production decline、 retarded growth, and even death among ducks. In order to know whether the virus could infect chickens or mammalians, BALB/c mouse and SPF chicken were infected artificially with Tembusu virus Fengxian2010isolate (FX2010). No virus replication was detected in the organs of the mice injected FX2010intramuscular or intraperitoneal injection. Virus replication was detected in the lungs and nasal tissue of the mice injected----FX2010intranasally. When inoculated intracerebral with FX2010, the mice got sick and lost their weight significantly, the virus replication was detected in the brains and lungs. The more viruses inoculated, the more serious the symptoms appeared. Moreover, although the virus titers decreased significantly in lungs8days after mice inoculated intracerebral, the virus titers were still high in brains. Two-week-old SPF chickens were injected with FX2010intramuscularly and intranasally. The chickens injected intranasally grew slowly, while the chickens injected intramuscularly grew normally when compared with the control chickens. When intranasally inoculated with FX2010, the laying hens excreted green feces, and egg production declined. No chicken died, indicated that FX2010was low pathogenic to chicken.To develop a DNA vaccine against duck Tembusu disease, recombinant plasmids pCAGGS-E and pCAGGS-OptiE were constructed by inserting E gene and coden-optimized E genes into pCAGGS vector. E protein was expressed in293T cells transfected with pCAGGS-E and pCAGGS-OptiE and proved by IFA and Western blot assay. The pCAGGS-OptiE expressed more E proteins than pCAGGS-E in transfected293T cells. Both pCAGGS-OptiE and pCAGGS-E could stimulate mouse immuno-system to produce specific antibodies against duck Tembusu virus, although pCAGGS-OptiE was more efficiency than pCAGGS-E. This experiment provided primary dates for DNA vaccine against the disease caused by duck Tembusu virus.In conclusion, this study we described partial characteristics of H9N2AIVs and duck Tembusu virus, and developed primary vaccines against the diseases caused by H9N2AIVs and duck Tembusu virus. The results would be helpful in the prevention the two diseases.更多还原