2株鸭源 H11N9亚型禽流感病毒的基因遗传进化及致病性分析

为了解从湖南省洞庭湖区鸭群中分离的2株 H11N9亚型禽流感病毒变异特点、进化规律及生物学特性,本研究对2株H11N9亚型禽流感病毒的HA、NA序列进行同源性和遗传进化分析,并用2株毒株对SPF鸡进行致病性试验。结果显示,本试验分离到2株 H11N9亚型禽流感毒株的 HA裂解位点均没有多个连续的碱性氨基酸插入,属于低致病性毒株;HA基因的受体结合位点均非常保守,具有典型的禽源性特征;NA基因序列与在周边国家野鸟中分离的H11N9亚型毒株的氨基酸同源性较高;鼻腔接种SPF鸡后,均能使鸡感染并通过喉头或泄殖腔排毒,但感染的鸡均不表现明显的临床症状,并且不能使同居鸡感染排毒。     

悬浮培养MDCK细胞制备的H9亚型禽流感灭活疫苗免疫剂量试验

为研究由悬浮培养的MDCK细胞制备的H9亚型AIV抗原所制备的灭活疫苗的免疫剂量,在生物反应器中悬浮培养MDCK细胞,接种H9亚型禽流感病毒WD98和HN04种毒,收获抗原液,以2种抗原液分别制备2个毒株的单价油佐剂灭活疫苗。每种疫苗均按照0.3、0.2、0.1、0.05mL/只(107.5 EID50/只~105.52 EID50/只)的剂量接种21日龄SPF鸡,免疫接种后3周采血测定AIV的HI抗体,采血后分别用与制苗株同源的H9亚型AIV种毒静脉注射攻毒,测定疫苗保护率。2种疫苗不同剂量免疫接种后21d的AIV HI抗体水平达到3.8log2~5.6log2,对照组鸡AIV HI抗体均为0log2。攻毒试验证实,以105.52 EID50/只~107.5 EID50/只剂量进行免疫接种,免疫鸡只获得了80%以上的保护。按照兽药典H9亚型禽流感疫苗攻毒保护率90%为合格的标准,悬浮培养MDCK细胞制备的H9亚型禽流感疫苗的最小免疫接种剂量为106.82 EID50/只。     

一株野鸟源H9N2亚型禽流感病毒的全基因组序列分析及致病性研究

为进一步了解2014年分离自我国南方野鸟粪便中的一株H9N2亚型禽流感病毒(AIV)Wide Bird/Hu N/SC1400/2014(H9N2)(WB/400/14)的生物学特性,本研究对其进行全基因组序列测定、进化分析及SPF鸡、SPF鸭和BALB/c小鼠的感染性试验。序列分析显示:该分离株的HA裂解位点基序为333PAASDR↓GL340,其中不存在多个连续的碱性氨基酸,符合低致病性禽流感病毒(LPAIV)氨基酸序列特征。该分离株不同基因片段来源较复杂,分别与H9、H6、H4、H1、H11、H10、H3等多种亚型的LPAIV同源性较高,呈现明显的多样性。感染性试验显示,WB/400/14不能够在SPF鸡和小鼠体内有效复制,但病毒感染SPF鸭后能够在部分脏器中检测到病毒的存在,并且感染鸭能通够过咽喉和泄殖腔同时向外排毒,而同居感染鸭仅通过泄殖腔向外排毒,表明分离株在SPF鸭群中具有良好的水平传播能力。本研究为AIV的监测和防控提供实验依据。     

Pathogenicity of a Strain of H5N6 Subtype Avian Influenza Virus in Ducks and Mice

To investigate the pathogenicity of A/Hero/Guangdong/C1/2013(H5N6), an AIV strain isolated from Heron on ducks and mice, pathogenicity of this new virus strain and changing of histopathology as well as a preliminary study on its biological characteristics were studied by virus challenges test via intranasal and eye-drop and in chicken via jugular vein injection.Results showed that the EID₅₀ of this strain of virus was 10^-8.16/0.1 mL in embryonated chicken eggs and the intravenous inoculation of pathogenic index (IVPI) was 2.76.In ducks and mice, the 50% lethal doses (LD₅₀) of it were determined to be 10^-4.0/0.2 mL and 10^-4.67/0.05 mL, respectively.Symptoms of infection included loss of appetite, depression, swollen head and tears after being infected with 10⁶ EID₅₀ per duck by intranasal and eye-drop administration.Most of ducks died 4 to 7 days post-infection, liver, lung and kidney still eliminated toxicants at day 7 post-infection.Anatomy showed symptoms of pericardial effusion, pulmonary congestion and kidney enlargement, while pathological section showed pathological change like karyopycnosis and inflammatory cell infiltration in heart, liver, kidney and spleen.Mice developed symptoms of infection like loss of appetite, depression, shaggy coat and ruffled coat after being infected with 5×10⁵ EID₅₀ per mouse by intranasal and eye-drop administration.Most of the infected mice died 5 to 7 days post-infection and only liver still eliminated toxicants at day 7 post-infection.Although organ anatomy showed no obvious pathological changes, pathological section showed pathological changes like karyopycnosis and inflammatory cell infiltration in heart, kidney and lung.Our research demonstrated that this H5N6 subtype AIV had a strong pathogenicity and could be defined as a highly pathogenic AIV strain as its IVPI was greater than 1.2.Our work laid a theory foundation for study, prevention and control of H5N6 subtype AIV.     

The Expression of Long Noncoding RNA (H19) in Mouse Skeletal Muscle Development

This experiment was intend to study the changes of long non-coding RNA (H19) expression levels in skeletal muscle development and regeneration,and lay the foundation of its mechanism reach in skeletal muscle development.C2C12 cell line and ICR mice were used as experimental material,bioinformatics assay was used to exploit its non-coding character and low conservatism in different species,and the expressions of H19 in C2C12 cell differentiation,skeletal muscle development and the phase of muscle regeneration were detected by quantitative Real-time PCR (qRT-PCR).The results showed that,H19 expression levels in postnatal mouse skeletal muscle decreased with increasing age;during C2C12 cell differentiation,H19 mRNA increased gradually,and then maintained a high level;The expression of H19 was maintained at a high level through days 4 to 6 after injury.In consideration of its express character in C2C12 cell differentiation and skeletal muscle damage repair model,H19 may play an important role in promoting myogenesis and skeletal muscle regeneration.     

Isolation and Identification of one Strain of H9 Subtype Avian Influenza Virus and Study on its Biological Characteristics

In 2013,one case of suspected H9 subtype avian influenza occurred in a chicken farm of Jilin povince.Clinical samples were collected from the diseased farm,inoculated into the allantoic cavity of 9-day-old SPF chicken embryo,and then one strain of virus was isolated.The results of HA test,HI test and molecular biology test all showed that the isolate belonged to H9 subtype avian influenza virus (AIV).The HA cleavage site of the isolate was RSSR↓GLF,which was consisted with the molecular characteristic of low pathogenic AIV.The HA peptide chain had 9 potential glycosylation sites which were same as other isolates of recent years.The isolate had 8 receptor binding sites,including 234 receptor binding site by glutamine (Q) mutating into threonine (T).The phylogenetic tree revealed the isolate belonged to Eurasian lineages and it had far genetic relationship with the earliest domestic isolate (A/Chicken/Beijing/1/94(H9N2)),but had close genetic relationship with the representative strain (A/Chicken/Guangxi/55/2005(H9N2)) of major epidemic branch since 2007.We prepared an inactivated oil-emulsion vaccine of the isolate,and then vaccinated SPF chicken.21 days after vaccination,the HI titer of chicken serum antibody reached up to 10log2.The result suggested the isolate had good immunogenicity.     

禽流感疾病防治疫苗发展现状

高致病性禽流感最近在我周边国家及我国部分地区暴发流行,引致养禽业巨大经济损失,禽流感病毒血清型众多、不同毒株间毒力差异大,而且低致病性毒株能突变为高致病性毒株,给其防制带来了巨大困难。目前,与其他病毒性疾病相同,禽流感的防制尚无特别有效的方法,接种疫苗是预防禽流感发生与传播的最有效手段。随着禽流感病毒多种亚型的发现,以及基础免疫学理论、分子生物学及生物技术的发展,科研人员已研发出了针对禽流感的数种疫苗。除了应用较为普遍的全病毒灭活疫苗外,对多种新型疫苗的研发也有了较大的进展。     

融水香鸭禽流感母源抗体的消长变化研究

为确定融水香鸭禽流感疫苗的首免日龄,本试验对融水香鸭雏鸭进行母源抗体的检测,对其母源抗体的消长变化进行研究。结果显示,融水香鸭雏鸭母源抗体水平较高,抗体持续期较长,在1日龄即达最高值,随着日龄的增加而下降,至13日龄时效价降低到3.9 log2,其母源抗体达标率为70%,至17日龄时仍维持在3.9 log2,抗体保护合格率下降到了60%,至21日龄时降低到3.5log2,抗体合格率下降到了40%。根据融水香鸭禽流感母源抗体的消长规律,建议雏鸭的首免接种时间为13~17日龄,最佳的首免日龄为13日龄。     

某牛场粪源大肠杆菌0157∶H7的分离鉴定

为了解重庆市某牛场牛群大肠杆菌O157∶H7的感染情况,我们以牛场犊牛为研究对象,采集其新鲜粪便,经LB培养基增菌培养、免疫磁珠富集后,在山梨醇麦康凯琼脂上划线培养,最后PCR扩增eaeA基因目的片段,以此分离鉴定粪源大肠杆菌O157∶H7。结果显示：在所采集的39份犊牛粪便样品中,分离得到一株符合大肠杆菌O157∶H7生长特点且含有eaeA基因的菌株。     

PA-X基因的长度变化对H3N2犬流感病毒复制能力和致病性的影响

H3N2犬流感病毒(canine influenza virus, CIV)已在中国多地的犬群中流行,是禽流感跨宿主感染并形成新分支的近期案例。研究表明,PA-X基因与甲型流感病毒适应新宿主的能力相关,且其长度能够影响甲型流感病毒的复制及致病能力。为了解PA-X基因的长度变化对H3N2 CIV复制能力及致病力的影响,本研究利用H3N2 CIV的8质粒操作系统,拯救了三株重组H3N2 CIV毒株：PA-X基因表达大小为232个氨基酸多肽的亲本病毒CIV＿PA-X＿232;对PA编码区第191、192位氨基酸的密码子进行改造,PA-X基因不表达蛋白的重组病毒CIV＿PA-X＿Knock;对PA+1编码区第232位氨基酸进行突变,PA-X基因表达大小为252个氨基酸多肽的重组病毒CIV＿PA-X＿252。通过比较3株重组病毒的聚合酶活性,在MDCK细胞中的复制效率及对小鼠致病性的差异,来评价表达不同长度的PA-X基因对H3N2 CIV的影响。结果显示,CIV＿PA-X＿252和CIV＿PA-X＿Knock的聚合酶活性显著(P<0.05)高于CIV＿PA-X＿232,且CIV＿PA-X＿...