以金磁微球为载体的H5N1 AIV免疫PCR检测方法的建立

【目的】将高特异性的抗原-抗体反应与高灵敏性的PCR扩增技术相结合,建立快速检测低浓度H5N1禽流感病毒(Avianinfluenzavirus,AIV)的免疫PCR方法。【方法】以pUC19为模板,采用5′端标记有生物素的引物进行PCR扩增,制备含有生物素的报告DNA分子。以金磁微球为固相吸附载体,通过亲和素-生物素的桥联作用,将含有生物素的报告DNA分子与生物素标记的抗AIVH5N1血凝素蛋白的检测抗体分子连接,H5N1AIV与检测抗体结合后,体外扩增报告DNA,间接放大低含量病毒信号,建立可有效检测微量H5N1AIV的免疫PCR方法。对建立的免疫PCR方法的最适报告DNA浓度、最适链亲和素工作浓度、灵敏度和特异性进行确定和评价。【结果】建立的免疫PCR方法最适报告DNA质量浓度为1ng/mL,最适链亲和素工作质量浓度为20ng/mL,该方法可成功检测到10-4EID50/mL的H5N1AIV,而且特异性良好。【结论】建立的以金磁微球为吸附载体的免疫PCR是一种灵敏度高、特异性好的检测微量H5N1AIV的方法。     

禽流感病毒表面抗原HA基因转化小麦的初步研究

为了建立利用小麦生产禽流感口服疫苗的方法,选择禽流感病毒H5N1亚型表面抗原HA基因,与谷类作物启动子构建小麦表达载体,经基因枪法转入我国小麦栽培品种郑9023幼胚,在含5 mg/L草铵膦愈伤诱导培养基和分化培养基及含4 mg/L草铵膦生根培养基上筛选后,PCR鉴定获得转HA基因植株,Southern杂交分析转基因T1...     

3种检测方法在野鸟禽流感监测中的应用与比较

分别应用血凝抑制试验(HI)、反转录聚合酶链式反应(RT-PCR)与病毒分离3种检测方法对260份野鸟样品开展禽流感检测。结果表明,用HI方法检出禽流感样品57份,阳性检出率为21.92%;RT-PCR方法没有检出禽流感样品,阳性检出率为0;病毒分离方法检出禽流感样品15份,阳性检出率为5.77%。     

Prevalence of Influenza A Virus in Exhibition Swine during Arrival at Agricultural Fairs

The exhibition swine at agricultural fairs provides a critical human–swine interface that allows for the bidirectional transmission of influenza A virus (IAV). Previous IAV surveillance at the end of fairs has resulted in frequent detection of IAV‐infected swine; little is known, however, about the frequency with which swine arrive at fairs already infected with IAV. We investigated the IAV prevalence among exhibition swine entering fairs to better understand the epidemiology of IAV in this unique human–swine interface. In 2014, snout wipes were collected from 3547 swine during the first day of nine agricultural exhibitions in Indiana and Ohio. Samples were screened for IAV using rRT‐PCR and positive samples were inoculated into cultured cells for virus isolation. The overall IAV prevalence detected among swine arriving at exhibitions was 5.3% (188/3547) via rRT‐PCR and 1.5% (53/3547) via virus isolation, with IAV being detected and recovered from swine at 5 of the 9 exhibitions. Within the fairs with IAV‐positive swine, the individual exhibition IAV prevalence ranged from 0.2% (1/523) to 34.4% (144/419) using rRT‐PCR and 0.2% (1/523) to 10.3% (43/419) with virus isolation. Single IAV subtypes were detected at three of the fairs but subtype diversity was detected among the pigs at two fairs as both H1N1 and H3N2 were recovered from incoming swine. At two of the exhibitions, a temporal relationship was observed between the order of the individual swine in sampling and the associated IAV rRT‐PCR results, indicating the fomite transmission of IAV through common contact surfaces may occur. With the knowledge that a small proportion of swine arrive at fairs shedding IAV, resources should be directed towards preventive strategies focused on limiting transmission during fairs to protect swine and humans during exhibitions.     

马流感病毒血凝素基因甲病毒复制子重组表达质粒的构建及其免疫效力评价

为评价马流感病毒(EIV)HA基因核酸免疫效果,本研究以甲病毒复制子载体pSFV1CS分别构建了表达EIV H3N8亚型的美洲型和欧洲型HA基因的重组真核表达质粒。并将其转染293T细胞,经间接免疫荧光鉴定表明HA基因获得表达;以重组质粒免疫的BALB/c鼠能够检测到特异性抗体产生,而且HI抗体水平持续升高,同时小鼠体内IFN-γ、IL-4分泌水平也有所升高。攻毒后小鼠表现轻度临床症状,但病毒分离和RT-PCR均未检测到病毒。上述结果表明,该重组质粒pSFV1CS-EIV-HA具有良好的免疫原性并且可以诱导免疫动物产生较高免疫应答的能力。     

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＿...     

Protective efficacy of an H5/H7 trivalent inactivated vaccine produced from Re-11, Re-12, and H7-Re2 strains against challenge with different H5 and H7 viruses in chickens

We developed an H5/H7 trivalent inactivated vaccine by using Re-11, Re-12, and H7-Re2 vaccine seed viruses, which were generated by reverse genetics and derived their HA genes from A/duck/Guizhou/S4184/2017(H5 N6)(DK/GZ/S4184/17)(a clade 2.3.4.4 d virus), A/chicken/Liaoning/SD007/2017(H5 N1)(CK/LN/SD007/17)(a clade 2.3.2.1 d virus), and A/chicken/Guangxi/SD098/2017(H7 N9)(CK/GX/SD098/17), respectively. The protective efficacy of this novel vaccine and that of the recently used H5/H7 bivalent inactivated vaccine against different H5 and H7 N9 viruses was evaluated in chickens. We found that the H5/H7 bivalent vaccine provided solid protection against the H7 N9 virus CK/GX/SD098/17, but only 50–60% protection against different H5 viruses. In contrast, the novel H5/H7 trivalent vaccine provided complete protection against the H5 and H7 viruses tested. Our study underscores the importance of timely updating of vaccines for avian influenza control.     

H5N1亚型高致病性禽流感病毒A/goose/Guangdong/1/96株反向基因操作系统的建立

 A/goose/Guangdong/1/96（GSGD/1/96）是中国分离的第1株H5N1亚型禽流感病毒，它不仅是97香港感染并致人死亡的H5N1亚型流感病毒HA基因供体株，而且是中国目前已报到的H5亚型流感病毒分离株的共同祖先。本研究建立了该病毒的8质粒反向基因操作系统，并通过细胞转染成功拯救了该病毒（R-GSGD/1/96）。R-GSGD/1/96在对SPF鸡和 Balb/c小鼠的致病性方面保持了与亲本野毒(W-GSGD/1/96)一致的生物学特性，即对鸡都是高致病性毒株，R-GSGD/1/96与W-GSGD/1/96的静脉致病指数分别为2.01和2.10；救获病毒与野生病毒一样，尽管106EID50经鼻腔感染小鼠后1～2 d内能从肺脏检测到低滴度的病毒，但不能在小鼠体内成功复制。GSGD/1/96反向基因操作系统的成功建立为进一步开展中国高致病性禽流感病毒分离株的生物学特性、遗传衍化及结构与其功能关系研究奠定了基础。     

Suppression of influenza virus infection by the orf virus isolated in Taiwan

Orf virus (ORFV), a member of parapoxvirus, is an enveloped virus with genome of double-stranded DNA. ORFV causes contagious pustular dermatitis or contagious ecthyma in sheep and goats worldwide. In general, detection of viral DNA and observing ORFV virion in tissues of afflicted animals are two methods commonly used for diagnosis of orf infection; however, isolation of the ORFV in cell culture using virus-containing tissue as inoculum is known to be difficult. In this work, the ORFV (Hoping strain) isolated in central Taiwan was successfully grown in cell culture. We further examined the biochemical characteristic of our isolate, including viral genotyping, viral mRNA and protein expression. By electron microscopy, one unique form of viral particle from ORFV infected cellular lysate was demonstrated in the negative-stained field. Moreover, immunomodulating and anti-influenza virus properties of this ORFV were investigated. ORFV stimulated human monocytes (THP-1) secreting proinflammatory cytokines IL-8 and TNF-α. And, pre-treatment of ORFV-infected cell medium prevents A549 cells from subsequent type A influenza virus (IAV) infection. Similarly, mice infected with ORFV via both intramuscular and subcutaneous routes at two days prior to IAV infection significantly decreased the replication of IAV. In summary, the results of a current study indicated our Hoping strain harbors the immune modulator property; with such a bio-adjuvanticity, we further proved that pre-exposure of ORFV protects animals from subsequent IAV infection.     

H9N2禽流感病毒全基因组密码子使用偏好性及影响因素分析

【目的】探究H9N2禽流感病毒(AIV)全基因组的密码子使用偏好性及影响因素。【方法】选取2010—2018年国内H9N2 AIV流行毒株的全基因组为研究对象,分析其碱基组成特性、最优密码子、密码子使用偏好性的影响因素以及病毒对宿主密码子使用模式的适应性。【结果】H9N2 AIV的全基因组中AU含量高于GC。大部分最优密码子以A或U结尾,有效密码子数(ENC)平均值为52.86,提示存在密码子使用偏好性但偏好性较低。密码子使用偏好性主要受到突变压力和自然选择的共同作用,其中自然选择(所占比例为61.79%～76.15%)作用大于突变压力(所占比例为23.85%～38.21%)。H9N2 AIV对人Homo sapiens的密码子适应指数平均值为0.739～0.741,提示H9N2AIV禽流感病毒可能已适应人类的密码子使用模式。【结论】本研究为H9N2 AIV的基因进化分析、已有疫苗的密码子优化和新型疫苗(密码子去优化疫苗)研制提供了理论依据。