Systemic immune responses to an inactivated,whole H9N2 avian influenza virus vaccine using class B CpG oligonucleotides in chickens

Commercial vaccines against avian influenza viruses (AIV) in chickens consist mainly of inactivated AIV, requiring parenteral administration and co-delivery of an adjuvant. Limitations in T helper 1 or T helper 2 biased responses generated by these vaccines emphasize the need for alternative, more efficacious adjuvants. The Toll-like receptor (TLR) 21 ligand, CpG oligodeoxynucleotides (ODN), has been established as immunomodulatory in chickens. Therefore, the objective of this study was to investigate the adjuvant potential of high (20 μg) and low (2 μg) doses of CpG ODN 2007 (CpG 2007) and CpG ODN 1826 (CpG 1826) when administered to chickens with a formalin-inactivated H9N2 AIV. Antibody responses in sera were evaluated in 90 specific pathogen free (SPF) chickens after intramuscular administration of vaccine formulations at 7 and 21 days post-hatch. Antibody responses were assessed based on haemagglutination inhibition (HI) and virus neutralization (VN) assays; virus-specific IgM and IgY antibody responses were evaluated by ELISA. The results suggest that the vaccine formulation containing low dose CpG 2007 was significantly more effective at generating neutralizing (both HI and VN) responses than formulations with high or low doses of CpG 1826 or high dose CpG 2007. Neutralizing responses elicited by low dose CpG 2007 significantly exceeded those generated by a squalene-based adjuvanted vaccine formulation during peak responses. A significantly higher IgM response was elicited by the formulation containing low dose CpG 2007 compared to high and low doses of 1826. Although the low dose of CpG 2007 elicited a higher IgY response than CpG 1826, the difference was not statistically significant. In conclusion, 2 μg of CpG 2007 is potentially promising as a vaccine adjuvant when delivered intramuscularly with inactivated H9N2 virus to chickens. Future studies may be directed at determining the mucosal antibody responses to the same vaccine formulations.     

寡核苷酸芯片技术在甲型流感及登革病毒检测分型中的初步应用

目的 建立寡核苷酸芯片检测技术,对甲型流感病毒及登革病毒进行早期检测及分型.方法 根据Genebank中病毒基因组序列的保守区域,设计病毒的特异性检测探针,制备病毒检测芯片;利用限制性显示技术标记样品中的病毒靶序列,将标记产物与芯片杂交、清洗、扫描及结果分析.结果 该方法可以正确检测到细胞培养所获得的甲型(H1N1,H3N2)流感病毒和登革四型病毒.结论 寡核苷酸芯片技术可应用于多种病毒及其亚型的早期检测.     

流行性感冒病毒裂解疫苗甲醛含量的探讨

为了探索流行性感冒 (流感 )病毒裂解疫苗中灭活剂甲醛的最适浓度 ,单价病毒液采用不同浓度的甲醛进行灭活后 ,分别跟踪测定单价病毒液和配制的疫苗液中血凝素 (HA)含量的变化。结果显示 :用 0 0 1%甲醛灭活的单价病毒液和配制的疫苗液 ,HA含量下降明显 ;用 0 0 0 4 %甲醛灭活的单价病毒液和配制的疫苗液 ,HA含量则较为稳定。提示流感疫苗宜采用较低浓度的甲醛进行灭活。     

养阴生津方抗菌、抗病毒作用的实验研究

目的观察养阴生津方对金黄色葡萄球菌和流感病毒的药理作用.方法抗菌实验采用试管法、平板法;抗病毒实验采用体内法,以小鼠流感病毒FM1株滴鼻感染正常小鼠,胃饲药物水提取液,观察给药前后肺部病变的变化.结果养阴生津方的最低抑金黄色葡萄球菌浓度(MIC)为15.6mg/mL,最低杀菌浓度(MBD)为31.2mg/mL;养阴生津方对小鼠肺指数升高有显著抑制作用(P＜0.001),抑制率在20.38%～24.08%之间.结论养阴生津方具有抗金黄色葡萄球菌及抗流感病毒的作用.     

流感病毒RNA聚合酶亚基PA6片断的克隆及表达

目的将流感病毒RNA聚合酶PA6亚基片段进行亚克隆、表达,获得重组的亚克隆多肽,为进一步研究PA6亚基功能奠定基础。方法以PA亚基cDNA为模板,用PCR方法扩增出PA6片段,应用定向克隆策略将扩增后片段与高效表达载体pQE32重组,阳性克隆重组质粒经酶切及测序鉴定,IPTG诱导各亚克隆系高效表达,优化表达条件。结果PCR法扩增出402bp的片段,酶切及测序鉴定获得正确的重组质粒,在E.coliM15中表达得到相对分子量约为17KD的重组蛋白,获得重组多肽。结论成功地亚克隆及表达了流感病毒RNA聚合酶PA6亚基。     

Replication of respiratory viruses, particularly influenza virus, rhinovirus, and coronavirus in HuH7 hepatocarcinoma cell line

Detection of viral antigens and isolation methods has long been used for the diagnosis of respiratory virus infections. The objective was to determine the ability of HuH7 cells to support the replication of prototype and wild strains of respiratory viruses. The cell culture-adapted strains of influenza viruses A and B, parainfluenza viruses 1-4, respiratory syncytial viruses A and B, both strains of the human metapneumoviruses, numerous rhinoviruses, most of the adenoviruses, coronaviruses 229E and OC43, and a number of enteroviruses (poliovirus type 3, coxsackie virus B1, echovirus type 30) replicate in HuH7. The kinetic study of the replication of influenza A and B viruses showed that there were infected cells in HuH7 and MDCK lines as early as 24 hr post-infection. However, the replication of influenza A and B viruses was more rapid and intense on MDCK cells than on HuH7 cells. During the three winters of 1999-2000, 2000-2001, and 2001-2002, of the 1,226 (23.3%) direct fluorescent assay-positive nasal aspirates from children admitted to hospital, 788 were positive for respiratory syncytial virus, 228 for influenza virus, 133 for parainfluenza virus, and 77 for adenovirus. Of the 4,032 direct fluorescent assay-negative nasal aspirates, 571 virus isolates were identified by using HuH7 cell culture; 272 rhinoviruses, 100 influenza viruses A and B, 85 enteroviruses, 40 adenoviruses, 35 coronaviruses, 31 parainfluenza viruses, and 10 respiratory syncytial viruses. Interestingly, 100/328 (30.5%) influenza viruses A and B, 40/189 (21.1%) adenoviruses, and 31/164 (19%) parainfluenza viruses type 1-3, not detected by direct fluorescent assay, were identified by isolation in HuH7 cell culture.     

清肺饮在狗肾传代细胞中抗流感病毒活性的研究

目的研究清肺饮对A/PR/8/34(H1 N1)(甲型流感病毒)、B/汉防/194/95(乙型流感病毒)的预防、治疗作用.方法运用病毒的组织细胞培养技术,采用细胞病变(CPE)抑制试验、血凝滴度法(HA)、血球吸咐法(HD),研究清肺饮粗提物对流感病毒的直接作用、预防作用和流感病毒感染后的治疗作用.结果(1)对流感病毒的预防作用和直接灭活作用:清肺饮组半数组织培养感染(TCID50)比病毒对照组低2 Log以上,且与药物浓度呈正相关;(2)对狗肾传代细胞(MDCK细胞)感染流感病毒后的治疗作用,其TCID50比病毒对照组低1.5 Log以上.结论清肺饮具有直接抑制A/PR/8/34(H1 N1)、B/汉防/194/95的作用,且呈明显量效关系;对这两型病毒具有显著预防、治疗作用.     

成都某部一起感染Yamagata系乙型流感病毒血凝素基因分析

目的 分析成都某部感染乙型流感病毒遗传进化与血凝素（HA）基因突变位点。方法 通过犬肾上皮细胞（MDCK细胞）体外分离患者咽拭子标本流感病毒毒株,用PCR获取乙型流感病毒HA基因并测序,与NCBI数据库在线比对并利用MEGA 6.06软件构建系统发育进化树,分析突变位点。结果 通过MDCK细胞接种,分离出1株乙型流感病毒株,以感染病例咽拭子核酸及分离毒株的核酸为模板进行PCR扩增得到1 755 bp全长HA基因,获得的序列提交至GenBank数据库,获得基因登录号为MH236281。通过在线比对及系统发育树构建,该病例感染的病毒为Yamagata系乙型流感病毒。与乙型流感病毒Yamagata系的代表毒株Influenza B/Yamagata/16/88（GenBank No.M36105）相比,HA基因点突变碱基为57个;与世界卫生组织（WHO）推荐的疫苗株Influenza B/Utah/08/2014（GenBank No.KU592766）相比,突变碱基数为20个。进一步对HA1氨基酸突变位点进行分析,与四川地区往年流行株相比均发生了不同程度的突变,其中与2010年四川温江的分离株（GenBank No.KP461138）相比突变位点较少,仅有4处点突变;与WHO推荐的疫苗株Influenza B/Utah/08/2014相比,有2个氨基酸位点发生了变异,分别为L176Q和M255V,但突变没有处于HA1上的抗原决定簇区域。其中176位点是一个全新的突变,以往四川流行毒株、Yamagata系的代表毒株Influenza B/Yamagata/16/88及WHO推荐的疫苗株Influenza B/Utah/08/2014的HA1 176位点均为亮氨酸（L）,而本研究病例感染的乙型流感毒株HA1 176位点突变为谷氨酰胺（Q）。结论 成都地区驻地部队2017-2018流行季感染的乙型流感病毒HA基因已发生一些突变,但突变尚未造成抗原性的改变。     

H3N2猪流感病毒诱导的小鼠急性肺损伤与SOD、NO、MDA和OH·变化的相关性

目的: 探讨超氧化物歧化酶(SOD)活性以及一氧化氮(NO)、丙二醛(MDA)和羟自由基(OH·)含量的变化与H3N2猪流感病毒诱导的小鼠肺损伤的关系。方法: 选用6-8周龄、SPF级BALB/c小鼠80只,随机分为H3N2病毒感染急性肺损伤组和模拟感染对照组,每组各40只。在感染后的第3、5、7、10和14 d,每组处死小鼠6只,做如下处理:其中2只小鼠取肺组织进行HE染色,观察肺组织的病理学变化;剩余4只小鼠处死,收集血液分离血清;然后进行支气管肺泡灌洗,收集支气管肺泡灌洗液(BALF)。测BALF 和血清内SOD活性以及NO、MDA和OH·含量。结果: 病毒感染小鼠肺脏 组织学变化表现为以严重的肺泡间质水肿、炎性细胞渗出、出血为特征的弥漫性肺泡损伤;与模拟感染对照组相比,感染组BALF与血清内的NO、MDA和OH·的含量均明显增加,差异显著;感染组SOD活性与对照组相比显著下降。BALF内SOD、NO、MDA和OH·的变化幅度明显大于血清的变化。结论: 感染小鼠血清和BALF内NO、MDA和OH·显著升高,表明在H3N2猪流感病毒诱导的小鼠肺损伤过程中上述自由基可能发挥重要作用。     

纳米银对流感病毒H3N2的抑制作用

目的探讨纳米银（NaAg）对流感病毒H3N2的抑制作用。方法采用血球凝集试验、MTT分析法、血球吸附试验和鸡胚培养法,探讨NaAg对流感病毒H3N2的抑制作用。结果NaAg／H3N2组和H3N2对照组血球凝集试验效价分别为〈1：4和1：1024（P〈0．001）;NaAg溶液在MD-25细胞上最大无毒浓度为25μg／ml;H3N2在MD-25细胞上的细胞半数感染浓度（TCID50）为10^3.5／0．1ml。等体积的50μg/mlNaAg与40TCID50流感病毒H3N2溶液室温充分混和作用2h后感染MD-25细胞,细胞存活率为（96．77±2．07）％;20TCID。流感病毒H3N2感染MD-25细胞的细胞存活率为（33．09±1．48）％（P〈0．001）。结论NaAg对流感病毒H3N2具有明显的抑制作用。