甲型H1N1流感病毒疫苗株高适应性MDCK细胞的筛选及鉴定

目的筛选甲型H1N1流感病毒疫苗株高适应的MDCK单克隆细胞株,用于培养生产流感病毒疫苗,为细胞代替鸡胚生产制备流感病毒疫苗提供保证。方法通过有限稀释法将MDCK细胞进行单克隆化,扩大培养建立单克隆化细胞库,通过血凝和TCID50筛选甲型H1N1流感病毒疫苗株的高适应性单克隆化细胞株,并鉴定所获得的细胞株细胞表面NeuAcα2,6 Gal的丰度。结果共制备了97株单克隆化MDCK细胞,经过甲型H1N1流感病毒疫苗株的筛选,共筛选到2株高适应甲型H1N1流感病毒疫苗株的MDCK单克隆化细胞株,其TCID50分别为6.68 log10 TCID50/ml和6.77 log10 TCID50/ml,其表面NeuAcα2,6 Gal的丰度明显提高。结论成功培养了MDCK单克隆细胞株,经筛选获得的单克隆细胞株其血凝滴度,TCID50都比普通MDCK细胞有明显提高,为细胞培养生产流感病毒疫苗奠定了基础。     

2010年广州市甲型H1N1流感病毒分离株HA基因变异分析

目的比较2010年广州市分离到的甲型H1N1流感病毒血凝素（HA）基因和2009年中国大陆甲型H1N1流感病毒HA基因的变异情况,为甲型H1N1流感的监测和防控提供理论依据。方法收集2010年广州市有发热和呼吸道症状病人的咽拭子标本,用H1N1流感特异性引物进行PCR检测,扩增分离到的H1N1病毒HA片段,测序后与2009年的H1N1毒株进行比对和分析,并用生物信息学方法对抗原位点和糖基化位点进行分析。结果共收集到426份标本,甲型流感阳性211份,其中H1N1流感4株,与2009年分离的甲型H1N1流感相比,有12个氨基酸碱基位点发生了有意义突变,其中6个位点位于抗原位点上;4株毒株HA基因145位氨基酸都发生了变异;其中2株毒株在第180位氨基酸位点的抗原位点发生了变异。进化分析表明4株毒株与2009年中国大陆分离的8株毒株进化关系较远。结论 2010年广州市甲型H1N1毒株与2009年相比发生了较大变异。HA基因145位和180位氨基酸位点变异对H1N1毒株抗原变异有重要意义。本文分离的A/Guangdong/ZS03/2010（H1N1）和A/Guangdong/ZS01/2010（H1N1）毒株可能已经发生了抗原性漂移。     

抗禽流感(H5N1)免疫球蛋白制备及体外抑制甲型流感病毒FM1株的试验研究

目的:制备抗禽流感(H5N1)卵划黄免疫球蛋白(IgY),探讨其体外阻断甲型流感病毒FM1株的作用.方法:对经过禽流感疫苗免疫后产生并富集在卵黄中的卵黄免疫球蛋白进行提纯,用辛酸-硫酸氨二步盐析-凝胶色谱法进行纯化,利用细胞病变(CPE)抑制实验,在狗肾细胞系(MDCK)检测抗禽流感(H5N1)免疫球蛋白无毒浓度(TD0)范围及阻断甲型流感病毒FM1株攻击MDCK细胞的最小阻断浓度.结果:抗禽流感免疫球蛋白对DMCK的无毒浓度为1.764 mg/ml,当浓度为0.082 8mg/ml对流感病毒仍有阻断作用.结论:利用辛酸-硫酸氨二步盐析-凝胶色谱法成功制备卵黄免疫球蛋白,抗禽流感(H5N1)免疫球蛋白体外对流感病毒有较强的抑制作用.     

甲1型流感病毒新分离株HA基因的序列分析

目的 研究新分离的H1N1亚型流感病毒株的HA1基因序列。方法 甲型流感病毒通过鸡胚增殖后提取RNA、逆转录合成cDNA,经PCR扩增和产物纯化构建重组质粒,用双脱氧链终止法进行核苷酸序列测定;并进行基因特性分析。结果 新分离到的3株流感病毒株（H1N1）HA1区基因长度为981bp,编码327个氨基酸;与A/桂防/10/94和A/Bayern/07/95（H1N1）标准株比较其同源性分别为92.8%和91.3%,丢失了第130位氨基酸和304位糖基化位点;新分离的3株甲型流感病毒（H1地）标准株比较其同源性分别为92.8%和91.3%,丢失了第130位氨基酸和304位糖基化位点,新分离的3株甲型流感病毒株（H1N1）HA1区氨基酸同源性高达98%;A/桂防/10/94和A/Bayern/07/95(H1N1)毒株HN1氨基酸的同源性高达96%。结论 新分离到的3株H1N1毒株HA编码氨基酸不同于A/Baydrn/07/95(H1N1)和A/桂防/10/94（H1N1）标准株,它们可能为新的甲型流感病毒变异性。     

惠州市流感病原学监测结果分析

目的对我市流感样病例标本进行病原学检测,分析其病原学特点,为流感防控提供病原学依据。方法采用MDCK细胞（狗肾细胞）培养法分离流感病毒,用血凝抑制试验对病毒株进行分型鉴定。结果从哨点医院采集的520份标本中共分离出流感病毒4l株,分离率为7．9％,以新甲型H1N1为主。其中新甲型H1N126株（63．4％）,H3N2型5株（12．2％）,B（Yamagata）型1株（2．4％）．B（Victoria）型9株（22．0％）。流感流行高峰出现在春季的1—3月和秋季的8-9月。健康人群血清中流感抗体的阳性率不高,最高为新甲型H1N1抗体阳性率41．9％,最低为B（Yamagata）的抗体阳性率,仅8．1％。对2010年2株新甲型H1N1进行基因测序,结果显示甲型H1N1基因未发生变异,暂时不会造成大的流行。结论惠州市流感病毒的流行时间有明显的季节性,活动相对平缓,新甲型H1N1流感病毒是春季的优势毒株,下半年逐渐转变为H3N2型流感病毒。     

两质粒系统重配甲型H1N1流感病毒株的研究

目的构建两质粒冷适应流感病毒拯救系统,提高流感病毒的重配效率。方法本研究通过构建克隆载体pfastbac△plh HTB,将流感病毒A/Ann Arbor/6/60(H2N2)6个内部基因(PB2、PB1、PA、NP、M、NS)的双向转录表达元件定向克隆入此载体,获得pfastbac△plh HTBP6。同时将流感病毒A/California/7/2009(H1N1)表面基因(HA、NA)双向转录表达元件定向克隆入p ENTR-1A质粒,获得p ENTR-1AP2。将2个重组质粒共转染MDCK/COSI细胞,细胞上清接种10日龄SPF鸡胚,收取尿囊液,血凝实验筛选阳性株,并进行全面鉴定。结果本研究利用两质粒系统成功重配甲型H1N1流感病毒株,命名为TRG A/California/07/2009(H1N1)Vca,重配病毒株传代后HA效价为2~8,形态符合流感病毒典型特征,大小80~120 nm,具有冷适应、温度敏感表型。结论两质粒流感病毒拯救系统为高效重配流感疫苗株提供了新策略。     

流感病毒致小鼠急性肺损伤模型建立及利巴韦林干预作用研究

目的 建立A/FM/1/47/(H1N1)流感病毒感染诱导的小鼠急性肺损伤模型,并使用利巴韦林对其进行治疗,观察其保护机制.方法 将30只13-15g的昆明(KM)小鼠随机分为三组(正常组、模型组和利巴韦林组),每组10只,模型组与利巴韦林组采用H1N1流感病毒经鼻腔接种,利巴韦林组小鼠配以药物治疗,定时称量各组小鼠的体质量、观察记录小鼠存活状态,连续观察15d;另取36只13-15g的KM小鼠,如上随机分为三组,每组12只,模型组与利巴韦林组采用H1N1流感病毒经鼻腔接种,利巴韦林组小鼠配以药物治疗,感染后第6d测量肺系数、肺湿/干重比、观察肺病理组织学变化、动脉血气分析、血清细胞因子含量和肺部病毒载量.结果 实验结果显示,流感病毒滴鼻感染可诱发小鼠病毒性肺损伤.利巴韦林可延长感染小鼠生存时间,降低肺水肿,改善低氧血症,抑制炎性细胞的分泌,抑制病毒在体内的复制.结论 本研究利用流感病毒感染小鼠的病毒性肺损伤,分析了利巴韦林对流感病毒诱发的病毒性肺损伤的保护作用.该模型对进一步开发抑制流感病毒性肺损伤的新药有重要意义.     

2009年泉州市H1N1流感监测及基因特性分析

目的 了解2009年泉州地区H1N1流感监测情况,分析泉州市H1N1流感病毒的HA和NA基因特征,探讨该病毒的遗传变异及分子特性.方法 对泉州市H1N1流感监测期间的病人咽拭子采用real-time RT-PCR方法检测病毒核酸,MDCK细胞培养进行病毒分离、鉴定,并提取其中2株代表性毒株病毒RNA;采用RT-PCR扩增病毒HA和NA基因,纯化产物进行核苷酸序列测定;用DNAStar Megalign软件进行序列分析.结果 1020份咽拭子中有200份为H1N1流感病毒核酸阳性,70份季节性流感病毒核酸阳性,其中53份为H3N2亚型,14份为H1N1亚型,3份为B型,并分离到29株甲型H1N1流感病毒株.HA基因经核苷酸序列测定显示,该毒株与北美流行株高度同源,由HA基因核苷酸序列推导的氨基酸系列与疫苗株A/Brisbane/59/2007相比,有22个位于抗原决定簇的氨基酸位点发生变异,但受体结合特异性仍为人样受体.NA基因耐药性位点分析,显示对达菲药物依然敏感.结论 2009年泉州市H1N1流感流行毒株与北美流行株高度同源,相对于疫苗代表株出现了HA蛋白抗原性的改变.     

2009年泉州市流感监测结果分析

目的了解泉州市2009年流感活动情况及其型别特征。结论通过流行病学和病原学监测,对泉州市流感流行情况进行分析。结果 2009年全市流感样病例占就诊人数的0.26%～2.34%;全年采集监测标本1392份,经RT-PCR法检测样本阳性率为22.34%;经MDCK细胞分离流感毒株134株,其中季节性H3N2和HINl型各占60.45%和11.19%,甲型H1N1流感占21.64%,B型占6.70%。结论 2009年泉州市流感春夏季以季节性H3N2亚型流感为优势毒株,秋冬季以甲型H1N1流感为流行株,需加强泉州市流感监测和流感病毒抗原变异研究。     

2004-2008年我国流行的A型流感病毒抗原性及HA1基因特性的研究

目的 了解我国2004-2008年A(H1N1、H3N2)型流感病毒流行情况、抗原性和基因特性变异关系,了解疫苗株与我国流行株之间抗原性变化情况.方法 选择2004年以来我国分离的A(H1N1、H3N2)型流感病毒进行抗原性及HA1区基因序列,通过比对HA1蛋白位点变异情况,分析我国流感病毒抗原性及基因特性变化情况.结果 A(H1N1)亚型流感毒株抗原性2004-2007年分离的A(H1N1)亚型流感病毒的抗原性与疫苗株A/New Caledonia/20/1999(H1N1)类似;2008年我国流行的A(H1N1)亚型毒株的抗原性与2008-2009年北半球的流感疫苗株A/Brisben/59/2007(H1N1)类似.2004-2005年分离的A(H3N2)亚型流感病毒的抗原性与疫苗株A/Fujian/411/12002(H3N2)比较发生了变异;2006-2007年我国流行的H3N2毒株与A/Wiscansin/67/2006(H3N2)类似,2008年我国流行的H3N2毒株与疫苗株A/Brisben/10/2006(H3N2)类似.结论 2004-2008年我国流行的A(H1N1、H3N2)亚型流感病毒的抗原性和基因特性发生了改变.     

High prevalence of amantadine-resistant influenza A virus isolated in Gyeonggi Province, South Korea, during 2005–2010

Amantadine resistance among influenza A viruses was investigated in South Korea in 2005–2010. Of 308 influenza A viruses examined, 229 had the S31N substitution in the M2 protein. The frequency of amantadine resistance was 30 %, 100 %, and 76 % in influenza A/H1N1, pandemic A/H1N1 2009(A/H1N1pdm), and A/H3N2 subtypes, respectively. The amantadine-resistant influenza A/H1N1pdm and A/H3N2 viruses were circulating continuously from 2008 to 2009 and from 2005 to 2006, respectively. Amantadine resistance among influenza A viruses increased dramatically during the 5-year study period, and this has diminished the usefulness of this class of drugs.     

Molecular analysis of amantadine-resistant influenza A (H1N1 pdm09) virus isolated from slum dwellers of Dhaka,Bangladesh

Influenza is a highly contagious viral infection associated with excessive hospitalizations and deaths throughout the world. Continuous antigenic shift and drift is not only responsible for this devastating effect of influenza but also causes ineffectiveness of antiviral drugs and vaccines. In this study, we investigated the effectiveness of ribavirin, oseltamivir, and amantadine drugs in vitro against nine influenza A isolates collected during June 2012–August 2013 from different slums in Dhaka city. The effectiveness of these drugs was determined by measuring the inhibition of virus-induced cytopathic effect on MDCK cells through MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Our data showed that all nine influenza isolates (6 H1N1 pdm09 and 3 H3N2 subtypes) were completely susceptible to ribavirin (The 50% effective concentrations, EC₅₀ 3.0 µg/ml) and oseltamivir (EC₅₀ 0.35 µg/ml). When influenza A infection was challenged with amantadine drug, eight out of nine isolates (88%) demonstrated susceptibility to amantadine drug (EC₅₀ 0.30 µg/ml) while one H1N1 pdm09 isolate exhibited higher EC₅₀ value (>10 µg/ml) beyond the cell tolerance level of drug (>5 µg/ml). Genetic analysis of transmembrane matrix protein 2 (M2), which is a target for the amantadine drug and vital for viral replication, showed a substitution of amino acid at position 31(S31 N) of that amantadine-resistant isolate indicating the possible reason of amantadine drug resistance.     

Neuraminidase inhibitor susceptibility of swine influenza A viruses isolated in Germany between 1981 and 2008

European swine influenza A viruses donated the matrix protein 2 as well as the neuraminidase (NA) gene to pandemic influenza A (H1N1) viruses that emerged in 2009. As a result, the latter became amantadine resistant and neuraminidase inhibitor (NAI) susceptible. These recent developments reflecting the close connection between influenza A virus infection chains in humans and pigs urge an antiviral surveillance within swine influenza A viruses. Here, NAI susceptibility of 204 serologically typed swine influenza A viruses of subtypes H1N1, H1N2, and H3N2 circulating in Germany between 1981 and 2008 was analyzed in chemiluminescence-based NA inhibition assays. Mean 50% inhibitory concentrations of oseltamivir and zanamivir indicate a good drug susceptibility of tested viruses. As found for human isolates, the oseltamivir and zanamivir susceptibility was subtype-specific. So, swine influenza A (H1N1) viruses were just as susceptible to oseltamivir as to zanamivir. In contrast, swine H1N2 and H3N2 influenza A viruses were more sensitive to oseltamivir than to zanamivir. Furthermore, reduction in plaque size and virus spread by both drugs was tested with selected H1N1 and H1N2 isolates in MDCK cells expressing similar amounts of α2.3- and α2.6-linked sialic acid receptors. Data obtained in cell culture-based assays for H1N1 isolates correlated with that from enzyme inhibition assays. But, H1N2 isolates that are additionally glycosylated at Asn158 and Asn163 near the receptor-binding site of hemagglutinin (HA) were resistant to both NAI in MDCK cells. Possibly, these additional HA glycosylations cause a misbalance between HA and NA function that hampers or abolishes NAI activity in cells.     

2009甲型H1N1流感流行期间北京急性呼吸道感染儿童常见呼吸道病毒的研究

目的了解2009至2010年甲型H1N1流感大流行期间北京地区急性呼吸道感染患儿中,除流感病毒外的其他几种常见呼吸道病毒感染的流行情况。方法设计并建立检测包括呼吸道合胞病毒（RSV）A/B亚型,副流感病毒（PIV）1、2、3型,腺病毒（ADV）和人博卡病毒（HBoV）在内的多重实时荧光PCR方法,并应用该方法对2009年6月至2010年2月甲型H1N1流感大流行期间,对首都儿科研究所附属儿童医院就诊的急性呼吸道感染患儿中甲型H1N1流感病毒检测阴性的咽拭子标本进行上述呼吸道病毒的核酸检测。结果新建立的多重RT-PCR方法最低可检测的目标基因含量为10～300拷贝数,未见与其他非目标病毒的交叉反应。本研究共检测标本849份,病毒检测总阳性率为39.0%（331份）,5岁以下占87.6%（290/331份）。各病毒的检测阳性率分别为RSV-A亚型1.4%（12份）,RSV-B亚型8.4%（71份）,PIV-1型8.2%（70份）,PIV-2型0.5%（4份）,PIV-3型3.9%（33份）,ADV13.9%（118份）,HBoV2.7%（23份）。RSV检出以B亚型为主（85.5%）,流行高峰在2009年11月至2010年2月。PIV检出以PIV-1型及PIV-3型为主,PIV-1型的流行高峰在2009年7月至2009年10月,PIV-2型及PIV-3型的流行高峰在2009年6～9月。ADV病毒在研究期间均有较高检出率（平均为13.9%）,HBoV的流行高峰在2009年9～12月。结论除流感病毒外,ADV、RSV-B及PIV可能也是2009甲型H1N1流感流行期间引起儿童急性呼吸道感染的重要病原。比较以往的资料可见各病原在2009甲型H1N1流感流行期间的流行季节性及检出率基本未受到新型流感病毒的影响。     

广州市甲型H1N1流感暴发疫点与监测人群病毒抗体检测

目的通过分析广州市甲型H1N1流感暴发疫点与监测人群病毒的抗体水平,了解甲型H1N1流感的流行趋势,为预防甲型H1N1流感疫情提供科学依据。方法应用红细胞血凝抑制（HI）方法检测流感甲型H1N1抗体,对比分析1570名疫区学生与1326名监测人群血清标本H1N1流感病毒的抗体水平。结果疫区学生甲型H1N1流感病毒感染率、流感罹患率分别为32.17%、22.23%,明显高于市区监测人群的22.62%、15.38%（P=0.000,P=0.000）。疫点学生与市区监测人群甲型H1N1流感隐性感染率分别为9.94%、7.24%,差异有统计学意义（P=0.012）。在已感染甲型H1N1流感病毒的疫点学生和市区监测人群中,甲型H1N1流感隐性感染率（30.89%、32.00%）差异无统计学意义（P=0.754）。疫点人群显性感染者的抗体滴度明显高于隐性感染者（t=4.701,P=0.000）,监测人群中显性感染与隐性感染者的抗体滴度无显著差异（t=0.248,P=0.804）。结论疫点学生甲型H1N1流感隐性感染率明显高于监测人群。提示隐性感染人群具有潜在的传染力,应加强隐性感染者的监测。     

Antiviral activity of arbidol, a broad-spectrum drug for use against respiratory viruses, varies according to test conditions

The therapeutic activity of arbidol was investigated against representatives of seven different virus families. Its 50% median effective concentration (EC₅₀) was 0.22–11.8 µg/ml (0.41–22 nM). Therapeutic indices of 91 were obtained for type 1 poliovirus and 1.9–8.5 for influenza A and B, human paramyxo‐3, avian infectious bronchitis‐, and Marek's disease viruses. Arbidol was more inhibitory for influenza A/Aichi/2/68 (H3N2) virus than rimantadine or amantadine (EC₅₀ 10 vs. >15 and >31.6 µg/ml); greater inhibition occurred when end‐points were expressed as TCID₅₀s. For respiratory syncytial virus (RSV), a reduction in plaque size but not number was observed. However, when the drug was added to infected cultures (≥5 µg/ml), a 3‐log reduction in titer occurred. Arbidol did not inhibit directly influenza A/Aichi/2/68 hemagglutinin (HA) or neuraminidase (NA) activity, but inhibition of fusion between the viral envelope and chicken red blood cells occurred when added at 0.1 µg/ml prior to infection. Arbidol induced changes to viral mRNA synthesis of the PB2, PA, NP, NA, and NS genes in MDCK cultures infected with influenza A/PR/8/34. There was no indirect evidence of enhancement of interferon‐α by arbidol following infection with A/Aichi/2/68. Arbidol neither reduced lung viral titers nor caused a significant reduction of lung consolidation in BALB/c mice after administration by the oral and intraperitoneal (i.p.) routes and intranasal challenge with influenza A/Aichi/2/68. A small reduction in lung consolidation, but not viral titer, occurred after i.p. administration and subsequent challenge with RSV. The results indicate the potential of arbidol as a broad‐spectrum respiratory antiviral drug. J. Med. Virol. 84:170–181, 2011. © 2011 Wiley Periodicals, Inc.     

2009—2010年广州甲型H1N1流感的流行特征分析

目的分析甲型H1N1流感在广州的流行特征。方法 2009年5月至2010年4月,每周分别从广州四家监测医院采集流感样症状患者的嗽口液或咽拭子,采用real-time PCR方法鉴定甲型H1N1流感病毒;在广州5大城区采集不同年龄的人群血清,通过血凝抑制实验检测血清中甲型H1N1流感抗体;对其时间和年龄的分层进行分析。结果全年共采集嗽口液或咽拭子3580份,甲型H1N1流感核酸检测阳性530份,阳性率为14.8%,阳性率最高的月份出现在10～12月,为36.5%～42.3%,阳性率最高的年龄组为5～岁和15～岁组,为18.5%和24.2%,最低的年龄组为≥60岁组,为4.1%。人群血清标本共采集1527份,甲型H1N1流感抗体阳性439份,阳性率为28.7%,阳性率最高的年龄组为5～岁和15～岁组,为36.1%和39.5%,最低的年龄组为≥60岁组,为8.6%。结论甲型H1N1流感的流行期发生在10～12月,流行时间短,感染的主要人群是学龄组人群,而老年人群的感染率相对较低。预防流感的工作重点应放在学校内。     

Comparison of colorimetric,fluorometric, and visual methods for determining anti-influenza (H1N1 and H3N2) virus activities and toxicities of compounds

Methods have been developed previously for rapid evaluation of compounds for antiviral activity in 96-well microplates, which include visual quantitation of antiviral activity based upon inhibition of virus-induced cytopathic effect (CPE) or by less subjective colorimetric or fluorometric means. In the present studies we compared a number of colorimetric (crystal violet, MTT [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide], and neutral red) and fluorometric (Alamar Blue, bisbenzimide [Hoechst 33258], fluorescein diacetate, and rhodamine 6G) methods to visual scoring of antiviral activity in influenza A virus infections in Madin Darby canine kidney (MDCK) cells. Toxicity determinations using these same methods were also made for anti-influenza inhibitors and other compounds known to inhibit cell proliferation. Against influenza A/Texas/36/91 (H1N1) and A/Sydney/05/97 (H3N2) viruses, visual scoring and dye or stain methods produced results that were not significantly different from each other in deriving 50% virus-inhibitory concentrations (EC(50) values) for six anti-influenza compounds (amantadine, rimantadine, ribavirin, RWJ-270201 [BCX-1812], oseltamivir carboxylate, and zanamivir), with the exception of Alamar Blue which quantified lower EC(50) values than expected. In uninfected replicating cells, the visual and Alamar Blue methods underestimated the 50% cytotoxic concentrations (IC(50) values) of ribavirin, 1-beta-D-arabinofuranosylcytosine, and 6-azauridine, but more accurately assessed the toxicities of amantadine, rimantadine, and cycloheximide. Visual scoring, coupled with the use of one of these dyes or stains except Alamar Blue, can be used to accurately and rapidly quantify the anti-influenza virus activities and toxicities of potential new influenza virus inhibitors. These methods should also be applicable to evaluating antiviral effects against other lytic virus infections.