广州2006年乙型流感病毒血凝素和神经氨酸酶基因特性分析

为了解2006年广州地区流行的乙型流感病毒株血凝素(HA)和神经氨酸酶(NA)的基因特性,选择病原学监测病毒株和暴发性疫情病毒株,提取病毒RNA并逆转录为cDNA,通过PCR方法扩增乙型流感病毒HA和NA全长基因,将扩增的DNA片段接入T-A克隆载体进行测序,并使用DNAStar软件对测序结果进行分析。结果显示:不同来源的流感病毒株HA的同源性为99%以上,都属于Victoria系;不同来源的病毒株NA同源性为98%以上。HA和NA的种系发生树分析表明:病原学监测毒株同源性更接近,而暴发性疫情毒株的同源性则相对较为分散。所有毒株与WHO推荐的2005~2006年度疫苗株B/Shanghai/361/2002的同源性只有88.9%~89.7%,说明该年度的流感疫苗对乙型流感不能提供最佳的保护。     

甲3型流行性感冒病毒血凝素的pH特征

本文报告,pH9.6碳酸缓冲液对甲3型流感病毒的血凝滴度有明显降低作用,对甲1型和乙型仅有轻微影响,对甲2型的影响则介于两者之间。用不同pH的碳酸缓冲液、磷酸缓冲液及盐水等,测定甲3型流感病毒的血凝滴度,结果表明高pH对其有明显影响。分别具有甲3、甲2或甲1血凝素的重组株,在pH9.6碳酸缓冲液中,其血凝素的稳定性也和上述结果一样,即具有甲3血凝素的重组株,其血凝素对pH9.6碳酸缓冲液最敏感;甲1重组株的血凝素较稳定:而具有甲2血凝素的重组株则介于两者之间。利用此pH特征测定新分离的经血凝抑制试验鉴定为甲3型和乙型流感病毒,得到同样结果,因此有可能利用此pH特征对新分离的甲3型流感病毒进行初步鉴定。     

科研快讯

《科学》:"万能"流感疫苗研究获进展由斯克里普斯研究所和荷兰Crucell疫苗研究所的科学家们领导的一个研究小组发现了三种可保护小鼠抵御2种主要的乙型流感病毒株的人类抗体。相关成果发表在《科学》(Science)杂志上。乙型流感病毒被认为相较于甲型流感病毒不那么危险,由于它们突变形成致命流行病毒株的     

甲型流行性感冒病毒自然温度敏感株基因损害定位

采用重组试验和聚丙烯酰胺凝胶电泳(PAGE)技术,把晚期甲3型流感病毒自然ts突变株齐防79-39的ts损害定位在膜蛋白(M)基因上。但互补试验表明,齐防79-39与M基因损害的WSN标准株ts51可以发生互补,这是基因内互补的一个证据。PAGE技术证实,新甲1型流感病毒自然ts株津防77-78的M基因上确有损害。互补试验证明齐防79-39属于一个互补组,而津防77-78与ts51同属于另一个互补组。 本文结果还表明,晚期甲3型齐防79-39的ts损害基因可能是由甲3型野毒株自发突变所产生,而并非通过在自然界与新甲1型重组而获得。     

南京市2011年乙型流感血凝素基因分子特征分析

[目的]分析2011年南京市乙型流感病毒的血凝素(HA)分子学特征.[方法]选择7株2011年南京市不同时间段有代表性的乙型流感毒株进行HA基因序列测定,通过生物信息学方法对HA分子学特征进行分析.[结果]7株乙型流感毒株分为两个系,4株为Victoria,3株为Yamagata;与2011年度疫苗株相比,Victoria和Yamagata系毒株分别在抗原位点146、197和116、198发生了氨基酸替换;其中197和198位点分别是Victoria和Yamagata毒株的受体结合位点,由于上述位点的替换使得Victoria系/Yamagata系毒株分别在197/196位增加了一个潜在的糖基化位点.[结论]2011年南京市乙型流感Victoria 系和Yamagata系病毒同时存在,Victoria/Yamagata毒株197/198位点的氨基酸替换,值得做进一步的探讨.     

乙型流感病毒河北株的HA1基因序列及种系发生的分析

对１９８９年春在河北保定分离的两株乙型流感病毒进行了抗原性、ＨＡ＿１基因序列和种系发生分析,与不同期的国内外代表株比较结果表明,自１９８８年以来乙型流感病毒变异较快,Ｂ／河北／５３／８９株的ＨＡ＿１基因序列与Ｂ／挪威／１／８５株相比,其氨基酸的同源性为９４．５２％,与同期的Ｂ／香港／２０／８９株同源性为９７．１２％。种系发生分析结果,从１９８８至１９８９年乙型流感病毒出现了５个支系,同期在保定分离的两株病毒,在同源替代中分别属于两个支系。日本国基本每隔两年出现一次乙型流感的流行优势型,其发生频度与甲型流感相似。国内少见乙型的流行优势型,可能和使用的分离病毒材料有关,日本用ＭＤＣＫ细胞比国内用鸡胚对乙型流感病毒的分离阳性率高。     

稳定表达乙型流感病毒血凝素蛋白细胞的构建

获得稳定表达乙型流感病毒血凝素蛋白的HEK-293-HA细胞株。采用RT-PCR方法扩增乙型流感病毒的HA基因,将其克隆至真核表达载体pcDNA3.1(+)上,构建pcDNA3.1(+)-HA重组质粒。将已鉴定正确的pcDNA3.1(+)-HA质粒与辅助质粒PLV-EF1a-EGFP(2A)Puro通过脂质体介导法共转染HEK293细胞,经嘌呤霉素筛选后得到重组细胞株HEK293-HA,通过流式细胞术法(FCM)来检测细胞中HA的表达情况。所得到的重组细胞经扩大培养后再连续培养15代,采用间接免疫荧光法(IFA)和蛋白免疫印迹法(WB)来检测HA蛋白表达的稳定性。结果表明重组质粒pcDNA3.1(+)-HA经双酶切及测序鉴定正确;共获得3株高表达阳性细胞株。细胞扩大培养后连续传代培养15代后进行Western blot和IFA检测结果表明,重组细胞的HA蛋白得到稳定的表达。已成功获得了能稳定表达乙型流感病毒血凝素蛋白的HEK-293-HA细胞,可为乙型流感病毒HA蛋白的进一步研究提供良好的基础。     

2021-2022年嘉兴市乙型流感病毒序列进化分析

本研究分析了嘉兴市2021-2022年乙型流感病毒基因进化特征。首先采集2021-2022年嘉兴市流感样病例咽拭子标本进行流感病毒核酸检测、病毒分离。然后从嘉兴市2021-2022年流感分离株中共选取27株乙型流感病毒代表毒株进行全基因组高通量测序。最后利用生物信息学软件从核苷酸、氨基酸及分子层面对流感毒株进行分子特征分析。2021年嘉兴市共检测1362例流感样病例标本,阳性标本数98例,阳性率为7.20%,均为乙型流感病毒Victoria系（Influenza B virus Lineage Victoria, BV）。2022年嘉兴市共检测1318例流感样病例标本,阳性标本数335例,其中BV系89例,阳性率为6.75%,H3N2亚型246例,阳性率为18.66%。与疫苗株相比,2021年血凝素（haemagglutinin,HA）核苷酸与氨基酸序列相似性为98.92%±0.24%、98.55%±0.21%,神经氨酸酶（neuraminidase,NA）核苷酸与氨基酸序列相似性为99.25%±0.13%、99.37%±0.22%;2022年HA核苷酸与氨基酸序列相似性为99.12%...     

板蓝根水提物S-03体外抑制甲、乙型流感病毒感染的实验研究

以水提法分离制备板蓝根水提物S-03分析其基本化学成分,并在狗肾细胞(MDCK)上分别接种人甲1、3型和乙型流感病毒标准株、临床分离株以及禽流感病毒,采用空斑减少和免疫荧光及血凝抑制实验方法,在预防、治疗和直接作用三种试验模式下探讨S-03体外对流感病毒的抑制作用。研究结果表明S-03的主要化学成分为糖类,多糖所占总重的比例最高。S-03体外抗病毒药效显示:①预防模式:对各型流感病毒均无抑制作用;②治疗和直接作用模式:对不同亚型流感病毒均有一定程度的抑制作用,且直接作用(SI=2.5~16)效果优于治疗模式(SI=2.2~5.8);③血凝抑制试验:对不同亚型的人流感病毒血凝素有不同程度抑制作用(最低抑制浓度为3.12~25 mg/mL),并且对禽流感病毒(H6N2、H7N3、H9N2)的血凝素也有一定的抑制作用(最低抑制浓度25~50 mg/mL),其作用机制可能为抑制流感病毒表面的血凝素(HA),从而阻止病毒感染。     

儿童乙型流感病毒感染特点及防治的研究进展

乙型流感病毒是引起流行性感冒(简称流感)发生和流行的主要病原体之一,近年来流感高发的季节感染率呈上升趋势,儿童是乙型流感病毒感染的易感、高危群体。乙型流感病毒虽然抗原变异性较弱,但仍可引起暴发流行,严重威胁患儿的健康,因此对于儿童乙型流感病毒感染的防治至关重要。本文综合近年来国内外相关文献,从流行病学特征、临床表现、实验室检查、乙型流感病毒感染的药物治疗、免疫疗法等角度出发对乙型流感病毒感染特点及治疗进展予以综述,对乙型流感病毒进行长期有效的监测,了解儿童乙型流感病毒的感染特点,为儿童乙型流感的预防及临床诊断与防治提供理论参考。     

Differences in RNA patterns of influenza A viruses.

Analysis of the segmented RNAs of influenza A viruses by electrophoresis on polyacrylamide urea slab gels has provided a method for sharper resolution of the number and migration rates of different segments than previously has been possible. Using this system, the RNA genome of influenza A/WSN (HON1) virus can be separated into seven to nine separate bands, depending on whether virus is obtained after high or low multiplicity of infection, and the genome of influenza A/PR/8 (HON1) virus can be resolved into eight bands, six of which migrate differently from comparable RNA bands of WSN virus. Comparision of the RNA patterns produced by influenza A/PR/8 (HON1) and A/England/42/72 (H8n2) virus also reveals major differences in migration speeds of different bands, and analysis of the RNAs of the RNAs of an HON2 recombinant virus derived from these two strains permits the identification of RNA segments which have been derived from one particular parent. By extension of these techniques, it may be possible to define which RNA segment codes for each viral protein and to analyze recombinant strains to identify which genes have been derived from each of its parents.     

流行性感冒病毒重组株京生75-29 R2 T1及冷适应株31-广的基因分析

用聚丙烯酰胺凝胶电泳方法分析了流行性感冒病毒重组株京生75-29R2 T1(H3N2)及冷适应株31-广(H3N2)的RNA及多肽。重组株京生75-29R2 T1的HA及M基因系来自流行病毒亲本株/甲/北京/29/75(H3N2),而P_2、NA、NP及NS基因则来自温度敏感母株福R3(H2N2)。流行病毒株甲/穗/03/68(H3N2)在低温条件下经鸡胚尿囊腔传递24代而获得的冷适应疫苗毒株31-广(H3N2)其基因型与野毒株一致。     

M protein (M1) of influenza virus: antigenic analysis and intracellular localization with monoclonal antibodies.

A panel of 16 monoclonal antibodies recognizing M protein (M1) of influenza virus was generated. Competition analyses resulted in localization of 14 monoclonal antibodies to three antigenic sites. Three monoclonal antibodies localized to site 1B recognized a peptide synthesized to M1 (residues 220 to 236) with enzyme-linked immunosorbent assay titers equivalent to or greater than that seen with purified M1; therefore, site 1B is located near the C terminus of M1. Sites 2 and 3 localize to the N-terminal half of M1. Antigenic variation of M proteins was seen when the monoclonal antibodies were tested against 14 strains of type A influenza viruses. Several monoclonal antibodies showed specific recognition of A/PR/8/34 and A/USSR/90/77 M proteins and little or no reactivity for all other strains tested. Immunofluorescence analysis with the monoclonal antibodies showed migration of M protein to the nucleus during the replicative cycle and demonstrated association of M protein with actin filaments in the cytoplasm. Use of a vaccinia virus recombinant containing the M-protein gene demonstrated migration of M protein to the nucleus in the absence of synthesis of gene products from other influenza virus RNA segments.     

流感病毒感染诱导MDCK细胞凋亡的研究

用荧光染色、DNA凝胶电泳等方法检测了A型流感病毒株A1／京防86－1和B型流感病毒株B/沪防93－1诱导狗肾体代细胞(MDCKcells)的凋亡情况,并采用MTT法和流式细胞仪比较了这2株病毒对MMCK细胞的毒力和凋亡诱导能力水平。结果显示：病毒感染6h后,细胞DNA发生断裂,病毒感染12h后,可见明显的染色质凝聚;在一定范围内,细胞凋亡强度表现出明显的时间和剂量依赖关系;并且,A型流感病毒株的毒力和调亡诱导能力均强于B型流感病毒株。实验结果表明：流感病毒感染引起的细胞死亡主要是通过调亡实现的,毒力不同的流感病毒株诱导细胞调亡的能力不同。     

Virucidal effect of sodium p-chloromercuribenzoate on influenza viruses attributable to inhibition of virus particle-associated RNA-dependent RNA polymerase.

Sodium p-chloromercuribenzoate (PCMB) caused a noticeable reduction of infectivity of prototype strains of type A and Lee strain of type B influenza viruses at concentrations of 100 and 200 mug/ml, respectively, after an incubation at 37 C for 60 min. The virucidal effect on A/AA/2/60 (H2N2) strain was dependent on the concentration of the drug and temperature as well as on the time of incubation. The reagent exerted this effect at a concentration which induced little change in the hemagglutinating and neuraminidase activities of the virus. PCMB inhibited by 50% the virus particle-associated RNA polymerase activity of all prototype strains of type A influenza virus at about 2 mug/ml and that of Lee strain of type B influenza virus at 8.5 mug/ml. Other sulfhydryl reagent such as phenylmercuric nitrate also exhibited virucidal effect on A/AA/2/60 virus which paralleled their inhibition of the virus particle-associated RNA polymerase activity. From these results it was considered likely that the virucidal action of PCMB on influenza viruses was attributable to inhibition of the virus particle-associated RNA polymerase activity.     

A reassortment-incompetent live attenuated influenza virus vaccine for protection against pandemic virus strains

Although live-attenuated influenza vaccines (LAIV) are safe for use in protection against seasonal influenza strains, concerns regarding their potential to reassort with wild-type virus strains have been voiced. LAIVs have been demonstrated to induce enhanced mucosal and cell-mediated immunity better than inactivated vaccines while also requiring a smaller dose to achieve a protective immune response. To address the need for a reassortment-incompetent live influenza A virus vaccine, we have designed a chimeric virus that takes advantage of the fact that influenza A and B viruses do not reassort. Our novel vaccine prototype uses an attenuated influenza B virus that has been manipulated to express the ectodomain of the influenza A hemagglutinin protein, the major target for eliciting neutralizing antibodies. The hemagglutinin RNA segment is modified such that it contains influenza B packaging signals, and therefore it cannot be incorporated into a wild-type influenza A virus. We have applied our strategy to different influenza A virus subtypes and generated chimeric B/PR8 HA (H1), HK68 (H3), and VN (H5) viruses. All recombinant viruses were attenuated both in vitro and in vivo, and immunization with these recombinant viruses protected mice against lethal influenza A virus infection. Overall, our data indicate that the chimeric live-attenuated influenza B viruses expressing the modified influenza A hemagglutinin are effective LAIVs.     

Virucidal Effect of Sodium p-Chloromercuribenzoate on Influenza Viruses Attributable to Inhibition of Virus Particle-Associated RNA-Dependent RNA Polymerase

Sodium p-chloromercuribenzoate (PCMB) caused a noticeable reduction of infectivity of prototype strains of type A and Lee strain of type B influenza viruses at concentrations of 100 and 200 μg/ml, respectively, after an incubation at 37 C for 60 min. The virucidal effect on A/AA/2/60 (H₂N₂) strain was dependent on the concentration of the drug and temperature as well as on the time of incubation. The reagent exerted this effect at a concentration which induced little change in the hemagglutinating and neuraminidase activities of the virus. PCMB inhibited by 50% the virus particle-associated RNA polymerase activity of all prototype strains of type A influenza virus at about 2 μg/ml and that of Lee strain of type B influenza virus at 8.5 μg/ml. Other sulfhydryl reagent such as phenylmercuric nitrate also exhibited virucidal effect on A/AA/2/60 virus which paralleled their inhibition of the virus particle-associated RNA polymerase activity. From these results it was considered likely that the virucidal action of PCMB on influenza viruses was attributable to inhibition of the virus particle-associated RNA polymerase activity.