小鼠感染流感病毒后可抵抗致死性流感病毒攻击的研究

目的 BALB/c小鼠初次感染流感病毒(A/California/7/2009(H1N1)(pCA07)和A/Guangzhou/333/99(H9N2)(GZ333))后,用流感病毒鼠肺适应株A/PR/8/34(H1N1)(PR8) 10倍致死剂量攻击,观察攻毒后小鼠的反应.方法 BALB/c小鼠150只,分成3组,空白对照组PBS滴鼻,实验组分别感染甲型H1N1流感病毒pCA07和禽源H9N2病毒GZ333;感染56 d后用10倍致死剂量的鼠肺适应株流感病毒PR8攻击两个实验组和对照组小鼠,比较PR8病毒攻击后,病毒载量和抗体水平,以及对小鼠存活率的影响.结果 感染过pCA07和GZ333的小鼠在致死病毒PR8攻击后全部存活,并分别在病毒攻击6d和9d后小鼠肺组织中检测不到攻击病毒.对初次感染病毒的抗体水平在病毒PR8攻击后迅速升高,在保持初次感染病毒抗体的同时能够产生针对PR8病毒的抗体.结论 不同亚型流感病毒感染小鼠后可以提供交叉保护.     

Detection of influenza C virus but not influenza D virus in Scottish respiratory samples

BackgroundA newly proposed genus of influenza virus (influenza D) is associated with respiratory disease in pigs and cattle. The novel virus is most closely related to human influenza C virus and can infect ferrets but infection has not been reported in humans.ObjectivesTo ascertain if influenza D virus can be detected retrospectively in patient respiratory samples.Study design3300 human respiratory samples from Edinburgh, Scotland, covering the period 2006–2008, were screened in pools of 10 by RT-PCR using primers capable of detecting both influenza C and D viruses.ResultsInfluenza D was not detected in any sample. Influenza C was present in 6 samples (0.2%), compared with frequencies of 3.3% and 0.9% for influenza A and B viruses from RT-PCR testing of respiratory samples over the same period. Influenza C virus was detected in samples from individuals <2 years or >45 years old, with cases occurring throughout the year. Phylogenetic analysis of nearly complete sequences of all seven segments revealed the presence of multiple, reassortant lineages.ConclusionWe were unable to detect viruses related to influenza D virus in human respiratory samples. Influenza C virus was less prevalent than influenza A and B viruses, was associated with mild disease in the young (<2 years) and old (>45 years) and comprised multiple, reassortant lineages. Inclusion of influenza C virus as part of a diagnostic testing panel for respiratory infections would be of limited additional value.     

Long term persistence of IgE anti-influenza virus antibodies in pediatric and adult serum post vaccination with influenza virus vaccine

The production of IgE specific to different viruses (HIV-1, Parvovirus B19, Parainfluenza virus, Varicella Zoster Virus), and the ability of IgE anti-HIV-1 to suppress HIV-1 production in vitro, strongly suggest an important role for IgE and/or anti viral specific IgE in viral pathogenesis. Nevertheless, the presence and persistence of IgE anti-Influenza virus antibodies has not been studied. Total serum IgE and specific IgE and IgG anti-Influenza virus antibodies were studied in children (N=3) (m/f 14-16 y/o) and adults (N=3) (m/f, 41-49 y/o) 2-20 months after vaccination with Influenza virus (Flumist^® or Fluzone^®), as well as in non-vaccinated children (N=2). (UniCAP total IgE Fluoroenzymeimmunoassay, ELISA, Immunoblot). We found that serum of vaccinated children and adults contained IgE and IgG anti-Influenza virus antibodies approaching two years post vaccination. Non-vaccinated children did not make either IgE or IgG anti-Influenza antibodies. Similar levels of IL-2, IFN-γ, IL-4, and IL-10 cytokines were detected in serum of vaccinated compared with non vaccinated subjects (p>0.05), as well as between vaccinated adults compared with vaccinated children and non vaccinated subjects (p>0.05). Vaccinated children and adults continue to produce IgE anti-Influenza virus antibodies long term post vaccination. The long term production of IgE anti-Influenza virus antibodies induced by vaccination may contribute to protective immunity against Influenza.     

2006年中国流感病毒抗原性及基因特性研究

目的分析2006年中国季节性流感的流行状况，以及病毒的抗原性和基因变异情况。方法对来自流感监测网络的毒株进行单向血凝抑制试验，在此基础上选择不同时间、地点分离的毒株进行血凝素基因的序列测定，然后分析其基因特性。结果2006年我国同时流行A型（H1N1亚型、H3N2亚型）和B型流感病毒。H1N1亚型毒株和B型Victoria系流感病毒为优势毒株。对H1N1亚型毒株的HA1区序列比较发现，2006年分离的毒株与A，湖北洪山／53／2005（H1N1）比较，在192、193、196、198位发生氨基酸替换的毒株．这些位点位于抗原决定簇的B区。H3N2亚型毒株与A，云南，1145／2005（H3N2）比较，在142、144位发生氨基酸替换。我国流行的B型流感毒株无论是Victoria系和Yamagata系毒株的抗原性均没有发生变异，与2005--2006年我国的流行株B／shenzhen／155／2005、B／tianjin／144／2005类似。结论2006年中国流行的H1N1亚型和H3N2亚型流感病毒的抗原性及基因特性已经发生改变；B型流感病毒的抗原性和基因特性没有改变。     

In vitro immunomodulatory activity of celastrol against influenza A virus infection

Context: The influenza A virus (IAV) causes severe respiratory disease that remains a leading reason for morbidity and mortality. Previous studies have indicated that influenza complications in addition to viral replication are due to overproduction of pro-inflammatory cytokines. Therefore, a new compound is needed to be used with current antiviral drugs to modulate overproduction of pro-inflammatory cytokines in IAV infection.

Objective: This study investigated the effect of celastrol on mRNA expression and concentration levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα) and interleukin-6 (IL6) that are induced by influenza A/Puerto Rico/8/34 (H1N1; PR8) in Madin-Darby Canine Kidney (MDCK) cells. The effect of this compound on virus titration and viral mRNA expression was also investigated.

Methods: Confluent MDCK cells were infected with influenza virus (H1N1; PR8) and treated with celastrol at different concentrations. After incubation, mRNA expression and concentrations of TNFα and IL6 were investigated using real-time polymerase chain reaction (PCR) and ELISA. The viral mRNA expression and virus titration were investigated using real-time PCR and 50% tissue culture infectious dose (TCID₅₀) assay, respectively.

Results: mRNA expression and concentrations of TNFα and IL6 increased significantly in control virus compared to cell control, and decreased significantly when compared with control virus after celastrol treatment. Viral mRNA expression and virus titration did not decrease after celastrol treatment.

Conclusion: Due to reducing mRNA expression and concentrations of TNFα and IL6, celastrol can serve as a suitable choice to control cytokine-induced inflammation in IAV infection, and therefore it can be used with current antiviral drugs.     

多重RT-PCR方法检测甲型流感病毒

目的建立一种快速、敏感、特异的多重RT-PCR,同时检测甲型流感病毒中的3个分型:甲型H1N1流感病毒,季节性H1N1流感病毒,季节性H3N2流感病毒,并将此方法应用到实验室流感病毒核酸检测技术中。方法利用甲型流感病毒3个分型病毒的引物,在同一个RT-PCR反应体系中,对疑似流感咽拭子标本进行检测。结果多重RT-PCR对甲型流感病毒中分型病毒有较高的灵敏度和特异性,可直接从疑似流感标本中同时进行甲型流感病毒分型检测。结论此实验中采用的多重RT-PCR具有与常规RT-PCR一样的特异性和敏感度,而且比普通RT-PCR和病毒分离法更快速,也更简便。     

Recombinant parainfluenza virus 5 (PIV5) expressing the influenza A virus hemagglutinin provides immunity in mice to influenza A virus challenge

Parainfluenza virus type 5 (PIV5), formerly known as simian virus 5 (SV5), is a non-segmented negative strand RNA virus that offers several advantages as a vaccine vector. PIV5 infects many cell types causing little cytopathic effect, it replicates in the cytoplasm of infected cells, and does not have a DNA phase in its life cycle thus avoiding the possibility of introducing foreign genes into the host DNA genome. Importantly, PIV5 can infect humans but it is not associated with any known human illness. PIV5 grows well in tissue culture cells, including Vero cells, which have been approved for vaccine production, and the virus can be obtained easily from the media. To test the feasibility of using PIV5 as a live vaccine vector, the hemagglutinin (HA) gene from influenza A virus strain A/Udorn/72 (H3N2) was inserted into the PIV5 genome as an extra gene between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Recombinant PIV5 containing the HA gene of Udorn (rPIV5-H3) was recovered and it replicated similarly to wild type PIV5, both in vitro and in vivo. The HA protein expressed by rPIV5-H3-infected cells was incorporated into the virions and addition of the HA gene did not increase virus virulence in mice. The efficacy of rPIV5-H3 as a live vaccine was examined in 6-week-old BALB/c mice. The results show that a single dose inoculation provides broad and considerable immunity against influenza A virus infection.     

Inhibition of influenza A virus replication by influenza B virus nucleoprotein: an insight into interference between influenza A and B viruses

Given that co-infection of cells with equivalent titers of influenza A and B viruses (FluA and FluB) has been shown to result in suppression of FluA growth, it is possible that FluB-specific proteins might hinder FluA polymerase activity and replication. We addressed this possibility by individually determining the effect of each gene of FluB on the FluA polymerase assay and found that the nucleoprotein of FluB (NP_FluB) inhibits polymerase activity of FluA in a dose-dependent manner. Mutational analyses of NP_FluB suggest that functional NP_FluB is necessary for this inhibition. Slower growth of FluA was also observed in MDCK cells stably expressing NP_FluB. Further analysis of NP_FluB indicated that it does not affect nuclear import of NP_FluA. Taken together, these findings suggest a novel role of NP_FluB in inhibiting replication of FluA, providing more insights into the mechanism of interference between FluA and FluB and the lack of reassortants between them.     

用反向遗传技术产生冷适应致弱的重组A型人流感病毒的研究

目的 以鸡胚高度适应株A/PR/8/34株为重组流感病毒骨架,利用反向遗传技术拯救冷适应致弱的重组A型人流感病毒.方法 对冷适应、温度敏感、减毒的A/Ann Arbor/6/60(H2N2)流感病毒株的PB2基因片段,进行了全基因序列合成,同时人工引入PB2265(N265S)氨基酸的突变.PB2基因片段通过与改造后的转录/表达载体pAD3000连接,构建PB2基因的拯救载体.该重组质粒与PR8进行"7 1"组合的病毒拯救,共转染COS-1细胞.结果 经测序获得序列准确的拯救质粒pMDV-A-PB2,利用反向遗传技术成功拯救出了具有血凝性(1×25)的冷适应的重组A型流感病毒.结论 利用反向遗传技术成功拯救冷适应致弱的重组A型人流感病毒,该系统为深入研究甲型人流感病毒的基因功能和新型疫苗研发奠定了基础.     

甲型流感病毒核蛋白基因的克隆表达及纯化

目的　将甲型流感病毒核蛋白(NP)基因克隆到原核表达载体进行可溶性融合表达,制备纯化的病毒核蛋白,为制备甲型流感病毒单克隆抗体提供材料。方法　提取甲型流感病毒RNA ,设计引物,RT PCR扩增NP基因,利用基因工程的手段,将甲型流感病毒的NP基因在大肠埃希菌中进行融合表达,并将表达产物进行亲和层析。结果　成功构建了甲型流感病毒NP基因原核表达载体,经亲和层析制备了较高纯度的目的表达产物。结论　通过合理控制发酵时间、生长温度和诱导物浓度,制备了较为理想的可溶性甲型流感病毒核蛋白。     

Reduced incorporation of the influenza B virus BM2 protein in virus particles decreases infectivity

BM2 is the fourth integral membrane protein encoded by the influenza B virus genome. It is synthesized late in infection and transported to the plasma membrane from where it is subsequently incorporated into progeny virus particles. It has recently been reported that BM2 has ion channel activity and may be the functional homologue of the influenza A virus M2 protein acting as an ion channel involved in viral entry. Using a reverse genetic approach it was not possible to recover virus which lacked BM2. A recombinant influenza B virus was generated in which the BM2 AUG initiation codon was mutated to GUG. This decreased the efficiency of translation of BM2 protein such that progeny virions contained only 1/8 the amount of BM2 seen in wild-type virus. The reduction in BM2 incorporation resulted in a reduction in infectivity although there was no concomitant decrease in the numbers of virions released from the infected cells. These data imply that the incorporation of sufficient BM2 protein into influenza B virions is required for infectivity of the virus particles.     

维百氏康空气健康剂抗流感病毒初步研究

目的 进行维百氏康空气健康剂抗流感病毒实验研究方法将不同稀释度的样品与100 TCID50的病毒液等量混合,37℃中和30 min,取适量接种于含维持液的细胞孔内,一式3孔,设病毒对照、细胞对照和不同稀释度样品对照,置37℃ CO2培养箱,逐日观察CPE,当病毒对照组CPE出现++++时终止试验,测定血凝程度.结果样品原液的1∶60,1∶120,1∶240,1∶480可100%灭活甲型及乙型流感病毒.原液的1∶960和1∶1920可灭活25%～50%甲型及乙型流感病毒.结论为维百氏康空气健康剂产品的研发提供了抗流感病毒试验依据.     

Establishment of a cynomolgus macaque model of influenza B virus infection

Pathogenicity of influenza B virus was examined in cynomolgus macaques to establish a macaque model suitable for vaccine and antiviral drug development. We prepared influenza B viruses for inoculation with minimal passages after isolation from patients. Macaques inoculated with influenza B virus showed higher body temperature than that before infection for 6 to 12 days. Virus was detected in nasal, tracheal, and bronchial samples until 6 days after inoculation followed by an increase in neutralizing antibody. High levels of IL-6 and TNF-α in nasal swabs from the infected macaques were correlated with fever. Symptoms and duration of the viral replication would be sufficient to evaluate efficacy of vaccines and antiviral agents. In addition, measurement of immune responses including antibody and cytokine production would provide an immunological rationale in efficacy of vaccines and antiviral agents. The results suggest that cynomolgus macaques are appropriate model animals for research of influenza B virus.     

Serologic evidence of exposure to influenza D virus among persons with occupational contact with cattle

BackgroundInfluenza D virus (IDV), a novel influenza virus with proposed classification: family Orthomyxoviridae, genus Influenzavirus D, species Influenza D virus, has been associated with influenza-like illness in cattle and swine. More recently, anti-IDV antibodies have also been detected in small ruminants. A seroprevalence of approximately 1.3% has been estimated for the general human population.ObjectivesTo gain insights on the zoonotic potential of IDV to human adults with occupational exposure to cattle in north central Florida.StudyA cross-sectional serological study was performed on human serum samples from 35 cattle-exposed and 11 non-cattle-exposed adults to screen for IDV antibodies using hemagglutination inhibition (HI) and microneutralization (MN) assays.ResultsA seroprevalence of 91% was detected via HI assay, and 97% by MN assay among individuals working with cattle in Florida. Among non-cattle-exposed individuals, seropositivity determined via MN assay (only) was lower (18%).ConclusionsIDV poses a zoonotic risk to cattle-exposed workers, based on detection of high seroprevalence (94–97%). Whereas it is still unknown whether IDV causes disease in humans, our studies indicate that the virus may be an emerging pathogen among cattle-workers.     

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。     

Interaction of porcine conventional dendritic cells with swine influenza virus

Swine influenza virus (SwIV) causes sub-acute or acute respiratory infections on swine farms and pigs can act as “mixing vessels” for new influenza strains. Knowledge of the immune response of SwIV in its natural host, pigs, is very limited. Dendritic cells (DCs) mediate the induction of immunity to pathogens, but their interaction with SwIV has not been fully characterized. Thus, porcine bone marrow derived DCs (poBMDCs) were exposed to a circulating strain of H3N2 SwIV in vitro. Infection of poBMDCs resulted in structures resembling influenza virus inside poBMDCs in vesicles and also free in cytoplasm. Viral progeny was undetectable in supernatant but limited replication was detected in the first 8 h after infection. However, viral particles from infected-poBMDCs were able to induce cytopathic effect in susceptible cells only when cell-to-cell interaction was favoured. The data generated in our studies reveal the particular interaction of H3N2 SwIV with conventional DCs.     

Receptor specificity of H5 influenza virus escape mutants

The binding of viruses to synthetic polyacrylamide (PAA)-based sialylglycoconjugates was used to characterize the receptor specificities of antibody escape mutants of the influenza virus A/Mallard/Pennsylvania/10218/84 (H5N2). The sialylglycoconjugates that were used carried identical terminal Neu5Ac2–3Gal moieties but differed in the structure of the next saccharide residue(s). Our data show that mutations in the vicinity of the haemagglutinin (HA) receptor-binding site (RBS) effect the recognition of the third saccharide residue and change the affinity pattern of binding. The affinity of the majority of the escape mutants for sialyl receptors increased compared to the parental strain.     

我国人群中一种新重配的H1N2亚型流感病毒株的发现

１９９６年１月太原铁路卫生防疫站从上感患者中分离到３株流感病毒。经血清学鉴定，它们不同于１９８９和１９９２年所发现的Ｈ１Ｎ２亚型毒株，其ＨＡ的抗原性类似于Ａ／ＰＲ／８／３４（Ｈ１Ｎ１）病毒，而明显不同于当前人群中流行的Ｈ１Ｎ１亚型毒株。病毒粒不同基因节段迁移率比较表明，它们的１～４基因节段迁移率接近于Ａ／ＰＲ／８／３４（Ｈ１Ｎ１）毒株，５～６基因节段迁移率类似于Ａ／武汉／３５９／９５（Ｈ３Ｎ２）病毒，而７～８两节段既不同于Ａ／ＰＲ／８／３４（Ｈ１Ｎ１），又不同于Ａ／武汉／３５９／９５（Ｈ３Ｎ２）病毒。故可认为它们是一种新重配的Ｈ１Ｎ２亚型毒株。     

流行性感冒病毒裂解疫苗的安全性和免疫原性研究

目的 研究流行性感冒(流感)病毒裂解疫苗的安全性和免疫原性。方法 根据国家食品药品监督管理局2002SL0043号《新药临床研究申请批件》的要求，选择876例观察对象按随机双盲法分成试验组和对照组，观察局部反应和全身反应。用微量血凝抑制(H1)试验检验抗体应答反应，计算抗体阳转率、保护率及几何平均滴度(GMT)增长倍数。结果 试验疫苗和对照疫苗间全身和局部不良反应发生率差异无显著意义；两组间H1抗体阳转率、保护率及H3N2亚型和B型H1抗体GMT增长倍数差异无显著意义；然而，两种疫苗H1N1亚型的H1抗体GMT增长倍数间差异有显著意义。结论 两种疫苗均具有很好的安全性和免疫原性。