Protocatechuic Acid,a Novel Active Substance against Avian Influenza Virus H9N2 Infection

Influenza virus H9N2 subtype has triggered co-infection with other infectious agents, resulting in huge economical losses in the poultry industry. Our current study aims to evaluate the antiviral activity of protocatechuic acid (PCA) against a virulent H9N2 strain in a mouse model. 120 BALB/c mice were divided into one control group, one untreated group, one 50 mg/kg amantadine hydrochloride-treated group and three PCA groups treated 12 hours post-inoculation with 40, 20 or 10 mg/kg PCA for 7 days. All the infected animals were inoculated intranasally with 0.2 ml of a A/Chicken/Hebei/4/2008(H9N2) inoculum. A significant body weight loss was found in the 20 mg/kg and 40 mg/kg PCA-treated and amantadine groups as compared to the control group. The 14 day survivals were 94.4%, 100% and 95% in the PCA-treated groups and 94.4% in the amantadine hydrochloride group, compared to less than 60% in the untreated group. Virus loads were less in the PCA-treated groups compared to the amantadine-treated or the untreated groups. Neutrophil cells in BALF were significantly decreased while IFN-γ, IL-2, TNF-α and IL-6 decreased significantly at days 7 in the PCA-treated groups compared to the untreated group. Furthermore, a significantly decreased CD4+/CD8+ ratio and an increased proportion of CD19 cells were observed in the PCA-treated groups and amantadine-treated group compared to the untreated group. Mice administered with PCA exhibited a higher survival rate and greater viral clearance associated with an inhibition of inflammatory cytokines and activation of CD8+ T cell subsets. PCA is a promising novel agent against bird flu infection in the poultry industry.     

Novel Avian Influenza A (H7N9) Virus Induces Impaired Interferon Responses in Human Dendritic Cells

In March 2013 a new avian influenza A(H7N9) virus emerged in China and infected humans with a case fatality rate of over 30%. Like the highly pathogenic H5N1 virus, H7N9 virus is causing severe respiratory distress syndrome in most patients. Based on genetic analysis this avian influenza A virus shows to some extent adaptation to mammalian host. In the present study, we analyzed the activation of innate immune responses by this novel H7N9 influenza A virus and compared these responses to those induced by the avian H5N1 and seasonal H3N2 viruses in human monocyte-derived dendritic cells (moDCs). We observed that in H7N9 virus-infected cells, interferon (IFN) responses were weak although the virus replicated as well as the H5N1 and H3N2 viruses in moDCs. H7N9 virus-induced expression of pro-inflammatory cytokines remained at a significantly lower level as compared to H5N1 virus-induced “cytokine storm” seen in human moDCs. However, the H7N9 virus was extremely sensitive to the antiviral effects of IFN-α and IFN-β in pretreated cells. Our data indicates that different highly pathogenic avian viruses may show considerable differences in their ability to induce host antiviral responses in human primary cell models such as moDCs. The unexpected appearance of the novel H7N9 virus clearly emphasizes the importance of the global influenza surveillance system. It is, however, equally important to systematically characterize in normal human cells the replication capacity of the new viruses and their ability to induce and respond to natural antiviral substances such as IFNs.     

两株H9N2亚型禽流行性感冒病毒HA基因序列分析

禽流感(AI)是由A型流行性感冒(流感)病毒引起的一种严重危害禽类健康的传染性疾病.禽类感染禽流感病毒(AIV)后,症状可表现为非显性感染,亚临诊感染,或轻度呼吸道疾病,产蛋量降低,直至急性全身致死性疾病等多种形式.     

Genome Sequence of a Novel Reassortant H3N2 Avian Influenza Virus in Southern China

The distribution and prevalence of H3 subtype influenza viruses in avian and mammalian hosts constitutes a potential threat to both human and avian health. We report a complete genome sequence of a novel reassortant H3N2 avian influenza virus. Phylogenetic analysis showed that HA and NA showed the highest sequence homologies with those of A/white-backed munia/Hong Kong/4519/2009 (H3N2). However, the internal genes had the highest sequence homologies with those of H6 and H7 subtypes. The data provide further evidence of the existence of a natural reassortant H3N2 strain in southern China.     

Two DNA Aptamers against Avian Influenza H9N2 Virus Prevent Viral Infection in Cells

New antiviral therapy for pandemic influenza mediated by the H9N2 avian influenza virus (AIV) is increasingly in demand not only for the poultry industry but also for public health. Aptamers are confirmed to be promising candidates for treatment and prevention of influenza viral infections. Thus, we studied two DNA aptamers, A9 and B4, selected by capillary electrophoresis-based systemic evolution of ligands by exponential enrichment (CE-SELEX) procedure using H9N2 AIV purified haemagglutinin (HA) as target. Both aptamers had whole-virus binding affinity. Also, an enzyme-linked aptamer assay (ELAA) confirmed binding affinity and specificity against other AIV subtypes. Finally, we studied aptamer-inhibitory effects on H9N2 AIV infection in Madin–Darby canine kidney (MDCK) cells and quantified viral load in supernatant and in cell with quantitative PCR (qPCR). Our data provide a foundation for future development of innovative anti-influenza drugs.     

共表达新城疫病毒F基因和H9亚型禽流感病毒HA基因的重组鸡痘病毒

应用RT PCR扩增出新城疫病毒F4 8E8株融合蛋白 (F)基因 ,将其克隆入pGEM Teasyvector构建重组质粒pGEM TF并进行测序确证。分别从pGEM T和pUCHA切下F基因和H9亚型禽流感病毒F株 (A chicken china F 1 998)血凝素 (HA)基因 ,通过一系列分子生物学操作步骤插入到质粒pFPV7S中的鸡痘病毒基因组复制非必需片段构建重组质粒p7SHF ,其中F基因和HA基因分别由鸡痘病毒启动子PE L和合成启动子PS调控。最后将P1 1 LacZ报告基因表达盒插入质粒p7SHF获得转移载体pFPVHF ,用以转染已预先感染鸡痘病毒 2 82E4疫苗株的鸡胚成纤维细胞 (CEF)。通过在含有X Gal的营养琼脂上连续挑选蓝色病毒蚀斑获得并纯化重组病毒。PCR和Southernblot检测证实了F基因和HA基因已插入鸡痘病毒的基因组 ;间接免疫荧光试验结果表明重组病毒能够同时正确表达HA和F蛋白。     

用8质粒病毒拯救系统产生H9N2/WSN重组A型流行性感冒病毒

把禽流行性感冒(流感)病毒A／Chicken／Shanghai／F／98(H9N2)的血凝素(HA)和神经氨酸酶(NA)基因cDNA克隆至polⅠ-pol Ⅱ双向转录和表达载体pHW2000,用这两种质粒与8质粒病毒拯救系统中流感病毒A／WSN／33(H1N1)6个内部基因cDNA的质粒组合(6 2重排),共转染COS-1细胞,产生了能在鸡胚中高滴度增殖的H9N2／、WSN重组病毒。用A／WSN／33的8个基因cDNA质粒作对照,也产生了转染子病毒。经过EID50测定和MDCK感染实验,新基因型H9N2／WSN病毒感染鸡胚的能力强(EID50为10^-11／0．2m1),而且对鸡胚的毒力弱,在不加胰酶的情况下不使MDCK细胞产牛病变。经电镜观察,两个转染子病毒的形态与野生型流感病毒相似。反向遗传操作技术的建立,为对禽流感病毒基因功能和疫苗构建等方面的研究提供了新的手段。     

Genetic Characterization of a Novel Recombinant H5N2 Avian Influenza Virus Isolated from Chickens in Tibet

In this report, a novel H5N2 avian influenza virus (AIV) was isolated from chickens in Tibet in 2010, western China. Phylogenetic analysis demonstrated that it was a natural reassortant between H9N2 and H5N1 subtypes. It is of note that this virus has an HP genotype with HA, PB2, M, and NS genes homologous to those of A/peregrine falcon/Hong Kong/2142/2008(H5N1)-like HPAIV isolated from dead wild birds. Publishing this genome information will contribute to the investigation of avian influenza epidemiology and to further research of AIV''s biological properties.     

Antigenic Mapping of the Hemagglutinin of an H9N2 Avian Influenza Virus Reveals Novel Critical Amino Acid Positions in Antigenic Sites

H9N2 influenza virus is undergoing extensive genetic and antigenic evolution, warranting detailed antigenic mapping of its hemagglutinin (HA). Through examining antibody escape mutants of an Asian avian H9N2 virus, we identified 9 critical amino acid positions in H9 antigenic sites. Five of these positions, 164, 167, 168, 196, and 207, have not been reported previously and, thus, represent novel molecular markers for monitoring the antigenic change of H9N2 virus.     

H9N2禽流感病毒反向遗传系统转录/表达载体的构建和验证

设计带有BsmBI、BsaⅠ或AarⅠ酶切位点的引物,用RT PCR扩增H9N2亚型禽流感病毒(AIV)的8个基因全长片段,克隆入双向转录/表达载体pHW2000,并在PB2、PB1和NA基因中共引入了3个沉默突变标签.将其2个表面基因(HA和NA基因)加上任意1个内部基因,而其它5个内部基因来自A/WSN/33,进行了6种3+5组合形式的基因重排,把相应组合的转录/表达质粒共转染COS-1细胞,均产生了预期组合、有感染性的H9N2亚型流感病毒,表明亲缘关系遥远的流感病毒可以互相获取基因片段产生重组病毒,提示表面结构基因和单个内部基因不足以限制H9N2 AIV在哺乳动物细胞上的宿主范围,同时也验证了构建的8个转录/表达载体均能有效工作,为进一步研究H9N2亚型AIV基因结构与功能、AIV与宿主之间的关系打下了基础.     

H9N2禽流感病毒反向遗传系统转录／表达载体的构建和验证

设计带有BsmBI、BsaI或AarI酶切位点的引物,用RT-PCR扩增H9N2亚型禽流感病毒(AIV)的8个基因全长片段,克隆入双向转录/表达载体pHW2000,并在PB2、PB1和NA基因中共引入了3个沉默突变标签。将其2个表面基因(HA和NA基因)加上任意1个内部基因,而其它5个内部基因来自A/WSN/33,进行了6种3 5组合形式的基因重排,把相应组合的转录/表达质粒共转染COS-1细胞,均产生了预期组合、有感染性的H9N2亚型流感病毒,表明亲缘关系遥远的流感病毒可以互相获取基因片段产生重组病毒,提示表面结构基因和单个内部基因不足以限制H9N2AIV在哺乳动物细胞上的宿主范围,同时也验证了构建的8个转录/表达载体均能有效工作,为进一步研究H9N2亚型AIV基因结构与功能、AIV与宿主之间的关系打下了基础。     

The Therapeutic Effect of Pamidronate on Lethal Avian Influenza A H7N9 Virus Infected Humanized Mice

A novel avian influenza virus H7N9 infection occurred among human populations since 2013. Although the lack of sustained human-to-human transmission limited the epidemics caused by H7N9, the late presentation of most patients and the emergence of neuraminidase-resistant strains made the development of novel antiviral strategy against H7N9 in urgent demands. In this study, we evaluated the potential of pamidronate, a pharmacological phosphoantigen that can specifically boost human Vδ2-T-cell, on treating H7N9 virus-infected humanized mice. Our results showed that intraperitoneal injection of pamidronate could potently decrease the morbidity and mortality of H7N9-infected mice through controlling both viral replication and inflammation in affected lungs. More importantly, pamidronate treatment starting from 3 days after infection could still significantly ameliorate the severity of diseases in infected mice and improve their survival chance, whereas orally oseltamivir treatment starting at the same time showed no therapeutic effects. As for the mechanisms underlying pamidronate-based therapy, our in vitro data demonstrated that its antiviral effects were partly mediated by IFN-γ secreted from human Vδ2-T cells. Meanwhile, human Vδ2-T cells could directly kill virus-infected host cells in a perforin-, granzyme B- and CD137-dependent manner. As pamidronate has been used for osteoporosis treatment for more than 20 years, pamidronate-based therapy represents for a safe and readily available option for clinical trials to treat H7N9 infection.     

A Combination of Serological Assays to Detect Human Antibodies to the Avian Influenza A H7N9 Virus

Human infection with avian influenza A H7N9 virus was first identified in March 2013 and represents an ongoing threat to public health. There is a need to optimize serological methods for this new influenza virus. Here, we compared the sensitivity and specificity of the hemagglutinin inhibition (HI), microneutralization (MN), and Western blot (WB) assays for the detection of human antibodies against avian influenza A (H7N9) virus. HI with horse erythrocytes (hRBCs) and a modified MN assay possessed greater sensitivity than turkey erythrocytes and the standard MN assay, respectively. Using these assays, 80% of tested sera from confirmed H7N9 cases developed detectable antibody to H7N9 after 21 days. To balance sensitivity and specificity, we found serum titers of ≥20 (MN) or 160 (HI) samples were most effective in determining seropositive to H7N9 virus. Single serum with HI titers of 20–80 or MN titer of 10 could be validated by each other or WB assay. Unlike serum collected from adult or elderly populations, the antibody response in children with mild disease was low or undetectable. These combinations of assays will be useful in case diagnosis and serologic investigation of human cases.     

H9N2禽流感病毒M2 DNA疫苗初免-蛋白加强免疫的保护效果研究

流感病毒M2(基质蛋白2)是A型流感病毒的一个高度保守的蛋白。由于其免疫原性较弱,本研究采用M2 DNA疫苗初免-蛋白加强的策略来考察M2的免疫保护效果。制备A/Chicken/Jiangsu/07/2002(H9N2)流感病毒的M2 DNA疫苗以及经大肠杆菌表达的去除M2跨膜区的M2蛋白即sM2。以SPF级BALB/c小鼠为模型,电击法免疫M2 DNA疫苗,滴鼻法免疫sM2蛋白,免疫间隔三周,并于末次免疫后三周以致死量5LD50流感病毒H9N2攻击小鼠,通过检测小鼠存活率、体重丢失率、肺部病毒滴度及IgG抗体水平等指标来评价免疫的保护效果。实验结果表明,基于M2的疫苗采用DNA疫苗初免蛋白加强免疫二次的免疫程序能诱导较高的特异性抗体,明显减轻小鼠流感病症,提供完全的保护。     

Potential Geographic Distribution of the Novel Avian-Origin Influenza A (H7N9) Virus

Background

In late March 2013, a new avian-origin influenza virus emerged in eastern China. This H7N9 subtype virus has since infected 240 people and killed 60, and has awakened global concern as a potential pandemic threat. Ecological niche modeling has seen increasing applications as a useful tool in mapping geographic potential and risk of disease transmission.

Methodology/Principals

We developed two datasets based on seasonal variation in Normalized Difference Vegetation Index (NDVI) from the MODIS sensor to characterize environmental dimensions of H7N9 virus. One-third of well-documented cases was used to test robustness of models calibrated based on the remaining two-thirds, and model significance was tested using partial ROC approaches. A final niche model was calibrated using all records available.

Conclusions/Significance

Central-eastern China appears to represent an area of high risk for H7N9 spread, but suitable areas were distributed more spottily in the north and only along the coast in the south; highly suitable areas also were identified in western Taiwan. Areas identified as presenting high risk for H7N9 spread tend to present consistent NDVI values through the year, whereas unsuitable areas show greater seasonal variation.     

Quantitative Characterization of Glycan-Receptor Binding of H9N2 Influenza A Virus Hemagglutinin

Avian influenza subtypes such as H5, H7 and H9 are yet to adapt to the human host so as to establish airborne transmission between humans. However, lab-generated reassorted viruses possessing hemagglutinin (HA) and neuraminidase (NA) genes from an avian H9 isolate and other genes from a human-adapted (H3 or H1) subtype acquired two amino acid changes in HA and a single amino acid change in NA that confer respiratory droplet transmission in ferrets. We previously demonstrated for human-adapted H1, H2 and H3 subtypes that quantitative binding affinity of their HA to α2→6 sialylated glycan receptors correlates with respiratory droplet transmissibility of the virus in ferrets. Such a relationship remains to be established for H9 HA. In this study, we performed a quantitative biochemical characterization of glycan receptor binding properties of wild-type and mutant forms of representative H9 HAs that were previously used in context of reassorted viruses in ferret transmission studies. We demonstrate here that distinct molecular interactions in the glycan receptor-binding site of different H9 HAs affect the glycan-binding specificity and affinity. Further we show that α2→6 glycan receptor-binding affinity of a mutant H9 HA carrying Thr-189→Ala amino acid change correlates with the respiratory droplet transmission in ferrets conferred by this change. Our findings contribute to a framework for monitoring the evolution of H9 HA by understanding effects of molecular changes in HA on glycan receptor-binding properties.     

H9N2亚型禽流感病毒广东分离株的全基因克隆及序列分析

本研究以一株2006年广东省分离的H9N2亚型禽流感病毒A/Chicken/Guangdong/HL/2006(H9N2)(简称Ck/GD/HL/06)为研究对象,用RT-PCR法扩增病毒基因组各片段(包括5′端和3′端的非编码区序列),将扩增片段进行克隆、测序并与参考毒株的相应序列进行比较分析,绘制各基因片段的系统发生树。分析结果表明,Ck/GD/HL/06株的HA基因同1997年中国香港鸭源毒株Dk/HK/Y280/97(H9N2)在同一进化分支,从HA的糖基化位点、受体结合位点等综合分析,该毒株HA基因未发生明显的变异,符合我国大陆H9亚型禽流感病毒的特点。HA的226位氨基酸残基为亮氨酸(Leu),具有同哺乳动物SAα,2-6受体结合的特性。Ck/GD/HL/06的PB1、PA和NP基因,同2004年越南分离的人源高致病性H5N1亚型流感病毒A/VietNam/1203/2004(H5N1)株(简写A/VN/1203/04)的核苷酸序列一致性分别是93.8%、95%和96.8%,在先前的研究中未见有类似特性毒株的报道,而这种特性H9N2亚型AIV的出现,是否会增加在重组过程中产生新的高致病性H5N1亚型AIV的可能性,是值得我们关注的一个问题,也提醒在我国华南地区应更加重视防控H9N2亚型AIV,做好长期对H9N2亚型AIV监控及分子流行病学调查的工作。