Antibodies elicited by Newcastle disease virus-vectored H7N9 avian influenza vaccine are functional in activating the complement system

H7N9 subtype avian influenza virus poses a great challenge for poultry industry.Newcastle disease virus(NDV)-vectored H7N9 avian influenza vaccines(NDV_(vec)H7N9)are effective in disease control because they are protective and allow mass administration.Of note,these vaccines elicit undetectable H7N9-specific hemagglutination-inhibition(HI)but high IgG antibodies in chickens.However,the molecular basis and protective mechanism underlying this particular antibody immunity remain unclear.Herein,immunization with an NDV_(vec)H7N9 induced low anti-H7N9 HI and virus neutralization titers but high levels of hemagglutinin(HA)-binding IgG antibodies in chickens.Three residues(S150,G151 and S152)in HA of H7N9 virus were identified as the dominant epitopes recognized by the NDV_(vec)H7N9 immune serum.Passively transferred NDV_(vec)H7N9 immune serum conferred complete protection against H7N9 virus infection in chickens.The NDV_(vec)H7N9 immune serum can mediate a potent lysis of HA-expressing and H7N9 virus-infected cells and significantly suppress H7N9 virus infectivity.These activities of the serum were significantly impaired after heat-inactivation or treatment with complement inhibitor,suggesting the engagement of the complement system.Moreover,mutations in the 150-SGS-152 sites in HA resulted in significant reductions in cell lysis and virus neutralization mediated by the NDV_(vec)H7N9 immune serum,indicating the requirement of antibody-antigen binding for complement activity.Therefore,antibodies induced by the NDV_(vec)H7N9 can activate antibody-dependent complement-mediated lysis of H7N9 virus-infected cells and complement-mediated neutralization of H7N9 virus.Our findings unveiled a novel role of the complement in protection conferred by the NDV_(vec)H7N9,highlighting a potential benefit of engaging the complement system in H7N9 vaccine design.

2014-2018年新疆人感染H7N9禽流感分子流行病学特征分析

目的分析2014-2018年新疆14例人感染H7N9禽流感病例的分子流行病学特征,为防控及治疗提供科学依据。方法提取核酸后采用高通量基因测序技术获得基因组序列,并用生物信息学分析软件对基因组序列进行同源性分析、进化分析、变异位点分析及同源建模。结果14例人感染H7N9禽流感病毒HA基因核苷酸同源性介于97.39%~100%,氨基酸同源性介于98.38%~100%;NA基因核苷酸同源性介于97.73%~100%,氨基酸同源性介于97.73%~100%,均属于长江三角洲谱系和低致病性禽流感病毒,分属于2个亚分支,与A/Hunan/02650/2016疫苗株最为接近。HA蛋白G186V、T160A全部发生了变异,Q226L中13株病毒发生了变异,NA蛋白4个关键的神经氨酸酶抑制剂耐药位点全部未发生变异。M2蛋白S31N和V27A位点全部发生了突变,PB1蛋白T368V位点全部发生了突变,PA蛋白位点K356R全部发生了突变。结论新疆H7N9禽流感病毒具有双受体结合性,对金刚烷胺类药物耐药,对神经氨酸酶抑制剂敏感,可在疾病发生早期及时使用神经氨酸酶抑制剂。需加强禽流感病毒基因组变化的监测,做好基因突变的应对措施。

抗H7N9流感病毒的单克隆抗体与胃组织的交叉反应研究

目的筛选鉴定与胃组织交叉反应的抗H7N9流感病毒的单克隆抗体,初步探讨流感病毒感染会引发肠道相关疾病的可能致病机制。方法利用H7N9流感病毒制备了大量的单克隆抗体,对制备的单克隆抗体运用ELISA方法进行亚型和效价测定,免疫组化对制备的单克隆抗体与组织的反应性进行筛选,巢式PCR分子生物学技术克隆单克隆抗体轻重链可变区序列。结果制备的单克隆抗体H7N9-71和H7N9-78均能与H7N9抗原特异性结合,抗体效价达1∶10~5,免疫组化结果表明H7N9-71、H7N9-78与人的胃组织能发生特异性反应,进一步的研究发现这两株单抗与小鼠胃组织能特异性结合。轻重链可变区序列结果证实二者为两株不同的单抗。结论H7N9流感病毒可能与胃组织之间存在异嗜性抗原,提示流感病毒感染可能与胃肠道疾病的发生存在某些关联。

中国H7N9禽流感遗传演化趋势及防控措施

H7N9禽流感病毒(AIV)感染人类的情况最先出现在中国,而且具有很高的突变性。2013年首次在人类身上分离出的是低致病性H7N9禽流感病毒,发展到2017年,已经出现高致病性H7N9禽流感病毒,严重威胁动物和人类的生命健康安全,同时,还会对社会的稳定和经济发展有一定的影响。综述通过NCBI(National Center for Biotechnology Information)和GISAID数据库收集了中国国内2001~2021年共4641条有关H7N9的数据,拟通过流行病学的角度来说明H7N9禽流感病毒在中国的发生、发展以及在时间、空间和种群间的分布情况,同时总结中国对H7N9所引起的大流行所采取的防控措施,以期为后续的疫情防控措施规划提供依据。

Genetic and biological properties of H9N2 avian influenza viruses isolated in central China from2020 to 2022

The H9N2 subtype of avian influenza virus(AIV)is widely prevalent in poultry and wild birds globally,and has become the predominant subtype circulating in poultry in China.The H9N2 AIV can directly or indirectly(by serving as a"donor virus")infect humans,posing a significant threat to public health.Currently,there is a lack of in-depth research on the prevalence of H9N2 viruses in Shanxi Province,central China.In this study,we isolated 14 H9N2 AIVs from October 2020 to April 2022 in Shanxi Province,and genetic analysis revealed that these viruses belonged to 7 different genotypes.Our study on animals revealed that the H9N2 strains we identified displayed high transmission efficiency among chicken populations,and exhibited diverse replication abilities within these birds.These viruses could replicate efficiently in the lungs of mice,with one strain also demonstrating the capacity to reproduce in organs like the brain and kidneys.At the cellular level,the replication ability of different H9N2 strains was evaluated using plaque formation assays and multi-step growth curve assays,revealing significant differences in the replication and proliferation efficiency of the various H9N2 viruses at the cellular level.The antigenicity analysis suggested that these isolates could be classified into 2 separate antigenic clusters.Our research provides crucial data to help understand the prevalence and biological characteristics of H9N2 AIVs in central China.It also highlights the necessity of enhancing the surveillance of H9N2 AIVs.

2022年湖南省家禽H7N9亚型禽流感监测与分析

为了解湖南省家禽(鸡、鸭、鹅)H7N9亚型禽流感的免疫抗体水平及其流行情况,2022年在全省14个市州,采用配额抽样方法,在种禽场、商品代场、散养户和活禽市场共采集46112份禽血清和53246份禽拭子样品,通过血凝抑制试验、荧光定量PCR方法分别进行免疫抗体和病毒核酸检测。结果显示:H7N9亚型禽流感免疫抗体平均场群合格率为93.27%,平均个体合格率为90.60%,仅在活禽市场的1份鸡拭子样品中检测到病毒核酸阳性;鸡H7N9亚型禽流感免疫抗体平均场群合格率和个体合格率(94.66%和91.51%)均高于水禽(83.26%和81.86%);种禽场的免疫抗体平均场群合格率和个体合格率最高,活禽市场最低,两者差异具有统计学意义(P<0.05);全省14个市州的H7N9亚型禽流感免疫抗体平均场群合格率和个体合格率均在70%以上。结果表明:2022年湖南省家禽H7N9亚型禽流感免疫效果较好,在饲养场禽群中也未检出病原,发生大规模疫情的风险较低,但在活禽市场中仍检出病原,且水禽以及散养户和活禽市场禽群的免疫抗体水平略低,疫情散发风险依然存在。建议进一步加强对H7N9亚型禽流感的免疫监测以及动物检疫监管工作,不断提升公共卫生安全防护水平。

鸽H9N2亚型禽流感病毒的分离鉴定及致病性试验

H9N2亚型禽流感病毒属于A型流感病毒,目前H9N2亚型禽流感病毒不仅可以感染家禽,而且可以感染猪、猫等哺乳动物,甚至出现人类感染现象。因此,H9N2亚型流感病毒不仅影响畜禽的健康养殖,同时也具有一定的公共卫生学意义。近年来鸽群感染流感病毒时有报道,这其中包括H5N1、H7N9以及H9N2亚型禽流感病毒。虽然H9N2亚型禽流感毒株的致病性相对于H5和H7亚型较低,但是该亚型病毒在我国家禽中存在较为普遍,且目前从鸽子已分离到重组的H9N2毒株,该毒株不仅引起鸽子感染而且具有优先与人类呼吸道表面受体结合的能力,因此加强流感病毒在鸽群中流行情况具有重要的现实意义。

H7N9亚型禽流感病毒HA基因mRNA的构建

H7N9亚型禽流感病毒(AIV)持续给家禽及人类健康造成威胁。为构建H7N9亚型AIV(简称H7N9 AIV)mRNA疫苗,本研究以AIV分离株A/chicken/Yunnan/SD024/2021(H7N9)(简称CK/YN/SD024/2021)的HA基因为目的基因、选择来自人β-球蛋白和非洲爪蟾β-球蛋白的不同编码基因序列作为HA基因的非翻译区(UTR),构建了中间转录质粒pGEM-YN和pXT7-YN,以2个质粒为模板通过体外转录制备了mRNA mH7-YN-pGEM和mH7-YN-pXT7;将2种mRNA转染HEK293T细胞,western blot鉴定结果显示2种mRNA转染的细胞均可检测到约为70 ku的HA蛋白条带和50 ku的HA1蛋白条带;通过间接免疫荧光试验(IFA)确定最适转染剂量和HEK293T细胞中HA蛋白的动态表达,结果显示,3μg mRNA转染106个细胞并于转染24 h后荧光信号强度均最强;将2种mRNA转染鸡胚成纤维细胞(CEF),均可通过IFA检测到HA蛋白的表达。以上结果表明,本研究正确构建了2种含H7N9 AIV HA基因的mRNA,且其均能够在HEK293T细胞和CEF中正确表达HA蛋白,3μg mRNA转染HEK293T细胞可获得最佳的蛋白表达水平,转染24 h后HA蛋白的表达水平达到最高。本研究基于UTR序列的差异首次正确构建了两种能在哺乳动物细胞和禽类细胞中高效表达H7N9 AIV HA蛋白的mRNA,为禽流感mRNA疫苗的研发和免疫效力评估奠定了良好的物质和技术基础。

What clinicians should know to fight against the novel avian-origin influenza A （H7N9） virus？

China has markedly increased infectious disease surveillance efforts after the outbreak of severe acute respiratory syndrome （SARS） in 2003.＇ When the first pneumonia cases with unknown etiology from Shanghai were reported to the National Health and Family Planning Commission and the China Center of Disease Control and Prevention （CDC） in March 2013, our Chinese doctors and scientists show more confident to face the emerging infectious disease than 10 years ago.

稳定表达H7N9亚型禽流感病毒HA蛋白的293T细胞株构建及应用

为获得用于评价H7N9亚型禽流感疫苗诱导的抗体Fc免疫效应的靶细胞,研究采用慢病毒包装与感染技术构建一株稳定表达H7N9亚型禽流感病毒(H7N9 AIV)HA蛋白的细胞株,并以该细胞株为靶细胞建立H7N9亚型禽流感疫苗免疫血清补体介导的细胞毒(ADCML)活性的检测方法。结果显示成功包装了一株重组慢病毒,滴度可达108 FU/mL;构建了稳定表达H7N9 AIV HA蛋白的293T细胞株,HA蛋白主要在细胞膜上表达。基于新城疫病毒(NDV)载体的H7N9亚型禽流感疫苗免疫鸡后,诱导产生的HA结合性IgG抗体滴度为2180。以稳定细胞株为靶细胞,载体疫苗血清诱导的细胞死亡水平显著高于空载体与未免疫鸡血清(P<0.001);补体灭活后,载体疫苗血清诱导的细胞死亡水平显著降低(P<0.001)。研究表明,基于NDV载体的H7N9亚型禽流感疫苗免疫鸡血清具有较强的ADCML活性,稳定表达H7N9 AIV HA蛋白的细胞株可作为评价H7N9亚型禽流感疫苗免疫血清Fc免疫效应的靶细胞。

鸡鸭H7N9禽流感和新城疫抗体水平检测结果分析

为掌握衢州市衢江区鸡鸭H7N9禽流感和新城疫的免疫效果,笔者从2019至2021年连续3年对辖区内的规模养殖场及散养户鸡和鸭开展免疫抗体水平检测。检测结果表明,鸡H7N9禽流感、鸡新城疫及鸭H7N9禽流感的抗体合格率分别为93.91%、79.75%及90.96%,均达到国家规定合格率,其中鸡H7N9禽流感的抗体合格率显著高于鸡新城疫的抗体合格率(P=0.014);规模场的新城疫抗体合格率为88.37%显著高于散养户的抗体合格率61.15%(P=0.012);H7N9禽流感抗体合格率与新城疫抗体合格率在上半年和下半年间没有显著差异,说明季节对抗体合格率的影响较小。

Human infection with a novel avian-origin influenza A （H7N9） virus： serial chest radiographic and CT findings

Background Rapidly progressive pneumonia infection with H7N9 virus is a novel disease,and limited information is available concerning serial chest radiographic and computed tomography （CT） findings.The aim of this study was to evaluate the changes in serial radiologic findings in patients with H7N9 pneumonia.Methods The two institutional ethics review boards approved this retrospective study.This study included 10 patients with H7N9 pneumonia.All patients underwent chest radiologic examinations at different time points.Serial radiologic images were systematically analyzed.Results All patients showed abnormal results on initial chest radiography and CT.The initial radiographic abnormalities were unilateral （n=9） and bilateral （n=1）,including ground-glass opacities （GGOs） （n=5） and consolidation （n=5）.The initial CT findings consisted of unilateral （n=6） and bilateral （n=4）,including consolidation （n=10）,GGOs （n=10）,reticular opacities （n=2）,and pleural effusion （n=3）.Follow-up radiologic findings showed rapid development of consolidation or GGOs within two weeks after illness onset.Pneumomediastinum with secondary subcutaneous emphysema and pneumothorax were noted in two patients.Follow-up high resolution computed tomography （HRCT） after two weeks showed slow improvement in both size and opacity of the lesions.On HRCT after discharge,patients had substantial residual lesions such as irregular linear opacities,reticular opacities,parenchymal bands,traction bronchiectasis,and cystic lesions.Conclusions The most common radiologic findings at presentation are multifocal or diffuse areas of consolidation and GGOs in H7N9 pneumonia.HRCT in sequence can show more changes in rapid progression of disease and a slow decrease of both size and opacity of the lesions plays an important role in the evaluation of H7N9 pneumonia.

Risk assessment on the epidemics of human infection with a novel avian influenza A (H7N9) virus in Jiangsu Province, China

A novel avian influenza A （H7N9） virus was discovered in February 2013 in China and has resulted in more than 100 comfirmed human infections including 26 fatal cases as of May 2, 2013. The situation raises many ur- gent questions and global public health concerns. In this study, epidemiologic characteristics of infected human cases in Jiangsu province were analyzed and risk assessment was undertaken based on the information available. Briefly, it is highly unlikely that a pandemic of human infection with avian influenza A （HTN9） virus will happen in Jiangsu Province in the near future. Iia the end, some measures are recommended to prevent the situation from becoming worse.

Bedside chest radiography of novel influenza A （H7N9） virus infections and follow-up findings after short-time treatment

Background Influenza A （H7Ng） virus infections were first observed in China in March 2013.This type virus can cause severe illness and deaths,the situation raises many urgent questions and global public health concerns.Our purpose was to investigate bedside chest radiography findings for patients with novel influenza A （H7Ng） virus infections and the followup appearances after short-time treatment.Methods Eight hospitalized patients infected with the novel influenza A （H7Ng） virus were included in our study.All of the patients underwent bedside chest radiography after admission,and all had follow-up bedside chest radiography during their first ten days,using AXIOM Aristos MX and/or AMX-Ⅳ portable X-ray units.The exposure dose was generally 90 kV and 5 mAs,and was slightly adjusted according to the weight of the patients.The initial radiography data were evaluated for radiological patterns （ground glass opacity,consolidation,and reticulation）,distribution type （focal,multifocal,and diffuse）,lung zones involved,and appearance at follow-up while the patients underwent therapy.Results All patients presented with bilateral multiple lung involvement.Two patients had bilateral diffuse lesions,three patients had unilateral diffuse lesions of the right lobe with multifocal lesions of the left lobe,and the remaining three had bilateral multifocal lung lesions.The lesions were present throughout bilateral lung zones in three patients,the whole right lung zone in three patients with additional involvement in the left middle and/or lower lung zone（s）,both lower and middle lung zones in one patient,and the right middle and lower in combination with the left lower lung zones in one patient.The most common abnormal radiographic patterns were ground glass opacity （8/8）,and consolidation （8/8）.In three cases examined by CT we also found the pattern of reticulation in combination with CT images.Four patients had bilateral and four had unilateral pleural effusion.After a short period of treatment the pneumonia in one patient had significantly improved and three cases demonstrated disease progression.In four cases the severity of the pneumonia fluctuated.Conclusions In patients with influenza A （H7N9） virus infection,the distribution of the lung lesions are extensive,and the disease usually involves both lung zones.The most common imaging findings are a mixture of ground glass opacity and consolidation.Pleural effusion is common.Most cases have a poor short-time treatment response,and seem to have either rapid progressive radiographic deterioration or fluctuating radiographic changes.Chest radiography is helpful for evaluating patients with severe clinical symptoms and for follow-up evaluation.

Clinical Study of Mesenchymal Stem Cell Treatment for Acute Respiratory Distress Syndrome Induced by Epidemic Influenza A(H7N9) Infection: A Hint for COVID-19 Treatment

2013年,H7N9病毒在哺乳动物宿主之间迅速传播,并具有人与人之间传播的风险。患者表现为重症肺炎、急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)和呼吸衰竭。间充质干细胞(mesenchymal stem cell,MSC)移植在病毒性肺炎的治疗中有广阔的前景,并于2013年作为应急性治疗方法来治疗H7N9感染的ARDS。该研究在单中心开展,为开放式临床研究。根据自愿和知情同意的原则,17例H7N9感染的ARDS患者移植同种异体经血来源的MSC作为治疗组,44例H7N9感染的ARDS患者作为对照组。与对照组相比,MSC移植显著降低了ARDS病死率(MSC治疗组和对照组死亡率分别为17.6%和54.5%)。此外,在对4名MSC移植患者进行为期5年的随访中,未发现MSC移植产生有害影响。总体而言,该研究结果表明MSC移植显著提高了H7N9感染的ARDS的存活率,并为开展临床前和临床应用MSC治疗H7N9感染的ARDS的研究提供了理论基础。由于H7N9和新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)临床表现相似(如ARDS和呼吸衰竭),并继发多器官功能障碍,因此基于MSC的疗法可能为COVID-19所致的ARDS患者获益。

Characterization of Avian Influenza A(H7N9) Virus Prevalence in Humans and Poultry in Huai′an,China：Molecular Epidemiology,Phylogenetic,and Dynamics Analyses

Objective To trace the source of human H7N9 cases in Huai＇an and elucidate the genetic characterization of Huai＇an strains associated with both humans and birds in live poultry market.Methods An enhanced surveillance was implemented when the first human H7N9 case was confirmed in Huai＇an.Clinical specimens,cloacal swabs,and fecal samples were collected and screened by real-time reverse transcription-polymerase chain reaction（RT-PCR） for H7N9 virus.The positive samples were subjected to further RT-PCR and genome sequencing.The phylodynamic patterns of H7N9 virus within and separated from Huai＇an and evolutionary dynamics of the virus were analyzed.Results Six patients with H7N9 infection were previously exposed to live poultry market and presented symptoms such as fever（〉38.0 °C） and headaches.Results of this study support the hypothesis that live poultry markets were the source of human H7N9 exposure.Phylogenetic analysis revealed that all novel H7N9 viruses,including Huai＇an strains,could be classified into two distinct clades,A and B.Additionally,the diversified H7N9 virus circulated in live poultry markets in Huai＇an.Interestingly,the common ancestors of the Huai＇an H7N9 virus existed in January 2012.The mean nucleotide substitution rates for each gene segment of the H7N9 virus were（3.09-7.26）×10-3 substitutions/site per year（95% HPD：1.72×10-3 to 1.16×10-2）.Conclusion Overall,the source of exposure of human H7N9 cases in Huai＇an was live poultry market,and our study highlights the presence of divergent genetic lineage of H7N9 virus in both humans and poultry specimens in Huai＇an.

Development of a real-time RT-PCR method for the detection of newly emerged highly pathogenic H7N9 influenza viruses

In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China. Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin（HA） cleavage site, leading to the emergence of a highly pathogenic virus. The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza. Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus. One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established. Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity. The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs. Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples. Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.

H7N9 influenza-the laboratory presentations: A letter to editor

To the editor,1.Introduction to new H7N9 bird flu Influenza virus infection is an important group of respiratory infectious disease.Several groups of influenza viruses can cause human infections.However,in the recent years,there are many newly emerging influenza infections,which have never been previously seen in human.Crossing species from animal to human lead

A Suspected Person-to-person Transmission of Avian Influenza A （H7N9） Case in Ward

Since throat swab specimens obtained from three adult Chinese patients were confirmed as an avian-origin influenza A （H7N9） virus by local Centers for Disease Control and Prevention （CDC） in 2013,many confirmed cases have been reported in Mainland of China.

Clinical variability in onset of influenza A （H7N9） infection

Clinical information of infections with the novel avian influenza A （H7N9） have been described elsewhere,but clinical variability, especially changes ot mental status in the onset of infection, has rarely been reported in patients infected with H7N9. In this report, the initial clinical manifestations of three confirmed cases are summarized. Two of the patients were in critical condition. In addition,