Research progress in human infection with avian influenza H7N9 virus

Since the identification of the novel reassortant avian influenza A(H7N9) virus in China in 2013, until Jun 30, 2017, the virus has caused five epidemic waves leading to a total of 1,552 human infections, with a fatality rate of about 40%. In the spring of2017, highly pathogenic avian influenza(HPAI) H7N9 virus emerged and has caused 25 human infections. The HPAI H7N9 virus has some biological differences from the LPAI one, such as its multiple basic amino acid residues on HA leading to its independence on trypsin for replication. The pathogenicity of the HPAI H7N9 virus to experimental animals or humans is still unclear. A(H7N9) vaccine development for pandemic preparedness is ongoing, including the reassortment(H7N9/PR8)reverse genetic based vaccine, the virus like particle(VLP) vaccine, the intranasal live attenuated influenza vaccine(LAIV),the non-adjuvant Vero cell culture-derived inactivated whole-virus vaccine, the MDCK culture-derived vaccine, the H7 DNA vaccine and the recombinant replicative H7N9 virus \(H7N9-53 TM\) vaccine. Five neuramidinase resistant sites of A(H7N9)virus isolated from patients have been reported. Some alternative drugs have been studied, such as DAS181(Fludase), ribavirin,troglitazone and minocycline. Persistent surveillance and enhanced global control are essential to fight against human infections with A(H7N9) virus.

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.

Conservation of T cell epitopes between seasonal influenza viruses and the novel influenza A H7N9 virus

A novel avian influenza A(H7N9) virus recently emerged in the Yangtze River delta and caused diseases, often severe, in over 130 people. This H7N9 virus appeared to infect humans with greater ease than previous avian influenza virus subtypes such as H5N1 and H9N2. While there are other potential explanations for this large number of human infections with an avian influenza virus, we investigated whether a lack of conserved T-cell epitopes between endemic H1N1 and H3N2 influenza viruses and the novel H7N9 virus contributes to this observation. Here we demonstrate that a number of T cell epitopes are conserved between endemic H1N1 and H3N2 viruses and H7N9 virus. Most of these conserved epitopes are from viral internal proteins. The extent of conservation between endemic human seasonal influenza and avian influenza H7N9 was comparable to that with the highly pathogenic avian influenza H5N1. Thus, the ease of inter-species transmission of H7N9 viruses(compared with avian H5N1 viruses) cannot be attributed to the lack of conservation of such T cell epitopes. On the contrary, our findings predict significant T-cell based cross-reactions in the human population to the novel H7N9 virus. Our findings also have implications for H7N9 virus vaccine design.

The protective effects of a D-tetra-peptide hydrogel adjuvant vaccine against H7N9 influenza virus in mice

Repeated waves of influenza virus H7N9 epidemics after 2013 have caused severe influenza in humans,with mortality reaching approximately 40%–50%.To prevent possible pandemics,the development of highly effective vaccines against influenza virus H7N9 is highly desired.In the present study,by taking advantage of the D-tetra-peptide adjuvant(G^(D)F^(D)F^(D)Y),we reported a simple method to prepare H7N9 vaccines.Naproxen(Npx),with good anti inflammatory and broad anti-viral effects,was employed as an N-terminal capping group to construct a hydrogel precursor,Npx-G^(D)F^(D)F^(D)Y.The hydrogel adjuvant was prepared using a routine heating cooling protocol and the final vaccine was ready after mixing with the split A/Zhejiang/DTID-ZJU01/2013(H7N9)antigen by vortexing.Compared with the traditional Al(OH)_(3) adjuvant vaccine and the split vaccine,our hydrogel adjuvant vaccine showed the best preventive effects against H7N9 infection.A mechanistic study illustrated that higher antibody responses and variations in cytokine expression might account for its increased protective effects.Our strategy demonstrated the advantages of a peptide hydrogel adjuvant in the application of vaccines against H7N9 and demonstrated its potential application in vaccines against emerging threats from other viruses.

H7N9禽流感病毒在小鼠体内的适应性

目的进一步探讨H7N9禽流感病毒对哺乳动物的感染与致病能力,并对其分子机制进行研究。方法以病毒滴鼻感染小鼠,在BALB/c小鼠肺组织中连续传代,通过病毒全基因组测序及比对探寻适应的分子机制。结果禽流感H7N9 A/Anhui/1/2013病毒,经过在小鼠体内进行9次传代后,对鼠肺适应株与野生型毒株进行基因比对,发现适应株HA基因,NA基因以及PA亚基共发生了4个有义突变。结论禽流感H7N9 A/Anhui/1/2013病毒经连续传代后基因发生突变,但对病毒的毒力以及致病力影响仍需后续实验验证。

Computational analysis of antigenic epitopes of avian influenza A (H7N9) viruses

Influenza virus can rapidly change its antigenicity, via mutation in the hemagglutinin(HA) protein, to evade host immunity. The emergence of the novel human-infecting avian H7N9 virus in China has caused widespread concern. However, evolution of the antigenicity of this virus is not well understood. Here, we inferred the antigenic epitopes of the HA protein from all H7 viruses, based on the five well-characterized HA epitopes of the human H3N2 virus. By comparing the two major H7 phylogenetic lineages, i.e., the Eurasian lineage and the North American lineage, we found that epitopes A and B are more frequently mutated in the Eurasian lineage, while epitopes B and C are more frequently mutated in the North American lineage. Furthermore, we found that the novel H7N9 virus(derived from the Eurasian lineage) isolated in China in the year 2013, contains six frequently mutated sites on epitopes that include site 135, which is located in the receptor binding domain. This indicates that the novel H7N9 virus that infects human may already have been subjected to gradual immune pressure and receptor-binding variation. Our results not only provide insights into the antigenic evolution of the H7 virus but may also help in the selection of suitable vaccine strains.

Environmental connections of novel avian-origin H7N9 influenza virus infection and virus adaptation to the human

A novel H7N9 influenza A virus has been discovered as the causative identity of the emerging acute respiratory infection cases in Shanghai,China.This virus has also been identified in cases of infection in the neighboring area Hangzhou City in Zhejiang Province.In this study,epidemiologic,clinical,and virological data from three patients in Hangzhou who were confirmed to be infected by the novel H7N9 influenza A virus were collected and analyzed.Human respiratory specimens and chicken feces from a contacted free market were tested for influenza virus by real-time reverse transcription PCR(RT-PCR) and sequencing.The clinical features of the three cases were similar featured with high fever and severe respiratory symptoms;however,only one of the patients died.A certain degree of diversity was observed among the three Hangzhou viruses sequenced from human samples compared with other reported H7N9 influenza A viruses.The sequences of the novel avian-origin H7N9 influenza viruses from Hangzhou City contained important amino acid substitutions related to human adaptation.One of the Hangzhou viruses had gained a novel amino acid substitution(Q226I) in the receptor binding region of hemagglutinin.More importantly,the virus sequenced from the chicken feces had a 627E substitution in the PB2 protein instead of the mammalian-adapted 627K substitution that was found in the PB2 proteins from the Hangzhou viruses from the three patients.Therefore,the newly-emerging H7N9 virus might be under adaptation pressure that will help it "jump" from avian to human hosts.The significance of these substitutions needs further exploration,with both laboratory experiments and extensive field surveillance.

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.

Human infection with avian influenza A（H7N9） virus in Shanghai： current status and future trends

Avian influenza A（H7N9） virus is one subgroup among the larger group of H7 viruses,which normally circulate among birds.The H7N9 subtype of avian influenza viruses has not been known to infect humans until only recently.On March 31,2013,China confirmed the first three human cases of novel avian influenza A（H7N9）infection in Shanghai and Anhui,two of these patients died.1 As of February 27,2014,367 laboratory-confirmed human cases have been reported from 15 provinces/municipalities in China's Mainland,

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.

2014 update of the Chinese guidelines for diagnosis and treatment of avian influenza A（H7N9） virus infection

The National Health and Family Planning Commission of China published an updated guidance （2014 version） on clinical management in Chinese on January 26, 2014.The guidelines come as human cases of avian influenza A（H7N9） virus infection undergo a seasonal spike.

Bayesian evolutionary analysis for emerging infectious disease:an exemplified application for H7N9 avian influenza viruses

Dear Editor,Ever since the first human infection with H7N9 avian influenza virus（AIV）was reported in China in March 2013,there have been five H7N9 AIV pandemics in humans.Wave5 began earlier than the previous four waves,spread to more districts and counties in affected provinces,and had more confirmed cases（Wang et al.,2017）.

Generation and application of replication-competent Venus-expressing H5N1,H7N9,and H9N2 influenza A viruses

The generation and application of replication-competent influenza A virus （IAV） expressing a reporter gene represent a valuable tool to elucidate the mechanism of viral pathogenesis and establish new coun- termeasures to combat the threat of influenza. Here, replication-competent 1AVs with a neuraminidase （NA） segment harboring a fluorescent reporter protein, Venus, were generated in the background of H5N1, H7N9, and H9N2 influenza viruses, the three subtypes of viruses with imminent pandemic poten- tial. All three reporter viruses maintained virion morphology, replicated with similar or slightly reduced titers relative to their parental viruses, and stably expressed the fluorescent signal for at least two pas- sages in embryonated chicken eggs. As a proof of concept, we demonstrated that these reporter viruses, used in combination with a high-content imaging system, can serve as a convenient and rapid tool for the screening of antivirals and host factors involved in the virus life cycle. Moreover. the reporter viruses demonstrated similar growth properties and tissue tropism as their parental viruses in mice, among which the HTN9 NA-Venus virus could potentially be used in ex vivo studies to better understand H7N9 pathogenesis or to develop novel therapeutics.

Evolution of Influenza A H7N9 Virus with an Emphasis on Gene Constellation

The H7 subtype avian influenza viruses, including H7N2, HTN3 and HTN7, have posed a public health threat worldwide. Except one H7N7 fatal case in the Netherlands in 2003, the other H7 human cases have resulted in self-limiting conjunc- tivitis or mild upper respiratory illness.

新型H7N9禽流感病毒的病原学研究进展

2013年初在中国长三角地区首次发现一种新的H7N9禽流感病毒可导致人的感染和死亡。该病毒是由H7、N9以及H9N2禽流感病毒重配而成,病毒传播到其他地区之后仍与当地的H9N2病毒不断重配,产生不同的基因型。H7N9禽流感病毒特殊的双受体结合特性是其突破种属屏障感染人的重要机制,也是该病毒比H5N1禽流感病毒更容易感染人的原因,这种受体特性的分子基础主要与病毒HA蛋白的Q226L和G186V突变有关。H7N9病毒对禽类不致病,但人感染后的病死率超过30%。人群没有免疫力、病毒感染所导致的免疫病理以及病毒在肺部组织的高效复制是导致人感染H7N9病毒后高病死率的重要原因。雪貂动物模型研究也表明该病毒能够在雪貂中有效复制和接触传播。因此,H7N9禽流感病毒导致流感大流行的风险不容忽视,对动物和人群中H7N9禽流感病毒的监测不容松懈。

H7N9禽流感患者外周血T淋巴细胞亚群及细胞因子检测

目的探讨H7N9禽流感患者外周血细胞因子变化。方法收集H7N9禽流感患者外周血,用流式细胞仪检测T淋巴细胞亚群百分率,ELISA法检测干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)、白细胞介素-4(IL-4)及白细胞介素-10(IL-10)细胞因子水平,并与正常对照组进行比较分析。结果 10例患者外周血CD4+、CD8+T淋巴细胞百分率为25.26(15.83~33.34)、15.01(9.23~20.33),较对照组明显减少,差异有统计学意义(P<0.05)。Th2型T淋巴细胞为2.31(0.40~4.59),较对照组明显增高,差异有统计学意义(P<0.05)。外周血细胞因子IFN-γ、TNF-α、IL-10水平分别为35.51(18.54~46.23)ng/L、40.65(23.12~65.87)ng/L和5.34(3.25~6.32)ng/L,均明显高于对照组,差异有统计学意义(P<0.05)。而IL-4水平为6.11(2.51~8.14)ng/L,与对照组比较差异无统计学意义(P>0.05)。结论 H7N9禽流感患者存在T淋巴细胞亚群及Th1、Th2免疫失调,多种细胞因子上调,参与机体免疫损伤。

广东省2013—2018年人感染H7N9和H5N6禽流感流行病学及临床特征分析

目的分析广东省2013—2018年人感染H7N9及H5N6禽流感病例的监测数据及临床资料.方法以2013—2018年人感染H7N9及H5N6禽流感确诊病例为对象,对疫情的基本情况、三间分布、暴露史、聚集性及临床特征等进行统计分析.结果发现人感染H7N9病例259例,病死率为38.6%;人感染H5N6病例8例,病死率为62.5%.病例主要发生在11月至次年4月,主要集中在珠三角地区.H7N9病例多见于男性,H5N6病例女性居多.245例H7N9病例(94.6%)及7例H6N6(87.5%)有直接或间接活禽接触史.广东省H7N9病例有6起聚集性病例.H7N9及H5N6病例多为重症病例,从发病到使用奥司他韦时间的中位数均在5 d以上.结论广东省人感染H7N9及H5N6禽流感的流行特征和临床特征不同,应做好不同亚型人禽流感病例的早期发现和干预,防止疫情的传播和流行.

Human T-cell immunity against the emerging and re-emerging viruses

Over the past decade, we have seen an alarming number of high-profile outbreaks of newly emerging and re-emerging viruses.Recent outbreaks of avian influenza viruses, Middle East respiratory syndrome coronaviruses, Zika virus and Ebola virus present great threats to global health. Considering the pivotal role of host T-cell immunity in the alleviation of symptoms and the clearance of viruses in patients, there are three issues to be primarily concerned about T-cell immunity when a new virus emerges: first, does the population possess pre-existing T-cells against the new virus through previous infections of genetically relevant viruses; second, does a proper immune response arise in the patients to provide protection through an immunopathogenic effect; lastly, how long can the virus-specific immune memory persist. Herein, we summarize the current updates on the characteristics of human T-cell immunological responses against recently emerged or re-emerged viruses, and emphasize the necessity for timely investigation on the T-cell features of these viral diseases, which may provide beneficial recommendations for clinical diagnosis and vaccine development.

甲型流感病毒基因组的进化与变异

甲型流感病毒对人类社会造成严重危害,而快速变异的特点使得预防控制工作极为复杂。目前的研究大多聚焦于部分亚型的进化及突变,缺少能系统反映甲型流感病毒基因组的整体研究工作。本文结合生物信息学与病毒学研究最新成果,针对甲型流感病毒全基因组8条片段的进化特点及突变对功能的影响进行全面综述,从而揭示近年来对人类社会中造成严重影响的H1N1、H3N2、H5N1与H7N9等亚型流感病毒的起源与进化,有助于进一步提高对甲型流感病毒的认识和抵御潜在流感大流行的能力。