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.

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.

Diversity of the RNA Polymerase in the H7N9 Influenza A Virus

A novel influenza virus of the H7N9 subtype has infected more than 350 people in China since 19 February 2013. Evolutionary analysis indicates that the virus is a reassortant originated from H7, N9 and H9N2 avian influenza viruses, and bears some amino acids associated with mammalian receptor binding, raising concern over the possibility of a new influenza pandemic. Besides HA and NA, the mutation of the polymerase is known to have an important role in virulence, host adaptation and transmissibility in mammalians. In this article, the annotation of the polymerase protein domain associated with molecular function has been highlighted, suggesting the combination of RNA polymerase of H7N9 viruses is still not stable for host adaptation. In addition, the mutation hallmarks in polymerase gene of H7N9 are compared, providing the potential determinants of the evolution in the H7N9 influenza A virus.

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.

Multiple RT-PCR Detection of H5,H7,and H9 Subtype Avian Influenza Viruses and Newcastle Disease Virus

Objective:This paper focuses on the multiple detection RT-PCR technology of H5,H7,AND H9 subtype avian influenza viruses and Newcastle disease virus,and points out the specific detection methods and detection procedures of avian influenza and Newcastle disease virus.Methods:The genes of Newcastle disease virus carrying out the HA gene sequence of H5,H7 and H9 subtype AIV in GenBank were used to establish a strategy for simultaneous detection of three subtypes of avian influenza virus and Newcastle disease virus.Results:The results showed that the program can detect and distinguish H5,H7 and H9 subtype avian influenza viruses and Newcastle disease virus at one time.Conclusion:Multiple RT-PCR detection method has high detection sensitivity and can detect and determine different subtypes of avian influenza virus and Newcastle disease virus quickly and accurately,therefore,it has a crucial role in the detection and control of avian influenza H5,H7 and H9 subtypes and Newcastle disease.

Ventilator management for acute respiratory distress syndrome associated with avian infl uenza A(H7N9) virus infection: A case series

BACKGROUND: Data on the mechanical ventilation(MV) characteristics and radiologic features for the cases with H7 N9-induced ARDS were still lacking.METHODS: We describe the MV characteristics and radiologic features of adult patients with ARDS due to microbiologically confirmed H7 N9 admitted to our ICU over a 3-month period.RESULTS: Eight patients(mean age 57.38±16.75; 5 male) were diagnosed with H7 N9 in the first quarter of 2014. All developed respiratory failure complicated by acute respiratory distress syndrome(ARDS), which required MV in ICU. The baseline APACHE II and SOFA score was 11.77±6.32 and 7.71±3.12. The overall CT scores of the patients was 247.68±34.28 and the range of CT scores was 196.3–294.7. The average MV days was 14.63±6.14, and 4 patients required additional rescue therapies for refractory hypoxemia. Despite these measures, 3 patients died.CONCLUSION: In H7 N9-infected patients with ARDS, low tidal volume strategy was the conventional mode. RM as one of rescue therapies to refractory hypoxemia in these patients with serious architectural distortion and high CT scores, which could cause further lung damage, may induce bad outcomes and requires serious consideration. Prone ventilation may improve mortality, and should be performed at the early stage of the disease, not as a rescue therapy.

Distribution of Avian Influenza A Viruses in Poultry-Related Environment and Its Association with Human Infection in Henan, 2016 to 2017

Objective To survey avian influenza A viruses(AIVs) in the environment and explore the reasons for the surge in human H7 N9 cases.Methods A total of 1,045 samples were collected from routine surveillance on poultry-related environments and 307 samples from human H7 N9 cases-exposed environments in Henan from 2016 to2017. The nucleic acids of influenza A(Flu A), H5, H7, and H9 subtypes were detected by real-time polymerase chain reaction.Results A total of 27 H7 N9 cases were confirmed in Henan from 2016 to 2017, 24 had a history of live poultry exposure, and 15 had H7 N9 virus detected in the related live poultry markets(LPMs). About 96%(264/275) Flu A positive-environmental samples were from LPMs. H9 was the main AIV subtype(10.05%) from routine surveillance sites with only 1 H7-positive sample, whereas 21.17% samples were H7-positive in H7 N9 cases-exposed environments. Samples from H7 N9 cases-exposed LPMs(47.56%)had much higher AIVs positive rates than those from routine surveillance sites(12.34%). The H7+H9 combination of mixed infection was 78.18%(43/55) of H7-positive samples and 41.34%(43/104) of H9-positive samples.Conclusion The contamination status of AIVs in poultry-related environments is closely associated with the incidence of human infection caused by AIVs. Therefore, systematic surveillance of AIVs in LPMs in China is essential for the detection of novel reassortant viruses and their potential for interspecies transmission.

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.

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.

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.

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.

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.

Discovery process, clinical characteristics, and treatment of patients infected with avian influenza virus （H7N9） in Shanghai

During the spring of 2013,a novel avian-origin influenza A （H7N9） virus emerged and spread in Shanghai.On May 9,2013,33 lab-confirmed cases （2 children and 31 adults） have been reported,among whom 13 died.Among the 31 adult cases of H7N9 avian influenza A virus infections in humans,80.6% （25/31） were older men （average age 65 years）.Clear history of poultry exposure was found in eight patients （8/33,24.3%） and the remaining 21 cases had been suspected poultry exposure.Clustered cases were found in two families （two cases from a father and his son,and the other two cases from a wife and her husband）.However,no evidence of human-to-human transmission had been identified.Avian influenza is one of the most dangerous contagions in poultry worldwide,and avian influenza A virus is the major pathogen responsible.Transmission from poultry to humans has now been proven.Some gene segments of the strain have mutated,and further mutations might result in human-to-human transmission.

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,

Novel human H7N9 influenza virus in China

Outbreaks of H7N9 avian influenza in humans in 5 provinces and 2 municipalities of China have reawakened concern that avian influenza viruses may again cross species barriers to infect the human population and thereby initiate a new influenza pandemic.Evolutionary analysis shows that human H7N9 influenza viruses originated from the H9N2,H7N3 and H11N9 avian viruses,and that it is as a novel reassortment influenza virus.This ar­ticle reviews current knowledge on 11 subtypes of influenza A virus from human which can cause human infec­tions.

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.

Effect of chlorine dioxide on avian influenza A(H7N9)virus

Avian influenza remains a threat to human wellbeing.Hypochlorite derivatives are commonly used as disinfectants to prevent the spread of the disease.The World Health Organization(WHO)has listed chlorine dioxide(ClO_(2))as an A1‐level,safe,and efficient disinfectant.In this study,we tested the efficacy of ClO_(2),in aqueous solution and gas forms,against avian influenza A(H7N9)virus.The virus suspension was mixed with ClO_(2) aqueous solutions of various concentrations and for various time intervals.Aliquots of the mixture were then serially diluted,and the 50%tissue culture infective dose(TCID50)was measured with a hemagglutination test on MDCK cells.ClO_(2) gas produced from generators was introduced in a chamber containing the virus suspension in a Petri dish.The infective activity of the surviving virus was measured by the hemagglutination test.An aqueous solution of ClO_(2) at 126μg/mL for 15 s was effective given that no surviving virus was detected with the hemagglutination test.ClO_(2) gas at>5μL/L sustained for 1 h inactivated the virus effectively,while at 2.5μL/L for 1 h,it only partially inactivated the virus.ClO_(2) as gas or aqueous solution at a certain concentration is effective in inactivating the H7N9 virus,and can be applied for the decontamination and disinfection of environments.

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.