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.

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.

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.

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.

Genetic Variation Analysis on the Whole Genomic Sequence of a H9N2 Subtype Avian Influenza Virus Isolate

A Objective3 This study was to understand the genetic variation characters of the H9N2 subtype avian influenza virus isolate （A/Chicken/ Hebei/WD/98, abbreviated as WD98） by comparing with other reference strains. I-Method3 Eight complete genes were amplified by RT-PCR and sequenced. The homology and genetic evolution relationship were analyzed between these sequences and that of the seven reference strains. [Result] The whole genomic sequence of WD98 strain was 91.1% -95.8% homologous to that of seven reference strains tested. This isolate shared the highest homology （95.8%） to D/HK/Y280/97 and the lowest homology （91.1% ） to C/Pak/2/99. The HA cleavage site of the WD98 strain was R-S-S-R G, and the 226th amino acid at receptor-binding site was Gin. [ Condmion] WD98 strain belongs to mildly pathogenic avian in- fluenza virus and may not infect human. The genetic relationship is the closest between A/Chicken/Hebei/wD/98 and A/duck/HongKong/Y280/ 97, both of which belong to the sub-line of A/Chicken/Beijing/1/94 in Eurasian line. And A/Chicken/Hebei/WD/98 and A/Chicken/Beijing/1/94 are genetically distant within the same sub-line.

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.

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.

Pathogenesis and Immunogenicity of an Avian H9N2 Influenza Virus Isolated from Human

Objective To investigate the pathogenesis and immunogenicity of H9N2 influenza virus A/Guangzhou/333/99 （a reassortant of G1 and G9 viruses isolated from a female patient in 1999） in a mouse model of infection.Methods Mice were infected with increasing virus titers.Viral load in the lungs and trachea was determined by EID50 assay.Pulmonary histopathology was assessed by hematoxylin‐eosin staining.Anti‐HI antibody titers and T‐cell responses to viral HA were determined by ELISPOT and confirmed by flow cytometry.Results Mice presented a mild syndrome after intranasal infection with A/Guangzhou/333/99 （H9N2） influenza virus.Virus was detected in the trachea and lungs of mice harvested on days 3,6,and 9 post‐infection.A T‐cell response to viral HA was detected on day 6 and H9 HA‐specific CD 4＋ T‐cells predominated.Seroconversion was detected after 14 days and antibody persisted for at least 28 weeks.Conclusion Our results suggest that H9N2 （A/Guangzhou/333/99） can replicate in the murine respiratory tract without prior adaptation,and both humoral and cell‐mediated immunity play an important role in the immune response.

Phylogenetic and Molecular Analysis of an H7N7 Avian Influenza Virus Isolated in East Dongting Lake in 2012

Objective In March 2012, an H7N7 subtype avian influenza virus （AIV） named A/wild goose/Dongting/PC0360/2022 （H7N7） （DT/PC0360） was recovered from a wild goose in East Dongting Lake. We performed whole-genome sequencing of the isolate, and analyzed the phylogenetic and molecular characterization. Methods RNA was extracted from environment samples （including fecal samples from wild bird or domestic ducks, and water samples） for detecting the presence of Influenza A Virus targeting Matrix gene, using realtime RT-PCR assay. The positive samples were performed virus isolation with embryonated eggs. The subtype of the isolates were identified by RT-PCR assay with the HI-HI6 and N1-N9 primer set. The whole-genome sequencing of isolates were performed. Phylogenetic and molecular characterizations of the eight genes of the isolates were analyzed. Results Our results suggested that all the eight gene segments of DT/PC0360 belonged to the Eurasian gene pool, and the HA gene were belonged to distinct sublineage with H7N9 AIV which caused outbreaks in China's Mainland in 2013. The hemagglutinin cleavage site of HA of DT/PC0360 showed characterization of low pathogenic avian influenza virus. Conclusion Strengthening the surveillance of AlVs of wild waterfowl and poultry in this region is vita for our knowledge of the ecology and mechanism of transmission to prevent an influenza pandemic.

Establishment and Application of Digital RT-PCR Assay for Detection of Avian Influenza Virus H9 Subtype

A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of the two methods for H9 were determined by gradient dilution using the same pair of primers and probes. Both methods were able to detect 104 times diluted H9 pathogens, while digital RT-PCR could detect H9 in single droplets, and its sensitivity was higher than real-time quantitative RT-PCR. At the same time, the specificities of both methods were very strong, with no amplification reactions for H3N2, H4N2, H6N2. The reproducibility of the two methods were also good. Digital RT-PCR has a higher sensitivity than real-time quantitative RT-PCR and could play an important role in the rapid detection of H9 subtype influenza virus.

Pathogenicity and amino acid sequences of hemagglutinin cleavage site and neuraminidase stalk of differently passaged H9N2-avian influenza virus in broilers

Low pathogenic Avian Influenza (AI) virus has the ability to evolve to high pathogenic viruses resulting in significant economic losses in the poultry sector. This study aims at assessing the impact of H9N2 viral passaging in broilers and its relatedness to pathogenicity and amino acid (a.a) sequences of the hemagglutinin (HA) cleavage site and neuraminidase (NA) stalk. The original H9N2 AI virus (P0) was used to challenge ten-21 days old broilers. Individual recovery of H9N2 virus from homogenates of trachea, lungs and airsacs was attempted in 9 days old chicken embryos, as a conclusion of the first passage (P1). Tracheal isolates of H9N2 were passaged for a second (P2) and a third (P3) time in broilers, followed by a similar embryonic recovery procedure. The a.a. sequence of a part of HA1 cleavage site and Neuraminidase stalk were compared among the differently passaged viruses;an assessement of the relatedness of the determined a.a. sequences to the pathogenicity in broilers, based on frequency of mortality, morbidity signs, gross and microscopic lesions at 3 days post challenge with the P1, P2, and P3-H9N2, is concluded. An increase in certain morbidity signs and specific lesions was observed in P2- and P3-H9N2 challenged broilers compared to birds challenged with P1-H9N2. A conserved R-S-S-R amino acid sequence at the HA1 cleavage site was observed in the differently passaged H9N2, associated with a variability in the NA stalk-a.a sequences. The passaging of the low pathogenic H9N2 virus in broilers leads to a trend of increase in pathogenicity, manifested in higher frequency of morbidity signs, and of specific gross and microscopic lesions of the examined organs. This passaging was associated with a conserved a.a. sequence of the hemaglutinin cleavage site and a variability in the sequence of the neuraminidase stalk. A detailed study of the potential of the detected variability in the neuraminidase stalk of H9N2 in induction of a higher pathogenicity in broilers will be the subject of future investigations.

Receptor binding specificity and sequence comparison of a novel avian-origin H7N9 virus in China

Avian influenza such as H5N1 could infect humans and cause great health concern due to its high mortality rate. In March 2013, a novel reassortant avian-origin influenza A (H7N9) virus was found in several patients with severe respiratory illness in China. Since then, at least 82 people in China have been infected with this new virus and 17 have died from this virus. The question of how these people were infected with this virus and whether this virus will spread among people remains an urgent topic for research. This study took an early investigation of this virus ?by comparing the collected viral genome sequences ?of 2013 H7N9 inChinaagainst those of previous avian H7N9 and examined the receptor binding specificity of this new virus. This virus was found to be very different from the previous avian H7N9 viruses and surprisingly many of the internal proteins of 2013 H7N9 from the avian and human hosts in China were either identical or similar. Our analysis of the HA protein of this virus implied that the current strains of 2013 H7N9 inChina displayed avian type receptors as their primary binding preference and human type receptors as secondary. For pandemic risk assessment, we also detected 23 mutations, including a few well known for host adaptation, in the HA1 domain of the HA protein from this virus. Each mutation was quantified for its impact on the recaptor binding selection using a bioinformatics approach. Collectively these current mutations tended to decrease the HA binding affinity for avian type recaptors and increase that for human type receptors, which could enhance the ability of this virus to infect humans.

Preparation of Monoclonal Antibodies against Hemagglutinin of Avian Influenza Virus H9 Subtype by Plasmid Immunization

Avian influenza has caused enormous economic losses to poultry industry. To develop kits for rapid diagnosis of avian influenza virus （AIV） H9 subtype, 8-week-old Balb/c mice were administered with pcDNA3.1 （ ＋ ） carrying hemagglutinin （HA） gene of AIV H9 subtype. After cell fusion, one positive hybridoma cell strain was screened out by hemagglutination inhibition assay （ HI ）, and another positive hybddoma call strain was screened out by ELISA. After subcloning 3 times, the two cell strains could still secret antibodies against the HA of AIV H9 subtype. The mono- clonal antibodies did not react with Newcastle disease virus, AIV H5 subtype and duck adenovirus A. Their subtypes were IgG2b with kappa light chain. These two hybridoma cell strains may play an important role in rapid diagnosis and early-warning surveillance of AIV H9 subtype.

Effects of Trypsin on Proliferation of Avian Influenza Virus H9N2 Subtype in MDCK Cells

[Objective] The study aims to determine the optimal concentration of trypsin for the proliferation of avian influenza virus （AIV） H9N2 subtype in Madin- Darby canine kidney （MDCK） cells. [Method] Three AIV H9 subtype isolates were inoculated on MDCK cells respectively. Then, DMEM containing different concentrations of trypsin as maintenance media were added to MDCK monolayer cells. The cytopathic effect （CPE） was observed once every 24 h, and the HA titer of the supematant was measured by HA assay. [Result] When the trypsin concentration was 10 -20 μg/ml in DMEM, the HA titer of virus culture reached 7 log2 （1：128）. Almost all cells were cytopathic after 96 h post inoculation with 1：1 000 or 1：10 000 dilution of AIV culture, and the virus titer reached a peak after 72 -96 h. [ Conclusion] The optimal concentration of trypsin is 10 -20 pg/ml for proliferation of AIV H9N2 subtype in MDCK cells.

The cloning of non-structural-1 (NS1) gene of H9N2 subtype of avian influenza virus in pGEX-4T-1 and pMAL-c2X plasmids and expression in <i>Escherichia coli</i>DH5<i>α</i>strain

Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including trade embargoes and interfering with serologic surveillance in differentiation between infected and vaccinated animals (DIVA strategy). Several distinct DIVA strategies have been presented to conquer these limitations. In this study, the open reading frame of NS1 gene of a H9N2 subtype of AI virus was amplified by polymerase chain reaction. After extraction and purification of NS1 gene from agarose gel, it was inserted into two different pGEX-4T-1 and pMAL-c2X plasmids and transferred in DH5α strain of Escherichia coli by using electroporation procedure. The E. coli colonies possessing recombinant NS1 gene were screened using PCR, restriction mapping and sequencing analysis. The expressed rNS1 protein was purified using affinity chromatography based on MBP (pMAL- c2X) and GST (pGEX-4T-1). The MBP-NS1 and GST- NS1 proteins on SDS-PAGE had bands with molecular weight of 68 and 52 kDa respectively. Western blotting with MBP-NS1 protein showed positive reaction using antisera obtained from chickens challenged with a H9N2 subtype strain. But, the most sera prepared from H9N2 vaccinated chickens were negative in WB. These findings indicated that the MBP-rNS1 protein of 26 kDa expressed by pMAL-c2X plasmid can be used in a DIVA for differentiation of AI infected and vaccinated chickens.

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.

Effect of Different Culture Media on the Proliferation of Avian Influenza Virus H9 Subtypes in MDCK Cells

[Objective] To screen the best culture media for the proliferation of avian influenza virus （AIV） H9 subtypes in MDCK cells. [Method] The DMEM containing 10% （V/V） newborn calf serum, low-serum containing medium （ MEM-MD-611 ） and serum-free medium （SFE4Mega） were used to culture the MDCK monolayer ceils, which were then inoculated with different dilutions of AIV H9 subtypes, and the 3 kinds of media were al- so used as the maintenance solution to culture the virus. The cytopathic changes were observed at every 24 h, and the HA titers of the culture su- pernatants were also determined. [ Result] After culturing for 72 -96 h, the HA titers of the serum-free media were higher than that of low-serum culture media, while the HA titers were higher in the low-serum media than in the serum containing media. [ Conclusion] The 3 kinds of media can all used for the proliferation of AIV_ but the low-serum culture medium （MEM-MD-611 ） and serum-free medium （SFE4Meaa3 are preferred.

Sequence and phylogenetic analysis of hemagglutinin genes of H9N2 influenza viruses isolated from chicken in China from 2013 to 2015

H9N2 avian influenza virus（AIV） infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin（HA） gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in Gen Bank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216 L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.

Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A(H5N1) Virus

Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients.The genetic basis,biological functions,and epitopes of the obtained Bn Abs were assessed and modeled.Results Two Bn Abs,2-12 D5,and 3-37 G7.1,were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset.Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity(ADCC)activity.Albeit derived from distinct Ab lineages,i.e.,V^H1-69-D2-15-JH^4(2-12D5)and V^H1-2-D3-9-JH^5(3-32 G7.1),the Bn Abs were directed toward CR6261-like epitopes in the HA stem,and HA2 I45 in the hydrophobic pocket was the critical residue for their binding.Signature motifs for binding with the HA stem,namely,IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs,were also observed in 2-12D5 and 3-32 G7.1,respectively.Conclusions Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus.The HA stem epitopes targeted by the Bn Abs,and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.