Activation Dynamics and Immunoglobulin Evolution of Pre-existing and Newly Generated Human Memory B cell Responses to Influenza Hemagglutinin.

Vaccine-induced memory B cell responses to evolving viruses like influenza A involve activation of pre-existing immunity and generation of new responses. To define the contribution of these two types of responses, we analyzed the response to H7N9 vaccination in H7N9-naive adults. We performed comprehensive comparisons at the single-cell level of the kinetics, Ig repertoire, and activation phenotype of established pre-existing memory B cells recognizing conserved epitopes and the newly generated memory B cells directed toward H7 strain-specific epitopes. The recall response to conserved epitopes on H7 HA involved a transient expansion of memory B cells with little observed adaptation. However, the B cell response to newly encountered epitopes was phenotypically distinct and generated a sustained memory population that evolved and affinity matured months after vaccination. These findings establish clear differences between newly generated and pre-existing memory B cells, highlighting the challenges in achieving long-lasting, broad protection against an ever-evolving virus.

PB2 residue 473 contributes to the mammalian virulence of H7N9 avian influenza virus by modulating viral polymerase activity via ANP32A.

Since the first human infection reported in 2013, H7N9 avian influenza virus (AIV) has been regarded as a serious threat to human health. In this study, we sought to identify the virulence determinant of the H7N9 virus in mammalian hosts. By comparing the virulence of the SH/4664 H7N9 virus, a non-virulent H9N2 virus, and various H7N9-H9N2 hybrid viruses in infected mice, we first pinpointed PB2 as the primary viral factor accounting for the difference between H7N9 and H9N2 in mammalian virulence. We further analyzed the in vivo effects of individually mutating H7N9 PB2 residues different from the closely related H9N2 virus and consequently found residue 473, alongside the well-known residue 627, to be critical for the virulence of the H7N9 virus in mice and the activity of its reconstituted viral polymerase in mammalian cells. The importance of PB2-473 was further strengthened by studying reverse H7N9 substitutions in the H9N2 background. Finally, we surprisingly found that species-specific usage of ANP32A, a family member of host factors connecting with the PB2-627 polymorphism, mediates the contribution of PB2 473 residue to the mammalian adaption of AIV polymerase, as the attenuating effect of PB2 M473T on the viral polymerase activity and viral growth of the H7N9 virus could be efficiently complemented by co-expression of chicken ANP32A but not mouse ANP32A and ANP32B. Together, our studies uncovered the PB2 473 residue as a novel viral host range determinant of AIVs via species-specific co-opting of the ANP32 host factor to support viral polymerase activity.IMPORTANCEThe H7N9 avian influenza virus has been considered to have the potential to cause the next pandemic since the first case of human infection reported in 2013. In this study, we identified PB2 residue 473 as a new determinant of mouse virulence and mammalian adaptation of the viral polymerase of the H7N9 virus and its non-pathogenic H9N2 counterparts. We further demonstrated that the variation in PB2-473 is functionally linked to differential co-opting of the host ANP32A protein in supporting viral polymerase activity, which is analogous to the well-known PB2-627 polymorphism, albeit the two PB2 positions are spatially distant. By providing new mechanistic insight into the PB2-mediated host range determination of influenza A viruses, our study implicated the potential existence of multiple PB2-ANP32 interfaces that could be targets for developing new antivirals against the H7N9 virus as well as other mammalian-adapted influenza viruses.

Passive immunotherapies for the next influenza pandemic.

Influenzavirus is among the most relevant candidates for a next pandemic. We review here the phylogeny of former influenza pandemics, and discuss candidate lineages. After briefly reviewing the other existing antiviral options, we discuss in detail the evidences supporting the efficacy of passive immunotherapies against influenzavirus, with a focus on convalescent plasma.

Safety and Immunogenicity of a Delayed Heterologous Avian Influenza A(H7N9) Vaccine Boost Following Different Priming Regimens: A Randomized Clinical Trial.

BACKGROUND: Influenza A (H7N9) has caused multiple disease waves with evidence of strain diversification. Optimal influenza A (H7N9) prime-boost vaccine strategies are unknown. METHODS: We recruited participants who had received monovalent inactivated A/Shanghai/2/2013 (H7N9) vaccine (MIV) approximately 5 years earlier, as follows: MIV with MF59 (MF59 × 2 group), MIV with AS03 (AS03 × 2 group), unadjuvanted MIV (No Adj group), MIV with MF59 or AS03 followed by unadjuvanted MIV (Adjx1 group), and A/H7-naive (unprimed group). Participants were randomized to receive 1 dose of AS03-adjuvanted or unadjuvanted A/Hong Kong/125/2017 (H7N9) MIV and were followed for safety and immunogenicity using hemagglutination inhibition (HAI) and neutralizing antibody assays. RESULTS: We enrolled 304 participants: 153 received the adjuvanted boost and 151 received the unadjuvanted boost. At 21 days postvaccination, the proportion of participants with HAI antibody titers against the boosting vaccine strain of ≥40 in the adjuvanted and unadjuvanted arms, respectively, were 88% and 49% in MF59 × 2 group, 89% and 75% in AS03 × 2 group, 59% and 20% in No Adj group, 94% and 55% in Adjx1group, and 9% and 11% in unprimed group. CONCLUSIONS: Serologic responses to a heterologous A(H7N9) MIV boost were highest in participants primed and boosted with adjuvant-containing regimens. CLINICAL TRIALS REGISTRATION: NCT03738241.

A Phase 2 Clinical Trial to Evaluate the Safety, Reactogenicity, and Immunogenicity of Different Prime-Boost Vaccination Schedules of 2013 and 2017 A(H7N9) Inactivated Influenza Virus Vaccines Administered With and Without AS03 Adjuvant in Healthy US Adults.

INTRODUCTION: A surge of human influenza A(H7N9) cases began in 2016 in China from an antigenically distinct lineage. Data are needed about the safety and immunogenicity of 2013 and 2017 A(H7N9) inactivated influenza vaccines (IIVs) and the effects of AS03 adjuvant, prime-boost interval, and priming effects of 2013 and 2017 A(H7N9) IIVs. METHODS: Healthy adults (n = 180), ages 19-50 years, were enrolled into this partially blinded, randomized, multicenter phase 2 clinical trial. Participants were randomly assigned to 1 of 6 vaccination groups evaluating homologous versus heterologous prime-boost strategies with 2 different boost intervals (21 vs 120 days) and 2 dosages (3.75 or 15 µg of hemagglutinin) administered with or without AS03 adjuvant. Reactogenicity, safety, and immunogenicity measured by hemagglutination inhibition and neutralizing antibody titers were assessed. RESULTS: Two doses of A(H7N9) IIV were well tolerated, and no safety issues were identified. Although most participants had injection site and systemic reactogenicity, these symptoms were mostly mild to moderate in severity; injection site reactogenicity was greater in vaccination groups receiving adjuvant. Immune responses were greater after an adjuvanted second dose, and with a longer interval between prime and boost. The highest hemagglutination inhibition geometric mean titer (95% confidence interval) observed against the 2017 A(H7N9) strain was 133.4 (83.6-212.6) among participants who received homologous, adjuvanted 3.75 µg + AS03/2017 doses with delayed boost interval. CONCLUSIONS: Administering AS03 adjuvant with the second H7N9 IIV dose and extending the boost interval to 4 months resulted in higher peak antibody responses. These observations can broadly inform strategic approaches for pandemic preparedness. Clinical Trials Registration. NCT03589807.

Evolution and Antigenic Differentiation of Avian Influenza A(H7N9) Virus, China.

We characterized the evolution and molecular characteristics of avian influenza A(H7N9) viruses isolated in China during 2021-2023. We systematically analyzed the 10-year evolution of the hemagglutinin gene to determine the evolutionary branch. Our results showed recent antigenic drift, providing crucial clues for updating the H7N9 vaccine and disease prevention and control.

Evaluating the epizootic and zoonotic threat of an H7N9 low-pathogenicity avian influenza virus (LPAIV) variant associated with enhanced pathogenicity in turkeys.

Between 2013 and 2017, the A/Anhui/1/13-lineage (H7N9) low-pathogenicity avian influenza virus (LPAIV) was epizootic in chickens in China, causing mild disease, with 616 fatal human cases. Despite poultry vaccination, H7N9 has not been eradicated. Previously, we demonstrated increased pathogenesis in turkeys infected with H7N9, correlating with the emergence of the L217Q (L226Q H3 numbering) polymorphism in the haemagglutinin (HA) protein. A Q217-containing virus also arose and is now dominant in China following vaccination. We compared infection and transmission of this Q217-containing 'turkey-adapted' (ty-ad) isolate alongside the H7N9 (L217) wild-type (wt) virus in different poultry species and investigated the zoonotic potential in the ferret model. Both wt and ty-ad viruses demonstrated similar shedding and transmission in turkeys and chickens. However, the ty-ad virus was significantly more pathogenic than the wt virus in turkeys but not in chickens, causing 100 and 33% mortality in turkeys respectively. Expanded tissue tropism was seen for the ty-ad virus in turkeys but not in chickens, yet the viral cell receptor distribution was broadly similar in the visceral organs of both species. The ty-ad virus required exogenous trypsin for in vitro replication yet had increased replication in primary avian cells. Replication was comparable in mammalian cells, and the ty-ad virus replicated successfully in ferrets. The L217Q polymorphism also affected antigenicity. Therefore, H7N9 infection in turkeys can generate novel variants with increased risk through altered pathogenicity and potential HA antigenic escape. These findings emphasize the requirement for enhanced surveillance and understanding of A/Anhui/1/13-lineage viruses and their risk to different species.

Antigenic Characterization of Human Monoclonal Antibodies for Therapeutic Use against H7N9 Avian Influenza Virus.

Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,500 human infections and the culling of millions of poultry. Despite large-scale poultry vaccination, H7N9 AIVs continue to circulate among poultry in China and pose a threat to human health. Previously, we isolated and generated four monoclonal antibodies (mAbs) derived from humans naturally infected with H7N9 AIV. Here, we investigated the hemagglutinin (HA) epitopes of H7N9 AIV targeted by these mAbs (L3A-44, K9B-122, L4A-14, and L4B-18) using immune escape studies. Our results revealed four key antigenic epitopes at HA amino acid positions 125, 133, 149, and 217. The mutant H7N9 viruses representing escape mutations containing an alanine-to-threonine substitution at residue 125 (A125T), a glycine-to-glutamic acid substitution at residue 133 (G133E), an asparagine-to-aspartic acid substitution at residue 149 (N149D), or a leucine-to-glutamine substitution at residue 217 (L217Q) showed reduced or completely abolished cross-reactivity with the mAbs, as measured by a hemagglutination inhibition (HI) assay. We further assessed the potential risk of these mutants to humans should they emerge following mAb treatment by measuring the impact of these HA mutations on virus fitness and evasion of host adaptive immunity. Here, we showed that the L4A-14 mAb had broad neutralizing capabilities, and its escape mutant N149D had reduced viral stability and human receptor binding and could be neutralized by both postinfection and antigen-induced sera. Therefore, the L4A-14 mAb could be a therapeutic candidate for H7N9 AIV infection in humans and warrants further investigation for therapeutic applications. IMPORTANCE Avian influenza virus (AIV) H7N9 continues to circulate and evolve in birds, posing a credible threat to humans. Antiviral drugs have proven useful for the treatment of severe influenza infections in humans; however, concerns have been raised as antiviral-resistant mutants have emerged. Monoclonal antibodies (mAbs) have been studied for both prophylactic and therapeutic applications in infectious disease control and have demonstrated great potential. For example, mAb treatment has significantly reduced the risk of people developing severe disease with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In addition to the protection efficiency, we should also consider the potential risk of the escape mutants generated by mAb treatment to public health by assessing their viral fitness and potential to compromise host adaptive immunity. Considering these parameters, we assessed four human mAbs derived from humans naturally infected with H7N9 AIV and showed that the mAb L4A-14 displayed potential as a therapeutic candidate.

Amino acid mutations PB1-V719M and PA-N444D combined with PB2-627K contribute to the pathogenicity of H7N9 in mice.

H7N9 subtype avian influenza viruses (AIVs) cause 1567 human infections and have high mortality, posing a significant threat to public health. Previously, we reported that two avian-derived H7N9 isolates (A/chicken/Eastern China/JTC4/2013 and A/chicken/Eastern China/JTC11/2013) exhibit different pathogenicities in mice. To understand the genetic basis for the differences in virulence, we constructed a series of mutant viruses based on reverse genetics. We found that the PB2-E627K mutation alone was not sufficient to increase the virulence of H7N9 in mice, despite its ability to enhance polymerase activity in mammalian cells. However, combinations with PB1-V719M and/or PA-N444D mutations significantly enhanced H7N9 virulence. Additionally, these combined mutations augmented polymerase activity, thereby intensifying virus replication, inflammatory cytokine expression, and lung injury, ultimately increasing pathogenicity in mice. Overall, this study revealed that virulence in H7N9 is a polygenic trait and identified novel virulence-related residues (PB2-627K combined with PB1-719M and/or PA-444D) in viral ribonucleoprotein (vRNP) complexes. These findings provide new insights into the molecular mechanisms underlying AIV pathogenesis in mammals, with implications for pandemic preparedness and intervention strategies.

DC-SIGN and Galectin-3 individually and collaboratively regulate H5N1 and H7N9 avian influenza A virus infection via interaction with viral envelope hemagglutinin protein.

DC-SIGN and Galectin-3 are two different lectins and have been reported to participate in regulation of several virus infections. WHO has pointed that H5N1 and H7N9 avian influenza viruses (AIVs) play continuous threats to global health. AIV hemagglutinin (HA) protein-a highly glycosylated protein-mediates influenza infection and was proposed to have DC-SIGN and Gal3 interactive domains. This study aims to address the individual and collaborative roles of DC-SIGN and Gal3 toward AIVs infection. Firstly, A549 cells with DC-SIGN expression or Gal3-knockdown, via lentiviral vector-mediated CD209 gene expression or LGALS-3 gene knockdown, respectively were generated. Quantitative reverse transcription PCR (qRT-PCR) results indicated that DC-SIGN expression and Gal3 knockdown in A549 cells significantly promoted and ameliorated HA or NP gene expression, respectively after H5N1 and H7N9-reverse genetics (RG) virus postinfections (P < 0.05). Similar results observed in immunoblotting, indicating that DC-SIGN expression significantly facilitated H5N1-RG and H7N9-RG infections (P < 0.05), whereas Gal3 knockdown significantly reduced both viral infections (P < 0.05). Furthermore, we found that DC-SIGN and Gal3 co-expression significantly enhanced infectivity of both H5N1-RG and H7N9-RG viruses (P < 0.01) and higher regulatory capabilities by DC-SIGN and Gal3 in H5N1-RG than H7N9-RG were noted. The promoting effect mainly relied on exogenous Gal3 and DC-SIGN directly interacting with the HA protein of H5N1 or H7N9 AIVs, subsequently enhancing virus infection. This study sheds light on two different lectins individually and collaboratively regulating H5N1 and H7N9 AIVs infection and suggests that inhibitors against DC-SIGN and Gal3 interacting with HA could be utilized as alternative antiviral strategies.

Specific Monoclonal Antibodies Targeting Unique HA Epitopes Block H7N9 Influenza A Viral Replication.

The H7N9 subtype influenza A viruses pose a serious threat to public health, and there is still a lack of vaccines or drugs for humans against H7N9 influenza viruses. In this study, we screened two monoclonal antibodies (MAbs), 4H1E8 and 7H9A6, that specifically recognize the hemagglutinin (HA) protein of H7N9 influenza virus and display highly neutralizing activity against H7N9 virus. The epitopes recognized by two MAbs are nearly all conserved within all known H7 subtypes. Characteristic identification showed that two MAbs have high avidity for the HA protein but no hemagglutinin inhibition activity or antibody-dependent cellular cytotoxicity. Mechanistically, the 4H1E8 and 7H9A6 antibodies inhibit the pH-dependent conformational change of HA and block the HA-mediated membrane fusion. More importantly, 4H1E8 and 7H9A6 exhibit promising prophylactic and therapeutic effects against lethal challenge with H7N9 virus. Moreover, 4H1E8- and 7H9A6-treated mice displayed inhibition of pulmonary viral replication and reduced lung lesions after viral challenge. Together, these findings indicate that antibodies 4H1E8 and 7H9A6 recognize unique epitopes in the HA protein and possess the neutralizing activity and protective efficacy against the H7N9 influenza A viruses. IMPORTANCE In 2013, H7N9 influenza viruses appeared in China and other countries resulting in more than 1,500 individual infections or death. There are still limited studies on vaccines or drugs for humans against H7N9 influenza viruses. Alternative approaches against H7N9 virus infection need to be developed. Here, we identified two monoclonal antibodies (4H1E8 and 7H9A6) that possess neutralizing activity by blocking the pH-dependent HA-mediated membrane fusion. Additionally, the two monoclonal antibodies protect mice against the H7N9 virus challenge prophylactically or therapeutically. Therefore, our study demonstrates that 4H1E8 and 7H9A6 could be used for the prevention and treatment of the H7N9 influenza virus, and the conserved epitopes we identified may contribute to the development of a broad H7N9 vaccine and provide insights into unique antiviral approaches.

The Origin of Internal Genes Contributes to the Replication and Transmission Fitness of H7N9 Avian Influenza Virus.

H9N2 avian influenza viruses (AIVs) have donated internal gene segments during the emergence of zoonotic AIVs, including H7N9. We used reverse genetics to generate A/Anhui/1/13 (H7N9) and three reassortant viruses (2:6 H7N9) which contained the hemagglutinin and neuraminidase from Anhui/13 (H7N9) and the six internal gene segments from H9N2 AIVs belonging to (i) G1 subgroup 2, (ii) G1 subgroup 3, or (iii) BJ94 lineages, enzootic in different regions throughout Asia. Infection of chickens with the 2:6 H7N9 containing G1-like H9N2 internal genes conferred attenuation in vivo, with reduced shedding and transmission to contact chickens. However, possession of BJ94-like H9N2 internal genes resulted in more rapid transmission and significantly elevated cloacal shedding compared to the parental Anhui/13 H7N9. In vitro analysis showed that the 2:6 H7N9 with BJ94-like internal genes had significantly increased replication compared to the Anhui/13 H7N9 in chicken cells. In vivo coinfection experiments followed, where chickens were coinfected with pairs of Anhui/13 H7N9 and a 2:6 H7N9 reassortant. During ensuing transmission events, the Anhui/13 H7N9 virus outcompeted 2:6 H7N9 AIVs with internal gene segments of BJ94-like or G1-like H9N2 viruses. Coinfection did lead to the emergence of novel reassortant genotypes that were transmitted to contact chickens. Some of the reassortant viruses had a greater replication in chicken and human cells compared to the progenitors. We demonstrated that the internal gene cassette determines the transmission fitness of H7N9 viruses in chickens, and the reassortment events can generate novel H7N9 genotypes with increased virulence in chickens and enhanced zoonotic potential. IMPORTANCE H9N2 avian influenza viruses (AIVs) are enzootic in poultry in different geographical regions. The internal genes of these viruses can be exchanged with other zoonotic AIVs, most notably the A/Anhui/1/2013-lineage H7N9, which can give rise to new virus genotypes with increased veterinary, economic and public health threats to both poultry and humans. We investigated the propensity of the internal genes of H9N2 viruses (G1 or BJ94) in the generation of novel reassortant H7N9 AIVs. We observed that the internal genes of H7N9 which were derivative of BJ94-like H9N2 virus have a fitness advantage compared to those from the G1-like H9N2 viruses for efficient transmission among chickens. We also observed the generation of novel reassortant viruses during chicken transmission which infected and replicated efficiently in human cells. Therefore, such emergent reassortant genotypes may pose an elevated zoonotic threat.

Coinfection of Chickens with H9N2 and H7N9 Avian Influenza Viruses Leads to Emergence of Reassortant H9N9 Virus with Increased Fitness for Poultry and a Zoonotic Potential.

An H7N9 low-pathogenicity avian influenza virus (LPAIV) emerged in 2013 through genetic reassortment between H9N2 and other LPAIVs circulating in birds in China. This virus causes inapparent clinical disease in chickens, but zoonotic transmission results in severe and fatal disease in humans. To examine a natural reassortment scenario between H7N9 and G1 lineage H9N2 viruses predominant in the Indian subcontinent, we performed an experimental coinfection of chickens with A/Anhui/1/2013/H7N9 (Anhui/13) virus and A/Chicken/Pakistan/UDL-01/2008/H9N2 (UDL/08) virus. Plaque purification and genotyping of the reassortant viruses shed via the oropharynx of contact chickens showed H9N2 and H9N9 as predominant subtypes. The reassortant viruses shed by contact chickens also showed selective enrichment of polymerase genes from H9N2 virus. The viable "6+2" reassortant H9N9 (having nucleoprotein [NP] and neuraminidase [NA] from H7N9 and the remaining genes from H9N2) was successfully shed from the oropharynx of contact chickens, plus it showed an increased replication rate in human A549 cells and a significantly higher receptor binding to α2,6 and α2,3 sialoglycans compared to H9N2. The reassortant H9N9 virus also had a lower fusion pH, replicated in directly infected ferrets at similar levels compared to H7N9 and transmitted via direct contact. Ferrets exposed to H9N9 via aerosol contact were also found to be seropositive, compared to H7N9 aerosol contact ferrets. To the best of our knowledge, this is the first study demonstrating that cocirculation of H7N9 and G1 lineage H9N2 viruses could represent a threat for the generation of novel reassortant H9N9 viruses with greater virulence in poultry and a zoonotic potential. IMPORTANCE We evaluated the consequences of reassortment between the H7N9 and the contemporary H9N2 viruses of the G1 lineage that are enzootic in poultry across the Indian subcontinent and the Middle East. Coinfection of chickens with these viruses resulted in the emergence of novel reassortant H9N9 viruses with genes derived from both H9N2 and H7N9 viruses. The "6+2" reassortant H9N9 (having NP and NA from H7N9) virus was shed from contact chickens in a significantly higher proportion compared to most of the reassortant viruses, showed significantly increased replication fitness in human A549 cells, receptor binding toward human (α2,6) and avian (α2,3) sialic acid receptor analogues, and the potential to transmit via contact among ferrets. This study demonstrated the ability of viruses that already exist in nature to exchange genetic material, highlighting the potential emergence of viruses from these subtypes with zoonotic potential.

Mendelian randomization study on the causal effect of serum IgA levels on H7N9 avian influenza A virus susceptibility.

Avian influenza A viruses (IAVs) that cross the species barrier to infect humans have the potential to initiate a new pandemic. However, the host factors influencing avian IAV infection remain poorly understood. To address this knowledge gap, we conducted a two-sample Mendelian randomization (MR) analysis by integrating our in-house genome-wide association study (GWAS) of avian IAV H7N9 susceptibility (with 217 cases and 116 controls) with the largest GWAS of serum IgA levels to date (sample size 41 263). Using the inverse-variance weighted (IVW) method, we discovered that genetically decreased serum IgA levels were associated with an increased risk of H7N9 infection (ß = -2.528, 95% confidence interval [CI]: -4.572 to -0.484; p = 0.015). Consistent results were obtained from three other MR methods, including robust IVW estimation (ß = -2.506, 95% CI: -4.109 to -0.902; p = 0.002), generalized summary-data-based MR (GSMR) (ß = -2.238, 95% CI: -4.106 to -0.602; p = 0.019), and MR-pleiotropy residual sum and outlier (MR-PRESSO) (ß = -2.528, 95% CI: -4.396 to -0.892; p = 0.026). In conclusion, our analysis provided compelling evidence support a causal relationship between genetically predicted serum IgA levels and avian IAV H7N9 susceptibility.

Avian influenza A virus H7N9 in China, a role reversal from reassortment receptor to the donator.

Reassortment can introduce one or more gene segments of influenza A viruses (IAVs) into another, resulting in novel subtypes. Since 2013, a new outbreak of human highly pathogenic avian influenza has emerged in the Yangtze River Delta (YRD) and South-Central regions of China. In this study, using Anhui province as an example, we discuss the possible impact of H7N9 IAVs on future influenza epidemics through a series of gene reassortment events. Sixty-one human H7N9 isolates were obtained from five outbreaks in Anhui province from 2013 to 2019. Bioinformatics analyses revealed that all of them were characterized by low pathogenicity and high human or mammalian tropism and had introduced novel avian influenza A virus (AIV) subtypes such as H7N2, H7N6, H9N9, H5N6, H6N6, and H10N6 through gene reassortment. In reassortment events, Anhui isolates may donate one or more segments of HA, NA, and the six internal protein-coding genes for the novel subtype AIVs. Our study revealed that H7N9, H9N2, and H5N1 can serve as stable and persistent gene pools for AIVs in the YRD and South-Central regions of China. Novel AIV subtypes might be generated continuously by reassortment. These AIVs may have obtained human-type receptor-binding abilities from their donors and prefer binding to them, which can cause human epidemics through accidental spillover infections. Facing the continual threat of emerging avian influenza, constant monitoring of AIVs should be conducted closely for agricultural and public health.

Influenza H7N9 virus disrupts the monolayer human brain microvascular endothelial cells barrier in vitro.

Influenza H7N9 virus causes human infections with about 40% case fatality rate. The severe cases usually present with pneumonia; however, some present with central nervous system complications. Pneumonia syndrome is attributed to the cytokine storm after infection with H7N9, but the pathogenic mechanism of central nervous system complications has not been clarified. This study used immortalized human brain microvascular endothelial cells hCMEC/D3 to simulate the blood-brain barrier. It demonstrated that H7N9 virus could infect brain microvascular endothelial cells and compromise the blood-brain barrier integrity and permeability by down-regulating the expression of cell junction-related proteins, including claudin-5, occludin, and vascular endothelial (VE)-cadherin. These results suggested that H7N9 could infect the blood-brain barrier in vitro and affect its functions, which could be a potential mechanism for the pathogenesis of H7N9 viral encephalopathy.

Reintroduction of highly pathogenic avian influenza A H7N9 virus in southwestern China.

Highly pathogenic (HP) avian influenza A H7N9 virus has emerged in China since 2016. In recent years, it has been most prevalent in northern China. However, several strains of HP H7N9 reappeared in southwestern China (Yunnan Province) in 2021. As a result, we are wondering if these viruses have re-emerged in situ or been reintroduced. Here, we present phylogenetic evidence that the HP H7N9 viruses isolated in Yunnan emigrated from northern to southwestern China in 2020. The northern subregion of China has become a novel epicenter in HP H7N9 dissemination. Meanwhile, a cleavage motif re-emerged due to the T341I mutation, implying a parallel evolution. This cross-region transmission, which originated in non-adjacent provinces and traveled a great geographic distance in an unknown way, indicates that HP H7N9 dissemination did not halt in 2020, even under the shadow of the COVID-19 pandemic. Additional surveillance studies in poultry are required to determine the HP H7N9 virus's geographic distribution and spread.

Screening and identification of specific cluster miRNAs in N2a cells infected by H7N9 virus.

This study aims to screen and identify specific cluster miRNAs of H7N9 virus-infected N2a cells and explore the possible pathogenesis of these miRNAs. The N2a cells are infected with H7N9 and H1N1 influenza viruses, and the cells are collected at 12, 24 and 48 h to extract total RNA. To sequence miRNAs and identify different virus-specific miRNAs, high-throughput sequencing technology is used. Fifteen H7N9 virus-specific cluster miRNAs are screened, and eight of them are included in the miRBase database. These cluster-specific miRNAs regulate many signaling pathways, such as the PI3K-Akt signaling pathway, the RAS signaling pathway, the cAMP signaling pathway, actin cytoskeleton regulation and cancer-related genes. The study provides a scientific basis for the pathogenesis of H7N9 avian influenza, which is regulated by miRNAs.

Dynamic PB2-E627K substitution of influenza H7N9 virus indicates the in vivo genetic tuning and rapid host adaptation.

Avian-origin influenza viruses overcome the bottleneck of the interspecies barrier and infect humans through the evolution of variants toward more efficient replication in mammals. The dynamic adaptation of the genetic substitutions and the correlation with the virulence of avian-origin influenza virus in patients remain largely elusive. Here, based on the one-health approach, we retrieved the original virus-positive samples from patients with H7N9 and their surrounding poultry/environment. The specimens were directly deep sequenced, and the subsequent big data were integrated with the clinical manifestations. Unlike poultry/environment-derived samples with the consistent dominance of avian signature 627E of H7N9 polymerase basic protein 2 (PB2), patient specimens had diverse ratios of mammalian signature 627K, indicating the rapid dynamics of H7N9 adaptation in patients during the infection process. In contrast, both human- and poultry/environment-related viruses had constant dominance of avian signature PB2-701D. The intrahost dynamic adaptation was confirmed by the gradual replacement of 627E by 627K in H7N9 in the longitudinally collected specimens from one patient. These results suggest that host adaptation for better virus replication to new hosts, termed "genetic tuning," actually occurred in H7N9-infected patients in vivo. Notably, our findings also demonstrate the correlation between rapid host adaptation of H7N9 PB2-E627K and the fatal outcome and disease severity in humans. The feature of H7N9 genetic tuning in vivo and its correlation with the disease severity emphasize the importance of testing for the evolution of this avian-origin virus during the course of infection.

Serological Evidence of Human Infection With Avian Influenza A(H7N9) Virus: A Systematic Review and Meta-analysis.

BACKGROUND: The extent of human infections with avian influenza A(H7N9) virus, including mild and asymptomatic infections, is uncertain. METHODS: We performed a systematic review and meta-analysis of serosurveys for avian influenza A(H7N9) virus infections in humans published during 2013-2020. Three seropositive definitions were assessed to estimate pooled seroprevalence, seroconversion rate, and seroincidence by types of exposures. We applied a scoring system to assess the quality of included studies. RESULTS: Of 31 included studies, pooled seroprevalence of A(H7N9) virus antibodies from all participants was 0.02%, with poultry workers, close contacts, and general populations having seroprevalence of 0.1%, 0.2%, and 0.02%, respectively, based on the World Health Organization (WHO)-recommended definition. Although most infections were asymptomatic, evidence of infection was highest in poultry workers (5% seroconversion, 19.1% seroincidence per 100 person-years). Use of different virus clades did not significantly affect seroprevalence estimates. Most serological studies were of low to moderate quality and did not follow standardized seroepidemiological protocols or WHO-recommended laboratory methods. CONCLUSIONS: Human infections with avian influenza A(H7N9) virus have been uncommon, especially for general populations. Workers with occupational exposures to poultry and close contacts of A(H7N9) human cases had low risks of infection.