The host tropism of current zoonotic H7N9 viruses depends mainly on an acid-labile hemagglutinin with a single amino acid mutation in the stalk region.

The incidence of human infection by zoonotic avian influenza viruses, especially H5N1 and H7N9 viruses, has increased. Current zoonotic H7N9 avian influenza viruses (identified since 2013) emerged during reassortment of viruses belonging to different subtypes. Despite analyses of their genetic background, we do not know why current H7N9 viruses are zoonotic. Therefore, there is a need to identify the factor(s) responsible for the extended host tropism that enables these viruses to infect humans as well as birds. To identify H7N9-specific amino acids that confer zoonotic properties on H7N9 viruses, we performed multiple alignment of the hemagglutinin (HA) amino acid sequences of A/Shanghai/1/2013 (H7N9) and A/duck/Zhejiang/12/2011(H7N3) (a putative, non- or less zoonotic HA donor to the zoonotic H7N9 virus). We also analyze the function of an H7N9 HA-specific amino acid with respect to HA acid stability, and evaluated the effect of acid stability on viral infectivity and virulence in a mouse model. HA2-116D, preserved in current zoonotic H7N9 viruses, was crucial for loss of HA acid stability. The acid-labile HA protein in H7 viruses played an important role in infection of human airway epithelial cells; HA2-116D contributed to infection and replication of H7 viruses. Finally, HA2-116D served as a H7 virulence factor in mice. These results suggest that acid-labile HA harboring HA2-116D confers zoonotic characteristics on H7N9 virus and that future novel zoonotic avian viruses could emerge from non-zoonotic H7 viruses via acquisition of mutations that remove HA acid stability.

Characterization of Conserved Evolution in H7N9 Avian Influenza Virus Prior Mass Vaccination.

Vaccination is crucial for the prevention and mitigation of avian influenza infections in China. The inactivated H7N9 vaccine, when administered to poultry, significantly lowers the risk of infection among both poultry and humans, while also markedly decreasing the prevalence of H7N9 detections. Highly pathogenic (HP) H7N9 viruses occasionally appear, whereas their low pathogenicity (LP) counterparts have been scarcely detected since 2018. However, these contributing factors remain poorly understood. We conducted an exploratory investigation of the mechanics via the application of comprehensive bioinformatic approaches. We delineated the Yangtze River Delta (YRD) H7N9 lineage into 5 clades (YRD-A to E). Our findings highlight the emergence and peak occurrence of the LP H7N9-containing YRD-E clade during the 5th epidemic wave in China's primary poultry farming areas. A more effective control of LP H7N9 through vaccination was observed compared to that of its HP H7N9 counterpart. YRD-E exhibited a tardy evolutionary trajectory, denoted by the conservation of its genetic and antigenic variation. Our analysis of YRD-E revealed only minimal amino acid substitutions along its phylogenetic tree and a few selective sweep mutations since 2016. In terms of epidemic fitness, the YRD-E was measured to be lower than that of the HP variants. Collectively, these findings underscore the conserved evolutionary patterns distinguishing the YRD-E. Given the conservation presented in its evolutionary patterns, the YRD-E LP H7N9 is hypothesized to be associated with a reduction following the mass vaccination in a relatively short period owing to its lower probability of antigenic variation that might affect vaccine efficiency.

Neu5Gc binding loss of subtype H7 influenza A virus facilitates adaptation to gallinaceous poultry following transmission from waterbirds.

Between 2013 and 2018, the novel A/Anhui/1/2013 (AH/13)-lineage H7N9 virus caused at least five waves of outbreaks in humans, totaling 1,567 confirmed human cases in China. Surveillance data indicated a disproportionate distribution of poultry infected with this AH/13-lineage virus, and laboratory experiments demonstrated that this virus can efficiently spread among chickens but not among Pekin ducks. The underlying mechanism of this selective transmission remains unclear. In this study, we demonstrated the absence of Neu5Gc expression in chickens across all respiratory and gastrointestinal tissues. However, Neu5Gc expression varied among different duck species and even within the tissues of the same species. The AH/13-lineage viruses exclusively bind to acetylneuraminic acid (Neu5Ac), in contrast to wild waterbird H7 viruses that bind both Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The level of Neu5Gc expression influences H7 virus replication and facilitates adaptive mutations in these viruses. In summary, our findings highlight the critical role of Neu5Gc in affecting the host range and interspecies transmission dynamics of H7 viruses among avian species.IMPORTANCEMigratory waterfowl, gulls, and shorebirds are natural reservoirs for influenza A viruses (IAVs) that can occasionally spill over to domestic poultry, and ultimately humans. This study showed wild-type H7 IAVs from waterbirds initially bind to glycan receptors terminated with N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). However, after enzootic transmission in chickens, the viruses exclusively bind to Neu5Ac. The absence of Neu5Gc expression in gallinaceous poultry, particularly chickens, exerts selective pressure, shaping IAV populations, and promoting the acquisition of adaptive amino acid substitutions in the hemagglutinin protein. This results in the loss of Neu5Gc binding and an increase in virus transmissibility in gallinaceous poultry, particularly chickens. Consequently, the transmission capability of these poultry-adapted H7 IAVs in wild water birds decreases. Timely intervention, such as stamping out, may help reduce virus adaptation to domestic chicken populations and lower the risk of enzootic outbreaks, including those caused by IAVs exhibiting high pathogenicity.

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.

A temperature compensated fiber probe for highly sensitive detection in virus gene biosensing.

This research presents an innovative reflective fiber optic probe structure, mutinously designed to detect H7N9 avian influenza virus gene precisely. This innovative structure skillfully combines multimode fiber (MMF) with a thin-diameter seven-core photonic crystal fiber (SCF-PCF), forming a semi-open Fabry-Pérot (FPI) cavity. This structure has demonstrated exceptional sensitivity in light intensity-refractive index (RI) response through rigorous theoretical and experimental validation. The development of a quasi-distributed parallel sensor array, which provides temperature compensation during measurements, has achieved a remarkable RI response sensitivity of up to 532.7 dB/RIU. The probe-type fiber optic sensitive unit, expertly functionalized with streptavidin, offers high specificity in detecting H7N9 avian influenza virus gene, with an impressively low detection limit of 10-2 pM. The development of this biosensor marks a significant development in biological detection, offering a practical engineering solution for achieving high sensitivity and specificity in light-intensity-modulated biosensing. Its potential for wide-ranging applications in various fields is now well-established.

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.

Microfluidic paper-based chemiluminescence sensing platform based on functionalized CaCO3 for time-resolved multiplex detection of avian influenza virus biomarkers.

Multiplex detection can enhance diagnostic precision and improve diagnostic efficiency, providing important assistance for epidemiological investigation and epidemic prevention. There is a great need for multi-detection sensing platforms to accurately diagnose diseases. Herein, we reported a µPAD-based chemiluminescence (CL) assay for ultrasensitive multiplex detection of AIV biomarkers, based on three DNAzyme/Lum/PEI/CaCO3. Three time-resolved CL signals were sequentially generated with detection limits of 0.32, 0.34, and 0.29 pM for H1N1, H7N9, and H5N1, respectively, and with excellent selectivity against interfering DNA. The recovery test in human serum displayed satisfactory analysis capabilities for complex biological samples. The µPAD-based CL assay achieved multiplex detection within 70 s, with a high time resolution of 20 s. The proposed strategy has the advantages of low cost, high sensitivity, good selectivity, and wide time resolution, the µPAD-based CL assay has shown great potential in the early and accurate diagnosis of diseases.

Pathogenicity and transmission of novel highly pathogenic H7N2 variants originating from H7N9 avian influenza viruses in chickens.

The H7 subtype avian influenza viruses are circulating widely worldwide, causing significant economic losses to the poultry industry and posing a serious threat to human health. In 2019, H7N2 and H7N9 co-circulated in Chinese poultry, yet the risk of H7N2 remained unclear. We isolated and sequenced four H7N2 viruses from chickens, revealing them as novel reassortants with H7N9-derived HA, M, NS genes and H9N2-derived PB2, PB1, PA,NP, NA genes. To further explore the key segment of pathogenicity, H7N2-H7N9NA and H7N2-H9N2HA single-substitution were constructed. Pathogenicity study showed H7N2 isolates to be highly pathogenic in chickens, with H7N2-H7N9NA slightly weaker than H7N2-Wild type. Transcriptomic analysis suggested that H7N9-derived HA genes primarily drove the high pathogenicity of H7N2 isolates, eliciting a strong inflammatory response. These findings underscored the increased threat posed by reassorted H7N2 viruses to chickens, emphasizing the necessity of long-term monitoring of H7 subtype avian influenza viruses.

R229I substitution from oseltamivir induction in HA1 region significantly increased the fitness of a H7N9 virus bearing NA 292K.

The proportion of human isolates with reduced neuraminidase inhibitors (NAIs) susceptibility in highly pathogenic avian influenza (HPAI) H7N9 virus was high. These drug-resistant strains showed good replication capacity without serious loss of fitness. In the presence of oseltamivir, R229I substitution were found in HA1 region of the HPAI H7N9 virus before NA R292K appeared. HPAI H7N9 or H7N9/PR8 recombinant viruses were developed to study whether HA R229I could increase the fitness of the H7N9 virus bearing NA 292K. Replication efficiency was assessed in MDCK or A549 cells. Neuraminidase enzyme activity and receptor-binding ability were analyzed. Pathogenicity in C57 mice was evaluated. Antigenicity analysis was conducted through a two-way HI test, in which the antiserum was obtained from immunized ferrets. Transcriptomic analysis of MDCK infected with HPAI H7N9 24hpi was done. It turned out that HA R229I substitution from oseltamivir induction in HA1 region increased (1) replication ability in MDCK(P < 0.05) and A549(P < 0.05), (2) neuraminidase enzyme activity, (3) binding ability to both α2,3 and α2,6 receptor, (4) pathogenicity to mice(more weight loss; shorter mean survival day; viral titer in respiratory tract, P < 0.05; Pathological changes in pneumonia), (5) transcriptome response of MDCK, of the H7N9 virus bearing NA 292K. Besides, HA R229I substitution changed the antigenicity of H7N9/PR8 virus (>4-fold difference of HI titre). It indicated that through the fine-tuning of HA-NA balance, R229I increased the fitness and changed the antigenicity of H7N9 virus bearing NA 292K. Public health attention to this mechanism needs to be drawn.

Development of a Fully Protective Pandemic Avian Influenza Subunit Vaccine in Insect Pupae.

In this study, we pioneered an alternative technology for manufacturing subunit influenza hemagglutinin (HA)-based vaccines. This innovative method involves harnessing the pupae of the Lepidoptera Trichoplusia ni (T. ni) as natural biofactories in combination with baculovirus vectors (using CrisBio® technology). We engineered recombinant baculoviruses encoding two versions of the HA protein (trimeric or monomeric) derived from a pandemic avian H7N1 virus A strain (A/chicken/Italy/5093/99). These were then used to infect T. ni pupae, resulting in the production of the desired recombinant antigens. The obtained HA proteins were purified using affinity chromatography, consistently yielding approximately 75 mg/L of insect extract. The vaccine antigen effectively immunized poultry, which were subsequently challenged with a virulent H7N1 avian influenza virus. Following infection, all vaccinated animals survived without displaying any clinical symptoms, while none of the mock-vaccinated control animals survived. The CrisBio®-derived antigens induced high titers of HA-specific antibodies in the vaccinated poultry, demonstrating hemagglutination inhibition activity against avian H7N1 and human H7N9 viruses. These results suggest that the CrisBio® technology platform has the potential to address major industry challenges associated with producing recombinant influenza subunit vaccines, such as enhancing production yields, scalability, and the speed of development, facilitating the global deployment of highly effective influenza vaccines.

Evolution of H7N9 highly pathogenic avian influenza virus in the context of vaccination.

Human infections with the H7N9 influenza virus have been eliminated in China through vaccination of poultry; however, the H7N9 virus has not yet been eradicated from poultry. Carefully analysis of H7N9 viruses in poultry that have sub-optimal immunity may provide a unique opportunity to witness the evolution of highly pathogenic avian influenza virus in the context of vaccination. Between January 2020 and June 2023, we isolated 16 H7N9 viruses from samples we collected during surveillance and samples that were sent to us for disease diagnosis. Genetic analysis indicated that these viruses belonged to a single genotype previously detected in poultry. Antigenic analysis indicated that 12 of the 16 viruses were antigenically close to the H7-Re4 vaccine virus that has been used since January 2022, and the other four viruses showed reduced reactivity with the vaccine. Animal studies indicated that all 16 viruses were nonlethal in mice, and four of six viruses showed reduced virulence in chickens upon intranasally inoculation. Importantly, the H7N9 viruses detected in this study exclusively bound to the avian-type receptors, having lost the capacity to bind to human-type receptors. Our study shows that vaccination slows the evolution of H7N9 virus by preventing its reassortment with other viruses and eliminates a harmful characteristic of H7N9 virus, namely its ability to bind to human-type receptors.

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.

Avian influenza virus cross-infections as test case for pandemic preparedness: From epidemiological hazard models to sequence-based early viral warning systems.

Pandemic preparedness starts with an early warning system of viruses with a pandemic potential. Based on information collected in a multitude of surveys, hazard models were developed identifying influenza viruses presenting a pandemic threat. Scores are attributed for 10 viral traits by expert panels which identified avian influenza viruses (AIV) belonging to subtypes H7N9 and H5N1 as representing the greatest pandemic risk. In 2013, more than 100 human cases infected with AIV H7N9 were observed in China. Case fatality rate (CFR) was high (27%), but the human-to-human transmission rate was low and by serological evidence H7N9 did not spread widely. Nevertheless, until 2019 more than 1500 H7N9 patients were identified characterized by a high CFR of 39%. Serology demonstrated that mild infections with H7N9 were widespread. In 2003, more than 400 people experienced AIV H7N7 cross-infection causing mainly conjunctivitis during a large poultry epidemic in The Netherlands. Between 1996 and 2019, a total of 881 human infections with avian H5N1 viruses were documented showing a CFR of 52%. Outbreaks were centred on South East Asia and showed close associations with epizootics in poultry. Mutations predisposing to human cross-infections were identified in the haemagglutinin (HA) and the RNA polymerase subunit PB2 of human H7N9 isolates. Human H5N1 isolates showed mutations in the receptor binding domain of HA and transmission in mammals could be obtained by as few as four additional aa changes introduced experimentally. Researchers have defined viral point mutations in HA, PB2 and the nucleoprotein NP that allowed AIV to cross the species barrier to mammals with respect to receptor recognition, RNA replication and escape from innate immunity respectively. Based on this insight a sequence-based early warning system for AIV preadapted to human transmission could be envisioned. Mink farms and live poultry markets are prime targets for such sequencing efforts.

Probenecid Inhibits Influenza A(H5N1) and A(H7N9) Viruses In Vitro and in Mice.

Avian influenza (AI) viruses cause infection in birds and humans. Several H5N1 and H7N9 variants are highly pathogenic avian influenza (HPAI) viruses. H5N1 is a highly infectious bird virus infecting primarily poultry, but unlike other AIs, H5N1 also infects mammals and transmits to humans with a case fatality rate above 40%. Similarly, H7N9 can infect humans, with a case fatality rate of over 40%. Since 1996, there have been several HPAI outbreaks affecting humans, emphasizing the need for safe and effective antivirals. We show that probenecid potently inhibits H5N1 and H7N9 replication in prophylactically or therapeutically treated A549 cells and normal human broncho-epithelial (NHBE) cells, and H5N1 replication in VeroE6 cells and mice.

Immunoinformatic based designing of potential immunogenic novel mRNA and peptide-based prophylactic vaccines against H5N1 and H7N9 avian influenza viruses.

Influenza viruses are the most common cause of serious respiratory illnesses worldwide and are responsible for a significant number of annual fatalities. Therefore, it is crucial to look for new immunogenic sites that might trigger an effective immune response. In the present study, bioinformatics tools were used to design mRNA and multiepitope-based vaccines against H5N1 and H7N9 subtypes of avian influenza viruses. Several Immunoinformatic tools were employed to extrapolate T and B lymphocyte epitopes of HA and NA proteins of both subtypes. The molecular docking approach was used to dock the selected HTL and CTL epitopes with the corresponding MHC molecules. Eight (8) CTL, four (4) HTL, and Six (6) linear B cell epitopes were chosen for the structural arrangement of mRNA and of peptide-based prophylactic vaccine designs. Different physicochemical characteristics of the selected epitopes fitted with suitable linkers were analyzed. High antigenic, non-toxic, and non-allergenic features of the designed vaccines were noted at a neutral physiological pH. Codon optimization tool was used to check the GC content and CAI value of constructed MEVC-Flu vaccine, which were recorded to be 50.42% and 0.97 respectively. the GC content and CAI value verify the stable expression of vaccine in pET28a + vector. In-silico immunological simulation the MEVC-Flu vaccine construct revealed a high level of immune responses. The molecular dynamics simulation and docking results confirmed the stable interaction of TLR-8 and MEVC-Flu vaccine. Based on these parameters, vaccine constructs can be regarded as an optimistic choice against H5N1 and H7N9 strains of the influenza virus. Further experimental testing of these prophylactic vaccine designs against pathogenic avian influenza strains may clarify their safety and efficacy.Communicated by Ramaswamy H. Sarma.

Recombinant duck enteritis virus bearing the hemagglutinin genes of H5 and H7 influenza viruses is an ideal multivalent live vaccine in ducks.

Due to the fact that many avian influenza viruses that kill chickens are not lethal to ducks, farmers are reluctant to use avian influenza inactivated vaccines on ducks. Large numbers of unvaccinated ducks play an important role in the transmission of avian influenza viruses from wild birds to domestic poultry, creating a substantial challenge to vaccination strategies for avian influenza control. To solve this problem, we constructed a recombinant duck enteritis virus (DEV), rDEV-dH5/H7, using a live attenuated DEV vaccine strain (vDEV) as a vector. rDEV-dH5/H7 carries the hemagglutinin gene of two H5 viruses [GZ/S4184/17 (H5N6) (clade 2.3.4.4 h) and LN/SD007/17 (H5N1) (clade 2.3.2.1d)] and an H7 virus [GX/SD098/17 (H7N9)]. These three hemagglutinin genes were stably inherited in rDEV-dH5/H7 and expressed in rDEV-dH5/H7-infected cells. Animal studies revealed that rDEV-dH5/H7 and vDEV induced similar neutralizing antibody responses and protection against lethal DEV challenge. Importantly, rDEV-dH5/H7 induced strong and long-lasting hemagglutinin inhibition antibodies against different H5 and H7 viruses and provided complete protection against challenges with homologous and heterologous highly pathogenic H5 and H7 influenza viruses in ducks. Our study shows that rDEV-dH5/H7 could serve as an ideal live attenuated vaccine to protect ducks against infection with lethal DEV and highly pathogenic avian influenza viruses.

Hemagglutinin affects replication, stability and airborne transmission of the H9N2 subtype avian influenza virus.

H9N2 subtype avian influenza virus (AIV) can transmit by direct as well as airborne contacts. It has been widespread in poultry and continued to contribute to zoonotic spillover events by providing its six internal genes for the reassortment of novel influenza viruses (eg, H7N9) that infect poultry and humans. Compared to H7N9, H9N2 virus displays an efficient airborne transmissibility in poultry, but the mechanisms of transmission difference have been insufficiently studied. The Hemagglutinin (HA) and viral polymerase acidic protein (PA) have been implicated in the airborne transmission of influenza A viruses. Accordingly, we generated the reassortant viruses of circulating airborne transmissible H9N2 and non-airborne transmissible H7N9 viruses carrying HA and/or PA gene. The introduction of the PA gene from H7N9 into the genome of H9N2 virus resulted in a reduction in airborne transmission among chickens, while the isolated introduction of the HA gene segment completely eliminated airborne transmission among chickens. We further showed that introduction of HA gene of non-transmissible H7N9 did not influence the HA/NA balance of H9N2 virus, but increased the threshold for membrane fusion and decreased the acid stability. Thus, our results indicate that HA protein plays a key role in replication, stability, and airborne transmission of the H9N2 subtype AIV.

Mutations in HA and PA affect the transmissibility of H7N9 avian influenza virus in chickens.

Low pathogenic (LP) H7N9 avian influenza virus (AIV) emerged in 2013 and had spread widely over several months in China, experienced a noteworthy reduction in isolation rate in poultry and human since 2017. Here, we examined the transmission of H7N9 viruses to better understand viral spread and dissemination mechanisms. Three out of four viruses (2013-2016) could transmit in chickens through direct contact, and airborne transmission was confirmed in the JT157 (2016) virus. However, we did not detect the transmission of the two 2017 viruses, WF69 and AH395, through either direct or airborne exposure. Molecular analysis of genome sequence of two viruses identified eleven mutations located in viral proteins (except for matrix protein), such as PA (K362R and S364N) and HA (D167N, H7 numbering), etc. We explored the genetic determinants that contributed to the difference in transmissibility of the viruses in chickens by generating a series of reassortants in the JT157 background. We found that the replacement of HA gene in JT157 by that of WF69 abrogated the airborne transmission in recipient chickens, whereas the combination of HA and PA replacement led to the loss of airborne and direct contact transmission. Failure with contact transmission of the viruses has been associated with the emergence of the mutations D167N in HA and K362R and S364N in PA. Furthermore, the HA D167N mutation significantly reduced viral attachment to chicken lung and trachea tissues, while mutations K362R and S364N in PA reduced the nuclear transport efficiency and the PA protein expression levels in both cytoplasm and nucleus of CEF cells. The D167N substitution in HA reduced the H7N9 viral acid stability and avian-like receptor binding, while enhanced human-like receptor binding. Further analysis revealed these mutants grew poorly in vitro and in vivo. To conclude, H7N9 AIVs that contain mutations in the HA and PA protein reduced the viral transmissibility in chicken, and may pose a reduced threat for poultry but remain a heightened public health risk.

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.

Characterization of A/H7 influenza virus global antigenic diversity and key determinants in the hemagglutinin globular head mediating A/H7N9 antigenic evolution.

IMPORTANCE: A/H7 avian influenza viruses cause outbreaks in poultry globally, resulting in outbreaks with significant socio-economical impact and zoonotic risks. Occasionally, poultry vaccination programs have been implemented to reduce the burden of these viruses, which might result in an increased immune pressure accelerating antigenic evolution. In fact, evidence for antigenic diversification of A/H7 influenza viruses exists, posing challenges to pandemic preparedness and the design of vaccination strategies efficacious against drifted variants. Here, we performed a comprehensive analysis of the global antigenic diversity of A/H7 influenza viruses and identified the main substitutions in the hemagglutinin responsible for antigenic evolution in A/H7N9 viruses isolated between 2013 and 2019. The A/H7 antigenic map and knowledge of the molecular determinants of their antigenic evolution add value to A/H7 influenza virus surveillance programs, the design of vaccines and vaccination strategies, and pandemic preparedness.