H5N1 Avian Influenza Viruses Research Articles

Heat-Denatured Lysozyme is a Novel Potential Non-alcoholic Disinfectant Against Respiratory Virus.

Respiratory diseases are significant recurrent threats to global public health. Since the 1918 Spanish flu pandemic, seasonal influenza viruses continue to cause epidemics around the world each year. More recently, the COVID-19 global pandemic conducted a public health crisis with more than 6 million deaths and it also severely affected the global economy. Due to the phenomenon that people get infection from objects carrying viruses, it has aroused people's attention to home disinfection. As there is no ideal existing common domestic disinfectant, new and safer antiviral disinfectants are urgently needed. Lysozyme is a natural antibacterial agent widespread in nature and widely used in healthcare and food industry because of is recognized safety. Recently, it has been shown that thermally denatured lysozyme has the ability to kill murine norovirus and hepatitis A virus. In our study, we also demonstrated that heat-denatured lysozyme (HDLz) had an antiviral effect against H1N1 influenza A virus, and we optimized its antiviral activities by testing different heating denaturation conditions, to generalize this property, using pseudotype virus neutralization assay, we found that HDLz can also inhibit the entry of H5N1, H5N6, and H7N1 avian influenza viruses as well as SARS-CoV and SARS-CoV-2 particles in cell with IC50 at the ng/mL range. Finally, using western blot analysis, we provide evidence that HDLz polymerization correlates with antiviral effect, which may be a precious possible quality control test. Altogether, our data support HDLz as a powerful anti-respiratory virus disinfectant as a sole or additive of current disinfectants to reduce concentration of toxic component.

Comparison of replication competence between two highly pathogenic avian influenza viruses H5N1 and H5N8 in vitro

Comparison of replication competence between two highly pathogenic avian influenza viruses H5N1 and H5N8 in vitro

We need to keep an eye on avian influenza.

H5N1 avian influenza viruses of the A/goose/Guangdong/1/96 lineage have been circulating in wild birds for many years, have inflicted significant economic losses on the poultry industry and have caused zoonotic infections since 1997. However, the recent spread of avian H5N1 virus to the Americas and its detection in mammals have raised concerns about its pandemic potential. Recent evidence of transmission of avian H5N1 viruses among mammals raises concerns about their pandemic potential. We need to increase our awareness and preparedness, and should also be thinking about safe farming practices to prevent the emergence of concerning pathogens in the future.

Genetic characterization and pathogenicity of H7N9 highly pathogenic avian influenza viruses isolated from South China in 2017.

Since 2017, the new H7N9 highly pathogenic avian influenza viruses (HPAIVs) have been responsible for more than 200,000 cases of chicken infection and more than 120,000 chicken deaths in China. Our previous study found that the Q26 was chicken-origin H7N9 HPAIV. In this study, we analyzed the genetic characterization of Q24, Q65, Q66, Q85, and Q102 H7N9 avian influenza viruses isolated from Guangdong, China in 2017. Our results showed that these viruses were highly pathogenic and belonged to two different genotypes, which suggested they occurred genetic reassortant. To investigate the pathogenicity, transmission, and host immune responses of H7N9 virus in chickens, we selected Q24 and Q26 viruses to inoculate chickens. The Q24 and Q26 viruses killed all inoculated chickens within 3 days and replicated effectively in all tested tissues. They were efficiently transmitted to contact chickens and killed them within 4 days through direct contact. Furthermore, we found that the expressions of several immune-related genes (e.g., TLR3, TLR7, MDA5, MAVS, IFN-β, IL-6, IL-8, OAS, Mx1, MHC I, and MHC II) were upregulated obviously in the lungs and spleen of chickens inoculated with the two H7N9 viruses at 24 h post-inoculation (HPI). Among these, IL-6 and IFN-β in lungs were the most upregulated (by 341.02-381.48-fold and 472.50-500.56-fold, respectively). These results suggest that the new H7N9 viruses isolated in 2017, can replicate and transmit effectively and trigger strong immune responses in chickens, which helps us understand the genetic and pathogenic variations of H7N9 HPAIVs in China.

Pathogenicity, transmissibility, and immunogenicity of recombinant H9N2 avian influenza viruses based on representative viruses of Southeast China

H9N2 is currently the main subtype of avian influenza in China. In order to use reverse genetics to rapid preparation of seed strains for vaccine production, and intend to prevent and control the H9N2 subtype epidemic strains of avian influenza virus (AIV). In this study, we successfully rescued 2 H9N2 recombinant viruses based on the representative viruses of Southeast China and confirmed by RT-PCR and sequencing. Genetic stability, pathogenicity, transmissibility, and antigenicity of 2 recombinant viruses were evaluated. Compared to the FZ1, the growth kinetics of H9N2(HA+NA)/PR8 showed no significant difference, H9N2(HA+NA+M+PB1)/PR8 was slightly lower. Our study also confirmed 2 recombinant viruses had good genetic stability after 10 passages but possessed lower pathogenicity than FZ1. Although both recombinant viruses led to seroconversion in all inoculated birds on 14 dpi, they complete loss of viral transmission of the virus to contact birds. In addition, birds were immunized via hypodermic route by inactivated vaccines of H9N2(HA+NA)/PR8, H9N2(HA+NA+M+PB1)/PR8 and wild-type virus with a single dose, and the results showed that the hemagglutination inhibition titers on 21 dpv were 10.5, 9.6, and 10.5 log2, respectively. And recombinant viruses both provided a certain protection against wild-type virus challenge. In conclusion, these data indicated that 2 recombinant viruses will be expected to be used as inactivated vaccines to controlling the spread of H9N2 subtype AIV even have potential application for attenuated viral vaccines, which provides a reference for the prevention and control of influenza virus pandemics.

Phylogenetic and epidemiological characteristics of H9N2 avian influenza viruses in Shandong Province, China from 2019 to 2021

H9N2 avian influenza virus (AIV) has widely circulated in poultry worldwide and sporadic infections in humans and mammals. During our surveillance of chicken from 2019 to 2021 in Shandong Province, China, we isolated 11 H9N2 AIVs. Phylogenetic analyses showed that the 8 gene segments of the 11 isolates were closely related to several sublineages of Eurasian lineage: BJ/94-like clades (HA and NA genes), G1-like clades (PB2 and M genes), and SH/F/98-like clades (PB1, PA, NP and NS genes). The isolates showed mutation sites that preferentially bind to human-like receptors (HA) and mammalian fitness sites (PB2, PB1 and PA), as well as mutations in antigen and drug resistance sites. Moreover, studies with mice revealed 4 isolates with varying levels of pathogenicity. The average antibody titer of the H9N2 AIVs was 8.60 log2. Based on our results, the epidemiological surveillance of H9N2 AIVs should be strengthened.

That H9N2 avian influenza viruses circulating in different regions gather in the same live-poultry market poses a potential threat to public health.

H9N2 avian influenza viruses are endemic and persistent in China, but those that are prevalent in different provinces are also causes of wide epidemics, related to the spread of wild birds and the cross-regional trade in live poultry. For the past 4 years, beginning in 2018, we have sampled a live-poultry market in Foshan, Guangdong, in this ongoing study. In addition to the prevalence of H9N2 avian influenza viruses in China during this period, we identified isolates from the same market belonging to clade A and clade B, which diverged in 2012-2013, and clade C, which diverged in 2014-2016, respectively. An analysis of population dynamics revealed that, after a critical divergence period from 2014 to 2016, the genetic diversity of H9N2 viruses peaked in 2017. Our spatiotemporal dynamics analysis found that clade A, B, and C, which maintain high rates of evolution, have different prevalence ranges and transmission paths. Clades A and B were mainly prevalent in East China in the early stage, and then spread to Southern China, becoming epidemic with clade C. Strains from different regions converge at the same live-poultry market to communicate, which may be one reasons the H9N2 viruses are difficult to eradicate and increasingly dominant throughout China. Selection pressure and molecular analysis have demonstrated that single amino acid polymorphisms at key receptor binding sites 156, 160, and 190 under positive selection pressure, suggesting that H9N2 viruses are undergoing mutations to adapt to new hosts. Live-poultry markets are important because people who visit them have frequent contact with poultry, H9N2 viruses from different regions converge at these markets and spread through contact between live birds and humans, generating increased risks of human exposure to these viruses and threatening public health safety. Thus, it is important to reducing the cross-regional trade of live poultry and strengthening the monitoring of avian influenza viruses in live-poultry markets to reduce the spread of avian influenza viruses.

Experimental Pathogenicity of H9N2 Avian Influenza Viruses Harboring a Tri-Basic Hemagglutinin Cleavage Site in Sonali and Broiler Chickens.

Low-pathogenic avian influenza (LPAI) H9N2 virus is endemic in Bangladesh, causing huge economic losses in the poultry industry. Although a considerable number of Bangladeshi LPAI H9N2 viruses have been molecularly characterized, there is inadequate information on the pathogenicity of H9N2 viruses in commercial poultry. In this study, circulating LPAI H9N2 viruses from recent field outbreaks were characterized, and their pathogenicity in commercial Sonali (crossbred) and broiler chickens was assessed. Phylogenetic analysis of currently circulating field viruses based on the hemagglutinin (HA) and neuraminidase (NA) gene sequences revealed continuous circulation of G1 lineages containing the tri-basic hemagglutinin cleavage site (HACS) motif (PAKSKR*GLF) at the HA protein. Both the LPAI susceptible Sonali and broiler chickens were infected with selected H9N2 isolates A/chicken/Bangladesh/2458-LT2/2020 or A/chicken/Bangladesh/2465-LT56/2021 using intranasal (100 µL) and intraocular (100 µL) routes with a dose of 106 EID50/mL. Infected groups (LT_2-So1 and LT_56-So2; LT_2-Br1 and LT_56-Br2) revealed no mortality or clinical signs. However, at gross and histopathological investigation, the trachea, lungs, and intestine of the LT_2-So1 and LT_56-So2 groups displayed mild to moderate hemorrhages, congestion, and inflammation at different dpi. The LT 2-Br1 and LT 56-Br2 broiler groups showed nearly identical changes in the trachea, lungs, and intestine at various dpi, indicating no influence on pathogenicity in the two commercial bird species under study. Overall, the prominent lesions were observed up to 7 dpi and started to disappear at 10 dpi. The H9N2 viruses predominantly replicated in the respiratory tract, and higher titers of virus were shed through the oropharyngeal route than the cloacal route. Finally, this study demonstrated the continuous evolution of tri-basic HACS containing H9N2 viruses in Bangladesh with a low-pathogenic phenotype causing mild to moderate tracheitis, pneumonia, and enteritis in Sonali and commercial broiler chickens.

Amino Acid Variation at Hemagglutinin Position 193 Impacts the Properties of H9N2 Avian Influenza Virus.

Despite active control strategies, including the vaccination program in poultry, H9N2 avian influenza viruses possessing mutations in hemagglutinin (HA) were frequently isolated. In this study, we analyzed the substitutions at HA residue 193 (H3 numbering) of H9N2 and investigated the impact of these mutations on viral properties. Our study indicated that H9N2 circulating in the Chinese poultry have experienced frequent mutations at HA residue 193 since 2013, with viruses that carried asparagine (N) being replaced by those with alanine (A), aspartic acid (D), glutamic acid (E), glycine (G), and serine (S), etc. Our results showed the N193G mutation impeded the multiple cycles of growth of H9N2, and although most of the variant HAs retained the preference for human-like receptors as did the wild-type N193 HA, the N193E mutation altered the preference for both human and avian-like receptors. Furthermore, these mutations substantially altered the antigenicity of H9N2 as measured by both monoclonal antibodies and antisera. In vivo studies further demonstrated that these mutations showed profound impact on viral replication and transmission of H9N2 in chicken. Viruses with D, E, or S at residue 193 acquired the ability to replicate in lungs of the infected chickens, whereas virus with G193 reduced its transmissibility in infected chickens to those in direct contact. Our findings demonstrated that variations at HA residue 193 altered various properties of H9N2, highlighting the significance of the continued surveillance of HA for better understanding of the etiology and effective control of H9N2 in poultry. IMPORTANCE H9N2 are widespread and have sporadically caused clinical diseases in humans. Extensive vaccinations in poultry helped constrain H9N2; however, they might have facilitated the evolution of the virus. It is therefore of importance to monitor the variation of the circulating H9N2 and evaluate its risk to both veterinary and public health. Here, we found substitutions at position 193 of HA from H9N2 circulated since 2013 and assessed the impact of several mutations on viral properties. Our data showed these mutations resulted in substantial antigenic change. N193E altered the binding preference of HA for human-like to both avian and human-like receptors. More importantly, N193G impaired the growth of H9N2 and its transmission in chickens, whereas mutations from N to D, E, and S enhanced the viral replication in lungs of chickens. Our study enriched the knowledge about H9N2 and may help implement an effective control strategy for H9N2.

Phenotypic effects of mutations observed in the neuraminidase of human origin H5N1 influenza A viruses

Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential.

Natural variant R246K in hemagglutinin increased zoonotic characteristics and renal inflammation in mice infected with H9N2 influenza virus

Considered a potential pandemic candidate, the widespread among poultry of H9N2 avian influenza viruses across Asia and North Africa pose an increasing threat to poultry and human health. The massive epidemic of H9N2 viruses has expanded the host range; however, the molecular basis and characteristic underlying the transmission to poultry and mammals remains unclear. Our previous study has proved that some natural mutations in the HA gene enhanced the binding ability of the H9N2 virus to α-2,6 SA receptors. Here, we systematically analyzed the impact of these natural mutations on zoonotic characteristics and the pathogenicity of H9N2 AIVs in poultry and mammals. Our study demonstrated that mutation R246K increased the replication in human lung epithelial cells in vitro. Mutation R246K increased the virus shedding of oropharyngeal swabs during early-stage infection in chickens. Moreover, mutation R246K displayed stronger pH stability and pathogenicity in mice. The strong renal tropism and inflammatory response may accelerate the pathogenicity. In summary, we found that natural variation R246K in HA of prevalent H9N2 in China promoted the transmissibility in chicken and accelerate the pathogenicity in mice, posing a great concern for zoonotic and pandemic emergence.

Bidirectional Movement of Emerging H5N8 Avian Influenza Viruses Between Europe and Asia via Migratory Birds Since Early 2020.

Migratory birds play a critical role in the rapid spread of highly pathogenic avian influenza (HPAI) H5N8 virus clade 2.3.4.4 across Eurasia. Elucidating the timing and pattern of virus transmission is essential therefore for understanding the spatial dissemination of these viruses. In this study, we surveyed >27,000 wild birds in China, tracked the year-round migration patterns of 20 bird species across China since 2006, and generated new HPAI H5N8 virus genomic data. Using this new data set, we investigated the seasonal transmission dynamics of HPAI H5N8 viruses across Eurasia. We found that introductions of HPAI H5N8 viruses to different Eurasian regions were associated with the seasonal migration of wild birds. Moreover, we report a backflow of HPAI H5N8 virus lineages from Europe to Asia, suggesting that Europe acts as both a source and a sink in the global HPAI virus transmission network.

Risk assessment of the newly emerged H7N9 avian influenza viruses

ABSTRACT Since the first human case in 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1500 human infections with a mortality rate of approximately 40%. Despite large-scale poultry vaccination regimes across China, the H7N9 AIVs continue to persist and evolve rapidly in poultry. Recently, several strains of H7N9 AIVs have been isolated and shown the ability to escape vaccine-induced immunity. To assess the zoonotic risk of the recent H7N9 AIV isolates, we rescued viruses with hemagglutinin (HA) and neuraminidase (NA) from these H7N9 AIVs and six internal segments from PR8 virus and characterized their receptor binding, pH of fusion, thermal stability, plaque morphology and in ovo virus replication. We also assessed the cross-reactivity of the viruses with human monoclonal antibodies (mAbs) against H7N9 HA and ferret antisera against H7N9 AIV candidate vaccines. The H7N9 AIVs from the early epidemic waves had dual sialic acid receptor binding characteristics, whereas the more recent H7N9 AIVs completely lost or retained only weak human sialic acid receptor binding. Compared with the H7N9 AIVs from the first epidemic wave, the 2020/21 viruses formed larger plaques in Madin-Darby canine kidney (MDCK) cells and replicated to higher titres in ovo, demonstrating increased acid stability but reduced thermal stability. Further analysis showed that these recent H7N9 AIVs had poor cross-reactivity with the human mAbs and ferret antisera, highlighting the need to update the vaccine candidates. To conclude, the newly emerged H7N9 AIVs showed characteristics of typical AIVs, posing reduced zoonotic risk but a heightened threat for poultry.

Comparative Surface Electrostatics and Normal Mode Analysis of High and Low Pathogenic H7N7 Avian Influenza Viruses.

Influenza A viruses are rarely symptomatic in wild birds, while representing a higher threat to poultry and mammals, where they can cause a variety of symptoms, including death. H5 and H7 subtypes of influenza viruses are of particular interest because of their pathogenic potential and reported capacity to spread from poultry to mammals, including humans. The identification of molecular fingerprints for pathogenicity can help surveillance and early warning systems, which are crucial to prevention and protection from such potentially pandemic agents. In the past decade, comparative analysis of the surface features of hemagglutinin, the main protein antigen in influenza viruses, identified electrostatic fingerprints in the evolution and spreading of H5 and H9 subtypes. Electrostatic variation among viruses from avian or mammalian hosts was also associated with host jump. Recent findings of fingerprints associated with low and highly pathogenic H5N1 viruses, obtained by means of comparative electrostatics and normal modes analysis, prompted us to check whether such fingerprints can also be found in the H7 subtype. Indeed, evidence presented in this work showed that also in H7N7, hemagglutinin proteins from low and highly pathogenic strains present differences in surface electrostatics, while no meaningful variation was found in normal modes.

Avian H7N9 influenza viruses are evolutionarily constrained by stochastic processes during replication and transmission in mammals.

H7N9 avian influenza viruses (AIVs) have caused over 1,500 documented human infections since emerging in 2013. Although wild-type H7N9 AIVs can be transmitted by respiratory droplets in ferrets, they have not yet caused widespread outbreaks in humans. Previous studies have revealed molecular determinants of H7N9 AIV host switching, but little is known about potential evolutionary constraints on this process. Here, we compare patterns of sequence evolution for H7N9 AIV and mammalian H1N1 viruses during replication and transmission in ferrets. We show that three main factors-purifying selection, stochasticity, and very narrow transmission bottlenecks-combine to severely constrain the ability of H7N9 AIV to effectively adapt to mammalian hosts in isolated, acute spillover events. We find rare evidence of natural selection favoring new, potentially mammal-adapting mutations within ferrets but no evidence of natural selection acting during transmission. We conclude that human-adapted H7N9 viruses are unlikely to emerge during typical spillover infections. Our findings are instead consistent with a model in which the emergence of a human-transmissible virus would be a rare and unpredictable, though highly consequential, 'jackpot' event. Strategies to control the total number of spillover infections will limit opportunities for the virus to win this evolutionary lottery.

Determining the Protective Efficacy of Toll-Like Receptor Ligands to Minimize H9N2 Avian Influenza Virus Transmission in Chickens.

Low-pathogenicity avian influenza viruses (AIV) of the H9N2 subtype can infect and cause disease in chickens. Little is known about the efficacy of immune-based strategies for reducing the transmission of these viruses. The present study investigated the efficacy of Toll-like receptor (TLR) ligands (CpG ODN 2007 and poly(I:C)) to reduce H9N2 AIV transmission from TLR-treated seeder (trial 1) or inoculated chickens (trial 2) to naive chickens. The results from trial 1 revealed that a low dose of CpG ODN 2007 led to the highest reduction in oral shedding, and a high dose of poly(I:C) was effective at reducing oral and cloacal shedding. Regarding transmission, the recipient chickens exposed to CpG ODN 2007 low-dose-treated seeder chickens showed a maximum reduction in shedding with the lowest number of AIV+ chickens. The results from trial 2 revealed a maximum reduction in oral and cloacal shedding in the poly(I:C) high-dose-treated chickens (recipients), followed by the low-dose CpG ODN 2007 group. In these two groups, the expression of type I interferons (IFNs), protein kinase R (PKR), interferon-induced transmembrane protein 3 (IFITM3), viperin, and (interleukin) IL-1β, IL-8, and 1L-18 was upregulated in the spleen, cecal tonsils and lungs. Hence, TLR ligands can reduce AIV transmission in chickens.

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.

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.

Pathogenicity and Transmissibility of Clade 2.3.4.4h H5N6 Avian Influenza Viruses in Mammals.

Avian influenza viruses (AIVs) have the potential for cross-species transmission and pandemics. In recent years, clade 2.3.4.4 H5N6 AIVs are prevalent in domestic poultry, posing a threat to the domestic poultry industry and public health. In this study, two strains of H5N6 AIVs were isolated from chickens in Hebei, China, in 2019: A/chicken/Hebei/HB1907/2019(H5N6) and A/chicken/Hebei/HB1905/2019(H5N6). Phylogenetic analysis showed that both viral HA genes clustered in the 2.3.4.4h clade. Receptor binding analysis showed that the HB1905 strain preferentially binds to α-2,3-linked sialic acid (SA) receptors, while the HB1907 strain preferentially binds to α-2,3- and α-2,6-linked sialic acid (SA) receptors. During early infection, the HB1907 strain is highly replicable in MDCK cells, more so than the HB1905 strain. Pathogenicity assays in mice showed that both viruses could replicate in the lungs without prior adaptation, with HB1907 being more highly pathogenic in mice than the HB1905 strain. Significantly, both the HB1905 and HB1907 strains can be transmitted through direct contact among guinea pigs, but the transmission efficiency of the HB1907 strain through contact between guinea pigs is much greater than that of the HB1905 strain. These results strengthen the need for ongoing surveillance and early warning of H5N6 AIVs in poultry.

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.