Emergence, Evolution, and Biological Characteristics of H10N4 and H10N8 Avian Influenza Viruses in Migratory Wild Birds Detected in Eastern China in 2020.

H10Nx influenza viruses have caused increasing public concern due to their occasional infection of humans. However, the genesis and biological characteristics of H10 viruses in migratory wild birds are largely unknown. In this study, we conducted active surveillance to monitor circulation of avian influenza viruses in eastern China and isolated five H10N4 and two H10N8 viruses from migratory birds in 2020. Genetic analysis indicated that the hemagglutinin (HA) genes of the seven H10 viruses were clustered into the North American lineage and established as a novel Eurasian branch in wild birds in South Korea, Bangladesh, and China. The neuraminidase (NA) genes of the H10N4 and H10N8 viruses originated from the circulating HxN4 and H5N8 viruses in migratory birds in Eurasia. We further revealed that some of the novel H10N4 and H10N8 viruses acquired the ability to bind human-like receptors. Animal studies indicated that these H10 viruses can replicate in mice, chickens, and ducks. Importantly, we found that the H10N4 and H10N8 viruses can transmit efficiently among chickens and ducks but induce lower HA inhibition (HI) antibody titers in ducks. These findings emphasized that annual surveillance in migratory waterfowl should be strengthened to monitor the introduction of wild-bird H10N4 and H10N8 reassortants into poultry. IMPORTANCE The emerging avian influenza reassortants and mutants in birds pose an increasing threat to poultry and public health. H10 avian influenza viruses are widely prevalent in wild birds, poultry, seals, and minks and pose an increasing threat to human health. The occasional human infections with H10N8 and H10N3 viruses in China have significantly increased public concern about the potential pandemic risk posed by H10 viruses. In this study, we found that the North American H10 viruses have been successfully introduced to Asia by migratory birds and further reassorted with other subtypes to generate novel H10N4 and H10N8 viruses in eastern China. These emerging H10 reassortants have a high potential to threaten the poultry industry and human health due to their efficient replication and transmission in chickens, ducks, and mice.

The PB2 co-adaptation of H10N8 avian influenza virus increases the pathogenicity to chickens and mice.

Avian influenza (AI) is an important zoonotic disease, which can be transmitted across species barriers to other hosts, especially humans, posing a serious threat to the poultry industry and public health. In recent years, human cases infected with the H10N8, H9N2, and H7N9 of avian influenza viruses (AIVs) have been identified frequently as have the internal genes of H7N9 and H10N8, which are derived from H9N2 viruses. The adaptive mutation of the PB2 gene is an important way for the H10N8, H9N2, and H7N9 AIVs to spread across species to adapt to new hosts. Several well-known adaptive mutations in the PB2 gene, such as E627K, D701N, and A588V, significantly enhanced the virulence of the AIVs in mammals. However, the co-adaptation of AIVs to avian and mammalian hosts is rarely studied. In this study, we found that the mutations of PB2-I292V, PB2-R389K, PB2-A588V, PB2-T598M/V, PB2-L648V, and PB2-T676M substitutions significantly increased after 2012. In addition, in our previous studies, we found that the human-origin and avian-origin of H10N8 AIVs with very high homology also have these six mutation differences in PB2 gene, and the avian-origin H10N8 strain known as JX102 with all the key amino acids on the PB2 protein in the pre-evolutionary stage, so JX102 was chosen as a model strain. Among them, PB2-A588V significantly enhanced the activity of polymerase in avian and mammalian cells. Notably, animal experiments showed that PB2-A588V substitution increased the pathogenicity and transmissibility in chickens and the virulence of mice. The combined mutations of PB2-F6 (including PB2-I292V, PB2-R389K, PB2-A588V, PB2-T598M, PB2-L648V, and PB2-T676M) obtained higher adaptability of AIVs in avians and mammals than that of the single mutation of PB2-A588V, which suggested that the PB2 588 site is a key co-adaptation site and that synergies with other mutation sites can further enhance this co-adaptability. The results of this study show that the emergence of co-adaptation not only increases the threat to avians and mammals but may also contribute to a pandemic among avians and cross the interspecies barrier to mammals.

Evidence of H10N8 influenza virus infection among swine in southern China and its infectivity and transmissibility in swine.

Infection with a novel H10N8 influenza virus in humans was first described in China in December 2013, which raised concerns related to public health. This novel virus was subsequently confirmed to have originated from a live poultry market. However, whether this virus can infect other mammals remains unclear. In the present study, antibody specific for H10N8 influenza virus was detected in swine herds in southern China during serological monitoring for swine influenza virus. The pathogenicity and transmissibility of this H10N8 influenza virus to swine was examined. The results showed that swine are susceptible to infection with human-origin H10N8 influenza virus, which causes viral shedding, severe tissue lesions, and seroconversion, while infection with avian-origin H10N8 influenza virus causes only seroconversion and no viral shedding. Importantly, human-origin H10N8 influenza virus can inefficiently be transmitted between swine and cause seroconversion through direct contact. This study provides a new perspective regarding the ecology of H10N8 influenza virus and highlights the importance of epidemiological monitoring of the H10N8 influenza virus in different animal species, which will be helpful for preventing and controlling future infections by this virus.

mRNA vaccines against H10N8 and H7N9 influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase 1 randomized clinical trials.

BACKGROUND: We evaluated safety and immunogenicity of the first mRNA vaccines against potentially pandemic avian H10N8 and H7N9 influenza viruses. METHODS: Two randomized, placebo-controlled, double-blind, phase 1 clinical trials enrolled participants between December 2015 and August 2017 at single centers in Germany (H10N8) and USA (H7N9). Healthy adults (ages 18-64 years for H10N8 study; 18-49 years for H7N9 study) participated. Participants received vaccine or placebo in a 2-dose vaccination series 3 weeks apart. H10N8 intramuscular (IM) dose levels of 25, 50, 75, 100, and 400 µg and intradermal dose levels of 25 and 50 µg were evaluated. H7N9 IM 10-, 25-, and 50-µg dose levels were evaluated; 2-dose series 6 months apart was also evaluated. Primary endpoints were safety (adverse events) and tolerability. Secondary immunogenicity outcomes included humoral (hemagglutination inhibition [HAI], microneutralization [MN] assays) and cell-mediated responses (ELISPOT assay). RESULTS: H10N8 and H7N9 mRNA IM vaccines demonstrated favorable safety and reactogenicity profiles. No vaccine-related serious adverse event was reported. For H10N8 (N = 201), 100-µg IM dose induced HAI titers ≥ 1:40 in 100% and MN titers ≥ 1:20 in 87.0% of participants. The 25-µg intradermal dose induced HAI titers > 1:40 in 64.7% of participants compared to 34.5% of participants receiving the IM dose. For H7N9 (N = 156), IM doses of 10, 25, and 50 µg achieved HAI titers ≥ 1:40 in 36.0%, 96.3%, and 89.7% of participants, respectively. MN titers ≥ 1:20 were achieved by 100% in the 10- and 25-µg groups and 96.6% in the 50-µg group. Seroconversion rates were 78.3% (HAI) and 87.0% (MN) for H10N8 (100 µg IM) and 96.3% (HAI) and 100% (MN) in H7N9 (50 µg). Significant cell-mediated responses were not detected in either study. CONCLUSIONS: The first mRNA vaccines against H10N8 and H7N9 influenza viruses were well tolerated and elicited robust humoral immune responses. ClinicalTrials.gov NCT03076385 and NCT03345043.

A sandwich ELISA for detecting the hemagglutinin of avian influenza A (H10N8) virus.

Novel influenza A virus (H10N8) infected human with fatality in China during 2013-2014. It is important to detect such nonprevalent subtype influenza A virus in clinic and regular surveillance in the early stage for effective control and prevention from the potential pandemic. Unavailability of convenient rapid diagnosis for this subtype virus in resources-limited setting is an obstacle for timely recognizing human case. In the present study, a panel of mouse H10 specific monoclonal antibodies (mAbs) was generated, two of which were used to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for detecting the hemagglutinin of avian influenza A (H10N8) virus. ELISA results showed high sensitivity with the lowest detection limit of 0.5HAU/50 µL for live virus, which laid a foundation for clinic use as a promising diagnostic methodology.

Genetic and biological characterization of H9N2 avian influenza viruses isolated in China from 2011 to 2014.

The genotypes of the H9N2 avian influenza viruses have changed since 2013 when almost all H9N2 viruses circulating in chickens in China were genotype 57 (G57) with the fittest lineage of each gene. To characterize the H9N2 variant viruses from 2011 to 2014, 28 H9N2 influenza viruses were isolated from live poultry markets in China from 2011-2014 and were analyzed by genetic and biological characterization. Our findings showed that 16 residues that changed antigenicity, two potential N-linked glycosylation sites, and one amino acid in the receptor binding site of the HA protein changed significantly from 2011-2014. Moreover, the HA and NA genes in the phylogenetic tree were mainly clustered into two independent branches, A and B, based on the year of isolation. H9N2 virus internal genes were related to those from the human-infected avian influenza viruses H5N1, H7N9, and H10N8. In particular, the NS gene in the phylogenetic tree revealed genetic divergence of the virus gene into three branches labeled A, B, and C, which were related to the H9N2, H10N8, and H7N9 viruses, respectively. Additionally, the isolates also showed varying levels of infection and airborne transmission. These results indicated that the H9N2 virus had undergone an adaptive evolution and variation from 2011-2014.

Avian influenza viruses (AIVs) H9N2 are in the course of reassorting into novel AIVs.

In 2013, two episodes of influenza emerged in China and caused worldwide concern. A new H7N9 avian influenza virus (AIV) first appeared in China on February 19, 2013. By August 31, 2013, the virus had spread to ten provinces and two metropolitan cities. Of 134 patients with H7N9 influenza, 45 died. From then on, epidemics emerged sporadically in China and resulted in several victims. On November 30, 2013, a 73-year-old woman presented with an influenza-like illness. She developed multiple organ failure and died 9 d after the onset of disease. A novel reassortant AIV, H10N8, was isolated from a tracheal aspirate specimen that was obtained from the patient 7 d after onset. This case was the first human case of influenza A subtype H10N8. On 4 February, 2014, another death due to H10N8 avian influenza was reported in Jiangxi Province, China.

Rhesus Macaque Myeloid-Derived Suppressor Cells Demonstrate T Cell Inhibitory Functions and Are Transiently Increased after Vaccination.

Myeloid-derived suppressor cells (MDSCs) are major regulators of T cell responses in several pathological conditions. Whether MDSCs increase and influence T cell responses in temporary inflammation, such as after vaccine administration, is unknown. Using the rhesus macaque model, which is critical for late-stage vaccine testing, we demonstrate that monocytic (M)-MDSCs and polymorphonuclear (PMN)-MDSCs can be detected using several of the markers used in humans. However, whereas rhesus M-MDSCs lacked expression of CD33, PMN-MDSCs were identified as CD33+ low-density neutrophils. Importantly, both M-MDSCs and PMN-MDSCs showed suppression of T cell proliferation in vitro. The frequency of circulating MDSCs rapidly and transiently increased 24 h after vaccine administration. M-MDSCs infiltrated the vaccine injection site, but not vaccine-draining lymph nodes. This was accompanied by upregulation of genes relevant to MDSCs such as arginase-1, IDO1, PDL1, and IL-10 at the injection site. MDSCs may therefore play a role in locally maintaining immune balance during vaccine-induced inflammation.

Avian influenza viruses (AIVs) H9N2 are in the course of reassorting into novel AIVs / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

In 2013, two episodes of influenza emerged in China and caused worldwide concern. A new H7N9 avian influenza virus (AIV) first appeared in China on February 19, 2013. By August 31, 2013, the virus had spread to ten provinces and two metropolitan cities. Of 134 patients with H7N9 influenza, 45 died. From then on, epidemics emerged sporadically in China and resulted in several victims. On November 30, 2013, a 73-year-old woman presented with an influenza-like illness. She developed multiple organ failure and died 9 d after the onset of disease. A novel reassortant AIV, H10N8, was isolated from a tracheal aspirate specimen that was obtained from the patient 7 d after onset. This case was the first human case of influenza A subtype H10N8. On 4 February, 2014, another death due to H10N8 avian influenza was reported in Jiangxi Province, China.

Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses.

Recently, the World Health Organization confirmed 120 new human cases of avian H7N9 influenza in China resulting in 37 deaths, highlighting the concern for a potential pandemic and the need for an effective, safe, and high-speed vaccine production platform. Production speed and scale of mRNA-based vaccines make them ideally suited to impede potential pandemic threats. Here we show that lipid nanoparticle (LNP)-formulated, modified mRNA vaccines, encoding hemagglutinin (HA) proteins of H10N8 (A/Jiangxi-Donghu/346/2013) or H7N9 (A/Anhui/1/2013), generated rapid and robust immune responses in mice, ferrets, and nonhuman primates, as measured by hemagglutination inhibition (HAI) and microneutralization (MN) assays. A single dose of H7N9 mRNA protected mice from a lethal challenge and reduced lung viral titers in ferrets. Interim results from a first-in-human, escalating-dose, phase 1 H10N8 study show very high seroconversion rates, demonstrating robust prophylactic immunity in humans. Adverse events (AEs) were mild or moderate with only a few severe and no serious events. These data show that LNP-formulated, modified mRNA vaccines can induce protective immunogenicity with acceptable tolerability profiles.

The 150-Loop Restricts the Host Specificity of Human H10N8 Influenza Virus.

Adaptation of influenza A viruses to new hosts are rare events but are the basis for emergence of new influenza pandemics in the human population. Thus, understanding the processes involved in such events is critical for anticipating potential pandemic threats. In 2013, the first case of human infection by an avian H10N8 virus was reported, yet the H10 hemagglutinin (HA) maintains avian receptor specificity. However, the 150-loop of H10 HA, as well as related H7 and H15 subtypes, contains a two-residue insert that can potentially block human receptor binding. Mutation of the 150-loop on the background of Q226L and G228S mutations, which arose in the receptor-binding site of human pandemic H2 and H3 viruses, resulted in acquisition of human-type receptor specificity. Crystal structures of H10 HA mutants with human and avian receptor analogs, receptor-binding studies, and tissue staining experiments illustrate the important role of the 150-loop in H10 receptor specificity.

Induction of protective immunity against influenza A/Jiangxi-Donghu/346/2013 (H10N8) in mice.

Human infections with A/Jiangxi-Donghu/346/2013 (H10N8) virus have raised concerns about its pandemic potential. In order to develop a vaccine against this virus, the immunogenicity of its haemagglutinin protein was evaluated in mice. Using both whole-virion and recombinant subunit protein vaccines, we showed that two doses of either vaccine elicited neutralizing antibody responses. The protective efficacy of the vaccine-induced responses was assessed using a reverse-genetics-derived H10 reassortant virus on the A/Puerto Rico/8/34 (H1N1) backbone. The reassortant virus replicated efficiently in the respiratory tract of unvaccinated mice whereas vaccinated mice were completely protected from challenge, with no detectable viral load in the lower respiratory tract. Finally, the serum neutralizing antibody responses elicited by the H10 vaccines also exhibited cross-neutralizing activity against three heterologous wild-type H10 viruses. Collectively, these findings demonstrate that different vaccine platforms presenting the H10 haemagglutinin protein induce protective immunity.

Identification of Epitopes for Neutralizing Antibodies Against H10N8 Avian Influenza Virus Hemagglutinin.

Objective To develop neutralizing monoclonal antibodies (MAbs) against H10N8 avian influenza virus hemagglutinin and to identify the binding sites. Methods MAbs against hemagglutinin of H10N8 avian influenza virus were developed by genetic engineering. Neutralizing MAbs were screened by microneutralization assay,and then tested by enzyme-linked immunosorbent assay and Western blot to identity the binding sites.The homology modeling process was performed using Discovery Studio 3.5 software,while the binding epitopes were analyzed by BioEdit software. Results One MAb that could neutralize the H10N8 pseudovirus was obtained and characterized. Analysis about epitopes suggested that the antibody could bind to the HA1 region of hemagglutinin,while the epitopes on antigen were conserved in H10 subtypes.Conclusions One neutralizing antibody was obtained by this research.The MAb may potentially be further developed as a pre-clinical candidate to treat avian influenza H10N8 virus infection.

Mono- and quadri-subtype virus-like particles (VLPs) containing H10 subtype elicit protective immunity to H10 influenza in a ferret challenge model.

Avian-origin influenza represents a global public health concern. In 2013, the H10N8 virus caused documented human infections for the first time. Currently, there is no approved vaccine against H10 influenza. Recombinant virus-like particles (VLPs) represent a promising vaccine approach. In this study, we evaluated H10 VLPs containing hemagglutinin from H10N8 virus as an experimental vaccine in a ferret challenge model. In addition, we evaluated quadri-subtype VLPs co-localizing H5, H7, H9 and H10 subtypes. Both vaccines elicited serum antibody that reacted with the homologous H10 derived from H10N8 virus and cross-reacted with the heterologous H10N1 virus. Quadri-subtype vaccine also elicited serum antibody to the homologous H5, H7, and H9 antigens and cross-reacted with multiple clades of H5N1 virus. After heterologous challenge with the H10N1 virus, all vaccinated ferrets showed significantly reduced titers of replicating virus in the respiratory tract indicating protective effect of vaccination with either H10 VLPs or with quadri-subtype VLPs.

PB2-588 V promotes the mammalian adaptation of H10N8, H7N9 and H9N2 avian influenza viruses.

Human infections with avian influenza H7N9 or H10N8 viruses have been reported in China, raising concerns that they might cause human epidemics and pandemics. However, how these viruses adapt to mammalian hosts is unclear. Here we show that besides the commonly recognized viral polymerase subunit PB2 residue 627 K, other residues including 87E, 292 V, 340 K, 588 V, 648 V, and 676 M in PB2 also play critical roles in mammalian adaptation of the H10N8 virus. The avian-origin H10N8, H7N9, and H9N2 viruses harboring PB2-588 V exhibited higher polymerase activity, more efficient replication in mammalian and avian cells, and higher virulence in mice when compared to viruses with PB2-588 A. Analyses of available PB2 sequences showed that the proportion of avian H9N2 or human H7N9 influenza isolates bearing PB2-588 V has increased significantly since 2013. Taken together, our results suggest that the substitution PB2-A588V may be a new strategy for an avian influenza virus to adapt mammalian hosts.

Preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein.

Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150-200nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation.

Deep sequencing reveals the viral adaptation process of environment-derived H10N8 in mice.

The H10N8 virus was isolated from the water of Dongting Lake, China. Mice were infected while showing no obvious symptoms and replication was restricted to the lungs. When the wild-type virus was serially passaged in the lungs of mice, the resulting viruses became lethal and capable of replication in many other organs. This offered an applicable model for the exploration of viral genome gradual mutation during adaptation in mice. The different passage viruses from mice lung lavage were named P1, P3, P5, and P7, respectively. We sequenced the four viruses using next-generation sequencing (NGS) to analyze the dynamics of the H10N8 viral genome, polymorphism, and amino acid mutation of related proteins. We aimed to demonstrate how a mutant strain of low pathogenicity could become lethal to mice. Using Illumina high-throughput data, we detected the gradual mutations of F277S, C278Q, F611S and L653P in the polymerase acidic (PA) protein, and of L207V and E627K in the PB2 protein during adaptation. Interestingly, many amino acid sites mutated quickly; the others did so more slowly and remained in a heterozygous state for several generations. The PA amino acids S277 and Q278 have previously been found in clinical wild-type strains, including the human-H10N8 isolate in 2013. This demonstrates that the wild-type H10N8 virus had mutated to adapt to mammalian hosts. These data provide important reference information for influenza virus research.

Hemagglutinin Stalk- and Neuraminidase-Specific Monoclonal Antibodies Protect against Lethal H10N8 Influenza Virus Infection in Mice.

UNLABELLED: Between November 2013 and February 2014, China reported three human cases of H10N8 influenza virus infection in the Jiangxi province, two of which were fatal. Using hybridoma technology, we isolated a panel of H10- and N8-directed monoclonal antibodies (MAbs) and further characterized the binding reactivity of these antibodies (via enzyme-linked immunosorbent assay) to a range of purified virus and recombinant protein substrates. The H10-directed MAbs displayed functional hemagglutination inhibition (HI) and neutralization activity, and the N8-directed antibodies displayed functional neuraminidase inhibition (NI) activity against H10N8. Surprisingly, the HI-reactive H10 antibodies, as well as a previously generated, group 2 hemagglutinin (HA) stalk-reactive antibody, demonstrated NI activity against H10N8 and an H10N7 strain; this phenomenon was absent when virus was treated with detergent, suggesting the anti-HA antibodies inhibited neuraminidase enzymatic activity through steric hindrance. We tested the prophylactic efficacy of one representative H10-reactive, N8-reactive, and group 2 HA stalk-reactive antibody in vivo using a BALB/c challenge model. All three antibodies were protective at a high dose (5 mg/kg). At a low dose (0.5 mg/kg), only the anti-N8 antibody prevented weight loss. Together, these data suggest that antibody targets other than the globular head domain of the HA may be efficacious in preventing influenza virus-induced morbidity and mortality. IMPORTANCE: Avian H10N8 and H10N7 viruses have recently crossed the species barrier, causing morbidity and mortality in humans and other mammals. Although these reports are likely isolated incidents, it is possible that more cases may emerge in future winter seasons, similar to H7N9. Furthermore, regular transmission of avian influenza viruses to humans increases the risk of adaptive mutations and reassortment events, which may result in a novel virus with pandemic potential. Currently, no specific therapeutics or vaccines are available against the H10N8 influenza virus subtype. We generated a panel of H10- and N8-reactive MAbs. Although these antibodies may practically be developed into therapeutic agents, characterizing the protective potential of MAbs that have targets other than the HA globular head domain will provide insight into novel antibody-mediated mechanisms of protection and help to better understand correlates of protection for influenza A virus infection.

Identification of Epitopes for Neutralizing Antibodies Against H10N8 Avian Influenza Virus Hemagglutinin / 中国医学科学院学报

Objective To develop neutralizing monoclonal antibodies (MAbs) against H10N8 avian influenza virus hemagglutinin and to identify the binding sites. Methods MAbs against hemagglutinin of H10N8 avian influenza virus were developed by genetic engineering. Neutralizing MAbs were screened by microneutralization assay,and then tested by enzyme-linked immunosorbent assay and Western blot to identity the binding sites.The homology modeling process was performed using Discovery Studio 3.5 software,while the binding epitopes were analyzed by BioEdit software. Results One MAb that could neutralize the H10N8 pseudovirus was obtained and characterized. Analysis about epitopes suggested that the antibody could bind to the HA1 region of hemagglutinin,while the epitopes on antigen were conserved in H10 subtypes.Conclusions One neutralizing antibody was obtained by this research.The MAb may potentially be further developed as a pre-clinical candidate to treat avian influenza H10N8 virus infection.

Rapid Detection of Subtype H10N8 Influenza Virus by One-Step Reverse Transcription-Loop-Mediated Isothermal Amplification Methods.

We developed hemagglutinin- and neuraminidase-specific one-step reverse transcription-loop-mediated isothermal amplification assays for detecting the H10N8 virus. The detection limit of the assays was 10 copies of H10N8 virus, and the assays did not amplify nonspecific RNA. The assays can detect H10N8 virus from chicken samples with high sensitivity and specificity, and they can serve as an effective tool for detecting and monitoring H10N8 virus in live poultry markets.