A Single Amino Acid Substitution at Residue 218 of Hemagglutinin Improves the Growth of Influenza A(H7N9) Candidate Vaccine Viruses.

The potential avian influenza pandemic remains a threat to public health, as the avian-origin influenza A(H7N9) virus has caused more than 1,560 laboratory-confirmed human infections since 2013, with nearly 40% mortality. Development of low-pathogenic candidate vaccine viruses (CVVs) for vaccine production is essential for pandemic preparedness. However, the suboptimal growth of CVVs in mammalian cells and chicken eggs is often a challenge. By introducing a single adaptive substitution, G218E, into the hemagglutinin (HA), we generated reassortant A(H7N9)-G218E CVVs that were characterized by significantly enhanced growth in both cells and eggs. These G218E CVVs retained the original antigenicity, as determined by a hemagglutination inhibition assay, and effectively protected ferrets from lethal challenge with the highly pathogenic parental virus. We found that the suboptimal replication of the parental H7 CVVs was associated with impeded progeny virus release as a result of strong HA receptor binding relative to weak neuraminidase (NA) cleavage of receptors. In contrast, the G218E-mediated growth improvement was attributed to relatively balanced HA and NA functions, resulted from reduced HA binding to both human- and avian-type receptors, and thus facilitated NA-mediated virus release. Our findings revealed that a single amino acid mutation at residue 218 of the HA improved the growth of A(H7N9) influenza virus by balancing HA and NA functions, shedding light on an alternative approach for optimizing certain influenza CVVs.IMPORTANCE The circulating avian influenza A(H7N9) has caused recurrent epidemic waves with high mortality in China since 2013, in which the alarming fifth wave crossing 2016 and 2017 was highlighted by a large number of human infections and the emergence of highly pathogenic avian influenza (HPAI) A(H7N9) strains in human cases. We generated low-pathogenic reassortant CVVs derived from the emerging A(H7N9) with improved virus replication and protein yield in both MDCK cells and eggs by introducing a single substitution, G218E, into HA, which was associated with reducing HA receptor binding and subsequently balancing HA-NA functions. The in vitro and in vivo experiments demonstrated comparable antigenicity of the G218E CVVs with that of their wild-type (WT) counterparts, and both the WT and the G218E CVVs fully protected ferrets from parental HPAI virus challenge. With high yield traits and the anticipated antigenicity, the G218E CVVs should benefit preparedness against the threat of an A(H7N9) influenza pandemic.

Avian influenza virus A H7N9 infects multiple mononuclear cell types in peripheral blood and induces dysregulated cytokine responses and apoptosis in infected monocytes.

Most patients with avian influenza A H7N9 virus (H7N9) infection suffer from severe illness, accompanied by dysregulated cytokine/chemokine response, delayed viral clearance and impaired neutralizing antibody response. Here, we evaluated the role of peripheral blood mononuclear cells (PBMCs) in the pathogenesis of H7N9 infection using an ex vivo infection model. H7N9 infected a significantly higher percentage of PBMCs (23.9 %) than those of avian influenza A H5N1 virus (H5N1) (12.3 %) and pandemic H1N1 virus (pH1N1) (5.5 %) (P<0.01). H7N9 infected significantly more B and T lymphocytes than H5N1. When compared with pH1N1, H7N9-infected PBMCs had significantly higher mRNA levels of proinflammatory cytokines and type I interferons (IFNs) at 6 h post-infection (p.i.), but significantly lower levels of IFN-γ and IP-10 at 12 h p.i. Among the PBMCs, CD14+ monocytes were most permissive to H7N9 infection. The percentage of infected CD14+ monocytes was significantly higher for H7N9 than that of pH1N1, but not significantly different from that of H5N1. H7N9-infected monocytes showed higher expression of MIP-1α, MIP-1ß and RANTES than that of pH1N1 at 6 h p.i. H7N9- but not pH1N1-infected monocytes died rapidly via apoptosis. Furthermore, pH1N1- but not H7N9-infected monocytes showed increased expression of the monocyte activation and differentiation markers. Unlike pH1N1, H7N9 showed similar PBMC/monocyte cytokine/chemokine expression profile, monocyte cell death and expression of activation/differentiation markers to H5N1. Besides proinflammatory cytokine activation leading to a cytokine storm, impaired IFN-γ production, rapid monocytic death and lack of monocyte differentiation may affect the ability of H7N9-infected innate immune cells to recruit protective adaptive immunity.

Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses.

A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. IMPORTANCE: Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage are known to be present during severe human infections, the role of pulmonary endothelial cells in the pathogenesis of avian influenza virus infections is largely unknown. By comparing human seasonal influenza strains to avian influenza viruses, we provide greater insight into the interaction of influenza virus with human pulmonary endothelial cells. We show that human influenza virus infection is blocked during the early stages of virus entry, which is likely due to the relatively high expression of the host antiviral factors IFITMs (interferon-induced transmembrane proteins) located in membrane-bound compartments inside cells. Overall, this study provides a mechanism by which human endothelial cells limit replication of human influenza virus strains, whereas avian influenza viruses overcome these restriction factors in this cell type.

Susceptibility of chickens, quail, and pigeons to an H7N9 human influenza virus and subsequent egg-passaged strains.

H7N9 human influenza virus A/Anhui/1/2013 (Anhui2013) showed low pathogenicity in chickens, quail, and pigeons, with quail being the most susceptible among the species tested. IVPIE1-1, which was recovered from a dead chicken after intravenous inoculation of Anhui 2013, had broader tissue tropism in chickens than did the original inoculum, as well as amino acid substitutions in the polymerase acidic gene and neuraminidase gene segments, but its pathogenicity was not enhanced. Viruses obtained after passage of Anhui 2013 in 10- and 14-day-old embryonated eggs showed rapid accumulation of amino acid substitutions at the receptor-binding site of the hemagglutinin protein. Two strains obtained through egg passage, 10E4/14E17 and 10E4/10E13, replicated better in intranasally infected chickens than did the original Anhui 2013 strain, yet the new isolates showed low pathogenicity in chickens despite their amino acid substitutions. The increased virus replication in chickens of 10E4/14E17 and 10E4/10E13 was not correlated with temperature-sensitive replication, given that virus replication was suppressed at increased temperatures. The existence of highly susceptible hosts, such as quail, which permit asymptomatic infection, facilitates increased mutation of the virus through amino acid substitution at the receptor-binding site, and this might be one of the mechanisms underlying the prolonged circulation of H7N9 influenza virus.

The Hemagglutinin Stem-Binding Monoclonal Antibody VIS410 Controls Influenza Virus-Induced Acute Respiratory Distress Syndrome.

Most cases of severe influenza are associated with pulmonary complications, such as acute respiratory distress syndrome (ARDS), and no antiviral drugs of proven value for treating such complications are currently available. The use of monoclonal antibodies targeting the stem of the influenza virus surface hemagglutinin (HA) is a rapidly developing strategy for the control of viruses of multiple HA subtypes. However, the mechanisms of action of these antibodies are not fully understood, and their ability to mitigate severe complications of influenza has been poorly studied. We evaluated the effect of treatment with VIS410, a human monoclonal antibody targeting the HA stem region, on the development of ARDS in BALB/c mice after infection with influenza A(H7N9) viruses. Prophylactic administration of VIS410 resulted in the complete protection of mice against lethal A(H7N9) virus challenge. A single therapeutic dose of VIS410 given 24 h after virus inoculation resulted in dose-dependent protection of up to 100% of mice inoculated with neuraminidase inhibitor-susceptible or -resistant A(H7N9) viruses. Compared to the outcomes in mock-treated controls, a single administration of VIS410 improved viral clearance from the lungs, reduced virus spread in lungs in a dose-dependent manner, resulting in a lower lung injury score, reduced the extent of the alteration in lung vascular permeability and protein accumulation in bronchoalveolar lavage fluid, and improved lung physiologic function. Thus, antibodies targeting the HA stem can reduce the severity of ARDS and show promise as agents for controlling pulmonary complications in influenza.

Pro-inflammatory cytokine dysregulation is associated with novel avian influenza A (H7N9) virus in primary human macrophages.

Since March 2013, more than 500 laboratory-confirmed human H7N9 influenza A virus infection cases have been recorded, with a case fatality rate of more than 30%. Clinical research has shown that cytokine and chemokine dysregulation contributes to the pathogenicity of the H7N9 virus. Here, we investigated cytokine profiles in primary human macrophages infected with the novel H7N9 virus, using cytokine antibody arrays. The levels of several pro-inflammatory cytokines, particularly TNF-α, were increased in H7N9-infected macrophages. Induction of the transcriptional and translational levels of the pro-inflammatory cytokines by H7N9 virus seemed to be intermediate between those induced by highly pathogenic avian H5N1 and pandemic human H1N1 viruses, which were detected by ELISA and real-time quantitative PCR, respectively. Additionally, compared with H5N1, the upregulation of pro-inflammatory cytokines caused by H7N9 infection occurred rapidly but mildly. Our results identified the overall profiles of cytokine and chemokine induction by the H7N9 influenza virus in an in vitro cell-culture model, and could provide potential therapeutic targets for the control of severe human H7N9 disease.

The nucleoprotein of newly emerged H7N9 influenza A virus harbors a unique motif conferring resistance to antiviral human MxA.

UNLABELLED: Interferon-induced Mx proteins show strong antiviral activity against influenza A viruses (IAVs). We recently demonstrated that the viral nucleoprotein (NP) determines resistance of seasonal and pandemic human influenza viruses to Mx, while avian isolates retain Mx sensitivity. We identified a surface-exposed cluster of amino acids in NP of pandemic A/BM/1/1918 (H1N1), comprising isoleucine-100, proline-283, and tyrosine-313, that is essential for reduced Mx sensitivity in cell culture and in vivo. This cluster has been maintained in all descendant seasonal strains, including A/PR/8/34 (PR/8). Accordingly, two substitutions in the NP of PR/8 [PR/8(mut)] to the Mx-sensitive amino acids (P283L and Y313F) led to attenuation in Mx1-positive mice. Serial lung passages of PR/8(mut) in Mx1 mice resulted in a single exchange of tyrosine to asparagine at position 52 in NP (in close proximity to the amino acid cluster at positions 100, 283, and 313), which partially compensates loss of Mx resistance in PR/8(mut). Intriguingly, the NP of the newly emerged avian-origin H7N9 virus also contains an asparagine at position 52 and shows reduced Mx sensitivity. N52Y substitution in NP results in increased sensitivity of the H7N9 virus to human Mx, indicating that this residue is a determinant of Mx resistance in mammals. Our data strengthen the hypothesis that the human Mx protein represents a potent barrier against zoonotic transmission of avian influenza viruses. However, the H7N9 viruses overcome this restriction by harboring an NP that is less sensitive to Mx-mediated host defense. This might contribute to zoonotic transmission of H7N9 and to the severe to fatal outcome of H7N9 infections in humans. IMPORTANCE: The natural host of influenza A viruses (IAVs) are aquatic birds. Occasionally, these viruses cross the species barrier, as in early 2013 when an avian H7N9 virus infected humans in China. Since then, multiple transmissions of H7N9 viruses to humans have occurred, leaving experts puzzled about molecular causes for such efficient crossing of the species barrier compared to other avian influenza viruses. Mx proteins are known restriction factors preventing influenza virus replication. Unfortunately, some viruses (e.g., human IAV) have developed some resistance, which is associated with specific amino acids in their nucleoproteins, the target of Mx function. Here, we demonstrate that the novel H7N9 bird IAV already carries a nucleoprotein that overcomes the inhibition of viral replication by human MxA. This is the first example of an avian IAV that is naturally less sensitive to Mx-mediated inhibition and might explain why H7N9 viruses transmitted efficiently to humans.

Assessment of Antiviral Properties of Peramivir against H7N9 Avian Influenza Virus in an Experimental Mouse Model.

The H7N9 influenza virus causes a severe form of disease in humans. Neuraminidase inhibitors, including oral oseltamivir and injectable peramivir, are the first choices of antiviral treatment for such cases; however, the clinical efficacy of these drugs is questionable. Animal experimental models are essential for understanding the viral replication kinetics under the selective pressure of antiviral agents. This study demonstrates the antiviral activity of peramivir in a mouse model of H7N9 avian influenza virus infection. The data show that repeated administration of peramivir at 30 mg/kg of body weight successfully eradicated the virus from the respiratory tract and extrapulmonary tissues during the acute response, prevented clinical signs of the disease, including neuropathy, and eventually protected mice against lethal H7N9 influenza virus infection. Early treatment with peramivir was found to be associated with better disease outcomes.

Novel H7N9 influenza virus shows low infectious dose, high growth rate, and efficient contact transmission in the guinea pig model.

The zoonotic outbreak of H7N9 subtype avian influenza virus that occurred in eastern China in the spring of 2013 resulted in 135 confirmed human cases, 44 of which were lethal. Sequencing of the viral genome revealed a number of molecular signatures associated with virulence or transmission in mammals. We report here that, in the guinea pig model, a human isolate of novel H7N9 influenza virus, A/Anhui/1/2013 (An/13), is highly dissimilar to an H7N1 avian isolate and instead behaves similarly to a human seasonal strain in several respects. An/13 was found to have a low 50% infectious dose, grow to high titers in the upper respiratory tract, and transmit efficiently among cocaged guinea pigs. The pH of fusion of the hemagglutinin (HA) and the binding of virus to fixed guinea pig tissues were also examined. The An/13 HA displayed a relatively elevated pH of fusion characteristic of many avian strains, and An/13 resembled avian viruses in terms of attachment to tissues. One important difference was seen between An/13 and both the H3N2 human and the H7N1 avian viruses: when inoculated intranasally at a high dose, only the An/13 virus led to productive infection of the lower respiratory tract of guinea pigs. In sum, An/13 was found to retain fusion and attachment properties of an avian influenza virus but displayed robust growth and contact transmission in the guinea pig model atypical of avian strains and indicative of mammalian adaptation.

Role of poultry in the spread of novel H7N9 influenza virus in China.

UNLABELLED: The recent outbreak of H7N9 influenza in China has resulted in many human cases with a high fatality rate. Poultry are the likely source of infection for humans on the basis of sequence analysis and virus isolations from live bird markets, but it is not clear which species of birds are most likely to be infected and shedding levels of virus sufficient to infect humans. Intranasal inoculation of chickens, Japanese quail, pigeons, Pekin ducks, Mallard ducks, Muscovy ducks, and Embden geese with 10(6) 50% egg infective doses of the A/Anhui/1/2013 virus resulted in infection but no clinical disease signs. Virus shedding was much higher and prolonged in quail and chickens than in the other species. Quail effectively transmitted the virus to direct contacts, but pigeons and Pekin ducks did not. In all species, virus was detected at much higher titers from oropharyngeal swabs than cloacal swabs. The hemagglutinin gene from samples collected from selected experimentally infected birds was sequenced, and three amino acid differences were commonly observed when the sequence was compared to the sequence of A/Anhui/1/2013: N123D, N149D, and L217Q. Leucine at position 217 is highly conserved for human isolates and is associated with α2,6-sialic acid binding. Different amino acid combinations were observed, suggesting that the inoculum had viral subpopulations that were selected after passage in birds. These experimental studies corroborate the finding that certain poultry species are reservoirs of the H7N9 influenza virus and that the virus is highly tropic for the upper respiratory tract, so testing of bird species should preferentially be conducted with oropharyngeal swabs for the best sensitivity. IMPORTANCE: The recent outbreak of H7N9 influenza in China has resulted in a number of human infections with a high case fatality rate. The source of the viral outbreak is suspected to be poultry, but definitive data on the source of the infection are not available. This study provides experimental data to show that quail and chickens are susceptible to infection, shed large amounts of virus, and are likely important in the spread of the virus to humans. Other poultry species can be infected and shed virus but are less likely to play a role of transmitting the virus to humans. Pigeons were previously suggested to be a possible source of the virus because of isolation of the virus from several pigeons in poultry markets in China, but experimental studies show that they are generally resistant to infection and are unlikely to play a role in the spread of the virus.

Residues 41V and/or 210D in the NP protein enhance polymerase activities and potential replication of novel influenza (H7N9) viruses at low temperature.

BACKGROUND: The influenza A (H7N9) virus emerged in the spring of 2013 in China. It contained six internal genes from Y280-like H9N2 viruses, which have co-circulated with G1-like lineage viruses throughout poultry in China. Accompanied with continuous reassortment among H7N9 and H9N2 viruses in poultry, it is possible for H7N9 viruses to acquire internal genes of G1-lineage viruses. Thus, it is important to evaluate potential impact of G1-like viruses on the H7N9 viruses. FINDINGS: We used in vitro assays of polymerase activities and growth kinetics to evaluate the potential contribution of G1-like virus genes to the replication abilities of H7N9 viruses. Two mutations in the NP protein (41V and/or 210D) could enhance H7N9 RNP activities, especially at low temperature (33°C, which is similar to the temperature of human upper respiratory tract). Meanwhile, G1 viruses with V41I or D210E substitutions exhibited poor growth ability in the early infection stage at low temperature. The D210E substitution also reduced the replication ability of G1 virus at 12 and 24 hour post infection at 37°C. In both tested temperatures, V41I could compensate for the defective virus replication induced by the D210E mutation. CONCLUSIONS: Mutations 41V and/or 210D in the NP protein conferred improved RNP activity in H7N9 viruses and promoted the replication ability of H9N2 viruses, particularly at lower temperature. Substitutions at these two positions may promote the replication ability of H7N9 viruses in low temperature and thus might contribute to viral transmissibility. While these two residues have not yet been observed in H7N9 viruses, attention should be devoted to these two residues.

The R292K mutation that confers resistance to neuraminidase inhibitors leads to competitive fitness loss of A/Shanghai/1/2013 (H7N9) influenza virus in ferrets.

BACKGROUND: Neuraminidase (NA) inhibitors are the only licensed therapeutic option for human zoonotic H7N9 infections. An NA-R292K mutation that confers broad-spectrum resistance to NA inhibitors has been documented in H7N9 patients after treatment. METHODS: We evaluated the transmission potential of a human influenza A H7N9 isolate with a NA-R292K mutation in the ferret model followed by genotyping assay to monitor its competitive fitness in vivo. RESULTS: Plaque-purified A/Shanghai/1/2013 wild-type and NA-R292K viruses transmitted at comparable efficiency to direct or respiratory droplet contact ferrets. In ferrets inoculated with the plaque-purified A/Shanghai/1/2013 NA-R292K virus with dominant K292 (94%), the resistant K292 genotype was outgrown by the wild-type R292 genotype during the course of infection. Transmission of the resistant K292 genotype was detected in 3/4 direct contact and 3/4 respiratory droplet contact ferrets at early time points but was gradually replaced by the wild-type genotype. In the respiratory tissues of inoculated or infected ferrets, the wild-type R292 genotype dominated in the nasal turbinate, whereas the resistant K292 genotype was more frequently detected in the lungs. CONCLUSIONS: The NA inhibitor-resistant H7N9 virus with the NA-R292K mutation may transmit among ferrets but showed compromised fitness in vivo while in competition with the wild-type virus.

H7N9 Influenza Virus Containing a Polybasic HA Cleavage Site Requires Minimal Host Adaptation to Obtain a Highly Pathogenic Disease Phenotype in Mice.

Low pathogenic avian influenza (LPAI) H7N9 viruses have recently evolved to gain a polybasic cleavage site in the hemagglutinin (HA) protein, resulting in variants with increased lethality in poultry that meet the criteria for highly pathogenic avian influenza (HPAI) viruses. Both LPAI and HPAI variants can cause severe disease in humans (case fatality rate of ~40%). Here, we investigated the virulence of HPAI H7N9 viruses containing a polybasic HA cleavage site (H7N9-PBC) in mice. Inoculation of mice with H7N9-PBC did not result in observable disease; however, mice inoculated with a mouse-adapted version of this virus, generated by a single passage in mice, caused uniformly lethal disease. In addition to the PBC site, we identified three other mutations that are important for host-adaptation and virulence in mice: HA (A452T), PA (D347G), and PB2 (M483K). Using reverse genetics, we confirmed that the HA mutation was the most critical for increased virulence in mice. Our study identifies additional disease determinants in a mammalian model for HPAI H7N9 virus. Furthermore, the ease displayed by the virus to adapt to a new host highlights the potential for H7N9-PBC viruses to rapidly acquire mutations that may enhance their risk to humans or other animal species.

Human matriptase/ST 14 proteolytically cleaves H7N9 hemagglutinin and facilitates the activation of influenza A/Shanghai/2/2013 virus in cell culture.

BACKGROUND: Influenza is a zoonotic disease that infects millions of people each year resulting in hundreds of thousands of deaths, and in turn devastating pandemics. Influenza is caused by influenza viruses, including influenza A virus (IAV). There are many subtypes of IAV but only a few seem to be able to adapt to humans and to cause disease. In 2013, an H7N9 IAV subtype emerged in China that does not cause clinical symptoms in its chicken host but leads to severe infections when transmitted into humans. Since 2013, there have been six epidemic waves of H7N9 with 1567 laboratory-confirmed human infections and 615 deaths. Pathogenicity of IAV is complex, but a crucial feature contributing to virulence is the activation of the hemagglutinin (HA) fusion protein by host proteases that triggers membrane fusion and leads to subsequent virus propagation. METHODS: 293T, VERO, and MDCK cells were used to conduct Western blot analysis, immunofluorescence assays, and pseudoparticle and live virus infections, and to evaluate H7N9 HA cleavage-activation. RESULTS/CONCLUSIONS: We show that human matriptase/ST 14 is able to cleave H7N9 HA. Cleavage of H7N9 HA expressed in cell culture results in fusogenic HA and syncytia formation. In infection studies with viral pseudoparticles carrying matriptase/ST 14-activated H7N9 HA, we observed a high infectivity of cells. Finally, human matriptase/ST 14 also activated H7N9 live virus which resulted in high infectivity. Our data demonstrate that human matriptase/ST 14 is a likely candidate protease to promote H7N9 infections in humans.

SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses.

Viruses possessing class I fusion proteins require proteolytic activation by host cell proteases to mediate fusion with the host cell membrane. The mammalian SPINT2 gene encodes a protease inhibitor that targets trypsin-like serine proteases. Here we show the protease inhibitor, SPINT2, restricts cleavage-activation efficiently for a range of influenza viruses and for human metapneumovirus (HMPV). SPINT2 treatment resulted in the cleavage and fusion inhibition of full-length influenza A/CA/04/09 (H1N1) HA, A/Aichi/68 (H3N2) HA, A/Shanghai/2/2013 (H7N9) HA and HMPV F when activated by trypsin, recombinant matriptase or KLK5. We also demonstrate that SPINT2 was able to reduce viral growth of influenza A/CA/04/09 H1N1 and A/X31 H3N2 in cell culture by inhibiting matriptase or TMPRSS2. Moreover, inhibition efficacy did not differ whether SPINT2 was added at the time of infection or 24 h post-infection. Our data suggest that the SPINT2 inhibitor has a strong potential to serve as a novel broad-spectrum antiviral.

Identification of key hemagglutinin residues responsible for cleavage, acid stability, and virulence of fifth-wave highly pathogenic avian influenza A(H7N9) viruses.

We previously demonstrated that despite no airborne transmissibility increase compared to low pathogenic avian influenza viruses, select human isolates of highly pathogenic avian influenza A(H7N9) virus exhibit greater virulence in animal models and a lower threshold pH for fusion. In the current study, we utilized both in vitro and in vivo approaches to identify key residues responsible for hemagglutinin (HA) intracellular cleavage, acid stability, and virulence in mice. We found that the four amino acid insertion (-KRTA-) at the HA cleavage site of A/Taiwan/1/2017 virus is essential for HA intracellular cleavage and contributes to disease in mice. Furthermore, a lysine to glutamic acid mutation at position HA2-64 increased the threshold pH for HA activation, reduced virus stability, and replication in mice. Identification of a key residue responsible for enhanced acid stability of A(H7N9) viruses is of great significance for future surveillance activities and improvements in vaccine stability.

Identification and characterization of GLDC as host susceptibility gene to severe influenza.

Glycine decarboxylase (GLDC) was prioritized as a candidate susceptibility gene to severe influenza in humans. The higher expression of GLDC derived from genetic variations may confer a higher risk to H7N9 and severe H1N1 infection. We sought to characterize GLDC as functional susceptibility gene that GLDC may intrinsically regulate antiviral response, thereby impacting viral replication and disease outcome. We demonstrated that GLDC inhibitor AOAA and siRNA depletion boosted IFNß- and IFN-stimulated genes (ISGs) in combination with PolyI:C stimulation. GLDC inhibition and depletion significantly amplified antiviral response of type I IFNs and ISGs upon viral infection and suppressed the replication of H1N1 and H7N9 viruses. Consistently, GLDC overexpression significantly promoted viral replication due to the attenuated antiviral responses. Moreover, GLDC inhibition in H1N1-infected BALB/c mice recapitulated the amplified antiviral response and suppressed viral growth. AOAA provided potent protection to the infected mice from lethal infection, comparable to a standard antiviral against influenza viruses. Collectively, GLDC regulates cellular antiviral response and orchestrates viral growth. GLDC is a functional susceptibility gene to severe influenza in humans.

SMRT sequencing revealed the diversity and characteristics of defective interfering RNAs in influenza A (H7N9) virus infection.

Influenza defective interfering (DI) particles are replication-incompetent viruses carrying large internal deletion in the genome. The loss of essential genetic information causes abortive viral replication, which can be rescued by co-infection with a helper virus that possesses an intact genome. Despite reports of DI particles present in seasonal influenza A H1N1 infections, their existence in human infections by the avian influenza A viruses, such as H7N9, has not been studied. Here we report the ubiquitous presence of DI-RNAs in nasopharyngeal aspirates of H7N9-infected patients. Single Molecule Real Time (SMRT) sequencing was first applied and long-read sequencing analysis showed that a variety of H7N9 DI-RNA species were present in the patient samples and human bronchial epithelial cells. In several abundantly expressed DI-RNA species, long overlapping sequences have been identified around at the breakpoint region and the other side of deleted region. Influenza DI-RNA is known as a defective viral RNA with single large internal deletion. Beneficial to the long-read property of SMRT sequencing, double and triple internal deletions were identified in half of the DI-RNA species. In addition, we examined the expression of DI-RNAs in mice infected with sublethal dose of H7N9 virus at different time points. Interestingly, DI-RNAs were abundantly expressed as early as day 2 post-infection. Taken together, we reveal the diversity and characteristics of DI-RNAs found in H7N9-infected patients, cells and animals. Further investigations on this overwhelming generation of DI-RNA may provide important insights into the understanding of H7N9 viral replication and pathogenesis.

Evaluation of novel disposable bioreactors on pandemic influenza virus production.

Since 1997, the highly pathogenic influenza H5N1 virus has spread from Hong Kong. According to the WHO bulletin report, the H5N1 virus is a zoonotic disease threat that has infected more than 850 humans, causing over 450 deaths. In addition, an outbreak of another new and highly pathogenic influenza virus (H7N9) occurred in 2013 in China. These highly pathogenic influenza viruses could potentially cause a worldwide pandemic. it is crucial to develop a rapid production platform to meet this surge demand against any possible influenza pandemic. A potential solution for this problem is the use of cell-based bioreactors for rapid vaccine production. These novel bioreactors, used for cell-based vaccine production, possess various advantages. For example, they enable a short production time, allow for the handling highly pathogenic influenza in closed environments, and can be easily scaled up. In this study, two novel disposable cell-based bioreactors, BelloCell and TideCell, were used to produce H5N1 clade II and H7N9 candidate vaccine viruses (CVVs). Madin-Darby canine kidney (MDCK) cells were used for the production of these influenza CVVs. A novel bench-scale bioreactor named BelloCell bioreactor was used in the study. All culturing conditions were tested and scaled to 10 L industrial-scale bioreactor known as TideCell002. The performances of between BelloCell and TideCell were similar in cell growth, the average MDCK cell doubling time was slightly decreased to 25 hours. The systems yielded approximately 39.2 and 18.0 µg/ml of HA protein with the 10-liter TideCell002 from the H5N1 clade II and H7N9 CVVs, respectively. The results of this study not only highlight the overall effectiveness of these bioreactors but also illustrate the potential of maintaining the same outcome when scaled up to industrial production, which has many implications for faster vaccine production. Although additional studies are required for process optimization, the results of this study are promising and show that oscillating bioreactors may be a suitable platform for pandemic influenza virus production.

An avian influenza virus A(H7N9) reassortant that recently emerged in the United States with low pathogenic phenotype does not efficiently infect swine.

In 2017, outbreaks of low and highly pathogenic avian H7N9 viruses were reported in four States in the United States. In total, over 270 000 birds died or were culled, causing significant economic loss. The potential for avian-to-swine transmission of the U.S. avian H7N9 was unknown. In an experimental challenge in swine using a representative low pathogenic H7N9 (A/chicken/Tennessee/17-007431-3/2017; LPAI TN/17) isolated from these events, no infectious virus in the upper and minimal virus in the lower respiratory tract was detected, nor was lung pathology or evidence of transmission in pigs observed, indicating that the virus cannot efficiently infect swine.