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

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

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

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

Influenza A virus infection disrupts the function of syncytiotrophoblast cells and contributes to adverse pregnancy outcomes.

Pregnancy heightens susceptibility to influenza A virus (IAV) infection, thereby increasing the risk of severe pneumonia and maternal mortality. It also raises the chances of adverse outcomes in offspring, such as fetal growth restriction, preterm birth, miscarriage, and stillbirth in offsprings. However, the underlying mechanisms behind these effects remain largely unknown. Syncytiotrophoblast cells, crucial in forming the placental barrier, nutrient exchange and hormone secretion, have not been extensively studied for their responses to IAV. In our experiment, we used Forskolin-treated BeWo cells to mimic syncytiotrophoblast cells in vitro, and infected them with H1N1, H5N1 and H7N9 virus stains. Our results showed that syncytiotrophoblast cells, with their higher intensity of sialic acid receptors, strongly support IAV infection and replication. Notably, high-dose viral infection and prolonged exposure resulted in a significant decrease in fusion index, as well as gene and protein expression levels associated with trophoblast differentiation, ß-human chorionic gonadotropin secretion, estrogen and progesterone biosynthesis, and nutrient transport. In pregnant BALB/c mice infected with the H1N1 virus, we observed significant decreases in trophoblast differentiation and hormone secretion gene expression levels. IAV infection also resulted in preterm labor, fetal growth restriction, and increased maternal and fetal morbidity and mortality. Our findings indicate that IAV infection in syncytiotrophoblastic cells can result in adverse pregnancy outcomes by altering trophoblast differentiation, suppressing of ß-hCG secretion, and disrupting placental barrier function.

Competitive Endogenous RNA Network Activates Host Immune Response in SARS-CoV-2-, panH1N1 (A/California/07/2009)-, and H7N9 (A/Shanghai/1/2013)-Infected Cells.

The global outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still ongoing, as is research on the molecular mechanisms underlying cellular infection by coronaviruses, with the hope of developing therapeutic agents against this pandemic. Other important respiratory viruses such as 2009 pandemic H1N1 and H7N9 avian influenza virus (AIV), influenza A viruses, are also responsible for a possible outbreak due to their respiratory susceptibility. However, the interaction of these viruses with host cells and the regulation of post-transcriptional genes remains unclear. In this study, we detected and analyzed the comparative transcriptome profiling of SARS-CoV-2, panH1N1 (A/California/07/2009), and H7N9 (A/Shanghai/1/2013) infected cells. The results showed that the commonly upregulated genes among the three groups were mainly involved in autophagy, pertussis, and tuberculosis, which indicated that autophagy plays an important role in viral pathogenicity. There are three groups of commonly downregulated genes involved in metabolic pathways. Notably, unlike panH1N1 and H7N9, SARS-CoV-2 infection can inhibit the m-TOR pathway and activate the p53 signaling pathway, which may be responsible for unique autophagy induction and cell apoptosis. Particularly, upregulated expression of IRF1 was found in SARS-CoV-2, panH1N1, and H7N9 infection. Further analysis showed SARS-CoV-2, panH1N1, and H7N9 infection-induced upregulation of lncRNA-34087.27 could serve as a competitive endogenous RNA to stabilize IRF1 mRNA by competitively binding with miR-302b-3p. This study provides new insights into the molecular mechanisms of influenza A virus and SARS-CoV-2 infection.

SPINK6 inhibits human airway serine proteases and restricts influenza virus activation.

SPINK6 was identified in human skin as a cellular inhibitor of serine proteases of the KLK family. Airway serine proteases are required to cleave hemagglutinin (HA) of influenza A viruses (IAVs) to initiate an infection in the human airway. We hypothesized that SPINK6 may inhibit common airway serine proteases and restrict IAV activation. We demonstrate that SPINK6 specifically suppresses the proteolytic activity of HAT and KLK5, HAT- and KLK5-mediated HA cleavage, and restricts virus maturation and replication. SPINK6 constrains the activation of progeny virions and impairs viral growth; and vice versa, blocking endogenous SPINK6 enhances HA cleavage and viral growth in physiological-relevant human airway organoids where SPINK6 is intrinsically expressed. In IAV-infected mice, SPINK6 significantly suppresses viral growth and improves mouse survival. Notably, individuals carrying the higher SPINK6 expression allele were protected from human H7N9 infection. Collectively, SPINK6 is a novel host inhibitor of serine proteases in the human airway and restricts IAV activation.

Limited onward transmission potential of reassortment genotypes from chickens co-infected with H9N2 and H7N9 avian influenza viruses.

The segmented genome of influenza A virus has conferred significant evolutionary advantages to this virus through genetic reassortment, a mechanism that facilitates the rapid expansion of viral genetic diversity upon influenza co-infections. Therefore, co-infection of genetically diverse avian influenza viruses in poultry may pose a significant public health risk in generating novel reassortants with increased zoonotic potential. This study investigated the reassortment patterns of a Pearl River Delta-lineage avian influenza A(H7N9) virus and four genetically divergent avian influenza A(H9N2) viruses upon co-infection in embryonated chicken eggs and chickens. To characterize "within-host" and "between-host" genetic diversity, we further monitored the viral genotypes that were subsequently transmitted to contact chickens in serial transmission experiments. We observed that co-infection with A(H7N9) and A(H9N2) viruses may lead to the emergence of novel reassortant viruses in ovo and in chickens, albeit with different reassortment patterns. Novel reassortants detected in donor chickens co-infected with different combinations of the same A(H7N9) virus and different A(H9N2) viruses showed distinct onward transmission potential to contact chickens. Sequential transmission of novel reassortant viruses was only observed in one out of four co-infection combinations. Our results demonstrated different patterns by which influenza viruses may acquire genetic diversity through co-infection in ovo, in vivo, and under sequential transmission conditions.

Long noncoding RNA#45 exerts broad inhibitory effect on influenza a virus replication via its stem ring arms.

A growing body of evidence suggests the pivotal role of long non-coding RNA (lncRNA) in influenza virus infection. Based on next-generation sequencing, we previously demonstrated that Lnc45 was distinctively stimulated by H5N1 influenza virus in mice. In this study, we systematically investigated the specific role of Lnc45 during influenza A virus (IAV) infection. Through qRT-PCR, we first demonstrated that Lnc45 is highly up-regulated by different subtypes of IAV strains, including H5N1, H7N9, and H1N1 viruses. Using RNA-FISH and qRT-PCR, we then found that Lnc45 can translocate from nuclear to cytoplasm during H5N1 virus infection. In addition, forced Lnc45 expression dramatically impeded viral replication of H1N1, H5N1, and H7N9 virus, while abolish of Lnc45 expression by RNA interference favored replication of these viruses, highlighting the potential broad antiviral activity of Lnc45 to IAV. Correspondingly, overexpression of Lnc45 inhibited viral polymerase activity and suppressed IAV-induced cell apoptosis. Moreover, Lnc45 significantly restrained nuclear aggregation of viral NP and PA proteins during H5N1 virus infection. Further functional study revealed that the stem ring arms of Lnc45 mainly mediated the antiviral effect. Therefore, we here demonstrated that Lnc45 functions as a broad-spectrum antiviral factor to inhibit influenza virus replication probably through inhibiting polymerase activity and NP and PA nuclear accumulation via its stem ring arms. Our study not only advances our understanding of the complexity of the IAV pathogenesis but also lays the foundation for developing novel anti-IAV therapeutics targeting the host lncRNA.

Rare variant MX1 alleles increase human susceptibility to zoonotic H7N9 influenza virus.

Zoonotic avian influenza A virus (IAV) infections are rare. Sustained transmission of these IAVs between humans has not been observed, suggesting a role for host genes. We used whole-genome sequencing to compare avian IAV H7N9 patients with healthy controls and observed a strong association between H7N9 infection and rare, heterozygous single-nucleotide variants in the MX1 gene. MX1 codes for myxovirus resistance protein A (MxA), an interferon-induced antiviral guanosine triphosphatase known to control IAV infections in transgenic mice. Most of the MxA variants identified lost the ability to inhibit avian IAVs, including H7N9, in transfected human cell lines. Nearly all of the inactive MxA variants exerted a dominant-negative effect on the antiviral function of wild-type MxA, suggesting an MxA null phenotype in heterozygous carriers. Our study provides genetic evidence for a crucial role of the MX1-based antiviral defense in controlling zoonotic IAV infections in humans.

Virus Adaptation Following Experimental Infection of Chickens with a Domestic Duck Low Pathogenic Avian Influenza Isolate from the 2017 USA H7N9 Outbreak Identifies Polymorphic Mutations in Multiple Gene Segments.

In March 2017, highly pathogenic (HP) and low pathogenic (LP) avian influenza virus (AIV) subtype H7N9 were detected from poultry farms and backyard birds in several states in the southeast United States. Because interspecies transmission is a known mechanism for evolution of AIVs, we sought to characterize infection and transmission of a domestic duck-origin H7N9 LPAIV in chickens and genetically compare the viruses replicating in the chickens to the original H7N9 clinical field samples used as inoculum. The results of the experimental infection demonstrated virus replication and transmission in chickens, with overt clinical signs of disease and shedding through both oral and cloacal routes. Unexpectedly, higher levels of virus shedding were observed in some cloacal swabs. Next generation sequencing (NGS) analysis identified numerous non-synonymous mutations at the consensus level in the polymerase genes (i.e., PA, PB1, and PB2) and the hemagglutinin (HA) receptor binding site in viruses recovered from chickens, indicating possible virus adaptation in the new host. For comparison, NGS analysis of clinical samples obtained from duck specimen collected during the outbreak indicated three polymorphic sides in the M1 segment and a minor population of viruses carrying the D139N (21.4%) substitution in the NS1 segment. Interestingly, at consensus level, A/duck/Alabama (H7N9) had isoleucine at position 105 in NP protein, similar to HPAIV (H7N9) but not to LPAIV (H7N9) isolated from the same 2017 influenza outbreak in the US. Taken together, this work demonstrates that the H7N9 viruses could readily jump between avian species, which may have contributed to the evolution of the virus and its spread in the region.

Avian Influenza H7N9 Virus Adaptation to Human Hosts.

Avian influenza virus A (H7N9), after circulating in avian hosts for decades, was identified as a human pathogen in 2013. Herein, amino acid substitutions possibly essential for human adaptation were identified by comparing the 4706 aligned overlapping nonamer position sequences (1-9, 2-10, etc.) of the reported 2014 and 2017 avian and human H7N9 datasets. The initial set of virus sequences (as of year 2014) exhibited a total of 109 avian-to-human (A2H) signature amino acid substitutions. Each represented the most prevalent substitution at a given avian virus nonamer position that was selectively adapted as the corresponding index (most prevalent sequence) of the human viruses. The majority of these avian substitutions were long-standing in the evolution of H7N9, and only 17 were first detected in 2013 as possibly essential for the initial human adaptation. Strikingly, continued evolution of the avian H7N9 virus has resulted in avian and human protein sequences that are almost identical. This rapid and continued adaptation of the avian H7N9 virus to the human host, with near identity of the avian and human viruses, is associated with increased human infection and a predicted greater risk of human-to-human transmission.

Spatial and temporal analysis of human infection with the avian influenza A (H7N9) virus in China and research on a risk assessment agent-based model.

OBJECTIVES: From 2013 to 2017, the avian influenza A (H7N9) virus frequently infected people in China, which seriously affected the public health of society. This study aimed to analyze the spatial characteristics of human infection with the H7N9 virus in China and assess the risk areas of the epidemic. METHODS: Using kernel density estimation, standard deviation ellipse analysis, spatial and temporal scanning cluster analysis, and Pearson correlation analysis, the spatial characteristics and possible risk factors of the epidemic were studied. Meteorological factors, time (month), and environmental factors were combined to establish an epidemic risk assessment proxy model to assess the risk range of an epidemic. RESULTS: The epidemic situation was significantly correlated with atmospheric pressure, temperature, and daily precipitation (P < 0.05), and there were six temporal and spatial clusters. The fitting accuracy of the epidemic risk assessment agent-based model for lower-risk, low-risk, medium-risk, and high-risk was 0.795, 0.672, 0.853, 0.825, respectively. CONCLUSIONS: This H7N9 epidemic was found to have more outbreaks in winter and spring. It gradually spread to the inland areas of China. This model reflects the risk areas of human infection with the H7N9 virus.

Genetic and biological properties of H7N9 avian influenza viruses detected after application of the H7N9 poultry vaccine in China.

The H7N9 avian influenza virus (AIV) that emerged in China have caused five waves of human infection. Further human cases have been successfully prevented since September 2017 through the use of an H7N9 vaccine in poultry. However, the H7N9 AIV has not been eradicated from poultry in China, and its evolution remains largely unexplored. In this study, we isolated 19 H7N9 AIVs during surveillance and diagnosis from February 2018 to December 2019, and genetic analysis showed that these viruses have formed two different genotypes. Animal studies indicated that the H7N9 viruses are highly lethal to chicken, cause mild infection in ducks, but have distinct pathotypes in mice. The viruses bound to avian-type receptors with high affinity, but gradually lost their ability to bind to human-type receptors. Importantly, we found that H7N9 AIVs isolated in 2019 were antigenically different from the H7N9 vaccine strain that was used for H7N9 influenza control in poultry, and that replication of these viruses cannot, therefore, be completely prevented in vaccinated chickens. We further revealed that two amino acid mutations at positions 135 and 160 in the HA protein added two glycosylation sites and facilitated the escape of the H7N9 viruses from the vaccine-induced immunity. Our study provides important insights into H7N9 virus evolution and control.

The PB1 protein of influenza A virus inhibits the innate immune response by targeting MAVS for NBR1-mediated selective autophagic degradation.

Influenza A virus (IAV) has evolved various strategies to counteract the innate immune response using different viral proteins. However, the mechanism is not fully elucidated. In this study, we identified the PB1 protein of H7N9 virus as a new negative regulator of virus- or poly(I:C)-stimulated IFN induction and specifically interacted with and destabilized MAVS. A subsequent study revealed that PB1 promoted E3 ligase RNF5 to catalyze K27-linked polyubiquitination of MAVS at Lys362 and Lys461. Moreover, we found that PB1 preferentially associated with a selective autophagic receptor neighbor of BRCA1 (NBR1) that recognizes ubiquitinated MAVS and delivers it to autophagosomes for degradation. The degradation cascade mediated by PB1 facilitates H7N9 virus infection by blocking the RIG-I-MAVS-mediated innate signaling pathway. Taken together, these data uncover a negative regulatory mechanism involving the PB1-RNF5-MAVS-NBR1 axis and provide insights into an evasion strategy employed by influenza virus that involves selective autophagy and innate signaling pathways.

Identifying novel amino acid substitutions of hemagglutinin involved in virulence enhancement in H7N9 virus strains.

BACKGROUND: To identify site-specific features of amino acid substitutions that confer enhanced H7N9 virulence in humans, we independently generated mammalian-adapted variants of A/Anhui/1/2013 (AH-H7N9) and A/Shanghai/2/2013 (SH-H7N9) by serial passaging in Madin-Darby canine kidney (MDCK) cells. METHODS: Virus was respectively extracted from cell culture supernatant and cells, and was absolutely quantified by using real-time polymerase chain reaction. Viral RNAs were extracted and subjected to sequencing for identifying mutations. Then, site-specific mutations introduced by viral passaging were selected for further constructing HA7 or NA9 mutant plasmids, which were used to generate recombinant viruses. The interaction between the recombinant HA and receptors, H7N9-pseudotyped viruses and receptors were detected. RESULTS: Both subtypes displayed high variability in replicative capability and virulence during serial passaging. Analysis of viral genomes revealed multiple amino acid mutations in the hemagglutinin 7 (HA7) (A135T [AH-H7N9], T71I [SH-H7N9], T157I [SH-H7N9], T71I-V223I [SH-H7N9], T71I-T157I-V223I [SH-H7N9], and T71I-T157I-V223I-T40I [SH-H7N9]), and NA9 (N171S [AH-H7N9] and G335S [AH-H7N9]) proteins in various strains of the corresponding subtypes. Notably, quite a few amino acid substitutions indeed collectively strengthened the interactions between H7N9 strains and sialic acid receptors. Moreover, some of the amino acid substitutions identified were highly and specifically cytopathogenic to MDCK cells. CONCLUSIONS: This study demonstrated that AH-H7N9 and SH-H7N9 subtypes can acquire enhanced receptor affinity for sialic receptors through novel amino acid substitutions. Such changes in affinitive interactions are conferred by site-specific mutations of HA7 proteins that affect the virulence and pathology of the virus strain, and/or limited compatibility between the host and the virus strain.

V292I mutation in PB2 polymerase induces increased effects of E627K on influenza H7N9 virus replication in cells.

Characterization of host adaptation markers among human isolates is important for recognizing the potential for cross-species transmission in avian influenza A viruses. Here, we studied two new potential adaptive mutations, V292I and D740A, in the PB2 protein that were identified by a multi-factor regression model. The study shows that the prevalence of the PB2-V292I mutation is increased in H7N9 influenza viruses isolated from both humans and birds over the past 6 years. The phylogenetic tree showed that influenza A/H7N9 has a lineage based on the strains containing PB2-292I. Polymerase complexes containing PB2-292I/627K derived from H7N9 exhibit increased polymerase activity. PB2-292I coupled with 627K also enhances viral transcription and replication in cells, whereas PB2-292I alone did not show the same effect in the H7N9 virus. However, PB2-740A only had a limited prevalence in 2013, and the change from D to A in PB2-740A may have a negative effect on the replication of the H7N9 virus in cells.

The Dynamics of the Ferret Immune Response During H7N9 Influenza Virus Infection.

As the recent outbreak of SARS-CoV-2 has highlighted, the threat of a pandemic event from zoonotic viruses, such as the deadly influenza A/H7N9 virus subtype, continues to be a major global health concern. H7N9 virus strains appear to exhibit greater disease severity in mammalian hosts compared to natural avian hosts, though the exact mechanisms underlying this are somewhat unclear. Knowledge of the H7N9 host-pathogen interactions have mainly been constrained to natural sporadic human infections. To elucidate the cellular immune mechanisms associated with disease severity and progression, we used a ferret model to closely resemble disease outcomes in humans following influenza virus infection. Intriguingly, we observed variable disease outcomes when ferrets were inoculated with the A/Anhui/1/2013 (H7N9) strain. We observed relatively reduced antigen-presenting cell activation in lymphoid tissues which may be correlative with increased disease severity. Additionally, depletions in CD8+ T cells were not apparent in sick animals. This study provides further insight into the ways that lymphocytes maturate and traffic in response to H7N9 infection in the ferret model.

SRSF10 inhibits the polymerase activity and replication of avian influenza virus by regulating the alternative splicing of chicken ANP32A.

Compared with mammalian ANP32A, most avian-coded ANP32A contains a 33 amino acids insertion (ch-ANP32A-33) or a 29 amino acids insertion (ch-ANP32A-29), which can rescue the mammalian-restricted avian influenza virus polymerase activity, with ch-ANP32A-33 exhibiting a more potent phenotype. The alternative splicing of 3' splice sites (SSs) of chicken ANP32A intron 4 generates full-length ch-ANP32A-33 and truncated ch-ANP32A-29. In this study, we found a splicing regulatory cis-element that affected the alternative splicing of 3' SSs by block-scanning mutagenesis. RNA affinity purification and mass spectrometry showed that the SRSF10 bound to the splicing cis-element and the binding was further identified and confirmed by RIP experiment. Overexpression of SRSF10 changed the ratio of the two chicken ANP32A transcripts with the increased ch-ANP32A-29 and the decreased ch-ANP32A-33. The knockdown of both of the ch-ANP32A-33 and ch-ANP32A-29 was harmful to avian influenza virus polymerase activity in DF-1 cells, but the restoration and increasement of only ch-ANP32A-29 could not completely rescue the activity of avian influenza virus polymerase. Overexpression of SRSF10 negatively affected the polymerase activity and replication of avian influenza virus, and the expression of ch-ANP32A-33 could partially recover the decrease of polymerase activity of avian influenza virus. By contrast, SRSF10 had weak inhibition on the polymerase activity of mammalian adapted influenza virus and had no effect on the replication of mammalian adapted influenza virus. Taken together, we demonstrated that SRSF10 acts as a negative regulator in polymerase activity and replication of avian influenza virus by binding to the splicing cis-element to regulate the alternative splicing of chicken ANP32A intron 4 for the reduced ch-ANP32A-33 and increased ch-ANP32A-29.

Low replicative fitness of neuraminidase inhibitor-resistant H7N9 avian influenza a virus with R292K substitution in neuraminidase in cynomolgus macaques compared with I222T substitution.

Human cases of H7N9 influenza A virus infection have been increasing since 2013. The first choice of treatment for influenza is neuraminidase (NA) inhibitors (NAIs), but there is a concern that NAI-resistant viruses are selected in the presence of NAIs. In our previous study, an H7N9 virus carrying AA substitution of threonine (T) for isoleucine (I) at residue 222 in NA (NA222T, N2 numbering) and an H7N9 virus carrying AA substitution of lysine (K) for arginine (R) at residue 292 in NA (NA292K, N2 numbering) were found in different macaques that had been infected with A/Anhui/1/2013 (H7N9) and treated with NAIs. In the present study, the variant with NA292K showed not only resistance to NAIs but also lower replication activity in MDCK cells than did the virus with wild-type NA, whereas the variant with NA222T, which was less resistant to NAIs, showed replication activity similar to that of the wild-type virus. Next, we examined the pathogenicity of these H7N9 NAI-resistant viruses in macaques. The variants caused clinical signs similar to those caused by the wild-type virus with similar replication potency. However, the virus with NA292K was replaced within 7 days by that with NA292R (same as the wild-type) in nasal samples from macaques infected with the virus with NA292K, i.e. the so-called revertant (wild-type virus) became dominant in the population in the absence of an NAI. These results suggest that the clinical signs observed in macaques infected with the NA292K virus are caused by the NA292K virus and the NA292R virus and that the virus with NA292K may not replicate continuously in the upper respiratory tract of patients without treatment as effectively as the wild-type virus.

Induction of Cross-Reactive and Protective Antibody Responses After DNA Vaccination With MHCII-Targeted Stem Domain From Influenza Hemagglutinin.

Novel and more broadly protective vaccines against influenza are needed to efficiently meet antigenic drift and shift. Relevant to this end, the stem domain of hemagglutinin (HA) is highly conserved, and antibodies specific for epitopes located to the stem have been demonstrated to be able to confer broad protection against various influenza subtypes. However, a remaining challenge is to induce antibodies against the poorly immunogenic stem by vaccination strategies that can be scaled up for prophylactic vaccination of the general population. Here, we have developed DNA vaccines where the conserved stem domain of HA from influenza A/PR/8/34 (H1N1) and A/Shanghai/2/2013 (H7N9) was targeted toward MHC class II molecules on antigen-presenting cells (APC) for increased immunogenicity. Each of these vaccines induced antibodies that cross-reacted with other subtypes in the corresponding phylogenetic influenza groups. Importantly, when mixing the MHCII-targeted stem domains from H1N1 and H7N9 influenza viruses into one vaccine bolus, we observed broad protection against candidate stains from both phylogenetic groups 1 and 2.

A System Based-Approach to Examine Host Response during Infection with Influenza A Virus Subtype H7N9 in Human and Avian Cells.

Although the influenza A virus H7N9 subtype circulates within several avian species, it can also infect humans with a severe disease outcome. To better understand the biology of the H7N9 virus we examined the host response to infection in avian and human cells. In this study we used the A/Anhui/1/2013 strain, which was isolated during the first wave of the H7N9 epidemic. The H7N9 virus-infected both human (Airway Epithelial cells) and avian (Chick Embryo Fibroblast) cells, and each infected host transcriptome was examined with bioinformatic tools and compared with other representative avian and human influenza A virus subtypes. The H7N9 virus induced higher expression changes (differentially regulated genes) in both cell lines, with more prominent changes observed in avian cells. Ortholog mapping of differentially expression genes identified significant enriched common and cell-type pathways during H7N9 infections. This data confirmed our previous findings that different influenza A virus subtypes have virus-specific replication characteristics and anti-virus signaling in human and avian cells. In addition, we reported for the first time, the new HIPPO signaling pathway in avian cells, which we hypothesized to play a vital role to maintain the antiviral state of H7N9 virus-infected avian cells. This could explain the absence of disease symptoms in avian species that tested positive for the presence of H7N9 virus.