PB2 residue 473 contributes to the mammalian virulence of H7N9 avian influenza virus by modulating viral polymerase activity via ANP32A.

Since the first human infection reported in 2013, H7N9 avian influenza virus (AIV) has been regarded as a serious threat to human health. In this study, we sought to identify the virulence determinant of the H7N9 virus in mammalian hosts. By comparing the virulence of the SH/4664 H7N9 virus, a non-virulent H9N2 virus, and various H7N9-H9N2 hybrid viruses in infected mice, we first pinpointed PB2 as the primary viral factor accounting for the difference between H7N9 and H9N2 in mammalian virulence. We further analyzed the in vivo effects of individually mutating H7N9 PB2 residues different from the closely related H9N2 virus and consequently found residue 473, alongside the well-known residue 627, to be critical for the virulence of the H7N9 virus in mice and the activity of its reconstituted viral polymerase in mammalian cells. The importance of PB2-473 was further strengthened by studying reverse H7N9 substitutions in the H9N2 background. Finally, we surprisingly found that species-specific usage of ANP32A, a family member of host factors connecting with the PB2-627 polymorphism, mediates the contribution of PB2 473 residue to the mammalian adaption of AIV polymerase, as the attenuating effect of PB2 M473T on the viral polymerase activity and viral growth of the H7N9 virus could be efficiently complemented by co-expression of chicken ANP32A but not mouse ANP32A and ANP32B. Together, our studies uncovered the PB2 473 residue as a novel viral host range determinant of AIVs via species-specific co-opting of the ANP32 host factor to support viral polymerase activity.IMPORTANCEThe H7N9 avian influenza virus has been considered to have the potential to cause the next pandemic since the first case of human infection reported in 2013. In this study, we identified PB2 residue 473 as a new determinant of mouse virulence and mammalian adaptation of the viral polymerase of the H7N9 virus and its non-pathogenic H9N2 counterparts. We further demonstrated that the variation in PB2-473 is functionally linked to differential co-opting of the host ANP32A protein in supporting viral polymerase activity, which is analogous to the well-known PB2-627 polymorphism, albeit the two PB2 positions are spatially distant. By providing new mechanistic insight into the PB2-mediated host range determination of influenza A viruses, our study implicated the potential existence of multiple PB2-ANP32 interfaces that could be targets for developing new antivirals against the H7N9 virus as well as other mammalian-adapted influenza viruses.

Genetic Characterization of Novel H7Nx Low Pathogenic Avian Influenza Viruses from Wild Birds in South Korea during the Winter of 2020-2021.

Zoonotic infection with avian influenza viruses (AIVs) of subtype H7, such as H7N9 and H7N4, has raised concerns worldwide. During the winter of 2020-2021, five novel H7 low pathogenic AIVs (LPAIVs) containing different neuraminidase (NA) subtypes, including two H7N3, an H7N8, and two H7N9, were detected in wild bird feces in South Korea. Complete genome sequencing and phylogenetic analysis showed that the novel H7Nx AIVs were reassortants containing two gene segments (hemagglutinin (HA) and matrix) that were related to the zoonotic Jiangsu-Cambodian H7 viruses causing zoonotic infection and six gene segments originating from LPAIVs circulating in migratory birds in Eurasia. A genomic constellation analysis demonstrated that all H7 isolates contained a mix of gene segments from different viruses, indicating that multiple reassortment occurred. The well-known mammalian adaptive substitution (E627K and D701N) in PB2 was not detected in any of these isolates. The detection of multiple reassortant H7Nx AIVs in wild birds highlights the need for intensive surveillance in both wild birds and poultry in Eurasia.

Genetic Characterization and Pathogenicity of H7N7 and H7N9 Avian Influenza Viruses Isolated from South Korea.

H7 low pathogenic avian influenza viruses (LPAIVs) can mutate into highly pathogenic avian influenza viruses (HPAIVs). In addition to avian species, H7 avian influenza viruses (AIVs) also infect humans. In this study, two AIVs, H7N9 (20X-20) and H7N7 (34X-2), isolated from the feces of wild birds in South Korea in 2021, were genetically analyzed. The HA cleavage site of the two H7 Korean viruses was confirmed to be ELPKGR/GLF, indicating they are LPAIVs. There were no amino acid substitutions at the receptor-binding site of the HA gene of two H7 Korean viruses compared to that of A/Anhui/1/2013 (H7N9), which prefer human receptors. In the phylogenetic tree analysis, the HA gene of the two H7 Korean viruses shared the highest nucleotide similarity with the Korean H7 subtype AIVs. In addition, the HA gene of the two H7 Korean viruses showed high nucleotide similarity to that of the A/Jiangsu/1/2018(H7N4) virus, which is a human influenza virus originating from avian influenza virus. Most internal genes (PB2, PB1, PA, NP, NA, M, and NS) of the two H7 Korean viruses belonged to the Eurasian lineage, except for the M gene of 34X-2. This result suggests that active reassortment occurred among AIVs. In pathogenicity studies of mice, the two H7 Korean viruses replicated in the lungs of mice. In addition, the body weight of mice infected with 34X-2 decreased 7 days post-infection (dpi) and inflammation was observed in the peribronchiolar and perivascular regions of the lungs of mice. These results suggest that mammals can be infected with the two H7 Korean AIVs. Our data showed that even low pathogenic H7 AIVs may infect mammals, including humans, as confirmed by the A/Jiangsu/1/2018(H7N4) virus. Therefore, continuous monitoring and pathogenicity assessment of AIVs, even of LPAIVs, are required.

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.

Intra-species sialic acid polymorphism in humans: a common niche for influenza and coronavirus pandemics?

The ongoing COVID-19 pandemic has led to more than 159 million confirmed cases with over 3.3 million deaths worldwide, but it remains mystery why most infected individuals (∼98%) were asymptomatic or only experienced mild illness. The same mystery applies to the deadly 1918 H1N1 influenza pandemic, which has puzzled the field for a century. Here we discuss dual potential properties of the 1918 H1N1 pandemic viruses that led to the high fatality rate in the small portion of severe cases, while about 98% infected persons in the United States were self-limited with mild symptoms, or even asymptomatic. These variations now have been postulated to be impacted by polymorphisms of the sialic acid receptors in the general population. Since coronaviruses (CoVs) also recognize sialic acid receptors and cause severe acute respiratory syndrome epidemics and pandemics, similar principles of influenza virus evolution and pandemicity may also apply to CoVs. A potential common principle of pathogen/host co-evolution of influenza and CoVs under selection of host sialic acids in parallel with different epidemic and pandemic influenza and coronaviruses is discussed.

The virulence modulator PA-X protein has minor effect on the pathogenicity of the highly pathogenic H7N9 avian influenza virus in mice.

PA-X is a novel discovered accessory protein encoded by the PA mRNA of the influenza A virus. Accumulated studies have demonstrated the crucial role of this protein in regulating the virulence of various subtypes of influenza virus, including H1N1, H5N1, H9N2, H1N2, H3N8 and H3N2 virus. However, the role of PA-X protein in regulating the virulence of the highly pathogenic avian H7N9 virus was unknown. In this study, we firstly generated two recombinant H7N9 viruses which have lower PA-X expression level than the parental H7N9 virus. We then systematically compared their difference in virus replication, polymerase activity, virulence and virus-induced host immune responses in mice. The results showed that the PA-X deficient viruses significantly increased viral replication in madin darby canine kidney cells and slightly increased viral replication in mouse lung. In addition, loss of PA-X expression significantly increased viral polymerase activity and alleviated the host-shutoff activity mediated by the parental PA protein. However, in contrast with the usual function of PA-X in regulating the virulence in different subtype influenza virus, no obvious effect on viral virulence in mice was observed by H7N9 PA-X protein. Furthermore, among the 12 kinds of cytokines and 2 kinds of complement derived components that we tested, the PA-X deficiency viruses only induced significantly higher expression levels of MX1 than the parental virus. Altogether, these results showed that PA-X has little effect on viral virulence and viral induced innate immune response of the H7N9 subtype virus. Our study adds further information for the growing understanding of the complexity of PA-X in regulating viral virulence and host innate immune response of different influenza virus.

Mink is a highly susceptible host species to circulating human and avian influenza viruses.

Pandemic influenza, typically caused by the reassortment of human and avian influenza viruses, can result in severe or fatal infections in humans. Timely identification of potential pandemic viruses must be a priority in influenza virus surveillance. However, the range of host species responsible for the generation of novel pandemic influenza viruses remains unclear. In this study, we conducted serological surveys for avian and human influenza virus infections in farmed mink and determined the susceptibility of mink to prevailing avian and human virus subtypes. The results showed that farmed mink were commonly infected with human (H3N2 and H1N1/pdm) and avian (H7N9, H5N6, and H9N2) influenza A viruses. Correlational analysis indicated that transmission of human influenza viruses occurred from humans to mink, and that feed source was a probable route of avian influenza virus transmission to farmed mink. Animal experiments showed that mink were susceptible and permissive to circulating avian and human influenza viruses, and that human influenza viruses (H3N2 and H1N1/pdm), but not avian viruses, were capable of aerosol transmission among mink. These results indicate that farmed mink could be highly permissive "mixing vessels" for the reassortment of circulating human and avian influenza viruses. Therefore, to reduce the risk of emergence of novel pandemic viruses, feeding mink with raw poultry by-products should not be permitted, and epidemiological surveillance of influenza viruses in mink farms should be urgently implemented.

Mutations during the adaptation of H7N9 avian influenza virus to mice lungs enhance human-like sialic acid binding activity and virulence in mice.

The first avian H7N9 influenza outbreak in spring of 2013 emerged in an unprecedented transmission from infected poultry to humans in the Yangtze delta area, eastern China, posing a dual challenge to public health and poultry industry. However, the mechanism for how avian H7N9 influenza virus adapts to mammalian hosts has not been clearly understood. Here, to identify adaptive changes that confer enhanced virulence of H7N9 virus in mammals, we generated a mouse-adapted H7N9 variant virus (S8) by serial lung-to-lung passages of the wild-type SDL124 virus in mice and compared their phenotype in vivo and in vitro. Sequence analysis showed that the two viruses differed by 27 amino acids distributed among six genes, containing changes in PB2 (E627K, D701N) and HA (Q226L) genes. The 50% mouse lethal dose (MLD50) of S8 reduced about 500 folds, to be moderately pathogenic to mice when compared to that of low pathogenic wild-type SDL124. Moreover, S8 replicated efficiently in mouse lungs and displayed expanded tissue tropism, and induced a greater degree of pulmonary edema and higher level of inflammatory cell infiltration in bronchoalveolar lavage fluids than SDL124 did. Interestingly, the mouse adapted S8 virus obtained strong affinity for human-like (SAα-2,6 Gal) receptor during the adaptation in mice. Correspondingly, compared with SDL124 virus, S8 virus showed higher replication efficiency in mammalian cells, whereas lower replication ability in avian cells. Taken together, these findings suggest that these mutations synergistically elevate the ability of H7N9 virus to disseminate to multiple organs and subsequently enhance the virulence of H7N9 virus in mammalian hosts.

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.

Experience and practice of the Emergency Operations Center, Chinese Center for Disease Control and Prevention: a case study of response to the H7N9 outbreak.

BACKGROUND: Emergency Operations Center (EOC) is a place to provide response to public health emergencies. Chinese Center for Disease Control and Prevention (China CDC)'s EOC was officially established in 2016, which has been the core department for the public health emergencies and risk response. In recent years, we have been continuously improving the function of EOC through many incidents. In the study, we hope to share the construction status, operation management experience of China CDC's EOC and the response process in the human avian influenza A (H7N9) outbreak. MAIN TEXT: The China CDC's EOC mainly focus on building the five core elements including sites/places and facilities, information and data, plans and procedures, training and exercises, and logistics. Based on summarizing previous emergency response, the China CDC's EOC established its own incident management and the standardized response procedures. The event-specific data, context-specific data and event management data could be obtained through various source. The logistics department of the EOC also provides comprehensive support. The well-trained staff is another necessary conditions for its operation. Through sharing the response process of H7N9 outbreak, it further explains the EOC's functions in the five phases of outbreak response, such as the formulation of the incident response framework, monitoring, personnel dispatch and resource mobilization. CONCLUSIONS: The EOC contributes to faster and more efficient responses during emergencies which enable a greater reduction in morbidity and mortality. Compared with the traditional incident response process, under the command and coordination of China CDC's EOC, each group involved in the response has a clearer goal, responsibilities and tasks at each stage. Meanwhile, each group also gave full play to its own expertise and advantages. As a whole, incident response tended to be more specialized and precise, which generally improves the efficiency of incident response. However, different countries and regions have different response processes to the events. We still suggested that appropriate emergency operation plan should be made according to the complexity of incident response in the region when constructing response mechanism, through our experience. And the China CDC's EOC is still at growing and groping phase.

Pathobiological Origins and Evolutionary History of Highly Pathogenic Avian Influenza Viruses.

High-pathogenicity avian influenza (HPAI) viruses have arisen from low-pathogenicity avian influenza (LPAI) viruses via changes in the hemagglutinin proteolytic cleavage site, which include mutation of multiple nonbasic to basic amino acids, duplication of basic amino acids, or recombination with insertion of cellular or viral amino acids. Between 1959 and 2019, a total of 42 natural, independent H5 (n = 15) and H7 (n = 27) LPAI to HPAI virus conversion events have occurred in Europe (n = 16), North America (n = 9), Oceania (n = 7), Asia (n = 5), Africa (n = 4), and South America (n = 1). Thirty-eight of these HPAI outbreaks were limited in the number of poultry premises affected and were eradicated. However, poultry outbreaks caused by A/goose/Guangdong/1/1996 (H5Nx), Mexican H7N3, and Chinese H7N9 HPAI lineages have continued. Active surveillance and molecular detection and characterization efforts will provide the best opportunity for early detection and eradication from domestic birds.

Analysis of the circRNAs expression profile in mouse lung with H7N9 influenza A virus infection.

Influenza A virus is a single-stranded RNA virus that can cause great mortality and economic loss worldwide. Circular RNAs (circRNAs) are non-coding RNAs that have been shown to have important functions in the regulation of biological processes. However, their functions during the influenza A virus infection process remain unclear. Herein, RNA sequencing technology was used to identify circRNAs expressed in mouse lungs during infection with H7N9/PB2-627 K/701D (H7N9/Wild-type) virus and PB2 mutant viruses (H7N9/PB2-627E/701D and H7N9/PB2-627E/701 N). We identified 7126 circRNAs at different genomic locations during H7N9 influenza virus and its mutant virus infections, of which 186 were differentially expressed. Enrichment analysis revealed that the differentially expressed circRNAs were associated with the viral infection process. Our study shows that circRNA expression profiles were altered following H7N9 influenza A virus infection and the differentially expressed circRNAs may have an important immune-regulating function during viral infection.

Computational predicting the human infectivity of H7N9 influenza viruses isolated from avian hosts.

The genome composition of a given avian influenza virus is the primary determinant of its potential for cross-species transmission from birds to humans. Here, we introduce a viral genome-based computational tool that can be used to evaluate the human infectivity of avian isolates of influenza A H7N9 viruses, which can enable prediction of the potential risk of these isolates infecting humans. This tool, which is based on a novel class weight-biased logistic regression (CWBLR) algorithm, uses the sequences of the eight genome segments of an H7N9 strain as the input and gives the probability of this strain infecting humans (reflecting its human infectivity). We examined the replication efficiency and the pathogenicity of several H7N9 avian isolates that were predicted to have very low or high human infectivity by the CWBLR model in cell culture and in mice, and found that the strains with high predicted human infectivity replicated more efficiently in mammalian cells and were more infective in mice than those that were predicted to have low human infectivity. These results demonstrate that our CWBLR model can serve as a powerful tool for predicting the human infectivity and cross-species transmission risks of H7N9 avian strains.

Glycosylation generates an efficacious and immunogenic vaccine against H7N9 influenza virus.

Zoonotic avian influenza viruses pose severe health threats to humans. Of several viral subtypes reported, the low pathogenic avian influenza H7N9 virus has since February 2013 caused more than 1,500 cases of human infection with an almost 40% case-fatality rate. Vaccination of poultry appears to reduce human infections. However, the emergence of highly pathogenic strains has increased concerns about H7N9 pandemics. To develop an efficacious H7N9 human vaccine, we designed vaccine viruses by changing the patterns of N-linked glycosylation (NLG) on the viral hemagglutinin (HA) protein based on evolutionary patterns of H7 HA NLG changes. Notably, a virus in which 2 NLG modifications were added to HA showed higher growth rates in cell culture and elicited more cross-reactive antibodies than did other vaccine viruses with no change in the viral antigenicity. Developed into an inactivated vaccine formulation, the vaccine virus with 2 HA NLG additions exhibited much better protective efficacy against lethal viral challenge in mice than did a vaccine candidate with wild-type (WT) HA by reducing viral replication in the lungs. In a ferret model, the 2 NLG-added vaccine viruses also induced hemagglutination-inhibiting antibodies and significantly suppressed viral replication in the upper and lower respiratory tracts compared with the WT HA vaccines. In a mode of action study, the HA NLG modification appeared to increase HA protein contents incorporated into viral particles, which would be successfully translated to improve vaccine efficacy. These results suggest the strong potential of HA NLG modifications in designing avian influenza vaccines.

Comparison of patients hospitalized with COVID-19, H7N9 and H1N1.

BACKGROUND: There is an urgent need to better understand the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for that the coronavirus disease 2019 (COVID-19) continues to cause considerable morbidity and mortality worldwide. This paper was to differentiate COVID-19 from other respiratory infectious diseases such as avian-origin influenza A (H7N9) and influenza A (H1N1) virus infections. METHODS: We included patients who had been hospitalized with laboratory-confirmed infection by SARS-CoV-2 (n = 83), H7N9 (n = 36), H1N1 (n = 44) viruses. Clinical presentation, chest CT features, and progression of patients were compared. We used the Logistic regression model to explore the possible risk factors. RESULTS: Both COVID-19 and H7N9 patients had a longer duration of hospitalization than H1N1 patients (P < 0.01), a higher complication rate, and more severe cases than H1N1 patients. H7N9 patients had higher hospitalization-fatality ratio than COVID-19 patients (P = 0.01). H7N9 patients had similar patterns of lymphopenia, neutrophilia, elevated alanine aminotransferase, C-reactive protein, lactate dehydrogenase, and those seen in H1N1 patients, which were all significantly different from patients with COVID-19 (P < 0.01). Either H7N9 or H1N1 patients had more obvious symptoms, like fever, fatigue, yellow sputum, and myalgia than COVID-19 patients (P < 0.01). The mean duration of viral shedding was 9.5 days for SARS-CoV-2 vs 9.9 days for H7N9 (P = 0.78). For severe cases, the meantime from illness onset to severity was 8.0 days for COVID-19 vs 5.2 days for H7N9 (P < 0.01), the comorbidity of chronic heart disease was more common in the COVID-19 patients than H7N9 (P = 0.02). Multivariate analysis showed that chronic heart disease was a possible risk factor (OR > 1) for COVID-19, compared with H1N1 and H7N9. CONCLUSIONS: The proportion of severe cases were higher for H7N9 and SARS-CoV-2 infections, compared with H1N1. The meantime from illness onset to severity was shorter for H7N9. Chronic heart disease was a possible risk factor for COVID-19.The comparison may provide the rationale for strategies of isolation and treatment of infected patients in the future.

The Effects of Genetic Variation on H7N9 Avian Influenza Virus Pathogenicity.

Since the H7N9 avian influenza virus emerged in China in 2013, there have been five seasonal waves which have shown human infections and caused high fatality rates in infected patients. A multibasic amino acid insertion seen in the HA of current H7N9 viruses occurred through natural evolution and reassortment, and created a high pathogenicity avian influenza (HPAI) virus from the low pathogenicity avian influenza (LPAI) in 2017, and significantly increased pathogenicity in poultry, resulting in widespread HPAI H7N9 in poultry, which along with LPAI H7N9, contributed to the severe fifth seasonal wave in China. H7N9 is a novel reassorted virus from three different subtypes of influenza A viruses (IAVs) which displays a great potential threat to public health and the poultry industry. To date, no sustained human-to-human transmission has been recorded by the WHO. However, the high ability of evolutionary adaptation of H7N9 and lack of pre-existing immunity in humans heightens the pandemic potential. Changes in IAVs proteins can affect the viral transmissibility, receptor binding specificity, pathogenicity, and virulence. The multibasic amino acid insertion, mutations in hemagglutinin, deletion and mutations in neuraminidase, and mutations in PB2 contribute to different virological characteristics. This review summarized the latest research evidence to describe the impacts of viral protein changes in viral adaptation and pathogenicity of H7N9, aiming to provide better insights for developing and enhancing early warning or intervention strategies with the goal of preventing highly pathogenic IAVs circulation in live poultry, and transmission to humans.

Dynamic PB2-E627K substitution of influenza H7N9 virus indicates the in vivo genetic tuning and rapid host adaptation.

Avian-origin influenza viruses overcome the bottleneck of the interspecies barrier and infect humans through the evolution of variants toward more efficient replication in mammals. The dynamic adaptation of the genetic substitutions and the correlation with the virulence of avian-origin influenza virus in patients remain largely elusive. Here, based on the one-health approach, we retrieved the original virus-positive samples from patients with H7N9 and their surrounding poultry/environment. The specimens were directly deep sequenced, and the subsequent big data were integrated with the clinical manifestations. Unlike poultry/environment-derived samples with the consistent dominance of avian signature 627E of H7N9 polymerase basic protein 2 (PB2), patient specimens had diverse ratios of mammalian signature 627K, indicating the rapid dynamics of H7N9 adaptation in patients during the infection process. In contrast, both human- and poultry/environment-related viruses had constant dominance of avian signature PB2-701D. The intrahost dynamic adaptation was confirmed by the gradual replacement of 627E by 627K in H7N9 in the longitudinally collected specimens from one patient. These results suggest that host adaptation for better virus replication to new hosts, termed "genetic tuning," actually occurred in H7N9-infected patients in vivo. Notably, our findings also demonstrate the correlation between rapid host adaptation of H7N9 PB2-E627K and the fatal outcome and disease severity in humans. The feature of H7N9 genetic tuning in vivo and its correlation with the disease severity emphasize the importance of testing for the evolution of this avian-origin virus during the course of infection.

Limited Tryptic Digestion-Isotope Dilution Mass Spectrometry (LTD-IDMS): A Reagent-Free Analytical Assay To Quantify Hemagglutinin of A(H5N1) Vaccine Material.

Avian influenza viruses, such as A(H5N1) and A(H7N9), are primary public health concerns due to their pandemic potential. Influenza vaccines represent the most effective response to this threat especially with timely provision. The current pandemic response timelines require a substantial period for strain-specific reference antigen and sera preparation for use with single-radial immunodiffusion (SRID), the accepted vaccine potency assay. To address this time lag, the isotope dilution mass spectrometry (IDMS) method was developed to quantify the absolute hemagglutinin (HA, the main influenza antigen) amount in the vaccine without the need for purified, inactivated, and calibrated virus reference antigens. However, an additional challenge in determining potency is to differentiate between vaccine antigens in their most potent form from other less potent, stressed antigen forms. The limited trypsin digestion (LTD) method has been developed and does not require strain-specific full-length reference antigens or antibodies; instead, stressed HA is selectively degraded, leaving the more potent form to be measured. LTD, followed by precipitation and IDMS, allows for efficient differentiation between potent and significantly less potent HA for vaccine release and potency testing across the vaccine's shelf life. In this study, we tested the LTD-IDMS assay on A(H5N1) vaccine material that had been stressed by low pH, heat, and multiple freeze-thaw cycles. The results showed that the LTD-IDMS method effectively quantified the potent HA in A(H5N1) vaccine material with results comparable to SRID. As such, it shows great promise to complement and potentially replace SRID in a pandemic when strain-specific reagents may not be readily available.

Profile and generation of reduced neuraminidase inhibitor susceptibility in highly pathogenic avian influenza H7N9 virus from human cases in Mainland of China, 2016-2019.

Human infections with highly pathogenic avian influenza (HPAI) H7N9 virus were detected in late 2016. We examined the drug resistance profile of 30 HPAI H7N9 isolates from Mainland of China (2016-2019). Altogether, 23% (7/30) carried neuraminidase inhibitors (NAIs) - resistance mutations, and 13% (4/30) displayed reduced susceptibility to NAIs in neuraminidase (NA) inhibition test. An HPAI H7N9 reassortment virus we prepared was passaged with NAIs for 10 passages. Passage with zanamivir induced an E119G substitution in NA, whereas passage with oseltamivir induced R292K and E119V substitutions that simulated that seen in oseltamivir -treated HPAI H7N9 cases, indicating that the high frequency of resistant strains in the HPAI H7N9 isolates is related to NAIs use. In presence of NAIs, R238I, A146E, G151E and G234T substitutions were found in HA1 region of HA. No amino acid mutations were found in the internal genes of the recombinant virus.

Genetic and antigenic characterization of H5 and H7 avian influenza viruses isolated from migratory waterfowl in Mongolia from 2017 to 2019.

The circulation of highly pathogenic avian influenza viruses (HPAIVs) of various subtypes (e.g., H5N1, H5N6, H5N8, and H7N9) in poultry remains a global concern for animal and public health. Migratory waterfowls play important roles in the transmission of these viruses across countries. To monitor virus spread by wild birds, active surveillance for avian influenza in migratory waterfowl was conducted in Mongolia from 2015 to 2019. In total, 5000 fecal samples were collected from lakesides in central Mongolia, and 167 influenza A viruses were isolated. Two H5N3, four H7N3, and two H7N7 viruses were characterized in this study. The amino acid sequence at hemagglutinin (HA) cleavage site of those isolates suggested low pathogenicity in chickens. Phylogenetic analysis revealed that all H5 and H7 viruses were closely related to recent H5 and H7 low pathogenic avian influenza viruses (LPAIVs) isolated from wild birds in Asia and Europe. Antigenicity of H7Nx was similar to those of typical non-pathogenic avian influenza viruses (AIVs). While HPAIVs or A/Anhui/1/2013 (H7N9)-related LPAIVs were not detected in migratory waterfowl in Mongolia, sporadic introductions of AIVs including H5 and H7 viruses into Mongolia through the wild bird migration were identified. Thus, continued monitoring of H5 and H7 AIVs in both domestic and wild birds is needed for the early detection of HPAIVs spread into the country.