On-Site and Visual Detection of the H5 Subtype Avian Influenza Virus Based on RT-RPA and CRISPR/Cas12a.

Avian influenza viruses (AIVs) of the H5 subtype rank among the most serious pathogens, leading to significant economic losses in the global poultry industry and posing risks to human health. Therefore, rapid and accurate virus detection is crucial for the prevention and control of H5 AIVs. In this study, we established a novel detection method for H5 viruses by utilizing the precision of CRISPR/Cas12a and the efficiency of RT-RPA technologies. This assay facilitates the direct visualization of detection results through blue light and lateral flow strips, accurately identifying H5 viruses with high specificity and without cross-reactivity against other AIV subtypes, NDV, IBV, and IBDV. With detection thresholds of 1.9 copies/µL (blue light) and 1.9 × 103 copies/µL (lateral flow strips), our method not only competes with but also slightly surpasses RT-qPCR, demonstrating an 80.70% positive detection rate across 81 clinical samples. The RT-RPA/CRISPR-based detection method is characterized by high sensitivity, specificity, and independence from specialized equipment. The immediate field applicability of the RT-RPA/CRISPR approach underscores its importance as an effective tool for the early detection and management of outbreaks caused by the H5 subtype of AIVs.

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.

Impairment of Left Ventricular Function in the Depressed Chinese Miniature Swine Model by Cardiovascular Magnetic Resonance Feature-Tracking: A Preliminary Study.

PURPOSE: Individuals with depression have an increased risk of cardiovascular disease, and more often have a poor prognosis with cardiovascular disease. This study aimed to investigate the impact of depression on Left Ventricular (LV) alterations using Cardiovascular Magnetic Resonance Featuretracking (CMR-FT). METHODS: Seven anesthetized, healthy Chinese miniature swine were included in the study. Basic data, including CMR scans at baseline and after 14 days of depression modeling, were collected. Behavioral tests, including the Open-field Test (OFT), Sucrose Preference Test (SPT), and measurements of the time taken to consume a specific amount of food and sugar, were conducted to assess the success of the depression models. CMR cine images were acquired and CVI software was employed to analyze Global Longitudinal Strain (GLS), Global Circumferential Strain (GCS), and Global Radial Strain (GRS). Late Gadolinium Enhancement (LGE) imaging was used to detect myocardial infarction and/or scar. RESULTS: The outcomes demonstrated successful depression modeling, indicated by reduced scores in the OFT and SPT, as well as an extended time to intake food and sugar compared to baseline. However, no significant differences were observed in LV End-diastolic Volume (LVEDV), LV Endsystolic Volume (LVESV), LV Ejection Fraction (LVEF), LV End-diastolic Myocardial Mass (LVMASSED), and Cardiac Output (CO) before and after modeling. Regarding LV global strain parameters, there was a downward trend in GRS (25.35% ± 6.9% vs. 22.86% ± 6.4%, P=0.021), GCS (-16.71% ± 4.2% vs. -14.78% ± 2.3%, P=0.043), and GLS (-17.66% ± 2.9% vs. -14.53% ± 2.5%, P=0.056), respectively, after modeling. GRS and GCS were significantly reduced after modeling compared to baseline. CONCLUSION: The study suggests that depression may contribute to early LV systolic dysfunction, particularly affecting LV GCS and GRS.

Myocardial deformation characteristics assessed by cardiovascular magnetic resonance feature tracking in a healthy Chinese population.

Purpose: To explore global/regional myocardial deformation across various layers, vascular distributions, specific levels and distinct walls in healthy individuals using cardiovascular magnetic resonance feature tracking (CMR-FT). Methods: We selected a cohort of 55 healthy participants and CMR cine images were used to obtain the left ventricular (LV) peak longitudinal, circumferential, radial strains (LS, CS, RS). The characteristics of normal LV strain in various layers (endocardium, myocardium, epicardium), territories [left anterior descending artery (LAD), left circumflex artery (LCX), and right coronary artery (RCA)], levels (basal, middle, apical) and walls (anterior, septum, inferior, lateral) were compared. Results: The absolute values of the LV global LS and CS gradually decreased from endocardium to epicardium. The absolute LV global RS (65.7 ± 47.7%) was maximum relative to LS (-22.0 ± 10.8%) and CS (-22.8 ± 7.7%). The absolute values of the LCX territorial strain were the largest compared with the LAD and RCA territorial strains. Regional RS, endo-CS and endo-LS gradually increased from the basal to the apical level. The LV lateral walls had the highest strain values (CS, LS, and RS). Conclusions: Variations in normal LV strain values across various layers, territories, levels, and walls were observed, suggesting the necessity for careful clinical interpretation of these strain values. These findings also partially revealed the complexity of normal cardiac mechanics.

Evolution of H7N9 highly pathogenic avian influenza virus in the context of vaccination.

Human infections with the H7N9 influenza virus have been eliminated in China through vaccination of poultry; however, the H7N9 virus has not yet been eradicated from poultry. Carefully analysis of H7N9 viruses in poultry that have sub-optimal immunity may provide a unique opportunity to witness the evolution of highly pathogenic avian influenza virus in the context of vaccination. Between January 2020 and June 2023, we isolated 16 H7N9 viruses from samples we collected during surveillance and samples that were sent to us for disease diagnosis. Genetic analysis indicated that these viruses belonged to a single genotype previously detected in poultry. Antigenic analysis indicated that 12 of the 16 viruses were antigenically close to the H7-Re4 vaccine virus that has been used since January 2022, and the other four viruses showed reduced reactivity with the vaccine. Animal studies indicated that all 16 viruses were nonlethal in mice, and four of six viruses showed reduced virulence in chickens upon intranasally inoculation. Importantly, the H7N9 viruses detected in this study exclusively bound to the avian-type receptors, having lost the capacity to bind to human-type receptors. Our study shows that vaccination slows the evolution of H7N9 virus by preventing its reassortment with other viruses and eliminates a harmful characteristic of H7N9 virus, namely its ability to bind to human-type receptors.

Assessment of the antigenic evolution of a clade 6B.1 human H1N1pdm influenza virus revealed differences between ferret and human convalescent sera.

BACKGROUND: Influenza viruses continually acquire mutations in the antigenic epitopes of their major viral antigen, the surface glycoprotein haemagglutinin (HA), allowing evasion from immunity in humans induced upon prior influenza virus infections or vaccinations. Consequently, the influenza strains used for vaccine production must be updated frequently. METHODS: To better understand the antigenic evolution of influenza viruses, we introduced random mutations into the HA head region (where the immunodominant epitopes are located) of a pandemic H1N1 (H1N1pdm) virus from 2015 and incubated it with various human sera collected in 2015-2016. Mutants not neutralized by the human sera were sequenced and further characterized for their haemagglutination inhibition (HI) titers with human sera and with ferret sera raised to H1N1pdm viruses from 2009 to 2015. FINDINGS: The largest antigenic changes were conferred by mutations at HA amino acid position 187; interestingly, these antigenic changes were recognized by human, but not by ferret serum. H1N1pdm viruses with amino acid changes at position 187 were very rare until the end of 2018, but have become more frequent since; in fact, the D187A amino acid change is one of the defining changes of clade 6B.1A.5a.1 viruses, which emerged in 2019. INTERPRETATION: Our findings indicate that amino acid substitutions in H1N1pdm epitopes may be recognized by human sera, but not by homologous ferret sera. FUNDING: This project was supported by funding from the NIAID-funded Center for Research on Influenza Pathogenesis (CRIP, HHSN272201400008C).

A sequence-based machine learning model for predicting antigenic distance for H3N2 influenza virus.

Introduction: Seasonal influenza A H3N2 viruses are constantly changing, reducing the effectiveness of existing vaccines. As a result, the World Health Organization (WHO) needs to frequently update the vaccine strains to match the antigenicity of emerged H3N2 variants. Traditional assessments of antigenicity rely on serological methods, which are both labor-intensive and time-consuming. Although numerous computational models aim to simplify antigenicity determination, they either lack a robust quantitative linkage between antigenicity and viral sequences or focus restrictively on selected features. Methods: Here, we propose a novel computational method to predict antigenic distances using multiple features, including not only viral sequence attributes but also integrating four distinct categories of features that significantly affect viral antigenicity in sequences. Results: This method exhibits low error in virus antigenicity prediction and achieves superior accuracy in discerning antigenic drift. Utilizing this method, we investigated the evolution process of the H3N2 influenza viruses and identified a total of 21 major antigenic clusters from 1968 to 2022. Discussion: Interestingly, our predicted antigenic map aligns closely with the antigenic map generated with serological data. Thus, our method is a promising tool for detecting antigenic variants and guiding the selection of vaccine candidates.

From Short- to Long-Range Chiral Recognition on Surfaces: Chiral Assembly and Synthesis.

Research on chiral behaviors of small organic molecules at solid surfaces, including chiral assembly and synthesis, can not only help unravel the origin of the chiral phenomenon in biological/chemical systems but also provide promising strategies to build up unprecedented chiral surfaces or nanoarchitectures with advanced applications in novel nanomaterials/nanodevices. Understanding how molecular chirality is recognized is considered to be a mandatory basis for such studies. In this review, a series of recent studies in chiral assembly and synthesis at well-defined metal surfaces under ultra-high vacuum conditions are outlined. More importantly, the intrinsic mechanisms of chiral recognition are highlighted, including short/long-range chiral recognition in chiral assembly and two main strategies to steer the reaction pathways and modulate selective synthesis of specific chiral products on surfaces.

Key Amino Acid Residues That Determine the Antigenic Properties of Highly Pathogenic H5 Influenza Viruses Bearing the Clade 2.3.4.4 Hemagglutinin Gene.

The H5 subtype highly pathogenic avian influenza viruses bearing the clade 2.3.4.4 HA gene have been pervasive among domestic poultry and wild birds worldwide since 2014, presenting substantial risks to human and animal health. Continued circulation of clade 2.3.4.4 viruses has resulted in the emergence of eight subclades (2.3.4.4a-h) and multiple distinct antigenic groups. However, the key antigenic substitutions responsible for the antigenic change of these viruses remain unknown. In this study, we analyzed the HA gene sequences of 5713 clade 2.3.4.4 viruses obtained from a public database and found that 23 amino acid residues were highly variable among these strains. We then generated a series of single-amino-acid mutants based on the H5-Re8 (a vaccine seed virus) background and tested their reactivity with a panel of eight monoclonal antibodies (mAbs). Six mutants bearing amino acid substitutions at positions 120, 126, 141, 156, 185, or 189 (H5 numbering) led to reduced or lost reactivity to these mAbs. Further antigenic cartography analysis revealed that the amino acid residues at positions 126, 156, and 189 acted as immunodominant epitopes of H5 viruses. Collectively, our findings offer valuable guidance for the surveillance and early detection of emerging antigenic variants.

Evaluation of left ventricular diastolic function in patients with coronary microvascular dysfunction via cardiovascular magnetic resonance feature tracking.

Background: Coronary microvascular dysfunction (CMD) has been suggested to be one of the pathologic mechanisms contributing to heart failure with preserved left ventricular ejection fraction (LVEF) and left ventricular (LV) diastolic dysfunction. We therefore aimed to evaluate LV diastolic function in patients with CMD using cardiovascular magnetic resonance feature tracking (CMR-FT). Methods: We prospectively enrolled 115 patients referred to cardiology clinics for chest pain assessment who subsequently underwent coronary computed tomography angiogram and stress perfusion CMR. CMD was defined as the presence of subendocardial inducible ischemia detected through visual assessment. LV diastolic function was evaluated using CMR-derived volume-time curves and CMR-FT parameters. The former included early peak filling rate (PFR) and time to PFR; the latter included LV global/regional peak longitudinal diastolic strain rate (LDSR), circumferential diastolic strain rate (CDSR), and radial diastolic strain rate (RDSR). Results: A total of 92 patients with 1,312 segments were eventually included. Of these, 19 patients were classified as non-CMD (48.8±11.2 years; 63.2% male) and 73 as with CMD (52.3±11.9 years; 54.8% male). The LVEFs were similar and preserved in both groups (P=0.266). At the per-patient level, no differences were observed in PFR, time to PFR, or LV global diastolic strain rates between the two groups. At the per-segment level, 51% (665/1,312) of the myocardial segments were classified as CMD, whereas 49% (647/1,312) were classified as non-CMD. CMD segments showed significantly lower regional CDSR (P=0.019) and RDSR (P=0.006) compared with non-CMD segments. Conclusions: Despite normal LV ejection fraction in CMD patients, decreased LV diastolic function in CMD myocardial segments indicates early diastolic impairment.

The SUMO-interacting motif in NS2 promotes adaptation of avian influenza virus to mammals.

Species differences in the host factor ANP32A/B result in the restriction of avian influenza virus polymerase (vPol) in mammalian cells. Efficient replication of avian influenza viruses in mammalian cells often requires adaptive mutations, such as PB2-E627K, to enable the virus to use mammalian ANP32A/B. However, the molecular basis for the productive replication of avian influenza viruses without prior adaptation in mammals remains poorly understood. We show that avian influenza virus NS2 protein help to overcome mammalian ANP32A/B-mediated restriction to avian vPol activity by promoting avian vRNP assembly and enhancing mammalian ANP32A/B-vRNP interactions. A conserved SUMO-interacting motif (SIM) in NS2 is required for its avian polymerase-enhancing properties. We also demonstrate that disrupting SIM integrity in NS2 impairs avian influenza virus replication and pathogenicity in mammalian hosts, but not in avian hosts. Our results identify NS2 as a cofactor in the adaptation process of avian influenza virus to mammals.

Influenza H3 hemagglutinin vaccine with scrambled immunodominant epitopes elicits antibodies directed toward immunosubdominant head epitopes.

Vaccination is the most effective countermeasure to reduce the severity of influenza. Current seasonal influenza vaccines mainly elicit humoral immunity targeting hemagglutinin (HA). In particular, the amino acid residues around the receptor-binding site in the HA head domain are predominantly targeted by humoral immunity as "immunodominant" epitopes. However, mutations readily accumulate in the head domain due to high plasticity, resulting in antigenic drift and vaccine mismatch, particularly with influenza A (H3N2) viruses. A vaccine strategy that targets more conserved immunosubdominant epitopes is required to attain a universal vaccine. Here, we designed an H3 HA vaccine antigen with various amino acids at immunodominant epitopes of the HA head domain, termed scrambled HA (scrHA). In ferrets, scrHA vaccination induced lower serum neutralizing antibody levels against homologous virus compared with wild-type (WT) HA vaccination; however, similar levels of moderately neutralizing titers against antigenically distinct H3N2 viruses were observed. Ferrets vaccinated with scrHA twice and then challenged with homologous or heterologous virus showed the same level of reduced virus shedding in nasal swabs as WT HA-vaccinated animals but reduced body temperature increase, whereas WT HA-vaccinated ferrets exhibited body temperature increases similar to those of mock-vaccinated animals. scrHA elicited antibodies against HA immunodominant and -subdominant epitopes at lower and higher levels, respectively, than WT HA vaccination, whereas antistalk antibodies were induced at the same level for both groups, suggesting scrHA-induced redirection from immunodominant to immunosubdominant head epitopes. scrHA vaccination thus induced broader coverage than WT HA vaccination by diluting out the immunodominancy of HA head epitopes. IMPORTANCE Current influenza vaccines mainly elicit antibodies that target the immunodominant head domain, where strain-specific mutations rapidly accumulate, resulting in frequent antigenic drift and vaccine mismatch. Targeting conserved immunosubdominant epitopes is essential to attain a universal vaccine. Our findings with the scrHA developed in this study suggest that designing vaccine antigens that "dilute out" the immunodominancy of the head epitopes may be an effective strategy to induce conserved immunosubdominant epitope-based immune responses.

HbA1c is related to microcirculation blood perfusion in patients with coronary microvascular disease using stress perfusion cardiac magnetic resonance: An observational study.

BACKGROUND: In coronary microvascular disease (CMD) patients, the incidence of major adverse cardiovascular events (MACEs) in patients with myocardial perfusion reserve index (MPRI) ≤ 1.47 is three times higher than that in MPRI > 1.47. We investigated whether the increase of glycated hemoglobin A1c (HbA1c) could increase the risk of MPRI ≤1.47 in diabetic and non-diabetic patients. METHODS: From November 2019, patients with ischemic symptoms but without obstructive coronary disease were screened. Use MPRI measured by stress perfusion cardiac magnetic resonance (CMR) to reflect microcirculation blood perfusion, and MPRI <2.5 were included. The patients were divided into two groups based on MPRI was greater or <1.47. The risk factors for CMD were explored using logistic regression analysis. RESULTS: A total of 80 patients with an MPRI of 1.69 ± 0.79 were included. CMD patients with an MPRI of ≤1.47(n = 33) were higher than MPRI of >1.47(n = 47) in age, presence of diabetes mellitus, fasting blood glucose levels and HbA1c levels (P < 0.05). In non-diabetic patients, increased HbA1c was associated with the risk of MPRI≤1.47 (OR = 0.017, 95%CI: 0.050-1.107, P = 0.045). Compared with non-diabetic patients with HbA1c < 6.0, non-diabetic patients with HbA1c ≥ 6.0 increased the risk of MPRI of ≤1.47 (OR = 0.219, 95%CI: 0.069-0.697, P = 0.010). In diabetic patients, HbA1c was not associated with the risk of MPRI of ≤1.47 (OR = 1.043, 95%CI: 0.269, 4.044, P = 0.952). And compared with non-diabetic patients with HbA1c <6.0, diabetic patients with HbA1c <6.0 (OR = 0.917, 95%CI: 0.233-3.610, P = 0.901) or ≥6.0 (OR = 0.326, 95%CI: 0.073-1.446, P = 0.140), the risk of MPRI ≤ 1.47 was not further increased. CONCLUSIONS: In non-diabetic patients, elevated HbA1c is related to MPRI≤1.47(a value increased incidence of MACEs). Therefore, in patients with undiagnosed diabetes, early management of glycosylated hemoglobin is very important. TRIAL REGISTRATION: This clinical trial has been registered in the Chinese clinical Trial Registry with an identifier: ChiCTR1900025810.

Highly Selective On-Surface Reactions of Aryl Propiolic Acids via Decarboxylative Coupling.

Aryl propiolic acids are introduced as a new class of monomers in the field of on-surface chemistry to build up poly(arylenebutadiynylenes) through decarboxylative Glaser coupling. As compared to aryl alkynes that are routinely used in the on-surface Glaser coupling, it is found that the decarboxylative coupling occurs at slightly lower temperature and with excellent selectivity. Activation occurs through decarboxylation for the propiolic acids, whereas the classical Glaser coupling is achieved through alkyne CH activation, and this process shows poor selectivity. The efficiency of the decarboxylative coupling is documented by the successful polymerization of bis(propiolic acids) as monomers. It is also found that the new activation mode is compatible with aryl bromide functionalities, which allows the formation of unsymmetric metal-organic polymers on the surface by chemoselective sequential reactions. All transformations are analyzed by a scanning tunneling microscope and are further studied by density functional theory calculations.

Genetic analysis and biological characterization of H10N3 influenza A viruses isolated in China from 2014 to 2021.

The H10 subtypes of avian influenza viruses pose a continual threat to the poultry industry and human health. The sporadic spillover of H10 subtypes viruses from poultry to humans is represented by the H10N8 human cases in 2013 and the recent H10N3 human infection in 2021. However, the genesis and characteristics of the recent reassortment H10N3 viruses have not been systemically investigated. In this study, we characterized 20 H10N3 viruses isolated in live poultry markets during routine nationwide surveillance in China from 2014 to 2021. The viruses in the recent reassortant genotype acquired their hemagglutinin (HA) and neuraminidase (NA) genes from the duck H10 viruses and H7N3 viruses, respectively, whereas the internal genes were derived from chicken H9N2 viruses as early as 2019. Receptor-binding analysis indicated that two of the tested H10N3 viruses had a higher affinity for human-type receptors than for avian-type receptors, highlighting the potential risk of avian-to-human transmission. Animal studies showed that only viruses belonging to the recent reassortant genotype were pathogenic in mice; two tested viruses transmitted via direct contact and one virus transmitted by respiratory droplets in guinea pigs, though with limited efficiency. These findings emphasize the need for enhanced surveillance of H10N3 viruses.

Continued evolution of the Eurasian avian-like H1N1 swine influenza viruses in China.

Animal influenza viruses continue to pose a threat to human public health. The Eurasian avian-like H1N1 (EA H1N1) viruses are widespread in pigs throughout Europe and China and have caused human infections in several countries, indicating their pandemic potential. To carefully monitor the evolution of the EA H1N1 viruses in nature, we collected nasal swabs from 103,110 pigs in 22 provinces in China between October 2013 and December 2019, and isolated 855 EA H1N1 viruses. Genomic analysis of 319 representative viruses revealed that these EA H1N1 viruses formed eight different genotypes through reassortment with viruses of other lineages circulating in humans and pigs, and two of these genotypes (G4 and G5) were widely distributed in pigs. Animal studies indicated that some strains have become highly pathogenic in mice and highly transmissible in ferrets via respiratory droplets. Moreover, two-thirds of the EA H1N1 viruses reacted poorly with ferret serum antibodies induced by the currently used H1N1 human influenza vaccine, suggesting that existing immunity may not prevent the transmission of the EA H1N1 viruses in humans. Our study reveals the evolution and pandemic potential of EA H1N1 viruses and provides important insights for future pandemic preparedness.

ARNT Inhibits H5N1 Influenza A Virus Replication by Interacting with the PA Protein.

Increasing evidence suggests that the polymerase acidic (PA) protein of influenza A viruses plays an important role in viral replication and pathogenicity. However, information regarding the interaction(s) of host factors with PA is scarce. By using a yeast two-hybrid screen, we identified a novel host factor, aryl hydrocarbon receptor nuclear translocator (ARNT), that interacts with the PA protein of the H5N1 virus. The interaction between PA and human ARNT was confirmed by co-immunoprecipitation and immunofluorescence microscopy. Moreover, overexpression of ARNT downregulated the polymerase activity and inhibited virus propagation, whereas knockdown of ARNT significantly increased the polymerase activity and virus replication. Mechanistically, overexpression of ARNT resulted in the accumulation of PA protein in the nucleus and inhibited both the replication and transcription of the viral genome. Interaction domain mapping revealed that the bHLH/PAS domain of ARNT mainly interacted with the C-terminal domain of PA. Together, our results demonstrate that ARNT inhibits the replication of the H5N1 virus and could be a target for the development of therapeutic strategies against H5N1 influenza viruses.

Influenza Viruses Suitable for Studies in Syrian Hamsters.

Several small animal models, including mice, Syrian hamsters, guinea pigs, and ferrets are used to study the pathogenicity, transmissibility, and antigenicity of seasonal and pandemic influenza viruses. Moreover, animal models are essential for vaccination and challenge studies to evaluate the immunogenicity and protective efficacy of new vaccines. However, authentic human influenza viruses do not always replicate efficiently in these animal models. Previously, we developed a high-yield A/Puerto Rico/8/34 (PR8-HY) vaccine virus backbone that conferred an increased virus yield to several seasonal influenza vaccines in eukaryotic cells and embryonated chicken eggs. Here, we show that this PR8-HY genetic backbone also increases the replication of several seasonal influenza viruses in Syrian hamsters compared to the authentic viruses. Therefore, the PR8-HY backbone is useful for animal studies to assess the biological properties of influenza viral HA and NA.

Global dissemination of H5N1 influenza viruses bearing the clade 2.3.4.4b HA gene and biologic analysis of the ones detected in China.

H5N1 avian influenza viruses bearing the clade 2.3.4.4b hemagglutinin gene have been widely circulating in wild birds and are responsible for the loss of over 70 million domestic poultry in Europe, Africa, Asia, and North America since October 2020. During our routine surveillance, 13 H5N1 viruses were isolated from 26,767 wild bird and poultry samples that were collected between September 2021 and March 2022 in China. To investigate the origin of these Chinese isolates and understand their genetic relationship with the globally circulating H5N1 viruses, we performed a detailed phylogenic analysis of 233 representative H5N1 strains that were isolated from 28 countries. We found that, after they emerged in the Netherlands, the H5N1 viruses encountered complicated gene exchange with different viruses circulating in wild birds and formed 16 genotypes. Genotype one (G1) was predominant, being detected in 22 countries, whereas all other genotypes were only detected in one or two continents. H5N1 viruses of four genotypes (G1, G7, G9, and G10) were detected in China; three of these genotypes have been previously reported in other countries. The H5N1 viruses detected in China replicated in mice, with pathogenicity varying among strains; the G1 virus was highly lethal in mice. Moreover, we found that these viruses were antigenically similar to and well matched with the H5-Re14 vaccine strain currently used in China. Our study reveals the overall picture of H5N1 virus evolution and provides insights for the control of these viruses.