Evolutionary dynamics and comparative pathogenicity of clade 2.3.4.4b H5 subtype avian influenza viruses, China, 2021-2022.

The recent concurrent emergence of H5N1, H5N6, and H5N8 avian influenza viruses (AIVs) has led to significant avian mortality globally. Since 2020, frequent human-animal interactions have been documented. To gain insight into the novel H5 subtype AIVs (i.e., H5N1, H5N6 and H5N8), we collected 6102 samples from various regions of China between January 2021 and September 2022, and identified 41 H5Nx strains. Comparative analyses on the evolution and biological properties of these isolates were conducted. Phylogenetic analysis revealed that the 41 H5Nx strains belonged to clade 2.3.4.4b, with 13 related to H5N1, 19 to H5N6, and 9 to H5N8. Analysis based on global 2.3.4.4b viruses showed that all the viruses described in this study were likely originated from H5N8, exhibiting a heterogeneous evolutionary history between H5N1 and H5N6 during 2015-2022 worldwide. H5N1 showed a higher rate of evolution in 2021-2022 and more sites under positive selection pressure in 2015-2022. The antigenic profiles of the novel H5N1 and H5N6 exhibited notable variations. Further hemagglutination inhibition assay suggested that some A(H5N1) viruses may be antigenically distinct from the circulating H5N6 and H5N8 strains. Mammalian challenge assays demonstrated that the H5N8 virus (21GD001_H5N8) displayed the highest pathogenicity in mice, followed by the H5N1 virus (B1557_H5N1) and then the H5N6 virus (220086_H5N6), suggesting a heterogeneous virulence profile of H5 AIVs in the mammalian hosts. Based on the above results, we speculate that A(H5N1) viruses have a higher risk of emergence in the future. Collectively, these findings unveil a new landscape of different evolutionary history and biological characteristics of novel H5 AIVs in clade 2.3.4.4b, contributing to a better understanding of designing more effective strategies for the prevention and control of novel H5 AIVs.

Effect of soybean polysaccharide and soybean oil on gelatinization and retrogradation properties of corn starch.

This investigation stems from the wide interest in mitigating starch retrogradation, which profoundly impacts the quality of starch-based food, garnering significant attention in the contemporary food industry. Our study delves into the intricate dynamics of soluble soybean polysaccharide (SSPS) and soybean oil (SO) when added individually or in combination to native corn starch (NCS), offering insights into the gelatinization and retrogradation phenomena. We observed that SSPS (0.5 %, w/w) hindered starch swelling, leading to an elevated gelatinization enthalpy change (∆H) value, while SO (0.5 %, w/w) increased ∆H due to its hydrophobicity. Adding SSPS and/or SO concurrently reduced the viscosity and storage modulus (G') of starch matrix. For the starch gel (8 %, w/v) after refrigeration, SSPS magnified water-holding capacity (WHC) and decreased hardness through hydrogen bonding with starch, while SO increased hardness with limited water retention. Crucially, the combination of SSPS and SO maximized WHC, minimized hardness, and significantly inhibited starch retrogradation. The specific ratio of SSPS to SO was found to significantly influence the starch properties, with a 1:1 ratio resulting in the most desirable quality for application in starch-based foods. This study offers insights for utilizing polysaccharides and lipids in starch-based food products to extend shelf life.

Mastering textural control in multi-polysaccharide gels: Effect of κ-carrageenan, konjac glucomannan, locust bean gum, low-acyl gellan gum, and sodium alginate.

To comprehend the intricate interplay of five common food polysaccharides, κ-Carrageenan (KC), konjac glucomannan (KGM), locust bean gum (LBG), low-acyl gellan gum (LAG), and sodium alginate (SA), within composite polysaccharide gels, widely employed for textural modulation and flavor enhancement. This study systematically modulates the quantities of these five polysaccharides to yield six distinct multi-polysaccharide gels. The unique impact of each polysaccharide on the overall quality of composite gels were studied by thermostability, microstructure, water-holding capacity (WHC), texture, and sensory attributes. The findings unequivocally manifest the phenomenon of thermoreversible gelation in all composite gels, except for the KC-devoid sample, which displayed an inability to solidify. Notably, KGM, LBG, and LAG emerged as pivotal enhancers of the network structure in these composite gels, while SA was identified as a promotor of layered structure, resulting in a reduction of surface hardness. Leveraging principal component analysis (PCA) to analyzed 14 critical evaluation parameters of the five multi-polysaccharide gels, revealing the order as follows: KC > KGM > SA > LAG > LBG. These findings would imparts valuable insights into the pragmatic utilization of multi-polysaccharide gels for the development of food products (e.g. Bobo balls in milk tea) with tailored textural and sensory attributes.

Fabrication and characterization of complex coacervates utilizing gelatin and carboxymethyl starch.

BACKGROUND: Modified polysaccharides have greatly expanded applications in comparison with native polysaccharides due to their improved compatibility and interactions with proteins and active compounds in food-related areas. Nonetheless, there is a noticeable dearth of research concerning the utilization of carboxymethyl starch (CMS) as a microcapsule wall material in food processing, despite its common use in pharmaceutical delivery. The development of an economical and safe embedding carrier using CMS and gelatin (GE) holds immense importance within the food-processing industry. In this work, the potential of innovative coacervates formed by the combination of GE and CMS as a reliable, stable, and biodegradable embedding carrier is evaluated by turbidity measurements, thermogravimetric analysis (TGA), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and rheological measurements. RESULTS: The results indicate that GE-CMS coacervates primarily resulted from electrostatic interactions and hydrogen bonding. The optimal coacervation was observed at pH 4.6 and with a GE/CMS blend ratio of 3:1 (w/w). However, the addition of NaCl reduced coacervation and made it less sensitive to temperature changes (35-55 °C). In comparison with individual GE or CMS, the coacervates exhibited higher thermal stability, as shown by TGA. X-ray diffraction analysis shows that the GE-CMS coacervates maintained an amorphous structure. Rheological testing reveals that the GE-CMS coacervates exhibited shear-thinning behavior and gel-like properties. CONCLUSION: Overall, attaining electroneutrality in the mixture boosts the formation of a denser structure and enhances rheological properties, leading to promising applications in food, biomaterials, cosmetics, and pharmaceutical products. © 2023 Society of Chemical Industry.

Analysis of mRNA m6A modification and mRNA expression profiles in middle ear cholesteatoma.

Introduction: Middle ear cholesteatoma is characterized by the hyperproliferation of keratinocytes. In recent decades, N6-methyladenosine (m6A) modification has been shown to play an essential role in the pathogenesis of many proliferative diseases. However, neither the m6A modification profile nor its potential role in the pathogenesis of middle ear cholesteatoma has currently been investigated. Therefore, this study aimed to explore m6A modification patterns in middle ear cholesteatoma. Materials and methods: An m6A mRNA epitranscriptomic microarray analysis was performed to analyze m6A modification patterns in middle ear cholesteatoma tissue (n = 5) and normal post-auricular skin samples (n = 5). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to predict the potential biological functions and signaling pathways underlying the pathogenesis of middle ear cholesteatoma. Subsequently, m6A modification levels were verified by methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) in middle ear cholesteatoma tissue and normal skin samples, respectively. Results: A total of 6,865 distinctive m6A-modified mRNAs were identified, including 4,620 hypermethylated and 2,245 hypomethylated mRNAs, as well as 9,162 differentially expressed mRNAs, including 4,891 upregulated and 4,271 downregulated mRNAs, in the middle ear cholesteatoma group relative to the normal skin group. An association analysis between methylation and gene expression demonstrated that expression of 1,926 hypermethylated mRNAs was upregulated, while expression of 2,187 hypomethylated mRNAs and 38 hypermethylated mRNAs was downregulated. Moreover, GO analysis suggested that differentially methylated mRNAs might influence cellular processes and biological behaviors, such as cell differentiation, biosynthetic processes, regulation of molecular functions, and keratinization. KEGG pathway analysis demonstrated that the hypermethylated transcripts were involved in 26 pathways, including the Hippo signaling pathway, the p53 signaling pathway, and the inflammatory mediator regulation of transient receptor potential (TRP) channels, while the hypomethylated transcripts were involved in 13 pathways, including bacterial invasion of epithelial cells, steroid biosynthesis, and the Hippo signaling pathway. Conclusion: Our study presents m6A modification patterns in middle ear cholesteatoma, which may exert regulatory roles in middle ear cholesteatoma. The present study provides directions for mRNA m6A modification-based research on the epigenetic etiology and pathogenesis of middle ear cholesteatoma.

That H9N2 avian influenza viruses circulating in different regions gather in the same live-poultry market poses a potential threat to public health.

H9N2 avian influenza viruses are endemic and persistent in China, but those that are prevalent in different provinces are also causes of wide epidemics, related to the spread of wild birds and the cross-regional trade in live poultry. For the past 4 years, beginning in 2018, we have sampled a live-poultry market in Foshan, Guangdong, in this ongoing study. In addition to the prevalence of H9N2 avian influenza viruses in China during this period, we identified isolates from the same market belonging to clade A and clade B, which diverged in 2012-2013, and clade C, which diverged in 2014-2016, respectively. An analysis of population dynamics revealed that, after a critical divergence period from 2014 to 2016, the genetic diversity of H9N2 viruses peaked in 2017. Our spatiotemporal dynamics analysis found that clade A, B, and C, which maintain high rates of evolution, have different prevalence ranges and transmission paths. Clades A and B were mainly prevalent in East China in the early stage, and then spread to Southern China, becoming epidemic with clade C. Strains from different regions converge at the same live-poultry market to communicate, which may be one reasons the H9N2 viruses are difficult to eradicate and increasingly dominant throughout China. Selection pressure and molecular analysis have demonstrated that single amino acid polymorphisms at key receptor binding sites 156, 160, and 190 under positive selection pressure, suggesting that H9N2 viruses are undergoing mutations to adapt to new hosts. Live-poultry markets are important because people who visit them have frequent contact with poultry, H9N2 viruses from different regions converge at these markets and spread through contact between live birds and humans, generating increased risks of human exposure to these viruses and threatening public health safety. Thus, it is important to reducing the cross-regional trade of live poultry and strengthening the monitoring of avian influenza viruses in live-poultry markets to reduce the spread of avian influenza viruses.

Comparative Analyses of the Exopalaemon carinicauda Gut Bacterial Community and Digestive and Immune Enzyme Activity during a 24-Hour Cycle.

The change in life activities throughout a cycle of approximately 24 h is called the circadian rhythm. Circadian rhythm has an important impact on biological metabolism, digestion, immunity, and other physiological activities, but the circadian rhythm of crustaceans has rarely been studied. In this study, the activity of digestive enzymes (α-amylase, trypsin, and lipase) and immune enzymes (superoxide dismutase, lysozyme, and catalase), as well as the circadian rhythm of the intestinal bacterial community of Exopalaemon carinicauda, were studied. The results showed that the digestive and immune enzyme activities of E. carinicauda changed significantly (p < 0.05) at four time points throughout the day by one-way ANOVA analysis, with the highest value at 24:00 and the lowest value at 12:00. The highest values of alpha diversity and richness were observed in the 24:00 samples, which were significantly higher than those in the other groups (p < 0.05). The principal coordinate analysis (PCoA) results obviously showed that the samples from the same sampling time had higher similarity in the bacterial community structure. Candidatus hepatoplasma had the highest abundance among the intestinal microorganisms at 24:00, and Marinomonas had the highest abundance at 12:00. This study contributed to the understanding of digestive enzyme activity, immune enzyme activity, and the circadian rhythm of the intestinal bacterial community structure in E. carinicauda. It will play an important role in optimizing feeding times and improving digestion and nutrient utilization for E. carinicauda. The results of this study provide a basis for further study on the physiological mechanism of diurnal variation of intestinal flora in crustaceans.

A Characterization and an Evolutionary and a Pathogenicity Analysis of Reassortment H3N2 Avian Influenza Virus in South China in 2019-2020.

Seasonal H3N2 influenza virus has always been a potential threat to public health. The reassortment of the human and avian H3N2 influenza viruses has resulted in major influenza outbreaks, which have seriously damaged human life and health. To assess the possible threat of the H3N2 avian influenza virus to human health, we performed whole-genome sequencing and genetic evolution analyses on 10 H3N2 field strains isolated from different hosts and regions in 2019-2020 and selected representative strains for pathogenicity tests on mice. According to the results, the internal gene cassettes of nine strains had not only undergone reassortment with the H1, H2, H4, H6, and H7 subtypes, which circulate in poultry and mammals, but also with H10N8, which circulates in wild birds in the natural environment. Three reassorted strains were found to be pathogenic to mice, of these one strain harboring MP from H10N8 showed a stronger virulence in mice. This study indicates that reassorted H3N2 AIVs may cross the host barrier to infect mammals and humans, thereby, necessitating persistent surveillance of H3N2 AIVs.

Treatment of Recurrent Nasopharyngeal Carcinoma: A Sequential Challenge.

Recurrent nasopharyngeal carcinoma (NPC), which occurs in 10-20% of patients with primary NPC after the initial treatment modality of intensity-modulated radiation therapy (IMRT), is one of the major causes of death among NPC patients. Patients with recurrent disease without distant metastases still have a chance to be saved, but re-treatment often carries more serious toxicities or higher risks. For this group of patients, both otolaryngologists and oncologists are committed to developing more appropriate treatment regimens that can prolong patient survival and improve survival therapy. Currently, there are no international guidelines for the treatment of patients with recurrent NPC. In this article, we summarize past publications on clinical research and mechanistic studies related to recurrent NPC, combined with the experience and lessons learned by our institutional multidisciplinary team in the treatment of recurrent NPC. We propose an objective protocol for the treatment of recurrent NPC.

Exosomes Derived hsa-miR-4669 as a Novel Biomarker for Early Predicting the Response of Subcutaneous Immunotherapy in Pediatric Allergic Rhinitis.

Purpose: Subcutaneous immunotherapy (SCIT) is an effective treatment for pediatric allergic rhinitis (AR), but its efficacy fluctuates among individuals. This study aims to identify the profile of serum exosomes derived microRNAs (miRNAs) and evaluate their capacities to early predict SCIT efficacy in pediatric AR. Patients and Methods: High-throughput sequencing was applied to identify the miRNA of serum exosomes in AR children. GO enrichment and KEGG pathway analysis were performed to enrich the biological annotations of target mRNAs of miRNAs. Then we validated differentially expressed miRNAs in two independent cohorts by RT-qPCR. Logistic regression and receiver operating characteristic curve (ROC) were applied to evaluate the abilities of identified miRNAs in predicting the efficacy of SCIT in AR children. Results: A total of 812 miRNAs were detected in the serum exosomes, including 16 upregulated and 14 downregulated. Differentially expressed genes are enriched in the biological process of developmental process and regulation of cellular process, and gathered in pathways such as the signaling pathways regulating pluripotency of stem cells and the Wnt signaling pathway. In the first validation cohort, hsa-miR-4669 (P=0.009) and hsa-miR-4686 (P=0.032) were significantly downregulated in the effective group than the ineffective group, while hsa-miR-3196 (P=0.015) was upregulated. In the second cohort, hsa-miR-4669 level (P<0.0001) was downregulated in the effective group than the ineffective group. In addition, logistic regression revealed that hsa-miR-4669 level was correlated with the visual analogue scale (r=0.323, P=0.001) and total nasal symptoms score (r=0.269, P =0.007). ROC curve highlighted that hsa-miR-4669 level exhibited a reliable accuracy in predicting SCIT efficacy in pediatric AR (AUC=0.785). Conclusion: Serum exosomes derived miRNA were associated with the efficacy of SCIT. Serum exosomes derived hsa-miR-4669 might serve as a novel biomarker for early predicting the response of SCIT in AR children.

Predictive Value of Nasal Nitric Oxide and Serum NOS2 Levels in the Efficacy of Subcutaneous Immunotherapy in Pediatric Patients with Allergic Rhinitis.

Background: Subcutaneous immunotherapy (SCIT) is an effective therapy for allergic rhinitis (AR), but some AR patients still do not benefit from it. Nasal nitric oxide (nNO) and inducible nitric oxide synthase (iNOS/NOS2) act important roles in AR. This study aims to explore the abilities of serum NOS2 and nNO in predicting the clinical efficacy of SCIT in AR patients. Methods: We recruited 40 healthy controls (HCs) and 120 AR patients in this study. Serum NOS2 and nNO levels were compared between the two groups. In the AR group, patients underwent and finished 1-year of SCIT, and divided into the effective and ineffective groups, and the relationships between serum NOS2 and nNO levels and efficacy of SCIT were evaluated. Results: The serum NOS2 and nNO levels were higher in AR patients than HCs. In the effective group, the serum NOS2 and nNO levels were increased than the ineffective group. ROC curves presented that a combination of serum NOS2 and nNO exhibited promising predictive ability in predicting the clinical efficacy of SCIT. Conclusions: Serum NOS2 and nNO levels were enhanced in AR patients and might affect the efficacy of SCIT. The combined use of serum NOS2 and nNO levels could be a reliable and useful method for predicting the clinical efficacy of SCIT.

Novel Reassortant Avian Influenza A(H5N6) Virus, China, 2021.

Although reports of human infection with influenza A(H5N6) increased in 2021, reports of similar H5N6 virus infection in poultry are few. We detected 10 avian influenza A(H5N6) clade 2.3.4.4b viruses in poultry from 4 provinces in China. The viruses showed strong immune-escape capacity and complex genetic reassortment, suggesting further transmission risk.

Salvage Endoscopic Skull Base Surgery: Another Treatment Option After Immunotherapy for Recurrent Nasopharyngeal Carcinoma.

Background: Advanced recurrent nasopharyngeal carcinoma (NPC) is a relatively common nasopharyngeal skull base disease for which there is no uniform treatment modality. Not all patients are satisfied with the efficacy of immunotherapy with or without chemotherapy. Methods: This study included patients who underwent salvage endoscopic skull base nasopharyngectomy after immunotherapy between February 2017 and June 2021. Patient survival information was analyzed. Relevant publications were retrieved from five databases from December 1, 2011 to December 1, 2021. The outcomes of patients with advanced recurrent NPC who received programmed death 1 (PD-1) immunotherapy were collected and analyzed. Results: Nine patients who underwent skull base surgery, all of whom had previously undergone PD-1 immunotherapy, were included in this study. The 2-year overall survival (OS) and progression-free survival (PFS) rates of these patients were 25% and 29.2%, respectively. Eight publications involving 688 patients with advanced recurrent NPC were also included in this study. The combined complete response (CR), partial response (PR), and stable disease (SD) values were 2%, 23%, and 29%, respectively. The combined DCR included the three disease conditions, CR, PR, and SD, with a value of 53%. PD-1 monotherapy was more effective than PD-1 combination chemotherapy. Conclusions: PD-1 immunotherapy may improve the remission rate in patients with recurrent NPC. Salvage endoscopic skull base nasopharyngectomy may be another option for patients with poor immunotherapeutic outcomes. For patients with advanced recurrent NPC, better evidence-based medical data are needed to determine whether they should receive immunotherapy before or after surgery.

Hypoxia stress affects the physiological responses, apoptosis and innate immunity of Kuruma shrimp, Marsupenaeus japonicus.

For commercial aquatic animals, hypoxia phenomenon often occurs in live transport and aquaculture. In previous studies, much interest has been focused on antioxidant enzyme activities and could not present the complexities. The multifaceted responses, especially considering physiological indexes, histological structure, cell apoptosis, and immune pathways, are still unknown. In this study, we investigated the comprehensive hypoxic responses of Marsupenaeus japonicus. The results showed that the physiological indexes showed time-dependent changes upon hypoxia stress. Hypoxia stress led to significant tissue damage and cell apoptosis in the gill and hepatopancreas. Compared with the control group, the apoptosis index (AI) of the 12 h hypoxic treatment increased significantly (p < 0.05) in the gills and hepatopancreas. Comparative transcriptome analysis identified 900 and 1400 differentially expressed genes (DEGs) in the gill and hepatopancreas, respectively. Several DEGs were related to the lysosome, glycolysis/gluconeogenesis, citrate cycle, and apoptosis, and seven of them were validated using quantitative real-time PCR. This study provided valuable clues to understanding the mechanisms underlying the hypoxic responses of M. japonicus.

Emergence of novel avian origin H7N9 viruses after introduction of H7-Re3 and rLN79 vaccine strains to China.

In early 2021, roughly 6 months after the H7N9 H7-Re3 and H7N9 rLN79 vaccine strains were introduced into China, we monitored a number of H7N9 subtype avian influenza viruses, which could have escaped vaccine-induced immunity in live poultry markets (LPMs) in Yunnan, Hebei, Shanxi and Guangdong provinces, China. To investigate whether these viruses were a novel H7N9 variant of highly pathogenic avian influenza (HPAI) virus and whether they had the potential for further spread, we characterized the genetic evolution, antigenic divergence and pathogenicity of the viruses in the context of vaccine immunity. The results show further diversification in the HA gene of newly isolated HPAI H7N9 viruses compared with antigenic variants that emerged after the period of 2017-2019. There were clear antigenic differences between current vaccines and these viruses, and SPF broilers under vaccine protection could not resist virus challenges. Our study demonstrates that the current vaccine has insufficient protective capacity against the novel H7N9 variants under experimental conditions. A novel H7N9 immune escape virus has emerged. Faced with potential outbreaks, we should strengthen surveillance and update vaccine strains.

Microarray analysis and functional prediction of differentially expressed circular RNAs in acquired middle ear cholesteatoma.

BACKGROUND: Middle ear cholesteatoma is characterized by hyper-proliferation of keratinocytes. Circular RNA (circRNA) plays an essential role in the pathogenesis of many proliferative diseases. However, the role of circRNA in the etiopathogenesis of middle ear cholesteatoma is rarely investigated so far. We aimed to investigate the differential expression profiling of circRNAs between acquired middle ear cholesteatoma and normal skin, and to identify potential circRNAs contributing to the etiopathogenesis of middle ear cholesteatoma. Microarray analysis and functional prediction were performed to investigate the circRNA expression profiling between middle ear cholesteatoma and normal skin. Validation of differentially expressed circRNAs was conducted by qRT-PCR. Prediction of m6A modification was also carried out. RESULTS: Microarray analysis displayed that totally 93 up-regulated and 85 down-regulated circRNAs were identified in middle ear cholesteatoma. Through validation, expressions of hsa_circRNA_104327 and hsa_circRNA_404655 were significantly higher, while hsa_circRNA_000319 was significantly down-regulated in cholesteatoma. GO classification, KEGG pathway, and ceRNA network analyses suggested that these differentially expressed circRNAs might play important roles in the etiopathogenesis of middle ear cholesteatoma. Prediction of m6A modification exhibited that hsa_circRNA_000319 possessed 4 m6A sites with very high confidence, and hsa_circRNA_404655 had 3 m6A sites with high confidence. CONCLUSIONS: Our study revealed that these differentially expressed circRNAs might contribute to the etiopathogenesis of middle ear cholesteatoma. Further researches should be conducted to investigate the exact mechanism of these differentially expressed circRNAs in the etiopathogenesis of middle ear cholesteatoma. Targeting on these circRNAs may provide a new strategy for middle ear cholesteatoma therapy in the future.

Identification of Novel Biomarkers for Evaluating Disease Severity in House-Dust-Mite-Induced Allergic Rhinitis by Serum Metabolomics.

The aim of this study was to identify differences in serum metabolomics profiles of house-dust-mite (HDM)-induced allergic rhinitis (AR) patients compared to controls and to explore novel biomarkers reflecting disease severity. Serum samples were collected from 29 healthy controls and HDM-induced 72 AR patients, including 30 mild patients (MAR) and 42 moderate to severe AR patients (MSAR). Metabolomics detection was performed, and orthogonal partial least square discriminate analysis was applied to assess the differences between AR patients and controls and for subgroups based on disease severity. These analysis results successfully revealed distinct metabolite signatures which distinguished MAR patients and MSAR patients from controls. MSAR patients also could be discriminated from MAR patients based on their metabolic fingerprints. Most observed metabolite changes were related to glycine, serine, and threonine metabolism, pyrimidine metabolism, sphingolipid metabolism, arginine and proline metabolism, and fatty acid metabolism. Levels of sarcosine, sphingosine-1-phosphate, cytidine, and linoleic acid significantly correlated with the total nasal symptom score and visual analogue scale in AR patients. These results suggest that metabolomics profiling may provide novel insights into the pathophysiological mechanisms of HDM-induced AR and contribute to its evaluation of disease severity.

Accelerated Evolution of H7N9 Subtype Influenza Virus under Vaccination Pressure.

No avian H7N9 outbreaks have occurred since the introduction of H7N9 inactivated vaccine in the fall of 2017. However, H7N9 is still prevalent in poultry. To surveil the prevalence, genetic characteristics, and antigenic changes of H7N9, over 7000 oropharyngeal and cloaca swab specimens were collected from live poultry markets and farms in 15 provinces of China from 2017 to 2019. A total of 85 influenza virus subtype H7N9 strains were isolated and 20 representative strains were selected for genetic analysis and antigenicity evaluation. Results indicated the decreased prevalence of low-pathogenic H7N9 strains while highly-pathogenic H7N9 strains became dominated since the introduction of vaccine. Phylogenetic analysis showed that strains from 2019 formed an independent small branch and were genetically distant to strains isolated in 2013-2018. Analysis of key amino acid sites showed that the virus strains may adapt to the host environment evolutionally through mutation. Our analysis predicted additional potential glycosylation sites for HA and NA genes in the 2019 strains. Sequence analysis of HA gene in strains isolated from 2018 to 2019 showed that there were an increased nucleotide substitution rate and an increased mutation rate in the first and second nucleotides of coding codons within the open reading frame. The hemagglutination inhibition (HI) assay showed that H7-Re1 and H7-Re2 exhibited a lower HI titer for isolates from 2019, while H7-Re3 and rLN79 showed a high HI titer. The protective effect of the vaccine decreased after 15 months of use. Overall, under vaccination pressure, the evolution of influenza virus subtype H7N9 has accelerated.

Genetic Evolution Characteristics of Genotype G57 Virus, A Dominant Genotype of H9N2 Avian Influenza Virus.

This study aimed to investigate the genetic evolution of the H9N2 avian influenza virus (AIV). Whole genome phylogenetic trees were constructed based on 306 H9N2 avian influenza strains collected in China from 2014 to 2019. The results showed that eight gene sequences were clustered separately according to their dominant clades, and a total of 10 genotypes were identified (seven of which were novel types). Among them, G57 genotype was confirmed as the most prevalent genotype with a frequency of 94%. In China, the G57 genotype of H9N2 first emerged in 2007, and then became the most common genotype in 2013. Therefore, the nucleotide substitution rates of G57 genotype in HA and NA genes collected from 2007 to 2019 were estimated, and the positive selection pressure sites in the same data set were measured. Taking 2013 as the boundary, the time period was divided into two periods: 2007-2012 and 2013-2019. From 2007 to 2012, multiple genotypes coexisted and could bear the pressures from both nature and environment; while G57 genotype was still in the adaptation stage, subjected to less selection pressure and in the process of slow evolution. However, from 2013 to 2019, G57 became the dominant genotype, and most of the external pressure reacted on it. Moreover, G57 genotype showed better adaptability than other genotypes. From 2013 to 2019, the nucleotide substitution rates of the HA gene were increased, and the positive selection pressures on HA and NA genes were stronger compared to those from 2007 to 2012. To sum up, the absolutely dominant G57 genotype exhibited a relatively constant genotype frequency and experienced adaptive evolution and natural selection simultaneously during the monitoring period. Therefore, urgent attention and diligent surveillance of H9N2 avian influenza virus are becoming increasingly important.