Genetic and biological properties of H9N2 avian influenza viruses isolated in central China from2020 to 2022

The H9N2 subtype of avian influenza virus(AIV)is widely prevalent in poultry and wild birds globally,and has become the predominant subtype circulating in poultry in China.The H9N2 AIV can directly or indirectly(by serving as a"donor virus")infect humans,posing a significant threat to public health.Currently,there is a lack of in-depth research on the prevalence of H9N2 viruses in Shanxi Province,central China.In this study,we isolated 14 H9N2 AIVs from October 2020 to April 2022 in Shanxi Province,and genetic analysis revealed that these viruses belonged to 7 different genotypes.Our study on animals revealed that the H9N2 strains we identified displayed high transmission efficiency among chicken populations,and exhibited diverse replication abilities within these birds.These viruses could replicate efficiently in the lungs of mice,with one strain also demonstrating the capacity to reproduce in organs like the brain and kidneys.At the cellular level,the replication ability of different H9N2 strains was evaluated using plaque formation assays and multi-step growth curve assays,revealing significant differences in the replication and proliferation efficiency of the various H9N2 viruses at the cellular level.The antigenicity analysis suggested that these isolates could be classified into 2 separate antigenic clusters.Our research provides crucial data to help understand the prevalence and biological characteristics of H9N2 AIVs in central China.It also highlights the necessity of enhancing the surveillance of H9N2 AIVs.

In silico evaluation of kuwanon compounds as antiviral agents targeting H9N2 influenza virus

Background:The threat of avian influenza a subtype avian influenza A(H9N2)virus remains a significant concern,necessitating the exploration of novel antiviral agents.This study employs network pharmacology and computational analysis to investigate the potential of kuwanons,a natural compounds against H9N2 influenza virus.Methods:Leveraging comprehensive databases and bioinformatics tools,we elucidate the molecular mechanisms underlying Kuwanons pharmacological effects against H9N2 influenza virus.Network pharmacology identifies H9N2 influenza virus targets and compounds through integrated protein-protein interaction and Kyoto Encyclopedia of Genes and Genomes analyses.Molecular docking studies were performed to assess the binding affinities and structural interactions of Kuwanon analogues with key targets,shedding light on their potential inhibitory effects on viral replication and entry.Results:Compound-target network analysis revealed complex interactions(120 nodes,163 edges),with significant interactions and an average node degree of 2.72.Kyoto Encyclopedia of Genes and Genomes analysis revealed pathways such as Influenza A,Cytokine-cytokine receptor interaction pathway in H9N2 influenza virus.Molecular docking studies revealed that the binding free energy for the docked ligands ranged between-5.2 and-9.4 kcal/mol for the human interferon-beta crystal structure(IFNB1,Protein Data Bank:1AU1)and-5.4 and-9.6 kcal/mol for Interleukin-6(IL-6,PDB:4CNI).Conclusion:Our findings suggest that kuwanon exhibits promising antiviral activity against H9N2 influenza virus by targeting specific viral proteins,highlighting its potential as a natural therapeutic agent in combating avian influenza infections.

Stop H5N1 influenza in US cattle now

The relentless march of a highly pathogenic avian influenza virus(HPAIV)strain,known as H5N1,to become an unprecedented panzootic continues unchecked.The leap of H5N1 clade 2.3.4.4b from Eurasia and Africa to North America in 2021 and its further spread to South America and the Antarctic have exposed new avian and mammalian populations to the virus and led to outbreaks on an unrivaled scale.The virus has infected wild birds across vast geographic regions and caused wildlife deaths in some of the world's most biodiverse ecosystems.

Antibodies elicited by Newcastle disease virus-vectored H7N9 avian influenza vaccine are functional in activating the complement system

H7N9 subtype avian influenza virus poses a great challenge for poultry industry.Newcastle disease virus(NDV)-vectored H7N9 avian influenza vaccines(NDV_(vec)H7N9)are effective in disease control because they are protective and allow mass administration.Of note,these vaccines elicit undetectable H7N9-specific hemagglutination-inhibition(HI)but high IgG antibodies in chickens.However,the molecular basis and protective mechanism underlying this particular antibody immunity remain unclear.Herein,immunization with an NDV_(vec)H7N9 induced low anti-H7N9 HI and virus neutralization titers but high levels of hemagglutinin(HA)-binding IgG antibodies in chickens.Three residues(S150,G151 and S152)in HA of H7N9 virus were identified as the dominant epitopes recognized by the NDV_(vec)H7N9 immune serum.Passively transferred NDV_(vec)H7N9 immune serum conferred complete protection against H7N9 virus infection in chickens.The NDV_(vec)H7N9 immune serum can mediate a potent lysis of HA-expressing and H7N9 virus-infected cells and significantly suppress H7N9 virus infectivity.These activities of the serum were significantly impaired after heat-inactivation or treatment with complement inhibitor,suggesting the engagement of the complement system.Moreover,mutations in the 150-SGS-152 sites in HA resulted in significant reductions in cell lysis and virus neutralization mediated by the NDV_(vec)H7N9 immune serum,indicating the requirement of antibody-antigen binding for complement activity.Therefore,antibodies induced by the NDV_(vec)H7N9 can activate antibody-dependent complement-mediated lysis of H7N9 virus-infected cells and complement-mediated neutralization of H7N9 virus.Our findings unveiled a novel role of the complement in protection conferred by the NDV_(vec)H7N9,highlighting a potential benefit of engaging the complement system in H7N9 vaccine design.

Multiplex PCR Assay Establishment for Detection of H5,H9 Subtypes Avian Influenza Virus and Duck Tembusu Virus

The paper aimed to establish a rapid multiplex PCR assay for detection of 1-15, H9 subtype avian influenza virus （VIA） and duck Tembusu virus （DT- MUV）. According to the HA gene sequences of I-L5 and H9 AIV and NS5 gene of DTMUV in C, enBank, three sets of primer specific to these three kinds of viruses were designed, respectively. The multiplex PCR for simultaneous detection of AIV （ HS, H9 ） and DTMUV was established, and was applied in clinical. The speci- ficity and sensitivity of the multiplex PCR were tested. The results showed that the assay could specifically amplify the gene fragments of I-IS AIV （380 bp）, H9 AIV （ 732 bp）, and DTMUV （250 bp ）, and were all negative for other duck viruses. This method showed the sensitivity of 533 pg/μL for H5 MV, 56 pg/μL for I-I9, and 6.6 ng/μL for DTMUV. Additionally, the detection results of 200 clinical samples indicated that this multiplex PCR method was a rapid, sensitive and svecific tool for clinical sample detection.

The cloning of non-structural-1 (NS1) gene of H9N2 subtype of avian influenza virus in pGEX-4T-1 and pMAL-c2X plasmids and expression in <i>Escherichia coli</i>DH5<i>α</i>strain

Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including trade embargoes and interfering with serologic surveillance in differentiation between infected and vaccinated animals (DIVA strategy). Several distinct DIVA strategies have been presented to conquer these limitations. In this study, the open reading frame of NS1 gene of a H9N2 subtype of AI virus was amplified by polymerase chain reaction. After extraction and purification of NS1 gene from agarose gel, it was inserted into two different pGEX-4T-1 and pMAL-c2X plasmids and transferred in DH5α strain of Escherichia coli by using electroporation procedure. The E. coli colonies possessing recombinant NS1 gene were screened using PCR, restriction mapping and sequencing analysis. The expressed rNS1 protein was purified using affinity chromatography based on MBP (pMAL- c2X) and GST (pGEX-4T-1). The MBP-NS1 and GST- NS1 proteins on SDS-PAGE had bands with molecular weight of 68 and 52 kDa respectively. Western blotting with MBP-NS1 protein showed positive reaction using antisera obtained from chickens challenged with a H9N2 subtype strain. But, the most sera prepared from H9N2 vaccinated chickens were negative in WB. These findings indicated that the MBP-rNS1 protein of 26 kDa expressed by pMAL-c2X plasmid can be used in a DIVA for differentiation of AI infected and vaccinated chickens.

Multiple RT-PCR Detection of H5,H7,and H9 Subtype Avian Influenza Viruses and Newcastle Disease Virus

Objective:This paper focuses on the multiple detection RT-PCR technology of H5,H7,AND H9 subtype avian influenza viruses and Newcastle disease virus,and points out the specific detection methods and detection procedures of avian influenza and Newcastle disease virus.Methods:The genes of Newcastle disease virus carrying out the HA gene sequence of H5,H7 and H9 subtype AIV in GenBank were used to establish a strategy for simultaneous detection of three subtypes of avian influenza virus and Newcastle disease virus.Results:The results showed that the program can detect and distinguish H5,H7 and H9 subtype avian influenza viruses and Newcastle disease virus at one time.Conclusion:Multiple RT-PCR detection method has high detection sensitivity and can detect and determine different subtypes of avian influenza virus and Newcastle disease virus quickly and accurately,therefore,it has a crucial role in the detection and control of avian influenza H5,H7 and H9 subtypes and Newcastle disease.

Complete Genome Sequencing and Genetic Variation Analysis of Two H9N2 Subtype Avian Influenza Virus Strains

[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 genes were obtained by using RT-PCR,and these sequences were analyzed with that of six H9N2 subtype avian influenza isolates in homology comparison and genetic evolution relation.[Result] The results showed that the nucleotide sequence of entire gene of the strain shared 91.1%-95.4% homology with other seven reference strains,and PG08 shared the highest homology 91.3% with C/BJ/1/94;ZD06 shared the highest homology 92.3% with D/HK/Y280/97.HA cleavage sites of two H9N2 subtype avian influenza virus isolated strains were PARSSR/GLF,typical of mildly pathogenic avian influenza virus.[Conclusion] Phylogenetic tree for entire gene of eight strains showed that the genetic relationship was the closest between ZD06 and C/Pak/2/99 strains,which belonged to the Eurasian lineage;PG08 shared the highest homology 91.3% with ZD06,it may be the product of gene rearrangements of other sub-lines.

Sequence Analysis of HA Genes from Three H9N2 Subtype Avian Influenza Viruses

[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/ liu/00 were amplified by RT-PCR and then sequenced. [ Result] The results of phylogenetic analysis showed that A/Chicken/Beijing/xu/00, A/ Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 shared the nucleotide homologies of 84.8% （ Dk/HK/Y439/97 ） -98.0% （ Ck/GX17/00 ）, 85.1% （Dk/HK/Y439/97） - 99.1% （ Ck/GXl 7/00）, 90.7% （ Ck/BJ/3/01 ） - 99.1% （Ck/GX17/00） with the isolates from Hongkong and other are- as of Chinese Mainland respectively. At the same time, the analysis of amino acid indicated that the three isolates belonged to low pathogenic H9N2 isolates of avian origin. The 226^th amino acid of them were L （ Leu）, suggesting their high binding affinity to human cells. There were seven glyco- sylation sites in HA protein, five from HA1 and two from HA2. [ Cenclusien] By analysis at molecular level, it could be concluded that A/Chicken/ Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 were low pathogenic H9N2 isolates of avian origin.

H5N1和H9N2禽流感病毒HA抗原性对比分析和基因免疫研究

根据H9N2和H5N1亚型禽流感病毒血凝素基因序列,设计合成全基因扩增引物,将RT-PCR扩增2个毒株的HA基因进行序列测定,对推导的氨基酸序列通过MIF Bioinformatics软件进行抗原表位分析和糖基化位点进行分析。构建2个基因的真核表达质粒,检测2个毒株的HA基因疫苗免疫后外周血中HA特异性抗体的效价,并比较HA2个亚型免疫应答特异性的差异。结果表明,2株毒株禽流感病毒血凝素抗原表位和糖基化位点存在较大差异,重组质粒免疫后成功地诱导了体液免疫反应。两种亚型禽流感的HA基因免疫诱导的对本亚型病毒的抗体应答水平明显高于另一亚型。

丹参酮ⅡA通过miR-155-5p激活SIRT1-AMPK通路改善H9c2心肌细胞缺血/再灌注损伤

目的:探究丹参酮ⅡA(TⅡA)通过miR-155-5p激活沉默信息调节因子1(SIRT1)-腺苷酸活化蛋白激酶(AMPK)通路改善H9c2心肌细胞缺血/再灌注(I/R)损伤的机制。方法:体外培养H9c2细胞并建立I/R损伤模型,造模完成后将H9c2细胞随机分为模型组、TⅡA组、TⅡA+miR-NC组、TⅡA+miR-155-5p mimics组,转染后分别加入10μmol/L TⅡA进行干预,并以添加DMSO的H9c2细胞作为对照组。qRT-PCR检测miR-155-5p表达水平;MTT法分析细胞增殖能力;流式细胞术评估细胞凋亡情况;ELISA测定TNF-α、IL-4、IL-10、IL-17、乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)水平;Western blot检测SIRT1、AMPK、p-AMPK蛋白水平。结果:与对照组相比,模型组miR-155-5p表达升高,细胞活力下降,凋亡率及TNF-α、IL-17、LDH、MDA表达升高,IL-4、IL-10、SOD、SIRT1、p-AMPK表达减少(P<0.05);与模型组比较,TⅡA组miR-155-5p表达降低,细胞活力增强,凋亡率及TNF-α、IL-17、LDH、MDA表达下降,IL-4、IL-10、SOD、SIRT1、p-AMPK表达升高(P<0.05);与TⅡA组和TⅡA+miR-NC组比较,TⅡA+miR-155-5p mimics组miR-155-5p表达升高,细胞活力降低,凋亡率上升,TNF-α、IL-17、LDH、MDA表达上升,IL-4、IL-10、SOD、SIRT1、p-AMPK表达降低(P<0.05)。结论:TⅡA可通过下调miR-155-5p改善H9c2心肌细胞I/R损伤,其机制可能与激活SIRT1-AMPK通路有关。

Sequence Comparison and Analysis of HA Gene of Four H9N2 Avian Influenza Virus Isolates

[ Objective] To determine the HA gene sequences of four H9N2 Avian influenza virus （AIV） strains and carry out comparative analysis so as to understand the difference and variation pattern of each strain from the angle of molecular biology and to know the distribution and epidemic law of H9N2 AIV. [Method] One pair of primers was designed referring to HA gene sequences of H9N2 AIV. The HA genes of A/Chicken/Hebei/WD/98 （H9N2; WD98 for short）, A/Chicken/Hebei/ZD/04 （H9N2; ZD04 for short））, A/Chicken/Beijing/MY/06 （H9N2; MY06 for short） ）, and A/Chicken/Beijing/PG/08 （H9N2; PG08 for short）） were amplified, cloned and sequenced. Then the HA gene sequences of these strains were compared with that of 10 H9N2 AIV stains in GenBank. [Result] The ORF of HA genes of the four strains was 1 683 bp in size, encoding 516 amino acids. The HA gene sequences of the four strains, WD98, MY06, PG08, and ZD04, were 82.6% -95.1%, 83.0% -99.0%, 82.7% -95.5%, and 81.3% -95.7% homologous to that of the 10 H9N2 AIV stains, respectively. And the homology of amino acid was respectively 86.6% -96.3%, 86.6% -97.9%, 87.0% -97.1%, and 86.9% -97.3%. [ Conclusion] The HA gene has greatly high homology among different strains.

Cloning and Phylogenetic Analysis of NS1 Genes from Different Isolates of H9N2 Subtype Duck Influenza Virus

[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007（H9N2） （A3 for short）, A/Duck/FJ/301/2007 （H9N2） （C1 for short）, A/Duck/NH/306/2007（H9N2） （ D6 for short）, A/duck/SS/402/2007（H9N2） （ E2 for short）, and a strain named A/duck/ZC/2007（H9N2） （L1 for short） from a muscovy duck died of avian influenza virus （AIV）, were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 （chicken, 2003）. By comparison with the NS1 gene sequences of L1, AF523514 （duck）, AY664743 （chicken） and EF155262.1 （quail） using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 （ R→Q）, 70, 71 （ KE→EG）, 86 （ A→S）, 124 （V→M） and 225 （ S→N）, and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase （PKR） binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.

Study on Piezoelectric Immunosensor for the Detection of H9-subtype Avian Influenza Virus

[Objective] The aim is to develop the piezoelectric immunosensor to detect H9-subtype avian influenza virus（AIV）.[Method] The immunosensor chip was constructed by self-assembling mercaptopmpionic acid（MPA） to be monolayer on the silver-coated electrode of quartz crystal and coupling the monoclonal antibody to H9 subtype AIV with N-ethy-N＇-（3-dimethyl aminopropyl）carbodiimide hydrochloride（EDC） and N-hydroxysuccinimide（NHS）.The immunosensor to detect H9 subtype AIV was established.[Result] The results showed that the immunosensor displayed better specificity to H9 AIV and had no response to H5AIV and NDV when it was used for detection.The sensitivity test indicated the detection sensitivity for the H9 subtype AIV could reach 20-100 EID50.[Conclusion] The research provided a foundation for further research on the immunosensor for detecting AIV and it could be a new approach to detect other related viruses.

贵州省正安县不同养殖模式下鸡H5、H7和H9亚型禽流感血清学调查

为了监测并评价贵州省正安县各养殖模式禽流感的免疫效果,采用血凝试验与血凝抑制试验检测血清中H5、H7、H9亚型禽流感病毒(AIV)的抗体情况,2020—2022年累计采集贵州省正安县鸡血样660份,为做好禽流感监测预警与防控工作奠定基础。结果表明,2020—2022年,鸡H5抗体阳性率分别为81.00%、79.20%、76.30%,H7抗体阳性率分别为74.00%、77.90%、82.00%,H9抗体阳性率分别为78.64%、70.00%、84.00%。禽流感H5、H7、H9免疫抗体合格率均达到国家标准。

H5N1 influenza viruses： outbreaks and biological properties

All known subtypes of influenza A viruses are maintained in wild waterfowl, the natural reservoir of these viruses. Influenza A viruses are isolated from a variety of animal species with varying morbidity and mortality rates. More importantly, influenza A viruses cause respiratory disease in humans with potentially fatal outcome. Local or global outbreaks in humans are typically characterized by excess hospitalizations and deaths. In 1997, highly pathogenic avian influenza viruses of the H5N1 subtype emerged in Hong Kong that transmitted to humans, resulting in the first documented cases of human death by avian influenza virus infection. A new outbreak started in July 2003 in poultry in Vietnam, Indonesia, and Thailand, and highly pathogenic avian H5N1 influenza viruses have since spread throughout Asia and into Europe and Africa. These viruses continue to infect humans with a high mortality rate and cause worldwide concern of a looming pandemic. Moreover, H5N1 virus outbreaks have had devastating effects on the poultry industries throughout Asia. Since H5N1 virus outbreaks appear to originate from Southern China, we here examine H5N1 influenza viruses in China, with an emphasis on their biological properties.

H5N6 influenza virus infection, the newest influenza

The most recent new emerging infection is the H5N6 inl uenza virus infection. This infection has just been reported from China in early May 2014. The disease is believed to be a cross species infection. All indexed cases are from China. Of interest, the H5N6 inl uenza virus is the primary virus for avian. The avian H5N6 inl uenza virus in avian population is a low virulent strain. However, the clinical manifestation in human seems severe. In this mini-review, the authors summarize and discuss on this new emerging inl uenza.

An Overview of the Highly Pathogenic H5N1 Influenza Virus

Since the first human case of H5N1 avian influenza virus infection was reported in 1997, this highly pathogenic virus has infected hundreds of people around the world and resulted in many deaths. The ability of H5N1 to cross species boundaries, and the presence of polymorphisms that enhance virulence, present challenges to developing clear strategies to prevent the pandemic spread of this highly pathogenic avian influenza (HPAI) virus. This review summarizes the current understanding of, and recent research on, the avian influenza H5N1 virus, including transmission, virulence, pathogenesis, clinical characteristics, treatment and prevention.

Improvement of the efficacy of influenza vaccination (H5N1) in chicken by using extract of Cochinchina momordica seed (ECMS)

Seeds of a Chinese traditional medicine plant, Cochinchina momordica were used in the present study for the improvement of influenza vaccine （HSN 1） in chicken. Crude extraction from Cochinchina momordica seed （ECMS） was obtained by ethanol extraction method. In experiment No. 1, two weeks old chickens were immunized with influenza vaccine （HSN1） alone or combined with ECMS （5, 10, 20, 40 and 80 μg/dose）. Serum IgG antibody levels （by ELISA） as well as effects on dally weight gain were measured on 0, 7, 14 and 28th day after immunization. Results revealed that all ECMS groups numerically increased the antibody levels while 10 and 20 μg/dose groups significantly （P〈0.05） enhanced total IgG antibody on day 28, when compared with control. Average daily weight gain was also significantly higher in 20 μg/dose ECMS group. Adjuvant effect was also confirmed in experiment No. 2 when chickens were immunized with 20 μg/dose ECMS and antibody titer was measured through hemagglutination inhibition （HI）. It is concluded that ECMS has potential to improve the immune responses and deserve further study as an adjuvant.

The miR-9-5p/CXCL11 pathway is a key target of hydrogen sulfide-mediated inhibition of neuroinflammation in hypoxic ischemic brain injury

We previously showed that hydrogen sulfide(H2S)has a neuroprotective effect in the context of hypoxic ischemic brain injury in neonatal mice.However,the precise mechanism underlying the role of H2S in this situation remains unclear.In this study,we used a neonatal mouse model of hypoxic ischemic brain injury and a lipopolysaccharide-stimulated BV2 cell model and found that treatment with L-cysteine,a H2S precursor,attenuated the cerebral infarction and cerebral atrophy induced by hypoxia and ischemia and increased the expression of miR-9-5p and cystathionineβsynthase(a major H2S synthetase in the brain)in the prefrontal cortex.We also found that an miR-9-5p inhibitor blocked the expression of cystathionineβsynthase in the prefrontal cortex in mice with brain injury caused by hypoxia and ischemia.Furthermore,miR-9-5p overexpression increased cystathionine-β-synthase and H2S expression in the injured prefrontal cortex of mice with hypoxic ischemic brain injury.L-cysteine decreased the expression of CXCL11,an miR-9-5p target gene,in the prefrontal cortex of the mouse model and in lipopolysaccharide-stimulated BV-2 cells and increased the levels of proinflammatory cytokines BNIP3,FSTL1,SOCS2 and SOCS5,while treatment with an miR-9-5p inhibitor reversed these changes.These findings suggest that H2S can reduce neuroinflammation in a neonatal mouse model of hypoxic ischemic brain injury through regulating the miR-9-5p/CXCL11 axis and restoringβ-synthase expression,thereby playing a role in reducing neuroinflammation in hypoxic ischemic brain injury.