Control of highly pathogenic avian influenza through vaccination

The stamping-out strategy has been used to control highly pathogenic avian influenza viruses in many countries,driven by the belief that vaccination would not be successful against such viruses and fears that avian influenza virus in vaccinated birds would evolve more rapidly and pose a greater risk to humans.In this review,we summarize the successes in controlling highly pathogenic avian influenza in China and make suggestions regarding the requirements for vaccine selection and effectiveness.In addition,we present evidence that vaccination of poultry not only eliminates human infection with avian influenza virus,but also significantly reduces and abolishes some harmful characteristics of avian influenza virus.

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.

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.

Establishment of a humanized ST6GAL1 mouse model for influenza research

Background:This study aimed to construct and characterize a humanized influenza mouse model expressing hST6GAL1.Methods:Humanized fragments,consisting of the endothelial cell-specific K18 promoter,human ST6GAL1-encoding gene,and luciferase gene,were microinjected into the fertilized eggs of mice.The manipulated embryos were transferred into the oviducts of pseudopregnant female mice.The offspring were identified using PCR.Mice exhibiting elevated expression of the hST6GAL1 gene were selectively bred for propagation,and in vivo analysis was performed for screening.Expression of the humanized gene was tested by performing immunohistochemical(IHC)analysis.Hematologic and biochemical analyses using the whole blood and serum of humanized hST6GAL1 mice were performed.Results:Successful integration of the human ST6GAL1 gene into the mouse genome led to the overexpression of human SiaT ST6GAL1.Seven mice were identified as carrying copies of the humanized gene,and the in vivo analysis indicated that hST6GAL1gene expression in positive mice mirrored influenza virus infection characteristics.The IHC results revealed that hST6GAL1 was expressed in the lungs of humanized mice.Moreover,the hematologic and biochemical parameters of the positive mice were within the normal range.Conclusion:A humanized influenza mouse model expressing the hST6GAL1 gene was successfully established and characterized.

Risk assessment on the epidemics of human infection with a novel avian influenza A (H7N9) virus in Jiangsu Province, China

A novel avian influenza A （H7N9） virus was discovered in February 2013 in China and has resulted in more than 100 comfirmed human infections including 26 fatal cases as of May 2, 2013. The situation raises many ur- gent questions and global public health concerns. In this study, epidemiologic characteristics of infected human cases in Jiangsu province were analyzed and risk assessment was undertaken based on the information available. Briefly, it is highly unlikely that a pandemic of human infection with avian influenza A （HTN9） virus will happen in Jiangsu Province in the near future. Iia the end, some measures are recommended to prevent the situation from becoming worse.

Distribution of Avian Influenza A Viruses in Poultry-Related Environment and Its Association with Human Infection in Henan, 2016 to 2017

Objective To survey avian influenza A viruses(AIVs) in the environment and explore the reasons for the surge in human H7 N9 cases.Methods A total of 1,045 samples were collected from routine surveillance on poultry-related environments and 307 samples from human H7 N9 cases-exposed environments in Henan from 2016 to2017. The nucleic acids of influenza A(Flu A), H5, H7, and H9 subtypes were detected by real-time polymerase chain reaction.Results A total of 27 H7 N9 cases were confirmed in Henan from 2016 to 2017, 24 had a history of live poultry exposure, and 15 had H7 N9 virus detected in the related live poultry markets(LPMs). About 96%(264/275) Flu A positive-environmental samples were from LPMs. H9 was the main AIV subtype(10.05%) from routine surveillance sites with only 1 H7-positive sample, whereas 21.17% samples were H7-positive in H7 N9 cases-exposed environments. Samples from H7 N9 cases-exposed LPMs(47.56%)had much higher AIVs positive rates than those from routine surveillance sites(12.34%). The H7+H9 combination of mixed infection was 78.18%(43/55) of H7-positive samples and 41.34%(43/104) of H9-positive samples.Conclusion The contamination status of AIVs in poultry-related environments is closely associated with the incidence of human infection caused by AIVs. Therefore, systematic surveillance of AIVs in LPMs in China is essential for the detection of novel reassortant viruses and their potential for interspecies transmission.

Pathogenesis and Immunogenicity of an Avian H9N2 Influenza Virus Isolated from Human

Objective To investigate the pathogenesis and immunogenicity of H9N2 influenza virus A/Guangzhou/333/99 （a reassortant of G1 and G9 viruses isolated from a female patient in 1999） in a mouse model of infection.Methods Mice were infected with increasing virus titers.Viral load in the lungs and trachea was determined by EID50 assay.Pulmonary histopathology was assessed by hematoxylin‐eosin staining.Anti‐HI antibody titers and T‐cell responses to viral HA were determined by ELISPOT and confirmed by flow cytometry.Results Mice presented a mild syndrome after intranasal infection with A/Guangzhou/333/99 （H9N2） influenza virus.Virus was detected in the trachea and lungs of mice harvested on days 3,6,and 9 post‐infection.A T‐cell response to viral HA was detected on day 6 and H9 HA‐specific CD 4＋ T‐cells predominated.Seroconversion was detected after 14 days and antibody persisted for at least 28 weeks.Conclusion Our results suggest that H9N2 （A/Guangzhou/333/99） can replicate in the murine respiratory tract without prior adaptation,and both humoral and cell‐mediated immunity play an important role in the immune response.

Effects of closing and reopening live poultry markets on the epidemic of human infection with avian influenza A virus

Live poultry markets（LPMs） are crucial places for human infection of influenza A（H7N9 virus）.In Yangtze River Delta,LPMs were closed after the outbreak of human infection with avian influenza A（H7N9） virus,and then reopened when no case was found.Our purpose was to quantify the effect of LPMs＇ operations in this region on the transmission of influenza A（H7N9） virus.We obtained information about dates of symptom onset and locations for all human influenza A（H7N9） cases reported from Shanghai,Jiangsu and Zhejiang provinces by May 31,2014,and acquired dates of closures and reopening of LPMs from official media.A two-phase Bayesian model was fitted by Markov Chain Monte Carlo methods to process the spatial and temporal influence of human cases.A total of 235 cases of influenza A（H7N9） were confirmed in Shanghai,Jiangsu and Zhejiang by May 31,2014.Using these data,our analysis showed that,after LPM closures,the influenza A（H7N9） outbreak disappeared within two weeks in Shanghai,one week in Jiangsu,and one week in Zhejiang,respectively.Local authorities reopened LPMs when there was no outbreak of influenza A（H7N9）,which did not lead to reemergence of human influenza A（H7N9）.LPM closures were effective in controlling the H7N9 outbreak.Reopening of LPM in summer did not increase the risk of human infection with H7N9.Our findings showed that LPMs should be closed immediately in areas where the H7N9 virus is confirmed in LPM.When there is no outbreak of H7N9 virus,LPMs can be reopened to satisfy the Chinese traditional culture of buying live poultry.In the long term,local authorities should take a cautious attitude in permanent LPM closure.

H7N9 avian influenza with first manifestation of occipital neuralgia:A case report

BACKGROUND Most of the first symptoms of avian influenza are respiratory symptoms,and cases with occipital neuralgia as the first manifestation are rarely reported.CASE SUMMARY A middle-aged patient complaining of paroxysmal pain behind the ear was admitted to our hospital.The patient’s condition changed rapidly,and high fever,unexpected respiratory failure,and multiple organ failure developed rapidly.The patient was diagnosed with H7N9 avian influenza based on etiology.CONCLUSION We believe that the etiology of occipital neuralgia is complex and could be the earliest manifestation of severe diseases.The possibility of an infectious disease should be considered when occipital neuralgia is accompanied by fever.Avian influenza is one of these causative agents.

Development and Characterization of Monoclonal Antibody Specific to Nuclear Protein of Avian Influenza Virus Type A

Five monoclonal antibodies（Mabs） to nuclear protein of avain influenza virus（AIV） were developed by syncretizing SP 2/0 and the spleen cells from BALB of mice immuized with H9 subtype AIV. Specificity of these Mabs were identified by immunofluorescent assay（IFA） and enzyme linked immunosorbent assay （ELISA）. These five Mabs which were named as AIV-NP-2C3, AIV-NP-6A5, AIV-NP-3 H9, AIV-NP-7B4, AIV-NP-2H4 could react with all viruses of AIV-H9 strains in tests. The result of Western blotting showed that only the 60 ku protein antigen of AIV-H9 could be recognized by the Mabs but never recognized by New castle disease virus, REV and infectious bursa disease virus. The result of preliminary application showed that avian influenza viruses could be deetected bv Mabs in IFA and ELISA. All these Mabs will probably play important roles in preventing and monitoring avian influenza viruses.

Designing Primers for H5 and H7 Subtypes of Avian Influenza Virus and Multiplex RT-PCR Amplification

[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.

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.

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.

候鸟迁徙与禽流感(Avian Influenza,AI)传播

2003年以来,亚洲许多国家接连爆发了高致病性禽流感,对家禽养殖业已造成了巨大损失.通过分析禽流感和禽流感疫情暴发的季节性和区域性特点与鸟类迁徙特点的关系.我们认为鸟类迁徙在禽流感的传播中起重要作用.

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.

Avian influenza and gut microbiome in poultry and humans:A“One Health”perspective

A gradual increase in avian influenza outbreaks has been found in recent years.It is highly possible to trigger the next human pandemic due to the characteristics of antigenic drift and antigenic shift in avian influenza virus(AIV).Although great improvements in understanding influenza viruses and the associated diseases have been unraveled,our knowledge of how these viruses impact the gut microbiome of both poultry and humans,as well as the underlying mechanisms,is still improving.The“One Health”approach shows better vitality in monitoring and mitigating the risk of avian influenza,which requires a multi-sectoral effort and highlights the interconnection of human health with environmental sustainability and animal health.Therefore,monitoring the gut microbiome may serve as a sentinel for protecting the common health of the environment,animals,and humans.This review summarizes the interactions between AIV infection and the gut microbiome of poultry and humans and their potential mechanisms.With the presented suggestions,we hope to address the current major challenges in the surveillance and prevention of microbiome-related avian influenza with the“One Health”approach.

The role of wild goose(Anser) populations of Russia and theTibet Plateau in the spread of the avian influenza virus

Wild birds of the orders Anseriformes and Charadriiformes represent a natural reservoir of low pathogenic avian influenza(LPAI) viruses(family Orthomyxoviridae).Wild geese(order Anseriformes)relating to waterfowls undertake extensive migration flights reaching thousands of kilometers.Isolation of the avian influenza virus(AIV) from wild geese is quite low or absent.The aims of this study are to monitor the AIV in different wild goose species,nesting on Russian territory and the Tibet Plateau and to analyze the derived data for the purpose of determining the role of these wild bird species in spreading pathogens.In our study 3245 samples from nine wild goose species in nine regions of Russia and on the territory of the Tibet Plateau(the Xizang Autonomous Region) were tested and no AIV were detected.Our study shows the non-essential role of wild geese in the spread of the AIV over long distances and reaches theconclusion that geese are probably not natural reservoirs for the primary viruses.However,further inquiry of AIV in wild goose populations is required.Studies of wild geese and AIV ecology will allow us to obtainmore information about pathogen-host relationships and to make arrangements for the maintenance ofwild goose populations.

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.

Astragalus polysaccharide enhances immunity and inhibits H9N2 avian influenza virus in vitro and in vivo

This study investigated the humoral immunization of Astragalus polysaccharide （APS） against HgN2 avian influenza virus （H9N2 AIV） infection in chickens. The effects of APS treatment on H9N2 infection was evaluated by an Mqq- [3（4, 5-dimethylthiazol-2-yl）-2, 3-diphenyl tetrazolium bromide] assay and analysis of MFIC and cytokine mRNA expression. The effect on lymphocyte and serum antibody titers in vivo was also investigated. IL-4, IL-6, IL-10, LITAF, IL-12 and antibody titers to H9N2 AIV wet enhanced in the first week after APS treatment. The results indicated that APS treatment reduces H9N2 AIV replication and promotes early humoral immune responses in young chickens.This study investigated the humoral immunization of Astragalus polysaccharide （APS） against HgN2 avian influenza virus （H9N2 AIV） infection in chickens. The effects of APS treatment on HgN2 infection was evaluated by an M]q- [3（4, 5-dimethylthiazol-2-yl）-2, 3-diphenyl tetrazolium bromide] assay and analysis of MHC and cytokine mRNA expression. The effect on lymphocyte and serum antibody titers in vivo was also investigated. IL-4, IL-6, IL-10, LITAF, IL-12 and antibody titers to PIgN2 AIV were enhanced in the first week after APS treatment. The results indicated that APS treatment reduces HgN2 AIV replication and promotes early humoral immune responses in young chickens.

Development of a reverse-transcription loop-mediated isothermal amplification assay to detect avian influenza viruses in clinical specimens

In recent years,the avian influenza has brought not only serious economic loss to the poultry industry in China but also a serious threat to human health because of the avian influenza virus(AIV) gene recombination and reassortment.Until now,traditional RT-PCR,fluorescence RT-PCR and virus isolation identification have been developed and utilized to detect AIV,but these methods require high-level instruments and experimental conditions,not suitable for the rapid detection in field and farms.In order to develop a rapid,sensitive and practical method to detect and identify AIV subtypes,4 specific primers to the conserved region of AIV M gene were designed and a loop-mediated isothermal amplification(RT-LAMP) method was established.Using this method,the M gene of H1–H16 subtypes of AIV were amplified in 30 min with a water bath and all 16 H subtypes of AIV were able to be visually identified in presence of fluorescein,without cross reaction with other susceptible avian viruses.In addition,the detection limit of the common H1,H5,H7,and H9 AIV subtypes with the RT-LAMP method was 0.1 PFU(plaque-forming unit),which was 10 times more sensitive than that using the routine RT-PCR.Further comparative tests found that the positivity rate of RT-LAMP on detecting clinical samples was 4.18%(14/335) comparing with 3.58%(12/335) from real-time RT-PCR.All these results suggested that the RT-LAMP method can specifically detect and identify AIV with high sensitivity and can be considered as a fast,convenient and practical method for the clinic test and epidemiological investigation of AIV.