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.

Development and Assessment of Two Highly Pathogenic Avian Influenza(HPAI) H5N6 Candidate Vaccine Viruses for Pandemic Preparedness

Objective In China, 24 cases of human infection with highly pathogenic avian influenza(HPAI) H5 N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5 N6 candidate vaccine viruses(CVVs).Methods In accordance with the World Health Organization(WHO) recommendations, we constructed two reassortant viruses using reverse genetics(RG) technology to match the two different epidemic H5 N6 viruses. We performed complete genome sequencing to determine the genetic stability. We assessed the growth ability of the studied viruses in MDCK cells and conducted a hemagglutination inhibition assay to analyze their antigenicity. Pathogenicity attenuation was also evaluated in vitro and in vivo.Results The results showed that no mutations occurred in hemagglutinin or neuraminidase, and both CVVs retained their original antigenicity. The replication capacity of the two CVVs reached a level similar to that of A/Puerto Rico/8/34 in MDCK cells. The two CVVs showed low pathogenicity in vitro and in vivo, which are in line with the WHO requirements for CVVs.Conclusion We obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics,which may aid in our preparedness for a potential H5N6 pandemic.

Characterization of Highly Pathogenic Avian Influenza H5N1 Viruses Isolated from Domestic Poultry in China

The highly pathogenic avian influenza （HPAI） H5N1 virus has caused several outbreaks in domestic poultry. Despite great efforts to control the spread of this virus, it continues to evolve and poses a substantial threat to public health because of a high mortality rate. In this study, we sequenced whole genomes of eight H5N1 avian influenza viruses isolated from domestic poultry in eastern China and compared them with those of typical influenza virus strains. Phylogenetic analyses showed that all eight genomes belonged to clade 2.3.2.1 and clade 7.2, the two main circulating clades in China. Viruses that clustered in clade 2.3.2.1 shared a high degree of homology with H5N1 isolates located in eastern Asian. Isolates that clustered in clade 7.2 were found to circulate throughout China, with an east-to-west density gradient. Pathogenicity studies in mice showed that these isolates replicate in the lungs, and clade 2.3.2.1 viruses exhibit a notably higher degree of virulence compared to clade 7.2 viruses. Our results contribute to the elucidation of the biological characterization and pathogenicity of HPAI H5N1 viruses.

Development of a real-time RT-PCR method for the detection of newly emerged highly pathogenic H7N9 influenza viruses

In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China. Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin（HA） cleavage site, leading to the emergence of a highly pathogenic virus. The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza. Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus. One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established. Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity. The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs. Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples. Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.

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.

Establishment of a Risk Assessment Framework for Analysis of the Spread of Highly Pathogenic Avian Influenza

To evaluate the risk of highly pathogenic avian influenza （HPAI） in China's Mainland, a risk assessment framework was built. Risk factors were determined by analyzing the epidemic data using the brainstorming method; the analytic hierarchy process was designed to weigh risk factors, and the integrated multicriteria analysis was used to evaluate the final result. The completed framework included the risk factor system, data standards for risk factors, weights of risk factors, and integrated assessment methods. This risk assessment framework can be used to quantitatively analyze the outbreak and spread of HPAI in China's Mainland.

Assessment of the Level of Knowledge of the Nature of Highly Pathogenic Avian Influenza Demonstrated by the Nigerian Veterinary Laboratory Staff Involved in HPAI Diagnosis in Nigeria

The study was designed to evaluate the level of knowledge of Nigerian Veterinary Laboratory Staff on the nature of Highly Pathogenic Avian Influenza (HPAI) disease using structured questionnaires. The study comprised the Staff of National Veterinary Research Institute (NVRI) and five reference Veterinary Teaching Hospitals (VTH) designated for HPAI diagnosis. A total of 69 questionnaires were distributed to the laboratory staff. Questions on the general nature of the disease such as the cause, signs, mode of transmission, methods of identification, lesions, control and prevention, etc. were asked. The results showed that 77.38% of the staff answered all the questions correctly indicating their considerable knowledge of the HPAI disease. Considerable percentage of the staff listed correctly the equipment used for serology (36.23%) and RT-PCR (31.88%). Interestingly only 13.04% of the staff listed correctly the equipment used in rapid tests despite the fact that they are simpler and recommended for all P2 laboratories. In conclusion, the veterinary laboratory staff assessed demonstrated a significant level of knowledge on HPAI diagnosis;however, most of their laboratories lack the structure, organization, funds and basic facilities required for effective HPAI diagnosis.

Genetic and biological properties of H10Nx influenza viruses in China

H10 subtype avian influenza viruses(AIV)have been circulating in China for 40 years.H10 AIVs in China have expanded their host range from wild birds to domestic poultry and mammals,even human.Most of the H10 subtype AIVs reported in China were isolate from the southeast part.We isolated an H10N3 AIV,A/Chicken/Liaoning/SY1080/2021(SY1080),from live poultry market(LPM)in Liaoning Province of the Northeast China.SY1080replicated efficiently in mice lungs and nasal turbinates without prior adaptation.We systematically compared SY1080 with other H10 subtype isolates in China.Phylogenetic analysis showed that SY1080 and most of the H10strains belonged to the Eurasian lineage.H10 AIVs in China have formed 63 genotypes.SY1080 as well as the H10N3 strains from human infections belonged to G60 genotype.H10Nx AIV acquired multiple mammalian adaptive and virulence related mutations during circulation and the recent reassortants derived internal genes from chicken H9N2 AIVs.The H10Nx subtypes AIVs posed potential threat to public health.These results suggested we should strengthen the surveillance and evaluation of H10 subtype strains.

The Evidence of Clade 7.1 Avian Influenza Virus (H5N1) in Qinghai Lake

The highly pathogenic influenza A virus subtype H5N1 spread throughout Asia since 2003, reached to Europe in 2005, and the Middle East, as well as Africa and caused a global concern for a potential pandemic threat last decade. A Clade 2.3.2 H5N1 virus became dominate in the Qinghai Lake region in 2009 with sporadic mammal cases of infection and transferred to Russia and Europe through wild migratory birds. Currently, HPAI H5N1 of clades 2.3.4, 2.3.2, and 7 are the dominant co-circulating H5N1 viruses in poultry in Asia. 2.3.2 Clade is dominant in wild birds through the world whereas there is no evident data about Clade 7 circulation in wild birds. We detected HPAI H5N1 virus of Clade 7.1 in Qinghai Lake, that closely related to Shanxi-like and Vietnam viruses co-circulating in poultry. This is the first report of Clade 7.1 H5N1 in wild birds. Based on phylogenetic analyses, the virus can be originated from Clade 7.1 virus gene pool that spread in Vietnam and Chinese poultry and could spread with migratory birds to Qinghai Lake. The Qinghai Lake continues to be significant hotspot for H5N1 surveillance since the regular outbreaks occurred there in wild birds and mammals. Based on these facts and findings, the related researchers should pay more attention to the Qinghai Lake basin as significant hotspot for H5N1 avian influenza surveillance since the regular H5N1 outbreaks occurred there in wild birds with sporadic mammal cases of infection.

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.

Highly pathogenic avian influenza H5N1 Clade 2.3.2.1c virus in migratory birds,2014–2015

A novel Clade 2.3.2.1c H5N1 reassortant virus caused several outbreaks in wild birds in some regions of China from late 2014 to 2015.Based on the genetic and phylogenetic analyses,the viruses possess a stable gene constellation with a Clade 2.3.2.1c HA,a H9N2-derived PB2 gene and the other six genes of Asian H5N1-origin.The Clade 2.3.2.1c H5N1 reassortants displayed a high genetic relationship to a human H5N1 strain(A/Alberta/01/2014).Further analysis showed that similar viruses have been circulating in wild birds in China,Russia,Dubai(Western Asia),Bulgaria and Romania(Europe),as well as domestic poultry in some regions of Africa.The affected areas include the Central Asian,East Asian-Australasian,West Asian-East African,and Black Sea/Mediterranean flyways.These results show that the novel Clade 2.3.2.1c reassortant viruses are circulating worldwide and may have gained a selective advantage in migratory birds,thus posing a serious threat to wild birds and potentially humans.

Case report for human infection with a highly pathogenic avian influenza A(H5N6) virus in Beijing, China 2019

Bird infections with highly pathogenic avian influenza A(H5N6) viruses have been identified since 2014. With very limited occasion, the virus could sporadically spilled over to infect humans. It has been recognized that all human infections were within southern region of China's Mainland until the case reported here in Beijing in Aug. 2019. This was the first human case infected with highly pathogenic avian influenza A(H5N6) virus in northern China. The infection was confirmed by real-time RT-PCR assay. The whole genome sequences were obtained from clinical sample. Genetic characteristics of the virus were identified similar to those of previous avian influenza A(H5N6) viruses, retaining the main features of the avian influenza virus.

Knowledge,attitudes and practices towards avian influenza A(H5N1) among Cambodian women:A cross-sectional study

Objective:To measure highly pathogenic avian influenza(HPAI)-related knowledge,attitudes, and practices(KAPs) among Cambodian women.Methods:This cross-sectional study selected 246 married women aged between 18-55 years who had backyard poultry and lived at least one year in the areas of the survey through multi-stage cluster sampling.An average score of correct answers was generated to evaluate respondents’ knowledge(Good/Poor).attitudes(Positive/ Negative),and practices(Good/Bad).Results:We reported that about half of the respondents had good knowledge and good practices and four-fifth of them had positive attitudes towards HPAl.Odds ratios(ORs) and 95%confidence intervals(CIs) were estimated through a logistic regression model to explore contributing factors that raise their KAP levels.Most of the sources were significant in increasing knowledge of the respondent,like television(OR=l.6.95%CI=1.0- 2.7),radio(OR=2.5.95%CI=l.3-4.9).leaflets/booklets(OR=2.1,95%CI=1.2-3.9),school students (OR=18.4.95%CI=2.4-142.9).village health volunteers(OR=4.5.95%CI=2.2-10.9) etc.Factors such as television(OR=3.7.95%CI=2.1-6.4).leaflets/booklets(OR=2.6,95%CI7=1.4-5.1).and public health staff(OR=2.2,95%CI=1.2-4.1) had similar influence on practices.Although,we found similar effect on raising the attitudes of the responded,it was not significant.Conclusions: We report a satisfactory level of positive attitudes,and moderate level of knowledge and practices related to HPAI among Cambodian women.Raising KAPs through television,radio and other medias may be more efficient than using usual information,education and communication materials to prevent HPAI.

Low Pathogenic Avian Influenza A(H5N7) Virus Isolated from a Domestic Duck in Dongting Lake Wetland of China,2016

Dear Editor,The avian influenza virus (AIV) H5N7 was first isolated from wild birds in North America in 2001 (Spackman et al.2007), and information on only 25 strains of this virus has been deposited in the Global Initiative on Sharing All Influenza Data (GISAID-http://platform.gisaid.org/epi3/frontend#493de3) database until October 30, 2018. Twenty viruses were identified in the United States from 2001 to2017.

International chicken trade and increased risk for introducing or reintroducing highly pathogenic avian influenza A (H5N1) to uninfected countries

Every year billions of chickens are shipped thousands of miles around the globe in order to meet the ever increasing demands for this cheap and nutritious protein source.Unfortunately,transporting chickens internationally can also increase the chance for introducing zoonotic viruses,such as highly pathogenic avian influenza A(H5N1)to new countries.Our study used a retrospective analysis of poultry trading data from 2003 through 2011 to assess the risk of H5N1 poultry infection in an importing country.We found that the risk of infection in an importing country increased by a factor of 1.3(95%CI:1.1e1.5)for every 10-fold increase in live chickens imported from countries experiencing at least one H5N1 poultry case during that year.These results suggest that the risk in a particular country can be significantly reduced if imports from countries experiencing an outbreak are decreased during the year of infection or if biosecurity measures such as screening,vaccination,and infection control practices are increased.These findings show that limiting trade of live chickens or increasing infection control practices during contagious periods may be an important step in reducing the spread of H5N1 and other emerging avian influenza viruses.

Surveillance for highly pathogenic avian influenza in wild birds in the USA

As part of the USA’s National Strategy for Pandemic Influenza,an Interagency Strategic Plan for the Early Detection of Highly Pathogenic H5N1 Avian Influenza in Wild Migratory Birds was developed and implemented.From1April2006 through 31 March 2009,261946 samples fromwild birds and 101457 wild bird fecalsamples were collected in the USA;no highly pathogenic avian influenza was detected.The United States Department of Agriculture,and state and tribal cooperators accounted for 213115(81%)of the wild bird samples collected;31,27,21 and 21%of the samples were collected from theAtlantic,Pacific,Central and Mississippi flyways,respectively.More than 250 species of wild birds in all 50 states were sampled.The majority of wild birds(86%)were dabbling ducks,geese,swans and shorebirds.The apparent prevalence of low pathogenic avian influenza viruses during biological years 2007 and 2008 was 9.7 and 11.0%,respectively.The apparent prevalence of H5 and H7 subtypes across all species sampled were 0.5 and 0.06%,respectively.The pooled fecal samples(n=101539)positive for low pathogenic avian influenza were 4.0,6.7 and 4.7%for biological years 2006,2007 and 2008,respectively.The highly pathogenic early detection system for wild birds developed and implemented in the USA represents the largest coordinated wildlife disease surveillance system ever conducted.This effort provided evidence that wild birds in the USA were free of highly pathogenic avian influenza virus(given the expected minimum prevalence of 0.001%)at the 99.9%confidence level during the surveillance period.

Assessment of vaccination strategies against highly pathogenic avian influenza in China

Vaccination for highly pathogenic avian influenza(HPAI)has been implemented in China for a decade,however,the virus is still present in poultry.A series of recombinant vaccines,Re-1 to Re-7,have been developed and used,and Re-8 will also be used in clinical settings to prevent the prevailing flu strains.The question remains,when can China eradicate the disease?Here,we review the epidemiology of H5 HPAI along with the development,usage and problems of vaccines.Further suggestions for controlling the disease in China are provided.

基于HPAIV实验活动的实验室生物风险分析方法的建立

高致病禽流感病毒(HPAIV)的实验活动必须在生物安全三级(BSL-3)实验室开展,对HPAIV开展生物风险分析,是保证实验室生物安全工作的前提。结合相关标准和华南农业大学兽医学院的工作实际,建立了针对HPAIV实验活动的实验室生物风险分析方法。

HPAIV感染小鼠外周血白细胞表面CD54及其受体的检测

目的研究外周血白细胞表面CD54及受体CD11b／CD18在小鼠高致病性禽流感病毒（highly pathogenic avian influenza virus，HPAIV）感染中的作用。方法18～20g普通健康昆明小鼠60只，随机分为实验组和对照组，应用荧光标记的抗小鼠CD54、CD11b、CD18单抗，采用全血直接免疫荧光流式细胞术，对不同时相实验组和对照组小鼠外周血白细胞表面粘附分子（adhesion molecules，AMS）CD11b、CD18、CD54的表达含量进行测定。结果实验组小鼠外周血白细胞表面CD11b、CD18、CD54的阳性细胞百分率（PPC）和／或平均荧光强度（MFI）与对照组比较普遍上调（P〈0．05或P〈0．01）。结论AMS在HPAIV感染中发挥了重要作用。