Strengthened Monitoring of H5 Avian Influenza Viruses in External Environment in Hubei, 2018

The contamination status of H5 avian influenza viruses and distribution of subtypes of H5N1 and H5N6 in poultry-related environment of Hubei areas were investigated.Urban and rural live poultry markets,poultry farms,intensive livestock farms and other monitoring types of 103 counties in 17 cities were selected in Hubei.Wiping samples from cage surface,wiping samples from chopping board,fecal specimens and other environmental samples were collected and tested by real-time RT-PCR using primers and probes of influenza A,avian influenza of H5,N1 and N6 from December 2017 to March 2018.The avian influenza virus positive rate was compared among different monitoring sites,samples,time and regions.Totally,7132 environmental samples were collected in 1634 monitoring points with a positive rate of 2.24%.The positive rate of H5 avian influenza virus was the highest in urban and rural live poultry markets(3.44%,x^2=61.329,P<0.05)in 6 monitoring sites and wiping samples from chopping board(5.46%,x^2=67.072,P<0.05)in 6 sample types.H5N6 avian influenza viruses were detected more in eastern than western Hubei,and H5N6 avian influenza viruses were detected only in Xiangyang city of western Hubei.There were important high-risk places of human infection with H5 avian influenza virus in urban and rural live poultry markets and the poultry slaughtering plants.H5N6 has been the predominant subtype of H5 avian influenza viruses in the eastern and western Hubei and H5N6 avian influenza viruses were still present in a few areas of Hubei.Outbreaks of human H5N1 and H5N6 avian influenza remain at risk in Hubei province.

Development of Neural Network for BLSOM Clustering of HA Genes of Avian Influenza Viruses Isolated in Guangdong Province

A neural network classification method,and a batch-learning self-organizing map(BLSOM),was established using trinucleotide and tetranucleotide in the hemagglutinin gene sequences of 25 avian influenza viruses isolated in Guangdong Province. Statistical analysis and normalization of the fragment number were done and MATLAB function was used to simulate the human brain thinking for self-organizing learning. When the number of training steps was 100 and above,the strains could be successfully clustered. H_1,H_3,H_5,H_7 and H_9 subtype strains fell within different classes,respectively,and the HA gene cluster map of H_3N_2 and H_7N_9strains was quite similar,suggesting that these strains shared the same origin; H_5N_1 strain was quite different in different years; H_1N_1 and H_9N_2 strains could be clustered into one group,indicating the natural recombinant variation in the two kinds of viruses,thereby providing a reference for high-risk strain screening and traceability.

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.

A duplex RT-PCR assay for detection of H9 subtype avian influenza viruses and infectious bronchitis viruses

H9 s ubtype avian influenza virus(AIV) and infectious bronchitis virus(IBV) are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction(RT-PCR) assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR(d RT-PCR) was established. Two primer sets target the hemagglutinin(HA) gene of H9 AIV and the nucleocapsid(N) gene of IBV, respectively. Spec ific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the d RT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×10~1, 1.5×10~1 and 1.5×10~1 50% egg infective doses(EID_(50)) m L^(–1), respectively. The concordance rates between the d RT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the d RT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and survei llance of H9 AIVs and IBVs.

Phylogenetic and Molecular Analysis of an H7N7 Avian Influenza Virus Isolated in East Dongting Lake in 2012

Objective In March 2012, an H7N7 subtype avian influenza virus(AIV) named A/wild goose/Dongting/PC0360/2012(H7N7)(DT/PC0360) was recovered from a wild goose in East Dongting Lake. We performed whole-genome sequencing of the isolate, and analyzed the phylogenetic and molecular characterization. Methods RNA was extracted from environment samples(including fecal samples from wild bird or domestic ducks, and water samples) for detecting the presence of Influenza A Virus targeting Matrix gene, using realtime RT-PCR assay. The positive samples were performed virus isolation with embryonated eggs. The subtype of the isolates were identified by RT-PCR assay with the H1-H16 and N1-N9 primer set. The whole-genome sequencing of isolates were performed. Phylogenetic and molecular characterizations of the eight genes of the isolates were analyzed. Results Our results suggested that all the eight gene segments of DT/PC0360 belonged to the Eurasian gene pool, and the HA gene were belonged to distinct sublineage with H7N9 AIV which caused outbreaks in China's Mainland in 2013. The hemagglutinin cleavage site of HA of DT/PC0360 showed characterization of low pathogenic avian influenza virus. Conclusion Strengthening the surveillance of AIVs of wild waterfowl and poultry in this region is vital for our knowledge of the ecology and mechanism of transmission to prevent an influenza pandemic.

Detection of avian influenza virus H9N_(2) based on self-driving and self-sensing microcantilever piezoelectric sensor

In this article, we used the self-excitation and self-inductance characteristics of polyvinylidene fluoride(PVDF) piezoelectric materials, combined with the powerful signal processing and calculation analysis capabilities of integrated circuits, for the first time to explore a set of microcantilever sensor "readout system" without additional driver(self-driving) and can realize self-sensing external signal(self-sensing).It was successfully applied to the unlabeled detection of avian influenza virus(AIV) H9N_(2). The specific force of the antigen-antibody complexes on the surface of the microcantilever leads to the change of the stress of the cantilever, which drives the constructed detection device, and does not require an additional excitation source to drive it, that is, the self-driving part. At the same time, due to the movement of piezoelectric charges in the film caused by the positive piezoelectric effect of the PVDF film, self-inductive charges are generated on the surface of the sensor dielectric. The charge signal is converted into a voltage signal, and the sensing part is completed, that is, self-sensing. The immunosensor has a linear range of100-1000 ng/m L with a detection limit of 2.9 ng/m L. The method will also open up a new avenue for the detection of other analytes based on antigen-antibody responses.

Genetic and biological characteristics of the globally circulating H5N8 avian influenza viruses and the protective efficacy offered by the poultry vaccine currently used in China

The H5N8 avian influenza viruses have been widely circulating in wild birds and are responsible for the loss of over 33 million domestic poultry in Europe, Russia, Middle East, and Asia since January 2020. To monitor the invasion and spread of the H5N8 virus in China, we performed active surveillance by analyzing 317 wild bird samples and swab samples collected from 41,172 poultry all over the country. We isolated 22 H5N8 viruses from wild birds and 14 H5N8 viruses from waterfowls. Genetic analysis indicated that the 36 viruses formed two different genotypes: one genotype viruses were widely detected from different wild birds and domestic waterfowls;the other genotype was isolated from a whopper swan. We further revealed the origin and spatiotemporal spread of these two distinct H5N8 virus genotypes in 2020 and 2021. Animal studies indicated that the H5N8 isolates are highly pathogenic to chickens, mildly pathogenic in ducks, but have distinct pathotypes in mice. Moreover, we found that vaccinated poultry in China could be completely protected against H5N8 virus challenge. Given that the H5N8 viruses are likely to continue to spread in wild birds, vaccination of poultry is highly recommended in high-risk countries to prevent H5N8 avian influenza.

Endemicity of H9N2 and H5N1 avian influenza viruses in poultry in China poses a serious threat to poultry industry and public health

The H9N2 and H5N1 avian in fluenza viruses(AIVs) have been circulating in poultry in China and become endemic since 1998 and 2004, respectively.Currently, they are prevalent in poultry throughout China. This endemicity makes them actively involved in the emergence of the novel lineages of other subtypes of in fluenza viruses, such as the well-known viruses of the highly pathogenic avian in fluenza(HPAI) H5N2 and the2013 novel H7N7, H7N9 and H10N8 subtypes, thereby threatening both the poultry industry and public health.Here, we will review brie fly the prevalence and evolution,pathogenicity, transmission, and disease control of these two subtypes and also discuss the possibility of emergence of potentially virulent and highly transmissible AIVs to humans.

LabVIEW-based impedance biosensing system for detection of avian influenza virus

In order to detect the multiple avian influenza viruses(AIVs)rapidly,specifically and sensitively,a LabVIEW and microelectrode array-based impedance biosensor was developed and demonstrated.A laptop with LabVIEW software was used to generate excitation signals at different frequencies with an audio card and measure the impedance of target viruses through a data acquisition card.The audio card of the laptop was used as a function generator,while a data acquisition card was used for data communication.A virtual instrument was programmed with LabVIEW to provide a platform for impedance measurement,data processing,and control.Six interdigitated microelectrodes were placed at the bottom of six wells on a microplate to form six sensors for different AIVs and controls.Then,AIV specific ligands were immobilized on the microelectrode surface to capture target viruses.To enhance the sensitivity,AIV specific aptamers conjugated gold nanoparticles and thiocyanuric acid were employed to form a network structure and used as an amplifier.Results of the measured impedance were compared with a commercial IM6 impedance analyzer,and the error was less than 5%.The developed biosensor was portable with the sensitivity and specificity for applications to on-site or in-field rapid screening of avian influenza viruses.

Analysis of human infectious avian influenza virus:Hemagglutinin genetic characteristics in Asia and Africa from 2004 to 2009

In the present study,we used nucleotide and protein sequences of avian influenza virus H5N1,which were obtained in Asia and Africa,analyzed HA proteins using ClustalX1.83 and MEGA4.0,and built a genetic evolutionary tree of HA nucleotides.The analysis revealed that the receptor specificity amino acid of A/HK/213/2003,A/Turkey/65596/2006 and etc mutated into QNG,which could bind withá-2,3 galactose andá-2,6 galactose.A mutation might thus take place and lead to an outbreak of human infections of avian influenza virus.The mutations of HA protein amino acids from 2004 to 2009 coincided with human infections provided by the World Health Organization,indicating a“low–high–highest–high–low”pattern.We also found out that virus strains in Asia are from different origins:strains from Southeast Asia and East Asia are of the same origin,whereas those from West Asia,South Asia and Africa descend from one ancestor.The composition of the phylogenetic tree and mutations of key site amino acids in HA proteins reflected the fact that the majority of strains are regional and long term,and virus diffusions exist between China,Laos,Malaysia,Indonesia,Azerbaijan,Turkey and Iraq.We would advise that pertinent vaccines be developed and due attention be paid to the spread of viruses between neighboring countries and the dangers of virus mutation and evolution.

The challenges of avian influenza virus:mechanism,epidemiology and control

Early 2009, eight human infection cases of H5N1 highly pathogenic avian influenza (HPAI) virus, with 5 death cases, were reported in China. This again made the world alert on a possible pandemic worldwide, probably caused

Two special topics on the avian influenza virus and on epigenetics,have drawn much attention

Several excellent well-organized reviews and research papers on two special topics, "The challenges of avian influenza virus: mechanism, epidemiology, and control" and "Molecular

Conserved peptides enhance immune efficiency of inactive vaccines against emerging avian influenza viruses in chicken

Avian influenza viruses(AIVs) such as H5N1 and H7N9 are a great threat to poultry economics and public health. Vaccination can effectively inhibit the spread of AIV in poultry, which is also a viable strategy for controlling virus transmission from poultry to human. Adjuvants that are commonly used in current inactivated vaccines to provide stronger anti-AIV immune responses are often limited in their capacity to quantitatively induce both humoral and cellular immune responses. Herein, we assessed the levels of immune responses generated by a vaccine formulation comprising inactivated H5N1 antigen and synthetic peptides covering conserved CD4^+, CD8^+ T cell, and B cell epitopes. We found that the synthetic peptides enhanced the antibody responses against conserved influenza virus antigen M2 e. Notably, the hemagglutination inhibition test results indicated that the peptides significantly augmented the antibody responses of inactivated H5N1 antigen even in the 1/10 or 1/5 dose group, in the identical antibody level as antigen alone used at the full dose. This indicates that the peptide can significantly reduce the use of inactivated virus, lowering the cost of the vaccine. Moreover, the peptides increased the transcript levels of interleukin-4 and interferon-γcytokines in chicken peripheral blood mononuclear cells, which may facilitate both humoral and cellular immune responses. Our data suggest that this peptide combined with inactivated H5N1 antigen enhances both the humoral and cellular immune responses,which may benefit the prediction and design of synthetic peptide-based adjuvants for vaccines in chicken.

Bayesian evolutionary analysis for emerging infectious disease:an exemplified application for H7N9 avian influenza viruses

Dear Editor,Ever since the first human infection with H7N9 avian influenza virus(AIV)was reported in China in March 2013,there have been five H7N9 AIV pandemics in humans.Wave5 began earlier than the previous four waves,spread to more districts and counties in affected provinces,and had more confirmed cases(Wang et al.,2017).Human infections

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.

Risk factors for avian influenza virus in backyard poultry flocks and environments in Zhejiang Province,China:a crosssectional study

Background:Human infection of avian influenza virus(AIV)remains a great concern.Although live poultry markets are believed to be associated with human infections,ever more infections have been reported in rural areas with backyard poultry,especially in the fifth epidemic of H7N9.However,limited information is available on backyard poultry infection and surrounding environmental contamination.Methods:Two surveillance systems and a field survey were used to collect data and samples in Zhejiang Province.In total,4538 samples were collected by surveillance systems and 3171 from the field survey between May 2015 and May 2017,while 352 backyard poultry owners were interviewed in May 2017 by questionnaire to investigate factors influencing the prevalence of avian influenza A virus and other AIV subtypes.RT-PCR was used to test the nucleic acids of viruses.ArcGIS 10.1 software was used to generate maps.Univariate and logistic regression analyses were conducted to identify risk factors for AIV infection.Results:Of the 428 poultry premises observed by the surveillance system,53(12.38%)were positive for influenza A virus.Of the 352 samples from poultry premises observed by field survey,13(3.39%)were positive for influenza A virus.The prevalence of AIV was unevenly distributed and the dominant subtype differed among cities.Eastern(Shaoxing and Ningbo)and southern(Wenzhou)cities exhibited a higher prevalence of AIV(16.33,8.94,and 7.30%respectively).Contamination of AIV subtypes was most severe in January,especially in 2016(23.26%,70/301).The positive rate of subtype H5/H7/H9 was 2.53%(115/4538).Subtype H5 was the least prevalent,while subtypes H7 and H9 had similar positivity rates(1.50 and 1.32%respectively).Poultry flocks and environmental samples had a similar prevalence of AIV(4.46%vs 5.06%).The type of live birds was a risk factor and the sanitary condition of the setting was a protective factor against influenza A contamination.Conclusions:AIV subtypes were prevalent in backyard poultry flocks and surrounding environments in Zhejiang Province.The types of live birds and sanitary conditions of the environment were associated with influenza A contamination.These findings shine a light on the characteristics of contamination of AIV subtypes and emphasize the importance of reducing AIV circulation in backyard poultry settings.

Distribution of avian influenza viruses according to environmental surveillanee during 2014-2018, China

Background:Recurrent infections of animal hosts with avian influenza viruses(AIVs)have posted a persistent threat.It is very important to understand the avian influenza virus distribution and characteristics in environment associated with poultry and wild bird.The aim of this study was to analyze the geographic and seasonal distributions of AIVs in the 31 provinces,municipalities and autonomous region(PMA)of China,compare the AIVs prevalence in different col-lecting sites and sampling types,analyze the diversity of AIVs subtypes in environment.

Genomic characterizations of H4 subtype avian influenza viruses from live poultry markets in Sichuan province of China, 2014–2015

Dear Editor,Avian influenza viruses (AIVs) have posed a serious threat to poultry production and public health. To date, more than fourteen AIV subtypes that are able to infect human beings have been documented. Also, it is suggested that new subtypes may be reported in the future, owing to the migration of wild birds and live poultry transportation (Gao, 2018).Poultry may act as a potential incubator for novel subtypes of avian influenza virus (Bi et al., 2016a; Bi et al., 2016b; Liu et al., 2014a; Su et al., 2017). Up to date, the H7N9 AIV emerged in February 2013 has caused 1,567 human cases,with a fatality rate of 39.2%(http://www.who.int/influenza/

A new and simple method for count and identification of avian influenza viruses in water

To detect and enrich avian influenza viruses from environmental water samples, a modified VIRADEN (viruses’ adsorption-enumeration) method was established. Avian

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.