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.

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.

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.

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

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.

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.

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.

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.

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.

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/PC0360/2022 （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 HI-HI6 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 AlVs of wild waterfowl and poultry in this region is vita for our knowledge of the ecology and mechanism of transmission to prevent an influenza pandemic.

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.

Sequence and phylogenetic analysis of hemagglutinin genes of H9N2 influenza viruses isolated from chicken in China from 2013 to 2015

H9N2 avian influenza virus（AIV） infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin（HA） gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in Gen Bank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216 L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.

Genetic Variation Analysis on the Whole Genomic Sequence of a H9N2 Subtype Avian Influenza Virus Isolate

A Objective3 This study was to understand the genetic variation characters of the H9N2 subtype avian influenza virus isolate （A/Chicken/ Hebei/WD/98, abbreviated as WD98） by comparing with other reference strains. I-Method3 Eight complete genes were amplified by RT-PCR and sequenced. The homology and genetic evolution relationship were analyzed between these sequences and that of the seven reference strains. [Result] The whole genomic sequence of WD98 strain was 91.1% -95.8% homologous to that of seven reference strains tested. This isolate shared the highest homology （95.8%） to D/HK/Y280/97 and the lowest homology （91.1% ） to C/Pak/2/99. The HA cleavage site of the WD98 strain was R-S-S-R G, and the 226th amino acid at receptor-binding site was Gin. [ Condmion] WD98 strain belongs to mildly pathogenic avian in- fluenza virus and may not infect human. The genetic relationship is the closest between A/Chicken/Hebei/wD/98 and A/duck/HongKong/Y280/ 97, both of which belong to the sub-line of A/Chicken/Beijing/1/94 in Eurasian line. And A/Chicken/Hebei/WD/98 and A/Chicken/Beijing/1/94 are genetically distant within the same sub-line.

Subtype Identification of Avian Influenza Virus on DNA Microarray

We have developed a rapid microarray-based assay for the reliable detection of H5, H7 and H9 subtypes of avian influenza virus （AIV）. The strains used in the experiment were A/Goose/Guangdong/1/96 （H5N1）, A/African starling/983/79 （H7N1） and A/Turkey/Wiscosin/1/66 （H9N2）. The capture DNAs clones which encoding approximate 500-bp avian influenza virus gene fragments obtained by RT-PCR, were spotted on a slide-bound microarray. Cy5-labeled fluorescent cDNAs, which generated from virus RNA during reverse transcription were hybridized to these capture DNAs. These capture DNAs contained multiple fragments of the hemagglutinin and matrix protein genes of AIV respectively, for subtyping and typing AIV. The arrays were scanned to determine the probe binding sites. The hybridization pattern agreed approximately with the known grid location of each target. The results show that DNA microarray technology provides a useful diagnostic method for AIV.

The Protection Efficacity of DNA Vaccine Encoding Hemagglutinin of H5 Subtype Avian Influenza Virus

The DNA vaccine pCIHA5 encoding hemagglutinin can protect SPF chicken against lethal H5N1 avian influenza virus challenge. The more characters about its protection efficacity were studied. The protective rates in 10, 40, 70, 100 and 150 μg groups immunized with pCIHA5 were 12.5 (1/8), 58.3 (7/12), 72.7 (8/11), 50.0 (6/12) and 66.7% (8/12), respectively. The protective rates in 5, 20, 35 and 50 μg groups were 145.5 (5/11), 58.3 (7/12), 58.3 (7/12) and 91.7% (11/12), respectively. The 70, 100 and 5 μg groups have virus shedding of 1/8, 2/6 and 1/5. Though the inactived oil-emulsion vaccine has high HI antibody titers and 100% protective rate, the AGP antibody could be detected after vaccination. Results show that the pCIHA5 is fit to boost by intramuscular injection. This would be useful to the study on gene engineering vaccine of avian influenza virus.

HA Gene Variation Analysis of H9N2 Sub-type Avian Influenza Virus from Three Strains in Different Times

HA Gene of H9N2. sub-type avian influenza virus from three strains in different times was amplified, purified and then sequenced. The results of its sequence analysis showed that the whole length of the amplified HA Gene was 1 683 bp, encoding 560 amino acids. The amino acid sequence of three virulent strains at cleavage site was R-S-S-R, which was low-pathogenicity strain. According to the amino acid sequence of the isolated strains, there were 7 potential glycosylation sites, and the receptor-binding site was the specific sequence of the avian-derived influenza virus. Amino acids on the left edge of receptor-binding site were all NGQQG, while amino acids on the right edge of receptor-binding site were GTSKA. From the comparative sequence analysis of HA Gene from some referenced strains, the results indicated that nucleotide and amino acid homology between isolated strains and referenced strains was higher. Evolutionary tree analysis showed that three strains were all Eurasian species, and there was a close relationship with the representative strains of A / duck / Hong Kong/Y280/97.

Immune Efficacy of a Recombinant Fowlpox Virus Co-Expressing HA and NA Genes of Avian Influenza Virus in SPF Chickens

A recombinant fowlpox virus co-expressing Haemagglutinin (HA) and Neuraminidase (NA)named as rFPV-HA-NA was produced by HA and NA gene of A/Goose/Guangdong/3/96(H5N1)isolate of avian influenza virus recombined into the genome of fowlpox virus. In thisstudy, to evaluate its ability of protecting chickens against challenge with a lethaldose of highly pathogenic isolates of avian influenza virus, eight-week-old specific-pathogenic-free (SPF) chickens were vaccinated with recombinant virus or the wildtypefowlpox virus by wing-web puncture. After challenge 4 weeks with 10 LD50 highly pathogenicavian influenza virus H5N1 and H7N1 isolate, all chickens vaccinated with recombinantvirus were protected, while the chickens vaccinated with the wildtype fowlpox virus orunvaccinated controls experienced 100% mortality respectively following challenge. Thiscomplete protection was accompanied by the high levels of specific antibody response tothe respective components of the recombinant virus.