We developed a method for accurate quantification of the intact virus particles in inactivated avian influenza virus feedstocks. To address the problem of impurities interference in the detection of inactivated avian influenza virus feedstocks by direct high performance size exclusion chromatography (HPSEC), we firstly investigated polyethylene glycol (PEG) precipitation and ion exchange chromatography (IEC) for H5N8 antigen purification. Under the optimized conditions, the removal rate of impurity was 86.87% in IEC using DEAE FF, and the viral hemagglutination recovery was 100%. HPSEC was used to analyze the pretreated samples. The peak of 8.5-10.0 min, which was the characteristic adsorption of intact virus, was analyzed by SDS-PAGE and dynamic light scattering. It was almost free of impurities and the particle size was uniform with an average particle size of 127.7 nm. After adding antibody to the IEC pretreated samples for HPSEC detection, the characteristic peak disappeared, indicating that IEC pretreatment effectively removed the impurities. By coupling HPSEC with multi-angle laser scattering technique (MALLS), the amount of intact virus particles in the sample could be accurately quantified with a good linear relationship between the number of virus particles and the chromatographic peak area (R²=0.997). The established IEC pretreatment-HPSEC-MALLS assay was applied to accurate detection of the number of intact virus particles in viral feedstocks of different subtypes (H7N9), different batches and different concentrations, all with good applicability and reproducibility, Relative standard deviation < 5%, n=3.
Animals ; Reproducibility of Results ; Influenza A Virus, H7N9 Subtype ; Influenza in Birds ; Chromatography, Gel ; Virion ; Lasers

Objective: To analyze the genetic characteristics of the first human infection with the G4 genotype of Eurasian avian H1N1 swine influenza virus (EA H1N1 SIV) in Shaanxi Province. Methods: The patient's throat swab samples were collected, and MDCK cells were inoculated for virus isolation to obtain the virus strain. The whole genome deep sequencing method was used to obtain the eight gene segments of the isolated strain. The nucleotide homology analysis was conducted through the Blast program in the GenBank database, and a phylogenetic tree was constructed to analyze the genetic characteristics of the virus. Results: The throat swab specimens of the case were confirmed as EA H1N1 SIV in the laboratory, and the isolated strain was named A/Shaanxi-Weicheng/1351/2022(H1N1v). Homology analysis found that the PB2, NP, HA, NA, and M genes of this isolate had the highest nucleotide homology with A/swing/Beijing/0301/2018 (H1N1), about 98.29%, 98.73%, 97.41%, 97.52%, and 99.08%, respectively. The phylogenetic tree showed that the isolate belonged to G4 genotype EA H1N1 SIV, with PB2, PB1, PA, NP and M genes from pdm/09 H1N1, HA and NA genes from EA H1N1, and NS gene from Triple-reassortant H1N1. The cleavage site of the HA protein was IPSIQSR↓G, which was the molecular characteristic of the low pathogenic influenza virus. No amino acid mutations associated with neuraminidase inhibitors were found in the NA protein. PB2 protein 701N mutation, PA protein P224S mutation, NP protein Q357K mutation, M protein P41A mutation, and NS protein 92D all indicated its enhanced adaptability to mammals. Conclusion: The patient is the first human infection with G4 genotype EA H1N1 SIV in Shaanxi province. The virus is low pathogenic, but its adaptability to mammals is enhanced. Therefore, it is necessary to strengthen the monitoring of such SIVs.
Swine ; Humans ; Animals ; Influenza A Virus, H1N1 Subtype/genetics* ; Phylogeny ; Genotype ; Influenza A virus ; China ; Birds ; Mammals

Objective: To analyze the genetic characteristics of the first human infection with the G4 genotype of Eurasian avian H1N1 swine influenza virus (EA H1N1 SIV) in Shaanxi Province. Methods: The patient's throat swab samples were collected, and MDCK cells were inoculated for virus isolation to obtain the virus strain. The whole genome deep sequencing method was used to obtain the eight gene segments of the isolated strain. The nucleotide homology analysis was conducted through the Blast program in the GenBank database, and a phylogenetic tree was constructed to analyze the genetic characteristics of the virus. Results: The throat swab specimens of the case were confirmed as EA H1N1 SIV in the laboratory, and the isolated strain was named A/Shaanxi-Weicheng/1351/2022(H1N1v). Homology analysis found that the PB2, NP, HA, NA, and M genes of this isolate had the highest nucleotide homology with A/swing/Beijing/0301/2018 (H1N1), about 98.29%, 98.73%, 97.41%, 97.52%, and 99.08%, respectively. The phylogenetic tree showed that the isolate belonged to G4 genotype EA H1N1 SIV, with PB2, PB1, PA, NP and M genes from pdm/09 H1N1, HA and NA genes from EA H1N1, and NS gene from Triple-reassortant H1N1. The cleavage site of the HA protein was IPSIQSR↓G, which was the molecular characteristic of the low pathogenic influenza virus. No amino acid mutations associated with neuraminidase inhibitors were found in the NA protein. PB2 protein 701N mutation, PA protein P224S mutation, NP protein Q357K mutation, M protein P41A mutation, and NS protein 92D all indicated its enhanced adaptability to mammals. Conclusion: The patient is the first human infection with G4 genotype EA H1N1 SIV in Shaanxi province. The virus is low pathogenic, but its adaptability to mammals is enhanced. Therefore, it is necessary to strengthen the monitoring of such SIVs.
Swine ; Humans ; Animals ; Influenza A Virus, H1N1 Subtype/genetics* ; Phylogeny ; Genotype ; Influenza A virus ; China ; Birds ; Mammals

Objective: To analyze the characteristics of low pathogenic H3, H4 and H6 subtypes of avian influenza viruses in environment related avian influenza viruses in China from 2014 to 2021. Methods: Surveillance sites were located in 31 provinces, autonomous region and municipalities to collect environmental samples related to avian influenza, detect the nucleic acid detection of influenza A virus, isolate virus, deeply sequence, analyze pathogenicity related molecular sites, and determine the distribution and variation characteristics of common H3, H4 and H6 subtypes of avian influenza virus in different regions, places and sample types. Results: A total of 388 645 samples were collected. The positive rate of low pathogenic H3 (0.56‰) and H6 (0.53‰) was higher than that of H4 (0.09‰). The positive rate of H4 subtype virus in live poultry market was higher than that in other places, and the difference was statistically significant. The positive rate of H3 and H6 subtypes in sewage samples was higher than that in other samples, and the difference was statistically significant. The positive rate of H3, H4 and H6 viruses in the south was higher than that in the north, and the difference was statistically significant. December was the most active time for virus. The analysis of pathogenicity related molecular sites showed that H3, H4 and H6 subtypes of viruses combined with avian influenza virus receptors, and some gene sites related to increased pathogenicity had mutations. Conclusion: The H3, H4 and H6 subtypes of low pathogenic avian influenza viruses have a high isolation positive rate in the live poultry market and sewage. The distribution of the three subtypes of viruses has obvious regional and seasonal characteristics, and the genetic characteristics still show the feature of low pathogenic avian influenza.
Humans ; Animals ; Influenza in Birds/epidemiology* ; Sewage ; Phylogeny ; Influenza A virus/genetics* ; Poultry ; China/epidemiology*

BACKGROUND: The new emerging avian influenza A H7N9 virus, causing severe human infection with a mortality rate of around 41%. This study aims to provide a novel treatment option for the prevention and control of H7N9. METHODS: H7 hemagglutinin (HA)-specific B cells were isolated from peripheral blood plasma cells of the patients previously infected by H7N9 in Jiangsu Province, China. The human monoclonal antibodies (mAbs) were generated by amplification and cloning of these HA-specific B cells. First, all human mAbs were screened for binding activity by enzyme-linked immunosorbent assay. Then, those mAbs, exhibiting potent affinity to recognize H7 HAs were further evaluated by hemagglutination-inhibiting (HAI) and microneutralization in vitro assays. Finally, the lead mAb candidate was selected and tested against the lethal challenge of the H7N9 virus using murine models. RESULTS: The mAb 6-137 was able to recognize a panel of H7 HAs with high affinity but not HA of other subtypes, including H1N1 and H3N2. The mAb 6-137 can efficiently inhibit the HA activity in the inactivated H7N9 virus and neutralize 100 tissue culture infectious dose 50 (TCID50) of H7N9 virus (influenza A/Nanjing/1/2013) in vitro, with neutralizing activity as low as 78 ng/mL. In addition, the mAb 6-137 protected the mice against the lethal challenge of H7N9 prophylactically and therapeutically. CONCLUSION The mAb 6-137 could be an effective antibody as a prophylactic or therapeutic biological treatment for the H7N9 exposure or infection.
Animals ; Antibodies, Monoclonal/therapeutic use* ; Antibodies, Neutralizing/therapeutic use* ; Antibodies, Viral ; Hemagglutinins ; Humans ; Influenza A Virus, H1N1 Subtype ; Influenza A Virus, H3N2 Subtype ; Influenza A Virus, H7N9 Subtype ; Influenza Vaccines ; Influenza in Birds ; Influenza, Human/prevention & control* ; Mice

The conserved hemagglutinin (HA) stem region of avian influenza virus (AIV) is an important target for designing broad-spectrum vaccines, therapeutic antibodies and diagnostic reagents. Previously, we obtained a monoclonal antibody (mAb) (5D3-1B5) which was reactive with the HA stem epitope (aa 428-452) of H7N9 subtype AIV. To systematically characterize the mAb, we determined the antibody titers, including the HA-binding IgG, hemagglutination-inhibition (HI) and virus neutralizing (VN) titers. In addition, the antigenic epitope recognized by the antibody as well as the sequence and structure of the antibody variable region (VR) were also determined. Moreover, we evaluated the cross-reactivity of the antibody with influenza virus strains of different subtypes. The results showed that the 5D3-1B5 antibody had undetectable HI and VN activities against H7N9 virus, whereas it exhibited strong reactivity with the HA protein. Using the peptide-based enzyme-linked immunosorbent assay and biopanning with a phage-displayed random peptide library, a motif with the core sequence (⁴³¹W-⁴³³Y-⁴³⁷L) in the C-helix domain in the HA stem was identified as the epitope recognized by 5D3-1B5. Moreover, the mAb failed to react with the mutant H7N9 virus which contains mutations in the epitope. The VR of the antibody was sequenced and the complementarity determining regions in the VR of the light and heavy chains were determined. Structural modeling and molecular docking analysis of the VR verified specific binding between the antibody and the C-helix domain of the HA stem. Notably, 5D3-1B5 showed a broad cross-reactivity with influenza virus strains of different subtypes belonging to groups 1 and 2. In conclusion, 5D3-1B5 antibody is a promising candidate in terms of the development of broad-spectrum virus diagnostic reagents and therapeutic antibodies. Our findings also provided new information for understanding the epitope characteristics of the HA protein of H7N9 subtype AIV.
Animals ; Antibodies, Monoclonal ; Antibodies, Viral ; Hemagglutinin Glycoproteins, Influenza Virus/genetics* ; Hemagglutinins ; Influenza A Virus, H7N9 Subtype ; Influenza in Birds ; Molecular Docking Simulation

The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log₂, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.
Animals ; Antibodies, Viral ; Chickens ; HN Protein/metabolism* ; Newcastle Disease/prevention & control* ; Newcastle disease virus/metabolism* ; Oryza/genetics*

Aged ; Animals ; Arcobacter/genetics* ; Chickens ; Diarrhea/microbiology* ; Drug Resistance, Bacterial/genetics* ; Genes, Bacterial ; Gram-Negative Bacterial Infections/veterinary* ; Humans ; Male ; Meat ; Microbial Sensitivity Tests ; Phylogeny ; Poultry Diseases/microbiology* ; Virulence ; Virulence Factors/genetics*

To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek's disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.
Animals ; Chick Embryo ; Chickens ; Chromosomes, Artificial, Bacterial ; DNA, Recombinant ; Herpesvirus 2, Gallid/genetics* ; Marek Disease

The avian influenza A (H7N9) virus is a zoonotic virus that is closely associated with live poultry markets. It has caused infections in humans in China since 2013. Five waves of the H7N9 influenza epidemic occurred in China between March 2013 and September 2017. H7N9 with low-pathogenicity dominated in the first four waves, whereas highly pathogenic H7N9 influenza emerged in poultry and spread to humans during the fifth wave, causing wide concern. Specialists and officials from China and other countries responded quickly, controlled the epidemic well thus far, and characterized the virus by using new technologies and surveillance tools that were made possible by their preparedness efforts. Here, we review the characteristics of the H7N9 viruses that were identified while controlling the spread of the disease. It was summarized and discussed from the perspectives of molecular epidemiology, clinical features, virulence and pathogenesis, receptor binding, T-cell responses, monoclonal antibody development, vaccine development, and disease burden. These data provide tools for minimizing the future threat of H7N9 and other emerging and re-emerging viruses, such as SARS-CoV-2.
Animals ; COVID-19 ; China/epidemiology* ; Humans ; Influenza A Virus, H7N9 Subtype ; Influenza in Birds/epidemiology* ; Influenza, Human/prevention & control* ; Poultry ; SARS-CoV-2