Background: influenza viruses cause Avian influenza (AI) is a serious infectious disease belonging to type A Orthomyxovirus. A viral RNA synthesis is due to an interaction of the nucleoprotein (NP) with the viral polymerase. In the present study, we have evaluated the immunogenicity of Avian influenza virus nucleoprotein. Materials & Methods: An influenza virus N9H2 subtype A/Chicken Iran/259/2014 was selected. In order to perform electrophoresis and purification of nucleoprotein, the protein concentration of the samples was determined. SDS-PAGE and Native-PAGE electrophoresis on polyacrylamide gel were done and differences in gel bands in two methods were compared. Purification of the virus nucleoprotein performed by electroelution method and purified nucleoprotein were assayed by ELISA to obtain the produced antibody titers. Ouchterlony and western blot tests were used for final approval. Results: By determining the molecular weight of each polypeptides, the molecular weights of H9N2 proteins were ranged from 30 to 140kDa and the molecular weight of the nucleoprotein was 60kDa. The nucleoprotein was purified by electroelution method. Ouchterlony showed that in serum dilution of 1: 2 and 1: 4, the sediment lines were formed between the serum and the NP antigen. The NP-ELISA enables rapid serological diagnosis in 1: 20. Finally, the western blot test confirmed the 60kDa nucleoprotein band. Conclusion: The nucleoprotein which was purified by electroelution retained its antigenic property and it could be applied in diagnostic kits.

The humoral immunization potential of panax ginseng polysaccharide (GPS) against H9N2 Avian influenza virus (H9N2 AIV) in chickens was investigated. The effects of GPS treatment before and during H9N2 AIV infection were determined in chicken embryo fibroblast (CEF) by MTT (3 (4, 5-dimethylthiazol-2-yl) -2, 3-diphenyl tetrazolium bromide) assays and quantitative RT-PCR analysis of MHC and cytokine expression. The percentages of CD3+, CD4+and CD8+T cells in peripheral blood lymphocytes and serum antibody titers were examined in vivo. High expressions of MHCII and the cytokines IL-2, IL-4, and IL-10 were observed in CEF treated with GPS before and during H9N2 infection. These results indicated that the antiviral activity of GPS was enhanced by pretreatment of CEF and that GPS promotes early humoral immune responses in young chickens.

Avian influenza virus (AIV) is classified in the genus of influenza virus A within the Orthomyxoviridae family. The aim of this study was the cloning and expression of NSI gene belongs to an Avian influenza virus isolate (AiChicken/Iran/AH-l/06), serotype H9N2 from a broiler chicken flock in Ahvaz, with respiratory signs and mortality. The NS 1 protein is used for the DIVA test (Differentiating Infected from Vaccinated Animals) in the form of an ELISA test. For this purpose several nucleotide sequence of the NS 1 coding region (from nucleotide 27 to 680) of Avian influenza virus subtype H9N2 retrieved extracted from the Gene bank in order to design the required primers. The NS 1 gene was subsequently amplified by RT - PCR. The vector, pMAL-c2X and the amplified NSI gene, both were digested by EcoRI and Pstl restriction endonucleases and ligated by T4 DNA ligase. The construct was then transformed into BL strain of E. coli and finally the purified recombinant vector was used for expression of NS 1 protein. The 66.4 KDa protein was identified by both SDS-PAGE and Western blotting. Observation of expressed protein with the expected molecular weight in SDS-PAGE and a positive reaction with Avian influenza virus H9N2 infected chicks sera by Western blotting revealed a successful expression of the segment ofNSl gene in Eicoli.

In August 1998, the Avian influenza (AI) outbreak appeared in one of the layer farms in Tehran province. Birds on the affected farm exhibited respiratory infection and reduction of egg production with very low mortality. An agar gel precipitation (AGP) test using AN antiserum revealed the presence of the virus in the samples from the affected birds. The sera collected from the surviving birds of the affected flocks also showed the presence of antibodies against AIV by AGP test. An Avian influenza virus H9N2 subtype was isolated in embryonated eggs from the tracheal and clocal swabs as well as pooled visceral organs of the affected birds and coded Alchicken/Iran/259/1998/H9N2.Virulence testing of the low passage virus was undertaken at the time of the outbreak in specific pathogen free (SPF) chickens. The experimentally infected chickens showed significant clinical signs and recovered gradually without any death. The isolated virus was characterized as nHPAI on the basis of pathotyping studies in SPF chickens. It is the first report on isolation of AIV from the chicken flocks in Iran.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the rapid and efficient large scale screening of antibodies to Avian influenza virus (AIV) infection in chicken. Antigen was a whole-purified influenza virus produced from the H9N2 subtype. Optimum dilution for goat anti-chicken conjugate to be used in the ELISA was 1:1000, as determined by signal-to-noise ratio. The antigen concentration was 0.375μg of protein per well, as determined by checkerboard titration. The sensitivity of the ELISA was compared with hemmaglutination inhibition test under field exposure. After testing of 656 field sera, the correlation coefficient for the results of two tests was significant (r=0.929, P<0.001). Testing 8 standard antisera of various subtypes (H1, H2, H4, H5, H6, H7, H9 and H10) of AIV and AIV antibody positive and negative sera determined specificity of the ELISA. Antisera to all 8-hemmaglutinin subtypes were strongly positive. Both the sensitivity and specificity of the ELISA was compared to the other test. Thus, the ELISA was able to detect specific AIV antibodies and suitable for screening large numbers of samples in diagnostic laboratories.  

Background: In recent years, various poultry diseases have posed risks to this industry. Respiratory and infectious diseases are the most common diseases. According to previous findings, influenza disease is recognized as a significant life-threatening disease in the industry of poultry worldwide. influenza virus type A, which belongs to the family Orthomyxoviridae, is responsible for a serious infectious disease. H9N2 AVI subtype circulates in the poultry worldwide, causing significant economic losses and infections in humans, also domestic and wild animals. Objectives: The purpose of the present experimental research was monoclonal antibodies (MAbs) production in contradiction of the NP of Avian influenza virus (AIV) H9N2 subtype to detect AIV antigens and antibodies in the Department of Proteomics and Biochemistry, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran. in 2017. Methods: The conserved protein of NP from H9N2A/Chicken/Iran/259/2014 virus, with 60 kDa molecular weight, was isolated using the electroelution method. The purified protein was applied for Australian BALB/c mice immunization. After evaluating immunization by ELISA assay, the spleen of immunized mice was isolated and hybridized with SP2/0 myeloma cell. Next, the hybridized cell was cultured, and clone soups were collected after 15 days to examine antibodies via ELISA assay. The produced antibodies using Western blotting and antibody isotype kits were characterized. Results: Thirty antibody-producing clones were examined for reactivity against Nucleoprotein (NP), the antigen, at 1  g/mL concentration. According to the ELISA assay of antibody titers, two (3/F10 and 2/D7) out of 30 antibodies were bound to the antigen with titer 0. 863 and 1. 641, respectively. Two hybrid clones, 2D7 and 3F10, which produced anti-NP antibodies, were isolated and cultured. Characterizing of the produced antibodies usingWestern blotting was performed using H9N2 virus; finally, two clone soups (3/F10 and 2/D7) reacted with the NP virus protein. According to the isotyping of antibodies produced by 3F10 and 2D7 clones, 3F10 clone produced IgG1 with a  chain, and IgG1 concentration was 1. 997, based on ELISA assay. Also, 2D7 clone produced IgG2a with a  chain, and IgG2a concentration was obtained 1. 951. Conclusions: According to the findings of our study, the produced antibody might be used in the diagnosis of influenza.

Background: Avian influenza virus (AIV) belongs to the family of Orthomyxoviruses typeAandcauses Avian influenza (AI) infectious disease. Currently, serological diagnostic techniques such as agar gel propagation (AGP), hemagglutination inhibition, and enzymelinked immunosorbent assay (ELISA) are considered as important tools for the antibodies detection against viral antigens. Due to antigenic variation in the surface of AIV glycoproteins (hemagglutinin and neuraminidase), these proteins cannot be used in serological tests. Development of assays to detect AI surface glycoproteins is problematic because a great variety of combinations of these subtypes are found in nature. The internal antigen determinants on the nucleoprotein (NP) are highly conserved within influenza viruses, making this protein more appropriate for a serological test. Objectives: In the experimental present study, an effectual method was expanded to purify NP of H9N2 AIV based on Electroelution method. Methods: AIV strain A/flash chicken/Iran/772/1998 (H9N2) was acquired from the Department of Avian influenza Reference Laboratory, Razi Vaccine and Serum Research Institute, Iran, about 2 cc, in 2017. Nucleoprotein of AIV (H9N2) was purified by an efficient and simple modified method directly from native polyacrylamide gel electrophoresis (PAGE) according to the Electroelution method. The purified protein concentration was defined by the Lowry method, and the purifiedNPprotein (60 KDa) was examined by Tricine-SDS-PAGE. Results: The protein concentration of the virus solution was 4. 62 mg/mL by the Lowry method. The purified Nucleoprotein concentration was 0. 296 mg/mL by Lowry method and the Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed only a 60-KDa protein band in the gel. Conclusions: The current technique was simple and rapid and made it possible to isolate NP from the H9N2 virus. The Nucleoprotein antigen is an appropriate candidate potential to detect antibodies against all subtypes of AIV and used as the main target antigen for the diagnosis of influenza virus due to its very high scale of sequence preserved among exist strains.