Search

Avian influenza (AI), caused by H9N2 subtype avian influenza virus (AIV), poses a serious threat to poultry farming and public health due to its transmissibility and pathogenicity. The PB2 protein is a major component of the viral RNA polymerase complex. It is of great importance to identify the antigenic determinants of the PB2 protein to explore the function of the PB2 protein. In this study, the PB2 sequence of H9N2 subtype AIV, from 1090 to 1689 bp, was cloned and expressed. The recombinant PB2 protein with cutting gel was used to immunize BALB/c mice. After cell fusion, the hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the PB2 protein were screened by indirect ELISA and western blotting, and the antigenic epitopes of mAbs were identified by constructing truncated overlapping fragments in the PB2 protein of H9N2 subtype AIV. The results showed that three hybridoma cell lines (4B7, 4D10, and 5H1) that stably secreted mAbs specific to the PB2 protein were screened; the heavy chain of 4B7 was IgG2α, those of 4D10 and 5H1 were IgG1, and all three mAbs had kappa light chain. Also, the minimum B-cell epitope recognized was 475LRGVRVSK482 and 528TITYSSPMMW537. Homology analysis showed that these two epitopes were conserved among the different subtypes of AIV strains and located on the surface of the PB2 protein. The above findings provide an experimental foundation for further investigation of the function of the PB2 protein and developing monoclonal antibody-based diagnostic kits. [ABSTRACT FROM AUTHOR]

Simple Summary: H9N2 subtype avian influenza virus (AIV), which is mild but widespread, has been a concern due to the potential threat to poultry farming and public safety. AIV-infected poultry exhibit a complex range of various types of disease, which, coupled with the fact that AIV viruses are prone to genetic mutation and remodeling and exhibit a very high degree of variability, further increases the difficulty of immunization and diagnosis of avian influenza. We focused on the PB1 protein, a vital part of the virus's ability to replicate. In this study, a truncated PB1 protein was designed and expressed in the prokaryotic expression system. Four hybridoma cells secreting the antibody specific to PB1 protein were screened using mouse immunization and cell fusion techniques. Two B cell antigenic determinants were identified by gradually truncating protein expression, and were conserved across different flu strains and located on the surface of PB1 protein. These findings could help make better vaccines against the H9N2 virus and might be important for controlling the spread of bird flu and protecting both animals and humans. Recently, low pathogenic avian influenza virus (LPAIV), including H9N2 subtype, has been common clinical epidemic strains, and is widely distributed globally. The PB1 protein is a key component of the viral RNA polymerase complex (vRNP), and is vital to viral transcription and translation. In this study, to investigate the antigenic determinants in the PB1 protein, the truncated PB1 sequence (1bp-735bp) from H9N2 subtype AIV was amplified with PCR, and expressed in plasmid pET-28a (+). After purification, the recombinant PB1 protein was used to immunize BALB/c mice. Following immunization, hybridoma cells producing PB1-specific monoclonal antibodies were generated through the fusion of splenic lymphocytes with SP2/0 cells. Then, four stable hybridoma cell lines (5F12, 5B3, 2H9, and 3E6) were screened using indirect ELISA and Western blotting. Furthermore, two antigenic sites, 67NPIDGPLPED76 and 97ESHPGIFENS106, were identified through the construction of truncated overlapping fragments of the PB1 protein. These sites were conserved among 28 AIV strains, and were located on the PB1 protein surface. The findings offer a theoretical reference for the development and improvement of H9N2 vaccines and offer biological materials for virus detection during AIV infection mechanisms. [ABSTRACT FROM AUTHOR]

H9N2 subtype of avian influenza virus (AIV) is primarily a bird virus, which is widespread in clinical avian disease, and reported in cases of human infection. As one of the surface proteins of AIV, the neuraminidase (NA) protein plays an important role mainly in viral budding. However, vaccine development and detection methods for NA of H9N2 AIVs are in urgent clinical need. In this study, a truncated NA gene (205–900 bp) was cloned from the NA sequence of H9N2 strain, and then expressed using pET-28a (+) vector. This purified recombinant NA protein was used to immunize BALB/c mice, and the monoclonal antibodies were screened through the indirect enzyme-linked immunosorbent assay (ELISA). Next, eight prokaryotic expression vectors were constructed for epitope identification. After cell fusion, three hybridoma cell lines producing the antibodies special to NA protein were screened by ELISA, western blotting, and indirect immunofluorescence; these were named 1B10, 2B6, and 5B2, respectively. Epitope scanning techniques were used to identify three B-cell epitopes recognized by these three monoclonal antibodies, 196KNATASIIYDGMLVD210, 210DSIGSWSKNIL220 and 221RTQESECVCI230. The subsequent homology analysis revealed the three epitopes were highly conserved in H9N2 AIV strains. The structural predictions of the antigenic epitopes indicated that all three epitopes were located in the catalytic region of NA. These results provide a basis for studying the function of the NA protein of H9N2 AIV and technical support for the development of a universal detection method based on anti-NA monoclonal antibodies. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "Identification of Unique and Conserved Neutralizing Epitopes of Vestigial Esterase Domain in HA Protein of the H9N2 Subtype of Avian Influenza Virus." The correction addresses an error in Figure 4A of the original publication, where two duplicated images were mistakenly included. The corrected figure has been provided, and the original publication has been updated accordingly. The article is available as an open access article under the Creative Commons Attribution license. [Extracted from the article]

The H9N2 subtype of avian influenza virus (AIV) has been reported to infect not only birds, but also humans. The hemagglutinin (HA) protein is the main surface antigen of AIV and plays an important role in the viral infection. For treatment strategies and vaccine development, HA protein has been an important target for the development of broadly neutralizing antibodies against influenza A virus. To investigate the vital target determinant cluster in HA protein in this work, HA gene was cloned and expressed in the prokaryotic expression vector pET28a. The spleen lymphocytes from BALC/c mice immunized with the purified recombinant HA protein were fused with SP2/0 cells. After Hypoxanthine-Aminopterin-Thymidine (HAT) medium screening and indirect ELISA detection, six hybridoma cell lines producing anti-HA monoclonal antibodies were screened. The gradually truncated HA gene expression and western blotting were used to identify their major locations in epitopes specific to these monoclonal antibodies. It was found that the epitopes were located in three areas: 112NVENLEEL119, 117EELRSLFS124, and 170PIQDAQ175. Epitope 112NVENLEEL119 has a partial amino acid crossover with 117EELRSLFS124, which is located in the vestigial esterase domain "110-helix" of HA, and the monoclonal antibody recognizing these epitopes showed the neutralizing activity, suggesting that the region 112NVENLEELRSLFS124 might be a novel neutralizing epitope. The results of the homology analysis showed that these three epitopes were generally conserved in H9N2 subtype AIV, and will provide valuable insights into H9N2 vaccine design and improvement, as well as antibody-based therapies for treatment of H9N2 AIV infection. [ABSTRACT FROM AUTHOR]

Avian influenza caused by H9N2 subtype avian influenza virus (AIV) poses a great threat to the healthy development of the poultry industry. Vimentin is closely related to intracellular lipid metabolism, which plays an important role during the viral infection process. However, the function of lipid metabolism and vimentin on H9N2 AIV replication is unclear. In this paper, the cholesterol level and 3-hydroxy-3-methylglutaryl coenzyme a reductase (HMGCR) phosphorylation were investigated in vimentin knockout (KO) and human cervical carcinoma cells (HeLa) cell with or without AIV infection. The results showed that compared to the control group without infected with H9N2 subtype AIV, the cholesterol contents were significantly increased, while HMGCR phosphorylation level was reduced in both KO and HeLa cell after virus infection. Furthermore, viral replication was significantly inhibited in the cells treated with the cholesterol inhibitor lovastatin. Compared with the control group, adenylate activated protein kinase (AMPK), a kinase regulating HMGCR enzymatic activity was inhibited in both KO and HeLa cells in the infected virus group, and AMPK phosphorylation levels were significantly lower in KO HeLa cell than that of HeLa cells. Additionally, after MβCD treatment, viral hemagglutinin (HA) gene level was significantly decreased in HeLa cells, while it was significantly increased in KO HeLa cells. In addition, vimentin expression was significantly increased in MβCD-treated HeLa cells with the viral infection and returned to normal levels after exogenous cholesterol to backfill the MβCD-treated cells. Therefore, the disruption of lipid rafts during the binding phase of viral invasion of cells significantly reduced viral infection. These studies indicated that the lipid rafts and cholesterol levels might be critical for H9N2 subtype AIV infection of human-derived cells and that vimentin might play an important role in the regulation of lipids on viral replication, which provided an important antiviral target against influenza virus. [ABSTRACT FROM AUTHOR]

Avian Influenza (AI) caused by the H9N2 subtype of the avian influenza virus (AIV) poses a serious threat to both the poultry industry and to public health safety. NP is one of the major structural proteins in influenza viruses. B-cell determinants located on NP proteins have attracted increasing attention. In this study, based on the NP sequence of the H9N2 (A/chicken/Shandong/LY1/2017) strain, the truncated NP gene (71 AA–243 AA) was cloned and prokaryotically expressed in a pET-28a (+) vector. BALB/c mice were immunized with a purified recombinant of an NP protein to prepare a monoclonal antibody against NP proteins. The prokaryotic expression of four overlapping fragments, NP-N-96, NP-C-103, NP-C-54 and NP-C-49, were used to recognize an antigenic epitope of the NP protein. The results show that, after cell fusion, one hybridoma cell clone secreted the antibody specific to the NP protein, following screening with ELISA and indirect immunofluorescence, which is named the 4F5 monoclonal antibody (mAb). Western blotting on the overlapping fragments showed that the 230FQTAAQRA237 motif was identified as the minimal motif recognized by 4F5mAb, which was represented as the linear B-cell epitope of the NP protein. Homology analysis of this epitope shows that it was highly conserved in 18 AIVs analyzed in this study, and the epitope prediction results indicate that the epitope may be located on the surface of the NP protein. These results provide a strong experimental basis for studying the function of the NP protein of the H9N2 AIV and also strong technical support for the development of a universal assay based on an anti-NP monoclonal antibody. [ABSTRACT FROM AUTHOR]

CpG oligodeoxynucleotides (CpG ODN) present adjuvant activities for antigen proteins, which can induce humoral and cellular immune responses to antigens. However, the immunomodulatory functions of CpG ODNs with different sequences are very different. In this paper, six CpG ODNs with different sequences were designed based on CpG2007 as a template. Through the screening of CEF cells in vitro, the stimulating activity of CpG ODNs was determined. Then, two selected CpG ODN sequence backbones were modified by substituting the oxygen with sulfur (S-CpG) and verifying the immune activity. Next, to prove the feasibility of S-CpG as an immune potentiator, two immune models with or without white oil adjuvant were prepared in 20-day-old chicken vaccinations. The screening experiment in vitro showed that the inducing roles of CpG ODN 4 and 5 could strongly stimulate various immune-related molecular expressions. Additionally, CpG ODN 4 and 5 with sulfation modification significantly induced various cytokines' expressions. Furthermore, CpG ODN 4 and 5 induced the strongly humoral and cellular immune responses during vaccination, in which white oil, as an adjuvant, could significantly improve the immune effect of CpG ODN. These results provide an important experimental basis for exploring the structural characteristics and vaccine immunity of CpG ODN. [ABSTRACT FROM AUTHOR]

The infection and replication of avian influenza virus (AIV) in host cells is a complex biological process that involves the transport of viral genes through the host cell's transport systems. Actin, microtubules and vimentin are known to facilitate transport of endosomes to the perinuclear region, but the biological role of Keratin, another intermediate filament, in viral transport during AIV replication is not well understood. In this study, the viral NS2 protein was used as the target protein to identify the potential interacting proteins following GST-Pulldown method and protein mass spectrometry. It was discovered that Keratin10 interacted with NS2. Subsequently, it was found AIV infection did not affect the gene level or protein level of keratin10 in HeLa cells, but when Keratin10 was knocked down, the expressions of viral NP mRNA and protein were reduced, and the generation of offspring virus also was also decreased. Furthermore, in early viral infection, Keratin10 could aggregate and co-localize with NP proteins, suggesting that Keratin10 might be connected to early viral transport. Additionally, it was demonstrated that Keratin10 co-localized with Lamp1 and that AIV particles were trapped in late endosomes/Lysosomes after Keratin10 was knocked down. Finally, it was discovered that the knocking down Keratin10 in HeLa cells led to an increase in the acidic pH of endosomes and lysosomes, which prevented AIV from undergoing fusion and uncoating, and then inhibited the process of the viral infection. Overall, the results suggested that Keratin10 might play the critical role in the release of vRNPs from LEs/Ls and can affect the generation of offspring virus. The study provides the novel insights into the role of Keratin10 in the process of AIV infection and transmission, which may have implications for developing new strategies to against AIV infections. • Keratin10 knockdown is first reported to inhibit AIV infection in HeLa cells. • Keratin 10 co-locates with AIV and Lamp-1in the early infection stage. • vRNPs are restricted to LEs/Ls and cannot be released after KRT10 knockdown. • Knockdown of Keratin 10 affect the acidic pH of LEs/Ls. [ABSTRACT FROM AUTHOR]