The molecular evolutionary characteristics of new isolated H9N2 AIV from East China and the function of vimentin on virus replication in MDCK cells

Abstract

Background: The low pathogenic H9N2 AIV caused the serious impact on the poultry industry and public safety. Our purpose was to investigate the molecular evolutionary characteristics of the new isolated H9N2 virus and investigate the intracellular target protein of H9N2 AIV replication in sensitive cells. Methods: AIV A/chicken/Shandong/LY1/2017 (H9N2) was isolated from the cloaca of the healthy chicken in Shandong, and the full-length eight gene segments of this isolated H9N2 AIV were amplified by RT-PCR and analyzed. MDCK cells were used as the target cell model, and VOPBA assay and LC-MS/MS were carried out to identify the virus-binding protein of H9N2 AIV. MDCK cells were pre-treated with the special antibody and siRNA, and treated with H9N2 AIV to detect the virus replication. Additionally, Vimentin-pcDNA3.0 was successfully constructed, and transinfected into MDCK cells, and then H9N2 AIV mRNA was detected with RT-PCR. Results: Phylogenetic analysis revealed that HA, NA, PB2, PB1, PA, NP and M seven genes of the isolated H9N2 AIV were derived from A/Chicken/Shanghai/F/98, while NS gene was derived from A/Duck/Hong Kong/Y439/97. The cleavage site sequence of HA gene of the isolated H9N2 AIV was a PARSSR G pattern, and the left side sequence (224~229) of receptor binding site was NGQQGR pattern, which were similar to that of A/Chicken/Shanghai/F/98. Following VOPBA assay, we found one protein of about 50KDa binding to H9N2 AIV, and the results of LC-MS/MS analysis proved that vimentin was the vital protein binding to H9N2 AIV. The pre-incubation of the specific antibody and siRNA decreased the viral RNA level in MDCK cells treated with H9N2 AIV. Furthermore, we found that over-expressed vimentin increased H9N2 AIV replication in MDCK cells.

Combination Treatment with the Vimentin-Targeting Antibody hzVSF and Tenofovir Suppresses Woodchuck Hepatitis Virus Infection in Woodchucks

“…Furthermore, the presence of surface VIM/vi-VIM is not restricted to infection with hepadnaviruses, as other viruses, such as human severe acute respiratory syndrome-related coronavirus (SARS-CoV), Japanese encephalitis virus (JEV), and enterovirus 71 (EV-A71), also induce this filamentous protein at least in vitro [ 23 , 63 , 64 , 65 ]. Surface VIM/vi-VIM is further used by SARS-CoV, JEV, EV-A71, porcine reproductive and respiratory syndrome virus, dengue virus, H9N2 subtype avian influenza virus, Chandipura virus, and cowpea mosaic virus as a receptor or at least as a co-receptor for cell attachment and entry by endocytosis, and in many studies viral infection can be reduced or even inhibited in vitro by using antibodies against VIM presented at the cell surface [ 62 , 64 , 66 , 67 , 68 , 69 , 70 , 71 ]. Although the exact mechanism of surface VIM/vi-VIM induction needs to be elucidated, infection with rather diverse viruses apparently changes the conformation of intracellular VIM thereby allowing its presentation on the cell surface.…”

Transcriptomic analysis of spleen B cell revealed the molecular basis of bursopentin on B cell differentiation

“…BP5 was synthesized by GenScript Biotechnology Co., Ltd (Nanjing, China), and the purity determined by HPLC and mass spectrometry was 98.8%. Avian influenza virus strain A / chicken/Shandong/Ly1/2017(H9N2) was stored in our laboratory [20]. Virus was harvested from the allantoic fluid of 9-day chicken embryos, and the hemagglutination titer of the virus was 2 9 .…”

Identification of NP Protein-Specific B-Cell Epitopes for H9N2 Subtype of Avian Influenza Virus

“…The H9N2 (A/chicken/Shandong/LY1/2017) strain was isolated and preserved in our laboratory [14], which was injected into a 9-day-old SPF chicken embryo allantoic cavity, and the allantoic fluid was collected and stored at −80 • C. SP2/0 cells were cultured in RPMI-1640 medium (Vicente Biotechnology Nanjing Co., Ltd., Nanjing, China) with 20% fetal bovine serum (FBS), 1% penicillin and 100 µg/mL streptomycin at 37 • C and 5% CO 2 .…”

“…To investigate the structural characteristics of NP proteins [14], the antigenicity was analyzed, as shown in Figure 1A. It was observed that the region between 71 and 243 AA in the NP protein possessed an apparent antigenic index, hydrophobicity and surface probability (Figure 1A, marked by the red box), which suggests that this region in the BP protein might have good antigenic characteristics and a B-cell determinant site.…”

“…McGee and Huang reported that the NP [147][148][149][150][151][152][153][154][155] epitope was a T-cell epitope that could trigger an NP-specific CTL response [27]. Noisumdaeng et al identified two conserved T-cell epitopes of NP proteins, NP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and NP [411][412][413][414][415][416][417][418][419][420][421][422][423][424][425][426][427][428][429][430] , from the sera of recovered patients [28]. Additionally, Bethny Morrissey et al identified four HA epitopes of the H3N2 subtype of avian influenza virus by mass spectrometry using HA monoclonal antibodies and found that an NP peptide, 230 FQTAAQR 236 , could cross-react with HA monoclonal antibodies, in which 230 FQTAAQR 236 was speculated to be a possi...…”