Replication Efficiency of Influenza A Virus H9N2: A Comparative Analysis Between Different Origin Cell Types

Abstract

Background: Efficient isolation and detection of low pathogenic avian influenza viruses from surveillance samples continues to be a high priority. Currently, the new cell lines are considered for supporting the replication to high virus strains titers. Objectives: The replication efficiency of a low pathogenic avian influenza virus in different origin cells was evaluated under different conditions. Materials and Methods: Chicken embryo fibroblast (CEF) cell and human alveolar epithelial cell line (A549) were infected with H9N2 at a multiplicity of infection of 0.1. The amount of infectious virus released into the cell culture supernatants at various post-infection time intervals were tittered by tissue culture infectious dose (TCID50) assay. The impact of these cells adaptation was investigated by determination the virus genes nucleotide sequences. Results: The influenza virus infectivity was not significant difference in these cells in the presence of trypsin. The results of fusion assay and determination of cellular protease confirmed that A549 cells support virus entry with or without supplemental trypsin. However, the H9N2 virus showed lower titer and infectivity in the trypsin-free infected A549 cells within longer time. The comparative sequence analysis indicated several simultaneously nucleotide substitutions were occurred in NA of the virus replicated in A549 cells resulted in two fixed amino acid changes at positions G320 to A and G414 to A up to the fifth passage. Conclusions: After seven consecutive passages of both cell cultures, the H9N2 virus showed similar antigenicity, also no change on viral titer level and virus replication behavior in adaptation was found. The results highlighted the use of A549 cells for efficient virus isolation.

In Silico Analysis of HA2/Mx Chimera Peptide for Developing an Adjuvanted Vaccine to Induce Immune Responses Against Influenza Viruses

“…However development HA-based vaccine is often difficult to attain due to emergence variants that have undergone sufficient antigenic drift in HA1 globular head to evade existing antibody responses. 13,14 It has been shown that HA2 is highly conserved as compared to HA1 subunit and antibodies recognizing stalk domain of the subunit neutralize the virus and provide sufficiently protection against infection and do cross-react with the HA of other subtypes. [15][16][17] Generally enhanced and directed immune responses to viral vaccine can be achieved by using adjuvants.…”

Establishment of MDCK/FX Cell for Efficient Replication of Influenza Viruses

“…Trypsin causes large nonspecific loss of viruses' infectivity under incubation conditions similar to FX. Thus, the factor is much more effective and specific in cleavage activation than in trypsin (13,25). The potential of FX to cleave HA and promote replication of influenza viruses is confirmed by our study in which maximum viral titers were reached one day earlier in MDCK/FX with comparative MDCK cells.The impact of FX on H9N2 virus replication kinetics upon engineered expression in BHK-21 cell line has been evaluated (16).…”

Necrotic Response to Low Pathogenic H9N2 Influenza Virus in Chicken Hepatoma Cells

“…Proteolytic activation of HA is essential for the entry of influenza viruses into the target cells, and also to trigger the dynamic infectivity process. The HA cleavage site of H9N2 viruses is a monobasic motif, which is cleaved extracellularly by trypsin, and trypsin-like proteases in the cells lining the respiratory tract, resulting in localized infections ( 1 , 2 , 17 ). In addition to those enzymes, plasminogen, a blood-derived protease, may cleave HA of influenza viruses and promote replication of the viruses outside respiratory tissues ( 18 , 19 ).…”