Selection and Characterization of Chicken Polyclonal Antibody Resistant Isolates of Influenza A Virus

Aquatic birds are the natural reservoir of influenza A virus and the known human strains of influenza have their origin in birds. The major surface proteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA), constitute the major immunogenic components of the virus. Polyclonal antibodies generated against these proteins provide protection from virus infection and disease in human. Retrospective studies conducted with influenza samples isolated from infected humans and birds have shown accumulation of mutations in specific regions of HA and NA proteins, that are readily recognized by host antibody. Several studies have demonstrated the selection of viral polymerase introduced mutations by the host immune system, which in turn leads to the evolution of influenza virus both in animal models and in vitro. Similar studies had not been undertaken with birds, hence we choose to test the effect of chicken polyclonal antibody on virus growth in a cell culture model using chicken polyclonal IgY immune serum. We hypothesized that chicken antibody may have a role in influencing the evolution of influenza virus by selecting for changes in surface proteins. For this study, A/California/07/2004 (H3N2) x A/Puerto Rico/8/34 which was a reassortant strain of influenza A virus referred as (CalX-Wt), was sequentially passaged in Madin-Darby canine kidney (MDCK) cell line in the presence of a pre-determined concentration of chicken antiserum raised against the same virus. Viral outgrowths obtained, designated as IgY-selected isolates, were characterized by plaque assay, focus forming assay (FFA), and hemagglutination inhibition assay (HAI) for changes in phenotypic characteristics. Changes in genotype were also determined via cloning and sequencing. IgY-selected isolates and CalX-Wt were also compared for their differences in the modulation of host cell apoptosis. Results showed that some of the IgY-selected isolates had significantly larger median focus size than CalX-Wt, which was corroborated by plaque assay. While most IgY-selected isolates had similar HAI sensitivity as CalX-Wt. Sequencing of the HA gene and whole genome of representative IgY-selected isolates and CalX-Wt resulted in the identification of mutations in the HA. NA, and PB2 genes. Ability to induce apoptosis was measured using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and revealed that lgY-selected isolates induced lower levels of apoptosis in MDCK cells than CalX-Wt. IgY-selected isolates also induced a different pattern of gene expression in HeLa cell line from that of CalX-Wt in real-time PCR analysis. Hence, we conclude that in addition to changes in surface proteins, replicative fitness of the virus may have a role in influenza evolution. A study was also conducted to compare the sensitivity of plaque assay and FFA in estimating viral titer, and results showed FFA to be more sensitive in determining viral titer than plaque assay.