Detection of Highly Pathogenic Avian Influenza H5N1 Viral Load by Real-Time RT-PCR in Tissues of Experimentally Infected Chickens

Abstract

This study reports the highly pathogenic avian influenza (HPAI) H5N1 virus load estimation in different organs of chickens following experimental infection. The TaqMan probe based quantitative real-time reverse transcriptase PCR (qRT-PCR) assay was optimized for quantification of HPAI virus RNA in tissues collected from experimentally infected chickens. Conserved region in the matrix gene of avian influenza virus served as target for the primers and TaqMan probe. A recombinant plasmid containing the matrix protein gene amplicon was constructed for a quantitative estimation of copy numbers of the target gene. Quantification of avian influenza virus RNA was accomplished using a standard curve generated from ten-fold serial dilutions of IVT RNA generated from recombinant plasmid containing matrix gene. High viral RNA load was detected in spleen, brain and lung indicating enormous replication of virus in these tissues. However, spleen showed significantly higher viral RNA load (P<0.03) over other organs.