Evaluation and Attempted Optimization of Avian Embryos and Cell Culture Methods for Efficient Isolation and Propagation of Low Pathogenicity Avian Influenza Viruses

Surveillance of wild bird populations for avian influenza viruses (AIV) contributes to our understanding of AIV evolution and ecology. Both real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) and virus isolation in embryonating chicken eggs (ECE) are standard methods for detecting AIV in swab samples from wild birds, but AIV detection rates are higher with RRT-PCR than isolation in ECE. In this study we tested duck embryos, turkey embryos, and multiple cell lines for AIV growth as compared to ECE for improved isolation and propagation of AIV for isolates representing all 16 hemagglutinin subtypes. There were no differences in low pathogenicity AIV (LPAIV) propagation titers in duck or turkey embryos compared to ECE. The replication efficiency of LPAIV was lower in each of the cell lines tested compared to ECE. LPAIV titers were 1-3 log mean tissue-culture infective doses (TCID50) lower in Madin-Darby canine kidney (MDCK), primary chicken embryo kidney (CEK), and primary chicken embryo fibroblast (CEF) cell cultures, and 3-5 log TCID50 lower in chicken bone marrow macrophage (HD11), chicken fibroblast (DF-1), and mink lung epithelial (Mv1Lu) cells than the corresponding mean embryo infective doses (EID50) in ECE. The quail fibroblast (QT-35) and baby hamster kidney (BHK-21) cell lines produced titers 5-7 log TCID50 less than EID50 in ECE. Overall, ECEs were the most efficient system for growth of LPAIV. However, the savings in time and resources incurred with the use of the MDCK, CEK, and CEF cultures would allow a higher volume of samples to be processed with the same fiscal and financial resources, thus being potentially advantageous despite the lower replication efficiency and lower isolation rates.