Title: Evaluation and optimization of avian embryos and cell culture methods for efficient isolation and propagation of avian influenza viruses Authors
Submitted to: International Symposium on Avian Influenza
Publication Type: Abstract Only
Publication Acceptance Date: January 11, 2009
Publication Date: April 5, 2009
Citation: Moresco, K.A., Swayne, D.E., Stallknecht, D.E. 2009. Evaluation and optimization of avian embryos and cell culture methods for efficient isolation and propagation of avian influenza viruses [abstract]. Abstracts of the 7th International Symposium on Avian Influenza, April 5-8, 2009, Athens, Georgia. p. 93. Technical Abstract: 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. Growth of low pathogenicity (LP) AIV strains representing all 16 hemagglutinin (HA) subtypes obtained from various poultry and wild bird species were tested in each system. There were no differences in 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 1-2 log ID50 lower in Madin-Darby canine kidney (MDCK), primary chicken embryo kidney (CEK), and primary chicken embryo fibroblast (CEF) cell cultures and 3-4 log ID50 lower in chicken bone marrow macrophage (HD11) and mink lung epithelial (Mv1Lu) cells. The quail fibroblast (QT-35), baby hamster kidney (BHK-21), and chicken fibroblast (DF-1) cell lines produced titers 4-6 log ID50 less than ECE. To determine efficiency for primary isolation, oropharyngeal and cloacal swabs from experimentally inoculated Laughing Gulls were examined. LPAIV isolation rates in ECE were similar to RRT-PCR detection rates when RRT-PCR+ results were confirmed by conventional reverse transcriptase polymerase chain reaction (RT-PCR) for the NS1 gene. The same samples will be grown in select cell cultures to determine which lines could be useful for isolating LPAIV from surveillance samples. The isolation and/or detection rates will be compared to those from the ECE and RRT-PCR methods.