development and sequence analysis of a cold-adapted strain of influenza a/new caledonia/20/1999(h1n1) virus

Background and aims: vaccination is the most effective method to prevent influenza infection. among the available vaccines, cold-adapted live-virus vaccines are suitable approach that have been produced and evaluated for recent years in few countries. the goal of this project was to derivate a cold adapted variant of the influenza a/new caledonia/20/1999(h1n1). materials and methods: influenza a/new caledonia/20/1999(h1n1) was adapted to grow at 25°c by gradually decreasing the incubation temperature through the sequential passages in embryonic eggs. the viral genome extracted from the starting seed and the last round of passage at 25°c was amplified by rt-pcr. the amplified cdna fragments were subjected to sequencing determination bi-directionally. sequence data were aligned to find mutated positions. results: sequence analysis showed totally six cases of point mutations that five of them resulted in amino acid substitutions and one of them was a silent mutation. these substitutions of one amino acid occurred in pb2, pa, np proteins and two amino acid changes in ha protein sequence. conclusion: the variant of cold adapted strain made here could be used as a master donor to generate attenuated reassortant influenza vaccine viruses.