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.