Susceptibility of influenza mutants to Tamiflu® and molecular diagnostics of FMD virus -

Abstract

The two aims of avian influenza research were to optimize the antiviral activity of Oseltamivir in chick embryos against an initial avian influenza-H9N2 virus (P0), and to apply the optimized protocol for studying the drug susceptibility of four H9N2 mutants (M1, M2, M3, and M4), emerged by passaging the P0 strain in mature chicken hosts. The Oseltamivir delivered into the embryonated eggs, at a concentration of 0.27mg-100µl-9-day-old chicken embryo, against each of seven density levels of the P0 strain, ranging between 1.2x10⁻⁵ and 2.0 Hemagglutination (HA) units, showed its highest efficacy in reduction of viral propagation (95percent reduction in HA titer) (P0.05), when the inoculum level contained a minimum HA units of 1.2x10⁻⁵. The application of the 1.2x10⁻⁵ HA units in inocula for the evaluation of Oseltamivir-antiviral effect against the four H9N2 mutants revealed an emergence of one resistant mutant (M1), associated with two neighboring point mutations in its neuraminidase (N) amino acid (aa) sequence at positions 46 and 47. The other three mutants maintained their variable sensitivity to Oseltamivir, resulting in the following percent reduction in HA titers: M2 (82.9percent), M3 (61.5percent) and M4 (100.0percent). The different number and positions of point mutations in neuraminidase of the four mutants seems to determine the susceptibility of H9N2 virus to Oseltamivir. The standardization of the RT-PCR method for amplification of the conserved 5’ UTR gene of FMD virus using variable RNA concentrations and cycling conditions was performed on different fractions of FMD vaccines, and two field isolates (FMD1 and FMD2) delivered by the Ministry of Agriculture. The RNA amount was set in a volume of 4µL at 20 ng-µL and the optimal RT-PCR cycling conditions were as follows: 45 minutes at 50°C, followed by 15 minutes at 95°C, then by 30 cycles of 94°C for 1 minute, 55°C for 1 minute, 72°C for 2 minutes, one cycle at 72°C for 7 minutes.