Molecular diagnostics of Avian influenza virus

Abstract

The success of supervizing an infectious disease depends on the ability for speedy detection and characterization of the cause and the forming of a corresponding system for examining the success of control implemented in order to prevent a recurrence of the disease. Since influenza viruses continue to circle, causing significant morbidity and mortality both among the human population and among animals all over the world, it is essential to secure the timely identification and monitoring of the strains that are in circulation. The speedy detection and characterization of new highly-virulent varieties is one of the priorities of the World Health Organization monitoring network. The implementation of molecular methods has an increasingly significant role in diagnostics and the monitoring of the influenza virus. Among a large number of molecular methods, the one particularly in use is the reverse transcription-polimerase chain reaction (PT-PCR). Technological progress in the area of the conducting of molecular methods has enabled that we can prove, in one day, using the RT-PCR method even very small quantities of the infective agent in a sample. In an obtained PCR product, we can relatively easily establish the nucleotide sequence, a detailed analysis and molecular epidemiology of the circulating strains. The molecular diagnostics procedure (RT-PCR) is based on the correct choice or designing of primers depending on the desired knowledge. In order to obtain a specific diagnosis of influenza A, B or C, primers are used which multiply internal genes, such as the nucleoprotein (NP) or matrix gene (M), because these are genes that are highly conserved among the virus types. In the event that we are interested in the subtype of influenza A, after obtaining a positive reaction, primers for genes of surface antigens are selected, such as hemagglutinin. Following the correct detection of the H subtype, it is possible to establish the virus virulence through the direct sequencing of the PCR product. The possibility of typization using molecular methods is based on the big difference at the amino acid and nucleotide levels between different HA subtypes (from 20- 74%), while the differences between strains of the same HA subtype are relatively small (0- 9%). The basic advantage in the detection and typization of influenza viruses using the RTPCR method is that it saves time. Namely, it can be performed directly from the samples taken in the field, and the result can be obtained within the same day, contrary to conventional methods that take 7 to 10 days. The obtained PCR product can also be sequenced immediately, which can provide an answer to the possible virulent potential of the isolate and its further spreading. The establishment of changes in the HA gene sequence can provide us with the information about the direction of the development of the genetic drift. The paper will describe in detail the possibilities for the implementation of molecular methods in diagnostics and typization, in fact, in the molecular epizootiology of avian influenza.