Attenuation of Viruses by Targetted Mutagenesis
Development of a New Method to Obtain Apathogen Viruses via Targeted Mutagenesis
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
Institute of General Genetics and Cytology, Kazakstan, Almaty
- The Republican Government Enterprise on the basic of economic control rights “Research Institute for Biological Safety Problems” , Kazakstan, Gvardeiski
Project summaryThe aim of the project is to obtain stable apathogen influenza viruses that are the most perspective ones to develop universal new generation vaccines. Influenza is one of the most widespread and non-controlled infection diseases causing significant damage to health of the population and to the economy. According to the WHO, influenza and other severe respiratory infections take the third place among leading reasons of mortality (3.905.000), morbidity (394 million) and work inability in the world (WHO Report, 1997). Combating influenza should be based, first of all, on wide use of specific prophylaxis including utilization both alive and inactivated vaccines. But all vaccine preparations developed by present time have so-called “antigen retardation” related to high variability of the virus that excludes universality of their action. The problem of universal vaccine design has not been solved completely even for genetically more stable viruses, like morbilli (measles) and smallpox (variola) [Getmanova T.N. et al 1998]. Thus, attenuation of these viruses even using such classical methods as long lasting subinoculation (passage) in animals and tissues was accompanied by loss of infection ability and immunogeneity. Reduction of virus virulent ability through passages at low temperature does not change their genetic structure and does not exclude reversion of the changed properties [Ramig R. Bukrinskaya A.G., Akhmatullina N.B. 1990]. In order to obtain weakly pathogen viruses ts-mutants have been used, but they are not sufficiently stable. Besides, the issue of relationship between the ts-index and pathogeneity it is not completely solved. Therefore, any attempt to get virus of influenza with changed RNA is rather actual.
The approach to design weakly pathogen virus influenza that is offered will make impact on progress in development of applied and theoretical genetics and biotechnology. The approach is based on the feature of virus genome structure, that consists of several RNA threads acting in the form of discrete nuclei-protein complexes (RNP). This genome structure gets the possibility to affect separate parts of the genome with mutagene factors and to obtain site-specific mutations in selected genes without touching the whole genome. Therefore, in the range of targeted mutagenesis methods to obtain valuable breeds of agricultural plants and industrial strains of microbes etc., the new method for obtaining apathogene viruses will appear. Besides, localization and identification of genes or genome parts that are responsible for pathogeneity of influenza virus will expand understanding of genetic nature of this crucial property of all types of pathogens (parasites, fungi, bacteria, viruses etc.) affecting higher organisms.
At last, we plan to employ method of polymeraze chain reaction (PCR) for identification of mutations in inpidual genes. Absence of amplification of treated (modified) separate genes or fragments of virus RNA will be the true proof of a mutation in the fragment. In such tests arrangement methods of molecular biology will find new application and will participate in solution of biotechnology problems.
In research activity on the said problem qualified experts (Professors and Ph.D.) will participate and they will use both the results of own experiments and achievements of the world science regarding to features and mechanisms of mutation process. Scientific background includes proofs (first time in the world) of infection ability of RNP of influenza virus and sub-sell fractions of infected cells and the possibility of mutations via influence on sub-sell fractions, containing RNP sum. These data were confirmed by research activity with other viruses, in particular, with virus of NewCasle disease (Asadullaev et al 1984; Kilburn E., 1986).
Expected results: a method of induction of local mutation on influenza virus will be developed and a strain with reduced pathogeneity that is prospective for creation of universal vaccines of new generation will be obtained.
Methodology: analytical approach using features of molecular and genetic structure of the genome of influenza virus will be employed for targeted mutagenesis in order to obtain apathogen viruses.
The activity on the theme will start from elaboration of an approach to targeted modification of inpidual genes of influenza virus. For this purpose it is planned to carry out fractionating and separation of different classes of RNP of influenza virus, and re-association of biologically active RNP preparation.
Further it is planned to elaborate the conditions for mutagen treatment of separate RNP classes, selection of new reassortants, determination of their infection ability, haemagglutination and antigen properties and electrophoresis characteristics.