Molecular characterization of rabies virus strains in N, G, ir G-L intergenic regions

It is important to genetically characterize RABV from practical aspect was well, as since 2006 Lithuania undergoes a long-term wildlife vaccination against rabies program that is coordinated by European Commission. Although due to this program in recent years the number of rabies cases decreased dramatically, it still occurs in restricted regions of Lithuania. Besides, the genetic characterization is considered important for establishing more effective strategy of rabies control and prevention and the ultimate eradication of this disease in Lithuania. This study attempts to respond to this shortage of molecular epidemiological data by analyzing Lithuanian RABV strains collected between 2005 and 2009 and obtained from various regions of the country. The aim of the present study was to characterize rabies virus strains prevalent in Lithuania using molecular methods in N, G and G-L intergenic regions. To achieve this aim, the objectives were as follows: 1. To determine Lithuanian RABV partial nucleoprotein gene sequences and perform phylogenetic analysis; 2. To characterize glycoprotein coding region of rabies virus strains, perform phylogenetic analysis and determine genetic differences of Lithuanian RABV strains; 3. Determine sequences of RABV strains prevalent in Lithuania in G-L intergenic region and compare them with other referent rabies virus sequences circulating around the world; 4. Compare vaccine and Lithuanian field rabies virus strains and evaluate deduced amino acid antigenic profiles in rabies virus nucleoprotein and glycoprotein regions; 5. Use and evaluate rapid diagnostic methods (nested RT-PCR, one step RT-PCR, classical sequencing and pyrosequencing) for rabies virus identification or typing. In this study for the first time Lithuanian rabies virus strains were analyzed in detail using molecular methods. It is important to note that there are no data available describing Lithuanian rabies virus strains in scientific literature. For molecular characterization of RABV in clinical samples modified diagnostic primers, as well as nested RT-PCR, one step RT-PCR, pyrosequencing methods were used, which until present study were not applied in Lithuania. Although at the molecular biology level, rabies virus strains are widely described, but in this study we were the first to determine sequences, perform molecular characterization and conduct phylogenetic analysis of partial nucleoprotein, glycoprotein and G-L intergenic regions of RABV strains prevalent in Lithuania. We compared the data of newly obtained RABV nucleotide sequences with the data of various biotypes of rabies virus available in public databases from various countries of Europe and around the world, as well as laboratory and vaccine strains. Characterization of rabies virus strains at the molecular level, may give reliable prognostic information, are important in order to give suggestions for effective means of prevention from this disease. Importantly, in international database GenBank 12 partial nucleoprotein coding region nucleotide sequences were published under the accession numbers from EU616715 to EU616726, which are publicly available for other researches and in the future could serve as a template for designing of specific oligonucleotide primers for rabies diagnostic. It was proved, to the best of our knowledge, for the first time that Lithuanian red fox and raccoon dogs’ strains were practically identical. In addition, molecular characterization enables to differentiate RABV vaccine strains (including SAD Bern, RV-97, used for oral vaccination of wildlife) from field strains. Such differentiation is very important in order to detect possible reversion of vaccine strains to wild-type strains. Thus, our study is significant and supplement existing data with data from Lithuania, the country in which the spread of RABV infection among animals is one of the largest in European Union and gives opportunities for more detailed further investigations. Conclusions: 1. Rabies virus strains prevalent in Lithuania in nucleoprotein coding region belongs to first lyssavirus genotype, North East Europe (NEE) phylogenetic group and does not show Lithuanian geographic region or species specificity. 2. Lithuanian rabies virus strains nucleotide sequences coding glycoprotein also grouped in North East Europe (NEE) phylogenetic group, in which among Lithuanian strains was determined statistically significant grouping into 5 separate groups, that had unique change in antigenic site III, 338 amino acid position. 3. All analysed rabies virus strains in G-L intergenic region formed 5 phylogenetic groups as well, which had a statistically significant bootstrap value and differences of nucleotide sequences of 8 % permits to use this region for molecular characterization of very similar RABV strains. 4. Rabies virus strains prevalent among wild and domestic animals and vaccine strains in phylogenetic trees of nucleoprotein, glycoprotein and G-L intergenic regions distributed in different, statistically significantly delimited groups. 5. Deduced amino acid Jameson-Wolf antigenic profiles of RABV nucleoprotein region strains prevalent in Lithuania were different from SAD group vaccine strains in high and low antigenic sites, though antigenic sites of glycoprotein ectodomen region were similar. 6. One step RT-PCR and pyrosequencing results of 44 rabies virus strains proved that this method could be successfully used for diagnosis and classification of wild and domestic rabies virus strains circulating in Lithuania