Comparing the genetic typing methods for effective surveillance and rabies control in Georgia

Abstract

A full nucleoprotein gene sequencing of 68 isolates collected from passive rabies surveillance system in Georgia between 2015 and 2016 identified two distinct dog rabies phylogroups, GEO_V1 and GEO_V2, which both belonged to the cosmopolitan dog clade. GEO_V1 was found throughout the country and was further divided into four sub-phylogroups that overlapped geographically; GEO_V2 was found in the southeast region and was closely related to dog rabies in Azerbaijan. A sequence analysis of the full N gene, partial nucleoprotein gene of N-terminal and C-terminal, and the amplicon sequences of pan-lyssavirus RT-qPCR LN34 showed that all four sequencing approaches provided clear genetic typing results of canine rabies and could further differentiate GEO_V1 and GEO_V2. The phylogenetic analysis results vary and were affected by the length of the sequences used. Amplicon sequencing of the LN34 assay positive samples provided a rapid and cost-effective method for rabies genetic typing, which is important for improving rabies surveillance and canine rabies eradication globally.

Keywords: LN34 assay; canine rabies; genetic diversity; molecular epidemiology; rabies typing; surveillance.

Figure 1

(A) A Bayesian phylogenetic tree of the complete N gene was reconstructed using 107 complete N gene sequences. The branches of Georgian phylogroups are outlined in different colors. The GEO_V1 in blue was dispersed across the country and further split into four sub-groups; only nodes with a posterior support value higher than 0.6 leading to the Georgian phylogroups are indicated in this figure. Nodes with posterior support <0.6 are not indicated. The number in the scale bar is the number of substitutions per site. (B) Map generated using Microreact. Dots outlined in Georgia's geopolitical map represents the number of samples collected and the colors show the phylogroup/sub-group to which they belong. Sample locations were randomly assigned to region level.

Figure 2

Bayesian phylogenetic analysis of Georgia isolates based on 400 bp. of the N-terminal and C-terminal of the nucleoprotein gene. (A) N-terminal phylogenetic tree, with phylogroups/sub-groups highlighted in different colors. Leaves labeled with blue in GEO_V1 were obtained in this study and other isolates from Turkey, Russia, and Azerbaijan were obtained from the public domain. GEO_V2 with red labels were part of this study; this phylogroup included isolates from Azerbaijan. GEO_V3 in purple was detected in the 1980s in Georgia and Turkey but not in this study. (B) C-terminal phylogenetic tree; blue- and red-labeled tips indicate sequences obtained in this study. In both phylogenetic trees, only nodes with a posterior support value higher than 0.6 that differentiate the Georgian phylogroups are indicated; nodes with posterior support <0.6 are not indicated. The number in the scale bar is the number of substitutions per site.

Figure 3

Shows the trimmed alignment (115 bp) of 41 RABV sequences and three other lyssaviruses. The length of the sequence corresponds to the amplicon obtained by the LN34 assay. Selected RABV samples from Georgia representing each phylogroup were chosen for pattern matches. The color on the side indicates the phylogroups determined by the complete and partial N gene analysis. Highlighted nucleotides in the alignment indicate the nucleotide changes that differentiate the phylogroups.