Genomic Surveillance of Rabies Virus in Georgian Canines

Abstract

Rabies is a fatal zoonosis that is considered a re-emerging infectious disease. Although rabies remains endemic in canines throughout much of the world, vaccination programs have essentially eliminated dog rabies in the Americas and much of Europe. However, despite the goal of eliminating dog rabies in the European Union by 2020, sporadic cases of dog rabies still occur in Eastern Europe, including Georgia. To assess the genetic diversity of the strains recently circulating in Georgia, we sequenced seventy-eight RABV-positive samples from the brain tissues of rabid dogs and jackals using Illumina short-read sequencing of total RNA shotgun libraries. Seventy-seven RABV genomes were successfully assembled and annotated, with seventy-four of them reaching the coding-complete status. Phylogenetic analyses of the nucleoprotein (N) and attachment glycoprotein (G) genes placed all the assembled genomes into the Cosmopolitan clade, consistent with the Georgian origin of the samples. An amino acid alignment of the G glycoprotein ectodomain identified twelve different sequences for this domain among the samples. Only one of the ectodomain groups contained a residue change in an antigenic site, an R264H change in the G5 antigenic site. Three isolates were cultured, and these were found to be efficiently neutralized by the human monoclonal antibody A6. Overall, our data show that recently circulating RABV isolates from Georgian canines are predominantly closely related phylogroup I viruses of the Cosmopolitan clade. Current human rabies vaccines should offer protection against infection by Georgian canine RABVs. The genomes have been deposited in GenBank (accessions: OQ603609-OQ603685).

Keywords: RABV; canine; dog; genomics; jackal; lyssaviruses; neutralization; next-generation sequencing; phylogeny; rabies.

Figure 1

Phylogenetic tree of Georgian lyssavirus N gene sequences. A total of 140 sequences with 1353 nucleotide sites were included, and the log-likelihood of the consensus tree was −9845. The tree is rooted at the midpoint. The labels of samples collected in Georgia with geographical metadata are colored by subregion: Kakheti (green), Kvemo Kartli (purple), Samgrelo (orange), Tbilisi (blue), Guria (pink), Adjara (red), Shida Kartli (teal). (A) Full phylogenetic tree (the area shadowed in blue is zoomed in the B panel). (B) Zoomed in view of the branching area of the tree containing the RABV-GEO samples. (C) Map of the country of Georgia, indicating regions from which samples were collected (adaptation of “File: Regions of Georgia (country).svg” by Nordwestern, licensed under CC BY-SA 4.0). Colors were coded to match samples in (A,B).

Figure 2

Phylogenetic tree of Georgian lyssavirus G gene sequences. A total of 110 sequences with 1578 nucleotide sites were included, representing the available NCBI G sequences relevant to geographic/clade distribution. The log-likelihood of the consensus tree was −12,159. The tree is rooted at the midpoint. The labels of samples collected in this study are colored green. (A) Full phylogenetic tree (the area shadowed in blue is zoomed in the B panel). (B) Zoomed in view of the branching area of the tree containing the RABV-GEO samples.

Figure 3

Amino acid sequence of the G protein ectodomain of the Georgian ECTO1 group. (A) The amino acid sequence of the RABV G ectodomain is shown, using the sequence derived from the viruses of the ECTO1 group. The antigenic sites are underlined. Amino acids that vary between different ECTO groups are indicated in red. (B) Amino acid changes in different ECTO groups with respect to the reference (ECTO1). The positions of the amino acids in the ectodomain are indicated in superscript.

Figure 4

Neutralizing activity of anti-ABLV G human mAb A6 against RABV-GEO isolates. RABV-GEO viruses 6, 9, and 20 were isolated from primary canine brain tissue, followed by one round of in vitro passaging. Virus neutralization was measured by incubation of viral supernatants with human mAb A6, followed by infection of N2a cells. Viral foci were identified by staining with anti-Rabies G. Graph shows percent virus neutralization over a series of 5-fold dilutions of mAb A6.