Molecular characterization of cryptically circulating rabies virus from ferret badgers, Taiwan

Abstract

After the last reported cases of rabies in a human in 1959 and a nonhuman animal in 1961, Taiwan was considered free from rabies. However, during 2012-2013, an outbreak occurred among ferret badgers in Taiwan. To examine the origin of this virus strain, we sequenced 3 complete genomes and acquired multiple rabies virus (RABV) nucleoprotein and glycoprotein sequences. Phylogeographic analyses demonstrated that the RABV affecting the Taiwan ferret badgers (RABV-TWFB) is a distinct lineage within the group of lineages from Asia and that it has been differentiated from its closest lineages, China I (including isolates from Chinese ferret badgers) and the Philippines, 158-210 years ago. The most recent common ancestor of RABV-TWFB originated 91-113 years ago. Our findings indicate that RABV could be cryptically circulating in the environment. An understanding of the underlying mechanism might shed light on the complex interaction between RABV and its host.

Keywords: Melogale moschata subaurantiaca; Rabies; Taiwan; ferret badger; origin; phylogeography; viruses.

Figure 1

Collection sites of rabies-positive Taiwan ferret badgers (TWFB), Taiwan. Solid circles marked with 1–3 represent the collection sites of the first 3 rabies-positive animals. Triangles represent the collection sites of other rabies virus (RABV) sequences included in this study. Crosses represent the most diverged lineages of rabies virus from Taiwan ferret badgers (TWFB, TW1614, and TW1955), shown in Figure 5, panel B, Appendix, and the easternmost cross represents the isolate from a shrew, TW1955.

Figure 2

Phylogenetic relationships of 27 rabies virus (RABV) genomes constructed by maximum-likelihood method. Numbers close to the nodes were from 1,000 bootstrap replications. The tree was rooted with RABV from bats and raccoons. Three major groups, Asia, Cosmopolitan, and India, are strongly supported, as indicated (17). There are 4 major lineages within the group from Asia, including previously recognized China I, China II (16), Southeast Asia, and RABV from Taiwan ferret badgers (TWFB). RABVs derived from Chinese ferret badgers (CNFB) are clustered with China I, indicating that RABVs of TWFB and CNFB are of independent origin. Scale bar indicates nucleotide substitutions per site.

Figure 3

Sliding window analysis of rabies virus (RABV) genetic variations between Taiwan ferret badgers and China I, China II, and Chinese ferret badgers (CNFB). The genomic organization of RABV is shown at the bottom with nucleotide positions on the x-axis. The thick horizontal lines indicate conserved regions across lyssaviruses. N, nucleoprotein; P, phosphoprotein; M, matrix protein; G, glycoprotein; L, virion-associated RNA polymerase.

Figure 4

Phylogenetic relationships of major rabies virus groups based on (A) nucleoprotein and (B) glycoprotein gene sequences. The trees were constructed by the maximum-likelihood method based on the general time-reversible nucleotide substitution model. Numbers close to the node are from 100 bootstrap replications. The definition of major clades was based on the work of He et al. (16) and Bourhy et al. (17). Scale bar indicates nucleotide substitutions per site.

Figure 5

Maximum-likelihood trees of rabies virus based on (A) nucleoprotein and (B) glycoprotein gene sequences. Numbers on the branches are estimated divergences (above) and their 95% highest posterior density (below). The divergence time between different viral lineages and time to the most recent common ancestor of virus isolates were estimated by using an established Bayesian Markov chain Monte Carlo approach implemented in BEAST version 1.7 (21). The substitution rate was assumed to be 4.3 × 10–4 (range 3.1–6.6 × 10–4) or 2.3 × 10–4 (range 1.1–3.6 × 10–4)/sites/year (hatched numbers) for nucleoprotein (N) and 3.9 × 10–4 (1.2–6.5 × 10–4)/sites/year for glycoprotein (G) genes (17,23). The analysis was performed by using the general time-reversible model of nucleotide substitution, assuming an uncorrelated lognormal molecular clock (22). TW1955 is the only isolate from a shrew that is very close to TW1614 collected from the same area, suggesting that TW1955 was a spillover from a ferret badger (see also Figure 1). Scale bar indicates nucleotide substitutions per site.