Molecular inferences suggest multiple host shifts of rabies viruses from bats to mesocarnivores in Arizona during 2001-2009

Abstract

In nature, rabies virus (RABV; genus Lyssavirus, family Rhabdoviridae) represents an assemblage of phylogenetic lineages, associated with specific mammalian host species. Although it is generally accepted that RABV evolved originally in bats and further shifted to carnivores, mechanisms of such host shifts are poorly understood, and examples are rarely present in surveillance data. Outbreaks in carnivores caused by a RABV variant, associated with big brown bats, occurred repeatedly during 2001-2009 in the Flagstaff area of Arizona. After each outbreak, extensive control campaigns were undertaken, with no reports of further rabies cases in carnivores for the next several years. However, questions remained whether all outbreaks were caused by a single introduction and further perpetuation of bat RABV in carnivore populations, or each outbreak was caused by an independent introduction of a bat virus. Another question of concern was related to adaptive changes in the RABV genome associated with host shifts. To address these questions, we sequenced and analyzed 66 complete and 20 nearly complete RABV genomes, including those from the Flagstaff area and other similar outbreaks in carnivores, caused by bat RABVs, and representatives of the major RABV lineages circulating in North America and worldwide. Phylogenetic analysis demonstrated that each Flagstaff outbreak was caused by an independent introduction of bat RABV into populations of carnivores. Positive selection analysis confirmed the absence of post-shift changes in RABV genes. In contrast, convergent evolution analysis demonstrated several amino acids in the N, P, G and L proteins, which might be significant for pre-adaptation of bat viruses to cause effective infection in carnivores. The substitution S/T₂₄₂ in the viral glycoprotein is of particular merit, as a similar substitution was suggested for pathogenicity of Nishigahara RABV strain. Roles of the amino acid changes, detected in our study, require additional investigations, using reverse genetics and other approaches.

Figure 1. Bayesian tree of viruses, included in the study, based on the entire coding region of the G gene (1572 nuc).

Lineage abbreviations: EF-W1 and EF-W2 – Eptesicus fuscus, with predominantly western distribution; MYu – Myotis yumanensis; LX – Lasiurus xanthinus; LS – Lasiurus seminolus; LC – Lasiurus cinereus; LB – Lasiurus borealis; PS – Perimyotis subflavus; LN – Lasionycteris noctivagans; LI – Lasiurus intermedius; TB – Tadarida brasiliensis; DR – Desmodus rotundus; MYsp – Myotis spp; PH – Parastrellus hesperus; AP – Antrozous pallidus; EF-E1 and EF-E2 – Eptesicus fuscus, with predominantly eastern and central distribution; EFu – Eptesicus furinalis; SCSK – south-central skunk; RAC – North-American Raccoon; MexSK-1 – Mexican skunk, variant 1; SE Asia 1, 2 and 3 – diverse dog RABV lineages circulating in the South-East Asia; Africa-2 – dog RABV lineage from the central and western Africa; CASK – California skunk; MexSK-2 – Mexican skunk, variant 2; TXFX – Texas gray fox; COY – coyote; DOG LA – dog RABV from Latin America; AZFX – Arizona gray fox; NCSK – north-central skunk; EUR – fox viruses from moderate latitudes of Eurasia; Aftrca-1 – dog RABV, broadly distributed in Africa; Africa-3 – mongoose RABV from southern Africa; Arctic – Arctic RABV from Eurasia and North America; Arctic-like – Arctic-like RABV from southern and eastern Asia.

Figure 2. Bayesian tree of the EF-W1 lineage.

Skunk viruses are colored in red, fox viruses are colored in blue, other mesocarnivoran viruses are colored in green; bat viruses are black. Posterior probabilities are indicated at key nodes.

Figure 3. Reconstruction of post-host shift amino acid changes.

Left: Bayesian tree of EF-W1 lineage, based on the entire coding region of G gene (1572 nuc). The branches involved in host-shift are marked with red. The amino acid changes occurred within these branches were defined “post-host shift” changes. Other branches that lead to terrestrial mammal RABV are marked with green. Right: the magnification of the three clusters involved in the Flagstaff host shifts. For clarity, the branch length did not reflect substitutions per site. The branches with non-synonymous changes are marked with pink, and numbers of changes are shown above and below. For more details see Table 2.