Elimination of Enzootic Dog RVs from the United States
The domestic dog–coyote RV variant lineage was found in a total of 122 samples from either dogs or coyotes. Most (118) of these were found in Texas during 1991–2004 (, ; Appendix Figure
). The overall genetic identity among members of this lineage in the period analyzed was 98.9%. This lineage was monophyletic with an extinct enzootic dog rabies lineage distributed in central Mexico and west-central Mexico until the year 2000. An average identity value of 97.6% was noticed between these 2 lineages (). A consensus amino acid change at position 134 (V replaced by I) within the nucleoprotein gene was observed in both lineages. However, a unique amino acid change at position 426 (S replaced by T) was characteristic for the domestic dog–coyote RVs. Similarly, the Sonora dog lineage, distributed along the western United States–Mexico border until 1994, was monophyletic with extant lineages circumscribed southward in west-central Mexico, southeastern Mexico (Chiapas), and Central America (, ). The overall genetic distance among samples within this lineage was 0.017 (98.3% identity). When compared with its closest relatives in southeastern Mexico and Central America, the identity value was 97.7% (,). An amino acid change at position 40 within the nucleoprotein gene (C replaced by S), common to dog-related rabies enzootics in Africa and Asia (Africa 2 and 3, arctic fox and Arctic-like, plus all dog-related lineages in Asia and India), was found in this lineage. Within the United States, the Sonora dog lineage was detected in 2 dogs, 2 coyotes, and 1 bobcat in counties bordering northeast Mexico in 1993, 1995, and 1996, respectively, and in 1 dog in San Diego, California, in 1990.
Figure 1 Neighbor-joining phyogenetic tree reconstructed by using entire nucleoprotein sequences that show the consensus topology observed with maximum-likehood and Bayes methods (www.mrbayes.net). The hierarchy encompassing phylogroups, clades, groups, lineages, (more ...)
Figure 2 Current distribution of major rabies virus (RV) lineages associated with terrestrial carnivores and dogs in the United States and Mexico. Translocation movements proposed on the basis of the phylogenetic analysis (bidirectional arrows in colors) and confirmed (more ...)
The domestic dog–coyote RV variant and Sonora dog lineage enzootics were expected to be related because of their geographic proximity (). However, the phylogenetic data demonstrated that the Sonora dog lineage originated from independent dog rabies enzootics translocated from Central America, Honduras, Nicaragua, and Michoacán in west-central Mexico (, ).
The Texas gray fox RV variant was detected in Texas only, from 8 dogs and 3 coyotes during 1991–1995, and in 20 coyotes in the western part of Texas during 2007 (Appendix Figure
). Members of the Texas gray fox variant or lineage presented a consensus amino acid change within the nucleoprotein gene at position 247 (K replaced by R) and an overall identity value of 99.1%. In 1986, a rabid gray fox from Arizona was found with the Arizona gray fox RV variant. This variant was also found circulating in northwestern Mexico (,); 3 consensus amino acid changes within the nucleoprotein gene at positions 9 (K replaced by R), 13 (Q replaced by H), and 421 (I replaced by V) characterized Arizona gray fox RV variant samples among all other extinct and extant enzootic canine-related variants and lineages circulating throughout the world. The average identity value for members of the Arizona gray fox RV variant lineage was 98.8%.
The Texas gray fox RV variants are enzootic to Texas, and the Arizona gray fox RV variants are enzootic to Arizona and northwestern Mexico, respectively; average identity value between variants is 92.9% (). Distinctive consensus amino acid and nucleotide changes at the nucleoprotein gene have become fixed and have remained stable over time in these RVs circulating in gray fox populations in the southern United States. All lineages were monophyletic with historic RV (Flury high egg passage and low egg passage, CA human 1954, and NY dog 1949) obtained from virus repositories or vaccine strains that were originally recovered from canine rabies cases that occurred in the United States before dog rabies elimination (). The topology in group II () details the circulation of at least 2 canine rabies lineages representing different dog rabies enzootics (before dog rabies elimination in the United States): one closely related to the Texas gray fox variant circumscribed in California (CA human 1954) and the other widely circulating over the East Coast, related to the Flury and NY dog sequences. This group also encompassed surrogates of enzootic dog rabies in Europe, which indicated an epidemiologic link between dog rabies epizootics on both continents ().
The Puerto Rico mongoose RV variant was found in 2 mongooses and 1 dog. This lineage presented a homogeneous identity value of 99.1% and had common amino acid changes with Africa 1b and Africa 3 at position 254 (R replaced by K) and with the Arctic-like and Street-Alabama-Dufferin B19 RV at position 135 (S replaced by P), consistent with an Old World origin of these viruses. A characteristic amino acid change for this lineage was found at position 181 (I replaced by V). This variant was monophyletic with 2 North American lineages: the north-central skunk and the west Canada skunk RV. These skunk lineages, and the Puerto Rico mongoose lineage, were also monophyletic with Street-Alabama-Dufferin B19 derivatives and with a California skunk lineage, integrating group VI, which presented a low bootstrap value (). Nonetheless, group VI was consistent when different methods for phylogenetic reconstruction were used (maximum likelihood and Bayes data are available on request). Skunk RVs in the north-central United States, southwestern Canada, and California were monophyletic with Street-Alabama-Dufferin B19 and Pasteur virus strains, both surrogates for enzootic dog rabies before its elimination in the United States and Europe, respectively. These results clearly illustrate the association of enzootic skunk rabies with historic dog rabies enzootics ().
To underscore the importance of transcontinental translocation of dog-related rabies into the United States and also to illustrate that such phenomena still occur in the 21st century, during 2007 we studied a rabid dog imported from India into Washington state and then transported into Alaska. The infecting strain was determined to be the arctic fox–like variant, which is mainly enzootic in dog populations in northern India and central Asia ().
Conversely, interspecies transmission of rabies into domestic dogs and wildlife was also observed in this study. Two dogs, 1 from Texas and the other from New Mexico, were typed as the Tadarida brasiliensis RV variant, whereas 1 coyote from Texas was identified with the south-central skunk RV variant. These findings illustrate that the interspecies transmission of rabies is a process that also may occur with regularity.
Canine and Wildlife Rabies in the United States within a Historical and Global Context
To better understand dog rabies epizootiology in the United States, we also conducted a global analysis. Samples were divided into 2 main phylogroups, 1 associated with terrestrial carnivores and the other associated with bat rabies in the Americas. The terrestrial phylogroup was integrated by the cosmopolitan clade (CosC) that comprised at least 8 monophyletic groups (I to VIII, ). CosC encompassed 2–4 lineages representing independent enzootics associated with not only different species of Canidae (domestic dogs, jackals, wolves, gray foxes, red foxes, arctic foxes, raccoon dogs, Corsac foxes, crab-eating foxes, coyotes) but also with other species of terrestrial carnivores (mainly skunks, such as the Baja California skunk in Mexico and California skunks in the United States). Each of these groups consistently presented at least 1 lineage associated with rabies enzootics in domestic dogs (either extinct or extant). The remaining lineages were associated with rabies enzootics in terrestrial carnivores. The exceptions were groups VII and VIII, associated with either domestic dogs (Africa 4) or the Baja California spotted skunk Spilogale putorious (). These phylogenetic patterns were consistent throughout Europe, Africa, and the Americas, where enzootic dog rabies was introduced (). In addition, several other independent dog-related RV lineages were geographically circumscribed in different countries of the Americas, such as Argentina, Colombia, Bolivia, Peru, Ecuador, Nicaragua, and the Dominican Republic, and belong to the CosC as well (only partial nucleoprotein sequences available; tree available from the authors on request).
The average genetic distance among all members within CosC was 0.071%, equivalent to 93% average identity. The highest genetic distance observed between groups pertaining to this clade was 0.104 (89.6% average identity) and occurred between group III and group VIII (). Meanwhile, the lowest genetic distance observed was between group I and group II 0.073 (92.7% average identity), followed by the same genetic distance of 0.074 (92.6%) between groups IV–V, IV–VI, and V–VI (). The terrestrial phylogroup also encompassed independent groups and lineages associated with enzootic dog rabies in Asia (this group was the most divergent with an average genetic distance of 0.126, 87.4%, encompassing lineages from China, Thailand, Vietnam, and India), Africa (Africa 2, group IX with an average genetic distance of 0.079, 92.1%), the arctic fox and the arctic fox–like lineages (Arctic/Arctic-like group IX, with an average genetic distance of 0.044, 95.6%), and the African mongoose lineage (Africa 3, with an average genetic distance of 0.021, 97.9%).
Conversely, the bat phylogroup comprised RV lineages and variants associated with independent rabies enzootics maintained by different species of bats and 2 independent enzootics established in raccoons (raccoon variant) and skunks (south-central US skunk and Mexican north-central skunk), which may represent autochthonous RV lineages in the New World (, ). These lineages have an average genetic distance of 0.155 (84.5%) to 0.158 (84.7%) when compared with RV lineages associated with the terrestrial carnivore clade.