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.

Author Summary

Host shifts of the rabies virus (RABV) from bats to carnivores are important for our understanding of viral evolution and emergence, and have significant public health implications, particularly for the areas where “terrestrial” rabies has been eliminated. In this study we addressed several rabies outbreaks in carnivores that occurred in the Flagstaff area of Arizona during 2001–2009, and caused by the RABV variant associated with big brown bats (Eptesicus fuscus). Based on phylogenetic analysis we demonstrated that each outbreak resulted from a separate introduction of bat RABV into populations of carnivores. No post-shift changes in viral genomes were detected under the positive selection analysis. Trying to answer the question why certain bat RABV variants are capable for host shifts to carnivores and other variants are not, we developed a convergent evolution analysis, and implemented it for multiple RABV lineages circulating worldwide. This analysis identified several amino acids in RABV proteins which may facilitate host shifts from bats to carnivores. Precise roles of these amino acids require additional investigations, using reverse genetics and animal experimentation. In general, our approach and the results obtained can be used for prediction of host shifts and emergence of other zoonotic pathogens.

Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent in Thailand, causing a huge impact on the country's swine industry. Yet the diversity and origin of these Thai PRRSVs remained vague. In this context, we collected all the Thai PRRSV sequences described earlier and incorporated them into the global diversity. The results indicated that PRRSVs in Thailand were originated from multiple introductions involving both Type 1 and Type 2 PRRSVs. Many of the introductions were followed by extensive geographic expansion, causing regional co-circulation of diverse PRRSV variants in three major pig-producing provinces. Based on these results, we suggest (1) to avoid blind vaccination and to apply vaccines tailor-made for target diversity, (2) to monitor pig importation and transportation, and (3) to implement a better biosecurity to reduce horizontal transmissions as three potentially effective strategies of controlling PRRS in Thailand.

Type 2 (or North American-like) porcine reproductive and respiratory syndrome virus (PRRSV) was first recorded in 1987 in the United States and now occurs in most commercial swine industries throughout the world. In this study, we investigated the epidemiological and evolutionary behaviors of type 2 PRRSV. Based on phylogenetic analyses of 8,624 ORF5 sequences, we described a comprehensive picture of the diversity of type 2 PRRSVs and systematically classified all available sequences into lineages and sublineages, including a number of previously undescribed lineages. With the rapid growth of sequence deposition into the databases, it would be technically difficult for veterinary researchers to genotype their sequences by reanalyzing all sequences in the databases. To this end, a set of reference sequences was established based on our classification system, which represents the principal diversity of all available sequences and can readily be used for further genotyping studies. In addition, we further investigated the demographic histories of these lineages and sublineages by using Bayesian coalescence analyses, providing evolutionary insights into several important epidemiological events of type 2 PRRSV. Moreover, by using a phylogeographic approach, we were able to estimate the transmission frequencies between the pig-producing states in the United States and identified several states as the major sources of viral spread, i.e., “transmission centers.” In summary, this study represents the most extensive phylogenetic analyses of type 2 PRRSV to date, providing a basis for future genotyping studies and dissecting the epidemiology of type 2 PRRSV from phylogenetic perspectives.