Enzootic Transmission of Yellow Fever Virus, Venezuela

Phylogenetic analysis of yellow fever virus (YFV) strains isolated from Venezuela strongly supports YFV maintenance in situ in Venezuela, with evidence of regionally independent evolution within the country. However, there is considerable YFV movement from Brazil to Venezuela and between Trinidad and Venezuela.

Y ellow fever virus (YFV) is the prototype species for the genus Flavivirus. Historically, YFV is one of the most important human arboviral pathogens. It continues to cause large sporadic epidemics in Africa but typically emerges as epizootics among nonhuman primates in South America with or without associated human cases (1)(2)(3)(4)(5). YFV emergence is cyclical; outbreaks occur ≈7-10 years apart. Several phylogenetic studies have shown that YFV is locally maintained during these interepizootic periods in Peru (6), Brazil (7), and Trinidad (4). These studies also have indicated that the virus undergoes regionally independent evolution within some countries (6).
YFV has caused sporadic outbreaks in Venezuela; the most recently documented epizootic/epidemic occurred in 2005 (8). Although endemic to Venezuela, YFV has very rarely been isolated and characterized, and partial sequences have been determined only for 4 strains. Venezuela is located between Trinidad and Brazil, which have contributed major evidence for the enzootic maintenance of YFV in South America. Thus, sequencing Venezuelan YFV strains over a wide geographic area and temporal distribution might be valuable to test the hypothesis of local maintenance in Venezuela and to determine whether the virus moves regularly between Trinidad and Venezuela or between Venezuela and Brazil. Understanding the maintenance and spread of YFV in South America also is critical for developing effective surveillance and prevention strategies.

The Study
The sporadic emergence of YFV in the Americas has been strongly associated with infection of red howler monkeys (Alouatta seniculus), which are particularly susceptible to disease. As exemplified in this study, nonhuman primate surveillance targeting this species remains an efficient strategy for monitoring enzootic YFV activity. Isolates made during a surveillance study aimed at investigating the ecology of infectious diseases in Venezuelan nonhuman primates were detected by cell culture and passaged once in Vero cells before sequencing, as previously described (4). Sequences were manually aligned in Se-Al (http://tree.bio.ed.ac.uk/software/seal/) and confirmed as nonrecombinant by using Recombination Detection Program (RDP4) (10). We obtained phylodynamic and phylogeographic estimates using Bayesian inference as implemented in BEAST v1.8.0 (11,12). We assessed the extent of geographic structuring using Bayesian tip-significance testing (13) based on the Markov chain Monte Carlo phylogenies estimated in BEAST. A Bayesian phylogeny was also inferred in Mr. Bayes (14) by using the general time reversible (GTR+I+Γ 4 ) model for the complete open reading frame sequences.
Results of Bayesian tip-significance testing showed statistically significant geographic clustering among Venezuelan YFV strains. The association index, parsimony score, and maximum monophyletic clade statistics provided strong support (p<0.01) that strains from Venezuela cluster by location, suggesting that YFV is maintained for long periods within Venezuela. Similar results have been shown for Peru (6), Brazil, and Trinidad (4). The high posterior probabilities, >0.99, observed at all nodes that delineate Venezuelan strains further support these conclusions ( Figure 1).
We also found evidence of regionally independent evolution within Venezuela, as indicated by the existence of 2 phylogenetically distinct Venezuelan clades with posterior probabilities >0.99 (Figures 1, 2). The clade containing a 2004 strain (2A) from Guárico and a 2010 strain (10A) from Monagas represented all but one of the sequences from eastern Venezuela (i.e., east of Caracas; Table 1). The 7 other strains were collected on the western  Table 1). Two YFV strains were collected in 2004 from eastern and western Venezuela. Despite their nearly synchronous collections in 2004, these sequences fell into distinct clades in the maximum clade credibility (MCC) phylogeny, indicating population subdivision. Although we cannot rule out sampling bias, these data suggest in situ evolution of YFV in Venezuela and regionally independent evolution in distinct geographic foci within the country. The mechanism promoting this population subdivision among YFV strains is unclear and requires further investigation. Further studies on the ecology of the areas where these viruses were isolated might help explain the observed population subdivision.

Enzootic Transmission of Yellow Fever Virus, Venezuela
Although YFV had been maintained in situ for several years within Venezuela, our phylogeographic results indicated YFV movement between Brazil, Trinidad, and   This cluster of Trinidad isolates was previously used as evidence of enzootic YFV maintenance within Trinidad during interepizootic periods (4). Incorporating our new sequences now shows movement between Trinidad and eastern Venezuela. In our phylogeny, strain 1A is the sister lineage of the 2008-09 Trinidad epizootic strains, with the 1995 Trinidadian isolate lying basal to these sequences, with high posterior probabilities ( Figure 1). The position of the 2004 Venezuelan 1A sequence possibly reflects importation from Trinidad, which implies that enzootic YFV circulation in Trinidad is not isolated epidemiologically but gave rise to exportation of YFV to Venezuela. Substitutions resulting from nonsynonymous, synapomorphic mutations that define sequences in a clade/lineage are highlighted at relevant nodes. Two substitutions (NS2b A98T and NS5 A200T) occurred in earlier isolates from Brazil. The capsid A79V and NS5 A200T substitutions include the Brazilian isolate BeH655417, which lies directly basal to the Venezuela isolates. This indicates that these substitutions were probably present in the YFV progenitor when it was introduced into Venezuela. Furthermore, substitutions NS2a V10I, NS2a S184T, and NS2b A98T all appear to have arisen after YFV was introduced to Venezuela, further supporting enzootic YFV maintenance there. Taxon/tip labels include year of isolation, strain names and country where the virus was isolated. Scale bar indicates percentage of nucleotide sequence divergence. In the absence of more dense sampling, whether bidirectional YFV movement occurs between Venezuela and Trinidad is unclear. Given the proximity and boating traffic between these countries, substantial mixing between their YFV populations would not be surprising. Isolation and sequencing of additional YFV isolates from eastern Venezuela are needed to further evaluate movement between Trinidad and Venezuela.
Complete genomes were sequenced for 5 representative Venezuelan YFV strains from eastern and western Venezuela ( Table 1). Comparison of nucleotide and amino acid similarities showed a high degree of conservation across YFV genes ( Table 2). The 9 strains selected for comparisons represent the full spectrum of known YFV genetic diversity (Table 2; Figure 2). The most diverse genes shared >97% aa sequence identity, with >99.9% aa sequence identity for most proteins, even though these strains were collected >30 years apart ( Table 2). The Bayesian Markov chain Monte Carlo phylogeny based on complete open reading frame sequences strongly supported the inferred maximum clade credibility tree, with all Venezuelan sequences grouping together with strong posterior support. The eastern and western Venezuelan strains grouped separately, with strong support in all of our inferred phylogenies. A total of 5 substitutions delineated the Venezuelan sequences ( Figure 2).

Conclusions
Our phylogeographic analysis supports in situ evolution of YFV within Venezuela, as well as regionally independent evolution within the country. Brazil was identified as the major source of YFV introductions into Venezuela, and sequence analysis showed that considerable YFV movement may occur between Trinidad and Venezuela. Results of our Bayes factor test for non-zero rates also support the epidemiologic link between Venezuela, Brazil, and Trinidad ( Figure 1, panel B). The sequences generated in our study fill a major gap in the geographic sampling of YFV.