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Enzootic transmission of yellow fever virus, Venezuela.

Yellow 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 (1-5). YFV emergence is cyclical; outbreaks occur [approximately equal to] 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. We sequenced a prM/E gene fragment of 10 YFV isolates from 4 locations within Venezuela, spanning 6 years (2004-2010; Table 1). Additionally, we sequenced complete genomes for 5 representative isolates for comparison with 12 previously determined genomic sequences (4,9).

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+[[GAMMA].sub.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 (2 A) from Guarico 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 side of Venezuela (including Portuguesa, Apure, and Barinas States) (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.

Although YFV had been maintained in situ for several years within Venezuela, our phylogeographic results indicated YFV movement between Brazil, Trinidad, and Venezuela. Brazil is the major source of YFV introductions into Venezuela, accounting for introductions of Venezuelan strains sampled in 1959, 1961, 1998, and more recently (i.e., independent introductions in 2004 in eastern and western Venezuela; Figure 1). The basal location of a 2004 sequence from the Brazilian Amazon within the western Venezuelan 2004-2007 clade (Figure 1) suggests that the 2005 Venezuelan epizootic/epidemic was initiated by an imported progenitor from the Brazilian Amazon Basin that later evolved independently within Venezuela. Our estimate of the most recent common ancestor for the node containing the 2004-2007 Venezuelan sequences was 2001 (95% highest posterior density 1997-2003), suggesting that the ancestral lineage existed for [approximately equal to] 3 years in Venezuela before its detection in nonhuman primates in 2004.

[FIGURE 1 OMITTED]

The 2004 Venezuelan (1A) intermediary descendent between the 1995 and 2009 Trinidad sequences is noteworthy. 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.

[FIGURE 2 OMITTED]

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.

DOI: http://dx.doi.org/10.3201/eid2101.140814

Acknowledgments

We thank Lola Bravo, Cinda Martmez, and Humberto Montanez for help with virus isolations.

This work was supported by a grant from the National Institute of Allergy and Infectious Disease through the Western Regional Center of Excellence for Biodefense and Emerging Infectious Disease Research, National Institutes of Health grant U54 AIO57156. A.J.A. was supported by the McLaughlin Fellowship Fund. J.C.N. was supported by El Fondo Nacional de Ciencia, Tecnologia e Innovacion, Mision Ciencia 2008000911-4, Ministerio de Ciencia y Tecnologia.

The GenBank accession numbers for the YFV sequences derived in this study are KM388814-KM388823.

References

(1.) Monath TP. Yellow fever. In: Monath TP, editor. The arboviruses: ecology and epidemiology. Vol. 5. Boca Raton (FL): CRC Press. 1989. p. 139-231.

(2.) Van der Stuyft P, Gianella A, Pirard M, Cespedes J, Lora J, Peredo C, et al. Urbanisation of yellow fever in Santa Cruz, Bolivia. Lancet. 1999;353:1558-62. http://dx.doi.org/10.1016/S0140- 6736(99)03291-2

(3.) Figueiredo LT. The Brazilian flaviviruses. Microbes Infect. 2000;2:1643-9. http://dx.doi.org/10.1016/S1286-4579(00)01320-4

(4.) Auguste AJ, Lemey P, Pybus OG, Suchard MA, Salas RA, Adesiyun AA, et al. Yellow fever virus maintenance in Trinidad and its dispersal throughout the Americas. J Virol. 2010;84: 9967-77. http://dx.doi.org/10.1128/JVI.00588-10

(5.) de Souza RP, Foster PG, Sallum MA, Coimbra TL, Maeda AY, Silveira VR, et al. Detection of a new yellow fever virus lineage within the South American genotype I in Brazil. J Med Virol. 2010;82:175-85. http://dx.doi.org/10.1002/jmv.21606

(6.) Bryant J, Wang H, Cabezas C, Ramirez G, Watts D, Russell K, et al. Enzootic transmission of yellow fever virus in Peru. Emerg Infect Dis. 2003;9:926-33. http://dx.doi.org/10.3201/eid0908.030075

(7.) Vasconcelos PF, Bryant JE, da Rosa TP, Tesh RB, Rodrigues SG, Barrett AD. Genetic divergence and dispersal of yellow fever virus, Brazil. Emerg Infect Dis. 2004;10:1578-84. http://dx.doi.org/10.3201/eid1009.040197

(8.) Munoz-Rodriguez M, Arrivillaga J, Navarro J-C. Casos de fiebre amarilla en Portuguesa, Venezuela: un brote selvatico espurio? Revista Biomedica. 2010;21:163-77.

(9.) Nunes MR, Palacios G, Cardoso JF, Martins LC, Sousa EC Jr, de Lima CP, et al. Genomic and phylogenetic characterization of Brazilian yellow fever virus strains. J Virol. 2012;86:13263-71. http://dx.doi.org/10.1128/JVI.00565-12

(10.) Martin DP. Recombination detection and analysis using RDP3. Methods Mol Biol. 2009;537:185-205. http://dx.doi.org/10.1007/978-1-59745-251-9_9

(11.) Drummond AJ, Suchard MA, Xie D, Rambaut A. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol. 2012;29:1969-73. http://dx.doi.org/10.1093/molbev/mss075

(12.) Lemey P, Rambaut A, Drummond AJ, Suchard MA. Bayesian phylogeography finds its roots. PLOS Comput Biol. 2009;5:e1000520. http://dx.doi.org/10.1371/journal.pcbi.1000520

(13.) Parker J, Rambaut A, Pybus OG. Correlating viral phenotypes with phylogeny: accounting for phylogenetic uncertainty. Infect Genet Evol. 2008;8:239-46. http://dx.doi.org/10.1016/j.meegid.2007.08.001

(14.) Huelsenbeck JP, Ronquist F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics. 2001;17:754-5. http://dx.doi.org/10.1093/bioinformatics/17.8.754

Author affiliations: University of Texas Medical Branch, Galveston, Texas, USA (A.J. Auguste, N.A. Bergren, S.C. Weaver); Rega Institute, Leuven, Belgium (P. Lemey); Ciudad Universitaria, Caracas, Venezuela (D. Giambalvo, M. Moncada, D. Moron, R. Hernandez); and Universidad Central de Venezuela, Caracas (J.-C. Navarro)

Dr. Auguste is a postdoctoral fellow in the Department of Pathology, University of Texas Medical Branch, Galveston, Texas. His research interests include understanding the ecologic and evolutionary factors involved in emergence, dispersal, and maintenance of arboviruses and the genetic and structural characterization of novel arboviruses.

Address for correspondence: Scott C. Weaver, Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555-0610, USA; email: sweaver@utmb.edu
Table 1. Yellow fever virus strains sequenced in the
study and their metadata, Venezuela *

Isolate ID            Source          Location

1A               Red howler monkey    Monagas
2A ([dagger])    Red howler monkey    Guarico
3A               Red howler monkey   Portuguesa
4A               Red howler monkey   Portuguesa
5A                     Human         Portuguesa
6A ([dagger])          Human         Portuguesa
7A                     Human         Portuguesa
8A ([dagger])    Red howler monkey    Barinas
9A ([dagger])    Red howler monkey     Apure
10A ([dagger])   Red howler monkey    Monagas

Isolate ID        Year of     Passage      GenBank
                 collection   history   accession no.

1A                  2004      Vero 2      KM388819
2A ([dagger])       2004      Vero 2      KM388817
3A                  2005      Vero 2      KM388820
4A                  2005      Vero 2      KM388821
5A                  2005      Vero 2      KM388822
6A ([dagger])       2005      Vero 2      KM388814
7A                  2005      Vero 2      KM388823
8A ([dagger])       2006      Vero 2      KM388818
9A ([dagger])       2007      Vero 2      KM388815
10A ([dagger])      2010      Vero 2      KM388816

* ID, identification. Red howler monkey, Alouatta seniculus species.

([dagger]) Complete genome sequences determined.

Table 2. Nucleotide and amino acid divergence among individual
YFV genes of 9 representative YFV strains compared to the
TVP11767 * strain ([dagger])

          Strain, nucleotide (amino acid) divergence, %

           BeH622205      BeAR51300      BeH423602
Genes      (B, 2000)     8 (B, 1992)     (B, 1984)

Capsid     98.3 (100)     98.0 (100)     97.2 (100)
PreM/M    97.7 (99.4)     96.7 (100)    91.2 (98.1)
E          98.2 (100)     96.3 (100)    89.6 (98.6)
NS1       97.9 (99.7)     97.3 (100)    90.5 (99.4)
NS2A&B     97.4 (100)    96.2 (99.4)    90.4 (99.4)
NS3        97.4 (100)     96.7 (100)     88.4 (100)
NS4A&B     98.2 (100)    96.8 (99.7)    90.3 (98.7)
NS5       98.3 (99.8)    96.6 (99.8)    91.1 (99.1)

          Strain, nucleotide (amino acid) divergence, %

           BeH413820      BeH394880      BeH655417
Genes      (B, 1983)      (B, 1981)      (B, 2002)

Capsid    91.2 (97.4)     96.3 (100)    97.2 (99.1)
PreM/M     97.3 (100)     97.3 (100)    96.2 (99.4)
E          96.9 (100)     97.5 (100)     96.6 (100)
NS1       97.5 (99.7)     98.1 (100)    96.8 (99.7)
NS2A&B     96.8 (100)     97.6 (100)    95.8 (99.1)
NS3        97.4 (100)     97.4 (100)     96.3 (100)
NS4A&B    97.1 (99.7)     97.8 (100)    97.1 (99.7)
NS5       96.8 (99.8)     98.0 (100)     96.6 (100)

          Strain, nucleotide (amino acid) divergence, %

          6A (V, 2005)   9A (V, 2007)       10A
Genes                                    (V, 2010)

Capsid    97.2 (99.1)    97.2 (99.1)     96.6 (100)
PreM/M     97.7 (100)     97.7 (100)     97.7 (100)
E          97.2 (100)     97.0 (100)     97.6 (100)
NS1        98.7 (100)     98.4 (100)     98.6 (100)
NS2A&B     97.2 (100)    96.9 (99.4)     97.6 (100)
NS3        97.4 (100)     97.4 (100)     96.7 (100)
NS4A&B    98.1 (99.7)    98.0 (99.7)     98.2 (100)
NS5        98.4 (100)     98.1 (100)     98.2 (100)

* First South American complete genome sequence to be published.
TVP11767 was isolated in 2009 from an Alouatta seniculus specimen
in Trinidad.

([dagger]) YFV, yellow fever virus; B, Brazil; V, Venezuela;
M, membrane; E, envelope; NS, nonstructural.
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Title Annotation:DISPATCHES
Author:Auguste, Albert J.; Lemey, Philippe; Bergren, Nicholas A.; Giambalvo, Dileyvic; Moncada, Maria; Moro
Publication:Emerging Infectious Diseases
Geographic Code:3VENE
Date:Jan 1, 2015
Words:2449
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