Enzootic Activity of Pixuna and Rio Negro Viruses (Venezuelan Equine Encephalitis complex) in a Neotropical Region of Argentina

Introduction

T he Venezuelan Equine Encephalitis (VEE) complex comprises 14 subtypes and varieties, as well as seven different related Alphavirus species that are members of the Family Togaviridae (Weaver et al. 2000). Epidemiologically, they are classified as enzootic and epizootic viruses and comprise a serocomplex of antigenically related alphaviruses (Young and Johnson 1969). Enzootic viruses (subtypes ID, IE, and IF, and subtypes II–VI) are transmitted among rodent reservoirs by mosquitoes in sylvatic or swamp habitats, and only occasionally produce human disease. Epizootic viruses (subtypes IAB and IC) emerge periodically in regions of Central America and northern areas of South America (Venezuela, Colombia), causing outbreaks that involve humans and horses, with high morbidity and mortality rates (Powers et al. 1997). Both enzootic and epizootic VEE complex virus strains have been detected in different mosquito genera: Culex (Cx.), Aedes (Ae.), Mansonia (Ma.), and Psorophora (Ps.) (Mitchell et al. 1985), and Ae. and Ps. (Carrara et al. 2007), respectively.

In Argentina, enzootic strains identified as Rio Negro virus (AG80–663) (RNV), associated with acute febrile illness in humans (in Formosa province) (Contigiani et al. 1999), have been isolated from several mosquito species (Chaco province) (Mitchell et al. 1985) and from rodents. In addition, serological studies indicate the presence of human antibodies against more than one enzootic subtype (I and VI) in neotropical areas of Argentina (Cámara et al. 2003). For this reason, surveillance activities were carried out to detect circulation of VEE complex viruses in Chaco province (Argentina).

Materials and Methods

During December 2003 and from February to April 2004, mosquitoes were collected using CDC light traps supplemented with CO₂ in Monte Alto (27°26′38″S, 58°55′3″W) and Resistencia (27°27′76″S, 58°59′79″W) (Chaco Province). Monte Alto is a rural area that belongs to semi-humid Chaco biome, with few anthropogenic changes and livestock activity, 500 m away from Rio Negro River; sampling activities in Resistencia (capital city of Chaco Province) were carried out at campus of National University of Northeast. Collected mosquitoes were transported alive under cold conditions to the laboratory; pooled by species and place of collection and date, in lots of 1 to 58 unengorged individuals; and stored at −70°C until they were processed. Each pool for viral testing was triturated in a cold mortar with sterile minimum essential medium supplemented with 10% fetal bovine serum and 1% gentamicin, and clarified by centrifugation at 11400 g for 30 min. The supernatant was poured into individual screw-cap vials and frozen at −70°C, until RNA detection by RT-Nested PCR assay was performed.

Viral RNA was extracted with Trizol reagent (Invitrogen BRL; Life Technologies, Rockville, MD) according to the manufacturer's instructions. Detection of a 195 bp fragment of the nsP4 region of Alphavirus was performed by RT-Nested PCR (Sánchez-Seco et al. 2001). Positive alphavirus samples were purified using Quiaquick Gel Extraction Kit (Quiagen, Valencia, CA) and submitted to direct nucleotide sequencing reaction in both directions. Obtained sequences were submitted to the GenBank, and relatedness of newly characterized sequences was assessed by Basic Local Alignment Search Tool (BLAST). Phylogenetic analysis was constructed with Mega version 4 software using the Maximum Parsimony method (Tamura et al. 2007). Minimum infection rates (MIR) were calculated using PooledInfRate software version 3.0 (Biggerstaff 2008).

Results and Discussion

A total of 2057 mosquitoes (99 pools) belonging to 33 species were analyzed for Alphavirus detection. Five mosquito pools were positive: three from Monte Alto and two from Resistencia. The MIRs obtained showed more viral activity in Monte Alto (rural area) than in Resistencia (urban area) (Table 1). Phylogenetic analysis showed that four sequences clustered with RNV and one with Pixuna Virus (PIXV) (subtype IV) (Fig. 1). Although Ochlerotatus (Oc.) scapularis (31.7%), Cx. bidens (14.7%), and Ma. titillans (11.3%) were the most abundant species analyzed, RNV genome was detected in Cx. coronator, Cx. maxi, Ps. cingulata, and Ps. spp., and PIXV genome in Oc. hastatus oligopistus. None of these species had been previously found to be infected in Argentina (Mitchell et al. 1985). Moreover, Mitchell et al. isolated 12 RNV strains from Cx. (Melanoconion) delpontei, with high MIRs (range 1.3–2.5), indicating its possible role as an enzootic vector for this virus. In the present study, few mosquito pools from that species were analyzed, and no viruses were detected. These data could indicate changes in ecoepidemiological patterns for RNV in this region over time.

OPEN IN VIEWER

FIG. 1.

Phylogenetic tree showing evolutionary history inferred using the Maximum Parsimony method (Close-Neighbor-Interchange algorithm). The number of replicate trees (≥50%) in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.

Previous studies in Argentina have detected enzootic RNV in mosquitoes of the neotropical region of the country (Mitchell et al. 1985). Our results confirm that this virus is still circulating there. However, PIXV (VEE complex subtype IV) had never been detected in Argentina; this is the first report of its molecular detection in the country. This virus has been isolated in Belem (Brazil) in 1961 (Shope et al. 1964) and is still poorly characterized. There is no evidence of its association with human and animal diseases reported in the literature.

All these data demonstrate the circulation of more than one enzootic subtype of VEE complex viruses in Argentina, which could lead to recombination of strains, resulting in the emergence of new pathogenic strains (by interaction between them or by genetic mechanisms of adaptation to new hosts). For this reason, we consider it important to enhance the surveillance in the entire mentioned region.