Isolation of Tacaribe virus, a Caribbean arenavirus, from host-seeking Amblyomma americanum ticks in Florida

Abstract

Arenaviridae are a family of single stranded RNA viruses of mammals and boid snakes. Twenty-nine distinct mammalian arenaviruses have been identified, many of which cause severe hemorrhagic disease in humans, particularly in parts of sub-Saharan Africa, and in Central and South America. Humans typically become infected with an arenavirus through contact with excreta from infected rodents. Tacaribe virus (TCRV) is an arenavirus that was first isolated from bats and mosquitoes during a rabies surveillance survey conducted in Trinidad from 1956 to 1958. Tacaribe virus is unusual because it has never been associated with a rodent host and since that one time isolation, the virus has not been isolated from any vertebrate or invertebrate hosts. We report the re-isolation of the virus from a pool of 100 host-seeking Amblyomma americanum (lone star ticks) collected in a Florida state park in 2012. TCRV was isolated in two cell lines and its complete genome was sequenced. The tick-derived isolate is nearly identical to the only remaining isolate from Trinidad (TRVL-11573), with 99.6% nucleotide identity across the genome. A quantitative RT-PCR assay was developed to test for viral RNA in host-seeking ticks collected from 3 Florida state parks. Virus RNA was detected in 56/500 (11.2%) of surveyed ticks. As this virus was isolated from ticks that parasitize humans, the ability of the tick to transmit the virus to people should be evaluated. Furthermore, reservoir hosts for the virus need to be identified in order to develop risk assessment models of human infection.

Figure 1. Field site locations in Central Florida, USA.

Ticks were trapped for virus isolation at San Felasco Hammock State Preserve (center) in 2012. In addition to San Felasco Hammock, ticks were trapped at two additional sites in 2013 for screening by RT-qPCR for TCRV vRNA. Image produced with USGS National Map Viewer (http://viewer.nationalmap.gov/viewer/).

Figure 2. Non-infected Vero E6 control flasks (A) and Vero E6 flasks inoculated with tick homogenate (B) at day 13 post-inoculation.

Figure 3. PCR products generated from Vero E6 cell supernates using pan-New World virus primers and Tacaribe primers.

Bands in lanes 2 and 5 resulting in a 694 bp amplicon were generated using primers 1010C and 1696R as previously described by Bowen et al., 1997 . Bands in lanes 3, 4 (light load) and 6 resulting in a 316 bp amplicon were generated using primers TACV-F and TACV-R as previously described by Cogswell-Hawkinson et al., 2012 . Lane 1 contains a 100 bp ladder and lane 7 contains the non-template control. The gel was stained with ethidium bromide.

Figure 4. Transmission electron microscopy image of an arenavirus, identified by PCR as Tacaribe virus, in Vero E6 cells at magnifications of 33000x and 66000x (inset).

Arrows indicate budding viruses that are emerging from the surface of a granulated Vero E6 cell.

Figure 5. Arenavirus phylogenetic relationship inferred by MEGA software based on partial nucleotide sequences of the N gene of 18 NWVs and amplicons generated from PCR positive ticks.

Arenaviruses are listed by name and immediately followed by the GenBank accession number of the strain used in this analysis. Numbers 112, 124, 126, 127, 129, 136 and 149 represent individual ticks with the greatest number of virus copies detected by RT-qPCR. The evolutionary history was inferred using the UPGMA method. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site.