2014- The Tacaribe virus (Arenaviridae) was never known to be in any species of ticks (only in fruit bats in Trinidad) until it was discovered recently in 0%- 25.1% of lone star ticks in three different state parks in central Florida. No one has a clue what the natural reservoir host is that keeps it alive- they never found it.

Discovered after 60 +/- years of no known existence anywhere, the virus causes bleeding under the skin, bleeding from eyes, nose, ears, etc., shock, kidney failure, seizures and coma that often ends in death. No known treatment, no vaccine. (If you watch the “Last Ship” series on TV- it seems to be similar to how they portray that virus.)

News article and the corresponding scientific article are below.

WIKI Info

• Tacaribe virus (New World) complex:
  • Amapari virus
  • Chapare virus
  • Flexal virus
  • Guanarito virus
  • Junin virus
  • Latino virus
  • Machupo virus
  • Oliveros virus
  • Paraná virus
  • Patawa virus
  • Pichinde virus
  • Pirital virus
  • Sabiá virus
  • Tacaribe virus
  • Tamiami virus
  • Whitewater Arroyo virus

Ocala Post

Gainesville, Florida — University of Florida researchers have discovered a rare virus that belongs to the family Arenaviridae. Some arenaviruses are associated with severe hemorrhagic disease and has had a significant mortality in people in South America and sub-Saharan Africa. The virus was thought to have vanished four decades ago.

Dr. Katherine Sayler, and her team of researchers, found the virus in Lone Star ticks that were collected from Manatee, O’Leno, and San Felasco State Parks.

Researchers said that out of all the Lone Star ticks trapped, collected, and cultured from North Central Florida, 10 percent of the ticks were infected with the Tacaribe virus.

Tacaribe virus (TCRV) was first isolated from 11 Artibeus species bats captured in Trinidad in the 1950s during a rabies virus surveillance program. Despite significant effort, no evidence of infection of other mammals, mostly rodents, was found, suggesting that no other vertebrates harbored TCRV. For this reason, it was hypothesized that TCRV was naturally hosted by Artibeus bats. This particular virus has not been found in rodents as other similar arenaviruses associated with causing hemorrhagic fever.

“This finding is exciting because it expands the range in which these viruses might be circulating in the environment,” Dr. Sayler said in a university news release. “It also raises some really interesting questions about human risk,” she added.

“We never thought we would find an arenavirus in a tick. These types of viruses are usually transmitted by rodents,” Dr. Sayler noted.

Although the Tacaribe virus had previously been found in bats, recent studies indicate that bats are not the natural reservoir host, and efforts to find the virus in other mammals have failed.

“We still don’t know which animal is the natural host of this virus, and whether ticks have harbored the virus for a long time, or if this is something new,” Dr. Sayler said. “Without knowing if local rodents are a major reservoir of the virus, the extent that Floridians are sickened by the virus, and whether ticks can transmit the virus to humans, it makes it hard for us to know if and when there would be an outbreak. Clearly, much more work must be done.”

According to the Centers for Disease Control, Lone Star ticks are aggressive human biters and do carry Lyme disease.

“We need to use some of the tools that we have at UF to determine if the virus has been around and circulating for a long time, or if it has been absent from Florida and was introduced,” Dr. Sayler said. “Health care professionals should also be aware of the potential tick-transmitted pathogens that occur besides the one that causes Lyme disease. Medical doctors can’t be aware of every emerging tropical disease, but if we have greater awareness of emerging diseases, we can move forward from a proactive surveillance effort instead of from a reactive effort, when there is suddenly a huge outbreak and a crisis situation,” she added.

Arenaviruses can in fact cause hemorrhagic fever. Most arenavirus infections cause a flu-like illness that starts within a week of exposure. The initial symptoms are often mild, but may quickly worsen.

Symptoms of viral hemorrhagic fever include fatigue, fever, weakness, dizziness, and muscle aches; patients with more severe infections show bleeding under the skin, internal organs, or even from bodily orifices like the mouth, eyes, or ears. Some patients develop severe diarrhea that may also be bloody, and severely ill patients present with shock, delirium, seizures, kidney failure, and coma that often ends in death.

Patients with viral hemorrhagic fevers usually receive only supportive therapy; there is no other established cure for viral hemorrhagic fevers.

According to Dr. Sayler, the cultures taken from the ticks tested 99 percent identical to the only remaining sample from a virus found in Trinidad in the late 1950s.

Dr. Sayler said that people who are bitten by a tick should try to save the tick for further analysis by a medical professional. If possible, put the tick in a ziplock-type bag and place it in a freezer if you cannot get it to a doctor immediately.

Dr. Sayler said she would continue her work at UF, which would include searching for more isolates of the virus for genetic comparison.

“We need to figure out what mammals have it here in Florida and how it got here,” Dr. Sayler said.

This is the first time this virus has been found in the United States.

Link Here

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PLOS ONE

Citation: Sayler KA, Barbet AF, Chamberlain C, Clapp WL, Alleman R, Loeb JC, et al. (2014) Isolation of Tacaribe Virus, a Caribbean Arenavirus, from Host-Seeking Amblyomma americanum Ticks in Florida. PLoS ONE 9(12): e115769. doi:10.1371/journal.pone.0115769

Editor: Jens H. Kuhn, Division of Clinical Research, United States of America

Received: August 19, 2014; Accepted: November 26, 2014; Published: December 23, 2014

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

Funding: The University of Florida College of Veterinary Medicine internal “bridge grant” awarded to Dr. Anthony Barbet was used to fund this project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Arenaviridae are a family of rodent and boid snake-associated, single stranded RNA viruses that currently (as of December, 2014) includes only one genus, Arenavirus. Twenty-nine distinct arenaviruses have been identified, many of which cause severe hemorrhagic fever (HF) in humans [1]. At least ten of these viruses are associated with human disease in many parts of the world including western Africa, Argentina, Bolivia, Venezuela and Brazil [2].

Specifically, areanviruses have caused significant mortality in humid pampas of central Argentina, and multiple Old World arenaviruses cause morbidity in endemic foci in sub-Saharan Africa [3].

These viruses are divided into two serogroups based on shared antigens and geographic distribution:(a) Lymphocytic Choriomeningitis-Lassa virus (LCM-LAS) serocomplex viruses, or the Old World arenaviruses, and (b) Tacaribe serocomplex viruses, or the New World arenaviruses, (NWV) [4].

Though LCM virus has a world-wide distribution, viruses of the LCM-LAS complex are found primarily in Africa, whereas the Tacaribe complex viruses are found in North and South America.

The Tacaribe complex includes the following viruses: Tacaribe (TCR), Allpahuayo (ALL), Amapari (AMA), Bear Canyon Virus (BC), Chapare (CHP), Cupixi (CPX), Flexal (FLE), Guanarito (GTO), Junin (JUN), Latino (LAT), Machupo (MAC), Oliveros (OLV), Paraná (PAR), Pichinde (PIC), Pirital (PIR), Sabiá (SAB), Tamiami (TAM), and Whitewater Arroyo (WWA).

Heretofore, the only NWV recognized by the International Committee on the Taxonomy of Viruses to occur in North America were the TAM, BC, and WWA viruses.

The NWV are further subdivided into phylogenetic clades: A, B and C, and a recombinant clade. Although TCRV has been associated with one non-fatal laboratory acquired infection (mentioned briefly in reference 14), it is grouped in Clade B with the most pathogenic viruses, such as JUNV and MACV [5].

Arenavirus genomes are fairly simple, consisting of two ssRNA segments which encode four genes [6]. The large (L) segment contains genes for the RNA-dependent RNA polymerase (L protein) and the small RING-domain containing Z protein. The small (S) genome segment contains genes for the glycoprotein precursor (GPC) and the nucleoprotein (NP) of the virus.

The placement of TCRV within phylogenetic clade B is based on the sequences of its GPC and NP genes, which encode proteins that are involved in host cell invasion [2], [4].

TCRV has been associated with a non-fatal, febrile laboratory acquired infections of humans, but is most closely related to JUNV, which is associated with up to 30% mortality in infected patients [7].

The virus is an important model organism for highly pathogenic Clade B viruses and has been investigated as a vaccine candidate for Argentine hemorrhagic fever [8]–[10].

In addition, TCRV provides an excellent model system for identifying the determinants of pathogenesis of the lethal arenaviruses, without the need for BSL-4 containment facilities [11].

The natural reservoirs of mammalian arenaviruses are specific rodent species, in which the viruses are most often maintained through a largely asymptomatic, persistent infection.

Among rodents, arenaviruses are reported to be transmitted by both horizontal and congenital routes [12]–[14].

The geographic range of each arenavirus is typically thought to be determined by the range of its reservoir, and humans are most often infected through contact with excreta from these rodents [12].

TCRV is an arenavirus for which a rodent host has never been specified. Instead, it had been isolated from two species of Artibeus fruit bats in Trinidad from 1956 to 1958 during a rabies surveillance survey.

It was also isolated from a single pool of 344 mosquitoes in 1956. For ten years following the initial isolation from mosquitoes, over 1 million mosquitoes of various species from Trinidad tested negative for the virus [13].

Recent experimental evidence indicates that bats are not the natural reservoir host for TCRV following virus inoculation into the Jamaican fruit bat [14], and extensive efforts to identify the virus in other mammals, mostly rodents, have failed [13].

Although 19 TCRV isolates were obtained from fruit-eating bats in the 1950s, only one, TRVL-11573, remains, leaving much about the ecology and epidemiology of the virus unknown. All other isolates obtained from bats were accidentally destroyed.

TRVL-11573 has been extensively passaged, and is the only laboratory strain in existence for animal models of infection and for genetic comparison.

Here, we preliminarily analyzed the genome sequence of the Florida isolate of Tacaribe virus derived from a pool of 100 adult host-seeking ticks. In addition, we determined the prevalence of TCRV vRNA in host-seeking Amblyomma americanum (lone star ticks; LST) collected in North Central Florida.

Methods

Tick collection and processing

One hundred (100) adult host-seeking A. americanum were collected from San Felasco State Park, west of Gainesville, FL, USA, in March of 2012 (29°42′33.189″ N latitude and 82°27′23.213″ W longitude.).

Tick trapping was performed using 1 m2 flannel drags in combination with carbon dioxide-baited traps as previously described [15]. All tick trapping was performed in accordance with the Florida Department of Environmental Protection Research and Collection Permit #05231210.

Only host-seeking tick species common in Florida were collected because these species are most likely to attach to a person and take a blood meal. No protected or endangered species were sampled in this study. The ticks were identified according to standard taxonomic keys and immature ticks were removed from the sample pool.

The sample pool was subsequently stored frozen at −80°C until homogenization for virus isolation. A 10% w/v tick homogenate was prepared for virus isolation as described in Sang et al. [16].

In 2013, ticks were collected using the same methods as 2012 from two additional Florida state parks: Manatee Springs State Park in Chiefland, Florida (29° 29′47.401″ N, 82°58′4.429″ W) and O'Leno State Park in High Springs, Florida (29°55′11.863″ N and 82° 35′15.427″ W), to determine if the virus could be detected in other locations in North Central Florida (Fig.1).

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/).

doi:10.1371/journal.pone.0115769.g001

Cell cultures

A variety of cell lines were tested to improve the chances of isolating mammalian viruses, as some viruses replicate in particular cell lines. Cell lines A549 (CCL-185), BHK-21 (CCL-10), HeLa (CCL-2), LLC-MK2 (CCL-7), MDCK, (CCL-34), Mv1 Lu (CCL-64), Neuro-2a (CCL-131), NIH/3 T3 (CRL-1658), and Vero E6 (CRL-1586) were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA), and propagated as monolayers at 37°C and 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) (Mediatech, Inc., Manassas, VA, USA) or Eagle's Minimal Essential Medium (EMEM) (Invitrogen, Carlsbad, CA, USA), as appropriate per cell line.

DMEM and EMEM were supplemented with 2 mM L-Alanyl-L-Glutamine (GlutaMAX, Invitrogen, Carlsbad, CA, USA.), antibiotics [PSN; 50 µg/ml penicillin, 50 µg/ml streptomycin, 100 µg/ml neomycin (Invitrogen, Carlsbad, CA, USA)], and 10% (v/v) low IgG, heat-inactivated gamma-irradiated fetal bovine serum (HyClone, Logan, UT, USA). EMEM was also supplemented with sodium pyruvate (Invitrogen Corp.) and non-essential amino acids (Hyclone, Logan, UT, USA).

Virus isolation

Cell lines were grown in 25-cm2 cell culture flasks to 80% confluency. After rinsing with cell growth media, the cells were inoculated with 0.5 ml of clarified tick homogenate prepared in DMEM with 10% serum and antibiotics.

The inoculated cells were subsequently incubated at 37°C and observed daily for signs of cytopathic effects (CPE). The cultures were re-fed every 3 days, and blind-passaged every 2 weeks for a total of 6 blind passages before being considered negative for virus isolation. Non-infected cells were maintained and re-fed in parallel.

Detection and Identification of TCRV RNA and virus particles

Impeded cell growth without overt CPE became apparent after the fourth blind-passage in both Neuro-2a and Vero E6 cell lines inoculated with tick homogenate, suggesting the presence of an infectious agent.

In contrast, non-inoculated (negative control) Neuro-2a and Vero E6 cells replicated without signs of CPE. Multiple viral-based PCR primer sets were utilized for the detection of viral nucleic acids extracted from the spent media of both inoculated and non-inoculated Neuro-2a and Vero E6 cells (S1 Table).

Amplicons were purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA, USA), and plasmid clones of the amplicons were obtained using a TOPO-TA cloning kit according to the manufacturer's instructions (Life Technologies, Carlsbad, CA, USA).

Plasmids from a total of 6 clones were bi-directionally sequenced at the University of Florida Interdisciplinary Center for Biotechnology Research. In parallel, electron microscopy was performed.

Electron Microscopy

Inoculated Vero E6 cells were harvested, washed in 0.1 M phosphate buffer and pelleted by centrifugation at 2,500 rpm for 20 minutes. The supernatant was removed and the pellet resuspended in 2.5% gluteraldehyde in 0.1 M phosphate buffer and allowed to fix at 4°C overnight.

Fixed cells were submitted to the Electron Microscopy Lab at the Malcolm Randall Veterans Affairs Medical Center, North Florida-South Georgia Veterans Health System, for processing and performance of transmission electron microscopy.

Isolation of RNA from infected cell cultures

A QIAamp Viral RNA-mini kit (Qiagen) was used to extract RNA from liberated virions in the media of Vero E6 and Neuro-2a cells that had been inoculated with tick extract. Similarly, RNA was also extracted from Influenza B virus particles in the media of MDCK cells infected with that virus (non-template control), and from the spent media of non-inoculated cells of the same type grown in parallel under the same conditions (negative control).

The RNA extracts were immediately stored at −80°C until cDNA could be generated using Accuscript High Fidelity 1st strand cDNA kit (Agilent Technologies, Santa Clara, CA, USA) using both random 9-mers and TCRV-1 primer (see S2 Table for primer sequence).

Genome sequencing

Complete genome sequencing was accomplished using primer-walking PCR of the L and S segments of the virus. Virus isolation and genome sequencing by RT-PCR took place in separate laboratories, in separate buildings, using different equipment.

To generate overlapping PCR products of approximately 800 bp in length, 28 oligonucleotide primers were used: TCRV-1 through 18 for the L segment of the virus (S2 Table) and primers TCRV-19 through 28 for the S segment (S3 Table). RT-PCR was performed with 2.5 uL cDNA template in 50 uL reactions using Phusion High-Fidelity PCR master mix (New England Biolabs, Ipswich, MA, USA) per the manufacturer's instructions.

To obtain the 5′ and 3′ ends of the viral L and S genomes, 5′ and 3′ systems for the Rapid Amplification of cDNA Ends (RACE) were used per the manufacturer's protocols (Life Technologies, Carlsbad, CA, USA). Nucleotide sequences from bidirectionally Sanger-sequenced amplicons were assembled in Sequencher DNA sequence analysis software v2.1 (Gene Codes, Ann Arbor, MI, USA).

Complete contigs were translated into protein sequences using EMBOSS Transeq and Sixpack. Comparisons of the protein sequences of this isolate and TRVL-11573 (GenBank M20304 & J04340) were completed using ExPASy SIM tool.

RNA isolation from ticks

Adult A. americanum were stored in molecular grade 70% ethanol at -80°C before they were processed for RNA extraction (Fisher Scientific, Hampton, NH, USA). On ice, individual ticks were submerged in 10% bleach solution for 10 minutes, followed by three washes with 70% molecular grade ethanol and a final wash with molecular grade water.

Ticks were rapidly dissected on an ice-cold RNAase/DNAase free sterile Petri dish with sterile instruments (Genesee Scientific, San Diego, CA, USA). Salivary glands and midguts of adult ticks were removed and immediately placed into RNAlater stabilization reagent (Qiagen).

RNA was extracted from tick midguts and salivary glands using Quick-RNA MiniPrep (Zymo Research, Irvine, CA, USA). RNA was quantified by fluorometric quantitation (Qubit 2.0 Fluorometer, Life Technologies, Grand Island, NY, USA).

RT-qPCR

Forward primer TCRV-1 (S2 Table) was used as described above to generate full-length viral cDNAs. When reactions were complete, cDNA samples were stored at −20°C until needed for RT-qPCR.

A third designated laboratory was used for performing RT-qPCR; the third laboratory was separate from the laboratories in which the virus was isolated and separate from the laboratory in which nucleic acids were recovered from LSTs. TaqMan-based qPCR was performed with 5 uL of cDNA template in a total volume of 25 uL.

The reactions were performed using the QuantiTect Probe PCR master mix (Qiagen) as follows: 95°C for 15 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 60 seconds. The reaction mixture contained 600 µM of each primer and the probe at a concentration of 200 µM. TCRV-specific primers and FAM-labeled probe were designed to be highly specific for a 100 nucleotide region of the NP gene.

A previously reported a TCRV-specific forward primer was used [14] with a TCRV-specific reverse primer designed for this study: 5′-TGACGACAGACCTGGAAGT-3′ and the dual-labeled TaqMan probe: 5′-FAM-CCAGCCATCACCTGACAAACACAG-TAMRA-3′​.Standards were generated using ten-fold serial dilutions of N gene-recombinant plasmids. The assay sensitivity was determined using limited dilutions of cDNA generated from culture-isolated viral RNA spiked into a constant amount of tick genomic DNA from a virus negative tick. Assay results were analyzed using MJ Opticon Monitor software, version 3.1 (Bio-Rad Laboratories, Hercules, CA, USA). Molecular-grade water was used as a negative control.

Results

Impeded cell growth without overt CPE became apparent in both Neuro-2a and Vero E6 cell lines inoculated with tick homogenate after the fourth blind-passage. As noted by phase-contrast microscopy, the monolayer of inoculated flasks was subconfluent, whereas non-infected cells grew to confluency (Fig. 2). To gain clues on the identity of the infectious agent, universal primers specific for different DNA and RNA virus families were tested used in PCR or RT-PCR analyses.

Among the family-specific primers, only RT-PCR primers capable of amplifying the North or South American arenaviruses produced amplicons from RNA extracted from virions in the spent media of infected Neuro-2a and Vero E6 cells. This indicated an arenavirus had been isolated.

Subsequently, RT-PCR performed with TCRV-specific primers generated a product of the expected size. In contrast, no amplicons were formed from RNA that had been extracted from non-infected (negative control) cells (Fig. 3). Primers capable of amplifying the homologous region of LCMV-LASV viruses did not result in a product, ruling out the presence of any Old World viruses.

To determine if a single species or quasi-species was present in infected cultures, both resultant amplicons visible in Fig. 3 were cloned. Basic local alignment search tool (BLAST) analyses of these clones revealed alignment to TCRV glycoprotein and nucleoprotein mRNAs (GenBank: M20304). There was 99% identity to 316 nucleotides and 98% identity to 694 nucleotides, to plasmid clones generated with TCRV-specific and pan-NWV primers, respectively.

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

doi:10.1371/journal.pone.0115769.g002

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 [5]. 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 [14]. Lane 1 contains a 100 bp ladder and lane 7 contains the non-template control. The gel was stained with ethidium bromide.

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Additionally, electron microscopy of fixed cells revealed the presence of virus particles with dimensions and features typical of arenaviruses (Fig. 4). Collectively, changes in the growth patterns of inoculated cell cultures, screening of the infected cells for viral RNA by RT-PCR, cloning and sequence analyses of amplicons, and electron microscopy revealed the presence of a NWV, specifically TCRV, had been isolated.

The full genome sequence of virus isolate was derived by primer-walking and 5′ and 3′ RACE (described above, Methods), and the complete S and L segment sequences were submitted to GenBank (KF923400 and KF923401, respectively).

The genome of our TCVR isolate had an overall identity of 99.3% to the prototype virus from Trinidad at the genome level. It was surprising to observe such a high level of conservation between two geographically and temporally distant isolates of the virus, as each of the four genes of the virus were 96.8-100% identical to TRVL-11573 isolate at the protein level (Table 1).

Interestingly, the gap in the GPC that we observed due to the deletion of 12 amino acids at residues 123–134 in the published sequence [17] has been previously observed by other investigators [18] (S1 Fig.). Furthermore, our isolate deviated from the reference sequence at the four-amino acid region GPPT found at residues 389–392 of the NP gene.

This deviation from the reference was previously reported by Harmon et al. in 2013 [1]. This group reported that the viral stocks obtained from the National Institutes of Health Biodefense and Emerging Infectious Diseases repository deviated from the reference sequence at this particular four-amino acid region, GPPT (GenBank KC329849) versus DLQL (GenBank NC004293), respectively, which was identical to the deviation that we report here (S2 Fig.).

Furthermore, this group found that the TCRV NP gene had 98.2% identity to the reference published in GenBank, perhaps due to sequencing errors. Although errors may be present in sequences of TRVL-11573 accessioned in the early 1990's (M203304 and NC004293), the sequence of the Florida isolate reported here does deviate from any of the sequences reported previously in GenBank.

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.

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Table 1. Sequence identity of the Florida isolate of Tacaribe virus and the prototype virus.

doi:10.1371/journal.pone.0115769.t001

Next, to provide further evidence of this arenavirus in the local A. americanum population, ticks were collected from February through July of 2013 and screened for the presence of vRNA using a RT-qPCR assay which targets the nucleocapsid gene of TCRV.

A total of 500 host-seeking ticks were collected from three state parks located in North Central Florida, including the original field site where ticks were trapped for virus isolation attempts (Fig. 1).

Collection site and pool size tested from each park was based on host-seeking tick availability.

TCRV vRNA was detected in 0–25.1% of the ticks, depending on the site (Table 2). Overall, a total of 56/500 ticks (11.2%) had detectable quantities of TCRV vRNA. RT-qPCR positive ticks with the highest viral load were selected for conventional RT-PCR of a longer target and directly sequenced as described above.

PCR products generated directly from ticks were used for genetic analysis. Amplicons generated from TCRV RNA positive ticks were nearly identical, providing further evidence of the presence of the virus in ticks in north central Florida (Fig. 5).

This finding indicates that the virus was persistent in the local environment over a two year period.

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.

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