Identification of animal hosts of Fort Sherman virus, a New World zoonotic orthobunyavirus

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH. 1Institute of Virology, CharitéUniversitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany 2Federal University of Bahia, Salvador, Brazil 3Bahia State Agricultural Defense Agency, Salvador, Brazil 4German Centre for Infection Research (DZIF), Associated Partner Site Charité, Berlin, Germany 5Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, Sechenov University, Moscow, Russia


| INTRODUC TI ON
Orthobunyaviruses are arthropod-borne viruses associated with zoonotic transmission and animal disease worldwide. Known vectors include mosquitoes, midges, bugs and ticks (Elliott, 2014). Currently, 88 species, gathered in at least 18 serogroups (Elliott, 2014), are assigned to the Orthobunyavirus genus in the Peribunyaviridae family (Abudurexiti et al., 2019). Human infection is generally asymptomatic but can cause febrile disease and severe acute neurological disease, exemplified by viruses from the Bunyamwera serogroup such as Maguari virus (MAGV) and Cachey Valley virus (CVV) (Calisher & Sever, 1995;Campbell et al., 2006;Wilson et al., 2017). Besides humans, data from serological studies point to a wide vertebrate host range of orthobunyaviruses including horses, cattle, goat, sheep, bison, caribou, grizzly bear, moose and deer (Calisher et al., 1986).
Fort Sherman virus (FSV) was isolated in 1985 from a US soldier with acute febrile disease based in Panama. The virus was antigenically related to MAGV, CVV, and the mosquito-associated Tensaw virus (TENV) and assigned to the Bunyamwera serogroup (Mangiafico, Sanchez, Figueiredo, LeDuc, & Peters, 1988). FSVlike viruses were previously isolated in Argentina from Culicidae spp. mosquitos in the 60s (strain CbaAr426) and 80s (strain AG83-

| MATERIAL AND ME THODS
A total of 192 sera were collected from peri-domestic animals including 50 horses, 50 cattle, 57 sheep and 35 goats during 2014-2018 in northeastern Brazil (Table 1). Animals were sampled during veterinary surveillance activities in different farms that were 200-600 km distant from each other. Animals were handled following procedures approved by the animal ethics committee of the Federal University of Bahia with the authorization no. 55/2017. Viral RNA was extracted using the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche Life Sciences). Samples were screened for orthobunyavirus RNA using a broadly reactive and highly sensitive RT-PCR assay targeting the viral L gene (Lambert & Lanciotti, 2009). Viral RNA from serum and cell supernatant was quantified using a strain-specific real-time RT-PCR with the following primers FSHV-rtF (5′-TGTTGGTGATTGTGCATATATTGG), FSHV-rtR (5′-GGCGGACAACCATGTTTAATACT) and the probe FSHV-rtP (5′-ATCTAGCCAGTAGGTTATCTGCCACGCAGC), labelled with fluorescein amidite (FAM) at the 5′-end and a dark quencher at the 3′-end. The assay was controlled by photometrically quantified in vitro-transcribed RNA controls generated from synthesized DNA fragments (IDT) containing a T7-RNA polymerase promoter region as described previously (Drexler et al., 2009). Thermocycling involved reverse transcription at 55°C for 20 min followed by 94°C for 3 min and then 45 cycles of 94°C for 15 s and 58°C for 30 s. RT-PCR was done using the OneStep SuperScript III RT-PCR kit (Thermo Fisher) with 5 µl RNA input and reaction components according to the manufacturer's instructions.
Virus was isolated from the PCR-positive horse serum diluted at 1:10 and 1:100 and inoculated onto Vero E6 cells maintained at 37°C and cultivated in DMEM. After 1 hr of incubation, the inoculum was removed and replaced by medium supplemented with 5% foetal calf serum (FCS), 1% penicillin/Streptomycin (100 U/ml) (PS, Thermo Fisher) and 1% non-essential amino acids. Infected cells were passaged three times every 7 days and controlled daily for cytopathic effect (CPE) and increases in FSV RNA via real-time RT-PCR. FSV stocks were produced by propagation in Vero E6 cells as described above. Cells and supernatant were harvested 3 days post-infection, centrifuged at 2000 x g for 10 min and titrated via plaque assay in Vero E6 cells.
For FSV growth kinetics, we compared cell lines from primate  Beyond the Brazilian FSV, viruses used for PRNT were the well-characterized CVV strain 6V633 (Dunlop et al., 2018) and a BUNV prototype strain (GenBank accession number: X14383). Titres were calculated using a logistic regression function in Graphpad prism 6 (GraphPad software, www.graph pad.com).

TA B L E 1 Sampling table
Deep sequencing was done using MiSeq reagent v2 chemistry

| RE SULTS
The broadly reactive RT-PCR detected one viremic horse out of 192 peri-domestic animals (overall detection rate 0.5%; 95% confidence interval (CI), 0.01-3.2; sampling coordinates 12°08′54″S 44°59′33″W). In horses, the detection rate was 2.0% (95% CI, 0.01-11.5) ( Table 1 and Figure 1a). The viral RNA concentration in that serum as determined by strain-specific real-time RT-PCR was low at 6.2 × 10 3 RNA copies/ml.  Table 3). In sum, the serologic data confirmed a wide FSV animal host range and suggested that viruses closely related to the Panamanian FSV prototype strain or CVV might also be circulating in Brazil.
The two Argentinian mosquito-derived FSV strains were isolated in Aedes albifasciatus and Psorophora varinervis (Groseth et al., 2017)   Our results show that FSV is circulating over a wide geographic range among South American horses. Interestingly, the viremic horse reported here was clinically healthy, which is different from the fatal neurologic disease reported in Argentina (Tauro et al., 2015). Different courses of FSV infection are consistent with CVV that has been isolated from both symptomatic and asymptomatic horses (Calisher et al., 1986). While our serological data suggest a broad vertebrate host range of FSV beyond horses, antibodies elicited by antigenically related Bunyamwera serogroup viruses may cross-react among each other (Hunt & Calisher, 1979;Johnson et al., 2014), and to date, no isolation or molecular detection of FSV has been reported for vertebrates other than horses. Therefore, our serological data should be interpreted with caution until further proof by direct FSV detection.

| D ISCUSS I ON
The reassortant FSV prototype strain originated from Panama and consists of FSV-related L and S segments with a CVV-related M segment. Because CVV circulates in Northern America (Calisher et al., 1986;Waddell et al., 2019), and our data show wide circulation of FSV in Southern America, it seems feasible that a reassortant virus would emerge in Central America, where the two subcontinents are connected. Since orthobunyaviruses show frequent reassortment events (Briese, Calisher, & Higgs, 2013), it will be interesting to see whether this potential geography-driven reassortment pattern can also be observed in other American orthobunyaviruses in future studies.
The orthobunyavirus M-derived glycoproteins are responsible for attachment and cellular entry and therefore major components of the viral host range (Elliott, 2014 (Tauro et al., 2012). Altogether, it seems likely that both the reassortant and the non-reassortant FSV strains should be included in differential diagnostics of both humans and other animals with compatible symptoms.