sign in sign up
<<
Home , Haemadipsidae, Haemagogus, Hippoboscidae, Hippoboscoidea, Lebombo virus, Lutzomyia

Article of the (Diptera: ) Relatives of Medically and Agriculturally Important “Bat-Associated” Viruses

María M. Ramírez-Martínez 1,† , Andrew J. Bennett 2,3,†, Christopher D. Dunn 2, Thomas M. Yuill 2 and Tony L. Goldberg 2,*

1 Departamento de Ciencias de la Salud y Ecología Humana, Universidad de Guadalajara, Guadalajara, Autlán CP 48900, Mexico; [email protected] 2 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin–Madison, Madison, WI 53706, USA; [email protected] (A.J.B.); [email protected] (C.D.D.); [email protected] (T.M.Y.) 3 Genomics and Bioinformatics Department, Biological Defense Research Directorate, Naval Medical Research Center–Frederick, Fort Detrick, Frederick, MD 21702, USA * Correspondence: [email protected]; Tel.: +1-608-890-2618 † These authors contributed equally to this work.

Abstract: Bat flies (Hippoboscoidea: and Streblidae) are obligate hematophagous ec- toparasites of . We collected streblid bat flies from the (México) and the Old World (Uganda), and used metagenomics to identify their viruses. In México, we found méjal   (; Vesiculovirus), Amate virus (: ), and two unclassified viruses of invertebrates. Méjal virus is related to emerging zoonotic encephalitis viruses and to the agricultur- Citation: Ramírez-Martínez, M.M.; ally important vesicular stomatitis viruses (VSV). Amate virus and its sister taxon from a bat are Bennett, A.J.; Dunn, C.D.; Yuill, T.M.; most closely related to - and -borne , suggesting a previously unrecognized Goldberg, T.L. Bat Flies of the Family orbivirus cycle involving bats and bat flies. In Uganda, we found mamucuso virus Streblidae (Diptera: Hippoboscoidea) Host Relatives of Medically and (: Orthobunyavirus) and two unclassified viruses (a rhabdovirus and an invertebrate Agriculturally Important virus). Mamucuso virus is related to encephalitic viruses of and to viruses from nycteribiid “Bat-Associated” Viruses. Viruses bat flies and louse flies, suggesting a previously unrecognized orthobunyavirus transmission cycle 2021, 13, 860. https://doi.org/ involving hippoboscoid . Bat fly virus transmission may be neither strictly -borne nor 10.3390/v13050860 strictly vertical, with opportunistic feeding by bat flies occasionally leading to zoonotic transmission. Many “bat-associated” viruses, which are ecologically and epidemiologically associated with bats but Academic Editor: A. Lorena Passarelli rarely or never found in bats themselves, may actually be viruses of bat flies or other bat ectoparasites.

Received: 25 March 2021 Keywords: chiroptera; bat fly; hippoboscoidea; streblidae; nycteribiidae; rhabdoviridae; vesiculovirus; Accepted: 4 May 2021 reoviridae; orbivirus; peribunyaviridae; orthobunyavirus Published: 8 May 2021

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- iations. Bat flies (Hippoboscoidea: Nycteribiidae and Streblidae) are obligate hematophagous ectoparasites that are highly adapted to life on bats [1]. The Nycteribiidae occur mainly in the Old World, have lost their eyes and wings over evolutionary time, and have a small thorax relative to their abdomen (due to the loss of flight muscles), giving them a “spider-like” appearance [1–3]. In contrast, the Streblidae have a worldwide distribution Copyright: © 2021 by the authors. (but with most occurring in the tropics) and have greater morphological variation Licensee MDPI, Basel, Switzerland. Nycteribiidae This article is an open access article than the [1,4]. The wings of streblid bat flies can be normal (relative to other distributed under the terms and dipterans), reduced, or absent, the eyes can be small or absent, and the general body form conditions of the Creative Commons can be flattened laterally or dorsoventrally [4]. The Nycteribiidae and Streblidae are thought Attribution (CC BY) license (https:// to have originated during a major period of bat diversification between 50 and 30 million creativecommons.org/licenses/by/ years ago [5]. 4.0/).

Viruses 2021, 13, 860. https://doi.org/10.3390/v13050860 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 860 2 of 15

Nycteribiid bat flies host apicomplexan parasites of the Polychromophilus, the cause of “bat malaria” [6,7], and bacteria of the genus Bartonella, some of which are zoonotic [8,9]. Wolkberg and Kaeng Khoi viruses (Perbunyaviridae: Orthobunyavirus) were isolated from the nycteribiid bat flies Eucampsipoda africana Theodor, 1955 and E. sundaica Theodor, 1955 in South Africa and China, respectively [10–12], and Mahlapitsi virus (Re- oviridae: Orthoreovirus) was isolated from E. africana in South Africa [13]. Members of our group have reported a surprising diversity of ledanteviruses (Rhabdoviridae: Ledantevirus) related to zoonotic agents in nycteribiid bat flies from Uganda, with flies from individual bats hosting multiple divergent viral variants [14]. Fewer reports exist about microbes in streblid bat flies. Bartonella sp. bacteria have been detected in streblid bat flies in México [15] and globally [8], and dengue virus (: Flavivirus) has been detected in streblid bat flies on vampire bats (Desmodus rotundus Geoffroy, 1810) in México [16]. In our previous studies of nycteribiid bat flies [14,17], we hypothesized that highly co-evolved bat ectoparasites might host “bat-associated” viruses, defined as viruses that are ecologically and epidemiologically linked to bats but have rarely or never been found infecting bats. Such “bat-associated” viruses may actually be hosted not by bats themselves, but by other organisms that associate closely with bats in nature, such as bat ectoparasites. Here, we explore this idea further by examining streblid bat flies from the New World (México) and the Old World (Uganda). Our data expand the known diversity of viruses in- fecting bat flies and show that streblid bat flies do, indeed, host relatives of “bat-associated” viruses important to and health.

2. Materials and Methods 2.1. Study Sites and Sampling In México, we trapped bats in 3 locations in the southern part of Jalisco State: Villa Purificación (19◦4206.9600 N, 104◦36018.3300 E at 510 m elevation; semi-evergreen ); Jalo- cote (19◦48055.9500 N, 104◦24037.0500 E at 1162 m elevation; dry deciduous forest with ripar- ian vegetation); and Las Joyas Scientific Research Station (19◦35010.2900 N, 104◦16027.4500 E at 1950 m elevation; temperate high mountain forest) (Figure1). Trapping occurred in 2018 during the months of January, February and April, respectively. We set mist nets (Avinet, Portland, ME, USA) at each location and opened them for four hours beginning at sunset. We gently removed netted bats and placed them into in cloth bags (one bat per bag) for pro- cessing. We collected bat flies using entomological tweezers and preserved them in 0.5 mL RNAlater nucleic acid stabilization buffer (Thermo Fisher Scientific, Waltham, MA, USA) inside 1.2 mL cryovials (one vial per bat). After processing, we released bats on site. In Uganda, we trapped bats in Kamwenge District at Ngogo research station within Kibale National Park (0◦2907.9700 N, 30◦23021.9100 E at 1550 m elevation; semideciduous montane forest) in July 2017, as previously described [14,17–19] (Figure1). Briefly, we set mist nets at a large hollow tree roost and captured bats as they exited at dusk. We handled bats and collected bat flies as described above, and we released bats on site after processing. Viruses 2021, 13, 860 3 of 15 Viruses 2021, 13, x FOR PEER REVIEW 3 of 16

450000 480000 510000 540000 570000 600000 209596 209650 209704 209758 209812 209866

ab 56670 56714 56759 56804 56849 56894 56939 56984

2

1 4

n 3 a e c

O n ia P d ac In if ic O c ea n

N N

W E W E

2050000 2075000 2100000 2125000 2150000 2175000 2200000 2225000 S S

. 450000 480000 510000 540000 570000 600000 209596 209650 209704 209758 209812 209866 Figure 1.1. MapsMaps ofof study study sites. sites. (a )(a Sampling) Sampling locations locations in Jalisco, in Jalisco, México México (green (green area, area, shown shown with insets with ofinsets México of México and Jalisco). and SitesJalisco). in MSiteséxico in are México 1: Villa are Purificaci 1: Villa óPurificación,n, 2: Jalocote, 2:and Jalocote, 3: Las and Joyas 3: ScientificLas Joyas Research Scientific Station. Research (b )Station. Sampling (b) location Sampling in location in Uganda (green, with inset of Uganda in Africa) in Kibale National Park (4), near the Ngogo Research Station. Uganda (green, with inset of Uganda in Africa) in Kibale National Park (4), near the Ngogo Research Station. Numbers Numbers surrounding maps are latitudes and longitudes (UTM). surrounding maps are latitudes and longitudes (UTM).

2.2. Imaging of Bat Flies We captured stacked images of bat flies flies using a a KY-F75U KY-F75U digital camera camera (JVC, (JVC, Yoko- Yoko- hama, Japan) attached to a a Z16 Z16 APO APO dissecti dissectingng microscope microscope (Leica (Leica Microsystems, Microsystems, Wetzlar, Wetzlar, Germany) affixed affixed with with gooseneck gooseneck illuminators illuminators (Ace (Ace 1, Schott 1, Schott Corporation, Corporation, San SanDiego, Diego, CA, andCA, Intralux and Intralux 5000–1, 5000–1, Volpi VolpiGroup, Group, Schlieren, Schlieren, Switzerland). Switzerland). Images Imagesconsisted consisted of 15 stacked of 15 layersstacked assembled layers assembled using Auto using Montage Auto Montage Pro software Pro software v5.01.005 v5.01.005 (Synoptics (Synoptics LTD, Cam- LTD, bridge,Cambridge, UK). UK).

2.3. DNA Barcoding of Bat Flies We PCR-amplifiedPCR-amplified and sequenced a 636-bp portion of the mitochondrial cytochrome oxidase subunit 1 (cox1) . We firstfirst surface-sterilizedsurface-sterilized bat fliesflies using dilute bleach [[20]20] and homogenized them in 0.5 mL Hanks’ balanced salt solution in PowerBead PowerBead tubes con- con- taining 2.382.38 mmmm metalmetal beads beads (Qiagen, (Qiagen, Hilden, Hilden Germany)., Germany). We We then then centrifuged centrifuged 50 µ50L ofµL the of theresulting resulting homogenates homogenates at 10,000 at 10,000×× g for g 10for min 10 min and and extracted extracted DNA DNA from from pelleted pelleted material ma- terialusing using the DNeasy the DNeasy blood and tissueand tissue kit (Qiagen, kit (Qiagen, Hilden, Hilden, Germany). Germany). We We PCR-amplified PCR-ampli- 0 0 fiedcox1 cox1 using using barcoding barcoding primers primers LCO1490 LCO1490 (5 (5-GGTCAACAAATCATAAAGATATTGG-3′-GGTCAACAAATCATAAAGATATTGG-) 0 0 3and′) and HC02198 HC02198 (5 -TAAACTTCAGGGTGACCAAAAAATCA-3(5′-TAAACTTCAGGGTGACCAAAAAATCA-3)[21′) [21]] and and the the HotStarTaq HotStar- ◦ TaqPCR PCR system system (Qiagen, (Qiagen, Hilden, Hilden, Germany), Germany), with thewith following the following cycling cycling parameters: parameters: 95 C for95 15 min; 35 cycles of 94 ◦C for 30 s, 50 ◦C for 30 s, 72 ◦C for 1 min; 72 ◦C for 10 min, and °C for 15 min.; 35 cycles◦ of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 1 min; 72 °C for 10 min, andan indefinite an indefinite soak soak at 4 C.at We4 °C. electrophoresed We electrophoresed PCR products PCR products on 1% agaroseon 1% agarose gels stained gels withstained ethidium with ethidium bromide, bromide, excised amplicons,excised amplicons, gel-purified gel-purified them using them the using Zymoclean the Zymo- Gel cleanDNA Gel Recovery DNA KitRecovery (Zymo Kit Research, (Zymo Irvine,Research, CA, Irvine, USA), CA, and USA), submitted and purifiedsubmitted amplicons purified ampliconsfor Sanger for sequencing Sanger sequencing in both directions in both directions on 3730xl on Genetic 3730xl Analyzers Genetic Analyzers (ThermoFisher, (Ther- Waltham, MA, USA) at the University of Wisconsin-Madison Biotechnology Center. We moFisher, Waltham, MA, USA) at the University of Wisconsin-Madison Biotechnology Viruses 2021, 13, 860 4 of 15

proofread and assembled chromatograms using Sequencher 4.10.1 (Gene Codes, Ann Arbor, MI, USA) and queried the resulting sequences against DNA sequences of streblid bat flies available in GenBank database using the blastx homology searching algorithm [22].

2.4. Metagenomics and Phylogenetics We processed bat flies for virus characterization using previously described meth- ods [14,17]. Briefly, we extracted total nucleic acids from bat fly homogenates prepared as described above using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). We used the Superscript IV system (Thermo Fisher, Waltham, MA, USA) with random hexam- ers to reverse transcribe RNA to cDNA, and we prepared DNA libraries using the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA). We sequenced libraries on a MiSeq instrument (V2 chemistry, 300 cycle kit; Illumina, San Diego, CA, USA). We quality-trimmed reads to ≥Q30 and length ≥50 and then assembled them de novo into contiguous sequences (contigs) using CLC Genomics Workbench version 20.1 (CLC Bio, Aarhus, Denmark). To characterize viruses, we queried raw reads and assembled contigs at the nucleotide and deduced amino acid sequence levels against sequences of viruses in GenBank using blastn and blastx, respectively [22], employing methods previously described for identifying viruses in bat flies [14,17]. To infer the phylogenetic positions of identified viruses, we aligned polymerase nucleotide sequences with related sequences in GenBank using a codon-based version of the Prank algorithm [23] with Gblocks [24] applied to remove poorly aligned regions, implemented in TranslatorX [25]. We then inferred phylogenies using PhyML 3.056 [26] with smart model selection [27] and 1000 bootstrap replicates to assess statistical confidence in clades. We displayed resulting phylogenetic trees using FigTree v1.4.4. We measured viral concentrations as viral reads per million total reads per kilobase of target sequence (vRPM/kb), which is a metagenomic proxy for viral load that has been validated by comparison to quantitative real time polymerase chain reaction [28–30]. We calculated this measure for each virus in each sample by mapping reads to the polymerase gene sequence (or obtained portion thereof, when available) of each identified virus at 90% sequence identity then log10 transforming the results for ease of visualization.

3. Results 3.1. Bat Identification We processed 12 bat fly samples (pools of between 1 and 6 flies per bat) from 9 species of bats in México and 12 bat fly samples from 1 species of bat in Uganda (Table S1). All bat flies analyzed had the characteristic morphology of the Streblidae (Figure2). Sequences of the cox1 gene derived from the 24 samples matched five streblid genera, including Aspidoptera, Megistopoda, Trichobioides, and in México, and Nycterophilia in Uganda (Table S1). In total, six samples from México were >99% identical to published strebid sequences, enabling species-level taxonomic assignment (Aspidoptera phyllostomatis Perty 1833, Megistopoda aranea Coquillett, 1899, and Trichobius yunkeri Wenzel, 1966; Table S1). Other sequences from México and all sequences from Uganda were between 97.64% and 80.82%, identical to published strebid sequences, such that we could not make definitive taxonomic assignments (Table S1). Furthermore, these more divergent sequences were, in many cases, phylogenetically intermediate between published sequences of known streblid species (data not shown), suggesting that these specimens represent un-sequenced or novel species. Viruses 2021, 13, 860 5 of 15 Viruses 2021, 13, x FOR PEER REVIEW 5 of 16

Viruses 2021, 13, x FOR PEER REVIEW 5 of 16

Figure 2. Images of streblid bat flies from México and Uganda. (a,b) The Méxican specimen (a: Figure 2. Images of streblid bat flies from México and Uganda. (a,b) The Méxican specimen (a: dorsal, Figuredorsal, 2.b :Images ventral; of 2.5× streblid magnification bat flies from and Méxicoa patch sizeand ofUganda. 16) was (a,b collected) The Méxican from a Parnell’sspecimen mus- (a: b: ventral; 2.5× magnification and a patch size of 16) was collected from a Parnell’s mustached bat dorsal,tached bbat: ventral; (Pternotus 2.5× parnellii magnification Gray, 1843) and aat patch Estación size Científicaof 16) was Las collected Joyas, fromSierra a deParnell’s Manantlán, mus- (tachedPternotusJalisco, bat on parnellii (17Pternotus January,Gray, parnellii 2018. 1843) ( c,dGray, at)Estaci the 1843) Ugandanón at Cient Estación specimenífica LasCientíficaJoyas, (c: dorsal, SierraLas Joyas,d: deventral; Manantl Sierra 4.0× deá n, magnificationManantlán, Jalisco, on 17 JanuaryJalisco,and a patch on 2018. 17 size January, (c, dof) the18) 2018. was Ugandan collected(c,d) the specimen Ugandanfrom a (Noack’sc: specimen dorsal, roundleafd: ( ventral;c: dorsal, bat 4.0 (dHipposideros:× ventral;magnification 4.0× ruber magnification Noack, and a patch 1893) sizeandat Ngogo, ofa patch 18) wasKamwenge size collected of 18) District,was from collected a on Noack’s 1 July, from roundleaf2017. a Noack’s Scale bat roundleafbars (Hipposideros = 1 mm. bat (Hipposideros ruber Noack, ruber 1893) Noack, at Ngogo, 1893) Kamwengeat Ngogo, Kamwenge District, on District, 1 July 2017. on 1 Scale July, bars2017. = Scale 1 mm. bars = 1 mm. 3.2. Virus Characterization 3.2.3.2. VirusVirus CharacterizationCharacterization Average sequencing depth was 1,018,337 (standard deviation 306,682) reads per bat Average sequencing depth was 1,018,337 (standard deviation 306,682) reads per bat fly fly sampleAverage after sequencing quality trimming, depth was with 1,018,337 average (standard length of deviation 126.3 base 306,682) pairs (standard reads per devi- bat sample after quality trimming, with average length of 126.3 base pairs (standard deviation flyation sample 9.9). Fromafter quality these data, trimming, we identified with average four viruses length ofin 126.3bat flies base from pairs México (standard (Table devi- 1). 9.9). From these data, we identified four viruses in bat flies from México (Table1). M éjal ationMéjal 9.9). virus From (Rhabdoviridae these data,: Vesiculoviruswe identified) isfour most viruses closely in relatedbat flies to from Jurona México virus, (Table isolated 1). virus (Rhabdoviridae: Vesiculovirus) is most closely related to Jurona virus, isolated from Méjalfrom mosquitoesvirus (Rhabdoviridae in : andVesiculovirus of unknown) is mostmedical closely significance related to[31] Jurona (Figure virus, 3). The isolated other mosquitoes in Brazil and of unknown medical significance [31] (Figure3). The other fromviruses mosquitoes in this clade in Brazil are vectored and of unknown by mosquitoes medical and significance sand flies, [31] and (Figure Chandipura 3). The virus,other viruses in this clade are vectored by mosquitoes and sand flies, and Chandipura virus, Piry virusesPiry virus, in thisand cladeIshafan are virus vectored can infect by mosquitoes , the and former sand two flies, causing and Chandipura febrile illness virus, [32– virus, and Ishafan virus can infect humans, the former two causing febrile illness [32–34]. Piry34]. Thisvirus, clade, and Ishafan in turn, virus is most can closelyinfect humans, related tothe the former clade two of vesiculoviruses causing febrile illnesscontaining [32– This clade, in turn, is most closely related to the clade of vesiculoviruses containing the 34].the vesicularThis clade, stomatitis in turn, isviruses most (VSV;closely Figure related 3), to which the clade are important of vesiculoviruses containingpathogens vesicular stomatitis viruses (VSV; Figure3), which are important livestock pathogens that thethat vesicular occasionally stomatitis infect virusespeople [35,36].(VSV; Figure 3), which are important livestock pathogens occasionally infect people [35,36]. that occasionally infect people [35,36]. Perinet virus (mosquito, Madagascar, 2008, NC_025394) PerinetPiry virus virus (opossum, (mosquito, Brazil, Madagascar, 1960, NC_038286) 2008, NC_025394) PiryIsfahan virus virus (opossum, (sand fly, Brazil, Iran, 1960, 1975, NC_038286) NC_020806) Chandipura virus (human, India, 2004, NC_020805) 99 Isfahan virus (sand fly, Iran, 1975, NC_020806) ChandipuraMalpais Spring virus virus (human, (mosquito, India, USA,2004, 1985, NC_020805) NC_025364) 99 100 MalpaisMéjal virus Spring (streblid virus (mosquito, bat fly, México, USA, 1985, 2018, NC_025364) MW798173) 100 MéjalJurona virus virus (streblid (mosquito, bat Brazil, fly, México, 1962, NC_039206) 2018, MW798173) 76 83 Cocal virus (mites, Trinidad and Tobago, 1961, NC_028255) 60 Jurona virus (mosquito, Brazil, 1962, NC_039206) 76 83 CocalVesicular virus stomatitis (mites, Trinidad Alagoas and virus Tobago, (, 1961,Brazil, NC_028255) 1964, NC_025353) 100 60 VesicularMaraba virus stomatitis (sand Alagoasfly, Brazil, virus 1983, (mule, NC_025255) Brazil, 1964, NC_025353) 99 100 100 MarabaVesicular virus stomatitis (sand fly,Indiana Brazil, virus 1983, (, NC_025255) USA, 1998, NC_038236) 99 Morreton virus (sand fly, , 1986, NC_034508) 100 Vesicular stomatitis Indiana virus (horse, USA, 1998, NC_038236) 99 MorretonCarajas virus virus (sand (sand fly, fly, Brazil, Colombia, 1983, 1986, NC_038285) NC_034508) 99 CarajasVesicular virus stomatitis (sand fly,New Brazil, Jersey 1983, virus NC_038285) (cow, Honduras, 1984, NC_024473) 100 Radi virus (sand fly, Italy, 1982, NC_038287) 76 Vesicular stomatitis New Jersey virus (cow, Honduras, 1984, NC_024473) 100 RadiYug Bogdanovacvirus (sand fly, virus Italy, (sand 1982, fly, NC_038287) Serbia, 1976, NC_025378) 76 YugAmerican Bogdanovac bat vesiculovirus virus (sand (bat, fly, Serbia,USA, 2008, 1976, NC_022755) NC_025378) American bat vesiculovirus (bat, USA, 2008, NC_022755) 0.2 0.2 Figure 3. Maximum likelihood phylogenetic tree of vesiculoviruses based on L (polymerase) gene sequences. Virus names FigureFigureare followed 3. 3.Maximum Maximum (in parentheses) likelihoodlikelihood by phylogeneticphylogenetic host, country, tree tree year of of vesiculoviruses vesiculoviruses of collection, and based based GenBank on on L L (polymerase) (polymerase) accession number. gene gene sequences. sequences. Méjal virus, VirusVirus identified names names arearein followedthe followed present (in (in study, parentheses) parentheses) is shown by by in host, boldhost, country, typecountry, with year yearsilhouette of of collection, colle ofction, streblid and and GenBank bat GenBank fly. Numbers accession accession beside number. number. branches Mé Méjaljal virus,represent virus, identified identified statistical in inconfidence the present in study,clades is based shown on in 1000 bold boot typestrap with replicates;silhouette ofonly streblid bootstrap bat fly. values Numbers ≥50% beside are shown. branches Scale represent bar = nucleotide statistical the present study, is shown in bold type with silhouette of streblid bat fly. Numbers beside branches represent statistical confidencesubstitutions in perclades site. based on 1000 bootstrap replicates; only bootstrap values ≥50% are shown. Scale bar = nucleotide confidence in clades based on 1000 bootstrap replicates; only bootstrap values ≥50% are shown. Scale bar = nucleotide substitutions per site. substitutions per site. Viruses 2021, 13, 860 6 of 15 Viruses 2021, 13, x FOR PEER REVIEW 6 of 16

Additionally, in MMéxicoéxico we found two two cl closelyosely related related variants variants of of Amate Amate virus virus (Reo- (Re- oviridaeviridae: :OrbivirusOrbivirus).). The The genus genus OrbivirusOrbivirus containscontains several import importantant pathogens,pathogens, most notably thethe globallyglobally emergingemerging andand economicallyeconomically significantsignificant agriculturalagricultural pathogenpathogen bluetongue virusvirus [[37,38]37,38] (Figure(Figure4 4).). Amate Amate virus virus is is mostmost closelyclosely related related (based (based on on segment segment 1)1) to an unnamed virus from a black-bearded black-bearded tomb tomb bat bat ( (TaphozousTaphozous melanopogon Temminck,Temminck, 1841)1841) inin China (GenBank MH144552),MH144552), togethertogether formingforming aa cladeclade thatthat isis most closely relatedrelated to the mosquito- and tick-borne orbiviruses (Figure(Figure3 3),), somesome ofof whichwhich areare zoonoticzoonotic [[39,40].39,40]. The other two two viruses viruses from from Mexican Mexican bat bat flies flies (jalimé (jalim iflavirusé iflavirus 1 and 1 andjalimé jalim partiti-likeé partiti-like virus virus1) are 1)members are members of viral of groups viral groups known known only from only broad from metagenomic broad metagenomic surveys surveysof inverte- of invertebrates (Table1;[41]). brates (Table 1; [41]).

63 CHeRI orbivirus (white-tailed deer, USA, 2017, MK903619) Guangxi orbivirus (, China, 2015, NC_040478) Peruvian horse sickness virus (horse, , 1997, NC_007748) 100 Yunnan orbivirus (mosquito, China, <2005, NC_007656) 99 Mobuck virus (white-tailed deer, USA, 2012, NC_022626) Yonaguni orbivirus (cattle, Japan, 2015, LC533329) 56 100 Koyama Hill virus (mosquito, Japan, 2011, AB894484) 100 Stretch Lagoon orbivirus (mosquito, Australia, 2002, NC_012754) 94 (mosquito, USA, 1969, NC_024503) 83 54 Kammavanpettai virus (, India, 1963, MH327935) 100 Parry's Lagoon virus (mosquito, Australia, 2010, KU724110) Corriparta virus (mosquito, Australia, 1960, NC_038568) 55 Skunk River virus (mosquito, USA, 2007, MK100569) 99 Sathuvachari virus (bird, India, 1963, KC432629) Big Cypress virus (mosquito, USA, 2014, MK105769) 53 100 Lipovnik virus (tick, Slovakia, 1963, HM543475) 92 Tribeč virus (tick, Slovakia, 1963, HQ266581) 100 Kemerovo virus (tick, Russia, 1968, KC288130) 74 100 Great Island virus (tick, Canada, 1971, NC_014522) 99 Okhotskiy virus (tick, Sweden, 2016, MN830241) Wad Medani virus (tick, Sudan, 1952, NC_027533) 89 Chenuda virus (tick, Egypt, 1954, NC_027534) 90 Bukakata orbivirus (bat, Uganda, 2013, MK359215) 100 Fomédé virus (bat, Guinea, 1978, MK359225) Chobar Gorge virus (tick, Nepal, 1970, NC_027553) 99 Bluetongue virus (, USA, 1953, NC_006023) 51 Epizootic hemorrhagic disease virus (white-tailed deer, USA, 1955, NC_013396) 85 Pata virus (mosquito, Central African Republic, 1968, JQ070386) 98 100 Tibet orbivirus (mosquito, Tibet, 2013, KU754026) Orbivirus SX-2017a (mosquito, China, 2007, NC_033782) 61 Japanaut virus (mosquito, Papua New Guinea, 1965, MK359245) 96 Wallal virus (mosquito, Australia, 2010, NC_022553) 100 Eubenangee virus (mosquito, Australia, 1963, NC_038592) 67 Warego virus (mosquito, Australia, 1969, NC_038614) Changuinola virus (rat, Brazil, 1961, NC_022639) 89 74 (mosquito, Uganda, 1959, NC_038604) 50 Equine encephalosis virus (horse, South Africa, 1967, NC_038574) (mosquito, South Africa, 1956, NC_038594) 99 94 African horsesickness virus (horse, Morocco, 1656, NC_006021) 100 Palyam virus (cattle, Australia, 1970, NC_005990) 99 Chuzan virus (cattle, China, 2013, KT887180) 99 Ife virus (bat, Nigeria, 1971, MK359235) Letea virus (snake, Romania, 2014, MN873683) Ninarumi virus (mosquito, Peru, 2009, MF094119) 100 Amate virus (streblid bat fly, Mexico, 2018, MW798171) 100 Amate virus (streblid bat fly, Mexico, 2018, MW798172) Orbivirus sp. (bat, China, 2015, MH144552) St. Croix River virus (tick, USA, 1993, NC_005997) 0.2 FigureFigure 4.4. MaximumMaximum likelihoodlikelihood phylogeneticphylogenetic treetree ofof orbivirusesorbiviruses basedbased onon VP1VP1 (polymerase)(polymerase) genegene sequences.sequences. Virus namesnames areare followedfollowed (in(in parentheses)parentheses) byby host,host, country,country, yearyear ofof collection,collection, andand GenBankGenBank accessionaccession number.number. TheThe twotwo closelyclosely relatedrelated variants of Amate virus (98.7% nucleotide identity in the VP1 gene) identified in the present study, are shown in bold type variants of Amate virus (98.7% nucleotide identity in the VP1 gene) identified in the present study, are shown in bold with silhouette of streblid bat fly. Numbers beside branches represent statistical confidence in clades based on 1000 boot- type with silhouette of streblid bat fly. Numbers beside branches represent statistical confidence in clades based on 1000 strap replicates; only bootstrap values ≥50% are shown. Scale bar = nucleotide substitutions per site. bootstrap replicates; only bootstrap values ≥50% are shown. Scale bar = nucleotide substitutions per site. We identified three viruses in bat flies from Uganda (Table 1). Mamucuso virus We identified three viruses in bat flies from Uganda (Table1). Mamucuso virus ( Peri- (Peribunyaviridae: Orthobunyavirus) is a member of a genus containing vector-borne path- bunyaviridae: Orthobunyavirus) is a member of a genus containing vector-borne pathogens ogens known to cause severe encephalitis in humans and other mammals (Figure 3) [42– known to cause severe encephalitis in humans and other mammals (Figure3)[ 42–44]. 44]. Within the orthobunyaviruses, mamucuso virus is part of a subclade of viruses from Within the orthobunyaviruses, mamucuso virus is part of a subclade of viruses from nycteribiid bat fliesflies and louse fliesflies (based on the L segment), also containing the mosquito- vectored human pathogen Nyando virus [42] (Figure 5). This clade is most closely related Viruses 2021, 13, 860 7 of 15

Viruses 2021, 13, x FOR PEER REVIEW 7 of 16

vectored human pathogen Nyando virus [42] (Figure5). This clade is most closely related toto thethe cladeclade containingcontaining BwambaBwamba virus,virus, aa highly highly prevalent prevalent cause cause of of human human febrile febrile illness illness acrossacross Africa Africa [ [42,45],42,45], andand thethe lessless commoncommon PongolaPongola virus,virus, which which also also causescauses human human febrile febrile illnessillness [[42,46].42,46]. The The clade clade containing containing mamucuso mamucuso virus virus is equally is equally closely closely related related to the to glob- the globallyally expanding expanding and andneuropathogenic neuropathogenic California California serogroup serogroup of orthobunyaviruses of orthobunyaviruses [42]. [ 42An-]. Anotherother virus virus from from Ugandan Ugandan bat batflies, flies, kamu kamu rhabdovirus rhabdovirus 1, appears 1, appears to be toa divergent be a divergent mem- memberber of the of family the family RhabdoviridaeRhabdoviridae, although, although only a only small a smallfragment fragment (609 bases) (609 bases) of the ofnucle- the nucleocapsidocapsid gene genecould could be obtained be obtained (Table (Table 1). The1). other The other virus virus from fromUgandan Ugandan bat flies, bat flies,kamu kamutoti-like toti-like virus virus1, is a 1, member is a member of a viral of a viralgroup group known known onlyonly from from broad broad metagenomic metagenomic sur- surveysveys of ofinvertebrates invertebrates (Table (Table 1;1 [41]).;[41]).

85 Snowshoe hare virus (snowshoe hare, USA, 1959, EU2036780) 100 Chatanga virus (mosquito, Russia, 1987, KT288299) 62 La Crosse virus (human, USA, 1978, NC_004108) California encephalitis virus (mosquito, USA, 1943, KX817312) 100 San Angelo virus (mosquito, USA, 1958, NC_043637) 100 Tahyna virus (mosquito, Czechoslovakia, 1964, KF361881) Lumbo virus (mosquito, Mozambique, 1962, NC_043632) 97 99 Jamestown Canyon virus (mosquito, USA, 1977, MH900530) 100 Inkoo virus (mosquito, Russia, 1989, KT288284) 56 South River virus (mosquito, USA, 1960, KX817336) 100 100 99 Serra do Navio virus (mosquito, Brazil, 1966, NC_043641) Keystone virus (mosquito, USA, 1964, NC_043629) Melao virus (mosquito, Trinidad and Tobago, 1955, NC_043634) 99 100 Achiote virus (mosquito, Panama, 1976, KY555808) 100 Infirmatus virus (mosquito, USA, 2008, KY569262) Trivittatus virus (mosquito, USA, 2016, KX891321) 100 Pongola virus (mosquito, Kenya, 2006, KJ867181) 100 Bwamba virus (human, Uganda, 1937, NC_034479) 99 Mamucuso virus (streblid bat fly, Uganda, 2018, XXXXXXX) 99 Wuhan louse fly virus 1 (louse fly, China, 2012, KM817663) 66 Kaeng Khoi virus (nycteribiid bat fly, China, 2012, MF170066) 100 Wolkberg virus (nycteribiid bat fly, South Africa, 2013, KX470563 Nyando virus (mosquito, Ethiopia, 1963, KJ867196) Mojui dos Campos virus (bat, Brazil, 1976, KJ867202) 99 Alajuela virus (mosquito, Panama,1963, KM272186) 81 Gamboa virus (mosquito, Panama, 1962, KM272180) 100 Pueblo Viejo virus (mosquito, , 1975, KX900438) 100 Calchaqui virus (mosquito, Argentina, 1982, KM272183) Koongol virus (mosquito, Australia, 1960, KP792669) 0.2 Figure 5. Maximum likelihood phylogenetic tree of orthobunyaviruses based on L (polymerase) gene sequences. Virus names Figure 5. Maximum likelihood phylogenetic tree of orthobunyaviruses based on L (polymerase) gene sequences. Virus are followed (in parentheses) by host, country, year of collection, and GenBank accession number. Mamucuso virus, identified names are followed (in parentheses) by host, country, year of collection, and GenBank accession number. Mamucuso virus, in the present study, is shown in bold type with silhouette of streblid bat fly. Numbers beside branches represent statistical confidenceidentified in clades, in the presentbased on study, 1000 is bootst shownrap in replicates; bold type withonly silhouettebootstrap ofvalues streblid ≥50% bat are fly. shown. Numbers Scale beside bar = branches nucleotide represent substi- tutionsstatistical per site. confidence in clades, based on 1000 bootstrap replicates; only bootstrap values ≥50% are shown. Scale bar = nucleotide substitutions per site.

3.3. Virus Prevalence and Load All bat fly samples analyzed harbored at least one virus, with an average of 2.1 vi- ruses per sample in México, 2.7 viruses per sample in Uganda, and 2.4 viruses per sample overall. Prevalence of the 7 viruses identified in México and Uganda varied widely (Figure 6), ranging from 2/12 (16.7%) for méjal virus and jalimé iflavirus 1 to 12/12 (100%) for kamu toti-like virus 1. Prevalence of 11/12 (91.7%) for Amate virus and mamucuso virus, 10/12 (83.3%) for jalimé partiti-like virus 1, and 8/12 (66.7%) for kamu rhabdovirus 1 were intermediate. Similarly, viral loads varied over four orders of magnitude among viruses and samples, from 0 to 4.54 log10 vRPM/kb (Figure 4). Kamu toti-like virus 1 had the high- est average viral load across positive samples (3.42 log10 vRPM/kb) followed by jalimé iflavirus 1 (1.93), jalimé partiti-like virus 1 (1.47), méjal virus (1.39), amate virus (1.32), kamu rhabdovirus 1 (1.12), and mamucuso virus (1.10). Viruses 2021, 13, 860 8 of 15

Table 1. Viruses identified in streblid bat flies from México and Uganda.

Country Virus 1 Accession Closest Match 2 Family 3 Genus 3 % Identity 4 Orbivirus sp (Taphozous melanopogon México Amate virus MW798171MW798172 dsRNA, segmented Reoviridae Orbivirus 68.65 (78, 0.0) bat, China, 2018, MH144552) Jurona virus (Haemagogus sp. México Méjal virus MW798173 −ssRNA, linear Rhabdoviridae Vesiculovirus 68.60 (91, 0.0) mosquito, Brazil, 1962, NC_039206) Varroa destructor virus 2 (Varroa México Jalimé iflavirus 1 MW798174 +ssRNA, linear destructor mite, Israel, 2014, Iflaviridae unclassified 72.04 (42, 3e-53) NC_040601) Wuhan virus 24 (unspecified México Jalimé partiti-like virus 1 MW798175 dsRNA unclassified unclassified 68.42 (85, 7e-109) insects, China, 2013, KX884086) Wuhan Louse Fly Virus 1 Uganda Mamucuso virus MW798176 −ssRNA, segmented (unidentified Hippoboscoidea, Peribunyaviridae Orthobunyavirus 69.45 (96, 0.0) China, 2012, KM817663) American bat vesiculovirus Uganda Kamu rhabdovirus 1 MW798177 −ssRNA, linear (Eptesicus fuscus bat, USA, 2008, Rhabdoviridae unclassified 72.90 (22, 9e-13) JX569193) Hubei toti-like virus 22 (unspecified Uganda Kamu toti-like virus 1 MW798178 dsRNA unclassified unclassified 76.79 (5, 2e-9) insects, China, 2013, KX882954) 1 Etymology of virus names is as follows. “Amate” refers to a culturally important tree (Ficus obtusifolia) native to México in which many of the sampled bats roost. “Méjal” is a hybridization of “México” and “Jalisco.” “Jalimé” is a hybridization of “Jalisco” and “México.” “Mamucuso” refers to a culturally important tree (Ficus mucuso) native to Uganda, with “ma” referring to the local Tooro word for “mother” (the bats were sampled from a large, hollow specimen of this tree species known locally as the “mother mucuso”). “Kamu” is a hybridization of “Kamwenge” and “Uganda.”. 2 Closest match in the GenBank nucleotide database (source, location, date and accession number of closest match in parentheses). 3 Family and genus of the closest match in the GenBank database. 4 Percent nucleotide sequence identity to the closest match in the GenBank database (query cover and E-value in parentheses). Viruses 2021, 13, 860 9 of 15

3.3. Virus Prevalence and Load All bat fly samples analyzed harbored at least one virus, with an average of 2.1 viruses per sample in México, 2.7 viruses per sample in Uganda, and 2.4 viruses per sample overall. Prevalence of the 7 viruses identified in México and Uganda varied widely (Figure6) , ranging from 2/12 (16.7%) for méjal virus and jalimé iflavirus 1 to 12/12 (100%) for kamu toti-like virus 1. Prevalence of 11/12 (91.7%) for Amate virus and mamucuso virus, 10/12 (83.3%) for jalimé partiti-like virus 1, and 8/12 (66.7%) for kamu rhabdovirus 1 were intermediate. Similarly, viral loads varied over four orders of magnitude among viruses and samples, from 0 to 4.54 log10 vRPM/kb (Figure4 ). Kamu toti-like virus 1 had the highest average viral load across positive samples (3.42 log10 vRPM/kb) followed by jalimé Viruses 2021, 13, x FOR PEER REVIEWiflavirus 1 (1.93), jalimé partiti-like virus 1 (1.47), méjal virus (1.39), amate8 virusof 16 (1.32), kamu rhabdovirus 1 (1.12), and mamucuso virus (1.10).

México Uganda virus virus ID v01 v02 v03 v04 ID v05 v06 v07 MX01 UG01 MX02 UG02 MX03 UG03 MX04 UG04 MX05 UG05 MX06 UG06 MX07 UG07 MX08 UG08 MX09 UG09 MX10 UG10 MX11 UG11 MX12 UG12

0 4.54 log vRPM/kb 10

Figure 6. Heatmap of viral loads in streblid bat flies from México and Uganda. Data are log10 viral Figure 6. Heatmap of viral loads in streblid bat flies from México and Uganda. Data are log10 viral 6 readsreads per 10 106 totaltotal reads reads per per kilobase kilobase of target of target sequence sequence for each for virus each in virus each inbat each fly (MX01-MX12 bat fly (MX01-MX12 and UG01-UG12): méjal virus (v01), Amate virus (v02), jalimé iflavirus 1 (v03), jalimé partiti-like andvirus UG01-UG12): 1 (v04), mamucuso méjal virus virus (v05), (v01), kamu Amate rhabdo virusvirus (v02),1 (v06), jalim and ékamuiflavirus toti-like 1 (v03), virus 1 jalim (v07).é partiti-like virus 1 (v04), mamucuso virus (v05), kamu rhabdovirus 1 (v06), and kamu toti-like virus 1 (v07).

4. Discussion Our results show that New World and Old World streblid bat flies host viruses related to medically and agriculturally important pathogens. In streblids from México, we found méjal virus, a rhabodovirus in the genus Vesiculovirus. The closest known relative of méjal virus is Jurona virus, isolated in 1962 from mosquitoes in Brazil but with no clear association with disease [47]. However, vesiculoviruses in the clade to which méjal virus belongs (Figure3) infect humans and can cause disease, as has been shown for Chandipura virus, an emerging pathogen that causes encephalitis in people (mainly children) in India [34], Piry virus, which has caused serious febrile illness in humans infected through laboratory accidents [33], and Ishafan virus, which seroprevalence studies show has infected up to 86% of people in communities in Iran [32]. Other vesiculoviruses are important to animal health and agriculture. Notably, the vesicular stomatitis viruses (VSV) are zoonoses in México, South and Central America and occasionally infect livestock in the United States Viruses 2021, 13, 860 10 of 15

to cause epizootics of vesicular stomatitis, an economically important disease resembling foot and mouth disease [35]. In streblids from México, we also found Amate virus, a reovirus in the genus Orbivirus. Orbiviruses are genetically and antigenically diverse human and animal that are emerging globally [39,40]. Notably, bluetongue virus is a major emerging pathogen of wild and domestic worldwide that can also infect and is transmitted either by hematophagous midges of the genus or, in the case of certain emerging variants, horizontally [37]. Amate virus and its sister taxon, an incompletely described orbivirus from a black-bearded tomb bat (T. melanopogon) in China, are notably divergent from the tick-borne and mosquito borne orbiviruses (Figure4), suggesting a lineage of bat-infecting orbiviruses with bat flies as vectors. Other bat-associated orbiviruses included in Figure3 include Japanaut virus from a southern blossom bat ( Syconycteris crassa Thomas, 1895) and culicine mosquitoes in New Guinea [48], Ife virus from a straw-colored fruit bat (Eidolon helvum Kerr, 1792) in Nigeria, Cameroon, and the Central African Republic [49], and the sister taxa Fomédé virus and Bukakata orbivirus from a dwarf slit-faced bat (Nycteris nana Andersen, 1912) in Guinea [50], and an Egyptian fruit bat (Rousettus aegyptiacus leachii Smith, 1892) in Uganda [51], respectively. Heramatsu orbivirus was also isolated from an eastern long-fingered bat (Myotis macrodactylus Temminck, 1840) in Japan [52] but remains incompletely sequenced. Interestingly, Bukakata virus and Fomédé virus cluster within the tick-born orbivirus clade (Figure3), and serum neutralization assays of 54 bat samples from Bolivia identified one M. nigricans Schinz, 1821, and one Noctilio labialis Hershkovitz, 1949, to have been exposed to Matucaré virus, another incompletely sequenced tick-borne orbivirus [53]. Bat , like bat flies, are specialized for life on bats [54,55], such that orbivirus transmission through bat tick vectors could explain these observations. In streblid bat flies from Uganda, we found mamucuso virus, a peribunyavirus in the genus Orthobunyavirus. Orthobunyaviruses are a large group of arboviruses with a worldwide and expanding distribution that cause severe central nervous system disease in [42–44]. Mamucuso virus is part of a group of viruses associated with bats and their vectors (Figure5). The closest relative of mamucuso virus is Wuhan louse fly virus 1, found in a louse fly (Hippoboscoidea: ), which are obligate hematophagous ectoparasites closely related to bat flies [56]. Mamucuso virus’s next nearest relatives are Kaeng Khoi virus and Wolkberg virus (Figure5), both of which infect nycteribiid bat flies [10–12], with Kaeng Khoi virus being apparently pathogenic in bats [57]. Interestingly, Kaeng Khoi virus has also been isolated from bat bugs (Stricticimex parvus Ueshima) in Thailand, suggesting that it has a broad vector range among hematophagous [58]. Nyando virus, the only mosquito-vectored virus in the mamucuso virus clade and the sole member of its serogroup, causes febrile illness and is highly prevalent (based on serology) in equatorial African countries, including Uganda [59]. Mojui de Campos virus, isolated from an adult bat (species not specified) in Brazil in 1975, was not found in 31,645 mosquitoes of six genera collected from the same location at the same time [60]. Thus, our results and those of others support the existence of a group of Orthobunyavirus that are transmitted by hippoboscoid flies rather than by mosquitoes [59]. In addition to the vesiculoviruses, orbiviruses, and orthobunyaviruses, we found four RNA viruses from unclassified genera (Table1) that are most likely vertically transmitted insect viruses characteristic of the highly diverse invertebrate “virosphere” [41,61]. A possible exception is kamu rhabdovirus 1, a divergent rhabdovirus that we could not place phylogenetically due to limited sequence data but that may ultimately cluster with other medically important rhabdoviruses. Like many invertebrates, therefore, streblid bat flies host viruses from medically important lineages and other viral taxa specific to invertebrates [62]. In México, we sampled 3 sites and 9 species of bats, parasitized by 4 genera of bat flies in which we found 4 viruses (Table1 and Table S1) and an average of 2.1 viruses per sample. In Uganda, we sampled 1 site and 1 species of bat parasitized by a single genus (and putative species) of bat fly in which we nevertheless found 3 viruses (Table1 and Viruses 2021, 13, 860 11 of 15

Table S1) and an average of 2.7 viruses per sample. Prevalence and loads varied markedly but were comparable among viruses in both countries. Thus, despite greater geographic range, bat diversity, and bat fly diversity sampled in México than in Uganda, the richness and diversity of viruses identified was similar in both countries. These results support our previous findings that nycteribiid bat flies from a few or even a single location can host a greater diversity of viruses than would be predicted from the bats they parasitize [14]. The imperfect specificity of viruses for bat flies and of bat flies for bats in México was surprising (Figure6; Table S1). Our study sites in M éxico and Uganda are both biodiversity “hot spots” where host switching by bat flies may occur due, for example, to multispecies bat roosting [14,63,64], perhaps explaining these observations. In the case of the orbiviruses and orthobunyaviruses, phylogenetic trees imply the existence of heretofore unrecognized transmission cycles involving bats and bat flies. The tightly co-evolved relationships between bat flies and their bat hosts may predispose the of “hyperparasitism,” or “ of parasites”, in which symbionts such as the viruses documented herein become adapted to the codependent life history of host and parasite [65]. The evolution of hyperparasitism has been documented for nycteribiid bat flies and fungi [66] and for nycteribiid bat flies and rhabdoviruses of the genus Ledantevirus [14]. Our results suggest that communities of hyperparasitic viruses have also co-evolved with streblid bat flies and exist on at least two continents. If so, bat flies may be “missing ecological links” between bats and other mammals with respect to viral transmission [14,65]. Many viruses are called “bat associated” because they are ecologically associated with bats but have rarely or never been found in bats themselves. The viruses we have documented in streblid bat flies support the notion that some “bat associated” viruses may actually be viruses of bat ectoparasites. Bat flies take blood meals from bats nearly constantly [1,5,67], such that transmission of bat fly viruses to bats might occur even if transmission is inefficient. Bat flies also bite humans opportunistically (as do other Hippoboscoidea) [1,56,67], suggesting a mechanism for zoonotic transmission. Several zoonotic rhabdoviruses, for example, have rarely or never been found in bats but they or their relatives have been found bat flies [14,17]. Viruses of bat flies may, therefore, not fall neatly into categories, such as “vector-borne” or “vertically transmitted”. Rather, they may be adapted to modes of transmission along a spectrum from vertical (fly to fly) to vector-borne (fly to to fly) to directly transmitted (bat to bat, or bat to other mammal). An example of a virus with a similarly heterogeneous mode of transmission is La Crosse virus, a relative of mamucuso virus within the California serogroup of orthobunyaviruses (Figure5), which is maintained in nature through a combination of vector-borne and transovarial transmission [68]. Determining whether the viruses discovered here infect bats, bat flies, or both, and with what efficiency, would necessitate additional study and would likely require experimental infections. Our results also suggest an explanation for the enigmatic epidemiology of vesicular stomatitis. Despite the economic costs of vesicular stomatitis, the ecological mechanism by which the virus moves from Central or to the western USA remains unknown [35,69]. In Colombia, Alagoas serotype vesicular stomatitis virus has been isolated from naturally infected phlebotomine sand flies ( spp.) and can be transmitted through biting and transovarially under experimental conditions [70]. In the eastern USA (Ossabaw Island, Georgia), VSV New Jersey has been found at relatively high titer in L. shannoni Dyar [71,72], and L. shannoni can transmit the virus to through biting and to a small percentage of progeny via transovarial transmission under experimental conditions [73]. However, in the western USA (New Mexico and Colorado), sandflies have been found only in small numbers on or near livestock ranches with histories of vesicular stomatitis [74], and no relationship between sand flies and VSV has been established in this region. Furthermore, there is no evidence of which we are aware that VSV is repeatedly transported in suspect vectors moving unaided from endemic areas in Mexico, Central or South America to the western USA. Viruses 2021, 13, 860 12 of 15

We speculate that vesiculoviruses such as VSV might enter the USA on migratory bats and their ectoparasites and then become locally established in alternative vectors such as sand flies. Bat migration is an underappreciated mechanism for the long-distance transport of bat-associated pathogens [75]. Such a scenario could explain why VSV outbreaks in the southwestern United States occur in summer [69], coincident with the northward migration of certain bats (e.g., the Mexican free-tailed bat, Tadarida brasiliensis mexicana Saussure, 1860) into the USA to establish breeding colonies [76], as well as how the virus might initially be transmitted to livestock (e.g., via bat flies feeding on alternative hosts). In support of this idea, Malpais Spring virus, within the méjal virus clade (Figure3), is another vector-borne virus that infects wild and feral ungulates in New Mexico, USA, where VSV outbreaks also occur [31,77]. Our study has certain inherent limitations. First, we could only examine a small number of bat flies from México and Uganda. The global diversity of streblid bat fly viruses is, therefore, likely much higher than we have documented here. Second, identifying viruses using metagenomics does indicate whether those viruses actively replicate in bat flies. Certain bat flies had very high viral loads with sequences yielding complete viral with deep coverage, suggesting replication (Figure6), but experimental infection studies would be necessary to demonstrate replication definitively. Third, it is unclear whether the viruses we describe in streblid bat flies infect bats, either through classical vector-borne transmission or through alternative, heterogeneous transmission cycles, as described above. Unfortunately, matched samples from parasitized bats were not available for analysis. Despite these caveats, our data strongly suggest that streblid bat flies are underappre- ciated reservoirs of viral diversity. Bat flies occupy a unique niche that predisposes them towards infection with, and transmission of, viruses with ecological and evolutionary links to bats. In this light, we note that bats host other types of highly co-evolved ectoparasites, such as bat fleas, bat mites, bat ticks, and even bat [54,55,78]. These parasites may also host hyperparasitic viruses of relevance to human and animal health. Investigating the full taxonomic range of bat ectoparasites may therefore shed new light on the ecology, evolution and epidemiology of enigmatic “bat associated” viruses.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/v13050860/s1, Table S1: Classification of streblid bat flies based on mitochondrial cytochrome oxidase subunit I DNA sequences. Author Contributions: Conceptualization, M.M.R.-M., A.J.B. and T.L.G.; methodology, M.M.R.-M., A.J.B., C.D.D. and T.L.G.; formal analysis, A.J.B. and T.L.G.; writing—original draft preparation, M.M.R.-M. and T.L.G.; writing—review and editing, M.M.R.-M., A.J.B., C.D.D., T.M.Y. and T.L.G.; funding acquisition, A.J.B. and T.L.G. All authors have read and agreed to the published version of the manuscript. Funding: Please add: This research was funded by the Universidad de Guadalajara (M.M.R.M), University of Wisconsin-Madison Global Health Institute Graduate Research Award Program (A.J.B.) and by the University of Wisconsin-Madison John D. MacArthur Professorship Chair (T.L.G). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Animal Use Statement: The study was approved by the Institutional Animal Care and Use Commit- tees of Universidad de Guadalajara and the University of Wisconsin-Madison (Protocol V005734-R01). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data supporting reported results can be found herein. DNA sequence data are available in GenBank (MW792194-MW792215 and MW798171-MW798178). Acknowledgments: We thank M.P. Ibarra López, L.I. Iñiguez Dávalos and the students who partici- pated in field work at Las Joyas Scientific Research Station. We also thank the Las Joyas Scientific Research Station staff and the land owners in México who kindly allowed us to conduct field work Viruses 2021, 13, 860 13 of 15

on their properties. We thank the administration and staff of Makerere University Biological Field Station, Ngogo Research Station, and the Kibale EcoHealth Project for assistance with field work in Uganda. We also thank the Uganda Wildlife Authority and the Uganda National Council for Science and Technology for kindly granting permission to conduct this research in Uganda. We thank Dan Young and the University of Wisconsin-Madison Department of Entomology for assistance with bat fly imaging. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Dick, C.W.; Patterson, B.D. Bat flies: Obligate ectoparasites of bats. In Micromammals and Macroparasites; Morand, S., Krasnov, B.R., Poulin, R., Eds.; Springer: New York, NY, USA, 2006. 2. Peterson, B.V.; Wenzel, R.L. Nycteribiidae. In Manual of Nearctic Diptera; McAlpine, J.F., Peterson, B.V.E., Shewell, G.E., Teskey, H.J., Vockeroth, J.R., Wood, D.M., Eds.; Research Branch, Agriculture Canada: Ottawa, ON, Canada, 1987; Volume 2, pp. 1283–1291. 3. Graciolli, G.; Dick, C.W.; Guerrero, R. Family Nycteribiidae. Zootaxa 2016, 4122, 780–783. [CrossRef][PubMed] 4. Dick, C.W.; Graciolli, G.; Guerrero, R. Family Streblidae. Zootaxa 2016, 4122, 784–802. [CrossRef] 5. Dittmar, K.; Morse, S.F.; Dick, C.W.; Patterson, B.D. Bat fly evolution from the to the present (Hippoboscoidea, Streblidae and Nycteribiidae). In Parasite Diversity and Diversification: Evolutionary Ecology Meets Phylogenetics; Morand, S., Krasnov, B.R., Littlewood, T., Eds.; Cambridge University Press: Cambridge, UK, 2015; pp. 246–264. 6. Obame-Nkoghe, J.; Rahola, N.; Bourgarel, M.; Yangari, P.; Prugnolle, F.; Maganga, G.D.; Leroy, E.M.; Fontenille, D.; Ayala, D.; Paupy, C. Bat flies (Diptera: Nycteribiidae and Streblidae) infesting cave-dwelling bats in Gabon: Diversity, dynamics and potential role in Polychromophilus melanipherus transmission. Parasit. Vectors 2016, 9, 333. [CrossRef] 7. Gardner, R.A.; Molyneux, D.H. Polychromophilus murinus: A malarial parasite of bats: Life-history and ultrastructural studies. Parasitology 1988, 96, 591–605. [CrossRef] 8. Morse, S.F.; Olival, K.J.; Kosoy, M.; Billeter, S.; Patterson, B.D.; Dick, C.W.; Dittmar, K. Global distribution and genetic diversity of Bartonella in bat flies (Hippoboscoidea, Streblidae, Nycteribiidae). Infect. Genet. Evol. 2012, 12, 1717–1723. [CrossRef][PubMed] 9. Billeter, S.A.; Hayman, D.T.; Peel, A.J.; Baker, K.; Wood, J.L.; Cunningham, A.; Suu-Ire, R.; Dittmar, K.; Kosoy, M.Y. Bartonella species in bat flies (Diptera: Nycteribiidae) from western Africa. Parasitology 2012, 139, 324–329. [CrossRef] 10. Xu, Z.; Yang, W.; Feng, Y.; Li, Y.; Fu, S.; Li, X.; Song, J.; Zhang, H.; Zhang, Y.; Liu, W.J.; et al. Isolation and Identification of a highly divergent Kaeng Khoi virus from bat flies (Eucampsipoda sundaica) in China. Vector Borne Zoonotic Dis. 2019, 19, 73–80. [CrossRef] 11. Van Vuren, P.J.; Wiley, M.R.; Palacios, G.; Storm, N.; Markotter, W.; Birkhead, M.; Kemp, A.; Paweska, J.T. Isolation of a novel orthobunyavirus from bat flies (Eucampsipoda africana). J. Gen. Virol. 2017, 98, 935–945. [CrossRef] 12. Feng, Y.; Li, Y.; Fu, S.; Li, X.; Song, J.; Zhang, H.; Yang, W.; Zhang, Y.; Pan, H.; Liang, G. Isolation of Kaeng Khoi virus (KKV) from Eucampsipoda sundaica bat flies in China. Virus Res. 2017, 238, 94–100. [CrossRef] 13. Van Vuren, P.J.; Wiley, M.; Palacios, G.; Storm, N.; McCulloch, S.; Markotter, W.; Birkhead, M.; Kemp, A.; Paweska, J.T. Isolation of a novel fusogenic orthoreovirus from Eucampsipoda africana bat flies in South Africa. Viruses 2016, 8, 65. [CrossRef][PubMed] 14. Bennett, A.J.; Paskey, A.C.; Kuhn, J.H.; Bishop-Lilly, K.A.; Goldberg, T.L. Diversity, transmission, and cophylogeny of ledante- viruses (Rhabdoviridae: Ledantevirus) and nycteribiid bat flies parasitizing Angolan soft-furred fruit bats in Bundibugyo District, Uganda. Microorganisms 2020, 8, 750. [CrossRef] 15. Moskaluk, A.E.; Stuckey, M.J.; Jaffe, D.A.; Kasten, R.W.; Aguilar-Setien, A.; Olave-Leyva, J.I.; Galvez-Romero, G.; Obregon- Morales, C.; Salas-Rojas, M.; Garcia-Flores, M.M.; et al. Molecular detection of Bartonella species in blood-feeding bat flies from México. Vector Borne Zoonotic Dis. 2018, 18, 258–265. [CrossRef] 16. Abundes-Gallegos, J.; Salas-Rojas, M.; Galvez-Romero, G.; Perea-Martinez, L.; Obregon-Morales, C.Y.; Morales-Malacara, J.B.; Chomel, B.B.; Stuckey, M.J.; Moreno-Sandoval, H.; Garcia-Baltazar, A.; et al. Detection of Dengue virus in bat flies (Diptera: Streblidae) of common vampire bats, Desmodus rotundus, in Progreso, Hidalgo, Mexico. Vector Borne Zoonotic Dis. 2018, 18, 70–73. [CrossRef] 17. Goldberg, T.L.; Bennett, A.J.; Kityo, R.; Kuhn, J.H.; Chapman, C.A. Kanyawara virus: A novel Rhabdovirus infecting newly discovered nycteribiid bat flies infesting previously unknown pteropodid bats in Uganda. Sci. Rep. 2017, 7, 5287. [CrossRef] 18. Bennett, A.J.; Paskey, A.C.; Ebinger, A.; Pfaff, F.; Priemer, G.; Hoper, D.; Breithaupt, A.; Heuser, E.; Ulrich, R.G.; Kuhn, J.H.; et al. Relatives of rubella virus in diverse mammals. Nature 2020, 586, 424–428. [CrossRef] 19. Bennett, A.J.; Goldberg, T.L. Pteropine orthoreovirus in an Angolan soft-furred fruit bat (Lissonycteris angolensis) in Uganda dramatically expands the global distribution of an emerging bat-borne respiratory virus. Viruses 2020, 12, 740. [CrossRef] 20. Binetruy, F.; Dupraz, M.; Buysse, M.; Duron, O. Surface sterilization methods impact measures of internal microbial diversity in ticks. Parasit. Vectors 2019, 12, 268. [CrossRef] 21. Folmer, O.; Black, M.; Hoeh, W.; Lutz, R.; Vrijenhoek, R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 1994, 3, 294–299. 22. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410. [CrossRef] 23. Löytynoja, A. Phylogeny-aware alignment with PRANK. Methods Mol. Biol. 2014, 1079, 155–170. Viruses 2021, 13, 860 14 of 15

24. Castresana, J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 2000, 17, 540–552. [CrossRef] 25. Abascal, F.; Zardoya, R.; Telford, M. TranslatorX: Multiple alignment of nucleotide sequences guided by amino acid translations. Nucleic Acids Res. 2010, 38, W7–W13. [CrossRef] 26. Guindon, S.; Dufayard, J.F.; Lefort, V.; Anisimova, M.; Hordijk, W.; Gascuel, O. New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst. Biol. 2010, 59, 307–321. [CrossRef] 27. Lefort, V.; Longueville, J.E.; Gascuel, O. SMS: Smart model selection in PhyML. Mol. Biol. Evol. 2017, 34, 2422–2424. [CrossRef] 28. Toohey-Kurth, K.; Sibley, S.D.; Goldberg, T.L. Metagenomic assessment of adventitious viruses in commercial bovine sera. Biol. J. Int. Assoc. Biol. Stand. 2017, 47, 64–68. [CrossRef] 29. Negrey, J.D.; Thompson, M.E.; Langergraber, K.E.; Machanda, Z.P.; Mitani, J.C.; Muller, M.N.; Otali, E.; Owens, L.A.; Wrangham, R.W.; Goldberg, T.L. Demography, life-history trade-offs, and the gastrointestinal virome of wild chimpanzees. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2020, 375, 20190613. [CrossRef] 30. Richard, J.C.; Leis, E.; Dunn, C.D.; Agbalog, R.; Waller, D.; Knowles, S.; Putnam, J.; Goldberg, T.L. Mass mortality in freshwater mussels (Actinonaias pectorosa) in the Clinch River, USA, linked to a novel densovirus. Sci. Rep. 2020, 10, 14498. [CrossRef] 31. Vasilakis, N.; Widen, S.; da Rosa, A.P.T.; Wood, T.G.; Walker, P.J.; Holmes, E.C.; Tesh, R.B. Malpais spring virus is a new species in the genus Vesiculovirus. Virol. J. 2013, 10, 69. [CrossRef] 32. Tesh, R.; Saidi, S.; Javadian, E.; Loh, P.; Nadim, A. Isfahan virus, a new vesiculovirus infecting humans, gerbils, and sandflies in Iran. Am. J. Trop. Med. Hyg. 1977, 26, 299–306. [CrossRef] 33. De Souza, W.M.; Acrani, G.O.; Romeiro, M.F.; Júnior, O.R.; Tolardo, A.L.; de Andrade, A.A.; da Silva Gonçalves Vianez Júnior, J.L.; de Almeida Medeiros, D.B.; Nunes, M.R.; Figueiredo, L.T. Complete genome sequence of Piry vesiculovirus. Arch. Virol. 2016, 161, 2325–2328. [CrossRef] 34. Sapkal, G.N.; Sawant, P.M.; Mourya, D.T. Chandipura viral encephalitis: A brief review. Open Virol. J. 2018, 12, 44–51. [CrossRef] 35. Rodriguez, L.L. Emergence and re-emergence of vesicular stomatitis in the United States. Virus Res. 2002, 85, 211–219. [CrossRef] 36. Kumar, B.; Manuja, A.; Gulati, B.R.; Virmani, N.; Tripathi, B.N. Zoonotic viral diseases of equines and their impact on human and animal health. Open Virol. J. 2018, 12, 80–98. [CrossRef][PubMed] 37. Maclachlan, N.J.; Zientara, S.; Wilson, W.C.; Richt, J.A.; Savini, G. Bluetongue and epizootic hemorrhagic disease viruses: Recent developments with these globally re-emerging arboviral infections of . Curr. Opin. Virol. 2019, 34, 56–62. [CrossRef] 38. Alkhamis, M.A.; Aguilar-Vega, C.; Fountain-Jones, N.M.; Lin, K.; Perez, A.M.; Sanchez-Vizcaino, J.M. Global emergence and evolutionary dynamics of bluetongue virus. Sci. Rep. 2020, 10, 21677. [CrossRef][PubMed] 39. Attoui, H.; Mohd Jaafar, F. Zoonotic and emerging orbivirus infections. Rev. Sci. Tech. 2015, 34, 353–361. [CrossRef] 40. Ruder, M.G.; Lysyk, T.J.; Stallknecht, D.E.; Foil, L.D.; Johnson, D.J.; Chase, C.C.; Dargatz, D.A.; Gibbs, E.P. Transmission and epidemiology of bluetongue and epizootic hemorrhagic disease in North America: Current perspectives, research gaps, and future directions. Vector Borne Zoonotic Dis. 2015, 15, 348–363. [CrossRef] 41. Shi, M.; Lin, X.D.; Tian, J.H.; Chen, L.J.; Chen, X.; Li, C.X.; Qin, X.C.; Li, J.; Cao, J.P.; Eden, J.S.; et al. Redefining the invertebrate RNA virosphere. Nature 2016, 540, 539–543. [CrossRef] 42. Evans, A.B.; Peterson, K.E. Throw out the map: Neuropathogenesis of the globally expanding California serogroup of orthobun- yaviruses. Viruses 2019, 11, 794. [CrossRef] 43. Edridge, A.W.D.; van der Hoek, L. Emerging orthobunyaviruses associated with CNS disease. PLoS Negl. Trop. Dis. 2020, 14, e0008856. [CrossRef] 44. Hughes, H.R.; Adkins, S.; Alkhovskiy, S.; Beer, M.; Blair, C.; Calisher, C.H.; Drebot, M.; Lambert, A.J.; de Souza, W.M.; Marklewitz, M.; et al. ICTV virus profile: Peribunyaviridae. J. Gen. Virol. 2020, 101, 1–2. [CrossRef] 45. Tomori, O.; Monath, T.P.; Lee, V.; Fagbami, A.; Fabiyi, A. Bwamba virus infection: A sero-survey of veterbrates in five ecological zones in Nigeria. Trans. R. Soc. Trop. Med. Hyg. 1974, 68, 461–465. [CrossRef] 46. Kalunda, M.; Lwanga-Ssozi, C.; Lule, M.; Mukuye, A. Isolation of Chikungunya and Pongola viruses from patients in Uganda. Trans. R. Soc. Trop. Med. Hyg. 1985, 79, 567. [CrossRef] 47. Shope, R.E.; de Andrade, A.H.; Bensabath, G.; Causey, O.R.; Humphrey, P.S. The epidemiology of EEE WEE, SLE and turlock viruses, with special reference to , in a tropical rain forest near Belem, Brazil. Am. J. Epidemiol. 1966, 84, 467–477. [CrossRef] 48. Schnagl, R.D.; Holmes, I.H. Electron microscopy of Japanaut and Tilligerry viruses: Two proposed members of the orbivirus group. Aust. J. Biol. Sci. 1975, 28, 425–432. [CrossRef] 49. Kemp, G.E.; Le Gonidec, G.; Karabatsos, N.; Rickenbach, A.; Cropp, C.B. IFE: A new African orbivirus isolated from Eidolon helvum bats captured in Nigeria, Cameroon and the Central African Republic. Bull. Soc. Pathol. Exot. Fil. 1987, 81, 40–48. 50. Boiro, I.; Fidarov, F.M.; Lomonossov, N.N.; Linev, M.B.; Bachkirsov, V.N.; Inapogui, A. Isolation of the Fomédé virus from Chiroptera, Nycteris nana, in the Republic of Guinea. Bull. Soc. Pathol. Exot. Fil. 1985, 79, 180–182. 51. Fagre, A.C.; Lee, J.S.; Kityo, R.M.; Bergren, N.A.; Mossel, E.C.; Nakayiki, T.; Nalikka, B.; Nyakarahuka, L.; Gilbert, A.T.; Peterhans, J.K.; et al. Discovery and characterization of Bukakata orbivirus (Reoviridae:Orbivirus), a novel virus from a Ugandan bat. Viruses 2019, 11, 209. [CrossRef] 52. Miura, T.; Kitaoka, M. Viruses isolated from bats in Japan. Arch. Virol. 1977, 53, 281–286. [CrossRef] 53. Justines, G.; Kuns, M.L. Matucare virus, a new agent recovered from Ornithodoros (Alectorobius) boliviensis. Am. J. Trop. Med. Hyg. 1970, 19, 697–702. [CrossRef] Viruses 2021, 13, 860 15 of 15

54. Dick, C.W.; Gettinger, D.; Gardner, S.L. Bolivian ectoparasites: A survey of bats (Mammalia: Chiroptera). Comp. Parasitol. 2007, 74, 372–377. [CrossRef] 55. Whitaker, J.O.J.; Ritzi, C.M.; Dick, C.W. Collecting and preserving ectoparasites for ecological study. In Ecological and Behavioral Methods for the Study of Bats, 2nd ed.; Kunz, T.H., Parsons, S., Eds.; Johns Hopkins University Press: Baltimore, MD, USA, 2009; pp. 806–827. 56. Reeves, W.R.; Lloyd, J.E. Louse Flies, Keds, and Bat Flies (Hippoboscoidea). In Medical and Veterinary Entomology, 3rd ed.; Mullen, G.R., Durden, L.A., Eds.; Academic Press: London, UK, 2019; pp. 421–438. 57. Osborne, J.C.; Rupprecht, C.E.; Olson, J.G.; Ksiazek, T.G.; Rollin, P.E.; Niezgoda, M.; Goldsmith, C.S.; An, U.S.; Nichol, S.T. Isolation of Kaeng Khoi virus from dead Chaerephon plicata bats in Cambodia. J. Gen. Virol. 2003, 84, 2685–2689. [CrossRef] 58. Williams, J.E.; Imlarp, S.; Top, F.H., Jr.; Cavanaugh, D.C.; Russell, P.K. Kaeng Khoi virus from naturally infected bedbugs (Cimicidae) and immature free-tailed bats. Bull. World Health Organ. 1976, 53, 365–369. 59. Groseth, A.; Mampilli, V.; Weisend, C.; Dahlstrom, E.; Porcella, S.F.; Russell, B.J.; Tesh, R.B.; Ebihara, H. Molecular characterization of human pathogenic bunyaviruses of the Nyando and Bwamba/Pongola virus groups leads to the genetic identification of Mojui dos Campos and Kaeng Khoi virus. PLoS Negl. Trop. Dis. 2014, 8, e3147. [CrossRef] 60. Pinheiro, F.P.; Travassos da Rosa, A.P. Mojui dos Campos. In International Catalogue of Arboviruses Including Certain Other Viruses of Vertebrates, 3rd ed.; Karabatsos, N., Ed.; American Society of Tropical Medicine and Hygiene: San Antonio, TX, USA, 1985; pp. 695–696. 61. Zhang, Y.Z.; Shi, M.; Holmes, E.C. Using metagenomics to characterize an expanding virosphere. Cell 2018, 172, 1168–1172. [CrossRef][PubMed] 62. Zhang, Y.Z.; Chen, Y.M.; Wang, W.; Qin, X.C.; Holmes, E.C. Expanding the RNA virosphere by unbiased metagenomics. Annu. Rev. Virol. 2019, 6, 119–139. [CrossRef] 63. Téllez, H.L.A.; Iñiguez-Dávalos, L.I.; Olvera-Vargas, M.; Vargas-Contreras, J.A.; Herrera-Lizaola, O.A. Bats associated to caves in Jalisco, Mexico. THERYA 2018, 9, 29–40. 64. Horton, D.L.; Breed, A.C.; Arnold, M.E.; Smith, G.C.; Aegerter, J.N.; McElhinney, L.M.; Johnson, N.; Banyard, A.C.; Raynor, R.; Mackie, I.; et al. Between roost contact is essential for maintenance of European bat lyssavirus type-2 in Myotis daubentonii bat reservoir: ‘The Swarming Hypothesis’. Sci. Rep. 2020, 10, 1740. 65. Parratt, S.R.; Laine, A.L. The role of hyperparasitism in microbial pathogen ecology and evolution. ISME J. 2016, 10, 1815–1822. [CrossRef] 66. Haelewaters, D.; Hiller, T.; Dick, C.W. Bats, bat flies, and fungi: A case of hyperparasitism. Trends Parasitol. 2018, 34, 784–799. 67. Wenzel, R.L.; Tipton, V.J. Some relationships between mammal hosts and their ectoparasites. In Ectoparasites of Panama; Wenzel, R.L., Tipton, V.J., Eds.; Field Museum of Natural History: Chicago, IL, USA, 1966; pp. 677–723. 68. Watts, D.M.; Pantuwatana, S.; DeFoliart, G.R.; Yuill, T.M.; Thompson, W.H. Transovarial transmission of LaCrosse virus (California encephalitis group) in the mosquito, Aedes triseriatus. Science 1973, 182, 1140–1141. [CrossRef] 69. Rozo-Lopez, P.; Drolet, B.S.; Londoño-Renteria, B. Vesicular stomatitis virus transmission: A comparison of incriminated vectors. Insects 2018, 9, 190. [CrossRef] 70. Tesh, R.B.; Boshell, J.; Modi, G.B.; Morales, A.; Young, D.G.; Corredor, A.; de Carrasquilla, C.F.; de Rodriguez, C.; Walters, L.L.; Gaitan, M.O. Natural infection of humans, , and phlebotomine sand flies with the Alagoas serotype of vesicular stomatitis virus in Colombia. Am. J. Trop. Med. Hyg. 1987, 36, 653–661. [CrossRef] 71. Corn, J.L.; Comer, J.A.; Erickson, G.A.; Nettles, V.F. Isolation of vesicular stomatitis virus New Jersey serotype from phlebotomine sand flies in Georgia. Am. J. Trop. Med. Hyg. 1990, 42, 476–482. [CrossRef] 72. Comer, J.A.; Corn, J.L.; Stallknecht, D.E.; Landgraf, J.G.; Nettles, V.F. Titers of vesicular stomatitis virus, New Jersey serotype, in naturally infected male and female (Diptera: ) in Georgia. J. Med. Entomol. 1992, 29, 368–370. [CrossRef] 73. Comer, J.A.; Tesh, R.B.; Modi, G.B.; Corn, J.L.; Nettles, V.F. Vesicular stomatitis virus, New Jersey serotype: Replication in and transmission by Lutzomyia shannoni (Diptera: Psychodidae). Am. J. Trop. Med. Hyg. 1990, 42, 483–490. [CrossRef] 74. Schmidtmann, E.T.; Craig, M.E.; English, L.M.; Herrero, M.V. Sampling for sand flies (Diptera: Psychodidae) among prairie colonies on ranches with histories of vesicular stomatitis in new Mexico and Colorado. J. Med. Entomol. 2002, 39, 680–684. [CrossRef] 75. Fleming, T.H. Bat Migration. Encycl. Anim. Behav. 2019, 605–610. [CrossRef] 76. Wiederholt, R.; Lopez-Hoffman, L.; Cline, J.; Medellín, R.A.; Cryan, P.; Russell, A.; McCracken, G.; Diffendorfer, J.; Semmens, D. Moving across the border: Modeling migratory bat populations. Ecosphere 2013, 4, 114. [CrossRef] 77. Clark, G.G.; Calisher, C.H.; Crabbs, C.L.; Canestorp, K.M.; Tesh, R.B.; Bowen, R.A.; Taylor, D.E. Malpais spring virus: A new vesiculovirus from mosquitoes collected in New Mexico and evidence of infected indigenous and exotic ungulates. Am. J. Trop. Med. Hyg. 1988, 39, 586–592. [CrossRef] 78. Huang, T.; Liu, Z.; Gong, X.; Wu, T.; Liu, H.; Deng, J.; Zhang, Y.; Peng, Q.; Zhang, L.; Liu, Z. Vampire in the darkness: A new genus and species of land exclusively bloodsucking cave-dwelling bats from China (Hirudinda: : Haemadipsidae). Zootaxa 2019, 4560, 257–272. [CrossRef]