Phylogenetic characterization of Orthobunyaviruses isolated from Trinidad shows evidence of natural reassortment.

The genus Orthobunyavirus is a diverse group of viruses in the family Peribunyaviridae, recently classified into 20 serogroups, and 103 virus species. Although most viruses within these serogroups are phylogenetically distinct, the absence of complete genome sequences has left several viruses incompletely characterized. Here we report the complete genome sequences for 11 orthobunyaviruses isolated from Trinidad, French Guiana, Guatemala, and Panama that were serologically classified into six serogroups and 10 species. Phylogenetic analyses of these 11 newly derived sequences indicate that viruses belonging to the Patois, Capim, Guama, and Group C serocomplexes all have a close genetic origin. We show that three of the 11 orthobunyaviruses characterized (belonging to the Group C and Bunyamwera serogroups) have evidence of histories of natural reassortment through the M genome segment. Our data also suggests that two distinct lineages of Group C viruses concurrently circulate in Trinidad and are transmitted by the same mosquito vectors. This study also highlights the importance of complementing serological identification with nucleotide sequencing when characterizing orthobunyaviruses.

ICTV Virus Taxonomy Profile: Rhabdoviridae 2022.

The family Rhabdoviridae comprises viruses with negative-sense (-) RNA genomes of 10-16 kb. Virions are typically enveloped with bullet-shaped or bacilliform morphology but can also be non-enveloped filaments. Rhabdoviruses infect plants or animals, including mammals, birds, reptiles, amphibians or fish, as well as arthropods, which serve as single hosts or act as biological vectors for transmission to animals or plants. Rhabdoviruses include important pathogens of humans, livestock, fish or agricultural crops. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Rhabdoviridae, which is available at ictv.global/report/rhabdoviridae.

Negeviruses Reduce Replication of Alphaviruses during Coinfection.

Negeviruses are a group of insect-specific viruses (ISVs) that have been found in many arthropods. Their presence in important vector species led us to examine their interactions with arboviruses during coinfections. Wild-type negeviruses reduced the replication of several alphaviruses during coinfections in mosquito cells. Negev virus (NEGV) isolates were also used to express green fluorescent protein (GFP) and anti-chikungunya virus (CHIKV) antibody fragments during coinfections with CHIKV. NEGV expressing anti-CHIKV antibody fragments was able to further reduce replication of CHIKV during coinfections, while reductions of CHIKV with NEGV expressing GFP were similar to titers with wild-type NEGV alone. These results are the first to show that negeviruses induce superinfection exclusion of arboviruses and to demonstrate a novel approach to deliver antiviral antibody fragments with paratransgenic ISVs. The ability to inhibit arbovirus replication and express exogenous proteins in mosquito cells makes negeviruses a promising platform for control of arthropod-borne pathogens. IMPORTANCE Negeviruses are a group of insect-specific viruses (ISVs), viruses known to infect only insects. They have been discovered over a wide geographical and species range. Their ability to infect mosquito species that transmit dangerous arboviruses makes negeviruses a candidate for a pathogen control platform. Coinfections of mosquito cells with a negevirus and an alphavirus demonstrated that negeviruses can inhibit the replication of alphaviruses. Additionally, modifying Negev virus (NEGV) to express a fragment of an anti-CHIKV antibody further reduced the replication of CHIKV in coinfected cells. This is the first evidence to demonstrate that negeviruses can inhibit the replication of important arboviruses in mosquito cells. The ability of a modified NEGV to drive the expression of antiviral proteins also highlights a method for negeviruses to target specific pathogens and limit the incidence of vector-borne diseases.

Hayes Yard virus: a novel ephemerovirus isolated from a bull with severe clinical signs of bovine ephemeral fever is most closely related to Puchong virus.

Bovine ephemeral fever is a vector-borne disease of ruminants that occurs in tropical and sub-tropical regions of Africa, Asia and Australia. The disease is caused by a rhabdovirus, bovine ephemeral fever virus (BEFV), which occurs as a single serotype globally. Although several other closely related ephemeroviruses have been isolated from cattle and/or arthropods, only kotonkan virus from Nigeria and (tentatively) Mavingoni virus from Mayotte Island in the Indian Ocean have been previously associated with febrile disease. Here, we report the isolation of a novel virus (Hayes Yard virus; HYV) from blood collected in February 2000 from a bull (Bos indicus) in the Northern Territory of Australia. The animal was suffering from a severe ephemeral fever-like illness with neurological involvement, including recumbency and paralysis, and was euthanised. Histological examination of spinal cord and lung tissue identified extensive haemorrhage in the dura mata with moderate perineuronal oedema and extensive emphysema. HYV displayed cone-shaped morphology, typical of rhabdoviruses, and was found to be most closely related antigenically to Puchong virus (PUCV), isolated in 1965 from mosquitoes in Malaysia. Analysis of complete genome sequences of HYV (15 025 nt) and PUCV (14 932 nt) indicated that each has a complex organisation (3' N-P-M-G-GNS-α1-α2-ß-γ-L 5') and expression strategy, similar to that of BEFV. Based on an alignment of complete L protein sequences, HYV and PUCV cluster with other rhabdoviruses in the genus Ephemerovirus and appear to represent two new species. Neutralising antibody to HYV was also detected in a retrospective survey of cattle sera collected in the Northern Territory.

Genomic characterization of Changuinola viruses from Panama: evidence for multiple genome segment reassortment.

The complete coding sequences of five divergent strains of Changuinola virus (CGLV), collected over a 16-year period in Panama, were determined, using viral metagenomics. Each strain had 10 RNA segments that encoded structural and non-structural proteins with amino acid identities ranging from 33 to 99% with sequences of other 15 members of the Changuinola virus (Reoviridae: Orbivirus) species group. Genetic analyses of the five Panamanian virus strains revealed probable reassortment among multiple segments of the viruses.

Experimental yellow fever virus infection in the squirrel monkey (Saimiri spp.) I: gross anatomical and histopathological findings in organs at necropsy.

BACKGROUND: Non-human primates contribute to the spread of the yellow fever virus (YFV) and the establishment of transmission cycles in endemic areas. OBJECTIVE: To describe the severe histopathological aspects of YFV infection, 10 squirrel monkeys were infected with YFV and blood, brain, liver, kidney, spleen, heart, lung, lymph node and stomach were collected at 1-7, 10, 20 and 30 days post-infection (dpi). METHODS: Histopathological analysis and detection of the genome and viral antigens and neutralising antibodies were performed by RT-PCR, immunohistochemistry and neutralisation test, respectively. FINDINGS: Only one animal died from the experimental infection. The genome and viral antigens were detected in all investigated organs (1-30 dpi) and the neutralising antibodies from seven to 30 dpi. The brain contained perivascular haemorrhage (6 dpi); in the liver, midzonal haemorrhage and lytic necrosis (6 dpi) were observed. The kidney had bleeding in the Bowman's capsule and tubular necrosis (6 dpi). Pyknotic lymphocytes were observed in the spleen (1-20 dpi), the lung had haemorrhage (2-6 dpi), in the endocardium it contained nuclear pyknosis and necrosis (2-3 dpi) and the stomach contained blood in the lumen (6 dpi). MAIN FINDINGS: Squirrel monkeys reliably reproduced the responses observed in human cases of yellow fever and, therefore, constitute an excellent experimental model for studies on the pathophysiology of the disease.

Large-Scale Complete-Genome Sequencing and Phylodynamic Analysis of Eastern Equine Encephalitis Virus Reveals Source-Sink Transmission Dynamics in the United States.

Eastern equine encephalitis virus (EEEV) has a high case-fatality rate in horses and humans, and Florida has been hypothesized to be the source of EEEV epidemics for the northeastern United States. To test this hypothesis, we sequenced complete genomes of 433 EEEV strains collected within the United States from 1934 to 2014. Phylogenetic analysis suggested EEEV evolves relatively slowly and that transmission is enzootic in Florida, characterized by higher genetic diversity and long-term local persistence. In contrast, EEEV strains in New York and Massachusetts were characterized by lower genetic diversity, multiple introductions, and shorter local persistence. Our phylogeographic analysis supported a source-sink model in which Florida is the major source of EEEV compared to the other localities sampled. In sum, this study revealed the complex epidemiological dynamics of EEEV in different geographic regions in the United States and provided general insights into the evolution and transmission of other avian mosquito-borne viruses in this region.IMPORTANCE Eastern equine encephalitis virus (EEEV) infections are severe in horses and humans on the east coast of the United States with a >90% mortality rate in horses, an ∼33% mortality rate in humans, and significant brain damage in most human survivors. However, little is known about the evolutionary characteristics of EEEV due to the lack of genome sequences. By generating large collection of publicly available complete genome sequences, this study comprehensively determined the evolution of the virus, described the epidemiological dynamics of EEEV in different states in the United States, and identified Florida as one of the major sources. These results may have important implications for the control and prevention of other mosquito-borne viruses in the Americas.

Correction: Correction: A Novel Rhabdovirus Associated with Acute Hemorrhagic Fever in Central Africa.

[This corrects the article DOI: 10.1371/journal.ppat.1002924.].

Genomic characterisation of Cuiaba and Charleville viruses: arboviruses (family Rhabdoviridae, genus Sripuvirus) infecting reptiles and amphibians.

Viruses in the family Rhabdoviridae are ecologically very diverse, infecting mammals, birds, reptiles, fish, plants and a wide range of other terrestrial and aquatic invertebrates. The genus Sripuvirus currently comprises five viruses that appear to circulate in transmission cycles involving reptiles and sandflies. Here, we report an analysis of the complete coding sequences of Cuiaba virus (CUIV), isolated from an amphibian (Bufo marinus) collected in Brazil, and Charleville virus (CHVV), isolated from sandflies (Phlebotomus sp.) and lizards (Gehyra australis), collected in Australia. CUIV and CHVV cluster phylogenetically with the sripuviruses in maximum likelihood trees generated from complete L protein (RdRp) sequences. They also share with sripuviruses unique features in genome organisation, including an additional gene (U1) between the matrix protein (M) gene and glycoprotein (G) gene, and an alternative long open reading frame near the start of the G ORF that encodes a predicted transmembrane protein. In view of their phylogenetic relationships, similar genome organisations and similar ecological characteristics, we propose the assignment of CUIV and CHVV as novel members of the genus Sripuvirus.

Terrestrial Bird Migration and West Nile Virus Circulation, United States.

Host migration and emerging pathogens are strongly associated, especially with regard to zoonotic diseases. West Nile virus (WNV), a mosquitoborne pathogen capable of causing severe, sometimes fatal, neuroinvasive disease in humans, is maintained in highly mobile avian hosts. Using phylogeographic approaches, we investigated the relationship between WNV circulation in the United States and the flight paths of terrestrial birds. We demonstrated southward migration of WNV in the eastern flyway and northward migration in the central flyway, which is consistent with the looped flight paths of many terrestrial birds. We also identified 3 optimal locations for targeted WNV surveillance campaigns in the United States-Illinois, New York, and Texas. These results illustrate the value of multidisciplinary approaches to surveillance of infectious diseases, especially zoonotic diseases.

ICTV Virus Taxonomy Profile: Rhabdoviridae.

The family Rhabdoviridae comprises viruses with negative-sense (-) single-stranded RNA genomes of 10.8-16.1 kb. Virions are typically enveloped with bullet-shaped or bacilliform morphology but can also be non-enveloped filaments. Rhabdoviruses infect plants and animals including mammals, birds, reptiles and fish, as well as arthropods which serve as single hosts or act as biological vectors for transmission to animals or plants. Rhabdoviruses include important pathogens of humans, livestock, fish and agricultural crops. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Rhabdoviridae, which is available at www.ictv.global/report/rhabdoviridae.

Recombinant Isfahan Virus and Vesicular Stomatitis Virus Vaccine Vectors Provide Durable, Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge.

The demonstrated clinical efficacy of a recombinant vesicular stomatitis virus (rVSV) vaccine vector has stimulated the investigation of additional serologically distinct Vesiculovirus vectors as therapeutic and/or prophylactic vaccine vectors to combat emerging viral diseases. Among these viral threats are the encephalitic alphaviruses Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV), which have demonstrated potential for natural disease outbreaks, yet no licensed vaccines are available in the event of an epidemic. Here we report the rescue of recombinant Isfahan virus (rISFV) from genomic cDNA as a potential new vaccine vector platform. The rISFV genome was modified to attenuate virulence and express the VEEV and EEEV E2/E1 surface glycoproteins as vaccine antigens. A single dose of the rISFV vaccine vectors elicited neutralizing antibody responses and protected mice from lethal VEEV and EEEV challenges at 1 month postvaccination as well as lethal VEEV challenge at 8 months postvaccination. A mixture of rISFV vectors expressing the VEEV and EEEV E2/E1 glycoproteins also provided durable, single-dose protection from lethal VEEV and EEEV challenges, demonstrating the potential for a multivalent vaccine formulation. These findings were paralleled in studies with an attenuated form of rVSV expressing the VEEV E2/E1 glycoproteins. Both the rVSV and rISFV vectors were attenuated by using an approach that has demonstrated safety in human trials of an rVSV/HIV-1 vaccine. Vaccines based on either of these vaccine vector platforms may present a safe and effective approach to prevent alphavirus-induced disease in humans.IMPORTANCE This work introduces rISFV as a novel vaccine vector platform that is serologically distinct and phylogenetically distant from VSV. The rISFV vector has been attenuated by an approach used for an rVSV vector that has demonstrated safety in clinical studies. The vaccine potential of the rISFV vector was investigated in a well-established alphavirus disease model. The findings indicate the feasibility of producing a safe, efficacious, multivalent vaccine against the encephalitic alphaviruses VEEV and EEEV, both of which can cause fatal disease. This work also demonstrates the efficacy of an attenuated rVSV vector that has already demonstrated safety and immunogenicity in multiple HIV-1 phase I clinical studies. The absence of serological cross-reactivity between rVSV and rISFV and their phylogenetic divergence within the Vesiculovirus genus indicate potential for two stand-alone vaccine vector platforms that could be used to target multiple bacterial and/or viral agents in successive immunization campaigns or as heterologous prime-boost agents.

Maguari Virus Associated with Human Disease.

Despite the lack of evidence for symptomatic human infection with Maguari virus (MAGV), its close relation to Cache Valley virus (CVV), which does infect humans, remains a concern. We sequenced the complete genome of a MAGV-like isolate (OBS6657) obtained from a febrile patient in Pucallpa, Ucayali, Peru, in 1998. To facilitate its classification, we generated additional full-length sequences for the MAGV prototype strain, 3 additional MAGV-like isolates, and the closely related CVV (7 strains), Tlacotalpan (1 strain), Playas (3 strains), and Fort Sherman (1 strain) viruses. The OBS6657 isolate is similar to the MAGV prototype, whereas 2 of the other MAGV-like isolates are located on a distinct branch and most likely warrant classification as a separate virus species and 1 is, in fact, a misclassified CVV strain. Our findings provide clear evidence that MAGV can cause human disease.

Clinical Manifestations of Punta Toro Virus Species Complex Infections, Panama, 2009.

An investigation in Panama found that Punta Toro virus species complex (PTVs) may contribute to febrile illnesses with symptoms mirroring those of dengue fever. However, further studies are needed to determine if PTV infection causes only a mild disease or if it can have more serious manifestations in some patients.

Characterization of five unclassified orthobunyaviruses (Bunyaviridae) from Africa and the Americas.

The Bunyaviridae family is made up of a diverse range of viruses, some of which cause disease and are a cause for concern in human and veterinary health. Here, we report the genomic and antigenic characterization of five previously uncharacterized bunyaviruses. Based on their ultrastructure, antigenic relationships and phylogenomic relationships, the five viruses are classified as members of the Orthobunyavirus genus. Three are viruses in the California encephalitis virus serogroup and are related to Trivittatus virus; the two others are most similar to the Mermet virus in the Simbu serogroup, and to the Tataguine virus, which is not currently assigned to a serogroup. Each of these five viruses was pathogenic to newborn mice, indicating their potential to cause illness in humans and other animals.

Divergent Viruses Discovered in Arthropods and Vertebrates Revise the Evolutionary History of the Flaviviridae and Related Viruses.

UNLABELLED: Viruses of the family Flaviviridae are important pathogens of humans and other animals and are currently classified into four genera. To better understand their diversity, evolutionary history, and genomic flexibility, we used transcriptome sequencing (RNA-seq) to search for the viruses related to the Flaviviridae in a range of potential invertebrate and vertebrate hosts. Accordingly, we recovered the full genomes of five segmented jingmenviruses and 12 distant relatives of the known Flaviviridae ("flavi-like" viruses) from a range of arthropod species. Although these viruses are highly divergent, they share a similar genomic plan and common ancestry with the Flaviviridae in the NS3 and NS5 regions. Remarkably, although these viruses fill in major gaps in the phylogenetic diversity of the Flaviviridae, genomic comparisons reveal important changes in genome structure, genome size, and replication/gene regulation strategy during evolutionary history. In addition, the wide diversity of flavi-like viruses found in invertebrates, as well as their deep phylogenetic positions, suggests that they may represent the ancestral forms from which the vertebrate-infecting viruses evolved. For the vertebrate viruses, we expanded the previously mammal-only pegivirus-hepacivirus group to include a virus from the graceful catshark (Proscyllium habereri), which in turn implies that these viruses possess a larger host range than is currently known. In sum, our data show that the Flaviviridae infect a far wider range of hosts and exhibit greater diversity in genome structure than previously anticipated. IMPORTANCE: The family Flaviviridae of RNA viruses contains several notorious human pathogens, including dengue virus, West Nile virus, and hepatitis C virus. To date, however, our understanding of the biodiversity and evolution of the Flaviviridae has largely been directed toward vertebrate hosts and their blood-feeding arthropod vectors. Therefore, we investigated an expanded group of potential arthropod and vertebrate host species that have generally been ignored by surveillance programs. Remarkably, these species contained diverse flaviviruses and related viruses that are characterized by major changes in genome size and genome structure, such that these traits are more flexible than previously thought. More generally, these data suggest that arthropods may be the ultimate reservoir of the Flaviviridae and related viruses, harboring considerable genetic and phenotypic diversity. In sum, this study revises the traditional view on the evolutionary history, host range, and genomic structures of a major group of RNA viruses.

Evolution of genome size and complexity in the rhabdoviridae.

RNA viruses exhibit substantial structural, ecological and genomic diversity. However, genome size in RNA viruses is likely limited by a high mutation rate, resulting in the evolution of various mechanisms to increase complexity while minimising genome expansion. Here we conduct a large-scale analysis of the genome sequences of 99 animal rhabdoviruses, including 45 genomes which we determined de novo, to identify patterns of genome expansion and the evolution of genome complexity. All but seven of the rhabdoviruses clustered into 17 well-supported monophyletic groups, of which eight corresponded to established genera, seven were assigned as new genera, and two were taxonomically ambiguous. We show that the acquisition and loss of new genes appears to have been a central theme of rhabdovirus evolution, and has been associated with the appearance of alternative, overlapping and consecutive ORFs within the major structural protein genes, and the insertion and loss of additional ORFs in each gene junction in a clade-specific manner. Changes in the lengths of gene junctions accounted for as much as 48.5% of the variation in genome size from the smallest to the largest genome, and the frequency with which new ORFs were observed increased in the 3' to 5' direction along the genome. We also identify several new families of accessory genes encoded in these regions, and show that non-canonical expression strategies involving TURBS-like termination-reinitiation, ribosomal frame-shifts and leaky ribosomal scanning appear to be common. We conclude that rhabdoviruses have an unusual capacity for genomic plasticity that may be linked to their discontinuous transcription strategy from the negative-sense single-stranded RNA genome, and propose a model that accounts for the regular occurrence of genome expansion and contraction throughout the evolution of the Rhabdoviridae.

Eastern equine encephalitis in Latin America.

BACKGROUND: The eastern equine encephalitis (EEE) and Venezuelan equine encephalitis (VEE) viruses are pathogens that infect humans and horses in the Americas. Outbreaks of neurologic disease in humans and horses were reported in Panama from May through early August 2010. METHODS: We performed antibody assays and tests to detect viral RNA and isolate the viruses in serum samples from hospitalized patients. Additional cases were identified with enhanced surveillance. RESULTS: A total of 19 patients were hospitalized for encephalitis. Among them, 7 had confirmed EEE, 3 had VEE, and 1 was infected with both viruses; 3 patients died, 1 of whom had confirmed VEE. The clinical findings for patients with EEE included brain lesions, seizures that evolved to status epilepticus, and neurologic sequelae. An additional 99 suspected or probable cases of alphavirus infection were detected during active surveillance. In total, 13 cases were confirmed as EEE, along with 11 cases of VEE and 1 case of dual infection. A total of 50 cases in horses were confirmed as EEE and 8 as VEE; mixed etiologic factors were associated with 11 cases in horses. Phylogenetic analyses of isolates from 2 cases of equine infection with the EEE virus and 1 case of human infection with the VEE virus indicated that the viruses were of enzootic lineages previously identified in Panama rather than new introductions. CONCLUSIONS: Cases of EEE in humans in Latin America may be the result of ecologic changes that increased human contact with enzootic transmission cycles, genetic changes in EEE viral strains that resulted in increased human virulence, or an altered host range. (Funded by the National Institutes of Health and the Secretaría Nacional de Ciencia, Tecnología e Innovación, Panama.).

Eilat virus host range restriction is present at multiple levels of the virus life cycle.

UNLABELLED: Most alphaviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates, including birds, rodents, equids, humans, and nonhuman primates. This ability of most alphaviruses to infect arthropods and vertebrates is essential for their maintenance in nature. Recently, a new alphavirus, Eilat virus (EILV), was described, and in contrast to all other mosquito-borne viruses, it is unable to replicate in vertebrate cell lines. Investigations into the nature of its host range restriction showed the inability of genomic EILV RNA to replicate in vertebrate cells. Here, we investigated whether the EILV host range restriction is present at the entry level and further explored the viral factors responsible for the lack of genomic RNA replication. Utilizing Sindbis virus (SINV) and EILV chimeras, we show that the EILV vertebrate host range restriction is also manifested at the entry level. Furthermore, the EILV RNA replication restriction is independent of the 3' untranslated genome region (UTR). Complementation experiments with SINV suggested that RNA replication is restricted by the inability of the EILV nonstructural proteins to form functional replicative complexes. These data demonstrate that the EILV host range restriction is multigenic, involving at least one gene from both nonstructural protein (nsP) and structural protein (sP) open reading frames (ORFs). As EILV groups phylogenetically within the mosquito-borne virus clade of pathogenic alphaviruses, our findings have important evolutionary implications for arboviruses. IMPORTANCE: Our work explores the nature of host range restriction of the first "mosquito-only alphavirus," EILV. EILV is related to pathogenic mosquito-borne viruses (Eastern equine encephalitis virus [EEEV], Western equine encephalitis virus [WEEV], Venezuelan equine encephalitis virus [VEEV], and Chikungunya virus [CHIKV]) that cause severe disease in humans. Our data demonstrate that EILV is restricted both at entry and genomic RNA replication levels in vertebrate cells. These findings have important implications for arbovirus evolution and will help elucidate the viral factors responsible for the broad host range of pathogenic mosquito-borne alphaviruses, facilitate vaccine development, and inform potential strategies to reduce/prevent alphavirus transmission.

Comprehensive molecular detection of tick-borne phleboviruses leads to the retrospective identification of taxonomically unassigned bunyaviruses and the discovery of a novel member of the genus phlebovirus.

UNLABELLED: Until the recent emergence of two human-pathogenic tick-borne phleboviruses (TBPVs) (severe fever with thrombocytopenia syndrome virus [SFTSV] and Heartland virus), TBPVs have been neglected as causative agents of human disease. In particular, no studies have addressed the global distribution of TBPVs, and consequently, our understanding of the mechanism(s) underlying their evolution and emergence remains poor. In order to provide a useful tool for the ecological and epidemiological study of TBPVs, we have established a simple system that can detect all known TBPVs, based on conventional reverse transcription-PCR (RT-PCR) with degenerate primer sets targeting conserved regions of the viral L genome segment. Using this system, we have determined that several viruses that had been isolated from ticks decades ago but had not been taxonomically identified are novel TBPVs. Full-genome sequencing of these viruses revealed a novel fourth TBPV cluster distinct from the three known TBPV clusters (i.e., the SFTS, Bhanja, and Uukuniemi groups) and from the mosquito/sandfly-borne phleboviruses. Furthermore, by using tick samples collected in Zambia, we confirmed that our system had enough sensitivity to detect a new TBPV in a single tick homogenate. This virus, tentatively designated Shibuyunji virus after the region of tick collection, grouped into a novel fourth TBPV cluster. These results indicate that our system can be used as a first-line screening approach for TBPVs and that this kind of work will undoubtedly lead to the discovery of additional novel tick viruses and will expand our knowledge of the evolution and epidemiology of TBPVs. IMPORTANCE: Tick-borne phleboviruses (TBPVs) have been largely neglected until the recent emergence of two virulent viruses, severe fever with thrombocytopenia syndrome virus and Heartland virus. Little is known about the global distribution of TBPVs or how these viruses evolved and emerged. A major hurdle to study the distribution of TBPVs is the lack of tools to detect these genetically divergent phleboviruses. In order to address this issue, we have developed a simple, rapid, and cheap RT-PCR system that can detect all known TBPVs and which led to the identification of several novel phleboviruses from previously uncharacterized tick-associated virus isolates. Our system can detect virus in a single tick sample and novel TBPVs that are genetically distinct from any of the known TBPVs. These results indicate that our system will be a useful tool for the surveillance of TBPVs and will facilitate understanding of the ecology of TBPVs.