Abstract
Viruses in the family Bunyaviridae infect a wide range of plant, insect, and animal hosts. Tick-borne bunyaviruses in the Phlebovirus genus, including Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) in China, Heartland virus (HRTV) in the United States, and Bhanja virus in Eurasia and Africa have been associated with acute febrile illness in humans. Here we sought to characterize the growth characteristics and genome of Lone Star virus (LSV), an unclassified bunyavirus originally isolated from the lone star tick Amblyomma americanum. LSV was able to infect both human (HeLa) and monkey (Vero) cells. Cytopathic effects were seen within 72 h in both cell lines; vacuolization was observed in infected Vero, but not HeLa, cells. Viral culture supernatants were examined by unbiased deep sequencing and analysis using an in-house developed rapid computational pipeline for viral discovery, which definitively identified LSV as a phlebovirus. De novo assembly of the full genome revealed that LSV is highly divergent, sharing <61% overall amino acid identity with any other bunyavirus. Despite this sequence diversity, LSV was found by phylogenetic analysis to be part of a well-supported clade that includes members of the Bhanja group viruses, which are most closely related to SFSTV/HRTV. The genome sequencing of LSV is a critical first step in developing diagnostic tools to determine the risk of arbovirus transmission by A. americanum, a tick of growing importance given its expanding geographic range and competence as a disease vector. This study also underscores the power of deep sequencing analysis in rapidly identifying and sequencing the genomes of viruses of potential clinical and public health significance.
Highlights
Bunyaviridae is the largest family of viruses, with over 350 species that infect a broad range of hosts including plants, arthropods, and vertebrate animals [1]
The titers of infectious Lone Star virus (LSV) produced in HeLa and Vero cell culture at 72 hpi in plaque-forming units (PFU) per milliliter were 1.96106 PFU/ml and 1.26106 PFU/ml, respectively
As the alignable reads represented less than 50% overall coverage of the genome (Fig. 2A), the full LSV genome was subsequently recovered by 3 rounds of 15-cycle de novo assembly using a single ‘‘seed’’ corresponding to an identified read for each of the presumptive L, M, and S segments (Fig. 2B)
Summary
Bunyaviridae is the largest family of viruses, with over 350 species that infect a broad range of hosts including plants, arthropods, and vertebrate animals [1]. In 2011, a new bunyavirus in the Phlebovirus genus, named Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV), was reported as the cause of an outbreak of severe febrile illness in China [5,6,7]. The discovery of Heartland virus (HRTV), a new, putatively tick-borne phlebovirus distinct from LSV and associated with two human cases of critical febrile illness from Missouri, was reported [8]. The pathogenic spectrum of the BHAV group viruses has not yet been fully defined, Bhanja virus has been associated with febrile illness with central nervous system involvement in both laboratory and naturally infected cases [5,6,7,11,12,13]
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