Defining the virosphere of neglected hematophagous arthropods

PROJECT SUMMARY
Hematophagous arthropods transmit numerous viruses, many of which are serious human pathogens. To combat
the growing public health impact of arthropod-transmitted viruses, many metagenomics studies have been
performed to characterize the arthropod virome. The importance of proactive virus discovery cannot be
understated because it allows risk assessments to be performed and viral diagnostic assays and control strategies
to be developed before virus spillover occurs. Most metagenomics studies designed to identify novel arthropod-
associated viruses have focused on mosquitoes, with other hematophagous arthropods being largely neglected.
Another limitation of many previous studies is that virus isolation was not attempted, with newly discovered
viruses known only from sequence data, which has impeded their phenotypic and serological characterization. The
overall goal of this grant application is to define the virosphere of understudied hematophagous arthropods and to
identify and characterize novel viruses capable of vertebrate cell replication. Two specific aims have been designed
to achieve this goal. In specific aim 1, a diverse range blood-feeding arthropods (ticks, midges, sandflies, blackflies,
and fleas) will be assayed for novel and previously recognized viruses by unbiased high-throughput sequencing
(UHTS). These experiments will be performed using homogenates already in our possession and prepared from
>37,000 arthropods from North America. Total RNA will be directly extracted from every homogenate and viral
sequences will be identified using UHTS and an established bioinformatics pipeline, allowing the virosphere of the
arthropods to be defined. Additionally, the codon and dinucleotide frequencies of every novel virus will be
determined to provide insight into their host ranges and to identify those most likely to replicate in vertebrate
cells. In this regard, vertebrates and invertebrates preferentially have certain codon and dinucleotide usage biases
and the preferences of RNA viruses often mimic those of their hosts. In specific aim 2, an aliquot of each
homogenate will be inoculated onto human and nonhuman primate cells then two blind passages will be
performed. Total RNA will be extracted from all final culture passages and analyzed by UHTS and bioinformatics in
order to specifically identify vertebrate-infecting viruses. If an isolate is not recovered but the homogenate is
predicted to contain a vertebrate-infecting virus, as determined in the codon and dinucleotide frequency analysis,
virus isolation will be attempted using additional vertebrate cell lines. The in vitro replication kinetics and yields of
every novel virus capable of vertebrate cell replication will be determined. The studies proposed in this grant
application provide the groundwork for future in vivo experiments, where viruses capable of vertebrate cell
replication will be further characterized. Experimental infections will be performed with vertebrate animals and
arthropods to assess viral virulence and to identify competent vector species, respectively. Future experiments will
also be performed to determine the incidence and seroprevalence of select viruses in human and vertebrate animal
populations.

Grant Number: 5R03AI177172-02
NIH Institute/Center: NIH
Principal Investigator: Bradley Blitvich