Phenotypic characterization of T’Ho virus and the development of tools for its serologic diagnosis

Iowa State University, Ames IA

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Abstract

PROJECT SUMMARY T’Ho virus is a poorly characterized flavivirus recently discovered in Culex quinquefasciatus mosquitoes in the Yucatan Peninsula of Mexico. The genome of T’Ho virus was fully sequenced but an isolate was not recovered by suckling mouse brain inoculation or by virus isolation in vertebrate or mosquito cell cultures. Genome sequence alignments revealed that the closest known relatives of T’Ho virus are mosquito-transmitted flaviviruses associated with life-threatening human disease. Rocio virus, a BSL-3 pathogen, is the closest known relative, followed by Ilhéus, St. Louis encephalitis, Japanese encephalitis and West Nile viruses. Because T’Ho virus is most closely related to known human pathogens, it too could be a cause of human disease. Clinical and serological studies need to be performed to investigate this issue. There is also an important need to perform in vivo experimental infections to identify competent vertebrate host and vector species. The ability to perform these experiments is severely restricted by the unavailability of an isolate. To address this issue, recombinant technology will be used to create infectious viruses that can be used for T’Ho virus research and diagnosis. The first objective is to generate recombinant T’Ho virus and characterize its in vitro host range and replication kinetics. Based on its close phylogenetic relationship with flaviviruses that cycle between mosquitoes and vertebrate hosts, it is hypothesized that T’Ho virus replicates in both mosquito and vertebrate cells. The second objective is to create a chimeric virus that can be used in BSL-2 laboratories for the detection of neutralizing antibodies to T’Ho virus. Because T’Ho virus is closely related to several BSL-3 pathogens, it could eventually be classified as a BSL-3 agent. However, many arbovirus laboratories, particularly those in Latin America, lack BSL-3 facilities. Therefore, a chimeric virus will be generated by substituting the major structural protein genes of Zika virus, a BSL-2 pathogen, with the corresponding region of T’Ho virus, producing a virus that can be used in BSL-2 laboratories. It is hypothesized that the aforementioned genetic exchange is functionally compatible and will generate a chimeric virus that forms plaques in vertebrate cell monolayers. The proposed studies provide the foundation for many future experiments. Vertebrate animals and mosquitoes can be experimentally inoculated with the recombinant virus to identify competent reservoir hosts and vectors of T’Ho virus, providing insight into its transmission cycle. The recombinant virus will allow researchers to monitor humans and vertebrate animals in Mexico and elsewhere in the Americas for antibodies to T’Ho virus by plaque reduction neutralization test (PRNT). The PRNT is the gold- standard serologic technique for the diagnosis of flavivirus infections and it requires live virus. The chimeric virus can be used when BSL-3 facilities are unavailable. Flaviviruses are known for their serologic cross-reactivity; thus, it is likely that antibodies to T’Ho virus can bind to and neutralize other flaviviruses. This raises the possibility that some human and vertebrate animals previously tested by PRNT in serologic investigations in Latin America contained antibodies to T’Ho virus but were misdiagnosed. It is important that infectious T’Ho virus is available so it can be included in PRNTs and considered in the differential diagnosis.