Isolation of Imperial Valley Virus and Sapphire II Virus in Argas cooleyi from Imperial Valley, California through Cloning

An Orbivirus, Imperial Valley Virus (IVV), was isolated from Argas cooleyi ticks collected in Imperial Valley, California from cliff swallow nests. This was verified by Whole Genome Sequencing (WGS) and an Electron Microscope image. WGS revealed complete sequences of a second virus, Sapphire II virus, from the family Nairoviridae, genus Orthonairovirus. This project’s purpose was to isolate the two viruses in the tick pool homogenates by using different cell cultures and cloning. This will help with identification and phenotypic characterization of the viruses. It will also help determine if one is naturally dominant in a host. Various passages of the tick pool homogenates were performed for three different tick pools. The passages were done using the following three different cell lines: V76 (African green monkey kidney cells), BHK (Baby hamster kidney cells), and RK (rabbit kidney cells). PCR and RNA extraction determined whether the viruses were present in the different passages and if they were successfully isolated. Microscopy was used to visualize the cells and the cytopathic effect (CPE). Cloning of plaques and serial dilution microwells were used to isolate the viruses. A key result of this experiment was the two viruses were successfully isolated, however the different cell lines provided no natural isolation. It was also determined that IVV causes CPE, however, Sapphire II doesn’t cause CPE in cells. This innovative result changes the identification process of tick viruses because Sapphire II virus, regardless of CPE, needs to be tested by PCR for a proper diagnosis.