Complete genome sequencing of Kaisodi virus isolated from ticks in India belonging to Phlebovirus genus, family Phenuiviridae

An unknown virus was repeatedly isolated from hard tick (Haemaphysalis spinigera) during a proactive arbovirus survey in ticks conducted in 1957, in India. The virus remained uncharacterized for a long time. The passages of this virus in different vertebrate and invertebrate cells along with human and monkey-derived cell culture showed no cytopathic effect. It was identified later to be a member of Kaisodi group among Phlebovirus genus in the family Phenuiviridae (Order: Bunyavirales) by serological methods. Due to its genomic diversity, sequencing of this virus was a challenge for a while. In this study, we were able to sequence the complete genome of this virus isolate using next-generation sequencing (NGS) platform. The unknown virus was identified to be Kaisodi virus (KASDV) using NGS analysis. De novo genome assembly derived three genomic segments for the KASDV which encode for RNA-dependent RNA polymerase, glycoprotein precursor, and nucleoprotein. Functional as well as conserved domains for Kaisodi serogroup viruses were predicted and compared to a known representative of the genus Phlebovirus. The phylogenetic tree revealed its closeness to Silverwater virus, of Kaisodi serogroup with nucleotide (69%, 62%, and 61%) and amino acid (52%, 51%, and 62%) identity for L, M, and S segment, respectively. The study demonstrates the presence of a conserved motif ((72)TRGNK(76)) around the RNA binding motif region in tick-borne phleboviruses. The intergenic region encompassing the S segment of Kaisodi serogroup was GC-rich whereas the other Phlebovirus had AT-rich genome. KASDV has the largest intergenic region and larger loops, suggesting stem-loops formed due to larger loops as a possible factor for instability and cause of transcription termination. This paper also describes the real-time RT-PCR and RT-PCR assays developed and used for the detection of KASDV RNA in ticks from Karnataka, Kerala and Maharashtra State, India. The KASDV positivity observed in the recently collected tick pools indicates that the KASDV, isolated from Karnataka state in 1957, is also circulating in the adjoining Kerala state. On the basis of the current study, it should be possible to develop diagnostic assays which would facilitate an in-depth field survey exploring the veterinary and medical significance of KASDV.