Inadvertent Detection of Rift Valley Fever Virus by Metagenomic Sequencing in Febrile Patients Seeking Health Care at Marigat Sub-District Hospital, Baringo County, Kenya

Abstract

Abstract Background Rift Valley Fever (RVF) outbreaks occur following unusually heavy rainfall and flooding, which increase the breeding activities of mosquitoes that transmit the RVF virus (RVFV). Such conditions occurred from May to June 2018 in Wajir and Marsabit counties, northeastern Kenya. In December 2019, a blood sample that had been collected in June 2018 from a febrile child attending a hospital 648 km away from Wajir town produced a few short sequences that mapped to RVFV by shotgun metagenomic next-generation sequencing (mNGS). The agnostic mNGS was part of a pathogen discovery exercise that aimed to identify viral pathogens of concern that are missed by targeted testing for endemic fever-causing. Methodology Following identification of sequence reads that mapped to RVFV, we expanded subsequent testing by reverse transcriptase real time PCR (RT-qPCR) for RVFV to 44 serum samples, including the test case, collected between June and September 2018. Positive samples were further analyzed by shotgun metagenomics using a pathogen agnostic discovery pipeline that involves converting the RNA to cDNA, followed by random amplification using viral genome sequencing primers. The generated products were used for library preparation and subsequently sequenced on Illumina MiSeq. Genome assembly was performed using the ngs_mapper pipeline, while lineage classification and phylogeny were performed using rvfv typing tool v1 and phyml v3, respectively. Results Of the 44 specimens, three, including the index sample tested positive at cycle threshold (Ct) values of 19.3, 34.3 and 31 (index sample). The whole genome of RVFV comprising the large (L), medium (M), and small (S) segments was obtained from the serum sample with the lowest Ct value. The other samples had partial sequences of the L segment. The genomes were classified as C lineage. Phylogeny drawn from the whole genome sample clustered in a clade comprising sequences obtained from the 2017 human RVFV outbreak in Uganda and the 2021 cattle outbreak in Kiambu, Central Kenya. Conclusion This study used unbiased pathogen detection to identify presence of RVFV in the community living in Baringo County, Kenya that would otherwise have gone undocumented. Based on these data, RT-qPCR test for RVFV has been included in our routine testing panel for febrile illness.