Using Multiplex Amplicon PCR Technology to Efficiently and Timely Generate Rift Valley Fever Virus Sequence Data for Genomic Surveillance
Abstract
Rift Valley fever (RVF) is a febrile vector-borne disease endemic in Africa and continues to spread in new territories. It is a climate-sensitive disease mostly triggered by abnormal rainfall patterns. The disease is associated with high mortality and morbidity in both humans and livestock. RVF is caused by the Rift Valley fever virus (RVFV) of the genus Phlebovirus in the family Phenuiviridae. It is a tripartite RNA virus with three genomic segments: small (S), medium (M) and large (L). Pathogen genomic sequencing is becoming a routine procedure and a powerful tool for understanding the evolutionary dynamics of infectious organisms, including viruses. Inspired by the utility of amplicon-based sequencing demonstrated in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Ebola, Zika and West Nile viruses, we report an RVFV sample preparation based on amplicon multiplex polymerase chain reaction (amPCR) for template enrichment and reduction of background host contamination. The technology can be implemented rapidly to characterize and genotype RVFV during outbreaks in a near-real-time manner. To achieve this, we designed 74 multiplex primer sets covering the entire RVFV genome to specifically amplify the nucleic acid of RVFV in clinical samples from an animal tissue. Using this approach, we demonstrate achieving complete RVFV genome coverage even from samples containing a relatively low viral load. We report the first primer scheme approach of generating multiplex primer sets for a tripartite virus which can be replicated for other segmented viruses.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Feb 09, 2023
- Source ID
- 10.3390/v15020477
Entities
People
- Alan Christoffels
- Amos A. Onwong’a
- Bernard Bett
- Collins Muli
- Edward Kiritu
- Isidore Nsengimana
- James Akoko
- John Juma
- Kristina Roesel
- Paul O. Dobi
- Rachael W. Gachogo
- Reuben Mwangi
- Richard Nyamota
- Rosemary Sang
- Samson L. Konongoi
- Samuel O. Oyola
- Shebbar Osiany
Organizations
- Defense Threat Reduction Agency
- International Livestock Research Institute
- Jomo Kenyatta University of Agriculture and Technology
- Kenya Medical Research Institute
- Ministry of Agriculture and Livestock Development
- University of Cape Town
- University of the Western Cape