Using Multiplex Amplicon PCR Technology to Efficiently and Timely Generate Rift Valley Fever Virus Sequence Data for Genomic Surveillance

John Juma; Samson L Konongoi; Isidore Nsengimana; Reuben Mwangi; James Akoko; Richard Nyamota; Collins Muli; Paul O Dobi; Edward Kiritu; Shebbar Osiany; Amos A Onwong'a; Rachael W Gachogo; Rosemary Sang; Alan Christoffels; Kristina Roesel; Bernard Bett; Samuel O Oyola

doi:10.3390/v15020477

Using Multiplex Amplicon PCR Technology to Efficiently and Timely Generate Rift Valley Fever Virus Sequence Data for Genomic Surveillance

Viruses. 2023 Feb 9;15(2):477. doi: 10.3390/v15020477.

Authors

John Juma^{1

2}, Samson L Konongoi^{1

3}, Isidore Nsengimana^{4

5}, Reuben Mwangi¹, James Akoko¹, Richard Nyamota¹, Collins Muli¹, Paul O Dobi¹, Edward Kiritu¹, Shebbar Osiany¹, Amos A Onwong'a^{6

7}, Rachael W Gachogo⁸, Rosemary Sang³, Alan Christoffels², Kristina Roesel¹, Bernard Bett¹, Samuel O Oyola¹

Affiliations

¹ International Livestock Research Institute (ILRI), Nairobi P.O. Box 30709, Kenya.
² South African National Bioinformatics Institute (SANBI), University of the Western Cape, Cape Town 7535, South Africa.
³ Centre for Virus Research, Kenya Medical Research Institute (KEMRI), Nairobi P.O. Box 54840, Kenya.
⁴ Department of Microbiology, Parasitology and Biotechnology, Sokoine University of Agriculture, Morogoro 3019, Tanzania.
⁵ Rwanda Inspectorate, Competition and Consumer Protection Authority, Kigali P.O. Box 375, Rwanda.
⁶ Department of Veterinary Services, Ministry of Agriculture, Livestock and Fisheries, Nairobi P.O. Box 30028, Kenya.
⁷ Department of Medical Laboratory Sciences, Jomo Kenyatta University of Agriculture and Technology (JKUAT), Nairobi 00200, Kenya.
⁸ Division of Immunology, Department of Human Pathology, University of Cape Town, Cape Town 7925, South Africa.

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.

Keywords: Rift Valley fever; amplicon; culture; enrichment; genome; multiplex PCR.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
COVID-19 Testing
COVID-19*
Genomics
Humans
Multiplex Polymerase Chain Reaction
Rift Valley Fever*
Rift Valley fever virus* / genetics
SARS-CoV-2 / genetics
Zika Virus Infection*
Zika Virus*

Grants and funding

The research reported in this publication was supported through a research-for-development project, Boosting Uganda’s Investments in Livestock Development (BUILD), led by the International Livestock Research Institute (ILRI) and funded by the German government through the Federal Ministry of Economic Cooperation and Development (BMZ), Grant No. BMZ001201. Additional funding was received from the Rockefeller Foundation and the Africa CDC through a sub-grant award to S.O.O., the CGIAR Research Programs on Livestock and Agriculture for Nutrition Health and the Department of Defense’s Defense Threat Reduction Agency, Grant No. HDTRA11910031, supporting the project ‘Co-infection with Rift Valley fever virus, Brucella spp. and Coxiella burnetii in humans and animals in Kenya: Disease burden and ecological factors’. We also acknowledge the CGIAR Fund Donors (https://www.cgiar.org/funders (accessed on 7 November 2022)).