Universal primers for rift valley fever virus whole-genome sequencing

Abstract

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease causing acute hemorrhagic fever. Accurate identification of mutations and phylogenetic characterization of RVF virus (RVFV) require whole-genome analysis. Universal primers to amplify the entire RVFV genome from clinical samples with low copy numbers are currently unavailable. Thus, we aimed to develop universal primers applicable for all known RVFV strains. Based on the genome sequences available from public databases, we designed eight pairs of universal PCR primers covering the entire RVFV genome. To evaluate primer universality, four RVFV strains (ZH548, Kenya 56 (IB8), BIME-01, and Lunyo), encompassing viral phylogenetic diversity, were chosen. The nucleic acids of the test strains were chemically synthesized or extracted via cell culture. These RNAs were evaluated using the PCR primers, resulting in successful amplification with expected sizes (0.8-1.7 kb). Sequencing confirmed that the products covered the entire genome of the RVFV strains tested. Primer specificity was confirmed via in silico comparison against all non-redundant nucleotide sequences using the BLASTn alignment tool in the NCBI database. To assess the clinical applicability of the primers, mock clinical specimens containing human and RVFV RNAs were prepared. The entire RVFV genome was successfully amplified and sequenced at a viral concentration of 10⁸ copies/mL. Given the universality, specificity, and clinical applicability of the primers, we anticipate that the RVFV universal primer pairs and the developed method will aid in RVFV phylogenomics and mutation detection.

Figure 1

Position of the designed universal primers and the expected amplicons alongside the genome of RVFV. The graphical image of the (A) L segment, (B) M segment, and (C) S segment of the RVFV genome shows the length and gene contents of the RNA genome. The localization of expected PCR amplicons from the eight universal primers designed in this study are indicated as blue solid lines.

Figure 2

Experimental amplification products of RVFV universal primers. Agarose gel electrophoresis image of PCR amplicons from the four test strains of RVFV. (A) ZH548, (B) Kenya 56 (IB8), (C) BIME-01, and (D) Lunyo.

Figure 3

Phylogenetic tree based on the whole-genome sequences of RVFV. Maximum likelihood tree of the (A) L segment, (B) M segment, and (C) S segment. Tree analysis was performed using RAxML version 8.2.10 and evaluated using 100,000 bootstrap replicates. Lineage names followed the nomenclature of Grobbelaar et al..

Figure 4

Comparison of primer efficiency among the eight pairs of universal primers measured by sequencing read depth coverage. The MinION sequencing read depth coverage is indicated in the y axis, and the genome region amplified by the eight primer pairs (four pairs in the L segment, two pairs in the M segment, and two pairs in the S segment) is indicated in the x axis. *, S1 product encodes the N gene (738 bp) and S2 product encodes the NSs gene (798 bp).