Illumina-based sequencing of the complete genomes of two Rift Valley Fever Virus strains isolated from zoonotic outbreaks in Africa and Saudi Arabia

Rift Valley Fever Virus (RVFV) is a mosquito-borne pathogen that causes periodic disease outbreaks in Africa and the Arabian Peninsula with devastating effects on agriculture and human health. RVFV is a member of the... [ view full abstract ]

Rift Valley Fever Virus (RVFV) is a mosquito-borne pathogen that causes periodic disease outbreaks in Africa and the Arabian Peninsula with devastating effects on agriculture and human health. RVFV is a member of the Bunyaviridae family (genus Phlebovirus), characterized by a tripartite, negative- or ambisense, single-stranded RNA genome composed of large (L), medium (M) and small (S) segments. RVFV infects sheep, goats, and cattle, and results in characteristic abortion storms, fetal deformities, and significant mortality in young animals. In humans, RVFV can cause severe disease, including blindness and fatal hemorrhagic fever. Widespread mosquito-vector competence of RVFV makes it a palpable threat for introduction into non-endemic areas, and there are currently no fully licensed human or livestock vaccines available in the US. Therefore, it is necessary to understand strain-specific factors that drive RVFV virulence for the development of improved biosurveillance, infection models, and therapeutic strategies. In the current study, we determined the complete genome sequences of two RVFV strains isolated from Africa and the Arabian Peninsula, respectively, to identify sequence variations that may be responsible for observed differences in virulence. The Kenya-128B-15 (Ken06) strain was isolated from a mosquito during the Kenya 2006/2007 outbreak, and the SA01-1322 (SA01) strain was isolated from a mosquito during the Saudi Arabia 2000/2001 outbreak. Both RVFV strains were responsible for significant human and livestock mortalities, and were used to establish infection models in sheep and cattle. Both strains produced early onset viremia accompanied by typical clinical signs of RVFV infection. Interestingly, the Ken06 strain resulted in higher levels of viremia in both sheep and cattle, with more severe pathology compared to the SA01 strain. To determine sequence variations, which might be involved in strain-specific phenotypes, the complete genomes of both strains were determined using next-generation sequencing technology. RNA was extracted from the Ken06 and SA01 viruses propagated in cell culture. Each of the three genome segments were amplified in two parts to obtain the full genome sequence; the untranslated regions alone (UTRs) were amplified using a T7 RNA ligase strategy, and the complete viral RNA and UTRs were reverse transcribed, followed by amplification with gene-specific primers. The sequencing library was prepared from 1 ng DNA for each strain using the Nextera XT-DNA library preparation kit (Illumina, CA) and sequencing was performed using standard protocols on the Illumina MiSeq, with 150-bp paried end reads. Genome sequence analysis of SA01 and Ken06 revealed nucleotide and amino acid variations in the L, M, and S segments. At the amino acid level, Ken06 strain manifested 11 aa substitutions (compared to SA01) in the polymerase protein, 2 in the non-structural NSm protein, 8 in the Gn glycoprotein, 2 in the Gc glycoprotein, and 5 in the nonstructural NSs protein. The complete genome sequences of these two RVFV strains are necessary for continued studies into the genetic basis for RVFV virulence using reverse genetics as well as the evolution and adaptation of this agriculturally important zoonotic pathogen.