Comparative study of two Rift Valley fever virus field strains originating from Mauritania

Abstract

Rift Valley fever (RVF) is one of the major viral arthropod-borne diseases in Africa. In recent decades, RVF virus (RVFV), the causative agent of RVF, has been responsible for multiple outbreaks in West Africa with important consequences on human and animal health. In particular, an outbreak occurred in 2010 after heavy rains in the desertic region of Adrar, Mauritania. It was characterized by the appearance of severe clinical signs among dromedary camels. Another one occurred in 2013-2014 across Senegal and the southern part of Mauritania. In this study, we characterized two RVFV field strains isolated during these two outbreaks. The first strain, MRU25010-30, was isolated from a camel (2010) while the second, MRU2687-3, was isolated from a goat (2013). By deep-sequencing and rapid amplification of cDNA-ends by polymerase chain reaction, we successfully sequenced the complete genome of these two RVFV strains as well as the reference laboratory strain ZH548. Phylogenetic analysis showed that the two field viruses belong to two different RVFV genetic lineages. Moreover, we showed that MRU25010-30 replicates more efficiently in various in vitro cell culture models than MRU2687-3 and ZH548. In vivo, MRU25010-30 caused rapid death of BALB/c mice and proved to be more virulent than MRU2687-3, regardless of the route of inoculation (subcutaneous or intranasal). The virulence of MRU25010-30 is associated with a high viral load in the liver and serum of infected mice, while the death of mice infected with MRU2687-3 and ZH548 correlated with a high viral load in the brain. Altogether, the data presented in this study provide new avenues to unveil the molecular viral determinants that modulate RVFV virulence and replication capacity.

Fig 1. Phylogenetic trees and protein sequences analysis of MRU25010-30 and MRU2687-3 strains.

(A) Phylogenetic analysis of the S, M, and L segments based on the classification of Bird and colleagues [25]. The trees were generated by the Maximum likelihood GTR model with 500 bootstraps. MRU25010-30, MRU2687-3, and ZH548 are in bold text and indicated by an asterisk. Genetic lineages (A-G) originally proposed by Bird and colleagues are represented for each segment. (B) Schematic representation of the amino acid residue substitutions between the consensus sequences of MRU25010-30 and MRU2687-3. L protein (top panel), full length polyprotein of segment M (middle panel) and, NSs and N proteins of segment S (bottom panel) are presented. p78 is shown in dark grey, NSm in white, Gn in black, and Gc in light grey. Position and amino acid residues of MRU25010-30 (left) and MRU2687-3 (right) strains are indicated.

Fig 2. In vitro growth properties of MRU25010-30, MRU2687-3, and ZH548 strains.

HepaRG, A549, and A549Npro cells were infected at MOI 0.01, while hiPSC differentiated into neural cells were infected with 2x10⁴ PFU (approximately a MOI of 0.1). Supernatants were analysed by TCID₅₀ method at indicated time pi and titres are expressed as TCID₅₀/ml of supernatant. In conventional cell lines, this experiment was repeated three times independently, each time either in duplicate or in triplicate. Experiment in hiPSC differentiated into neural cells was repeated twice independently (MRU25010-30 and MRU2687-3) or performed once (ZH548) using hiPSC obtained from two donors (MRU25010-30 and MRU2687-3: n = 40 and n = 38 at 24h pi, respectively, and n = 24 at 48h pi; ZH548: n = 16 at 24h pi, and n = 8 at 48h pi, n corresponding to the number of supernatant analysed). Error bars represent standard deviations around the mean value. Kruskal-Wallis statistical analyses were performed at each time point, and statistical significance is presented as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

Fig 3. Kaplan-Meier survival curves of BALB/c mice infected by MRU25010-30, MRU2687-3, and ZH548 strains.

6–8 weeks old female BALB/c mice were infected subcutaneously (SC) or intranasally (IN) with 10¹ or 10³ PFU of the indicated RVFV strains (n = 12 for MRU25010-30 and MRU2687-3 strains, n = 6 for ZH548 and n = 5 for PBS). Survival curves were analysed using the Gehan–Breslow–Wilcoxon test (ns p>0.05; * p<0.05; ** p<0.01; *** p<0.001).

Fig 4. RVFV RNAemia kinetics in BALB/c mice.

6–8 weeks old female BALB/c mice were infected intranasally (IN) or subcutaneously (SC) with 10¹ or 10³ PFU of the indicated RVFV strain. Levels of viral RNA were measured in sera of infected mice by RT-qPCR targeting segment M. Values are expressed in viral RNA copy number per ml of serum. Sera were collected at days 3, 6, and 10 (circles) as well as in euthanised mice (inverted triangles). MRU25010-30 (n = 12) is indicated in red, MRU2687-3 (n = 12) in green, and ZH548 (n = 6) in blue.

Fig 5. Levels of viral RNA and titres in the liver and brain of BALB/c infected mice.

Liver and brain were collected from euthanised mice and levels of viral RNA measured. Values are expressed as viral RNA copy number per g of liver or brain (left). Positives samples for viral RNA were also titrated by plaque assays and viral titres are expressed as PFU/g (right). Triangles represent animal euthanised between D3 and D6 pi, squares between D7 and D10 pi, and circled dots between D11 and D15 pi. Surviving mice at D15 are represented with dots.

Fig 6. Levels of RVFV RNA in the serum, liver, and brain of BALB/c mice at early stages of infection.

6–8 weeks old female BALB/c mice were infected intranasally (IN) or subcutaneously (SC) with 10³PFU of MRU25010-30 (red), MRU2687-3 (green) or ZH548 (blue). Levels of viral RNA were measured by RT-qPCR targeting segment M and values are expressed as viral RNA copy number per ml (serum) or per g (liver and brain). Sera were collected at days 1, 2, and 3 (MRU25010-30, n = 3), days 3 and 6 (MRU2687-3, n = 3 but in SC-10³ group where one mouse died at D6 pi), and days 2, 3, and 6 (ZH548, n = 3).