Host-dependence of in vitro reassortment dynamics among the Sathuperi and Shamonda Simbuviruses

Abstract

Orthobunyaviruses are arboviruses (Arthropod Borne Virus) and possess multipartite genomes made up of three negative RNAs corresponding to the small (S), medium (M) and large (L) segments. Reassortment and recombination are evolutionary driving forces of such segmented viruses and lead to the emergence of new strains and species. Retrospective studies based on phylogenetical analysis are able to evaluate these mechanisms at the end of the selection process but fail to address the dynamics of emergence. This issue was addressed using two Orthobunyaviruses infecting ruminants and belonging to the Simbu serogroup: the Sathuperi virus (SATV) and the Shamonda virus (SHAV). Both viruses were associated with abortion, stillbirth and congenital malformations occurring after transplacental transmission and were suspected to spread together in different ruminant and insect populations. This study showed that different viruses related to SHAV and SATV are spreading simultaneously in ruminants and equids of the Sub-Saharan region. Their reassortment and recombination potential was evaluated in mammalian and in insect contexts. A method was set up to determine the genomic background of any clonal progeny viruses isolated after in vitro coinfections assays. All the reassortment combinations were generated in both contexts while no recombinant virus was isolated. Progeny virus populations revealed a high level of reassortment in mammalian cells and a much lower level in insect cells. In vitro selection pressure that mimicked the host switching (insect-mammal) revealed that the best adapted reassortant virus was connected with an advantageous replicative fitness and with the presence of a specific segment.

Keywords: Orthobunyavirus; arbovirus; reassortment; recombination; viral selection.

Figure 1.

Viral Neutralization Test against SHAV and SATV performed in animal sera collected in northeastern Nigeria. Results are expressed in Log2 ED50 against SATV and against SHAV. Results are considered positive if Log2 ED50 > 2. Four ruminant species (Cattle, Goats, Sheep and Camels) and two equine species (Horses and Donkeys) were analysed. Results obtained for 20 individuals of each animal species are presented. When identical serologic results were observed for several animals, the number of repeats is indicated above the dot.

Figure 2.

Comparison of the sequences and growth kinetics of the two Simbuviruses SHAV and SATV. (a) Similarity plot based on the aligned genome sequences of SHAV and SATV. The plot was performed using the Recombination Analysis Tool (RAT) [48]. Segments S, M and L are indicated by double arrows and encoded proteins are illustrated below. Percentage of nucleotide and protein identity for each segment and each encoded protein are annotated. (b) Growth kinetics of the two viruses SHAV and SATV in mammalian and in insect cells. Titers, expressed in TCID₅₀/mL, were determined after 2, 12, 24, 36, 48 and 72 h post infection at MOI 0.01. SATV growth kinetics are illustrated by black lines and SHAV growth kinetics are indicated by grey lines. Growth curves were tested in BHK-21 cells (square lines) or in insect KC cells (diamond lines).

Figure 3.

Method used to discriminate progeny viruses. (a) Schematic representation of primers used to discriminate both extremities of each segment. Universal primers are represented by white arrows, SATV specific primers are represented by black arrows and SHAV specific primers are represented by grey arrows. (b) Electrophoresis patterns of discriminative PCRs. Each segment (S, M and L) of both viruses (SHAV and SATV) were discriminated at both extremities (3′gRNA and 5′gRNA). On the left: SHAV and on the right: SATV. MW: Molecular Weight (Smart ladder, Eurogentec).

Figure 4.

Distribution of progeny viruses obtained after co-infection experiment in BHK-21 mammalian (a) and insect (b) cells (passage 0). Gray boxes represent frequency of progeny viruses obtained after co-infection at MOI 0.01 and black boxes represent frequency of progeny viruses obtained after co-infection at MOI 10. Parental and reassortant viruses and theirs respective genotypes (S, M, L) are indicated below each box and are illustrated by schematic viruses with coloured segments (white for SHAV segment and black for SATV segment). Error bars represent the confidence intervals of each virus frequency (α = 0.05).

Figure 5.

Growth kinetics of parental and reassortant viruses in BHK-21 cells. Titers, expressed in TCID₅₀/mL, were determined after 0, 2, 12, 24, 36 and 48 h post infection at MOI 0.01 in BHK-21 cell line. Error bars represent standard deviation calculated by using triplicate experiments carried out for each parental or reassortant virus.

Figure 6.

Plaque size assays of parental and reassortant viruses. (a) Examples of plaques obtained after infection of BHK-21 cells with dilutions of SHAV, SATV and the six different combinations of reassortants (R1 to R6). (b) Schematic representation of plaque size induced by the parental or the reassortant viruses. Plaque size value of the parental strains SHAV and SATV were fixed at 4 and 2 respectively. Error bars represent standard deviation calculated by using three different sets of viruses. An ANOVA test was performed to compare the six reassortants together. A Tukey test was performed to compare each reassortant against every other reassortant. * represents significant difference with P < .05. (c) Comparison of the average plaque sizes induced by the different reassortant viruses according to the parental origin of each segment. Error bars represent standard deviation calculated by using three different viruses harbouring this segment. * represents significant difference with P < .05 by using Welch’s t-test.

Figure 7.

Dynamics of reassortment observed during 10 successive infections by alternating the insect and the mammalian context. (a) Schematic representation of the competition assay where the initial viral population was alternatively propagated in insect and mammalian cells during 10 passages. The number of characterized progeny viruses for each tested MOI are indicated for each analysed passage (#1, #2, #3, #4, #9 and #10). (b) Representation of the viral population identified for the two tested MOI: 0.01 (grey boxes) and 10 (black boxes). Parental and reassortant viruses and their respective genotypes (S, M, L) are indicated below each box. Error bars represent the confidence intervals of each virus frequency (α = 0.05).

Figure 8.

Dynamics of reassortment observed during 10 successive infections in mammalian or in insect cells. (a) Schematic representation of the competition assay where the initial viral populations were propagated in insect or in mammalian cells during 10 passages. The number of characterized progeny viruses for each tested MOI are indicated for each analysed passage (#1, #2, #5 and #10). (b) Representation of the viral population identified in the two BHK-21 (left panel) and KC (right panel) cell lines and for the two tested MOI: 0.01 (grey boxes) and 10 (black boxes). Parental and reassortant viruses and their respective genotypes (S, M, L) are indicated below each box. Error bars represent the confidence intervals of each virus frequency (α = 0.05).