First dating of a recombination event in mammalian tick-borne flaviviruses

Abstract

The mammalian tick-borne flavivirus group (MTBFG) contains viruses associated with important human and animal diseases such as encephalitis and hemorrhagic fever. In contrast to mosquito-borne flaviviruses where recombination events are frequent, the evolutionary dynamic within the MTBFG was believed to be essentially clonal. This assumption was challenged with the recent report of several homologous recombinations within the Tick-borne encephalitis virus (TBEV). We performed a thorough analysis of publicly available genomes in this group and found no compelling evidence for the previously identified recombinations. However, our results show for the first time that demonstrable recombination (i.e., with large statistical support and strong phylogenetic evidences) has occurred in the MTBFG, more specifically within the Louping ill virus lineage. Putative parents, recombinant strains and breakpoints were further tested for statistical significance using phylogenetic methods. We investigated the time of divergence between the recombinant and parental strains in a Bayesian framework. The recombination was estimated to have occurred during a window of 282 to 76 years before the present. By unravelling the temporal setting of the event, we adduce hypotheses about the ecological conditions that could account for the observed recombination.

Figure 1. Analytical framework for the BEAST analyses.

Inference 1 analyses the variation in substitution rates across the genome for the 28 full length ORFs. The inferred posterior probability distributions of meanRates for individual genes are set as priors for inferences 2 and 3. Inference 2 dates the recombination event based on the 28 full length ORFs. Dates are evaluated separately for the recombinant region and for the non-recombinant sequences. Inference 3 gathers priors information for root height, tMRCA(W-TBE) and tMRCA(LIV & SSEV) used for the next inference. The alignment for inference 3contains the same 28 sequences, with the deletion of E-gene and the recombinant region. Inference 4 refines estimates for tMRCA(W-TBEV) based on 161 E-sequences. Prior distribution for meanRate parameter is derived from the literature. Priors for tMRCA(W-TBEV) and tMRCA(LIV & SSEV) were obtained from BEAST inference 3.

Figure 2. Split-graph constructed by the neighbor-net method based on 41 complete genomes of TBEV, LIV, LGTV and OHFV.

The split-graph focuses on phylogenetic relationships within the Tick-borne encephalitis virus species. The three TBEV subtypes are highlighted in color and the positions of all prototype strains are indicated in bold. Two Russian strains (886–84 and 178–79) were not assigned to any main subtype at this stage. A larger split between LIV and SSEV (in yellow) suggests a recombination event.

Figure 3. Most likely phylogram from the maximum likelihood analysis of the partitioned NS3-gene.

The partitions of NS3 correspond to (a) the recombinant fragment nt 1320–1524, (b) nt 1–1319 concatenated with nt 1525–1866. Numbers above nodes indicate branch support (bootstrap support ≥70%/Bayesian posterior probability ≥0.90). Asterisks (*) mark nodes that that are not recovered by Bayesian inference. Bars show different amounts of substitution for the in-group and out-group taxa. The position and branch support of LIV are indicated in red. “M” and “m” refers to the divergences of the major parent and minor parents respectively, while “r” points to the recombination event in the tree. Genbank accessions are indicated in parentheses.

Figure 4. Effect of mean rate variation across the genome and consequences on date estimations.

(a) Posterior probability distribution for the mean substitution rate (meanRate) parameter for individual genes across the full-length coding genome. (b) Root antiquity (root height) and (c) tMRCA(W-TBEV) inferred from individual genes for the 28 full-length coding sequences.

Figure 5. Maximum Clade Credibility tree from BEAST inference 4.

The general tree (a) summarizes the entire TBEV species evolutionary history. A paraphyletic group branching at the basis of the far Eastern lineage is addressed by the informal name X-TBEV. It contains Ural Siberian, Central Siberian and Transbaikal strains with characteristic E-gene motifs . The enlarged chronogram (b) focuses on the relationships within W-TBE, LIV, SSEV, GGEV, TSEV cluster. Grey bars at nodes represent 95% HDP credibility intervals. tMRCA are indicated above branches. Nodes investigated for tMRCAs in both analyses of the 163 E-sequences and with the datasets derived from the 28 complete nucleotide genomes are highlighted with red bullets. Asterisks refer to nodes supported by PP>0.90. Nodes with letters are mentioned in the text and in Table 4. Genbank accessions are indicated in parentheses.