# New Insights into the Biology of the Emerging Tembusu Virus
## 2 Tembusu Virus
### Phylogeny  
The TMUV members belong to the _Flaviviridae_ family, genus _Flavivirus_[9]. Based on the vector type, the Flavivirus genus is divided into four different groups: (1) mosquito-borne, (2) tick-borne, (3) insect-specific and (4) unknown vector flaviviruses [20]. Mosquito-borne flaviviruses (MBF) can be subdivided into _Aedes-_ and _Culex-_ transmitted viruses. _Aedes-_borne viruses, including DENV, YFV and Zika virus (ZIKV), are generally associated with hemorrhagic fevers and non-human primate ancestors, and those transmitted by the _Culex_ genus are associated with meningoencephalitis diseases and bird reservoirs such as JEV, WNV and TMUV [21]. _Culex-_transmitted MBF are classified depending on the serology in two groups, the Japanese encephalitis serocomplex including JEV and WNV, and the Ntaya serocomplex including Ntaya virus and TMUV. The TMUV group is genetically distinct within the Ntaya serocomplex and includes homologous strains that were initially considered as single species, including Sitiawan virus [5], BYD virus [4], Perak virus [3], duck egg drop syndrome virus (DEDSV) [4; 22] and duck Tembusu virus (DTMUV) [23]. These viruses are now phylogenetically linked in different sub-clusters within the TMUV group (Figure 2). The first TMUV was isolated from _Cx. tritaeniorhynchus_ in Malaysia in 1955 [24] and represents the TMUV strain that is the closest to other members of the Ntaya serogroup. Virus strains related to the TMUV group were then reported in the 1990s in mosquitoes (refer to dedicated section below) in the north of Kuala Lumpur, in Sitiawan and in Sarawak, on Kalimantan island (Borneo) in Malaysia and in Thailand [1; 2; 25]. From our phylogenetic analysis with the available sequences, all strains isolated until 2000, except one identified in Thailand in 1992, are grouped in the TMUV cluster and are closely related to viruses belonging to the Ntaya serogroup. Interestingly, a recent strain isolated in Taiwan from _Cx. annulus_ and _Cx. tritaeniorhynchus_ mosquitoes was grouped in the cluster TMUV, despite the fact that no TMUV has ever been reported in Taiwan [26]. Long-distance travel via migratory birds followed by a low-level maintenance in wild birds in Taiwan may explain the recent identification of TMUV in Taiwan [27; 28]. A similar spreading mode was observed for other flaviviruses such as JEV and avian influenza viruses. Figure 2: Phylogenetic analysis of TMUV strains. Non-concatenated nucleic acid sequences of the partial E genes from sequences available in the GenBank database were aligned using MEGA7. Sites that could not be unambiguously aligned were excluded, and divergent regions were excluded from subsequent analyses. Phylogenetic trees were generated using the GTR+G substitution model using PhyML software [29] and edited with FigTree v1.4.4 software (2006–2018, Source code available from: [[http://github.com/rambaut/tigtree/](http://github.com/rambaut/tigtree/)]([http://github.com/rambaut/tigtree/](http://github.com/rambaut/tigtree/))). The bootstrap method was used to measure the robustness of nodes with 1000 iterations. * represents bootstrap values with a score higher than 80%. Identified TMUV strains are differentially colored in the tree depending on country origin. Annotations on the right denote TMUV cluster. All sequence accession numbers used in this review are shown in Supplementary Table S1. Since the beginning of the 21 st century, TMUV circulation has been reported in SEA and China [30]. However, these viral isolates were found to be phylogenetically separated from the TMUV cluster and have been grouped in three different lineages comprising strains either isolated in Malaysia, in Thailand or in China. A virus isolated in 2007 in Thailand formed a monophyletic lineage with two strains isolated in Malaysia in 2012 and was named Cluster 1 in our phylogenetic tree (Figure 2) [3]. In 2010, for the first time, TMUV was reported in China and caused an outbreak in egg-laying ducks [23].