# New Insights into the Biology of the Emerging Tembusu Virus
## 4 Clinical Features and Pathogenicity
### Cell Biology Tools  
To ensure its transmission, TMUV must replicate in mosquito vectors prior to a vertebrate host (Figure 4). In parallel with virus isolation in embryonated chicken eggs, C6/36 mosquito cell lines were rapidly used for virus isolation [5; 25]. However, the lack of information on the virus in the vertebrate host has led to the use of primary cells and cell lines to investigate the biology of the virus (Table 3).  
Different types of avian cells have been used to evaluate TMUV biology and include BK3 cells, a transformed chicken B lymphocyte cell line, the chicken macrophage-derived cell line HD-11 and the DF1 cell line, isolated from chicken embryo fibroblasts. Besides specific usage for research, chicken embryo fibroblasts and duck and chicken eggs are routinely used for virus isolation and maintenance [3; 4; 5; 26; 30; 93; 94].  
TMUV is also able to replicate in a wide range of non-avian cells which are used to assess the impact of TMUV in mammalian hosts. The primary cells and cell lines used in TMUV investigation are summarized in Table 3. Human cell lines were used to evaluate tropism of the virus and determine molecular mechanisms involved in TMUV infection. The HEK293T cell line and a mouse primary cell line (MEF) were used to investigate the innate immune response and how TMUV evades the immune response [95] (see specific paragraph). A recent investigation on TMUV cell tropism was carried out using various human cell lines. The authors pointed out the susceptibility of liver, kidney and nerve cell lines at various degrees, while the lung, muscle, B cells, T cells and monocytes were largely resistant to the infection [96]. All these reports indicate that TMUV is able to replicate in a wide range of vertebrate cells. Results suggest that targeted organs are similar in mammals and birds. However, knowledge on pathogenesis in human cells is parceled, and further investigation is needed. Furthermore, while TMUV is transmitted via mosquitoes' bite, there is a need for more information on the biology in mosquitoes as only in vitro studies with C6/36 cells have been conducted.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline  & **Name** & **Organism, Tissue** & **Type** & **Reference** \\ \hline \multirow{4}{*}{Avian} & DF-1 & Chicken, embryo fibroblast & Cell line & [3; 26; 93] \\ \cline{2-5}  & HD11 & Chicken, bone marrow macrophage & Cell line & [97] \\ \cline{2-5}  & DEF & Duck embryonic fibroblast &
\begin{tabular}{c} Primary cells from \\ 9-day-old duck embryos \\ \end{tabular} & [10; 98] \\ \cline{2-5}  & goose PBMCs & Goose blood & Primary cell & [99] \\ \cline{2-5}  & LSCC-BK3 & Chicken, B lymphocyte & Cell line & [5] \\ \hline \multirow{4}{*}{Mammalian} & MARC145 & Monkey kidney & Cell line & [5] \\ \cline{2-5}  & VERO & African green monkey kidney & Cell line & [3; 5; 26; 58; 95] \\ \cline{2-5}  & BHK-21 & Hamster, kidney & Cell line & [10; 22; 26] \\ \cline{2-5}  & CPK & Porcine, kidney & Cell line & [5] \\ \cline{2-5}  & MEF & Mouse embryonic fibroblast & Primary cells & [98] \\ \cline{2-5}  & A549 & Human, epithelial lung & Cell line & [95; 96] \\ \cline{2-5}  & HeLa & Human, epithelial cervix & Cell line & [95] \\ \cline{2-5}  & SH-SY5Y & Human, epithelial bone marrow & Cell line & [95; 96] \\ \cline{2-5}  & HEK293T & Human, epithelial kidney & Cell line & [96; 98] \\ \cline{2-5}  & HUH7 & Human, liver & Cell line & [96; 98] \\ \cline{2-5}  & RD & Human rhabdomyosarcoma cell & Cell line & [96] \\ \cline{2-5}  & SUP-T1 & Human T cell lymphoblastic lymphoma cell & Cell line & [96] \\ \cline{2-5}  & Z-138 & Human B cell non-Hodgkin’s lymphoma cell & Cell line & [96] \\ \cline{2-5}  & U937 & Human histiocytic lymphoma cell & Cell line & [96] \\ \cline{2-5}  & imHC & Human hepatocyte-like cell & Derived cell from hiPSC & [96] \\ \hline Insect & C6/36 & _Aedes albopictus_, larva & Cell line & [22; 26; 58] \\ \hline \hline \end{tabular}
\end{table}
Table 3: Cell models used for TMUV investigations.