Cell-to-Cell Spread of Dengue Viral RNA in Mosquito Cells

Abstract

Dengue virus (DENV) is an important mosquito-borne arbovirus that is particularly prevalent in tropical and subtropical areas of the world. The virus is generally ingested with a blood meal, replicates in host tissues, and disseminates into salivary glands for transmission to the next host. Membrane-bound vacuoles carrying DENV particles have been documented in mosquito cells and play a role in the cell-to-cell transmission of DENV2. C189 is one member of the tetraspanin family and generally increases its expression as one component of the vacuoles (C189-VCs) within C6/36 cells infected with DENV2. In the present study, we have further demonstrated via sucrose gradient centrifugation as well as magnetic immune isolation (MI) that the RNA of DENV2 was eventually carried by C189-VCs. In addition, viral RNA was shown to spread from donor to recipient cells in a coculture assay even when 20 mM NH4Cl was added to inhibit virus replication in the culture. In an alternate assay using the transwell system, viral RNA was only detected in recipient cells in the absence of 40 mM NH4Cl, suggesting that cell-cell contact is required for the intercellular spread of DENV2. In turn, the formation of viral synapse (VS) derived from aggregates of viral particles was frequently observed at sites of cell contact. Taken together, the formation of C189-VCs in C6/36 cells is induced by DENV2 infection, which may serve as a vehicle for transferring virions and also viral RNA to neighboring cells by cell-to-cell transmission after cell-cell contact. This finding provides insight into the understanding of viral spread between mosquito cells. It may also elucidate the benign persistent infection in mosquito cells and efficient dissemination of DENV infection within a mosquito vector.

Tetraspanins as Potential Therapeutic Candidates for Targeting Flaviviruses

“…This study further observed increased cell-to-cell transmission of DENV at the site where the donor cell directly interacts with the recipient cell (7). A recent study has shown that tetraspanin C189 containing vacuoles mediate cell-to-cell spread of viral RNA within DENV2infected C6/36 cells (See Table 1) (43). Magnetic immune isolation (MI) and sucrose gradient centrifugation showed that DENV2 RNA is transmitted by C189-VCs from donor to recipient cells.…”

“…Magnetic immune isolation (MI) and sucrose gradient centrifugation showed that DENV2 RNA is transmitted by C189-VCs from donor to recipient cells. Viral RNA was only detected in recipient cells in transwell system, indicating that cell-cell contact is essential for DENV2 intercellular spread (43).…”

“…C189-VCs served as carriers for transmission of viral RNA and virions to neighboring cells after established contact, suggesting persistent infection in mosquito cells and well-organized dissemination of DENV2-infection within mosquito vector (43). Delineating the role of arthropod tetraspanins is an interesting avenue to investigate the mechanisms of viral transmission.…”

Life as a Vector of Dengue Virus: The Antioxidant Strategy of Mosquito Cells to Survive Viral Infection

“…DENV infection is generally initiated by cell-free viruses in the mosquito midgut, likely followed by the cell-to-cell mode of dissemination to their destination, i.e., salivary glands. In cultures of mosquito cells with DENV2 infection, virions and viral RNA carried by vacuoles (VCs) were demonstrated to transfer from one cell to its neighbors via the cell-to-cell transmission mode [ 70 , 71 ]. During the transmission process of this virus, a novel tetraspanin called C189 is induced and is incorporated as a membrane component of VCs (C189-VCs) formed in response to DENV infection in mosquito cells [ 72 ].…”

“…The VC is presumed to serve as a vehicle which carries the virus from one cell to its neighbors. Moreover, a “viral synapse” (VS) located in the intercellular space is frequently formed [ 71 ], which was also observed at the interface between donor and recipient cells during cell-to-cell transmission of HIV [ 65 ]. The formation of VCs in humans may play a role of helping the virus evade humoral immunity, resulting in rapid transmission and continued infection of the host [ 73 ].…”