Replication of not-known-vector flaviviruses in mosquito cells is restricted by intracellular host factors rather than by the viral envelope proteins

Abstract

Chimeric yellow fever virus 17D (YFV-17D) and dengue virus type 2 (DENV2) carrying the surface proteins of Modoc virus (MODV), a not-known-vector (NKV) flavivirus, replicated efficiently in mammalian (Vero-B) and mosquito (C6/36) cells, whereas MODV failed to replicate in mosquito cells. Transfection of C6/36 cells with MODV RNA did not result in virus replication; however, transfection of these mosquito cells with YFV-17D or DENV2 RNA did. The inability of NKV viruses (such as MODV) to infect and replicate in arthropod cells is thus not determined by the viral envelope, but by a post-entry event.Flaviviruses cause a variety of diseases, including encephalitis and haemorrhagic fevers. The genus Flavivirus contains (i) viruses that are transmitted by mosquitoes or ticks (arthropod-borne) and (ii) viruses with no known vector (NKV) (Lindenbach & Rice, 2001). Many flaviviruses have evolved unique strategies to adapt to the special requirements of their specific target cells (reviewed recently by Fernandez-Garcia et al., 2009). Permissivity to flavivirus infection is determined by many different hostcell factors that may either be required for efficient infection of mammalian and arthropod cells (host susceptibility factors, HSFs) or restrict infection in certain cells (host resistance factors, HRFs) (Krishnan et al., 2008;Sessions et al., 2009). For example, .40 candidate HSFs have been identified that are required for growth of the mosquito-borne dengue virus (DENV) type 2 in arthropod, but not in mammalian, cells (Sessions et al., 2009). It is largely unknown which viral determinants are responsible for host tropism and vector specificity, i.e. (i) why do certain flaviviruses infect mosquitoes and others only ticks and (ii) why are NKV flaviviruses not able to infect either ticks or mosquitoes? The ability or inability of flaviviruses to infect cultured cells derived from mosquitoes reflects the natural vector-virus relationship. Whilst the mosquito cell line C6/36 is highly susceptible to infection with mosquito-borne viruses such as yellow fever virus (YFV) and DENV, these cells are not infectable with several tick-borne viruses, or with the NKV viruses Apoi virus or Modoc virus (MODV) (Lawrie et al., 2004;Leyssen et al., 2002). In general, the inability of NKV flaviviruses to infect mosquito cells may possibly be due to a failure of the virus to gain entry into the cell, or to a post-entry event. To study whether the viral envelope (glyco)proteins determine the (in)ability of flaviviruses to infect mosquito cells, we monitored the replication of (i) two mosquito-borne flaviviruses [YFV strain 17D (YFV-17D) and DENV type 2 New Guinea strain (DENV2)], (ii) an NKV flavivirus (MODV) and (iii) two chimeric flaviviruses derived from them, which consist of either the YFV-17D or the DENV2 genome in which the prM+E region or the AnchC+prM+E region was replaced by the corresponding region of MODV, respectively. (Fig. 1b, d). Surprisingly, the MOD/YFV chimera replicated readily in C6/36 cells, although less efficiently...

Structure Determination of the Top‐Loop of the Conserved 3′‐Terminal Secondary Structure in the Genome of Flaviviruses

“…Therefore the pentanucleotide motif in the chimeric MODV/YFV was mutated from CACAG to CUCAG, characteristic for NKV viruses. [6] Due to this mutation, the chimeric construct lost its ability to replicate in mosquito cells, providing strong evidence that the conserved pentanucleotide motif in vector-borne flaviviruses plays a crucial role in vector specificity.…”

“…A second construct was prepared by exchanging the genes encoding the prM and E glycoproteins of the mosquito-borne YFV for those of the murine Modoc virus, a flavivirus with NKV. [6] The resulting chimeric MODV/YFV virus still replicates in mosquito cells (as YFV), [6] despite the absence of the mosquito-born prM and E glycoproteins. Both constructs demonstrated other factors than these glycoproteins of NKV flaviviruses are responsible their vector specificity.…”

“…[5] However, exchanging the genes encoding the prM and E of the mosquito-borne yellow fever virus (YFV) for those of the murine Modoc virus, a flavivirus with NKV, resulted in a chimeric MODV/YFV virus that replicates in mosquito cells (as YFV). [6] This demonstrated that factors other than the envelope proteins of NKV flaviviruses are responsible for the fact that NKV viruses do not replicate in mosquito or tick cells.…”

“…One point mutation in the conserved 5'-CACAG-3' motif of the chimeric MODV/YFV to 5'-CUCAG-3' typical for NKV viruses, resulted in a virus that was no longer able to replicate in mosquito cells, but replication in eukaryotic Vero-cells was still possible. [6] These results indicate that the conserved pentanucleotide motif 5'-CACAG-3' contributes to the viral vector-specificity and give rise to a second hypothesis in which structural motifs in 3'-UTR of the viral To what extent small differences in RNA sequences (mutations) can have a profound impact on biology remains an intriguing question. This effect can be studied by using untranslated RNA regions as a model.…”

Chimeric Tick-Borne Encephalitis/Dengue Virus Is Attenuated inIxodes scapularisTicks andAedes aegyptiMosquitoes

“…These data were consistent with studies of other chimeric flaviviruses that are able to replicate in C6/36 cells, but unable to replicate or disseminate in Ae. aegypti (Bhatt et al 2000, Johnson et al 2003, Blaney et al 2004, 2007, Charlier et al 2010). …”