Most chimeric flaviviruses have been developed for vaccine purposes. In these studies, live-attenuated vaccine candidates were created by inserting specific genetic elements (typically the prM-E genes) of the flavivirus of interest into a full-length infectious cDNA backbone of another flavivirus such as the YFV vaccine vector, YFV-17D, or an attenuated strain of DENV [32–38]. The construction and characterization of chimeric flaviviruses has also provided critical information on the genetic elements that modulate the differential vector ranges, transmissibilities and disease phenotypes of divergent flaviviruses. Several of these studies have been performed using representative flaviviruses from the tick-borne and mosquito-borne groups [39–44]. More pertinent to this investigation are the few studies that describe the construction and characterization of viral chimeras between NKV and arthropod/vertebrate flaviviruses [45–47]. Five chimeric flaviviruses have now been created between viruses from these two groups. The first chimeric virus was generated by substituting the prM-E genes of an infectious YFV cDNA infectious clone with the homologous genes of MODV  and the second contains the prM-E genes of MODV in a DENV-2 backbone . Both chimeric viruses replicated in C6/36 cells indicating that the inability of NKV flaviviruses to infect mosquito cells is not mediated by the viral envelope but by a post-entry event. Two more chimeric viruses were constructed by replacing the conserved pentanucleotide sequence (CPS) or variable region (VR) of the 3’ UTR of a DENV-4 infectious clone with the corresponding region of MODV. Both viruses could infect C6/36 cells and adult mosquitoes at similar efficiencies to DENV-4 suggesting that the CPS and VR of mosquito/vertebrate flaviviruses are not required for mosquito infectivity. We too have successfully created a chimeric virus using a NKV and mosquito-borne flavivirus but, unlike the above studies, our virus was constructed using the vertebrate-specific virus as the backbone. The virus, designated MODV-WNV(prM-E), was created by replacing the prM-E genes of a MODV infectious clone with the corresponding sequences of WNV. MODV-WNV(prM-E) possesses the capacity to infect and replicate within vertebrate but not mosquito cell cultures indicating that there are sequence elements outside of the prM-E region that dictate the vertebrate-specific host range of MODV. However, it is important to note that the mosquito cells were cultured at a much lower temperature than the mammalian cells and thus, it is not known whether MODV-WNV(prM-E) was unable to infect mosquito cells due to a cell tropism restriction or a temperature-dependent restriction.
The fusion product designated fpMODV-CxFV(prM-E), which was created by replacing the prM and E genes of MODV with the homologous sequences of CxFV, failed to yield detectable virus. This finding is in contrast to the numerous studies that report the successful production of chimeric virus after the prM-E genes of one flavivirus are replaced with those of another [42, 48–54]. However, all of these studies were performed with flaviviruses that possess at least one common host. Indeed, although chimeric viruses have been created between viruses as divergent as tick- and mosquito-borne flaviviruses, and NKV and mosquito-borne flaviviruses, all viruses within these groups possess the ability to replicate within vertebrate cells. In contrast, ISFs and NKV flaviviruses do not possess a common host by virtue of their insect and vertebrate-specific phenotypes. Thus, the generation of chimeric viruses between ISFs and NKV flaviviruses may not be achievable or, at the very least, will prove extremely challenging because their genomes and resulting translation products may be fundamentally incompatible as a consequence of their evolutionary divergence and specialization to vastly different hosts.
Conserved complementary cyclization sequences reside within the capsid gene and 3’ UTR of the flavivirus genome. These sequences interact with one another to facilitate genome cyclization and are essential for viral replication [55, 56]. Thus, one explanation for the inability to produce infectious virus with the fusion products containing the C-prM-E genes of WNV and CxFV is because the genome cyclization elements within the 3’ UTR of MODV and the C gene of the alternate virus do not have sufficient complementary to support genome cyclization. In this regard, replacement of the 3’UTR of a DENV-4 infectious clone with the corresponding region of MODV also failed to produce virus . Virus was also unable to be recovered when both UTRs as well as the C gene of DENV-4 were replaced with the corresponding regions of either LGTV or MODV, despite the presence of complementary cyclization sequences [44, 47]. The authors speculated that infectious virus was not produced because fundamental incompatibilities exist between the UTRs and replication complexes of highly divergent (e.g. mosquito-borne, tick-borne and vertebrate-specific) flaviviruses. However, C-prM-E gene substitutions between divergent flaviviruses have occasionally proven successful; Pletnev and colleagues produced chimeric virus after replacing all three structural genes of DENV-4 with those of TBEV .
The inability to produce chimeric virus with fpMODV-CxFV(prM-E), fpMODV-CxFV(C-prM-E) and fpMODV-WNV(C-prM-E) is unlikely due to aberrant replication complex formation. Assembly of the viral replication complex should not have been impeded due to mismatches between the various viral and cellular proteins that interact during this process because no nonstructural gene substitutions were made. It is also unlikely that correct proteolytic processing of the chimeric polyproteins could not occur. Amino acid sequence alignments have shown that the predicted cleavage sites required for proteolytic cleavage of the CxFV and MODV polyproteins are similar to one another and to those of WNV and other dual-host flaviviruses [22, 57–59]. Although the junctions of all four constructs were sequenced and shown to contain no nucleotide errors, these constructs were not sequenced in their entirety and thus, we cannot dismiss the possibility that the non-viable constructs contained lethal mutations outside the junctions that occurred during one of the PCR amplifications. Another explanation why fpMODV-CxFV(C-prM-E) and fpMODV-WNV(C-prM-E) failed to produce virus is because the encapsidation signal sequence of MODV (which, as with all flaviviruses, remains to be identified ) is not recognized by the capsid proteins of WNV or CxFV.
The replication kinetics and yields of MODV-WNV(prM-E) in Vero cells were similar to those of MODV. These data suggest that genetic elements outside of the prM-E region dictate the in vitro replication profiles of NKV flaviviruses in vertebrate cells. Other studies have also shown that chimeric flaviviruses generated by prM-E gene substitutions exhibit replication kinetics and yields similar to the virus from which the nonstructural genes were derived but distinct from the virus that contributed the prM-E sequences [46, 51, 61]. For instance, the in vitro replication kinetics of a chimeric virus that possessed the prM-E genes of MODV in a YFV-17D backbone were similar to those of YFV-17D but distinct from MODV which reached a higher peak titer . Although the chimeric virus and MODV displayed similar in vitro replication kinetics, these two viruses exhibited differential plaque morphologies in Vero cells. MODV-WNV(prM-E) plaques were at least threefold larger than MODV plaques but approximately fourfold smaller than WNV plaques. These findings indicate that genetic elements both within and outside of the prM-E region modulate the plaque sizes of NKV flaviviruses. These findings differ from most other studies which compare the plaque sizes of chimeric flaviviruses generated by prM-E gene substitutions to those of both parental viruses. Usually prM-E gene substitutions generate chimeric viruses that produce plaques that are indistinguishable from one of the parental viruses [51, 62–64] or are smaller than both parental viruses [46, 65, 66]. However, replacement of the prM-E genes of JEV with those of DENV-4 produced a chimeric virus which, like our chimeric virus, exhibited an intermediate plaque phenotype; the chimeric virus produced plaques that were smaller than JEV but larger than DENV-4 in mammalian cells .
MODV-WNV(prM-E) did not always cause CPE in Vero cells, and the occurrence of CPE appeared dependent on the passage history of the virus. MODV-WNV(prM-E) was able to induce CPE after a single passage in Vero cells if it had first been cultured in BHK-21 cells. In contrast, CPE did not occur in Vero cells until the third passage when the virus had not been passaged in BHK-21 cells. One explanation for these findings is that MODV-WNV(prM-E) replicates more efficiently in BHK-21 cells as compared to Vero cells, possibly because it is a rodent cell line and most of the chimeric flaviviral genome was acquired from a virus with a natural host range that is apparently restricted to rodents. Alternatively, repeated passaging of the virus in Vero cells could have resulted in the accumulation of mutations that altered its ability to induce CPE in this cell type. In this regard, the C-prM-E gene sequence of chimeric virus derived from the original inoculum contained one non-synonymous mutation when compared to the corresponding regions of parental viruses while chimeric viruses that had undergone three passages in BHK-21 and/or Vero cells acquired three to four additional mutations in the structural gene region. Whether these mutations, or mutations that may have occurred elsewhere in the viral genome, altered the ability of the virus to induce CPE is not known but it does offer a likely explanation.
In summary, we report the first chimeric flavivirus to be constructed using a NKV flavivirus as the backbone. We also report the first attempts to create a chimeric flavivirus between an ISF and NKV flavivirus. Two constructs were generated, including one that contains the CxFV prM-E genes in a MODV backbone, but neither yielded detectable virus. Most success in the generation of chimeric flaviviruses has been achieved through prM-E gene substitutions. However, unlike our study, all previous studies were performed using flaviviruses that share a common host. These findings indicate that the successful generation of chimeric viruses between ISFs and NKV flaviviruses will prove extremely challenging due to the evolutionary divergence and differential host ranges of these viruses.