The genus Flavivirus
(family Flaviviridae) is comprised of 53 virus species of enveloped, single-stranded, positive-sense RNA viruses. Flaviviral genomes are approximately 11 kb in length and encode a single long open-reading frame that is cleaved into three structural (C, prM, E), and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins and two known protein variants (NS1′ and 2K peptide) [1
]. Phylogenetic analyses of flaviviruses have demonstrated clustering based on host preference range: insect-specific flaviviruses (ISFs), dual-host tick-borne flaviviruses (TBFVs), viruses with no known vector (NKV), or mosquito-borne (dual-host) flaviviruses (MBFVs) [4
]. However, a number of recent viruses have been described whose sequences cluster with the MBFVs phylogenetically but have an apparent insect-specific host restrictive phenotype. These viruses have tentatively been designated as “Unidentified Vertebrate Host Flaviviruses” (UVHF) [6
] and include representatives from wide geographic locations: Chaoyang virus (CHAOV) from the Republic of Korea [7
] and China [8
], Donggang virus (DONV) from China [9
], Barkedji virus from Senegal and Israel [10
], Nounane virus (NOUV) from Côte d’Ivoire [11
], Lammi (LAMV) and Ilomantsi (ILOV) viruses from Finland [12
], Nanay virus from Peru [14
], Marisma mosquito virus (MMV) from Spain [15
] and Italy [16
], and Nhumirim virus (NHUV) from Brazil [6
]. The preponderance of data suggests that these viruses are not capable of infecting vertebrate cells and the use of the term “Unidentified Vertebrate Host” has been chosen to serve as an antithetical term to “No Known Vector” for flaviviruses that have been described that have not demonstrated a capacity to replicate in invertebrate cells.
All described flaviviruses associated with human disease fall within the MBFV and TBFV host range groups including dengue virus (DENV1-4) [18
], yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV) [19
], St. Louis encephalitis virus (SLEV) [20
], and tick-borne encephalitis virus (TBEV) [21
]. These viruses cause millions of human infections each year ranging from mild febrile symptoms to fatal hemorrhagic/neurologic disease. The fact that UVHFs and ISFs often infect the same mosquito vectors used by dual host MBFs for transmission of these agents to humans and the relative genetic similarity between these flaviviruses led to the independent assessment by many research groups of the potential for replicative interference between ISFs and MBFs; however, despite the positive association between genetic relatedness of the infecting and superinfecting virus for the efficacy of superinfection exclusion (SIE) [22
], only one evaluation of interference has been reported between a UVHF and MBFV [6
Superinfection studies with MBFVs and ISFs have been performed in order to establish a preventative intervention strategy for blocking the transmission of agents of human diseases and in order to gain a better understanding of any additional factor(s) that could alter vector competence of mosquitoes in both enzootic and epizootic transmission cycles. A number of studies have assessed the relative potential of different ISFs [Culex flavivirus (CxFV) and Palm Creek virus (PCV)] to interfere with replication of flaviviruses of human health importance in cultured mosquito cells. The in vitro
interference between an ISF and WNV was supported by a study in which C6/36 cells previously inoculated with a CxFV from Colorado demonstrated reduced WNV titers at early time points post infection [23
]. Previous inoculation of C6/36 cells with PCV was found to have a significant effect on replication of both WNV and Murray Valley encephalitis virus [24
]. In contrast, Kent et al.
demonstrated that pre-inoculation of C6/36 cells with CxFV from Guatemala had no interfering effect on subsequent WNV replication [25
], and Kuwata et al.
observed no evidence of SIE of JEV and DENV2 derived from Culex tritaeniorhynchus
cells persistently infected with a Japanese strain of CxFV [26
]. Variable interference observed for WNV replication with prior infection of mosquito cells with PCV and CxFV could very likely be due to genetic differences between PCV and CxFV viruses or the WNV challenge (lineage 1a vs.
1b) viruses utilized.
Few in vivo
studies have addressed the importance of superinfection of MBFVs with ISFs, and no in vivo
assessments of cross-interference have been reported between MBFVs and UVHFs. Two studies have directly addressed the potential for SIE between CxFV and WNV. In one study, performed with colonized Culex pipiens
mosquitoes persistently infected with CxFV, a significantly lower dissemination rate with WNV was observed at days post-inoculation (dpi) seven; however, differences in the infection and transmission rates were not detected [23
]. In a study performed with sequentially infected Culex quinquefasciatus
, no differences in infection, dissemination, or transmission were observed. Interestingly, when these mosquitoes were co-inoculated with CxFV and WNV, a higher percentage of mosquitoes were observed to transmit WNV while no difference was observed in the other mosquito colony [25
]. These in vivo
studies indicate a definite potential for the positive and negative modulation of vector competence for a flavivirus of human health significance that is likely ISF virus- and mosquito strain-dependent.
Previous in vitro
experiments have demonstrated robust inhibition of WNV, SLEV, and JEV in mosquito cells previously or concurrently infected with the UVHF virus, NHUV [6
]. In order to assess the potential that UVHFs, more closely genetically related to MBFVs than ISFs, have a greater potential for SIE, Cx. quinquefasciatus
and Cx. pipiens
mosquitoes were co-inoculated with NHUV and WNV, and the relative capacity for transmission compared to WNV-only inoculated mosquitoes was compared. Results described herein indicate the potential for in vivo
SIE that results in reduced transmissibility of WNV. These data indicated the potential modulatory effect of certain pre-existing flaviviruses on the capacity for establishment of WNV superinfection in mosquitoes and highlights a potential method for blocking mosquito infection as a public health measure.
Data presented herein indicate productive infection and subsequent oral transmission of NHUV following intrathoracic inoculation in Cx. pipiens
and Cx. quinquefasciatus
mosquitoes; however, the relevance of the transmissibility of NHUV orally by salivation has yet to be determined in the context of its potential insect-specific host restrictive phenotype [6
]. However, the observation of NHUV in the saliva of inoculated Culex
spp. mosquitoes indicates that SIE mechanisms could be critical for blocking successful transmission of other flaviviruses at all stages of extrinsic incubation including the infection and escape from the salivary glands.
Attempts to grow NHUV in limited vertebrate cell lines have proven unsuccessful [6
]. The phylogenetic placement of NHUV with other UVHF viruses in close proximity to MBFs, coupled with the similar codon usage pattern of NHUV to MBFs, indicate that if the vertebrate host-restricted phenotype does limit replication of UVHFs to mosquitoes, this restriction could have occurred relatively recently [6
]. Despite a small sample size, vertical transmission of NHUV in F1 progeny of IT-inoculated female Culex
spp. mosquitoes was demonstrated herein. This was in direct contrast to studies of an ISF, CxFV, in which an IT-inoculated colony of Cx. pipiens
females, despite demonstrating viral RNA presence in the ovaries, failed to pass virus vertically. Interestingly, field-collected mosquitoes reared from isofemale lines that were found to be positive for CxFV were found to exhibit extremely high vertical infection and filial infection rates in their progeny [31
]. While the assessment of the mechanisms and efficiency of vertical transmission of NHUV should be repeated on a much larger scale, it does suggest that this virus uses a maintenance method demonstrated by many classical ISFs [23
Vector competence studies for WNV in Cx. quinquefasciatus
mosquitoes co-infected with NHUV were performed and described herein. The results demonstrating significant reduction in the transmissibility of WNV in Culex
mosquitoes co-inoculated with NHUV at later, rather than earlier, time points is interesting and portends that prior infection of mosquitoes with NHUV and an establishment of SIE mechanisms in salivary acinar cells critical for viral egress prior to exposure to WNV could result in an even more striking inhibition of WNV transmissibility. This notion is supported by the previous report that longer time intervals between initial infection and superinfection is critical for the magnitude of the inhibitory effects of SIE between DENV2 and DENV4 in vitro
]. Future studies focused on assessing serial infections and monitoring the relative replication profiles of both viruses in dually infected mosquitoes will be critical to address this as a potential factor altering vector competence of field mosquito populations.
Superinfection barriers have been described previously for numerous arthropod-borne viruses in arthropods (mosquitoes, ticks and culicoides) as well as in arthropod cells [35
]. For example, Aedes triseriatus
mosquitoes experimentally infected with LaCrosse virus have been shown to be resistant to infection with another closely-related bunyavirus, Snowshoe Hare virus, after approximately two days post-infection [35
]. Dual infections with Thogoto virus have been shown to establish resistance to co-infection for periods ranging between 1–10 days after the initial infection [36
]. These data could indicate that the retarded replication rate of secondary flaviviral infections could be the result of pre-established monopolization of cellular transcriptional and translational machinery or the competitive blockage of cellular receptors. Replicating WNV replicons in BHK cells superinfected with WNV and other flaviviruses have demonstrated that preoccupation of intracellular resources such as host transcriptional and/or translational complexes are a likely explanation for the inhibition of the secondarily infecting virus [22
]. It is interesting to note that despite previous studies that have demonstrated increased replication of the NS4A 2K peptide mutant (V9M) in the presence of a previous WNV infection both in vitro
] and in vivo
in mosquitoes [27
], no such moderation of the effects of SIE were observed with this mutant when the initial infecting virus was NHUV. The 2K mutant WNV was capable of replicating to approximately 100-fold higher titers in C6/36 cells than the wild type WNV strain, while no difference in viral growth was observed between the two viruses in C7-10 cells. This finding likely indicates that the in vitro
selection for this mutant capable of increased replicative efficiency at the RNA transcriptional level occurred in a cell-specific manner, afforded in the context of a homologous rather than heterologous superinfection.
The RNAi response in mosquitoes can serve as a significant selection pressure for diversification of viral genomes. WNV has been shown to incorporate mutations in its genome in regions of the viral genome commonly targeted by the RNAi response [42
]. High genetic identity between NHUV and WNV in these critical highly-targeted regions of the genome could facilitate the secondary inhibition observed in the studies presented herein; however, the finding that NHUV infection in C6/36 cells that lack a functional RNAi response [43
] demonstrated a robust SIE effect on WNV independent of NHUV infection timing compared to WNV infection [6
] indicates that in vitro
RNAi-mediated inhibition, at least in the case presented in this study, is likely not a mechanism of SIE. Enhanced viral growth of JEV and DENV2 was previously observed in Cx. tritaeniorhynchus
cells persistently infected with CxFV, indicating the potential that previous infection with heterologous ISFs could potentially enhance infection by secondary MBFs [26
]. An ecological association for this phenomenon was also represented by the finding of Newman et al.
reported in 2011 in which a significant positive association between WNV and CxFV infection in individual female mosquitoes was observed [32
]. One potential explanation for this phenomenon of secondary enhancement could be the suppression of an RNAi response by the initial ISF infection. The lack of enhanced WNV replication in RNAi-deficient C6/36 cells previously infected with CxFV [23
], contrasted by the finding that Cx. quinquefasciatus
inoculated with CxFV from Honduras exhibited higher WNV infection rates than non-CxFV inoculated mosquitoes [25
], supports this possible explanation.
Several potential mechanisms could explain the lack of heterologous agent exclusion observed between MBFs and ISFs compared to those observed herein between a MBF and UVHF: (i) competitive exclusion through the scavenging of dissimilar genetic templates is inefficient; (ii) poorly-conserved amino acid identity between these viruses could reduce interfering heterologous protein-protein interactions; (iii) recombination events that result in non-functional products during RNA replication are infrequent due to poor efficiency of template switching; (iv) codon usage patterns between ISFs and MBFs are dissimilar enough that availability of tRNAs is not an impediment to translational efficiency of the secondary infecting virus; and (v) alternative intracellular trafficking could result in reduced direct interactions between the competing viruses. In contrast, the much higher degree of genetic identity between NHUV and WNV (54.3% nt, 49.4% aa) vs.
between ISFs and WNV (34%–38% nt, 21%–26% aa) as well as the similar codon usage pattern [6
] and the potentially recent acquisition of a mosquito restrictive phenotype for which intracellular trafficking of UVHFs much more closely resembles that of MBFVs could minimize these SIE impediments between WNV/NHUV and account for the more robust interference observed herein. Infection with genetically-distinctive secondary alphaviruses have demonstrated induction of SIE in C6/36 cells while unrelated bunyaviruses or flaviviruses could establish infection in Sindbis-infected C6/36 cells [37
]. Furthermore, WNV replicon-expressing BHK cells were capable of blocking subsequent infection with a series of heterologous flaviviruses but were ineffectual in obstructing rhabdoviral or alphaviral superinfection [22
]. The level of genetic identity necessary for effective induction of SIE has not been thoroughly established for any viral system nor has the specific mechanism(s) been determined. Synthetic biological approaches should allow for an efficient means to specifically address the relationship between the genetic identity of a superinfecting virus and the level of observed SIE. The close genetic identity between UVHFs and MBFVs could provide a foundation for robust inhibition that has not been recognized previously in MBFV and ISF interference experiments. Furthermore, UVHFs could serve as excellent research models for the direct assessment of the genetic basis for interference that could potentially be employed as additional intervention strategies for preventing mosquito transmission of human disease agents.