RNA viruses form genetically complex populations of closely related variants within hosts; such populations are frequently referred to as a mutant swarm or as a quasispecies. In WNV, as with other RNA viruses (29
), the amount of genetic variation present in the mutant swarm is host dependent (15
): replication in mosquitoes tends to promote viral genetic diversity, and replication in vertebrates tends to limit this diversity. In this study, we examined mp20c, a WNV population that had replicated in mosquitoes for 20 consecutive passages and was highly genetically diverse and attenuated in mice. Consensus genome sequence data revealed that the nucleotide substitutions that accumulated during 20 mosquito passages were unremarkable (16
). However, a range of plaque sizes was observed when the titer of mp20c was determined on Vero cells. This range of plaque sizes led us to question whether minor genomic variants might be present within the mutant swarm and whether these might be capable of influencing the population phenotypes. Therefore, we examined two variants from the attenuated mp20c population that differed in plaque size and evaluated several phenotypes in order to gain insight into the relationships between individual and group phenotypes in WNV.
First, we isolated biological clones representing small-plaque and large-plaque phenotypes from the mixed population and characterized several phenotypes. Plaque sizes were consistent after a single Vero cell passage, indicating the stability of the phenotype and the suitability of the passaged stocks for further experiments. We found that the mouse mortality rate was variable following peripheral inoculation with a constant virus dose. Specifically, whereas the small-plaque variant produced no mortality, the large-plaque variant produced 50% mortality. The mortality rate of the mixed population was 25%. These results establish that the SP3v1 small-plaque variant is attenuated in mice. This finding was not particularly surprising: previous work has shown that the sp phenotype in WNV is associated with attenuation (7
). Slightly more surprising was the finding that mp20c caused an intermediate (25%) degree of mortality compared to sp3v1 (0%) and lp1v1 (50%). We had expected that the pathogenic potential of a virus population such as mp20c would be dominated by that of its most pathogenic component. This clearly was not the case, suggesting that, as has been reported for vesicular stomatitis virus (9
), high-fitness WNV variants (here we directly equate fitness with pathogenesis in mice) can be “suppressed” through replication along with a genetically diverse mutant swarm containing lower-fitness variants.
Molecular clones were then constructed to define the viral genetic determinants of the sp and attenuated phenotype. After sequencing the sp and lp biological clones, two candidate attenuating mutations (substitution of Thr for Met in NS4A [NS4aM64T] and an Asp deletion in NS3 [NS3Δ483]) were identified and engineered into a cDNA clone of WNV. We found that NS3Δ483, but not NS4aM64T, was specifically associated with both the sp and mouse-attenuated phenotypes and with decreased replication in mouse L929 cells. This result suggested that less efficient in vivo replication in mice might lead to attenuation. Accordingly, we evaluated the possibility that replication and/or spread in mice might differ between NS3Δ483 and WT WNV.
Examination of viral loads in mouse sera, lymph nodes, and spleen demonstrated that NS3Δ483 replicated and spread similarly to WT WNV, suggesting that a frank deficit in replication efficiency in vivo is unlikely to produce the attenuation observed in mice. However, NS3Δ483 was cleared from circulation more efficiently than WT WNV, and neuroinvasion was less efficient in mice infected by NS3Δ483 than in those infected by WT WNV. Collectively, these observations led us to conclude that NS3Δ483 was more effectively controlled by the host than was WT WNV.
The deleted residue, Asp483, is located within the RNA helicase domain of the NS3 protein and is highly conserved among the encephalitic flaviviruses WNV, Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) but not in dengue virus (DEN) or yellow fever virus (YFV) (34
), suggesting that it may be an important determinant of neuroinvasion and/or neurovirulence. Specifically, it may influence the efficiency of unwinding double-stranded RNA (dsRNA) replicative intermediates. The presence of higher concentrations of intracellular dsRNA could lead to enhanced interferon activation through TLR3-dependent signaling. Alternatively, the overall slowed rate of RNA replication might render the virus more susceptible to the antiviral state that develops in vertebrate hosts. Therefore, we measured interferon production in mice that had been infected by mutant and wild-type viruses and assessed the in vitro
sensitivity of the viruses to interferon in Vero cells. In mice, NS3Δ483 did not induce significantly different levels of type I interferon compared to WT. Upon in vitro
treatment of cells with type I IFN, NS3Δ483 WNV was more sensitive than WT WNV, resulting in 10-fold-lower viral production. Taken together, these results led us to conclude that the NS3Δ483 mutation renders the virus more susceptible to the antiviral state that develops in the animal during the course of WNV infection but does not influence initial replication or interferon induction. Furthermore, we propose a model in which the individual phenotype of greater susceptibility to type I IFN (NS3Δ483) contributes to the phenotype of the quasispecies (mp20c). In this model, all genotypes contribute to the initial antiviral state. Once the antiviral state is induced, the NS3Δ483 mutant is more effectively controlled than the more resistant genotypes, such as the WT genotype. With reduced viral production for the more susceptible genotypes (NS3Δ483), viral infection in the animal as a whole was better controlled, resulting in partial attenuation of the quasispecies (mp20c).
Finally, since the NS3Δ483 mutation arose during prolonged replication in mosquitoes, we tested whether this mutation might be important in balancing WNV fitness between vertebrates, where it appears to be required for interferon resistance, and mosquitoes, which lack an interferon response. To accomplish this, we used a previously reported system for estimating competitive fitness in vivo
that takes advantage of a genetically marked WNV competitor virus (13
). NS3Δ483 was effectively outcompeted by WT WNV in chickens, demonstrating that the RNA helicase function is a major fitness determinant in these hosts. In mosquitoes, we found that NS3Δ483 competed moderately effectively at 7 days after infection but not at 14 days. These results indicate that this determinant within the RNA helicase domain of NS3 is a fitness determinant in both vertebrate and invertebrate components of the WNV transmission cycle but is more important in vertebrates than in mosquitoes. Interestingly, a major mosquito innate antivirus defense (RNA interference [RNAi]) also targets double-stranded RNA via the RNase III enzyme Dicer. It follows that RNA helicase activity should also be associated with fitness in mosquitoes and that defects in helicase function would lead to fitness declines because intracellular concentrations of viral dsRNA replicative intermediates are higher, which could lead to Dicer-mediated cleavage of greater efficiency. Our data on fitness in mosquitoes support this observation. Moreover, the studies we report highlight the complex relationships that exist between individual and group phenotypes in highly variable populations such as RNA viruses and demonstrate that phenotypically significant variants may be undetectable through conventional approaches to obtaining the virus consensus sequence. Finally, our studies have shown that the WNV RNA helicase is both a fitness determinant of natural hosts (mosquitoes and birds) and a determinant of virus virulence in mice.