Greta Van Slyke

Cooperative interactions in the West Nile virus mutant swarm.

BackgroundRNA viruses including arthropod-borne viruses (arboviruses) exist as highly genetically diverse mutant swarms within individual hosts. A more complete understanding of the phenotypic correlates of these diverse swarms is needed in order to equate RNA swarm breadth and composition to specific adaptive and evolutionary outcomes.ResultsHere, we determined clonal fitness landscapes of mosquito cell-adapted West Nile virus (WNV) and assessed how altering the capacity for interactions among variants affects mutant swarm dynamics and swarm fitness. Our results demonstrate that although there is significant mutational robustness in the WNV swarm, genetic diversity also corresponds to substantial phenotypic diversity in terms of relative fitness in vitro. In addition, our data demonstrate that increasing levels of co-infection can lead to widespread strain complementation, which acts to maintain high levels of phenotypic and genetic diversity and potentially slow selection for individual variants. Lastly, we show that cooperative interactions may lead to swarm fitness levels which exceed the relative fitness levels of any individual genotype.ConclusionsThese studies demonstrate the profound effects variant interactions can have on arbovirus evolution and adaptation, and provide a baseline by which to study the impact of this phenomenon in natural systems.

Quantification of intrahost bottlenecks of West Nile virus in Culex pipiens mosquitoes using an artificial mutant swarm

Mosquito-borne viruses are predominantly RNA viruses which exist within hosts as diverse mutant swarms. Defining the way in which stochastic forces within mosquito vectors shape these swarms is critical to advancing our understanding of the evolutionary and adaptive potential of these pathogens. There are multiple barriers within a mosquito which a viral swarm must traverse in order to ultimately be transmitted. Here, using artificial mutant swarms composed of neutral variants of West Nile virus (WNV), we tracked changes to swarm breadth over time and space in Culex pipiens mosquitoes. Our results demonstrate that all variants have the potential to survive intrahost bottlenecks, yet mean swarm breadth decreases during both midgut infection and transmission when starting populations contain higher levels of minority variants. In addition, WNV swarms are subject to temporal sweeps which act to significantly decrease intrahost diversity over time. Taken together, these data demonstrate the profound effects that stochastic forces can have in shaping arboviral mutant swarms.

Sequence-Specific Fidelity Alterations Associated with West Nile Virus Attenuation in Mosquitoes.

High rates of error-prone replication result in the rapid accumulation of genetic diversity of RNA viruses. Recent studies suggest that mutation rates are selected for optimal viral fitness and that modest variations in replicase fidelity may be associated with viral attenuation. Arthropod-borne viruses (arboviruses) are unique in their requirement for host cycling and may necessitate substantial genetic and phenotypic plasticity. In order to more thoroughly investigate the correlates, mechanisms and consequences of arbovirus fidelity, we selected fidelity variants of West Nile virus (WNV; Flaviviridae, Flavivirus) utilizing selection in the presence of a mutagen. We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity. Both deep-sequencing and in vitro biochemical assays confirmed strain-specific differences in both fidelity and mutational bias. WNV fidelity variants demonstrated host-specific alterations to replicative fitness in vitro, with modest attenuation in mosquito but not vertebrate cell culture. Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes. Taken together, these studies (i) demonstrate the importance of allosteric interactions in regulating mutation rates, (ii) establish that mutational spectra can be both sequence and strain-dependent, and (iii) display the profound phenotypic consequences associated with altered replication complex function of flaviviruses.

The evolution of virulence of West Nile virus in a mosquito vector: implications for arbovirus adaptation and evolution

BackgroundVirulence is often coupled with replicative fitness of viruses in vertebrate systems, yet the relationship between virulence and fitness of arthropod-borne viruses (arboviruses) in invertebrates has not been evaluated. Although the interactions between vector-borne pathogens and their invertebrate hosts have been characterized as being largely benign, some costs of arbovirus exposure have been identified for mosquitoes. The extent to which these costs may be strain-specific and the subsequent consequences of these interactions on vector and virus evolution has not been adequately explored.ResultsUsing West Nile virus (WNV) and Culex pipiens mosquitoes, we tested the hypothesis that intrahost fitness is correlated with virulence in mosquitoes by evaluating life history traits following exposure to either non-infectious bloodmeals or bloodmeals containing wildtype (WNV WT) or the high fitness, mosquito-adapted strain, WNV MP20 derived from WNV WT. Our results demonstrate strain-specific effects on mosquito survival, fecundity, and blood feeding behavior. Specifically, both resistance to and infection with WNV MP20, but not WNV WT, decreased survival of Cx. pipiens and altered fecundity and bloodfeeding such that early egg output was enhanced at a later cost.ConclusionsAs predicted by the trade-off hypothesis of virulence, costs of infection with WNV MP20 in terms of survival were directly correlated to viral load, yet resistance to infection with this virulent strain was equally costly. Taken together, these results demonstrate that WNV MP20 infection decreases the transmission potential of Cx. pipiens populations despite the increased intrahost fitness of this strain, indicating that a virulence-transmission trade-off in invertebrates could contribute significantly to the adaptive and evolutionary constraint of arboviruses.

Point mutations in the West Nile virus (Flaviviridae; Flavivirus) RNA-dependent RNA polymerase alter viral fitness in a host-dependent manner in vitro and in vivo

The West Nile virus (WNV) genome contains a single RNA-dependent RNA polymerase (RdRp) gene, which is responsible for replication of the viral genome and, as such, is an important target for antiviral therapy. Viral RdRps are known to lack proofreading capabilities and as a result viruses such as WNV exist as a mixture of viral genotypes within an infection, enabling the virus to readily emerge and adapt to new host environments. To test the consequences of subtle structural alterations remote from the RdRp active-site, the following single point mutations were engineered in the WNV NS5 RdRp coding region: T363N, A365N, and T537I; these mutations were selected in an effort to stabilize the secondary structural elements near the rNTP binding pocket of the RdRp. Mutant viruses were tested in vitro on Vero, C6/36, Culex tarsalis and DF-1 cell types and in vivo in one day old chickens and Culex pipiens mosquitoes. Plaque morphology was affected by each mutation and growth and RNA replication kinetics were altered as well. Our results demonstrate that subtle alteration of the RdRp protein away from the active site can have a significant overall biological effect on WNV fitness, and that this effect can be host-dependent.

Exposure to West Nile Virus Increases Bacterial Diversity and Immune Gene Expression in Culex pipiens

Complex interactions between microbial residents of mosquitoes and arboviruses are likely to influence many aspects of vectorial capacity and could potentially have profound effects on patterns of arbovirus transmission. Such interactions have not been well studied for West Nile virus (WNV; Flaviviridae, Flavivirus) and Culex spp. mosquitoes. We utilized next-generation sequencing of 16S ribosomal RNA bacterial genes derived from Culex pipiens Linnaeus following WNV exposure and/or infection and compared bacterial populations and broad immune responses to unexposed mosquitoes. Our results demonstrate that WNV infection increases the diversity of bacterial populations and is associated with up-regulation of classical invertebrate immune pathways including RNA interference (RNAi), Toll, and Jak-STAT (Janus kinase-Signal Transducer and Activator of Transcription). In addition, WNV exposure alone, without the establishment of infection, results in similar alterations to microbial and immune signatures, although to a lesser extent. Multiple bacterial genera were found in greater abundance in WNV-exposed and/or infected mosquitoes, yet the most consistent and notable was the genus Serratia.

High-Definition Mapping of Four Spatially Distinct Neutralizing Epitope Clusters on RiVax, a Candidate Ricin Toxin Subunit Vaccine

ABSTRACT RiVax is a promising recombinant ricin toxin A subunit (RTA) vaccine antigen that has been shown to be safe and immunogenic in humans and effective at protecting rhesus macaques against lethal-dose aerosolized toxin exposure. We previously used a panel of RTA-specific monoclonal antibodies (MAbs) to demonstrate, by competition enzyme-linked immunosorbent assay (ELISA), that RiVax elicits similar serum antibody profiles in humans and macaques. However, the MAb binding sites on RiVax have yet to be defined. In this study, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes on RiVax recognized by nine toxin-neutralizing MAbs and one nonneutralizing MAb. Based on strong protection from hydrogen exchange, the nine MAbs grouped into four spatially distinct epitope clusters (namely, clusters I to IV). Cluster I MAbs protected RiVaxs α-helix B (residues 94 to 107), a protruding immunodominant secondary structure element known to be a target of potent toxin-neutralizing antibodies. Cluster II consisted of two subclusters located on the “back side” (relative to the active site pocket) of RiVax. One subcluster involved α-helix A (residues 14 to 24) and α-helices F-G (residues 184 to 207); the other encompassed β-strand d (residues 62 to 69) and parts of α-helices D-E (154 to 164) and the intervening loop. Cluster III involved α-helices C and G on the front side of RiVax, while cluster IV formed a sash from the front to back of RiVax, spanning strands b, c, and d (residues 35 to 59). Having a high-resolution B cell epitope map of RiVax will enable the development and optimization of competitive serum profiling assays to examine vaccine-induced antibody responses across species.

Spatial location of neutralizing and non-neutralizing B cell epitopes on domain 1 of ricin toxin’s binding subunit

Ricin toxin’s binding subunit (RTB) is a galactose-/N-acetylgalactosamine (Gal/GalNac)-specific lectin that mediates uptake and intracellular trafficking of ricin within mammalian cells. Structurally, RTB consists of two globular domains, each divided into three homologous sub-domains (α, β, γ). In this report, we describe five new murine IgG monoclonal antibodies (mAbs) against RTB: MH3, 8A1, 8B3, LF1, and LC5. The mAbs have similar binding affinities (KD) for ricin holotoxin, but displayed a wide range of in vitro toxin-neutralizing activities. Competition ELISAs indicate that the two most potent toxin-neutralizing mAbs (MH3, 8A1), as well as one of the moderate toxin-neutralizing mAbs (LF1), recognize distinct epitopes near the low affinity Gal recognition domain in RTB subdomain 1α. Evaluated in a mouse model of systemic ricin challenge, all five mAbs afforded some benefit against intoxication, but only MH3 was protective. However, neither MH3 nor 24B11, another well-characterized mAb against RTB subdomain 1α, could passively protect mice against a mucosal (intranasal) ricin challenge. This is in contrast to SylH3, a previously characterized mAb directed against an epitope near RTB’s high affinity Gal/GalNac recognition element in sub-domain 2γ, which protected animals against systemic and mucosal ricin exposure. SylH3 was significantly more effective than MH3 and 24B11 at blocking ricin attachment to host cell receptors, suggesting that mucosal immunity to ricin is best imparted by antibodies that target RTB’s high affinity Gal/GalNac recognition element in subdomain 2γ, not the low affinity Gal recognition domain in subdomain 1α.

Mutagen resistance and mutation restriction of St. Louis encephalitis virus

The error rate of the RNA-dependent RNA polymerase (RdRp) of RNA viruses is important in maintaining genetic diversity for viral adaptation and fitness. Numerous studies have shown that mutagen-resistant RNA virus variants display amino acid mutations in the RdRp and other replicase subunits, which in turn exhibit an altered fidelity phenotype affecting viral fitness, adaptability and pathogenicity. St. Louis encephalitis virus (SLEV), like its close relative West Nile virus, is a mosquito-borne flavivirus that has the ability to cause neuroinvasive disease in humans. Here, we describe the successful generation of multiple ribavirin-resistant populations containing a shared amino acid mutation in the SLEV RdRp (E416K). These E416K mutants also displayed resistance to the antiviral T-1106, an RNA mutagen similar to ribavirin. Structural modelling of the E416K polymerase mutation indicated its location in the pinky finger domain of the RdRp, distant from the active site. Deep sequencing of the E416K mutant revealed lower genetic diversity than wild-type SLEV after growth in both vertebrate and invertebrate cells. Phenotypic characterization showed that E416K mutants displayed similar or increased replication in mammalian cells, as well as modest attenuation in mosquito cells, consistent with previous work with West Nile virus high-fidelity variants. In addition, attenuation was limited to mosquito cells with a functional RNA interference response, suggesting an impaired capacity to escape RNA interference could contribute to attenuation of high-fidelity variants. Our results provide increased evidence that RNA mutagen resistance arises through modulation of the RdRp and give further insight into the consequences of altered fidelity of flaviviruses.The error rate of the RNA-dependent RNA polymerase (RdRp) of RNA viruses is important in maintaining genetic diversity for viral adaptation and fitness. Numerous studies have shown that mutagen-resistant RNA virus variants display amino acid mutations in the RdRp and other replicase subunits, which in turn exhibit an altered fidelity phenotype affecting viral fitness, adaptability, and pathogenicity. St. Louis encephalitis virus (SLEV), like its close relative West Nile virus (WNV), is a mosquito-borne flavivirus which has the ability to cause neuroinvasive disease in humans. Here, we describe the successful generation of multiple ribavirin-resistant populations containing a shared amino acid mutation in the SLEV RdRp (E416K). These E416K mutants also displayed resistance to the antiviral T-1106, an RNA mutagen similar to ribavirin. Structural modeling of the E416K polymerase mutation indicate its location in the pinky finger domain of the RdRp, distant from the active site. Deep-sequencing of the E416K mutant revealed lower genetic diversity than wildtype SLEV after growth in both vertebrate and invertebrate cells. Phenotypic characterization showed E416K mutants displayed similar or increased replication in mammalian cells, as well as modest attenuation in mosquito cells, consistent with previous work with WNV high-fidelity variants. In addition, attenuation was limited to mosquito cells with a functional RNA interference (RNAi) response, suggesting an impaired capacity to escape RNAi could contribute to attenuation of high-fidelity variants. Our results provide increased evidence that RNA mutagen resistance arises through modulation of the RdRp and gives further insight into the consequences of altered fidelity of flaviviruses.

Vertebrate attenuated West Nile virus mutants have differing effects on vector competence in Culex tarsalis mosquitoes

Previous mutational analyses of naturally occurring West Nile virus (WNV) strains and engineered mutant WNV strains have identified locations in the viral genome that can have profound phenotypic effect on viral infectivity, temperature sensitivity and neuroinvasiveness. We chose six mutant WNV strains to evaluate for vector competence in the natural WNV vector Culex tarsalis, two of which contain multiple ablations of glycosylation sites in the envelope and NS1 proteins; three of which contain mutations in the NS4B protein and an attenuated natural bird isolate (Bird 1153) harbouring an NS4B mutation. Despite vertebrate attenuation, all NS4B mutant viruses displayed enhanced vector competence by Cx. tarsalis. Non-glycosylated mutant viruses displayed decreased vector competence in Cx. tarsalis mosquitoes, particularly when all three NS1 glycosylation sites were abolished. These results indicate the importance of both the NS4B protein and NS1 glycosylation in the transmission of WNV by a significant mosquito vector.