Structural evolution of an immune evasion determinant shapes pathogen host tropism.

Modern infectious disease outbreaks often involve changes in host tropism, the preferential adaptation of pathogens to specific hosts. The Lyme disease-causing bacterium Borrelia burgdorferi (Bb) is an ideal model to investigate the molecular mechanisms of host tropism, because different variants of these tick-transmitted bacteria are distinctly maintained in rodents or bird reservoir hosts. To survive in hosts and escape complement-mediated immune clearance, Bb produces the outer surface protein CspZ that binds the complement inhibitor factor H (FH) to facilitate bacterial dissemination in vertebrates. Despite high sequence conservation, CspZ variants differ in human FH-binding ability. Together with the FH polymorphisms between vertebrate hosts, these findings suggest that minor sequence variation in this bacterial outer surface protein may confer dramatic differences in host-specific, FH-binding-mediated infectivity. We tested this hypothesis by determining the crystal structure of the CspZ-human FH complex, and identifying minor variation localized in the FH-binding interface yielding bird and rodent FH-specific binding activity that impacts infectivity. Swapping the divergent region in the FH-binding interface between rodent- and bird-associated CspZ variants alters the ability to promote rodent- and bird-specific early-onset dissemination. We further linked these loops and respective host-specific, complement-dependent phenotypes with distinct CspZ phylogenetic lineages, elucidating evolutionary mechanisms driving host tropism emergence. Our multidisciplinary work provides a novel molecular basis for how a single, short protein motif could greatly modulate pathogen host tropism.

Phylogeographic reconstruction of the emergence and spread of Powassan virus in the northeastern United States.

Powassan virus is an emerging tick-borne virus of concern for public health, but very little is known about its transmission patterns and ecology. Here, we expanded the genomic dataset by sequencing 279 Powassan viruses isolated from Ixodes scapularis ticks from the northeastern United States. Our phylogeographic reconstructions revealed that Powassan virus lineage II was likely introduced or emerged from a relict population in the Northeast between 1940 and 1975. Sequences strongly clustered by sampling location, suggesting a highly focal geographical distribution. Our analyses further indicated that Powassan virus lineage II emerged in the northeastern United States mostly following a south-to-north pattern, with a weighted lineage dispersal velocity of ~3 km/y. Since the emergence in the Northeast, we found an overall increase in the effective population size of Powassan virus lineage II, but with growth stagnating during recent years. The cascading effect of population expansion of white-tailed deer and I. scapularis populations likely facilitated the emergence of Powassan virus in the northeastern United States.

Bourbon Virus Transmission, New York, USA.

In July 2019, Bourbon virus RNA was detected in an Amblyomma americanum tick removed from a resident of Long Island, New York, USA. Tick infection and white-tailed deer (Odocoileus virginianus) serosurvey results demonstrate active transmission in New York, especially Suffolk County, emphasizing a need for surveillance anywhere A. americanum ticks are reported.

Role of Anopheles Mosquitoes in Cache Valley Virus Lineage Displacement, New York, USA.

Cache Valley virus (CVV) is a mosquitoborne virus that infects livestock and humans. We report results of surveillance for CVV in New York, USA, during 2000-2016; full-genome analysis of selected CVV isolates from sheep, horse, humans, and mosquitoes from New York and Canada; and phenotypic characterization of selected strains. We calculated infection rates by using the maximum-likelihood estimation method by year, region, month, and mosquito species. The highest maximum-likelihood estimations were for Anopheles spp. mosquitoes. Our phylogenetic analysis identified 2 lineages and found evidence of segment reassortment. Furthermore, our data suggest displacement of CVV lineage 1 by lineage 2 in New York and Canada. Finally, we showed increased vector competence of An. quadrimaculatus mosquitoes for lineage 2 strains of CVV compared with lineage 1 strains.

Reversion to ancestral Zika virus NS1 residues increases competence of Aedes albopictus.

Both mosquito species-specific differences and virus strain -specific differences impact vector competence. Previous results in our laboratory with individual populations of N. American mosquitoes support studies suggesting Aedes aegypti are more competent than Ae. albopictus for American Zika virus (ZIKV) strains and demonstrate that U.S. Ae. albopictus have higher competence for an ancestral Asian ZIKV strain. A982V, an amino acid substitution in the NS1 gene acquired prior to the American outbreak, has been shown to increase competence in Ae. aegypti. We hypothesized that variability in the NS1 could therefore contribute to species-specific differences and developed a reverse genetics system based on a 2016 ZIKV isolate from Honduras (ZIKV-WTic) to evaluate the phenotypic correlates of individual amino acid substitutions. In addition to A982V, we evaluated G894A, which was acquired during circulation in the Americas. Reversion of 982 and 894 to ancestral residues increased infectivity, transmissibility and viral loads in Ae. albopictus but had no effect on competence or replication in Ae. aegypti. In addition, while host cell-specific differences in NS1 secretion were measured, with significantly higher secretion in mammalian cells relative to mosquito cells, strain-specific differences in secretion were not detected, despite previous reports. These results demonstrate that individual mutations in NS1 can influence competence in a species-specific manner independent of differences in NS1 secretion and further indicate that ancestral NS1 residues confer increased competence in Ae. albopictus. Lastly, experimental infections of Ifnar1-/- mice demonstrated that these NS1 substitutions can influence viral replication in the host and, specifically, that G894A could represent a compensatory change following a fitness loss from A982V with some viral genetic backgrounds. Together these data suggest a possible role for epistatic interactions in ZIKV fitness in invertebrate and vertebrate hosts and demonstrate that strains with increased transmission potential in U.S. Ae. albopictus could emerge.

Heartland Virus Transmission, Suffolk County, New York, USA.

During 2018, Heartland virus RNA was detected in an Amblyomma americanum tick removed from a resident of Suffolk County, New York, USA. The person showed seroconversion. Tick surveillance and white-tailed deer (Odocoileus virginianus) serosurveys showed widespread distribution in Suffolk County, emphasizing a need for disease surveillance anywhere A. americanum ticks are established or emerging.

Agnostic Framework for the Classification/Identification of Organisms Based on RNA Post-Transcriptional Modifications.

We propose a novel approach for building a classification/identification framework based on the full complement of RNA post-transcriptional modifications (rPTMs) expressed by an organism at basal conditions. The approach relies on advanced mass spectrometry techniques to characterize the products of exonuclease digestion of total RNA extracts. Sample profiles comprising identities and relative abundances of all detected rPTM were used to train and test the capabilities of different machine learning (ML) algorithms. Each algorithm proved capable of identifying rigorous decision rules for differentiating closely related classes and correctly assigning unlabeled samples. The ML classifiers resolved different members of the Enterobacteriaceae family, alternative Escherichia coli serotypes, a series of Saccharomyces cerevisiae knockout mutants, and primary cells of the Homo sapiens central nervous system, which shared very similar genetic backgrounds. The excellent levels of accuracy and resolving power achieved by training on a limited number of classes were successfully replicated when the number of classes was significantly increased to escalate complexity. A dendrogram generated from ML-curated data exhibited a hierarchical organization that closely resembled those afforded by established taxonomic systems. Finer clustering patterns revealed the extensive effects induced by the deletion of a single pivotal gene. This information provided a putative roadmap for exploring the roles of rPTMs in their respective regulatory networks, which will be essential to decipher the epitranscriptomics code. The ubiquitous presence of RNA in virtually all living organisms promises to enable the broadest possible range of applications, with significant implications in the diagnosis of RNA-related diseases.

West Nile virus is predicted to be more geographically widespread in New York State and Connecticut under future climate change.

The effects of climate change on infectious diseases are a topic of considerable interest and discussion. We studied West Nile virus (WNV) in New York (NY) and Connecticut (CT) using a Weather Research and Forecasting (WRF) model climate change scenario, which allows us to examine the effects of climate change and variability on WNV risk at county level. We chose WNV because it is well studied, has caused over 50,000 reported human cases, and over 2200 deaths in the United States. The ecological impacts have been substantial (e.g., millions of avian deaths), and economic impacts include livestock deaths, morbidity, and healthcare-related expenses. We trained two Random Forest models with observational climate data and human cases to predict future levels of WNV based on future weather conditions. The Regional Model used present-day data from NY and CT, whereas the Analog Model was fit for states most closely matching the predicted future conditions in the region. Separately, we predicted changes to mosquito-based WNV risk using a trait-based thermal biology approach (Mosquito Model). The WRF model produced control simulations (present day) and pseudo-global warming simulations (future). The Regional and Analog Models predicted an overall increase in human cases of WNV under future warming. However, the Analog Model did not predict as strong of an increase in the number of human cases as the Regional Model, and predicted a decrease in cases in some counties that currently experience high numbers of WNV cases. The Mosquito Model also predicted a decrease in risk in current high-risk areas, with an overall reduction in the population-weighted relative risk (but an increase in area-weighted risk). The Mosquito Model supports the Analog Model as making more realistic predictions than the Regional Model. All three models predicted a geographic increase in WNV cases across NY and CT.

West Nile Virus fidelity modulates the capacity for host cycling and adaptation.

The fidelity of flaviviruses is thought to be tightly regulated for optimal fitness within and between hosts. West Nile virus (WNV) high-fidelity (HiFi) mutations V793I and G806R within the RNA-dependent RNA polymerase, and low-fidelity (LoFi) mutation T248I within the methyltransferase, were previously shown to attenuate infectivity and replicative fitness in Culex mosquitoes and Culex tarsalis (CXT) cells but not in mammalian cells. We hypothesized that fidelity alterations would modify adaptation and maintenance in a host-specific manner. To test this hypothesis, wild-type (WT), HiFi (V793I/G806R) and LoFi (T248I) variants were sequentially passaged eight times in avian (PDE) or mosquito cells, or alternately between the two. Initial characterization confirmed that fidelity mutants are attenuated in mosquito, but not avian, cells. Deep sequencing revealed mutations unique to both cell lines and fidelity mutants, including ENV G1378A, a mutation associated with avian cell adaptation. To characterize maintenance and adaptation, viral outputs were monitored throughout passaging and viral fitness was assessed. The results indicate that fidelity mutants can at times recover fitness during mosquito cell passage, but remain attenuated relative to WT. Despite similar initial fitness, LoFi mutants were impaired during sequential passage in avian cells. Conversely, HiFi mutants passaged in avian cells showed increased adaptation, suggesting that increased fidelity may be advantageous in avian hosts. Although some adaptation occurred with individual mutants, the output titres of fidelity mutants were on average lower and were often lost during host switching. These data confirm that arbovirus fidelity is likely fine-tuned to maximize survival in disparate hosts.

Vector Competence of Aedes caspius and Ae. albopictus Mosquitoes for Zika Virus, Spain.

We assessed the vector competence of Aedes caspius and Aedes albopictus mosquitoes in Spain for the transmission of Zika virus. Whereas Ae. albopictus mosquitoes were a competent vector, Ae. caspius mosquitoes were unable to transmit Zika virus. We also identified high levels of vertical transmission of Zika virus in Ae. albopictus mosquitoes.

Large-Scale Complete-Genome Sequencing and Phylodynamic Analysis of Eastern Equine Encephalitis Virus Reveals Source-Sink Transmission Dynamics in the United States.

Eastern equine encephalitis virus (EEEV) has a high case-fatality rate in horses and humans, and Florida has been hypothesized to be the source of EEEV epidemics for the northeastern United States. To test this hypothesis, we sequenced complete genomes of 433 EEEV strains collected within the United States from 1934 to 2014. Phylogenetic analysis suggested EEEV evolves relatively slowly and that transmission is enzootic in Florida, characterized by higher genetic diversity and long-term local persistence. In contrast, EEEV strains in New York and Massachusetts were characterized by lower genetic diversity, multiple introductions, and shorter local persistence. Our phylogeographic analysis supported a source-sink model in which Florida is the major source of EEEV compared to the other localities sampled. In sum, this study revealed the complex epidemiological dynamics of EEEV in different geographic regions in the United States and provided general insights into the evolution and transmission of other avian mosquito-borne viruses in this region.IMPORTANCE Eastern equine encephalitis virus (EEEV) infections are severe in horses and humans on the east coast of the United States with a >90% mortality rate in horses, an ∼33% mortality rate in humans, and significant brain damage in most human survivors. However, little is known about the evolutionary characteristics of EEEV due to the lack of genome sequences. By generating large collection of publicly available complete genome sequences, this study comprehensively determined the evolution of the virus, described the epidemiological dynamics of EEEV in different states in the United States, and identified Florida as one of the major sources. These results may have important implications for the control and prevention of other mosquito-borne viruses in the Americas.

Vertical Transmission of Zika Virus by Aedes aegypti and Ae. albopictus Mosquitoes.

To determine the potential role of vertical transmission in Zika virus expansion, we evaluated larval pools of perorally infected Aedes aegypti and Ae. albopictus adult female mosquitoes; ≈1/84 larvae tested were Zika virus-positive; and rates varied among mosquito populations. Thus, vertical transmission may play a role in Zika virus spread and maintenance.

Effects of Zika Virus Strain and Aedes Mosquito Species on Vector Competence.

In the Western Hemisphere, Zika virus is thought to be transmitted primarily by Aedes aegypti mosquitoes. To determine the extent to which Ae. albopictus mosquitoes from the United States are capable of transmitting Zika virus and the influence of virus dose, virus strain, and mosquito species on vector competence, we evaluated multiple doses of representative Zika virus strains in Ae. aegypti and Ae. albopictus mosquitoes. Virus preparation (fresh vs. frozen) significantly affected virus infectivity in mosquitoes. We calculated 50% infectious doses to be 6.1-7.5 log 10 PFU/mL; minimum infective dose was 4.2 log 10 PFU/mL. Ae. albopictus mosquitoes were more susceptible to infection than Ae. aegypti mosquitoes, but transmission efficiency was higher for Ae. aegypti mosquitoes, indicating a transmission barrier in Ae. albopictus mosquitoes. Results suggest that, although Zika virus transmission is relatively inefficient overall and dependent on virus strain and mosquito species, Ae. albopictus mosquitoes could become major vectors in the Americas.

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.

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.

Increased replicative fitness of a dengue virus 2 clade in native mosquitoes: potential contribution to a clade replacement event in Nicaragua.

UNLABELLED: The four dengue virus (DENV) serotypes (DENV serotype 1 [DENV-1] to DENV-4) are transmitted by Aedes aegypti and A. albopictus mosquitoes, causing up to 390 million DENV infections worldwide each year. We previously reported a clade replacement of the DENV-2 Asian-American genotype NI-1 clade by the NI-2B clade in Managua, Nicaragua. Here, we describe our studies of the replicative ability of NI-1 and NI-2B viruses in an A. aegypti cell line (Aag2) and A. aegypti mosquitoes reared from eggs collected in Managua. In coinfection experiments, several different pairs of NI-1 and NI-2B clinical isolates were used to infect Aag2 cells or blood-fed A. aegypti mosquitoes. Results consistently showed a significant replicative advantage of NI-2B over NI-1 viruses early after infection in vitro, and in mosquitoes, NI-2B viruses attained a higher replicative index than NI-1 isolates 3 to 7 days postinfection (dpi). At 7 dpi, NI-2B viruses displayed a significantly higher replicative index in legs and salivary glands; however, this advantage was lost by 14 and 21 dpi. We also found that the percentage of mosquitoes in which NI-2B viruses were dominant was significantly higher than that in which NI-1 viruses were dominant on day 7 but not at later time points. Taken together, these data demonstrate that clade NI-2B holds a replicative advantage over clade NI-1 early in infection that wanes at later time points. This early fitness advantage of NI-2B viruses over NI-1 viruses in the native vector, A. aegypti, suggests a shorter extrinsic incubation period for NI-2B viruses, which could have contributed to the clade replacement event in Managua. IMPORTANCE: Dengue virus (DENV), one of the most medically important arthropod-borne viruses, is transmitted to humans by Aedes aegypti and A. albopictus mosquitoes in tropical and subtropical regions worldwide. Dengue epidemics continue to increase in frequency, geographic range, and severity and are a major public health concern. This is due to globalization, unplanned urbanization, and climate change, as well as host genetics and immune responses and viral genetic changes. DENV consists of four serotypes, in turn composed of genotypes and genetically distinct clades. What drives the frequent replacement of a previously circulating DENV clade by another is unclear. Here, we investigate the replicative fitness of two clades of DENV serotype 2 in Aedes aegypti cells and mosquitoes collected from the region where the viruses circulated and conclude that increased replicative fitness could have contributed to a DENV clade replacement event in Nicaragua. These findings provide insight into vector-driven evolution of DENV epidemics.

Consequences of in vitro host shift for St. Louis encephalitis virus.

Understanding the potential for host range shifts and expansions of RNA viruses is critical to predicting the evolutionary and epidemiological paths of these pathogens. As arthropod-borne viruses (arboviruses) experience frequent spillover from their amplification cycles and are generalists by nature, they are likely to experience a relatively high frequency of success in a range of host environments. Despite this, the potential for host expansion, the genetic correlates of adaptation to novel environments and the costs of such adaptations in originally competent hosts are still not characterized fully for arboviruses. In the studies presented here, we utilized experimental evolution of St. Louis encephalitis virus (SLEV; family Flaviviridae, genus Flavivirus) in vitro in the Dermacentor andersoni line of tick cells to model adaptation to a novel invertebrate host. Our results demonstrated that levels of adaptation and costs in alternate hosts are highly variable among lineages, but also that significant fitness increases in tick cells are achievable with only modest change in consensus genetic sequence. In addition, although accumulation of diversity may at times buffer against phenotypic costs within the SLEV swarm, an increased proportion of variants with an impaired capacity to infect and spread on vertebrate cell culture accumulated with tick cell passage. Isolation and characterization of a subset of these variants implicates the NS3 gene as an important host range determinant for SLEV.

The effect of temperature on life history traits of Culex mosquitoes.

Climatic changes forecasted in the coming years are likely to result in substantial alterations to the distributions and populations of vectors of arthropod-borne pathogens. Characterization of the effect of temperature shifts on the life history traits of specific vectors is needed to more accurately define how such changes could impact the epidemiological patterns of vector-borne disease. Here, we determined the effect of temperatures including 16, 20, 24, 28, and 32 degreeC on development time, immature survival, adult survival, mosquito size, blood feeding, and fecundity of both field and colonized populations of the Culex mosquitoes Culex pipiens L, Culex quinquefasciatus Say, and Culex restuans Theobald. Our results demonstrate that temperature significantly affects all of these traits, yet also that the extent of this effect is at times incongruent among temperatures, as well as being population and species-specific. Comparisons of colonized mosquitoes with field populations generally demonstrate decreased adult and immature survival, increased blood feeding and egg production, and significant variation in the effects of temperature, indicating that such colonies are not fully representative of natural populations. Results with field populations in general indicate that increases in temperature are likely to accelerate mosquito development, and that this effect is greater at temperatures below 24 degreeC, but also that temperature significantly increases mortality. Among field populations, Cx. restuans were most affected by temperature increases, with decreased longevity relative to other species and significant increases in adult and immature mortality measured with each incremental temperature increase. Despite the unique climates characteristic of the geographic ranges ofCx. quinquefasciatus and Cx. pipiens, evidence of significant species-specific adaptation to temperature ranges was not seen. Taken together, these results indicate that geographic region, as well as species and population differences, must be considered when measuring the effect of temperature on vector populations.

Direct Evidence of Powassan Virus Vertical Transmission in Ixodes scapularis in Nature.

Powassan virus (POWV) is a tick-borne flavivirus endemic in North America and Russia. Experimental infections with POWV have confirmed horizontal, transstadial, vertical, and cofeeding transmission routes for potential virus maintenance. In the field, vertical transmission has never been observed. During New York State tick-borne pathogen surveillance, POWV RNA and/or infectious POWV was detected in five pools of questing Ixodes scapularis larvae. Additionally, engorged female I. scapularis adults were collected from hunter-harvested white-tailed deer (Odocoileus virginianus) in a region with relatively high tick infection rates of POWV and allowed to oviposit under laboratory conditions. POWV RNA was detected in three female adult husks and one pool of larvae from a positive female. Infectious virus was isolated from all three RNA-positive females and the single positive larval pool. The detection of RNA and infectious virus in unfed questing larvae from the field and larvae from replete females collected from the primary tick host implicates vertical transmission as a potential mechanism for the maintenance of POWV in I. scapularis in nature, and elucidates the potential epidemiological significance of larval ticks in the transmission of POWV to humans.

Aedes albopictus saliva contains a richer microbial community than the midgut.

BACKGROUND: Past findings demonstrate that arthropods can egest midgut microbiota into the host skin leading to dual colonization of the vertebrate host with pathogens and saliva microbiome. A knowledge gap exists on how the saliva microbiome interacts with the pathogen in the saliva. To fill this gap, we need to first define the microbial composition of mosquito saliva. METHODS: The current study aimed at analyzing and comparing the microbial profile of Aedes albopictus saliva and midgut as well as assessing the impact of Zika virus (ZIKV) infection on the midgut and saliva microbial composition. Colony-reared Ae. albopictus strains were either exposed to ZIKV infectious or noninfectious bloodmeal. At 14 ays postinfection, the 16S V3-V4 hypervariable rRNA region was amplified from midgut and saliva samples and sequenced on an Illumina MiSeq platform. The relative abundance and diversity of midgut and saliva microbial taxa were assessed. RESULTS: We observed a richer microbial community in the saliva compared with the midgut, yet some of the microbial taxa were common in the midgut and saliva. ZIKV infection did not impact the microbial diversity of midgut or saliva. Further, we identified Elizabethkingia spp. in the Ae. albopictus saliva. CONCLUSIONS: This study provides insights into the microbial community of the Ae. albopictus saliva as well as the influence of ZIKV infection on the microbial composition of its midgut and saliva. The identification of Elizabethkingia spp., an emerging pathogen of global health significance, in Ae. albopictus saliva is of medical importance. Future studies to assess the interactions between Ae. albopictus saliva microbiome and ZIKV could lead to novel strategies for developing transmission barrier tools.