Vector competence of Aedes aegypti, Culex tarsalis, and Culex quinquefasciatus from California for Zika virus.

Zika virus (ZIKV) has emerged since 2013 as a significant global human health threat following outbreaks in the Pacific Islands and rapid spread throughout South and Central America. Severe congenital and neurological sequelae have been linked to ZIKV infections. Assessing the ability of common mosquito species to transmit ZIKV and characterizing variation in mosquito transmission of different ZIKV strains is important for estimating regional outbreak potential and for prioritizing local mosquito control strategies for Aedes and Culex species. In this study, we evaluated the laboratory vector competence of Aedes aegypti, Culex quinquefasciatus, and Culex tarsalis that originated in areas of California where ZIKV cases in travelers since 2015 were frequent. We compared infection, dissemination, and transmission rates by measuring ZIKV RNA levels in cohorts of mosquitoes that ingested blood meals from type I interferon-deficient mice infected with either a Puerto Rican ZIKV strain from 2015 (PR15), a Brazilian ZIKV strain from 2015 (BR15), or an ancestral Asian-lineage Malaysian ZIKV strain from 1966 (MA66). With PR15, Cx. quinquefasciatus was refractory to infection (0%, N = 42) and Cx. tarsalis was infected at 4% (N = 46). No ZIKV RNA was detected in saliva from either Culex species 14 or 21 days post feeding (dpf). In contrast, Ae. aegypti developed infection rates of 85% (PR15; N = 46), 90% (BR15; N = 20), and 81% (MA66; N = 85) 14 or 15 dpf. Although MA66-infected Ae. aegypti showed higher levels of ZIKV RNA in mosquito bodies and legs, transmission rates were not significantly different across virus strains (P = 0.13, Fisher's exact test). To confirm infectivity and measure the transmitted ZIKV dose, we enumerated infectious ZIKV in Ae. aegypti saliva using Vero cell plaque assays. The expectorated plaque forming units PFU varied by viral strain: MA66-infected expectorated 13±4 PFU (mean±SE, N = 13) compared to 29±6 PFU for PR15-infected (N = 13) and 35±8 PFU for BR15-infected (N = 6; ANOVA, df = 2, F = 3.8, P = 0.035). These laboratory vector competence results support an emerging consensus that Cx. tarsalis and Cx. quinquefasciatus are not vectors of ZIKV. These results also indicate that Ae. aegypti from California are efficient laboratory vectors of ancestral and contemporary Asian lineage ZIKV

Chikungunya virus populations experience diversity- dependent attenuation and purifying intra-vector selection in Californian Aedes aegypti mosquitoes.

Chikungunya virus (Togaviridae, Alphavirus; CHIKV) is a mosquito-borne global health threat that has been transmitted transiently in the southeastern United States. A primary CHIKV mosquito vector, Aedes aegypti, was recently established in the populous state of California, but the vector competence of Californian mosquitoes is unknown. Explosive CHIKV epidemics since 2004 have been associated with the acquisition of mosquito-adaptive mutations that enhance transmission by Ae. aegypti or Ae. albopictus. As a highly mutable RNA virus, CHIKV has the potential for extensive and rapid genetic diversification in vertebrate hosts and mosquito vectors. We previously demonstrated that expansion of CHIKV diversity in cell culture allows for greater adaptability to novel selection pressures, and that CHIKV fidelity variants are able to diversify more than wildtype (WT) CHIKV in mice. The evolution of intra-vector CHIKV populations and the correlation between CHIKV population diversity and infectivity and transmissibility in mosquitoes has not yet been studied. Here, we address these gaps in knowledge via experimental infection of Ae. aegypti from California with WT and fidelity variant CHIKV. We show that Ae. aegypti from California are highly competent vectors for CHIKV. We also report that CHIKV fidelity variants diversify more than WT in mosquitoes and exhibit attenuated infectivity at the level of the midgut. Furthermore, we demonstrate that intra-vector populations of CHIKV are subjected to purifying selection in mosquito bodies, and sequences of non-coding CHIKV regions are highly conserved. These findings will inform public health risk assessment for CHIKV in California and improve our understanding of constraints to CHIKV evolution in mosquitoes

Infecting (bodies), disseminating (legs+wings) and transmitted (saliva) ZIKV RNA levels 14 or 15 days after <i>Ae</i>. <i>aegypti</i> orally ingested one of three Asian lineage ZIKV strains.

<p>Each dot represents the mean log₁₀ ZIKV genome copies per tissue or saliva sample from an individual <i>Ae</i>. <i>aegypti</i>. <i>Ae</i>. <i>aegypti</i> from Los Angeles, California, USA, were fed on viremic mice infected with ZIKV from Malaysia 1966 (MA66), Puerto Rico 2015 (PR15) or Brazil 2015 (BR15). Mosquitoes exposed to BR15 were assayed 15 dpf, MA66 and PR15 cohorts were assayed 14 dpf. Each dot represents the mean of two or more RT-qPCR technical replicates. The dashed lines represent the limits of detection (LOD). Dots below dashed line represent tested samples with an undetectable Ct or a Ct value of >38. The LOD for saliva was lower than for tissues because RNA was extracted from a higher proportion of the saliva sample volume. Asterisks show significant differences in means across groups of the same tissue type, <i>P</i><0.01, ANOVA, Tukey post-hoc test. No asterisk indicates no significant difference across groups.</p

Individual mosquito ZIKV RNA levels in body, legs+wings, and saliva of <i>Aedes aegypti</i> from Los Angeles, CA, USA.

<p><i>Ae</i>. <i>aegypti</i> from Los Angeles ingested blood from viremic ZIKV-infected interferon receptor deficient mice that had been inoculated with different ZIKV strains. Each colored box represents an individual mosquito sample showing the magnitude of ZIKV RNA detected in each body (B), legs+wings (LW), and saliva (S). The red to blue color scale shows high (red) to low (blue) ZIKV RNA levels. Samples with no detectable ZIKV RNA are colored white. ZIKV BR15-infected mosquitoes were not tested 21 dpf.</p

ZIKV infection, dissemination, and transmission rates in California <i>Aedes</i> and <i>Culex</i> mosquitoes 14 and 21 days post ingestion of blood from viremic mice.

<p>ZIKV infection, dissemination, and transmission rates in California <i>Aedes</i> and <i>Culex</i> mosquitoes 14 and 21 days post ingestion of blood from viremic mice.</p

Density distributions of unvaccinated and vaccinated specimen collection dates by day since symptom onset.

Day 0 on the x-axis denotes self-reported day of symptom onset. Negative values for days indicate specimen collection prior to symptom onset. Symptom onset data were available for n = 6,871 unvaccinated cases and n = 5,522 vaccinated cases. Two-sided K-S test: p = 0.0012; median days since symptom onset were 2.4 for both unvaccinated and vaccinated cases. (TIF)</p

Vaccinated vs. Unvaccinated.

Vaccinated vs. Unvaccinated.</p

Concordance between self-reported vaccination status and records in public health vaccine registries.

Individuals were considered fully vaccinated based on vaccine registry (WIR/WEDSS) data if the registries indicated receipt of a final vaccine dose at least 14 days prior to submitting the sample used in our analysis. For individuals whose vaccination status could not be verified in the registry, self-reported data collected at the time of testing were used. Individuals were considered unvaccinated based on self-report only if there was an explicit declaration of unvaccinated status in the self-reported data. Individuals were considered fully vaccinated based on self-report if they fulfilled all of the following criteria: (1) indicated that they had received a COVID vaccine prior to testing; (2) indicated that they did not require another vaccine dose; and (3) reported a date of last vaccine dose that was at least 14 days prior to testing. Specimens lacking data on vaccination status were excluded from the study. Specimens from partially vaccinated individuals (incomplete vaccine series, or A. Of 20,431 specimens with vaccination status available from at least one source, 5,078 specimens had data available from both sources. Under-reporting of full vaccination status in self-reports 1,064/6,142 or 17%) was more common than over-reporting (409/5,487 or 7.4%). B. N1 Ct values for SARS-CoV-2-positive specimens grouped by vaccination status for individuals whose vaccination status was determined by vaccine registry or by self-reported data. (TIF)</p

Infectious SARS-CoV-2 detected in the majority of fully vaccinated individuals with low Ct values.

Infectiousness was determined for a subset of N1 Ct-matched specimens with Ct (TIF)</p