Chikungunya Virus Replication in Salivary Glands of the Mosquito Aedes albopictus

Chikungunya virus (CHIKV) is an emerging arbovirus transmitted to humans by mosquitoes such as Aedes albopictus. To be transmitted, CHIKV must replicate in the mosquito midgut, then disseminate in the hemocele and infect the salivary glands before being released in saliva. We have developed a standardized protocol to visualize viral particles in the mosquito salivary glands using transmission electron microscopy. Here we provide direct evidence for CHIKV replication and storage in Ae. albopictus salivary glands.     

Multiple Salivary Proteins from Aedes aegypti Mosquito Bind to the Zika Virus Envelope Protein

Aedes aegypti mosquitoes are important vectors of several debilitating and deadly arthropod-borne (arbo) viruses, including Yellow Fever virus, Dengue virus, West Nile virus and Zika virus (ZIKV). Arbovirus transmission occurs when an infected mosquito probes the host’s skin in search of a blood meal. Salivary proteins from mosquitoes help to acquire blood and have also been shown to enhance pathogen transmission in vivo and in vitro. Here, we evaluated the interaction of mosquito salivary proteins with ZIKV by surface plasmon resonance and enzyme-linked immunosorbent assay. We found that three salivary proteins AAEL000793, AAEL007420, and AAEL006347 bind to the envelope protein of ZIKV with nanomolar affinities. Similar results were obtained using virus-like particles in binding assays. These interactions have no effect on viral replication in cultured endothelial cells and keratinocytes. Additionally, we found detectable antibody levels in ZIKV and DENV serum samples against the recombinant proteins that interact with ZIKV. These results highlight complex interactions between viruses, salivary proteins and antibodies that could be present during viral transmissions.     

实验感染登革热病毒的蚊虫涎腺荧光抗体染色检查

本文通过对接种感染和经口感染登革热病毒的白纹伊蚊的头压片和涎腺荧光抗体染色的结果比较,选定以涎腺阳性率作为比较不同蚊种和蚊株媒介效能的一个指标。其优点为阳性率高,荧光亮度强,结果清晰易判断,无非特异荧光。采用冻麻状态解剖后,1人1小时可解剖20～30只蚊虫的涎腺,成功率在95％以上。     

Evaluation of Aedes aegypti,Aedes albopictus,and Culex quinquefasciatus Mosquitoes Competence to Oropouche virus Infection

The emergence of new human viral pathogens and re-emergence of several diseases are of particular concern in the last decades. Oropouche orthobunyavirus (OROV) is an arbovirus endemic to South and Central America tropical regions, responsible to several epidemic events in the last decades. There is little information regarding the ability of OROV to be transmitted by urban/peri-urban mosquitoes, which has limited the predictability of the emergence of permanent urban transmission cycles. Here, we evaluated the ability of OROV to infect, replicate, and be transmitted by three anthropophilic and urban species of mosquitoes, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus. We show that OROV is able to infect and efficiently replicate when systemically injected in all three species tested, but not when orally ingested. Moreover, we find that, once OROV replication has occurred in the mosquito body, all three species were able to transmit the virus to immunocompromised mice during blood feeding. These data provide evidence that OROV is restricted by the midgut barrier of three major urban mosquito species, but, if this restriction is overcome, could be efficiently transmitted to vertebrate hosts. This poses a great risk for the emergence of permanent urban cycles and geographic expansion of OROV to other continents.     

Investigation of Biological Factors Contributing to Individual Variation in Viral Titer after Oral Infection of Aedes aegypti Mosquitoes by Sindbis Virus

The mechanisms involved in determining arbovirus vector competence, or the ability of an arbovirus to infect and be transmitted by an arthropod vector, are still incompletely understood. It is well known that vector competence for a particular arbovirus can vary widely among different populations of a mosquito species, which is generally attributed to genetic differences between populations. What is less understood is the considerable variability (up to several logs) that is routinely observed in the virus titer between individual mosquitoes in a single experiment, even in mosquitoes from highly inbred lines. This extreme degree of variation in the virus titer between individual mosquitoes has been largely ignored in past studies. We investigated which biological factors can affect titer variation between individual mosquitoes of a laboratory strain of Aedes aegypti, the Orlando strain, after Sindbis virus infection. Greater titer variation was observed after oral versus intrathoracic infection, suggesting that the midgut barrier contributes to titer variability. Among the other factors tested, only the length of the incubation period affected the degree of titer variability, while virus strain, mosquito strain, mosquito age, mosquito weight, amount of blood ingested, and virus concentration in the blood meal had no discernible effect. We also observed differences in culture adaptability and in the ability to orally infect mosquitoes between virus populations obtained from low and high titer mosquitoes, suggesting that founder effects may affect the virus titer in individual mosquitoes, although other explanations also remain possible.     

Genetic Adaptation by Dengue Virus Serotype 2 to Enhance Infection of Aedes aegypti Mosquito Midguts

Dengue viruses (DENVs), serotypes 1–4, are arthropod-borne viruses transmitted to humans by mosquitoes, primarily Aedes aegypti. The transmission cycle begins when Ae. aegypti ingest blood from a viremic human and the virus infects midgut epithelial cells. In studying viruses derived from the DENV2 infectious clone 30P-NBX, we found that when the virus was delivered to female Ae. aegypti in an infectious blood meal, the midgut infection rate (MIR) was very low. To determine if adaptive mutations in the DENV2 envelope (E) glycoprotein could be induced to increase the MIR, we serially passed 30P-NBX in Ae. aegypti midguts. After four passages, a single, non-conservative mutation in E protein domain II (DII) nucleotide position 1300 became dominant, resulting in replacement of positively-charged amino acid lysine (K) at position 122 with negatively-charged glutamic acid (E; K122E) and a significantly-enhanced MIR. Site directed mutagenesis experiments showed that reducing the positive charge of this surface-exposed region of the E protein DII correlated with improved Ae. aegypti midgut infection.     

白纹伊蚊经实验感染后涎腺中基孔肯雅病毒和乙型脑炎病毒抗原检测

用基孔肯雅和乙型脑炎病毒经口感染白纹伊蚊，采用免疫荧光试验对雌蚊涎腺作病毒抗原检查，结果于感染后第８￣１４天基孔肯雅的阳性为５０％￣９１．６７％，乙型脑炎为７０％￣８０％。试验认为，白纹伊蚊在感染后第６￣８天即可传播基孔肯雅和乙型脑炎病毒。     

High-Throughput Method for Detection of Arbovirus Infection of Saliva in Mosquitoes Aedes aegypti and Ae. albopictus

Vector competence refers to the ability of a vector to acquire, maintain, and transmit a pathogen. Collecting mosquito saliva in medium-filled capillary tubes has become the standard for approximating arbovirus transmission. However, this method is time-consuming and labor-intensive. Here we compare the capillary tube method to an alternative high-throughput detection method the collection of saliva on paper cards saturated with honey, with (FTA card) and without (filter paper) reagents for the preservation of nucleic acid for Aedes aegypti and Aedes albopictus mosquitoes infected with two emerging genotypes of the chikungunya virus (CHIKV). Model results showed that the Asian genotype CHIKV dissemination in the harvested legs of both Ae. aegypti and Ae. albopictus increased the odds of females having a positive salivary infection and higher salivary viral titers, while for the IOL genotype the same effect was observed only for Ae. aegypti. Of the three tested detection methods, the FTA card was significantly more effective at detecting infected saliva of Ae. aegypti and Ae. albopictus females than the capillary tube and filter paper was as effective as the capillary tube for the Asian genotype. We did not find significant effects of the detection method in detecting higher viral titer for both Asian and IOL genotypes. Our results are discussed in light of the limitations of the different tested detection methods.     

The Effect of Temperature on Wolbachia-Mediated Dengue Virus Blocking in Aedes aegypti

Dengue fever, caused by dengue virus (DENV), is endemic in more than 100 countries. The lack of effective treatment of patients and the suboptimal efficacies of the tetravalent vaccine in trials highlight the urgent need to develop alternative strategies to lessen the burden of dengue fever. Wolbachia pipientis, an obligate intracellular bacterium, is being developed as a biocontrol strategy against dengue because it limits the replication of the DENV in the mosquito vector, Aedes aegypti. However, several recent studies have demonstrated the sensitivity of pathogens, vectors, and their symbionts to temperature. To understand how the tripartite interactions between the mosquito, DENV, and Wolbachia may change under different temperature regimes, we assessed the vector competence and transmission potential of DENV-infected mosquitoes reared at a common laboratory setting of a constant 25°C and at two diurnal temperature settings with mean of 25°C and 28°C and a fluctuating range of 8°C (±4°C). Temperature significantly affected DENV infection rate in the mosquitoes. Furthermore, temperature significantly influenced the proportion of mosquitoes that achieved transmission potential as measured by the presence of virus in the saliva. Regardless of the temperature regimes, Wolbachia significantly and efficiently reduced the proportion of mosquitoes achieving infection and transmission potential across all the temperature regimes studied. This work reinforces the robustness of the Wolbachia biocontrol strategy to field conditions in Cairns, Australia, and suggests that similar studies are required for local mosquito genotypes and field relevant temperatures for emerging field release sites globally.     

Expressing the Pro-Apoptotic Reaper Protein via Insertion into the Structural Open Reading Frame of Sindbis Virus Reduces the Ability to Infect Aedes aegypti Mosquitoes

Arboviruses continue to threaten a significant portion of the human population, and a better understanding is needed of the determinants of successful arbovirus infection of arthropod vectors. Avoiding apoptosis has been shown to be one such determinant. Previous work showed that a Sindbis virus (SINV) construct called MRE/rpr that expresses the Drosophila pro-apoptotic protein Reaper via a duplicated subgenomic promoter had a reduced ability to orally infect Aedes aegypti mosquitoes at 3 days post-blood meal (PBM), but this difference diminished over time as virus variants containing deletions in the inserted reaper gene rapidly predominated. In order to further clarify the effect of midgut apoptosis on disseminated infection in Ae. aegypti, we constructed MRE/rprORF, a version of SINV containing reaper inserted into the structural open reading frame (ORF) as an in-frame fusion. MRE/rprORF successfully expressed Reaper, replicated similarly to MRE/rpr in cell lines, induced apoptosis in cultured cells, and caused increased effector caspase activity in mosquito midgut tissue. Mosquitoes that fed on blood containing MRE/rprORF developed significantly less midgut and disseminated infection when compared to MRE/rpr or a control virus up to at least 7 days PBM, when less than 50% of mosquitoes that ingested MRE/rprORF had detectable disseminated infection, compared with around 80% or more of mosquitoes fed with MRE/rpr or control virus. However, virus titer in the minority of mosquitoes that became infected with MRE/rprORF was not significantly different from control virus. Deep sequencing of virus populations from ten mosquitoes infected with MRE/rprORF indicated that the reaper insert was stable, with only a small number of point mutations and no deletions being observed at frequencies greater than 1%. Our results indicate that expression of Reaper by this method significantly reduces infection prevalence, but if infection is established then Reaper expression has limited ability to continue to suppress replication.     

The Effect of Fluctuating Incubation Temperatures on West Nile Virus Infection in Culex Mosquitoes

Temperature plays a significant role in the vector competence, extrinsic incubation period, and intensity of infection of arboviruses within mosquito vectors. Most laboratory infection studies use static incubation temperatures that may not accurately reflect daily temperature ranges (DTR) to which mosquitoes are exposed. This could potentially compromise the application of results to real world scenarios. We evaluated the effect of fluctuating DTR versus static temperature treatments on the infection, dissemination, and transmission rates and viral titers of Culex tarsalis and Culex quinquefasciatus mosquitoes for West Nile virus. Two DTR regimens were tested including an 11 and 15 °C range, both fluctuating around an average temperature of 28 °C. Overall, no significant differences were found between DTR and static treatments for infection, dissemination, or transmission rates for either species. However, significant treatment differences were identified for both Cx. tarsalis and Cx. quinquefasciatus viral titers. These effects were species-specific and most prominent later in the infection. These results indicate that future studies on WNV infections in Culex mosquitoes should consider employing realistic DTRs to reflect interactions most accurately between the virus, vector, and environment.     

Brazilian Populations of Aedes aegypti Resistant to Pyriproxyfen Exhibit Lower Susceptibility to Infection with Zika Virus

Zika virus (ZIKV) infection has caused devastating consequences in Brazil as infections were associated with neurological complications in neonates. Aedes aegypti is the primary vector of ZIKV, and the evolution of insecticide resistance (IR) in this species can compromise control efforts. Although relative levels of phenotypic IR in mosquitoes can change considerably over time, its influence on vector competence for arboviruses is unclear. Pyriproxyfen (PPF)-resistant populations of Ae. aegypti were collected from five municipalities located in Northeast of Brazil, which demonstrated different resistance levels; low (Serrinha, Brumado), moderate (Juazeiro do Norte, Itabuna), and high (Quixadá). Experimental per os infection using ZIKV were performed with individuals from these populations and with an insecticide susceptible strain (Rockefeller) to determine their relative vector competence for ZIKV. Although all populations were competent to transmit ZIKV, mosquitoes derived from populations with moderate to high levels of IR exhibited similar or lower susceptibility to ZIKV infection than those from populations with low IR or the susceptible strain. These observations suggest an association between IR and arbovirus infection, which may be attributable to genetic hitchhiking. The use of PPF to control Brazilian Ae. aegypti may be associated with an indirect benefit of reduced susceptibility to infection, but no changes in disseminated infection and transmission of ZIKV among PPF-resistant phenotypes.     

Effect of temperature on the vector efficiency of Aedes aegypti for dengue 2 virus

The effect of temperature on the ability of Aedes aegypti to transmit dengue (DEN) 2 virus to rhesus monkeys was assessed as a possible explanation for the seasonal variation in the incidence of dengue hemorrhagic fever in Bangkok, Thailand. In two laboratory experiments, a Bangkok strain of Ae. aegypti was allowed to feed upon viremic monkeys infected with DEN-2 virus. Blood-engorged mosquitoes were separated into two groups and retained at constant temperatures. Virus infection and transmission rates were determined for Ae. aegypti at intervals ranging from 4 to 7 days during a 25-day incubation period. Results of the first experiment for mosquitoes infected with a low dose of DEN-2 virus and maintained at 20, 24, 26, and 30 degrees C, indicated that the infection rate ranged from 25% to 75% depending on the incubation period. However, DEN-2 virus was transmitted to monkeys only by Ae. aegypti retained at 30 degrees C for 25 days. In the second experiment, the infection rate for Ae. aegypti that ingested a higher viral dose, and incubated at 26, 30, 32, and 35 degrees C ranged from 67% to 95%. DEN-2 virus was transmitted to monkeys only by mosquitoes maintained at greater than or equal to 30 degrees C. The extrinsic incubation period was 12 days for mosquitoes at 30 degrees C, and was reduced to 7 days for mosquitoes incubated at 32 degrees C and 35 degrees C. These results imply that temperature-induced variations in the vector efficiency of Ae. aegypti may be a significant determinant in the annual cyclic pattern of dengue hemorrhagic fever epidemics in Bangkok.     

Temperature-Mediated Effects on Mayaro Virus Vector Competency of Florida Aedes aegypti Mosquito Vectors

Mayaro virus (MAYV) is an emerging mosquito-borne arbovirus and public health concern. We evaluated the influence of temperature on Aedes aegypti responses to MAYV oral infection and transmission at two constant temperatures (20 °C and 30 °C). Infection of mosquito tissues (bodies and legs) and salivary secretions with MAYV was determined at 3, 9, 15, 21, and 27 days post ingestion. At both temperatures, we observed a trend of increase in progression of MAYV infection and replication kinetics over time, followed by a decline during later periods. Peaks of MAYV infection, titer, and dissemination from the midgut were detected at 15 and 21 days post ingestion at 30 °C and 20 °C, respectively. Mosquitoes were able to transmit MAYV as early as day 3 at 30 °C, but MAYV was not detectable in salivary secretions until day 15 at 20 °C. Low rates of MAYV in salivary secretions collected from infected mosquitoes provided evidence supporting the notion that a substantial salivary gland barrier(s) in Florida Ae. aegypti can limit the risk of MAYV transmission. Our results provide insights into the effects of temperature and time on the progression of infection and replication of MAYV in Ae. aegypti vectors.     

Sindbis Virus Infection in Non-Blood-Fed Hibernating Culex pipiens Mosquitoes in Sweden

A crucial, but unresolved question concerning mosquito-borne virus transmission is how these viruses can remain endemic in regions where the transmission is halted for long periods of time, due to mosquito inactivity in, e.g., winter. In northern Europe, Sindbis virus (SINV) (genus alphavirus, Togaviridae) is transmitted among birds by Culex mosquitoes during the summer, with occasional symptomatic infections occurring in humans. In winter 2018–19, we sampled hibernating Culex spp females in a SINV endemic region in Sweden and assessed them individually for SINV infection status, blood-feeding status, and species. The results showed that 35 out of the 767 collected mosquitoes were infected by SINV, i.e., an infection rate of 4.6%. The vast majority of the collected mosquitoes had not previously blood-fed (98.4%) and were of the species Cx. pipiens (99.5%). This is the first study of SINV overwintering, and it concludes that SINV can be commonly found in the hibernating Cx. pipiens population in an endemic region in Sweden, and that these mosquitoes become infected through other means besides blood-feeding. Further studies on mosquito ecology and viral interactions are needed to elucidate the mechanisms of the persistence of these viruses over winter.     

Neotropical Sylvatic Mosquitoes and Aedes aegypti Are Not Competent to Transmit 17DD Attenuated Yellow Fever Virus from Vaccinated Viremic New World Non-Human Primates

Beside humans, thousands of non-human primates (NHPs) died during the recent outbreak caused by the yellow fever virus (YFV) in Brazil. Vaccination of NHPs against YFV with the YF 17DD attenuated virus has emerged as a public health strategy, as it would reduce sylvatic transmission while also preserving endangered susceptible species. The hypothesis of establishing an uncontrolled transmission of this attenuated virus in nature was raised. We assessed vector competence of four sylvatic mosquito species, Haemagogus leucocelaenus, Haemagogus janthinomys/capricornii, Sabethes albiprivus, and Sabethes identicus, as well as the urban vector Aedes aegypti for YF 17DD attenuated vaccine virus when fed directly on eleven viremic lion tamarins or artificially challenged with the same virus. No infection was detected in 689 mosquitoes engorged on viremic lion tamarins whose viremia ranged from 1.05 × 10³ to 6.61 × 10³ FFU/mL, nor in those artificially taking ≤ 1 × 10³ PFU/mL. Low viremia presented by YF 17DD-vaccinated New World NHPs combined with the low capacity and null dissemination ability in sylvatic and domestic mosquitoes of this attenuated virus suggest no risk of its transmission in nature. Thus, vaccination of captive and free-living NHPs against YFV is a safe public health strategy.     

Body Size and Wing Shape Measurements as Quality Indicators of Aedes aegypti Mosquitoes Destined for Field Release

There is increasing interest in rearing modified mosquitoes for mass release to control vector-borne diseases, particularly Wolbachia-infected Aedes aegypti for suppression of dengue. Successful introductions require release of high quality mosquitoes into natural populations. Potential indicators of quality are body size and shape. We tested to determine if size, wing/thorax ratio, and wing shape are associated with field fitness of Wolbachia-infected Ae. aegypti. Compared with field-collected mosquitoes, released mosquitoes were larger in size, with lower size variance and different wing shape but similar in wing-thorax ratio and its associated variance. These differences were largely attributed to nutrition and to a minor extent to wMel Wolbachia infection. Survival potential of released female mosquitoes was similar to those from the field. Females at oviposition sites tended to be larger than those randomly collected from BG-Sentinel traps. Rearing conditions should thus aim for large size without affecting wing/thorax ratios.