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.     

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.     

Distribution of Mosquito Larvae on Kosrae Island,Kosrae State,the Federated States of Micronesia

Surveys of mosquito larvae were carried out in six areas of Kosrae Island, Kosrae State, the Federated States of Micronesia in December 2009 and June 2012. A total of 962 larvae of six species were collected from 106 natural and artificial habitats. They were identified as Aedes aegypti, Ae. albopictus, Ae. marshallensis, Culex quinquefasciatus, Cx. annulirostris, and Cx. kusaiensis. This is the first report from Kosrae Island for three of these species—Ae. marshallensis, Cx. quinquefasciatus, and Cx. annulirostris. The most abundant species was Ae. albopictus, followed by Ae. marshallensis, and these two species were found in all areas. Relatively large numbers of Cx. quinquefasciatus and Cx. kusaiensis were found in five areas. Fewer Cx. annulirostris were found, and only in three areas. Aedes aegypti larvae were collected from a single habitat at Tafunsak in 2009. To prevent the outbreak of dengue fever, environmental management should focus on the destruction, alteration, disposal and recycling of containers that produce larger numbers of adult Aedes mosquitoes.     

Detection of Persistent Chikungunya Virus RNA but not Infectious Virus in Experimental Vertical Transmission in Aedes aegypti from Malaysia

Vertical transmission may contribute to the maintenance of arthropod-borne viruses, but its existence in chikungunya virus (CHIKV) is unclear. Experimental vertical transmission of infectious clones of CHIKV in Aedes aegypti mosquitoes from Malaysia was investigated. Eggs and adult progeny from the second gonotrophic cycles of infected parental mosquitoes were tested. Using polymerase chain reaction (PCR), 56.3% of pooled eggs and 10% of adult progeny had detectable CHIKV RNA, but no samples had detectable infectious virus by plaque assay. Transfected CHIKV RNA from PCR-positive eggs did not yield infectious virus in BHK-21 cells. Thus, vertical transmission of viable CHIKV was not demonstrated. Noninfectious CHIKV RNA persists in eggs and progeny of infected Ae. aegypti, but the mechanism and significance are unknown. There is insufficient evidence to conclude that vertical transmission exists in CHIKV, as positive results reported in previous studies were almost exclusively based only on viral RNA detection.     

Ovicidal activity of three insect growth regulators against Aedes and Culex mosquitoes

Interspecific variations in the susceptibility of freshly and embryonated eggs of Aedes albopictus, Ae. aegypti, Ae. atropalpus and Culex pipiens were tested against three classes of insect growth regulators (IGRs) including ecdysone agonist (azadirachtin), chitin synthesis inhibitor (diflubenzuron) and juvenile hormone analog (pyriproxyfen) at 0.001, 0.01, 0.1 and 1.0 ppm concentrations. Egg hatching inhibition was dose dependent, the highest being at 1.0 ppm concentration for freshly laid eggs of Ae. albopictus (pyriproxyfen: 80.6%, azadirachtin: 42.9% and diflubenzuron: 35.8%). Aedes aegypti showed lower egg hatching inhibition when exposed to pyriproxyfen (47.3%), azadirachtin (15.7%) and diflubenzuron (25.5%). Freshly laid eggs of Cx. pipiens were most susceptible to diflubenzuron. Aedes atropalpus eggs were tolerant to all three classes of IGRs. Embryonated eggs of Ae. albopictus, Ae. aegypti, Ae. atropalpus and Cx. pipiens were resistant to pyriproxyfen, azadirachtin and diflubenzuron than freshly laid eggs. The median desiccation time (DT₅₀) of Ae. atropalpus eggs was maximum (5.1 h) as compared to Ae. aegypti (4.9 h), Ae. albopictus (3.9 h) or Cx. pipiens (1.7 h) eggs. Insignificant relationship between the rates of desiccation and egg hatching inhibition suggests other factors than physical providing eggs the ability to tolerate exposures to various IGRs. Egg hatching inhibition was due to the alteration in embryonic development caused by IGRs. Changes in the egg shell morphology and abnormal egg hatching from the side of the egg wall instead of operculum, was observed at higher concentrations of diflubenzuron. Morphological and physiological variations in eggs may be the key factor to influence the ovicidal efficacy of IGRs. The present data provide a base line for the improvement of the ovicidal efficacy of the insecticide and its formulation.     

Imperviousness as a predictor for infestation levels of container-breeding mosquitoes in a focus of dengue and Saint Louis encephalitis in Argentina

Dengue and Saint Louis encephalitis virus are among the most important emerging viruses transmitted by mosquitoes at the global scale, and from 2009 onward both diseases have reached temperate Argentina. To test whether the urbanization level can be used as a predictor for the infestation levels of container-breeding mosquito vectors, we searched for Aedes aegypti and Culex pipiens in 8400 water-filled containers from 14 cemeteries of Buenos Aires Province and we used generalized linear models to relate positive containers with the impervious area quantified inside (internal PIA) and outside (external PIA) cemeteries. The best model for Ae. aegypti explained 91% of the variability and included the season, the internal PIA and the external PIA at 1 km as a quadratic function, showing a parabolic response peaking in ∼75%. Regarding the infestation levels of Cx. pipiens, the final model explained 75% of the variability and included only the season. In view of these results, the percentage of impervious area efficiently predicted the infestation levels of Ae. aegypti but not of Cx. pipiens. Considering the worldwide relevance of the former in dengue transmission, the simple quantification of imperviousness proposed herein provides a helpful basis for vector surveillance and control in urbanized areas.     

Chikungunya Virus Infection

Chikungunya virus (CHIKV) is an alphavirus transmitted by mosquitoes, mostly Aedes aegypti and Aedes albopictus. After half a century of focal outbreaks of acute febrile polyarthralgia in Africa and Asia, the disease unexpectedly spread in the past decade with large outbreaks in Africa and around the Indian Ocean and rare autochthonous transmission in temperate areas. This emergence brought new insights on its pathogenesis, notably the role of the A226V mutation that improved CHIKV fitness in Ae. albopictus and the possible CHIKV persistence in deep tissue sanctuaries for months after infection. Massive outbreaks also revealed new aspects of the acute stage: the high number of symptomatic cases, unexpected complications, mother-to-child transmission, and low lethality in debilitated patients. The follow-up of patients in epidemic areas has identified frequent, long-lasting, rheumatic disorders, including rare inflammatory joint destruction, and common chronic mood changes associated with quality-of-life impairment. Thus, the globalization of CHIKV exposes countries with Aedes mosquitoes both to brutal outbreaks of acute incapacitating episodes and endemic long-lasting disorders.     

Vector Competence of the Invasive Mosquito Species Aedes koreicus for Arboviruses and Interference with a Novel Insect Specific Virus

The global spread of invasive mosquito species increases arbovirus infections. In addition to the invasive species Aedes albopictus and Aedes japonicus, Aedes koreicus has spread within Central Europe. Extensive information on its vector competence is missing. Ae. koreicus from Germany were investigated for their vector competence for chikungunya virus (CHIKV), Zika virus (ZIKV) and West Nile virus (WNV). Experiments were performed under different climate conditions (27 ± 5 °C; 24 ± 5 °C) for fourteen days. Ae. koreicus had the potential to transmit CHIKV and ZIKV but not WNV. Transmission was exclusively observed at the higher temperature, and transmission efficiency was rather low, at 4.6% (CHIKV) or 4.7% (ZIKV). Using a whole virome analysis, a novel mosquito-associated virus, designated Wiesbaden virus (WBDV), was identified in Ae. koreicus. Linking the WBDV infection status of single specimens to their transmission capability for the arboviruses revealed no influence on ZIKV transmission. In contrast, a coinfection of WBDV and CHIKV likely has a boost effect on CHIKV transmission. Due to its current distribution, the risk of arbovirus transmission by Ae. koreicus in Europe is rather low but might gain importance, especially in regions with higher temperatures. The impact of WBDV on arbovirus transmission should be analyzed in more detail.     

Avian Communal Roosts as Amplification Foci for West Nile Virus in Urban Areas in Northeastern United States

West Nile virus (WNV) perpetuates in an enzootic transmission cycle involving Culex mosquitoes and virus-competent avian hosts. In the northeastern United States, the enzootic vectors, Cx. pipiens and Cx. restuans, feed preferentially on American robins (Turdus migratorius), suggesting a key role for this bird species in the WNV transmission cycle. We examined the role of American robin communal roosts as virus amplification foci in greater New Haven, Connecticut. Robin communal roosts were located by radio tracking. After mid-August, when most robins were using the roosts, Cx. pipiens and Cx. restuans fed often on robins and were significantly more infected with WNV at communal roosts than at non-roosting sites. We also identified 6.4% human-derived blood meals in Aedes vexans in communal roosts. Our results indicate that communal roosts act as late-season amplification foci facilitating transmission to humans because of high infection rates, high abundance, and feeding patterns of enzootic and bridge vectors.     

First dengue virus detection in Aedes albopictus from Delhi, India: its breeding ecology and role in dengue transmission

Objective To report dengue virus and its disease transmission in Aedes albopictus in the National Capital Territory of Delhi, India. Methods Monthly Aedes surveys were carried out in 126 urban localities of Delhi in 2008 and 2009. Pools of all three species of Aedes mosquitoes were tested for Dengue virus (DENV) using an antigen‐capture enzyme‐linked immunosorbent assay. Results Aedes aegypti was the most prevalent species, breeding throughout the year. Aedes albopictus was found in 9.52% of surveyed localities including the central urban part of Delhi, in March and from August to October. Aedes albopictus and Aedes vittatus are adapting to breed in manmade containers in the urban areas of Delhi in addition to their natural habitats of bamboo bushes and rock pits. Of the 229 pools of Ae. aegypti and 34 pools of Ae. albopictus tested, 10.5% and 11.76% were positive for dengue virus, respectively. No dengue virus infection was recorded in Ae. vittatus. Conclusion This is the first report of dengue virus in Ae. albopictus from north India. Because DENV was detected in Ae. albopictus, which adapted to manmade containers, both its spread and transmission dynamics should be checked.     

Larvicidal and repellent activity of tetradecanoic acid against Aedes aegypti (Linn.) and Culex quinquefasciatus (Say.) (Diptera:Culicidae)

ObjectiveTo investigate the larvicidal and repellent efficacy of tetradecanoic acid against Aedes aegypti (Ae. aegypti) L. and Culex quinquefasciatus (Cx. quinquefasciatus) Say (Diptera: Culicidae).MethodsLarvicidal efficacy of tetradecanoic acid was tested at various concentrations against the early third instar larvae of Ae. aegypti and Cx. quinquefasciatus. The repellent activity was determined against two mosquito species at three concentrations viz., 1.0,2.5 and 5.0 ppm under the laboratory conditions.ResultsThe tetradecanoic acid was found to be more effective against Cx. quinquefasciatus than Ae. aegypti larvae. The LC₅₀ values were 14.08 ppm and 25.10 ppm, respectively. Tetradecanoic acid showed lesser repellency against Ae. aegypti and Cx. quinquefasciatus. The highest repellency was observed in higher concentration of 5.0 mg/cm² provided 100% protection up to 60 and 90 min against Ae. aegypti and Cx. quinquefasciatus respectively.ConclusionsFrom the results it can be concluded the tetradecanoic acid is a potential for controlling Cx. quinquefasciatus and Ae. aegypti mosquitoes.     

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.     

The History of Dengue Outbreaks in the Americas

Dengue is a viral disease usually transmitted by Aedes aegypti mosquitoes. Dengue outbreaks in the Americas reported in medical literature and to the Pan American Health Organization are described. The outbreak history from 1600 to 2010 was categorized into four phases: Introduction of dengue in the Americas (1600–1946); Continental plan for the eradication of the Ae. aegypti (1947–1970) marked by a successful eradication of the mosquito in 18 continental countries by 1962; Ae. aegypti reinfestation (1971–1999) caused by the failure of the mosquito eradication program; Increased dispersion of Ae. aegypti and dengue virus circulation (2000–2010) characterized by a marked increase in the number of outbreaks. During 2010 > 1.7 million dengue cases were reported, with 50,235 severe cases and 1,185 deaths. A dramatic increase in the number of outbreaks has been reported in recent years. Urgent global action is needed to avoid further disease spread.     

Low Aedes aegypti Vector Competence for Zika Virus from Viremic Rhesus Macaques

Despite worldwide efforts to understand the transmission dynamics of Zika virus (ZIKV), scanty evaluation has been made on the vector competence of Aedes aegypti fed directly on viremic human and non-human primates (NHPs). We blood-fed Ae. aegypti from two districts in Rio de Janeiro on six ZIKV infected pregnant rhesus macaques at several time points, half of which were treated with Sofosbuvir (SOF). Mosquitoes were analyzed for vector competence after 3, 7 and 14 days of incubation. Although viremia extended up to eight days post monkey inoculation, only mosquitoes fed on the day of the peak of viremia, recorded on day two, became infected. The influence of SOF treatment could not be assessed because the drug was administered just after mosquito feeding on day two. The global infection, dissemination and transmission rates were quite low (4.09%, 1.91% and 0.54%, respectively); no mosquito was infected when viremia was below 1.26 × 10⁵ RNA copies/mL. In conclusion, Ae. aegypti vector competence for ZIKV from macaques is low, likely to be due to low viral load and the short duration of ZIKV viremia in primates suitable for infecting susceptible mosquitoes. If ZIKV infection in human and macaques behaves similarly, transmission of the Zika virus in nature is most strongly affected by vector density.     

Incrimination of Aedes (Stegomyia) hensilli Farner as an Epidemic Vector of Chikungunya Virus on Yap Island,Federated States of Micronesia, 2013

Two species of Aedes (Stegomyia) were collected in response to the first chikungunya virus (CHIKV) outbreak on Yap Island: the native species Ae. hensilli Farner and the introduced species Ae. aegypti (L.). Fourteen CHIKV-positive mosquito pools were detected. Six pools were composed of female Ae. hensilli, six pools were composed of female Ae. aegypti, one pool was composed of male Ae. hensilli, and one pool contained female specimens identified as Ae. (Stg.) spp. Infection rates were not significantly different between female Ae. hensilli and Ae. aegypti. The occurrence of human cases in all areas of Yap Island and the greater number of sites that yielded virus from Ae. hensilli combined with the ubiquitous distribution of this species incriminate Ae. hensilli as the most important vector of CHIKV during the outbreak. Phylogenic analysis shows that virus strains on Yap are members of the Asia lineage and closely related to strains currently circulating in the Caribbean.     

Wolbachia Do Not Induce Reactive Oxygen Species-Dependent Immune Pathway Activation in Aedes albopictus

Aedes albopictus is a major vector of dengue (DENV) and chikungunya (CHIKV) viruses, causing millions of infections annually. It naturally carries, at high frequency, the intracellular inherited bacterial endosymbiont Wolbachia strains wAlbA and wAlbB; transinfection with the higher-density Wolbachia strain wMel from Drosophila melanogaster led to transmission blocking of both arboviruses. The hypothesis that reactive oxygen species (ROS)-induced immune activation plays a role in arbovirus inhibition in this species was examined. In contrast to previous observations in Ae. aegypti, elevation of ROS levels was not observed in either cell lines or mosquito lines carrying the wild-type Wolbachia or higher-density Drosophila Wolbachia strains. There was also no upregulation of genes controlling innate immune pathways or with antioxidant/ROS-producing functions. These data suggest that ROS-mediated immune activation is not an important component of the viral transmission-blocking phenotype in this species.     

Effect of Dengue-2 Virus Infection on Protein Expression in the Salivary Glands of Aedes aegypti Mosquitoes

Dengue virus (DENV) is the most important mosquito-transmitted flavivirus that is transmitted throughout the tropical and subtropical regions of the world. The primary mosquito vector of DENV in urban locations is Aedes aegypti. Key to understanding the transmission of DENV is the relationship between pathogen and vector. Accordingly, we report our preliminary characterization of the differentially expressed proteins from Ae. aegypti mosquitoes after DENV infection. We investigated the virus–vector interaction through changes in the proteome of the salivary glands of mosquitoes with disseminated DENV serotype 2 (DENV-2) infections using two-dimensional gel electrophoresis and identification by mass spectrometry. Our findings indicate that DENV-2 infection in the Ae. aegypti salivary gland alters the expression of structural, secreted, and metabolic proteins. These changes in the salivary gland proteome highlight the virally influenced environment caused by a DENV-2 infection and warrant additional investigation to determine if these differences extend to the expectorated saliva.     

Densovirus Oil Suspension Significantly Improves the Efficacy and Duration of Larvicidal Activity against Aedes albopictus

Aedes albopictus is the sole vector for various mosquito-borne viruses, including dengue, chikungunya, and Zika. Ecofriendly biological agents are required to reduce the spread of these mosquito-borne infections. Mosquito densoviruses (MDVs) are entomopathogenic mosquito-specific viruses, which can reduce the capacity of isolated vectors and decrease mosquito-borne viral disease transmission. However, their variable pathogenicity restricts their commercial use. In the present study, we developed a series of novel larvicide oil suspensions (denoted Bacillus thuringiensis (Bti) oil, Ae. albopictus densovirus (AalDV-5) oil, and a mixture of AalDV-5+Bti oil), which were tested against Ae. albopictus larvae under experimental semi-field and open-field conditions. The effect of AalDV-5 on non-target species was also evaluated. The combined effect of AalDV-5+Bti was greater than that of individual toxins and was longer lasting and more persistent compared with the laboratory AalDV-5 virus strain. The virus was quantified on a weekly basis by quantitative polymerase chain reaction (qPCR) and was persistently detected in rearing water as well as in dead larvae. Wildtype densovirus is not pathogenic to non-target organisms. The present findings confirm the improved effect of a mixed microbial suspension (AalDV-5+Bti oil) larvicide against Ae. albopictus. The development and testing of these products will enable better control of the vector mosquitoes.