Variable effects of Wolbachia on alphavirus infection in Aedes aegypti.

Wolbachia pipientis (=Wolbachia) has promise as a tool to suppress virus transmission by Aedes aegypti mosquitoes. However, Wolbachia can have variable effects on mosquito-borne viruses. This variation remains poorly characterized, yet the multimodal effects of Wolbachia on diverse pathogens could have important implications for public health. Here, we examine the effects of somatic infection with two strains of Wolbachia (wAlbB and wMel) on the alphaviruses Sindbis virus (SINV), O'nyong-nyong virus (ONNV), and Mayaro virus (MAYV) in Ae. aegypti. We found variable effects of Wolbachia including enhancement and suppression of viral infections, with some effects depending on Wolbachia strain. Both wAlbB- and wMel-infected mosquitoes showed enhancement of SINV infection rates one week post-infection, with wAlbB-infected mosquitoes also having higher viral titers than controls. Infection rates with ONNV were low across all treatments and no significant effects of Wolbachia were observed. The effects of Wolbachia on MAYV infections were strikingly strain-specific; wMel strongly blocked MAYV infections and suppressed viral titers, while wAlbB did not influence MAYV infection. The variable effects of Wolbachia on vector competence underscore the importance of further research into how this bacterium impacts the virome of wild mosquitoes including the emergent human pathogens they transmit.

Vector competence of selected North American Anopheles and Culex mosquitoes for Zika virus.

Zika virus (ZIKV) is a vector-borne flavivirus that has caused recent outbreaks associated with serious disease in infants and newborns in the Americas. Aedes mosquitoes are the primary vectors for ZIKV, but little is known about the diversity of mosquitoes that can transmit ZIKV in North America. We chose three abundant North American mosquito species (Anopheles freeborni, Anopheles quadrimaculatus, and Culex tarsalis) and one known vector species (Aedes aegypti), fed them blood meals supplemented with a recent outbreak ZIKV strain, and tested bodies, legs, and saliva for infectious ZIKV. ZIKV was able to infect, disseminate, and be transmitted by Aedes aegypti. However, Anopheles freeborni, Anopheles quadrimaculatus, and Culex tarsalis were unable to be infected. We conclude that these species are unlikely to be involved in ZIKV transmission in North America. However, we should continue to examine the ability for other mosquito species to potentially act as ZIKV vectors in North America.

Wolbachia effects on Rift Valley fever virus infection in Culex tarsalis mosquitoes.

Innovative tools are needed to alleviate the burden of mosquito-borne diseases, and strategies that target the pathogen are being considered. A possible tactic is the use of Wolbachia, a maternally inherited, endosymbiotic bacterium that can (but does not always) suppress diverse pathogens when introduced to naive mosquito species. We investigated effects of somatic Wolbachia (strain wAlbB) infection on Rift Valley fever virus (RVFV) in Culex tarsalis mosquitoes. When compared to Wolbachia-uninfected mosquitoes, there was no significant effect of Wolbachia infection on RVFV infection, dissemination, or transmission frequencies, nor on viral body or saliva titers. Within Wolbachia-infected mosquitoes, there was a modest negative correlation between RVFV body titers and Wolbachia density, suggesting that Wolbachia may slightly suppress RVFV in a density-dependent manner in this mosquito species. These results are contrary to previous work in the same mosquito species, showing Wolbachia-induced enhancement of West Nile virus infection rates. Taken together, these results highlight the importance of exploring the breadth of pathogen modulations induced by Wolbachia.

Vector competence of Anopheles and Culex mosquitoes for Zika virus.

Zika virus is a newly emergent mosquito-borne flavivirus that has caused recent large outbreaks in the new world, leading to dramatic increases in serious disease pathology including Guillain-Barre syndrome, newborn microcephaly, and infant brain damage. Although Aedes mosquitoes are thought to be the primary mosquito species driving infection, the virus has been isolated from dozens of mosquito species, including Culex and Anopheles species, and we lack a thorough understanding of which mosquito species to target for vector control. We exposed Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes to blood meals supplemented with two Zika virus strains. Mosquito bodies, legs, and saliva were collected five, seven, and 14 days post blood meal and tested for infectious virus by plaque assay. Regardless of titer, virus strain, or timepoint, Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes were refractory to Zika virus infection. We conclude that Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes likely do not contribute significantly to Zika virus transmission to humans. However, future studies should continue to explore the potential for other novel potential vectors to transmit the virus.

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.

Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes.

Over evolutionary time, Wolbachia has been repeatedly transferred between host species contributing to the widespread distribution of the symbiont in arthropods. For novel infections to be maintained, Wolbachia must infect the female germ line after being acquired by horizontal transfer. Although mechanistic examples of horizontal transfer exist, there is a poor understanding of factors that lead to successful vertical maintenance of the acquired infection. Using Anopheles mosquitoes (which are naturally uninfected by Wolbachia) we demonstrate that the native mosquito microbiota is a major barrier to vertical transmission of a horizontally acquired Wolbachia infection. After injection into adult Anopheles gambiae, some strains of Wolbachia invade the germ line, but are poorly transmitted to the next generation. In Anopheles stephensi, Wolbachia infection elicited massive blood meal-induced mortality, preventing development of progeny. Manipulation of the mosquito microbiota by antibiotic treatment resulted in perfect maternal transmission at significantly elevated titers of the wAlbB Wolbachia strain in A. gambiae, and alleviated blood meal-induced mortality in A. stephensi enabling production of Wolbachia-infected offspring. Microbiome analysis using high-throughput sequencing identified that the bacterium Asaia was significantly reduced by antibiotic treatment in both mosquito species. Supplementation of an antibiotic-resistant mutant of Asaia to antibiotic-treated mosquitoes completely inhibited Wolbachia transmission and partly contributed to blood meal-induced mortality. These data suggest that the components of the native mosquito microbiota can impede Wolbachia transmission in Anopheles. Incompatibility between the microbiota and Wolbachia may in part explain why some hosts are uninfected by this endosymbiont in nature.

Wolbachia enhances West Nile virus (WNV) infection in the mosquito Culex tarsalis.

Novel strategies are required to control mosquitoes and the pathogens they transmit. One attractive approach involves maternally inherited endosymbiotic Wolbachia bacteria. After artificial infection with Wolbachia, many mosquitoes become refractory to infection and transmission of diverse pathogens. We evaluated the effects of Wolbachia (wAlbB strain) on infection, dissemination and transmission of West Nile virus (WNV) in the naturally uninfected mosquito Culex tarsalis, which is an important WNV vector in North America. After inoculation into adult female mosquitoes, Wolbachia reached high titers and disseminated widely to numerous tissues including the head, thoracic flight muscles, fat body and ovarian follicles. Contrary to other systems, Wolbachia did not inhibit WNV in this mosquito. Rather, WNV infection rate was significantly higher in Wolbachia-infected mosquitoes compared to controls. Quantitative PCR of selected innate immune genes indicated that REL1 (the activator of the antiviral Toll immune pathway) was down regulated in Wolbachia-infected relative to control mosquitoes. This is the first observation of Wolbachia-induced enhancement of a human pathogen in mosquitoes, suggesting that caution should be applied before releasing Wolbachia-infected insects as part of a vector-borne disease control program.

Effects of larval rearing temperature on immature development and West Nile virus vector competence of Culex tarsalis.

BACKGROUND: Temperature is known to induce changes in mosquito physiology, development, ecology, and in some species, vector competence for arboviruses. Since colonized mosquitoes are reared under laboratory conditions that can be significantly different from their field counterparts, laboratory vector competence experiments may not accurately reflect natural vector-virus interactions. METHODS: We evaluated the effects of larval rearing temperature on immature development parameters and vector competence of two Culex tarsalis strains for West Nile virus (WNV). RESULTS: Rearing temperature had a significant effect on mosquito developmental parameters, including shorter time to pupation and emergence and smaller female body size as temperature increased. However, infection, dissemination, and transmission rates for WNV at 5, 7, and 14 days post infectious feeding were not consistently affected. CONCLUSIONS: These results suggest that varying constant larval rearing temperature does not significantly affect laboratory estimates of vector competence for WNV in Culex tarsalis mosquitoes.

Requirement of glycosylation of West Nile virus envelope protein for infection of, but not spread within, Culex quinquefasciatus mosquito vectors.

Most of sequenced West Nile virus (WNV) genomes encode a single N-linked glycosylation site on their envelope (E) proteins. We previously found that WNV lacking the E protein glycan was severely inhibited in its ability to replicate and spread within two important mosquito vector species, Culex pipiens and Cx. tarsalis. However, recent work with a closely related species, Cx. pipiens pallens, found no association between E protein glycosylation and either replication or dissemination. To examine this finding further, we expanded upon our previous studies to include an additional Culex species, Cx. quinquefasciatus. The non-glycosylated WNV-N154I virus replicated less efficiently in mosquito tissues after intrathoracic inoculation, but there was little difference in replication efficiency in the midgut after peroral infection. Interestingly, although infectivity was inhibited when WNV lacked the E protein glycan, there was little difference in viral spread throughout the mosquito. These data indicate that E protein glycosylation affects WNV-vector interactions in a species-specific manner.

Larval nutritional stress does not affect vector competence for West Nile virus (WNV) in Culex tarsalis.

In some mosquito species the conditions experienced by larvae during development have been shown to lead to changes in susceptibility to various arboviruses in the adult female. Since laboratory mosquitoes are generally reared under ideal conditions, mosquito vector competence experiments in the laboratory may not accurately reflect vector?virus relationships in nature. We examined the consequences of larval nutritional stress on Culex tarsalis vector competence for West Nile virus (WNV). Larval nutrition deprivation resulted in increased development time, decreased pupation and emergence rates, and smaller adult female body size. However, infection, dissemination, and transmission rates for WNV at 5, 7, and 14 days postfeeding were not consistently affected. These results suggest that larval nutritional rearing protocols are not a major factor in laboratory estimates of WNV vector competence in Cx. tarsalis.