Joseph R. Fauver

Vector Competence of American Mosquitoes for Three Strains of Zika Virus

In 2015, Zika virus (ZIKV; Flaviviridae; Flavivirus) emerged in the Americas, causing millions of infections in dozens of countries. The rapid spread of the virus and the association with disease outcomes such as Guillain-Barré syndrome and microcephaly make understanding transmission dynamics essential. Currently, there are no reports of vector competence (VC) of American mosquitoes for ZIKV isolates from the Americas. Further, it is not clear whether ZIKV strains from other genetic lineages can be transmitted by American Aedes aegypti populations, and whether the scope of the current epidemic is in part facilitated by viral factors such as enhanced replicative fitness or increased vector competence. Therefore, we characterized replication of three ZIKV strains, one from each of the three phylogenetic clades in several cell lines and assessed their abilities to be transmitted by Ae. aegypti mosquitoes. Additionally, laboratory colonies of different Culex spp. were infected with an American outbreak strain of ZIKV to assess VC. Replication rates were variable and depended on virus strain, cell line and MOI. African strains used in this study outcompeted the American strain in vitro in both mammalian and mosquito cell culture. West and East African strains of ZIKV tested here were more efficiently transmitted by Ae. aegypti from Mexico than was the currently circulating American strain of the Asian lineage. Long-established laboratory colonies of Culex mosquitoes were not efficient ZIKV vectors. These data demonstrate the capacity for additional ZIKV strains to infect and replicate in American Aedes mosquitoes and suggest that neither enhanced virus replicative fitness nor virus adaptation to local vector mosquitoes seems likely to explain the extent and intensity of ZIKV transmission in the Americas.

Genomic epidemiology reveals multiple introductions of Zika virus into the United States

Zika virus (ZIKV) is causing an unprecedented epidemic linked to severe congenital abnormalities. In July 2016, mosquito-borne ZIKV transmission was reported in the continental United States; since then, hundreds of locally acquired infections have been reported in Florida. To gain insights into the timing, source, and likely route(s) of ZIKV introduction, we tracked the virus from its first detection in Florida by sequencing ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We show that at least 4 introductions, but potentially as many as 40, contributed to the outbreak in Florida and that local transmission is likely to have started in the spring of 2016—several months before its initial detection. By analysing surveillance and genetic data, we show that ZIKV moved among transmission zones in Miami. Our analyses show that most introductions were linked to the Caribbean, a finding corroborated by the high incidence rates and traffic volumes from the region into the Miami area. Our study provides an understanding of how ZIKV initiates transmission in new regions.

Impact of simultaneous exposure to arboviruses on infection and transmission by Aedes aegypti mosquitoes

The recent emergence of both chikungunya and Zika viruses in the Americas has significantly expanded their distribution and has thus increased the possibility that individuals may become infected by more than one Aedes aegypti-borne virus at a time. Recent clinical data support an increase in the frequency of coinfection in human patients, raising the likelihood that mosquitoes could be exposed to multiple arboviruses during one feeding episode. The impact of coinfection on the ability of relevant vector species to transmit any of these viruses (that is, their vector competence) has not been determined. Thus, we here expose Ae. aegypti mosquitoes to chikungunya, dengue-2 or Zika viruses, both individually and as double and triple infections. Our results show that these mosquitoes can be infected with and can transmit all combinations of these viruses simultaneously. Importantly, infection, dissemination and transmission rates in mosquitoes are only mildly affected by coinfection.

West African Anopheles gambiae mosquitoes harbor a taxonomically diverse virome including new insect-specific flaviviruses, mononegaviruses, and totiviruses.

Anopheles gambiae are a major vector of malaria in sub-Saharan Africa. Viruses that naturally infect these mosquitoes may impact their physiology and ability to transmit pathogens. We therefore used metagenomics sequencing to search for viruses in adult Anopheles mosquitoes collected from Liberia, Senegal, and Burkina Faso. We identified a number of virus and virus-like sequences from mosquito midgut contents, including 14 coding-complete genome segments and 26 partial sequences. The coding-complete sequences define new viruses in the order Mononegavirales, and the families Flaviviridae, and Totiviridae. The identification of a flavivirus infecting Anopheles mosquitoes broadens our understanding of the evolution and host range of this virus family. This study increases our understanding of virus diversity in general, begins to define the virome of a medically important vector in its natural setting, and lays groundwork for future studies examining the potential impact of these viruses on anopheles biology and disease transmission.

Genetic Drift during Systemic Arbovirus Infection of Mosquito Vectors Leads to Decreased Relative Fitness during Host Switching

The emergence of mosquito-borne RNA viruses, such as West Nile virus (WNV), is facilitated by genetically complex virus populations within hosts. Here, we determine whether WNV enzootic (Culex tarsalis, Cx. quinquefasciatus, and Cx. pipiens) and bridge vectors (Aedes aegypti) have differential impacts on viral mutational diversity and fitness. During systemic mosquito infection, WNV faced stochastic reductions in genetic diversity that rapidly was recovered during intra-tissue population expansions. Interestingly, this intrahost selection and diversification was mosquito species dependent with Cx. tarsalis and Cx. quinquefasciatus exhibiting greater WNV divergence. However, recovered viral populations contained a preponderance of potentially deleterious mutations (i.e., high mutational load) and had lower relative fitness in avian cells compared to input virus. These findings demonstrate that the adaptive potential associated with mosquito transmission varies depending on the mosquito species and carries a significant fitness cost in vertebrates.

Experimental Evolution of an RNA Virus in Wild Birds: Evidence for Host-Dependent Impacts on Population Structure and Competitive Fitness

Within hosts, RNA viruses form populations that are genetically and phenotypically complex. Heterogeneity in RNA virus genomes arises due to error-prone replication and is reduced by stochastic and selective mechanisms that are incompletely understood. Defining how natural selection shapes RNA virus populations is critical because it can inform treatment paradigms and enhance control efforts. We allowed West Nile virus (WNV) to replicate in wild-caught American crows, house sparrows and American robins to assess how natural selection shapes RNA virus populations in ecologically relevant hosts that differ in susceptibility to virus-induced mortality. After five sequential passages in each bird species, we examined the phenotype and population diversity of WNV through fitness competition assays and next generation sequencing. We demonstrate that fitness gains occur in a species-specific manner, with the greatest replicative fitness gains in robin-passaged WNV and the least in WNV passaged in crows. Sequencing data revealed that intrahost WNV populations were strongly influenced by purifying selection and the overall complexity of the viral populations was similar among passaged hosts. However, the selective pressures that control WNV populations seem to be bird species-dependent. Specifically, crow-passaged WNV populations contained the most unique mutations (~1.7× more than sparrows, ~3.4× more than robins) and defective genomes (~1.4× greater than sparrows, ~2.7× greater than robins), but the lowest average mutation frequency (about equal to sparrows, ~2.6× lower than robins). Therefore, our data suggest that WNV replication in the most disease-susceptible bird species is positively associated with virus mutational tolerance, likely via complementation, and negatively associated with the strength of selection. These differences in genetic composition most likely have distinct phenotypic consequences for the virus populations. Taken together, these results reveal important insights into how different hosts may contribute to the emergence of RNA viruses.

Rapid and specific detection of Asian- and African-lineage Zika viruses.

A rapid, specific, sensitive, and inexpensive method has been developed that detects RNA from a Zika virus strain associated with the current outbreak. LAMP shines light on Zika virus Rapid and simple assays to detect infectious agents are key to tracking emerging epidemics. Chotiwan et al. describe a loop-mediated amplification (LAMP) assay that detects Zika virus RNA in human biofluids such as serum and semen as well as in mosquitoes, the insect vector that transmits the disease. This approach successfully distinguished the Asian-lineage Zika virus, associated with the current outbreak in the Americas, from the African-lineage Zika virus. This LAMP assay should enable tracking of the Asian-lineage strain as it moves into new geographical locations. A key advantage of this approach is detection without the need for RNA purification or copying RNA into DNA. Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers.

American Aedes vexans Mosquitoes are Competent Vectors of Zika Virus

Starting in 2013–2014, the Americas have experienced a massive outbreak of Zika virus (ZIKV) which has now reached at least 49 countries. Although most cases have occurred in South America and the Caribbean, imported and autochthonous cases have occurred in the United States. Aedes aegypti and Aedes albopictus mosquitoes are known vectors of ZIKV. Little is known about the potential for temperate Aedes mosquitoes to transmit ZIKV. Aedes vexans has a worldwide distribution, is highly abundant in particular localities, aggressively bites humans, and is a competent vector of several arboviruses. However, it is not clear whether Ae. vexans mosquitoes are competent to transmit ZIKV. To determine the vector competence of Ae. vexans for ZIKV, wild-caught mosquitoes were exposed to an infectious bloodmeal containing a ZIKV strain isolated during the current outbreak. Approximately 80% of 148 mosquitoes tested became infected by ZIKV, and approximately 5% transmitted infectious virus after 14 days of extrinsic incubation. These results establish that Ae. vexans are competent ZIKV vectors. Their relative importance as vectors (i.e., their vectorial capacity) depends on feeding behavior, longevity, and other factors that are likely to vary in ecologically distinct environments.

Temporal and Spatial Variability of Entomological Risk Indices for West Nile Virus Infection in Northern Colorado: 2006–2013

Abstract West Nile virus (WNV) is enzootic in northern Colorado. Annual surveillance activities in Fort Collins, CO, include collecting female Culex mosquitoes and testing them for the presence of WNV RNA in order to calculate 1) Culex female abundance, 2) WNV infection rate, and 3) the vector index (VI). These entomological risk indices inform public policy regarding the need for emergency adulticiding. Currently, these are calculated on a citywide basis. In this study, we present descriptive data from historical surveillance records spanning 2006–2013 to discern seasonal and yearly patterns of entomological risk for WNV infection. Also, we retrospectively test the hypothesis that entomological risk is correlated with human transmission risk and is heterogeneous within the City of Fort Collins. Four logistically relevant zones within the city were established and used to test this hypothesis. Zones in the eastern portion of the city consistently had significantly higher Culex abundance and VI compared with zones in the west, leading to higher entomological risk indicators for human WNV infection in the east. Moreover, the relative risk of a reported human case of WNV infection was significantly higher in the eastern zones of the city. Our results suggest that a more spatially targeted WNV management program may better mitigate human risk for WNV infection in Fort Collins, and possibly other cities where transmission is enzootic, while at the same time reducing pesticide use.

Maporal Hantavirus Causes Mild Pathology in Deer Mice (Peromyscus maniculatus).

Rodent-borne hantaviruses can cause two human diseases with many pathological similarities: hantavirus cardiopulmonary syndrome (HCPS) in the western hemisphere and hemorrhagic fever with renal syndrome in the eastern hemisphere. Each virus is hosted by specific reservoir species without conspicuous disease. HCPS-causing hantaviruses require animal biosafety level-4 (ABSL-4) containment, which substantially limits experimental research of interactions between the viruses and their reservoir hosts. Maporal virus (MAPV) is a South American hantavirus not known to cause disease in humans, thus it can be manipulated under ABSL-3 conditions. The aim of this study was to develop an ABSL-3 hantavirus infection model using the deer mouse (Peromyscus maniculatus), the natural reservoir host of Sin Nombre virus (SNV), and a virus that is pathogenic in another animal model to examine immune response of a reservoir host species. Deer mice were inoculated with MAPV, and viral RNA was detected in several organs of all deer mice during the 56 day experiment. Infected animals generated both nucleocapsid-specific and neutralizing antibodies. Histopathological lesions were minimal to mild with the peak of the lesions detected at 7–14 days postinfection, mainly in the lungs, heart, and liver. Low to modest levels of cytokine gene expression were detected in spleens and lungs of infected deer mice, and deer mouse primary pulmonary cells generated with endothelial cell growth factors were susceptible to MAPV with viral RNA accumulating in the cellular fraction compared to infected Vero cells. Most features resembled that of SNV infection of deer mice, suggesting this model may be an ABSL-3 surrogate for studying the host response of a New World hantavirus reservoir.