Mexican mosquitoes: overcoming barriers for dengue and Zika virus infection

2017 Fall.Includes bibliographical references.The mosquito transmitted arboviruses cause an important burden of disease worldwide. In Latin America dengue disease is endemic with more than 1 million dengue fever cases reported yearly. In addition to dengue, chikungunya and Zika viruses have been also circulating since their introduction in 2014 and 2015 respectively. For a mosquito-borne infection to occur susceptible humans, the mosquito vector and the virus should coincide. This dissertation was focused in the mosquito vector and its ability to acquire, maintain and then transmit the virus, termed vector competence. The vector competence was a fundamental measure for the research chapters in which we studied different aspects on the interactions between Aedes aegypti and Aedes albopictus mosquitoes and Dengue-2 and Zika viruses. This dissertation includes three research chapters which were based on the following specific aims. Specific aim 1: Determine the patterns of gene flow and vector competence for DENV-2 of Aedes aegypti from around the Mexican Neovolcanic Axis. It was previously reported that the intersection of the Neovolcanic axis (NVA) with the Gulf of Mexico coast in the state of Veracruz acts as a discrete barrier to gene flow among Ae. aegypti populations north and south of the NVA. These collections also differed in their vector competence (VC) for Dengue virus serotype 2 (DENV-2). Therefore, the goal of the present study was to determine if the same patterns remained 8 years later in collections from 2012. For which haplotype variation for the mitochondrial ND4 and the nuclear genes Dicer-2 and Argonaute-2 was analyzed for north and south of the NVA mosquito populations. Also, the VC of those populations for DENV-2 was determined (Chapter 2). Specific aim 2: Profile the microRNA response of Aedes aegypti midguts to DENV-2 exposure and DENV-2 infection. The microRNA pathway has been found to modulate important physiological mechanisms in mosquito vectors. Therefore in the context of DENV infection, miRNA modulation may provide information about key genes that are important for infection. Differential expression patterns of miRNAs from mosquito midguts upon infection have been unexplored. Therefore, we explored on the involvement of the miRNA pathway in persistently DENV-2 infected mosquitoes, for which DENV-2 virus was detected at 14 days post-infection (dpi). Two comparisons were included in the study. In the first group, DENV-2 infected midguts that produced a disseminated infection (did not have a midgut escape barrier) were contrasted with those that were given a non-infectious blood meal. Also, we included a comparison group from a subset of mosquitoes from the same cohort that were exposed to DENV-2 regardless of their midgut infection status contrasted to unexposed mosquitoes. Analysis of miRNA regulation in mosquitoes may help us to understand more about the intricate interactions between the virus and the vector host (Chapter 3). Specific aim 3: Assess the variation in competence for Zika virus transmission by Aedes aegypti and Aedes albopictus from Mexico. Previous studies have reported low Zika virus (ZIKV) transmission rates for the Asian lineage of ZIKV using mosquitoes from a wide geographical range from the Americas. Beside low transmission rates we hypothesized that VC is variable and is highly dependent upon the geographic origin of the mosquito populations. Hence, we analyzed the ZIKV transmission potential of recently colonized Aedes collections. Ten Ae. aegypti and three Ae. albopictus collections from different locations across Mexico were analyzed for ZIKV (strain PRVABC59 Asian genotype) vector competence at 7 and 14 dpi. We calculated the additive contribution of each of the four transmission barriers to ZIKV infection. In addition, we evaluated the contribution of both mosquito species to ZIKV transmission in areas where their distributions overlap (Chapter 4)

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

Impact of Extrinsic Incubation Temperature on Natural Selection During Zika Virus Infection of Aedes Aegypti and Aedes Albopictus

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C-40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of \u3e 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes

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

Susceptibility of South Texas Aedes aegypti to Pyriproxyfen

An integral part to integrated mosquito management is to ensure chemical products used for area-wide control are effective against a susceptible population of mosquitoes. Prior to conducting an intervention trial using an insect growth regulator, pyriproxyfen, in South Texas to control Aedes aegypti, we conducted a larval bioassay to evaluate baseline levels of susceptibility. We used seven serially-diluted doses ranging from 2.5 ppb to 6.3 × 10−4 ppb. We observed 100% inhibition emergence (IE) at even the lowest dose of 6.3 × 10−4 ppb in our susceptible reference colony of Ae. aegypti Liverpool. In our field strain of Ae. aegypti (F5 colonized from South Texas) we observed 79.8% IE at 6.3 × 10−4 ppb, 17.7% IE at 1.25 × 10−3 ppb, 98.7% IE at 1.25 × 10−2 ppb, and 100% emergence inhibition for the remainder of the doses. Given that commercial pyriproxyfen products are labeled for doses ranging to 50 ppb, we conclude that the field population sampled by this study are susceptible to this insect growth regulator

The Eco-Bio-Social Factors That Modulate Aedes aegypti Abundance in South Texas Border Communities

Aedes aegypti control requires dedicated resources that are usually scarce, limiting the reach and sustainability of vector control programs. This generates a need to focus on areas at risk of disease transmission and also understand the factors that might modulate local mosquito abundance. We evaluated the eco-bio-social factors that modulate indoor and outdoor relative abundance of female Ae. aegypti in communities of South Texas. We conducted housing quality and Knowledge Attitudes and Practices surveys in households that were part of a weekly mosquito surveillance program in November of 2017 and 2018. Our results showed widespread knowledge of mosquitoes and Zika virus by our participants. However, less than 35% considered them as serious problems in this region. The presence of window-mounted air conditioning units increased the risk of female mosquito relative abundance indoors. An increase in outdoor relative abundance was associated with larger properties and a higher number of children between 6 to 17 years of age. Interestingly, we observed that an increasing number of children <5 years of age modulated both indoor and outdoor relative abundance, with a 52% increase indoors and 30% decrease outdoors. The low perception of mosquito and disease risk highlights engagement needs for vector-borne disease prevention in this region. The identified risk factors can help guide public health officials in their efforts to reduce human and vector contact

The Eco-Bio-Social Factors That Modulate <em>Aedes aegypti</em> Abundance in South Texas Border Communities

Aedes aegypti control requires dedicated resources that are usually scarce, limiting the reach and sustainability of vector control programs. This generates a need to focus on areas at risk of disease transmission and also understand the factors that might modulate local mosquito abundance. We evaluated the eco-bio-social factors that modulate indoor and outdoor relative abundance of female Ae. aegypti in communities of South Texas. We conducted housing quality and Knowledge Attitudes and Practices surveys in households that were part of a weekly mosquito surveillance program in November of 2017 and 2018. Our results showed widespread knowledge of mosquitoes and Zika virus by our participants. However, less than 35% considered them as serious problems in this region. The presence of window-mounted air conditioning units increased the risk of female mosquito relative abundance indoors. An increase in outdoor relative abundance was associated with larger properties and a higher number of children between 6 to 17 years of age. Interestingly, we observed that an increasing number of children <5 years of age modulated both indoor and outdoor relative abundance, with a 52% increase indoors and 30% decrease outdoors. The low perception of mosquito and disease risk highlights engagement needs for vector-borne disease prevention in this region. The identified risk factors can help guide public health officials in their efforts to reduce human and vector contact

Cell fusing agent virus (Flavivirus) infection in Aedes aegypti in Texas: seasonality, comparison by trap type, and individual viral loads

South Texas has experienced local transmission of Zika virus and of other mosquito-borne viruses such as chikungunya virus and dengue virus in the last decades. Using a mosquito surveillance program in the Lower Rio Grande Valley (LRGV) and San Antonio, TX, from 2016 to 2018, we detected the presence of an insect-specific virus, cell fusing agent virus (CFAV), in the Aedes aegypti mosquito population. We tested 6,326 females and 1,249 males from the LRGV and 659 females from San Antonio for CFAV by RT-PCR using specific primers. Infection rates varied from 0 to 261 per 1,000 mosquitoes in the LRGV and 115 to 208 per 1,000 in San Antonio depending on the month of collection. Infection rates per 1,000 individuals appeared higher in females collected from BG Sentinel 2 traps compared to Autocidal Gravid Ovitraps, but the ratio of the percentage of infected pools did not differ by trap type. The natural viral load in individual males ranged from 1.25 x 102 to 5.50 x 106 RNA copies and in unfed females from 5.42 x 103 to 8.70 x 106 RNA copies. Gravid females were found to harbor fewer viral particles than males and unfed females

Phylogenetic tree of 29 Zika virus isolates.

<p>The tree was generated by Neighbor-Joining methods using the Tamura-Nei genetic distance model (bootstrapped 1,000 times). Similar results were obtained using maximum parsimony or maximum likelihood algorithms. Strains used in this study are shown in bold; distinct lineages are East African (MR766), West African (41525), and Asian (PRVABC59).</p

Mexican <i>Aedes aegypti</i> have increased vector competence for African strains of ZIKV.

<p>Mosquitoes were fed a blood-meal containing 1.6x10⁷ PFU of different ZIKV. Mosquitoes were then held for 7 or 14 days (n = 48 each time point for each group) and then (A) Infection rate (% of mosquitoes with virus in their bodies), (B) Dissemination Rate (% of mosquitoes, regardless of infection status, with virus in their legs), (C) Transmission Rate (% of mosquitoes, regardless of infection status, with virus in their saliva) and (D) Transmission (D) Rate (% of mosquitoes with a disseminated infection, with virus in their saliva) were collected. * Indicates p<0.05, ** p<0.01, and *** p<0.001 by two-tailed Fisher’s exact test. n = 126–144 for each group at each time point.</p