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 > 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.

A mutation-mediated evolutionary adaptation of Zika virus in mosquito and mammalian host

Zika virus (ZIKV) caused millions of infections during its rapid and expansive spread from Asia to the Americas from 2015 to 2017. Here, we compared the infectivity of ZIKV mutants with individual stable substitutions which emerged throughout the Asian ZIKV lineage and were responsible for the explosive outbreaks in the Americas. A threonine (T) to alanine (A) mutation at the 106th residue of the ZIKV capsid (C) protein facilitated the transmission by its mosquito vector, as well as infection in both human cells and immunodeficient mice. A mechanistic study showed that the T106A substitution rendered the C a preferred substrate for the NS2B-NS3 protease, thereby facilitating the maturation of structural proteins and the formation of infectious viral particles. Over a complete “mosquito-mouse-mosquito” cycle, the ZIKV C-T106A mutant showed a higher prevalence of mosquito infection than did the preepidemic strain, thus promoting ZIKV dissemination. Our results support the contribution of this evolutionary adaptation to the occasional widespread reemergence of ZIKV in nature.

Entomovirological Surveillance in Schools: Are They a Source for Arboviral Diseases Transmission?

Surveillance and control activities for virus-transmitting mosquitoes have primarily focused on dwellings. There is little information about viral circulation in heavily trafficked places such as schools. We collected and analyzed data to assess the presence and prevalence of dengue, chikungunya, and Zika viruses in mosquitoes, and measured Aedes indices in schools in Medellín (Colombia) between 2016–2018. In 43.27% of 2632 visits we collected Aedes adults, creating 883 pools analyzed by RT-PCR. 14.27% of pools yielded positive for dengue or Zika (infection rates of 1.75–296.29 for Aedes aegypti). Ae. aegypti was more abundant and had a higher infection rate for all studied diseases. Aedes indices varied over time. There was no association between Aedes abundance and mosquito infection rates, but the latter did correlate with cases of arboviral disease and climate. Results suggest schools are important sources of arbovirus and health agencies should include these sites in surveillance programs; it is essential to know the source for arboviral diseases transmission and the identification of the most population groups exposed to these diseases to research and developing new strategies.

Prevalence of asymptomatic P. falciparum gametocyte carriage among school children in Mbita, Western Kenya and assessment of the association between gametocyte density, multiplicity of infection and mosquito infection prevalence.

Background: Asymptomatic Plasmodium falciparum gametocyte carriers are reservoirs for sustaining transmission in malaria endemic regions. Gametocyte presence in the host peripheral blood is a predictor of capacity to transmit malaria. However, it does not always directly translate to mosquito infectivity. Factors that affect mosquito infectivity include, gametocyte sex-ratio and density, multiplicity of infection (MOI), and host and vector anti-parasite immunity. We assess the prevalence of gametocyte carriage and some of its associated risk factors among asymptomatic schoolchildren in Western Kenya and to further analyse the association between gametocyte density, multiplicity of infection (MOI) and mosquito infection prevalence. Methods: P. falciparum parasite infections were detected by RDT (Rapid Diagnostic Test) and microscopy among schoolchildren (5-15 years old). Blood from 37 microscopy positive gametocyte carriers offered to laboratory reared An. gambiae s.l. mosquitoes. A total of 3395 fully fed mosquitoes were screened for Plasmodium sporozoites by ELISA. P. falciparum was genotyped using 10 polymorphic microsatellite markers. The association between MOI and gametocyte density and mosquito infection prevalence was investigated. Results: A significantly higher prevalence of P. falciparum infection was found in males 31.54% (764/2422) (p-value < 0.001) compared to females 26.72% (657/2459). The microscopic gametocyte prevalence among the study population was 2% (84/4881). Children aged 5-9 years have a higher prevalence of gametocyte carriage (odds ratios = 2.1 [95% CI = 1.3–3.4], P = 0.002) as compared to children aged 10-15 years. After offering gametocyte positive blood to An. gambiae s.l. by membrane feeding assay, our results indicated that 68.1% of the variation in mosquito infection prevalence was accounted for by gametocyte density and MOI (R-SQR. = 0.681, p < 0.001). Conclusions: We observed a higher risk of gametocyte carriage among the younger children (5-9 years). Gametocyte density and MOI significantly predicted mosquito infection prevalence.

Probability of consolidation constrains novel serotype emergence in dengue fever virus

Since their first sequencing 40 years ago, Dengue virus (DENV) genotypes have shown extreme coherence regarding the serotype class they encode. Considering that DENV is a ribonucleic acid (RNA) virus with a high mutation rate, this behavior is intriguing. Here, we explore the effect of various parameters on likelihood of new serotype emergence. In order to determine the time scales of such an event, we used a Timed Markov Transmission Model to explore the influences of sylvatic versus peri-urban transmission, viral mutation rate, and vertical transmission on the probabilities of novel serotype emergence. We found that around 1 000 years are required for a new serotype to emerge, consistent with phylogenetic analysis of extant dengue serotypes. Furthermore, we show that likelihood of establishing chains of mosquito-human-mosquito infection, known as consolidation, is the primary factor which constrains novel serotype emergence. Our work illustrates the restrictions on and provides a mechanistic explanation for the low probability of novel dengue virus serotype emergence and the low number of observed DENV serotypes.

Impact of extrinsic incubation temperature on natural selection during Zika virus infection of Aedes aegypti

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 > 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimum 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 diurnally fluctuating temperatures which more faithfully 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. The effect of diurnal temperature was distinct and could not have been predicted from constant temperature-derived data. 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 diurnal temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology within mosquitoes, including major effects on the selective environment within mosquitoes.Author SummaryArthropod-borne viruses (arboviruses) have emerged in recent decades due to complex factors that include increases in international travel and trade, the breakdown of public health infrastructure, land use changes, and many other factors. Climate change also has the potential to shift the geographical ranges of arthropod vectors, consequently increasing the global risk of arbovirus infection. Changing temperatures may also alter the virus-host interaction, ultimately resulting in the emergence of new viruses and virus genotypes in new areas. Therefore, we sought to characterize how temperature (both constant and fluctuating) alters the ability of Aedes aegypti and Aedes albopictus to transmit Zika virus, and how it influences virus populations within mosquitoes. We found that intermediate temperatures maximize virus transmission compared to more extreme and fluctuating temperatures. Constant temperatures increased positive selection on virus genomes, while fluctuating temperatures strengthened purifying selection. Our studies provide evidence that in addition to altering VC, temperature significantly influences the selective environment within mosquitoes.

Individual-based dengue virus surveillance in Aedes aegypti mosquitoes concurrently collected with suspected patients in Tarlac City, Philippines

Background: Vector control measures are critical in the prevention and reduction of dengue virus (DENV) transmission. In this context, an effective vector control is reliant not only on the knowledge of mosquito abundance, but also on the timely and accurate detection of mosquito infection. Mosquito-based virus surveillance programs typically rely on a pool-based mosquito testing, although whether individual-based mosquito testing could represent a feasible alternative is not largely studied. Applying an individual-based mosquito testing approach, we conducted a one-month DENV surveillance of adult Aedes (Ae.) aegypti mosquitoes around households of suspected dengue patients during the 2015 dengue peak season in Tarlac City, Philippines to more accurately assess the mosquito infection rate and identify the DENV serotypes and genotypes concurrently co-circulating in mosquitoes and patients.Methods: We performed a one-step multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the simultaneous DENV detection and serotyping in patients and individual female Ae. aegypti mosquito. Additionally, we performed sequencing and phylogenetic analyses to further characterize the detected DENVs in mosquitoes and patients at the genotype level.Results: We collected a total of 583 adult Ae. aegypti mosquitoes; of which, we individually tested 359 female mosquitoes for the presence of DENV. Ten (2.8%) among the 359 female mosquitoes were positive for the presence of DENV. We detected DENV-1, DENV-2, and DENV-4 in the field-collected mosquitoes, which were consistent with the serotypes concurrently infecting patients. Sequencing and phylogenetic analyses of the detected DENVs based on the partial envelope (E) gene revealed three genotypes concurrently present in the sampled mosquitoes and patients during the study period, namely: DENV-1 genotype IV, DENV-2 Cosmopolitan genotype, and DENV-4 genotype II.Conclusions: In this study, we demonstrate the utility of the one-step multiplex real-time RT-PCR assay in the individual-based DENV surveillance of mosquitoes. Our findings reinforce the significance of detecting and monitoring virus activity in local mosquito populations, which is critical for dengue prevention and control activities.

Prevalence of asymptomatic P. falciparum gametocyte carriage in schoolchildren and assessment of the association between gametocyte density, multiplicity of infection and mosquito infection prevalence

Background: Malaria is a major public health threat in sub-Saharan Africa. Asymptomatic Plasmodium falciparum gametocyte carriers are potential infectious reservoirs for sustaining transmission in many malaria endemic regions. The aim of the study was to assess the prevalence of gametocyte carriage and some of its associated risk factors among asymptomatic schoolchildren in Western Kenya and further analyse the association between gametocyte density, multiplicity of infection (MOI) and mosquito infection prevalence. Methods: Rapid diagnostic tests were used to screen for P. falciparum parasite infection among schoolchildren (5-15 years old) and the results were verified using microscopy. Microscopy positive gametocyte carriers were selected to feed laboratory reared An. gambiae s.l. mosquitoes using membrane feeding method. Genomic DNA was extracted from dry blood spot samples and P. falciparum populations were genotyped using 10 polymorphic microsatellite markers. Assessment of the association between MOI and gametocyte density and mosquito infection prevalence was conducted. Results: A significantly higher prevalence of P. falciparum infection was found in males 31.54% (764/2422) (p-value < 0.001) compared to females 26.72% (657/2459). The microscopy gametocyte prevalence among the study population was 2% (84/4881). Children aged 5-9 years have a higher prevalence of gametocyte carriage (odds ratios = 2.1 [95% CI = 1.3–3.4], P = 0.002) as compared to children aged 10-15 years. After challenging An. gambiae s.l. by membrane feeding assay on gametocyte positive patient blood, our results indicate that 68.1% of the variation in mosquito infection prevalence is accounted for by gametocyte density and MOI (R-SQR. = 0.681, p < 0.001). Conclusions: Age was a significant risk factor for gametocyte carriage, as indicated by the higher risk of gametocyte carriage among the younger children (5-9 years). Gametocyte density and MOI statistically significantly predicted mosquito infection prevalence. Both of the variables added significantly to the prediction (p < 0.05).

Individual-based dengue virus surveillance in Aedes aegypti mosquitoes collected concurrently with suspected patients in Tarlac City, Philippines

Background: Vector control measures are critical in the prevention and reduction of dengue virus (DENV) transmission. In this context, effective vector control is reliant not only on knowledge of mosquito abundance, but also on the timely and accurate detection of mosquito infection. Mosquito-based virus surveillance programs commonly rely on pool-based mosquito testing, but whether individual-based mosquito testing could represent a feasible alternative is not largely studied. Applying an individual-based mosquito testing approach, we conducted a one-month DENV surveillance of adult Aedes aegypti mosquitoes around households of suspected dengue patients during the 2015 dengue peak season in Tarlac City, Philippines to more accurately assess the mosquito infection rate, and to identify the DENV serotypes and genotypes concurrently co-circulating in mosquitoes and patients. Methods: We performed a one-step multiplex real-time RT-PCR assay for the simultaneous detection and serotyping of DENV in patients and in individual female Ae. aegypti mosquito. Additionally, we performed sequencing and phylogenetic analyses to further characterize the detected DENVs in mosquitoes and patients at the genotype level. Results: We collected a total of 583 adult Ae. aegypti mosquitoes, of which we tested 359 female mosquitoes individually for the presence of the DENV. Ten mosquitoes (2.8%) from amongst 359 female mosquitoes were confirmed to be positive for the presence of the DENV. We detected DENV-1, DENV-2, and DENV-4 in the field-collected mosquitoes, which were consistent with the serotypes concurrently infecting patients. Sequencing and phylogenetic analyses of the detected DENVs based on the partial envelope (E) gene revealed three genotypes concurrently present in the sampled mosquitoes and patients during the study period, namely: DENV-1 genotype IV, DENV-2 Cosmopolitan genotype and DENV-4 genotype II. Conclusions: In this study, we demonstrate the utility of a one-step multiplex real-time RT-PCR assay in individual-based DENV surveillance of mosquitoes. Our findings reinforce the importance of detecting and monitoring virus activity in local mosquito populations, which is critical for dengue prevention and control activities.