Mosquito-transmitted Pathogen Research Articles

Comparative analysis of midgut bacterial communities in Chikungunya virus-infected and non-infected Aedes aegypti Thai laboratory strain mosquitoes

Chikungunya virus (CHIKV) poses a significant global health threat, re-emerging as a mosquito-transmitted pathogen that caused high fever, rash, and severe arthralgia. In Thailand, a notable CHIKV outbreak in 2019–2020 affected approximately 20,000 cases across 60 provinces, underscoring the need for effective mosquito control protocols. Previous studies have highlighted the role of midgut bacteria in the interaction between mosquito vectors and pathogen infections, demonstrating their ability to protect the insect from invading pathogens. However, research on the midgut bacteria of Aedes (Ae.) aegypti, the primary vector for CHIKV in Thailand remains limited. This study aims to characterize the bacterial communities in laboratory strains of Ae. aegypti, both infected and non-infected with CHIKV. Female mosquitoes from a laboratory strain of Ae. aegypti were exposed to a CHIKV-infected blood meal through membrane feeding, while the control group received a non-infected blood meal. At 7 days post-infection (dpi), mosquito midguts were dissected for 16S rRNA gene sequencing to identify midgut bacteria, and CHIKV presence was confirmed by E1-nested RT-PCR using mosquito carcasses. The study aimed to compare the bacterial communities between CHIKV-infected and non-infected groups. The analysis included 12 midgut bacterial samples, divided into three groups: CHIKV-infected (exposed and infected), non-infected (exposed but not infected), and non-exposed (negative control). Alpha diversity indices and Bray–Curtis dissimilarity matrix revealed significant differences in bacterial profiles among the three groups. The infected group exhibited an increased abundance of bacteria genus Gluconobacter, while Asaia was prevalent in both non-infected and negative control groups. Chryseobacterium was prominent in the negative control group. These findings highlight potential alterations in the distribution and abundance of gut microbiomes in response to CHIKV infection status. This study provides valuable insights into the dynamic relationship between midgut bacteria and CHIKV, underscoring the potential for alterations in bacterial composition depending on infection status. Understanding the relationships between mosquitoes and their microbiota holds promise for developing new methods and tools to enhance existing strategies for disease prevention and control. This research advances our understanding of the circulating bacterial composition, opening possibilities for new approaches in combating mosquito-borne diseases.

Battle of the Bites: The Effect of Sewage Effluent Exposure on Mosquitofish Biocontrol of Mosquitoes in Residential Louisiana.

Mosquitofish, Gambusia affinis, are eponymous larval mosquito predators. Their ability to colonize and survive in habitats that are uninhabitable by other potential predators allows them to naturally manage larval mosquito populations in most ground pools they are present in. However, effluent from residential onsite wastewater treatment systems (OWTSs) appears to limit the presence of fish predators. This is especially problematic in Louisiana, where regulations allow the discharge of OWTS effluent into open drainage conveyances. To determine the effect of effluent on the capacity of mosquitofish for biocontrol in contaminated areas, we assessed the body condition metrics of populations from two effluent-exposed sites and two sites not exposed to effluent, determined the lethal effect of effluent-contaminated drainage water on fish, and measured the prey consumption rates in the presence of effluent. Female fish collected from effluent-impacted sites had a reduced somatic body condition and most females examined displayed masculinized anal fins resembling the male gonopodium structure. This trait was not seen in fish collected from the control sites and has not yet been documented in association with OWTSs or in the state of Louisiana. Fish from the control sites survived at effluent-contaminated water levels < 70%, and the prey clearance rates increased with dilution. Onsite wastewater treatment system effluent has significant effects on both the short- and long-term persistence of mosquitofish, their body composition, reproductive health, and larval mosquito consumption. These effects likely release mosquito larvae from suppression and may increase the threat of mosquito-transmitted pathogens in effluent-contaminated locations.

The Shockwè trap: a human-baited exposure-free device for surveillance and behaviour studies of anthropophilic vectors.

Background: The human biting rate (MBR) and entomological inoculation rate (EIR) are common parameters routinely used to measure the risk of malaria transmission. Both parameters can be estimated using human landing catches (HLC). Although it is considered the gold-standard, HLC puts collectors at higher risk of infection with mosquito-transmitted pathogens. Methods: A novel exposure-free host-seeking mosquito electrocution trap, the Shockwè trap (SHK), was developed and its efficiency for monitoring mosquito community composition and abundance was compared with human landing catches (HLC) as the gold-standard. Field experiments were performed in Massavasse village, southern Mozambique. Simultaneous indoor and outdoor collections of nocturnal host-seeking mosquitoes were carried out using the SHK and HLC methods. The relative sampling efficiency of SHK was estimated as the ratio of the numbers of mosquitoes caught in SHK compared HLC. Proportionality and density-dependence between SHK and HLC catches were estimated by mean of Bayesian regression approaches. Results: A total of 69,758 and 27,359 host-seeking mosquitoes comprising nineteen species and four genera, were collected by HLC and SHK respectively. In general, SHK and HLC sampled similar numbers of mosquito species, with the exceptions of the least common species Aedes sudanensis, Ae. subargenteus, and Coquillettidia versicolor that were caught only by HLC. The relative sampling efficiency and proportionality between SHK and matched HLC catches varied greatly between species and collection site. However, all mosquitoes collected by SHK were unfed, confirming the Shockwè trap design's performance and reliability as a successful mosquito exposure free sampling approach. Conclusions: Results demonstrate that SHK is a safe and reliable human-exposure free device for monitoring the occurrence of a wide range of mosquito, including major malaria and arboviruses vector species. However, improvements are needed to increase its sampling efficiency for less abundant mosquito species.

Equine Polyclonal Antibodies Prevent Acute Chikungunya Virus Infection in Mice.

Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen that causes chikungunya disease (CHIK); the disease is characterized by fever, muscle ache, rash, and arthralgia. This arthralgia can be debilitating and long-lasting, seriously impacting quality of life for years. Currently, there is no specific therapy available for CHIKV infection. We have developed a despeciated equine polyclonal antibody (CHIKV-EIG) treatment against CHIKV and evaluated its protective efficacy in mouse models of CHIKV infection. In immunocompromised (IFNAR-/-) mice infected with CHIKV, daily treatment for five consecutive days with CHIKV-EIG administered at 100 mg/kg starting on the day of infection prevented mortality, reduced viremia, and improved clinical condition as measured by body weight loss. These beneficial effects were seen even when treatment was delayed to 1 day after infection. In immunocompetent mice, CHIKV-EIG treatment reduced virus induced arthritis (including footpad swelling), arthralgia-associated cytokines, viremia, and tissue virus loads in a dose-dependent fashion. Collectively, these results suggest that CHIKV-EIG is effective at preventing CHIK and could be a viable candidate for further development as a treatment for human disease.

Mosquito pericardial cells upregulate Cecropin expression after an immune challenge

Most mosquito-transmitted pathogens grow or replicate in the midgut before invading the salivary glands. Pathogens are exposed to several immunological factors along the way. Recently, it was shown that hemocytes gather near the periostial region of the heart to efficiently phagocytose pathogens circulating in the hemolymph. Nerveless, not all pathogens can be phagocyted by hemocytes and eliminated by lysis. Interestingly, some studies have shown that pericardial cells (PCs) surrounding periostial regions, may produce humoral factors, such as lysozymes. Our current work provides evidence that Anopheles albimanus PCs are a major producer of Cecropin 1 (Cec1). Furthermore, our findings reveal that after an immunological challenge, PCs upregulate Cec1 expression. We conclude that PCs are positioned in a strategic location that could allow releasing humoral components, such as cecropin, to lyse pathogens on the heart or circulating in the hemolymph, implying that PCs could play a significant role in the systemic immune response.

A human monoclonal antibody to neutralize all four serotypes of dengue virus derived from patients at the convalescent phase of infection

Dengue virus (DENV) is a prevalent mosquito-transmitted human pathogen, causing about 100 million cases of acute dengue fever and 21,000 deaths annually worldwide. Therapeutic neutralizing antibodies against dengue virus might be effective to treat severe dengue fever. Here, we showed that human monoclonal antibody (HMAb) 9C7 bound to all four intact serotypes of DENV but not to the recombinant envelope protein, suggesting HMAb 9C7 recognized a conformational epitope of the envelope protein. Taken together our results suggested that HMAb 9C7 neutralized all four serotypes of DENV in vitro and, for DENV-1, indicated activity at the pre- and post-attachment steps in the viral life cycle. HMAb 9C7 potently protected suckling mice from lethal challenge with all four serotypes of DENV. FcγRII-mediated uptake of immune complexes and antibody-dependent enhancement at low doses of the antibody were abolished by two Leu-to-Ala (9C7-LALA) mutations or deletion of nine amino acids (9C7-9del) in HMAb 9C7 Fc. Therefore, HMAb 9C7 represented a promising prophylactic and therapeutic agent against all four serotypes of DENV.

High DDT resistance without apparent association to kdr and Glutathione-S-transferase (GST) gene mutations in Aedes aegypti population at hotel compounds in Zanzibar

Global efforts to control Aedes mosquito-transmitted pathogens still rely heavily on insecticides. However, available information on vector resistance is mainly restricted to mosquito populations located in residential and public areas, whereas commercial settings, such as hotels are overlooked. This may obscure the real magnitude of the insecticide resistance problem and lead to ineffective vector control and resistance management. We investigated the profile of insecticide susceptibility of Aedes aegypti mosquitoes occurring at selected hotel compounds on Zanzibar Island. At least 100 adults Ae. aegypti females from larvae collected at four hotel compounds were exposed to papers impregnated with discriminant concentrations of DDT (4%), permethrin (0.75%), 0.05 deltamethrin (0.05%), propoxur (0.1%) and bendiocarb (0.1%) to determine their susceptibility profile. Allele-specific qPCR and sequencing analysis were applied to determine the possible association between observed resistance and presence of single nucleotide polymorphisms (SNPs) in the voltage-gated sodium channel gene (VGSC) linked to DDT/pyrethroid cross-resistance. Additionally, we explored the possible involvement of Glutathione-S-Transferase gene (GSTe2) mutations for the observed resistance profile. In vivo resistance bioassay indicated that Ae. aegypti at studied sites were highly resistant to DDT, mortality rate ranged from 26.3% to 55.3% and, moderately resistant to deltamethrin with a mortality rate between 79% to and 100%. However, genotyping of kdr mutations affecting the voltage-gated sodium channel only showed a low frequency of the V1016G mutation (n = 5; 0.97%). Moreover, for GSTe2, seven non-synonymous SNPs were detected (L111S, C115F, P117S, E132A, I150V, E178A and A198E) across two distinct haplotypes, but none of these were significantly associated with the observed resistance to DDT. Our findings suggest that cross-resistance to DDT/deltamethrin at hotel compounds in Zanzibar is not primarily mediated by mutations in VGSC. Moreover, the role of identified GSTe2 mutations in the resistance against DDT remains inconclusive. We encourage further studies to investigate the role of other potential insecticide resistance markers.

Chikungunya Virus Infects the Heart and Induces Heart-Specific Transcriptional Changes in an Immunodeficient Mouse Model of Infection.

Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen in family Togaviridae, genus Alphavirus. Although CHIKV is well known for its ability to cause debilitating rheumatoid-like arthritis, it has been also been observed to cause cardiovascular symptoms such as arrhythmias. Here, using samples from a previous study, we sequenced RNA from serum, kidney, skeletal muscle, and cardiac muscle from CHIKV- and mock-infected IFN-αR-/- mice using two sequencing techniques to investigate heart-specific changes in virus mutational profiles and host gene expression. Mutation rates were similar across muscle tissues although heart tissue carried heart-specific CHIKV minority variants, one of which had a coding change in the nsP3 gene and another in the 3'UTR. Importantly, heart-specific transcriptional changes included differential expression of genes critical for ion transport and muscle contraction. These results demonstrate that CHIKV replicates in the hearts of immunodeficient mice and induce heart-specific mutations and host responses with implications for cardiac pathologies.

Prevalence of arbovirus antibodies in young healthy adult population in Brazil

BackgroundThe emergence and re-emergence of infectious diseases are a cause for worldwide concern. The introduction of Zika and Chikungunya diseases in the Americas has exposed unforeseen medical and logistical challenges for public health systems. Moreover, the lack of preventive measures and vaccination against known and emerging mosquito-transmitted pathogens, and the occurrence of unanticipated clinical complications, has had an enormous social and economic impact on the affected populations. In this study, we aimed to measure the seroprevalence of endemic and emerging viral pathogens in military personnel stationed in Manaus, Amazonas state.MethodsWe measured the seropositivity of antibodies against 19 endemic and emerging viruses in a healthy military personnel group using a hemagglutination inhibition assay (HIA).ResultsOverall, DENV positivity was 60.4%, and 30.9% of the individuals reacted against ZIKV. Also, 46.6%, 54.7%, 51.3% and 48.7% individuals reacted against West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Ilheus virus (ILHV) and Rocio virus (ROCV), respectively. Individuals with high DENV HIA titer reacted more frequently with ZIKV or WNV compared to those with low HIA titers. Observed cross-reactivity between Flaviviruses varied depending on the virus serogroup. Additionally, 0.6% and 0.3% individuals were seropositive for Oropouche virus (OROV) and Catu virus (CATUV) from the family Peribunyaviridae, respectively. All samples were negative for Eastern Equine Encephalitis virus (EEEV), Western Equine Encephalomyelitis virus (WEEV), Mayaro virus (MAYV), Mucambo virus (MUCV) and CHIKV from the family Togaviridae.ConclusionsA high proportion of individuals in our high-risk population (~ 60%) lacked antibodies against major endemic and emerging viruses, which makes them susceptible for further infections. Military personnel serving in the Amazon region could serve as sentinels to strengthen global infectious disease surveillance, particularly in remote areas.Graphical abstract

Pan-protective anti-alphavirus human antibodies target a conserved E1 protein epitope

Alphaviruses are emerging, mosquito-transmitted pathogens that cause musculoskeletal and neurological disease in humans. Although neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses have not been reported. Here, we identify DC2.112 and DC2.315, two pan-protective yet poorly neutralizing human monoclonal antibodies (mAbs) that avidly bind to viral antigen on the surface of cells infected with arthritogenic and encephalitic alphaviruses. These mAbs engage a conserved epitope in domain II of the E1 protein proximal to and within the fusion peptide. Treatment with DC2.112 or DC2.315 protects mice against infection by both arthritogenic (chikungunya and Mayaro) and encephalitic (Venezuelan, Eastern, and Western equine encephalitis) alphaviruses through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions. These findings define a conserved epitope recognized by weakly neutralizing yet protective antibodies that could be targeted for pan-alphavirus immunotherapy and vaccine design.

The effect of urbanization and climate change on the mosquito population in the Pearl River Delta region of China.

The rising incidence of mosquito-borne diseases is a global concern. Changes in regional climate, due to urbanization and global greenhouse gas concentrations, may affect the ecology of mosquitoes and mosquito-transmitted pathogens. The effects of changes in climate on mosquito population dynamics are complex but critical and urgent for implementing more effective public health policies. This study quantified the impact of urbanization and global climate change on the population of the mosquito species, Culex quinquefasciatus, in the Pearl River Delta region in southern China-an area that has undergone substantial urbanization and is expected to experience changes in climate. This study employed a mechanistic model to simulate mosquito population dynamics under various greenhouse gas emission and land-cover change scenarios based on climate data provided by a state-of-the-art regional climate model. Our results show a 12.6% decrease in the annual mosquito population in newly urbanized areas and a 5.9% increase in the annual mosquito population in existing urban areas. Furthermore, changing climate conditions are projected to cause a 15-17% reduction in the total annual mosquito population; however, the change will not be uniform throughout the year. Peak months exhibit a reduction in population, whereas non-peak months show a significant increase. These findings suggest mosquito control strategies may need to be adjusted to respond to the impacts of urbanization and climate change on mosquito population dynamics to maximize effectiveness. Region specific, quantitative analyses of environmental impacts on mosquito-borne disease ecology, like this study, are needed to provide policy makers with a scientific reference to guide the formation of effective transmission intervention strategies.

The ZIKA Virus Delays Cell Death Through the Anti-Apoptotic Bcl-2 Family Proteins.

Zika virus (ZIKV) is an emerging human mosquito-transmitted pathogen of global concern, known to be associated with complications such as congenital defects and neurological disorders in adults. ZIKV infection is associated with induction of cell death. However, previous studies suggest that the virally induced apoptosis occurs at a slower rate compared to the course of viral production. In this present study, we investigated the capacity of ZIKV to delay host cell apoptosis. We provide evidence that ZIKV has the ability to interfere with apoptosis whether it is intrinsically or extrinsically induced. In cells expressing viral replicon-type constructions, we show that this control is achieved through replication. Finally, our work highlights an important role for anti-apoptotic Bcl-2 family protein in the ability of ZIKV to control apoptotic pathways, avoiding premature cell death and thereby promoting virus replication in the host-cell.

Sera-epidemiology of Dengue Virus Infection in Clinically Suspected Patients Attended in Dhaka Medical College Hospital During January to December 2016

Introduction: Dengue is now established as one of the most important Arboviral infection in tropical countries. As the epidemic continues worldwide, this Aedes mosquito-transmitted pathogen is considered a major re-emerging tropical disease and significant public health concern. The study was conducted to determine the sero-epidemiology and clinical picture of dengue virus infection among the clinically suspected patients attended in Dhaka Medical College Hospital during January to December 2016.Materials and Methods: The study was carried out on 145 clinically suspected Dengue patients attended in Dhaka Medical College Hospital from January to December 2016 who had fever with temperature &gt;39°C. Blood sample was collected during acute febrile phase of patients and separated sera were tested for NS1 dengue antigen and IgM antibodies using commercial test kits (NS1 by OMC Healthcare (Pvt.) Ltd &amp; IgM antibody by Omega Diagnostics Ltd.).Results: Of the total 145 patients 40 (27.6%) were positive for Dengue NS1 antigen and or IgM antibody test. Of the positive 40 patients, 18 (45%) were positive for NS1, 19 (47.5%) were positive for IgM and 3 (7.5%) were positive for both NS1 and IgM antibody. Major clinical manifestations were fever with arthralgia (79.5%) and retro-orbital pain (50%).Conclusion: Outbreak of Dengue fever is continuing every year and typical presentation of Dengue fever has changed. Arthralgia and retro orbital pain was the most common clinical feature in the present study instead of headache and rash with fever. A large number of suspected dengue cases were negative by Dengue tests for antigen NS1 and IgM antibody which suggests other organisms of similar clinical manifestations are becoming prevalent in Dhaka city.J Dhaka Medical College, Vol. 26, No.2, October, 2017, Page 111-116

Molecular aspects of Rift Valley fever virus and the emergence of reassortants.

Rift Valley fever phlebovirus (RVFV) is a mosquito-transmitted pathogen endemic to sub-Saharan Africa and the Arabian Peninsula. RVFV is a threat to both animal and human health and has costly economic consequences mainly related to livestock production and trade. Competent hosts and vectors for RVFV are widespread, existing outside of endemic countries including the USA. Thus, the possibility of RVFV spreading to the USA or other countries worldwide is of significant concern. RVFV (genus Phlebovirus) is comprised of an enveloped virion containing a three-segmented, negative-stranded RNA genome that is able to undergo genetic reassortment. Reassortment has the potential to produce viruses that are more pathogenic, easily transmissible, and that have wider vector or host range. This is especially concerning because of the wide use of live attenuated vaccine strains throughout endemic countries. This review focuses on the molecular aspects of RVFV, genetic diversity of RVFV strains, and RVFV reassortment.

A Chikungunya Virus trans-Replicase System Reveals the Importance of Delayed Nonstructural Polyprotein Processing for Efficient Replication Complex Formation in Mosquito Cells.

Chikungunya virus (CHIKV) is a medically important alphavirus that is transmitted by Aedes aegypti and Aedes albopictus mosquitoes. The viral replicase complex consists of four nonstructural proteins (nsPs) expressed as a polyprotein precursor and encompasses all enzymatic activities required for viral RNA replication. nsPs interact with host components of which most are still poorly understood, especially in mosquitos. A CHIKV trans-replicase system that allows the uncoupling of RNA replication and nsP expression was adapted to mosquito cells and subsequently used for analysis of universal and host-specific effects of 17 different nonstructural polyprotein (ns-polyprotein) mutations. It was found that mutations blocking nsP enzymatic activities as well as insertions of enhanced green fluorescent protein (EGFP) into different nsPs had similar effects on trans-replicase activity regardless of the host (i.e., mammalian or mosquito). Mutations that slow down or accelerate ns-polyprotein processing generally had no effect or reduced trans-replicase activity in mammalian cells, while in mosquito cells most of them increased trans-replicase activity prominently. Increased RNA replication in mosquito cells was counteracted by an antiviral RNA interference (RNAi) response. Substitution of the W258 residue in the membrane binding peptide of nsP1 resulted in a temperature-sensitive defect, in the context of both the trans-replicase and infectious CHIKV. The defect was compensated for by secondary mutations selected during passaging of mutant CHIKV. These findings demonstrate the value of alphavirus trans-replicase systems for studies of viral RNA replication and virus-host interactions.IMPORTANCE Chikungunya virus is an important mosquito-transmitted human pathogen. This virus actively replicates in mosquitoes, but the underlying molecular mechanisms and interactions of viral and host components are poorly understood. This is partly due to the lack of reliable systems for functional analysis of viral nonstructural polyproteins (ns-polyproteins) and nonstructural proteins (nsPs) in mosquito cells. Adaption of a CHIKV trans-replicase system allowed study of the effects of mutations in the ns-polyprotein on RNA replication in cells derived from mammalian and mosquito hosts. We found that a slowdown of ns-polyprotein processing facilitates replication complex formation and/or functioning in mosquito cells and that this process is antagonized by the natural RNAi defense system present in mosquito cells. The mosquito-adapted CHIKV trans-replicase system represents a valuable tool to study alphavirus-mosquito interactions at the molecular level and to develop advanced antiviral strategies.

Identification of Candidate Iron Transporters From the ZIP/ZnT Gene Families in the Mosquito Aedes aegypti.

Mosquito-transmitted viral pathogens, such as dengue and Zika, afflict tens of thousands of people every year. These viruses are transmitted during the blood-feeding process that is required for mosquito reproduction, the most important vector being Aedes aegypti. While vertebrate blood is rich in protein, its high iron content is potentially toxic to mosquitoes. Although iron transport and sequestration are essential in the reproduction of vector mosquitoes, we discovered that culicine mosquitoes lack homologs of the common iron transporter NRAMP. Using a novel cell-based screen, we identified two ZIP and one ZnT genes as candidate iron transporters in the mosquito A. aegypti, the vector of dengue, Zika, and chikungunya. We determined the organ-specific expression pattern of these genes at critical time points in early reproduction. The result indicates modulation of these genes upon blood feeding, especially a ZIP13 homolog that is highly up-regulated after blood feeding, suggesting its importance in iron mobilization during blood digestion and reproduction. Gene silencing resulted in differential iron accumulation in the midgut and ovaries. This study sets a foundation for further investigation of iron transport and control strategies of this viral vector.

Reverse genetic system, genetically stable reporter viruses and packaged subgenomic replicon based on a Brazilian Zika virus isolate.

Zika virus (ZIKV, genus Flavivirus) has emerged as a major mosquito-transmitted human pathogen, with recent outbreaks associated with an increased incidence of neurological complications, particularly microcephaly and the Guillain-Barré syndrome. Because the virus has only very recently emerged as an important pathogen, research is being hampered by a lack of reliable molecular tools. Here we report an infectious cDNA (icDNA) clone for ZIKV isolate BeH819015 from Brazil, which was selected as representative of South American ZIKV isolated at early stages of the outbreak. icDNA clones were assembled from synthetic DNA fragments corresponding to the consensus sequence of the BeH819015 isolate. Virus rescued from the icDNA clone had properties identical to a natural ZIKV isolate from South America. Variants of the clone-derived virus, expressing nanoluciferase, enhanced green fluorescent or mCherry marker proteins in both mammalian and insect cells and being genetically stable for multiple in vitro passages, were obtained. A ZIKV subgenomic replicon, lacking a prM- and E glycoprotein encoding region and expressing a Gaussia luciferase marker, was constructed and shown to replicate both in mammalian and insect cells. In the presence of the Semliki Forest virus replicon, expressing ZIKV structural proteins, the ZIKV replicon was packaged into virus-replicon particles. Efficient reverse genetic systems, genetically stable marker viruses and packaged replicons offer significant improvements for biological studies of ZIKV infection and disease, as well as for the development of antiviral approaches.

Epitope Addition and Ablation via Manipulation of a Dengue Virus Serotype 1 Infectious Clone.

Despite the clinical relevance, dengue virus (DENV) research has been hampered by the absence of robust reverse genetic systems to manipulate the viral serotypes for propagation and generation of mutant viruses. In this article, we describe application of an infectious clone system for DENV serotype 1 (DENV1). Similar to previous clones in both flaviviruses and coronaviruses, the approach constructs a panel of contiguous cDNAs that span the DENV genome and can be systematically and directionally assembled to produce viable, full-length viruses. Comparison of the virus derived from the infectious clone with the original viral isolate reveals identical sequence, comparable endpoint titers, and similar focus staining. Both focus-forming assays and percent infection by flow cytometry revealed overlapping replication levels in two different cell types. Moreover, serotype-specific monoclonal antibodies (MAbs) bound similarly to infectious clone and the natural isolate. Using the clone, we were able to insert a DENV4 type-specific epitope recognized by primate MAb 5H2 into envelope (E) protein domain I (EDI) of DENV1 and recover a viable chimeric recombinant virus. The recombinant DENV1 virus was recognized and neutralized by the DENV4 type-specific 5H2 MAb. The introduction of the 5H2 epitope ablated two epitopes on DENV1 EDI recognized by human MAbs (1F4 and 14C10) that strongly neutralize DENV1. Together, the work demonstrates the utility of the infectious clone and provides a resource to rapidly manipulate the DENV1 serotype for generation of recombinant and mutant viruses. IMPORTANCE Dengue viruses (DENVs) are significant mosquito-transmitted pathogens that cause widespread infection and can lead to severe infection and complications. Here we further characterize a novel and robust DENV serotype 1 (DENV1) infectious clone system that can be used to support basic and applied research. We demonstrate how the system can be used to probe the antigenic relationships between strains by creating viable recombinant viruses that display or lack major antibody epitopes. The DENV1 clone system and recombinant viruses can be used to analyze existing vaccine immune responses and inform second-generation bivalent vaccine designs.

Rearing of Culex spp. and Aedes spp. Mosquitoes.

Mosquito-transmitted pathogens cause major public health problems and contribute substantially to the global burden of disease. Aedes mosquitoes transmit dengue, Zika, yellow fever, and Chikungunya viruses; Culex mosquitoes transmit West Nile, Japanese encephalitis, and Saint Louis encephalitis viruses, among others. Experiments utilizing laboratory-reared colonized mosquitoes can address many issues such as vector biology, vector competence, vector-pathogen interaction, and vector control. The establishment of healthy and standardized mosquito colonies requires generation and implementation of protocols, attention to detail, and an understanding of the factors that affect mosquito fitness, such as temperature and humidity, nutrient quality and availability, population density, blood feeding and mating behavior, and egg-laying requirements. Here, we present a standard protocol for the rearing of Culex spp. and Aedes spp. mosquitoes and maintenance of the mosquito colony.

Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice.

Mosquito-transmitted pathogens are among the leading causes of severe disease and death in humans. Components within the saliva of mosquito vectors facilitate blood feeding, modulate host responses, and allow efficient transmission of pathogens, such as Dengue, Zika, yellow fever, West Nile, Japanese encephalitis, and chikungunya viruses, as well as Plasmodium parasites, among others. Here, we describe standardized methods to assess the impact of mosquito-derived factors on immune responses and pathogenesis in mouse models of infection. This protocol includes the generation of mosquito salivary gland extracts and intradermal inoculation of mouse ears. Ultimately, the information obtained from using these techniques can help reveal fundamental mechanisms of interaction between pathogens, mosquito vectors, and the mammalian host. In addition, this protocol can help establish improved infection challenge models for pre-clinical testing of vaccines or therapeutics that take into account the natural route of transmission via mosquitoes.