Metagenomic Analysis of the Virome of Mosquito Excreta.

Traditional screening for arboviruses in mosquitoes requires a priori knowledge and the utilization of appropriate assays for their detection. Mosquitoes can also provide other valuable information, including unexpected or novel arboviruses, nonarboviral pathogens ingested from hosts they feed on, and their own genetic material. Metagenomic analysis using next-generation sequencing (NGS) is a rapidly advancing technology that allows us to potentially obtain all this information from a mosquito sample without any prior knowledge of virus, host, or vector. Moreover, it has been recently demonstrated that pathogens, including arboviruses and parasites, can be detected in mosquito excreta by molecular methods. In this study, we investigated whether RNA viruses could be detected in mosquito excreta by NGS. Excreta samples were collected from Aedes vigilax and Culex annulirostris experimentally exposed to either Ross River or West Nile viruses and from field mosquitoes collected across Queensland, Australia. Total RNA was extracted from the excreta samples, reverse transcribed to cDNA, and sequenced using the Illumina NextSeq 500 platform. Bioinformatic analyses from the generated reads demonstrate that mosquito excreta provide sufficient RNA for NGS, allowing the assembly of near-full-length viral genomes. We detected Australian Anopheles totivirus, Wuhan insect virus 33, and Hubei odonate virus 5 and identified seven potentially novel viruses closely related to members of the order Picornavirales (2/7) and to previously described, but unclassified, RNA viruses (5/7). Our results suggest that metagenomic analysis of mosquito excreta has great potential for virus discovery and for unbiased arbovirus surveillance in the near future.IMPORTANCE When a mosquito feeds on a host, it ingests not only its blood meal but also an assortment of microorganisms that are present in the blood, thus acting as an environmental sampler. By using specific tests, it is possible to detect arthropod-borne viruses (arboviruses) like dengue and West Nile viruses in mosquito excreta. Here, we explored the use of next-generation sequencing (NGS) for unbiased detection of RNA viruses present in excreta from experimentally infected and field-collected mosquitoes. We have demonstrated that mosquito excreta provide a suitable template for NGS and that it is possible to recover and assemble near-full-length genomes of both arboviruses and insect-borne viruses, including potentially novel ones. These results importantly show the direct practicality of the use of mosquito excreta for NGS, which in the future could be used for virus discovery, environmental virome sampling, and arbovirus surveillance.

Development and Field Evaluation of a System to Collect Mosquito Excreta for the Detection of Arboviruses.

Mosquito-borne diseases are a major public health concern globally and early detection of pathogens is critical to implement vector management and control strategies. Existing methods for pathogen detection include screening sentinel animals for antibodies and analyzing mosquitoes for pathogen presence. While these methods are effective, they are also expensive, labor-intense, and logistically challenging. To address these limitations, a new method was developed whereby mosquito saliva is collected on honey-coated nucleic acid preservation cards which are analyzed by molecular assays for detection of pathogens. However, mosquitoes only expel small amounts of saliva when feeding on these cards, potentially leading to false negatives. Another bodily fluid that is expelled by mosquitoes in larger volumes than saliva is excreta, and recent laboratory experiments have demonstrated that a range of mosquito-borne pathogens can be detected in mosquito excreta. In the current study, we have modified light and passive mosquito traps to collect their excreta and assessed their efficacy in field evaluations. From these field-collections, we detected West Nile, Ross River, and Murray Valley encephalitis viruses. Our findings suggest that mosquito traps are easily modified to collect excreta and, that this system has the potential to enhance detection of pathogens.

Development, Optimization, and Field Evaluation of the Novel Collapsible Passive Trap for Collection of Mosquitoes.

Disease surveillance for mosquito-borne pathogens in remote areas can be challenging. Most traps used to collect mosquitoes either need a source of electricity or are bulky and inflexible, making transportation awkward. To reduce these issues we developed three Collapsible Passive Traps (CPTs) and conducted trials in Cairns, Australia to evaluate the optimal design for a CPT and compared them to traditionally-used traps such as Centers for Disease Control and Prevention (CDC) and Encephalitis Vector Surveillance (EVS) light traps. We found that two of the CPTs collected comparable numbers of mosquitoes and that one of the CPTs outperformed the CDC light trap in collecting Aedes species. Mosquitoes did not have to pass through a fan while entering the CPT, and thus were not damaged and were often alive. Our results suggest that the CPT can be an effective trap for mosquito surveillance, especially in remote areas.

Searching for the proverbial needle in a haystack: advances in mosquito-borne arbovirus surveillance.

Surveillance is critical for the prevention and control of mosquito-borne arboviruses. Detection of elevated or emergent virus activity serves as a warning system to implement appropriate actions to reduce outbreaks. Traditionally, surveillance of arboviruses has relied on the detection of specific antibodies in sentinel animals and/or detection of viruses in pools of mosquitoes collected using a variety of sampling methods. These methods, although immensely useful, have limitations, including the need for a cold chain for sample transport, cross-reactivity between related viruses in serological assays, the requirement for specialized equipment or infrastructure, and overall expense. Advances have recently been made on developing new strategies for arbovirus surveillance. These strategies include sugar-based surveillance, whereby mosquitoes are collected in purpose-built traps and allowed to expectorate on nucleic acid preservation cards which are submitted for virus detection. New diagnostic approaches, such as next-generation sequencing, have the potential to expand the genetic information obtained from samples and aid in virus discovery. Here, we review the advancement of arbovirus surveillance systems over the past decade. Some of the novel approaches presented here have already been validated and are currently being integrated into surveillance programs. Other strategies are still at the experimental stage, and their feasibility in the field is yet to be evaluated.

Modifying the Biogents Sentinel Trap to Increase the Longevity of Captured Aedes aegypti.

Aedes aegypti (L.) (Diptera: Culicidae) is a vector of viruses causing dengue, Zika, chikungunya, and yellow fever and subsequently pose a significant global threat to public health. While sampling live mosquitoes is useful for surveillance purposes, most traps targeting Aedes kill captured mosquitoes. The Biogents Sentinel (BGS) trap, the gold standard for capturing Ae. aegypti, is one such trap. In our study, we modified the BGS trap to increase the survival of captured Ae. aegypti by replacing the trap's catch bag with a catch pot that protects mosquitoes from desiccation by airflow. A sucrose-soaked sponge or nucleic acid preservative card can also be placed inside the pot to enhance mosquito survival and augment arbovirus detection. These modifications to the BGS significantly increased the longevity of mosquitoes captured with weekly survivals of 93% in a semifield structure and 86% in the field. These high survival rates resulted in 3.5 times more alive Ae. aegypti captured weekly in the modified BGS compared to the original BGS, despite 40% lower overall catch rates. These cheap and simple trap modifications facilitate easier specimen identification as well as experiments requiring live field-collected samples such as virus detection from mosquito saliva and excreta.