Mosquito excreta: A sample type with many potential applications for the investigation of Ross River virus and West Nile virus ecology

Abstract

BackgroundEmerging and re-emerging arthropod-borne viruses (arboviruses) cause human and animal disease globally. Field and laboratory investigation of mosquito-borne arboviruses requires analysis of mosquito samples, either individually, in pools, or a body component, or secretion such as saliva. We assessed the applicability of mosquito excreta as a sample type that could be utilized during studies of Ross River and West Nile viruses, which could be applied to the study of other arboviruses.Methodology/Principal findingsMosquitoes were fed separate blood meals spiked with Ross River virus and West Nile virus. Excreta was collected daily by swabbing the bottom of containers containing batches and individual mosquitoes at different time points. The samples were analyzed by real-time RT-PCR or cell culture enzyme immunoassay. Viral RNA in excreta from batches of mosquitoes was detected continuously from day 2 to day 15 post feeding. Viral RNA was detected in excreta from at least one individual mosquito at all timepoints, with 64% and 27% of samples positive for RRV and WNV, respectively. Excretion of viral RNA was correlated with viral dissemination in the mosquito. The proportion of positive excreta samples was higher than the proportion of positive saliva samples, suggesting that excreta offers an attractive sample for analysis and could be used as an indicator of potential transmission. Importantly, only low levels of infectious virus were detected by cell culture, suggesting a relatively low risk to personnel handling mosquito excreta.Conclusions/SignificanceMosquito excreta is easily collected and provides a simple and efficient method for assessing viral dissemination, with applications ranging from vector competence experiments to complementing sugar-based arbovirus surveillance in the field, or potentially as a sample system for virus discovery.

Arbovirus surveillance using FTATMcards in modified CO2‐baited encephalitis virus surveillance traps in the Northern Territory, Australia

“…Future work is needed to fully evaluate the sensitivity of the FTA TM card system is comparison to sentinel chickens. However, since this study was conducted, Ramirez et al (2018a) have found that detection of arboviruses in excreta under laboratory conditions was more sensitive compared to detection in saliva, and that excreta samples tested positive to arbovirus RNA within a much shorter time period following the ingestion of an infectious blood meal compared to mosquito saliva samples, where the intrinsic virus incubation period delayed rapid virus RNA detection. Using mosquito excreta for arbovirus detection has since been successfully tested in the field by Meyer et al (2019), with this method another potential alternative for rapid arbovirus detection.…”

Genetic, Morphological and Antigenic Relationships between Mesonivirus Isolates from Australian Mosquitoes and Evidence for Their Horizontal Transmission

“…However, confirmation of mesonivirus transmission via the saliva was recently provided in mosquito transmission studies [ 18 ]. The inability of mesoniviruses to replicate in vertebrate cells from a range of species suggests that a role for viral amplification in a vertebrate host is unlikely and thus, it is more likely that oral infection of mosquitoes occurs via co-feeding of an infected mosquito on the same source, whether that be virus-contaminated nectar or the simultaneous feeding on the same vertebrate [ 43 , 44 , 45 ].…”

Metagenomic Analysis of the Virome of Mosquito Excreta

“…Excreta samples were collected from groups of 5 Culex annulirostris or Aedes vigilax mosquitoes exposed to either WNV (Kunjin subtype [WNV KUN ]) or RRV, by swabbing a Parafilm disc placed in the bottom of each container ( 14 ). RNA extracted from all six excreta samples collected from groups of experimentally infected mosquitoes were positive for either WNV KUN or RRV by RT-rtPCR, with mean (± standard error of the mean [SEM]) cycle threshold ( C T ) values of 26.3 ± 0.4 and 26.8 ± 1.0, respectively.…”

“…Recently, arboviruses such as the dengue viruses (DENVs), Ross River virus (RRV), and West Nile virus (WNV) have been detected in mosquito excreta by reverse-transcription real-time PCR (RT-rtPCR) by us and others (13,14). Additionally, hepatitis B virus, which does not replicate in the mosquito, has been detected in mosquito excreta by RT-PCR and Southern blotting up to 72 h after the ingestion of an infectious blood meal (15).…”