Stability and detection of nucleic acid from viruses and hosts in mosquito blood meals

Monitoring the presence and spread of pathogens in the environment is of critical importance. Rapid detection of infectious disease outbreaks and prediction of their spread can facilitate early responses of health agencies and reduce the severity of outbreaks. Current sampling methods are sorely limited by available personnel and throughput. For instance, xenosurveillance utilizes captured arthropod vectors, such as mosquitoes, as sampling tools to access blood from a wide variety of vertebrate hosts. Next generation sequencing (NGS) of nucleic acid from individual blooded mosquitoes can be used to identify mosquito and host species, and microorganisms including pathogens circulating within either host. However, there are practical challenges to collecting and processing mosquitoes for xenosurveillance, such as the rapid metabolization or decay of microorganisms within the mosquito midgut. This particularly affects pathogens that do not replicate in mosquitoes, preventing their detection by NGS or other methods. Accordingly, we performed a series of experiments to establish the windows of detection for DNA or RNA from human blood and/or viruses present in mosquito blood meals. Our results will contribute to trap design for mosquito-based xenosurveillance, including sample stabilization and ideal time spent from collection to NGS processing.


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Mosquito-borne disease transmission represents a continued threat to human health and 42 imposes an immense economic burden on at-risk populations (1)(2)(3)(4)(5) an additional challenge. The ability to rapidly detect emerging and established pathogens in humans and zoonotic reservoirs could allow for timely intervention and the potential to reduce outbreak severity by scaling up treatment production and prevention regimes, establishing quarantines and/or limiting 49 contact with or culling afflicted animals. Recent studies have sampled wild mosquitoes to monitor the 50 DNA and RNA of vertebrate blood and associated pathogens (6, 7). This technique, termed 51 xenosurveillance, has been proposed for monitoring known and novel infectious diseases at (8-10).

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The nucleic acid of both captured mosquitoes and blood meals can be analyzed by several 53 approaches. PCR is an affordable method that has been used to screen for targeted hosts and 54 pathogens in mosquito populations, using specific primer sequences for the taxa of interest (11).

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Recently described techniques utilize oligonucleotide hybridization to screen for panels of clinically

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Nucleic acid content of mosquitoes fed with virus-containing blood meals was assessed by 89 qPCR. Each mosquito blood meal was estimated to constitute an inoculum of 1,000 PFU (18). The first 90 study included two RNA viruses, dengue virus 2 New Guinea strain C (DENV-2) and influenza A, which 91 were fed to mosquitos either individually (5*10 5 PFU / mL) or in combination (for a combined total of 92 5*10 5 PFU / mL). Mosquitoes from the control group were either unfed or fed blood without virus.

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DENV-2 is an arbovirus with tropism for Ae. aegypti, and was expected to be detectable by 94 qPCR once it infected midgut tissues and started replicating with a 7 to 10-day extrinsic incubation 95 period (19). In contrast, influenza A, a non-arbovirus and thus a proxy for xenosurveillance, was 96 expected to have a much shorter window of detection, as the qPCR signal was expected to decay after 97 the influenza A genome was digested or broken down. The combined feed was performed to determine whether the presence of more than one virus and/or an infected midgut might affect viral detection for 99 xenosurveillance. 4 or 5 mosquitoes from each feed condition and time point were collected, and RNA 100 was extracted for qPCR.

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We found that influenza was readily detected by qPCR immediately after feeding and for the 102 following 72 hours. However, there was little to no influenza signal at any time after four days post-feed 103 (Fig. 1A). The influenza qPCR signal was otherwise unaffected by the presence of DENV-2 (Fig. 1B).

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The DENV-2 decay pattern was similar to that observed for influenza. A dramatic reduction of viral 105 DENV-2 RNA signal was observed as early as 12 hours post feed, and little to no signal was apparent 106 after 24 hours (Fig. 1C). As expected, the RNA levels began to increase between 100-and 200-hours    130 2). We again found that signals for viral genomes of both SeV and HAdV-5 decayed appreciably 24 131 hours after feeding, and SeV was nearly undetectable by 48 hours. DNA and RNA from human cells 132 within the blood, based on GAPDH levels, decayed somewhat more quickly, with a near complete loss

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RNA were found to be significantly higher in mosquitoes that were held for 24 hours at 4°C (p=.0003),

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suggesting that the blood meal and contents were better preserved by cold temperature (Fig. 3). There 158 was no significant difference between the RNA levels of either GAPDH (p=0.2403) or SeV (p=0.0758) 159 recovered from mosquitoes stored from 12 to 36 hours at 4°C and from those directly collected after 12 160 hours at 27°C. We performed additional tests with bovine blood meals (Fig. 4)

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HAU/mL) and 800 HAU/mL stocks contained 10 9 and 5*10 7 PFU/mL, respectively, and the later was 202 used for all mosquito blood feed experiments.

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Whole heparinized bovine blood (Hemostat, Dixon, CA) was purchased and after starving for 12 hours 225 mosquitoes were fed via an artificial membrane feeder (Hemotek, Blackburn, UK). After feeding 7 to 14 226 fully engorged mosquitoes were transferred to each screened 50 mL tube. Tubes were held at 21° C or 227 4° C for 48 hours. At 48 hours mosquitoes were moved to -80°C until processed.

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All virus feeding experiments were performed with Ae. aegypti under containment conditions.

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Twelve hours prior to blood feeds, female mosquitoes were identified and removed from rearing cages.

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Approximately 60 to 80 females were aspirated into each feeding cup. Fresh whole human blood, 231 preserved with 1.8 mg/mL EDTA, was supplied by the Johns Hopkins University Parasite Core within 232 two days prior to feeding experiments. Blood was allowed to warm to room temperature and virus stock 233 was added such that the final concentration of virus was 5*10 5 PFU/mL, or 1,000 PFU per each 234 expected 2 µL blood meal. Parafilm-covered glass feeders were placed on each feeding cage and 235 warmed to 37°C. Cages of unfed 'control' mosquitoes were kept in the incubation chamber throughout 236 the feed. Each feeder was loaded with 300 µL of blood, with or without virus, and mosquitoes were 237 allowed to feed for up to 60 minutes before being knocked down and visibly fed mosquitoes sorted into

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GGATTGCTGATAAGAGGTTGGTG-3'). Real-time signal of blood feed nucleic acid was normalized to 269 the signal of mosquito ribosomal protein S7 or S17 (for Anopheles and Aedes, respectively).

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The use of xenosurveillance with blood-feeding arthropods presents an enticing possibility for 273 sampling a wide variety of animal and human blood in order to monitor for viruses and other pathogens.

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The breakdown of nucleic acids and the digestive activity in mosquito midguts limits the 281 potential window of detection for blood meals. Our blood feed time courses confirmed that DNA and 282 RNA (human, bovine, Influenza, Adenovirus, and Sendai virus) present in blood meals decayed within 283 several days of feeding, while mosquito nucleic acid levels remained constant (Fig 1, 2, 4).

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Interestingly, detection of viral genomes appeared to decay more slowly than that of human blood (Fig   285  2), perhaps as a consequence of the encapsidated viral genomes being better protected than cellular 286 DNA and RNA. However, this difference could also be due to relative transcript/genome abundance,

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and NGS would be a better platform to assess this effect.

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The optimal 24-to 36-hour window for detection of host blood and viral nucleic acid suggests 289 that mosquito traps without any preservative capabilities should be collected, at minimum, daily. Given 290 that mosquitoes will not necessarily enter the trap immediately after feeding, more regular trap 292 be extended to a longer period post-feeding (from 12 to 36 hours [ Fig. 3] and 0 to 48 hours [ Fig. 4