Field-deployable molecular diagnostic platform for arbovirus and Wolbachia detection in Aedes aegypti

Background Surveillance of mosquito infection status is critical for planning and deployment of proper mosquito control initiatives. Concurrently, Wolbachia is being widely used as a control method for arboviral transmission. Point-of-care (POC) detection assays are necessary for monitoring the infection prevalence and geographic range of viruses as well as Wolbachia in mosquito vector populations. We therefore assessed the novel qPCR bCUBE molecular diagnostic system as a tool for virus and Wolbachia detection. Results We developed a reliable, specific, and sensitive diagnostic assay for detecting Zika virus and dengue virus serotype 2 using the real-time qPCR platform bCUBE. With bCUBE-based qRT-PCR, both Wolbachia bacterium and virus RNA could be reliably detected in individually infected Ae. aegypti mosquitoes and in pools of 5, 10, or 15 mosquitoes. Conclusions The portable qPCR bCUBE diagnostic platform is capable of detecting Zika and dengue virus as well as Wolbachia in mosquitoes and therefore has potential as a practical field-deployable diagnostic test for vector-borne disease surveillance programs.


Introduction 49
Arthropod-borne diseases threaten over two-thirds of the global population and are exhibiting an 50 ongoing expansion of their geographic range and prevalence as a result of climate change, 51 urbanization, and globalization (1)(2)(3)(4). According to the World Health Organization, dengue virus 52 infection has increased 30-fold over the past 50 years and now affects nearly 100 million people, 53 primarily in the Americas and Asia (5, 6). Recent outbreaks of Zika virus have also led to 54 widespread concern because of the virus's ability to cause newborn malformations (7,8). outcomes. Aedes mosquitoes, with their aggressive blood-feeding behavior, have allowed for 62 efficient human-mosquito transmission of these arboviruses (3, 11). The geographical presence 63 of this mosquito vector has dramatically increased in the last few decades, leading to an 64 expanded transmission of these arboviruses (12)(13)(14). The lack of vaccines and treatment against 65 these arboviruses highlights the importance of mosquito control and surveillance strategies (15). 66 Current and future mosquito-targeted control strategies are, and will be, having a significant 67 epidemiological impact but also require mosquito and pathogen surveillance (15). Surveillance 68 of geographical distribution of the vector mosquitoes and the pathogens they carry is an essential 69 component of disease prevention and control. 70 71 Wolbachia pipientis is increasingly being used as a method for limiting arboviral transmission in 72 dengue and Zika virus-endemic countries. This endosymbiotic bacterium is passed from mother 73 to offspring and has been shown to suppress dengue and Zika virus transmission in Aedes 74 mosquitoes (16, 17). Field-release trials require continual monitoring of Wolbachia-infected Ae. 75 aegypti in mosquito populations (18-20). Real-time qPCR has been used as the primary method 76 for detecting Wolbachia genetic material in mosquitoes (21). 77 78 Accurate, rapid, and cost-effective mosquito and pathogen surveillance is critical for monitoring 79 infection prevalence and thereby mitigating transmission risk. Methods currently used for 80 arboviral detection include viral culture, antibody detection, antigen detection, and RNA 81 detection using quantitative real-time RT-PCR (qPCR). Currently, the qPCR method is the gold 82 standard because of its use of specific molecular markers (22,23 In the present study, we evaluated a novel and portable real-time PCR platform, bCUBE (Hyris, 104 Ltd), as a PCR-based arboviral detection method with potential for field-deployability. bCUBE 105 makes possible the genetic testing of biological samples in any setting, at any time, with real-106 time access to results on its dedicated cloud-based software platform. This technology is a 107 portable device, similar in size to a Bluetooth speaker, which is used for biological analysis in 108 several fields, including agricultural pest control. The device can be operated from a laptop, 109 tablet, or cellular device through an easy-to-use gateway and generates centralized data analysis 110 immediately after a reaction. The device is capable of performing thermocycling reactions such 111 as real-time PCR, as well as loop-mediated isothermal amplification (LAMP). The system can be 112 calibrated to distinguish between positive and negative samples in a single reaction with 113 predetermined conditions that have been established ahead of time in the laboratory. This 114 feature allows the bCUBE to be operated by individuals lacking in depth training in qPCR assays 115 and data analysis skills. We have now explored the use of bCUBE technology for detection of 116 both dengue and Zika virus in Aedes aegypti, optimizing and standardizing the sample 117 preparation method to be used with a commercially available one-step qRT-PCR assay kit. 75-80% humidity with a 12-h light/dark cycle. Mosquitoes were reared using a standard rearing 133 protocol, and colonies were maintained on Swiss Webster mice (Charles River Laboratories). 134 DENV2 and ZIKV infection of mosquitoes was carried out as previously described (37). Virus 135 supplemented blood was fed to mosquitoes using an artificial membrane glass feeder, and 136 infected mosquitoes were double-caged and incubated in a reach-in incubator under conditions 137 similar to those in the standard insectary chamber described above. Wolbachia wAlbB-infected 138 (41) ( Table 1). The primers were modified and optimized for bCUBE qPCR (Table 1). Primer 162 sequences were assessed with NCBI Blast to search for potential cross-reactivity against other 163 viruses, bacteria, and vectors. Previously established primers for wAlbB detection were used for 164 Wolbachia-infected Ae. aegypti (42). The sequences of the primers are summarized in Table 1. Previously established primers were used, as listed in Table 1  Melting curve analysis was performed at the end by cooling to 60°C, followed by heating to 215 95°C at 0.05°C/s. Automatic data analysis was generated with the Hyris data analysis platform. 216 Two technical replicates were performed on the platform for each biological replicate. Three 217 biological replicates were included for each assay. Transcriptase (Promega). Conventional PCR was used with the primer (Table 1)   The reliability of the portable bCUBE qPCR machine is equal to that of a laboratory-272

standard qPCR system 273
The reliability of the novel bCUBE real-time PCR system was validated by comparing the 274 system to a standard qPCR instrument (Applied Biosystems StepOnePlus, ABI). Our proof-of-275 principle studies used mosquito tissue homogenates (abdomen with the midgut, and head with 276 the thorax) in squash buffer that were tested with the mosquito housekeeping gene (ribosomal 277 protein S17, rps17). Aedes aegypti were dissected into crude compartments, including the 278 abdomen and head plus thorax. Identical samples were processed using both qPCR machines, 279 identical settings, and identical SYBR Green Master Mix and instructions (Applied Biosystems). 280 As shown in Fig. 1, the housekeeping Rps17 gene was detected in all 24 samples by both 281 machines. The cycle threshold (Ct) values for the Rps17 gene in Ae. aegypti were detected 282 significantly earlier in the bCUBE system than in the laboratory standard procedure, with a mean 283 difference of 2.40 Ct value (paired t-test, p-value < 0.0001). This result suggests that the bCUBE 284 qPCR is reliable in detecting a mosquito gene from a single mosquito tissue using a crude 285 sample-preparation method.

Fig. 1. Comparison of Ae. aegypti Rps17 gene detection in abdomen (ABD) and head plus 288 thorax (HT) samples using bCUBE and Applied Biosystems real-time PCR. Ae. aegypti 289
abdomens and head plus thoraces were collected, and gDNA was extracted using the squash 290 buffer method. The housekeeping gene Rps17 was amplified using the bCUBE and Applied 291 Biosystems real-time PCR systems. Cycle threshold (Ct) values are plotted. Statistical 292 significance was determined by Mann-Whitney test (****, p < 0.0001; Mann-Whitney). 293 294

bCUBE-based one-step qRT-PCR is sufficiently sensitive for DENV2 and ZIKV detection 295
We performed sensitivity studies on the bCUBE-based qPCR system based on absolute 296 quantification standard curve analysis. Cloned fragments of DENV2 and ZIKV were quantitated 297 for RNA copy number and serially diluted to establish a qPCR standard curve. End-point 298 limitations were set to < 30 for Zika virus, whereas dengue end-point limitations were set to < 29 299 based on standard curve ( Supplementary Fig. 1). Correlation coefficient (R 2 ) values were 0.99 300 for DENV2 and 0.98 for ZIKV. Based on the standard curve analyses, the limit of detection 301 range was 10 viral RNA copy numbers for DENV2 and ZIKV. Amplification above these values 302 was attributed to non-specific amplification. 303 304

bCUBE-based qRT-PCR is specific for DENV2 and ZIKV detection 305
The specificity of the ZIKV and DENV2 assays was then determined using a cross-reactivity 306 panel. To address challenges involving primer cross-reactivity with other arboviruses (46, 48) as 307 well as the tendency toward false-positive amplification in negative samples, we tested the 308 specificity of the assay against a panel of Flaviviruses and one Alphavirus. The frequently co-309 circulating arboviral RNA samples, obtained from BEI resources (Table 2)   The ZIKV and DENV2 primer pairs showed a high degree of specificity for amplification of 312 their respective virus RNAs ( NanoDrop for molecular weight and amplified on qRT-PCR with their own primer sequences. 320 321 322 bCUBE-based qRT-PCR is as accurate as plaque assay in detecting infectious viral RNA 323 PCR detection of non-infectious viral RNA is possible when viral RNA is not actively 324 replicating (49). Therefore, the viral RNA copy number and viral infectious RNA load can differ 325 in mosquitoes. We thus sought to evaluate the infection prevalence using bCUBE qRT-PCR and 326 plaque assays as opposed to titer comparisons. Three biological replicates of Ae. aegypti were 327 infected with ZIKV and DENV-2. Each replicate of infected mosquitoes was collected and 328 separated into two halves to compare infectious prevalence as determined by plaque assay and 329 bCUBE qPCR analysis. In each of the three replicates, no-significant differences were seen in 330 infection prevalence values for both ZIKV and DENV2 between the two methods. These results 331 suggest that bCUBE qRT-PCR analysis can accurately detect infectious ZIKV AND DENV2 332 (Fig. 2). 333 quantitated in samples containing single abdomen with midguts and head with thoraces of 351 infected mosquitoes (Fig. 3). Corresponding leg (L) samples were also collected on day 14. Viral RNA extraction was 359 performed using the squash method, followed by amplification using 1-step qPCR on the 360 bCUBE. Uninfected mosquitoes were not included.

362
Next we investigated detection of virus in leg samples of Ae. aegypti (Fig. 3). Forty-six leg 363 samples were collected from mosquitoes that were also analyzed for infection in the heads and 364 thoraces to confirm infectious status. We found significant differences between the Ct values for 365 the head with thorax samples versus the leg samples. For the leg samples, six false negative 366 sample results were obtained for DENV2, and two false negative results were obtained for 367 ZIKV. One false positive result was detected for ZIKV (Fig. 4). These results indicate that Aedes aegypti used as a negative control. Because of the false negative results, the DENV2 370 sensitivity of leg samples was 76% (95% CI, 54.87% to 90.64%) and the diagnostic specificity of 371 leg samples was 100% (95% CI, 83.89% to 100.00%). The sensitivity for ZIKV of leg samples 372 was 94.59% (95% CI, 81.81% to 99.34%), and the specificity was 88.89% (95% CI, 51.75% to 373 99.72%). The overall accuracy of the bCUBE qPCR platform for detecting leg samples was 374 93.48% for ZIKV (95% CI, 82.10 to 98.63%) and 86.96% for DENV2 (95% CI, 73.75% to 375 95.06%) (Fig. 4). 376 377 Fig. 4. Fig. 4. Viral concentration of individual head with thoraces compared to legs. 378 Infected Ae. aegypti (n=46) were collected, and identical samples of head with thorax and legs 379 were amplified for Zika virus (A) and DENV2 (B) using bCUBE-based qRT-PCR. Infection 380 status was compared (****, p<0.0001; paired two-tailed t-test). its infectious cargo is essential for planning adequate mosquito control to reduce transmission. 408 Endemic countries frequently lack surveillance resources for monitoring pathogen and vector 409 distribution. Therefore, cost-effective field-deployable pathogen detection methods can greatly 410 facilitate surveillance and control efforts. We sought to develop a method for detecting viral 411 pathogens in their Aedes aegypti vector with the potential for field deployment. Currently, 412 pathogen detection in the field is limited by expensive lab-based equipment and reactions and the 413 necessity for highly trained personnel to process samples. Real-time qPCR has long been 414 considered the gold standard for viral detection; however, inadequate access to sample 415 preparation methods and the requirement for bulky equipment render this method incompatible 416 with fieldwork. 417

418
As an alternative to laboratory PCR, we have now explored the use of bCUBE, a novel qPCR 419 platform, with a simple nucleic acid extraction method for detection of arboviruses and 420 Wolbachia in mosquito vectors. Actions were taken to limit the cost needed for analysis. We 421 used DNA dye binding qPCR commercial kits as an alternative to hydrolysis probe-based qPCR 422 kits. Furthermore, we extracted DNA/RNA using the squash buffer methodology as opposed to 423 expensive extraction kits. It is important to note that current bCUBE cartridges are restricted to We confirmed the reliability of the bCUBE by comparing its performance against laboratory 430 standards. Therefore, bCUBE is capable of performing real-time qPCR while overcoming the 431 barriers presented by the need for bulky equipment. We then employed a crude extraction 432 method using squash buffer to extract viral DENV2 and ZIKV RNA from mosquitoes for use in 433 the bCUBE system. To address challenges involving primer cross-reactivity with other 434 arboviruses (46, 48) as well as the tendency toward false-positive amplification in negative 435 samples, we tested the specificity of the assay against a panel of frequently co-circulating 436 arboviruses. The results confirmed the assay's specificity for DENV2 and ZIKV RNA, with the 437 potential to apply these primer pairs for field conditions. To negate any non-specific 438 amplification of the host's genetic material, we used a standard curve to determine the limit of 439 detection and subsequently, the cut off values for detecting ZIKV and DENV2. This allowed for 440 positive identification of viral RNA from infected mosquito samples while limiting the 441 amplification of false positive samples as a result of background noise. Following primer 442 optimization, we compared the bCUBE assay to plaque assays for determining infection 443 prevalence. Because qPCR is generally capable of detecting low RNA copy numbers in 444 individual samples, it is important to evaluate the assay's potential for detecting infectious viral 445 RNA rather than viral RNA that is not replicating (49, 51). No significant differences were seen 446 between the bCUBE and plaque assay for three biological replicates, suggesting that the bCUBE 447 assay is able to detect infectious viral RNA. 448

449
We evaluated the use of Ae. aegypti leg samples for viral detection. Some infected leg samples 450 turned out to be false negatives suggesting this sample type is unreliable for assaying virus. We have developed a highly sensitive qPCR method for detection and quantitation of DENV2 471 and ZIKV in single mosquitoes using the portable qPCR Hyris bCUBE platform. Our assay 472 allowed for sensitive and reliable DENV2 and ZIKV detection that make it a potential tool for 473 mosquito surveillance programs in endemic countries facing arboviral outbreaks. The detection 474 of Wolbachia in Ae. aegypti further demonstrates the platform's ability to be used as a multi-475