Detection limit of viruses in mosquitoes by one-step real-time reverse transcription quantitative PCR


 BackgroundMosquitoes are the deadliest animals in the world. Their ability to carry and spread diseases to humans causes millions of deaths every year. Due to the lack of efficient vaccines, the control of mosquito-borne diseases often relies on management of the vector. Traditional control methods such as source reduction and chemical insecticides, have proven not to be sufficient to prevent the proliferation and spread of mosquito populations. The sterile insect technique (SIT) is an additional control method that can be combined with other control tactics to suppress specific mosquito populations. The SIT requires the mass-rearing and release of sterile males that would induce sterility in the wild female population. Samples collected from the environment for laboratory colonization, as well as released males, should be free from mosquito-borne viruses (MBV). Therefore, efficient detection methods with defined detection limits for MBV are required. Although a one-step reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) method was developed to detect arboviruses in human and mosquito samples, its detection limit in mosquito samples has yet to be defined. MethodsWe evaluated the detection sensitivity of one step RT-qPCR for targeted arboviruses in large mosquito pools, using pools of non-infected mosquitoes of various sizes (165, 320 and 1600 mosquitoes) containing one infected mosquito body with defined virus titers of chikungunya virus (CHIKV), usutu virus (USUV), West Nile (WNV) virus and Zika virus (ZIKV).ResultsCHIK, USUV, ZIKV, and WNV virus were detected in all tested pools using the RT-qPCR assay. Moreover, in the largest mosquito pools (1600 mosquitoes), RT-qPCR was able to detect the targeted viruses using different total RNA quantities (10, 1, 0.1 ng per reaction) as a template. Correlating the virus titer with the total RNA quantity allowed predicting the maximum number of mosquitoes per pool in which the RT-qPCR can theoretically detect the virus infection. The corresponding equation uses a Ct value of 36 as a cut-off value for virus detection and a virus copy number of 108 for the positive mosquito body. Based on this formula, the detection limits of CHIK, USUV, ZIKV and WNV were 5.08 x 105, 8.74 x 106, 2.33 x 107, and 5.24 x 105, respectively.ConclusionMosquito borne viruses can be reliably detected by RT-qPCR assay in pools of mosquitoes exceeding 1000 specimens. This will represent an important step to expand pathogen-free colonies for mass-rearing sterile males for programmes that have an SIT component.


Introduction
Mosquitoes are a group of vectors that transmit an array of human viruses. Most of mosquito-borne viruses (MBVs) belong to the Flaviviridae, Togaviridae and Bunyaviridae families and can cause severe human diseases including haemorrhagic fever, biphasic fever, encephalitis, and meningitis [1]. Hundreds of millions of infections are caused by MBV annually [2]. Dengue virus (DENV), chikungunya virus (CHIKV), West Nile virus (WNV) and Zika virus (ZIKV) are the most prevalent arboviruses in the world [3]. Emerging arboviral infections have caused substantial public concerns in recent years [4]. For example, dengue viruses are estimated to infect around 400 million people per year, and over half of the world's population is at risk of the disease [5]. Chikungunya virus emerged from Africa in the mid − 2000s, spreading rst across India and Asia and then into the Americas in 2013 [6]. West Nile virus was rst isolated from a human in 1937. Since then, its distribution has expanded to all continents except the arctic regions [7]. Zika virus outbreaks occurred in the South Paci c in 2013 and in the Americas in 2015 [6]. Usutu virus (USUV) is an emerging mosquito-borne avivirus belonging to the Flaviviridae family [8], and is closely related to Murry Valley encephalitis virus and WNV [8]. USUV virus has been found to cocirculate with WNV in Europe and one asymptomatic blood donor in Austria was found to be potentially co-infected with both viruses [9,10].
No effective antiviral drugs or vaccines are currently available for most of the MBV except yellow fever virus, but they can be prevented by avoiding mosquito bites [11], and hence, effective mosquito-control methods are urgently needed. Traditional mosquito control methods include source reduction by removing the breeding sites and the use of chemical insecticides including the application of insecticidetreated nets (ITNs) and indoor residual spraying (IRS). Although these strategies, in addition to various biological control tactics (such the use of larvivorous sh in larval breeding sites) and personal protection measures have been effective in the past, they have shown limited sustainability, and they have not been able to prevent the proliferation and spread of mosquito populations and their associated MBVs. For the sterile insect technique (SIT), male insects are irradiated with ionising radiation that creates dominant lethal mutations in the sperm. Mating of sterile males with wild females will induce sterility in the wild female target insect population and this method has successfully suppressed or locally eradicated populations of selected insect pests [12,13]. It represents an additional control tactic that can be combined with other suppression tools for sustainable mosquito population management to protect human health and the environment. Combining the incompatible insect technique (IIT) with the SIT enabled suppression of eld populations of Aedes albopictus -the world's most invasive mosquito species -in two isolated villages in China [14]. Millions of factory-reared adult males were released in the eld to compete for, and mate with wild females, resulting in non-viable eggs. Inundative, sequential releases of competitive sterile males over many generations resulted in a signi cant reduction of the wild population.
The SIT requires the establishment of a mother colony from wild collected mosquitoes before up-scaling and ultimately mass-rearing to produce sterile males for releases. The collection of wild mosquitoes for this purpose holds the risk of initiating the mother colony with individuals that are infected with viruses, as MBVs are widespread in most regions. For this reason, a sensitive detection method is crucial to screen wild collected mosquitoes and ensure that samples are virus-free before establishing the mother colony in the insectary. Furthermore, periodic screening of the colonies and mass-reared material is important to ensure the absence of any accidental contamination of the colonies by any MBVs. Such screening as part of quality control in mass-rearing facilities is not only essential to ensure adequate biosafety for insectary staff, but also to ensure the general public that the sterile males released during SIT programmes are pathogen-free.
RT-qPCR is a highly sensitive and speci c assay for the identi cation and detection of several RNA viruses such as CHIKV [15], WNV [16], USUV [17] and ZIKV [18] and the detection limits for some of these viruses have been determined. The dengue virus is a single positive-stranded RNA virus in the genus Flavivirus and includes four DENV serotypes; DEN-1, DENV-2, DENV-3 and DENV-4. A real-time RT quantitative PCR has been developed to detect viral RNA of each DEN virus serotype. In single reactions and in fourplex reactions (containing four primer-probe sets in a single reaction mixture), standard dilutions of virus equivalent to 0.002 plaque forming unit (PFU) of DENV-2, DENV-3, and DENV-4 viruses were detected, and the limit of detection of DENV-1 virus was 0.5 equivalent PFU [19].
Previous studies of ZIKV detection indicate that viral concentrations vary between sample matrices, such as blood, urine, or saliva. In the case of urine and saliva, the lowest viral RNA detected was reported to be 10 2 copies/mL in urine and 40 copies/mL in saliva with their highest range being 2.68 × 10 3 copies/mL and 7.44 × 10 4 copies/mL, respectively [20].
Lanciotti et al., reported a rapid TaqMan assay for the detection of WNV in a variety of human clinical specimens and eld-collected mosquitoes. The RT-qPCR was speci c for WNV and detected 0.1 PFU with greater sensitivity than the traditional RT-qPCR method [16]. Nikolay et al. presented a quantitative realtime RT-qPCR assay for USUV based on conserved regions from Europe and Africa. The assay provides high analytical speci city for USUV and 60 copies/reaction for the RNA standard [17].
Although a number of molecular tests have been published for detecting MBVs, few have reported the use of these tests for detecting MBVs in mosquito samples. Sutherland et al., conducted a laboratory evaluation of the ability of commercial antigen-capture assays, the Rapid Analyte Measurement Platform (RAMPH) and the VecTestH wicking assay, as well as Real Time reverse transcriptase-polymerase chain reaction (RT-qPCR, Taqman) and Vero cell plaque assay to detect WNV in large mosquito pools. Real-Time PCR (Taqman) was the most sensitive, detecting WNV ribonucleic acid (RNA) in 100% of the samples containing a single infected mosquito in pool sizes of up to 500 mosquitoes. Mosquito body tissues minimally impacted the ability of RT-qPCR to detect WNV in a pool size of 500, reducing sensitivity to 0.6 log 10 PFU/ml [21].
This study aimed to establish detection limits of MBVs for the purpose of effective periodic screening of mosquito colonies and released insects in the context of applying the SIT against disease-transmitting mosquito populations. The purpose of this article was to determine the extent that pool size could be increased while still maintaining the ability to detect one infected individual.

Mosquito species
Aedes albopictus strains used in the present study were maintained in the bio-secure insectary of the Insect Pest Control Laboratory (IPCL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Seibersdorf, Austria. The non-infected mosquito strain used in the experiment was reared and maintained following the FAO/IAEA guidelines [22]. In brief, mosquito strains were kept under standard laboratory conditions at a temperature of 27 ± 1 °C, 60 ± 10% relative humidity, and a photoperiod of 12:12 (L:D) h including dusk (1 hour) and dawn (1 hour) transitional periods [22,23]. Adults were kept in standard 30 × 30 × 30 cm Bugdorm cages (Megaview Science Education Services Co. Ltd., Taichung, Taiwan) in an insectary deprived of natural light and continuously supplied with 10% wt: vol sucrose solution. Before total RNA extraction, adults were starved for 12 h to empty stomach content and stored at -80 °C.

Virus-infected Mosquito Samples
The project 'Research infrastructures for the control of vector-borne diseases' (lnfravec2, https://infravec2.eu/) provided Aedes aegypti, strain BORA, infected with CHIKV and ZIKVs, and Culex pipiens strain Gavà, infected with WNV and USUV viral strains. In brief, 5-7-day old females of the Ae. aegypti strain BORA were infected with CHIKV and ZIKV by feeding on an infectious blood-meal with virus titers of 1.5 × 10 7 and 1.02 × 10 7 plaque-forming units (PFU)/ml respectively. Virus titration was performed by the plaque assay for CHIKV and ZIKV and expressed in PFU/ml as previously described [24,25]. A Hemotek® system was used for feeding the adult females and engorged females were fed with 10% sucrose in a chamber incubated at 28 °C and 80% humidity for 14 days in the bio-secure insectary of the Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle, Institut de Recherche pour le Développement (IRD), Montpellier, France. In addition, Cx. pipiens females inoculated with WNV and USUV were prepared at the Centre de Recerca en Sanitat Animal, (Campus of Autonomous University of Barcelona) Barcelona, Spain. Culex pipiens females were inoculated intrathoracically with 1-2 µl per adult using a stock of WNV (7,52 log 10 TCID 50 /ml) and USUV (6,88 log 10 TCID 50 /ml). Virus titers were determined by a standard limiting dilution assay [26] using monolayers of cells employed for virus propagation. Viral titers were expressed in 50% tissue culture infectious dose (TCID 50 ) per ml. Injected females were kept at 28 °C and 80% humidity for 7 days. Bodies and heads were separated in individual tubes and homogenized in 500 µl of TRIzol and kept at -80 °C. Infected heads and bodies were delivered to the IPCL for RNA extraction and further analysis. The level of infection of infected mosquito bodies was estimated by evaluating the virus infection titer in the corresponding head using quantitative RT-qPCR. Only bodies of which the corresponding head showed high virus titer were considered infected and were used in spiking the non-infected mosquito samples.

Spiking Non-infected Mosquito Samples With Virus Infected Mosquitos
To determine the ability of the quantitative RT-qPCR assays to detect MBV, a single virus-infected mosquito Ae. aegypti infected with CHIKV or ZIKV and Cx. pipiens infected with WNV or USUV, was homogenized within large pools of uninfected adult mosquitoes, i.e. n = 165 (100 mg), n = 320 (200 mg) and n = 600 (1000 mg) and total RNA extracted. A pool consisting of only uninfected mosquitoes was included as a negative control. Negative control, 320 and 1600 mosquito pools were replicated twice.

Total Rna Extraction
Total RNA was extracted from mosquitoes using TRIzol™ Reagent (Invitrogen, CA, USA) according to the supplier's instructions. After grinding and homogenizing mosquito adult pools using liquid nitrogen, TRIzol reagent containing one single infected mosquito was added to the mosquito pool and the RNA extraction was carried out according to the supplier's instructions. For the negative control pool, TRIzol reagent was added after liquid nitrogen grinding. The total RNA pellet was resuspended in 20-1000 µl of RNase-free water and the volume was adjusted according to the mosquito pool size. The RNA samples quantity and quality were determined using Synergy™ H1 microplate reader (BioTek). The RNA samples were diluted to 10, 1, 0.1, 0.01, 0.001, 0.0001 ng/µl as a concentration that will consistently give the same amount per well in the RT-qPCR and were stored at -80 °C.

Evaluation Of Primers And Probes Speci city
To investigate the possibility of using multiple primers and probes sets to detect several viruses in the same reaction, we tested the speci city of each primers and probe sets to detect other MBVs. The total RNA extracted from uninfected mosquitoes was tested to ensure that there were no false positives caused by cross-reactions with the host-species. This step also determined any background signal generated due to primer cross-reactivity with mosquito-derived RNA.

Generation Standard Curves For The Rt-qpcr
To obtain a sustainable positive control for the detection of MBVs, the RNA sequences containing and anking the sequence regions of the virus speci c primers and probes of CHIKV, ZIKV, WNV and USUV were ampli ed using the primers listed in Table 1 and cloned into pGEM-T vector (Promega, USA). The SuperScript® III First-Strand Synthesis System for RT-qPCR (Invitrogen) was used to synthesize rststrand cDNA from puri ed poly(A) + or total RNA following the supplier's instructions. The targeted sequences were ampli ed by Taq PCR Master Mix (Qiagen) with the following PCR conditions: 5 min 94 °C, 35 cycles of 30 seconds 94 °C, 30 seconds 58 °C and 1 min 72 °C, and 10 min 72 °C. The PCR product was puri ed using the High Pure PCR Clean-up Micro Kit (Roche, Germany) and ligated to the pGEM-T vector (Promega, USA), following the supplier's instructions. The recombinant plasmids were transformed into DH5α competent bacteria (Invitrogen) following the supplier's instructions. The recombinant plasmids and the inserted sequences were con rmed by Sanger sequence (Euro ns Genomics) with the universal vector primers M13F_uni (-21) TGTAAAACGACGGCCAGT and M13R_rev (-29) CAGGAAACAGCTATGACC. Recombinant plasmids were ampli ed, and the quantity and quality were determined using Synergy™ H1 microplate reader (BioTek). The DNA copy number was estimated using NEBioCalculator (https://nebiocalculator.neb.com/#!/dsdnaamt), then seven concentrations with known DNA copy numbers/µl were prepared by serial dilutions and used to estimate the virus copy number in infected and non-infected mosquito samples. Sterile, nuclease-free water was used as a no template control (NTC), then tested in triplicates. One-step Real-time Rt-qpcr Mosquito-borne viruses speci c primers and Taqman probes previously reported to detect each speci c virus were synthesized by Euro ns Genomics with 5-FAM, HEX as the reporter dye for the probe. The details of the used primers and probes sequences and characteristics are shown in  [16,27,28]. Positive results were determined according to the ampli cation cycle at which the uorescence was detected above the threshold cycle (CT) relative uorescence unit (RFU) in the PCR baseline-subtracted RFU. Baseline thresholds for the four uorophores were determined with the CFX manager software in a series of reactions using the virus standard dilutions and then set for subsequent runs as auto calculated [29].

Calibration Curve
To ensure a sustainable positive control and prepare a DNA sample with known viral copy numbers, the viral sequence containing the primers and Taqman probe for each virus was ampli ed using the primers listed in Table 1 and cloned into a bacterial plasmid. The recombinant plasmids containing the inserted sequence of CHIKV, ZIKV, USUV and WNV where constructed, ampli ed, puri ed and the insert sequence was veri ed using Sanger sequence. The sequence analysis con rmed the presence of the targeted sequence of each virus which correctly matched with the virus sequence available in the sequence database. A blast alignment of the sequences showed similarity with the CHIKV (99%), ZIKV, USUV, and WNV genome (100%) (Supplementary Fig. 1). Using the puri ed plasmid of each virus, DNA concentration with a known copy number of 4.7 × 10 9 was prepared. Consequently, 10-fold serial dilutions in water were used to prepare 7 DNA concentrations with copy numbers ranging from 4.7 × 10 8 to 4.7 × 10 2 per ml, which were used to prepare the calibration curves for each virus primers and probes. Viral DNA detection was successful for all viruses and the standard curves exhibited linearity over seven orders of magnitude  (Fig. 1). The melting curve analysis showed one speci c curve for each virus.

Speci city Of Viral Primers And Probes
The results of the speci city of the primers and probes for ZIKV, USUV, WNV, and CHIKV are shown in Table 3. The results indicate that the four viruses were detected using their corresponding primers. The assays were speci c for the single target virus; no uorogenic signal was detected for other tested mosquito-borne viruses. No virus was detected in the Ae. albopictus negative samples. No cross-reaction between these four viruses were detected indicating the high speci city of the assay (Table 3).

Quanti cation of the viral copy number in mosquito heads and bodies
To ensure that the virus infected mosquito bodies were infected and had relatively homogenous virus copy numbers before using it to spike mosquito pools, the virus copy number was quanti ed in some randomly selected individual bodies with their corresponding heads. The results indicated that the virus was detected in both head and body and the quantity of the virus in the body and in the corresponding head was positively correlated (Fig. 2). The high regression coe cient (R 2 ) of 0.994 and 0.995 for CHIKV and WNV, respectively indicated that the assay is highly reproducible. However, R 2 values were rather low for USUV (0.725) and ZIKV (0.710). No head with low USUV copy number was detected. Mosquito bodies of the corresponding head with low CHIKV and WNV copy number (~ 10 3 ) exhibited low virus titers (0-10 2 ) indicating homogenous virus distribution between the body and the head of infected mosquito.
However, interestingly, mosquito bodies corresponding to heads with low ZIKV copy number (10 1 -10 2 ) exhibited high virus copy number (~ 10 8 ) indicated less abundance of the virus in the head compared to the body (Fig. 2).
The virus infection prevalence was evaluated in all virus inoculated mosquito heads (Fig. 3 to these heads were not used to spike the uninfected mosquito pools (Supplementary le 2). Based on the positive correlation between the virus copy number in the head and the body of infected mosquitoes, the virus copy number was evaluated in the head of all virus infected mosquitoes (Fig. 2, Supplementary le 2) and subsequently the virus copy number in the corresponding body was calculated as shown in Supplementary File 3. Positive bodies predicted with high virus titers were used in the virus detection limit experiment.

Determination Of Viral Detection Limit
The initial detection of MBVs in small pools of mosquitoes (< 100 mosquitoes) containing one virus infected mosquito body indicated the possibility to detect the target viruses (data not shown). Therefore, attempts were made to detect the virus in larger pools of 165, 320 and 1600 uninfected mosquitoes that contained one infected mosquito body. All tested viruses could be detected in all tested pools. For the largest pools of mosquitoes used (1600 mosquitoes spiked with one infected mosquito body), the results indicate the ability to detect CHIKV, WNV, ZIKV and USUV not only by using 10 ng total RNA as a template but also with lower concentrations i.e. 1 and 0.1 ng (Supplemental Fig. 4). Using the correlation between the virus copy number detected and the different quantities of total RNA, a formula was derived that was used to evaluate the detection limit for each virus (Fig. 4). The detection limits per reaction were 196.79, 190.90, 4.28 and 11.44 virus copy numbers for the CHIKV, WNV, ZIKV and USUV, respectively (Fig. 4). Based on the formula presented in Fig. 4, in theory, if mosquito pools were spiked with one positive mosquito containing 10 8 copy numbers of the virus and with a cut off Ct value of 36, it might be possible to detect MBVs in mosquito pools of 5.08 × 10 5 , 5.24 × 10 5 , 2.33 × 10 7 and 8.74 × 10 6 viral copy numbers for CHIKV, WNV, ZIKV and USUV, respectively (Supplementary le 5).

Discussion
As demonstrated with the successful management of several plant pests, the SIT has shown great potential for the area-wide management of mosquito populations and hence, the diseases these vectors transmit. The technique has many advantages as it is an environment-friendly and species-speci c control method; however it requires several prerequisites for its implementation [30,31]. One of these prerequisites is the need to establish a mass-rearing colony of the targeted mosquito species. The mother colony prior to up-scaling is often established from eld collected samples which might be infected with MBVs. Taking into account that MBVs can maintain infection for up to seven generations within a laboratory colony through vertical and horizontal transmission [32][33][34][35][36][37][38], this represents a serious concern for insectary staff handling the mosquito colonies and for the public living in target release sites should sterile female mosquitoes be released accidentally. To avoid such risks, initiating colonies from virus-free material collected from the eld is a prerequisite and regular screening of mosquito males and females is recommended to detect any infection in the colony even if infections rates are very low.
Although there are many different detection methods to detect MBVs, the use of RT-qPCR and cell culture were considered the most sensitive techniques [39], however the cell culture technique requires a Biosafety Level 3 (BSL 3) laboratory. The RT-qPCR can be done in a BSL2 laboratory, where the mosquito samples can be homogenized in virus deactivation solution, i.e. TRIzol or lysis buffer. Both techniques have the advantages of speed, speci city, and sensitivity for the detection of viral RNA, however, cell culture can only detect viable virus particles that can initiate infection and cause cytopathogenic effects (CPE) or they have speci c antibodies to detect them using uorescent focus-forming units (FFU) in the selected cell culture. Viruses that cannot infect these cell cultures or did not induce visible CPE or does not have speci c antibodies FFU detection will not be detected unless other techniques are used to con rm the presence of virus such as RT-qPCR and electron microscopy. RT-qPCR can detect not only viral RNA from viable virus particles but any viral RNA, i.e. mRNA that can be found in the mosquito samples and can be detected with the selected primers and probe sequence. This limitation can be reduced by using multiplex PCR where several sets of primers and probes can be used although this procedure reduces the sensitivity by one log as compared to the single primers methods [40]. In our study, due to the lack of a BSL3 laboratory, and the time e ciency of the RT-qPCR, this technique was used to detect MBVs.
The detection of MBVs in mosquito pools has been previously studied and the impact of the size of mosquito pools on detection of the virus is well documented [21,41]. Considering the low virus prevalence in wild mosquitoes, the use of the minimum infection rate (MIR) method was recommended to evaluate mosquito infection rates. In addition, it was shown that increasing the probability to detect MBVs will depend on the size of mosquito pools [42,43]. In this study, the infection rate in a mosquito mass-reared colony initiated from virus-free materials is expected to be lower than the infection rate in wild populations. Therefore, larger mosquito pools (320 and 1600 mosquito) were used. Taking into consideration the formula of Gu and Novak [42], the probability of detection of MBVs remain almost the same (0.634 ± 0.011) for the different pools used in this study, even though the mosquito infection rate was signi cantly different. The infection rate (following the spiking rate of one positive mosquito per pool) was 0.00625, 0.00313 and 0.00067 for the mosquito pools with 160, 320 and 1600 individuals, respectively. This indicates that a larger mosquito pool size compensates for a reduced infection rate and hence, maintains the probability of virus detection. This was con rmed by the detection of the MBVs in the largest mosquito pools (1600) used in this study. These results also agree with the prediction of Gu and Novak (2004), who showed that the detection of low levels of mosquito infections requires large samples (i.e. greater than 1600 mosquitoes for obtaining a higher probability of infection (0.8)).
Our data not only con rm the possibility of detecting MBVs in larger pools of uninfected mosquitoes (which were larger than the pool sizes tested in previous studies) [21,39,42,44], but they also indicate the theoretical possibility of detecting MBVs in even larger pools of 5.08 × 10 5 , 5.24 × 10 5 , 2.33 × 10 7 and 8,74 × 10 6 mosquitoes for CHIKV, WNV, ZIKV and USUV, respectively. These results agree with the results of Jupp et al., (2000), who reported the detection of the Rift Valley fever phlebovirus by RT-qPCR in a pool of 16,000 mosquitoes. The large size of screenable mosquito pools predicted in our study might be due to the improvement of the virus detection capacity, i.e. the optimization of RT-qPCR master mix, primers and probe quantity, or due to the difference in the sensitivity of the primers and the probe. It is also worth noting that the predicted size of the mosquito pools remains theoretical since using such large numbers of mosquitoes is not practical for the mosquito homogenization and RNA extraction process.

Conclusions
Based on the overall data presented in this study, it is recommended that eld-collected mosquitoes should be kept in a quarantine area and mosquito pools of up to 100 mosquitoes either from collected adults or emerged from collected eggs should be screened to detect any MBVs before initiating colony rearing in the insectary. In case the screening results turn out to be negative, up-scaling and expanding of the offspring of these mother colonies can be justi ed. Once a larger mass-rearing colony is established, pools of 1600 mosquitoes can be used to carry out routine screens as part of quality control and biosafety measures to con rm the absence of MBVs, and to assure the insectary staff and the public that both laboratory colonies and released mosquitoes cannot be associated with any risk of spreading MBVs in the environment during the implementation of SIT programmes for mosquito control. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Figure 1 Ampli cation and standard curves of serial dilution of plasmid containing the sequence targeted by the primers and probes for the RT-qPCR for CHIKV, WNV, USUV, ZIKV detection. The correlation between the relative orescent unit (RFU) and the Cycle threshold (Ct) in left and between the Vitus log 10 copy number and the Ct in right. Measurements were taken in triplicate. The regression equations and correlation coe cients (R) are given for each plot.

Figure 2
Correlation between the log10 virus titer in head and body of infected mosquito.  Correlation between the log10 copy number per reaction and the Cycle threshold (Ct) to determine the mosquito-borne virus detection limit based on the cut off of Ct value of 36.

Supplementary Files
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