A laboratory-based study to explore the use of honey-impregnated cards to detect chikungunya virus in mosquito saliva.

Mosquito control is implemented when arboviruses are detected in patients or in field-collected mosquitoes. However, mass screening of mosquitoes is usually laborious and expensive, requiring specialized expertise and equipment. Detection of virus in mosquito saliva using honey-impregnated filter papers seems to be a promising method as it is non-destructive and allows monitoring the viral excretion dynamics over time from the same mosquito. Here we test the use of filter papers to detect chikungunya virus in mosquito saliva in laboratory conditions, before proposing this method in large-scale mosquito surveillance programs. We found that 0.9 cm2 cards impregnated with a 50% honey solution could replace the forced salivation technique as they offered a viral RNA detection until 7 days after oral infection of Aedes aegypti and Aedes albopictus mosquitoes with CHIKV.

Introduction

Female mosquitoes are hematophagous arthropods that need blood for egg production and plant nectar as energy source for flying [1]. While blood is a key element for pathogen transmission, sugar source is pivotal for the survival of male mosquitoes and female fecundity [2]. Mosquitoes store sugar meals in large ventral diverticulum, the crop and sugar is poured out from time to time into the midgut for digestion and absorption [3]. When getting blood on a viremic host, a competent female mosquito ingests viral particles which penetrate inside midgut epithelial cells and replicate. Produced virions are released into the hemocele, where helped by the hemolymph, the virus reaches various internal organs including the salivary glands where it actively replicates. When the female mosquito has a subsequent blood meal, the virus is inoculated in the vertebrate host with the saliva delivered [4]. Once infected, the mosquito female remains infected for her entire life and able to transmit every time she bites [4]. Therefore, when feeding on a sugar source, saliva is excreted and if infectious, virus is expelled.

Mosquito-borne viruses have emerged during the past decades affecting millions of people [5]. Chikungunya hit a large belt of the tropical regions: islands of the Indian Ocean [6], Central Africa in 2007 [7], Americas in 2013 [8], and South Pacific region in 2014 [9]. Chikungunya virus (CHIKV; Alphavirus, Togaviridae) is transmitted by the anthropophilic mosquitoes Aedes aegypti and Aedes albopictus which serve as vectors in an epidemic cycle [10]. CHIKV of the East-Central-South African genotype harboring an amino acid change at the position 226 (Ala to Val) in the envelope glycoprotein E1 is preferentially transmitted by Ae. albopictus and has become the most common CHIKV genotype worldwide [6, 11, 12].

Vector surveillance for tracking mosquito-borne pathogens is a tool for an early detection of arboviruses, and a help in designing appropriate intervention strategies prior to onset of human illness. Detection of arbovirus circulation is usually labor intensive and costly, imposing intensive captures of mosquitoes and mass screening for arboviruses [13]. The low prevalence of infection in mosquitoes [14] and the rapid degradation of viruses are the main issues that make this method irrelevant. Thus sampling mosquito saliva can be a gold standard if implemented properly [15]. Honey-coated cards have been used successfully to detect arbovirus circulation (namely, West Nile virus, Ross River virus, Barmah Forest virus, Japanese encephalitis virus and CHIKV) [16-19]. Here we explore the use of honey-impregnated cards for detecting CHIKV in laboratory conditions before proposing it as a method suitable for a surveillance system of arboviruses.

Materials and methods

Ethic statements

Animals were housed in the Institut Pasteur animal facilities (Paris) accredited by the French Ministry of Agriculture for performing experiments on live rodents. Work on animals was performed in compliance with French and European regulations on care and protection of laboratory animals (EC Directive 2010/63, French Law 2013–118, February 6th, 2013). All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the Institut Pasteur (Ethics Committee #89 and registered under the reference APAFIS (Autorisation de Projet utilisant des Animaux à des FIns Scientifiques) #6573-201606l412077987 v2).

Mosquito species tested

Two laboratory colonies were used: (i) Ae. albopictus Providence originally collected in 2010 in La Providence on La Réunion Island and maintained since then in insectaries; this population was involved in major outbreaks of CHIKV [6], and (ii) Ae. aegypti Paea collected in Tahiti (French Polynesia) and maintained in the laboratory since 1994 [20]. Mosquitoes were reared in standardized laboratory conditions. After egg hatching, larvae were distributed in pans of 200 individuals and supplied with a yeast tablet in 1 liter of dechlorinated water. Immature stages are maintained at 25±1°C. Pupae were collected and placed in cages where adults emerged. Adults were fed with a 10% sucrose solution and kept at 28±1°C with a 16L:8D cycle and 80% relative humidity.

Preparation of filter papers

To determine the effects of storage conditions on the ability of filter papers to preserve nucleic acids, 50 μL of serial viral dilutions (corresponding to 5 to 5x106 viral particles) were spotted on honey-coated 0.9 cm2 heat-sterilized filter papers (Whatman, Piscataway, New Jersey). Honey (fir honey, France) solutions were proposed at 10%, 20%, or 50% prepared with distilled water. Cards of 0.9 cm2 were incubated for up to 7 days at 28°C and 70% humidity. At day 1, 2, 3 and 7, exposed cards were immersed in 500 μL of FBS (fetal bovine serum) for 1h at +4°C and stored at -80°C until examination. Two cards (replicates) were prepared per viral dilution and honey concentration.

Mosquito infections

Seven to ten day-old mosquito females were sorted at a rate of 60 individuals per box. For each species, five to six boxes were exposed to an infectious blood meal containing 1.4 mL of washed rabbit erythrocytes, 700 μL of CHIKV suspension and ATP at 1 mM as a phagostimulant. CHIKV strain (06.21) isolated from a patient on La Réunion Island in 2005 [6], belonging to the East-Central-South African genotype, was kindly provided by the French National Reference Center for Arboviruses; viral stocks were produced after two passages on C6/36 cells in T75 flasks. Briefly, subconfluent C6/36 cells were inoculated with 500 μL of viral suspension at a 0.1 MOI (multiplicity of infection) and incubated at 28°C. After 1 h of adsorption, 10 mL of Leibovitz’s L-15 medium (Life Technologies) complemented with 2% fetal bovine serum (FBS; Thermo Fisher Scientific), 1X of non-essential amino acids (NAA; Life Technologies), 1X of Penicillin-Streptomycin (Life Technologies) were added to the flask. After 2 days of incubation, the cell culture supernatant was harvested and the percentage of FBS adjusted to 10%. Aliquots were stored at -80°C until used. The titer of the blood meal was at 106.5 plaque-forming unit (pfu)/mL. Four Hemotek® (Hemotek Ltd, Blackburn, UK) feeders containing the infectious blood meal were prepared and each box was placed under a feeder for 15 min. Then mosquitoes fed through a piece of pork intestine covering the base of a feeder maintained at 37°C. After feeding, fully fed mosquitoes were isolated in cardboard containers and maintained with 10% sucrose under controlled conditions (28±1°C, relative humidity of 80%, 16h light:8 h dark cycle) for up to 7 days. After feeding, mosquitoes were individually isolated in 50 mL Falcon® tube closed with a mesh on the top. Females had then access to a 0.9 cm2 card impregnated with a 50% honey solution put on the mesh. Mosquitoes were maintained at 28°C and 70% humidity. To define whether the quantity of virus detected depends on contact time between cards and mosquitoes, cards were unchanged until day of examination or changed one day before examination. At 3 and 7 days post-exposure (dpe), honey-impregnated cards were prepared for RNA extraction and viral quantification. To compare with the standard technique of forced salivation [21], mosquitoes were examined at 3 and 7 days after infection; wings and legs were removed from each mosquito, and the proboscis was inserted into a 20 μL tip containing 5 μL FBS. After 30 min, saliva-containing FBS was expelled in 45 μL of Leibovitz L-15 medium (Life Technologies) for quantification. Three experimental infections (replicates) were performed per condition. Non-infected mosquitoes (exposed to non-infectious blood or culture media) were not tested.

Nucleic acid extraction and quantitative RT-PCR

Cards were immersed in 500 μL of Leibovitz L15 medium (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS) for 1h at +4°C. After homogenization and centrifugation, the supernatant containing viral particles was stored at -80°C until use. Samples were processed for RNA extraction using the Nucleospin® RNA II kit (Macherey-Nagel, Hoerdt, France) followed by one-step RT-PCR performed in a volume of 25 μL containing 3 μL RNA template, 12.5 μL 2X Brilliant SYBR Green I QPCR Master Mix (Stratagene), 1 μL sense (2.5 μM), 1 μL anti-sense (2.5 μM), 0.25 μL Fluorescein (1 μM), and 0.0625 μL Stratascript RT/RNAse block enzyme. Primers were selected in the E2 structural protein coding region [11]: sense Chik/E2/9018/+ (CACCGCCGCAACTACCG) and anti-sense Chik/E2/9235/- (GATTGGTGACCGCGGCA). The amplification program in a CFX96 Real-Time System (Biorad, CA) included: a reverse transcription at 50°C for 10 min, a step of reverse transcriptase inactivation at 95°C for 1 min followed by 40 cycles of 95°C 10 s and 60°C 30 s (annealing-extension step). After amplification, a melting curve was acquired to check the specificity of PCR products. PCR was performed in duplicate for each sample. Signals were normalized to the standard curve using 10-fold serial dilutions of viral RNA (101 to 108). Normalized data were used to measure the number of RNA copies according to the ΔCt analysis. The number of RNA copies detected corresponded to the quantity of viral particles tested (S1 Table).

Statistical analysis

To evaluate the ability of honey-impregnated cards to preserve nucleic acids, we first compared the proportion of samples with detected virus according to virus dilution, honey concentration and day post-infection (dpi) using chi-square tests. In a second step, in samples with virus detected, we compared RNA copies detected according to virus dilution, honey concentration and dpi using Kruskall-Wallis non-parametric tests. A linear regression model was used to evaluate the adjusted effects of virus dilutions, honey concentration, and dpi.

In experiments with mosquitoes, proportions of females showing transmission were compared between species, dpe and treatments using logistic regression models. Then, in mosquitoes showing transmission, the level of viral RNA detected was compared between species and dpe using Student’s t test, and between treatments using analysis of variance (ANOVA).

Statistical analyses were conducted using the Stata software (StataCorp LP, Texas, USA). p-values < 0.05 were considered significant. If necessary, the significance level of each test was adjusted based on the number of tests run, according to the sequential method of Bonferroni.

Results

Viral RNA detection on filter papers

Before conducting sugar-feeding experiments with mosquitoes, we determined the effects of several conditions on the ability of filter papers to preserve nucleic acids when impregnated with honey solutions provided at three different concentrations (10%, 20%, and 50%). We found that the proportion of honey-impregnated cards allowing virus detection did not vary according to the day post-infection (dpi) (chi-square test: p = 0.08) or honey concentration (10%, 20% or 50%) (chi-square test: p = 0.47). Fig 1a–1c showed the quantity of viral RNA detected on cards impregnated with different dilutions of honey (10%, 20%, 50%) and on which, were deposited different quantities of viral particles (5 to 5x106 pfu) (details in S2 Table). When only considering the samples with virus detected, the number of viral RNA detected on cards did not vary according to dpi (Kruskal-Wallis test: p = 0.10) but increased as the honey concentration increased (Kruskal-Wallis test: p = 0.037). Moreover, the number of viral RNA detected on cards decreased as the virus dilution increased (Kruskal-Wallis test: p <0.001). Using a linear regression and adjusting to the quantity of viral particles deposited and the dpi, we showed a significant effect of the honey concentration with a higher viral RNA detected on cards impregnated with a 50% honey dilution (logistic regression model: p < 0.001). One viral CHIKV RNA can be detected 7 days after depositing one viral particle on filter papers impregnated with a 50% honey solution (Fig 1c). Altogether, the number of viral RNA detected on filter papers did not vary according to the dpi but vary with the honey dilution; the 50% dilution of honey preserved better viral RNA on cards.

Fig 1

Viral RNA copies detected on 0.9 cm2 spotted with different quantities of CHIKV particles (5 to 5x106 pfu) and impregnated with honey solutions (10%, 20%, 50%), examined at day 1, 2, 3 and 7.

Different quantities of viral particles were deposited on 0.9 cm2 cards imbibed with honey solutions. After 1, 2, 3 and 7 days of incubation at 28°C, cards were immersed in 500 μL of FBS (fetal bovine serum) for 1h at +4°C. Samples were processed for RNA extraction and qRT-PCR. Two replicates were performed. Each dot represents one card; detailed information in S2 Table.

Detection of viral RNA on honey-impregnated filter papers placed in contact with mosquitoes orally infected with a CHIKV-infectious blood meal

To define whether filter papers can replace saliva collection by forced salivation, Ae. aegypti and Ae. albopictus previously exposed to an infectious blood meal were put individually in contact with filter papers prepared under two conditions (status unchanged and changed) and examined at 3 and 7 dpe; the status “unchanged” refers to filter papers that were placed on the top of the tube and kept as it is until the day of examination and the status “changed” to filter papers placed on the top of the tube that were changed one day before the examination. Filter papers were all impregnated with a 50% honey solution. When examining the transmission efficiencies (TE, corresponding to the proportion of mosquitoes with infectious saliva among mosquitoes tested), the three replicates were similar (Fisher’s exact test, p > 0.05) allowing to pool all individuals exposed to the same treatment (Table 1).

Table 1

Transmission efficiencies (%) of Aedes aegypti Paea and Aedes albopictus La Providence, 3 and 7 days after exposure to an infectious blood meal containing 106.5 pfu/mL of CHIKV.

Saliva were collected using the forced salivation technique and quantified by RT-qPCR. In brackets, number of mosquitoes tested.

	Species	Aedes aegypti Paea			Aedes albopictus La Providence
	Replicate	1	2	3	1	2	3
Card unchanged	Day3	30% (10)	55.55% (9)	27.27% (11)	0% (5)	50% (10)	71.42% (14)
	Mean (N)	36.66% (30)			51.72% (29)
	Day 7	50% (10)	50% (8)	80% (10)	60% (5)	75% (4)	42.85% (14)
	Mean (N)	60.71% (28)			52.17% (23)
Card changed	Day3	30% (10)	30% (10)	54.54% (11)	20% (5)	62.5% (8)	64.28% (14)
	Mean (N)	38.70% (31)			55.55% (27)
	Day 7	60% (10)	80% (5)	36.36% (11)	40% (5)	0% (2)	37.5% (8)
	Mean (N)	53.84% (26)			33.33% (15)
Saliva collection	Day3	35% (40)	35% (20)	55% (20)	- (0)	30% (20)	60% (20)
	Mean (N)	40% (80)			45.0% (40)
	Day 7	96.42% (28)	80% (20)	85% (20)	75% (8)	100% (20)	100% (20)
	Mean (N)	88.23% (68)			95.83% (48)

A total of 257 mosquitoes (among 445; 57.7%) were able to expectorate the virus. Dpe (3, 7) (p < 10−4) and treatments (card unchanged, card changed and saliva collection) (p = 0.0007) affected significantly TE while mosquito species (Ae. aegypti, Ae. albopictus) did not (p = 0.25) (S3 Table). After adjusting to dpe, treatments remained significantly correlated with TE (p < 10−4) and more specifically, for saliva collection (p = 0.001). When examining the number of viral RNA detected, loads were significantly higher at 7 dpe compared to 3 dpe (Student‘s test: p < 10−3) and did not vary according to the mosquito species (Student’s test: p = 0.42) and treatments (ANOVA: p = 0.75).

For Ae. aegypti mosquitoes, TEs calculated using filter papers did not increase with the dpe (3 and 7) whatever the status of filter papers (unchanged versus changed) (Fisher’s exact test, p > 0.05). However, saliva collection by forced salivation allowed detecting more mosquitoes with infectious saliva at 7 dpe (Fisher’s exact test, p < 10−4).

For Ae. albopictus, TEs did not increase with the dpe (Fisher’s exact test, p > 0.05) and as for Ae. aegypti, we detected more mosquitoes with infectious saliva by forced salivation (Fisher’s exact test, p < 10−4). Finally, Ae. aegypti and Ae. albopictus behaved similarly whatever the dpe and the way to detect saliva-excreted viruses (Fisher’s exact test, p > 0.05).

When analyzing the number of viral RNA expectorated by mosquitoes (Fig 2), neither the species, the dpi, nor the status of filter papers (unchanged and changed) affected significantly the quantities of viral RNA detected (Kruskal-Wallis test: p > 0.05) with only one exception, the number of viral RNA being higher in changed filter papers at 7 dpe for Ae. aegypti (Kruskal-Wallis test: p = 0.009).

Fig 2

CHIKV-infected saliva detected on cards, 3 and 7 days after exposure of Aedes aegypti (b, d) and Aedes albopictus (c, e) to an infectious blood meal. (a) Mosquitoes were exposed to a CHIKV infectious blood meal at 106.5 pfu/mL and maintained in individual tubes at 28°C. A 0.9 cm2 of 50% honey impregnated card was deposited on the top of tubes. Filter papers were (1) changed one day before examination or (2) kept unchanged until examination, and compared to the typical (3) saliva collection using the forced salivation technique. The number of viral RNA copies were estimated by qRT-PCR. Each dot represents an individual mosquito. Between 11 to 60 mosquitoes were analyzed for Ae. aegypti and 5 to 46 for Ae. albopictus. Three replicates were performed per condition. Bars indicate the mean. ns (non-significant) indicates the lack of statistical significance (p > 0.05).

Discussion

Our study shows that filter papers could replace the forced salivation technique to detect viral RNA in saliva of Ae. aegypti and Ae. albopictus. CHIKV RNA can be detected in saliva deposited on filter papers until 7 days after oral infection of mosquitoes. Moreover, viral CHIKV RNA can be detected 7 days after being deposited on filter papers impregnated with a 50% honey solution. To summarize, viral RNA can be detected on filter papers 14 days after oral infection of mosquitoes.

Our pilot experiment shows that the filter papers impregnated with a honey solution can preserve viral RNA until day 7 post infection. We are able to detect one viral RNA copy on filter papers coated with a 50% honey solution. This method has been used successfully to detect different arboviruses (e.g. West Nile virus (WNV), Ross River virus (RRV), Barmah Forest virus (BFV), Japanese encephalitis virus (JEV), and CHIKV) [16-19]. Its gives results comparable to those obtained using the standard forced salivation technique of mosquitoes [16]. An early warning of virus circulation prior detection in humans can be performed using immunologically naïve sentinel animals [22] and field-collected mosquitoes [13]. While sentinel animals raise some ethical concerns and restrictive constraints (methodological, financial), tracking arboviruses in mosquitoes have more advantages. However, mass screening of mosquitoes for arboviruses requires collecting thousands of mosquitoes that should be stored in appropriate conditions to preserve viral RNA and the infection rate of mosquitoes is usually low [13]. The use of filter papers to collect mosquito saliva exploits the biological need of mosquitoes to feed on a sugar source [16]; infectious mosquitoes will be brought to excrete the virus on filters papers during sugar feeding. This method being non-destructive, monitoring the viral excretion dynamics over time is made possible from the same mosquito. Using these papers in combination with trapping mosquitoes (e.g. using the BG-Sentinel traps) can be a promising tool for detecting mosquito-borne viruses. A 50% honey solution promotes viral RNA stability for up to 7 days, compatible with a surveillance system applicable on the field. One disadvantage of using filter papers is that the identity of the mosquito that has expectorated the virus, i.e. the potential vector cannot be determined.

Viral CHIKV RNAs are successfully detected from day 3 post-exposure with comparable quantities, whether the excreted virus is deposited on filter papers or excreted from mosquitoes. Successful viral transmission by a mosquito will occur if the virus overcomes at least two different anatomical barriers: the midgut and the salivary glands [23]. The midgut is the first barrier where the virus cannot penetrate inside the epithelial cell after being ingested with the infectious blood meal (i.e. midgut infection barrier) and/or the virus cannot escape into the hemocele after replication in epithelial cells, infecting different internal organs including the salivary glands (i.e. midgut escape barrier). The salivary glands correspond to the second barrier with also a salivary gland infection barrier and a salivary gland escape barrier. When the virus is detected in the expectorated mosquito saliva, it means that the mosquito is capable of transmission and the time interval between virus acquisition during blood feeding and the transmission is referred to as the extrinsic incubation period (EIP); EIP is the most critical parameter for virus transmission and will condition the choice of the vector control strategy [4]. Indeed, vector control will aim in reducing the mosquito lifespan to decrease the probability of successful transmission (e.g. for Ae. aegypti and Ae. albopictus, mean EIP for CHIKV is 2–3 days at 28°C [21], imposing to implement vector control measures promptly after human cases are detected). However, the forced salivation technique does not allow determining the physiological dose of virus delivered by mosquitoes when bite [21]. This method allows measuring accurately the vector competence of a mosquito species that must be easily reared and infected in laboratory conditions. These constraints exclude to study a number of mosquitoes not adapted to laboratory conditions.

In conclusion, we show that viral CHIKV RNA can be detected in mosquito saliva deposited on filter papers soaked with a honey solution. This method can advantageously replace the forced salivation technique for assessing vector competence in laboratory [24]. The use of honey-impregnated filter in field conditions may help to better monitor the risk of transmission and anticipate the emergence.

Detection sensitivity of CHIKV RNA copies by qRT-PCR.

50 μL of different dilutions of viral particles were processed for RNA extraction and quantification of RNA copies by qRT-PCR.

(PDF)

Click here for additional data file.

Quantities of RNA copies detected on cards impregnated with honey solutions (10%, 20%, 50%) and examined at different days (1, 2, 3 and 7) after spotting different quantities of CHIKV particles.

Two replicates (R1 and R2) were performed.

(PDF)

Click here for additional data file.

Comparison of transmission efficiencies between mosquito species, day post-exposure and treatments (logistic regression model).

(PDF)

Click here for additional data file.

8 Feb 2021

PONE-D-20-36874

A laboratory-based study to explore the use of honey‑impregnated cards to detect chikungunya virus in mosquito saliva

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Reviewer #1: In their manuscript ‘A laboratory-based study to explore the use of honey‑impregnated cards to detect chikungunya virus in mosquito saliva’, the authors examine the potential use of honey-infused filter papers as a diagnostic tool for CHIKV infection in Aedes aegypti and Aedes albopictus mosquitoes using experimental infection and salivation assays.

They determined that cards infused with 10%, 20% and 50% honey solutions are suitable for virus detection out to 7 days post-expectoration, and that the 50% treatment was most effective at preserving viral RNA. They then compare transmission efficiencies for both species, for cards against the traditional forced salivation assay and show that both approaches could be utilized, although a significantly greater proportion of positive samples were detected with the forced salivation approach. The study is a useful examination of a tool that could prove to be an important form of arboviral diagnostic in the future. However, in its current form, the manuscript is somewhat disorganized and key information necessary for readers to fully interpret the results, or repeat the assays is missing. Please see specific comments below:

1. Lines 51- 53 “CHIKV of the East-Central-South African genotype harboring an amino acid change at the position 226 (Ala to Val) in the envelope glycoprotein E1 is preferentially transmitted by Ae. albopictus (6, 11, 12).” – Given the inclusion of this statement, the authors should state whether the virus isolate used in the study has this genotype, and consider what that implies for their results.

2. Details that should be added to/clarified in the methods section:

• Methods – Were any uninfected controls to help gauge the false positive rate?

• Were the filter papers sterilized prior to experiments?

• How many replicates were performed for each experiment? There is some conflicting information. “Five to six batches of 60 females” – Does this mean five independent experimental infections were performed, or were all mosquitoes fed at one time but in five different containers?

• “CHIKV strain (06.21) was isolated from a patient on La Réunion Island in 2005 (6);viral stocks were produced after two passages on 101 C6/36 cells” – Does this mean that the virus was only ever passaged twice over 15 years? More specific details about viral isolate history, and pre-experimental passaging/storage are needed.

• Lines 111-112 - “with the standard technique of forced salivation (21), wings and legs were removed from each mosquito, and the proboscis was inserted into a 20 μL tip containing 5 μL FBS. – The phrasing is a little confusing, as it sounds like wings/legs were removed for mosquitoes feeding on the filter paper too. Should state the time points post-exposure when salivation experiments were performed.

3. The introduction would be better structured if the first and second paragraphs were swapped.

4. Lines 60-62 “Honey-coated cards have been used successfully to detect arbovirus circulation (namely, West Nile virus, Ross River virus, Barmah Forest virus, Japanese encephalitis virus and CHIKV) (16-19).” – what is different between this historical CHIKV study and the current study?

5. Cards were homogenized in 500uL of medium. Saliva samples in 5uL FBS were added to 45uL medium. This is a 10x difference in media volume, and could have led to differences in detection rates between the two approaches, depending on their relative sensitivity. Was this accounted for in downstream calculations and statistical comparisons.

6. The terms days post-exposure and days post-infection are both used throughout the manuscript. For the sake of consistency, only one should be used.

7. The Fig 2 legend does not do a good job of describing the figure and should be re-written. From the data in these panels, some treatment groups have a smaller sample size than anticipated based on what is described in the methods (one has an N of 3). The sample sizes for individual treatment groups should be described somewhere. Legends should also state whether data were pooled across replicates, or if they represent a single experiment.

8. The discussion section could be improved on in the following ways:

• The section overlooks the authors results which demonstrate that filter cards are less accurate at detecting virus in saliva than the forced salivation method. An objective discussion of the strengths and weaknesses of the two approaches is important.

• The EIP discussion from line 252 is very general and does not provide supporting evidence to the authors argument that honey-impregnated filter cards are a useful tool for arbovirus surveillance.

• Some key advantages of the filter cards are overlooked: they could be deployed in the field, left for a week and collected – a feeding station for surveillance, similar to sentinel birds for WNV. This would offer a direct measurement not feasible for other approaches. Honey promotes RNA stability, overcoming a major weakness with other detection methods.

9. Abstract – “Detection of virus in mosquito saliva using honey-impregnated filter papers seems to be a promising method.” – This sentence is vague. Why, specifically, is this a promising method?

10. Lines 17-18 “Mosquito control is implemented when arboviruses are detected in patients or in field collected mosquitoes.” – It is more commonly implemented pre-emptively in areas of known transmission.

11. Lines 162-163 - “and as the virus dilution increased (Kruskal-Wallis test: p <0.001).” – This reads as though more virus was detected when samples were more diluted, but the figure shows the opposite effect.

12. Line 169 – “50% preserving better viral RNA.” This is confusing and needs to be rephrased.

13. Lines 178-179 – “Detection of viral RNA on honey-impregnated filter papers proposed to CHIKV orally-infected mosquitoes” – This is confusing and needs to be rephrased.

14. Lines 180-183 “To define whether filter papers can replace saliva collection by forced salivation, Ae. aegypti and Ae. albopictus previously exposed to an infectious blood meal were put individually in contact with filter papers prepared under two conditions (status unchanged and changed) and examined at 3 and 7 dpe.” – This is a little difficult to understand, and it would be helpful if definitions for ‘changed’ and ‘unchanged’ were included in the text. In the legend for figure 2, the process is not well defined.

15. Line 212 “viral RNA excreted” – should read viral RNA expectorated

16. Lines 248-249 “This method has the advantage of being non-destructive as is the technique of forced salivation” – I think the phrasing here needs a little work as it makes it sound as though you are arguing that forced salivation is not destructive, even though it typically involves removing the legs and wings of the mosquitoes involved.

17. Table 1 – mean transmission efficiency units (% I assume) should be included in the table.

Reviewer #2: Mosquito-borne viruses, including Chikungunya virus (CHIKV), represent a permanent and increasing global public health threat. Prevention and control strategies for arboviral diseases include continuous surveillance of arboviruses transmitting mosquito populations. However, systematic detection of infected vectors on the field remains fastidious. In this work, Fourniol et al., assessed the use of honey impregned cards as a laboratory tool to routinely and efficiently detect CHIKV in mosquito saliva. By deposing controlled amounts of viruses on honey impregned cards, the authors determined an optimal honey concentration for a better recovery and conservation of viral RNA. They finally demonstrated that detection of viral RNA in mosquito saliva using honey cards was as efficient as the use of the well-established forced saliva method. Taken together, these results are of interest for field application in monitoring the risk of arboviruses transmission and may also inspire the development of similar assays in other vector insects transmitting different pathogens. While this work is of interest for the field, the paper would benefit of language improvement, proofreading, and authors should address the following questions/concerns:

1) The authors should clarify how their work is novel compared to the already published study that was using the same approach to detect CHIKV RNA in mosquito saliva (doi: 10.1073/pnas.1002040107).

2) As the transmission efficiency is mosquito dependent, it is rather complicated to compare the results obtained with the honey cards and the forced salivation without taking this parameter into account. Using forced salivation on the mosquitoes that were exposed to the honey cards to detect viral RNA would have allowed a better comparison between the different detection methods.

3) The authors should explain the aim of testing the honey cards in two distinct conditions (changed and unchanged).

4) Related to the Table 1: The authors indicated that the replicates obtained for each condition were similar (line 185). Could the authors confirm this statement as the variations for A. albopictus at Day 3 vary from 0 to 71 (card unchanged) and from 20 to 64.28 (card changed).

5) The titer of the virus is indicated as 106.5 pfu/mL. This annotation is quite unusual, could the authors confirm that they mean 3.16 x 106 pfu/mL?

6) The authors should provide, to the best extent possible, all the information in the figure legend and make table clearer so the reader can understand the figures:

a. In Figure 1, it is not clear what each dot represents. Indeed, if one dot represents one honey card then the authors should make visible on the graph all the data points, even when no viral RNA copies were detected. Another alternative would be to indicate above each virus dilution how many honey cards were tested.

b. In Table 1, it is not clear what the unbracketed number refer to.

c. In Table 2, it is not clear how the treatments were spread across the two different mosquito species.

d. In Table 2, it is difficult to assess what data comparison the p-values are referring to.

e. In Figure 2, statistic could be indicated even if p-values are not significant.

7) Please refer to the figures in the text when necessary.

a. No mention of Figure 1a, 1b.

b. Text in lane 199 and between lane 199-203 should refer to table S2.

**********

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3 Mar 2021

Answers to Reviewer #1

1) Lines 51- 53 “CHIKV of the East-Central-South African genotype harboring an amino acid change at the position 226 (Ala to Val) in the envelope glycoprotein E1 is preferentially transmitted by Ae. albopictus (6, 11, 12).” – Given the inclusion of this statement, the authors should state whether the virus isolate used in the study has this genotype, and consider what that implies for their results.

We understand and have added: “and has become the most common CHIKV genotype worldwide” in line 52, and “CHIKV strain (06.21) isolated from a patient on La Réunion Island in 2005 (6), belonging to the East-Central-South African genotype, was kindly provided by the French National Reference Center for Arboviruses;” in lines 100-102.

2) Details that should be added to/clarified in the methods section:

• Methods – Were any uninfected controls to help gauge the false positive rate?

To answer properly to this question, we need more details.

• Were the filter papers sterilized prior to experiments?

Filters have been sterilized before use. We have added in lines 89 “heat-sterilized filter papers”

• How many replicates were performed for each experiment? There is some conflicting information. “Five to six batches of 60 females” – Does this mean five independent experimental infections were performed, or were all mosquitoes fed at one time but in five different containers?

For more consistency, this section has been rearranged from line 97 to line 113: “Seven to ten day-old mosquito females were sorted at a rate of 60 individuals per box. For each species, five to six boxes were exposed to an infectious blood meal containing 1.4 mL of washed rabbit erythrocytes, 700 µL of CHIKV suspension and ATP at 1 mM as a phagostimulant. CHIKV strain (06.21) isolated from a patient on La Réunion Island in 2005 (6), belonging to the East-Central-South African genotype, was kindly provided by the French National Reference Center for Arboviruses; viral stocks were produced after two passages on C6/36 cells in T75 flasks. Briefly, subconfluent C6/36 cells were inoculated with 500 µL of viral suspension at a 0.1 MOI (multiplicity of infection) and incubated at 28°C. After 1 h of adsorption, 10 mL of Leibovitz’s L-15 medium (Life Technologies) complemented with 2% fetal bovine serum (FBS; Thermo Fisher Scientific), 1X of non-essential amino acids (NAA; Life Technologies), 1X of Penicillin-Streptomycin (Life Technologies) were added to the flask. After 2 days of incubation, the cell culture supernatant was harvested and the percentage of FBS adjusted to 10%. Aliquots were stored at -80°C until used. The titer of the blood meal was at 106.5 plaque-forming unit (pfu)/mL. Four Hemotek® (Hemotek Ltd, Blackburn, UK) feeders containing the infectious blood meal were prepared and each box was placed under a feeder for 15 min. Then mosquitoes fed through a piece of pork intestine covering the base of a feeder maintained at 37°C.”

• “CHIKV strain (06.21) was isolated from a patient on La Réunion Island in 2005 (6);viral stocks were produced after two passages on C6/36 cells” – Does this mean that the virus was only ever passaged twice over 15 years? More specific details about viral isolate history, and pre-experimental passaging/storage are needed.

The viral stock was constituted once and stored in aliquots until use. Details on the virus production has been added from line 102 to line 109: “viral stocks were produced after two passages on C6/36 cells in T75 flasks. Briefly, subconfluent C6/36 cells were inoculated with 500 µL of viral suspension at a 0.1 MOI (multiplicity of infection) and incubated at 28°C. After 1 h of adsorption, 10 mL of Leibovitz’s L-15 medium (Life Technologies) complemented with 2% fetal bovine serum (FBS; Thermo Fisher Scientific), 1X of non-essential amino acids (NAA; Life Technologies), 1X of Penicillin-Streptomycin (Life Technologies) were added to the flask. After 2 days of incubation, the cell culture supernatant was harvested and the percentage of FBS adjusted to 10%. Aliquots were stored at -80°C until used.”

• Lines 111-112 - “with the standard technique of forced salivation (21), wings and legs were removed from each mosquito, and the proboscis was inserted into a 20 μL tip containing 5 μL FBS. – The phrasing is a little confusing, as it sounds like wings/legs were removed for mosquitoes feeding on the filter paper too. Should state the time points post-exposure when salivation experiments were performed.

This missing information has been added in line 123: “mosquitoes were examined at 3 and 7 days after infection”

3) The introduction would be better structured if the first and second paragraphs were swapped.

We prefer to keep as it is: the first paragraph describes the mosquito physiology and its needs to get blood and sugar and the third paragraph states that we take advantage of these features to set up a surveillance system aiming at detecting in an anticipated manner any circulating viruses.

4) Lines 60-62 “Honey-coated cards have been used successfully to detect arbovirus circulation (namely, West Nile virus, Ross River virus, Barmah Forest virus, Japanese encephalitis virus and CHIKV) (16-19).” – what is different between this historical CHIKV study and the current study?

In Hall-Mendelin et al. (2010; PMID: 20534559), the design of experiments is different:

- Mosquitoes used are from a different region

- CHIKV strain was isolated from a patient in Australia and viral stocks were obtained after three passages on vertebrate (Vero) cells

- 12 days after experimental infection, mosquitoes were exposed to cards for 48 h

The authors found 75% (21/28) of mosquitoes that had expectorated the virus on honey-impregnated filters compared to 52% (14/27) of mosquitoes that have virus detected in saliva collected using a capillary tube. There was no significxant difference between the two methods.

5) Cards were homogenized in 500uL of medium. Saliva samples in 5uL FBS were added to 45uL medium. This is a 10x difference in media volume, and could have led to differences in detection rates between the two approaches, depending on their relative sensitivity. Was this accounted for in downstream calculations and statistical comparisons.

The dilution effect has been taken into account in our calculations and comparisons.

To quantify RNA copies in samples, signals were normalized to the standard curve using 10-fold serial dilutions of viral RNA (101 to 108). The number of RNA copies detected corresponded to the quantity of viral particles tested (see S1 Table).

6) The terms days post-exposure and days post-infection are both used throughout the manuscript. For the sake of consistency, only one should be used.

Dpe and dpi are two terms which are used in two different situations:

- dpe (days post-exposure ) refers to the numbers of days after exposure of mosquitoes to honey-impregnated cards

- dpi (days post-infection) corresponds to the number of days after depositing viral dilutions on cards

7) The Fig 2 legend does not do a good job of describing the figure and should be re-written. From the data in these panels, some treatment groups have a smaller sample size than anticipated based on what is described in the methods (one has an N of 3). The sample sizes for individual treatment groups should be described somewhere. Legends should also state whether data were pooled across replicates, or if they represent a single experiment.

We have added the following information in the legend of Fig. 2: “Each dot represents an individual mosquito. Between 11 to 60 mosquitoes were analyzed for Ae. aegypti and 5 to 46 for Ae. albopictus. Three replicates were performed per condition. Bars indicate the mean. ns (non-significant) indicates the lack of statistical significance (p > 0.05).”

8) The discussion section could be improved on in the following ways:

• The section overlooks the authors results which demonstrate that filter cards are less accurate at detecting virus in saliva than the forced salivation method. An objective discussion of the strengths and weaknesses of the two approaches is important.

• The EIP discussion from line 252 is very general and does not provide supporting evidence to the authors argument that honey-impregnated filter cards are a useful tool for arbovirus surveillance.

• Some key advantages of the filter cards are overlooked: they could be deployed in the field, left for a week and collected – a feeding station for surveillance, similar to sentinel birds for WNV. This would offer a direct measurement not feasible for other approaches. Honey promotes RNA stability, overcoming a major weakness with other detection methods.

The discussion section has been completely rearranged:

- the first paragraph describes the advantages of filter papers (non destructive, track viral dynamics in mosquitoes) and underlines the disadvantage of not being able to identify the vector species

- the second paragraph deals with the forced salivation technique used to measure the vector competence; the method is accurate but only examine mosquitoes which can be reared and infected in laboratory conditions.

9) Abstract – “Detection of virus in mosquito saliva using honey-impregnated filter papers seems to be a promising method.” – This sentence is vague. Why, specifically, is this a promising method?

As suggested, we have added details in the sentence: “Detection of virus in mosquito saliva using honey-impregnated filter papers seems to be a promising method as it is non-destructive and allows monitoring the viral excretion dynamics over time from the same mosquito.”

10) Lines 17-18 “Mosquito control is implemented when arboviruses are detected in patients or in field collected mosquitoes.” – It is more commonly implemented pre-emptively in areas of known transmission.

Usually, vector control is implemented when the virus is detected in patients or field-collected mosquitoes (it is at least what we do in France). The main concern is to limit the use of insecticides to avoid selecting insecticide-reisistance mosquitoes and chemical pollution.

11) Lines 162-163 - “and as the virus dilution increased (Kruskal-Wallis test: p <0.001).” – This reads as though more virus was detected when samples were more diluted, but the figure shows the opposite effect.

We found more virus on cards where we have deposited more virus and thus, where the dilution is the lowest. For a better understanding, we have rephrased as follows: “Moreover, the number of viral RNA detected on cards decreased as the virus dilution increased (Kruskal-Wallis test: p <0.001).” in lines 175-177.

12) Line 169 – “50% preserving better viral RNA.” This is confusing and needs to be rephrased.

We have replaced by “Altogether, the number of viral RNA detected on filter papers did not vary according to the dpi but vary with the honey dilution; the 50% dilution of honey preserved better viral RNA on cards.”

13) Lines 178-179 – “Detection of viral RNA on honey-impregnated filter papers proposed to CHIKV orally-infected mosquitoes” – This is confusing and needs to be rephrased.

We suggest: “Detection of viral RNA on honey-impregnated filter papers placed in contact with mosquitoes orally infected with a CHIKV-infectious blood meal”

14) Lines 180-183 “To define whether filter papers can replace saliva collection by forced salivation, Ae. aegypti and Ae. albopictus previously exposed to an infectious blood meal were put individually in contact with filter papers prepared under two conditions (status unchanged and changed) and examined at 3 and 7 dpe.” – This is a little difficult to understand, and it would be helpful if definitions for ‘changed’ and ‘unchanged’ were included in the text. In the legend for figure 2, the process is not well defined.

To be more accurate, we have added in lines 199-201: “the status “unchanged” refers to filter papers that were placed on the top of the tube and kept as it is until the day of examination and the status “changed” to filter papers placed on the top of the tube that were changed one day before the examination.”

15) Line 212 “viral RNA excreted” – should read viral RNA expectorated

It has been modified.

16) Lines 248-249 “This method has the advantage of being non-destructive as is the technique of forced salivation” – I think the phrasing here needs a little work as it makes it sound as though you are arguing that forced salivation is not destructive, even though it typically involves removing the legs and wings of the mosquitoes involved.

We have replaced by: “This method being non-destructive, monitoring the viral excretion dynamics over time is made possible from the same mosquito.”

17) Table 1 – mean transmission efficiency units (% I assume) should be included in the table.

% have been added.

Answers to Reviewer #2

1) The authors should clarify how their work is novel compared to the already published study that was using the same approach to detect CHIKV RNA in mosquito saliva (doi: 10.1073/pnas.1002040107).

In Hall-Mendelin et al. (2010; PMID: 20534559), authors (i) used Aedes aegypti and not Aedes albopictus, (ii) CHIKV orally-infected mosquitoes were placed in contact with honey-soaked cards at day 12 post-infection, (iii) cards were examined 2 days after. They found no significant difference between the proportion of mosquitoes that had expectorated the virus on honey-impregnated filters and the proportion of mosquitoes that have virus detected in saliva collected using a capillary tube.

Our study shows that filter papers can replace the forced salivation technique to detect viral RNA in saliva of Ae. aegypti and Ae. albopictus. CHIKV RNA can be detected in saliva deposited on filter papers until 7 days after oral infection of mosquitoes and RNA can be detected on filter papers coated with a 50% honey solution for up to 7 days after the virus has been deposited on the filter. It has been inserted in lines 250-253: “Moreover, viral CHIKV RNA can be detected 7 days after being deposited on filter papers impregnated with a 50% honey solution. To summarize, viral RNA can be detected on filter papers 14 days after oral infection of mosquitoes.”

2) As the transmission efficiency is mosquito dependent, it is rather complicated to compare the results obtained with the honey cards and the forced salivation without taking this parameter into account. Using forced salivation on the mosquitoes that were exposed to the honey cards to detect viral RNA would have allowed a better comparison between the different detection methods.

We agree with reviewer’s comment. The ideal experimental design would be to: (1) expose orally infected mosquitoes to filter papers and (2) immediately after to collect saliva using the forced salivation technique. The main problem is to make sure that the quantity virus expectorated by the mosquito using the method (1) will not interfere with the quantity obtained with the method (2).

3) The authors should explain the aim of testing the honey cards in two distinct conditions (changed and unchanged).

As suggested, we have inserted in lines 118-120: “To define whether the quantity of virus detected depends on contact time between cards and mosquitoes, cards were unchanged until day of examination or changed one day before examination.”

4) Related to the Table 1: The authors indicated that the replicates obtained for each condition were similar (line 185). Could the authors confirm this statement as the variations for A. albopictus at Day 3 vary from 0 to 71 (card unchanged) and from 20 to 64.28 (card changed).

The details of our statistical analysis are presented in the following table

Aedes aegypti Aedes albopictus

Pearson chi2 P Pearson chi2 P

Card unchanged Day 3 1.9922 0.369 7.5459 0.023

Day 7 2.4257 0.297 1.4450 0.486

Card changed Day 3 1.8022 0.406 3.1484 0.207

Day 7 2.8814 0.237 1.1625 0.559

Saliva collection Day 3 2.5000 0.287 3.6364 0.057

Day 7 3.3190 0.190 10.4348 0.005

Two P values are above 0.05 ‘in bold) and as such, are considered significantly different. However, when taking into count the number of tests and adjusting the significance level to the number of tests, these P values are below the significance threshold.

P (Aedes aegypti) from max to min P (Aedes albopictus from max to min Number of tests run Threshold

0,406 0,559 1 0,05

0,36 0,486 2 0,025

0,297 0,207 3 0,0125

0,287 0,057 4 0,00625

0,237 0,023 5 0,003125

0,19 0,005 6 0,0015625

We have added inlines 160-161: “If necessary, the significance level of each test was adjusted based on the number of tests run, according to the sequential method of Bonferroni.”

5) The titer of the virus is indicated as 106.5 pfu/mL. This annotation is quite unusual, could the authors confirm that they mean 3.16 x 106 pfu/mL?

Yes we confirm.

6) The authors should provide, to the best extent possible, all the information in the figure legend and make table clearer so the reader can understand the figures:

a. In Figure 1, it is not clear what each dot represents. Indeed, if one dot represents one honey card then the authors should make visible on the graph all the data points, even when no viral RNA copies were detected. Another alternative would be to indicate above each virus dilution how many honey cards were tested.

We have added in the ms in the legend of Fig. 1 “Each dot represents one card; detailed information in S2 Table.”

An S2 Table has been added to the ms.

b. In Table 1, it is not clear what the unbracketed number refer to.

They are percentages; % have been added.

c. In Table 2, it is not clear how the treatments were spread across the two different mosquito species.

This table has become the S3 Table.

The proportions of mosquitoes showing transmission were compared between species, day post-exposure (dpe) and treatments using logistic regression models.

Dpe (3, 7) and treatments affected significantly TE while mosquito species did not. Within “treatments”, mosquitoes are considered together without distinction of species.

d. In Table 2, it is difficult to assess what data comparison the p-values are referring to.

There is no Table 2. This table has become the S3 Table.

We have rearranged the table.

e. In Figure 2, statistic could be indicated even if p-values are not significant.

In the legend of Figure 2, we have indicated: “ns (non-significant) indicates the lack of statistical significance (p > 0.05).”

7) Please refer to the figures in the text when necessary.

a. No mention of Figure 1a, 1b.

It has been corrected.

b. Text in lane 199 and between lane 199-203 should refer to table S2.

“When examining the number of viral RNA detected, loads were significantly higher at 7 dpe compared to 3 dpe (Student‘s test: p < 10-3) and did not vary according to the mosquito species (Student’s test: p = 0.42) and treatments (ANOVA: p = 0.75)” did not refer to S3 Table.

Submitted filename: MS_Fourniol_Answers to Reviewers.doc

Click here for additional data file.

16 Mar 2021

PONE-D-20-36874R1

A laboratory-based study to explore the use of honey‑impregnated cards to detect chikungunya virus in mosquito saliva

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Most of my comments have been sufficiently addressed but two have not.

Initial question - #2a - Were any uninfected controls to help gauge the false positive rate?

Answer - To answer properly to this question, we need more details

Follow-up: I will clarify my comment: As a control in experimental infections, it’s worthwhile to include a mock infection treatment group - mosquitoes that were not exposed to CHIKV-infected blood, and were instead fed naïve blood, or blood plus sterile culture media. Mosquitoes from this treatment would then be allowed to salivate on the honey-impregnated cards in the same manner as CHIKV-exposed mosquitoes in order to assess the accuracy of the viral detection/quantification methodology. If a similar control group was included in the experiments, the results should be discussed in the paper. If no control group was included that should be specified in the methodology.

Initial question - #4 - Lines 60-62 “Honey-coated cards have been used successfully to detect arbovirus circulation (namely, West Nile virus, Ross River virus, Barmah Forest virus, Japanese encephalitis virus and CHIKV) (16-19).” – what is different between this historical CHIKV study and the current study?

In Hall-Mendelin et al. (2010; PMID: 20534559),

Answer - the design of experiments is different: -Mosquitoes used are from a different region

-CHIKV strain was isolated from a patient in Australia and viral stocks were obtained after three passages on vertebrate (Vero) cells

-12 days after experimental infection, mosquitoes were exposed to cards for 48 h

The authors found 75% (21/28) of mosquitoes that had expectorated the virus on honey-impregnated filters compared to 52% (14/27) of mosquitoes that have virus detected in saliva collected using a capillary tube. There was no significxant difference between the two methods.

Follow-up: These details need to be included somewhere in the introduction or discussion.

Reviewer #2: The authors have taken into consideration and addressed the majority of the comments provided in the first round of revision.

However, two minor comments remain to be fully addressed:

1) The authors should incorporate into the discussion few sentences to explain how they work complete the previous work from Hall-Mendelin et al., 2010 (doi: 10.1073/pnas.1002040107). This will help the readers to better understand the novelty of this paper.

2) While the authors have modified the table 1 to incorporate percentages, many of percent symboles are still missing. Could the authors carefully correct this point?

**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: No

Reviewer #2: Yes: Benjamin Voisin

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17 Mar 2021

Answers to Reviewer #1:

1) Initial question - #2a - Were any uninfected controls to help gauge the false positive rate?

Answer - To answer properly to this question, we need more details

Follow-up: I will clarify my comment: As a control in experimental infections, it’s worthwhile to include a mock infection treatment group - mosquitoes that were not exposed to CHIKV-infected blood, and were instead fed naïve blood, or blood plus sterile culture media. Mosquitoes from this treatment would then be allowed to salivate on the honey-impregnated cards in the same manner as CHIKV-exposed mosquitoes in order to assess the accuracy of the viral detection/quantification methodology. If a similar control group was included in the experiments, the results should be discussed in the paper. If no control group was included that should be specified in the methodology.

We understand but we did not expose mosquitoes to a non-infectious blood meal.

We have inserted this statement in line 128: “Non-infected mosquitoes (exposed to non-infectious blood or culture media) were not tested”.

1) Initial question - #4 - Lines 60-62 “Honey-coated cards have been used successfully to detect arbovirus circulation (namely, West Nile virus, Ross River virus, Barmah Forest virus, Japanese encephalitis virus and CHIKV) (16-19).” – what is different between this historical CHIKV study and the current study?

In Hall-Mendelin et al. (2010; PMID: 20534559),

Answer

- the design of experiments is different:

-Mosquitoes used are from a different region

-CHIKV strain was isolated from a patient in Australia and viral stocks were obtained after three passages on vertebrate (Vero) cells

-12 days after experimental infection, mosquitoes were exposed to cards for 48 h

The authors found 75% (21/28) of mosquitoes that had expectorated the virus on honey-impregnated filters compared to 52% (14/27) of mosquitoes that have virus detected in saliva collected using a capillary tube. There was no significxant difference between the two methods.

Follow-up: These details need to be included somewhere in the introduction or discussion.

As suggested, we have added in lines 261-263: “Its gives results comparable to those obtained using the standard forced salivation technique of mosquitoes (16)”.

Answers to Reviewer #2

1) The authors should incorporate into the discussion few sentences to explain how they work complete the previous work from Hall-Mendelin et al., 2010 (doi: 10.1073/pnas.1002040107). This will help the readers to better understand the novelty of this paper.

We have added in lines 261-263: “Its gives results comparable to those obtained using the standard forced salivation technique of mosquitoes (16)”.

2) While the authors have modified the table 1 to incorporate percentages, many of percent symboles are still missing. Could the authors carefully correct this point?

Thanks for noticing it. It has been corrected.

Submitted filename: MS_Fourniol_Answers to Reviewers-2_17 03 2021.doc

Click here for additional data file.

19 Mar 2021

A laboratory-based study to explore the use of honey‑impregnated cards to detect chikungunya virus in mosquito saliva

PONE-D-20-36874R2

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23 Mar 2021

PONE-D-20-36874R2

A laboratory-based study to explore the use of honey‑impregnated cards to detect chikungunya virus in mosquito saliva

Dear Dr. Failloux:

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