Entomo-virological surveillance of arboviruses in the Americas: prospects for the use of paper-based nucleic acid stabilization materials

Abstract

Arthropod-borne viruses (arboviruses) represent a major public health burden for people living or visiting tropical and subtropical countries, where an endemic/epidemic cycle has been successfully established for Orthoflavivirus denguei, Alphavirus chikungunya, and O. zikaense. The risk for the reemergence of other viruses in the urban cycle in the Americas, such as A. mayaro and O. flavi is also of major concern, which justifies the intensification of active surveillance in urban, rural, and sylvatic areas to determine the virus activity in different settings. Entomological surveillance consists mainly of collecting, identifying, and estimating the relative densities of the main disease-transmitting vector species, and more recently, estimating infection rates for the more relevant pathogens. The new approaches for monitoring arthropod vectors of medical importance pose new challenges, including maintaining the cold chain to preserve the pathogen infectivity or nucleic acid stability, especially for RNA genomes, such as those of the main arboviruses. Hence, alternatives such as mosquito traps equipped with solid-phase materials impregnated with honey baits for mosquito feeding, saliva expectoration, and nucleic acid stabilization have been gaining popularity as they can be used as an early warning system to provide arbovirus genetic information useful in molecular epidemiology studies. Here, we present the state of the art in the use of this approach and highlight the need for exploration of the potential of different available solid-phase materials for nucleic acid immobilization and stabilization during mosquito feeding on honey-baited traps. The current challenges for the successful implementation of feasible entomo-virological surveillance as a routine arbovirus surveillance tool in mosquitoes in urban and rural settings of endemic areas are also discussed.

Keywords

arboviruses entomo-virological surveillance FTA cards molecular detection mosquito saliva nucleic acid stabilization

Introduction

Arthropod-borne viruses (arboviruses) are disease-causing viruses biologically transmitted by several species of hematophagous arthropods (mainly Culicidae), which serve as vectors after feeding on viremic vertebrates. To date, more than 150 arboviruses may cause human diseases, among which mosquito-transmitted viruses such as Orthoflavivirus denguei (DENV), Alphavirus chikungunya (CHIKV), Orthoflavivirus zikaense (ZIKV), Orthoflavivirus flavi (YFV), Alphavirus mayaro (MAYV), Orthoflavivirus japonicum (JEV), and Orthoflavivirus nilense (WNV) have become the most important in terms of morbidity and mortality.¹ Together, they represent most vector-borne diseases that cause 17% of the global burden and expose over 80% of the population in endemic regions.² For this reason, the World Health Organization (WHO) has launched a strategy for integrated vector management³ and has encouraged the implementation of entomo-virological approaches⁴ as part of the public health response to the current⁵ and future socioeconomic burden of arboviral infections, which is expected to increase due to globalization, urbanization, and climate change.⁶

Given the complexity of the vector–virus–vertebrate host triad, there are surveillance approaches that gather information from different sources depending on the transmission cycles of the viruses, which can be divided into sylvatic (enzootic), epizootic, and urban (endemic/epidemic) cycles.^7–9 Epidemiological surveillance of arboviral diseases mainly relies on case notification from hospitals and health practitioners with a low rate of laboratory confirmation of infection due to insufficient laboratory testing in low-to middle-income countries and the fact that clinical signs and symptoms overlap with those caused by several other infectious agents, mainly in the tropical region.^7,8 Wild or domestic animals as sentinels placed in specific environments to be exposed to mosquito bites were historically used as a successful approach for arbovirus surveillance; however, limitations such as accessing the natural environments, the bioethical implications of the use of animals in experimentation, and the fact that several viruses could not infect certain vertebrate species have limited their current use.^7,8 Entomological surveillance consists primarily of collecting hematophagous arthropods, identification, and estimating densities or infestation rates as indirect measures of the risk of transmission of pathogens, such as arboviruses. When combined with virological or molecular approaches for the specific detection of arboviruses, the entomo-virological approach emerges as an early warning system for better assessing the transmission risk and providing information about the genetics and evolutionary dynamics of circulating viruses.^8,10–12 However, limitations, such as maintenance of the cold chain to preserve the virus infectivity for isolation success, and the viral genome integrity, need to be surpassed.

One of the ways that has been gaining ground for entomo-virological surveillance is the use of paper-based nucleic acid stabilization materials that act like matrices, which offer a rapid and simple way of collecting, stabilizing, and transporting the genetic material until it can be extracted.¹³ Common paper-based materials differ in their adsorption and desorption capacities, porosity, mechanical strength, surface elements, and functional groups, among others. Commercial names of validated materials that can successfully retain nucleic acids can be found as Whatman™ filter paper, FTA^® card, Fusion 5 membrane, silica membrane, and polyethersulfone membrane, where the FTA^® card has demonstrated its stabilizing properties.¹⁴ This article aims to gather information regarding the current use and prospects of paper-based nucleic acid retention and stabilization materials in entomo-virological surveillance, as it is considered a promising alternative to help the appropriate detection, diagnosis, and prevention of arboviruses circulating in the urban cycle, extending to the rural and sylvatic settings.

Relevance of mosquito-borne viruses in the Americas

Dengue is the fastest-growing mosquito-borne disease in the world and represents a major epidemiologic impact.^6,9 It is caused by any of the four distinct serotypes of DENV. DENV was first isolated almost 80 years ago and, as the distribution range of the main vectors increases,¹⁵ the number of reported cases has also been increasing over the past decades, becoming endemic in more than 120 countries, mainly in the tropical and subtropical regions of the Americas, Southeast Asia, and the Western Pacific, representing a risk for around 4 billion people.⁶ Even though vaccines have been recently approved, their use is limited, and each year there are at least 390 million infections and about 50,000 deaths.⁵ Other alternatives, such as the delivery of Aedes aegypti mosquitoes persistently infected with insect-specific endosymbiotic bacteria from the genus Wolbachia (wMel strain), have been used to reduce the number of mosquitoes able to transmit arboviruses in Colombia, Australia, Indonesia, Brazil, and other countries, with a significant impact in reducing DENV notifications in the areas of intervention, compared to the decade before Wolbachia-infected mosquitoes were released.^16,17 However, these approaches will need to be extended and accompanied by an intensified entomo-virological surveillance to prove their stability over time and to anticipate any unpredicted ecological effect.^16,18 Despite efforts, the number of affected people is expected to increase due to latitudinal expansion and optimal conditions for vector transmission driven by globalization and climate change, putting even more people at risk.⁶ Dengue cases in the Americas increased by 184% in 2024, compared to the same period in 2023, and about 360% compared to the average of the last 5 years, with the higher number of cases being reported by Brazil, Argentina, México, Colombia, and Paraguay.¹⁹

CHIKV, responsible for the Chikungunya fever, has been identified in four continents in over 110 countries, where it has infected more than 2 million people. The incidence has decreased after the reemergence peak in some areas, mainly due to the herd immunity acquired after the infection of most susceptible individuals in regions of epidemic circulation where the virus exhibited an extremely high attack rate.²⁰ Because symptoms can overlap with other viral, bacterial, or parasitic diseases, CHIKV can be easily and frequently misdiagnosed. Deaths and severe symptoms are usually related to coexisting health problems, and there have been cases where rheumatologic symptoms last for months and years.²¹ Ocular, cardiac, and neurological complications have been sporadically described concurrently with common symptoms.²² After a long interepidemic period, the Americas reported a rise in the number of cases in 2023, with 410,754 and 419 deaths.²³

ZIKV reemergence and epidemic spread in South America and the Caribbean in 2014–2015 affected around 90 countries and was declared a Public Health Emergency of International Concern after a causal link between the virus infection and the occurrence of congenital malformations (e.g., microcephaly) and other neurological impairments (e.g., Guillain–Barre syndrome, neuropathy, and myelitis) was established. ZIKV infection differs from Dengue and Chikungunya fever in its high proportion of asymptomatic cases, reaching 80% of infections.²⁴

The incidence of DENV, CHIKV, and ZIKV infections in the Americas is depicted in Figure 1.

Figure 1.

Accumulated number of cases of arbovirus infection in the Americas, 2024.

Entomological surveillance of arbovirus vectors

Arbovirus vector surveillance is a critical tool in public health. It collects data by measuring shifts in geographical distribution, quantifying key population indicators, and pinpointing areas of high-density infestation or increasing infectious populations. These data are used for informed decision-making, executing public health policies, and planning interventions when needed.²⁵

Although entomological surveillance often focuses on mosquitoes and arboviruses circulating in the urban cycle, monitoring of a wide range of arthropod vectors of public and animal health importance in urban, rural, and sylvatic settings—such as ticks, biting midges, and sandflies—is also crucial to understanding the dynamics of less frequent but potentially epidemic or emerging infectious agents.^26–28

The information generated through effective entomological surveillance—such as mosquito density, species composition, and infection rates—forms the basis for stratifying entomological risk and selecting appropriate vector control interventions. Once this stratification has been achieved, it enables a more targeted and cost-effective implementation of control strategies. Integrated strategies for vector control should involve environmental management, social mobilization, public education and behavior change, personal protection measures, physical barriers, larvicides, and biological or chemical approaches.²⁹ These traditional interventions have proven to be useful but require integration with other strategies to maximize their impact, opening the door for new biological innovations such as the use of genetically modified (GMM) or Wolbachia-infected mosquitoes; strategies that can cause mosquitoes’ sterility, lead them to cytoplasmic incompatibility, or make them refractory to viral infections. The introduction of these interventions requires a rigorous follow-up to adequately measure their impact on vector control.³⁰ Some gaps that can interfere with vector control in the Americas include limited entomological evidence to guide interventions, inadequate stratification and information management, a shortage of trained human resources, difficulties with insecticide procurement and equipment provision, and a lack of an organized, evidence-based, and integrated management across different environments and programs.³

In urban settings, mosquito surveillance includes monitoring of the different stages of mosquito development: eggs, larvae, pupae, and adults. Depending on the phase to be evaluated, it provides specific information to understand the vector’s behavior, patterns, and reproduction. Each stage provides advantages and limitations, but together, they provide complementary information to make better decisions.²⁹ In the context of the traditional entomological surveillance of Aedes aegypti, in urban settings in the Americas, several larval indices are commonly used. The most widely applied methods involve periodic household inspections to estimate infestation levels through indices such as the House Index (percentage of houses infested with larvae), Container Index (percentage of water-holding containers infested with larvae), and Breteau Index (number of positive containers per 100 inspected houses).^31–33 However, they are a poor indicator of adult production, have high costs, lack the sensitivity to detect subtle changes in vector behavior, depend on the technical capacity to search for larvae, and other factors, such as communication and community acceptance.^25,31 Other methods of estimation consist of measuring relative and absolute density. Relative density refers to the number of mosquitoes collected per unit of effort (e.g., per trap or per aspiration session), allowing for comparisons across time and locations. In contrast, absolute density, as defined by the Pan American Health Organization (PAHO), is an estimate of the number of mosquitoes present in a specific area, typically obtained through direct sampling techniques such as pupae per household or hectare, adult aspiration indoors and outdoors, or mark–release–recapture methods.²⁹

Mosquito traps have long been proposed as they can provide additional information such as positive individuals per trap, density or number of individuals per trap, characteristics of both female and male adults, data about populations, and give the possibility of integrating arbovirus surveillance with entomological surveillance, which can provide predictions as an early warning system for outbreaks and serotype/genotype introductions.^31,32 However, the effectiveness of some trap-based surveillance strategies depends on the capture of a sufficiently large number of mosquitoes to ensure statistically meaningful results, and many traps rely on batteries and CO₂ to enhance mosquito attraction, which increases operational costs and limits their use in remote or low-resource settings.³⁴

Incorporation of the virological component for a better assessment of the disease risk

Traditional indicators of entomologic surveillance have demonstrated limited utility in detecting high-risk populations for arbovirus transmission and anticipating outbreaks and epidemics.^35,36 Direct detection of arboviruses has been used to improve entomological surveillance since the foundation of arbovirology.³⁷ By regularly monitoring vector populations and reservoirs for the presence of viruses of medical importance, scientists in collaborating centers and national laboratories can help inform viral activity, enabling public health authorities to make decisions to prevent or control outbreaks promptly.⁴ Virological surveillance can also be useful for measuring the effectiveness of vector control interventions on transmission risk by monitoring changes in virus prevalence before and after their implementation.³⁴

Virus isolation, hemagglutination inhibition, complex fixation, neutralization tests, and immunofluorescence were the classical virological methods successfully used to discover and monitor several viruses associated with human diseases.³⁷ More recently, with the advent of molecular biology, isothermal (e.g., Loop-mediated isotermal AMPlification-LAMP, Nucleic Acid Sequence-Based Amplification-NASBA), Polymerase Chain Reaction-PCR-based (e.g., endpoint PCR, real-time PCR, and digital PCR), and DNA sequencing-based (e.g., di-deoxy Sanger sequencing and next-generation sequencing) methods have been implemented for the routine diagnostics and surveillance of arboviruses as part of national programs of disease control and prevention and for academic purposes.³⁸ The entomo-virological approach has been based on the detection of viral RNA in hundreds or thousands of mosquitoes, being recommended by health authorities to maximize the effectiveness of public health policies, as it can help with the identification and monitoring of vector-borne diseases³¹ (Figure 2).

Figure 2.

The entomo-virological approach for arbovirus surveillance.

The minimum infection rate (MIR) has been implemented, which is considered more appropriate than the traditional indices as it enables assessing the proportion of infected mosquitoes in a population, helping with the identification of high-risk areas, and more accurately measuring virus transmission over time. To measure this, adult mosquitoes or larvae are collected, pooled for the molecular detection of arbovirus, and the number of positive pools is divided by the total number of specimens and then multiplied by 1000. MIR is particularly useful when the infection rates are low and the pool sizes are large, which minimizes the possibility of overestimating the infection prevalence. However, this method assumes that only one infected individual exists in a positive pool, which becomes problematic when infection rates are high or pool sizes are too large.³⁹ In contrast with MIR, the maximum likelihood estimation (MLE) can also be used, especially when the infection rates are high and pools are large and constant or variable size, allowing a more accurate estimate of the infection rate since it relaxes the constraints of MIR.^39,40

Alternative approaches have been proposed for the evaluation of mosquito positivity to arboviruses, such as the processing of whole specimens at different stages of mosquito development (egg, larvae, pupae, and adult),¹⁰ and the independent examination of the mosquito body parts and fluids (head, thorax, legs, salivary glands, saliva, or even the excreta) in order to stablish the different levels of infection and retrieve additional information about the virus’s success in crossing the mosquito barriers.^41–44

Arbovirus detection in the whole body (including the midgut) can be the result of a recent mosquito feeding on a viremic source, but their detection in the head, salivary glands, or saliva (mainly explained by the successful crossing of the different mosquito barriers during the extrinsic period) offers important evidence about their potential transmission. The discovery that infected mosquitoes can expectorate arboviruses in the saliva while feeding on sucrose-baited pledgets has opened a new opportunity for the entomo-virological surveillance,⁴² as this is a noninvasive technique that offers multiple advantages, from avoiding manipulation of mosquitoes while they feed, reducing the cost by allowing to represent the saliva of multiple mosquitoes in a single membrane, to reducing logistic costs because the viral genetic material can be stabilized in field conditions, not relying on cold chains, for instance.³⁴

Molecular detection of arbovirus in mosquitoes’ saliva using paper-based nucleic acid stabilization materials

After demonstrating the efficient transmission of arboviruses through the use of the mosquitoes’ saliva expectorated during feeding on solid-phase matrices,^42,45 a new approach to entomo-virological studies emerged with a growing number of studies supporting its usefulness for pathogen surveillance.^46–48 This strategy has several advantages, including: (1) the possibility of having multiple mosquitoes feeding on the same card installed in a trap, achieving a reduction in the number of samples entering the molecular biology workflow, (2) the long-term stabilization of viral nucleic acid on nucleic acid stabilization materials (e.g., Flinders Technology Associates—FTA^® cards) without the need for a cold chain, facilitating its implementation as part of the routine activities of fieldwork technical teams for entomological surveillance in areas of endemic arbovirus circulation, and (3) the usefulness of virological information to demonstrate the circulation of arboviruses in specific vector populations, and their transmission potential (presence in saliva) as an early warning system that allows anticipating outbreaks.

FTA^® cards can be considered the gold standard for entomo-virological surveillance from mosquitoes’ saliva. These cards are composed of filter paper with a proprietary technology that allows the lysis and release of nucleic acids from cells, and their subsequent immobilization and preservation for months and even years, until they are ready for purification.⁴⁹ Since they can store blood, cultured cells, plasmids, and even solid tissues, a wide range of applications include food and agriculture testing, molecular biology, forensic, and biomedical sciences. FTA^® cards have demonstrated to be the best option for nucleic acid preservation when compared to other matrices, allowing the inactivation of infectious agents, avoiding subsequent microbial contamination of samples, and enabling the detection of multiple viruses.⁵⁰ When combined with sugar or honey baits, FTA^® cards have demonstrated their usefulness for collecting mosquitoes’ saliva for subsequent arbovirus detection through molecular methods.^51–54

Several studies performed in Australia, Brazil, French Guiana, Germany, and Italy have successfully detected arboviruses from FTA^® cards, including Orthoflavivirus usutuense (USUV),^47,48 Alphavirus rossriver (RRV),^13,45,55 Alphavirus barmah (BFV),^13,45,55 Orthoflavivirus murrayense (MVEV),⁵⁵ CHIKV,⁵⁴ ZIKV,⁵⁶ DENV,⁵⁷ Orthoflavivirus kokoberaorum (STRV),¹³ and WNV.^47,48,55 These studies report variable but promising rates of detection in field studies. While any of these studies confirmed that FTA^® card-based detection preceded human cases, several emphasized its value in early detection, particularly in endemic or high-risk areas.

Assisted methods for allowing mosquitoes to feed on the cards include the introduction of manually collected adult mosquitoes for a specific period in small flasks containing the honey-baited accompanied with a food dye to assess the feeding efficiency. Other methods comprise the use of electrical mosquito traps that depend on battery life, which are limited by electricity, potential damage, and extreme conditions such as increased temperatures and high humidity. Several standard traps, both passive and electrical, have been evaluated in protocols implementing mosquitoes’ feeding on honey-baited FTA^® cards.^46,52 The attractiveness of the traps could be enhanced by the production of CO₂ by fermentation reactions, sublimation of dry ice, or by adding semiochemicals like octenol.^34,58 In order to ensure active feeding on these cards, some traps have been developed or modified to prolong mosquito survival and then increase the probability of feeding.^8,59 The reported toxicity of FTA^® cards—significantly reducing mosquito survival without repellent effects—could be considered an additional advantage for their use as attractive toxic sugar baits.⁶⁰

Despite the inherent challenges associated with the use of FTA^® cards, including the cost, they represent a superior alternative to traditional entomo-virological approaches that involve the collection and analysis of mosquitoes and their tissues. Research efforts should be strategically directed toward the development or modification of other solid-phase materials that could warrant the stabilization, preservation, and safe handling of the genetic material.

Prospects of other membrane-based nucleic acid binding/stabilization materials for arbovirus surveillance

Preserving arbovirus genomic RNA in mosquito samples collected under field conditions is a challenge because of its labile nature, which makes it more susceptible to degradation than DNA.⁶¹ High temperatures, RNases derived from the microorganisms’ metabolism, and those generated as byproducts of mosquito death after collection can cause viral RNA to degrade quickly,^62,63 which has been partially overcome by warranting the cold chain, while samples are transported for RNA extraction and further processing in the laboratory. Although several RNA stabilization solutions have been developed for protecting RNA in unfrozen tissues, cells and cell-free samples,⁶⁴ and solid-phase nucleic acid extraction materials have shown a good performance for nucleic acid binding and subsequent elution due to their specific absorption and desorption properties,⁶⁵ the solid-phase RNA stabilization during sample collection, transportation, and storage before extraction continues to be a major challenge.

During the last decade, the possibility of using paper-based matrices for nucleic acid extraction has been explored due to their portability, low cost, time efficiency, environmental friendliness, and adaptability.^14,66 Although these materials have been implemented in the technical approaches for the diagnosis of arboviral diseases in humans (Table 1), a smaller proportion of materials have demonstrated their value in entomo-virological surveillance.^67,68

Table 1.

Summary of membrane-based nucleic acid immobilization/stabilization materials with current or prospective use in entomo-virological surveillance.

Membrane-based material (matrix)	Arbovirus detected	Type of samples	Ref.
FTA^® cards	DENV, CHIKV, ZIKV, other arboviruses	Blood, mosquitoes, mosquitoes’ saliva, mosquitoes’ excreta	13, 45, 50, 52, 54, 56, 68, 69 –71
Silica	DENV	Cell cultures	72
Fusion 5	DENV, CHIKV	Serum, plasma	73, 74
Polyethersulfone (PES)	DENV, CHIKV, ZIKV	Serum, blood	75, 76
Whatman filter paper	DENV, CHIKV, Encephalitic Viruses	Serum, plasma, blood, mosquitoes’ saliva, mosquitoes’ excreta	73, 77 –81
Cationic (Q) papers	DENV, CHIKV, ZIKV	Plasma, urine, mosquitoes	82, 83
Chromatography or cellulose paper	ZIKV	Plasma, urine, saliva	84, 85
Glass fibers	DENV, CHIKV	Serum, plasma	73
Nitrocellulose	DENV	Virus isolation, control RNA	86, 87
Nylon	DENV, ZIKV	Serum	88
Other filter papers	DENV, CHIKV, other arboviruses	Serum, plasma, blood, mosquitoes’ saliva, mosquitoes’ excreta	44, 67, 73, 80, 89 –93

CHIKV, Alphavirus chikungunya; DENV, Orthoflavivirus denguei; ZIKV, Orthoflavivirus zikaense.

A wide range of paper-based nucleic acid stabilization/binding materials could be explored soon for the stabilization, storage, and subsequent detection of arboviruses, such as DENV, CHIKV, and ZIKV, in mosquito’ samples collected in urban, rural, and sylvatic settings. Nucleic acid stabilization materials are based on the principles of nucleic acid preservation and the chemical or physical properties of the membranes. Additionally, the membrane needs to have a high binding capacity for nucleic acids, should be free of any contaminants that can degrade nucleic acids, particularly RNA, and needs to be able to stabilize the nucleic acids after being treated with different agents depending on the specific application and the nucleic acid nature.¹⁴

Silica membranes are silicone oxides that have become crystallized forming positively charged microporous structures with high binding affinity for nucleic acids under alkaline conditions and high salt concentration.⁹⁴ They can be integrated into paper-based matrices,⁹⁵ or glass fibers.⁹⁶ Silica matrices have been implemented in the most widely used and commercially available column-based RNA extraction and purification methods, but their properties for RNA stabilization have not been extensively explored.⁹⁷

Cellulose-based membranes, such as the Whatman^® filter paper #1 (Merck KGaA, Darmstadt, Alemania), are used to identify materials in routine applications with medium retention and flow rate. These membranes have been used for more than 50 years as a method for simple collection and storage of samples for later evaluation in screening tests, monitoring of drugs, analysis of genomes, and epidemiologic studies.⁹⁸ As it has been more commonly used to collect blood samples, the detection of arboviruses has been mainly oriented to virus surveillance in humans and animals. Nevertheless, at least a study focused on the detection of arboviruses in mosquitoes’ saliva and excreta included these filter papers as part of the feeding substrates.⁷⁷ Other filter papers from Whatman^® and other brands have been used to store dry blood spots and have allowed the detection of flaviviruses for up to 1 year storage at 37°C.^89,99

Fusion 5 is a proprietary single-layer matrix membrane commonly used in lateral flow test kits, which has also been explored for nucleic acid extraction, taking advantage of its positively charged glass fibers and the negative charge of the DNA and RNA.⁹⁶ Fusion 5 membranes were successfully used in RNA extraction and storage packets allowing the subsequent rRT-PCR detection of DENV, CHIKV, and OROV at comparable cycle threshold (Ct) values with the other two types of materials.⁷³ Another study successfully used Fusion 5 modified with chitosan for the extraction of DENV RNA from plasma samples.⁷⁴

Borosilicate glass fibers, such as the Whatman^® glass microfiber grade GF/D (Merck KGaA, Darmstadt, Alemania), are another promising material that demonstrated the best performance for DENV, CHIKV, and OROV RNA stabilization and recovery after exposure to room temperature for 35 days, compared to Fusion 5 and cellulose-based membranes.⁷³

Polyethersulfone (PES) membranes are composed of a polymer with uniform pores, controlled flow rates, retention of low protein, and purification qualities. As well as other membranes, its surface can be modified so it can be used for binding and extracting DNA, as was done by Baeumner and co-workers while detecting DENV in blood samples.¹⁰⁰ More recently, a biosensor with a modified PES membrane was evaluated to detect DENV in blood samples; it was low-cost and had high sensitivity and specificity.⁷⁵ PES has also been used to extract viral RNA from DENV, CHIKV, and ZIKV in a two-step protocol including a paper-chip device for detection on serum samples.⁷⁶

Cationic (Q) paper is another material successfully used to stabilize and recover arboviral nucleic acids from mosquito bodies. This paper, which is cheap and can be prepared in bulk, consists of cationic moieties formed by quaternary ammonium groups covalently attached to the surface by treatment with 2,3-epoxypropyltrimethylammonium chloride.⁸² Despite its prospects, there is a lack of studies exploring its performance in arboviral nucleic acid stabilization for molecular detection.

Nylon (polyamide), polyvinylidene difluoride, and nitrocellulose membranes have been classically used to immobilize macromolecules, including RNA, in widely used Northern blot protocols.¹⁰¹ Positively, negatively charged and neutral surfaces have been explored with differential performance. Once the RNA is transferred to the membrane by electrophoresis, it can be immobilized on the membrane using UV radiation or baking. UV light causes the nitrogenous bases to form covalent bonds with the amine groups on the surface of the membrane. Baking allows the removal of water from RNA, promoting hydrophobic contacts with the aromatic groups on the membrane.¹⁰¹ It is assumed that immobilization allows long-term preservation of RNA; however, current downstream protocols remain limited to the solid phase.

All the matrices discussed have been proven to stabilize or immobilize different arboviral genomes for their subsequent extraction and analysis, which means they show promise for use in field conditions if they evolve into matrices capable of preserving nucleic acids over time and under various storage conditions. Future studies could take advantage of matrices previously used for arbovirus diagnostics, as they are effective for stabilization and subsequent extraction of virus genetic material in other sample types. Studies could also compare the performance of the matrices and other stabilization methods, considering factors such as sample type, sample size, cost-effectiveness, and efficiency. In addition, they could focus on improving the sensitivity and specificity of existing paper-based materials by integrating them with nucleic acid stabilization techniques.

Paper-based matrices could be extremely useful for field research, emergency response, and routine nucleic acid testing, especially in resource-limited areas where these materials could offer a quick, accurate, lightweight, and durable method for sample handling from the field to the lab. More advantages could be reached if the paper facilitates steps of amplification and detection, for example, by the use of microfluidic chips and smartphone imaging,^84,102,103 which would decentralize even more the wet lab processes.

The present and prospects of metagenomic next-generation sequencing in viromic studies and its potential for entomo-virological surveillance

Effective surveillance is crucial for reducing the impact of arboviruses on human and animal health. Routine testing for endemic, emerging, and potentially new viruses is mandatory in the post-genomic era, and modern technologies can help to identify viruses without prior knowledge of the pathogen.

While the entomo-virological approach can provide early evidence of arbovirus activity in the mosquito populations and their potential transmission,^104,105 next-generation sequencing (NGS) technologies, when combined with metagenomic wet lab workflows and bioinformatic pipelines, have consolidated as the more robust approaches for the microbiome and virome characterization, pathogen identification, and genomic surveillance from several mosquitoes’ species,^106–112 providing detailed information about the viral genomes, their evolution and spread, as well as the accuracy of the diagnostic tests.^113,114 Despite its enormous potential, to the best of our knowledge, only a few studies have implemented the use of FTA^® cards for the successful characterization of the virome from mosquitoes’ saliva.^53,115

Concluding remarks

Arboviruses are disease-causing agents of major public health concern. Their surveillance requires approaches able to integrate epidemiological, entomological, and virological information to better assess the virus dynamics in the human and vector populations. Recent advances in methods have enabled the comprehensive detection of arboviruses through molecular methods from different sample types.

The integration of entomology and virology (entomo-virological surveillance) could help to stratify areas according to the real arbovirus transmission risk, increasing the accuracy of outbreak predictions, helping to prioritize and allocate resources for timely public health interventions.

A multidisciplinary, holistic system that incorporates advanced scientific methods, sophisticated surveillance tools, and low-cost traps coupled with attractive chemicals could improve arbovirus surveillance. Such traps have the greatest applicability for deployment in remote locations where entomological and arbovirus surveillance are difficult. The honey-baited system that collects mosquitoes’ saliva is a promising method because it is noninvasive, low cost, and easy to implement. While the FTA^® cards have demonstrated their usefulness for the collection and stabilization of RNA from mosquitoes’ saliva, new paper-based materials need to be further studied and optimized to find the best solution for this application.

Footnotes

Acknowledgements

To the Public Health Secretary of the Santa Marta district for their valuable collaboration during a decade of interdisciplinary work in entomovirology, and to the Program of Biomedical Engineering, Universidad CES, for the academic support to RP-F.

Declarations

ORCID iDs

Rosana Pérez-Franco

Daniel B. Ramírez-Osorio

Marlen Martínez-Gutiérrez

Gabriel Parra-Henao

Jose A. Usme-Ciro

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