Aptamers isolated against mosquito-borne pathogens

Abstract

Mosquito-borne diseases are a major threat to public health. The shortcomings of diagnostic tools, especially those that are antibody-based, have been blamed in part for the rising annual morbidity and mortality caused by these diseases. Antibodies harbor a number of disadvantages that can be clearly addressed by aptamers as the more promising molecular recognition elements. Aptamers are defined as single-stranded DNA or RNA oligonucleotides generated by SELEX that exhibit high binding affinity and specificity against a wide variety of target molecules based on their unique structural conformations. A number of aptamers were developed against mosquito-borne pathogens such as Dengue virus, Zika virus, Chikungunya virus, Plasmodium parasite, Francisella tularensis, Japanese encephalitis virus, Venezuelan equine encephalitis virus, Rift Valley fever virus and Yellow fever virus. Intrigued by these achievements, we carry out a comprehensive overview of the aptamers developed against these mosquito-borne infectious agents. Characteristics of the aptamers and their roles in diagnostic, therapeutic as well as other applications are emphasized.

Graphic abstract

Mosquito-borne infections have been identified as the leading cause of high mortality and morbidity worldwide (Hoenen et al. 2006; Price and Thio 2011; Siegel et al. 2016). Pathogens that cause mosquito-borne infections have a broad geographic range and can cause human illnesses at various levels of severity. Many of them also infect wild animals, including rodents, bats, birds and monkeys. In certain cases, animals act as a hidden viral reservoir in a sylvatic cycle (Kuno et al. 1998). The mosquitoes imbibe the pathogen when blood-feeding on an infected host to support the replication of the pathogen in their organism. As such, they deliver a sufficiently large inoculum of the pathogen into the host. In order to infect a recipient host, a specific level of viraemia is usually required. Thus, only vertebrate species that contain a distinct degree of viraemia are considered as the amplifying hosts (Bárdos and Rosický 1979). When infected mosquitoes spread the virus to humans, it creates a condition in which humans are unable to transmit the virus further and become dead-end hosts (Fig. 1) (Bowen and Calisher 1976; Walton et al. 1973). Transmission of mosquito-borne pathogens from male to female occurs during copulation [horizontal or venereal transmission (VT)] or even from female to the offspring [vertical or transovarial transmission (TOT)]. Under TOT conditions, the mosquito vector plays a role as a long-term reservoir of the virus. Some pathogens only have a few hosts and vectors, while others replicate in a large number of hosts and vectors. Aside from that, some have a widespread distribution, whereas others are restricted to a limited geographic area (Morales et al. 2017). The transmission rate is strongly influenced by factors such as the population density of mosquito vectors, vertebrate hosts, floods, droughts and habitats such as the wetlands, shallow water reservoirs or sewage systems (Hubálek 2008). The complex interplay of these multiple factors necessitates an early diagnosis of these diseases to cure and prevent further viral progression, thus saving the lives of the infected. Available serological based diagnostic methods are largely beleaguered by the disadvantages of the antibodies such as reversible denaturation property, large batch-to-batch variation and temperature insensitive. An alternative molecular recognition element (MRE) known as aptamer has many advantages that have the potential to alleviate these drawbacks.

The schematic diagram of pathogen transmission by the mosquito

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The term “aptamer” comes from the combination of Latin “aptus” which means “to fit” and the Greek “meros” which means “particle” (Ellington and Szostak 1990). Aptamers are single-stranded oligonucleotides that are capable of binding to their cognate target molecules with high affinity and specificity based on their unique structural conformations (Ellington and Szostak 1990; Tuerk and Gold 1990). The aptamer-target interactions are governed by hydrogen bonding, electrostatic interactions, van der Waals forces and shape complementarity (Gelinas et al. 2016; Kong and Byun 2013; Nomura et al. 2010; Sun et al. 2014; Zhou et al. 2016). Since RNA aptamers contain a 2′-OH group, they can form more diverse three-dimensional (3D) structures compared to DNA aptamers, making them more flexible in binding to a broad spectrum of targets (Butcher and Pyle 2011; Hendrix et al. 2005; Sun et al. 2014). On the other hand, the absence of a hydroxyl group at the 2′ position of the deoxyribose sugar makes DNA aptamers more stable than RNA, eventually increasing the shelf-life of DNA aptamers (Lakhin et al. 2013; Zhu et al. 2015). Aptamers are generated in vitro from a random oligonucleotide library through a repetitive amplification and selection process known as systematic evolution of ligands by exponential enrichment (SELEX) (Fig. 2) (Ellington and Szostak 1990; Tuerk and Gold 1990). In several studies, different SELEX methods have been explored and modified to isolate high-affinity aptamers. Affinity chromatography and magnetic bead-based SELEX, nitrocellulose membrane filtration-based SELEX and capillary electrophoresis-based SELEX are the few examples (Gopinath 2007; Mencin et al. 2014; Song et al. 2012).

The schematic diagram of SELEX: a library preparation, b incubation, c binding, d partitioning, e elution, f amplification/ssDNA generation/in vitro transcription, and g cloning and sequencing

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To date, many aptamers have been selected against a wide range of targets such as metal ions, hormones, antibiotics, vitamins, toxins, proteins, cells, tissues, bacteria and viruses (Chiu et al. 2018; Kwon et al. 2020a, b; Li et al. 2019; Raducanu et al. 2020; Song et al. 2017; Stoltenburg et al. 2016; Su et al. 2020; Wang et al. 2019; Ye et al. 2019; Zhang et al. 2020). Aptamers have a low molecular weight, which allows for rapid tissue penetration, as well as other benefits such as greater structural flexibility, lower immunogenicity, prolonged shelf life, reusability due to reversible denaturation property, no batch-to-batch variation, lower production cost and ease of synthesis (Breaker 1997; Kruspe et al. 2014; Mairal et al. 2008; Wang et al. 2015; Zhang et al. 2019; Zhou and Rossi 2017). In addition, aptamers can selectively distinguish between very similarly structured biomolecules such as caffeine and theophylline, which differ only by a methyl group (Hermann and Patel 2000; Jenison et al. 1994; Wrist et al. 2020). Owing to their superior properties, aptamers have received a great deal of attention in numerous applications ranging from diagnostics to therapeutics, targeted drug delivery and bio-sensing (Alshamaileh et al. 2017; Citartan et al. 2019; Khan et al. 2018; Shigdar et al. 2013). Due to the aforementioned factors, aptamers are considered as an alternative to antibodies (Fig. 3).

The illustration of the advantages of aptamers over antibodies

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The advantages of aptamers have propelled extensive research into developing these molecules against a broad range of mosquito-borne infectious agents. An overview of these aptamers against mosquito-borne infectious agents was provided in this current study. This is the first endeavor to compile and underscore all the existing aptamers for mosquito-borne infectious agents including Dengue virus (DENV), Zika virus (ZIKV), Chikungunya virus (CHIKV), Plasmodium parasite, Francisella tularensis, Japanese encephalitis virus (JEV), Venezuelan equine encephalitis virus (VEEV), Rift Valley fever virus (RVFV) and Yellow fever virus (YFV). Moreover, the epidemiology of mosquito-borne diseases is briefly highlighted in this review (Table 1). The majority of aptamers were developed for diagnostic purposes as potential substituents for antibody-based pathogen detection that are still beset with setbacks. This review also includes several aptamers that have undergone therapeutic functionality testing and other applications.

Globally, there are more than two billion people in tropical and subtropical areas that are at risk for dengue fever, with 50–100 million people infected and 24,000 deaths annually (Diamond and Pierson 2015). Dengue infection is caused by DENV, which is a member of the Flaviviridae family and the genus Flavivirus. Four closely related dengue serotypes (DENV types 1–4) cause human diseases including non-specific viral syndrome, fatal dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS) and even death if not treated appropriately. This virus is transmitted to humans through the bite of infected female Aedes mosquitoes (WHO 2009). The rising global spread of DENV and the lack of an approved vaccine or anti-viral therapeutic has prompted extensive research towards diagnostics as well as therapeutics (Guzman et al. 2010). DENV has an 11-kb positive single-stranded RNA (ssRNA) genome. Three structural proteins (capsid (C), membrane (M), and envelope (E)) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are encoded by the genome; with type I cap structure at the 5′-end. The protein translation is achieved with the aid of the host translation machinery (Mukhopadhyay et al. 2005; Potisopon et al. 2014; Whitehead et al. 2007; Zhou et al. 2007).

Current diagnostics of DENV are enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) (Poloni et al. 2010). ELISA requires antibodies, which are time-consuming to produce, often unstable and have batch-to-batch variation. On the other hand, RT-PCR is a complicated procedure that takes a long time and necessitates the use of a costly thermocycler. As such, many efforts have paved the way towards aptamer-based diagnostics of DENV. Fletcher et al. (2010) achieved a significant milestone by developing a biosensor utilizing an oligonucleotide linker module based on restriction endonuclease EcoRI in a complex with an aptamer. The linker has a stem region that is complementary to the target sequence, in this case, a specific region of the viral genome. In the presence of the virus, the linker binds to the specific region of the genome. The linker’s complementary sequence to the aptamer sequence allows the aptamer to be released from the EcoRI-bound complex. As such, the released EcoRI enzyme will rapidly cleave multiple signaling molecules, generating detectable signals proportional to the number of viral copies. This is the first sensor that relies on the ‘amplifying effect’ of the restriction enzyme for the detection of the virus. Besides that, Basso et al. (2019) used a hybrid nanomaterial formed by magnetic nanoparticles γ-Fe₂O₃ conjugated to gold nanoparticles modified with DNA aptamers on the surface to develop a simple and fast colorimetric immunosensor for the detection of DENV. Fourier-transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), quartz crystal microbalance (QCM), UV–Vis and localized surface plasmon resonance (SPR) were among the techniques used to characterize the biosensor. Colorimetric changes upon the aptamer-target binding allow rapid visual detection of the virus without the requirement of any special equipment. As the DENV’s structure resembles that of another virus from the same genus, the binding specificity of the aptamer was crosschecked against ZIKV and YFV. The aptamer did not cross-react with ZIKV or YFV according to the UV–Vis results. One possible challenge that the researchers might have to deal with is the ambiguity of the colorimetric result, which could stem from the equivocality of the naked-eye observatory-based interpretation. In an innovative aptamer design, Kwon et al. (2020a, b) created a star-shaped DNA architecture carrying five molecular beacon-like motifs constructed to display ten DENV E protein domain III (ED3)-targeting aptamers that are specific and precisely fit the spatial arrangement of ED3 clusters on the DENV viral surface. The researchers endeavored to elevate the sensing sensitivity by incorporating an element of avidity into the sensor by conflating multiple aptamers into one single unit. The construct showed a high binding avidity for the ED3 protein and is promising for the development of a DENV detection sensor that outperforms the existing gold standard methods such as ELISA and quantitative reverse transcription polymerase chain reaction (RT-qPCR). Recently, several aptasensors targeting NS1 protein were developed. In one study, aptamer against NS1 protein of DENV was used for the development of fluorescence aptasensor. The fluorescence emission of the NS1 aptamer labelled with 6-carboxyfluorescein (FAM) at the 5′-end was quenched due to the structural conformation change upon binding to the target NS1. A limit of detection (LOD) of 2.51 nM and 8.13 nM in buffer and serum, respectively were achieved (Mok et al. 2021). Junior et al. (2021) have also isolated aptamers against the NS1 protein of DENV and used them in an electrochemical sensor. The aptamers were immobilized on the surface of the gold electrode with the aid of 6-mercapto-1-hexanol (MCH) to form a self-assembled monolayer while blocking was achieved with BSA. The LOD achieved was down to 0.025 ng/mL, which is within the clinical range of NS1. The specificity of the sensor was also tested against the E protein of DENV, which showed insignificant interaction. These aptamers against NS1 proteins should also be extended to point-of-care diagnostic systems such as lateral flow assay.

Several DENV proteins were subjected to aptamer isolation. These aptamers unveil therapeutic properties by antagonizing the interaction of these viral proteins, which would otherwise facilitate pathogenesis. Chen et al. (2015) used SELEX to obtain a DNA aptamer, S15, which binds to DENV-2 E protein domain III (ED3) with a dissociation constant of 200 nM. The Quadfinder prediction and circular dichroism experimentation results revealed that S15 is capable of forming a parallel G-quadruplex. Both the quadruplex structure and the sequence at the 5′-end of the aptamer were predicted to be an important region for the binding against the targeted protein domain. Furthermore, NMR titration results indicated that the highly conserved loop between the β_A and β_B strands of ED3 serves as the binding region for the aptamer. Interestingly, the S15 aptamer was unable to bind to the denatured ED3 in the western blot assay, suggesting that the aptamer recognizes a conformational epitope instead of a linear epitope. Although S15 aptamer was isolated against DENV-2, the authors also discovered that it could neutralize all four DENV serotypes. Using a similar protein target, ED3, Gandham et al. (2014) identified DENTA-1, an ssDNA thioaptamer with a dissociation constant of 154 nM that binds to the antibody-binding site within ED3. In addition, Jung et al. (2017) have generated an RNase-resistant 2′-fluoro-modified RNA aptamer that directly binds to DENV-2 methyltransferase (MTase) and truncated it down to 45-mer RNA sequence without losing binding specificity. The authors have unleashed the therapeutic value of the aptamer, as it was found to disrupt the function of the DENV MTase, which is responsible for the sequential guanine N-7 methylation and the ribose 2′-O methylation of the type-I cap of the DENV RNA. The 45-mer aptamer not only showed binding against DENV2 MTase with the K_d of 28 ± 2.1 nM but also binds to DENV3 MTase with the K_d of 15.6 ± 1.03 nM and was able to disrupt its methylation activity.

Most of the aptamers used so for were directed against the DENV proteins. Targeting intracellular host protein was also proven to be useful in a DENV therapeutic intervention. Balinsky et al. (2013) proved that the addition of a nucleolin (NCL)-binding aptamer (AS1411) would disrupt the association between DENV C protein and multifunctional host protein NCL. NCL is a multifunctional cellular protein that plays a significant role in an array of different cellular processes, including ribosome biogenesis, protein transport, chromatin remodeling, translational regulation, RNA processing and stability. The AS1411 aptamer reduces DENV C protein colocalization, causes a significant reduction of viral titers following DENV infection. Furthermore, the authors demonstrated that the treatment with AS1411 affected the viral DENV C's migration characteristics. Apart from therapeutic and diagnostic uses, aptamer were also used in the identification of the viral RNA binding proteins involved in DENV replication. Dong et al. (2015) have devised a novel affinity purification strategy focused on the fusion of the streptavidin-binding RNA aptamer S1 sequence to the 3′ end of DENV 5′–3′ UTR RNA to isolate DENV-2 RNA-binding proteins (RBPs) from living mammalian cells. The streptavidin magnetic beads were coated with the S1 aptamer. This stratagem allowed the DENV 5′–3′ UTR RNA to isolate endogenous viral ribonucleoprotein (RNP) from the mammalian cell extract. This method led to the identification of several novel hosts DENV RBPs via liquid chromatography with tandem mass spectrometry (LC–MS). The outcome revealed RPS8, one of the RBPs discovered, which is linked to DENV replication. The protein identified can be targeted for therapeutic application in suppressing the pathogenesis of DENV. In summary, in the realm of DENV, aptamers have found various roles, ranging from diagnostics, therapeutics to viral protein studies.

In recent years, ZIKV, which is associated with Guillain–Barre’ syndrome in adults and microcephaly in newborns, has caused sporadic outbreaks (Calvet et al. 2016). The World Health Organization (WHO) declared it as a Public Health Emergency of International Concern in February 2016. ZIKV is an arthropod-borne positive ssRNA virus that belongs to Flavivirus genus of the Flaviviridae family. It is primarily transmitted via mosquito bites of Aedes aegypti (Ae. aegypti) (Sher et al. 2019). However, there have been reports of mother-to-child, blood and sexual transmission (Calvet et al. 2016; Saiz et al. 2016). The ZIKV reproductive cycle begins when the viral particle attaches to the host’s cytoplasmic membrane via an E protein that promotes endocytosis. The viral membrane then fuses with the endosomal membrane, releasing positive ssRNA into the cytoplasm of the host cell. After that, translation starts and the resulting polyprotein is cleaved into three structural (C, pre-membrane (prM) and E) and seven NS proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) (Zmurko et al. 2015).

Even in the case of ZIKV infection, aptamers have proven to be an expedient tool. Numerous aptamers were generated against a number of ZIKV proteins. In a study conducted by Lee and Zeng (2017), two aptamers (Aptamer 2 and 10) were selected after 7 rounds of SELEX cycles against ZIKV NS1 protein. Aptamer 2 showed the best binding affinity of 24 pM, whereas aptamer 10 showed the best binding specificity when compared to dengue NS1 serotypes. The LOD for ZIKV NS1 in the sandwich ELISA using aptamer-2-antibody pair were 0.1~1, 1~10 and > 10 ng/mL in buffer, 10% human serum and 100% human serum, respectively. In addition to Lee and Zeng’s research, ssDNA aptamers were developed using SELEX against ZIKV NS1 and NS5 proteins for sensing applications (Abalo et al. 2019; Alves et al. 2018; Morais et al. 2018, 2019). Aside from DNA and RNA, a peptide aptamer was also designed against ZIKV. Kim et al. (2018) used three bioinformatics methods, including BCPreds, ABCpred, and Bepipred, to devise a peptide aptamer against ZIKV E protein called Z_10.8 peptide (KRAVVSCAEA). The peptide aptamer selection was done based on several parameters such as binding affinity, root-mean-square deviation, the position of amine residue of lysine at the N-terminus and the position of the interactive sites in a docking analysis. The equilibrium dissociation constant estimated for the Z_10.8 peptide was 706.0 ± 177.9 nM. The diagnostic functionality of the aptamer was validated by fluorescence-linked sandwich immunosorbent assay (FLISA) and peptide-linked sandwich FLISA using ZIKV-spiked human serum and urine. The LOD for the sandwich FLISA was estimated at 1 × 10⁴, for the 50% cell culture infectious dose (TCID) 50 mL. Researchers have also shown that even the matrix effect of serum or urine did not affect the performance of Z_10.8-linked sandwich FLISA. Interestingly, human serum interfered lesser with the peptides as opposed when using antibodies. Moreover, the smaller size of the peptides makes them less liable to non-specific electrostatic attraction from the components of serum.

As label-free assays are equally as sensitive as label-based approaches, many studies are gradually moving towards these strategies. Dolai and Tabib-Azar (2019) have created a label-free sensor that operates using an aptamer specific against ZIKV C protein. The aptamer was thiolated and immobilized on the surface of the 433 MHz Lithium Niobate (LiNbO3) microbalance sensors. As compared to the standard 5 MHz quartz microbalance, the whole virus can be detected with a sensitivity of 370 Hz/ng, which is approximately 400 times better. Dolai and Tabib-Azar (2020) have shown the applicability of quartz crystal microbalance (QCM) as a label-free approach of virus sensing. Similar researchers have reported the development of a paper-based potentiometric sensor to detect the whole ZIKV based on standard printer papers functionalized with aptamers. The paper sensor is made up of two to three 10 mm paper segments with conducting silver paint contact patches on both ends. When ZIKV was added, the two silver paint contacts reproducibly became more negative, yielding a minimum sensitivity of 0.26 nV/ZIKV and a minimum detectable signal of 2.4 × 10⁷ ZIKV. Moreover, the authors also developed a proof-of-concept device using a liquid crystalline display (LCD) powered by the sensor to read the sensor output. In a colorimetric-based diagnostic application, aptamers specific for the ZIKV were conjugated to the surface of the gold nanoparticles. The aptamer-gold nanoparticles conjugate aggregate in the presence of the target ZIKV, causing the colour to change from red to blue. The LOD for live ZIKV was achieved at 1.0 × 10⁵ PFU (Bosak et al. 2019). Though many sensors of various mode of sensing were developed thus far, more efforts should be directed towards the development of point-of-care-based strategy, such as lateral flow assay.

Apart from using aptamers generated against ZIKV, attempts to use other aptamers specific against certain dyes are also useful for the diagnostic detection of ZIKV infection. For instance, Kikuchi et al. (2019) designed a split DNA aptamer (SDA) hybridization probe employing two DNA strands. The innovation in it is that the SDA shows low binding affinity to the dapoxyl dye in normal conditions. However, in the presence of a specific analyte, it changes its structural conformation to form a dye-binding site (dapoxyl), amplifying the fluorescence signal by up to 120-fold. The developed SDA could selectively detect a conserved region of the ZIKV after isothermal nucleic acid sequence-based amplification (NASBA) reaction, which is a nucleic acid amplification strategy that operates only at a single temperature and has the potential to replace real-time PCR (qPCR). The effort by the researchers is laudable, as this assay is operatable at room temperature, in light of the isothermal nucleic acid amplification assay. This can be incorporated into a mobile system such as lateral flow assay, which as a whole fulfills the criterion of a point-of-care system.

Chikungunya is an infectious disease caused by CHIKV, which was discovered in febrile human serum in Tanzania, Africa in 1953 (Robinson 1955). The common symptoms for CHIKV infections include fever, severe joint pain, muscle pain, joint swelling, fatigue and rash. Although it is not life-threatening, complex clinicopathological manifestations and increased number of mortalities were reported during the Indian Ocean outbreak in 2004 to 2005 (Enserink 2007). Subsequent to this outbreak, CHIKV has spread to almost all parts of the world; Africa, Asia, Europe and America (Kumar et al. 2008; Pialoux et al. 2007; Rezza et al. 2007). Mainly transmitted by Aedes species mosquitoes, CHIKV transmission cycles can be either man-mosquito-man (urban cycle) or animal-mosquito-man (sylvatic cycle). The sylvatic cycle involves forest-dwelling Aedes mosquitoes (Ae. furcifer, Ae. africanus, Ae. fulgens, A.luteocephalus, Ae. camptorhynchites) and non-human primates, which serve as reservoir hosts and are more common in Africa (Diallo et al. 1999). To date, these two mosquitoes (Ae. aegypti and Ae. albopictus) are the principal vectors involved in the urban transmission cycle and because of their wide distribution, the number of Chikungunya infection rise globally. With two open reading frames with the size of 11.8 kb that encodes for four NS proteins (NS1, NS2, NS3 and NS4) and five structural proteins (C, E3, E2, 6K and E1), CHIKV is a positive-sense ssRNA virus (Solignat et al. 2009). Since Chikungunya fever’s clinical symptoms are very similar to those of other tropical infections like DENV or leptospirosis, laboratory confirmation is in dire need. Virus isolation, molecular detection and antigen detection are the most appropriate test for samples collected between days 1 to 5 days from the illness onset. Meanwhile, samples collected after more than 5 days of illness onset preferably detect antibody (IgM and IgG) in the patient’s sample. This can be achieved by using ELISA, immunofluorescent assay (IFA), plaque reduction neutralization test (PRNT) and haemagglutination inhibition assay (HIA) (Pialoux et al. 2007). The current methods mentioned require high-end laboratory equipment such as a biosafety cabinet and fluorescence microscope, which are not usually available in laboratories with resource-limited setting. The urgency of CHIKV detection calls for a diagnostic system that is able to meet the ASSURED criteria suggested by WHO: affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to those who need it (Urdea et al. 2006). Several endeavors were made to fulfill this aspiration using aptamers as the MREs. Bruno et al. (2012) developed DNA aptamers against CHIKV by using CHIKV E1 peptide as the target in SELEX. ELASA was used to rank the binding affinity of the aptamers based on the average absorbance at 405 nm. They successfully reported that both the capture and the conjugate aptamers could be used to detect CHIKV E peptide in a lateral flow chromatographic assay, despite not reporting on the binding affinity of each aptamer. On the other hand, Saraf et al. (2019) developed a multiplex framework for ZIKV and CHIKV E proteins using an aptamer-conjugated microfluidic channel approach. This aptamer-based device is one of the diagnostic platforms that can be used without the need for any fluorescent reporter because it is modular, compact and simple to use. In this format, the capture aptamer is immobilized on the surface of the microfluidic channel and the protein captured is detectable by a secondary aptamer conjugated with gold nanoparticles (AuNPs). As an electron-transferring agent, the presence of AuNPs leads to the deposition of silver reagents over the surface of AuNPs. A silver staining technique was used in this study to further amplify the colour change. The intensity of the colour depends on the concentration of analyte-bound aptamer-AuNPs. Therefore, silver deposition increases as the number of AuNPs increases, leading to intense colour change. This method achieves a detection limit of 1 pM of viral target protein in phosphine-buffered saline and 10 pM in diluted calf blood. The signal amplification strategy that is based on the usage of AuNPs and the deposition of silver should be used as an exemplary assay in other virus detection as well.

Plasmodium parasite is the causative agent of malaria. Transmitted by mosquito Anopheles, there are five Plasmodium species known to infect humans such as P. falciparum, P. vivax, P. ovale, P. malariae as well as P. knowlesi (Ashley et al. 2018). Despite global efforts to eradicate malaria, there were still an estimated 229 million cases and 409,000 deaths in 87 malaria-endemic countries in 2019 (WHO 2020), where the majority of the cases were contributed by P. falciparum and P. vivax. The gold standard for malarial diagnosis is the examination of stained blood films on light microscopy but has setbacks as it is a laborious, tedious, time-intensive technique and requires trained personnel (Ashley et al. 2018). As an effective malarial treatment requires early diagnosis, malaria diagnosis has been pivoted towards the development of rapid diagnostic kits (RDTs). Nonetheless, the current antibody-centric RDTs suffer from several caveats such as cost and thermal instability at high storage temperatures especially in tropical and subtropical countries (Jorgensen et al. 2006; Rafael et al. 2006). Thus, it is imperative to search for an alternative MRE with a much lower cost of synthesis and higher thermal stability.

In pursuant to this, several aptamers were isolated against biomarkers such as var2CSA (Birch et al. 2015), thirteen-amino acid long peptide (Frith et al. 2018), glutamate dehydrogenase (GDH) (Singh et al. 2018), High Mobility Group Box 1 (HMGB1) (Joseph et al. 2019), lactate dehydrogenase (LDH) (Cheung et al. 2013, 2020; Jain et al. 2016a; Lee et al. 2012) and the whole P. falciparum-infected red blood cells (Birch et al. 2015; Oteng et al. 2020). Among all, Plasmodium lactate dehydrogenase stands out as the most important target, as evidenced by the highest number of aptamers generated against this biomarker. Several aptamers developed against Plasmodium lactate dehydrogenase named pL1, 2008s and P38 were employed on multiple platforms such as gold nanoparticles (Cheung et al. 2013, 2020; Jain et al. 2016a, 2016b; Lee et al. 2012), graphene oxide (Jain et al. 2016a), silver nanoclusters (Wang et al. 2016), molybdenum disulfide (MoS₂) nanosheet, magnetic microparticles (Kim and Searson 2017), DNA tweezer (Shiu et al. 2017) and DNA origami (Godonoga et al. 2016; Tang et al. 2018). Apart from the direct identification of LDH protein, signal production of the malarial diagnosis could also be carried out based on the enzymatic property of LDH that reduces pyruvate to l-lactate. Predicating on this enzymatic activity, an assay namely Aptamer-Tethered Enzyme Capture assay (APTEC) was developed, achieving a LOD of 4.9 ng/mL or 4.33 ± 1.66 pg/µL, as reported by two groups of researchers (Cheung et al. 2020; Dirkzwager et al. 2015). Most of the aptasensing platforms developed thus far were sensitive enough for the detection of the clinically relevant amount of LDH protein, which is around 3–15 pg/μL in plasma (Martin et al. 2009). In addition to the application of aptamers in the diagnosis of malaria, aptamers can also be used as therapeutic agents (Cui et al. 2015). For instance, Niles et al. (2009) demonstrated a successful inhibition of the growth of parasites and the formation of hemozoin by heme-binding aptamers. This inhibitory action of the aptamer is laudable as it is able to inhibit the accumulation of hemozoin, which is detrimental to red blood cells. Severe malaria is most likely caused by the sequestration of the infected erythrocytes by P. falciparum erythrocyte M protein 1 (PfEMP1), which serves as a key molecule in modulating the interaction of parasite and host (Flick and Chen 2004). Sequestration of parasitized erythrocytes from peripheral blood can be achieved by a cytoadherence strategy known as rosette formation, a phenomenon where uninfected erythrocytes agglutinate around parasitized erythrocytes (David et al. 1988). This cytoadherence was achievable by RNA aptamers generated by Barfod et al. (2009), whereby the aptamers generated showed adherence to the non-infected erythrocytes with a rosette disrupting capacity at 33 nM and with 100% disruption at 387 nM in a live cell assay. Nik Kamarudin et al. (2020) have isolated RNA aptamers that could potentially interrupt the interaction between PfEMP1 and CD36 receptor, a host endothelial surface protein. The researchers have proven the inhibitory action of the aptamer by promoting anti-cytoadherence, which could be useful for malaria adjunt therapy. Efforts should be intensified into bringing these aptamers into clinical trials to assess their efficacy for malaria adjunct therapy.

Francisella tularensis, which is a Gram-negative non-motile Coccobacillus, is the causative agent responsible for Tularemia infection (Mandell et al. 2005). Tularemia is transmitted to humans by insect bites, ingestion of contaminated food or water and contact with infected animals. Apart from that, the transmission of tularemia was also proven to take place by the intermediate Ae. egypti (Bäckman et al. 2015). Additionally, an enzootic cycle is associated with the pathogenesis involving wild animals such as rodents and blood-sucking insects (Ellis et al. 2002). Due to high infectivity and the ability to cause lethal disease by aerosol, it is categorized as a class ‘A’ agent by the Centers for Disease Control and Prevention (CDC). In fact, F. tularensis could have a massive impact on public health if it was exploited by terrorists as a possible biological weapon (Day et al. 2009; Rotz et al. 2002; Willke et al. 2009). While various PCR platforms are used to detect F. tularensis, it is important to note that the gold standard for confirming F. tularensis detection is in vitro cultivation, which requires growth on cysteine or thioglycolate enriched medium and incubation times of 2–4 days at 37 °C (Dennis et al. 2001; Ellis et al. 2002). The viability of PCR to inhibitors and the time-consuming nature of the culture process calls for the possible applications of aptamers.

Only a few studies have been focused on the development of aptamers against F. tularensis. Vivekananda and Kiel (2006) selected DNA aptamers specific to the commercially available Francisella tularensis subspecies japonica bacterial antigen. The sandwich aptamer-linked immobilized sorbent assay (ALISA) and dot blot assay results exhibited the specificity of the aptamer cocktail to tularemia bacterial antigens from three different subspecies japonica, holarctica and tularensis, but not to unrelated Bartonella henselae, which was used as a negative control. Tularemia bacterial antigen was detected at concentrations as low as 25 ng. Apart from that, Lamont et al. (2014) used aptamers to perform a two-step enrichment process for improved cultivation and detection of F. tularensis in lettuce and soil. The first process utilizes logarithmic-phase F. tularensis spent culture filtrate to supplement standard culture medium to enhance F. tularensis growth in the presence of residual bacteria from food and environmental matrices. Within the spent culture supernatant, ultra-performance liquid chromatography (UPLC)/MS analysis found several unique chemicals including carnosine, which had a matching m/z ratio. Carnosine is a chemical that can cause enhanced growth and biofilm formation in E. coli. At low inoculums, adding 0.625 mg/mL of carnosine to conventional F. tularensis medium increased the growth of F. tularensis by ten fold. The second procedure used a DNA aptamer cocktail capture assay to concentrate and isolate F. tularensis amidst other bacteria present in food and environmental matrices in order to further enrich F. tularensis cells. The researchers have shown that using the aptamer as the capturing agent prior to enrichment resulted in a detection range of 1–10⁶ CFU/mL, which is better than that of without any aptamer-based enrichment. The aptamer-based enrichment can also be used in a nucleic acid amplification assay such as PCR or RT-PCR, which can tremendously improve the LOD of F. tularensis.

Japanese encephalitis (JE) is an acute viral infection of the central nervous system that is caused by JEV, which belongs to the genus Flavivirus in the family Flaviviridae. Only 0.1 to 1% of JEV infections result in encephalitis, while the rest are asymptomatic or characterized by a mild febrile illness (Grossman et al. 1973). There are approximately 67,900 cases of JEV-induced encephalitis globally each year with fatality rate ranges from 20 to 30% (Campbell et al. 2011). The majority of JE epidemic cases occur throughout Asian and Oceanian countries, with over three billion people are at risk of JEV transmission (Campbell et al. 2011; Wang and Liang 2015). JEV is an enveloped virus of about 50 nm virion size and consists of a positive-sense ssRNA genome of approximately 11 kb in length, which encodes for three structural proteins (C, prM and E) and seven NS proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Desingu et al. 2017; Lu et al. 2017; Sumiyoshi et al. 1992). Initially, JEV is transmitted from pigs to mosquitoes (Barzon and Palù 2018; Imoto et al. 2010; Sasaki et al. 1982). When JEV-infected mosquitoes feed on human blood rather than their primary amplifying hosts, JEV replicates in the human skin and lymphoid organs. Next, JEV is transported by the bloodstream to the peripheral organs before crossing the blood–brain barrier to infect neurons and trigger infiltration of inflammatory cells (Myint et al. 2007; Pearce et al. 2018). Therefore, the WHO integrated vaccination programs into routine health control schedules in JEV-endemic countries to prevent JEV infection (Bharucha et al. 2020). These vaccines are both safe and reliable, offering long-lasting protection. However, it does not inhibit JEV circulation and may not be 100% effective, as demonstrated by Tandale et al. (2018).

The gold standard for JE diagnosis, which measures anti-JEV IgM and IgG antibodies in the blood serum or the cerebrospinal fluid is by PRNT (Hills et al. 2009). PRNT is laborious, time-consuming and necessitates a high level of biosafety. Another method is via ELISA, which suffers from the sensitivity of only 50–70% and cross-reactivity with other closely related Flaviviruses (Dubot-Pérès et al. 2015; Johnson et al. 2016; Maeda and Maeda 2013; Robinson et al. 2010). RT-qPCR assay is punctuated by the hurdle of the absence of JEV total RNA in blood and cerebrospinal fluid. Aptamer can be an elegant MRE that would be able to alleviate the disadvantages associated with the aforementioned assays (Bharucha et al. 2018; Nan et al. 2018; Pantawane et al. 2018; Saron et al. 2018).

So far, the only target used for aptamer selection was JEV MTase. JEV MTase catalyzes the methylation of the N-7 position of the guanine and the 2′-OH position of the first ribonucleotide in the viral genome (Ray et al. 2006). Han and Lee (2016) reported the first isolation of a 24-mer truncated RNA aptamer modified with 2'-O-methyl pyrimidines against the JEV MTase by using SELEX. The aptamer isolated bind specifically to JEV MTase with a high affinity (K_d ~ 16 nM) and is also able to inhibit both N-7 and 2′-OH-MTase activity of JEV MTase. JEV production and replication in cells were suppressed up to 80% compared to control. Moreover, the cellular positive and negative JEV genome RNA levels were decreased by 65% and 76%, respectively. In the therapeutic realm of JEV, the biggest challenge for the aptamer is the need to cross the blood–brain barrier, which consists of tight and adherent junction between adjacent endothelial cells (Chen and Liu 2012). This can be achieved by conjugating the aptamer with the aptamer against the transferrin receptor, which is a protein abundant on the endothelial cells of the blood–brain barrier (Li et al. 2020).

The Venezuelan equine encephalitis virus (VEEV) is an Alphavirus belonging to the Togaviridae family that causes epidemics and equine epizootics. It is made up of an approximately 11.4 kb positive-sense RNA genome that encodes for four NS proteins (NS1 to NS4) and a structural polyprotein that is proteolytically cleaved into the C and the E glycoproteins E2 and E1. The NS proteins play a major role in viral replication (Murphy, 1995; Rice and Strauss 1981; Strauss and Strauss 1986). Rodent hosts especially the cotton spiney rat and mosquito vectors such as Aedes and Culex mosquitoes play a role in the enzoonotic cycle of VEEV (Carrara et al. 2005, 2007; Deardorff and Weaver 2010; Deardorff et al. 2011; Ortiz et al. 2008; Smith et al. 2007). The diagnostic detection of VEEV involves the use of the PCR method and plaque assay (Linssen et al. 2000; Sahu et al. 1994). However, the usage of both the detection methods is limited during the viraemic phase of infection.

So far, only one aptamer was isolated for the diagnostic detection of VEEV. The target protein chosen for the isolation of aptamer was C protein. C protein is a viral protein that interacts with viral genomic RNA to form a nucleocapsid (NC) in the cytoplasm during viral assembly. Kang et al. (2007) reported a phosphorothioate RNA aptamer for the detection of the C protein of VEEV. The aptamer was modified with phosphorothioate at the 5′ end to improve the binding affinity of the aptamer. Due to its high binding affinity of 7.1 ± 2.4 nM, one particular aptamer, 16_1 aptamer, was identified as the best thioaptamer targeting the C protein of VEEV by chemiluminescence electrophoretic mobility shift assay (CL EMSA). Human cellular extracts were also used to evaluate the thioaptamer’s specificity, whereby in the gel shift assay, some band shifts were observed, which reflect on the possible non-specific interaction of the aptamer with the components of the human cellular extract. However, the researchers have not identified the proteins involved, thus this aptamer must be used cautiously in diagnostic applications that involve human cellular extracts. Besides C protein, another protein that can be targeted for aptamer selection in the future is NS1 protein, which plays an important role in pathogenesis, analogous to NS1 protein in DENV.

Rift Valley fever virus (RVFV), a Bunyavirus from the genus Phlebovirus, is the etiological agent of Rift Valley fever (RVF). RVF is widespread across a large portion of Africa (Bird et al. 2009; Daubney et al. 1931). Mosquito vectors that have been linked to RVFV transmission are believed to be of Culex and Aedes genera (Chevalier et al. 2010). The RVFV consists of a tripartite negative-sense ssRNA genome, which is referred to as large (L), medium (M) and small (S). The L portion contains the coding regions for the RNA-dependent RNA polymerase (Müller et al. 1994). Meanwhile, the S portion expresses the nucleoprotein (N). The M segment encodes for a glycosylated 78-kDa protein, a non-glycosylated 14-kDa protein, two E glycoproteins Gn and Gc in a single open reading frame (Suzich et al. 1990). Virus isolation, histopathology, antigen detection, antibody detection and nucleic acid-based molecular assays such as nested RT-PCR methods, qPCR, multiplex PCR-based macroarray assay, real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and recombinant polymerase amplification (RPA) are among the latest RVFV diagnostic strategies (Bird et al. 2007; Drosten et al. 2002; Euler et al. 2012; Fukushi et al. 2012; Garcia et al. 2001; Jr et al. 1989; Kortekaas et al. 2013; Meegan et al. 1989; Mwaengo et al. 2012; Odendaal et al. 2014; Roux et al. 2009; Sall et al. 2002; Swanepoel et al. 1986; Venter et al. 2014; Wal et al. 2012; Weidmann et al. 2008). Besides that, numerous serological diagnosis methods are also available such as virus neutralization assay, ELISA, Indirect Immunofluorescence, agar gel immunodiffusion (AGID), radioimmunoassays and complement fixation and HIA (Pepin et al. 2010). However, these methods are time-consuming, lack specificity and require skilled personnel.

Direct detection of RVFV with aptamers by targeting the specific biomarkers can be a feasible diagnostic method. Due to its multifunction and viral-specific nature, the N protein is considered as a novel biomarker for diagnostic and therapeutic purposes among the other proteins encoded by the genome. The N protein protects the viral genome from degradation and prevents the formation of double-stranded RNA during replication, which could ignite antiviral response from host (Ruigrok et al. 2011). As such, aptamer selection has largely centered on N protein. Ellenbecker et al. (2012) successfully isolated a specific RNA aptamer targeting RVFV N protein using SELEX. Interestingly, the BLAST analysis data revealed the presence of GAUU and/or pyrimidine/guanine motifs within the coding region of the genome, implying that N protein might interact with non-terminal viral RNA sequences during replication. The aptamer was truncated and it retained an affinity of 2.6 µM for the N protein. In another EllenBecker’s experiment, he and his colleagues have conducted an in silico approach to isolate RNA aptamers and compared them with the experimentally acquired aptamer, MBE87. To compare the predicted secondary structures of the known aptamers against RVFV-N protein, they have designed an algorithm that uses a distance matrix and multidimensional scaling. The overall in silico-generated RNA aptamers were further validated in vitro with filter binding assay. The in silico-generated aptamers, AS-1 and AS4, did not bind with the similar binding affinity as the MBE87 sequence, the aptamer that survived 16 rounds of selection in the previous experiment. However, AS-1 and AS4 bind to the N protein and inhibit the viral function. The presence of the GAUU motif in both the experimentally derived and in silico-derived aptamers was predicted to be responsible for the high binding affinity (Ellenbecker et al. 2015). So far, the only protein targeted for aptamer selection is N protein. The isolation of aptamers should also be expanded beyond the target N protein, for example by using NS protein or even E glycoprotein as the SELEX target.

Yellow fever virus (YFV) is a Flavivirus with a positive-strand RNA genome that codes for three structural proteins and seven NS proteins, analogous to DENV, ZIKV and JEV. Despite the application of effective vaccines since 1930s, the disease still lingers. As the early symptoms caused by YFV are not significant, the diagnosis of YFV remains a hurdle. The non-specificity of certain serological methods also remains a challenge. Early detection of the disease is important to take on-time medical action. The qPCR, RT-LAMP and helicase-dependent amplification assays (HDA) were established as molecular methods for the diagnosis. However, they require expensive lab instruments and also skilled personnel (Escadafal et al. 2014).

The methylated 5′ cap is essential for mRNA stability and translation efficiency (Furuichi and Shatkin 2000). The viral protease/helicase activities of (NS2B/NS3) and the MTase of NS5 are prominent YFV biomarkers. So far, only one aptamer has been isolated against YFV, which was by Falk and Weisblum (2014). The aptamer against YFV MTase was functionalized at its 5′ end with fluorescein (FL-dT10). The FL-dT10 showed binding to YFV MTase with a binding affinity of 231 nM. As a whole, the isolation of aptamer against proteins associated with YFV is very limited thus far, which can be due to the low frequency of occurrence of yellow fever globally.

The current diagnostic tools for viral infections especially the ones that are incumbent upon antibodies as the MREs have their limitations. These drawbacks are addressable with the usage of aptamers based on their advantages over antibodies. In the future, we believe that early detection of mosquito-borne diseases will greatly improve disease control. In this review, we have overviewed many studies demonstrating the potential usage of aptamers in mosquito-borne infectious diseases agents, as outlined in Table 2. However, there are lack of aptamers available for many other mosquito-borne pathogens such as West Nile virus, Filariasis, Saint Louis encephalitis, Western equine encephalitis, Eastern equine encephalitis, Ross River fever, Barmah Forest fever, La Crosse encephalitis and newly detected Keystone virus. Future studies should also center on generating aptamers against these rare mosquito-borne pathogens. In the aspect of therapeutics, many aptamers have yet to enter human clinical trials. Therefore, clinical trials are needed to assess the actual efficacy of aptamers in therapeutics prior to the administration of aptamer-based treatments in humans.

Future perspectives should focus on improving the stability of aptamers by incorporating chemical modifications that enhance the stability (Odeh et al. 2020). In silico approaches such as molecular docking and molecular dynamics could also be very useful to predict and characterize specific interactions between the aptamer and the target, in order to choose the best aptamer candidates (Navien et al. 2021). In addition, the incorporation of aptamers into portable biosensors could fulfill the requirements of point-of-care diagnostics (Guo et al. 2020). Apart from that, it is important to gain more insights into the basic knowledge of mosquito-borne infections that allows the visibility of novel biomarkers in order to select the best target candidates for the aptamer-based diagnostics. Finally, it is also essential to attract the attention of researchers, the public and industries for the transition of research to the stage of the development of marketable diagnostic prototypes (Ospina-villa et al. 2020). Taken together, aptamers are the dawning MREs, paving the trajectory towards a much more amenable diagnostic strategy of mosquito-borne pathogens.

Material submitted is original; all authors are in agreement to have the article published.

Citartan M and Tang TH were supported by USM Research University Grant (1001.CIPPT.8011095). Navien TN was supported by USM Fellowship (IPS/Fellowship2019/IPG).

Citartan M and Tang TH were supported by USM Research University Grant (1001.CIPPT.8011095). Navien TN was supported by USM Fellowship (IPS/Fellowship2019/IPG).

Affiliations

1. Advanced Medical & Dental Institute (AMDI), Universiti Sains Malaysia, Bertam, 13200, Kepala Batas, Penang, Malaysia

   Tholasi Nadhan Navien, Tzi Shien Yeoh, Andrew Anna, Thean-Hock Tang & Marimuthu Citartan

2. Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak (UNIMAS), 94300, Kota Samarahan, Sarawak, Malaysia

   Andrew Anna

Contributions

TNN: Conceptualization, Original Draft Writing, Review and Editing. TSY: Writing. AA: Writing. THT: Review, Editing and Supervision. MC: Conceptualization, Review, Editing and Supervision.

Corresponding authors

Correspondence to Thean-Hock Tang or Marimuthu Citartan.

Conflicts of interest

Author’s declared to not have any conflict of interest.

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The authors hereby consent to participate.

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Material submitted is original; all authors are in agreement to have the article published.

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