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Abstract

Appropriate diagnosis is the key factor for treatment of viral diseases. Time is the most important factor in rapidly developing and epidemiologically dangerous diseases, such as influenza, Ebola and SARS. Chronic viral diseases such as HIV-1 or HCV are asymptomatic or oligosymptomatic and the therapeutic success mainly depends on early detection of the infective agent. Over the last years, aptamer technology has been used in a wide range of diagnostic and therapeutic applications and, concretely, several strategies are currently being explored using aptamers against virus proteins. From a diagnostics point of view, aptamers are being designed as a bio-recognition element in diagnostic systems to detect viral proteins either in the blood (serum or plasma) or into infected cells. Another potential use of aptamers is for therapeutics of viral infections, interfering in the interaction between the virus and the host using aptamers targeting host-cell matrix receptors, or attacking the virus intracellularly, targeting proteins implicated in the viral replication cycle. In this paper, we review how aptamers working against viral proteins are discovered, with a focus on recent advances that improve the aptamers' properties as a real tool for viral infection detection and treatment.

Characteristic Advantage

Aptamers are produced by chemical synthesis Little or no batch to batch variation Aptamers can be modified increasing their stability Reporter molecules can be attached to aptamers at precise locations not involved in binding

Aptamers to Nucleocapsid Protein

Aptamers to Surface Glycoprotein (gp 120) Gp120 is essential for virus entry into cells as it plays a vital role in attachment to specific cell surface receptors mainly on helper T-cells. Several small aptamers containing G-quadruplex selected against gp120 have demonstrated antiviral activity [33] . The first of these molecules was the phosphorothioate 8-mer d(TTGGGGTT), named ISIS 5320, which forms a tetrameric G-quadruplex structure that binds the V3 loop of gp120 inhibiting virus entry [48] . Later on, Koizumi et al. synthesized a set of G-rich oligonucleotides and identified the hexadeosyribonucleotide d(TGGGAG), known as Hotoda's sequence, [49, 50] . Several authors have used the Hotoda's sequence as a lead sequence to make a series of modifications at the 5 and 3 ends of the molecule or mutations in the sequence that allowed to find molecules with high anti-HIV activity [50] [51] [52] [53] [54] [55] [56] .

Use of Aptamers for Delivery of Therapeutic Molecules

Aptamers can also serve as elements that selectively recognize and bind to defined cell types or tissues. By attaching drug molecules, the aptamers can be used to deliver cargo molecules to or into specific cells or tissues of interest [64] . In order to reach efficient RNAi activity, aptamer-siRNA conjugates must be successfully internalized and released into the cytoplasm where they can meet the RNAi machinery [65] . To improve the Dicer entry and processing of the siRNA, one pair of complementary guanosine and cytosine (GC)-rich "sticky bridge" sequences can be chemically appended to the 3 end of the aptamer and one of the siRNA strands, respectively. Both the aptamer and siRNA portions are chemically synthesized and subsequently annealed via "sticky bridge" [66] .

Introduction

Viruses are infectious agents that enter and replicate only inside the living cells of other organisms. Once the virus replicates inside the cell, it may remain dormant for long periods of time or be released immediately and attach to other healthy cells to begin the infection process again. Many diseases are caused by viruses such as the influenza, hepatitis, human immunodeficiency virus (HIV) or emerging viral diseases. While they differ in symptoms such as fever and weakness, some present no symptoms at all. Rapid and secure diagnosis of viral infections is a key factor for treatment of these diseases avoiding new spread.

Aptamers to CCR5

Aptamers to Nucleolin (NCL) Nucleolin (NCL) is a multifunctional cellular protein that is overexpressed in cancer cell membranes. NCL is involved in the very initial step of HIV-1 virion-cell recognition. NCL is in cellular fractions containing the HIV genome, viral matrix and reverse transcriptase in addition to in complexes with CD4 and CXCR4/CCR5 at the cell membrane. This supports the potential role of this protein in viral entry. In the absence of the cellular receptors as CD4 and CXCR4/CCR5, HIV-1 attachment can occur through coordinated interactions with heparan sulfate proteoglycans and cell-surface-expressed NCL.
AS1411 is a G-rich aptamer that form a stable G-quadruplex structure and displays antineoplastic properties both in vitro and in vivo [14] . The major molecular target of AS1411 is NCL. Perrone et al. tested whether the aptamer AS1411 was able to interfere with HIV-1 cellular entry using different HIV-1 strains, host cells and at various times post-infection [26] . The results demonstrated that AS1411 efficiently inhibited HIV-1 attachment/entry into the host cell in the absence of cytotoxicity at the tested doses.

Use of Aptamers for Delivery of Therapeutic Molecules

Through either covalent conjugation or physical assembly, different siRNA molecules have been successfully functionalized with aptamers against HIV-1 gp120 and the CD4 receptor to achieve targeted RNAi efficacy which relies on specific interactions between the aptamer and its receptor expressed on the targeted cells or tissue. Zhou et al. isolated RNA aptamers against the HIV-1(BaL) gp120 protein that were used to create a series of dual inhibitory function anti-gp120 aptamer-siRNA chimeras [67] . The authors also demonstrated that one of these anti-gp120 aptamer-siRNA chimera is specifically taken up by cells expressing HIV-1 gp120, and that the attached siRNA is processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells (PBMCs). Interestingly, both the aptamer and the siRNA portions in the aptamer-siRNA chimera have potent anti-HIV activities [68] . Finally, the authors tested the antiviral activities of these chimeric RNAs in a humanized mouse model with multilineage human hematopoiesis showing that treatment with either the anti-gp120 aptamer or the aptamer-siRNA chimera suppressed HIV-1 replication by several orders of magnitude and prevented the viral-induced helper CD4 + T cell decline [69] . In order to improve the utility of aptamers as siRNA delivery vehicles, the same authors chemically synthesized a modified gp120 aptamer that was complexed with three different siRNAs (HIV-1 tat/rev and two HIV-1 host cell proteins, CD4 and TNPO3), resulting in an effective delivery of siRNAs in vivo, knockdown of target mRNAs and potent inhibition of HIV-1 replication in a humanized mouse model [70] . Very interestingly, following ending of the aptamer-siRNA cocktail treatment, HIV levels rebounded facilitating a follow-up treatment with the aptamer cocktail of siRNAs that resulted in complete suppression of HIV-1 viral loads that extended several weeks beyond the final injection.

Introduction

Viruses cause different types of damage to the body which, if left untreated, can lead to death. There are many antiviral drugs that block the infection process at different stages. Some drugs prevent the virus from interacting with the healthy cell by blocking a receptor that helps internalize the virus into the cell. Other drugs inhibit the proliferation of the virus within the cell. The simultaneously use of several drugs affecting different processes increases the probability of recovery of the patient. Although some viral infections such as hepatitis or HIV remain latent for a long time current treatments control the virus and prevent further damage to the body. Viral infections usually produce an immune response in the host that eliminates the infecting virus. The same protective effect is produced by vaccines, which confer an artificially acquired immunity to the viral infection. However, some viruses including those that cause acquired immune deficiency syndrome (AIDS) and viral hepatitis evade these immune responses and result in chronic infections. Antibiotics have no effect on viruses, but several antiviral drugs have been developed. Because viruses use vital metabolic pathways within host cells to replicate, they are difficult to eliminate without using drugs that cause toxic effects to host cells in general. The most effective medical approaches to viral diseases are vaccinations to provide immunity to infection, and antiviral drugs that selectively interfere with viral replication.
Most of the antiviral drugs are nucleoside analogues which lack the hydroxyl groups. Viruses mistakenly incorporate these analogues into their genomes during replication and, in consequence, the newly synthesized DNA is inactive and the life-cycle of the virus is then halted. Some of the most frequently prescribed antiviral nucleoside analogues based-drugs are aciclovir for Herpes simplex virus infections and lamivudine for HIV and Hepatitis B virus infections [1] . During the last years, the nucleoside analogue drug ribavirin combined with interferon has been used for hepatitis C treatment [2] , although currently there is a more effective treatment that includes simeprevir available for patients with genotype 1 and genotype 4 [3] . The treatment of chronic carriers of the hepatitis B virus by means of a similar strategy using lamivudine has been developed [4] . Today, the first line treatment of choice includes one of three drugs: Peg-IFN, entecavir or tenofovir because of their greater power and because they produce a very low rate of resistance.

Aptamers against Human Immunodeficiency Virus (HIV)

Human immunodeficiency virus is a lentivirus (a subgroup of retrovirus) that causes HIV infection and over time AIDS [21] . HIV infects essential cells in the human immune system such as CD4 + T cells, macrophages, and dendritic cells. When CD4 + T cell numbers decline below a critical level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to opportunistic infections. HIV enters cells by endocytosis through the interaction of gp120 viral surface protein (SU) with CD4 host cell. The subsequent interaction of this complex with chemokine coreceptors produces a conformational change in viral protein gp41 that promotes the fusion of virion and target cell membranes leading to the release of HIV particles into the cell. Once inside the cell, viral uncoating generates the viral reverse transcription complex, and the reverse transcription gives the HIV preintegration complex (PIC). The PIC gets into the nucleus and the HIV DNA (provirus) is integrated into the cellular chromosome. Integration can lead to either latent or transcriptionally active forms of infection. The latent form gives the viral latency in cells that can replicate in new infected cells; this provirus can remain hidden during years or replicate and form new viral particles in any moment. The transcriptional active forms are transcript and translated forming new viral particles that dead the host cell and goes to infect new cells. Great efforts are being made to get sensitive, fast and simple diagnostic methods and effective therapies.

Aptamers against HBV

Hepatitis B virus (HBV) is a partially double-stranded DNA virus of the Hepadnaviridae family classified into eight genotypes from A to H. The main element of the viral particle of HBV virus and also the most characterized component is the hepatitis B surface antigen (HBsAg) [72] .

Aptamers to 5′ and 3′ Untranslated Regions (5′ and 3′UTR)

Non-translated 5′ and 3′ regions have highly conserved sequences and structured regions closely related with transcription and replication of the HCV virus. Specifically, 5′ end contains the Internal

Aptamers to 5 and 3 Untranslated Regions (5 and 3 UTR)

Non-translated 5 and 3 regions have highly conserved sequences and structured regions closely related with transcription and replication of the HCV virus. Specifically, 5 end contains the Internal Ribosome Entry Site (IRES) domain responsible of transcription initiation by ribosome recognition of HCV viral genome. However, 3 UTR includes a region essential for viral replication named cis-acting replication element (CRE).

Aptamers to Nonstructural Protein 3 (NS3)

Multifunctional enzyme NS3 is an essential protein for virus survival and it is considered a good target for the development of new antiviral-drugs. The protease activity of the protein is found in the N-terminal domain and the helicase activity is present in the C-terminal domain of the enzyme. During the last years, the group of Nishikawa has been working with different sets of RNA aptamers against the full-length or truncated NS3 protein to inhibit its dual-activity. Initially, they isolated NS3-specific aptamers that inhibited the protease activity (10-G1) or the dual activity, protease/helicase, of the enzyme NS3 (G6-16 and G6-19) [101, 102] . Afterward, new RNA aptamers against the helicase domain of NS3 protein, named G9-I, II and III, were able to inhibit protease activity in vitro [103] . Later on, the authors described the interaction between G9-I aptamer with Arg161/Arg130 residues in the truncated NS3 form as a putative target for protease activity inhibition [104] . To stabilize and protect G9 aptamers against exonuclease activity in vivo, Nishikawa et al. conjugated G9-II aptamer to the stem IV region of the Hepatitis delta virus (HDV) ribozyme. In addition, to allow nuclear export of the aptamer, the chimeric molecule HDV-G9-II (HA) was fused to a constitutive transport element (CTE) generating HAC molecule. Finally, the protease-inhibition capability of G9-II aptamer was checked, using HA and HAC expression vector in vivo [105, 106] . In order to inhibit the dual activity of NS3, a poly U tail in the 5 or 3 ends of G9-I aptamer (5 -14U-NEO-III or NEO-III-14U) was added. The two constructions were able to inhibit with high efficiency of the protease and helicase activities of NS3. Moreover, NEO-III-14U decreased the interaction between NS3 protein and the 3 end of the positive or negative sense HCV RNA and inhibited protease activity of NS3 in vivo [107] . Next, a new set of RNA aptamers were selected against the helicase domain using the truncated NS3 protein (NS3h) and the aptamer with greatest capability to deplete helicase activity in vitro was identified and named aptamer #5 [108] . Finally, a dual-functional aptamer named G925-s50 was designed using a truncated version of aptamer #5 plus G9-II aptamer linked by 50 mer poly(U) spacer. The designed molecule G925-s50 showed a significant inhibition of NS3 helicase-protease activity in vivo and is proposed by Nishikawa group as the best candidate for anti-HCV therapy [109] .

Aptamers against Human Papilloma Virus (HPV)

Human papillomavirus (HPV) is a DNA virus from the papillomavirus family. Most HPV infections cause no symptoms and resolve spontaneously, but some of them persist and result in warts or precancerous lesions which increase the risk of cancer of the cervix, vulva, vagina, penis, anus, mouth, or throat [119, 120] .

Aptamers to HPV in Diagnostics

Several proteins from human papillomavirus, particularly E6 and E7, promote tumor growth and malignant transformation and are frequently associated with cervical cancer. Thus, these proteins represent ideal targets for diagnostic and therapeutic strategies. Belyaeva et al. reported two RNA aptamers to E6, named F2 and F4, which induced apoptosis in cells derived from an HPV16-transformed cervical carcinoma. This aptamers were able to inhibit the interaction between E6 and PDZ1 from Magi1, with F2 being the most effective inhibitor, while none of them inhibited E6-p53 interaction or p53 degradation [121] .
Toscano-Garibay et al. isolated an aptamer (G5α3N.4) that exhibited specificity for E7 with a Kd comparable to aptamers directed to other small targets [122] that may be used for the detection of papillomavirus infection and cervical cancer. The same group characterized an RNA aptamer, named Sc5-c3, that recognized baculovirus-produced HPV-16 L1 virus-like particles (VLPs) with high specificity and affinity (Kd = 0.05 pM). This aptamer produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection [123] . Recently, Graham and Zarbl identified several DNA aptamers that have high affinity and specificity to the non-tumorigenic, revertant of HPV-transformed cervical cancer cells, which can be used to identify new biomarkers that are related to carcinogenesis produced by HPV [124] .

Aptamers to Herpes Simplex Virus (HSV)

Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) are two members of the herpesvirus family, Herpesviridae, that infect epithelial tissues before invading the nervous system, where it becomes latent. Unfortunately until now, it has not found any treatment to eradicate the virus [128] . Table 5 shows information on the aptamers described against HPV and HSV. Aptamer technology has been used by Corbin-Lickfett et al. to identify RNA sequences capable of being recognized by HSV-1 ICP27 protein, an important regulator for viral gene expression. After SELEX procedure, GC-rich RNA sequences were isolated, which did not form stable secondary structures [129] . With a therapeutic purpose, Gopinath et al isolated two RNA aptamers (aptamer-1 and aptamer-5) against the ectodomain of the gD protein of HSV-1, which plays an important role in viral entry to the host cells. These aptamers specifically bind to gD protein of HSV-1 with high affinity but not the gD protein of HSV-2. Furthermore, aptamer-1 efficiently blocked the interaction between the gD protein and the HSV-1 target cell receptor (HVEM) in a dose-dependent manner with a EC 50 in the nanomolar range. Anti-HSV-1 activity of aptamer-1 was analyzed by using plaque assays and the results showed that this aptamer efficiently inhibited viral entry. A shorter variant of aptamer-1 named mini-1 aptamer (44-mer) had at least as high an affinity, specificity, and ability to interfere with gD-HVEM interactions [130] . In a similar way, Moore et al. have reported the isolation and characterization of one aptamer, G7a, that binds the gD protein of HSV-2 and neutralizes infection through the Nectin1 and HVEM entry receptors with IC 50 of 20 nM [131] . Interestingly, aptamers that prevent HSV-2 infection may also reduce the morbidity associated with HIV-1 as HSV-2 is a major risk factor for the acquisition of HIV-1.

Aptamers to Influenza

Influenza is considered the most prevalent infectious disease in humans. Three emerging influenza viruses were responsible for major pandemics in the twentieth century: the 1918 Spanish influenza virus, the 1957 Asian influenza virus, and the 1968 Hong Kong influenza virus [132] . Indeed, the 1918 Spanish influenza virus was estimated to have killed 20-50 million people worldwide [133] . More recently, a highly pathogenic avian virus of the H5N1 subtype has produced sporadic infections in humans and, while it is associated with high rates of mortality, its poor transmission in humans prevented a more extensive spread among human populations. However, in 2009, a new influenza A virus of the H1N1 subtype emerged (pH1N1) that possessed high transmissibility but relatively low virulence, rapidly spreading across the entire globe and causing the first pandemic of the 21st century [134, 135] . Subsequently, 2013 witnessed the appearance of a new highly pathogenic avian virus of the H7N9 subtype in China [136] .
Influenza viruses are enveloped RNA virus of the family Orthomyxoviridae. The virion surface carries two membrane glycoprotein components, hemagglutinin (HA) and neuraminidase (NA) and, in the central core, the viral RNA (negative-sense) genome fragmented into eight single-stranded molecules and viral proteins that package and protects this RNA. Each segment contains one or two genes that code for the 15 viral proteins. Highly variable surface proteins, HA and NA, are used to classify influenza subtypes. The combination of hemagglutinin and neuraminidase mainly determines the host organism and the viral infectiousness. Currently, 18 HA and 11 NA types have been identified being the subtypes H1, H2 and H3, and N1 and N2 commonly found in humans.

Aptamers to Influenza Virus in Diagnostics

The detection rapid of influenza virus as well as the categorization of these viruses is particularly important due to the high risk of infection, the rapid propagation and the high frequency of mutation that often induces the arrival of new strains that can cause epidemics and even pandemics. An extensive review about the diagnostic strategies for influenza has been recently published [137] .
The recent advances in the development of rapid, automatic, point of care devices for the diagnosis and subtyping of influenza virus are sustained in two facts: (i) the rapid spread of influenza-associated H1N1 viruses that has caused serious concern in recent years; and (ii) H5N1 subtype of the avian influenza virus (AIV) caused the most lethal outbreaks of highly pathogenic avian influenza (HPAI) in poultry and fatal infections in human cases for over a decade. Thus, aptamers have been generated and found to be specific against these recent pandemic influenza viruses A/H1N1pdm [142] and H5N1 [143] .
Lee et al. developed an integrated microfluidic system that was used to screen a specific aptamer for the influenza A/H1N1 virus in an automated and highly efficient manner [144] . The selected aptamer showed a specific and sensitive detection of the influenza A/H1N1 virus, even in biological samples such as throat swabs. Later, they used a new approach for fluorescence-based detection of the influenza A H1N1 virus using a sandwich-based aptamer assay that is automatically performed on an integrated microfluidic system [145] . The entire detection process was shortened to 30 min using this chip-based system which is much faster than the conventional viral culture method. The limit of detection was significantly improved due to the high affinity and high specificity of the H1N1-specific aptamers. In addition, this two-aptamer microfluidic system had about 10 3 times higher sensitivity than the conventional serological diagnosis. The conformation of the aptamers changes in response to the solvent composition, including ion type and concentration, pH, and temperature. On the basis of this, Wang et al. have developed a microfluidic system that exploited the predictable change in conformation of the aptamer previously used in the group, exposed to different ion concentrations in order to detect multiple types of the influenza virus [146] . Thus, a single fluorescent-labelled aptamer is able to identify three different influenza viruses (influenza A H1N1, H3N2, and influenza B) at the same time, by modifying operating conditions, in 20 min. This chip-based aptamer-binding assay has several important advantages; it is rapid, accurate, and cheaper than multiple-aptamer screening.
Current methods for H5N1 AIV detection are virus isolation and RT-PCR that requires several days and expensive equipment and reagents. Rapid detection assays are also available (such as ELISA or immunochromatographic strips) but are less sensitive and specific. The alternative approach is biosensors technology, several biosensors have been developed to detect AIV among them biosensors using as probe aptamers (aptasensors) (reviewed in [147] . In the Li's lab, a highly specific DNA aptamer that can bind H5N1 virus with high affinity was selected. Using this aptamer, other authors have developed different aptasensors based on Surface Plasmon Resonance (SPR) [148] , a quartz crystal microbalance (QCM) aptasensor crosslinked polymer hydrogel [149] and several aptasensors based on impedance methods [150] [151] [152] . These aptasensors were able to detect H5N1 quickly and/or with more sensitivity than antibody-based biosensors.
The impedance-based aptasensor described Fu et al. has the lowest detection limit, however, it requires signal amplification with labels and a prolonged detection limit [150] . The impedance aptasensor with microfluidics chips has a lower detection limit than the SPR-based aptasensor [148] and the same sensitivity as the QCM aptasensor [149] , but the QCM-based aptasensors are not practical for in-field use due to the QCM's predisposition to environmental noise. The major advantage of the impedance aptasensor with gold nanoparticles for signal amplification described by Karash et al. is that it requires a small sample volume and is cheaper than the detection platforms based on QCM or that use interdigitated electrode microfluidic chips [152] . Recently, Nguyen et al. developed a sandwich-type SPR-based biosensor for the detection of H5Nx viruses using a pair of aptamers selected against a mixture of H5Nx whole viruses using Multi-GO SELEX [153] . The sensitivity of the dual aptamer-based system increased by more than 50-fold than for single-aptamers. In addition, the sensitivity was additionally enhanced when the secondary aptamer was conjugated with gold nanoparticles.

Aptamers to Influenza Virus as Antiviral Agents

Several aptamers against influenza virus have been developed for therapeutics purposes, mainly targeting hemagglutinin (reviewed in [5] ) (Figure 4) . These aptamers are able to inhibit the entry of the virus of the cells by blocking hemagglutinin activity. The common technique to measure the inhibitory activity of the aptamers in vitro is the hemagglutination inhibition assay. The model was more extensively used to test the effect of the aptamers on the viral infection involving the use of cell cultures, mainly Madin-Darby canine kidney (MDCK) cells. The cells are infected with the virus and incubated with the aptamers and the inhibition of viral infectivity is tested. Using these assays, DNA and RNA aptamers selected against HA from Influenza A virus [142, [154] [155] [156] [157] [158] or avian influenza virus [159] [160] [161] , able to significantly decrease the viral infection in cells, have been described. However, only a few studies have described aptamers capable of mediating a reduction in viral pathogenicity in mice models. Jeon et al. evaluated the effect of the administration intranasal of the A22 aptamer, a DNA aptamer selected against the HA-(91-261) peptide, in mice before, at the same time and after virus infection [154] . The aptamer-induced inhibition of viral infection was determined by prevention of weight loss, decrease of viral load in the lungs and restriction of the level of inflammation and cellular infiltration. A22 reduced up to 95% of infection in all the strains tested (H1N1, H2N2 and H3N2). A22 was most effective when administered concomitantly with the viral infection leading to 95% reduction in viral burden. The administration of A22 one day prior to infection (preventive treatment) was less effective, probably because the DNA is partially degraded. Interestingly, the treatment with A22 two days following the infection (therapeutic treatment) still leads to almost 95% reduction in viral titer in the lungs of the mice. In 2014, Musafia et al. used A22 aptamer as a starting point and the quantitative structure-activity relationship (QSAR) tool to produce aptamers with 10-15 times more potent antiviral activity in animal models than A22 aptamer. The binding of these aptamers to the virus (20 times higher than A22) may not necessarily be sequence-specific being the most important properties the aptamer length, 2D-loops and repeating sequences of C nucleotides [157] .
Another antiviral strategy is the inhibition of the enzymes involved in the viral replication, transcription and translation. The polymerase complex of Influenza virus catalyzes the viral replication and transcription. This heterotrimer is composed of three subunits named PA, PB1 and PB2 [164] [165] [166] . PA plays the role of an endonuclease, cleaving host mRNAs downstream of their mRNA cap structures, which are recognized and bound by PB2 [167] . The N-terminal of the PA subunit (PA N ), which holds the endonuclease activity site, is highly conserved among different subtypes of influenza virus, which suggests it is an attractive target in the development of anti-influenza agents. Yuan et al. selected DNA aptamers against both PA protein (three aptamers), and the PA N domain (six aptamers) of an H5N1 virus strain [168] . Four of the six PA N selected aptamers inhibited both endonuclease activity and H5N1 virus infection whereas the three PA-selected aptamers did not inhibit endonuclease activity and virus infection. Finally, one of the four effective aptamers, exhibited cross-protection against infections of H1N1, H5N1, H7N7, and H7N9 influenza viruses, with a 50% inhibitory concentration (IC 50 ) around 10 nM.
Pharmaceuticals 2016, 9, 78 19 of 33 aptamers, exhibited cross-protection against infections of H1N1, H5N1, H7N7, and H7N9 influenza viruses, with a 50% inhibitory concentration (IC50) around 10 nM. The association of the viral polymerase, bound to the cap, and eIF4GI may be involved in the preferential translation of viral mRNAs during influenza infection. In addition, the interaction of NS1, bound to a conserved 5untranslated region (UTR) element of the viral mRNA, with eIF4GI and PABP1 could promote the formation of a "closed loop" between the 5′ and 3′ ends of the viral mRNA; (4) RIG-I is a cytosolic receptor for non-self RNA that mediates immune responses against viral infections through IFNα/β production. Mitochondrial antiviral-signaling (MAVS) protein. Table 6 shows information on the aptamers described against influenza virus. Vaccination is a powerful approach to diminish the effects of influenza epidemics, but the use of antiviral drugs can also be very useful, particularly in delaying the spread of new pandemic viruses. Neuraminidase inhibitors like oseltamivir, laninamivir, zanamivir, and peramivir are commonly used as antiviral agents to treat influenza infection, especially in Japan. However, because of the rapid increases in drug-resistant influenza virus, it is essential to develop new antiviral drugs as an emerging strategy to block cellular factors important for the infective cycle. The advantage of blocking important cellular pathways for the virus inhibitory effect is that, in principle, it is not specific of influenza strain and the emergence of resistant virus is minimized. A limited number of aptamers targeting host cell factors have been described. Of these, the use of RIG-I as a target for aptamers to control viral infection should be emphasized [169] . RIG-I is a cytosolic receptor for nonself RNA that mediates immune responses against viral infections through IFNα/β production [170] . The use of a specific RIG-I aptamer that activates RIG-I efficiently blocks the replication of the Newcastle disease virus, vesicular stomatitis virus and influenza virus in infected cells, evidencing that aptamers targeting cellular factors can act as efficient antiviral agents [169] . The association of the viral polymerase, bound to the cap, and eIF4GI may be involved in the preferential translation of viral mRNAs during influenza infection. In addition, the interaction of NS1, bound to a conserved 5-untranslated region (UTR) element of the viral mRNA, with eIF4GI and PABP1 could promote the formation of a "closed loop" between the 5 and 3 ends of the viral mRNA; (4) RIG-I is a cytosolic receptor for non-self RNA that mediates immune responses against viral infections through IFNα/β production. Mitochondrial antiviral-signaling (MAVS) protein. Table 6 shows information on the aptamers described against influenza virus. Vaccination is a powerful approach to diminish the effects of influenza epidemics, but the use of antiviral drugs can also be very useful, particularly in delaying the spread of new pandemic viruses. Neuraminidase inhibitors like oseltamivir, laninamivir, zanamivir, and peramivir are commonly used as antiviral agents to treat influenza infection, especially in Japan. However, because of the rapid increases in drug-resistant influenza virus, it is essential to develop new antiviral drugs as an emerging strategy to block cellular factors important for the infective cycle. The advantage of blocking important cellular pathways for the virus inhibitory effect is that, in principle, it is not specific of influenza strain and the emergence of resistant virus is minimized. A limited number of aptamers targeting host cell factors have been described. Of these, the use of RIG-I as a target for aptamers to control viral infection should be emphasized [169] . RIG-I is a cytosolic receptor for non-self RNA that mediates immune responses against viral infections through IFNα/β production [170] . The use of a specific RIG-I aptamer that activates RIG-I efficiently blocks the replication of the Newcastle disease virus, vesicular stomatitis virus and influenza virus in infected cells, evidencing that aptamers targeting cellular factors can act as efficient antiviral agents [169] . However, aptamers directed against cellular factors that establish essential interactions with influenza virus proteins had not been reported before. The mRNAs of influenza virus possess a 5 cap structure and a 3 poly (A) tail that makes them structurally indistinguishable from cellular mRNAs. However, selective translation of viral mRNAs occurs in infected cells through a discriminatory mechanism, whereby viral polymerase and NS1 interact with components of the translation initiation complex, such as the eIF4GI and PABP1 proteins [171] [172] [173] . Thus, the inhibition of viral protein-translation factor interactions or their destabilization can be potentially used as an antiviral strategy. Recently, Rodriguez et al. studied whether two aptamers which bind hPABP1 with high affinity (ApPABP7 and ApPABP11) are able to act as anti-influenza drugs [174] . Both aptamers inhibit influenza virus replication of H1N1 or H3N2 subtypes at high and low multiplicity of infection and the viral polymerase-eIF4GI interaction. In addition, aptamer ApPABP11 inhibits the interactions between NS1 and eIF4GI or PABP1. These results indicate that aptamers targeting the host factors that interact with viral proteins may potentially have a broad therapeutic spectrum, reducing the appearance of escape mutants and resistant subtypes.

Aptamers against Other Emerging Viruses

An emergent virus is a virus that has adapted and emerged as a new disease/pathogenic strain, with attributes facilitating pathogenicity in a field not normally associated with that of virus. This includes viruses that are the cause of a disease which has notably increased in incidence; this is often a result of a wide variety of causes from both the influence of man and nature. Most emergent viruses can be categorized as zoonotic (an animal disease that can be transmitted to humans), and this has the advantage of possibly having several natural reservoirs for the disease.
Most of these viruses have newly appeared in a population or have existed but are rapidly increasing in incidence or geographic range and only recently aptamers against emergent viruses such as Rift Valley Fever, Tick-borne encephalitis, Dengue, Ebola viruses or other arboviruses have been developed [175] .

Aptamers to Rift Valley Fever Virus (RVFV)

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus (genus Phlebovirus) responsible for widespread outbreaks of severe disease such as hepatitis, encephalitis and hemorrhagic fever in humans [176] . The virus is endemic throughout much of the African continent. However, the emergence of RVFV in the Middle East, northern Egypt and the Comoros Archipelago has highlighted that the geographical range of RVFV may be increasing, and has led to the concern that an incursion into Europe may occur. At present, there is no licensed human vaccine [177] .
The nucleocapsid protein (N) of RVFV is an RNA binding protein required for the production of viable virus because of its involvement in several stages of viral replication. This protein protects the viral genome from degradation and prevents the formation of double stranded RNA intermediates during replication and transcription by encapsidating viral genomic and antigenomic RNA [178] . Ellenbecker et al. isolated RNA aptamers that bound N with high affinity and identified GAUU and pyrimidine/guanine motifs in their sequences, which are also present within the coding region of the RVFV genome. Furthermore, the authors developed a truncated RNA aptamer labeled with fluorescein using a fluorescence polarization (FP) system. Titration of N with the 3 -FAM-labeled RNA aptamer gave an apparent Kd of 2.6 µM. Competitive binding experiments were conducted with four different aptamers and the apparent Ki values were all in the~200 nM range. These data demonstrate that these aptamers might be used to construct a sensitive fluorescence based sensor of N binding with potential applications for drug screening and imaging methodologies [179] .

Aptamers to Tick-Borne Encephalitis Virus (TBEV)

Tick-borne encephalitis virus (TBEV) belongs to the family Flaviviridae, genus Flavivirus. This virus produces tick-borne encephalitis (TBE), an important emerging infectious disease that targets the central nervous system (CNS) [180] . There is currently no specific antiviral treatment for TBE because the specific immunoglobulin used in clinical practice has several disadvantages. The purpose of Kondratov et al. was to obtain an aptamer population against a fragment of the surface protein E of the TBEV, since it is available for aptamers outside of the host cell [181] . Authors showed that the treatment with the library of aptamers produced a TBEV neutralization index comparable with the results of neutralization of the commercial human immunoglobulin against tick-borne encephalitis (NPO Microgen, Russia). In addition, the enzyme immunoassay systems based on the immobilization of viral particles on antibodies are most commonly used for the TBEV diagnosis and the authors claim that protein E aptamers could substitute antibodies in these systems.

Aptamers to Dengue Virus (DENV)

Dengue viruses (DENVs) belong to the Flaviviridae family, and contain four serologically and genetically distinct viruses, termed DENV-1, DENV-2, DENV-3 and DENV-4. The envelope (E) protein plays an important role in viral infection but, however, there is no effective antibody for clinical treatment due to antibody dependent enhancement of infection. Chen et al. identified an aptamer (S15) that can bind to DENV-2 envelop protein domain III (ED3) with a high binding affinity. S15 aptamer was found to form a parallel quadruplex structure that together with the sequence on 5 -end were necessary for the binding activity to a highly conserved loop between βA and βB strands of ED3. Although S15 aptamer was selected against DENV-2, the authors demonstrated that this aptamer can neutralize the infections by all four serotypes of DENVs [182] .
The DENV capsid (C) protein functions as a structural component of the infectious virion but it may also have additional functions in the virus replicative cycle [183] . Balinsky et al. showed that the DENV C protein interacts and colocalizes with the multifunctional host protein nucleolin (NCL) and that this interaction can be disrupted by the addition of a NCL binding aptamer (AS1411), developed as AGRO100 by Aptamera (Louisville, KY, USA). Treatment of cells with AS1411 produced a significant reduction of viral titers after DENV infection. Moreover, the authors showed that treatment with AS1411 affected the migration characteristics of the viral capsid and identified a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics [184] .

Aptamers to Ebola Virus (EV)

Ebola virus belong to the genus Ebolavirus. Four of five known viruses in this genus cause a severe and often fatal hemorrhagic fever in humans and other mammals, known as Ebola virus disease (EVD). Ebola virus has caused the majority of human deaths from EVD, and is the cause of the 2013-2015 Ebola virus epidemic in West Africa.
In many cases, aptamers have been used as a technological and research tool to identify RNA sequences that are recognized by different virus proteins. The zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of Moloney murine leukemia virus (MLV), Sindbis virus (SIN) and Ebola virus [187] , by targeting the viral mRNAs for degradation and preventing the accumulation in the cytoplasm. With the aim to identify RNA sequences that could be a target of ZAP, Huang et al. used aptamer technology identifying G-rich RNA aptamers that contained conserved "GGGUGG" and "GAGGG" motifs in the loop region. Interestingly, overexpression of the aptamers significantly reduced ZAP's antiviral activity and the substitution of the conserved motifs of the aptamers significantly impaired their ZAP-binding ability and ZAP-antagonizing activity, suggesting that the RNA sequence is important for specific interaction between ZAP and the target RNA [188] .

Aptamers to Severe Acute Respiratory Syndrome (SARS)

Severe acute respiratory syndrome (SARS) is a disease caused by SARS coronavirus (SARS-CoV) that caused a pandemic pneumonia in 2002-2003, with a total of 8096 reported cases, including 774 deaths in 27 countries. SARS-CoV belongs to the Coronavirus genus in the Coronaviridae family and is an enveloped, positive-sense RNA virus with a genome of 27.9 kilobases. This RNA encodes two large polyproteins, pp1a and pp1ab, and four structural proteins including spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. Pp1a and pp1ab are proteolytically cleaved into 16 non-structural proteins (nsps) that form the viral replicase-transcriptase complex (reviewed in [189] ).
Only a few studies have been focused on the development of aptamers against SARS-CoV as antivirals, in spite of the fact that there is still no effective therapeutic treatment against the virus. Two studies have selected DNA and RNA aptamers against the non-structural nsp13 protein. This protein possesses NTPase, duplex RNA/DNA-unwinding and RNA-capping activities that are essential for viral replication and proliferation. These aptamers inhibited helicase activity with subnanomolar IC 50 , while the ATPase activity was not affected, suggesting that the aptamers may bind to the nucleic acid binding site of the helicase and block the unwinding activity [192, 193] . Table 7 shows information on the aptamers described against emerging viruses. [190, 191] Severe acute respiratory syndrome n.d. DNA/RNA non-structural nsp13 protein [192, 193] n.d. = not determined.

Perspectives

The success of treatment in viral diseases depends on the early detection of the infective agent. The most probable use of aptamers in virus diagnostics involves the development of more simple, fast and cheap diagnostics devices. One of these simple detection systems can be the Lateral Flow Immunoassays (LFIAs) which are currently used for qualitative monitoring in resource-limited or non-laboratory environments. The LFIA biosensing platform mainly comprises the sample pad and test pad, which is generally composed of nitrocellulose membrane, and provides a platform for both reaction and detection where the capturing molecules are antibodies [197] . Lateral Flow biosensing platform has been developed using an aptamer against hepatitis C virus (HCV) core antigen [83] and could be applied for HIV or emerging virus detection using aptamers against specific proteins. This method allows detection of viruses in endemic or transit of human areas. Another interesting approach to obtaining cheaper diagnostics/genotyping devices is using only one aptamer to detect several targets. From this point of view, the strategy by Wang et al. [146] , in which they use the conformational change of one aptamer exposed to different ion concentrations to detect multiple types of the influenza virus could be used for genotyping of other viruses such as HBV or HCV.
From a therapeutic point of view, aptamers offer a hopeful solution in viral diseases because they can target elements of the virus or the infected host cell easier than the antibodies mainly due to their small size. The potential design of aptamers working against different targets might block the virion penetration into the cells or inhibit enzymes responsible for viral replication or other critical processes. In the case of HIV-1, despite efficient antiretroviral therapy, eradication of latent HIV-1 provirus is challenging and requires novel biological insights and therapeutic strategies. For this aim, novel target proteins should be chosen in HIV reservoir organs for the isolation of aptamers that could be applied to drug delivery or targeting of nanoparticles loaded with drugs to obtain HIV transcriptional activation.
As already mentioned above, RIG-I has been used as a target for aptamers to control viral infection [169] . Recently, Olagnier et al. investigated the inhibitory effect of a RIG-I agonist on the replication of Dengue and Chikungunya viruses [198] . The authors demonstrated that RIG-I stimulation generated a protective antiviral response against both pathogens. It would be motivating to study the use of RIG-I aptamers developed by Hwang for Dengue and Chikungunya therapy. Likewise, the effect of the aptamers developed by Guerra et al. against PABP in decreasing replication of influenza virus [174, 199] could be studied in other viruses that also use the PABP of the infected cell [200] .

Aptamers to HIV as Antiviral Agents

At present, the treatment HIV-1/AIDS is by a combination of several antiretroviral drugs (cART), which can slow the progress of the disease and reduce the risk of death and disease complication, but it is not curative. Moreover, many patients do not tolerate cART because it has severe side effects, and it is too expensive for patients in developing countries. In this regard, aptamers have been considered an alternative or adjuvant to the chemical antiviral agents in cART to overcome these limitations. To date, highly specific, nucleic acid-based aptamers that target various parts of HIV-1 genomes, HIV-1 proteins (including HIV-1 protease (PR), reverse transcriptase (RT), nucleocapsid, gp120, and Gag) and cellular proteins (nucleolin, CD4 or CCR5) have been isolated and shown to effectively suppress viral replication to apply in HIV therapy [5, [25] [26] [27] (Figure 2 ). (1) The HIV viral particle has an inner capside containing ssRNA viral genome and integrase and retro-transcriptase proteins, mainly; (2) The outer envelope has gp120 and gp40 proteins involved in interaction with cellular receptors (CD4, CCR5, NLS) and fusion to cellular membrane; (3) In the cytoplasm the ssRNA viral genome is released and the retro-transcription step is produced; (4) The integrase protein binds to dsDNA viral genome by LTR ends sequences and other cellular proteins forming the pre-integration complex (PIC); (5) The PIC goes into nucleus through the nuclear pore and is integrated in the cellular genome by the integrase protein activity (provirus); (6) The viral RNAs are transcripted from proviral DNA and exported to cytoplasm to translate viral proteins as protease and a big pre-protein that are assembled to new RNA viral genomes and leave the cell with outer envelope from the cellular membrane; (7) After the budding viral particle, the proteases process the big pre-protein and get a mature viral particle.

Aptamers to HIV Genome

(2) The outer envelope has gp120 and gp40 proteins involved in interaction with cellular receptors (CD4, CCR5, NLS) and fusion to cellular membrane; (3) In the cytoplasm the ssRNA viral genome is released and the retro-transcription step is produced; (4) The integrase protein binds to dsDNA viral genome by LTR ends sequences and other cellular proteins forming the pre-integration complex (PIC); (5) The PIC goes into nucleus through the nuclear pore and is integrated in the cellular genome by the integrase protein activity (provirus); (6) The viral RNAs are transcripted from proviral DNA and exported to cytoplasm to translate viral proteins as protease and a big pre-protein that are assembled to new RNA viral genomes and leave the cell with outer envelope from the cellular membrane; (7) After the budding viral particle, the proteases process the big pre-protein and get a mature viral particle.

Aptamers to Reverse Transcriptase (RT)

Reverse transcriptase has two enzymatic activities, a DNA polymerase activity that can copy either a DNA or an RNA template, and an RNase H that cleaves RNA only if the RNA is part of an RNA/DNA duplex. The two enzymatic functions of RT, polymerase and RNase H, cooperate to convert the RNA into a double-stranded linear DNA [40] . DeStefano and Nair confirmed in vitro effectiveness of DNA aptamer, named 37NT, directed against the reverse transcriptase of HIV HXB2 strain. The aptamer competed with the natural template for the binding site in the enzyme, subsequently producing inhibition of the viral replication [41] . In parallel, Michalowski et al. identified three aptamers (RT5, RT6 and RT47) which contained a bimodular structure comprising a 5 -stem-loop module linked to a 3 -G-quadruplex. In addition, the authors demonstrated that this DNA aptamers inhibited RT from diverse primate lentiviruses with low nM IC 50 values [42] .

Aptamers to CCR5

HIV-1 commonly uses C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR-4) as co-receptors along with CD4 to enter target cells. Human CCR5 is an important co-receptor for macrophage-tropic virus expressed by T-cells and macrophages. Differences in CCR5 are associated with resistance or susceptibility to HIV-1. As an essential factor for viral entry, CCR5 has represented an attractive cellular target for the treatment of HIV-1. Thus, Zhou et al. have reported the selection of RNA aptamers against CCR5 using high throughput sequencing (HTS) to analyze the RNA pools from selection rounds 5 to 9. The individual sequences were classified into six major groups (Group 1-6). Group 2, 4 and 5 shared a conserved sequence, which is comprised of 10 nucleotides UUCGUCUG(U/G)G, named G3. The G3 activity was studied by a "prophylactic" HIV-1 experiment determining whether the aptamer would block HIV infectivity of R5 viruses in cell culture. The results showed that the G3 aptamer efficiently neutralized HIV-1 infectivity of R5 strains with IC 50 about 170~350 nM [25] .

Aptamers HBV in Diagnostics

One of the current objectives in the diagnostic of HBV is to develop a daily screening assay with a short period of detection between infection and recognition. Therefore, Suh et al. have developed a fast and low cost detection test based on competitive binding assay combined with fluorescence resonance energy transfer (FRET) [73] . The assay was built with an aptamer selected against the hepatitis B virus surface antigen (HBsAg), the best characterize and most frequently used HBV marker [74] . The described aptasensor was approximately 40-fold more sensitive than the conventional method. In 2015, a new set of three different DNA aptamers was selected against HBsAg and applied to develop a chemiluminescence platform. The new aptasensor was designed with aptamers-conjugated to magnetic nanoparticles reaching a detection limit five-fold better than the current enzyme-linked immunosorbent assay (ELISA) kits used in hospitals [75] .

Aptamers HCV in Diagnostics

Aptamer-based biosensors are a promising diagnostic platform to allow HCV infection detection in early stages or in immunosuppressed patients. Thus, different groups have developed diverse aptasensors to improve diagnostic assay of HCV infection. First, Lee et al. developed a biosensor prototype that specifically recognizes the HCV core protein from sera of an infected patient using selected RNA aptamers against core antigen. The HCV viral particles were retained by the 2 -F aptamers immobilized in a 96-well plate and detected by sequential steps with anti-core and Cy3-labeled secondary antibodies [80] . Later on, Chen et al. developed an early diagnostic assay based on sandwich-ELISA to recognize HCV viral proteins using biotin-labelled DNA aptamers against HCV Envelope glycoprotein E2. The obtained results from infected patients showed a good correlation between viral genome quantification assay, HCV antibody detection and sandwich-ELISA aptasensor [81] . Afterwards, Shi et al. developed a similar platform for early detection, coating the bottom of the well with C7, a DNA aptamer against HCV core labelled with biotin, and HCV-core antibody conjugated with horseradish peroxidase (HRP) is applied over the surface. The platform was applied to the detection of the protein in sera from HCV-infected patients and showed a proportional relationship between amplified RNA copies and HCV core protein concentration [82] . Moreover, Wang et al. designed a rapid, easy-to-use diagnostic platform composed of lateral flow strips treated with thiol-DNA aptamers against HCV core antigen. HCV ELISA assay and core aptamer lateral flow strips showed positive coincidence rates when compared with HCV RNA amplification assay [83] . In an effort to develop a diagnostic test to monitor the infectivity of HCV samples, Park et al. have designed an ELISA-like assay replacing the capture and detection antibodies for DNA aptamers selected against HCV E2. The Enzyme Linked Apto-Sorbent Assay (ELASA) has been described to be used for qualitative and quantitative analysis of virus in infected samples [84] . Further, two laboratories have developed label-free aptasensor to eliminate the labelling step and simplify the HCV detection method. Hwang et al. have described a highly sensitive label-free aptasensor based on nanomechanical microcantilevers. The biosensors were able to measure the surface stress due to the interaction between immobilized RNA aptamers and the HCV helicase [85] . On the other hand, Roh et al. have developed a label-free diagnostic platform to detect and quantify the presence of HCV polymerase NS5B viral protein using conjugated streptavidin-biotin RNA aptamers on an Octet biosensor [86] .

Aptamers to HCV as Antiviral Agents

Eradication of HCV disease is one of the main objectives of global public health. Currently, HCV infected patients are treated combining protease inhibitors, as Telaprevir (TVR) and Boceprevir (BOC), with pegylated-interferon and Ribavirin. However, the new direct-acting antivirals (DAA), TVR and BOC, generate a high rate of side effects, are too expensive and are also susceptible to new resistant viruses [87] . Therefore, it is necessary to develop new DAA treatments that are more effective and with fewer side effects than current therapies. To this end, different advances have been made based on aptamers against HCV and host cell proteins as therapy [88] (Figure 3 ). amplification assay [83] . In an effort to develop a diagnostic test to monitor the infectivity of HCV samples, Park et al. have designed an ELISA-like assay replacing the capture and detection antibodies for DNA aptamers selected against HCV E2. The Enzyme Linked Apto-Sorbent Assay (ELASA) has been described to be used for qualitative and quantitative analysis of virus in infected samples [84] . Further, two laboratories have developed label-free aptasensor to eliminate the labelling step and simplify the HCV detection method. Hwang et al. have described a highly sensitive label-free aptasensor based on nanomechanical microcantilevers. The biosensors were able to measure the surface stress due to the interaction between immobilized RNA aptamers and the HCV helicase [85] .
Eradication of HCV disease is one of the main objectives of global public health. Currently, HCV infected patients are treated combining protease inhibitors, as Telaprevir (TVR) and Boceprevir (BOC), with pegylated-interferon and Ribavirin. However, the new direct-acting antivirals (DAA), TVR and BOC, generate a high rate of side effects, are too expensive and are also susceptible to new resistant viruses [87] . Therefore, it is necessary to develop new DAA treatments that are more effective and with fewer side effects than current therapies. To this end, different advances have been made based on aptamers against HCV and host cell proteins as therapy [88] (Figure 3) .

Aptamers to 5 and 3 Untranslated Regions (5 and 3 UTR)

In 2003, Toulmé et al. decided to use subdomain IIId of IRES element as an antiviral target. They selected RNA aptamer and verified that isolated aptamers inhibit HCV translation in vitro and in cell culture [89] . In the same year, Kikuchi et al. isolated RNA aptamers capable of binding to the domain II of HCV IRES and showed that IRES-mediated in vitro translation was reduced from 20% to 40% by using the 2-02 aptamer [90] . Later, they isolated a new RNA aptamer population against the HCV IRES domains III-IV and corroborated that 3-07 aptamer had a high inhibitory effect on IRES-mediated translation in vitro and in vivo [91] . To improve the inhibitory effect of selected aptamers, they constructed two new molecules, named 0207 and 0702, composed by 2-02 and 3-07 aptamers linked by their ends. The fused aptamers recognized two different subdomains of IRES element and are at least 10 times more efficient than the parental aptamers in the inhibition of mRNA IRES-dependent translation in vitro [92] . Following with IRES as an anti-viral target, Romero-López et al. described an innovative in vitro selection method to isolate aptamers fused to a hammerhead ribozyme with capacity to inhibit RNA translation mediated by IRES. Selected chimeric aptamer-ribozymes were able to recognize the IRES element and cleavage the 5 end at nucleotide position 363 [93] . The success of combining two functional elements in the same molecule was shown in the selected chimeric molecule HH363-50. Thus, the aptamer-ribozyme chimera did anchor to domain IV of the IRES element and inhibited in vitro and in vivo IRES-mediated translation [94] . Therefore, recruitment of ribosomal particles mediated by the IRES element was inhibited by the chimera HH363-24 that prevented both translation and replication in a hepatic cell line [95] . Moreover, to avoid HCV genome replication, Konno et al. isolated RNA aptamers against the 3 end of the negative strand of the virus genome [96, 97] . Interestingly, a RNA aptamer, named AP30, was able to recognize this minus-IRES region and reduce positive-strand genomic RNA synthesis [96] . To inhibit HCV replication, Marton et al. selected RNA aptamers against CRE element that were able to repress replication of HCV replicon in hepatic cells [98] . Subsequently, two selected aptamers, P58 and P78, interact with subdomain 5BSL3.2 of the CRE element and produce a structural reorganization of the 3 end HCV genome and a significant decrease of HCV replication in vivo [99] .

Aptamers to Nonstructural Protein 5A (NS5A)

It has been reported that NS5A protein is essential for HCV production and replication. Recently, Yu et al. have isolated and characterized DNA aptamers against HCV NS5A protein. Particularly, selected aptamer NS5A-5 was able to inhibit HCV virus infection by prevention of protein-protein interactions between NS5A and core protein [110] .
Aptamers to Nonstructural Protein 5B (NS5B) HCV nonstructural protein 5B (NS5B) is a RNA-dependent RNA polymerase protein (RdRp) responsible to the generation of positive-sense genomic HCV RNA and negative-sense RNA template. Reduction of HCV NS5B polymerase activity affects HCV viral life cycle and is one of the main objectives to isolate aptamers against NS5B. Thus, Biroccio et al. identified specific RNA aptamers against a truncated protein NS5B-∆55 without the C-terminal region. One of the selected aptamers, B.2, blocked RNA transcription but not competed with the complex RdRp-RNA, using different binding site than RNA template to the NS5B protein [111] . In the same way, Bellecave et al. selected DNA aptamers against the NS5B viral protein. One of the chosen aptamers, 27v, competed with positive and negative sense HCV viral RNA to bind RdRp polymerase and blocked initiation and elongation steps of RNA transcription [112] . However, 127v aptamer partially competes to dissociate RdRp-RNA complex formation and only inhibited initiation steps of HCV transcription [113] . Moreover, interference of viral production and transcription inhibition of HCV virus was confirmed in vivo using 27v aptamer. Table 4 shows information on the aptamers described against HCV. Five years later, another set of RNA aptamers against NS5B protein were obtained by Lee et al. [114] . To avoid aptamer degradation, oligonucleotides were modified with 2 hydroxyl (R-OH) or fluoropyrimidines (R-F). The R-OH aptamers blocked RNA synthesis of HCV replicon in cell culture without emergence of virus escape mutant or cellular toxicity. On the other hand, R-F oligonucleotides were truncated and conjugated with cholesterol-or galactose-PEG molecules to allow direct and specific liver delivery into cells or tissue. Cholesterol-and Gal-PEG-R-F t2 conjugated aptamer blocked RNA synthesis of HCV genome [115] . The above mentioned aptamers were non-genotype-specific; however, Jones et al. described for the first time aptamers against NS5 protein that exclusively recognized and inhibited RNA-polymerase activity of HCV virus subtype 3a [116] .

Aptamers to Structural Proteins E1, E2 and Core

Envelope E1 and E2 glycoproteins are putative targets in therapy due to its role in HCV viral recognition to enter into hepatic cells. Chen et al. have isolated DNA aptamer against E2 glycoprotein. The selected aptamers have higher affinity to genotype 1a, 1b and 2a than others, and strongly prevented HCV viral infection in Huh7 5.1 cells [81] . Afterwards, Yang et al. described the potential antiviral action of DNA aptamers selected against E1E2 protein by HCV infection suppression in HuH7.5 cells without innate immune response action [117] . In the case of core, an essential protein for HCV viral assembly, Shi et al. have applied for therapy the above-mentioned aptamers in diagnostics. In Huh7.5 cells, the aptamers against HCV core protein repressed viral production as a result of defective assembly of virus particle without stimulation of innate immune response [82] .

Aptamers to Influenza Virus in Diagnostics

The antibodies are the most common probe used to detect either the viral particles or host antibodies developed during the infection. However, although in most cases antibodies are able to distinguish between influenza A and B, only a few antibodies that differentiate subtypes of Influenza A or B have been reported. Alternative probes for subtyping are the aptamers. Thus, aptamers to hemagglutinin (HA) have been successfully and broadly used for the development on sensors for influenza detection. HA is expressed in high amounts in the viral surface and is required for binding and fusion with the host cell. Currently, more than 40 DNA and RNA aptamers to HA have been described since 2004, selected to recombinant hemagglutinins (H1, H3, H5, H9 and Ha from virus B) and to whole viruses (H5N1) (reviewed in [138] ). Misono and Kumar selected an RNA aptamer against to HA of A/Panama/ 2007/1999 (H3N2) using SPR-based SELEX [139] . Gopinath et al. generated two RNA aptamers against intact influenza virus A/Panama/2007/1999 and HA of B/Johannesburg/05/1999. These RNA aptamers are able to discriminate among both A and B influenza viruses [140, 141] .

Aptamers to Influenza Virus as Antiviral Agents

Other aptamers targeting NS1 or the PA polymerase subunit 1 have also been studied. NS1 is a nonstructural protein of small size, between amino acids 230 and 238 and with a molecular weight of 26 kDa. In view of its interaction with both RNAs and viral and cellular proteins, NS1 has been implicated in many of the alterations that occur during influenza virus infection. Moreover, NS1 has anti-interferon (IFN) properties leading to the inhibition of the host's innate immunity [162] . Thus, the importance of NS1 in viral infection makes it an attractive therapeutic target. Woo et al. selected a DNA aptamer specific to NS1 that induced IFN-β production by inhibiting NS1 function. In addition, the selected aptamer was able to inhibit the viral replication without affecting cell viability [163] .

Aptamers to Dengue Virus (DENV)

From a technological point of view, aptamers have been used for efficient isolation of endogenously assembled viral RNA-protein complexes. Hence, Dong et al. developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs) using the streptavidin-binding aptamer S1 sequence that was inserted into the 3 end of dengue virus (DENV) 5 -3 UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. This strategy led to identify several novel host DENV RBPs by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which were further implicated in DENV replication [185] .

Aptamers to Ebola Virus (EV)

Viral protein 35 (VP35) is a multifunctional dsRNA binding protein that plays important roles in viral replication, innate immune evasion and pathogenesis. These multifunctional proteins offer opportunities to develop molecules that target distinct functional regions. With this purpose, Binning et al. used a combination of structural and functional data to determine regions of Ebola virus (EBOV) VP35 (eVP35) to target aptamer selection. Two distinct classes of aptamers were characterized based on their interaction properties to eVP35. These results revealed that the aptamers bind to distinct regions of eVP35 with high affinity (10-50 nM) and specificity. In addition, the authors showed that these aptamers compete with dsRNA for binding to eVP35 and disturb the eVP35-nucleoprotein (NP) interaction. Consistent with the ability to antagonize eVP35-NP interaction, select aptamers can inhibit the function of the EBOV polymerase complex reconstituted by expression of select viral proteins [186] .

Aptamers to HIV as Antiviral Agents

At present, the treatment HIV-1/AIDS is by a combination of several antiretroviral drugs (cART), which can slow the progress of the disease and reduce the risk of death and disease complication, but it is not curative. Moreover, many patients do not tolerate cART because it has severe side effects, and it is too expensive for patients in developing countries. In this regard, aptamers have been considered an alternative or adjuvant to the chemical antiviral agents in cART to overcome these limitations. To date, highly specific, nucleic acid-based aptamers that target various parts of HIV-1 genomes, HIV-1 proteins (including HIV-1 protease (PR), reverse transcriptase (RT), nucleocapsid, gp120, and Gag) and cellular proteins (nucleolin, CD4 or CCR5) have been isolated and shown to effectively suppress viral replication to apply in HIV therapy [5, [25] [26] [27] (Figure 2 ). diamond field-effect transistor (FET) technique [24] . These "apta-biosensors" have high sensibility and specificity but the devices are complex and expensive.

Perspectives

In the field of viral diseases, the number of drugs for treating these infections is very small and most of the available therapeutics are not very effective [194] . In addition, current diagnostic tools for viral infections are expensive and time consuming. These important diagnostic and therapeutic limitations have favored the development of aptamer-based systems, mainly because these show several interesting advantages in relation to antibodies. Thus, aptamers can recognize and/or inhibit target activity through specific and strong interactions superior to other biologics and small molecule therapeutics, with lower toxicity and immunogenicity profiles. In this sense, during the last years, aptamer technology is being used in a wide range of diagnostic and therapeutic applications associated with viral pathologies [195, 196] . It is significant that aptamers selected as specific anti-viral molecules are effective in infected cells, however, none of the selected antiviral aptamers entered into clinical trials. In conclusion, it is necessary to continue research studies and successfully develop clinical trials to establish the use of aptamers as antivirals.
Nanoparticles have been considered for a wide range of applications, both in soluble and insoluble forms. An advantage of soluble forms of nanoparticles is that they can encapsulate antibiotics/drugs and then release them when they reach the cellular environment, making them highly applicable for drug delivery systems. Studies on aptamer-based enhanced drug delivery have been reported for prostate cancer and lymphoblastic leukemia cells [201] . With this purpose, for HIV therapy, nanoparticles successfully loaded with the antiretroviral (ARV) drugs efficiently inhibited HIV-1 infection [202, 203] . These results showed the benefit of the nanoparticles' application to delivery of antiviral drugs to improve its bioavailability.
In conclusion, the use of aptamers in the development of diagnostic platforms or as therapeutic drugs is a promising alternative for the treatment of viral diseases.
17 section matches

Abstract

Methods: This study was conducted to determine the effects of ethyl acetate (45 L Ea), ethanol (45 L Et), and hexane (45 L H) leaf extracts of G. parvifolia on the infectivity of pseudorabies virus (PrV) in Vero cells. The antiviral effects of the extracts were determined by cytopathic effect (CPE), inhibition, attachment, and virucidal assays.
Background: Garcinia species contain bioactive compounds such as flavonoids, xanthones, triterpernoids, and benzophenones with antibacterial, antifungal, anti-inflammatory, and antioxidant activities. In addition, many of these compounds show interesting biological properties such as anti-human immunodeficiency virus activity. Garcinia parvifolia is used in traditional medicine. Currently, the antiviral activity of G. parvifolia is not known.

Background

There are several ways by which therapeutic compounds interfere with viral replication. The antiviral effects can either be through prevention of viral attachment to host cell, binding to enzymes responsible for transcription, and prevention of cleavage of viral particles [1] . Viruses mutate over time and develop resistance to antiviral drugs and therapeutic compounds [2] . Thus, there is a need to discover and develop antiviral agents that do not become ineffective over time owing to development of resistance by the virus. But the pipeline of new drugs is drying up. There would be a tremendous benefit by integrating combinations of modern drugs with traditional medicinal plant extracts that have been used as folk medicine to broaden the curing spectrum via generating synergistic effects.

Viral attachment

Extracts, 45 L Ea and 45 l Et showed the greatest dosedependent inhibition of viral attachment to Vero cells

Discussion

Although, there are a few studies that determined the antiviral effect of several Garcinia species [10, [40] [41] [42] , reports were almost null on G. parvifolia. Owing to the limited antiviral research that has been done on this species, this study aims to enlighten researchers on the effect of G. parvifolia on PrV-infected Vero cells. It was shown that G. parvifolia ethyl acetate (45 L Ea) and ethanol (45 L Et) extracts particularly, did not only reduce but in fact almost completely inhibited Vero cell plaque formation. The antiviral effects of the extracts are proposed to occur through several mechanisms. The extracts inhibited viral infection of Vero cells as shown by reduction in plaque formation when PrV was firstly incubated with the extracts before exposure to Vero cells. The extracts also inhibited attachment of the virus to the cells. When the virus was allowed to infect Vero cells, the infected cells treated with G. parvifolia extract, plaque formation was inhibited. Similarly, when Vero cells were treated with extract-virus mixture, plaque formation was reduced. These effects were most prominent with 45 L Ea treatment. This finding suggests that the extract did not only inhibit cell infection by herpesviruses but also prevents viral replication of infected cells. The antiviral effect of G. parvifolia extracts are deemed to be virucidal. The virucidal property is only true for the ethyl acetate (45 L Ea) and ethanol (45 L Et) extracts, and not for the non-polar hexane (45 L H) extract. These more polar extracts caused almost total inhibition of plaque formation by the PrV-infected cells.
To realize the potential of a plant product as an antiviral compound, its mechanism of activity must be ascertained. It is important to differentiate between viral particle inactivation or virucidal activity from antiviral activity. Direct viral particle inactivation is an early effect where the virus is inactivated before it infects the cells while antiviral activity is the killing or suppression of replication ability of the virus. It would be ideal in virus infections for the treatment drug to possess both virucidal and antiviral activities. In our study, both the G. parvifolia ethyl acetate (45 L Ea) and ethanol (45 L Et) extracts presented with good virucidal activities of > 90%, and the effects of the extracts are partially on the inhibition of viral attachment and adsorption into target cells. The G. parvifolia hexane (45 L H) extract did not show similar activity on Vero cells.
Clusianone had been isolated in abundance from the leaves of G. parvifolia through hexane extraction [34] . Recently, the anti-proliferative potential of structurally modified clusianone and its derivatives has been shown [35, 36] . A significant correlation was reported on the structure activity relationship of clusianone against Respiratory Carcinoma Cells via cytotoxicity assay [36] . The strong toxic effect of 45 L H extract towards Vero cells at minimal dose concentration, with CC50 of < 1.25 μg/mL, in comparison to 45 L Ea (CC50 of 237) and 45 L Et (555.0 μg/mL), could predict either a prominent cytotoxicity or the anti-proliferative effect of clusianone, Previously, primary screening isolated 20 xanthones from plants of the Guttiferae family that have inhibitory effects on human herpesvirus 4 (HHV-4) also known as the Epstein-Barr virus (EBV). Xanthones, particularly 1,3,7-trihydroxy-2-(3-methyl-2-butenyl) xanthone, dulxanthone-B and latisxanthone-C, seemed to significantly inhibit EBA early antigen (EBV-EA), one of the viral genes required for the initiation complex at the lytic origin of viral replication in Raji cells [37] . Mangiferin with a broad spectrum beneficial biological activities, was the first xanthone shown to be pharmacologically effective for the treatment of diseases caused by herpesvirus, [38] . One of the effects of the xanthones is the inhibition of HIV-1 reverse transcriptase. Among xanthones, prenylated xanthones is restricted to the plant species of the family Guttiferae [38] . Prenylated xanthones namely mangostin and y-mangostin, isolated from G. mangostana are active against HIV-1 protease, preventing proteolytic cleavage during retroviral replication [38] . A recent investigation submitted a set of 272 xanthones to molecular docking examination and the results suggested that the xanthones could be suitable key components of agents to possess antiviral properties [39] . In fact, prenyl groups have been predicted to be important in protein-protein binding because of their specialized prenyl-binding domains that facilitate attachment to cell membranes. Therefore, a few lead compounds and their derivatizations could be screened using a structural-based docking method, or high-throughput screening methods for serving as a proof of concept for feasible viral target.

Conclusion

The antiviral activity of the G. parvifolia extracts seemed to occur at several stages of the replication cycle. The multiple antiviral effects of the extracts are suggested to occur through the interference of viral attachment and traverse across cell membrane, cytoplasmic transport, and viral genome replication. Therefore, the antiviral effect of the G. parvifolia ethyl acetate (45 L Ea) and ethanol (45 L Et) extracts is suggested to occur through several mechanisms and not solely virucidal. This study suggests that the G. parvifolia extracts prevent viral replication in infected cells, particularly the 45 L Ea containing the flavonoids, tannins and phenolics which could be the constituents that are responsible for the potent antiviral activities. Therefore, future works will be emphasized on the pure compounds isolation from these 45 L Ea extract and to study the mechanisms of antiviral action triggered by these pure bioactives.

Pseudorabies virus (PrV)

The stock PrV strain amorphous inclusion protein (AIP) used in this study was an established virus at the Virology Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia. Quantitation of stock virus was conducted on Vero cells (ATCC No. CCL-81) by using the plaqueforming assay. The virus was stored at − 80°C.

50% tissue culture infectious dose (TCID 50 )

Vero cells were seeded into flat-bottom 96-well microtiter plates at 2 × 10 4 cells/well and incubated for 24 h under 5% CO 2 humidified atmosphere at 37°C. Serial dilution of virus stock (10 8 PFU/ml) was prepared in media with FBS. So the working stock was formed forth dilution that was 1 × 10 4 PFU/ml, then incubated with the Vero cells for 72 h. The cytopathic effect (CPE) and proportional distance (PD) were calculated using the following formula [23] ; PD = (% of wells infected at dilution > 50%) -(50% infection) / (% of wells infected at dilution > 50%) -(% of wells infected at dilutions < 50%). The tissue culture infectious dose 50% (TCID 50 ) is calculated by using the following formula: TCID 50 = 10 log total dilution > 50% -(1 × log h) .

Background

Traditional medicinal trees are evergreen, abundant and available year round in tropical regions. Local communities used various parts of these trees in their traditional practice because of their high nutritive values but yet some of their detailed medicinal properties remain unknown. The plant studied, Garcinia parvifolia produces cherry-like fruit which is locally known as "asam kandis" or "asam kundong" [3] , whilst the young leaves are sometimes eaten as a vegetable. The leaf extracts of this plant were screened against pseudorabies virus (PrV). It is a broad host range herpesvirus, causes fatal encephalitis in a wide variety of animal species except its natural host, the adult pig [4] [5] [6] [7] . Since PrV is not a human pathogen, it is safe to be used in a laboratory set-up. The virus can easily be grown in the laboratory thus it is practical and convenient to be used in the screening and development of antiviral drugs or compounds.
G. parvifolia which belongs to the family of Clusiaceae (Guttiferae), is native in tropical and subtropical countries of South East Asia such as here in Malaysia, Thailand, Brunei, and Indonesia [8, 9] . Garcinia is known to produce xanthones and benzophenones [9, 10] and many of these compounds show interesting biological activities including anti-human immunodeficiency virus activity [9, 10] . There are at least 300 distinct Garcinia species and many contains bioactive compounds to include flavonoids, xanthones, triterpernoids, and benzophenones with beneficial biological activities [11] [12] [13] [14] . The crude extracts of some parts of G. parvifolia have shown antiplasmodial, antioxidant, cytotoxic and antibacterial activities [15] . However, the antiviral properties of the G. parvifolia extract are not known. Since G. parvifolia has rather similar properties with other Garcinia sp, it potentially has antiviral activities and hence is of great interest to test in the current study. In this study, their leaf extracts were obtained by using either ethyl acetate, ethanol, or hexane and screened for the efficiency to inhibit PRV.

Pseudorabies virus quantification

Vero cells (1.6 × 10 5 cells) in RPMI 1640 containing 2% FBS were seeded in each well of a 24-well plate and incubated under 5% CO 2 humidified atmosphere at 37°C in for 24 h. The medium was discarded, replaced with fresh medium and plates were again incubated under 5% CO 2 humidified atmosphere at 37°C for 48 h. The virus was diluted with fresh RPMI 1640 with 2% FBS to obtain a working virus solution. One hundred microliters of virus suspension containing 1X10 7 PFU PrV was added to each containing Vero cells in 1 mL of 1% methylcellulose and 2% FBS and the plate incubated rocking for 1 h.

Plaque reduction assay

The experiment was performed according to the method described by Zhu et al. [25] with brief modifications. Confluent monolayer of Vero cells grown in 24-well culture plates were treated with 100 μl of 37.5, 75, 150, and 300 μg/mL for 45 L Ea; 25, 50, 100, and 200 μg/mL for 45 L Et and 2.5, 5, 10, 20, 40 μg/mL for 45 L H. Besides, 100 PFU per 100 μL PrV was added and incubated at 37°C for 90 min. The virus and extract mixture was discarded and 1 mL of 1% methylcellulose and 2% FBS mixture were added to the wells and the plate was incubated in 5% CO 2 humidified atmosphere at 37°C for 48 h. Infected cells were fixed with methanol and stained with 0.5% crystal violet solution for 30 min. The number of plaques was counted and 50% inhibition concentration (IC 50 ) calculated by the following formula: IC 50 = [1-(PFU Treatment /PFU Control )] × 100 where PFU treatment = PFU of treatment and PFU Control = PFU all control cells. The assay was done in quadruplicates.

Virucidal assay

The experiment was performed according to the method described by Carlucci et. al. [27] . 1 × 10 6 PFU PrV were mixed with 125, 250 or 500 μg/mL of 45 L Ea, 62.5, 125 or 250 μg/mL of 45 L Et, and 1.25, 2.5, 5.0 or 10 μg/mL of 45 L H and then incubated at 25°C for 6 h. One hundred microliters of virus suspension or extract was mixed with 100 μL RPMI media containing 2% FBS and added to confluent monolayer Vero cells in a 24-well plate and incubated at 37°C for 90 min. The incubating mixture was removed and replaced with 1 mL of 1% methyl cellulose and 2% FBS mixture and incubated under 5% CO 2 humidified atmosphere at 37°C for 48 h. Formed plaques were fixed with methanol and stained with 0.5% crystal violet solution for 30 min. The number of plaques was counted and residual virus infectivity was determined by the following formula: Plaque formation (PFU) = Number of plaques × (1/viral inoculation) × (1/diluted fold). The IC 50 was calculated using the formula described in the plaque reduction assay.

Discussion

This study focused on the three crude leaf extracts of G. parvifolia, namely 45 L H, 45 L Et, and 45 L Ea, which were extracted via ethyl acetate, ethanol and hexane solvents, respectively. The phytochemical constituents of these crude extracts were elucidated (Table 1 ). Based on the findings, 45 L Et contained all six phytochemical constituents tested and 45 L Ea contained five of them except saponin and the non-polar 45 L H did not contain saponin, tannin and phenolic contents. Among these, saponin and tannin are indeed very common secondary metabolites for plant kingdom, which possess good antioxidant activities. The presences of different classes of chemical constituents such as flavonoids, phenolics, terpenoids and steroids in 45 L are rather conformed to those isolated from different parts of G. parvifolia including leaves, twigs, latex, fruits and barks [8, 11] . In fact, flavonoids and terpenoids isolated from plants had been reported to contain antiviral properties, particularly against Chikungunya virus [28] and severe acute respiratory syndrome coronavirus [29] .
Among approaches used in the determination of antiviral activity of natural compounds is the inhibition of viral DNA replication and reverse transcriptase. Most natural antiviral agents may act only on a limited number of viruses, because the viruses are prone to mutations that render the compounds eventually becoming ineffective [43, 44] . Antiviral compound should be highly effective while showing minimal toxicity to normal cells and tissues. One way determining potential of antiviral compound is by calculating the SI. In this study, the SI value of ethyl acetate (45 L Ea) extract was higher than either the ethanol (45 L Et) or hexane (45 L H) extract, indicating it has more potent antiviral activity. The results also suggest that the non-polar hexane (45 L H) extract was most toxic to Vero cells among the three extracts, thus may not be a suitable candidate as antiviral agent. The difference of phytochemical constituents between 45 L Ea (showing the highest antiviral potency) and 45 L H was the additional flavonoids, tannins and phenolics, which were extracted by ethyl acetate solvent. It might be possible that these constituents are responsible for the potent antiviral activities. It is worthy to mention that flavonoids, tannins, and phenolics had been found very important to inhibit different stages in the HIV replication cycle, where three of them act at the virus adsorption stage; flavonoids and tannins disable the reverse transcription and phenolics stop the viral integration in the human genome [32] . Besides, more recently, flavonoids of plant origin were found effective to combat against Chikungunya virus [28] .
8 section matches

Abstract

Aptamers are nucleic acid-based ligands identified through a process of molecular evolution named SELEX (Systematic Evolution of Ligands by Exponential enrichment). During the last 10-15 years, numerous aptamers have been developed specifically against targets present on or associated with the surface of human cells or infectious pathogens such as viruses, bacteria, fungi or parasites. Several of the aptamers have been described as potent probes, rivalling antibodies, for use in flow cytometry or microscopy. Some have also been used as drugs by inhibiting or activating functions of their targets in a manner similar to neutralizing or agonistic antibodies. Additionally, it is straightforward to conjugate aptamers to other agents without losing their affinity and they have successfully been used in vitro and in vivo to deliver drugs, siRNA, nanoparticles or contrast agents to target cells. Hence, aptamers identified against cell surface biomarkers represent a promising class of ligands. This review presents the different strategies of SELEX that have been developed to identify aptamers for cell surface-associated proteins as well as some of the methods that are used to study their binding on living cells.

Introduction

A large number of medical biomarkers are expressed at the surface of human cells or infectious pathogens such as viruses, bacteria, fungi or parasites. The composition and dynamics of the cell surface determine how a cell or a pathogen can interact with its environment and are crucial to send and receive chemical signals, to transport metabolites, ions, or larger molecules, to attach to neighboring cells and the extracellular matrix, etc. In the context of disease, a mutation, deletion or over-expression of cell surface proteins is associated with many pathological states and membrane proteins are currently the target for more than half of the approved drugs [1] . Hence, there is a high demand for specific ligands against cell surface targets for fundamental research, but also for diagnosis, monitoring and treatment of diseases. So far, most of these ligands have been developed against proteins whereas only a few exist against carbohydrates and lipids. Two types of ligands have been developed: (1) small molecule drugs which are predominantly designed to bind the intracellular catalytic domain of membrane proteins, and (2) peptide-based ligands or antibodies which are designed to bind both intracellular and extracellular domains.

In Vivo SELEX

As mentioned previously, cell-SELEX can be performed to identify aptamers against specific cell surface biomarkers of a disease without any prior knowledge of the markers. However, the question remains whether cells in culture are relevant models for a disease. With the purpose of adapting the SELEX procedure to a complete physiological environment, SELEX has recently been performed in vivo to select aptamers recognizing intrahepatic colorectal cancer metastasis [99] . A 2'F-Py RNA library was injected intravenously in a mouse bearing a previously implanted hepatic tumour. Liver tumours were collected and RNA was subsequently extracted and amplified. The new population of 2'F-Py RNA was re-injected in a new mouse and the process repeated several times. Interestingly, evolved RNA pools demonstrated higher affinity for a tumour protein extract than a normal colon cell protein extract and the population of round 14 was cloned and sequenced. Interestingly, the target of one aptamer was determined to be p68, an intracellular RNA helicase known to be upregulated in colorectal cancer.

SELEX Directed against Purified Cell Surface Biomarkers

Two years later a SELEX was performed against the full length extracellular domain of CD4 produced from a mammalian expression system and immobilized on beads [12] . To remove sequences with ability to bind to other sites than CD4 in the selection matrix, the library was in each round of the selection pre-exposed to beads lacking CD4, and furthermore, two types of beads were used during selection. The first six rounds were performed against biotinylated CD4 captured on beads coated with streptavidin. Subsequently, another six rounds were performed against CD4 captured onto beads coated with a monoclonal anti-CD4 antibody. Interestingly, the selected aptamers were not only able to bind the recombinant ectodomain of CD4 but also the entire protein expressed on the surface of a mouse T cell line after transfection. In contrast, no measurable binding was observed for the non-transfected cell line lacking the expression of CD4. Hence, aptamers selected against a purified ectodomain of a membrane protein may also be able to recognize the protein in its native environment. Similar SELEX experiments have been performed successfully against other types of membrane proteins including G Protein Coupled-Receptors (GPCRs), Receptor Tyrosine Kinases (RTKs), and Tumour Necrosis Factor (TNF) receptors (see Table 1 ). In addition, similar strategies have also been applied to select aptamers against proteins from the surface of bacteria and parasites as well as from the envelope or capsid of viruses (see Table 1 ).

SELEX Directed against Whole Living Cells, Bacteria, Viruses and Parasites

Although proteins in plasma membrane extracts are in a more physiological environment compared with purified ectodomains, it is well known that the plasma membrane is a dynamic system composed of membrane constituents that are in close contact with each other as well as with the intra-and/or extra-cellular matrix. Therefore, several groups have gone one step further and applied SELEX directly on a whole living and functional biological system (see Table 3 ). This kind of SELEX was performed for the first time against Trypanosoma brucei parasites and Bacillus anthracis spores in 1999 [55, 56] . In both cases, the partitioning of bound from unbound sequences was done by centrifugation of parasites or bacteria spores. Interestingly, the SELEX against Trypanosoma brucei resulted in only a few types of aptamers in spite of the numerous potential targets present on the surface of the parasite suggesting the existence of dominant epitopes. Aptamers were predominantly selected against a target specifically localized in the flagellar pocket of the trypanosome while no aptamer was selected against the VSG protein, which is the most abundant polypeptide on the trypanosome surface. In 2000, SELEX was performed against the human cytomegalovirus using filtration [57] . In that case, aptamers were predominantly selected against the glycoprotein B and H, which represent the most abundant and exposed envelope proteins of the virus.
In 2001, SELEX was performed for the first time on a whole living mammalian cell line [58] . During the SELEX, the library was counter-selected in each round against N9 microglial cells before selecting against rat YPEN-1 endothelial cells. As the two cell types were non-adhering cells, unbound sequences were removed by centrifugation. The counter-selection was performed to favour aptamers against targets whose expression is linked to the endothelial phenotype. The pool was fluorescently labeled during the SELEX, which allowed the monitoring of enrichment of cell-binding aptamers using flow cytometry. After sequencing, most of the individual sequences tested showed affinity for the YPEN-1 EC cell line by flow cytometry, and some of them could bind microvessels in cryostat tissue sections of rat brain glioblastomas. Since then, several aptamer selections have been conducted with living cells and the strategy has been named cell-SELEX. While centrifugation is the partitioning method of choice for non-adherent cells, in the case of adherent cells, gentle plate washing is used. However, recently another method was described by Raddatz et al. who used fluorescence-activated cell sorting (FACS) to perform the partitioning of bound from unbound sequences [59] . The study demonstrated that FACS was a powerful method for selecting aptamers against living Burkitt lymphoma B cells particularly because it allows the elimination of dead cells, which usually display high non-specific binding for nucleic acids. 2'NH2-Py RNA T. brucei No ND 13 [94] As mentioned previously for SELEX against membrane compartments, SELEX against a living biological system often also results in the identification of aptamers for an unknown target. It can be difficult to identify the unknown target of an aptamer, but it opens a new avenue for the application of SELEX within biomarker identification and cell phenotyping. For example, the subtractive cell-based method described for the SELEX against the YPEN-1 endothelial cell line allowed the identification of aptamers against targets that are differentially expressed between different cell types. Furthermore, aptamers have also been selected with ability to distinguish cells on the basis of differentiation state [60] , distinguish cancer cells or virus infected cells from normal cells of the same origin [61] [62] [63] and discriminate for instance different lineages of hematopoietic cancers [64, 65] (see Table 3 ). For such aptamers, target identification can lead to the identification or validation of cell specific biomarkers. The strategy has been named AptaBiD for Aptamer-facilated Biomarker Discovery [66] . Several protocols have been used to isolate and identify the target protein of aptamers selected using cell-SELEX [58, [67] [68] [69] [70] . Basically, affinity chromatography is performed using biotinylated aptamer pre-incubated with a membrane protein extract, a total protein extract, or alternatively with living cells followed by cell lysis. Bound protein is subsequently recovered and separated or not by SDS-PAGE electrophoresis before being identified by mass spectrometry.

Introduction

Nucleic acid-based ligands, named aptamers, appear as appealing new ligands in this field and more than one hundred aptamers have been selected against cell surface targets during the past 10-15 years. Aptamers are generated by a molecular directed evolution approach from a library of 10 14 -10 15 oligonucleotides containing a region of random base composition [2, 3] . This technique is usually named SELEX (Systematic Evolution of Ligands by EXponential enrichment) and consists of repetitive cycles of selection and amplification ( Figure 1 ). During each cycle, oligonucleotides with affinity for a desired target are retained and amplified, leading to their enrichment in the pool which is finally sequenced to identify the aptamers. Since 1990, this strategy has been used to identify aptamers against a wide variety of targets from small molecules to peptides, proteins, nucleic acid-based structures (for reviews, see [4, 5] ). In many cases, aptamers have been shown to present high specificity and affinity as well as inhibitory or modulatory activity towards their targets [6] . Moreover, they seem to lack immunogenicity and can be chemically modified in order to improve their stability against nucleases, modify their pharmacokinetics or allow labelling. Due to their unique advantages, aptamers have been used in several applications from basic to applied research. For instance, aptamers have been used to study natural interactions between RNA and proteins, to regulate gene expression, to develop biosensors, to purify specific molecules, to inhibit the function of a protein and to develop drugs (for reviews, see [7] [8] [9] [10] ). A random pool of oligonucleotide candidates is incubated with a target (purified cell surface biomarker, membrane extract or whole living cell or organism) (1). Sequences which do not bind the target are removed by different partitioning methods (ex: affinity chromatography, filtration, centrifugation) (2) . Bound sequences are eluted (ex: urea, EDTA, competition with a ligand) (3) and amplified by PCR (or RT-PCR and in vitro transcription in the case of RNA libraries) (4). The selected pool can then enter a new round of selection. During these repetitive rounds of selection, the population evolves towards the sequences with the best affinity for the target. At the end of the process, sequences are cloned and sequenced to identify the aptamers (5).

Conclusions and Perspectives

Cell surface targeting aptamers can potentially be used for several applications. For instance, aptamers that bind to membrane proteins involved in disease can often inhibit or activate their targets leading to the development of new drugs. For example, a neutralizing anti-nucleolin aptamer is currently in phase IIb clinical trials for the treatment of Acute Myeloid Leukemia (AML) in combination with cytarabine [101] . Additionally, aptamers targeting cell surface proteins have been successfully used as targeting moieties to deliver contrast agents, nanoparticles encapsulating drugs, or siRNA to specific tissues in vivo [102, 103] . Furthermore, they are being used within the development of diagnostic assays in fields such as cancer, infectious disease, food safety and bioterrorism. Finally, a more recent area of interest for aptamer selections against membrane proteins is the use of methods such as cell-SELEX within cell surface biomarker discovery and cell phenotyping. As a consequence, selections of aptamers against cell surface biomarkers have a high potential use not only for fundamental research, but also for diagnosis, monitoring and treatment of diseases.
6 section matches

Introduction

Pathogenic strain identification using LC-MS/MS-based proteomics presents a crucial, yet highly challenging task. Many pathogenic strains feature significant phenotypic differences within a species with respect to pathogenicity, zoonotic potential, cell attachment and entry, host-virus interaction and clinical symptoms [1] [2] [3] . In a diagnostic context, strain-level knowledge is important to infer virulence 4, 5 and drug resistance 6 for appropriate therapy. However, inferring exact strain information from proteomic samples remains a difficult task, in particular when the taxonomic origin of a sample is unknown and when related strains feature high sequence similarity. In recent years, MALDI-TOF mass spectrometry has gained popularity as fast, sensitive and economical method for microbial biotyping. However, identifying strains using MALDI-TOF workflows is still very challenging and requires curated, often proprietary spectral databases 7 . Several commercial platforms for microbial biotyping down to the species or strain level are available based on MALDI and other technologies such as the Bruker MALDI Biotyper Systems 8 , the Bruker Strain typing with IR Biotyper 9 and the Ibis T5000 Universal Biosensor 10 . Several studies report on limitations of MALDI-TOF biotyping for strain-level identifications and advocate advancements towards MS/MS marker peptide detection and, consequently, the analysis was shifted to the MS/MS level 11, 12 . In these studies, however, the MS/MS-based protein identification was established using sequence databases that were already targeted or restricted to particular species or limited sets. In contrast, untargeted MS/MS typing approaches are limited to species level identification 6, 13 . However, in general MS/MS is preferred for the analysis of complex unpurified peptide mixtures as it is considered to provide more distinct and unambiguous peptide and protein identifications 14 and thus increased proteome resolution 15 as well as higher statistical confidence 16 . In particular, organisms with unknown taxonomic origin benefit from peptide sequence-based analysis, as MALDI-based biotyping is in comparison too prone to ambiguous identifications 17 . Furthermore, advances in instrumentation including higher resolution, mass accuracy and dynamic range increasingly allow for identification of the majority of all fragmented peptides 18 resulting in higher sensitivity, higher coverage of target proteomes and thus higher availability of distinctive features. Taking advantage of the vast amount of available protein sequences for MS/MS strain-level identification is challenging. On the one hand, constraining the search space may result in unidentified strains or incorrectly assigned taxa, in particular for non-model organisms 19 . On the other hand, applying large databases is not recommended either since it decreases peptide identification rates 20 and thus eventually impedes taxonomic inference 21 . Furthermore, with increasing database size sequence quality often decreases (e.g. when using the complete NCBI Protein in comparison to the NCBI RefSeq database) and contaminant sequences may occur more often 22 . Therefore, extended databases should only be used when necessary. However, strain-level identification of MS/MS spectra from samples with unclear taxonomic status requires an untargeted search against comprehensive databases holding as many strains as possible. A common and popular concept to handle increased search spaces is the application of multiple identification steps in general, independently of target application such as strain-level identification. These multi-stage identification search strategies are described by several different terms such as serial search 23 , multi-step 24 , iterative 25, 26 , multi-stage 27 , two-step [28] [29] [30] as well as cascade search 31 and they find application in proteomics, metaproteomics 26, 28 and proteogenomics 30, 32 . Most of these strategies do not only overlap in their objective of increasing the identification rate or identification confidence, but share methodological principles as well. This includes the concept of identifying primarily unassigned spectra using databases of increasing complexity (for instance, by employing altered digestion parameters, additional post-translational modifications or additional spectral and genomic databases) [24] [25] [26] [27] 31 as well as the recurring theme of database size reduction 24, 26, [28] [29] [30] 32 . In addition, some methods rely on spectral quality assessment to enhance subsequent identification steps 25, 32 or exhibit a focus on algorithmic runtime reduction 24 . Apart from database size, multi-proteome databases present an additional challenge for taxonomic assignment in the form of high sequence similarity between proteomes, in particular between related species and strains. These similarities give rise to taxonomically ambiguous, widespread and false assignments and thus need to be accounted for. Several methods exist that provide various strategies to account for ambiguous peptide spectrum matches due to sequence similarity. Dworzanski et al. apply a proteogenomic mapping approach in combination with discriminant analysis to infer most likely bacterial assignments 33 . The proteogenomic approach was further developed in BACid that applies several statistical measures to account for similarity, including the comparison of ratios of taxonomic differences and of unique peptides to known error rates, i.e. the noise levels 34, 35 . BACid has since been applied to and extended for samples of unknown origin 36 as well as mixtures of bacteria 36, 37 . With the focus on metaproteomic analysis, MEGAN 38 , UniPept 39 , MetaProteomeAnalyzer 40,41 and TCUP 6 rely on lowest common ancestor or most specific taxonomy approaches to assign peptides spectrum matches to taxonomic levels. MiCId uses a clustering approach based on peptidome similarity of taxa on different levels in combination with unified E-values to infer statistically significant representatives of clusters as identified or classified microorganisms 13, 42 . Tracz et al. use a direct assignment strategy by concatenating all proteins of a proteome into one pseudo-polyprotein and considering only the top-scored spectrum matches to a peptide as counts for bacterial candidates 43 . In contrast, Pipasic explicitly makes use of ambiguous peptide spectrum matches (PSMs) by applying abundance correction based on intensityweighted proteome similarities of organisms in metaproteomic samples 44 . While these methods share the objective of reducing uncertainty in taxonomic identification due to ambiguous assignments in the context of the protein inference issue 45 , their application for untargeted strain-level identification is not straightforward for several reasons. For example, unique peptides are not necessarily available for all candidate strains in the sequence databases for which the information given is often limited to species or higher taxonomic levels. In addition, the approaches rely on fully sequenced genomes (and thus uniform proteome coverage) or other forms of targeted, curated, inhouse or validated databases and are thus restricted to a predefined limited set of organisms, often with definite focus on bacteria. Furthermore, some methods do not scale well with immoderate large databases (i.e. high number of proteomes) with respect to computational or analytical performance. Previous multi-step procedures illustrate the effect of database size on identification confidence and the advantage of applying concise, prefiltered or specialized databases. Thus, we transfer the general concept of multi-step procedures to approach the increased search space necessary for untargeted and detailed taxonomic classification. We present TaxIt, an iterative workflow for untargeted strain-level identification of protein samples. By applying two separate identification steps for species-and strain-level classification, we circumvent the immediate need for a comprehensive database containing strain All rights reserved. No reuse allowed without permission.

Benchmarking Experiments

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/812313 doi: bioRxiv preprint Since Pipasic is sensitive to a high number of taxa, we limit the input to taxa with a minimum of two hits as well as to the 100 most abundant taxa. Expressed proteins per taxa are extracted according to the taxonomic classification and digested peptides with a minimum length of six amino acids are prepared using trypsin digestion 58 . PSMs and tryptic peptides are then passed to Pipasic to obtain corrected relative counts. We perform all three strategies on several viral and bacterial MS/MS spectra samples with available strainlevel knowledge. This includes a Cowpox virus (Brighton Red) strain (acquired in-house), an Avian infectious bronchitis virus (strain Beaudette CK) 59 and a Bacillus subtilis BSN238 60 that we will refer to as cowpox, bronchitis and bacillus sample, respectively. B. subtilis BSN238 is a transgenic organism resulting from horizontal gene transfer (HGT) of the DivIVA protein from Listeria monocytogenes strain EGD-e to Bacillus subtilis subsp. subtilis str. 168. Since B. subtilis BSN238 is neither yet present in the NCBI Taxonomy nor in the NCBI Protein database and only one protein is modified, we expect B. subtilis subsp. subtilis str. 168 to be selected as final strain candidate. Bacillus samples are examined twice, once completely with 28902 spectra (bacillus all) and once randomly reduced to 1000 spectra (bacillus 1k) to improve performance with respect to the vast bacterial search space. A detailed description of the cowpox sample acquisition and the search parameters for all samples is provided in Supplementary item 1.

Results

To demonstrate the potential of iterative strain-level identification, we compared TaxIt against classic comprehensive search strategies based on non-iterative taxonomic identification supported by either unique PSMs or the abundance similarity correction of Pipasic 44 . The final selections of the top taxa candidates for all samples and all three compared identification strategies are summarized in Table 1 . For the cowpox sample, identification results agree the most. TaxIt (Figure 2) and Pipasic (Supplementary item 1 - Figure S1 ) are both able to identify the expected Cowpox virus (Brighton Red) strain. However, unique PSMs are limited to the parent Cowpox virus species and not available at the strain level. For this reason, an incorrect identification of Bat astrovirus Hil GX bszt12 could occur based on a single unique PSM (Supplementary item 1 -Figure S1 ). The results also show that the original TaxIt counts are not an All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/812313 doi: bioRxiv preprint appropriate measure to distinguish the expected strain from competing candidates (Figure 2) . However, the weight-based correction method implemented in TaxIt resolves the present tie and emphasizes the correct Cowpox virus strain (Brighton Red).
In comparison, bronchitis and bacillus samples feature notable variability in proposed taxa candidates. For bronchitis, TaxIt could identify the expected Avian infectious bronchitis virus (strain Beaudette CK) strain. Despite applying a small correction based on weighting, however, final candidate selection is not limited to a single strain but additionally includes a closely related strain, namely Avian infectious bronchitis virus (strain Beaudette US) ( Figure 3A) . Nevertheless, TaxIt provides a highly constrained selection of candidates in the first place. In contrast, the Pipasic-based strategy results in considerably more initial candidates and eventually promotes an incorrect strain, namely Avian infectious bronchitis virus (strain 6/82) (Figure 3B ). Since no unique PSMs are available for the bronchitis sample, strain-level identification was not possible with this strategy.

Discussion

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/812313 doi: bioRxiv preprint incorrect species. For some samples, correct strains are observed as a top candidate even in the original counts, independently of database choice and prior to count adjustments. Nevertheless, all samples benefit either from the iterative and focused database usage, the count adjustment procedure or the reduced resource consumption while the outcome remains legitimate. It was shown that TaxIt improves taxonomic assignment already in the count-based ranking, independently of count adjustments, by limiting candidates to strains of a single species early on ( Figure 3) . For instance, the search of bronchitis samples against the NCBI Blast NR bacteria subspace results in a rather uniform distribution of original counts for Infectious bronchitis virus strains: the strains Beaudette, Beaudette CK and Beaudette UK are only slightly increased in comparison to other strains (Figure 3B, Original) . In contrast, the iterative approach results in less strain candidates of the same species focusing solely on Beaudette strains when selectively searching against Avian coronavirus strains in the secondary iteration (Figure 3A, Original) . This is a consequence of how and whether parental or multispecies proteins are associated with strains or taxa in general within the NCBI taxonomy. In case of the iterative approach, fewer mutual species or genus proteins are consulted in the strain identification iteration. However, the extent of this effect varies between samples, candidate strains or target databases and taxonomies, respectively. For instance, bacterial strain proteomes such as the Bacillus subtilis strains feature numerous directly assigned mutual protein sequences and thus result in an extended range of candidate strains of the same species (Supplementary item 1 -Figure S3 and S4) . Nevertheless, the restriction to a specific set of strain proteomes prevents manifold primary misassignments to distant species, genera or even phyla as can be observed for Pipasic-and unique-PSMsbased original counts, respectively, that cannot be sufficiently resolved after correction (Supplementary item 1 - Figure S3 and S4) . In general, the iterative and selective database usage ensures that final strain selection is limited to strain candidates of an appropriate species. Thus, it prevents false positive hits on distinct strains of other taxa including species, genera and phyla and allows for a more confident final strain candidate selection. Furthermore, uniform distributions and even consensus in original counts of strain candidates demonstrate the need and benefit of count adjustment methods. The implemented weighting procedure can resolve ties between strains such as in the TaxIt cowpox sample analysis (Figure 2 ) or at least amplifies the correct strain and increases the distance to competing candidates. TaxIt infers exactly one strain for the presented samples each expect for the bronchitis sample where the strains Beaudette CK and Beaudette UK cannot be differentiated. We observed that the corresponding PSMs are fully shared between the two proteomes. Although different proteins are available for each strain in general, peptide hits are either assigned to shared proteins of the parent strain Infectious bronchitis virus or to homologous proteins that differ only in identifier but not in sequence. Though a more granular taxonomic relation cannot be ascertained from the NCBI taxonomy, we expect the Beaudette strains to feature a considerably closer relationship as compared to other Infectious bronchitis virus strains. Therefore, we consider the draw between Beaudette strains as sufficiently appropriate strain identification. For the unique-PSMs-based strategy, we observed a poor availability of unique PSMs at the strain level. While the exploitation of purely unique features is a common theme for species-level identification, the low amount of unique PSMs in strains is insufficient for strain-level inference. However, the frequency of All rights reserved. No reuse allowed without permission.