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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.

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.

Aptamer to HIV Proteins

Aptamers to Protease (PR) During its life cycle, HIV must successfully complete several key steps in order to replicate in the host environment. One such critical step involves the processing of its Gag and Gag-Pol polyprotein precursors into their mature functional components during viral maturation. This function is provided by the virally encoded aspartyl protease (PR) and is thought to occur either during or immediately after budding [30, 31] . In its active form, this protein is a 22 kDa homodimer consisting of two 99-amino acid long subunits each of which contributes a catalytic aspartate residue to the active site. Inhibition the proteolytic activity of PR leads to the production of immature and non-infectious particles. Duclair et al. have developed RNA aptamers against viral protease that inhibited HIV replication in vitro [32] .

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 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] .

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.

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] .

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.
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Abstract

Chloroquine is a 4-aminoquinoline previously used in malaria therapy and now becoming an emerging investigational antiviral drug due to its broad spectrum of antiviral activities. To explore whether the low pH-dependency of influenza A viruses might affect the antiviral effects of chloroquine at clinically achievable concentrations, we tested the antiviral effects of this drug on selected human and avian viruses belonging to different subtypes and displaying different pH requirements. Results showed a correlation between the responses to chloroquine and NH 4 Cl, a lysosomotropic agent known to increase the pH of intracellular vesicles. Time-of-addition experiments showed that the inhibitory effect of chloroquine was maximal when the drug had been added at the time of infection and was lost after 2 h post-infection. This timing approximately corresponds to that of virus/cell fusion. Moreover, there was a clear correlation between the EC 50 of chloroquine in vitro and the electrostatic potential of the HA subunit (HA2) mediating the virus/ cell fusion process. Overall, the present study highlights the critical importance of a host cell factor such as intravesicular pH in determining the anti-influenza activity of chloroquine and other lysosomotropic agents.

Background

Some of us previously analysed the reported effects of chloroquine on replication of several viruses and concluded that the drug should be studied as a broad spectrum antiviral agent against emerging viral infections, being relatively well tolerated, cheap, and immediately available worldwide [9] . As a weak base capable of accumulating within cellular organelles, chloroquine appears to be capable of interfering with pH-dependent steps in the replication of several viruses. Other mechanisms of viral inhibition by chloroquine, such as inhibition of polynucleotidyl transferases have, however, been considered [7] . In 2003-2005, chloroquine was studied as a promising in vitro anti-SARS agent [9] [10] [11] and recently entered clinical trials against chikungunya fever [12] .
The broad-spectrum antiviral effects of chloroquine deserve particular attention in a time in which there are several cases of avian influenza A virus transmission to humans from poultry, and the availability of antiviral drugs is fundamental during preparation and evaluation of effective vaccines. Chloroquine inhibition of both type A and B influenza viruses was first described in the 1980s [13, 14] . The concentrations employed in these studies were however too high to allow a theoretical transposition to in-vivo settings. Anecdotal reports of clinical benefits derived from a related compound, i.e. quinine, date back to the Spanish influenza pandemic of 1918/19. However, it was not until last year that the anti-influenza virus effects of chloroquine at clinically achievable concentrations were studied, in view of a possible application of this drug in the clinical management of influenza [4, 15] . Investigations still have to be done on this topic. For example, the mechanisms of orthomyxovirus inhibition by chloroquine have been uncertain at the clinically achievable concentrations adopted in the most recent studies [4, 15] , as well as the effects of chloroquine on field isolates, including avian strains potentially transmittable to humans.
We here report the results of an initial evaluation of susceptibility to chloroquine of human and avian influenza A viruses. Susceptibility to chloroquine appears to be dependent on the pH requirements of the viruses and the electrostatic potential of haemagglutinin subunit 2 (HA2), which is involved in virus/cell fusion. Accordingly, the antiviral effects are exerted at an early step of virus replication.

Results

We first tested the effects of chloroquine on low-pathogenic (LP) A/Ck/It/9097/97 (H5N9) virus, isolated from poultry in Italy. We found that chloroquine dose-dependently inhibited the viral cytopathic effect with a 50% effective concentration (EC 50 ) of 14.38 μM, in cells infected with the H5N9 virus at approx. 10 4 50% tissue culture infecting doses (TCID 50 )/ml (Fig. 1a) . Although this value was rather high, some of the inhibitory concentrations matched the blood concentrations reported in individuals under acute antimalarial treatment (1-15 μM) . The inhib-itory effects were confirmed using quantitative reverse transcritptase real-time PCR (qRRT-PCR) (Fig. 1b) . Ooi et al. (2006) [15] recently reported that chloroquine inhibited human H3N2 and H1N1 viruses with EC 50 values in the range of 0.84 -3.60 μM. To investigate whether the discrepancies with the inhibitory values reported above were due to the type of virus or to the different conditions and methods adopted, we tested the effects of chloroquine on replication of recent human H3N2 and H1N1 viral isolates under conditions similar to those adopted for the H5N9 avian influenza virus. Using the test of inhibition of viral cytopathogenicity, we found that chloroquine inhibited the H3N2 virus (10 4 TCID 50 /ml) with an EC 50 of 1.53 μM (Fig.1c) . Inhibition was confirmed using qRRT-PCR, both under similar conditions and at lower MOIs (Fig.1d) , thus confirming the results of Ooi et al. (the assay adopted by these authors employs a lower MOI than routinely used by our group). Results obtained with H1N1 viruses (10 4 TCID 50 /ml) showed a similar drug susceptibility for the human strain (EC 50 = 1.26 μM), in full agreement with Ooi et al. [15] , but no response to clinically achievable drug concentrations in an avian strain (IC 50 > 20 μM; data not shown). These data suggest a more pronounced inhibitory effect of chloroquine on human H3N2 and H1N1 viruses than on avian H5N9 virus replication.
Since 1) chloroquine is thought to interfere with pHdependent steps of the life cycles of several viruses [9] , and 2) some of us reported different pH requirements in influenza A viruses infecting different avian species [15] , we investigated whether response of influenza viruses to chloroquine might depend on the different pH requirements of the human and avian viruses.
Thus, we analysed the response of human H3N2 virus (good chloroquine responder) and avian H5N9 virus (poor chloroquine responder) to ammonium chloride (NH 4 Cl; 40 mM), a lysosomotropic agent known to increase the pH of intracellular vesicles. Results showed a good response of the chloroquine-sensitive H3N2 virus to NH 4 Cl inhibition of viral cytopathogenicity (100% inhibition under conditions described above). Conversely, the lower chloroquine-sensitivity of H5N9 virus was associated with lack of response to NH 4 Cl (data not shown). This observation raised the hypothesis that cellular pH might be a critical factor for chloroquine inhibition of influenza virus.
To explore this possibility, the action of chloroquine was tested on two avian H7N3 viruses whose haemagglutinins (HAs) differ in two amino acid positions (i.e. residue 261 in the HA1 subunit and residue 161 in HA2, the latter being the HA subunit mediating the fusion process), and which display different pH requirements [16] . The two viral strains showed a marked discrepancy in the response to chloroquine. In particular, A/Mallard/It/43/01 (H7N3) virus, which had been shown to be relatively more independent from pH increase than A/Ty/It/220158/02 virus [16] , was insensitive to clinically achievable chloroquine concentrations (EC 50 > 20 μM). In contrast, chloroquine exerted some inhibitory effect on A/Ty/It/220158/02 replication (EC 50 = 14.39 μM), although the response of this virus to chloroquine was lower than that of H3N2 virus.
The different pH requirements of the two viruses were confirmed by the different responses to NH 4 Cl (0% inhibition for A/Mallard/It/43/01; 30% inhibition for A/Ty/It/ 220158/02). This result raises the hypothesis that chloroquine inhibits pH-dependent events involving HA.
To further explore this possibility, the isoelectric point was calculated for HAs of all viruses used in the present study, and the electrostatic potential was mapped on the protein surfaces of 3D models obtained by homology Figure 1 Inhibition of H5 and H3 influenza A virus replication by CQ in MDCK cells. Cells were incubated with chloroquine (CQ) after virus inoculation or mock-infection and tested for cell viability and viral RNA copies at 24 h post-infection. A) Viability of cells infected with A/Chicken/Italy/9097/97 (H5N9) and treated with increasing concentrations of CQ as detected by colorimetric test. Assays were performed as described in the text. The dotted line indicates inhibition of uninfected cell viability, the solid line indicates inhibition of infected cell viability. Results are presented as the curves that best fit the data points. B) Results of one representative experiment showing inhibition by CQ of A/Chicken/Italy/9097/97 viral RNA production. Virus infected MDCK cells were incubated for one day in the presence of 0, 5, 10, 20 or 25 μM chloroquine. Cell supernatants were used for viral RNA extraction and subjected to a quantitative real-time RT-PCR (qRRT-PCR) assay. Oseltamivir (OS; 20 nM) was used as a positive control. C and D) as in A and B), respectively, using A/Panama/2007/99-like (H3N2) virus. In D) both results obtained with inocula containing 10 4 and 10 3 TCID 50 /ml are reported. Results in B) and D) are displayed for purely representative reasons to show that there is inhibition of virus production, and cannot be compared with each other or with those in A) and C), due to the high intra-and inter-assay variability of the qRRT PCR assay (see Ref. [29] ). Fig. 2 ). Instead, no correlation was found with the isoelectric point of HA1 (P > 0.05; data not shown). Although the viruses studied here belonged to different subtypes, chloroquine-resistant viruses, independently of the subtype, showed a more marked negative surface potential of HA2 than chloroquine-sensitive viruses (Fig. 2) . Viruses with intermediate drug sensitivity showed intermediate characteristics (Fig. 2 ). We conclude that structural determinants in HA2 are associated with response of influenza A viruses to chloroquine.

Inhibition of H5 and H3 influenza A virus replication by CQ in MDCK cells

If the hypothesis of a pH dependent inhibitory action of chloroquine was correct, the timing of drug inhibition should match that of virus/cell fusion, an early step of virus replication occurring in endosomes and requiring a low pH (approx. pH 5-5.5) in several, but not all, influenza A viruses, as shown by previous studies [17] . As the assays for detection of antiviral effects adopted in the first Correlation between electric characteristics of haemagglutinin subunit 2 (HA2) and response to chloroquine of influenza A viruses Figure 2 Correlation between electric characteristics of haemagglutinin subunit 2 (HA2) and response to chloroquine of influenza A viruses. A) Correlation between EC 50 of chloroquine (CQ) on viral cytopathogenicity (presented as Log values, x axis) and isoelectric point of HA2 (pH value at which the protein is neutral; y axis). The line best fitting the data points is shown. Isoelectric points were calculated based on the protein sequence using the web interface in Ref. [35] . B-G) Theoretical three-dimensional models for HA2 subunits of the viruses adopted in the present study, shown in ranked order of sensitivity to chloroquine (from resistant to clinically achievable concentrations to fully sensitive). part of this study were designed to allow multiple cycles of viral replication, we designed time-of-addition experiments using the chloroquine-sensitive human H3N2 virus. Chloroquine was added during virus adsorption onto cells (i.e. time 0; T 0 ) and/or at 1, 2, 3 and 4 h postinfection (T 1-4 ) Using qRRT-PCR, we found that the inhibitory effect of chloroquine was highest when the drug was added at T 0 (inhibition of viral replication corresponding to 89,36%) and at T 1 (inhibition of replication corresponding to 15,53%), whereas the inhibitory activity was completely lost at T 2 .

Discussion

The results so far obtained suggest that chloroquine inhibits the replication of those influenza A viruses requiring low pH for proper fusion activation and that the antiviral effects occur at an early stage of viral replication. Supporting evidence comes from: 1) the more sustained inhibitory effect of chloroquine on those viruses whose haemagglutinins (HAs) were found to require low pH for their fusion activity, 2) common HA2 characteristics such as surface potential and isoelectric point in chloroquinesensitive viruses, and 3) time-of-addition experiments with H3N2 virus.
Chloroquine is a weak base that is known to affect acid vesicles leading to dysfunction of several proteins [9] , and has been shown to inhibit different viruses requiring a pH-dependent step for entry [19] [20] [21] [22] . The results of the time-of-addition experiments, performed in the present study using a recent epidemic isolate of human H3N2 virus, are consistent with chloroquine inhibition of pHdependent steps occurring at an early phase of influenza A virus replication.
Most influenza viruses enter target cells by fusion of the viral and cell membranes at the endosomal pH (approx. pH 5-5.5), although some virus variants can replicate well also at higher pH values [16, 17, 23] . In a previous paper, the differential growth sensitivity of two naturally occurring variants of an H7N3 virus, isolated from mallards and turkeys, to increased pH values was shown to correlate with different fusion properties [16] . Since chloroquine appears to mimic the effects observed on these two viruses when pH is increased, our data support the hypothesis that the step of virus replication inhibited by clinically relevant chloroquine concentrations is the low-pH dependent haemagglutinin-mediated virus/cell fusion, in agreement with the evidence obtained by other authors at much higher concentrations than those used in this study [13, 14] . The correspondence between antiviral effects and isoelectric point of HA2 (i.e. the HA subunit mediating the fusion process) is also consistent with this mechanism. Acidic pH in the endosomal compartment also activates the influenza virus ion channel, M2, that promotes the uncoating of influenza virus in endosomes. However, M2 involvement as a possible target of the antiviral effect of chloroquine is unlikely, since no aminoacid differences were observed in M2 trans-membrane region (the ion-channel domain) between the two avian H7N3 viruses with different chloroquine sensitivities, as already reported [16] .
Although a comprehensive study on the variation of fusion pH requirements of influenza A viruses of all HA subtypes and isolates from different hosts is not available, several authors have documented that the threshold pH, at which the HA conformational change and virus-cell fusion occur, is strain-specific [24] [25] [26] . Interestingly, the viruses showing highest chloroquine sensitivity also displayed the highest HA2 isoelectric points. Thus, a relation-ship between isoelectric point and response to pH is apparent. However, a broad study relating the surface electrostatic potential with inactivation by pH would be required to analyse the molecular details of the HA/pH interplay. Analyses of virus production before and after exposure to chloroquine and of the possible changes in HA2 surface potential in viruses rendered resistant to chloroquine after long exposure to the drug will also be necessary.

Virus yield assay

After 24 hours of incubation of the virus-infected cells with different concentrations of the test compounds, under the appropriate conditions, pooled aliquots of the supernatants containing free viruses were subjected to RNA extraction and qRRT-PCR.

Viral RNA sequencing

Hemagglutinin genes of H3N2 and H1N1 viruses were sequenced using gene-specific primers, as previously described [30] . Sequence data so far unpublished will be deposited in GenBank by the time of publication of the present article.

Molecular modelling

Three-dimensional models for the HAs of the viruses used in the present study were obtained by homology modelling, using the SWISS model web server [31, 32] , using, as templates, structures of matched subtype representatives deposited in the protein data bank (PDB) [33] . Hydrogens were added using VEGA-ZZ (University of Milan, Italy) [34] , and the structures were then visualised using the Swiss PDB Viewer (SPDBV) program (Swiss Institute of Bioinformatics) [31] .

Inhibition of H5 and H3 influenza A virus replication by CQ in MDCK cells

If this timing was correct, chloroquine should inhibit influenza A replication by a novel mechanism, and therefore exert additive effects in combination with oseltamivir, inhibiting neuraminidase activity at the late stages of viral replication cycle. To test this hypothesis, human H3N2 virus-infected cells were treated with different chloroquine concentrations in the presence or absence of oseltamivir (10 nM). The virus-infected cells were also incubated with oseltamivir alone, EC 50 = 20 nM. Isobologram analysis showed that the two drugs exerted an additive effect (sum of FICs = 1) (data not shown). This result provides further evidence that chloroquine inhibits viral replication by a mechanism different from that of one major anti-influenza drug.

Conclusion

Although association between variables cannot be considered to be equivalent to causation, the results of the present study strongly suggest that pH critically determines the antiviral activity of chloroquine by regulating virus/host cell interactions. The potential use of this compound as an antiinfluenza drug should take into consideration the possibility that even within the same subtype, different strains may present significantly divergent sensitivities to chloroquine as a consequence of their different pH requirements. Moreover, sensitivity to chloroquine may vary in different cell populations susceptible to influenza A virus infection, depending on different capabilities of endosome acidification. Mutations affecting the electrostatic potential of the the HA2 protein subunit of various isolates of the same virus could also be relevant. All these factors should be carefully evaluated when hypothesising a potential clinical utilisation of chloroquine against influenza A viruses.

Quantitative Real Time RT-PCR (qRRT-PCR)

A one-step qRRT-PCR assay was employed, which makes use of minor groove binder (MGB) probe technology, as previously described [29] . Briefly, viral RNA was extracted from infected cell supernatants (QIAmp Viral RNA Mini kit -Qiagen, GmbH, Hilden, Germany) and amplified by RRT-PCR using primers and probe targeting a highly conserved region of the matrix gene of influenza type A viruses. The influenza matrix RNA was also in vitro transcribed from the corresponding DNA template, cloned into a plasmid vector as previously described [29] and used as standard RNA to generate standard curves for quantification of the vRNA in cell supernatants.

Assay for measurement of antiviral activity

100 μl of 1.5 × 10 5 cells/ml MDCK cells in growth medium was seeded into each well in the 96-well microtiter plate. When the cell monolayer was confluent, the culture medium was removed and cells were washed twice with serum-free MEM. Then, 100 μl of the Type A influenza viruses under study containing 10 3 -10 4 TCID 50 were inoculated in wells and the plates incubated for 1 hour at 37°C in humidified air of 5% CO 2 . The viral suspension was removed and the cells were washed two times; fresh medium containing TPCK-trypsin and chloroquine at different concentrations or NH 4 Cl (40 mM) was then added to culture wells in triplicate. Antiviral activity and cytotoxicity measurements were based on the viability of cells that had been infected or mock infected with influenza viruses in the presence of various concentrations of the test compounds. One to three days, depending on the kinetics of cytopathogenicity, after infection the number of viable cells was quantified by a tetrazolium-salt-basedcolorimetric method (CellTiter 96 AQueous One Solution kit, Promega, The Netherlands).

Assessment of the effects of two drugs in combination

To measure the anti-influenza effects of chloroquine/oseltamivir drug combinations, cell pellets were resuspended in media containing increasing concentrations of the antimalarial in the presence or absence of oseltamivir. A fractional inhibitory concentration (FIC) was then calculated as the ratio: 50% effective concentration (EC 50 ) of drug A in combination with drug B/EC 50 of drug A alone. The effect was considered to be additive when the sum of FICs was between 0.8 and 1.2, as previously described [8] Time-of-addition assay Monolayers of MDCK cells in 96-well plates were infected with 100 μl of medium containing approximately 10 4 TCID 50 of H3N2 subtype. After 1 hour of adsorption, cell monolayers were washed twice with serum-free MEM and incubated in fresh medium containing TPCK-trypsin and chloroquine at a concentration of 10 μM. Chloroquine was added at the time of infection or at four different time points thereafter. Eight hours post-infection, a time point at which all progeny virus in the supernatants is derived from the first replication cycle, cell supernatants were collected, viral RNA was extracted and the antiviral activity was determined by using the qRRT-PCR described above.

Background

A second look at selected compounds is giving new life to several abandoned therapies and new applications for existing drugs [1] [2] [3] . One such example is provided by chloroquine, being dismissed from antimalarial treatment and finding new applications in the clinical management of autoimmune diseases, tumours and nonmalarial infections [4, 5] . The use of chloroquine in the clinical management of a viral infection was first consid-ered in the 1990s, on the basis of its effects on HIV-1 [6, 7] . The drug is now being tested as an investigational antiretroviral [8] .

Discussion

Chloroquine was found to inhibit a number of cellular processes, some of which do not depend on low pH but might anyway interfere with viral replication. For example, the drug was found to inhibit viral nucleotidyl transferases such as HIV-1 integrase [7] . If chloroquine inhibited influenza A RNA-dependent RNA polymerase, the timing of viral inhibition would not be consistent with that observed in the present study, because RNA replication occurs in the nucleus at later stages [18] .
Based on bioinformatic studies, it was recently hypothesized that chloroquine might inhibit UDP-N acetylglucosamine transferase [1] , a limiting enzyme in sialic acid synthesis. This specific issue has not been addressed here. Nonetheless, if the antiviral effect of chloroquine reported here were due to inhibition of UDP-N acetylglucosamine transferase, the drug should likely have antagonized the antiviral effect of the neuraminidase inhibitor oseltamivir (acting on detachment of sialic acid-bound virions from parent cells), rendering oseltamivir inhibition unnecessary. Instead, chloroquine was found in the present study to exert antiviral effects that were additive to those of oseltamivir.
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On March 29, 2013, a police officer (patient 1) was admitted to the emergency department of the University Medical Centre Hamburg-Eppendorf in Hamburg, Germany, because of ARDS. The patient was given mechanical ventilation; all diagnostic test results for pathogens commonly known to cause pneumonia were negative (www.virus-genomics.org/ supplementaries/EID1406.pdf). Although treatment with antimicrobial drugs was immediately initiated, the patient died 6 days later of multiple organ failure.
We did not detect viral pathogens in any samples. At the DNA level, most nonhost reads originated from nonpathogenic single-stranded DNA anelloviruses (7) . No RNA viruses were found, although influenza A(H3N2) virus was readily identified in a MiSeq analysis of a control BAL sample from a patient with a diagnosis of influenza A (PCR cycle threshold 32) (Figure) . Furthermore, pairwise BLAST analysis (http://blast.ncbi.nlm. nih.gov/Blast.cgi) did not reveal the presence of unknown sequence contigs that were shared among the patients, as would be expected in case of infection with a novel viral agent. Together with the confirmed C. psittaci infection in patient 2, the absence of a common pathogen signature strongly suggests that the cases were unrelated.
Virus isolation for EV-C117 by using Vero and H1-HeLa cells was unsuccessful. Through blastn and phylogenetic analyses, we also found that the previously identified EV-C strain HC90835 (EU697831, from Nepal) (8), EV-C104 strain CL-C22 (EU840734, EU840744, and EU840749, from Switzerland) (9) and a rhinovirus strain SE-10-028 (JQ417886, from South Korea), also belong to EV-C117 ( Figure, panel A) , indicating that EV-C117 is widely distributed geographically. Because a large proportion of EV infections are subclinical or mild (1), the prevalence of EV-C117 should be further estimated by using serologic investigations in general populations.
On April 5, a second police officer (patient 2) from the same county was admitted to the same emergency department because of ARDS. He was transferred to the intensive care unit and given mechanical ventilation. Similar to the situation for patient 1, diagnostic test results were negative, prompting the news media to suspect an outbreak of a novel or mutated virus (1, 2) . Especially because of simultaneous outbreaks of avian influenza and infections with Middle East respiratory syndrome coronavirus in other parts of the world, these reports caused serious concern among the public and health officials.
After the death of patient 1 and hospitalization of patient 2, we subjected nucleic acids extracted from bronchoalveolar lavage (BAL) specimens from both patients to NGS by using a MiSeq sequencer (www.illumina.com/ systems.ilmn). To enable rapid and unbiased detection of bacterial and viral agents, we did not enrich specific sequences. The entire workflow (www. virus-genomics.org/supplementaries/ EID1406.pdf) was completed within 50 hours.
At admission of patient 3, samples from all 3 patients were analyzed at greater read depth by using a HiSeq system. RNA sequencing identified commensal bacteria in all samples, but C. psittaci was present only in samples from patient 2 (Figure) . Likewise, subsequent high-throughput 16S rRNA sequencing (6), PCR, and serologic analysis unequivocally identified a C. psittaci infection in patient 2, but not in patients 1 or 3 (www. virus-genomics.org/supplementaries/ EID1406.pdf).
This study was supported in part by a project grant from the German Center for Infection Research to N.F. and A.G. EVs are classified into 12 species according to the molecular and antigenic properties of their viral capsid protein (VP1). To date, 7 species are known to infect humans, including EV-A to EV-D and rhinovirus A, B, and C (www.picornastudygroup.com/taxa/ species/species.htm) EV-C117 was a newly found EV-C genotype. It was identified in a nasopharyngeal sample from a hospitalized child, 3 years and 9 months of age, with community-acquired pneumonia in Lithuania in 2012 (2,3) . However, aside from this case, little is known about the prevalence and clinical significance of EV-C117. Here, we report the detection of EV-C117 in children in China and Mongolia with respiratory tract infections (RTIs).
During March 2007-March 2013, we screened for EV-C117 in respiratory samples from patients with RTIs in China and Mongolia, including nasopharyngeal aspirates collected from 3,108 children in China who had lower respiratory tract infections when they were admitted to Beijing Children's Hospital (4) and swab samples from 2,516 patients in Mongolia with influenza-like illness (online Technical Appendix Table 1 , wwwnc. cdc.gov/EID/article/20/6/13-1596-Techapp1.pdf). Respiratory viruses in samples from China were screened by using multiplex PCR and single PCR assays as described (4) . Samples from Mongolia were screened by using the FTD Respiratory Pathogens Multiplex Assay Kit (Fast-track Diagnostics, Luxembourg City, Luxembourg). EVpositive samples were further genotyped by using reverse transcription PCR (RT-PCR) and primers sequentially targeting the VP1 region (5), the 5′-untranslated region (5′-UTR)/VP4/ VP2 region (6) and the 5′-UTR (7). A 394-nt amplicon corresponding to the 5′-UTR of EVs was obtained from 10 children in China; a 598-nt amplicon corresponding to the 5′-UTR/ VP4/VP2 region was obtained by RT-PCR from 5 children in Mongolia. Blastn analysis (www.blast.ncbi. nlm.nih.gov/Blast.cgi) of PCR amplicons showed that only amplicons detected in 2 children from China (patients BCH096A and BCH104A) and 2 children from Mongolia (patients MGL126 and MGL208) had the highest similarity (95%-98%) to the EV-C117 prototype strain LIT22.
On April 29, a third police officer (patient 3) who had been sharing office space with patient 2, came to the hospital with symptoms of pneumonia. After antimicrobial drug treatment, his condition rapidly improved and the patient was discharged on day 7.
First-line analysis clearly identified Chlamydophila psittaci in the RNA sample from patient 2, but no sequences of obvious pathogenic origin were detected in samples from patient 1 (Figure) . C. psittaci, an intracellular bacterium, can be transmitted by inhaling aerosolized secretions or feces from infected birds (3). Person-toperson transmission of this bacterium is rare (4, 5) . Ornithosis, a disease characterized by severe pneumonia and influenza-like symptoms, might develop in persons infected with this bacterium. Because of the rarity of the disease, standard diagnostic panels usually do not include C. psittaci. After 11 days of antimicrobial drug treatment, the condition of patient 2 improved, and the patient was transferred to a general hospital ward.
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Preventive and avoidance behaviors

Over time, >85% of the participants used a face mask and immediately visited a doctor when experiencing influenza-like symptoms. More than 50% of the participants washed their hands >10 times every day throughout the survey period (p>0.05). As the pandemic progressed, a decreasing percentage of participants wore masks in public areas; avoided touching their mouth, nose, and eyes; took anti-influenza drugs; avoided crowded places; and took Traditional Chinese medicine to prevent virus infection (p<0.01) ( Table 2) .