Favipiravir Treatment Prolongs Survival in a Lethal BALB/c Mouse Model of Ebinur Lake Virus Infection.

Orthobunyavirus is the largest and most diverse genus in the family Peribunyaviridae. Orthobunyaviruses are widely distributed globally and pose threats to human and animal health. Ebinur Lake virus (EBIV) is a newly classified Orthobunyavirus detected in China, Russia, and Kenya. This study explored the antiviral effects of two broad-spectrum antiviral drugs, favipiravir and ribavirin, in a BALB/c mouse model. Favipiravir significantly improved the clinical symptoms of infected mice, reduced viral titer and RNA copies in serum, and extended overall survival. The median survival times of mice in the vehicle- and favipiravir-treated groups were 5 and 7 days, respectively. Favipiravir significantly reduced virus titers 10- to 100-fold in sera at all three time points compared to vehicle-treated mice. And favipiravir treatment effectively reduced the virus copies by approximately 10-fold across the three time points, relative to vehicle-treated mice. The findings expand the antiviral spectrum of favipiravir for orthobunyaviruses in vivo.

Study on aptamer based high throughput approach identifies natural ingredients against RGNNV.

Nervous necrosis virus (NNV) is one of the most destructive pathogens in marine fish aquaculture and is capable of infecting more than 50 fish species worldwide, which resulted in great economic losses. Effective drugs for managing NNV infection are urgently required. Medicinal plants have been known for thousands of years and benefit of medicinal plants against pathogens in aquaculture have emerged. Nowadays, the most commonly used method for detecting virus infection and assessing antiviral drugs efficacy is reverse transcription-quantitative real-time PCR. However, the application is limited on account of high reagent costs, complex time-consuming operations and long detection time. Aptamers have been widely applied in application of pathogens or diseases diagnosis and treatments because of high specificity, strong affinity, good stability, easy synthesized and low costs. This study aimed to establish an aptamer (GBN34)-based high-throughput screening (GBN34-AHTS) model for efficient selection and evaluation of natural ingredients against NNV infection. GBN34-AHTS is an expeditious rapid method for selecting natural ingredients against NNV, which is characterized with high-speed, dram, sensitive and accurate. AHTS strategy could reduce work intensity and experimental costs and shorten the whole screening cycle of effective ingredients. AHTS should be suitable for rapid selection of effective ingredients against other viruses, which is important for improving the prevention and controlling of aquatic diseases.

Single-Domain Antibodies as Therapeutics for Respiratory RNA Virus Infections.

Over the years, infectious diseases with high morbidity and mortality disrupted human healthcare systems and devastated economies globally. Respiratory viruses, especially emerging or re-emerging RNA viruses, including influenza and human coronavirus, are the main pathogens of acute respiratory diseases that cause epidemics or even global pandemics. Importantly, due to the rapid mutation of viruses, there are few effective drugs and vaccines for the treatment and prevention of these RNA virus infections. Of note, a class of antibodies derived from camelid and shark, named nanobody or single-domain antibody (sdAb), was characterized by smaller size, lower production costs, more accessible binding epitopes, and inhalable properties, which have advantages in the treatment of respiratory diseases compared to conventional antibodies. Currently, a number of sdAbs have been developed against various respiratory RNA viruses and demonstrated potent therapeutic efficacy in mouse models. Here, we review the current status of the development of antiviral sdAb and discuss their potential as therapeutics for respiratory RNA viral diseases.

The development of broad-spectrum antiviral medical countermeasures to treat viral hemorrhagic fevers caused by natural or weaponized virus infections.

The Joint Program Executive Office for Chemical, Biological, Radiological, and Nuclear Defense (JPEO-CBRND) began development of a broad-spectrum antiviral countermeasure against deliberate use of high-consequence viral hemorrhagic fevers (VHFs) in 2016. The effort featured comprehensive preclinical research, including laboratory testing and rapid advancement of lead molecules into nonhuman primate (NHP) models of Ebola virus disease (EVD). Remdesivir (GS-5734, Veklury, Gilead Sciences) was the first small molecule therapeutic to successfully emerge from this effort. Remdesivir is an inhibitor of RNA-dependent RNA polymerase, a viral enzyme that is essential for viral replication. Its robust potency and broad-spectrum antiviral activity against certain RNA viruses including Ebola virus and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) led to its clinical evaluation in randomized, controlled trials (RCTs) in human patients during the 2018 EVD outbreak in the Democratic Republic of the Congo (DRC) and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic today. Remdesivir was recently approved by the US Food and Drug Administration (FDA) for the treatment of COVID-19 requiring hospitalization. Substantial gaps remain in improving the outcomes of acute viral infections for patients afflicted with both EVD and COVID-19, including how to increase therapeutic breadth and strategies for the prevention and treatment of severe disease. Combination therapy that joins therapeutics with complimentary mechanisms of action appear promising, both preclinically and in RCTs. Importantly, significant programmatic challenges endure pertaining to a clear drug and biological product development pathway for therapeutics targeting biodefense and emerging pathogens when human efficacy studies are not ethical or feasible. For example, remdesivir's clinical development was facilitated by outbreaks of Ebola and SARS-CoV-2; as such, the development pathway employed for remdesivir is likely to be the exception rather than the rule. The current regulatory licensure pathway for therapeutics targeting rare, weaponizable VHF agents is likely to require use of FDA's established Animal Rule (21 CFR 314.600-650 for drugs; 21 CFR 601.90-95 for biologics). The FDA may grant marketing approval based on adequate and well-controlled animal efficacy studies when the results of those studies establish that the drug is safe and likely to produce clinical benefit in humans. In practical terms, this is anticipated to include a series of rigorous, well-documented, animal challenge studies, to include aerosol challenge, combined with human safety data. While small clinical studies against naturally occurring, high-consequence pathogens are typically performed where possible, approval for the therapeutics currently under development against biodefense pathogens will likely require the Animal Rule pathway utilizing studies in NHPs. We review the development of remdesivir as illustrative of the effort that will be needed to field future therapeutics against highly lethal, infectious agents.

Lethal mutagenesis as an antiviral strategy.

[Figure: see text].

Targeting the Virus Capsid as a Tool to Fight RNA Viruses.

Several strategies have been developed to fight viral infections, not only in humans but also in animals and plants. Some of them are based on the development of efficient vaccines, to target the virus by developed antibodies, others focus on finding antiviral compounds with activities that inhibit selected virus replication steps. Currently, there is an increasing number of antiviral drugs on the market; however, some have unpleasant side effects, are toxic to cells, or the viruses quickly develop resistance to them. As the current situation shows, the combination of multiple antiviral strategies or the combination of the use of various compounds within one strategy is very important. The most desirable are combinations of drugs that inhibit different steps in the virus life cycle. This is an important issue especially for RNA viruses, which replicate their genomes using error-prone RNA polymerases and rapidly develop mutants resistant to applied antiviral compounds. Here, we focus on compounds targeting viral structural capsid proteins, thereby inhibiting virus assembly or disassembly, virus binding to cellular receptors, or acting by inhibiting other virus replication mechanisms. This review is an update of existing papers on a similar topic, by focusing on the most recent advances in the rapidly evolving research of compounds targeting capsid proteins of RNA viruses.

IFN-λ1 Displays Various Levels of Antiviral Activity In Vitro in a Select Panel of RNA Viruses.

Type III interferons (lambda IFNs) are a quite new, small family of three closely related cytokines with interferon-like activity. Attention to IFN-λ is mainly focused on direct antiviral activity in which, as with IFN-α, viral genome replication is inhibited without the participation of immune system cells. The heterodimeric receptor for lambda interferons is exposed mainly on epithelial cells, which limits its possible action on other cells, thus reducing the likelihood of developing undesirable side effects compared to type I IFN. In this study, we examined the antiviral potential of exogenous human IFN-λ1 in cellular models of viral infection. To study the protective effects of IFN-λ1, three administration schemes were used: 'preventive' (pretreatment); 'preventive/therapeutic' (pre/post); and 'therapeutic' (post). Three IFN-λ1 concentrations (from 10 to 500 ng/mL) were used. We have shown that human IFN-λ1 restricts SARS-CoV-2 replication in Vero cells with all three treatment schemes. In addition, we have shown a decrease in the viral loads of CHIKV and IVA with the 'preventive' and 'preventive/therapeutic' regimes. No significant antiviral effect of IFN-λ1 against AdV was detected. Our study highlights the potential for using IFN-λ as a broad-spectrum therapeutic agent against respiratory RNA viruses.

Melatonin may decrease risk for and aid treatment of COVID-19 and other RNA viral infections.

A recent retrospective study has provided evidence that COVID-19 infection may be notably less common in those using supplemental melatonin. It is suggested that this phenomenon may reflect the fact that, via induction of silent information regulator 1 (Sirt1), melatonin can upregulate K63 polyubiquitination of the mitochondrial antiviral-signalling protein, thereby boosting virally mediated induction of type 1 interferons. Moreover, Sirt1 may enhance the antiviral efficacy of type 1 interferons by preventing hyperacetylation of high mobility group box 1 (HMGB1), enabling its retention in the nucleus, where it promotes transcription of interferon-inducible genes. This nuclear retention of HMGB1 may also be a mediator of the anti-inflammatory effect of melatonin therapy in COVID-19-complementing melatonin's suppression of nuclear factor kappa B activity and upregulation of nuclear factor erythroid 2-related factor 2. If these speculations are correct, a nutraceutical regimen including vitamin D, zinc and melatonin supplementation may have general utility for the prevention and treatment of RNA virus infections, such as COVID-19 and influenza.

Natural Products with Potential to Treat RNA Virus Pathogens Including SARS-CoV-2.

Three families of RNA viruses, the Coronaviridae, Flaviviridae, and Filoviridae, collectively have great potential to cause epidemic disease in human populations. The current SARS-CoV-2 (Coronaviridae) responsible for the COVID-19 pandemic underscores the lack of effective medications currently available to treat these classes of viral pathogens. Similarly, the Flaviviridae, which includes such viruses as Dengue, West Nile, and Zika, and the Filoviridae, with the Ebola-type viruses, as examples, all lack effective therapeutics. In this review, we present fundamental information concerning the biology of these three virus families, including their genomic makeup, mode of infection of human cells, and key proteins that may offer targeted therapies. Further, we present the natural products and their derivatives that have documented activities to these viral and host proteins, offering hope for future mechanism-based antiviral therapeutics. By arranging these potential protein targets and their natural product inhibitors by target type across these three families of virus, new insights are developed, and crossover treatment strategies are suggested. Hence, natural products, as is the case for other therapeutic areas, continue to be a promising source of structurally diverse new anti-RNA virus therapeutics.

Targeting viral genome synthesis as broad-spectrum approach against RNA virus infections.

Zoonotic spillover, i.e. pathogen transmission from animal to human, has repeatedly introduced RNA viruses into the human population. In some cases, where these viruses were then efficiently transmitted between humans, they caused large disease outbreaks such as the 1918 flu pandemic or, more recently, outbreaks of Ebola and Coronavirus disease. These examples demonstrate that RNA viruses pose an immense burden on individual and public health with outbreaks threatening the economy and social cohesion within and across borders. And while emerging RNA viruses are introduced more frequently as human activities increasingly disrupt wild-life eco-systems, therapeutic or preventative medicines satisfying the "one drug-multiple bugs"-aim are unavailable. As one central aspect of preparedness efforts, this review digs into the development of broadly acting antivirals via targeting viral genome synthesis with host- or virus-directed drugs centering around nucleotides, the genomes' universal building blocks. Following the first strategy, selected examples of host de novo nucleotide synthesis inhibitors are presented that ultimately interfere with viral nucleic acid synthesis, with ribavirin being the most prominent and widely used example. For directly targeting the viral polymerase, nucleoside and nucleotide analogues (NNAs) have long been at the core of antiviral drug development and this review illustrates different molecular strategies by which NNAs inhibit viral infection. Highlighting well-known as well as recent, clinically promising compounds, structural features and mechanistic details that may confer broad-spectrum activity are discussed. The final part addresses limitations of NNAs for clinical development such as low efficacy or mitochondrial toxicity and illustrates strategies to overcome these.

Azithromycin and glucosamine may amplify the type 1 interferon response to RNA viruses in a complementary fashion.

Previous research demonstrates that, in clinically relevant concentrations, azithromycin can boost the ability of RNA viruses to induce type 1 interferon by amplifying the expression and virally-mediated activation of MDA5. O-GlcNAcylation of MAVS, a down-stream target of MDA5, renders it more effective for type 1 interferon induction. High-dose glucosamine administration up-regulates O-GlcNAcylation by increasing the cellular pool of UDP-N-acetylglucosamine. Hence, it is proposed that joint administration of azithromycin and high-dose glucosamine, early in the course of RNA virus infections, may interact in a complementary fashion to aid their control by enhancing type 1 interferon induction.

Favipiravir, an anti-influenza drug against life-threatening RNA virus infections.

Favipiravir has been developed as an anti-influenza drug and licensed as an anti-influenza drug in Japan. Additionally, favipiravir is being stockpiled for 2 million people as a countermeasure for novel influenza strains. This drug functions as a chain terminator at the site of incorporation of the viral RNA and reduces the viral load. Favipiravir cures all mice in a lethal influenza infection model, while oseltamivir fails to cure the animals. Thus, favipiravir contributes to curing animals with lethal infection. In addition to influenza, favipiravir has a broad spectrum of anti-RNA virus activities in vitro and efficacies in animal models with lethal RNA viruses and has been used for treatment of human infection with life-threatening Ebola virus, Lassa virus, rabies, and severe fever with thrombocytopenia syndrome. The best feature of favipiravir as an antiviral agent is the apparent lack of generation of favipiravir-resistant viruses. Favipiravir alone maintains its therapeutic efficacy from the first to the last patient in an influenza pandemic or an epidemic lethal RNA virus infection. Favipiravir is expected to be an important therapeutic agent for severe influenza, the next pandemic influenza strain, and other severe RNA virus infections for which standard treatments are not available.

Antiviral Drug Targets of Single-Stranded RNA Viruses Causing Chronic Human Diseases.

Ribonucleic acid (RNA) viruses associated with chronic diseases in humans are major threats to public health causing high mortality globally. The high mutation rate of RNA viruses helps them to escape the immune response and also is responsible for the development of drug resistance. Chronic infections caused by human immunodeficiency virus (HIV) and hepatitis viruses (HBV and HCV) lead to acquired immunodeficiency syndrome (AIDS) and hepatocellular carcinoma respectively, which are one of the major causes of human deaths. Effective preventative measures to limit chronic and re-emerging viral infections are absolutely necessary. Each class of antiviral agents targets a specific stage in the viral life cycle and inhibits them from its development and proliferation. Most often, antiviral drugs target a specific viral protein, therefore only a few broad-spectrum drugs are available. This review will be focused on the selected viral target proteins of pathogenic viruses containing single-stranded (ss) RNA genome that causes chronic infections in humans (e.g. HIV, HCV, Flaviviruses). In the recent past, an exponential increase in the number of available three-dimensional protein structures (>150000 in Protein Data Bank), allowed us to better understand the molecular mechanism of action of protein targets and antivirals. Advancements in the in silico approaches paved the way to design and develop several novels, highly specific small-molecule inhibitors targeting the viral proteins.

R430: A potent inhibitor of DNA and RNA viruses.

Acyclovir (ACV) is an effective antiviral agent for treating lytic Herpes Simplex virus, type 1 (HSV-1) infections, and it has dramatically reduced the mortality rate of herpes simplex encephalitis. However, HSV-1 resistance to ACV and its derivatives is being increasingly documented, particularly among immunocompromised individuals. The burgeoning drug resistance compels the search for a new generation of more efficacious anti-herpetic drugs. We have previously shown that trans-dihydrolycoricidine (R430), a lycorane-type alkaloid derivative, effectively inhibits HSV-1 infections in cultured cells. We now report that R430 also inhibits ACV-resistant HSV-1 strains, accompanied by global inhibition of viral gene transcription and enrichment of H3K27me3 methylation on viral gene promoters. Furthermore, we demonstrate that R430 prevents HSV-1 reactivation from latency in an ex vivo rodent model. Finally, among a panel of DNA viruses and RNA viruses, R430 inhibited Zika virus with high therapeutic index. Its therapeutic index is comparable to standard antiviral drugs, though it has greater toxicity in non-neuronal cells than in neuronal cells. Synthesis of additional derivatives could enable more efficacious antivirals and the identification of active pharmacophores.

Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein.

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV can be sensed by host innate immunity to induce expression of interferons (IFNs) and a number of antiviral effectors. In this study, we found HCV infection induced the expression of neuralized E3 ubiquitin protein ligase 3 (NEURL3), a putative E3 ligase, in a manner that requires the involvement of innate immune sensing but is independent of the IFN action. Furthermore, we showed that NEURL3 inhibited HCV infection while it had little effect on other RNA viruses, including Zika virus (ZIKV), dengue virus (DENV), and vesicular stomatitis virus (VSV). Mechanistic studies demonstrated that NEURL3 inhibited HCV assembly by directly binding HCV envelope glycoprotein E1 to interfere with the E1/E2 heterodimerization, an important prerequisite for virion morphogenesis. Finally, we showed that knockout of NEURL3 significantly enhanced HCV infection. In summary, we identified NEURL3 as a novel inducible antiviral host factor that suppresses HCV assembly. Our results not only shed new insight into how host innate immunity acts against HCV but also revealed a new important biological function for NEURL3.IMPORTANCE The exact biological function of NEURL3, a putative E3 ligase, remains largely unknown. In this study, we found that NEURL3 could be upregulated upon HCV infection in a manner dependent on pattern recognition receptor-mediated innate immune response. NEURL3 inhibits HCV assembly by directly binding viral E1 envelope glycoprotein to disrupt its interaction with E2, an action that requires its Neuralized homology repeat (NHR) domain but not the RING domain. Furthermore, we found that NEURL3 has a pangenotypic anti-HCV activity and interacts with E1 of genotypes 2a, 1b, 3a, and 6a but does not inhibit other closely related RNA viruses, such as ZIKV, DENV, and VSV. To our knowledge, our study is the first report to demonstrate that NEURL3 functions as an antiviral host factor. Our results not only shed new insight into how host innate immunity acts against HCV, but also revealed a new important biological function for NEURL3.

The Antiviral Effects of Na,K-ATPase Inhibition: A Minireview.

Since being first described more than 60 years ago, Na,K-ATPase has been extensively studied, while novel concepts about its structure, physiology, and biological roles continue to be elucidated. Cardiac glycosides not only inhibit the pump function of Na,K-ATPase but also activate intracellular signal transduction pathways, which are important in many biological processes. Recently, antiviral effects have been described as a novel feature of Na,K-ATPase inhibition with the use of cardiac glycosides. Cardiac glycosides have been reported to be effective against both DNA viruses such as cytomegalovirus and herpes simplex and RNA viruses such as influenza, chikungunya, coronavirus, and respiratory syncytial virus, among others. Consequently, cardiac glycosides have emerged as potential broad-spectrum antiviral drugs, with the great advantage of targeting cell host proteins, which help to minimize resistance to antiviral treatments, making them a very promising strategy against human viral infections. Here, we review the effect of cardiac glycosides on viral biology and the mechanisms by which these drugs impair the replication of this array of different viruses.

Nucleosides for the treatment of respiratory RNA virus infections.

Influenza virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, and rhinoviruses are among the most common viruses causing mild seasonal colds. These RNA viruses can also cause lower respiratory tract infections leading to bronchiolitis and pneumonia. Young children, the elderly, and patients with compromised cardiac, pulmonary, or immune systems are at greatest risk for serious disease associated with these RNA virus respiratory infections. In addition, swine and avian influenza viruses, together with severe acute respiratory syndrome-associated and Middle Eastern respiratory syndrome coronaviruses, represent significant pandemic threats to the general population. In this review, we describe the current medical need resulting from respiratory infections caused by RNA viruses, which justifies drug discovery efforts to identify new therapeutic agents. The RNA polymerase of respiratory viruses represents an attractive target for nucleoside and nucleotide analogs acting as inhibitors of RNA chain synthesis. Here, we present the molecular, biochemical, and structural fundamentals of the polymerase of the four major families of RNA respiratory viruses: Orthomyxoviridae, Pneumoviridae/Paramyxoviridae, Coronaviridae, and Picornaviridae. We summarize past and current efforts to develop nucleoside and nucleotide analogs as antiviral agents against respiratory virus infections. This includes molecules with very broad antiviral spectrum such as ribavirin and T-705 (favipiravir), and others targeting more specifically one or a few virus families. Recent advances in our understanding of the structure(s) and function(s) of respiratory virus polymerases will likely support the discovery and development of novel nucleoside analogs.

Favipiravir as a potential countermeasure against neglected and emerging RNA viruses.

Favipiravir, also known as T-705, is an antiviral drug that has been approved in 2014 in Japan to treat pandemic influenza virus infections. The drug is converted intracellularly into its active, phosphoribosylated form, which is recognized as a substrate by the viral RNA-dependent RNA polymerase. Interestingly, besides its anti-influenza virus activity, this molecule is also able to inhibit the replication of flavi-, alpha-, filo-, bunya-, arena-, noro-, and of other RNA viruses, which include neglected and (re)emerging viruses for which no antiviral therapy is currently available. We will discuss the potential of favipiravir as a broad-spectrum countermeasure against infections caused by such neglected RNA viruses. Favipiravir has already been used off-label to treat patients infected with the Ebola virus and the Lassa virus. Because of the particular set-up of the clinical trials during these outbreaks, clear conclusions on the efficacy of favipiravir could not be made. For several viruses, it was demonstrated that the barrier of resistance development against favipiravir is high. Favipiravir has been shown to be well tolerated in healthy volunteers and in influenza virus-infected patients; however, caution is needed because of the teratogenic risks of this molecule. Because of its antiviral activity against different RNA viruses and its high barrier for resistance, the potential of favipiravir as a broad-spectrum antiviral seems promising, but safety and potency issues should be overcome before this drug or similar molecules could be used to treat large patient groups.

BCX4430 - A broad-spectrum antiviral adenosine nucleoside analog under development for the treatment of Ebola virus disease.

The adenosine nucleoside analog BCX4430 is a direct-acting antiviral drug under investigation for the treatment of serious and life-threatening infections from highly pathogenic viruses, such as the Ebola virus. Cellular kinases phosphorylate BCX4430 to a triphosphate that mimics ATP; viral RNA polymerases incorporate the drug's monophosphate nucleotide into the growing RNA chain, causing premature chain termination. BCX4430 is active in vitro against many RNA viral pathogens, including the filoviruses and emerging infectious agents such as MERS-CoV and SARS-CoV. In vivo, BCX4430 is active after intramuscular, intraperitoneal, and oral administration in a variety of experimental infections. In nonclinical studies involving lethal infections with Ebola virus, Marburg virus, Rift Valley fever virus, and Yellow Fever virus, BCX4430 has demonstrated pronounced efficacy. In experiments conducted in several models, both a reduction in the viral load and an improvement in survival were found to be related to the dose of BCX4430. A Phase 1 clinical trial of intramuscular administration of BCX4430 in healthy subjects is currently ongoing.

Generation and characterization of novel DNA aptamers against coat protein of grouper nervous necrosis virus (GNNV) with antiviral activities and delivery potential in grouper cells.

Nervous necrosis virus (NNV) infected larvae and juveniles of more than 50 fish species, resulting in mortality rates of greater than 95%. However, there is no efficient method to control NNV infections. Aptamers generated by selective evolution of ligands by exponential enrichment (SELEX) are short, single-stranded nucleic acid oligomers. They display a high degree of affinity and specificity for many targets, such as viruses and viral proteins. In this study, three novel DNA aptamers (A5, A10, and B11) that specifically target the coat protein (CP) of grouper nervous necrosis virus (GNNV) were selected using SELEX. Secondary structures and minimum free energy (ΔG) predictions indicated that these aptamers could form stable, secondary stem-loop structures. Electrophoretic mobility shift assays, enzyme-linked immunosorbent assays, Kd measurements, the co-localization of tetramethylrhodamine (TAMRA) labeled-aptamers with the CP and flow cytometry analysis revealed that these aptamers could specifically bind the CP with high (nanomolar) affinities. In addition, competition analysis suggested the aptamers shared some common CP binding sites with the anti-CP antibody. Moreover, all three aptamers did not show any cytotoxic effects in vitro or in vivo, and anti-viral analysis indicated the selected aptamers could inhibit NNV infection in vitro and in vivo. Compared with controls, mortality of GNNV-infected fish decreased by 40% and 80% after 10 days infection, when the GNNV was pre-incubated with the 1000 nM A10 and B11, respectively. TAMRA-labeled aptamers could bind to NNV virions and directly enter NNV-infected cells, suggesting they could be used as tracers to study the mechanism of viral infection, as well as for targeted therapy. This is the first time that aptamers targeting a viral protein of marine fish have been generated and characterized. These aptamers hold promise as diagnostic, therapeutic, and targeted drug delivery agents for controlling NNV infections.