The mucosal surfaces of all vertebrates have been exposed to similar evolutionary pressures for millions of years. In terrestrial vertebrates such as birds and mammals, the nasopharynx-associated lymphoid tissue (NALT) represents a first line of immune defence. Here we propose that NALT is an ancient arm of the mucosal immune system not restricted to terrestrial vertebrates. We find that NALT is present in rainbow trout and that it resembles other teleost mucosa-associated lymphoid tissues. Trout NALT consists of diffuse lymphoid cells and lacks tonsils and adenoids. The predominant B-cell subset found in trout NALT are IgT + B cells, similar to skin and gut. The trout olfactory organ is colonized by abundant symbiotic bacteria, which are coated by trout secretory immunoglobulin. Trout NALT is capable of mounting strong anti-viral immune responses following nasal delivery of a live attenuated viral vaccine. Our results open up a new tool for the control of aquatic infectious diseases via nasal vaccination.

Influenza viruses are respiratory pathogens that are responsible for both seasonal influenza epidemics and occasional influenza pandemics. The narrow therapeutic window of oseltamivir, coupled with the emergence of drug resistance, calls for the next-generation of antivirals. With our continuous interest in developing AM2-S31N inhibitors as oral influenza antivirals, we report here the progress of optimizing the in vitro pharmacokinetic (PK) properties of AM2-S31N inhibitors. Several AM2-S31N inhibitors, including compound 10b, were discovered to have potent channel blockage, single to submicromolar antiviral activity, and favorable in vitro PK properties. The antiviral efficacy of compound 10b was also synergistic with oseltamivir carboxylate. Interestingly, binding kinetic studies (K, K, and K) revealed several AM2-S31N inhibitors that have similar K values but significantly different K and K values. Overall, this study identified a potent lead compound (10b) with improved in vitro PK properties that is suitable for the in vivo mouse model studies.

Enterovirus D68 (EV-D68) is a viral pathogen that leads to severe respiratory illness and has been linked with the development of acute flaccid myelitis (AFM) in children. No vaccines or antivirals are currently available for EV-D68 infection, and treatment options for hospitalized patients are limited to supportive care. Here, we report the expression of the EV-D68 2A protease (2Apro) and characterization of its enzymatic activity. Furthermore, we discovered that telaprevir, an FDA-approved drug used for the treatment of hepatitis C virus (HCV) infections, is a potent antiviral against EV-D68 by targeting the 2Apro enzyme. Using a fluorescence resonance energy transfer-based substrate cleavage assay, we showed that the purified EV-D68 2Apro has proteolytic activity selective against a peptide sequence corresponding to the viral VP1-2A polyprotein junction. Telaprevir inhibits EV-D68 2Apro through a nearly irreversible, biphasic binding mechanism. In cell culture, telaprevir showed submicromolar-to-low-micromolar potency against several recently circulating neurotropic strains of EV-D68 in different human cell lines. To further confirm the antiviral drug target, serial viral passage experiments were performed to select for resistance against telaprevir. An N84T mutation near the active site of 2Apro was identified in resistant viruses, and this mutation reduced the potency of telaprevir in both the enzymatic and cellular antiviral assays. Collectively, we report for the first time the in vitro enzymatic activity of EV-D68 2Apro and the identification of telaprevir as a potent EV-D68 2Apro inhibitor. These findings implicate EV-D68 2Apro as an antiviral drug target and highlight the repurposing potential of telaprevir to treat EV-D68 infection. IMPORTANCE A 2014 EV-D68 outbreak in the United States has been linked to the development of acute flaccid myelitis in children. Unfortunately, no treatment options against EV-D68 are currently available, and the development of effective therapeutics is urgently needed. Here, we characterize and validate a new EV-D68 drug target, the 2Apro, and identify telaprevir—an FDA-approved drug used to treat hepatitis C virus (HCV) infections—as a potent antiviral with a novel mechanism of action toward 2Apro. 2Apro functions as a viral protease that cleaves a peptide sequence corresponding to the VP1-2A polyprotein junction. The binding of telaprevir potently inhibits its enzymatic activity, and using drug resistance selection, we show that the potent antiviral activity of telaprevir was due to 2Apro inhibition. This is the first inhibitor to selectively target the 2Apro from EV-D68 and can be used as a starting point for the development of therapeutics with selective activity against EV-D68.

Enterovirus D68 (EV-D68) is a respiratory viral pathogen that primarily infects children under the age of 8. Although EV-D68 infection typically leads to moderate to severe respiratory illnesses, recent years have seen increasing cases of EV-D68 triggered neurological complications such as the acute flaccid myelitis (AFM). There is currently no vaccine or antiviral available for EV-D68, we therefore aimed to develop potent and specific small molecule antivirals against EV-D68. In this study, we report our discovery of a viral capsid inhibitor R856932 that inhibits multiple contemporary EV-D68 strains with single-digit to submicromolar efficacy. Mechanistic studies have shown that the tetrazole compound R856932 binds to the hydrophobic pocket of viral capsid protein VP1, thereby preventing viral uncoating and releasing of viral genome in the infected cells. The mechanism of action of R856932 was confirmed by time-of-addition, western blot, RT-qPCR, viral heat inactivation, serial viral passage and reverse genetics experiments. A single mutation located at VP1, A129V, confers resistance against R856932. However, recombination virus encoding VP1-A129V appeared to have compromised fitness of replication compared to the wild type EV-D68 virus as shown by the competition growth assay. Overall, the hit compound identified in this study, R856932, represents a promising starting point with a confirmed mechanism of action that can be further developed into EV-D68 antivirals.

Transportation of live fish is a common practice among aquaculture facilities. Many studies have previously reported how transport elicits physiological stress responses and increases disease susceptibility in farmed fish. The aim of this work is to investigate the changes that the skin of rainbow trout (Oncorhynchus mykiss) experiences due to stress. Since NaCl is commonly added to transport water as a stress mitigator, the effects of salt addition on the skin mucosa and skin-associated bacteria were also examined. Three experimental groups (Control, post-transport no salt (PTNS) and post-transport with salt (PTS)) were analyzed in a 5-hour transport acute stress model. Results indicate that the skin mucosa and the skin-associated bacteria are affected by transport stress. Total numbers of culturable skin-associated bacteria increased by ~10-fold and ~50-fold in the PTS and PTNS groups, respectively. Compared to controls, MUC2 expression was increased by 5-fold and 2-fold in the PTNS and PTS groups, respectively. Claudin-7, 8d and 12 expression levels were higher in both PTNS and PTS groups whereas antimicrobial peptide gene expression was lower than controls. Expression of the anti-inflammatory cytokine TGF-β but not IL-1β, IL-6 and TNF-α was up-regulated 2-3 fold in both the PTS and PTNS groups. The addition of salt diminished some of the physiological responses measured including the numbers of skin-associated bacteria. The responses recorded here appeared to be efficient at controlling bacterial translocation since stress did not lead to significant presence of bacteria in the liver or spleen of rainbow trout. When examining the ability of skin mucus to inhibit or promote growth of the bacterial pathogen Vibrio anguillarum, the skin mucus of PTS trout was more efficient at inhibiting V. anguillarum growth (20% inhibition) compared to control or PTNS mucus (11-12% inhibition). Our data clearly indicate the skin and skin microbiota of rainbow trout undergo important physiological responses during stress. The reduction in the magnitude of the skin responses recorded when salt was added to the transport water explains a new mechanism by which salt is an effective stress mitigator in some fish species. Aquaculture specialists will benefit from the present study by taking into consideration the importance of skin health during live transport.

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Adamantanes such as amantadine (1) and rimantadine (2) are FDA-approved anti-influenza drugs that act by inhibiting the wild-type M2 proton channel from influenza A viruses, thereby inhibiting the uncoating of the virus. Although adamantanes have been successfully used for more than four decades, their efficacy was curtailed by emerging drug resistance. Among the limited number of M2 mutants that confer amantadine resistance, the M2-V27A mutant was found to be the predominant mutant under drug selection pressure, thereby representing a high profile antiviral drug target. Guided by molecular dynamics simulations, we previously designed first-in-class M2-V27A inhibitors. One of the potent lead compounds, spiroadamantane amine (3), inhibits both the M2-WT and M2-V27A mutant with IC50 values of 18.7 and 0.3 μM, respectively, in in vitro electrophysiological assays. Encouraged by these findings, in this study we further examine the in vitro and in vivo antiviral activity of compound 3 in inhibiting both amantadine-sensitive and -resistant influenza A viruses. Compound 3 not only had single to submicromolar EC50 values against M2-WT- and M2-V27A-containing influenza A viruses in antiviral assays, but also rescued mice from lethal viral infection by either M2-WT- or M2-V27A-containing influenza A viruses. In addition, we report the design of two analogs of compound 3, and one was found to have improved in vitro antiviral activity over compound 3. Collectively, this study represents the first report demonstrating the in vivo antiviral efficacy of inhibitors targeting M2 mutants. The results suggest that inhibitors targeting drug-resistant M2 mutants are promising antiviral drug candidates worthy of further development.

As the number of drug-resistant influenza viruses continues to increase, antivirals with novel mechanisms of action are urgently needed. Among the two classes of FDA-approved antiviral drugs, neuraminidase (NA) inhibitors, oseltamivir, zanamivir, and peramivir, are currently the only choice for the prevention and treatment of influenza virus infection. Due to the antigenic drift and antigenic shift, it will only be a matter of time before influenza viruses become completely resistant to these NA inhibitors. In pursuing the next generation of antiviral drugs with complementary mechanisms of action to those of the NA inhibitors, we have identified a natural product, cyclosporine A (CsA) (1), as a desired drug candidate. In this study, we discovered that CsA (1) and its analogs have broad-spectrum antiviral activity against multiple influenza A and B strains, including strains that are resistant to either NA or M2 inhibitors or both. Moreover, CsA (1) displays a high in vitro genetic barrier of drug resistance than oseltamivir carboxylate Mechanistic studies revealed that CsA (1) acts at the intermediate step of viral replication post viral fusion. Its antiviral mechanism is independent of inhibiting the isomerase activity of cyclophilin A (CypA), and CsA (1) has no effect on the viral polymerase activity The potent antiviral efficacy of CsA (1), coupled with the high in vitro genetic barrier of drug resistance and novel mechanism of action, renders CsA (1) a promising anti-influenza drug candidate for further development.