Identification of cap-dependent endonuclease inhibitors with broad-spectrum activity against bunyaviruses.

Viral hemorrhagic fevers caused by members of the order Bunyavirales comprise endemic and emerging human infections that are significant public health concerns. Despite the disease severity, there are few therapeutic options available, and therefore effective antiviral drugs are urgently needed to reduce disease burdens. Bunyaviruses, like influenza viruses (IFVs), possess a cap-dependent endonuclease (CEN) that mediates the critical cap-snatching step of viral RNA transcription. We screened compounds from our CEN inhibitor (CENi) library and identified specific structural compounds that are 100 to 1,000 times more active in vitro than ribavirin against bunyaviruses, including Lassa virus, lymphocytic choriomeningitis virus (LCMV), and Junin virus. To investigate their inhibitory mechanism of action, drug-resistant viruses were selected in culture. Whole-genome sequencing revealed that amino acid substitutions in the CEN region of drug-resistant viruses were located in similar positions as those of the CEN α3-helix loop of IFVs derived under drug selection. Thus, our studies suggest that CENi compounds inhibit both bunyavirus and IFV replication in a mechanistically similar manner. Structural analysis revealed that the side chain of the carboxyl group at the seventh position of the main structure of the compound was essential for the high antiviral activity against bunyaviruses. In LCMV-infected mice, the compounds significantly decreased blood viral load, suppressed symptoms such as thrombocytopenia and hepatic dysfunction, and improved survival rates. These data suggest a potential broad-spectrum clinical utility of CENis for the treatment of both severe influenza and hemorrhagic diseases caused by bunyaviruses.

First report of antiviral activity of nordihydroguaiaretic acid against Fort Sherman virus (Orthobunyavirus).

The genus Orthobunyavirus are a group of viruses within arbovirus, with a zoonotic cycle, some of which could lead to human infection. A characteristic of these viruses is their lack of antiviral treatment or vaccine for its prevention. The objective of this work was to study the in vitro antiviral activity of nordihydroguaiaretic acid (NDGA), the most important active compound of Larrea divaricata Cav. (Zigophyllaceae), against Fort Sherman virus (FSV) as a model of Orthobunyavirus genus. At the same time, the effect of NDGA as a lipolytic agent on the cell cycle of this viral model was assessed. The method of reducing plaque forming units on LLC-MK2 cells was used to detect the action of NDGA on CbaAr426 and SFCrEq231 isolates of FSV. NDGA did not show virucidal effect, but it had antiviral activity with a similar inhibition in both isolates, which was dose dependent. It was established that the NDGA has a better inhibition 1-h post-internalization (p.i.), showing a different behavior in each isolate, which was dependent upon the time p.i. Since virus multiplication is dependent on host cell lipid metabolism, the antiviral effect of NDGA has been previously related to its ability to disturb the lipid metabolism, probably by interfering with the 5-lipoxigenase (5-LOX) and the sterol regulatory element-binding proteins (SREBP) pathway. We determined by using caffeic acid, a 5-LOX inhibitor, that the inhibition of this enzyme negatively affected the FSV replication; and by means of resveratrol, a SREBP1 inhibitor, it was showed that the negative regulation of this pathway only had action on the SFCrEq231 reduction. In addition, it was proved that the NDGA acts intracellularly, since it showed the ability to incorporate into LLC-MK2 cells. The information provided in this work converts the NDGA into a compound with antiviral activity in vitro against FSV (Orthobunyavirus), which can be subjected to structural modifications in the future to improve the activity.

Novel Ionophores Active against La Crosse Virus Identified through Rapid Antiviral Screening.

Bunyaviruses are significant human pathogens, causing diseases ranging from hemorrhagic fevers to encephalitis. Among these viruses, La Crosse virus (LACV), a member of the California serogroup, circulates in the eastern and midwestern United States. While LACV infection is often asymptomatic, dozens of cases of encephalitis are reported yearly. Unfortunately, no antivirals have been approved to treat LACV infection. Here, we developed a method to rapidly test potential antivirals against LACV infection. From this screen, we identified several potential antiviral molecules, including known antivirals. Additionally, we identified many novel antivirals that exhibited antiviral activity without affecting cellular viability. Valinomycin, a potassium ionophore, was among our top targets. We found that valinomycin exhibited potent anti-LACV activity in multiple cell types in a dose-dependent manner. Valinomycin did not affect particle stability or infectivity, suggesting that it may preclude virus replication by altering cellular potassium ions, a known determinant of LACV entry. We extended these results to other ionophores and found that the antiviral activity of valinomycin extended to other viral families, including bunyaviruses (Rift Valley fever virus, Keystone virus), enteroviruses (coxsackievirus, rhinovirus), flavirivuses (Zika virus), and coronaviruses (human coronavirus 229E [HCoV-229E] and Middle East respiratory syndrome CoV [MERS-CoV]). In all viral infections, we observed significant reductions in virus titer in valinomycin-treated cells. In sum, we demonstrate the importance of potassium ions to virus infection, suggesting a potential therapeutic target to disrupt virus replication.

Antivirals in medical biodefense.

The viruses historically implicated or currently considered as candidates for misuse in bioterrorist events are poxviruses, filoviruses, bunyaviruses, orthomyxoviruses, paramyxoviruses and a number of arboviruses causing encephalitis, including alpha- and flaviviruses. All these viruses are of concern for public health services when they occur in natural outbreaks or emerge in unvaccinated populations. Recent events and intelligence reports point to a growing risk of dangerous biological agents being used for nefarious purposes. Public health responses effective in natural outbreaks of infectious disease may not be sufficient to deal with the severe consequences of a deliberate release of such agents. One important aspect of countermeasures against viral biothreat agents are the antiviral treatment options available for use in post-exposure prophylaxis. These issues were adressed by the organizers of the 16th Medical Biodefense Conference, held in Munich in 2018, in a special session on the development of drugs to treat infections with viruses currently perceived as a threat to societies or associated with a potential for misuse as biothreat agents. This review will outline the state-of-the-art methods in antivirals research discussed and provide an overview of antiviral compounds in the pipeline that are already approved for use or still under development.

Potassium is a trigger for conformational change in the fusion spike of an enveloped RNA virus.

Many enveloped viruses enter cells through the endocytic network, from which they must subsequently escape through fusion of viral and endosomal membranes. This membrane fusion is mediated by virus-encoded spikes that respond to the dynamic endosomal environment, which triggers conformational changes in the spikes that initiate the fusion process. Several fusion triggers have been identified and include pH, membrane composition, and endosome-resident proteins, and these cues dictate when and where viral fusion occurs. We recently reported that infection with an enveloped bunyavirus requires elevated potassium ion concentrations [K+], controlled by cellular K+ channels, that are encountered during viral transit through maturing endosomes. Here we reveal the molecular basis for the K+ requirement of bunyaviruses through the first direct visualization of a member of the Nairoviridae family, namely Hazara virus (HAZV), using cryo-EM. Using cryo-electron tomography, we observed HAZV spike glycoproteins within infectious HAZV particles exposed to both high and low [K+], which showed that exposure to K+ alone results in dramatic changes to the ultrastructural architecture of the virion surface. In low [K+], the spikes adopted a compact conformation arranged in locally ordered arrays, whereas, following exposure to high [K+], the spikes became extended, and spike-membrane interactions were observed. Viruses exposed to high [K+] also displayed enhanced infectivity, thus identifying K+ as a newly defined trigger that helps promote viral infection. Finally, we confirmed that K+ channel blockers are inhibitory to HAZV infection, highlighting the potential of K+ channels as anti-bunyavirus targets.

Modulation of Potassium Channels Inhibits Bunyavirus Infection.

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare, and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K(+)) channels to infect cells. Time of addition assays using K(+) channel modulating agents demonstrated that K(+) channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K(+) channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, two-pore domain K(+) channels (K2P) were identified as the K(+) channel family mediating BUNV K(+) channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

Antiviral activity of type I interferons and interleukins 29 and 28a (type III interferons) against Apeu virus.

Interferons (IFNs) are cytokines with important immunomodulatory activity in vertebrates. Although type I IFNs and interleukins (IL) 29 and 28a (type III IFNs) bind to different cellular receptors and have distinct structures, most of their biological activities are redundant. Apeu virus (APEUV) is a member of the Bunyaviridae family isolated from the Brazilian rain forest. In this paper we evaluated the antiviral activity of type I and type III IFNs against APEUV. All tested IFNs were able to induce an antiviral state against the virus in a dose-dependent way. The activity of type III IFNs did not need the presence of type I IFNs. Mixing both types of IFNs did not improve the biological activity of each type alone. The tested IFNs were also able to protect human peripheral blood mononuclear cells from infection. IFN alpha2, IFN beta, IL-29 and IL-28a induced the expression of 2',5'-oligoadenylate synthetase (2'5'OAS) and 6-16 genes. Although MxA gene was related to antiviral activity against Bunyaviruses, there was no induction of MxA in our model. We were able to show activity of type I and type III IFNs against a RNA virus, and that this activity is not dependent on MxA gene.

In vitro and in vivo studies of the Interferon-alpha action on distinct Orthobunyavirus.

Oropouche, Caraparu, Guama, Guaroa and Tacaiuma viruses (Orthobunyavirus genus) cause human febrile illnesses and/or encephalitis. To achieve a therapeutical agent to prevent and/or treat these diseases we evaluated the antiviral action of Interferon-alpha (IFN-alpha) on these orthobunyaviruses. In vitro results showed that all the studied orthobunyaviruses are susceptible to antiviral action of IFN-alpha, but this susceptibility is limited and dependent on both concentration of drug and treatment period. In vivo results demonstrated that IFN-alpha present antiviral action on Oropouche and Guaroa viruses when used as a prophylactic treatment. Moreover, a treatment initiated 3h after infection prevented the death of Guaroa virus infected-mice. Additionally, mortality of mice was related to the migration and replication of viruses in their brains. Our results suggest that IFN-alpha could be potentially useful in the prevention of diseases caused by Oropouche virus and in the prevention and/or treatment of diseases caused by Guaroa virus.

In vitro study of antiviral activity of mycophenolic acid on Brazilian orthobunyaviruses.

OBJECTIVE: Oropouche, Caraparu, Guama, Guaroa and Tacaiuma are ssRNA viruses that belong to the genus Orthobunyavirus and have been associated with human febrile illnesses and/or encephalitis. In this study, we evaluated the antiviral action of mycophenolic acid (MPA) on theseorthobunyaviruses to achieve a therapeutic agent to treat the diseases caused by these viruses. METHODS: The in vitro antiviral evaluation to MPA was done by using plaque assay at different periods of treatment. RESULTS: Results showed that MPA at a concentration of 10 microg/ml has significant antiviral activity on Tacaiuma virus when treatment was initiated either 24 h before or 2 h after viral infection. Moreover, MPA has an inhibitory effect on Guama virus replication, but only when treatment was initiated before cell infection. Addition of guanosine in the culture reverted the inhibitory effect of MPA on Tacaiuma and Guama viruses, suggesting that the antiviral activity of this substance was via depletion of the intracellular guanosine pool. CONCLUSION: Our results suggest that MPA would not be a good therapeutic agent to treat the diseases caused by Oropouche, Caraparu, Guama, Guaroa, and Tacaiuma viruses.

In vitro and in vivo studies of ribavirin action on Brazilian Orthobunyavirus.

Oropouche, Caraparu, Guama, Guaroa, and Tacaiuma are viruses (genus Orthobunyavirus) that cause human febrile illnesses and encephalitis. The goal of this study was to evaluate the antiviral action of ribavirin on these orthobunyaviruses to achieve a therapeutical agent to treat the diseases caused by these viruses. In vitro results showed that ribavirin (50 microg/mL) had antiviral activity only on the Tacaiuma virus. Addition of guanosine in the culture reversed the antiviral effect of ribavirin on Tacaiuma virus, suggesting that ribavirin inhibited this virus by reducing the intra-cellular guanosine pool. Moreover, ribavirin was not an effective drug in vivo because it was unable to inhibit the death of the mice or virus replication in the brain. The results suggest that ribavirin has no antiviral activity on the Oropouche, Caraparu, Guama, Guaroa, or Tacaiuma viruses; consequently, ribavirin would not be a good therapeutical agent to treat these arboviruses.

Virus inactivation by nucleic acid extraction reagents.

Many assume that common methods to extract viral nucleic acids are able to render a sample non-infectious. It may be that inactivation of infectious virus is incomplete during viral nucleic acid extraction methods. Accordingly, two common viral nucleic acid extraction techniques were evaluated for the ability to inactivate high viral titer specimens. In particular, the potential for TRIzol LS Reagent (Invitrogen Corp., Carlsbad, CA) and AVL Buffer (Qiagen, Valencia, CA) were examined to render suspensions of alphaviruses, flaviviruses, filoviruses and a bunyavirus non-infectious to tissue culture assay. The dilution series for both extraction reagents consistently caused cell death through a 100-fold dilution. Except for the DEN subtype 4 positive control, all viruses had titers of at least 10(6)pfu/ml. No plaques were detected in any extraction reagent plus virus combination in this study, therefore, the extraction reagents appeared to inactivate completely each of the high-titer viruses used in this study. These results support the reliance upon either TRIzol LS Reagent or AVL Buffer to render clinical or environmental samples non-infectious, which has implications for the handling and processing of samples under austere field conditions and low level containment.

Effect of triethylamine on the recovery of selected South American alphaviruses, flaviviruses, and bunyaviruses from mosquito (Diptera: Culicidae) pools.

We evaluated the effect of triethylamine (TEA) on the recovery of infectious virus from pools of mosquitoes for two South American alphaviruses (eastern equine encephalomyelitis and Venezuelan equine encephalomyelitis subtypes IIIC and ID), one flavivirus (Ilheus) and two bunyaviruses (Mirim [Guama group] and Itaqui [group C]). Mosquitoes were inoculated intrathoracically with virus, held for 7-10 d at 26 degrees C, and handled under one of four regimens before testing for the presence of virus by plaque assay. Mosquitoes were killed by freezing at - 70 degrees C for 3 min and tested immediately for the presence of virus; killed by freezing at -70 degrees C for 3 min and then held at room temperature for 1 h before testing for the presence of virus; anesthetized with TEA and assayed immediately for the presence of virus; or anesthetized with TEA and then held at room temperature for 1 h before being assayed for the presence of virus. For each of the viruses tested, viral titers in mosquitoes anesthetized with TEA were similar to those in mosquitoes killed by freezing at-70 degrees C. Likewise, there was no significant difference in viral titers in mosquitoes anesthetized with TEA and held at room temperature for 1 h or in mosquitoes frozen at -70 degrees C and held at room temperature for 1 h before being processed for virus by isolation. Triethylamine is advantageous for the handling of mosquitoes in a field environment. The elimination of the need for a cold chain, without compromising virus recovery, increases the feasibility of conducting research projects requiring the isolation of live virus from mosquitoes in remote tropical environments.

Characterization of murine Caraparu Bunyavirus liver infection and immunomodulator-mediated antiviral protection.

A rapid, peripheral disease model utilizing the Bunyavirus, Caraparu, was established in mice for the evaluation of antiviral therapy with immunomodulators. 4-6-week-old B6C3F1 female mice, inoculated intraperitoneally with virus, developed coagulative liver necrosis and died between 4-6 days after infection. This Caraparu disease model was relatively resistant to treatment with immunomodulators, such as ABMP, Ampligen, alpha-interferon (IFN-alpha) or beta-interferon (IFN-beta). However, a significant increase in median survival time (MST) was consistently observed upon treatment with gamma-interferon (IFN-gamma). The nucleoside analog--ribavirin--was highly effective against Caraparu virus in repeated treatment schedules begun on either day -1, day 0, or day +1 of infection. Ribavirin gave little protection when initiation of treatment was delayed until day +2. However, combined treatment with IFN-gamma, starting on day 0 and ribavirin starting on day +2, significantly reduced mortality.