Inactivation of yellow fever virus by WHO-recommended hand rub formulations and surface disinfectants.

Despite continued outbreaks of yellow fever virus (YFV) in endemic regions, data on its environmental stability or guidelines for its effective inactivation is limited. Here, we evaluated the susceptibility of the YFV 17D vaccine strain to inactivation by ethanol, 2-propanol, World Health Organization (WHO)-recommended hand rub formulations I and II, as well as surface disinfectants. In addition, two pathogenic strains were tested to compare inactivation kinetics by WHO-recommended hand rub formulations I and II. Furthermore, environmental stability of the vaccine strain was assessed. YFV 17D particles displayed infectivity half-life decay profiles of ~13 days at room temperature. Despite this extended environmental stability, YFV was efficiently inactivated by alcohols, WHO-recommended hand formulations, and four out of five tested surface disinfectants. These results are useful in defining disinfection protocols to prevent non-vector borne YFV transmission.

A yellow fever virus NS4B inhibitor not only suppresses viral replication, but also enhances the virus activation of RIG-I-like receptor-mediated innate immune response.

Flavivirus infection of cells induces massive rearrangements of the endoplasmic reticulum (ER) membrane to form viral replication organelles (ROs) which segregates viral RNA replication intermediates from the cytoplasmic RNA sensors. Among other viral nonstructural (NS) proteins, available evidence suggests for a prominent role of NS4B, an ER membrane protein with multiple transmembrane domains, in the formation of ROs and the evasion of the innate immune response. We previously reported a benzodiazepine compound, BDAA, which specifically inhibited yellow fever virus (YFV) replication in cultured cells and in vivo in hamsters, with resistant mutation mapped to P219 of NS4B protein. In the following mechanistic studies, we found that BDAA specifically enhances YFV induced inflammatory cytokine response in association with the induction of dramatic structural alteration of ROs and exposure of double-stranded RNA (dsRNA) in virus-infected cells. Interestingly, the BDAA-enhanced cytokine response in YFV-infected cells is attenuated in RIG-I or MAD5 knockout cells and completely abolished in MAVS knockout cells. However, BDAA inhibited YFV replication at a similar extent in the parent cells and cells deficient of RIG-I, MDA5 or MAVS. These results thus provided multiple lines of biological evidence to support a model that BDAA interaction with NS4B may impair the integrity of YFV ROs, which not only inhibits viral RNA replication, but also promotes the release of viral RNA from ROs, which consequentially activates RIG-I and MDA5. Although the innate immune enhancement activity of BDAA is not required for its antiviral activity in cultured cells, its dual antiviral mechanism is unique among all the reported antiviral agents thus far and warrants further investigation in animal models in future.

AT-752, a double prodrug of a guanosine nucleotide analog, inhibits yellow fever virus in a hamster model.

Yellow fever virus (YFV) is a zoonotic pathogen re-emerging in parts of the world, causing a viral hemorrhagic fever associated with high mortality rates. While an effective vaccine is available, having an effective antiviral against YFV is critical against unexpected outbreaks, or when vaccination is not recommended. We have previously identified AT-281, the free base of AT-752, an orally available double prodrug of a guanosine nucleotide analog, as a potent inhibitor of YFV in vitro, with a 50% effective concentration (EC50) of 0.31 µM. In hamsters infected with YFV (Jimenez strain), viremia rose about 4 log10-fold and serum alanine aminotransferase (ALT) 2-fold compared to sham-infected animals. Treatment with 1000 mg/kg AT-752 for 7 days, initiated 4 h prior to viral challenge, reduced viremia to below the limit of detection by day 4 post infection (pi) and returned ALT to normal levels by day 6 pi. When treatment with AT-752 was initiated 2 days pi, the virus titer and ALT dropped >2 log10 and 53% by day 4 and 6 pi, respectively. In addition, at 21 days pi, 70-100% of the infected animals in the treatment groups survived compared to 0% of the untreated group (p<0.001). Moreover, in vivo formation of the active triphosphate metabolite AT-9010 was measured in the animal tissues, with the highest concentrations in liver and kidney, organs that are vulnerable to the virus. The demonstrated in vivo activity of AT-752 suggests that it is a promising compound for clinical development in the treatment of YFV infection.

Inhibition of Orbivirus Replication by Fluvastatin and Identification of the Key Elements of the Mevalonate Pathway Involved.

Statin derivatives can inhibit the replication of a range of viruses, including hepatitis C virus (HCV, Hepacivirus), dengue virus (Flavivirus), African swine fever virus (Asfarviridae) and poliovirus (Picornaviridae). We assess the antiviral effect of fluvastatin in cells infected with orbiviruses (bluetongue virus (BTV) and Great Island virus (GIV)). The synthesis of orbivirus outer-capsid protein VP2 (detected by confocal immunofluorescence imaging) was used to assess levels of virus replication, showing a reduction in fluvastatin-treated cells. A reduction in virus titres of ~1.7 log (98%) in fluvastatin-treated cells was detected by a plaque assay. We have previously identified a fourth non-structural protein (NS4) of BTV and GIV, showing that it interacts with lipid droplets in infected cells. Fluvastatin, which inhibits 3-hydroxy 3-methyl glutaryl CoA reductase in the mevalonic acid pathway, disrupts these NS4 interactions. These findings highlight the role of the lipid pathways in orbivirus replication and suggest a greater role for the membrane-enveloped orbivirus particles than previously recognised. Chemical intermediates of the mevalonic acid pathway were used to assess their potential to rescue orbivirus replication. Pre-treatment of IFNAR(-/-) mice with fluvastatin promoted their survival upon challenge with live BTV, although only limited protection was observed.

Alkylated benzimidazoles: Design, synthesis, docking, DFT analysis, ADMET property, molecular dynamics and activity against HIV and YFV.

A series of alkylated benzimidazole derivatives was synthesized and screened for their anti-HIV, anti-YFV, and broad-spectrum antiviral properties. The physicochemical parameters and drug-like properties of the compounds were assessed first, and then docking studies and MD simulations on HIV-RT allosteric sites were conducted to find the possible mode of their action. DFT analysis was also performed to confirm the nature of the hydrogen bonding interaction of active compounds. The in silico studies indicated that the molecules behaved like possible NNRTIs. The nature - polar or non-polar and position of the substituent present at fifth, sixth, and N-1 positions of the benzimidazole moiety played an important role in determining the antiviral properties of the compounds. Among the various compounds, 2-(5,6-dibromo-2-chloro-1H-benzimidazol-1-yl)ethan-1-ol (3a) showed anti-HIV activity with an appreciably low IC50 value as 0.386 × 10-5µM. Similarly, compound 2b, 3-(2-chloro-5-nitro-1H-benzimidazol-1-yl) propan-1-ol, showed excellent inhibitory property against the yellow fever virus (YFV) with EC50 value as 0.7824 × 10-2µM.

Generation of a reporter yellow fever virus for high throughput antiviral assays.

Yellow fever virus (YFV), a member of the Flaviviridae family, is an arthropod-borne virus that can cause severe disease in humans with a lethality rate of up to 60%. Since 2017, increases in YFV activity in areas of South America and Africa have been described. Although a vaccine is available, named strain 17D (Theiler and Smith, 1937), it is contraindicated for use in the elderly, expectant mothers, immunocompromised people, among others. To this day there is no antiviral treatment against YFV to reduce the severity of viral infection. Here, we used a circular polymerase extension reaction (CPER)-based reverse genetics approach to generate a full-length reporter virus (YFVhb) by introducing a small HiBit tag in the NS1 protein. The reporter virus replicates at a similar rate to the parental YFV in HuH-7 cells. Using YFVhb, we designed a high throughput antiviral screening luciferase-based assay to identify inhibitors that target any step of the viral replication cycle. We validated our assay by using a range of inhibitors including drugs, immune sera and neutralizing single chain variable fragments (scFv). In light of the recent upsurge in YFV and a potential spread of the virus, this assay is a further tool in the development of antiviral therapy against YFV.

Development of antibody-based assays for high throughput discovery and mechanistic study of antiviral agents against yellow fever virus.

Despite the availability of a highly effective yellow fever virus (YFV) vaccine, outbreaks of yellow fever frequently occur in Africa and South America with significant mortality, highlighting the pressing need for antiviral drugs to manage future outbreaks. To support the discovery and development of antiviral drugs against YFV, we characterized a panel of rabbit polyclonal antibodies against the three YFV structural proteins and five non-structural proteins and demonstrated these antibody reagents in conjunction with viral RNA metabolic labeling, double-stranded RNA staining and membrane floatation assays as powerful tools for investigating YFV polyprotein processing, replication complex formation, viral RNA synthesis and high throughput discovery of antiviral drugs. Specifically, the proteolytic processing of the viral polyprotein can be analyzed by Western blot assays. The predominant nuclear localization of NS5 protein as well as the relationship between intracellular viral non-structural protein distribution and foci of YFV RNA replication can be revealed by immunofluorescence staining and membrane flotation assays. Using an antibody against YFV NS4B protein as an example, in-cell western and high-content imaging assays have been developed for high throughput discovery of antiviral agents. A synergistic antiviral effect of an YFV NS4B-targeting antiviral agent BDAA and a NS5 RNA-dependent RNA polymerase inhibitor (Sofosbuvir) was also demonstrated with the high-content imaging assay. Apparently, the antibody-based assays established herein not only facilitate the discovery and development of antiviral agents against YFV, but also provide valuable tools to dissect the molecular mechanism by which the antiviral agents inhibit YFV replication.

Insights into the antiviral activity of phospholipases A2 (PLA2s) from snake venoms.

Viruses are associated with several human diseases that infect a large number of individuals, hence directly affecting global health and economy. Owing to the lack of efficient vaccines, antiviral therapy and emerging resistance strains, many viruses are considered as a potential threat to public health. Therefore, researches have been developed to identify new drug candidates for future treatments. Among them, antiviral research based on natural molecules is a promising approach. Phospholipases A2 (PLA2s) isolated from snake venom have shown significant antiviral activity against some viruses such as Dengue virus, Human Immunodeficiency virus, Hepatitis C virus and Yellow fever virus, and have emerged as an attractive alternative strategy for the development of novel antiviral therapy. Thus, this review provides an overview of remarkable findings involving PLA2s from snake venom that possess antiviral activity, and discusses the mechanisms of action mediated by PLA2s against different stages of virus replication cycle. Additionally, molecular docking simulations were performed by interacting between phospholipids from Dengue virus envelope and PLA2s from Bothrops asper snake venom. Studies on snake venom PLA2s highlight the potential use of these proteins for the development of broad-spectrum antiviral drugs.

Phase 1 Trial of a Therapeutic Anti-Yellow Fever Virus Human Antibody.

BACKGROUND: Insufficient vaccine doses and the lack of therapeutic agents for yellow fever put global health at risk, should this virus emerge from sub-Saharan Africa and South America. METHODS: In phase 1a of this clinical trial, we assessed the safety, side-effect profile, and pharmacokinetics of TY014, a fully human IgG1 anti-yellow fever virus monoclonal antibody. In a double-blind, phase 1b clinical trial, we assessed the efficacy of TY014, as compared with placebo, in abrogating viremia related to the administration of live yellow fever vaccine (YF17D-204; Stamaril). The primary safety outcomes were adverse events reported 1 hour after the infusion and throughout the trial. The primary efficacy outcome was the dose of TY014 at which 100% of the participants tested negative for viremia within 48 hours after infusion. RESULTS: A total of 27 healthy participants were enrolled in phase 1a, and 10 participants in phase 1b. During phase 1a, TY014 dose escalation to a maximum of 20 mg per kilogram of body weight occurred in 22 participants. During phases 1a and 1b, adverse events within 1 hour after infusion occurred in 1 of 27 participants who received TY014 and in none of the 10 participants who received placebo. At least one adverse event occurred during the trial in 22 participants who received TY014 and in 8 who received placebo. The mean half-life of TY014 was approximately 12.8 days. At 48 hours after the infusion, none of the 5 participants who received the starting dose of TY014 of 2 mg per kilogram had detectable YF17D-204 viremia; these participants remained aviremic throughout the trial. Viremia was observed at 48 hours after the infusion in 2 of 5 participants who received placebo and at 72 hours in 2 more placebo recipients. Symptoms associated with yellow fever vaccine were less frequent in the TY014 group than in the placebo group. CONCLUSIONS: This phase 1 trial of TY014 did not identify worrisome safety signals and suggested potential clinical benefit, which requires further assessment in a phase 2 trial. (Funded by Tysana; ClinicalTrials.gov number, NCT03776786.).

Confronting the Multidimensional Challenges of Research in the Context of Emerging Infectious Diseases in Brazil: The Example of Yellow Fever.

In the most recent Brazilian yellow fever (YF) outbreak, a group of clinicians and researchers initiated in mid-January 2018 a considerable effort to develop a multicenter randomized controlled clinical trial to evaluate the effect of sofosbuvir on YF viremia and clinical outcomes (Brazilian Clinical Trials Registry: RBR-93dp9n). The approval of this protocol had urgency given the seasonal/short-lived pattern of YF transmission, large number of human cases, and epidemic transmission at the outskirts of a large urban center. However, many intricacies in the research regulatory and ethical submission systems in Brazil were indomitable even under such pressing conditions. By April 2018, we had enrolled 29 patients for a target sample size of 90 participants. Had enrollment been initiated 3 weeks earlier, an additional 31 patients could have been enrolled, reaching the prespecified sample size for the interim analysis. This recent experience highlights the urgent need to improve local preparedness for research in the setting of explosive outbreaks, as has been seen in the last few years in different countries.

Inactivation of yellow fever virus with amotosalen and ultraviolet A light pathogen-reduction technology.

BACKGROUND: The reemergence of yellow fever virus (YFV) in Africa and Brazil, and massive vaccine campaigns triggered to contain the outbreaks, have raised concerns over blood transfusion safety and availability with increased risk of YFV transfusion-transmitted infections (TTIs) by native and vaccine-acquired YFV. Blood donor deferral for 2 to 4 weeks following live attenuated YFV vaccination, and deferral for travel to endemic/epidemic areas, may result in blood donor loss and impact platelet component (PC) stocks. This study investigated the efficacy of INTERCEPT Blood System pathogen reduction (PR) with use of amotosalen and ultraviolet A (UVA) light to inactivate high levels of YFV in PCs. MATERIALS: Four units of apheresis platelets prepared in 35% plasma/65% platelet additive solution (PC-PAS) and 4 units of PC in 100% human plasma (PC-Plasma) were spiked with high infectious titers of YFV (YFV-17D vaccine strain). YFV-17D infectious titers were measured by plaque assay and expressed as plaque-forming units (PFU) before and after amotosalen/UVA treatment to determine log reduction. RESULTS: The mean YFV-17D infectious titers in PC before inactivation were 5.5 ± 0.1 log PFU/mL in PC-PAS and 5.3 ± 0.1 log PFU/mL in PC-Plasma. No infectivity was detected immediately after amotosalen/UVA treatment. CONCLUSION: The amotosalen/UVA PR system inactivated high titers of infectious YFV-17D in PC. This PR technology could reduce the risk of YFV TTI and help secure PC supplies in areas experiencing YFV outbreaks where massive vaccination campaigns are required.

[Development of inactivated cultural yellow fever vaccine].

INTRODUCTION: The only currently available live vaccine against yellow fever (YF) based on chicken embryos infected with an attenuated 17D strain of the YF virus is one of the most effective vaccine preparations. However, the live vaccine is associated with "viscerotropic syndrome" (approximately 0.4 cases per 100 000 vaccinated). Therefore, the development and introduction of highly purified inactivated vaccine against YF is intended to ensure the maximum safety of vaccination against one of the most common human viral diseases.Goals and objectives. Development and evaluation of immunogenicity of the cultural inactivated vaccine against YF at the laboratory model level. MATERIAL AND METHODS: Adaptation of 17D strain of YF virus to Vero cell culture, cultivation, removal of cellular DNA, inactivation with ß-propiolactone, concentration, chromatographic purification, determination of protein and antigen of YF virus, assessment of immunogenicity in mice in parallel with commercial live vaccine. RESULTS AND DISCUSSION: Immunogenicity: the determination of specific antibodies of class G (IgG) and virus neutralizing antibodies in the sera of immunized mice showed high level of antibodies exceeding that of immunized with commercial live vaccine. The optimal dose of antigen in the vaccine (total protein) was 50 µg/ml (5 µg/0.1 ml -dose and volume per 1 vaccination of mice). Thus, the laboratory version of cultural inactivated vaccine against YF is as effective (and even superior) as the commercial live vaccine. CONCLUSION: Laboratory version of cultural inactivated vaccine against YF, which is not inferior in immunogenicity (in animal model) to commercial live vaccine, has been developed.

Sofosbuvir inhibits yellow fever virus in vitro and in patients with acute liver failure.

INTRODUCTION AND OBJECTIVES: Direct antiviral agents (DAAs) are very efficient in inhibiting hepatitis C virus and might be used to treat infections caused by other flaviviruses whose worldwide detection has recently increased. The aim of this study was to verify the efficacy of DAAs in inhibiting yellow fever virus (YFV) by using drug repositioning (a methodology applied in the pharmaceutical industry to identify new uses for approved drugs). MATERIALS AND METHODS: Three DAAs were evaluated: daclatasvir, sofosbuvir and ledipasvir or their combinations. For in vitro assays, the drugs were diluted in 100% dimethyl sulfoxide. Vaccine strain 17D and a 17D strain expressing the reporter fluorescent protein were used in the assays. A fast and reliable cell-based screening assay using Vero cells or Huh-7 cells (a hepatocyte-derived carcinoma ell line) was carried out. Two patients who acquired yellow fever virus with acute liver failure were treated with sofosbuvir for one week as a compassionate use. RESULTS: Using a high-content screening assay, we verified that sofosbuvir presented the best antiviral activity against YFV. Moreover, after an off-label treatment with sofosbuvir, the two female patients diagnosed with yellow fever infection displayed a reduction in blood viremia and an improvement in the course of the disease, which was observed in the laboratory medical parameters related to disease evolution. CONCLUSIONS: Sofosbuvir may be used as an option for treatment against YFV until other drugs are identified and approved for human use. These results offer insights into the role of nonstructural protein 5 (NS5) in YFV inhibition and suggest that nonstructural proteins may be explored as drug targets for YFV treatment.

The Role of NS5 Protein in Determination of Host Cell Range for Yellow Fever Virus.

Yellow fever virus (YFV) tropism is restricted to human and nonhuman primates. The nonstructural protein 5 (NS5) protein of YFV binds to primate signal transducer and activator of transcription 2 (STAT2) and antagonizes interferon (IFN) signaling. However, YFV NS5 is unable to bind mouse STAT2 and antagonize murine IFN signaling. A similar observation has been made with the NS5 protein of both dengue virus (DENV) and Zika virus (ZIKV). However, the key difference between the NS5 protein of YFV and those of DENV and ZIKV is that YFV NS5 binds human STAT2 in an IFN-dependent manner. In human cells, IFN-I treatment induces K63-linked ubiquitination on lysine (K) 6 of YFV NS5, which is required for binding human STAT2. This IFN-induced ubiquitination of YFV NS5 is absent in murine cells resulting in the lack of binding of YFV NS5 and human STAT2 in murine cells. This highlights the importance of YFV NS5 ubiquitination in determining the host cell range for YFV.

Midgut barriers prevent the replication and dissemination of the yellow fever vaccine in Aedes aegypti.

BACKGROUND: To be transmitted to vertebrate hosts via the saliva of their vectors, arthropod-borne viruses have to cross several barriers in the mosquito body, including the midgut infection and escape barriers. Yellow fever virus (YFV) belongs to the genus Flavivirus, which includes human viruses transmitted by Aedes mosquitoes, such as dengue and Zika viruses. The live-attenuated YFV-17D vaccine has been used safely and efficiently on a large scale since the end of World War II. Early studies have shown, using viral titration from salivary glands of infected mosquitoes, that YFV-17D can infect Aedes aegypti midgut, but does not disseminate to other tissues. METHODOLOGY/PRINCIPAL FINDINGS: Here, we re-visited this issue using a panel of techniques, such as RT-qPCR, Western blot, immunofluorescence and titration assays. We showed that YFV-17D replication was not efficient in Aedes aegypti midgut, as compared to the clinical isolate YFV-Dakar. Viruses that replicated in the midgut failed to disseminate to secondary organs. When injected into the thorax of mosquitoes, viruses succeeded in replicating into midgut-associated tissues, suggesting that, during natural infection, the block for YFV-17D replication occurs at the basal membrane of the midgut. CONCLUSIONS/SIGNIFICANCE: The two barriers associated with Ae. aegypti midgut prevent YFV-17D replication. Our study contributes to our basic understanding of vector-pathogen interactions and may also aid in the development of non-transmissible live virus vaccines.

Nucleoside Analogs with Antiviral Activity against Yellow Fever Virus.

Yellow fever virus (YFV) is a human Flavivirus reemerging in parts of the world. While a vaccine is available, large outbreaks have recently occurred in Brazil and certain African countries. Development of an effective antiviral against YFV is crucial, as there is no available effective drug against YFV. We have identified several novel nucleoside analogs with potent antiviral activity against YFV with 50% effective concentration (EC50) values between 0.25 and 1 µM with selectivity indices over 100 in culture.

Structure of the yellow fever NS5 protein reveals conserved drug targets shared among flaviviruses.

Yellow fever virus (YFV) is responsible for devastating outbreaks of Yellow fever (YF) in humans and is associated with high mortality rates. Recent large epidemics and epizootics and exponential increases in the numbers of YF cases in humans and non-human primates highlight the increasing threat YFV poses, despite the availability of an effective YFV vaccine. YFV is the first human virus discovered, but the structures of several of the viral proteins remain poorly understood. Here we report the structure of the full-length NS5 protein, a key enzyme for the replication of flaviviruses that contains both a methyltransferase domain and an RNA dependent RNA polymerase domain, at 3.1 Šresolution. The viral polymerase adopts right-hand fold, demonstrating the similarities of the Yellow fever, Dengue and Zika polymerases. Together this data suggests NS5 as a prime and ideal target for the design of pan-flavivirus inhibitors.

Inactivation of yellow fever virus in plasma after treatment with methylene blue and visible light and in platelet concentrates following treatment with ultraviolet C light.

BACKGROUND: Yellow fever virus (YFV) is endemic to tropical and subtropical areas in South America and Africa, and is currently a major public health threat in Brazil. Transfusion transmission of the yellow fever vaccine virus has been demonstrated, which is indicative of the potential for viral transfusion transmission. An approach to manage the potential YFV transfusion transmission risk is the use of pathogen inactivation (PI) technology systems, such as THERAFLEX MB-Plasma and THERAFLEX UV-Platelets (Macopharma). We aimed to investigate the efficacy of these PI technology systems to inactivate YFV in plasma or platelet concentrates (PCs). STUDY DESIGN AND METHODS: YFV spiked plasma units were treated using THERAFLEX MB-Plasma system (visible light doses: 20, 40, 60, and 120 [standard] J/cm2 ) in the presence of methylene blue (approx. 0.8 µmol/L) and spiked PCs were treated using THERAFLEX UV-Platelets system (ultraviolet C doses: 0.05, 0.10, 0.15, and 0.20 [standard] J/cm2 ). Samples were taken before the first and after each illumination dose and tested for residual virus using a modified plaque assay. RESULTS: YFV infectivity was reduced by an average of 4.77 log or greater in plasma treated with the THERAFLEX MB-Plasma system and by 4.8 log or greater in PCs treated with THERAFLEX UV-Platelets system. CONCLUSIONS: Our study suggests the THERAFLEX MB-Plasma and the THERAFLEX UV-Platelets systems can efficiently inactivate YFV in plasma or PCs to a similar degree as that for other arboviruses. Given the reduction levels observed in this study, these PI technology systems could be an effective option for managing YFV transfusion-transmission risk in plasma and PCs.

Broad spectrum anti-flavivirus pyridobenzothiazolones leading to less infective virions.

We report the design, synthesis, and biological evaluation of a class of 1H-pyrido[2,1-b][1,3]benzothiazol-1-ones originated from compound 1, previously identified as anti-flavivirus agent. Some of the new compounds showed activity in low µM range with reasonable selectivity against Dengue 2, Yellow fever (Bolivia strain), and West Nile viruses. One of the most interesting molecules, compound 16, showed broad antiviral activity against additional flaviviruses such as Dengue 1, 3 and 4, Zika, Japanese encephalitis, several strains of Yellow fever, and tick-borne encephalitis viruses. Compound 16 did not exert any effect on alphaviruses and phleboviruses and its activity was maintained in YFV infected cells from different species. The activity of 16 appears specific for flavivirus with respect to other virus families, suggesting, but not proving, that it might be targeting a viral factor. We demonstrated that the antiviral effect of 16 is not related to reduced viral RNA synthesis or virion release. On the contrary, viral particles grown in the presence of 16 showed reduced infectivity, being unable to perform a second round of infection. The chemical class herein presented thus emerges as suitable to provide pan-flavivirus inhibitors.

Expression, purification and virucidal activity of two recombinant isoforms of phospholipase A2 from Crotalus durissus terrificus venom.

The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds. Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A2 (PLA2-CB). We showed previously the antiviral effect of PLA2-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer. The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.