Instrução de trabalho para colheita, conservação e transporte de amostras de esfregaço/fluído lesão para diagnóstico de Varíola dos Macacos (Monkeypox) / Work instructions for collecting, preserving and transporting swab samples/lesion fluid for Monkeypox diagnosisWork instructions for collecting, preserving and transporting swab samples/lesion fluid for Monkeypox diagnosis

Para o diagnóstico de varíola dos macacos (Monkeypox), amostras de esfregaço e fluído da lesão são as recomendadas para diagnóstico. Amostras de esfregaço nasofaríngeo, esfregaço orofaríngeo, biopsia, sangue total e soro são amostras complementares às de esfregaço/ fluído da lesão e podem ser colhidas para aumentar a capacidade de detecção. Nesta instrução de trabalho, o foco são amostras de esfregaço/fluído da lesão...

Pharmacokinetics and Efficacy of a Potential Smallpox Therapeutic, Brincidofovir, in a Lethal Monkeypox Virus Animal Model.

Smallpox, caused by Variola virus (VARV), was eradicated in 1980; however, VARV bioterrorist threats still exist, necessitating readily available therapeutics. Current preparedness activities recognize the importance of oral antivirals and recommend therapeutics with different mechanisms of action. Monkeypox virus (MPXV) is closely related to VARV, causing a highly similar clinical human disease, and can be used as a surrogate for smallpox antiviral testing. The prairie dog MPXV model has been characterized and used to study the efficacy of antipoxvirus therapeutics, including recently approved TPOXX (tecovirimat). Brincidofovir (BCV; CMX001) has shown antiviral activity against double-stranded DNA viruses, including poxviruses. To determine the exposure of BCV following oral administration to prairie dogs, a pharmacokinetics (PK) study was performed. Analysis of BCV plasma concentrations indicated variability, conceivably due to the outbred nature of the animals. To determine BCV efficacy in the MPXV prairie dog model, groups of animals were intranasally challenged with 9 × 105 plaque-forming units (PFU; 90% lethal dose [LD90]) of MPXV on inoculation day 0 (ID0). Animals were divided into groups based on the first day of BCV treatment relative to inoculation day (ID-1, ID0, or ID1). A trend in efficacy was noted dependent upon treatment initiation (57% on ID-1, 43% on ID0, and 29% on ID1) but was lower than demonstrated in other animal models. Analysis of the PK data indicated that BCV plasma exposure (maximum concentration [Cmax]) and the time of the last quantifiable concentration (AUClast) were lower than in other animal models administered the same doses, indicating that suboptimal BCV exposure may explain the lower protective effect on survival.IMPORTANCE Preparedness activities against highly transmissible viruses with high mortality rates have been highlighted during the ongoing coronavirus disease 2019 (COVID-19) pandemic. Smallpox, caused by variola virus (VARV) infection, is highly transmissible, with an estimated 30% mortality. Through an intensive vaccination campaign, smallpox was declared eradicated in 1980, and routine smallpox vaccination of individuals ceased. Today's current population has little/no immunity against VARV. If smallpox were to reemerge, the worldwide results would be devastating. Recent FDA approval of one smallpox antiviral (tecovirimat) was a successful step in biothreat preparedness; however, orthopoxviruses can become resistant to treatment, suggesting the need for multiple therapeutics. Our paper details the efficacy of the investigational smallpox drug brincidofovir in a monkeypox virus (MPXV) animal model. Since brincidofovir has not been tested in vivo against smallpox, studies with the related virus MPXV are critical in understanding whether it would be protective in the event of a smallpox outbreak.

Study of Antiviral Activity of Metabolites of a New Serratia species K-57 Strain.

The results of studies of a newly isolated Serratia species K-57 strain are presented. The strain is characterized by antiviral activity towards human influenza A/Aichi/2/68/H3N2, vaccinia, mouse smallpox, and herpes simplex-2 viruses. The detected characteristics of the strain, including the data on activities on nucleolytic enzymes, recommend it for the development of therapeutic and preventive antiviral drugs.

Drug Development against Smallpox: Present and Future.

Forty years after the last endemic smallpox case, variola virus (VARV) is still considered a major threat to humans due to its possible use as a bioterrorism agent. For many years, the risk of disease reemergence was thought to solely be through deliberate misuse of VARV strains kept in clandestine laboratories. However, recent experiments using synthetic biology have proven the feasibility of recreating a poxvirus de novo, implying that VARV could, in theory, be resurrected. Because of this new perspective, the WHO Advisory Committee on VARV Research released new recommendations concerning research on poxviruses that strongly encourages pursuing the development of new antiviral drugs against orthopoxviruses. In 2018, the U.S. FDA advised in favor of two molecules for smallpox treatment, tecovirimat and brincidofovir. This review highlights the difficulties to develop new drugs targeting an eradicated disease, especially as it requires working under the FDA "animal efficacy rule" with the few, and imperfect, animal models available.

Preliminary Screening and In Vitro Confirmation of Orthopoxvirus Antivirals.

The lack of antiviral drugs for the treatment of orthopoxvirus disease represents an unmet medical need, particularly due to the threat of variola virus (the causative agent of smallpox) as an agent of biowarfare or bioterrorism (Henderson, 283:1279-1282, 1999). In addition to variola, monkeypox, cowpox, and vaccinia viruses are orthopoxviruses of concern to human health (Lewis-Jones, 17:81-89, 2004). Smallpox vaccination, using the closely related vaccinia virus, is no longer provided to the general public leading to a worldwide population increasingly susceptible not only to variola but to monkeypox, cowpox, and vaccinia viruses as well. Orthopoxviruses share similar life cycles (Fenner et al., WHO, Geneva, 1988), and significant nucleotide and protein homology, and are immunologically cross-protective against other species within the genus, which was the basis of the highly successful vaccinia virus vaccine. These similarities also serve as the basis for screening for antivirals for dangerous pathogens such as variola and monkeypox virus using generally safer viruses such as cowpox and vaccinia. Methods for preliminary screening and initial characterization of potential orthopoxvirus antivirals in vitro, using vaccinia virus as a relatively safe surrogate for more pathogenic orthopoxviruses, are described herein. They include candidate identification in a viral cytopathic effect (CPE) assay as well as evaluation of the antiviral activity in inhibition assays to determine mean effective (or inhibitory) concentrations (EC50 or IC50). These assays were utilized in the identification and early characterization of tecovirimat (ST-246) (Yang et al., 79:13,139-13,149, 2005). These initial steps in identifying and characterizing the antiviral activity should be followed up with additional in vitro studies including specificity testing (for other orthopoxviruses and against other viruses), single-cycle growth curves, time of addition assays, cytotoxicity testing, and identification of the drug target.

The development and approval of tecoviromat (TPOXX®), the first antiviral against smallpox.

The classification of smallpox by the U.S. Centers for Disease Control and Prevention (CDC) as a Category A Bioterrorism threat agent has resulted in the U.S. Government investing significant funds to develop and stockpile a suite of medical countermeasures to ameliorate the consequences of a smallpox epidemic. This stockpile includes both vaccines for prophylaxis and antivirals to treat symptomatic patients. In this manuscript, we describe the path to approval for the first therapeutic against smallpox, identified during its development as ST-246, now known as tecovirimat and TPOXX®, a small-molecule antiviral compound sponsored by SIGA Technologies to treat symptomatic smallpox. Because the disease is no longer endemic, the development and approval of TPOXX® was only possible under the U.S. Food and Drug and Administration Animal Rule (FDA 2002). In this article, we describe the combination of animal model studies and clinical trials that were used to satisfy the FDA requirements for the approval of TPOXX ® under the Animal Rule.

The Role of Brincidofovir in Preparation for a Potential Smallpox Outbreak.

Smallpox (variola) virus is considered a Category A bioterrorism agent due to its ability to spread rapidly and the high morbidity and mortality rates associated with infection. Current recommendations recognize the importance of oral antivirals and call for having at least two smallpox antivirals with different mechanisms of action available in the event of a smallpox outbreak. Multiple antivirals are recommended due in large part to the propensity of viruses to become resistant to antiviral therapy, especially monotherapy. Advances in synthetic biology heighten concerns that a bioterror attack with variola would utilize engineered resistance to antivirals and potentially vaccines. Brincidofovir, an oral antiviral in late stage development, has proven effective against orthopoxviruses in vitro and in vivo, has a different mechanism of action from tecovirimat (the only oral smallpox antiviral currently in the US Strategic National Stockpile), and has a resistance profile that reduces concerns in the scenario of a bioterror attack using genetically engineered smallpox. Given the devastating potential of smallpox as a bioweapon, preparation of a multi-pronged defense that accounts for the most obvious bioengineering possibilities is strategically imperative.

[THE USE OF THE MODEL MOUSE ICR--VARIOLA VIRUS FOR EVALUATION OF ANTIVIRAL DRUG EFFICACY].

Mice of the ICR outbred population were infected intranasally (i/n) with the variola virus (VARV, strain Ind-3a). Clinical signs of the disease did not appear even at the maximum possible dose of the virus 5.2 lg PFU/head (plaque-forming units per head). In this case, 50% infective dose (ID50) of VARV estimated by the presence or absence of the virus in the lungs three days after infection (p.i.) was equal to 2.7 ± 0.4 lg PFU/head. Taking into account the 10% application of the virus in the lungs during the intranasal infection of the mice, it was adequate to 1.7 lg PFU/lungs. This indicates a high infectivity of the VARV for mice comparable to its infectivity for humans. After the i/n infection of mice with the VARV at a dose 30 ID50/ head the highest concentration of the virus detected in the lungs (4.9 ± 0.0 lg PFU/ml of homogenate) and in nasal cavity tissues (4.8 ± 0.0 lg PFU/ml) were observed. The pathomorphological changes in the respiratory organs of the mice infected with the VARV appeared at 3-5 days p.i., and the VARV reproduction noted in the epithelial cells and macrophages were noticed. When the preparations ST-246 and NIOCH-14 were administered orally at a dose of 60 µg/g of mouse weight up to one day before infection, after 2 hours, 1 and 2 days p.i., the VARV reproduction in the lungs after 3 days p.i. decreased by an order of magnitude. Thus, outbred ICR mice infected with the VARV can be used as a laboratory model of the smallpox when evaluating the therapeutic and prophylactic efficacy of the antismallpox drugs.

Using ICR and SCID mice as animal models for smallpox to assess antiviral drug efficacy.

The possibility of using immunocompetent ICR mice and immunodeficient SCID mice as model animals for smallpox to assess antiviral drug efficacy was investigated. Clinical signs of the disease did not appear following intranasal (i.n.) challenge of mice with strain Ind-3a of variola virus (VARV), even when using the highest possible dose of the virus (5.2 log10 p.f.u.). The 50 % infective doses (ID50) of VARV, estimated by the virus presence or absence in the lungs 3 and 4 days post-infection, were 2.7 ± 0.4 log10 p.f.u. for ICR mice and 3.5 ± 0.7 log10 p.f.u. for SCID mice. After i.n. challenge of ICR and SCID mice with VARV 30 and 50 ID50, respectively, steady reproduction of the virus occurred only in the respiratory tract (lungs and nose). Pathological inflammatory destructive changes were revealed in the respiratory tract and the primary target cells for VARV (macrophages and epithelial cells) in mice, similar to those in humans and cynomolgus macaques. The use of mice to assess antiviral efficacies of NIOCH-14 and ST-246 demonstrated the compliance of results with those described in scientific literature, which opens up the prospect of their use as an animal model for smallpox to develop anti-smallpox drugs intended for humans.

SiO2 nanoparticles as platform for delivery of 3'-triazole analogues of AZT-triphosphate into cells.

A system for delivery of analogues of AZT-triphosphates (AZT*TP) based on SiO2 nanoparticles was proposed. For this purpose, a simple and versatile method was developed for the preparation of SiO2∼dNTP conjugates using the 'click'-reaction between AZTTP and premodified nanoparticles containing the alkyne groups. The substrate properties of SiO2∼AZT*TP were tested using Klenow fragment and HIV reverse transcriptase. The 3'-triazole derivatives of thymidine triphosphate being a part of the SiO2∼AZT*TP nanocomposites were shown to be incorporated into the growing DNA chain. It was shown by confocal microscopy that the proposed SiO2∼AZT*TP nanocomposites penetrate into cells. These nanocomposites were shown to inhibit the reproduction of POX and Herpes viruses at nontoxic concentrations.

The efficacy and pharmacokinetics of brincidofovir for the treatment of lethal rabbitpox virus infection: a model of smallpox disease.

Brincidofovir (BCV) has broad-spectrum in vitro activity against dsDNA viruses, including smallpox, and is being developed as a treatment for smallpox as well as infections caused by other dsDNA viruses. BCV has previously been shown to be active in multiple animal models of smallpox. Here we present the results of a randomized, blinded, placebo-controlled study of the efficacy and pharmacokinetics of a novel, "humanized" regimen of BCV for treatment of New Zealand White rabbits infected with a highly lethal inoculum of rabbitpox virus, a well characterized model of smallpox. Compared with placebo, a dose-dependent increase in survival was observed in all BCV-treatment groups. Concentrations of cidofovir diphosphate (CDV-PP), the active antiviral, in rabbit peripheral blood mononuclear cells (PBMCs) were determined for comparison to those produced in humans at the dose proposed for treatment of smallpox. CDV-PP exposure in PBMCs from rabbits given BCV scaled to human exposures at the dose proposed for treatment of smallpox, which is also currently under evaluation for other indications. The results of this study demonstrate the activity of BCV in the rabbitpox model of smallpox and the feasibility of scaling doses efficacious in the model to a proposed human dose and regimen for treatment of smallpox.

In vitro efficacy of brincidofovir against variola virus.

Brincidofovir (CMX001), a lipid conjugate of the acyclic nucleotide phosphonate cidofovir, is under development for smallpox treatment using "the Animal Rule," established by the FDA in 2002. Brincidofovir reduces mortality caused by orthopoxvirus infection in animal models. Compared to cidofovir, brincidofovir has increased potency, is administered orally, and shows no evidence of nephrotoxicity. Here we report that the brincidofovir half-maximal effective concentration (EC50) against five variola virus strains in vitro averaged 0.11 µM and that brincidofovir was therefore nearly 100-fold more potent than cidofovir.

Preventing the return of smallpox: molecular modeling studies on thymidylate kinase from Variola virus.

Smallpox was one of the most devastating diseases in the human history and still represents a serious menace today due to its potential use by bioterrorists. Considering this threat and the non-existence of effective chemotherapy, we propose the enzyme thymidylate kinase from Variola virus (VarTMPK) as a potential target to the drug design against smallpox. We first built a homology model for VarTMPK and performed molecular docking studies on it in order to investigate the interactions with inhibitors of Vaccinia virus TMPK (VacTMPK). Subsequently, molecular dynamics (MD) simulations of these compounds inside VarTMPK and human TMPK (HssTMPK) were carried out in order to select the most promising and selective compounds as leads for the design of potential VarTMPK inhibitors. Results of the docking and MD simulations corroborated to each other, suggesting selectivity towards VarTMPK and, also, a good correlation with the experimental data.

Efficacy of tecovirimat (ST-246) in nonhuman primates infected with variola virus (Smallpox).

Naturally occurring smallpox has been eradicated but remains a considerable threat as a biowarfare/bioterrorist weapon (F. Fleck, Bull. World Health Organ. 81:917-918, 2003). While effective, the smallpox vaccine is currently not recommended for routine use in the general public due to safety concerns (http://www.bt.cdc.gov/agent/smallpox/vaccination). Safe and effective countermeasures, particularly those effective after exposure to smallpox, are needed. Currently, SIGA Technologies is developing the small-molecule oral drug, tecovirimat (previously known as ST-246), as a postexposure therapeutic treatment of orthopoxvirus disease, including smallpox. Tecovirimat has been shown to be efficacious in preventing lethal orthopoxviral disease in numerous animal models (G. Yang, D. C. Pevear, M. H. Davies, M. S. Collett, T. Bailey, et al., J. Virol. 79:13139-13149, 2005; D. C. Quenelle, R. M. Buller, S. Parker, K. A. Keith, D. E. Hruby, et al., Antimicrob. Agents Chemother., 51:689-695, 2007; E. Sbrana, R. Jordan, D. E. Hruby, R. I. Mateo, S. Y. Xiao, et al., Am. J. Trop. Med. Hyg. 76:768-773, 2007). Furthermore, in clinical trials thus far, the drug appears to be safe, with a good pharmacokinetic profile. In this study, the efficacy of tecovirimat was evaluated in both a prelesional and postlesional setting in nonhuman primates challenged intravenously with 1 × 10(8) PFU of Variola virus (VARV; the causative agent of smallpox), a model for smallpox disease in humans. Following challenge, 50% of placebo-treated controls succumbed to infection, while all tecovirimat-treated animals survived regardless of whether treatment was started at 2 or 4 days postinfection. In addition, tecovirimat treatment resulted in dramatic reductions in dermal lesion counts, oropharyngeal virus shedding, and viral DNA circulating in the blood. Although clinical disease was evident in tecovirimat-treated animals, it was generally very mild and appeared to resolve earlier than in placebo-treated controls that survived infection. Tecovirimat appears to be an effective smallpox therapeutic in nonhuman primates, suggesting that it is reasonably likely to provide therapeutic benefit in smallpox-infected humans.

Pharmacokinetic and pharmacodynamic modeling to determine the dose of ST-246 to protect against smallpox in humans.

Although smallpox has been eradicated, the United States government considers it a "material threat" and has funded the discovery and development of potential therapeutic compounds. As reported here, the human efficacious dose for one of these compounds, ST-246, was determined using efficacy studies in nonhuman primates (NHPs), together with pharmacokinetic and pharmacodynamic analysis that predicted the appropriate dose and exposure levels to provide therapeutic benefit in humans. The efficacy analysis combined the data from studies conducted at three separate facilities that evaluated treatment following infection with a closely related virus, monkeypox virus (MPXV), in a total of 96 NHPs. The effect of infection on ST-246 pharmacokinetics in NHPs was applied to humans using population pharmacokinetic models. Exposure at the selected human dose of 600 mg is more than 4-fold higher than the lowest efficacious dose in NHPs and is predicted to provide protection to more than 95% of the population.

In vitro characterization of a nineteenth-century therapy for smallpox.

In the nineteenth century, smallpox ravaged through the United States and Canada. At this time, a botanical preparation, derived from the carnivorous plant Sarracenia purpurea, was proclaimed as being a successful therapy for smallpox infections. The work described characterizes the antipoxvirus activity associated with this botanical extract against vaccinia virus, monkeypox virus and variola virus, the causative agent of smallpox. Our work demonstrates the in vitro characterization of Sarracenia purpurea as the first effective inhibitor of poxvirus replication at the level of early viral transcription. With the renewed threat of poxvirus-related infections, our results indicate Sarracenia purpurea may act as another defensive measure against Orthopoxvirus infections.

Evaluation of disease and viral biomarkers as triggers for therapeutic intervention in respiratory mousepox - an animal model of smallpox.

The human population is currently faced with the potential use of natural or recombinant variola and monkeypox viruses as biological weapons. Furthermore, the emergence of human monkeypox in Africa and its expanding environs poses a significant natural threat. Such occurrences would require therapeutic and prophylactic intervention with antivirals to minimize morbidity and mortality of exposed populations. Two orally-bioavailable antivirals are currently in clinical trials; namely CMX001, an ether-lipid analog of cidofovir with activity at the DNA replication stage and ST-246, a novel viral egress inhibitor. Both of these drugs have previously been evaluated in the ectromelia/mousepox system; however, the trigger for intervention was not linked to a disease biomarker or a specific marker of virus replication. In this study we used lethal, intranasal, ectromelia virus infections of C57BL/6 and hairless SKH1 mice to model human disease and evaluate exanthematous rash (rash) as an indicator to initiate antiviral treatment. We show that significant protection can be provided to C57BL/6 mice by CMX001 or ST-246 when therapy is initiated on day 6 post infection or earlier. We also show that significant protection can be provided to SKH1 mice treated with CMX001 at day 3 post infection or earlier, but this is four or more days before detection of rash (ST-246 not tested). Although in this model rash could not be used as a treatment trigger, viral DNA was detected in blood by day 4 post infection and in the oropharyngeal secretions (saliva) by day 2-3 post infection - thus providing robust and specific markers of virus replication for therapy initiation. These findings are discussed in the context of current respiratory challenge animal models in use for the evaluation of poxvirus antivirals.