Orthopoxvirus Infections Research Articles

Antiviral Activity of 4'-thioIDU and Thymidine Analogs against Orthopoxviruses

The search for effective therapies for orthopoxvirus infections has identified diverse classes of molecules with antiviral activity. Pyrimidine analogs, such as 5-iodo-2′-deoxyuridine (idoxuridine, IDU) were among the first compounds identified with antiviral activity against a number of orthopoxviruses and have been reported to be active both in vitro and in animal models of infection. More recently, additional analogs have been reported to have improved antiviral activity against orthopoxviruses including several derivatives of deoxyuridine with large substituents in the 5 position, as well as analogs with modifications in the deoxyribose moiety including (north)-methanocarbathymidine, and 5-iodo-4′-thio-2′-deoxyuridine (4′-thioIDU). The latter molecule has proven to have good antiviral activity against the orthopoxviruses both in vitro and in vivo and has the potential to be an effective therapy in humans.

Ectromelia virus infections of mice as a model to support the licensure of anti-orthopoxvirus therapeutics.

The absence of herd immunity to orthopoxviruses and the concern that variola or monkeypox viruses could be used for bioterroristic activities has stimulated the development of therapeutics and safer prophylactics. One major limitation in this process is the lack of accessible human orthopoxvirus infections for clinical efficacy trials; however, drug licensure can be based on orthopoxvirus animal challenge models as described in the “Animal Efficacy Rule”. One such challenge model uses ectromelia virus, an orthopoxvirus, whose natural host is the mouse and is the etiological agent of mousepox. The genetic similarity of ectromelia virus to variola and monkeypox viruses, the common features of the resulting disease, and the convenience of the mouse as a laboratory animal underscores its utility in the study of orthopoxvirus pathogenesis and in the development of therapeutics and prophylactics. In this review we outline how mousepox has been used as a model for smallpox. We also discuss mousepox in the context of mouse strain, route of infection, infectious dose, disease progression, and recovery from infection.

Animal Models of Orthopoxvirus Infection

Smallpox was one of the most devastating diseases known to humanity. Although smallpox was eradicated through a historically successful vaccination campaign, there is concern in the global community that either Variola virus (VARV), the causative agent of smallpox, or another species of Orthopoxvirus could be used as agents of bioterrorism. Therefore, development of countermeasures to Orthopoxvirus infection is a crucial focus in biodefense research, and these efforts rely on the use of various animal models. Smallpox typically presented as a generalized pustular rash with 30 to 40% mortality, and although smallpox-like syndromes can be induced in cynomolgus macaques with VARV, research with this virus is highly restricted; therefore, animal models with other orthopoxviruses have been investigated. Monkeypox virus causes a generalized vesiculopustular rash in rhesus and cynomolgus macaques and induces fatal systemic disease in several rodent species. Ectromelia virus has been extensively studied in mice as a model of orthopoxviral infection in its natural host. Intranasal inoculation of mice with some strains of vaccinia virus produces fatal bronchopneumonia, as does aerosol or intranasal inoculation of mice with cowpox virus. Rabbitpox virus causes pneumonia and fatal systemic infections in rabbits and can be naturally transmitted between rabbits by an aerosol route similar to that of VARV in humans. No single animal model recapitulates all known aspects of human Orthopoxvirus infections, and each model has its advantages and disadvantages. This article provides a brief review of the Orthopoxvirus diseases of humans and the key pathologic features of animal models of Orthopoxvirus infections.

A protein-based smallpox vaccine protects non-human primates from a lethal monkeypox virus challenge

Concerns about infections caused by orthopoxviruses, such as variola and monkeypox viruses, drive ongoing efforts to develop novel smallpox vaccines that are both effective and safe to use in diverse populations. A subunit smallpox vaccine comprising vaccinia virus membrane proteins A33, B5, L1, A27 and aluminum hydroxide (alum) ± CpG was administered to non-human primates, which were subsequently challenged with a lethal intravenous dose of monkeypox virus. Alum adjuvanted vaccines provided only partial protection but the addition of CpG provided full protection that was associated with a more homogeneous antibody response and stronger IgG1 responses. These results indicate that it is feasible to develop a highly effective subunit vaccine against orthopoxvirus infections as a safer alternative to live vaccinia virus vaccination.

Diazepam leads to enhanced severity of orthopoxvirus infection and immune suppression

Benzodiazepines are drugs widely used as tranquilizers and in various other indications. We treated Balb/c mice with diazepam and infected them with cowpox (CPXV) and vaccinia virus (VACV). Disease index, weight loss and the antibody response were determined. Additionally the influence of different benzodiazepines on the mitogen response of human peripheral blood lymphocytes and spleen cells was tested. Diazepam led to earlier disease onset, prolonged duration of symptoms, higher weight loss and overall disease index in VACV infected mice. CPXV infected mice developed poxviral skin lesions only after drug administration and a significant decrease in the specific antibody response was also observed. Diazepam and alprazolam also inhibited the proliferative response of human lymphocytes/spleen cells in vitro but did not show noteworthy apoptotic effects. It is surprising that even a single dose of diazepam has a profound influence on the immune system, sufficient to facilitate symptomatic infectious disease. These data provide first evidence that commonly used drugs like Valium ® may augment severity of rare poxvirus infections such as CPXV or monkeypox. As VACV is still used as life vaccine against smallpox there is also a risk of enhanced side effects or possible interference with the success of vaccination.

Rapid non-enzymatic extraction method for isolating PCR-quality camelpox virus DNA from skin

Molecular diagnostic investigations of orthopoxvirus (OPV) infections are performed using a variety of clinical samples including skin lesions, tissues from internal organs, blood and secretions. Skin samples are particularly convenient for rapid diagnosis and molecular epidemiological investigations of camelpox virus (CMLV). Classical extraction procedures and commercial spin-column-based kits are time consuming, relatively expensive, and require multiple extraction and purification steps in addition to proteinase K digestion. A rapid non-enzymatic procedure for extracting CMLV DNA from dried scabs or pox lesions was developed to overcome some of the limitations of the available DNA extraction techniques. The procedure requires as little as 10mg of tissue and produces highly purified DNA [OD260/OD280 ratios between 1.47 and 1.79] with concentrations ranging from 6.5 to16μg/ml. The extracted CMLV DNA was proven suitable for virus-specific qualitative and, semi-quantitative PCR applications. Compared to spin-column and conventional viral DNA extraction techniques, the two-step extraction procedure saves money and time, and retains the potential for automation without compromising CMLV PCR sensitivity.

A novel highly reproducible and lethal nonhuman primate model for orthopox virus infection.

The intentional re-introduction of Variola virus (VARV), the agent of smallpox, into the human population is of great concern due its bio-terroristic potential. Moreover, zoonotic infections with Cowpox (CPXV) and Monkeypox virus (MPXV) cause severe diseases in humans. Smallpox vaccines presently available can have severe adverse effects that are no longer acceptable. The efficacy and safety of new vaccines and antiviral drugs for use in humans can only be demonstrated in animal models. The existing nonhuman primate models, using VARV and MPXV, need very high viral doses that have to be applied intravenously or intratracheally to induce a lethal infection in macaques. To overcome these drawbacks, the infectivity and pathogenicity of a particular CPXV was evaluated in the common marmoset (Callithrix jacchus).A CPXV named calpox virus was isolated from a lethal orthopox virus (OPV) outbreak in New World monkeys. We demonstrated that marmosets infected with calpox virus, not only via the intravenous but also the intranasal route, reproducibly develop symptoms resembling smallpox in humans. Infected animals died within 1–3 days after onset of symptoms, even when very low infectious viral doses of 5×102 pfu were applied intranasally. Infectious virus was demonstrated in blood, saliva and all organs analyzed.We present the first characterization of a new OPV infection model inducing a disease in common marmosets comparable to smallpox in humans. Intranasal virus inoculation mimicking the natural route of smallpox infection led to reproducible infection. In vivo titration resulted in an MID50 (minimal monkey infectious dose 50%) of 8.3×102 pfu of calpox virus which is approximately 10,000-fold lower than MPXV and VARV doses applied in the macaque models. Therefore, the calpox virus/marmoset model is a suitable nonhuman primate model for the validation of vaccines and antiviral drugs. Furthermore, this model can help study mechanisms of OPV pathogenesis.

Safety and Pharmacokinetics of the Antiorthopoxvirus Compound ST-246 following Repeat Oral Dosing in Healthy Adult Subjects

ST-246, a novel compound that inhibits egress of orthopoxvirus from infected cells, is being evaluated as a treatment for pathogenic orthopoxvirus infections in humans. This phase I, double-blind, randomized, placebo-controlled, escalating multiple-dose study was conducted to determine the safety, tolerability, and pharmacokinetics of ST-246 administered as a single daily oral dose of 250, 400, or 800 mg for 21 days to nonfasting healthy human volunteers. ST-246 appeared to be well tolerated, with no serious adverse events (AEs). Headache, for which one subject in the 800-mg group discontinued the study, was the most commonly reported AE in all treatment groups. The multiple-dose pharmacokinetics of ST-246 was well characterized. The day 21 mean elimination half-lives were calculated at 18.8, 19.8, and 20.7 h for each of the 250-, 400-, and 800-mg/day dose groups, respectively. Steady state was reached by day 6 (within 3 to 5 half-lives), saturable absorption was observed at the 800-mg dose level, and the fraction of parent drug excreted in the urine was very low. Based on these results, administration of 400 mg/day ST-246 can be expected to provide plasma concentrations above the efficacious concentration demonstrated in nonhuman primate models in earlier studies.

Pulmonary Orthopoxvirus infection compromises a murine OVA peptide immune response in a dose-dependent manner (39.8)

Abstract Cowpox virus (CPXV), a close relative of variola virus that causes smallpox, can suppress the immune system through a large array of immunosuppressive gene products. We developed a murine model in which DO11.10 T cells specific for an OVA peptide were transferred into BALB/c mice to assess the impact of a pulmonary CPXV infection on DO11.10 T cell proliferation in lung draining lymph nodes following intranasal OVA peptide delivery. High and low-dose CPXV infections were compared. Both doses lead to clinical illness, but the high dose is lethal. A high-dose infection reduced DO11.10 T cell proliferation, but a low-dose infection did not. At the time that proliferation of T cells was assessed (6d),15±1% of lung dendritic cells (DCs) were infected at the high-dose, but only 5±1% of DCs at the low-dose. At both doses, infected and uninfected DCs had decreased expression of co-stimulatory molecules. We speculated that the lung microenvironment created by infection, rather than direct infection of the DCs, suppressed DC immune function. In support, we found that bronchoalveolar lavage fluid and supernatant derived from lung homogenates from infected mice suppressed the function of uninfected lung DCs in vitro. Furthermore, the suppressive activity was more highly concentrated in lungs from high-dose infected mice. These studies strongly suggest that orthopoxvirus infections create an immunosuppressive microenvironment that compromises the host pulmonary immune responses.

A Silent Enzootic of an Orthopoxvirus in Ghana, West Africa: Evidence for Multi-Species Involvement in the Absence of Widespread Human Disease

Human monkeypox has never been reported in Ghana, but rodents captured in forested areas of southern Ghana were the source of the monkeypox virus introduced into the United States in 2003. Subsequent to the outbreak in the United States, 204 animals were collected from two commercial trapping sites in Ghana. Animal tissues were examined for the presence of orthopoxvirus (OPXV) DNA using a real-time polymerase chain reaction, and sera were assayed for antibodies against OPXV. Animals from five genera (Cricetomys, Graphiurus, Funiscirus, and Heliosciurus) had antibodies against OPXV, and three genera (Cricetomys, Graphiurus, and Xerus) had evidence of OPXV DNA in tissues. Additionally, 172 persons living near the trapping sites were interviewed regarding risk factors for OPXV exposure, and their sera were analyzed. Fifty-three percent had IgG against OPXV; none had IgM. Our findings suggest that several species of forest-dwelling rodents from Ghana are susceptible to naturally occurring OPXV infection, and that persons living near forests may have low-level or indirect exposure to OPXV-infected animals, possibly resulting in sub-clinical infections.

Long‐lasting immunity by early infection of maternal‐antibody‐protected infants

Newborn higher vertebrates are largely immuno-incompetent and generally survive infections--including poxviruses--by maternal antibody protection. Here, we show that mice survived epidemics as adults only if exposed to lethal orthopoxvirus infections during infancy under the umbrella of maternal protective antibodies. This implies that both the absence of exposure to infection during early infancy or of effective vaccination renders the population highly susceptible to new or old re-emerging pathogens.

Puromycin-sensitive aminopeptidase: An antiviral prodrug activating enzyme

Cidofovir (HPMPC) is a broad-spectrum antiviral agent, currently used to treat AIDS-related human cytomegalovirus retinitis. Cidofovir has recognized therapeutic potential for orthopox virus infections, although its use is hampered by its inherent low oral bioavailability. Val-Ser-cyclic HPMPC (Val-Ser-cHPMPC) is a promising peptide prodrug which has previously been shown by us to improve the permeability and bioavailability of the parent compound in rodent models ( Eriksson et al., 2008. Molecular Pharmaceutics 5, 598–609). Puromycin-sensitive aminopeptidase was partially purified from Caco-2 cell homogenates and identified as a prodrug activating enzyme for Val-Ser-cHPMPC. The prodrug activation process initially involves an enzymatic step where the l-Valine residue is removed by puromycin-sensitive aminopeptidase, a step that is bestatin-sensitive. Subsequent chemical hydrolysis results in the generation of cHPMPC. A recombinant puromycin-sensitive aminopeptidase was generated and its substrate specificity investigated. The k cat for Val-pNA was significantly lower than that for Ala-pNA, suggesting that some amino acids are preferred over others. Furthermore, the three-fold higher k cat for Val-Ser-cHPMPC as compared to Val-pNA suggests that the leaving group may play an important role in determining hydrolytic activity. In addition to its ability to hydrolyze a variety of substrates, these observations strongly suggest that puromycin-sensitive aminopeptidase is an important enzyme for activating Val-Ser-cHPMPC in vivo. Taken together, our data suggest that puromycin-sensitive aminopeptidase makes an attractive target for future prodrug design.

Infecções humanas causadas por poxvirus relacionados ao vírus vaccinia no Brasil

Since 1999, human infection caused by Orthopoxvirus has been observed in at least eight Brazilian states, with the presence of vesicles that evolve to pustules and crusts, especially on the hands, arms and face, after contact with cows showing comparable lesions on the udder. In addition to the skin lesions, there have been descriptions of patients with axillary ganglionic reactions that are sometimes painful, along with fever, headache, fatigue, dehydration, anorexia, sudoresis, arthralgia and muscle pain. The condition evolves over a three to four-week period. Vulvar lesions and transmission within families have also been described. Molecular studies have shown that the poxviruses identified are genetically related to vaccinia virus samples that were used in vaccination campaigns in the past. Clinical specimens from 80 human infections were studied in the laboratory, and orthopoxvirus infections were confirmed in 68 cases. The lesions observed in these patients are presented and the implications of this zoonosis in Brazil are discussed.

Inhibition of Monkeypox virus replication by RNA interference

The Orthopoxvirus genus of Poxviridae family is comprised of several human pathogens, including cowpox (CPXV), Vaccinia (VACV), monkeypox (MPV) and Variola (VARV) viruses. Species of this virus genus cause human diseases with various severities and outcome ranging from mild conditions to death in fulminating cases. Currently, vaccination is the only protective measure against infection with these viruses and no licensed antiviral drug therapy is available. In this study, we investigated the potential of RNA interference pathway (RNAi) as a therapeutic approach for orthopox virus infections using MPV as a model. Based on genome-wide expression studies and bioinformatic analysis, we selected 12 viral genes and targeted them by small interference RNA (siRNA). Forty-eight siRNA constructs were developed and evaluated in vitro for their ability to inhibit viral replication. Two genes, each targeted with four different siRNA constructs in one pool, were limiting to viral replication. Seven siRNA constructs from these two pools, targeting either an essential gene for viral replication (A6R) or an important gene in viral entry (E8L), inhibited viral replication in cell culture by 65-95% with no apparent cytotoxicity. Further analysis with wild-type and recombinant MPV expressing green fluorescence protein demonstrated that one of these constructs, siA6-a, was the most potent and inhibited viral replication for up to 7 days at a concentration of 10 nM. These results emphasis the essential role of A6R gene in viral replication, and demonstrate the potential of RNAi as a therapeutic approach for developing oligonucleotide-based drug therapy for MPV and other orthopox viruses.

Novel Antiviral Therapy Based on Innate Immunity

Orthopox viruses include variola major (the etiologic agent for smallpox), vaccinia virus (VV), and monkeypox. Although the devastating effects of smallpox have been successfully controlled by vaccination with VV, vaccine programs have been discontinued, raising the population’s susceptibility to VV bioterrorism. Furthermore, immunocompromised individuals and those with atopic dermatitis are at risk for fatal complications of vaccination, such as progressive vaccinia and eczema vaccinatum, respectively (Cono et al., 2003). Despite the effectiveness of intravenous Ig and other antiviral therapies, treatment of orthopox infections is imperfect. The innate immune system produces antimicrobial compounds such as human β-defensins and cathelicidins, which exhibit antiviral activity against VV (Howell et al., 2007). In addition, innate immunity in patients with atopic dermatitis has been found to be deficient. This may, in part, predispose these individuals to develop eczema vaccinatum. Therefore, peptides such as human β-defensin-3 might represent a potential treatment for these adverse reactions; however, rapid degradation by endogenous tissue proteases occurs when peptides are introduced systemically. Structurally similar to antimicrobial peptides and able to selectively disrupt bacterial cell walls, a novel class of synthetic compounds called ceragenins (CSAs) shows promise as a therapeutic agent against orthopox virus infections (Ding et al., 2004). To elucidate the salutary properties of CSAs, Howell and colleagues (2009, this issue) conducted a series of in vitro and in vivo experiments to assess the ability of various CSAs to destroy VV, restore and protect keratinocytes, and limit systemic viral dissemination; they also studied the mechanisms by which CSAs might work. CSA-13 was found to be the most potent of the CSAs studied, exhibiting in vitro antiviral activity and efficacy as a topically applied cream. The effects of CSAs might be multimodal, affecting viral propagation by disrupting the viral envelope and stimulating the activity of other antimicrobial peptides against VV. Through the following questions, we examine this paper in greater detail. For discussion and brief answers, please refer to the supplementary information linked to the online version of the paper at http://www.nature.com/jid.

Multicolour, multiplex real-time PCR assay for the detection of human-pathogenic poxviruses

After eradication of variola virus, one of the most dangerous infectious diseases affecting mankind, today other poxviruses of different genera can cause infection in humans. These viruses include human-specific molluscipoxviruses as well as zoonotic orthopoxviruses and parapoxviruses. While non-variola orthopoxvirus infections mostly cause mild symptoms in immunocompetent persons, they can evoke severe disease in immunocompromised patients. Since the typical poxviral skin lesions are rarely diagnosed by physicians, PCR-based identification of suspected poxviruses is often required. To simplify the PCR-based diagnosis of human-pathogenic poxviruses, we established a multicolour multiplex real-time PCR that simultaneously detects and differentiates human-pathogenic poxviruses in one reaction. Using 5′ nuclease probes labelled with FAM for orthopoxviruses, VIC for parapoxviruses and FAM and VIC for molluscipoxviruses, respectively, amplification of poxviral DNA resulted in a genus-specific reporter-dye profile. Validation with 36 human clinical specimens and DNA of pathogens causing pox-like skin lesions demonstrated the specificity of the assay. Probit analysis revealed a limit of detection of 9.7, 22.08 and 28.1 copies/assay (95% CI) for molluscipoxvirus, orthopoxvirus and parapoxvirus DNA, respectively. The combinatorial multicolour strategy applied has the potential to be used in further applications targeting even more than three pathogens.

Activity of Certain 5-Substituted-4′-Thio Pyrimindine Nucleosides against Orthopoxvirus Infections

Activity of Certain 5-Substituted-4′-Thio Pyrimindine Nucleosides against Orthopoxvirus Infections

Activation of the PI3K/Akt Pathway Early during Vaccinia and Cowpox Virus Infections Is Required for both Host Survival and Viral Replication

Viral manipulation of the transduction pathways associated with key cellular functions such as actin remodeling, microtubule stabilization, and survival may favor a productive viral infection. Here we show that consistent with the vaccinia virus (VACV) and cowpox virus (CPXV) requirement for cytoskeleton alterations early during the infection cycle, PBK/Akt was phosphorylated at S473 [Akt(S473-P)], a modification associated with the mammalian target of rapamycin complex 2 (mTORC2), which was paralleled by phosphorylation at T308 [Akt(T308-P)] by PI3K/PDK1, which is required for host survival. Notably, while VACV stimulated Akt(S473-P/T308-P) at early (1 h postinfection [p.i.]) and late (24 h p.i.) times during the infective cycle, CPXV stimulated Akt at early times only. Pharmacological and genetic inhibition of PI3K (LY294002) or Akt (Akt-X and a dominant-negative form of Akt-K179M) resulted in a significant decline in virus yield (from 80% to >/=90%). This decline was secondary to the inhibition of late viral gene expression, which in turn led to an arrest of virion morphogenesis at the immature-virion stage of the viral growth cycle. Furthermore, the cleavage of both caspase-3 and poly(ADP-ribose) polymerase and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling assays confirmed that permissive, spontaneously immortalized cells such as A31 cells and mouse embryonic fibroblasts (MEFs) underwent apoptosis upon orthopoxvirus infection plus LY294002 treatment. Thus, in A31 cells and MEFs, early viral receptor-mediated signals transmitted via the PI3K/Akt pathway are required and precede the expression of viral antiapoptotic genes. Additionally, the inhibition of these signals resulted in the apoptosis of the infected cells and a significant decline in viral titers.

Cat-to-Human Orthopoxvirus Transmission, Northeastern Italy

To the Editor: Kurth et al (1) recently described a cowpoxvirus chain of transmission from rat to human through an elephant in Germany. Zoonotic cowpoxvirus infections are well known in Europe (2,3). This virus can infect many animal species; serologic evidence of infection may approach 10% in cats in western Europe (4,5). Zoonotic orthopoxvirus (OPV) infection has been reported in several European countries, but it is rare south of the Alps, and no extensive description of cases is available for Italy. We describe 2 cases of zoonotic OPV in Friuli, northeastern Italy, in veterinary personnel scratched by cats. In December 2005, a male veterinary student (patient A) who had been scratched by a cat with multiple cutaneous ulcerated lesions sought care at a local hospital; he had a lesion on his right hand, moderate fever, and malaise. Histopathologic findings from the cat indicated feline poxvirus infection. In July 2007, a female veterinarian (patient B) who lived in a different area of the same region and also had been scratched by a cat, sought care at the same hospital; she had a lesion close to the right sternoclavicular joint. On the basis of patients’ history of exposure and clinical presentation of the animals’ disease, zoonotic transmission of OPV infection was suspected. Vesicle fluid and, subsequently, crusts from the patients’ lesions, were sent to the virology laboratory of the reference center for poxvirus infections at National Institute for Infectious Diseases in Rome, where OPV diagnosis was based on electron microscopy, virus isolation, and detection of viral nucleic acid. In addition, serial blood samples were sent to the same laboratory for analysis of specific antibodies and cellular immune response. The viruses were almost identical, according to the partial sequence of the crmB gene ({type:entrez-nucleotide,attrs:{text:EF612710,term_id:148833293,term_text:EF612710}}EF612710 and {type:entrez-nucleotide,attrs:{text:FJ445748,term_id:215982785,term_text:FJ445748}}FJ445748) and on the complete sequence of the hemagglutinin gene ({type:entrez-nucleotide,attrs:{text:EF612709,term_id:148833291,term_text:EF612709}}EF612709 and {type:entrez-nucleotide,attrs:{text:FJ445747,term_id:215982783,term_text:FJ445747}}FJ445747). The hemagglutinin (Figure) and the partial crmB (not shown) sequences from each isolate formed a distinct cluster within the OPV genus; similar results were obtained from concatenated analysis of both genes (not shown). The identity/similarity scores of the complete nucleotide sequences of crmB and hemagglutinin genes from patient A, in relation to other OPVs, were, respectively, Ectromelia ({type:entrez-nucleotide,attrs:{text:AF012825,term_id:22123748,term_text:AF012825}}AF012825), 0.598 and 0.841; cowpox ({type:entrez-nucleotide,attrs:{text:X94355,term_id:30519405,term_text:X94355}}X94355), 0.933 and 0.927; vaccinia ({type:entrez-nucleotide,attrs:{text:AY678276,term_id:56713624,term_text:AY678276}}AY678276), 0.844 and 0.934; camelpox ({type:entrez-nucleotide,attrs:{text:AY009086,term_id:18025096,term_text:AY009086}}AY009086), 0.941 and 0.940; monkeypox ({type:entrez-nucleotide,attrs:{text:DQ011153,term_id:68448677,term_text:DQ011153}}DQ011153), 0.909 and 0.914; and variola ({type:entrez-nucleotide,attrs:{text:DQ437588,term_id:109725669,term_text:DQ437588}}DQ437588), 0.922 and 0.906. On the basis of these results, species assignment of the isolated OPVs was not possible. Preliminary sequence data on additional genes (ATI, A27L, and CBP) from patient A’s isolate supported the segregation of these OPVs in Italy from the other known OPV species. However, the available information is still not enough to infer whether the isolates from Italy belong to a novel or known OPV species. More extensive biologic and molecular characterization is in progress. Figure Phylogenetic tree of nucleotide sequences of the complete hemagglutinin open reading frame (930 bp) of orthopoxviruses (OPVs) isolated from the 2 patients described in this report and additional poxviruses available in GenBank (cowpox: {type:entrez-nucleotide,attrs:{text:AY902279,term_id:88659748,term_text:AY902279}} ... The 2 cases reported here, occurring >1 year apart, indicate that OPV is circulating in domestic, and possibly wild, local fauna; they underscore the need for physicians and veterinarians to become aware of the risk for OPV zoonoses. A surveillance program has been launched among local veterinary clinics to identify nonvaccinated veterinarians who have been exposed to OPV. A surveillance plan for cats at these clinics has also been started.

ST-246 Antiviral Efficacy in a Nonhuman Primate Monkeypox Model: Determination of the Minimal Effective Dose and Human Dose Justification

Therapeutics for the treatment of pathogenic orthopoxvirus infections are being sought. In the absence of patients with disease, animal models of orthopoxvirus disease are essential for evaluation of the efficacies of antiviral drugs and establishment of the appropriate dose and duration of human therapy. Infection of nonhuman primates (NHP) by the intravenous injection of monkeypox virus has been used to evaluate a promising therapeutic drug candidate, ST-246. ST-246 administered at 3 days postinfection (which corresponds to the secondary viremia stage of disease) at four different doses (from 100 mg/kg of body weight down to 3 mg/kg) once a day for 14 days was able to offer NHP 100% protection from a lethal infection with monkeypox virus and reduce the viral load and lesion formation. In NHP, the administration of ST-246 at a dose of 10 mg/kg/day for 14 days resulted in levels of blood exposure comparable to the levels attained in humans administered 400 mg in the fed state. These results suggest that administration of an oral dosage of 400 mg once daily for 14 days will be effective for the prevention or treatment of smallpox or monkeypox infections in humans.