Nitric oxide (NO) is extremely toxic to Clostridium botulinum, but its molecular targets are unknown. Here, we identify a heme protein sensor (SONO) that displays femtomolar affinity for NO. The crystal structure of the SONO heme domain reveals a previously undescribed fold and a strategically placed tyrosine residue that modulates heme-nitrosyl coordination. Furthermore, the domain architecture of a SONO ortholog cloned from Chlamydomonas reinhardtii indicates that NO signaling through cyclic guanosine monophosphate arose before the origin of multicellular eukaryotes. Our findings have broad implications for understanding bacterial responses to NO, as well as for the activation of mammalian NO-sensitive guanylyl cyclase.

To support the licensure of a new and safer vaccine to protect people against smallpox, a monkeypox model of infection in cynomolgus macaques, which simulates smallpox in humans, was used to evaluate two vaccines, Acam2000 and Imvamune, for protection against disease. Animals vaccinated with a single immunization of Imvamune were not protected completely from severe and/or lethal infection, whereas those receiving either a prime and boost of Imvamune or a single immunization with Acam2000 were protected completely. Additional parameters, including clinical observations, radiographs, viral load in blood, throat swabs, and selected tissues, vaccinia virus-specific antibody responses, immunophenotyping, extracellular cytokine levels, and histopathology were assessed. There was no significant difference (P > 0.05) between the levels of neutralizing antibody in animals vaccinated with a single immunization of Acam2000 (132 U/ml) and the prime-boost Imvamune regime (69 U/ml) prior to challenge with monkeypox virus. After challenge, there was evidence of viral excretion from the throats of 2 of 6 animals in the prime-boost Imvamune group, whereas there was no confirmation of excreted live virus in the Acam2000 group. This evaluation of different human smallpox vaccines in cynomolgus macaques helps to provide information about optimal vaccine strategies in the absence of human challenge studies.

Encapsulation of antibiotics may improve treatment of intracellular infections by prolonging antibiotic release and improving antibiotic uptake into cells. In this study, liposome-encapsulated ciprofloxacin for inhalation (CFI) was evaluated as a postexposure therapeutic for the treatment of Coxiella burnetii, the causative agent of Q fever. Intranasal treatment of male A/Jola (A/J) mice with CFI (50 mg/kg of body weight) once daily for 7 days protected mice against weight loss and clinical signs following an aerosol challenge with C. burnetii. In comparison, mice treated twice daily with oral ciprofloxacin or doxycycline (50 mg/kg) or phosphate-buffered saline (PBS) lost 15 to 20% body weight and exhibited ruffled fur, arched backs, and dehydration. Mice were culled at day 14 postchallenge. The weights and bacterial burdens of organs were determined. Mice treated with CFI exhibited reduced splenomegaly and reduced bacterial numbers in the lungs and spleen compared to mice treated with oral ciprofloxacin or doxycycline. When a single dose of CFI was administered, it provided better protection against body weight loss than 7 days of treatment with oral doxycycline, the current antibiotic of choice to treat Q fever. These data suggest that CFI has potential as a superior antibiotic to treat Q fever.

To evaluate new vaccines when human efficacy studies are not possible, the FDA's "Animal Rule" requires well-characterized models of infection. Thus, in the present study, the early pathogenic events of monkeypox infection in nonhuman primates, a surrogate for variola virus infection, were characterized. Cynomolgus macaques were exposed to aerosolized monkeypox virus (10 5 PFU). Clinical observations, viral loads, immune responses, and pathological changes were examined on days 2, 4, 6, 8, 10, and 12 postchallenge. Viral DNA (vDNA) was detected in the lungs on day 2 postchallenge, and viral antigen was detected, by immunostaining, in the epithelium of bronchi, bronchioles, and alveolar walls. Lesions comprised rare foci of dysplastic and sloughed cells in respiratory bronchioles. By day 4, vDNA was detected in the throat, tonsil, and spleen, and monkeypox antigen was detected in the lung, hilar and submandibular lymph nodes, spleen, and colon. Lung lesions comprised focal epithelial necrosis and inflammation. Body temperature peaked on day 6, pox lesions appeared on the skin, and lesions, with positive immunostaining, were present in the lung, tonsil, spleen, lymph nodes, and colon. By day 8, vDNA was present in 9/13 tissues. Blood concentrations of interleukin 1ra (IL-1ra), IL-6, and gamma interferon (IFN-␥) increased markedly. By day 10, circulating IgG antibody concentrations increased, and on day 12, animals showed early signs of recovery. These results define early events occurring in an inhalational macaque monkeypox infection model, supporting its use as a surrogate model for human smallpox. IMPORTANCEBioterrorism poses a major threat to public health, as the deliberate release of infectious agents, such smallpox or a related virus, monkeypox, would have catastrophic consequences. The development and testing of new medical countermeasures, e.g., vaccines, are thus priorities; however, tests for efficacy in humans cannot be performed because it would be unethical and field trials are not feasible. To overcome this, the FDA may grant marketing approval of a new product based upon the "Animal Rule," in which interventions are tested for efficacy in well-characterized animal models. Monkeypox virus infection of nonhuman primates (NHPs) presents a potential surrogate disease model for smallpox. Previously, the later stages of monkeypox infection were defined, but the early course of infection remains unstudied. Here, the early pathogenic events of inhalational monkeypox infection in NHPs were characterized, and the results support the use of this surrogate model for testing human smallpox interventions.