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SUMMARY Monkeypox (MPXV) and cowpox (CPXV) are emerging agents that cause severe human infections on an intermittent basis, and variola virus (VARV) has potential for use as an agent of bioterror. Vaccinia immune globulin (VIG) has been used therapeutically to treat severe poxvirus infections, but is in short supply. We generated a large panel of orthopoxvirus-specific human monoclonal Abs from immune subjects to investigate the molecular basis of broadly neutralizing antibody responses for diverse orthopoxviruses. Detailed analysis revealed the principal neutralizing antibody specificities that are cross-reactive for VACV, CPXV, MPXV and VARV and that are determinants of protection in murine challenge models. Optimal protection following respiratory or systemic infection required a mixture of Abs that targeted several membrane proteins, including proteins on enveloped and mature virion forms of virus. This work reveals orthopoxvirus targets for human Abs that mediate cross-protective immunity and identifies new candidate Ab therapeutic mixtures to replace VIG.

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The ubiquitin-proteasome system has a central role in the degradation of intracellular proteins and regulates a variety of functions. Viruses belonging to several different families utilize or modulate the system for their advantage. Here we showed that the proteasome inhibitors MG132 and epoxomicin blocked a postentry step in vaccinia virus (VACV) replication. When proteasome inhibitors were added after virus attachment, early gene expression was prolonged and the expression of intermediate and late genes was almost undetectable. By varying the time of the removal and addition of MG132, the adverse effect of the proteasome inhibitors was narrowly focused on events occurring 2 to 4 h after infection, the time of the onset of viral DNA synthesis. Further analyses confirmed that genome replication was inhibited by both MG132 and epoxomicin, which would account for the effect on intermediate and late gene expression. The virus-induced replication of a transfected plasmid was also inhibited, indicating that the block was not at the step of viral DNA uncoating. UBEI-41, an inhibitor of the ubiquitin-activating enzyme E1, also prevented late gene expression, supporting the role of the ubiquitin-proteasome system in VACV replication. Neither the overexpression of ubiquitin nor the addition of an autophagy inhibitor was able to counter the inhibitory effects of MG132. Further studies of the role of the ubiquitin-proteasome system for VACV replication may provide new insights into virus-host interactions and suggest potential antipoxviral drugs.The ubiquitin-proteasome system has a central role in the degradation of intracellular proteins and regulates a variety of functions (22). Proteins to be degraded are modified by the addition of multiple copies of the 76-amino-acid ubiquitin through the sequential activities of an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3) (4, 12). The degradation is mediated by the 26S proteasome, a large multiprotein complex containing trypsin-, chymotrypsin-, and postglutamyl peptidyl hydrolytic-like protease activities. In addition, ubiquitylation has nondegradative roles in DNA repair, transcriptional regulation, signal transduction, endocytosis, and intracellular trafficking (48). Viruses belonging to several families utilize or modulate the ubiquitin-proteasome system (2, 13). The inhibition of proteasomal degradation prevents the entry of influenza virus (23) and mouse hepatitis virus (54); the early postentry steps of minute virus of mice (44) and herpes simplex virus (7); and the genome replication or expression of human coxsackie 3B virus (27), adenovirus (5), cytomegalovirus (20), infectious bursal disease virus (26), and vesicular stomatitis virus (40). In some cases the effects may be secondary to the activation of a cellular stress response and signaling pathway (24, 40, 52). Proteasomal inhibitors have an indirect effect on retroviruses and rhabdoviruses by depleting free ubiquitin needed to modify proteins for budding (16).Vaccinia virus (VACV), the repre...

We describe the results of a prospective observational study of the clinical natural history of human monkeypox virus (MPXV) infections at the remote General Hospital in Kole, DRC (Kole hospital), the rainforest of the Congo River basin of the Democratic Republic of the Congo (DRC) from March 2007 until August 2011. The research was conducted jointly by the Institute National de Recherche Biomedical (INRB) and the US Army Medical Research Institute of Infectious Diseases (USAMRIID). The Kole hospital was one of the two previous WHO Monkeypox (MPX) study sites (1981-1986). The hospital is staffed by the Missionaries of Christ Jesus, a Spanish Order of Catholic Sisters who have continued the treatment of patients with a clinical diagnosis of MPXV infection. Of 244 patients admitted with a clinical diagnosis of MPXV infection, 216 were positive in both the Pan-Orthopox and MPX specific PCR. The cardinal observations of these 216 patients are summarized in this report. There were three deaths (3/216) among these hospitalized patients; fetal death occurred in 4 of 5 (80%) patients who were pregnant at admission. The most common complaints were rash (96.8%), malaise (85.2%), sore throat (78.2%), and lymphadenopathy/adenopathy (57.4%). The most common physical exam findings were MPX rash (99.5%) and lymphadenopathy (98.6%). Age group of less than 5 years had the highest lesion count. Primary household cases tended to have higher lesion counts than secondary or later same household cases. Of the 216 patients, 200 were tested for IgM & IgG antibodies (Abs) to Orthopoxviruses. All 200 patients had anti-orthopoxvirus IgG Abs; whereas 189/200 were positive for IgM. Patients with hypoalbuminemia had a high risk of severe disease. Patients with fatal disease had significantly higher maximum geometric mean values than survivors for the following variables, respectively: viral DNA in blood (DNAemia, p=0.0072); maximum lesion count (p=0.0025); day of admission mean AST and ALT (p=0.0002 and p = 0.0224, respectively, adjusted p-values).

Polyprotein processing is a major strategy used by many plant and animal viruses to maximize the number of protein products obtainable from a single open reading frame. In Sesbania mosaic virus, open reading frame-2 codes for a polyprotein that is cleaved into different functional proteins in cis by the N-terminal serine protease domain. The soluble protease domain lacking 70-amino-acid residues from the N terminus (⌬N70Pro, where Pro is protease) was not active in trans. Interestingly, the protease domain exhibited trans-catalytic activity when VPg (viral protein genome-linked) was present at the C terminus. Bioinformatic analysis of VPg primary structure suggested that it could be a disordered protein. Biophysical studies validated this observation, and VPg resembled "natively unfolded" proteins. CD spectral analysis showed that the ⌬N70Pro-VPg fusion protein had a characteristic secondary structure with a 230 nm positive CD peak. Mutation of Trp-43 in the VPg domain to phenylalanine abrogated the positive peak with concomitant loss in cis-and trans-proteolytic activity of the ⌬N70Pro domain. Further, deletion of VPg domain from the polyprotein completely abolished proteolytic processing. The results suggested a novel mechanism of activation of the protease, wherein the interaction between the natively unfolded VPg and the protease domains via aromatic amino acid residues alters the conformation of the individual domains and the active site of the protease. Thus, VPg is an activator of protease in Sesbania mosaic virus, and probably by this mechanism, the polyprotein processing could be regulated in planta.

SummaryA resident of interior Alaska, was diagnosed with an Orthopoxvirus infection. Phylogenetic analysis revealed it is a novel, previously undescribed Orthopoxvirus species. Phylogenetically, the virus is sister to recognized Old World orthopoxviruses, rather than North American Orthopoxvirus species.