Congenital Mpox Syndrome (Clade I) in Stillborn Fetus after Placental Infection and Intrauterine Transmission, Democratic Republic of the Congo, 2008.

We report the autopsy pathology findings of a 21-week stillborn fetus with congenital mpox syndrome that occurred in the Democratic Republic of the Congo in 2008. The fetus acquired mpox from the mother after intrauterine transplacental monkeypox virus transmission. We confirmed monkeypox virus infection in the mother, fetus, and placenta by using a monkeypox virus-specific quantitative PCR. Subtyping of the virus was not performed, but the mother and fetus were almost certainly infected with the clade I variant that was endemic in the Democratic Republic of the Congo at the time. Risk for intrauterine infection appears to differ between virus clades, but clinicians should be aware of potential for intrauterine monkeypox virus transmission among pregnant persons during ongoing and future mpox outbreaks.

Development of a specific MPXV antigen detection immunodiagnostic assay.

Human monkeypox (mpox) has recently become a global public health emergency; however, assays that detect mpox infection are not widely available, largely due to cross-reactivity within the Orthopoxvirus genus. Immunoassay development was largely confined to researchers who focus on biothreats and endemic areas (Central and West Africa) until the 2022 outbreak. As was noted in the COVID-19 pandemic, antigen detection assays, integrated with molecular assays, are necessary to help curb the spread of disease. Antigen-detecting immunoassays offer the advantage of providing results ranging from within min to h and in lateral flow formats; they can be deployed for point-of-care, home, or field use. This study reports the development of an mpox-specific antigen detection immunoassay developed on a multiplexed, magnetic-bead-based platform utilizing reagents from all research sectors (commercial, academic, and governmental). Two semi-quantitative assays were developed in parallel and standardized with infectious mpox virus (MPXV) cell culture fluid and MPXV-positive non-human primate (NHP) sera samples. These assays could detect viral antigens in serum, were highly specific toward MPXV as compared to other infectious orthopoxviruses (vaccinia virus, cowpox virus, and camelpox virus), and exhibited a correlation with quantitative PCR results from an NHP study. Access to a toolbox of assays for mpox detection will be key for identifying cases and ensuring proper treatment, as MPXV is currently a global traveler.

Clinical characterization and placental pathology of mpox infection in hospitalized patients in the Democratic Republic of the Congo.

We describe the results of a prospective observational study of the clinical natural history of human monkeypox (mpox) virus (MPXV) infections at the remote L'Hopital General de Reference de Kole (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 Mpox study sites (1981-1986). The hospital is staffed by a Spanish Order of Catholic Nuns from La Congregation Des Soeurs Missionnaires Du Christ Jesus including two Spanish physicians, who were members of the Order as well, were part of the WHO study on human mpox. Of 244 patients admitted with a clinical diagnosis of MPXV infection, 216 were positive in both the Pan-Orthopox and MPXV 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 3 of 4 patients who were pregnant at admission, with the placenta of one fetus demonstrating prominent MPXV infection of the chorionic villi. 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 mpox rash (99.5%) and lymphadenopathy (98.6%). The single patient without the classic mpox rash had been previously vaccinated against smallpox. 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 higher maximum geometric mean values than survivors for the following variables, respectively: viral DNA in blood (DNAemia); maximum lesion count; day of admission mean AST and ALT.

Design and Optimization of a Monkeypox virus Specific Serological Assay.

Monkeypox virus (MPXV), a member of the Orthopoxvirus (OPXV) genus, is a zoonotic virus, endemic to central and western Africa that can cause smallpox-like symptoms in humans with fatal outcomes in up to 15% of patients. The incidence of MPXV infections in the Democratic Republic of the Congo, where the majority of cases have occurred historically, has been estimated to have increased as much as 20-fold since the end of smallpox vaccination in 1980. Considering the risk global travel carries for future disease outbreaks, accurate epidemiological surveillance of MPXV is warranted as demonstrated by the recent Mpox outbreak, where the majority of cases were occurring in non-endemic areas. Serological differentiation between childhood vaccination and recent infection with MPXV or other OPXVs is difficult due to the high level of conservation within OPXV proteins. Here, a peptide-based serological assay was developed to specifically detect exposure to MPXV. A comparative analysis of immunogenic proteins across human OPXVs identified a large subset of proteins that could potentially be specifically recognized in response to a MPXV infection. Peptides were chosen based upon MPXV sequence specificity and predicted immunogenicity. Peptides individually and combined were screened in an ELISA against serum from well-characterized Mpox outbreaks, vaccinee sera, and smallpox sera collected prior to eradication. One peptide combination was successful with ~86% sensitivity and ~90% specificity. The performance of the assay was assessed against the OPXV IgG ELISA in the context of a serosurvey by retrospectively screening a set of serum specimens from the region in Ghana believed to have harbored the MPXV-infected rodents involved in the 2003 United States outbreak.

Maternal and Fetal Outcomes Among Pregnant Women With Human Monkeypox Infection in the Democratic Republic of Congo.

Human monkeypox is an endemic disease in rain-forested regions of central Democratic Republic of Congo. We report fetal outcomes for 1 of 4 pregnant women who participated in an observational study at the General Hospital of Kole (Sankuru Province), where 222 symptomatic subjects were followed between 2007 and 2011. Of the 4 pregnant women, 1 gave birth to a healthy infant, 2 had miscarriages in the first trimester, and 1 had fetal death, with the macerated stillborn showing diffuse cutaneous maculopapillary skin lesions involving the head, trunk and extremities, including palms of hands and soles of feet.

Susceptibility of Marmosets (Callithrix jacchus) to Monkeypox Virus: A Low Dose Prospective Model for Monkeypox and Smallpox Disease.

Although current nonhuman primate models of monkeypox and smallpox diseases provide some insight into disease pathogenesis, they require a high titer inoculum, use an unnatural route of infection, and/or do not accurately represent the entire disease course. This is a concern when developing smallpox and/or monkeypox countermeasures or trying to understand host pathogen relationships. In our studies, we altered half of the test system by using a New World nonhuman primate host, the common marmoset. Based on dose finding studies, we found that marmosets are susceptible to monkeypox virus infection, produce a high viremia, and have pathological features consistent with smallpox and monkeypox in humans. The low dose (48 plaque forming units) required to elicit a uniformly lethal disease and the extended incubation (preclinical signs) are unique features among nonhuman primate models utilizing monkeypox virus. The uniform lethality, hemorrhagic rash, high viremia, decrease in platelets, pathology, and abbreviated acute phase are reflective of early-type hemorrhagic smallpox.

Smallpox vaccine, ACAM2000: Sites and duration of viral shedding and effect of povidone iodine on scarification site shedding and immune response.

The U.S. Department of Defense vaccinates personnel deployed to high-risk areas with the vaccinia virus (VACV)-based smallpox vaccine. Autoinoculations and secondary and tertiary transmissions due to VACV shedding from the vaccination site continue to occur despite education of vaccinees on the risks of such infections. The objectives of this study were to investigate, in naïve smallpox vaccinees, (a) whether the vaccination site can remain contagious after the scab separates and (b) whether the application of povidone iodine ointment (PIO) to the vaccination site inactivates VACV without affecting the immune response. These objectives were tested in 60 individuals scheduled to receive smallpox vaccine. Thirty individuals (control) did not receive PIO; 30 subjects (treatment) received PIO starting on post-vaccination day 7. Counter to current dogma, this study showed that VACV continues to shed from the vaccination site after the scab separates. Overall viral shedding levels in the PIO group were significantly lower than those in the control group (p=0.0045), and PIO significantly reduced the duration of viral shedding (median duration 14.5 days and 21 days in the PIO and control groups, respectively; p=0.0444). At least 10% of control subjects continued to shed VACV at day 28, and 3.4% continued to shed the virus at day 42. PIO reduced the proportion of subjects shedding virus from the vaccination site from day 8 until days 21-23 compared with control subjects. Groups did not differ significantly in the proportion of subjects mounting an immune response, as measured by neutralizing antibodies, IgM, IgG, and interferon-gamma enzyme-linked immunospot assay. When applied to the vaccination site starting on day 7, PIO reduced viral shedding without altering the immune response. The use of PIO in addition to a semipermeable dressing may reduce the rates of autoinoculation and contact transmission originating from the vaccination site in smallpox-vaccinated individuals.

Progression of pathogenic events in cynomolgus macaques infected with variola virus.

Smallpox, caused by variola virus (VARV), is a devastating human disease that affected millions worldwide until the virus was eradicated in the 1970 s. Subsequent cessation of vaccination has resulted in an immunologically naive human population that would be at risk should VARV be used as an agent of bioterrorism. The development of antivirals and improved vaccines to counter this threat would be facilitated by the development of animal models using authentic VARV. Towards this end, cynomolgus macaques were identified as adequate hosts for VARV, developing ordinary or hemorrhagic smallpox in a dose-dependent fashion. To further refine this model, we performed a serial sampling study on macaques exposed to doses of VARV strain Harper calibrated to induce ordinary or hemorrhagic disease. Several key differences were noted between these models. In the ordinary smallpox model, lymphoid and myeloid hyperplasias were consistently found whereas lymphocytolysis and hematopoietic necrosis developed in hemorrhagic smallpox. Viral antigen accumulation, as assessed immunohistochemically, was mild and transient in the ordinary smallpox model. In contrast, in the hemorrhagic model antigen distribution was widespread and included tissues and cells not involved in the ordinary model. Hemorrhagic smallpox developed only in the presence of secondary bacterial infections - an observation also commonly noted in historical reports of human smallpox. Together, our results support the macaque model as an excellent surrogate for human smallpox in terms of disease onset, acute disease course, and gross and histopathological lesions.

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.

Experience with intravenous ribavirin in the treatment of hemorrhagic fever with renal syndrome in Korea.

Results of a clinical study using intravenous (IV) ribavirin for treating Department of Defense personnel with hemorrhagic fever with renal syndrome (HFRS) acquired in Korea from 1987 to 2005 were reviewed to determine the clinical course of HFRS treated with IV ribavirin. A total of 38 individuals enrolled in the study had subsequent serological confirmation of HFRS. Four of the 38 individuals received three or fewer doses of ribavirin and were excluded from treatment analysis. Of the remaining 34 individuals, oliguria was present in one individual at treatment initiation; none of the remaining 33 subjects developed oliguria or required dialysis. The mean peak serum creatinine was 3.46 mg/dl and occurred on day 2 of ribavirin therapy. Both the peak serum creatinine and the onset of polyuria occurred on mean day 6.8 of illness. Reversible hemolytic anemia was the main adverse event of ribavirin, with a >or=25% decrease in hematocrit observed in 26/34 (76.5%) individuals. While inability to adjust for all baseline variables prevents comparison to historical cohorts in Korea where oliguria has been reported in 39-69% cases and dialysis required in approximately 40% HFRS cases caused by Hantaan virus, the occurrence of 3% oliguria and 0% dialysis requirement in the treatment cohort is supportive of a previous placebo-controlled HFRS trial in China where IV ribavirin given early resulted in decreased occurrence of oliguria and decreased severity of renal insufficiency.

Recombinant modified vaccinia virus Ankara provides durable protection against disease caused by an immunodeficiency virus as well as long-term immunity to an orthopoxvirus in a non-human primate.

Recombinant and non-recombinant modified vaccinia virus Ankara (MVA) strains are currently in clinical trials as human immunodeficiency virus-1 (HIV) and attenuated smallpox vaccines, respectively. Here we tested the ability of a recombinant MVA delivered by alternative needle-free routes (intramuscular, intradermal, or into the palatine tonsil) to protect against immunodeficiency and orthopoxvirus diseases in a non-human primate model. Rhesus macaques were immunized twice 1 month apart with MVA expressing 5 genes from a pathogenic simian human immunodeficiency virus (SHIV)/89.6P and challenged intrarectally 9 months later with the pathogenic SHIV/89.6P and intravenously 2.7 years later with monkeypox virus. Irrespective of the route of vaccine delivery, binding and neutralizing antibodies and CD8 responses to SHIV and orthopoxvirus proteins were induced and the monkeys were successively protected against the diseases caused by the challenge viruses in unimmunized controls as determined by viral loads and clinical signs. These non-human primate studies support the clinical testing of recombinant MVA as an HIV vaccine and further demonstrate that MVA can provide long-term poxvirus immunity, essential for use as an alternative smallpox vaccine.

N-(3,3a,4,4a,5,5a,6,6a-Octahydro-1,3-dioxo-4,6- ethenocycloprop[f]isoindol-2-(1H)-yl)carboxamides: Identification of novel orthopoxvirus egress inhibitors.

A series of novel, potent orthopoxvirus egress inhibitors was identified during high-throughput screening of the ViroPharma small molecule collection. Using structure--activity relationship information inferred from early hits, several compounds were synthesized, and compound 14 was identified as a potent, orally bioavailable first-in-class inhibitor of orthopoxvirus egress from infected cells. Compound 14 has shown comparable efficaciousness in three murine orthopoxvirus models and has entered Phase I clinical trials.

Adjuvant-enhanced antibody responses to recombinant proteins correlates with protection of mice and monkeys to orthopoxvirus challenges.

Recombinant proteins are being evaluated as smallpox and monkeypox vaccines because of their perceived safety compared to live vaccinia virus. Previously, we demonstrated that three or more injections of a Ribi-type adjuvant with a combination of three proteins from the outer membranes of intracellular (L1 protein) and extracellular (A33 and B5 proteins) forms of vaccinia virus protected mice against a lethal intranasal challenge with vaccinia virus. Here, we compared several adjuvants and found that QS-21 and to a lesser extent alum+CpG oligodeoxynucleotides accelerated and enhanced neutralizing antibody responses to a mixture of L1 and A33 proteins, provided the highest ratio of IgG2a to IgG1 isotype response, and protected mice against disease and death after only two immunizations 3 weeks apart. In addition, monkeys immunized with recombinant vaccinia virus proteins and QS-21 developed neutralizing antibody to monkeypox virus and had reduced virus load, skin lesions, and morbidity compared to the non-immunized group following monkeypox virus challenge.

Cynomolgus macaque as an animal model for severe acute respiratory syndrome.

BACKGROUND: The emergence of severe acute respiratory syndrome (SARS) in 2002 and 2003 affected global health and caused major economic disruption. Adequate animal models are required to study the underlying pathogenesis of SARS-associated coronavirus (SARS-CoV) infection and to develop effective vaccines and therapeutics. We report the first findings of measurable clinical disease in nonhuman primates (NHPs) infected with SARS-CoV. METHODS AND FINDINGS: In order to characterize clinically relevant parameters of SARS-CoV infection in NHPs, we infected cynomolgus macaques with SARS-CoV in three groups: Group I was infected in the nares and bronchus, group II in the nares and conjunctiva, and group III intravenously. Nonhuman primates in groups I and II developed mild to moderate symptomatic illness. All NHPs demonstrated evidence of viral replication and developed neutralizing antibodies. Chest radiographs from several animals in groups I and II revealed unifocal or multifocal pneumonia that peaked between days 8 and 10 postinfection. Clinical laboratory tests were not significantly changed. Overall, inoculation by a mucosal route produced more prominent disease than did intravenous inoculation. Half of the group I animals were infected with a recombinant infectious clone SARS-CoV derived from the SARS-CoV Urbani strain. This infectious clone produced disease indistinguishable from wild-type Urbani strain. CONCLUSIONS: SARS-CoV infection of cynomolgus macaques did not reproduce the severe illness seen in the majority of adult human cases of SARS; however, our results suggest similarities to the milder syndrome of SARS-CoV infection characteristically seen in young children.

An orally bioavailable antipoxvirus compound (ST-246) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus Challenge.

ST-246 is a low-molecular-weight compound (molecular weight = 376), that is potent (concentration that inhibited virus replication by 50% = 0.010 microM), selective (concentration of compound that inhibited cell viability by 50% = >40 microM), and active against multiple orthopoxviruses, including vaccinia, monkeypox, camelpox, cowpox, ectromelia (mousepox), and variola viruses. Cowpox virus variants selected in cell culture for resistance to ST-246 were found to have a single amino acid change in the V061 gene. Reengineering this change back into the wild-type cowpox virus genome conferred resistance to ST-246, suggesting that V061 is the target of ST-246 antiviral activity. The cowpox virus V061 gene is homologous to vaccinia virus F13L, which encodes a major envelope protein (p37) required for production of extracellular virus. In cell culture, ST-246 inhibited plaque formation and virus-induced cytopathic effects. In single-cycle growth assays, ST-246 reduced extracellular virus formation by 10 fold relative to untreated controls, while having little effect on the production of intracellular virus. In vivo oral administration of ST-246 protected BALB/c mice from lethal infection, following intranasal inoculation with 10x 50% lethal dose (LD(50)) of vaccinia virus strain IHD-J. ST-246-treated mice that survived infection acquired protective immunity and were resistant to subsequent challenge with a lethal dose (10x LD(50)) of vaccinia virus. Orally administered ST-246 also protected A/NCr mice from lethal infection, following intranasal inoculation with 40,000x LD(50) of ectromelia virus. Infectious virus titers at day 8 postinfection in liver, spleen, and lung from ST-246-treated animals were below the limits of detection (<10 PFU/ml). In contrast, mean virus titers in liver, spleen, and lung tissues from placebo-treated mice were 6.2 x 10(7), 5.2 x 10(7), and 1.8 x 10(5) PFU/ml, respectively. Finally, oral administration of ST-246 inhibited vaccinia virus-induced tail lesions in Naval Medical Research Institute mice inoculated via the tail vein. Taken together, these results validate F13L as an antiviral target and demonstrate that an inhibitor of extracellular virus formation can protect mice from orthopoxvirus-induced disease.

Interferon alfacon1 is an inhibitor of SARS-corona virus in cell-based models.

Preliminary data examining interferon alfacon1 treatment of SARS-CoV (severe acute respiratory syndrome-corona virus)-infected patients suggests this therapy is well tolerated and of therapeutic benefit. We report herein that interferon alfacon1, has potent in vitro antiviral activity against SARS-CoV. In a cytopathic effect protection (CPE) assay, interferon alfacon1 inhibited the generation of CPE in a dose-dependent manner with an IC50 of 0.001 microg/ml, a clinically achievable level. Furthermore, interferon alfacon1 also demonstrated significant antiviral activity in yield reduction and plaque reduction assays. The in vitro antiviral activity of interferon alfacon1 against SARS-CoV suggests continued evaluation of interferon alfacon1 as a therapeutic treatment for patients infected with SARS-CoV.

Generation of eGFP expressing recombinant Zaire ebolavirus for analysis of early pathogenesis events and high-throughput antiviral drug screening.

Zaire ebolavirus causes large outbreaks of severe and usually fatal hemorrhagic disease in humans for which there is no effective treatment or cure. To facilitate examination of early critical events in viral pathogenesis and to identify antiviral compounds, a recombinant Zaire ebolavirus was engineered to express a foreign protein, eGFP, to provide a rapid and sensitive means to monitor virus replication in infected cells. This genetically engineered virus represents the first insertion of a foreign gene into ebolavirus. We show that Ebola-eGFP virus (EboZ-eGFP) infects known early targets of human infections and serves as an ideal model to screen antiviral compounds in less time than any previously published assay.

Exploring the potential of variola virus infection of cynomolgus macaques as a model for human smallpox.

Smallpox virus (variola) poses a significant threat as an agent of bioterrorism. To mitigate this risk, antiviral drugs and an improved vaccine are urgently needed. Satisfactory demonstration of protective efficacy against authentic variola will require development of an animal model in which variola produces a disease course with features consistent with human smallpox. Toward this end, cynomolgus macaques were exposed to several variola strains through aerosol and/or i.v. routes. Two strains, Harper and India 7124, produced uniform acute lethality when inoculated i.v. in high doses (10(9) plaque-forming units). Lower doses resulted in less fulminant, systemic disease and lower mortality. Animals that died had profound leukocytosis, thrombocytopenia, and elevated serum creatinine levels. After inoculation, variola was disseminated by means of a monocytic cell-associated viremia. Distribution of viral antigens by immunohistochemistry correlated with the presence of replicating viral particles demonstrated by electron microscopy and pathology in the lymphoid tissues, skin, oral mucosa, gastrointestinal tract, reproductive system, and liver. These particles resembled those seen in human smallpox. High viral burdens in target tissues were associated with organ dysfunction and multisystem failure. Evidence of coagulation cascade activation (D dimers) corroborated histologic evidence of hemorrhagic diathesis. Depletion of T cell-dependent areas of lymphoid tissues occurred, probably as a consequence of bystander apoptotic mechanisms initiated by infected macrophages. Elaboration of cytokines, including IL-6 and IFN-gamma, contribute to a cytokine storm formerly known as "toxemia." A more precise understanding of disease pathogenesis should provide targets for therapeutic intervention, to be used alone or in combination with inhibitors of variola virus replication.

The host response to smallpox: analysis of the gene expression program in peripheral blood cells in a nonhuman primate model.

Smallpox has played an unparalleled role in human history and remains a significant potential threat to public health. Despite the historical significance of this disease, we know little about the underlying pathophysiology or the virulence mechanisms of the causative agent, variola virus. To improve our understanding of variola pathogenesis and variola-host interactions, we examined the molecular and cellular features of hemorrhagic smallpox in cynomolgus macaques. We used cDNA microarrays to analyze host gene expression patterns in sequential blood samples from each of 22 infected animals. Variola infection elicited striking and temporally coordinated patterns of gene expression in peripheral blood. Of particular interest were features that appear to represent an IFN response, cell proliferation, immunoglobulin gene expression, viral dose-dependent gene expression patterns, and viral modulation of the host immune response. The virtual absence of a tumor necrosis factor alpha/NF-kappaB-activated transcriptional program in the face of an overwhelming systemic infection suggests that variola gene products may ablate this response. These results provide a detailed picture of the host transcriptional response during smallpox infection, and may help guide the development of diagnostic, therapeutic, and prophylactic strategies.

Interferon-beta 1a and SARS coronavirus replication.

A global outbreak of severe acute respiratory syndrome (SARS) caused by a novel coronavirus began in March 2003. The rapid emergence of SARS and the substantial illness and death it caused have made it a critical public health issue. Because no effective treatments are available, an intensive effort is under way to identify and test promising antiviral drugs. Here, we report that recombinant human interferon-beta 1a potently inhibits SARS coronavirus replication in vitro.