Deletion of the monkeypox virus inhibitor of complement enzymes locus impacts the adaptive immune response to monkeypox virus in a nonhuman primate model of infection.
ABSTRACT: Monkeypox virus (MPXV) is an orthopoxvirus closely related to variola virus, the causative agent of smallpox. Human MPXV infection results in a disease that is similar to smallpox and can also be fatal. Two clades of MPXV have been identified, with viruses of the central African clade displaying more pathogenic properties than those within the west African clade. The monkeypox inhibitor of complement enzymes (MOPICE), which is not expressed by viruses of the west African clade, has been hypothesized to be a main virulence factor responsible for increased pathogenic properties of central African strains of MPXV. To gain a better understanding of the role of MOPICE during MPXV-mediated disease, we compared the host adaptive immune response and disease severity following intrabronchial infection with MPXV-Zaire (n = 4), or a recombinant MPXV-Zaire (n = 4) lacking expression of MOPICE in rhesus macaques (RM). Data presented here demonstrate that infection of RM with MPXV leads to significant viral replication in the peripheral blood and lungs and results in the induction of a robust and sustained adaptive immune response against the virus. More importantly, we show that the loss of MOPICE expression results in enhanced viral replication in vivo, as well as a dampened adaptive immune response against MPXV. Taken together, these findings suggest that MOPICE modulates the anti-MPXV immune response and that this protein is not the sole virulence factor of the central African clade of MPXV.
Project description:Monkeypox virus (MPXV) is considered the most significant human public health threat in the genus Orthopoxvirus since the eradication of variola virus (the causative agent of smallpox). MPXV is a zoonotic agent endemic to forested areas of Central and Western Africa. In 2003, MPXV caused an outbreak in the United States due to the importation of infected African rodents, and subsequent sequential infection of North American prairie dogs (Cynomys ludovicianus) and humans. In previous studies, the prairie dog MPXV model has successfully shown to be very useful for understanding MPXV since the model emulates key characteristics of human monkeypox disease. In humans, percutaneous exposure to animals has been documented but the primary method of human-to-human MPXV transmission is postulated to be by respiratory route. Only a few animal model studies of MPXV transmission have been reported. Herein, we show that MPXV infected prairie dogs are able to transmit the virus to naive animals through multiple transmission routes. All secondarily exposed animals were infected with MPXV during the course of the study. Notably, animals secondarily exposed appeared to manifest more severe disease; however, the disease course was very similar to those of experimentally challenged animals including inappetence leading to weight loss, development of lesions, production of orthopoxvirus antibodies and shedding of similar levels or in some instances higher levels of MPXV from the oral cavity. Disease was transmitted via exposure to contaminated bedding, co-housing, or respiratory secretions/nasal mucous (we could not definitively say that transmission occurred via respiratory route exclusively). Future use of the model will allow us to evaluate infection control measures, vaccines and antiviral strategies to decrease disease transmission.
Project description:The vaccinia virus (VACV) E3 protein has been shown to be important for blocking activation of the cellular innate immune system and allowing viral replication to occur unhindered. Mutation or deletion of E3L severely affects viral host range and pathogenesis. While the monkeypox virus (MPXV) genome encodes a homologue of the VACV E3 protein, encoded by the F3L gene, the MPXV gene is predicted to encode a protein with a truncation of 37 N-terminal amino acids. VACV with a genome encoding a similarly truncated E3L protein (VACV-E3L?37N) has been shown to be attenuated in mouse models, and infection with VACV-E3L?37N has been shown to lead to activation of the host antiviral protein kinase R pathway. In this report, we present data demonstrating that, despite containing a truncated E3 homologue, MPXV phenotypically resembles a wild-type (wt) VACV rather than VACV-E3L?37N. Thus, MPXV appears to contain a gene or genes that can suppress the phenotypes associated with an N-terminal truncation in E3. The suppression maps to sequences outside F3L, suggesting that the suppression is extragenic in nature. Thus, MPXV appears to have evolved mechanisms to minimize the effects of partial inactivation of its E3 homologue.Poxviruses have evolved to have many mechanisms to evade host antiviral innate immunity; these mechanisms may allow these viruses to cause disease. Within the family of poxviruses, variola virus (which causes smallpox) is the most pathogenic, while monkeypox virus is intermediate in pathogenicity between vaccinia virus and variola virus. Understanding the mechanisms of monkeypox virus innate immune evasion will help us to understand the evolution of poxvirus innate immune evasion capabilities, providing a better understanding of how poxviruses cause disease.
Project description:Variola virus (VARV) caused smallpox, one of the most devastating human diseases and the first to be eradicated, but its deliberate release represents a dangerous threat. Virulent orthopoxviruses infecting humans, such as monkeypox virus (MPXV), could fill the niche left by smallpox eradication and the cessation of vaccination. However, immunomodulatory activities and virulence determinants of VARV and MPXV remain largely unexplored. We report the molecular characterization of the VARV- and MPXV-secreted type I interferon-binding proteins, which interact with the cell surface after secretion and prevent type I interferon responses. The proteins expressed in the baculovirus system have been purified, and their interferon-binding properties characterized by surface plasmon resonance. The ability of these proteins to inhibit a broad range of interferons was investigated to identify potential adaptation to the human immune system. Furthermore, we demonstrate by Western blot and activity assays the expression of the type I interferon inhibitor during VARV and MPXV infections. These findings are relevant for the design of new vaccines and therapeutics to smallpox and emergent virulent orthopoxviruses because the type I interferon-binding protein is a major virulence factor in animal models, vaccination with this protein induces protective immunity, and its neutralization prevents disease progression.
Project description:Monkeypox virus (MPXV) is a human pathogen that is a member of the Orthopoxvirus genus, which includes Vaccinia virus and Variola virus (the causative agent of smallpox). Human monkeypox is considered an emerging zoonotic infectious disease. To identify host factors required for MPXV infection, we performed a genome-wide insertional mutagenesis screen in human haploid cells. The screen revealed several candidate genes, including those involved in Golgi trafficking, glycosaminoglycan biosynthesis, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. We validated the role of a set of vacuolar protein sorting (VPS) genes during infection, VPS51 to VPS54 (VPS51-54), which comprise the Golgi-associated retrograde protein (GARP) complex. The GARP complex is a tethering complex involved in retrograde transport of endosomes to the trans-Golgi apparatus. Our data demonstrate that VPS52 and VPS54 were dispensable for mature virion (MV) production but were required for extracellular virus (EV) formation. For comparison, a known antiviral compound, ST-246, was used in our experiments, demonstrating that EV titers in VPS52 and VPS54 knockout (KO) cells were comparable to levels exhibited by ST-246-treated wild-type cells. Confocal microscopy was used to examine actin tail formation, one of the viral egress mechanisms for cell-to-cell dissemination, and revealed an absence of actin tails in VPS52KO- or VPS54KO-infected cells. Further evaluation of these cells by electron microscopy demonstrated a decrease in levels of wrapped viruses (WVs) compared to those seen with the wild-type control. Collectively, our data demonstrate the role of GARP complex genes in double-membrane wrapping of MVs necessary for EV formation, implicating the host endosomal trafficking pathway in orthopoxvirus infection.IMPORTANCE Human monkeypox is an emerging zoonotic infectious disease caused by Monkeypox virus (MPXV). Of the two MPXV clades, the Congo Basin strain is associated with severe disease, increased mortality, and increased human-to-human transmission relative to the West African strain. Monkeypox is endemic in regions of western and central Africa but was introduced into the United States in 2003 from the importation of infected animals. The threat of MPXV and other orthopoxviruses is increasing due to the absence of routine smallpox vaccination leading to a higher proportion of naive populations. In this study, we have identified and validated candidate genes that are required for MPXV infection, specifically, those associated with the Golgi-associated retrograde protein (GARP) complex. Identifying host targets required for infection that prevents extracellular virus formation such as the GARP complex or the retrograde pathway can provide a potential target for antiviral therapy.
Project description:Smallpox, caused by <i>Variola virus</i> (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. <i>Monkeypox virus</i> (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?×?10<sup>5</sup> plaque-forming units (PFU; 90% lethal dose [LD<sub>90</sub>]) 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 [<i>C</i> <sub>max</sub>]) and the time of the last quantifiable concentration (AUC<sub>last</sub>) were lower than in other animal models administered the same doses, indicating that suboptimal BCV exposure may explain the lower protective effect on survival.<b>IMPORTANCE</b> 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 <i>in vivo</i> against smallpox, studies with the related virus MPXV are critical in understanding whether it would be protective in the event of a smallpox outbreak.
Project description:Monkeypox virus (MPXV) is an emerging pathogen from Africa that causes disease similar to smallpox. Two clades with different geographic distributions and virulence have been described. Here, we utilized bioinformatic tools to identify genomic regions in MPXV containing multiple virulence genes and explored their roles in pathogenicity; two selected regions were then deleted singularly or in combination. In vitro and in vivo studies indicated that these regions play a significant role in MPXV replication, tissue spread, and mortality in mice. Interestingly, while deletion of either region led to decreased virulence in mice, one region had no effect on in vitro replication. Deletion of both regions simultaneously also reduced cell culture replication and significantly increased the attenuation in vivo over either single deletion. Attenuated MPXV with genomic deletions present a safe and efficacious tool in the study of MPX pathogenesis and in the identification of genetic factors associated with virulence.
Project description:Monkeypox is a zoonotic disease endemic to central and western Africa, where it is a major public health concern. Although Monkeypox virus (MPXV) and monkeypox disease in humans have been well characterized, little is known about its natural history, or its maintenance in animal populations of sylvatic reservoir(s). In 2003, several species of rodents imported from Ghana were involved in a monkeypox outbreak in the United States with individuals of three African rodent genera (Cricetomys, Graphiurus, Funisciurus) shown to be infected with MPXV. Here, we examine the course of MPXV infection in Cricetomys gambianus (pouched Gambian rats) and this rodent species' competence as a host for the virus. We obtained ten Gambian rats from an introduced colony in Grassy Key, Florida and infected eight of these via scarification with a challenge dose of 4X104 plaque forming units (pfu) from either of the two primary clades of MPXV: Congo Basin (C-MPXV: n = 4) or West African (W-MPXV: n = 4); an additional 2 animals served as PBS controls. Viral shedding and the effect of infection on activity and physiological aspects of the animals were measured. MPXV challenged animals had significantly higher core body temperatures, reduced activity and increased weight loss than PBS controls. Viable virus was found in samples taken from animals in both experimental groups (C-MPXV and W-MPXV) between 3 and 27 days post infection (p.i.) (up to 1X108 pfu/ml), with viral DNA found until day 56 p.i. The results from this work show that Cricetomys gambianus (and by inference, probably the closely related species, Cricetomys emini) can be infected with MPXV and shed viable virus particles; thus suggesting that these animals may be involved in the maintenance of MPXV in wildlife mammalian populations. More research is needed to elucidate the epidemiology of MPXV and the role of Gambian rats and other species.
Project description:Infectious diseases that originate from multiple wildlife hosts can be complex and problematic to manage. A full understanding is further limited by large temporal and spatial gaps in sampling. However, these limitations can be overcome, in part, by using historical samples, such as those derived from museum collections. Here, we screened over 1000 museum specimens collected over the past 120 years to examine the historical distribution and prevalence of monkeypox virus (MPXV) in five species of African rope squirrel (Funisciurus sp.) collected across Central Africa. We found evidence of MPXV infections in host species as early as 1899, half a century earlier than the first recognized case of MPXV in 1958, supporting the suggestion that historic pox-like outbreaks in humans and non-human primates may have been caused by MPXV rather than smallpox as originally thought. MPX viral DNA was found in 93 of 1038 (9.0%) specimens from five Funisciurus species (F. anerythrus, F. carruthersi, F. congicus, F. lemniscatus and F. pyrropus), of which F. carruthersi and pyrropus had not previously been identified as potential MPXV hosts. We additionally documented relative prevalence rates of infection in museum specimens of Funisciurus and examined the spatial and temporal distribution of MPXV in these potential host species across nearly a hundred years (1899-1993).
Project description:Identification of human monkeypox cases during 2005 in southern Sudan (now South Sudan) raised several questions about the natural history of monkeypox virus (MPXV) in Africa. The outbreak area, characterized by seasonally dry riverine grasslands, is not identified as environmentally suitable for MPXV transmission. We examined possible origins of this outbreak by performing phylogenetic analysis of genome sequences of MPXV isolates from the outbreak in Sudan and from differing localities. We also compared the environmental suitability of study localities for monkeypox transmission. Phylogenetically, the viruses isolated from Sudan outbreak specimens belong to a clade identified in the Congo Basin. This finding, added to the political instability of the area during the time of the outbreak, supports the hypothesis of importation by infected animals or humans entering Sudan from the Congo Basin, and person-to-person transmission of virus, rather than transmission of indigenous virus from infected animals to humans.
Project description:Healthcare-associated transmission of monkeypox has been observed on multiple occasions in areas where the disease is endemic. Data collected by the US Centers for Disease Control and Prevention (CDC) from an ongoing CDC-supported program of enhanced surveillance in the Tshuapa Province of the Democratic Republic of the Congo, where the annual incidence of human monkeypox is estimated to be 3.5-5/10,000, suggests that there is approximately one healthcare worker infection for every 100 confirmed monkeypox cases. Herein, we describe a study that commenced in February 2017, the intent of which is to evaluate the effectiveness, immunogenicity, and safety of a third-generation smallpox vaccine, IMVAMUNE®, in healthcare personnel at risk of monkeypox virus (MPXV) infection. We describe procedures for documenting exposures to monkeypox virus infection in study participants, and outline lessons learned that may be of relevance for studies of other investigational medical countermeasures in hard to reach, under-resourced populations.