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Rabies is a fatal encephalitic disease in humans and animals caused by lyssaviruses, most commonly rabies virus (RABV). Human antemortem diagnosis of rabies is a complex process involving multiple sample types and tests for the detection of antibodies, antigen (protein), and nucleic acids (genomic RNA). Serological diagnosis of human rabies includes the detection of either neutralizing or binding antibodies in the cerebrospinal fluid (CSF) or serum samples from unimmunized individuals without prior rabies vaccination or passive immunization with purified immunoglobulins. While neutralizing antibodies are targeted against the surface-expressed glycoprotein (G protein), binding antibodies to viral antigens are predominantly against the nucleoprotein (N protein), although there can be antibodies against all RABV-expressed proteins. To determine N protein-specific antibody responses in the CSF and serum during RABV infection, we developed an enzyme-linked immunosorbent assay (ELISA) with purified recombinant N protein expressed in E. coli. N protein-specific immunoglobulin (Ig) subtypes IgG and IgM were detected in the CSF or serum of previously diagnosed human rabies cases. In addition, anti-N protein seroconversion was demonstrated over the course of illness in individual rabies cases. We compared the N protein ELISA results to those of an indirect fluorescent antibody (IFA) test, the current binding antibody assay used in diagnosis, and show that our ELISA is consistent with the IFA test. Sensitivity and specificity of the N protein ELISA ranged from 78.38-100% and 75.76-96.77% with respect to the IFA results. Our data provide evidence for the use of an N protein ELISA as an additional option for the detection of RABV-specific IgG or IgM antibodies in human CSF or serum specimens.

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. Smallpox, caused by Variola virus (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. Monkeypox virus (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 5 plaque-forming units (PFU; 90% lethal dose [LD 90 ]) 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 [ C max ]) and the time of the last quantifiable concentration (AUC last ) were lower than in other animal models administered the same doses, indicating that suboptimal BCV exposure may explain the lower protective effect on survival. IMPORTANCE 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 in vivo against smallpox, studies with the related virus MPXV are critical in understanding whether it would be protective in the event of a smallpox outbreak.

Since the eradication of smallpox, there have been increases in poxvirus infections and the emergence of several novel poxviruses that can infect humans and domestic animals. In 2015, a novel poxvirus was isolated from a resident of Alaska. Diagnostic testing and limited sequence analysis suggested this isolate was a member of the Orthopoxvirus (OPXV) genus but was highly diverged from currently known species, including Akhmeta virus. Here, we present the complete 210,797 bp genome sequence of the Alaska poxvirus isolate, containing 206 predicted open reading frames. Phylogenetic analysis of the conserved central region of the genome suggested the Alaska isolate shares a common ancestor with Old World OPXVs and is diverged from New World OPXVs. We propose this isolate as a member of a new OPXV species, Alaskapox virus (AKPV). The AKPV genome contained host range and virulence genes typical of OPXVs but lacked homologs of C4L and B7R, and the hemagglutinin gene contained a unique 120 amino acid insertion. Seven predicted AKPV proteins were most similar to proteins in non-OPXV Murmansk or NY_014 poxviruses. Genomic analysis revealed evidence suggestive of recombination with Ectromelia virus in two putative regions that contain seven predicted coding sequences, including the A-type inclusion protein.

Monkeypox virus (MPXV) is a member of the genus Orthopoxvirus, endemic in Central and West Africa. This viral zoonosis was introduced into the United States in 2003 via African rodents imported for the pet trade and caused 37 human cases, all linked to exposure to MPXV-infected black-tailed prairie dogs (Cynomys ludovicianus). Prairie dogs have since become a useful model of MPXV disease, utilized for testing of potential medical countermeasures. In this study, we used recombinant MPXV containing the firefly luciferase gene (luc) and in vivo imaging technology to characterize MPXV pathogenesis in the black-tailed prairie dog in real time. West African (WA) MPXV could be visualized using in vivo imaging in the nose, lymph nodes, intestines, heart, lung, kidneys, and liver as early as day 6 post infection (p.i.). By day 9 p.i., lesions became visible on the skin and in some cases in the spleen. After day 9 p.i., luminescent signal representing MPXV replication either increased, indicating a progression to what would be a fatal infection, or decreased as infection was resolved. Use of recombinant luc+ MPXV allowed for a greater understanding of how MPXV disseminates throughout the body in prairie dogs during the course of infection. This technology will be used to reduce the number of animals required in future pathogenesis studies as well as aid in determining the effectiveness of potential medical countermeasures.

Monkeypox virus (MPXV) causes a smallpox-like disease in humans. Clinical and epidemiological studies provide evidence of pathogenicity differences between two geographically distinct monkeypox virus clades: the West African and Congo Basin. Genomic analysis of strains from both clades identified a ∼10 kbp deletion in the less virulent West African isolates sequenced to date. One absent open reading frame encodes the monkeypox virus homologue of the complement control protein (CCP). This modulatory protein prevents the initiation of both the classical and alternative pathways of complement activation. In monkeypox virus, CCP, also known as MOPICE, is a ∼24 kDa secretory protein with sequence homology to this superfamily of proteins. Here we investigate CCP expression and its role in monkeypox virulence and pathogenesis. CCP was incorporated into the West African strain and removed from the Congo Basin strain by homologous recombination. CCP expression phenotypes were confirmed for both wild type and recombinant monkeypox viruses and CCP activity was confirmed using a C4b binding assay. To characterize the disease, prairie dogs were intranasally infected and disease progression was monitored for 30 days. Removal of CCP from the Congo Basin strain reduced monkeypox disease morbidity and mortality, but did not significantly decrease viral load. The inclusion of CCP in the West African strain produced changes in disease manifestation, but had no apparent effect on disease-associated mortality. This study identifies CCP as an important immuno-modulatory protein in monkeypox pathogenesis but not solely responsible for the increased virulence seen within the Congo Basin clade of monkeypox virus.

Although orthopoxviruses (OPXV) are known to encode a majority of the genes required for replication in host cells, genome-wide genetic screens have revealed that several host pathways are indispensable for OPXV infection. Through a haploid genetic screen, we previously identified several host genes required for monkeypox virus (MPXV) infection, including the individual genes that form the conserved oligomeric Golgi (COG) complex. The COG complex is an eight-protein (COG1–COG8) vesicle tethering complex important for regulating membrane trafficking, glycosylation enzymes, and maintaining Golgi structure. In this study, we investigated the role of the COG complex in OPXV infection using cell lines with individual COG gene knockout (KO) mutations. COG KO cells infected with MPXV and vaccinia virus (VACV) produced small plaques and a lower virus yield compared to wild type (WT) cells. In cells where the KO phenotype was reversed using a rescue plasmid, the size of virus plaques increased demonstrating a direct link between the decrease in viral spread and the KO of COG genes. KO cells infected with VACV displayed lower levels of viral fusion and entry compared to WT suggesting that the COG complex is important for early events in OPXV infection. Additionally, fewer actin tails were observed in VACV-infected KO cells compared to WT. Since COG complex proteins are required for cellular trafficking of glycosylated membrane proteins, the disruption of this process due to lack of individual COG complex proteins may potentially impair the virus-cell interactions required for viral entry and egress. These data validate that the COG complex previously identified in our genetic screens plays a role in OPXV infection.

Smallpox, caused by the solely human pathogen Variola virus (VARV), was declared eradicated in 1980. While known VARV stocks are secure, smallpox remains a bioterrorist threat agent. Recent U.S. Food and Drug Administration approval of the first smallpox anti-viral (tecovirimat) therapeutic was a successful step forward in smallpox preparedness; however, orthopoxviruses can become resistant to treatment, suggesting a multi-therapeutic approach is necessary. Animal models are required for testing medical countermeasures (MCMs) and ideally MCMs are tested directly against the pathogen of interest. Since VARV only infects humans, a representative animal model for testing therapeutics directly against VARV remains a challenge. Here we show that three different humanized mice strains are highly susceptible to VARV infection, establishing the first small animal model using VARV. In comparison, the non-humanized, immunosuppressed background mouse was not susceptible to systemic VARV infection. Following an intranasal VARV challenge that mimics the natural route for human smallpox transmission, the virus spread systemically within the humanized mouse before mortality (~ 13 days post infection), similar to the time from exposure to symptom onset for ordinary human smallpox. Our identification of a permissive/representative VARV animal model can facilitate testing of MCMs in a manner consistent with their intended use.

Smallpox was the first human disease to be eradicated, through a concerted vaccination campaign led by the World Health Organization. Since its eradication, routine vaccination against smallpox has ceased, leaving the world population susceptible to disease caused by orthopoxviruses. In recent decades, reports of human disease from zoonotic orthopoxviruses have increased. Furthermore, multiple reports of newly identified poxviruses capable of causing human disease have occurred. These facts raise concerns regarding both the opportunity for these zoonotic orthopoxviruses to evolve and become a more severe public health issue, as well as the risk of Variola virus (the causative agent of smallpox) to be utilized as a bioterrorist weapon. The eradication of smallpox occurred prior to the development of the majority of modern virological and molecular biological techniques. Therefore, there is a considerable amount that is not understood regarding how this solely human pathogen interacts with its host. This paper briefly recounts the history and current status of diagnostic tools, vaccines, and anti-viral therapeutics for treatment of smallpox disease. The authors discuss the importance of further research to prepare the global community should a smallpox-like virus emerge.

Monkeypox virus (MPXV) is endemic within Africa where it sporadically is reported to cause outbreaks of human disease. In 2003, an outbreak of human MPXV occurred in the US after the importation of infected African rodents. Since the eradication of smallpox (caused by an orthopoxvirus (OPXV) related to MPXV) and cessation of routine smallpox vaccination (with the live OPXV vaccinia), there is an increasing population of people susceptible to OPXV diseases. Previous studies have shown that the prairie dog MPXV model is a functional animal model for the study of systemic human OPXV illness. Studies with this model have demonstrated that infected animals are able to transmit the virus to naive animals through multiple routes of exposure causing subsequent infection, but were not able to prove that infected animals could transmit the virus exclusively via the respiratory route. Herein we used the model system to evaluate the hypothesis that the Congo Basin clade of MPXV is more easily transmitted, via respiratory route, than the West African clade. Using a small number of test animals, we show that transmission of viruses from each of the MPXV clade was minimal via respiratory transmission. However, transmissibility of the Congo Basin clade was slightly greater than West African MXPV clade (16.7% and 0% respectively). Based on these findings, respiratory transmission appears to be less efficient than those of previous studies assessing contact as a mechanism of transmission within the prairie dog MPXV animal model.

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.