Variola Virus and Clade I Mpox Virus Differentially Modulate Cellular Responses Longitudinally in Monocytes During Infection

Abstract

Variola virus (VARV), the etiological agent of smallpox, had enormous impacts on global health prior to its eradication. In the absence of global vaccination programs, mpox virus (MPXV) has become a growing public health threat that includes endemic and nonendemic regions across the globe. While human mpox resembles smallpox in clinical presentation, there are considerable knowledge gaps regarding conserved molecular pathogenesis between these 2 orthopoxviruses. Thus, we sought to compare MPXV and VARV infections in human monocytes through kinome analysis. We performed a longitudinal analysis of host cellular responses to VARV infection in human monocytes as well as a comparative analysis to clade I MPXV-mediated responses. While both viruses elicited strong activation of cell responses early during infection as compared to later time points, several key differences in cell signaling events were identified and validated. These observations will help in the design and development of panorthopoxvirus therapeutics.

Variola virus (VARV), the etiological agent of smallpox, has had the greatest impact on humans throughout history of all infectious diseases and resulted in more than 500 million deaths during the 20th century alone [1]. The Smallpox Eradication Program, which ran from 1966 to 1980, resulted in the eradication of the disease. This was facilitated greatly by the lack of a zoonotic reservoir for VARV [1]. Following smallpox eradication, there has been increasing concern regarding the potential for reemergence of the virus from unidentified source material, whether natural or through nefarious means [2]. The generation of horsepox virus using gene synthesis and polio virus cDNA generation underscores these concerns [3–5]. In addition, in the decades following cessation of the global smallpox eradication program, waning immunity increases the risks associated with orthopoxviruses. In recent years, there has been significant increases in human disease caused by other orthopoxviruses, particularly mpox virus (MPXV), due to the increased proportion of an immune-naive population and encroachment into environments of MPXV hosts. Taken together, these factors contribute to the possibility of emerging or reemerging poxviruses that require large-scale prophylactic and therapeutic responses.

Human mpox (formerly monkeypox) is a previously neglected reemerging zoonotic disease caused by MPXV from the genus Orthopoxvirus. MPXV can cause severe illness in infected patients and is endemic in numerous countries across Central and West Africa. Historically, MPXV was described by 2 genetically, clinically, and geographically distinct clades, the Central African (or clade I) and the West African (or clade II) clades [6, 7]. MPXV infections and onward transmission within communities were not typically identified outside of endemic regions. Comparisons of the clade-specific viral proteomes suggested that differences in pathogenesis between the 2 clades were associated with proteins responsible for viral life cycle, immune evasion, host range determination, or virulence factors [8–13]. In 2022, a global outbreak of MPXV resulted in the declaration of a public health emergency of international concern by the World Health Organization (WHO) with ongoing chains of human-to-human transmission across multiple nonendemic global communities where MPXV has not previously been reported [14]. Following phylogenetic assessments of sequencing data acquired during the current global outbreak, the virus circulating in the global outbreak was found to be sufficiently distinct to prompt the proposal of a new naming system with the former Central African clade designated as clade I MPXV, the former West African clade as clade IIa MPXV, and the newly identified global isolates designated as clade IIb MPXV [15, 16]. Case fatality rates (CFR) have been variable across reports. For clade I, the CFR has typically ranged from 1% to 10% in unvaccinated individuals [17]. This high mortality could be caused partly by the lack of basic health care in areas where these outbreaks occurred. It should also be appreciated that this CFR may also be overestimated due to low rates of detection for mild cases. Reporting of CFRs for clade IIa have been far more limited, although recent observations from Nigeria found a CFR of 3.6% [18, 19]. For prophylaxis, Imvamune, a third-generation smallpox vaccine which was granted emergency authorization use against smallpox in Canada in 2013, is now available for use for protection against VARV, MPXV, and other orthopoxviruses. Governments have made the limited quantities of vaccine available to at-risk populations in the current outbreak. An open-label prospective cohort study of Imvamune in adult health care personnel at risk of mpox has been ongoing in the Democratic Republic of the Congo since 2017 [20]. Additionally, an antiviral, tecovirimat or TPOXX, which is approved for use in Canada against smallpox, is also being made available for use in the current outbreak despite a lack of available efficacy data in humans against MPXV. Currently, clinical trials are underway in the PALM project as well as the PLATINUM-UK and PLATINUM-CAN studies [21, 22].

While human mpox resembles smallpox in clinical presentation, there is a paucity of information on potential similarities in host responses to infection between these 2 viruses. Given the increasing frequency of human mpox and the recent global expansion of the geographic range for MPXV, it is prudent to identify novel therapeutic targets or treatment modalities for patients. Here, we sought to compare how infected cells respond to MPXV and VARV and identify common trends in responses or individual cell signaling networks that may represent unique drug targets for these viruses and other human orthopoxviruses. We used kinome analysis to characterize host responses to virus insult and then utilized kinase inhibitors and US Food and Drug Administration (FDA)-licensed kinase therapeutics to selectively target kinases and pathways to validate our observations and identify novel therapeutic targets for human orthopoxviruses.

METHODS

Biosafety Committee Approval

All experiments with VARV were performed at the US Centers for Disease Control and Prevention, Atlanta, GA (CDC), and were approved by the WHO Advisory Committee on Variola Virus Research. Experimental protocols were reviewed and approved at the CDC by the Institutional Biosecurity Board in accordance with the US Government Policy for Oversight of Life Sciences Dual Use Research of Concern.

Cell and Virus Conditions

Clade I MPXV (Zaire 79) and VARV strain Jap51_hrpr were used for all infections in this study. MPXV-GFP (Zaire 79) expressed green fluorescent protein (GFP) under the control of a synthetic early/late promoter [23, 24]. Virus stocks were quantified in a standard plaque assay as described previously [19]. Human THP-1 monocytes (American Type Culture Collection [ATCC] TIB-202R) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% (v/v) heat inactivated fetal bovine serum, 2 mm L-glutamine, and 1 mm sodium pyruvate. All cultures were maintained at 37°C in a humidified 5% (v/v) CO₂ incubator.

Viral Infection

Cells were plated in 6-well plates and rested for 24 hours prior to infection. Cells (1 × 10⁶ cells per condition) were infected with MPXV Zaire 79 at a multiplicity of infection of 3 or mock infected with an equivalent fraction of virus-free culture media. All MPXV infections were performed at the National Institutes of Health at biosafety level 3 (BSL3) in accordance with National Institutes of Health/US CDC Biosafety in Microbiological and Biomedical Laboratories guidelines, as well as in accordance with CDC Select Agent regulations. Infections with VARV BSH74 were performed in the BSL4 laboratory at the US CDC. Infections used the same conditions as those described for MPXV Zaire 79. Virus was incubated with host cells for 1 hour at 37°C with periodic rocking. Following incubation, monocytes were washed twice with phosphate-buffered saline, resuspended with fresh RPMI 1640 media with 2% (v/v) fetal bovine serum. Cells were harvested longitudinally for kinome analysis over a 24-hour period.

Kinome Peptide Array Analysis

Kinome peptide array analysis was performed as previously described [8, 25–27]. Virus-infected or mock-infected cells were harvested at 1, 2, 4, 8, and 24 hours postinfection by centrifugation at 1200 rpm for 10 minutes. Supernatants were removed and cell pellets were flash frozen and stored at −80°C until all time points were collected. Cell pellets were thawed on ice and treated with kinome lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and 1× Pierce Halt Protease and Phosphatase Inhibitor) with the addition of benzonase endonuclease (25 U; Sigma) for removal of nucleic acids from samples, and incubated for 20 minutes on ice with 4× gentle agitation to ensure exposure of all cells to buffer. Lysis samples were then transferred to fresh microcentrifuge tubes and cell debris was removed by centrifugation at 14 000 rpm for 10 minutes at 4°C. Activation mix (50% glycerol, 50 μM ATP, 60 mM MgCl₂, 0.05% Brij 35, and 0.25 mg/mL bovine serum albumin) was added to the equivalent amounts of the total protein (100 μg) for each sample, and total sample volumes were matched by the addition of kinome lysis buffer. PepChip Kinome peptide arrays (Pepscan Therapeutics) were spotted with samples and incubated for 2 hours at 37°C and 5% CO₂. After incubation, arrays were rinsed once with 1% Triton X-100 and once with deionized water. Arrays were stained using PRO-Q Diamond phosphoprotein stain (Invitrogen) for 1 hour with gentle agitation. Following staining, arrays were washed 3 times with kinome destain (20% acetonitrile and 50 mM sodium acetate pH 4.0) for 10 minutes. Arrays were washed a final time with deionized water for 10 minutes and dried by centrifugation. A PowerScanner microarray scanner (Tecan) with a 580-nm filter was used to image arrays and Array-Pro Analyzer version 6.3 software (Media Cybernetics) was used to collect signal intensity values. Intensity values for spots and background were collected for each array.

Pathway Overrepresentation and Gene Ontology Analysis

Kinome data processing was undertaken using the Platform for Integrated, Intelligent Kinome Analysis (PIIKA 2) software as previously described [8, 28]. Briefly, the phosphorylation of each peptide target on the arrays was calculated by subtracting background intensity from foreground intensity. The resulting data were transformed using the variance stabilization model and average intensities were taken over the transformed replicate intensities and subjected to hierarchical clustering analysis. The R package variance stabilization was used for the transformation. Peptide phosphorylations were subjected to paired t tests to compare their signal intensities under a treatment condition with those under the control condition. The preprocessed data was subjected to hierarchical clustering and principal component analysis to cluster treatments based on their kinome profiles. For hierarchical clustering, McQuitty + (1 − Pearson correlation) was used. Pathway overrepresentation analysis (ORA) and gene ontology analysis of differentially phosphorylated proteins were performed using InnateDB software as described previously [24, 27]. Output data were limited to peptides that demonstrated statistically significant changes in expression as compared to the respective time-matched mock controls, with phosphorylation fold change values of >1.5 and P values <.05.

Chemical Inhibitors

The US FDA-licensed drugs tested (sorafenib, everolimus, dabrafenib, cabozantinib, afatanib, selumetinib, trametinib, and miltefosine) were purchased from Selleck Chemicals. Additional kinase inhibitors tested were purchased from Enzo Scientific. The inhibitors were reconstituted according to the manufacturers’ recommendations in either water or dimethyl sulfoxide.

Cytotoxicity Assays

Kinase inhibitor cytotoxicity was determined using the Cytotox colorimetric assay, which measures the amount of lactate dehydrogenase (Promega) released from treated cells, following the manufacturer's instructions. Huh7 cells were incubated with each of the drugs for 24 hours in a 37°C incubator with 5% CO₂ using the inhibitory concentrations used in the assays below. The cell culture supernatants were then used in the Cytotox 96-well assay, and the absorbance at 490 nm was read with a M1000 Tecan plate reader.

Statistical Analyses

All numerical data are presented as mean ± standard error of the mean. Statistical analyses were performed using Prism 8. A P value of ≤.05 was considered statistically significant for all analyses.

RESULTS

Host Kinase Phosphorylation States Separate Into 2 Major Clusters Following VARV Infection

Previously, we have used kinome analysis to characterize host responses through activation of complex cell signaling pathways and biological networks, as well as individual kinases, in species-specific contexts and across a broad range of pathogens [25–28]. This included demonstration that clade I MPXV infection resulted in suppression of host inflammatory responses and modulation of apoptosis as compared to clade IIa MPXV in human THP-1 monocytes [8]. Given the paucity of available host-pathogen interaction information available for VARV following its eradication, we sought to determine how cells respond to VARV during the infectious cycle.

To enable comparison with our prior investigation, we employed THP-1 monocytes for our VARV infections with longitudinal sample collections over a 24-hour infection period. Hierarchical clustering diagrams of host kinome responses at each time point are presented in Figure 1A . Clustering of the VARV data following background subtraction showed 2 major clusters, labeled A and B in Figure 1B . Cluster A included both the 2- and 8-hour postinfection time points; cluster B included the remaining time points with subclustering of the 1- and 24-hour time points as well as clustering with the 4-hour postinfection time point. In contrast, longitudinal characterization of host kinome responses to clade I MPXV resulted in 2 clusters separated by the 8-hour postinfection time point in 1 cluster and all other time points in the second cluster (Figure 1C ). Mock-subtracted longitudinal MPXV clustering is presented in Figure 1D .

Direct comparison of MPXV and VARV kinome data sets resulted in 2 major clusters and 1 subcluster (Figure 1E ). The largest cluster included 2 subclusters: 1 that included all VARV data sets and a second subcluster consisting of the 4-, 8-, and 24-hour MPXV time points. The second cluster consisted of the 1- and 2-hour MPXV infection time points only. These data suggested that host kinome responses to MPXV early in the course of viral infection differed from those in response to VARV. However, later MPXV postinfection time points clustered more strongly with the VARV samples as compared to these early MPXV data sets. This provided evidence for commonalities in host responses mid- to late-infection cycle at the level of specific kinases or signaling pathways.

VARV and MPXV Differentially Activate Host Innate Immune Responses During Infection in Monocytes

While hierarchical clustering provides general insights into global similarities or differences between data sets, we next sought to determine whether infection with either virus differentially modulated cellular responses throughout the viral life cycle.

Early Host Responses to Infection

Our initial analysis of the MPXV and VARV kinome data sets suggested that the 1- and 2-hour postinfection time points for MPXV differed from all other MPXV postinfection time points, as well as from all VARV data sets (Figure 1E ). Thus, we first assessed how responses in infected monocytes to MPXV infection through pathway ORA. At the earliest time point (1 hour postinfection), activation of proinflammatory response pathways (interleukin 23 [IL-23], IL-27, tumor necrosis factor [TNF], and IL-6) were identified. In addition, insulin receptor-mediated signaling and PI3K/Akt signaling were also overrepresented (Supplementary Table 1A). At the 2-hour postinfection time point, transforming growth factor-β (TGF-β)–mediated signaling events were highly overrepresented and comprised the majority of all identified upregulated pathways (Supplementary Table 1B). Additionally, multiple pathways related to response to infection were identified.

Early to Late Time Point Transition

Given the results from our clustering analysis, we expected that host responses from 4 hours postinfection onward for both MPXV and VARV would differ strongly from the early MPXV host responses. Beginning at the 4-hour time point, relatively few host responses were identified through our pathway ORA including involvement of both CBL- and GPCR-mediated signaling events (Supplementary Table 2). A trend towards fewer upregulated signaling pathways from 4 hours postinfection onwards was also found for VARV (Supplementary Table 1). In contrast to the MPXV data, VARV continued to activate pathways related to viral infection responses at the 4-hour time point. These were absent at later time points.

Selective Targeting of Kinases Pre- or Postinfection Inhibits Clade I MPXV Infection

Kinases play a central role in the regulation of cellular responses through their control of cell signaling pathways. We employed a kinase inhibitor screen to identify kinases that had regulatory roles in orthopoxvirus infection and viral replication (Table 1). Monocytes were pretreated with kinase inhibitors for 1 hour prior to the addition of clade I MPXV-GFP and infection was assessed 24 hours later as compared to mock-treated infected cells. Multiple inhibitors had >50% inhibitory activity on MPXV-GFP infection at both 10 µM and 1 µM (Figure 2A ). Staurosporine, which is a panspecific inhibitor, had the strongest inhibitory activity with >80% inhibition at both concentrations tested. Tyrphostin 9, AG 1024, and Rottlerin, which target PGDRFK, IGF-1, and PKCδ, respectively, all had >50% inhibitor activity at both tested concentrations (and >70% inhibition at the highest tested concentration). PKC-412 and Ro 31-8220 mesylate, which target PKC, had strong inhibitory activity (>70%) at the highest tested concentration but substantially lower activity at the lowest tested concentration. Six compounds that had moderate activity (>50% inhibition) at the highest tested concentration included nutlin-3 (activation of p53), PP1 (Src family), KN-62 and KN-93 (CaMK II), 5-iodotubercidin (ERK2, AdK, CK1/2), and SU1498 (Flk1). Compounds targeting AKT/PI3K (Akt-X and LY294002), MLCK (ML-7 and ML-9), Roscovitine (CDK), and BAY 11-7082 (IKK pathway) all had ≤40% inhibitory activity at the highest tested concentration but low activity (≤20%) at 1 µM. Most tested compounds had relatively small effects on cell health at the highest test concentration with <20% reductions in cellular metabolism; only 5-iodotubercidin resulted in >20% reduction of metabolic activity (Figure 2A ).

We next assessed the retention of inhibitory activity when kinases were added to cells following virus absorption and uptake (1 hour after virus addition). Most of the compounds had weak to moderate inhibitory activity (approximately 40% or less) when added following virus infection (Figure 2B ). Staurosporine and 5-iodotubercidin retained the strongest activities with 82% and 67% inhibition, respectively (Figure 2B ). Multiple inhibitors targeting PKC retained >30% inhibitory activity including PKC-412, Ro 31-8220 mesylate, and Rottlerin. In contrast, many inhibitors retained very weak inhibitory activity (<10%) when added 1 hour postinfection and included those targeting AKT/PI3K (Akt-X, LY294002), IGF-1 (AG 1024), Src family members (PP1), MLCK (ML-7 and ML-9), Flk1 (SU1498), and JAK-3 (ZM 449829). These data provide insights on the involvement of various signaling pathways and kinases on the viral life cycle at binding/entry as well as downstream replication.

Following this, we assessed the inhibitory activities of 17 FDA-licensed kinase inhibitors for activity against clade I MPXV infection (Table 2). Inhibitors with >40% activity against MPXV included sorafenib, dabrafenib, cabozantinib, afatinib, axtinib, regorafenib, ponatinib, and crizotinib (Figure 2C ). This included multiple compounds that targeted PDGRF, VEGFR, Raf family members, c-MET, and BCR-ABL. Compounds targeting PI3K/AKT and ERK/MEK were relatively weakly inhibitory against clade I MPXV infection (approximately 20% or less). Crizotinib, which has multiple cellular targets (ALK, HGFR, c-MET, ROS1, RON), had the strongest inhibitory activity of all FDA-licensed compounds (78%). Ponatinib, which targets BCR-ABL, was the only other compound with >60% inhibitory activity on MPXV infection. Sorafenib was the only compound that retained >40% inhibitory activity under both treatment conditions (10 µM and 1 µM).

Crizotinib and Sorafenib Reduce VARV Infection in Monocytes

Building on our analysis of the inhibitory activities of FDA-licensed kinase inhibitors against clade I MPXV, we sought to characterize their activities during VARV infection. The 17 FDA-licensed kinase inhibitors tested against MPXV infection were tested against VARV and inhibitory effects were assessed by plaque assay. Of all inhibitors tested (n = 17), only 2 had any identifiable inhibitory activity against VARV—crizotinib and sorafenib (Figure 2D ); 15 of 17 had no effect on viral titers (data not shown). Pretreatment of monocytes with crizotinib, which had the strongest inhibitory activity against clade I MPXV (Figure 2B ), resulted in significant reduction in VARV titers as compared to the nontreated control. A significant reduction in infectious VARV production was also found with a 1-µM treatment although this activity was much weaker than at the 10-µM treatment. Sorafenib significantly reduced VARV infection at both tested concentrations with a >1 log decrease in viral titers at the 10-µM concentration. Sorafenib was the only compound to retain >40% inhibitory activity against MPXV at both 10-µM and 1-µM concentrations.

DISCUSSION

Smallpox was a serious illness with devastating effects on global health and accounted for an estimated 300 million deaths in the 20th century. With announcement of smallpox eradication in 1980, and cessation of the global vaccination program, there has been decreasing immunity against orthopoxviruses in the human population and a continually increasing immune-naive population. MPXV infections in humans, which was first identified in 1970, have increased in frequency in the period after global smallpox vaccination with continual impacts on public health in endemic regions of Central and West Africa [29–31]. However, the identification of broad clade IIb MPXV circulation across nonendemic regions of the globe in 2022 resulted in the declaration of a public health emergency by the WHO and tens of thousands of confirmed infections. While smallpox and human mpox share numerous similarities in clinical presentation, CFRs are lower for mpox, with MPXV clade-specific variabilities in the degree of discordance [17]. However, there is a dearth of available data comparing these 2 orthopoxviruses, specifically regarding host-pathogen interactions. We addressed this by characterizing host cellular responses to VARV and clade I MPXV through longitudinal interrogation of the host kinome.

As demonstrated by our kinome analysis, VARV and clade I MPXV infections in human monocytes, which are targeted by both viruses during infection, differentially modulate host cellular responses. VARV infection resulted in the induction of numerous innate immune-related cellular responses throughout the early course of infection, including activation of multiple pattern recognition pattern pathways (TLRs, RIG-I, proinflammatory cytokine-related pathways) as well as pathways related directly to infectious pathogens. There was a marked reduction in the overall number of activated pathways identified by ORA in later time points, including those related to infection. Interestingly, while the trend of greater early activation of cellular responses as compared to later postinfection time points was retained in our clade I MPXV samples, early responses differed quite significantly between the 2 viruses. Infectious pathogen-related pathways were upregulated early in MPXV infection; however, there was no overrepresentation of pattern recognition-related pathways as found during VARV infection. Furthermore, there were far fewer upregulated events found in the MPXV samples from 4 hours postinfection onwards with no significantly upregulated pathways identified at the 8-hour time point. This is similar to prior investigations of MPXV where clade I infections were associated with global suppression of cellular responses during infection [8, 13, 32]. Our data suggest that VARV follows a similar trend with early activation of cellular responses to infection followed by suppression of host responses throughout the course of infection. It is important to consider that our investigation was limited to monocytes so whether similar observations are found in additional cells or physiologic locations remains to be determined. However, multiple investigations of MPXV infection observed suppression of host responses across different cell types, suggesting that this may be a common feature of human orthopoxviruses.

Kinases are a primary target for drug design and development, second only to G-coupled protein receptors [33–35]. Indeed, kinases play a critical regulatory role in the generation of cellular responses, including during viral infection. Our kinase inhibitor data demonstrated that targeting of pathways or kinases identified in our kinome analysis decreased the magnitude of MPXV infection. Targeting of IGF-1, PDGFR, IKK members, PKC, and PI3K/AKT, which were identified as members across multiple signaling pathways in our pathway ORA, had inhibitory activities during MPXV infection. The observation that multiple inhibitors had strong reductions in activity against clade I MPXV infection when added following the adsorption and entry phase (1 hour postinfection) suggests a more important role in viral entry versus during the replication cycle or egress. Given the long-standing need for human orthopoxvirus antivirals or host-targeted therapeutic options, kinase inhibitors are an intriguing consideration. Our screening of FDA-licensed kinase inhibitors identifies candidates for further preclinical investigation in vivo. Multiple licensed inhibitors had strong and/or sustained inhibitory activity against clade I MPXV in vitro. Unsurprisingly, those inhibitors that have multiple cellular targets had the strongest inhibitory activities and these targets overlapped well with those identified in our kinome analysis. Intriguingly, only 2 of 17 inhibitors had any inhibitory effect on the production of infectious VARV progeny, as described in our results. As would be anticipated, crizotinib and sorafenib, which target multiple kinases identified in our VARV kinome data across multiple time points, had the strongest inhibitory activities against VARV. As mentioned previously, inhibition against VARV was reliant on assessment of infectious progeny through plaque assay due to the lack of a reporter construct for VARV, unlike our clade I MPXV-GFP. As such, the correlation between reduced fluorescence as compared to infectious virus titers should be considered. However, the ability to screen numerous inhibitors and/or drugs should be considered for drug target down-selection. While crizotinib and sorafenib both significantly decreased VARV infection in treated monocytes, it should be appreciated that only sorafenib decreased viral titers by >1 log. This demonstrates that there is a continuing need for dedicated investigations of orthopoxvirus therapeutics, including repurposed drugs. It is also prudent to consider that investigations of human orthopoxvirus host-targeted therapeutics must account for orthopoxvirus species-specific differences in host responses following infection. Furthermore, the physiological similarities in disease presentation associated with clade I MPXV and VARV infections in humans could also mask critical differences in underlying molecular mechanisms of pathogenesis.

Overall, our analysis provides the first direct assessment of host kinome responses to VARV infection as well as the first cell-based comparative of MPXV and VARV. Furthermore, this investigation demonstrates that VARV and MPXV have important differences in the early host response to infection in monocytes and the design and development of panorthopoxvirus therapeutics should consider these early differences across virus species in screening and investigational strategies.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Disclaimer. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services (DHHS) or of the institutions and companies affiliated with the authors.

Financial support. This work was supported by the Canadian Institutes of Health Research (Tier 2 Canada Research Chair, grant number 950-231498 to J. K.); and Canadian Institutes of Health Research (grant numbers 202209MRR-489062-MPX-CDAA-168421 and 202209PPE-491319-VVP-CDAA-168421). Funding to pay the Open Access publication charges for this article was provided by the Canadian Institutes of Health Research. This work was supported in part through Battelle Memorial Institute’s former prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I. V. W. performed this work as former employees of Tunnell Government Services (TGS), a subcontractor of Battelle Memorial Institute under the prime contract.

Supplement sponsorship. This article appears as part of the supplement “Mpox: Challenges and Opportunities Following the Global 2022 Outbreak,” sponsored by the Centers for Disease Control and Prevention (Atlanta, GA).

References

Author notes