A comprehensive review of monkeypox virus and mpox characteristics

1Monkeypox virus (MPXV) is the etiological agent of a zoonotic disease called monkeypox (mpox). It is a double-stranded DNA (dsDNA) virus belonging to Orthopoxvirus (OPXV) genus within the Poxviridae family and Chordopoxvirinae as the subfamily. Other members of this genus include Variola virus (VARV), Cowpox virus (CPXV), Vaccinia virus (VACV), Camelpox virus (CMLV), Taterapox virus (TATV) and Ectromelia virus (ECTV). MPXV is divided into Clade I and Clade II, with Clade II subclassified as Clade IIa and IIb. For five decades, MPXV was endemic in West and Central Africa, and exportation of the virus to non-endemic regions was rare. However, the incidence (since 2017) of mpox outside endemic regions has increased, and the epidemiological profile of the disease within endemic regions has changed. This may have led to the MPXV emergence and re-emergence in endemic countries in 2022. This paper will cover the current state of knowledge on the characteristics of MPXV and mpox, the infection biology, molecular pathogenesis, and evolution of MPXV as well as the clinical features, diagnosis, epidemiology, and therapeutic options against mpox. In addition, the review will critically interrogate and evaluate the contributions of viral, host, and anthropogenic factors to the emergence and reemergence of mpox across the globe.

2Before 1970, there was no documented report of human MPXV infection, although the virus had previously caused infections in monkeys and apes. Infections in monkeys were reported in laboratory/captive animals and were first identified in captive monkeys in Denmark in 1958. The first human mpox case emerged in a 9-month-old boy in the Democratic Republic of the Congo (DRC) in August 1970. Subsequently, six additional mpox cases were identified between September 1970 and April 1971 in Liberia, Sierra Leone and Nigeria. Since then, MPXV has been reported in several countries and is endemic in Benin, Cameroon, the Central African Republic, the DRC, Gabon, Ivory Coast, Liberia, Nigeria, the Republic of the Congo, Sierra Leone, and South Sudan.

3Figure 1 displays the global mpox outbreak timeline. Between 1970 and 2021, the cases have been sporadic and geographically limited within endemic regions. Notably, the DRC is the only country that continuously reports yearly cases of mpox with tropical rainforest regions accounting for 98.7% of all cases pre-2022. In Nigeria, sporadic cases were reported in the 1970s; however, re-emergence of the disease started in 2017 with an eleven-year-old boy as the index case in Bayelsa state. At the end of 2017, Nigeria recorded 88 cases, and during this outbreak, travel-related cases in non-endemic countries were reported, including the United Kingdom (UK), the United States of America (USA), Israel, and Singapore, between 2018 and 2021.

4The first mpox outbreak in a non-endemic country was reported in 2003 in the USA linked to importation of rodents from Ghana ( Figure 1 ). By the end of the outbreak, 47 people had been infected (10 probable and 37 confirmed cases). There were no other travel-related cases reported until 2018. Between 2018 to 2021, 11 travel-related mpox cases were recorded in the UK, Singapore, Israel, and the USA ( Figure 1 ). Of these, four resulted in secondary cases: one healthcare worker in the UK was infected by contaminated bedding, an adult and a child from a family from the UK had a travel history to Nigeria, and one traveler to Israel who had visited Nigeria in 2018. Between 2019 and 2021, a total of seven mpox outbreaks occurred outside Africa in Singapore, the UK and the USA ( Figure 1 ). All travel related cases originated in Nigeria, with high-throughput sequencing confirming it as Clade II. Between 2017 and October 30, 2022, a total of 830 cases were recorded in 33 out of 36 states in Nigeria.

5The current global mpox outbreak started in May 2022 and was declared a public health emergency of international concern on July 23, 2022. As of August 02, 2023, there were a total of 88,600 laboratory-confirmed cases and 152 deaths (case-fatality rate, 0.17%) across 113 countries including 106 countries that have not historically reported mpox ( Figures 2A-C ). The Americas recorded the highest number of cases during the 2022 mpox outbreak, with the USA (n = 29,513) and Brazil (n = 10,168) accounting for 48.32% of the total cases ( Figure 2C ). Other notable affected countries include Spain (n = 7,408), France (n = 4,110), Colombia (n = 3,880), the UK (n = 3,730), Germany (n = 3,673), Peru (n = 3,561), Mexico (n = 3,455), and Canada (n = 1,459). In Africa, Nigeria has the highest mpox cases with 634 cases.

6Despite the name, monkeypox, monkeys are not the genuine reservoir of MPXV. Several animals can naturally or experimentally be infected with MPXV ( Table 1 ), but the natural host reservoir remains elusive. While specific host-cell receptors are responsible for cell tropism, the specificity of MPXV is yet to be determined. Factors like the monkeypox inhibitor of complement enzymes (MOPICE) and complement control protein (CCP) can influence the viral cellular and tissue tropism. Nonetheless, a wide spectrum of tissue and host tropism is expected, which may explain the possibility of MPXV establishing animal reservoirs in non-endemic regions. Organs such as ovaries, kidneys, heart, brain, pancreas, liver, and lung have been identified as some of the tissue tropism for MPXV. However, specific virus ligands remain unidentified. The inability to identify specific virus ligands and cognate host receptors for MPXV tropism suggests that the virus uses many alternative ligands to successfully invade host cells or the host receptors have functional redundancy to the virus ligand. Spillover to humans (zoonotic transmission) might arise from the disruptions of the natural habitats of wild animals. This could occur via various routes, including aerosol, direct contact, and fomite transmission. It is believed that the MPXV outbreaks in Africa prior to 2022 occurred as a result of a spillover from animals to humans. Thus, there is a likelihood of MPXV being sustained in the spillover due to the wide geographical coverage of the MPXV hosts.

7MPXV has a long and complex genome of 196 Kbp - 211 Kbp with a conserved central region and variable inverted terminal repeats (ITR) ( Figure 3A ). Within MPXV clades, Clade I isolates have more uniform genome length (196 Kbp - 199 Kbp) than Clade II isolates (196 Kbp - 211 Kbp). The length of MXPV ITR varies between 6.5 Kbp to 17.5 Kbp. Our genomic comparison of MPXV clades (Clade I, n=23; Clade IIa, n=13 and Clade IIb, n=161) indicated similar gene synteny (excluding the ITR), but slight differences in the gene content among the clades due to some genes that are missing or truncated in either one or two clades. The results are consistent with a previous study which indicates that four genes (D14L, D15L, D16L and D17L) are missing and three genes (D4L, B14L and B15L) are truncated in Clade II. Furthermore, the homologue of VACV-Cop E5R is only absent in Clade I and three genes (K1R, homologues of VACV-Cop A47L and VACV-Cop B11R) are truncated. Another major difference is the gene content of their left terminal in which Clade IIa contains four genes (N3R, N2R, N1R and R1R) that are absent in Clade I and IIb. However, these four genes are present on the right terminal of all clades. Furthermore, Clade I and IIb have theD2L gene. Within Clade IIb, gene content of the lineage A and B.1 are similar.

8Our Bayesian phylogenetic (BI) analysis of 62 non-recombinant conserved genes from 197 MPXV isolates resolved MPXV into three monophyletic clades, namely Clade I (Congo Basin Clade), Clade IIa (West Africa Clade) and Clade IIb consisting majorly of human MPXV (hMPXV) isolated between 2017-2022 ( Figure 3B ). Clade IIb was further divided into lineages: A (n=11), A.1 (n=11), A.2 (n=1), A.3 (n=1), and B.1 (n=135) as assigned by the GISAID (Global Initiative on Sharing All Influenza Data) (https://gisaid.org/). MPXV tree topology reported here is similar to that of the trees reported previously. Isidro et al. reported that the transition from A.1/A.1.1 to B.1 is characterized by a long, divergent branch which suggests accelerated microevolution. The findings of this current study ( Figure 3B ) agree with the suggestion.

9Lineage A (hMPXV-1A) corresponds to the 2017–2019 outbreak, although it contains strains isolated after this time frame ( Figure 3B ) and this observation has been reported by others. A recent study showed that new Nigerian hMPXV genomes isolated in 2019-2020 were identified as belonging to the lineage A. Lineage B.1 contains most hMPXV genomes from 2022 ( Figure 3B ). It is poorly resolved, and its sub-lineages cannot be unequivocally assigned ( Figure 3B ). The low clade supports are probably due to very low genetic variability among isolates. Polytomy within lineage B.1 could be an indication of uncertainty in the relatedness of isolates or the belief that those isolates evolved independently from a single origin, although recombination cannot be excluded.

10Furthermore, molecular dating analysis was carried out on the 62 non-recombinant conserved genes of 197 MPXV isolates to estimate the evolutionary rate and the time of the Most Recent Ancestor (tMRCA) ( Figure 3C ). TempEst analysis showed temporal signal in the dataset (R=0.65). Maximum Clade Credibility (MCC) tree ( Figure 3C ) demonstrated that MPXV emerged at 1730 (95% high posterior density interval (HPD), 1663 – 1790) with a mean evolutionary rate estimated to be 5.68 × 10−6 (subs/site/year), with 95% HPD of 4.53 × 10−6 − 6.86 × 10−6 subs/site/year. A recent publication estimated the substitution rate to be 5 x 10-6 which is in agreement with our result, but previous estimations of the substitution rates of Clades I and IIa were smaller. Clades I, IIa, and IIb were estimated to have emerged in 1956, 1928, and 1938, respectively which is earlier by 33, 47, and 97 years as estimated by Forni et al.. Lineage A and B.1 were estimated to have a tMRCA of 1998 (95% HPD, 1990 - 2006) and 2016 (95% HPD, 2014 – 2018), respectively. Our dating analysis put the emergence of the B.1 lineage six years earlier than that estimated by Luna et al. and Nextclade (https://clades.nextstrain.org/). This discrepancy may be explained by molecular dating methods used (ML-TSP versus BI-MCC). We hypothesize that the emergence of lineage A about 1998 allowed enough time for lineage A evolution and that may explain the long divergent branch length from lineage A to B.1. Similarly, the emergence of lineage B.1 in 2016 in tandem with clustering of some 2022 isolates within the lineage A (2017-2019) support cryptic transmission of the B.1 viruses prior to the current 2022 outbreak in multiple non-endemic countries. A recent molecular clock analysis based on accumulation of APOBEC3 (apolipoprotein B mRNA editing enzyme, catalytic polypetide 3)-type mutations inferred that MPXV with APOBEC3 editing has been in circulation since at least 2016, and this is in agreement with our molecular dating reported herein although their interpretation differs from ours. While we infer that B.1 lineage emerged in 2016, O`Toole et al. concluded that APOBEC3-type mutations, a predictor of human-to-human transmission emerged in 2016. The close phylogenetic relationship between A.1 isolates from Nigeria (in particular EPI_ISL_15370077) with B.1 suggests a single origin for lineage B.1.

11Our mutational analysis revealed that lineage B.1 has 66-86 nucleotide substitutions (60 consensus) and 28-39 amino acid substitutions (26 consensus) compared to reference genome NC_063383.1, MPXV from 2018 ( Figures 3D, E ). Consensus substitutions predominantly affect genes responsible for host/immune modulation and viral replication/transcription ( Figure 3E ). B21R (OPG210) codes for a T cell suppressor; our analysis demonstrated that this open reading frame (ORF) contained three consensus amino acid substitutions (D209N, P722S, and M1741I). A18R (OPG145) contained three substitutions (E62K, R243Q, and E435K). Excluding G8R (OPG093) with two substitutions, all the other affected ORFs contained one consensus amino acid substitution each ( Figure 3E ). The impact of these substitutions on the pathogenicity, transmissibility, immune evasion, and host specificity of MPXV remains unknown. The E353K substitution within F13L (OPG057) affects the target for the antiviral agent, tecovirimat; however, functional studies have not revealed any effect on the efficacy of the drug. Wang et al. also reported three mutations in OPG210 protein in MPXV 2022 outbreak strains. Additionally, they showed that this protein together with other nine proteins (D2L‐like, OPG023, OPG047, OPG071, OPG105, OPG109, A27L‐like, OPG153, and OPG188 proteins) have more mutations compared to the other MPXV proteins.

12In contrast to our study, previous mutational studies have reported fewer consensus substitutions. They reported 46 shared mutations in MPXV genomes from 2022 outbreak compared with NC_063383. However the number of mutations identified here and in other studies is higher than one would expect for MPXV, based on the low substitution rate of OPXV. The accelerated evolution of MPXV has been attributed to the human APOBEC3. Since most mutations identified here ( Figure 3D ) and elsewhere were GA>AA and TC>TT mutations, which are compatible with APOBEC3 activity. APOBEC3 is a cytidine deaminase known to play important functions in innate anti-viral immunity. Evidence for the accumulation of APOBEC3 enriched substitutions in MPXV isolates has been reported since 2017, which corresponds to the detection of lineage A. Ever since, every year, there has been an increase in APOBEC3-like mutations in MPXV. This mutational pattern has become more pronounced as suggested from more recent isolates (B.1), ( Figure 3D ). The recent pattern of upsurge of APOBEC3 derived mutations may be an indication of a change in virus-host interaction such as sustained human-human transmission or a new route of infection. Gigante et al. suggested the possibility of recombination in MPXV following observation of three sequences that showed a chimeric pattern in their genomes, although assembly errors could be a plausible explanation. Recently, tandem repeats and linkage disequilibrium analysis provided evidence of natural recombination in lineage B.1.

13Poxvirus mature particles are ovoid or brick-shaped with surface tubules and have a characteristic dumbbell-shaped nucleoprotein core containing the viral genome. MPXV virions are ~200 nm in diameter and ~300 nm in length. Like other OPXVs, MPXV forms three distinct infectious virus particles: intracellular mature virus (IMV), cell associated enveloped virus (CEV) and extracellular enveloped virus (EEV) although CEV and EEV are morphologically and structurally indistinguishable. Morphogenesis and transmission of IMV, CEV, and EEV are described in Figure 4A.

14Observations of cynomolgus monkeys infected with MPXV gave the first indications of the pathogenic role of this virus with intramuscular injection of the virus leading to an intense inflammatory immune response (, 1969). The pathogenesis of MPXV has also been studied in other animals, including rabbits, rodents and prairie dogs. Studies in monkeys, mice, and prairie dogs demonstrated that Clade I viruses are more virulent than Clade II strains, which reflects the situation in humans. Except for disease severity, the clinical features of the two clades are similar.

15MPXV infection has an incubation period of 5–21 days and the most common symptoms for the 2022 human mpox outbreak in non-endemic regions based on 48,622 patients were skin lesions (95%), fever (58%), lymphadenopathy (53%), fatigue/asthenia (39%), myalgia (31%), and headache (30%). Regarding skin lesions, anogenital lesions were most frequent (66%), followed by lesions on the trunk/torso (48%), face/head (39%), and extremities (~30%). This is in contrast with previous human mpox outbreaks from 1980-2022 where skin lesions were most common on the face and extremities. Lymphadenopathy, which typically occurs 1-2 days before rash, is a distinct feature of MPXV which is used to distinguish it from smallpox and chickenpox. The morphological progression of the rash is macular, popular, vesicular, and pustular lesions. The crust formed by pustules desquamate after 1-2 weeks. In the current outbreak, inguinal lymphadenopathy was more frequent than cervical and axillary lymphadenopathy, whereas in previous outbreaks in endemic countries submandibular, cervical and axillary lymphadenopathy were more frequent. The differences in clinical symptoms between human mpox infections before 2022 and the current outbreak are probably the result of different virus strain (Clade I versus Clade II, respectively) and patient group (both female and male youngsters versus mainly adult men having sex with men). In men having sex with men (MSM), atypical clinical symptoms such as genital lesions and anal ulcers were observed.

16MPXV can enter the host via the oral/respiratory tract, infecting the oral and respiratory tract mucosae, with the upper, middle and lower airway epithelium as main targets for primary infection. MPXV can directly infect damaged skin, and replicate in keratinocytes, fibroblasts, and endothelial cells. From the initial infection sites, virus can spread to draining lymph nodes, where the virus can replicate. MPXV can subsequently reach the tonsils, the spleen, and the liver. Replication in these organs results in a second viraemia wave, enabling the virus to access distant organs such as the lung, kidneys, intestines, and skin and causing recognizable clinical manifestation.

17Comparison of the Clade I and II strains has provided an indication which viral gene products may be responsible for their difference in virulence. The D14L gene which encodes valosin-containing protein (VCP)-MPXV, also known as CCP or MOPICE, is the orthologue of VACV secreted complement C3b/C4b-binding protein VACV-Cop C3L. This protein is known to inhibit the complement and to contribute to virulence. Compared to the VACV protein, VCP-MPXV/MOPICE is truncated due to a single nucleotide deletion in the D14L gene leading to a stop codon. The gene is absent from Clade II strains due to ~10 kbp deletion. Deletion of the C3L gene caused VACV attenuation in infected animal models. Studies with Clade I MPXV with deleted D14L showed that intranasal infection of prairie dogs resulted in decreased morbidity and mortality. However, ablation of D14L did not significantly affect virus replication compared to animals infected with control Clade I virus. On the other hand, Clade II virus with inserted D14L gene did not have virulence compared to Clade I virus and no apparent effect on disease-associated mortality compared to control Clade II virus was observed. So, other factors besides VCP-MPXV/MOPICE are necessary to explain the difference in virulence between Clade I and II MPXV.

18Other candidate virulence genes include BR-203, BR-209, and the OPXV major histocompatibility complex class I–like protein orthologue (OMCP or N3R). BR-203 protein is an orthologue to the myxoma virus M-T4, which plays a role in avoiding apoptosis of infected lymphocytes, hence promoting viral spread within the host. Myxoma virus expressing C-terminal truncated M-T4 caused increased inflammatory response compared to rabbits infected with wildtype virus, whereas challenging with virus lacking M-T4 resulted in disease attenuation. Thus, BR-203 may have a dual function in protecting infected lymphocytes from apoptosis and in modulating the inflammatory response to virus infection.

19The BR-209 gene encodes a 326 aa interleukin-1β (IL-1β) binding protein, which prevents IL-1β from interacting with the IL-1 receptor. Mice intranasally infected with VACV lacking IL-1β binding protein developed a more severe illness than wildtype virus. Perturbing the IL-1 signaling pathway dampens the innate and acquired immunity, explaining the virulent action of the IL-1β binding protein. Interestingly, the BR-209 gene of Clade I strains has two ORFs that can encode a putative N-terminal protein fragment of 210 aa and a C-terminal protein fragment of 126 aa, whereas the Clade II strains encode a putative N-terminal 163 aa polypeptide and a C-terminal 132 aa fragment. It is unknown if any of the fragments function in a way similar to the full-length protein, nor if the differences in the length of the N-terminal fragments of Clade I versus Clade II strains of MPXV contribute to the differences in virulence.

20The OMCP gene (N3R) from Clade I strains isolated in the DRC carries a 628 bp deletion, which removes the 5’region of the N3R gene (which encodes the OMCP) and the N2R gene (encoding a 73 aa polypeptide with unknown function). OMPC functions as soluble natural killer group 2, member D receptor (NKG2D) ligand, possibly representing a strategy to avoid NK-cell-mediated killing. Based on the clinical metadata, it was suggested that the deletion is associated with increased human-to-human transmission and pathogenicity compared to Clade IIa and Clade IIb isolates.

21The Clade I strain also contains truncated orthologues of VACV-Cop E3L and VACV-Cop K3L, two proteins that function in interferon (IFN) resistance. Clade I and Clade II genomes differ also in VACV-CopH5R (the VACV orthologue encodes a late transcription factor, which plays a role in viral replication, transcription and morphogenesis, VACV-Cop A9L (VACV orthologue encodes a morphogenesis factor), VACV-Cop A50R (VACV orthologue encodes a DNA ligase)), and VACV-Cop A36R (playing role in actin tail formation)). The exact mechanisms by which these proteins may also contribute to differences in pathogenicity between Clade I and II strains remains elusive.

22Manipulation of signaling pathways promotes viral replication and determines disease outcomes, mainly by targeting cell growth and immune responses. MPXV infection suppresses expression of host genes whose products are implicated in regulation of histone expression, cytoskeletal rearrangements, cell cycle progression, IFN-associated genes, and signaling pathways such as the nuclear factor kappa B (NFκB), the mitogen-activated protein kinase (MAPK), and metabolic pathways.

23The NFκB pathway plays pivotal roles in inflammation and immunity (Shao-Cong ). NFκB is typically found as a cytosolic trimer of p65/p50 and the inhibitor protein IκB. Phosphorylation of IκB by the trimer IKKα/IKKβ/IKKγ results in the release and activation of p65/p50, which translocate to the nucleus and act as a transcription factor ( Figure 4B ). Ankyrin proteins from OPXVs can inhibit the NFκB pathway by interfering with different components of this pathway. MPXV genome encodes the ankyrin-like proteins J3L, D1L, D7L, D9L, O1L, C1L, B5R, B17R, N4R, and J1R, but their exact function remains to be determined. In addition, the MPXV BCL2-like proteins A47R, B13R, C6R, and P1L can prevent activation of the NFκB pathway. The MPXV B1R gene encodes the VACV Kelch-like protein A55, which inhibits the NFκB pathway and stimulates CD8+ T cell proliferation. However, it remains to be determined whether B1R exerts a similar function.

24The Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathway mediates cellular responses to cytokines and growth factors. H1L inactivates STAT1, and C6R blocks STAT2. Whether the MPXV H1L and D11L orthologues act similarly has not been investigated ( Figure 4B ).

25The VACV epidermal growth factor homologue VGF (C11R) usurps the epidermal growth factor receptor (EGFR) pathway to provoke cell proliferation and to stimulate efficient virus spread and pathogenesis. The D3R gene (OPG019) encodes an EGF homologue that might activate the EGFR pathway and enhance viral dissemination. VACV-Cop F11L also promotes viral spreading by inhibiting the RhoA signaling pathway. It is not known whether the MPXV orthologue C17L possesses the same function.

26Circumventing apoptosis is used by viruses to achieve productive replication. VACV F1L and N1L proteins can prevent apoptosis by inhibiting pro-apoptotic proteins BAK, BID, BAD, and BAX. It is unknown whether the MPXV C7L and P1L isologues have similar functions. Other MPXV genes that prevent apoptosis include B12R, B19R, D5R, and F3L. B12 is a serine protease and its VACV orthologue B13R has been shown to be a caspase inhibitor.

27Signal transduction is often mediated through a cascade of phosphorylation events. Kindrachuk et al. compared the phosphorylation pattern of host cell proteins after Clade I or Clade II MPXV infection. They found that Clade I MPXV infection down-regulated pathways related to cell proliferation and apoptosis as compared with Clade II MPXV. Differences in MPXV-induced posttranslational modification may explain the differences in virulence between MPXV clades.

28Human infection with MPXV is associated with increased levels of ILs, C-C motif chemokine ligand 2 (CCL2) and CCL5, and a significant decrease in tumor necrosis factor alpha (TNF-α), IFN-γ, and IL-2 and IL-12. MPXV infection provokes IgM and IgG antibodies, long-term persistence of residual IgG-memory B cells, and a rapid expansion of activated effector CD4+ and CD8+ T cells followed by a decrease over time. MPXV also interferes with adaptive immune responses of antiviral CD8 + and CD4 + T cell responses via inhibiting T cell receptor-mediated T cell activation.

29Because of the high homology between the MPXV genes and the corresponding OPXV orthologues, it is assumed and, in some cases, demonstrated that MPXV applies similar strategies to evade the host immune defense system. The Toll-like receptor (TLR) family functions as pattern recognition receptors, which recognize damage-associated molecular patterns such as viral dsRNA. Binding of viral dsRNA to specific TLR members triggers the expression of proinflammatory molecules involved in host anti-viral responses and subsequent activation of the adaptive immune defense system. The VAVC A46 protein inhibits the TLR4 signaling pathway. However, it is unknown whether the MPXV orthologue A47 has the same property, but the A47 protein has structural similarities to VACV protein A52R, which can inhibit the TLR3 and TLR4 signaling pathways, underscoring a role for MPXV A47 in interfering with TLR signaling. MPXV produces low levels of dsRNA intermediates, but whether these are recognized by TLR3 has not been investigated, although transcriptome analysis of MPXV-infected cells revealed repression of TLR3 target genes. dsRNA can also activate kinase R (PKR), which mediates phosphorylation of eIF2α, resulting in the inhibition of viral and cellular mRNA translation. VACV E3 and K3 are inhibitors of PKR, allowing VACV to evade an antiviral response. The N-terminal domain of VACV E3 protein is absolutely required for the interaction with dsRNA. MPXV F3 protein is the VACV E3 protein homologue with a 37 aa truncation at the amino terminus, suggesting that MPXV F3 does not bind dsRNA. However, MPXV can inhibit host immune responses, although a recombinant VACV expressing the MPXV F3L gene did not inhibit host PKR activation, suggesting that MPXV has evolved to encode for yet undiscovered proteins that compensate for the missing N-terminal amino acids of F3 in limiting host antiviral activities. As previously mentioned, MPXV CCP (D14L gene) is completely absent in Clade II isolates, whereas the Clade I strains are predicted to express CCP, albeit with a truncated fourth short consensus repeat. Loss of expression of CCP/MOPICE limited the adaptive immune response against MPXV infection in rhesus macaques.

30IFNs are main effectors of the innate immune response and can inhibit virus replication. The MPXV B16 protein (VACV B19 orthologue) is a secreted type I IFN inhibitor and suppresses the antiviral type I IFN-induced signaling pathway. VACV K7 abrogates IFN signaling by destabilizing IFN-regulated factor 3 (IRF3) and inhibits NFκB activation, whereas VACV H1 can block IFN signaling. Whether the MPXV D9 and H1 orthologues have the same function remains to be proven, but both proteins are predicted to interact with several cellular proteins of the immune system. VACV E3 perturbs IFN signaling by binding host Z-DNA binding protein 1 (ZBP1). The MPXV E3 orthologue (F3) perturbs the IFN pathway, but whether in a ZBP1-dependent manner needs to be established. The DNA-sensing receptor pathway cGAS/STING (cyclic GMP-AMP synthase/stimulator of interferon genes) can activate the IFN and NFκB pathways. The VACV B16 and B8 proteins block IFN signaling by operating as soluble IFN-α and IFN-γ receptors, respectively. The MPXV ORF B16R and B9R encode functional homologues. OPXVs encode poxins that degrade 2′,3′ cGAMP and thereby inhibit cGAS/STING signaling. Poxin is conserved in MPXV where it is fused to an additional C-terminal domain previously noted to have homology with human schlafen proteins (VACV B2R = MPXV B4R), hence MPXV may evade the immune system by targeting the cGAS/STING pathway.

31MPXV can dodge the immune system by targeting the antiviral cytokine TNFα and other immunomodulating molecules. The MPXV-encoded cytokine response-modifying protein B (CrmB; J2L or OPG002) functions as a decoy receptor for TNFα. The C-terminal domain of VACV CrmB can bind CCL28, CCL25, CXC motif chemokine ligand 12 (CXCL12), CXCL13 and CXCL14. It is unknown whether MPXV CrmB interacts with these chemokines, but the amino acid sequence of the corresponding domain in MPXV CrmB differs significantly.

32Natural killer (NK) cells and cytotoxic T cells (CTL) play a crucial role in eliminating viral infections. Although the number of NK cells expand significantly in peripheral blood and lymph nodes in MPXV-infected rhesus macaques, their migrating capacity was reduced, and several functions such as expression of chemokine receptors (including CCR5, CCR6 and CXCR3) and secretion of IFNγ and TNFα were impaired. The importance of NK cells in controlling MPXV viral load was demonstrated in the highly vulnerable to OPXV infection CAST/EiJ mouse strain owing to low numbers of NK cells. IL-15 treatment, known to increase the numbers of IFNγ-secreting NK cells and CD8+ T cells, protected CAST/EiJ mice from lethal MPXV infection even when both CD4+ and CD8+ T cells were depleted. This implies that the expanded NK cells were responsible for the protective effect.

33MPXV protein B10 avoids detection of virus-infected cells by CTL by impairing peptide loading and MHC-I trafficking within the endoplasmic reticulum. However, NK cells continually screen cells via NKG2D for the absence of MHC-I, thereby ensuring that the MHC system is not compromised. MPXV-infected cells with downregulated MHC-I expression overcomes detection by NK cells by secreting the OCMP protein that binds NKG2D and suppress the typical NKG2D-dependent NK cell lysis of cells that do not express MHC-I ( Figure 4C ). IL-18, an IFNγ-inducing factor, stimulates the synthesis of various cytokines and chemokines, regulates Th1 and Th2 cell responses, and activates NK and CTL. OPXVs block IL-18 activities by producing an IL-18 binding protein. The MPXV D6L gene encodes such IL-18 binding protein.

34The MPXV J3R and A41L genes encode chemokine binding proteins, which are assumed to destroy the chemokine concentration gradient resulting in decreased neutrophil migration in tissues infected with MPXV and thus reducing viral virulence and inflammatory response.

35The complement system, which forms an essential part of the innate immune system is activated early in MPXV infection in mice, and is crucial for viral control. MPXV MOPICE which from studies with VARV and VACV MOPICE was found to inhibit activation of the complement pathway. MOPICE modulates the antiviral immune response as observed by enhanced viral replication in vivo and dampened adaptive immune response in rhesus macaques infected with MPXV lacking MOPICE expression. Because of the absence of the D14L gene in the Clade II, the virus and virus-infected cells would be predicted to be susceptible to the host complement attack. However, the expression of MOPICE in Clade II did not increase its virulence, demonstrating that MOPICE is not the sole determinant of differences in viral virulence between the two clades.

36Less is known how MPXV may interfere with the adaptive immune system. MPXV interferes with adaptive immune responses of antiviral CD8 + and CD4 + T cell responses via inhibiting T cell receptor-mediated T cell activation.VACV A35 blocks immune priming of T lymphocytes by interfering with MHC class II-restricted antigen presentation. Moreover, infection studies in cells with MPXV lacking the A35R gene demonstrated that A35 inhibits production of cytokines and chemokines. MPXV A35 analogue, A37, might suppress presentation of viral antigens to immune cells and help the virus to evade the host immune defense system. However, MPXV does not seem to interfere with MHC expression or intracellular transport of MHC molecules.

37Surveillance data on mpox in endemic countries during different periods between 1970-2015 showed that 71%-83% of the disease occurred in children (<10 years of age) and 51%-67% in males. In contrast, the median age for the 2017-2018 outbreak in Nigeria was 29 years, with males accounting for 64% of the cases. However, for the 2022 multi-country outbreak, males accounted for 96.8% of the cases, and the median age was 34 years (Interquartile range: 29 – 41) (World Health Organization., 2022). In the African region, children (0-9 years of age) accounted for 23.08% of mpox cases compared to <1% in Europe and the Americas. Notably, the male-to-female ratio in Africa is also markedly lower than in other regions ( Figures 5A-C ).

38Before the 2022 outbreak, the overall pooled estimated case fatality rate (CFR) was 8.7% and varied by the clade of the virus. The pooled CFR for Clade II was 3.6% and 10.6% for Clade I. The 2022 outbreak has an estimated CFR of 0.08%. The low CFR in the current outbreak may be related to several factors, including the fact that the Clade II has a CFR of <1%, active surveillance, early diagnosis, and treatment.

39Over the past five decades, the secondary attack rate for mpox has been stable and ranges from 0% to 10.2%. Higher estimates were reported in the 2013 outbreak in the DRC (50% among 16 households) and Nigeria (71% in the 2017- 2018 outbreak from a single household). However, it is imperative to note that it is unclear how the high estimates were obtained and thus may likely be overestimated.

40Reproduction number (R0) is the number of secondary cases anticipated to develop from a single primary case in a naive population. While historical data on R0 is limited, published evidence from DRC’s active surveillance between 1980 and 1984 estimated R0 to be 0.8, implying that transmission is ineffective as a human-to-human epidemic is likely to die out. However, early estimations of the R0 in the 2022 outbreak in non-endemic countries showed a high variability ranging from 1.54 (Belgium) to 3.62 (Germany), with a median of 2.44. This suggests sustained human-to-human transmission, possibly due to several contributing factors, including different social and/or sexual behaviors, different MPXV variants, population density or other unknown causes. While the early estimations indicated sustained transmission, the outbreak has slowed down with case reductions. This could be due to behavior change, infection induced immunity, and vaccinations.

41Transmission of MPXV can occur from animal-to-human (zoonotic) and from human-to-human (interhuman). Zoonotic transmission usually happens through contact with an infected animal’s bodily fluid or through a bite or scratch. Exposure to animal reservoirs, especially in regions with deforestation enhancing animal-human contact, and uncooked meat products are major risk factors for zoonotic transmission. During the 2003 USA outbreak, exposure was classified as “non-invasive” (touching an infected animal) or “complex” (invasive bite from an ill animal; non-invasive exposure i.e., any exposure that did not break the skin). Patients with complex exposures were more likely to develop systemic illness compared to those with non-invasive exposure. Dewitt et al. predicted the MPXV mode of transmission in most 2022 studies to be caused by inter-human transmission ( Figure 5D ). Large respiratory droplets, bodily fluids, contaminated fomites, and viral shedding through feces are also considered risk factors for viral transmission. Airborne transmission of MPXV between animals in experimental settings has also been reported and MPXV was detected in upper respiratory samples, suggesting that interhuman transmission of MPXV via the airborne route may be possible. However, epidemiological observations do not support airborne transmission as the primary route of transmission. The virus can also cross the placenta, suggesting vertical transmission. At least 12 pregnant women have been infected during the 2022 outbreak, but vertical transmission was not observed in any case.

42MPXV has also been detected in human semen, and in archival testes tissue of crab-eating macaque, suggesting potential sexual transmission of the virus. Recent outbreak suggests that MSM subpopulation may also be at an increased risk.

43In addition, waning immunity against smallpox has been considered another potential risk factor for the disease. Evidence from early outbreaks in the 1980s showed that previous smallpox vaccination provided 85% protection against mpox. Most cases reported were among those born after vaccination ceased in 1980, and herd immunity has significantly decreased.

44Evidence from the literature suggests that those who are immunocompromised due to HIV and other underlying conditions are at increased risk of severe mpox disease. A high proportion of patients with mpox in the 2022 outbreak had concurrent HIV infection and sexually transmitted infections (STI). Mpox patients with HIV infection were more likely to be hospitalized than those without HIV infection. However, the evidence on the reason for hospitalization is limited, and it is unknown if it reflects a more severe mpox illness.

45Furthermore, co-infection with other conditions with rashes, such as a varicella-zoster virus (VZV), can occur. This herpesvirus causes chickenpox and shingles and is frequently misdiagnosed as mpox in regions where both diseases are endemic. Chickenpox is exclusive to humans and more common in younger age groups, and co-infection with mpox has been reported more commonly among children.

46Electron microscopy, immunohistochemical detection of MPXV proteins, and detectable levels of anti-OPXV IgM antibody during the period of 4 to 56 days after rash onset can be used to diagnose MPXV infection, but all methods are not specific. According to the Centers for Disease Control and Prevention (CDC), mpox cases should be confirmed by real-time polymerase chain reaction (qPCR) or Next-Generation sequencing and isolation of MPXV in culture from a clinical specimen. The F3L, E9L, B6R and J2R genes are all target of qPCR in MPXV diagnosis. The suspected mpox cases are characterized by fulfilling one of the epidemiological criteria (within 21 days of illness onset), as outlined by CDC.

47One of the crucial ways of controlling the spread of mpox is contact tracing. Individuals exposed to MPXV should be monitored for 21 days checking mpox symptoms, and those with suspected or confirmed mpox cases should be isolated to avoid infecting others. Velavan et al. predicted the mpox outbreak would not last provided that cases are well isolated through the contact tracing. Although much attention is given to human-human contact tracing, great efforts need to be put into animal-animal and animal-human contact tracing especially due to non-specificity of reservoir hosts for MPXV. Surveillance cannot be undermined in curtailing mpox as surveillance would provide more insight into the epidemiology of the disease. In Nigeria, the Outbreak Response Management and Analysis System (SORMAS) for mpox surveillance across portions of 8 states was implemented in November 2017 for the mpox outbreak. The use of the system increased the quantity of epidemiological data collected and the communication of aggregate case data.

48Although there is no specific vaccine for MPXV, the smallpox vaccines have been reported to give 85% cross-immunity against MPXV due to shared antigenic features.

49ACAM2000™ is a replication-competent vaccinia virus vaccine licensed by Food and Drug Administration (FDA) in August 2007 for smallpox prevention, and it is derived from a single clonal viral isolate from Dryvax which is a first-generation smallpox vaccine. As a second-generation attenuated vaccinia virus vaccine, ACAM2000™ has been recommended as MPXV post-exposure prophylaxis. Although high level of protection against mpox in animal models has been recorded, the safety of ACAM2000™ in humans is still of great concern as cardiac complications, and extremely painful and uncomfortable cutaneous reaction at the injection site have been associated with the vaccine. Therefore, the vaccine is no longer licensed by European Union.

50JYNNEOS (Imvamune or Imvanex) was approved by FDA in September 2019 for prevention of smallpox and mpox in adults aged >18years. JYNNEOS is the brand name of Modified Vaccinia virus Ankara Bavarian Nordic (MVA-BN) vaccine, a non-replicating third-generation attenuated vaccine. JYNNEOS is considered safer (with proven efficacy in animals and humans) than ACAM2000™. The Advisory Committee on Immunization Practices has recommended JYNNEOS as an alternative to ACAM2000™. Nonetheless, both vaccines (JYNNEOS and ACAM2000™) have been recommended for MPXV high-risk groups.

51LC16m8 is another potential vaccine for MPXV which is obtained by subjecting VACV lister to 36 serial passages at low temperature (30°C) in primary rabbit kidney cells. As a third-generation attenuated vaccine, LC16m8 has been shown to be protective against MPXV in animal models with lower neurotoxicity. The frameshift mutation in LC16m8’s major extracellular enveloped virion antigen (B5R) contributes to the vaccine replication competence and low virulence. Presently, LC16m8 is only licensed in Japan.

52Although there are no specific antivirals for mpox, some antivirals (tecovirimat, brincidofovir, cidofovir) have been explored. Tecovirimat (ST-246 or TPOXX®), 4-trifluoromethylphenol derivative, was approved (for smallpox) by FDA in 2018 and approved by European Medicines Agency in January 2022 for treatment of smallpox and cowpox. Tecovirimat inhibits VP37 (p37) protein of VACV by targeting the viral F13L gene and the CPXV homolog V016 gene. VP37, a highly conserved protein in OPXV genus, is required for viral maturation and release from the infected cell. Inhibition of VP37 prevents viral spread within an infected animal models. Goyal etal. recommended tecovirimat to be administered as first line of mpox treatment in pregnant and breastfeeding patients. A tecovirimat analogue (synthesized by the State Research Center of Virology and Biotechnology, Russia) has been highlighted as a promising antivirals against OPXV infections.

53Cidofovir (CDV or Vistide®) prodrug is an acyclic nucleoside phosphate that was approved by FDA in 1996 for the treatment of retinitis (caused by cytomegalovirus) in AIDS patients. The efficacy of CDV has been identified during the in vitro studies in MPXV-infected animals, but the clinical data of CDV efficacy against mpox in human are not available. Brincidofovir (CMX001 or hexadecyloxypropyl-cidofovir), a CDV derivative, was approved for smallpox treatment in 2021 by FDA, and it has lesser toxic effects than CDV. Evaluation of CMX001 efficacy and safety in human mpox through the clinical trials is needed.

54Due to inadequate genome surveillance data particularly in endemic regions, little information about MPXV recombination is available. However, there are some evidences of recombination between coinfecting or superinfecting OPXVs both in a laboratory setting and in nature.

55MPXV circulating in non-endemic regions where other OPXVs are endemic, for instance, CPXV in Europe and VACV-like in south America, and vaccination against mpox with JYNNEOUS or ACAM 2000 are scenarios for coinfection and superinfection between different species of OPXV. Thus, there remains a potential risk of recombination between MPXV and other OPXVs that may result in MPXVs with mosaic genomes and altered biological characteristics.

56Although there is insufficient evidence of MPXV being used for bioterrorism at the moment, many scientists have expressed concerns over its potential use for bioterrorism because there is a report that there was an attempt by the former Soviet Union to use MPXV as a bioweapon. The possibility of MPXV as a potential bioweapon due to its global reemergence and its clinical similarities with VARV has placed the virus on the global public health agenda. For example, the USA has made preparation for the possibility of smallpox virus as a potential bioterror biological by storing smallpox vaccines and antivirals after the 9/11 attack. Smallpox virus is in category A of the CDC list of bioterrorism agents. The risk of bioterrorism is heightened knowing that MPXV genomes can be synthesized from publicly available sequence data and life viruses (with or without further genetic modification) can be re-constituted. This has already been demonstrated with horsepox virus (HPXV), which shares the same genus as MPXV. Hence, a strict dual-use policy must be agreed upon and implemented in all laboratories across the globe.

57Sexual orientation (especially LGBQTI+ community) and racial are two stigmas associated with MPXV. The persistent narrative of the media alongside many scientists linking the mpox 2022 outbreak to Africa/West Africa/Nigeria is worrisome. Despite the mpox 2022 outbreak, which occurred outside Africa, the nomenclature and the geographical labels of MPXV strains still reference West African, even though the origin of this outbreak is still unresolved. Furthermore, high mpox cases were reported among MSM. This narrative portrays these men contracting MPXV because they engaged in sexual intercourse with fellow men, meanwhile the spread of the virus can occur regardless the sex. Vaccine inequality affects lower- and middle-income countries (LMICs). An unknown number of mpox cases in LMICs are not captured due to a shortage of resources like limited testing and surveillance capacity. As highlighted by Malta et al., the MPXV vaccines are presently accessible only in high-income countries (Canada, the USA, and the UK).

58MPXV has emerged and re-emerged for over five decades and yet not much is known about its virological profile and the characteristics of the disease it causes. In particular, the reservoir host of MPXV remains unknown, the viral, host and environmental factors that modulate the virus maintenance in the wild, animal-to-animal transmission, zoonotic transmission and reverse spillover are still a mystery. Neither are we closer to reliable prognostication of virus emergence and accurate modelling of the disease outcomes. Current and future studies should prioritize understanding the molecular basis of MPXV infection to develop effective drugs and vaccines against mpox as well as functional mutational studies that will shed insight into the dynamics of MPXV transmission across hosts. To improve tracking of MPXV, laboratories particularly in resource-poor countries should be equipped with genome-based surveillance capacity and capability. Lastly, MPXV and mpox affect human, animal, and ecosystem health. Thus, a One Health strategy is indispensable to the prevention and treatment of current and future outbreaks.

59MO: Conceptualization, Data curation, Investigation, Methodology, Supervision, Writing – original draft, Writing – review & editing. EA: Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. DK: Formal analysis, Methodology, Writing – original draft. DD: Data curation, Formal analysis, Investigation, Writing – original draft. FA: Investigation, Writing – original draft. OA: Formal analysis, Investigation, Writing – original draft. UM: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing.