2.1. Phylogenetic Description of MPox Virus

The monkeypox virus (MPXV) consists of a large, linear double-stranded DNA genome that falls under the genus orthopoxvirus (OPV), family poxviridae, and subfamily chordopoxvirinae [6]. This viral genome is about 200 kilobases long and contains approximately 200 genes, translating to the potential for encoding around 150-190 proteins [7]. It has around 190 open reading frames (ORFs) critical for functions such as replication, morphogenesis, immunomodulation, and pathogenesis [8]. There are two distinct classified clades, or genetic variants, of MPXV that are indigenous to Africa: Clade I (previously referred to as the Congo Basin or Central Africa Clade) and Clade II (West African Clade). Clade I MPXV is further categorized into six subgroups, designated I, II, III, IV, V, and more recently, VI, which emerged following an observational cohort study in Kamituga, DRC [6, 9]. Clade II is also divided into two subclades: IIa and IIb [10]. These MPXV clades display different geographic, epidemiologic, and clinical characteristics. The Clade IIa variant was responsible for localized outbreaks in Nigeria, Ivory Coast, Sierra Leone, Liberia, and the United States [10]. However, due to the MPXV outbreak occurring in 2022–2024, a new subclade, IIb, was identified as part of the genetic variants involved [11]. The Subclade IIb MPXV was predominantly identified in men who have sex with men (MSM) and among individuals who engage in sexual contact with infectious persons [12]. As a result, Clade IIb is primarily transmitted between humans through both sexual and nonsexual interactions with an infected person, whereas Clade I and IIa MPXV tend to spread less among humans and are mainly transmitted zoonotically through rodents and small mammals [1]. Clade I MPXV is linked to more severe disease presentations and has an elevated CFR averaging 10.6%, compared to Clade IIb, which has a lower CFR of approximately 3.6% [13]. Clade I is endemic to the DRC, Central African Republic, Republic of Congo, Sudan, Cameroon, and Gabon and possesses smallpox-like attributes with a high occurrence of viremia [10]. Clade IIa presents milder symptoms, reduced viremia and transmissibility, and a much lower CFR (around 1%–3%) in comparison with Clade I MPXV [14, 15]. Figure 2 below shows the relationship between Clade I and Clade II.

2.2. Genetic Features of Clade 1 MPox

Clade 1 of the MPox virus exhibits a distinctive genetic composition that sets it apart from other clades. In the ongoing MPox outbreak, clusters of transmission through heterosexual contact (which was not a common observation) have been recorded and linked to a novel sublineage termed Clade Ib, which has spread to countries like Sweden where MPox cases were not previously recorded [16]. Clinical reports have described Clade Ib symptoms as disseminated whole-body rash, long-lasting genital lesions and outcomes including pregnancy loss in pregnant women, with a CFR of up to 10% in children and 5% in adults [17]. Given these new disease outcomes, demographic patterns, modes of transmission, and international spread, it is imperative to elucidate the genetic factors in Clade I contributing to this increased pathogenicity.

Clade I contain several key structural and functional genes that play crucial roles in its virulence and pathogenicity. Notable among these is the C9L gene, the only Kelch-like protein in the MPXV genome that acts as an inhibitor of the host innate immune response [18, 19]. The I4L gene, which encodes a ribonucleotide reductase (RR) large subunit 1 protein, is involved in viral replication in nondividing cells [20]. Although further studies are needed to investigate the specific contribution of the A25R gene to MPXV virulence and pathogenicity, it is believed to be fundamental to MPXV’s ability to replicate its DNA into RNA [21]. The central location of A25R in the core region of the viral genome and its high conservation across various MPox strains highlights its critical role in the viral life cycle and, thus, can be an important potential target for vaccine development and drug therapy [22]. Similarly, the role of the L6R gene is not fully understood; however, it is predicted to be essential for facilitating viral DNA replication and transcription [23]. The presence of the A17L (a viral envelope protein) in vaccinia virus (VAVC), which is involved in host cell entry and fusion, plays a similar and essential role in MPXV in determining intracellular sites for cleavage, disulfide bond formation, and infection onset [24]. A recent study conducted by de Araujo et al. [25] identified numerous epitopes from the A28L protein and proposed them as candidates for a multi-epitope vaccine, highlighting their potential for immunogenicity. In silico analyses have also revealed that these epitopes could trigger both humoral and cellular immune responses, making them a promising component in the development of an immunogenic construct against MPXV. This follows that the genetic diversity in MPXV Clade I is integral to viral transmission and survival within the host as well as its ability to suppress immune responses, infect host cells, and replicate, underscoring the significance of these genes in the overall genomic architecture of MPXV Clade I.

2.3. Genetic Variability and Significant Mutations Differentiating Clade I

As the most virulent clade of the MPox virus, Clade I have been associated with more severe disease and transmission outcomes. Genetic variability is one of the prominent factors that influence the epidemiological behavior of the ongoing MPox Clade Ib outbreak. Notably, this Clade Ib outbreak has been self-sustaining, exhibiting unusual characteristics rarely observed in previous Clade I outbreaks (which were primarily due to zoonotic transmission), such as human-to-human transmission through heterosexual contact, nonsexual community spread to geographic areas not previously reported such as in Europe, Asia, and the Americas, and infecting over 99, 176 people, across varying age groups including children [26, 27]. This paper describes the specific mutations or genetic markers in MPXV Clade 1 that makes the epidemiological profile of this new sublineage outbreak distinct from other previously observed Clade I outbreaks. A mutational analysis reported by Masirika et al. [6] revealed that MPXV Clade I (most importantly Clade Ib) contain a set of highly mutative proteins including C9L [OPG047], I4L [OPG080], L6R [OPG105], A17L [OPG143], A25R [OPG151], A28L [OPG153], and B21R [OPG210]). In comparison with the MPXV Clade I genome (NC_ 003310), these mutational hotspots contain a number of consensus inframe deletions, frameshift variants, synonymous variants, and amino acid substitutions. The B21R (OPG210) protein, which codes a surface glycoprotein, was found to be associated with the highest number of mutations including two consensus frameshift variants (K431X and P726X), synonymous variants (A1207 and C1715), and nine amino acid substitutions (A475V, S533L, T713P, T723P, E1056D, A1339T, T1372A, V1725S, and D1862E).

When compared with the reference genome, B21R (OPG210) contains a total of four unique changes, two frameshift variants (K431X and A475V), and two amino acid substitutions (P726X and E1056D). C9L (OPGO47), which is a Kelch-like protein, was found to have an inframe deletion (DGMG483-486E) and one frameshift variant (D483X) with three consensus amino acid substitutions (L151H, V127E, and Y114N). This protein, in comparison with subgroup V (JX878417), was found to have a unique inframe deletion [27]. Fourteen rare genes (D2L, D4L, D17L, D18L, C3L, A26L, A27L, A48R, B15L, B1R, B18R, K1R, R1R, and N2R) not previously observed for Clade I MPXV were also present in the genome samples and were found to be unique to the ongoing MPox Clade Ib outbreak. These mutational variabilities are reported to be likely responsible for the sustained nature, unusual transmission routes, and high virulence of the current Clade 1 outbreak [28]. Recently, Zhang et al. [25] conducted a phylogenetic analysis incorporating 84 locally sequenced MPXV Clade I genomes from Beijing. One of these (identified as an imported infection) exhibited 76 nucleotide substitutions and 34 amino acid substitutions relative to the reference genome (NC_063383.1) and included changes such as C33332T (OPG050:R26Q), G37974A (OPG056:S354L), and C149963T (OPG0176:S52L), which were not present in the previous Beijing sequences. Other studies have shown that Clade I exhibit a moderate level of genetic diversity, with variability primarily concentrated in the B21R and A25L gene regions [27, 29]. This variability is attributed to a combination of point mutations and insertion–deletion events that have occurred over time [8, 30]. It is reasonable to suggest that specific mutations, such as those in the C9L and B21R genes, can be linked to changes in viral protein expression and function, which may impact Clade Ib virus’s ability to evade host immune responses and exhibit more severe disease outcomes.

2.4. Comparative Context Between the Clade I and Clade II

Several studies have highlighted the genomic differences between Clade I and Clade II MPXV (including subclades IIa and IIb) and the potential correlation to virulence differences. Nine proteins that varied by at least five amino acids between the two clades were reported by Likos et al. [14]. Based on orthologs in the VAVC, four of them (OPG110, OPG135, OPG180, and OPG164) were reported to play functional roles in the viral life cycle and five were predicted to be involved in immune evasion or host range, including complement activation and apoptosis regulation. Other investigations have found OPG195, B14R, and OPG032 (which vary between clades I and II) to be determinants of MPXV virulence. Chen et al. [21] discovered that virulence differences between Clade I and Clade II MPXV likely originated from genes found in the terminal regions of the viral genome. Of these genes, two are fragmented in Clade IIa MPXV (OPG195 and B14R) and one (D14L) is absent. D14L has been identified as a likely candidate for virulence differences as this gene is present in Clade I MPXV but absent in Clade II MPXV. Additional sequence differences have been reported in nonvirulence orthologs related to the viral life cycle between Clade I and recent Clade IIb viral genome sequences. In terms of functional roles of MPXV virulence determinants, B10R, which is believed to abrogate apoptosis in infected cells based on homology to the myxoma virus M-T4 protein, encodes a 221 amino acid (aa) protein in Clade I viruses but is fragmented in Clade IIb viruses [21]. This follows that MPXV virulence factors may have heterogeneous roles in clade-specific contexts. Thus, functional characterization of the roles of putative virulence proteins in Clade IIb MPXV needs to be further studied. While other clades exhibit higher genetic diversity in certain genomic regions, Clade I maintain a relatively stable genome structure, suggesting a different evolutionary trajectory compared to Clade II, which has shown some recombination events [31]. These comparative insights highlight the evolutionary distinctiveness of Clade I and its implications for epidemiological monitoring and control strategies. The degree of genetic diversity within Clade I reflects its adaptive evolution and provides insights into its transmission dynamics and potential for outbreak resurgence. Identifying these mutations can enhance the understanding of how Clade I interact with the host immune system, evades immune responses, and adapts to different environmental pressures. This is not only vital for more accurate epidemiological surveillance but also for informing vaccine development, antiviral therapies, and diagnostic tools. Table 2 provides an overview of the key differences between Clade I and Clade II MPXV.

2.5. Drivers of Evolution; Recombination, Gene Loss, and Gene Expression

Recent advances in genomic sequencing have provided critical insights into the evolutionary dynamics of MPox Clade I, enhancing knowledge of MPXV genetic diversity and adaptation. As a highly virulent clade, exhibiting more severe clinical manifestations, Clade I have been the focus of extensive research, particularly in regions where it is endemic. The application of high-throughput sequencing technologies has allowed for easy tracking of mutations, identification of genetic markers, and understanding of the evolutionary pressures shaping Clade I MPXV. Double-stranded DNA viruses such as MPXVs exhibit a slow rate of evolution. However, to adapt to human hosts, MPXVs undergo microevolution, involving amino acid point mutations [34]. To quickly gain the first insights into phylogenetic placement and evolutionary tendencies of the MPXV, Yu et al. [3] classified frequent amino acid mutations into four groups based on their dates of first appearance which were determined through evolutionary analysis. Evidently, the incidence of amino acid mutations conserved in the MPXV genomes over time increased significantly between 2021 and 2023. The occurrence of such multiple genetic changes within this short period, which is not frequently observed in OPVs or particularly in MPXVs, implies that the MPox virus has acquired enhanced rapid host adaptation and gained a fitness advantage that facilitates its ability to maintain human-to-human transmission [8,30]. This rapid sequence of alterations is probable to have played a critical role in MPXV host adaptation, spread in dynamic and changing environmental conditions, and consequently, the higher transmission and infection establishment of Clade Ib in the human population. Since 2017, MPXV Clade IIb genomes have been observed to consistently accumulate apolipoprotein B editing complex (APOBEC3)-type mutations, induced by host defense mechanisms. These molecules target the viral genome during replication when single strands are exposed. Either of these strands can undergo deamination, which progressively causes changes, such as cysteine conversion to thymine or guanine to adenine [34]. Although APOBEC3-mutated genomes have been reported to be nonviable, these genomes may persist and get transmitted. Constant evolution within the human population, due to the irreversibility of APOBEC3, can result in a potential decline in the virus fitness. The timeline for this process remains an area for further studies and additional evolutionary factors such as recombination could inhibit this decline and restore virus fitness [32]. Further research into the mechanisms governing genome evolution and the significance of gene functions is also crucial to gain a deeper understanding of the processes involved in MPXV evolution.

2.6. Recombination

Recombination is predicted to be one of the main drivers of poxvirus evolution [35]. Although poxviruses undergo high-frequency recombination during cell infection, no natural recombination events have been reported for MPXVs and little is known about the exact role of recombination in MPXV evolution [36]. Nevertheless, few studies have attempted to engineer recombinant MPXVs in the lab such as Goff et al. [29] who replaced the D14L gene with an EGFP-GPT cassette in the MPXV Clade I genome using homologous recombination to examine the involvement of the MOPICE (monkeypox inhibitor of complement enzymes) in MPXV pathogenesis. This generated a recombinant MPXV-producing green fluorescent protein to analyze MPXV infection in a monkey model. The ease of constructing recombinant MPXVs raises the possibility that such recombination can occur or may have occurred naturally between co-infecting MPXVs. Yeh et al. [33] reported the first natural recombination of the MPXV genome using SNP-dependent and SNP-independent analysis tools, including linkage disequilibrium (LD) and tandem repeat (TR) analysis, speculating that the progeny MPXV recombinants emerged from a single origin which gained mutations, evolved into different lineages, and then went through homologous recombination through multiple possible mechanisms. The resulting recombinants, however, had mosaic patterns of TRs and no defective MPXV virus was detected arising from a single infection.

2.7. Gene Loss and Gene Expansion

Another driver of poxvirus evolution is believed to be gene loss, and gene gain as is the main form of host adaptation [37]. Gene content and genome sequence length have an inverse relationship with pathogenicity [38]. MPXV Clade II have larger genomes, approximately between 197,566 and 197,792 bp, more than Clade I (∼196,850–196,959 bp) which contributes to the high virulence of Clade I MPXV when compared to Clade II [36]. It is not unreasonable to assume that poxviruses (MPXV in particular) adopt reductive evolution by gene loss to enhance their response to, and evade, host immune response mechanisms. Hatcher et al. [39] provide strong evidence that gene loss primarily occurs through the introduction of ESMs (early stop mutations) leading to truncations, fragmentations, and complete deletion of protein-coding regions (ORF). A study demonstrated that under intense selective pressure, poxviruses can implement temporary gene expansion to counteract host antiviral response [40]. For example, deletion of E3L in VAVC was observed to increase the copy number of K3L, expanding its genome size by up to 10% which inhibited the antiviral defenses of PKR (human protein kinase R). It is plausible that this gene expansion mechanism may occur in MPXV and be a driver of evolutionary adaptation. Next-generation genome sequencing and mutational analysis studies are vital for monitoring the emergence of new variants, predicting potential outbreaks, and to better understand the mechanisms driving pathogenicity and response to environmental changes, ultimately contributing to more effective public health strategies, and developing targeted interventions.