Since 2023, there have been a sharp increase in Mpox cases in the Democratic Republic of Congo caused by a new strain of Clade I virus. As of 23Sep2024, The World Health Organization (WHO) announced nearly 30,000 suspected Mpox cases have been reported across Africa in 2024, with the majority occurring in the Democratic Republic of Congo (DRC). This outbreak strain is different from the Mpox Clade IIb which caused a multi-country global outbreak that was declared by the WHO in 2022. There has been concern whether diagnostic tools developed in 2022 will be able to meet the needs for testing in the current outbreak.
Mpox virus genome is composed of linear, double stranded DNA roughly 200 kb in length and encodes an estimated 190 proteins (10.1016/j.vaccine.2023.12.086). The central core region of the MPXV genome is highly conserved and shares more than 90% sequence homology with other orthopoxviruses (like Variola virus which causes smallpox, Cowpox and Vaccinia virus). Flanking regions on the left and right side of the core are more variable and contain genes associated with virulence and host range. There are also inverted terminal repeats (ITRs) at both ends of the genome.
There are two distinct Clades of Mpox: Clade I (which was formerly called the Congo Basin clade- Central Africa) and Clade II (formerly called West African Clade). Clade II is further divided into subclades IIa and IIb. Clade I is generally associated with more severe symptoms and a higher case fatality rate of ~10%, while Clade IIa and IIb have lower case fatality rate of ~1 - 3%. (https://doi.org/10.1073/pnas.23016621 but see also DOI: 10.1016/S1473-3099(24)00564-4). Despite differences in transmission, symptoms and morbidity, Clade I and Clade II exhibit approximately 95% sequence identity across the genome (https://doi.org/10.1073/pnas.222041512). The current outbreak, which centered in the city of Kamituga, in DRC has been referred to as Kamituga strain and is now designated Clade Ib. Previously Clade I had exhibited very little human to human transmission, but this Clade Ib Kamituga strain is unique in that it is spreading through both sexual contact and community spread including children.
Key Findings from Kamituga strain Genome Sequencing
A recent publication (10.3855/jidc.20136) used NGS to obtain the complete viral genome sequence of samples from the Kamituga outbreak to try to understand the genetic basis for the unique transmission patterns as well as the genetic evolution of this strain. Samples were collected from lesion swabs in Kamituga hospital and DNA was extracted from six samples and used for Oxford Nanopore Technologies NGS sequencing. The reads were aligned to the Mpox Clade I reference genome (NC_003310- approximately 110X to 1100X coverage across the genome) to understand the mutation map of the new outbreak strain.
“Seven proteins (including C9L [OPG047], I4L [OPG080], L6R [OPG105], A17L [OPG143], A25R [OPG151], A28L [OPG153], and B21R [OPG210]) emerged as mutational hotspots with a number of consensus inframe deletions, frameshift variants, synonymous variants, and amino acids substitutions” .”. These seven proteins identified as mutation hotspots make sense as those driving the unique features of the Kamituga strain, as they are proteins involved in key processes related to transmission and virulence (Table 1). B21R (OPG210), which codes a surface glycoprotein, was found to be associated with the highest number of mutations” and “the number of mutations found to be consistent across all six samples.” “The B21R (OPG210) protein of the Kamituga strain contains a total of four unique changes, two frameshift variants (K431X and A475V), and two amino acid substitutions (P726X and E1056D)”. The major surface glycoprotein plays a role in direct fusion of virus with host cells and endocytosis, but these surface proteins also enhance the recognition and inactivation of Mpox virus by immune cells (doi: 10.1038/s41392-023-01675-2) explaining why this gene may be under significant evolutionary pressure.
Table 1: Genes identified as mutation hotspots in Masirika et al. and role the encoded protein plays in the viral life cycle.
The authors also observed deletion of the D14L (OPG032) gene in all six samples. This is interesting because the role of the D14L protein is controversial. D14L encodes a complement inhibitor also referred to as MOPICE (monkeypox inhibitor of complement enzymes) which is deleted in clade IIa/IIb viruses. However, using recombinant MPXV, Estep and co-workers showed that deletion of D14L from the Zaire-96 strain renders the virus more rather than less virulent, at least in a macaque model (10.1016/j.meegid.2022.105372, J. Virol. 2011;85:9527–9542.)
Overall, the study concluded that the six Mpox consensus sequences from the Kamituga outbreak exhibited 99.47 to 99.79% identity with the reference genome (NC_003310) based on the local pairwise sequence alignment. Therefore, the changes in transmission observed in the Kamituga Clade I outbreak result from a small number of mutations in proteins critical for cell entry, viral replication and immune evasion rather than a rapid large-scale change in sequences across the genome. Given the impact of mutations observed from Clade IIb outbreak in 2022 to the Clade I outbreak in 2024 are minor, diagnostic assays developed to detect the Clade IIb strain in 2022 will still be able to detect this Clade I strain.
Impact on Diagnostics
PCR-based assays are designed with primers and probes of highly conserved genes across both clades, so the very targeted mutations observed in the Kamituga outbreak (10.3855/jidc.20136) are not expected to affect detection by current diagnostic assays. For example, laboratory developed assays using the US CDC published primer/probe sequences (Monkeypox virus Generic Real-Time PCR Test” Doc. No. CDC-007-00217 Rev 2, and Non-variola Orthopoxvirus Generic Real-Time PCR Test) detect two targets: One is a highly conserved region of TNF receptor gene (J2L), located at the terminal inverted repeat region (ITR). This assay, known as G2R-G, is a “generic” assay because the region detected is conserved in all clades of Mpox (Journal of Virological Methods 169 (2010) 223–227). The other target, often referred to as “E9L-NVAR”, targets the highly conserved F8L gene, encoding the catalytic subunit of the family B DNA polymerase critical for viral replication. Figure 1 shows an alignment of the sequence target areas for GenBank: ON585038.1, Clade IIb from 2022 outbreak with MpxV/DRC/HGRK-1L/2024|EPI_ISL_18886301 Clade I from Kamituga hospital in 2024 outbreak (https://doi.org/10.1186/1471-2105-10-421). The Clade I outbreak strain from Kamituga hospital is almost a perfect match for the existing primers and probes, and the one mismatch observed in the G2R-G assay is not expected to significantly impact assay performance. 
Figure 1A: The Query sequence is GenBank: ON585038.1, Clade IIb from 2022 outbreak, and Sbjct sequence is with MpxV/DRC/HGRK-1L/2024|EPI_ISL_18886301 Clade I from Kamituga hospital in 2024. Yellow highlighting shows the target sequence for the G2R-G forward and reverse primers within the TNF receptor gene (J2L) in the terminal repeat regions. Blue highlighting indicates the probe target sequence. There is a single mismatch under the forward primer, which is not expected to significantly impact amplification. 
Figure 1B: The Query sequence is GenBank: ON585038.1, Clade IIb from 2022 outbreak, and Sbjct sequence is with MpxV/DRC/HGRK-1L/2024|EPI_ISL_18886301 Clade I from Kamituga hospital in 2024. Yellow highlighting shows the target sequence for the E9L NVAR forward and reverse primers within F8L polymerase gene. Blue highlighting indicates the probe target sequence. The primers and probe are a perfect match to the 2024 Kamituga outbreak strain.
A subset of assays with Emergency Use Authorization from the US FDA for Mpox diagnostics is shown in Table 2. The assays use dual-target detection, which mitigates risk of diagnostic failure due to a mutation in one target sequence. Additionally, the assays all target highly conserved gene regions not identified as mutation hotspots in the current outbreak. Although the cobas MPXV assay targets the B21R/B22R region, Roche confirmed in a Media release from 20AUG2024 that cobas MPXV test detects the latest mpox virus variants.
Table 2: Leading Emergency Use Authorized (EUA) Mpox diagnostics available in the United States.
LGC Diagnostic Tools Can Help
LGC offers Mpox and Non-Variola Orthopoxvirus ValuPanels based on CDC -designed primer/probe sequences. All primers and probes are HPLC purified and delivered in dried format at 20 nmol for each primer and 5 nmol for each probe. The probes are labelled with FAM dye and BHQ quencher. Probes and primers are delivered in separate tubes and are not premixed, allowing the user to optimize the assay to their test system.
LGC also offers AccuPlex Monkeypox Reference Material Kit for positive QC materials designed to ensure accurate results when testing Mpox with leading EUA diagnostics (Table 2). These reference materials are composed of recombinant mammalian virus, enabling full process QC from extraction to detection via reverse-transcription PCR assays. Additionally, the recombinant viruses in the reference material are non-infectious and replication deficient, ensuring safety during assay development and end-use QC applications. Although the reference material was designed to bear diagnostically important sequences from GenBank: ON585038.1 (a sequence from clade IIb, 2022 outbreak strain), the information provided here shows that these are the same sequences important for current and future Mpox diagnostics.
To learn more about the stability and performance of the AccuPlex Monkeypox Reference Material Kit, download the poster by Revathi Raman et al, presented at the 2023 AACC Annual Scientific Meeting.
