Poxviruses are large double-stranded DNA viruses, and recombination has long been recognized as a fundamental engine of their evolution. Laboratory studies dating back to the late 1980s demonstrated high levels of genetic exchange among poxvirus DNAs in infected cells, and comparative genomics of variola, the smallpox virus, revealed signatures of past recombination events in its evolutionary history. Recombination requires co-infection of a single cell by two genetically distinct viruses, after which the cellular and viral machinery can copy alternating templates, stitching together segments from each parent. In principle, mpox viruses from different clades could recombine wherever their transmission chains overlap — in a co-infected individual or, more plausibly given the epidemiology, in an animal reservoir host where viruses from distinct phylogeographic lineages might meet.

The Indian case came to light through the country’s mpox diagnostic network, which the Indian Council of Medical Research has been strengthening since the global outbreak of clade IIb virus began in 2022. The patient, who acquired the infection through travel, was managed clinically while specimens were forwarded for molecular testing and whole-genome sequencing. Initial real-time PCR screening could not have distinguished this virus from any other mpox infection, since standard assays target conserved genes shared across clades. Only when the researchers assembled the complete genome did the mosaic structure become apparent: stretches of sequence unambiguously derived from clade Ib, the lineage that emerged in Central Africa and spread internationally in 2024, alternating with stretches characteristic of clade IIb, the West African lineage behind the 2022 worldwide epidemic.

Detailed comparative analysis showed that the recombinant genome carries clade-defining mutations from both parental lineages. Clade-defining mutations are the fixed single-nucleotide changes that distinguish the major mpox clades Ia, Ib, IIa and IIb from one another; their simultaneous presence in a single genome, arranged in a block-like pattern along the chromosome, is the molecular fingerprint of recombination rather than convergent mutation. The team mapped 34 recombinant tracts — continuous segments of the genome inherited from one or the other parental clade — and identified 21 mutations that define the recombinant lineage itself. This pattern mirrors that of the UK recombinant virus, which likewise displayed 34 recombinant tracts and 21 recombinant lineage-associated mutations, a coincidence of architecture that points to a shared ancestry.

Phylogenetic analysis reinforced that conclusion. When individual recombinant tracts were extracted and placed on separate phylogenetic trees, each tract clustered with reference genomes from its parental clade, alternating between Ib and IIb positions depending on the segment examined. When whole recombinant genomes were compared with representative sequences from all four clades, the Indian and UK viruses formed a distinct group positioned intermediate between clades Ib and IIb. A phylogenetic network constructed with SplitsTree placed the recombinants in a characteristic box-like structure, a visual signature of reticulate evolution that arises when sequence relationships cannot be explained by a single branching tree. Statistical testing supported the inference of recombination among these viruses.

Beyond the phylogenetics, the team went a step further than most surveillance studies by isolating live virus. Clinical material was propagated in Vero CCL-81 cells, and cytopathic effects were observed by the third day after infection at the second passage, with the experiment repeated three times with consistent results. Virus isolation matters because it confirms that the recombinant genome was not an artefact of mixed sequencing reads from a co-infected patient but a viable, replicating virus. It also provides reagents for phenotypic characterization — growth kinetics, drug susceptibility, antigenicity — that sequence data alone cannot deliver. Both the clinical sample sequence and the isolate sequence were deposited publicly in the Pathoplexus database and the International Nucleotide Sequence Database Collaboration, under accession numbers that allow any laboratory to verify or extend the analysis.

The practical consequences for diagnostics are among the most important implications of the work. Several multiplex real-time PCR assays have been developed to differentiate mpox clades, exploiting clade-specific mutations to classify infections rapidly, and targeted assays exist specifically to detect the novel clade Ib lineage. A recombinant virus that combines genetic material from two clades can scramble such classifications: depending on which primer and probe sets are used, a single specimen might be typed as Ib, as IIb, or yield discordant results across targets. In the worst case, a diagnostic algorithm keyed to a clade Ib-specific mutation that falls inside a clade IIb-derived tract of the recombinant genome could produce a false negative for clade Ib classification. The authors outline a diagnostic workflow for the preliminary identification of suspected recombinant MPXV isolates, emphasizing that clade-typing assays should be interpreted alongside whole-genome sequencing whenever results are discordant or epidemiologically unexpected.

The broader epidemiological question raised by the finding is where and how these recombination events occurred. The Indian virus was imported through travel, and the UK virus was likewise detected in a traveller returning from Asia, yet both genomes cluster together, implying a common recombinant ancestor rather than two independent recombination events. That places the origin of this lineage in a location where clade Ib and clade IIb viruses circulate in proximity — a region where the 2024 clade Ib outbreak overlapped geographically with lingering clade IIb transmission. If such co-circulation is sustained, further recombinants can be expected. The properties of any recombinant cannot be assumed in advance: recombination can combine, dilute, or disrupt adaptive mutations affecting transmissibility, immune evasion, or antiviral susceptibility, and only phenotypic study of isolated viruses will resolve those questions.

For surveillance programmes, the lesson is that clade-labelled snapshots are no longer sufficient. The 2022 global mpox epidemic demonstrated how quickly a localized zoonotic spillover can become an international epidemic once a virus adapts to sustained human transmission, and the emergence of clade Ib in 2024 with higher reported severity underscored the cost of delayed recognition. Recombination adds a third dimension of unpredictability on top of mutation and geographic spread. Wastewater surveillance, which has successfully detected mpox in environmental samples, offers one complementary channel for tracking viral diversity at the population level, but it too depends on downstream sequencing to reveal recombinant structure. What the Indian and UK detections prove is that the global sequencing network, when properly resourced and connected to rapid data-sharing platforms such as Pathoplexus, can catch these events and alert the international community within the timeframe that matters for public health response.

The Indian study also illustrates the value of linking clinical care, laboratory diagnostics and research sequencing under a single coordinated response. State health authorities in Kerala coordinated disease control activities, clinical teams managed the patient, and the virology team at ICMR–NIV Pune performed molecular testing, genome sequencing, phylogenetic analysis and virus isolation as an integrated pipeline, supported by intramural funding and by the Department of Health Research’s programme to expand the national mpox laboratory network. The result is a complete characterization of a rare viral entity within weeks of detection — from bedside to genome to viable isolate to publicly shared sequence data. As mpox continues to circulate in humans and presumably in animal reservoirs, recombinant lineages such as this Ib/IIb mosaic will serve as markers of where distinct viral populations meet, and continued genomic surveillance, backed by standardized diagnostics capable of recognizing their mosaic genomes, will determine whether the world spots the next such virus before it spreads.

Subject of Research: Recombinant Clade Ib and IIb monkeypox virus identified in an imported mpox case in India

Article Title: Genomic surveillance of an imported recombinant Clade Ib and IIb monkeypox virus case in India

Article References: Sahay, R. R., Khurana, J., Moorkoth, A. P., PP, B., Shete, A. M., Yadav, S., Majumdar, T., Rani, S., Vijay, N., Mali, D., Kamble, S., Gavari, S., Gawande, P., Marathe, H., Acharya, A. K., Gupta, N., & Yadav, P. D. (2026). Genomic surveillance of an imported recombinant Clade Ib and IIb monkeypox virus case in India. Nature Microbiology. https://doi.org/10.1038/s41564-026-02508-y

Image Credits: AI Generated

DOI: 10.1038/s41564-026-02508-y

Keywords: mpox, monkeypox virus, recombination, Clade Ib, Clade IIb, genomic surveillance, whole-genome sequencing, phylogenetics, virus isolation, diagnostics, India, public health

The study was structured as a descriptive cross-sectional investigation of every patient who met the World Health Organization’s suspected-case criteria for mpox and was reported through the Khyber Pakhtunkhwa Integrated Disease Surveillance and Response System, known as KP-IDSRS. Over the study period, 137 suspected cases were flagged by the surveillance network. Of these, 29 were laboratory-confirmed using real-time polymerase chain reaction, the standard molecular diagnostic for orthopoxvirus infection. Samples with sufficiently low cycle threshold values, at or below 23, indicating high viral loads, were selected for whole-genome sequencing on an Illumina iSeq platform, allowing the researchers to reconstruct near-complete viral genomes for phylogenetic analysis.

The demographic profile of the 29 confirmed cases illustrates that mpox in this setting is not confined to any single population group. The median age of patients was 31 years, with ages ranging from an infant of 0.6 years to an adult of 80 years. Twenty patients, or 68.9 percent, were male, eight, or 27.6 percent, were female, and one patient, 3.4 percent, was transgender. This breadth of age and gender stands in contrast to the epidemiology seen in many high-income countries during the 2022 global outbreak, where cases were heavily concentrated among men who have sex with men, and suggests that in Pakistan the routes of introduction and transmission are more varied, driven largely by travel and household contact rather than by a single transmission network.

Classifying each case by likely origin, the investigators found that 13 infections, or 44.8 percent, were imported, meaning the patients acquired the virus abroad and brought it back to Pakistan. Five cases, 17.2 percent, were secondary transmission cases, representing people infected through contact with an imported case. Six cases, 20.7 percent, were classified as locally acquired with no direct epidemiological link to a known imported infection, and a further five, 17.2 percent, were deemed probable local infections. Taken together, the locally acquired and probable local categories account for more than a third of confirmed cases, providing clear evidence that imported mpox viruses have seeded chains of transmission within the province rather than simply arriving and dying out.

Clinically, the illness observed was characteristic of mpox but generally mild. A vesiculopustular rash, the hallmark dermal manifestation of orthopoxvirus infection in which fluid-filled vesicles evolve into pus-filled pustules before crusting over, was present in all 29 confirmed cases, a 100 percent frequency. Fever was recorded in 82.8 percent of patients, musculoskeletal pain in 37.9 percent, genital or perianal lesions in 34.5 percent, and sore throat in 24.1 percent. The presence of genital and perianal lesions in roughly a third of cases is notable because such lesions can be mistaken for other sexually transmitted infections, delaying diagnosis and isolation. Most patients recovered completely, but the study also records two deaths among the confirmed cases, a somber reminder that even in a largely mild outbreak, mpox carries real mortality risk, particularly for vulnerable patients.

The genomic component of the study is where its significance is most pronounced. Whole-genome sequencing of four representative cases identified both Clade IIb viruses, specifically of lineage A.2.1, and Clade Ib viruses. Mpox virus, an enveloped double-stranded DNA virus of the genus Orthopoxvirus, was long divided into two major clades, with the former Congo Basin or Clade I lineage generally associated with more severe disease and the West African or Clade II lineage with milder illness. The nomenclature has since been refined: Clade IIb denotes the lineage responsible for the sustained human-to-human global spread that began in 2022, while Clade Ib is a recently emerged branch of Clade I that has driven outbreaks in the Democratic Republic of the Congo and neighboring countries and prompted the WHO to declare a second mpox Public Health Emergency of International Concern in 2024. Detecting Clade Ib in a non-endemic country is therefore a marker of how far this newer lineage has traveled.

Phylogenetic analysis of the Pakistani genomes revealed close genetic relationships with sequences reported from within Pakistan itself and from the United States, Nepal, and Thailand. This pattern of clustering across widely separated countries is consistent with the well-established role of international travel in moving mpox virus between continents. Pakistan’s particular vulnerability stems from extensive labor migration to Gulf countries, which creates a constant flow of travelers between South Asia and regions where mpox introductions occur. Each returning traveler represents a potential introduction event, and with limited decentralized diagnostic capacity, infections can go unrecognized until secondary cases appear, exactly the pattern the epidemiological classifications in this study document.

The laboratory work underpinning these findings reflects a coordinated public health infrastructure operating under real constraints. Specimens were processed at the Khyber Pakhtunkhwa Public Health Reference Laboratory at Khyber Medical University in Peshawar, with sequencing support from the National Institutes of Health in Islamabad. The workflow, from standardized surveillance forms through real-time PCR confirmation to genome assembly and phylogenetic comparison against international databases, demonstrates that a provincial reference laboratory in a resource-limited setting can generate genomic data of direct global relevance. The authors acknowledge the WHO and the Khyber Pakhtunkhwa Health Department for supplying diagnostic kits and consumables, and note that the study received no formal external funding, underscoring how much of this surveillance was sustained through institutional commitment rather than dedicated program budgets.

The public health implications of co-circulating clades are substantial. When two genetically distinct lineages circulate simultaneously, each introduction event must be traced independently, and genomic surveillance becomes essential for distinguishing imported cases from established local transmission chains. A locally acquired Clade Ib case, in particular, signals that the newer lineage has achieved at least limited autonomous transmission in the province. The authors argue that this evidence makes a case for strengthened genomic surveillance, decentralized diagnostics that can confirm cases outside a single reference laboratory, and improved access to vaccines and therapeutics. Vaccines such as the Modified Vaccinia Ankara Bavarian Nordic product, which is used for mpox prevention in many countries, remain limited in availability across much of South Asia, and expanding access is a policy challenge that extends well beyond laboratory walls.

For the broader scientific community, the study adds an important data point to the global map of the ongoing mpox emergency. It shows that Clade Ib has reached South Asia and established detectable local transmission there, that Clade IIb lineage A.2.1 continues to circulate in the same region, and that a surveillance system built on WHO case criteria, rapid response teams, and provincial laboratory capacity can detect and characterize this diversity. As international travel continues to connect endemic and non-endemic regions, the Pakistani experience illustrates a pattern likely to recur elsewhere: imported cases are inevitable, but whether they fade out or ignite sustained transmission depends on how quickly diagnostics, contact tracing, vaccination, and genomic sequencing can be brought to bear. The findings from Khyber Pakhtunkhwa suggest that in much of the world, that capacity still needs deliberate investment before the next introduction arrives.

Subject of Research: Emergence and co-circulation of Clade Ib and Clade IIb mpox virus in a non-endemic region of Pakistan

Article Title: Emergence and co-circulation of Clade Ib and Clade IIb Mpox viruses in Khyber Pakhtunkhwa Pakistan, 2024–2026: clinical, genomic, and public health insights from a non-endemic region

Article References: Zia, A., Islam, A., Jamal, Z., Khan, M., Khattak, A. A., Manzoor, M., Umair, M., Muhammad, A., Mehmood Noor, S., Rehman, K., Zohaib, A., Khan, M. A., & Yousafzai, Y. M. (2026). Emergence and co-circulation of Clade Ib and Clade IIb Mpox viruses in Khyber Pakhtunkhwa Pakistan, 2024–2026: clinical, genomic, and public health insights from a non-endemic region. BMC Infectious Diseases. https://doi.org/10.1186/s12879-026-14542-6

Image Credits: AI Generated

DOI: 10.1186/s12879-026-14542-6

Keywords: mpox, Clade Ib, Clade IIb, orthopoxvirus, whole-genome sequencing, genomic surveillance, Pakistan, Khyber Pakhtunkhwa, travel-associated infections, public health, epidemiology, emerging infectious diseases