Monkeypox have a higher probability of evolution during the time frame while case numbers are low

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Vaccinations and lifestyle adjustments in at risk-populations have enabled public health authorities to gradually get a better handle on monkeypox in the U.S. However, a new study warns that it’s too soon to relax.

“Just because a disease like monkeypox appears controllable does not mean it will stay controllable,” said the paper’s lead author, University of Maryland Biology Assistant Professor Philip Johnson.

“Slowly simmering epidemics like monkeypox have a higher probability of evolution during the time frame while case numbers are low. This means that waiting until the number of cases is high again would give monkeypox the opportunity to adapt more substantially to humans.”

The paper, “Evolutionary consequences of delaying intervention for monkeypox,” was published in the medical journal The Lancet on September 21, 2022.

In the paper, Johnson and his co-authors cited other high-profile outbreaks exacerbated by pathogen evolution, including the recent Ebola virus outbreak (2013-16), and the delta and omicron variants of SARS-CoV-2. These evolutionary changes likely made the viruses more difficult to control.

“We expect zoonotic infections – diseases that originate from animals, like Ebola from bats and monkeypox from rodents – to be poorly adapted to people when they first jump between species,” Johnson said. “But given enough time, the pathogens can mutate just a little with each new transmission and become increasingly better at thriving in humans.”

The team hopes that its research will encourage policymakers to avoid complacency and procrastination when tackling seemingly “controllable” viruses like monkeypox. If implemented quickly and consistently enough throughout an epidemic’s lifecycle, control measures like contact tracing or vaccination may give public health authorities the best chance to fully eradicate the outbreak before significant evolution occurs.

In the long term, the team’s goal is to increase global responses to any zoonotic infection in its earliest iteration – when it is relatively cheaper and easier to control.

“Pathogen evolution can’t be stopped, but it can definitely be slowed by control measures,” Johnson said. “We have finite public health resources, meaning that we need more research to develop tools that can identify possible early-stage evolutionary adaptations and help guide control efforts to where they’ll be most effective.”


Monkeypox is a rare zoonotic disease that is caused by the MPXV from the Orthopoxvirus genus, which includes the variola virus, the causative agent of smallpox1,2,3. With an incubation period of 5–21 days, human disease typically begins with fever, myalgia, fatigue and headache, often followed by maculopapular rash at the site of primary infection that can spread to other parts of the body1. Although the natural reservoir of MPXV remains unknown, animals such as rodents and non-human primates may harbor the virus, leading to occasional spill-over events to humans1,2,3. MPXV is endemic in West and Central African countries, and the rare reports outside these regions have been associated with imports from those endemic countries1,2,3,4.

We are now facing the first multi-country outbreak without known epidemiological links to West or Central Africa1, with more than 2,500 confirmed cases reported worldwide as of 18 June 2022 (refs. 5,6), since the first confirmed case on 7 May 2022 in the United Kingdom4. Several measures are being recommended by international health authorities to contain MPXV transmission1, including the use of vaccines for selected close contacts of patients with monkeypox (post-exposure) and for groups at risk of occupational exposure to monkeypox (pre-exposure)7. The virus can be transmitted from human to human by close contact with lesions, body fluids, respiratory droplets and contaminated materials1,3, but the current epidemiological context poses some degree of uncertainty about the viral transmission dynamics and outbreak magnitude.

International sequencing efforts immediately began to characterize the outbreak-causing MPXV to identify its origin and track its dissemination. Genome data will also inform about the virus evolutionary trajectory, genetic diversity and phenotypic characteristics with relevance for guiding diagnostics, prophylaxis and research. Here we report the rapid application of high-throughput shotgun metagenomics to reconstruct the first genome sequences of the MPXV associated with the 2022 MPXV outbreak, providing valuable genomic and phylogenetic data on this emerging threat.

To rapidly get the first insights on phylogenetic placement and evolutionary trends of the 2022 outbreak-causing MPXV, we focused our analysis on a first outbreak-related MPXV genome sequence, publicly released on 20 May 2022 by Portugal8, as well as on additional sequences released in the National Center for Biotechnology Information (NCBI) before 27 May 2022, with 15 sequences in total (most of them from Portugal) (Supplementary Tables 1 and 2). The rapid integration of the first sequence into the global MPXV genetic diversity (Fig. 1) confirmed that the 2022 outbreak virus belongs to the MPXV clade 3 (within the formerly designated ‘West African’ clade, which also includes clade 2)9. MPXV from clades 2 and 3 are most commonly reported from western Cameroon to Sierra Leone and usually carries a <1% case–fatality ratio (CFR), in contrast with viruses from the clade 1 (formerly designated as ‘Central African’ or ‘Congo Basin’ clade)9, which are considered more virulent with a >10% CFR10,11.

All outbreak MPXV strains sequenced so far tightly cluster together (Fig. 1), suggesting that the ongoing outbreak has a single origin. The 2022 outbreak cluster (lineage B.1)9 forms a divergent branch descendant from a branch with viruses (lineage A.1)9 associated with the exportation of MPXV in 2018 and 2019 from an endemic country (Nigeria) to the United Kingdom, Israel and Singapore12,13, with genetic linkage to a large outbreak occurring in Nigeria in 2017–2018 (ref. 13) (Fig. 1). Given these findings and the MPXV historical epidemiology (rare cases in non-endemic countries), it is likely that the emergence of the 2022 outbreak resulted from importation(s) of this MPXV from an endemic country, with the MPXV detected in 2022 potentially representing the continuous circulation and evolution of the virus that caused the 2017–2018 Nigeria outbreak.

The recent release of an MPXV sequence from a 2021 travel-associated case from Nigeria to the United States (USA_2021_MD; accession no. ON676708)14 phylogenetically placed between 2018–2019 and 2022 sequences (Fig. 1) is aligned with such hypothesis. We cannot, however, exclude the hypothesis of a prolonged period of cryptic dissemination in humans or animals in a non-endemic country (for example, after the reported 2018–2019 importations). Silent human-to-human transmission (for example, due to underdiagnosis) seems less likely considering the known disease characteristics of the affected individuals, usually involving localized or generalized skin lesions1. Cryptic transmission in an animal host in a non-endemic country followed by a recent spill-over event is another hypothesis, even though, again, this would be somehow surprising as such a scenario has never been reported.

Altogether, current data points for a scenario of more than one introduction from a single origin, with superspreader event(s) (for example, saunas used for sexual encounters) and travel abroad likely triggering the rapid worldwide dissemination15,16. Considering the expected incubation period of 5–21 days3, limited sampling (including limited viral genotyping data for the first confirmed cases in 2022) and the fact that multiple cases were confirmed in several countries in a 3-week period1 after a first report on 7 May 2022 by the United Kingdom1, the identification of the index cases associated with such presumable several introductions can be challenging. For example, although the first confirmed case has been hypothesized as the index of the outbreak (due to travel from Nigeria to the United Kingdom on 3–4 May 2022 (refs. 1,3)), this scenario can be discarded as the earliest symptom onset dates for confirmed cases in Portugal and in the United Kingdom were in late April15,16.

a, MPXV global phylogeny showing that the 2022 outbreak cluster (lineage B.1) belongs to clade 3. Clade and lineage are designated according to the nomenclature proposed by Happi et al.9. b, Genetic diversity within the outbreak cluster, including the 15 sequences analyzed in this study (released in the NCBI before 27 May 2022). The deletion symbol (Δ) denotes a large deletion (11,335–12,247 in the MPXV-UK_P2-010 gene) shared by sequences segregating in a small subcluster. c, Outbreak phylogenetic tree updated with sequences available in the NCBI as of 15 June 2022 (provided during revision for more updated contextualization). The list of the sequences used in these phylogenetic analyses is detailed in Supplementary Table 2, and the alignments are provided as Supplementary Data.

Notably, the 2022 MPXV diverges from the related 2018–2019 viruses by a mean of 50 single-nucleotide polymorphisms (SNPs) (Figs. 1 and 2), which is far more (roughly 6–12-fold more) than one would expect considering previous estimates of the substitution rate for Orthopoxviruses (1–2 substitutions per genome per year)17. Such a divergent branch might represent accelerated evolution. Of note, among the 46 SNPs (24 non-synonymous, 18 synonymous and four intergenic) (Supplementary Table 3) separating the 2022 MPXV outbreak virus from the reference sequence (MPXV-UK_P2, 2018; GenBank accession no. MT903344.1), three amino acid changes (D209N, P722S and M1741I) occurred in the immunogenic surface glycoprotein B21 (MPXV-UK_P2-182)18. Serological studies have previously indicated that the monkeypox B21 protein might be an important antibody target with several key immunodominant epitopes18. As discussed previously19, fine inspection of the mutation profile of those 46 SNPs further revealed a strong mutational bias, with 26 (14 non-synonymous, ten synonymous and two intergenic) and 15 (nine non-synonymous and 16 synonymous) being GA > AA and TC > TT nucleotide replacements, respectively (Fig. 2 and Supplementary Table 3).

A tool (https://github.com/insapathogenomics/mutation_profile) was built to rapidly screen these and other mutation profiles. The observed (hyper)mutation signature might suggest the potential action of apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) enzymes in the viral genome editing20. Also, MPXV is A:T rich, so a mutation bias leading to further incorporation of A/T suggests the action of a non-random mutational driver, such as APOBEC3. In fact, APOBEC3 enzymes can be upregulated in response to viral infection, being capable of inhibiting a wide range of viruses by introducing mutations through deaminase and deaminase-independent mechanisms20,21. In some circumstances (for example, lower levels of deamination), APOBEC3-mediating mutations might not completely disrupt the virus, thus increasing the likelihood of producing hyper-mutated (but viable) variants with altered characteristics (for example, HIV immune escape variants)20,22.

The repertoire and level of APOBEC3 enzymes depend on the host species/tissue, and different enzymes display different preferences for the nucleotide or motif (such as dinucleotides or tetranucleotides) to be mutated20,23,24. For instance, the GA > AA and TC > TT nucleotide replacements observed in the 2022 outbreak MPXV were also found to be the preferred mutational pattern of human APOBEC3A enzymes (expressed in keratinocytes and skin) during genetic editing of human papillomavirus (HPV) in HPV1a plantar warts and HPV16 pre-cancerous cervical biopsies25. Whether the excess of mutations seen in the 2022 MPXV is a direct consequence of APOBEC3-mediated genome editing in the human host cannot be discerned at this stage.

Also, the putative APOBEC3 effect on MPXV evolution augments the uncertainty regarding the 2022 outbreak origins and introductions, in addition to the complexity of the epidemiological context. This raises the need for future studies focusing on the weight of APOBEC3 in MPXV diversification. In particular, functional studies assessing whether this mutational driver triggers MPXV adaptive evolution toward altered phenotypic features, such as enhanced transmissibility, are warranted.

Light-colored mutations represent the SNPs separating the MPXV 2022 outbreak cluster from the MPXV_UK_P2 (MT903344.1) reference sequence (Supplementary Table 3). Dark-colored mutations represent the genetic diversity within the outbreak cluster (Supplementary Table 4).

Further phylogenomic analysis revealed the first signs of microevolution of this virus during human-to-human transmission. Among the 15 outbreak sequences analyzed here, we detected the emergence of 15 SNPs (eight non-synonymous, four synonymous, two intergenic and one stop gained) (Fig. 2 and Supplementary Table 4). Notably, all SNPs also follow the same mutational bias, including eight GA > AA (six non-synonymous and two synonymous) and seven TC > TT (two non-synonymous, two synonymous, one stop gained and two intergenic) nucleotide replacements. This further suggests a continuous action of APOBEC3 during MPXV evolution. Among the seven phylogenetic branches directly descendant from the most recent ancestor of the MPXV outbreak strain (Fig. 1), we identified a subcluster (supported by two SNPs) of two sequences (PT0005 and PT0008, each with an additional SNP) that also share a 913-bp frameshift deletion in a gene coding for an ankyrin/host range (MPXV-UK_P2-010). Although gene loss events are not unexpected for orthopoxviruses (for example, variola virus has most likely undergone reductive evolution26), these were previously observed in the context of endemic MPXV circulation in Central Africa, being hypothesized to correlate with human-to-human transmission27.

Our data reveal additional clues of ongoing viral evolution and potential human adaptation. Most emerging SNPs in sequences from Portugal were not 100% fixed in the viral population (frequencies 75–95%), supporting the existence of viral intra-patient population diversity. Further inspection of minor intra-patient single-nucleotide variants (iSNVs) in Illumina samples led to the validation of 11 non-synonymous minor iSNVs (across five samples), again most with the ‘APOBEC3 signature’ (Supplementary Table 5). Notably, among the targeted viral transcripts, we highlight a few encoded proteins that are known to interact with host immune system, such as an MHC class II antigen presentation inhibitor28, an IFN-alpha/beta receptor glycoprotein29 and IL-1/TLR signaling inhibitor30. These and other proteins (Supplementary Tables 3–5) found to be targeted during the 2022 outbreak MPXV divergence and microevolution might constitute priority targets for future functional studies aiming to assess their potential role in adaptation.

In summary, our genomic and phylogenomic data provide insights into the evolutionary trajectory of the 2022 MPXV outbreak strain and shed light on potential mechanisms and targets of human adaptation. The observed accelerated evolution of this human MPXV, potentially driven by the APOBEC3 action, suggests that viral genome sequencing might provide sufficient resolution to track the transmission dynamics and outbreak spread, which seemed to be challenging for a presumably slow-evolving double-stranded DNA virus. Together with the adopted strategy of real-time data sharing, this study may help guide novel outbreak control measures and subsequent research directions.

reference link : https://www.nature.com/articles/s41591-022-01907-y


More information: Philip L F Johnson et al, Evolutionary consequences of delaying intervention for monkeypox, The Lancet (2022). DOI: 10.1016/S0140-6736(22)01789-5

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