Reversion Of Mutation Research Articles

Safety evaluation of vitamin K2 (menaquinone-7) via toxicological tests

This study aims to evaluate the safety of MK-7 produced by fermentation process using a Bacillus subtilis var. natto strain for human ingestion via acute oral toxicity, repeated dose 90-day oral toxicity, 28-day recovery test, and genotoxicity tests. The acute oral toxicity test results indicated that all subjects survived at the dose of 5000 mg/kg with no toxic effects. For the repeated dose 90-day oral toxicity test, MK-7 was administered to rats at 500, 1500, and 4500 mg/kg for 90 d. No abnormal findings were detected in clinical observations or in clinical pathological and histopathological examinations. The no-observed-adverse-effect level(NOAEL) was determined to be 4500 mg/kg/d, the maximum dose tested. For the evaluation of genotoxicity, reverse mutation, chromosomal aberration, and micronucleus tests were performed. In the reversion mutation test, vitamin K2 did not induce reversion in bacterial strains, and no chromosomal abnormality was observed in the chromosomal abnormality test using Chinese hamster lung cells. In the micronucleus test, micronuclei were not induced using ICR mouse bone marrow cells. All the toxicity test results suggest that vitamin K2 produced by fermentation processes using Bacillus subtilis var. natto induced no toxicological changes under the experimental conditions.

Abstract 1113 Mechanistic studies of incorporation of activated Islatravir by HIV-1 reverse transcriptase mutants

Abstract 1113 Mechanistic studies of incorporation of activated Islatravir by HIV-1 reverse transcriptase mutants

Mitochondrial haplotype and mito-nuclear matching drive somatic mutation and selection throughout ageing

Mitochondrial genomes co-evolve with the nuclear genome over evolutionary timescales and are shaped by selection in the female germline. Here we investigate how mismatching between nuclear and mitochondrial ancestry impacts the somatic evolution of the mitochondrial genome in different tissues throughout ageing. We used ultrasensitive duplex sequencing to profile ~2.5 million mitochondrial genomes across five mitochondrial haplotypes and three tissues in young and aged mice, cataloguing ~1.2 million mitochondrial somatic and ultralow-frequency inherited mutations, of which 81,097 are unique. We identify haplotype-specific mutational patterns and several mutational hotspots, including at the light strand origin of replication, which consistently exhibits the highest mutation frequency. We show that rodents exhibit a distinct mitochondrial somatic mutational spectrum compared with primates with a surfeit of reactive oxygen species-associated G > T/C > A mutations, and that somatic mutations in protein-coding genes exhibit signatures of negative selection. Lastly, we identify an extensive enrichment in somatic reversion mutations that ‘re-align’ mito-nuclear ancestry within an organism’s lifespan. Together, our findings demonstrate that mitochondrial genomes are a dynamically evolving subcellular population shaped by somatic mutation and selection throughout organismal lifetimes.

Convergent evolution of BRCA2 reversion mutations under therapeutic pressure by PARP inhibition and platinum chemotherapy

Reversion mutations that restore wild-type function of the BRCA gene have been described as a key mechanism of resistance to Poly(ADP-ribose) polymerase (PARP) inhibitor therapy in BRCA-associated cancers. Here, we report a case of a patient with metastatic castration-resistant prostate cancer (mCRPC) with a germline BRCA2 mutation who developed acquired resistance to PARP inhibition. Extensive genomic interrogation of cell-free DNA (cfDNA) and tissue at baseline, post-progression, and postmortem revealed ten unique BRCA2 reversion mutations across ten sites. While several of the reversion mutations were private to a specific site, nine out of ten tumors contained at least one mutation, suggesting a powerful clonal selection for reversion mutations in the presence of therapeutic pressure by PARP inhibition. Variable cfDNA shed was seen across tumor sites, emphasizing a potential shortcoming of cfDNA monitoring for PARPi resistance. This report provides a genomic portrait of the temporal and spatial heterogeneity of prostate cancer under the selective pressure of a PARP inhibition and exposes limitations in the current strategies for detection of reversion mutations.

Longitudinal profiling identifies co-occurring BRCA1/2 reversions, TP53BP1, RIF1 and PAXIP1 mutations in PARP inhibitor-resistant advanced breast cancer

Resistance to therapies that target homologous recombination deficiency (HRD) in breast cancer limits their overall effectiveness. Multiple, preclinically validated, mechanisms of resistance have been proposed, but their existence and relative frequency in clinical disease are unclear, as is how to target resistance. Longitudinal mutation and methylation profiling of circulating tumour (ct)DNA was carried out in 47 patients with metastatic BRCA1-, BRCA2- or PALB2-mutant breast cancer treated with HRD-targeted therapy who developed progressive disease-18 patients had primary resistance and 29 exhibited response followed by resistance. ctDNA isolated at multiple time points in the patient treatment course (before, on-treatment and at progression) was sequenced using a novel >750-gene intron/exon targeted sequencing panel. Where available, matched tumour biopsies were whole exome and RNA sequenced and also used to assess nuclear RAD51. BRCA1/2 reversion mutations were present in 60% of patients and were the most prevalent form of resistance. In 10 cases, reversions were detected in ctDNA before clinical progression. Two new reversion-based mechanisms were identified: (i) intragenic BRCA1/2 deletions with intronic breakpoints; and (ii) intragenic BRCA1/2 secondary mutations that formed novel splice acceptor sites, the latter being confirmed by invitro minigene reporter assays. When seen before commencing subsequent treatment, reversions were associated with significantly shorter time to progression. Tumours with reversions retained HRD mutational signatures but had functional homologous recombination based on RAD51 status. Although less frequent than reversions, nonreversion mechanisms [loss-of-function (LoF) mutations in TP53BP1, RIF1 or PAXIP1] were evident in patients with acquired resistance and occasionally coexisted with reversions, challenging the notion that singular resistance mechanisms emerge in each patient. These observations map the prevalence of candidate drivers of resistance across time in a clinical setting, information with implications for clinical management and trial design in HRD breast cancers.

Comprehensive genomic evaluation of advanced and recurrent breast cancer patients for tailored precision treatments

AimThe aim of this study was to investigate genetic alterations within breast cancer in the setting of recurrent or de novo stage IV disease.Patients and methods: This study included 22 patients with recurrent breast cancer (n = 19) and inoperable de novo stage IV breast cancer (n = 3). For next generation sequencing, FoundationOneCDx (F1CDx) (Foundation Medicine Inc., Cambridge, MA, USA) was performed in 21 patients and FoundationOneLiquid CDx was performed in 1 patient.ResultsMedian age was 62.9 years (range, 33.4–82.1). Pathological diagnoses of specimens included invasive ductal carcinoma (n = 19), invasive lobular carcinoma (n = 2), and invasive micropapillary carcinoma (n = 1). F1CDx detected a median of 4.5 variants (range, 1–11). The most commonly altered gene were PIK3CA (n = 9), followed by TP53 (n = 7), MYC (n = 4), PTEN (n = 3), and CDH1 (n = 3). For hormone receptor-positive patients with PIK3CA mutations, hormonal treatment plus a phosphoinositide 3-kinase inhibitor was recommended as the treatment of choice. Patients in the hormone receptor-negative and no human epidermal growth factor receptor 2 expression group had significantly higher tumor mutational burden than patients in the hormone receptor-positive group. A BRCA2 reversion mutation was revealed by F1CDx in a patient with a deleterious germline BRCA2 mutation during poly ADP ribose polymerase inhibitor treatment.ConclusionGuidance on tailored precision therapy with consideration of genomic mutations was possible for some patients with information provided by F1CDx. Clinicians should consider using F1CDx at turning points in the course of the disease.

Targeting BRCA and PALB2 in Pancreatic Cancer.

An important subgroup of pancreatic ductal adenocarcinomas (PDACs) harbor pathogenic variants in BRCA1, BRCA2, or PALB2. These tumors are exquisitely sensitive to platinum-based chemotherapy and patients may experience deep and durable responses to this treatment. PARP inhibitors offer potential respite from the cumulative toxicities of chemotherapy as they significantly extend progression-free survival compared to a chemotherapy holiday. Given the lack of proven survival benefit, the decision to use a maintenance PARP inhibitor rather than continue chemotherapy should be individualized. Interestingly, in both published clinical trials of maintenance PARP inhibitors, there is a striking range of interpatient benefit: Even in the platinum-sensitive setting, roughly 25% of tumors appear to be PARP inhibitor refractory (progressive disease within 2 months of starting treatment), 50% sustain moderate benefit (up to 2 years), and 25% are hyper-responsive (more than 2 years of benefit). This finding highlights the need to refine our understanding of which patients will respond to maintenance PARP inhibitors, both by being able to identify biallelic loss and by deepening our knowledge of resistance mechanisms and who develops them. Recent data supports that reversion mutations are common in PARP inhibitor refractory patients, but we have little understanding of the mechanisms that drive delayed resistance and long-term responses. Identifying which patients are more prone to certain mechanisms of resistance and tackling them with specific treatment strategies are areas of active investigation. Additionally, given that PARP inhibitors have limited overall efficacy for most patients, upfront combination strategies are an important future strategy.

Heritable Epigenetic Modification of BpIAA9 Causes the Reversion Mutation of Leaf Shapes in Betula pendula ‘Dalecarlica’

The European white birch, scientifically known as Betula pendula, and its variant, B. pendula ‘Dalecarlica’, are characterized by a lobed leaf margin that enhances their aesthetic appeal. However, the lobed leaf trait can undergo random and spontaneous reversion to the typical ovoid or cordate shape during asexual reproduction. Investigating and elucidating the molecular mechanisms underlying this unpredictable reversion mutation is essential for comprehending the birch leaf development process. In this research, we employed an ovate−leaf mutant derived from a lobed–leaf birch clone during plant tissue culture. We adopted a multi-omics approach, including whole-genome resequencing, transcriptome sequencing, and methylation profiling, to analyze and compare genomic variations and gene expression modifications. The study revealed that the 24 variant genes affected by 1464 SNP/InDel sites in the genome of the ovate−leaf mutant are not associated with leaf development. While the overall methylation level in the mutant’s genome closely resembles that of the lobed–leaf birch, ten genes exhibit differential methylation accompanied by differential expression. Transcriptome sequencing demonstrated that the differentially downregulated genes in the mutant are significantly enriched in the GO:0009733 (response to auxin) and GO:0009734 (auxin-activated signaling) pathways. Validation through McrBC−PCR and qRT−PCR confirmed differential methylation and expression of BpIAA9 in the reversion mutant. The elevated methylation level in the BpIAA9 promoter leads to reduced expression, resulting in changes in the expression of auxin-responsive genes. This, in turn, leads to a transcriptional downregulation enrichment effect in auxin-related pathways in the reversion mutant, ultimately inhibiting the regulation of leaf veins by auxin during their development.

Unravelling the molecular basis of PARP inhibitor resistance in prostate cancer with homologous recombination repair deficiency.

Prostate cancer is a disease with heterogeneous characteristics, making its treatability and curability dependent on the cancer's stage. While prostate cancer is often indolent, some cases can be aggressive and evolve into metastatic castration-resistant prostate cancer (mCRPC), which is lethal. A significant subset of individuals with mCRPC exhibit germline and somatic variants in components of the DNA damage repair (DDR) pathway. Recently, PARP inhibitors (PARPi) have shown promise in treating mCRPC patients who carry deleterious alterations in BRCA2 and 13 other DDR genes that are important for the homologous recombination repair (HRR) pathway. These inhibitors function by trapping PARP, resulting in impaired PARP activity and increased DNA damage, ultimately leading to cell death through synthetic lethality. However, the response to these inhibitors only lasts for 3-4 months, after which the cancer becomesPARPi resistant. Cancer cells can develop resistance to PARPi through numerous mechanisms, such as secondary reversion mutations in DNA repair pathway genes, heightened drug efflux, loss of PARP expression, HRR reactivation, replication fork stability, and upregulation of Wnt/Catenin and ABCB1 pathways. OvercomingPARPi resistance is a critical and complex process, and there are two possible ways to sensitize the resistance. The first approach is to potentiate thePARPi agents through chemo/radiotherapy and combination therapy, while the second approach entails targeting different signaling pathways. This review article highlights the latest evidence on the resistance mechanism ofPARPi in lethal prostate cancer and discusses additional therapeutic opportunities available for prostate cancer patients with DDR gene alterations who do not respond toPARPi.

OmeDDG: Improved Protein Mutation Stability Prediction Based on Predicted 3D Structures.

Determining changes in the protein's thermal stability following mutations is critical in protein engineering and understanding pathogenic missense mutations. Despite the development of various computational methods to predict the effects of single-point mutations, their accuracy remains limited. In this study, we propose a new computational method, OmeDDG, that more accurately predicts mutation-induced Gibbs free energy changes in protein folding (ΔΔG). OmeDDG takes the sequences of wild-type and mutant proteins as input, utilizes OmegaFold to obtain the 3D structure, employs a convolutional neural network to extract structural features, and combines them with protein mutation features and pretraining features to predict the stability of single-point mutations in proteins. We performed a comprehensive comparison between OmeDDG and other available prediction methods on four blind test datasets, confirming that OmeDDG can effectively enhance protein mutation prediction performance. Notably, on the antisymmetric dataset Ssym, OmeDDG achieves the best performance, demonstrating favorable antisymmetry with PCC = 0.79 and RMSE = 0.96 for forward mutations and PCC = 0.77 and RMSE = 0.97 for reverse mutant types.

Exploring Roles of the Polysaccharide Capsule in Pathogenesis of Hypervirulent Acinetobacter baumannii Clinical Isolate Lac-4.

The frequently multidrug-resistant bacterial pathogen Acinetobacter baumannii is a leading cause of nosocomial infections, including ventilator-associated pneumonia, such that the World Health Organization and US Centers for Disease Control and Prevention have declared it a top priority candidate for novel drug development. Nearly all clinical A. baumannii strains express a thick surface polysaccharide capsule that protects against desiccation, host defenses, and disinfectants. In this study, we investigated the contribution of the polysaccharide capsule to virulence caused by the A. baumannii clinical isolate Ab Lac-4, which is rare in its ability to cause pneumonia and disseminated sepsis in healthy mice. We assessed the role of the capsule in wildtype Lac-4 (WT) by generating a premature stop codon in wza, which codes for the polysaccharide export protein. The wza# mutant was hypersensitive to killing by complement, whole blood, and healthy human neutrophils compared to WT and a revertant mutant (wza-Rev). Furthermore, the wza# mutant was highly attenuated in murine sepsis and unable to disseminate from the lungs during pneumonia. This study reinforces the capsule as a key contributor to Ab Lac-4 hypervirulence.

Targeted inhibition of the ATR/CHK1 pathway overcomes resistance to olaparib and dysregulates DNA damage response protein expression in BRCA2MUT ovarian cancer cells

Olaparib is a PARP inhibitor (PARPi) approved for targeted treatment of ovarian cancer (OC). However, its efficacy is impeded by the inevitable occurrence of resistance. Here, we investigated whether the cytotoxic activity of olaparib could be synergistically enhanced in olaparib-resistant OC cells with BRCA2 reversion mutation by the addition of inhibitors of the ATR/CHK1 pathway. Moreover, we provide insights into alterations in the DNA damage response (DDR) pathway induced by combination treatments. Antitumor activity of olaparib alone or combined with an ATR inhibitor (ATRi, ceralasertib) or CHK1 inhibitor (CHK1i, MK-8776) was evaluated in OC cell lines sensitive (PEO1, PEO4) and resistant (PEO1-OR) to olaparib. Antibody microarrays were used to explore changes in expression of 27 DDR-related proteins. Olaparib in combination with ATR/CHK1 inhibitors synergistically induced a decrease in viability and clonogenic survival and an increase in apoptosis mediated by caspase-3/7 in all OC cells. Combination treatments induced cumulative alterations in expression of DDR-related proteins mediating distinct DNA repair pathways and cell cycle control. In the presence of ATRi and CHK1i, olaparib-induced upregulation of proteins determining cell fate after DNA damage (PARP1, CHK1, c-Abl, Ku70, Ku80, MDM2, and p21) was abrogated in PEO1-OR cells. Overall, the addition of ATRi or CHK1i to olaparib effectively overcomes resistance to PARPi exerting anti-proliferative effect in BRCA2MUT olaparib-resistant OC cells and alters expression of DDR-related proteins. These new molecular insights into cellular response to olaparib combined with ATR/CHK1 inhibitors might help improve targeted therapies for olaparib-resistant OC.

Small-scale mutations are infrequent as mechanisms of resistance in post-PARP inhibitor tumour samples in high grade serous ovarian cancer

While the introduction of poly-(ADP)-ribose polymerase (PARP) inhibitors in homologous recombination DNA repair (HR) deficient high grade serous ovarian, fallopian tube and primary peritoneal cancers (HGSC) has improved patient survival, resistance to PARP inhibitors frequently occurs. Preclinical and translational studies have identified multiple mechanisms of resistance; here we examined tumour samples collected from 26 women following treatment with PARP inhibitors as part of standard of care or their enrolment in clinical trials. Twenty-one had a germline or somatic BRCA1/2 mutation. We performed targeted sequencing of 63 genes involved in DNA repair processes or implicated in ovarian cancer resistance. We found that just three individuals had a small-scale mutation as a definitive resistance mechanism detected, having reversion mutations, while six had potential mechanisms of resistance detected, with alterations related to BRCA1 function and mutations in SHLD2. This study indicates that mutations in genes related to DNA repair are detected in a minority of HGSC patients as genetic mechanisms of resistance. Future research into resistance in HGSC should focus on copy number, transcriptional and epigenetic aberrations, and the contribution of the tumour microenvironment.

BRCA1/2 reversion mutations in a pan-cancer cohort.

Tumor sensitivity to platinum (Pt)-based chemotherapy and poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors is increased by homologous recombination deficiency-causing mutations; in particular, reversion mutations cause drug resistance by restoring protein function. Treatment response is predicted by breast cancer susceptibility gene 1/2 (BRCA1/2) mutations; however, BRCA1/2 reversion mutations have not been comprehensively studied in pan-cancer cohorts. We aimed to characterize BRCA1/2 reversion mutations in a large pan-cancer cohort of Japanese patients by retrospectively analyzing sequencing data for BRCA1/2 pathogenic/likely pathogenic mutations in 3738 patients with 32 cancer types. We identified somatic mutations in tumors or circulating cell-free DNA that could restore the ORF of adverse alleles, including reversion mutations. We identified 12 (0.32%) patients with somatic BRCA1 (n = 3) and BRCA2 (n = 9) reversion mutations in breast (n = 4), ovarian/fallopian tube/peritoneal (n = 4), pancreatic (n = 2), prostate (n = 1), and gallbladder (n = 1) cancers. We identified 21 reversion events-BRCA1 (n = 3), BRCA2 (n = 18)-including eight pure deletions, one single-nucleotide variant, six multinucleotide variants, and six deletion-insertions. Seven (33.3%) reversion deletions showed a microhomology length greater than 1 bp, suggesting microhomology-mediated end-join repair. Disease course data were obtained for all patients with reversion events: four patients acquired mutations after PARP-inhibitor treatment failure, two showed somatic reversion mutations after disease progression, following Pt-based treatment, five showed mutations after both treatments, one patient with pancreatic cancer and BRCA1 reversion mutations had no history of either treatment. Although reversion mutations commonly occur in BRCA-associated cancers, our findings suggest that reversion mutations due to Pt-chemotherapy might be correlated with BRCA1/2-mediated tumorigenesis even in non-BRCA-associated histologies.

Genomic stability of self-inactivating rabies.

Transsynaptic viral vectors provide means to gain genetic access to neurons based on synaptic connectivity and are essential tools for the dissection of neural circuit function. Among them, the retrograde monosynaptic ΔG-Rabies has been widely used in neuroscience research. A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, allows the long term genetic manipulation of neural circuits. However, the high mutational rate of the rabies virus poses a risk that mutations targeting the key genetic regulatory element in the SiR genome could emerge and revert it to a canonical ΔG-Rabies. Such revertant mutations have recently been identified in a SiR batch. To address the origin, incidence and relevance of these mutations, we investigated the genomic stability of SiR in vitro and in vivo. We found that "revertant" mutations are rare and accumulate only when SiR is extensively amplified in vitro, particularly in suboptimal production cell lines that have insufficient levels of TEV protease activity. Moreover, we confirmed that SiR-CRE, unlike canonical ΔG-Rab-CRE or revertant-SiR-CRE, is non-toxic and that revertant mutations do not emerge in vivo during long-term experiments.

P55-2 BRCA2 reversion mutations as a resistance mechanism of olaparib in patients with BRCA2-associated mCRPC

P55-2 BRCA2 reversion mutations as a resistance mechanism of olaparib in patients with BRCA2-associated mCRPC

Flavivirus genome recoding by codon optimisation confers genetically stable in vivo attenuation in both mice and mosquitoes.

Virus genome recoding is an attenuation method that confers genetically stable attenuation by rewriting a virus genome with numerous silent mutations. Prior flavivirus genome recoding attempts utilised codon deoptimisation approaches. However, these codon deoptimisation approaches act in a species dependent manner and were unable to confer flavivirus attenuation in mosquito cells or in mosquito animal models. To overcome these limitations, we performed flavivirus genome recoding using the contrary approach of codon optimisation. The genomes of flaviviruses such as dengue virus type 2 (DENV2) and Zika virus (ZIKV) contain functional RNA elements that regulate viral replication. We hypothesised that flavivirus genome recoding by codon optimisation would introduce silent mutations that disrupt these RNA elements, leading to decreased replication efficiency and attenuation. We chose DENV2 and ZIKV as representative flaviviruses and recoded them by codon optimising their genomes for human expression. Our study confirms that this recoding approach of codon optimisation does translate into reduced replication efficiency in mammalian, human, and mosquito cells as well as in vivo attenuation in both mice and mosquitoes. In silico modelling and RNA SHAPE analysis confirmed that DENV2 recoding resulted in the extensive disruption of genomic structural elements. Serial passaging of recoded DENV2 resulted in the emergence of rescue or adaptation mutations, but no reversion mutations. These rescue mutations were unable to rescue the delayed replication kinetics and in vivo attenuation of recoded DENV2, demonstrating that recoding confers genetically stable attenuation. Therefore, our recoding approach is a reliable attenuation method with potential applications for developing flavivirus vaccines.

Deciphering the Pathways to PARP Sensitivity in Pancreatic Cancer.

A recent article analyzed paired cell-free DNA among patients with platinum-sensitive BRCA- or PALB2-mutated pancreatic cancer who received maintenance olaparib. Reversion mutations were linked with worse outcomes. These types of paired correlative studies are needed to improve our understanding of drug resistance and guide future clinical trials. See related article by Brown et al., p. 5207.

Metastatic lung adenocarcinoma with BRCA2 mutation and longstanding disease control on olaparib, developing triple negative breast adenocarcinoma with additional BRCA2 reversion mutation: a case report

BackgroundThe BRCA2 gene is a well-known tumor suppressor gene implicated in breast and ovarian cancers. BRCA1/2 mutations can be sensitive to poly ADP-ribose polymerase (PARP) inhibitors such as olaparib. However, some of these patients develop resistance to this treatment and an essential factor contributing to acquired insensitivity is the occurrence of reversion mutations in the BRCA1/2 genes.Case presentationWe report the case of a 65-year-old Brazilian female patient who had previously been diagnosed with metastatic lung carcinoma carrying a BRCA2 mutation that had extended to the central nervous system. Following disease progression, olaparib was administered, resulting in a stabilizing effect on her condition for ~ 30 months. During a routine follow-up, a new triple-negative breast tumor was found. Genetic testing revealed the presence of two distinct BRCA2 gene mutations in the breast tumor. The original mutation (p.Val220Ilefs4) led to a frameshift, culminating in the production of a truncated and non-functional BRCA2 protein; the second mutation, K437fs22, rectified the reading frame of exon 11. Consequently, Rad51 could properly bind to BRCA2—an essential protein crucial for DNA repair. This restoration resulted in a functional BRCA2 protein, effectively elucidating the clinical resistance observed in the new breast tumor in this case.ConclusionsThis case report highlights the clinical significance of comprehensive next-generation sequencing analyses for lung adenocarcinomas, both at diagnosis and upon progression. Such analyses enable informed decisions regarding targeted therapies and facilitate a deeper comprehension of resistance mechanisms.

Rapid emergence and transmission of virulence-associated mutations in the oral poliovirus vaccine following vaccination campaigns

There is an increasing burden of circulating vaccine-derived polioviruses (cVDPVs) due to the continued use of oral poliovirus vaccine (OPV). However, the informativeness of routine OPV VP1 sequencing for the early identification of viruses carrying virulence-associated reversion mutations has not been directly evaluated in a controlled setting. We prospectively collected 15,331 stool samples to track OPV shedding from children receiving OPV and their contacts for ten weeks following an immunization campaign in Veracruz State, Mexico and sequenced VP1 genes from 358 samples. We found that OPV was genetically unstable and evolves at an approximately clocklike rate that varies across serotypes and by vaccination status. Overall, 61% (11/18) of OPV-1, 71% (34/48) OPV-2, and 96% (54/56) OPV-3 samples with available data had evidence of a reversion at the key 5’ UTR attenuating position and 28% (13/47) of OPV-1, 12% (14/117) OPV-2, and 91% (157/173) OPV-3 of Sabin-like viruses had ≥1 known reversion mutations in the VP1 gene. Our results are consistent with previous work documenting rapid reversion to virulence of OPV and underscores the need for intensive surveillance following OPV use.