Systematic In-Silico Evaluation of the Diagnostic Impact of Mpox Genome Variants in the Current Outbreak

Abstract

Background and Objective

The rapid rate at which the current mpox virus outbreak has spread across the globe has led the World Health Organization to declare it a Public Health Emergency of International Concern. Polymerase chain reaction-based methods are one of the cornerstones for effective molecular detection of viruses including mpox virus. Genetic variants in primer binding sites are known to impact the efficiency of polymerase chain reaction and therefore diagnosis. Here we have analyzed the genetic variants and their impact on efficient binding of oligonucleotides used in diagnostics.

Methods

In this study, we have systematically collected primers and probes used in the detection of mpox virus from published literature and public resources, and assessed the impact of primer binding region genetic variants in the detection of mpox virus by analysing the thermodynamic parameters, Gibbs free energy and melting temperature. These were calculated using the nearest neighbour method for variants in mpox virus genomes available and the deviation in parameters was computed with respect to the reference genome sequence.

Results

We have identified 170 genetic variations that fall within the oligo binding region in 1176 mpox virus genomes out of which five oligos showed at least a 2 °C decrease in melting temperature, which could potentially affect the diagnostic efficacy.

Conclusions

Our analysis shows the importance of continuous monitoring of mpox virus detection primer efficacy and provides the list of oligos with potentially reduced detection efficiency in the current mpox virus outbreak.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40291-022-00629-8.

Key Points

Genomic variant analysis of mpox virus suggests a large number of variants located within the diagnostic primer binding region.

Thermodynamic analysis of a mismatch sequence provides a list of potentially affected primers/probes by genomic variation.

Introduction

According to the Centers for Disease Control and Prevention [1], as of 2 September, 2022, the current mpox virus (MPXV) outbreak has affected 53,027 individuals across the world in 100 different countries. Of the total cases, 52,516 cases have been reported in locations that have historically never reported MPXV infections before, thereby highlighting the fact that rapid and accurate detection of pathogens is necessary for effective surveillance and reducing the impact of the disease outbreak. Since the detection of the first case of MPXV infection in a human in 1970 [2], several primers and probes have been studied and published. The rapid spread of the current outbreak has also led to the creation and availability of several commercial kits as well as Centers for Disease Control and Prevention-recommended primer sequences [3].

Mpox virus is a double-stranded DNA virus with a previously established low mutation frequency. However, a recent study has identified 46 common mutations among the strains observed in the current outbreak [4]. Other studies focusing on the virus strains that have been reported since 2017 have found the virus to have a mutation rate of around ten times higher than its standard mutation rate [5]. As the virus can evolve at rapid rates through the accumulation of such genetic variants, these genetic variants can also directly affect the efficacy of the primers and probes used in polymerase chain reaction assays. In this study, we have systematically analysed the genetic variants of the MPXV genomes and our analysis suggests that some genetic variations that correspond to the target sites for the probes/primers may have an impact on the efficiency of detection/diagnosis.

Materials and Methods

Genome Sequences

The MPXV genomes that were deposited in the Global Initiative on Sharing Avian Influenza Data database [6] from 27-05-2022 to 23-08-2022 were downloaded. In total, 1176 genomes were used for the analysis of a primer mismatch out of which 15 were of lineage A and the remaining 1158 sequences were of lineage B.1.

Primers and Probes Used in Molecular Assays

A list of primers and probes used across molecular assays to detect the MPXV was curated using PubMed and Google Scholar. All primers reported in PubMed were collected using the search term “monkey pox primer sequence”, while Google Scholar was queried to include all preprints reported in 2022 using the search terms “biorxiv”, “monkeypox” and “primer” as well as “medrxiv”, “monkeypox” and “primer” [7–36]. Additionally, primers recommended by the Centers for Disease Control and Prevention, World Health Organization and the US Food and Drug Administration were also collected, along with those reported in commercially available kits for MPXV detection. In addition, multiplexed amplicon-based whole genome sequencing primers mentioned in protocols.io [37] were also included for the analysis.

All primers/probes were processed and were given unique IDs. A compiled collection of primers/probes was mapped against the reference genome [38] (NC_063383.1) using BLASTN [39], and the start and end positions of the primer binding sites in the genome were extracted. Further, 163 multiplexed primer pairs described in protocols.io for amplicon sequencing of MPXV were also independently analysed for their efficiency in covering the complete genome.

Variant Calling and Thermodynamic Parameter Calculation

Genetic variants in the MPXV genomes were called using Nextclade [40] against the reference genome. Substitutions, deletions and insertions that fell under the primer/probe binding regions were identified, and the frequency of each genetic variant at the primer binding region was calculated for 1176 genomes downloaded from the Global Initiative on Sharing Avian Influenza Data database.

For the calculation of thermodynamic parameters such as Gibbs free energy (ΔG) and melting temperature (Tm), first the 1176 MPXV genomes were aligned against the reference genome using nextalign [40]. Upon multiple sequence alignment, 1083 MPXV genomes were found aligned whereas the remaining 93 genomes failed to align because of multiple insertion/deletion events. Primer binding sequences were then extracted from the 1083 aligned genomes and were used for further analysis. The ΔG of primers and MPXV sequences/reference were calculated by the nearest neighbour method using the following equation:

$\mathrm{\Delta }\mathrm{G}037(\mathrm{DNA}\mathrm{duplex})=\mathrm{\Delta }\mathrm{G}037(\mathrm{initiation})+\mathrm{\Delta }\mathrm{G}037(\mathrm{symmetry})+\sum \mathrm{\Delta }\mathrm{G}037(\mathrm{stack})+\mathrm{\Delta }\mathrm{G}0\mathrm{AT}(\mathrm{terminal}),$where ΔG0 37 (initiation) =1.96 kcal/mol, ΔG0 AT (terminal) = 0.05 kcal/mol, ΔG0 37 (symmetry) = 0.43 kcal/mol penalty is applied on self-complementary sequences and ∑ΔG0 37 (stack) is the summation of the ΔG0 37 of the adjacent bases.

Next, the percentage change in ΔG for each primer/probe was calculated from the ΔG of primer/probe binding to the reference genome. The frequency of MPXV genome with at least a 2% increase in ΔG from the reference genome was calculated to assess the usability of the oligos in the current outbreak.

For the calculation of the Tm difference, Tm_NN function from the R package TmCalculator [41] was used. The difference was estimated by subtracting the Tm of the extracted primers/probes sequences from that of the reference sequence for each oligo.

Results and Discussion

We compiled a total of 207 oligos through our literature survey, and upon the removal of duplicate primer sets we were left with 180 oligos. Further, upon mapping to the reference genome, 12 primers mapped to two regions as they were falling within the variable region in left/right of the MPXV genome, and hence both mapping positions were included in the analysis. Seventy-six out of 180 oligos did not map to the reference genome and upon blasting against all nucleotide databases of the National Center for Biotechnology Information, these oligos aligned mostly to MPXV complete genomes of West African and Congo basin sequences. The 114 oligos that had a perfect match on the reference genome were taken for further analysis. To obtain an overview of how many genetic variants fell within the oligo binding region, we combined the number of substitutions, deletions and insertions in the MPXV genome: in total, 170 genetic variants were present in 1176 MPXV genome sequences that fell within the 114 oligos that were analysed, with the variant frequency range from 0.0008 to 0.9719 (median 0.0008). Two out of the 170 genetic variants were present in at least 1% of the compared genomes, namely G2591A was present in 1143/1176 (97.19%) of genomes followed by G159277A, which was present in 12/1176 (1.02%) of genomes (Fig. 1). Similarly, genetic variants falling within 326 primers of multiplexed whole genome amplification primers from protocol.io were analysed in which 362 genetic variants were present within the primer binding regions, with the frequency range from 0.0008 to 0.9821 (median 0.0008) [Fig. 1 of the Electronic Supplementary Material [ESM]). Four of 362 genetic variants were observed in at least 1% of the genome namely C121320T (98.21%), G2591A (97.19%), G161296A (1.53%) and C38937T (1.10%).

Fig. 1.

Variant frequency in mpox virus genome sequences from the Global Initiative on Sharing Avian Influenza Data database. Red dots indicate variants within the detection primer/probe binding region

As a significant number of genetic variants were found within the oligo binding regions that are used in the detection or whole genome amplification of MPXV, we further analysed whether these genetic variants brought about changes in ΔG and Tm due to mismatches in genetic variants of MPXV genome and oligos as compared with the reference genome, as these thermodynamic parameters are good indicators of binding stability. An increase in ΔG and/or reduction in Tm by at least 2 °C as compared with the reference can lead to reduced binding stability and ultimately affect the PCR efficiency. Seventeen oligos that were used in Loop-mediated isothermal amplification assays were removed from this analysis and the ΔG for MPXV genome was calculated for the remaining 97 oligos. Interestingly, an increase of 2% ΔG was observed in 20/97 oligos. Of the 20, five oligos were observed in at least 1% of the 1083 MPXV genomes analysed. The same five oligos also had at least a 2 °C decrease in Tm as compared with the reference genome, out of which four had a more than a 5 °C deviation (Fig. 2). The frequency of genomes with reduced stability for each oligo is provided in Table 1 of the ESM. Similarly, 5/326 primers that were used for multiplex whole genome amplification displayed a 2% increase in ΔG in at least 1% of the genomes while four primers had at least a 5 °C decrease in Tm as compared with the reference genome (Fig. 2 of the ESM). These analyses indicate the requirement for constant monitoring of detection-evading MPXV variants for efficient surveillance of the disease. The frequency of genomes with reduced stability for each oligo used in whole genome amplification is provided in Table 2 of the ESM.

Fig. 2.

Frequency heat map of oligos with destabilised thermodynamic parameters with both a minimum 2% Δ Gibbs free energy increase and melting temperature differences in the mpox virus (MPXV) genome where MPXV lineage A, lineage B.1 and all sequences were described as MPX_A, MPX_B.1 and MPX_All, respectively

Conclusions

The recent severe acute respiratory syndrome coronavirus 2 pandemic has shown us the importance of efficient detection and genomic surveillance of outbreak potential pathogens in mitigating the effect of damage. In this short report, we examined all available oligos that were used for the detection or whole genome amplification of MPXV and analysed their detection efficiency using whole genome sequences available on the Global Initiative on Sharing Avian Influenza Data platform. Our analysis shows the importance of continuous monitoring of MXPV detection oligos that could be impacted by genetic variants. We also provide a list of frequencies of genomes with reduced stability for all analysed oligos for healthcare workers to make an informed decision in the choice of using oligos in PCR-based detection of MPXV in the current outbreak. While an exhaustive search of published oligos was performed for the analysis, our study is limited by the fact that available sequence data are not uniform across all affected countries/regions. This might lead to an unintentional bias towards data from countries/regions that perform extensive sequencing, and an under-representation of data from countries/regions that have not participated in extensive genomic surveillance of MPXV.

Supplementary Information

Below is the link to the electronic supplementary material.

Declarations

Funding

No funding has been acquired for this study.

Conflicts of interest/competing interests

Aastha Vatsyayan, Arvinden VR and Vinod Scaria have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

Data sharing is not applicable to this article as no datasets were generated or analysed during the current study. All data analysed during this study are included in this published article (and its supplementary information files).

Code availability

Not applicable.

Authors’ contributions

VS conceptualised, designed and supervised the study. AV collected the compendium of primers and probes. AVR performed the analysis and data visualisation. Both AV and AVR contributed towards compiling the manuscript.