DNA metagenomic analyzes of clinical samples from monkeypox virus-infected individuals

DNA metagenomic analyzes of clinical samples from monkeypox virus-infected individuals

In a recent study published in bioRxiv * Researchers analyzed monkeypox virus (MPXV) genome sequences obtained from 18 patients who acquired MPXV infection between June and July 2022.

Studie: Die Sequenzierung des Monkeypox-Virus infizierter Patienten zeigt virale Genome mit APOBEC3-ähnlicher Bearbeitung, Geninaktivierung und bakteriellen Erregern der Hautsuperinfektion. Bildquelle: Kateryna Kon/Shutterstock

*Important NOTE:bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, intended to guide clinical practice/health-related behavior, or treated as established information.

background

MPXV has a double-stranded deoxyribonucleic acid (DNA) genome and belongs to the family Poxviridae viruses and the genus Orthopoxviruses. The World Health Organization (WHO) has recognized three virus groups, named one to three, which include viruses that emerged in the 2017-2018 outbreak (Nigeria) and those involved in the current 2022 transnational outbreak.

As of 2022, all human MPXV infections imported from sub-Saharan Africa have occurred sporadically in non-endemic countries. Previous phylogenetic studies based on MPXV genomes published in Portugal and France in 2022 found that these viruses belong to group III and are likely to have a single origin. The epidemiological and clinical characteristics of the current epidemic also suggest that it is sexually transmitted.

In addition, these viruses include a lineage called B.1 that emerged in Europe between November 2021 and May 2022. The B.1 lineage diverged from its ancestral A.1 lineage by approximately 50 nucleotide substitutions, almost ten times more than expected.

About the study

In the present study, researchers used next-generation Illumina and Nanopore genome sequencing (NGS) technologies to perform DNA metagenomic analyzes of clinical samples from MPXV-infected individuals. They obtained MPXV genomes from genital skin lesions and rectal swabs from 18 MPXV-infected patients.

The investigators considered NGS for clinical samples with a quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) cycle threshold (CT) ≤25. Next, they performed metagenomic analyzes of all NGS runs. In addition, they mapped raw NGS reads against the MPXV genome GenBank accession number. ON563414.3, a 197,205 base pair genome of MPXV collected from a patient sample in Massachusetts, USA, in May 2022. The Nextclade tool v1.6.0 uses these genome samples as the B.1 reference genome.

For phylogenetic analyses, the team obtained MPXV genomes that were deposited in the “Global Initiative on Sharing All Influenza Data” (GISAID) database until August 22, 2022. They identified mutations common to those in the present study. The genome GenBank accession numbers 412 NC_063383 and ON563414 were added to root the phylogenetic tree. The National Agency for Medicines and Healthcare products Safety (ANSM) has approved NGS of MPXV genomes.

Study results

Researchers tested 307 patients for the presence of MPXV using real-time qPCR on skin lesions and stool and nasopharyngeal samples between May and July 2022. Specifically, they obtained 11 samples of skin lesions, including five on the penis, six on the rectum and one on the nasopharynx.

The mean number of raw NSG reads mapped to the genome GenBank accession number. ON563414.3 was 44,615 per sample, with a mean sequencing depth and mean genome coverage of 48 ± 41 reads or 98.0 ± 2.8%, respectively. The team found that all 18 MPXV genomes obtained in the present study belonged to the B.1 lineage of group III according to WHO classification, with two belonging to the B.1.1 sublineage. Compared to the reference MPXV genome ON563414.3, six of the 18 genomes were genetically identical, while 12 had at least one mutation and the mean nucleotide identity between these genomes was 99.8 ± 0.2% according to pairwise comparison calculations. These 18 genomes had an average of 3.3 ± 2.2 mutations compared to ON563414.3.

Of the total 36 different mutations that were present in at least one of the 18 genomes, 22 were nonsynonymous while 14 were synonymous. The 22 nonsynonymous mutations were nested in 20 genes scattered along the MPXV genome. These 20 genes encoded –

i) two ribonucleic acid (RNA) polymerase subunits;

ii) two early transcription factor subunits and one intermediate and one late transcription factor;

iii) a phospholipase D-like protein; And

iv) four ankyrin repeat-containing proteins.

In other words, these nonsynonymous mutations resulted in amino acid changes in viral proteins that are critical for virion replication and morphogenesis as well as virus-host interactions. Future studies should further investigate the influence of these mutations on the function of these proteins and the MPXV replication cycle.

Another important observation was a higher mutation rate than expected for orthopoxviruses based on a previous assessment, suggesting that MPXV may accelerate its evolution. According to the authors, a higher number of mutations between 66 and 73 amino acids in the MPXV genomes compared to the NC_063383.1 genome obtained from 304 human samples collected in Nigeria in August 2018 could be due to the effect of the apolipoprotein B mRNA editing family of catalytic polypeptide-like enzymes (APOBEC3).

Interestingly, the researchers also discovered NGS reads that identified S. aureus and S. pyogenes sequences in most MPX virus-positive clinical skin lesion samples. These two bacteria cause superinfections in MPXV cases; However, more data are needed to establish the prevalence of these two bacteria in association with MPX virus lesions.

The WHO only recommends that skin lesions be closely monitored for superinfections associated with cellulitis or abscesses. They also recommend superinfection treatment with antibiotics effective against methicillin-susceptible S. aureus and S. pyogenes. Likewise, the U.S. Centers for Disease Control and Prevention (CDC) recommends antibiotic treatment for people with secondary bacterial skin infections. Nevertheless, bacterial superinfections in skin lesions caused significant morbidity in the 2022 MPXV outbreak. Therefore, in such cases, close monitoring is required for timely administration of antibiotics.

Conclusions

The study highlights the need for close monitoring of multiple aspects of the genetic evolution and mutation patterns of MPXV clades that emerged in non-endemic countries with the 2022 outbreak and have spread globally. For example, studies should examine the role of gene losses in MPXV transmissibility and replication and that of APOBEC3 enzymes in the increased mutation rate observed in MPXV genomes.

In particular, the expression and deaminase activity of APOBEC3 enzymes changes in various infections and cancers. Most importantly, in addition to sequencing and characterizing MPXV genomes, bacterial pathogens of skin superinfections also need to be detected.

*Important NOTE:bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, intended to guide clinical practice/health-related behavior, or treated as established information.

Reference:

• Vorläufiger wissenschaftlicher Bericht.
  Colson, P. et al. (2022) „Die Sequenzierung des Monkeypox-Virus von infizierten Patienten zeigt virale Genome mit APOBEC3-ähnlicher Bearbeitung, Geninaktivierung und bakteriellen Erregern der Hautsuperinfektion.“ bioRxiv. doi: 10.1101/2022.09.26.509493. https://www.biorxiv.org/content/10.1101/2022.09.26.509493v1