DNA metagenomic analyses of clinical samples from Monkeypox virus-infected individuals

In a recent study posted to the bioRxiv* server, researchers analyzed genomic sequences of Monkeypox virus (MPXV) obtained from 18 patients who contracted MPXV infection between June and July 2022.

Background

MPXV has a double-stranded deoxyribonucleic acid (DNA) genome and belongs to the Poxviridae virus family and genus orthopoxvirus. The World Health Organization (WHO) has recognized three viral clades named one to three encompassing viruses originating in the 2017-2018 outbreak (Nigeria) and those involved in the current 2022 multicountry outbreak.

Until 2022, all human MPXV infections imported from sub-Saharan Africa have occurred in non-endemic countries sporadically. Previous phylogenetic studies based on MPXV genomes released in Portugal and France in 2022 revealed that these viruses belong to clade III and likely have a single origin. Also, the epidemiological and clinical features of the current epidemic indicate it is sexually transmitted.

Further, these viruses comprise a lineage named B.1, which emerged in Europe between November 2021 and May 2022. The B.1 lineage had diverged from its ancestral A.1 lineage by approximately 50 nucleotide substitutions, nearly 10 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 analyses of clinical samples from MPXV-infected individuals. They obtained MPXV genomes from genital skin lesions and rectal swabbing of 18 MPXV-infected patients.

The researchers deliberated NGS for clinical samples with a cycle threshold value (C_T) of the quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) ≤25. Next, they proceeded to metagenomic analyses of all NGS runs. Further, they mapped raw NGS reads against the MPXV genome GenBank Accession no. ON563414.3, a 197,205 base pair-long genome of MPXV retrieved from a patient sample in Massachusetts, United States, in May 2022. The Nextclade tool v1.6.0 uses these genome samples as a B.1 reference genome.

For phylogenetic analyses, the team retreived MPXV genomes deposited in the global initiative on sharing all Influenza Data (GISAID) database until 22 August 2022. They identified mutations common with those obtained in the present study. The genomes GenBank accession numbers 412 NC_063383 and ON563414 were added to root the phylogenetic tree. The National Agency for the Safety of Medicines and Health Products (ANSM) authorized NGS of MPXV genomes.

Study findings

The researchers tested 307 patients for the presence of MPXV by real-time qPCR between May and July 2022 using skin lesions and fecal and nasopharyngeal samples. More specifically, they obtained 11 samples from skin lesions, including five localized on the penis, six from the rectum, and one from a nasopharynx.

The mean number of raw NSG reads mapped on the genome GenBank Accession no. ON563414.3 was 44,615 per sample, with mean sequencing depth and mean genome coverage of 48±41 reads and 98.0±2.8%, respectively. The team observed that all 18 MPXV genomes obtained in the present study belonged to the B.1 lineage of clade III per WHO classification, with two belonging to the B.1.1 sublineage. Relative to the reference MPXV genome ON563414.3, six of the 18 genomes were genetically identical, while 12 harbored a minimum of one mutation, and the mean nucleotide identity between these genomes was 99.8±0.2%, per pairwise comparison computations. These 18 genomes had a mean of 3.3±2.2 mutations relative to ON563414.3.

Of the total 36 different mutations present in at least one of the 18 genomes, 22 were non-synonymous while 14 were synonymous. The 22 non-synonymous mutations were nested inside 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 non-synonymous mutations led to amino acid changes in viral proteins crucial for virion replication and morphogenesis, and virus-host interactions. Future studies should further investigate the impact of these mutations on the function of these proteins and the MPXV replication cycle.

Another key observation was a greater mutation rate than expected based on a previous assessment for orthopoxviruses, which suggested that MPXV might be accelerating 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 specimens collected in Nigeria in August 2018 could be due to the action of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3) family.

Intriguingly, the researchers also detected NGS reads identifying sequences from S. aureus and S. pyogenes in most MPX virus-positive clinical samples from skin lesions. These two bacteria cause superinfections in MPXV cases; however, more data is needed to establish the prevalence of these two bacteria in association with MPX virus lesions.

The WHO only recommends monitoring skin lesions for superinfection associated with cellulitis or abscess strictly. Also, they recommend superinfection treatment with antibiotics active against methicillin-sensitive S. aureus and S. pyogenes. Similarly, the US Centers for Disease Control and Prevention (CDC) recommends antibiotic treatment for people who have secondary bacterial skin infections. Nevertheless, in the 2022 MPXV outbreak, bacterial superinfections in skin lesions have caused substantial morbidity. Thus, it warrants close monitoring for prompt administration of antibiotics in such cases.

Conclusions

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

Notably, the expression and deaminase activity of the APOBEC3 enzymes alters during various infections and cancers. Most importantly, there is a need to detect bacterial agents of skin superinfections alongside sequencing and characterization of MPXV genomes.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.