In a recent study published on the bioRxiv* server, researchers analyzed genome sequences of Monkeypox virus (MPXV) obtained from 18 patients who contracted MPXV infection between June and July 2022.
Study: Monkeypox virus sequencing from infected patients reveals viral genomes with APOBEC3-like editing, gene inactivation, and bacterial agents of skin superinfection. Image credit: Kateryna Kon/Shutterstock
background
MPXV has a double-stranded deoxyribonucleic acid (DNA) genome and belongs to the Poxviridae virus family and the Orthopoxvirus genus. The World Health Organization (WHO) has recognized three viral clades named one through three that encompass the viruses originating in the 2017-2018 outbreak (Nigeria) and those involved in the current 2022 multinational outbreak.
Until 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 revealed that these viruses belong to clade III and probably have a single origin. In addition, the epidemiological and clinical characteristics of the current epidemic indicate that it is sexually transmitted.
Additionally, these viruses comprise a lineage called 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 metagenomic analyzes of DNA from clinical samples from MPXV-infected individuals. They obtained MPXV genomes from genital skin lesions and rectal exfoliation from 18 MPXV-infected patients.
The researchers deliberated NGS for clinical samples with a quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) cycle threshold (CT) value ≤25. They then proceeded to metagenomic analyzes of all NGS runs. In addition, they mapped the raw NGS reads against the MPXV genome GenBank Accession no. ON563414.3, a 197,205 bp MPXV genome recovered from a patient sample in Massachusetts, United States, May 2022. The Nextclade v1.6.0 tool uses these genome samples as the B reference genome. 1.
For the phylogenetic analyses, the team retrieved MPXV genomes deposited in the global initiative to share all influenza data (GISAID) until August 22, 2022. They identified common mutations with those obtained in the present study. GenBank genomes 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.
Results of the study
The researchers tested 307 patients for the presence of MPXV by real-time qPCR between May and July 2022 using skin lesions and faecal and nasopharyngeal samples. More specifically, they obtained 11 samples of skin lesions, including five located on the penis, six from the rectum, and one from the nasopharynx.
The average number of raw NSG reads mapped to the genome GenBank Accession no. ON563414.3 was 44,615 per sample, with an average sequencing depth and average genome coverage of 48 ± 41 reads and 98.0 ± 2.8%, respectively. The team observed that the 18 MPXV genomes obtained in the present study belonged to the B.1 lineage of clade III according to the 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 had at least one mutation, and the average nucleotide identity between these genomes was 99.8 ± 0.2% , for pairwise comparison calculations. These 18 genomes had an average of 3.3 ± 2.2 mutations relative to ON563414.3.
Of the total of 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 within 20 genes scattered throughout the MPXV genome. These 20 genes encode:
i) two ribonucleic acid (RNA) polymerase subunits;
ii) two early subunits of the transcription factor and one intermediate and one late;
iii) a phospholipase D-like protein; i
iv) four proteins containing ankyrin repeats.
In other words, these non-synonymous mutations caused 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 higher mutation rate than expected based on a previous assessment of orthopoxviruses, suggesting that MPXV may 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 mRNA editing of apolipoprotein B. Family of enzymatic catalytic polypeptides (APOBEC3).
Interestingly, the researchers also detected NGS reads identifying S. aureus and S. pyogenes sequences in the majority of MPX virus-positive clinical samples from skin lesions. These two bacteria cause superinfections in cases of MPXV; however, more data are needed to establish the prevalence of these two bacteria in association with MPX virus lesions.
The WHO only recommends strict monitoring of skin lesions due to superinfection associated with cellulitis or abscess. They also recommend the treatment of superinfection 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. However, in the 2022 MPXV outbreak, bacterial superinfections in skin lesions have caused substantial morbidity. Therefore, it warrants close monitoring for prompt administration of antibiotics in such cases.
Conclusions
The study highlights the need for close monitoring of various aspects of the genetic evolution and mutational patterns of MPXV clades that have emerged in non-endemic countries with the 2022 outbreak and spread worldwide. For example, 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.
In particular, the expression and deaminase activity of APOBEC3 enzymes is altered during various infections and cancers. Most importantly, there is a need to detect bacterial agents of skin superinfections along with sequencing and characterization of MPXV genomes.
*Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health-related behavior, or be treated as established information.