Viral whole genome study in the United States identifies ORF-disrupting mutations in monkeypox virus

By Dr. Chinta SidharthanNov 7 2022Reviewed by Aimee Molineux

In a recent study published in Viruses, researchers used whole genome sequencing to evaluate monkeypox virus samples from Washington and Ohio in the United States and identified 25 samples with open reading frame (ORF)-disrupting mutations.

Background

In early 2022, monkeypox outbreaks were reported from areas outside the endemic regions of West and Central Africa. The etiological agent of monkeypox is the monkeypox virus (MPXV), a double-stranded deoxyribonucleic acid (DNA) virus of the Orthopoxvirus genus belonging to the Poxviridae family. The monkeypox virus has a 197 kilobase pair (kb) genome, which codes for around 190 genes.

The genes related to viral replication are highly conserved, but the terminal regions largely encode proteins that modulate the host's immune system and are under selection pressure. Understanding the monkeypox virus mutations and changes in previously used target regions will improve diagnosis and therapeutic options and help limit the current monkeypox outbreak.

About the study

The present study extracted the DNA from lesion swab samples and performed whole genome sequencing to identify mutations. A reference strain (MA001) sequence was used to map genomic positions. Deletions were confirmed using polymerase chain reaction (PCR) with primers designed to flank the regions containing the deletions.

Additionally, droplet digital PCR was performed to evaluate copy number since the deletions included the inverted terminal repeats. The primer sets amplified the MPXVgp004/MPXVgp188 gene, which is present in both the inverted terminal repeat regions, and a part of the F3L gene, which is not found in the inverted terminal repeat regions.

Results

The results reported 25 clinical specimens with 11 different ORF-disrupting mutations. Twenty samples contained nonsense mutations in six different genes, and other mutations included a frameshift mutation, an insertion, and three large deletions.

Ten samples, which included samples from Washington and Ohio, had identical nonsense mutations in the MPXVgp004/MPXVgp188 ORFs found in the 5’ and 3’ inverted terminal repeats. Three samples also revealed nonsense mutations in an ORF encoding a host immune response attenuating protein. One nonsense mutation in the gene encoding the protein thymidylate kinase resulted in a non-functional protein. In contrast, other nonsense mutations were found in ORFs and one C-terminus region of proteins whose functions remain unknown.

In one of the Ohio samples, the ORF of the MPXVgp135 gene, which encodes a DNA-dependent ribonucleic acid (RNA) polymerase subunit, also revealed an insertion where an extra adenine was inserted in the adenine homopolymer resulting in a frameshift mutation.

In another sample from Ohio, a deletion of cytosine from the 11764^th position regarding MA001 caused a frameshift mutation in the MPXVgp010 gene, which resulted in a novel C terminus region comprising 48 amino acids. The same sample also contained a non-synonymous mutation that replaced the 324^th position methionine with isoleucine. Furthermore, a 913 base pair (bp) deletion in the MPXVgp010 gene in one of the Washington samples also resulted in a novel C terminus region.

Two other large deletions of 4205 bp and 6881 bp were found in Ohio and Washington samples, respectively. The 4205 bp deletion removed the MPXVgp011 ORF start codon and the MPXVgp012, MPXVgp013, and MPXVgp014 regions, which encode type I interferon antagonistic proteins. The deletion also created a novel C-terminus residue in the MPXVgp015 gene. The 6881 bp deletion eliminated the MPXVgp184 to MPXVgp188 regions and a 3’ region of the MPXVgp182/B21R gene resulting in a truncated ORF.

Additionally, the authors explored the functions of some of the deleted genes using published literature and the Pfam (protein families) database and found that MPXVgp184 was an apoptosis inhibitor. The MPXVgp187 gene was involved in inhibiting the host immune response by secreting a multiple histocompatibility complex-I like protein which binds to the natural-killer group 2, member D (NKG2D) and prevents natural killer cells from binding to infected cell ligands.

Furthermore, the deleted region of the MPXVgp182/B21R contained epitopes that were targeted for serodiagnostics, suggesting that the deletion was a result of section pressure for immune evasion.

The phylogenetic analysis reported that all the MPXV variants with these documented mutations belonged to the B.1 lineage that emerged during the 2022 outbreak, and did not represent multiple introductions of divergent strains. The results also indicated that all the strains with the nonsense mutation in the MPXVgp004/MPXVgp188 gene belonged to the B.1.8 lineage and the mutations seem to be fixed and transmissible.

Conclusions

Overall, the results indicated that most mutations in the MPXV genome occurred in the inverted terminal repeat regions and regions encoding proteins that modulate host tropism and host immune responses. Furthermore, the ORF-disrupting mutations seem to be limited to non-essential genes.

The mutations in the inverted terminal repeats and regions used as serodiagnosis target epitopes also highlight the need for finding more conserved targets for diagnosis and therapy.