Intra‐ and interhost genomic diversity of monkeypox virus

Abstract The impact and frequency of infectious disease outbreaks demonstrate the need for timely genomic surveillance to inform public health responses. In the largest known outbreak of mpox, genomic surveillance efforts have primarily focused on high‐incidence nations in Europe and the Americas, with a paucity of data from South‐East Asia and the Western Pacific. Here we analyzed 102 monkeypox virus (MPXV) genomes sampled from 56 individuals in Melbourne, Australia. All genomes fell within the 2022 MPXV outbreak lineage (B.1), with likely onward local transmission detected. We observed within‐host diversity and instances of co‐infection, and highlight further examples of structural variation and apolipoprotein B editing complex‐driven micro‐evolution in the current MPXV outbreak. Updating our understanding of MPXV emergence and diversification will inform public health measures and enable monitoring of the virus’ evolutionary trajectory throughout the mpox outbreak.


| INTRODUCTION
Mpox is a viral zoonotic disease caused by monkeypox virus (MPXV) belonging to the Orthopoxvirus genus, previously endemic to West and Central Africa. 1 A multicountry outbreak of MPXV was confirmed in May 2022 and was declared a public health emergency of international concern by the World Health Organization (WHO) in July 2022, marking the first time mpox has spread widely outside of traditionally endemic areas. [2][3][4][5][6][7] As of April 2023, there have been more than 86 000 confirmed cases globally during this outbreak, primarily reported from Europe and the Americas, although encompassing more than 110 countries globally. 8,9 Genomic analysis has played a role in the ongoing mpox outbreak, including the tracking of mpox spread and diversification, and importantly, identifying the emergence of lineages, which may have clinically relevant phenotypes. [10][11][12] Clades, lineages, and sublineages have been defined from publicly available sequence data and are updated regularly as MPXV continues to diverge. 13,14 Genomic sequencing has demonstrated two major clades of MPXV, with the current outbreak forming a distinct lineage (Clade IIb) within Clade II, although co-circulating lineages within Clade II have been identified in the Northern Hemisphere Among these, the vast majority have been associated with the B.1 lineage of Clade IIb. 10 Genomic studies have also implicated the host apolipoprotein B editing complex (APOBEC3) cytosine deaminase as a driver of ongoing MPXV evolution and human adaptation. 15 During the COVID-19 pandemic, genomic surveillance directly informed public health responses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic diversity, coupled with epidemiological data, enabled identification of transmission clusters, and in some settings, directly informed contact tracing efforts. 16 To date, there are limited data on the utility of genomic data for assessing local transmission of mpox. Recent studies suggest a degree of intrapatient diversity of MPXV genomes, with apparent genomic variation both within and between anatomical sites of infection. 5,11,17 This potential intrapatient genomic diversity, coupled with the slow mutation rate of orthopoxviruses (estimated at 1-2 single-nucleotide polymorphisms [SNPs] per year), may make accurate inferences of interpatient transmission challenging, and requires rigorous validation of the variants observed in the current outbreak strains. 18 In Australia, mpox is a nationally notifiable disease, with 144 cases (confirmed and probable) diagnosed in Australia as of May 4, 2023. 8 Unlike other parts of Australia, most cases of mpox in the state of Victoria are epidemiologically linked to local transmission, rather than incursions from overseas. Here, to better understand the utility of genomic data for informing public health responses we undertook sequencing of MPXV in Victoria. In this study, we describe the genetic diversity of the B.1 outbreak of mpox in Victoria, Australia. Our findings reveal evidence of multiple introductions of mpox, followed by sustained local transmission. Additionally, our analysis suggests the presence of within-host microevolution, which may be differentiated from co-infection with multiple strains. These findings underscore the significance of assessing within-host variation in future studies concerning the evolution and transmission of mpox.

| MPXV genomes reveal multiple introductions of mpox into Australia
To better understand the utility of genomic data for informing public health responses, we performed whole genome sequencing (WGS) on all positive MPXV samples collected between May 2022 and September 2022 in Victoria, Australia. In total, 102 genomes from 56 individuals, were successfully sequenced and included in analyses (Supporting Information: Appendix Figure 1). The median number of samples sequenced per patient was two, ranging from one to seven (Supporting Information: Figure 2 Figure 2). 19 There was also a significant correlation (R = 0.67, p < 0.0001) between sample C t value and consensus genome completeness relating to that sample (Supporting Information: Figure 2).
To assess the relatedness between genomes of MPXV in Victoria and those circulating internationally, we generated a maximum likelihood (ML) phylogeny. Following exclusion according to quality control criteria, 1105 genomes were included in this phylogeny (102 Victorian and 1003 publicly available) (Supporting Information: Data set 1). Applying recently described lineage designations, 13 Figure 4).

| Characteristics of interhost genomic diversity
Overall, we observed low genetic diversity among Australian sequences; variants are yet to become fixed. 11 As such, exploration of minor SNPs  Deletion type refers to the amount of gene deleted where "full" means the whole gene is deleted and "partial" means some of the gene was deleted.
75% in a single sample ( Figure 2B). There was an abundance of GA to AA (34/97, 35.1%) and TC to TT (46/97, 47.4%) major SNPs, indicative of APOBEC3 activity ( Figure 2C). However, minor SNPs were distributed more equally between mutation profiles ( Figure 2D). There were no major SNPs in MPXVgp083, a palmitoylated EEV membrane protein homologous to F13L in vaccinia virus and the putative target of the therapeutic antiviral tecovirimat currently used to treat mpox. 20

| Characteristics of intrahost genomic diversity
Establishing the diversity within a patient or between epidemiologically linked pairs can be informative in understanding viral evolutionary dynamics. 21  However, in 2/8 individuals, it was unclear whether intra-host variation was due to evolution, a co-infection, or another mechanism ( Figure 3D-G).

| Diversity in Victorian MPXV genomes include large-scale multigene deletions
In addition to APOBEC3 activity, the large-scale deletion and rearrangement of MPXV genetic material is another putative feature of MPXV diversification and evolution, and was observed in this data set (Supporting Information: Appendix and Figure 5). 22 11 The observation that fixed variants were commonly APOBEC-driven, whereas minor variants did not share this pattern, may suggest a selective advantage for GA > AA and/or TC > TT variation by way of adaptive codon usage or GC content, as described for other viruses. 28 The limited genetic diversity of MPXV makes it challenging to use sequence data alone for inter-patient transmission analyses or fine-resolution contact tracing of outbreaks, unlike the use of genomics for SARS-CoV-2 transmission. 16 In the early stages of an outbreak, the causative pathogen may not have diverged sufficiently to confidently use sequence data to infer chains of transmission. 29  SARS-CoV-2, which could be applied to MPXV going forward. 21,31,32 Gene loss, duplication, and rearrangements have been documented in orthopoxviruses and may be associated with viral pathogenicity and host range. [33][34][35] These are most common in the genomic termini, where non-essential and virulence genes are housed. 36 It has been suggested that gene loss may serve as a mechanism for orthopoxviruses to evolve or adapt to a new host. 37 Such mutations may indicate that MPXV is adapting to human-to-human transmission. In  the suspected deletions junctions was used to additionally confirm the deletions. PCR was used to amplify the suspected deletion regions using custom oligonucleotides (Supporting Information: Table 2).
Amplicons were purified using a QIAquick PCR purification kit (Qiagen) according to the manufacturer's instructions and sequenced using standard Sanger sequencing methods using the appropriate oligonucleotides (Supporting Information: