The largest monkeypox virus (MPXV) outbreak described so far in non-endemic countries was identified in May 2022 (refs. 1–6). In this study, shotgun metagenomics allowed the rapid reconstruction and phylogenomic characterization of the first MPXV outbreak genome sequences, showing that this MPXV belongs to clade 3 and that the outbreak most likely has a single origin. Although 2022 MPXV (lineage B.1) clustered with 2018–2019 cases linked to an endemic country, it segregates in a divergent phylogenetic branch, likely reflecting continuous accelerated evolution. An in-depth mutational analysis suggests the action of host APOBEC3 in viral evolution as well as signs of potential MPXV human adaptation in ongoing microevolution. Our findings also indicate that genome sequencing may provide resolution to track the spread and transmission of this presumably slow-evolving double-stranded DNA virus.

a new nomenclature system for the monkeypox virus (MPXV) clades and lineages was proposed by Happi et al. 1 that defined the Clade 1 (former Congo Basin) and Clades 2 and 3 (both comprising the former West African clade). Our study, published online in Nature Medicine on 24 June 2022, relied on this nomenclature. Later, on 12 August 2022, the World Health Organization, together with a group of global experts, revised and agreed on the new naming convention for MPXV clades in which the previously proposed Clades 1, 2 and 3 are now designated Clades I, IIa and IIb, respectively 2 . The initial proposal by Happi et al. was subsequently updated 1 as well as the final corresponding publication 3 . In this context, the published version of our article (which relied on such proposal) should be read according to this official

Background In a context of multiple Omicron lineages circulation, it is relevant to clarify the effect of vaccination and previous infections on the risk of infection and severe post-infection outcomes. Methods Using electronic health records and SARS-CoV-2 laboratory surveillance data, we conducted a case-case and a cohort study covering the period of Omicron BA.2/BA.5 lineage replacement in Portugal, to compare vaccine effectiveness of complete primary and booster dose against infection, COVID-19 hospitalization, and mortality. Variant classification was performed through whole-genome sequencing (WGS) or Spike Gene Target Failure (SGTF). Findings Between April 25 and June 10, 2022, within a total of 27702 collected samples, 55.5% were classified as BA.2 and the remaining as BA.5. We observed no evidence of reduced vaccine effectiveness for the primary complete vaccination (OR=1.07, CI95%:0.93-1.23) or booster dose vaccination (OR=0.96, CI95%:0.84-1.09) against BA.5 infection compared with BA.2. The protection against reinfection was inferior in BA.5 cases when compared with BA.2 (OR=1.44; CI95%:1.30-1.60). Among those infected with BA.5, booster vaccination was associated with 77% and 88% of reduction in risk of COVID-19 hospitalization and death, respectively, while higher risk reduction was found for BA.2 cases, with 93% and 94%, respectively. Interpretation This study shows that the SARS-CoV-2 Omicron BA.5 lineage is associated with higher odds of reinfection compared with Omicron BA.2, regardless of the vaccination status. Although less effective compared with BA.2, COVID-19 booster vaccination still offers substantial protection against severe outcomes following BA.5 infection.

The 2022 multi-country monkeypox outbreak concurrent with the ongoing COVID-19 pandemic has further highlighted the need for genomic surveillance and pathogen whole genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early human monkeypox virus infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the current outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there is an urgent need for a more sensitive and broadly applicable sequencing approach. Amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for SARS-CoV-2. Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented during the COVID-19 pandemic. We sequenced clinical samples that tested presumptive positive for monkeypox virus with amplicon-based and metagenomic sequencing approaches. Upon comparison, we found notably higher genome coverage across the virus genome, particularly in higher PCR cycle threshold (lower DNA titer) samples, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach. By sending out primer pool aliquots to laboratories across the United States and internationally, we validated the primer scheme in 12 public health laboratories with their established Illumina or Oxford Nanopore Technologies sequencing workflows and with different sample types across a range of Ct values. Our findings suggest that amplicon-based sequencing increases the success rate across a wider range of viral DNA concentrations, with the PCR Ct value threshold at which laboratories were able to achieve 80% genome coverage at 10X ranging between Ct 25-33. Therefore, it increases the number of samples where near-complete genomes can be generated and it provides a cost-effective and widely applicable alternative to metagenomics for continued human monkeypox virus genomic surveillance. Importantly, we show that the human monkeypox virus primer scheme can be used with currently implemented amplicon-based SARS-CoV-2 sequencing workflows, with minimal change to the protocol.

We estimated comparative primary and booster vaccine effectiveness (VE) of SARS-CoV-2 Omicron BA.5 and BA.2 lineages against infection and disease progression. During April–June 2022, we implemented a case–case and cohort study and classified lineages using whole-genome sequencing or spike gene target failure. For the case–case study, we estimated the adjusted odds ratios (aORs) of vaccination using a logistic regression. For the cohort study, we estimated VE against disease progression using a penalized logistic regression. We observed no reduced VE for primary (aOR 1.07 [95% CI 0.93–1.23]) or booster (aOR 0.96 [95% CI 0.84–1.09]) vaccination against BA.5 infection. Among BA.5 case-patients, booster VE against progression to hospitalization was lower than that among BA.2 case-patients (VE 77% [95% CI 49%–90%] vs. VE 93% [95% CI 86%–97%]). Although booster vaccination is less effective against BA.5 than against BA.2, it offers substantial protection against progression from BA.5 infection to severe disease.